Patent Application
20100009920
The present invention relates to lung cancer, more particularly the diagnosis and treatment thereof.
Lung cancer is one of the most common causes of cancer death worldwide, and non-small cell lung cancer (NSCLC) accounts for nearly 80% of those cases (Greenlee, R. T., et al., (2001) CA Cancer J Clin, 51: 15-36.). Small cell lung cancer (SCLC) comprises 15-20% of all lung cancers (Chute J P et al., (1999) J Clin Oncol.; 17:1794-801, Simon G R et al., (2003) Chest.; 123(1 Suppl):259S-271S). Although many genetic alterations associated with the development and progression of lung cancer have been reported, but precise molecular mechanisms remain unclear (Sozzi, G. Eur J Cancer, (2001) 37 Suppl 7.: S63-73.). Over the last decade, newly developed cytotoxic agents, including paclitaxel, docetaxel, gemcitabine, and vinorelbine, have emerged to offer multiple therapeutic choices for patients with advanced NSCLC; however, each of the new regimens can provide only modest survival benefits as compared to cisplatin-based therapies (Schiller, J. H. et al. (2002) N Engl J Med, 346: 92-8.; Kelly, K., et al. (2001) J Clin Oncol, 19: 3210-8.). Hence, the development of new therapeutic strategies, such as molecular-targeted agents and antibodies, and cancer vaccines, are eagerly anticipated.
Systematic analysis of expression levels of thousands of genes using cDNA microarray technology provides an effective approach for identifying unknown molecules involved in pathways of carcinogenesis, and can reveal candidate targets for the development of novel therapeutics and diagnostics. Attempts to isolate novel molecular targets for diagnosis, treatment and prevention of NSCLC by analyzing genome-wide expression profiles of NSCLC cells on a cDNA microarray containing 27,648 genes, using pure populations of tumor cells prepared from 101 lung cancer tissues by laser-capture microdissection, are ongoing (Kikuchi T, et al. Oncogene. 2003 Apr. 10; 22(14):2192-205.; Kikuchi T, et al. Int J. Oncol. 2006 April; 28(4):799-805.; Kakiuchi S, et al., Mol Cancer Res. 2003 May; 1(7):485-99.; Hum Mol. Genet. 2004 Dec. 15; 13(24):3029-43. Epub 2004 Oct. 20.; Taniwaki M, et al, Int J. Oncol. 2006 September; 29(3):567-75.). In the course of this genome wide cDNA microarray analysis, 642 up-regulated genes and 806 down-regulated genes have been identified as diagnostic markers and therapeutic targets for NSCLC (See WO 2004/31413, the contents of which are incorporated by reference herein).
Epidermal growth factor receptor (EGFR) plays a critical role in the growth and survival of human cancers in various tissues by stimulation of ligands such as EGF that, in turn, leads to autophosphorylation of EGFR and thus activates the EGFR signaling pathway. Through cDNA and tissue microarray analyses, TTK has been identified as over-expressed in the great majority of lung cancers and has further been shown to be associated with poor prognosis. Furthermore, suppression of endogenous TTK expression by treatment with siRNA has been shown to cause significant growth inhibition of non-small cell lung cancer cells. Screening of potential substrates for TTK kinase using a panel of antibodies against phospho-proteins related to cancer-cell signaling resulted in the identification of an EGFR as an intracellular target of TTK. It was further discovered that phosphorylation at Tyr-992 and Ser-967 of EGFR by TTK occurred independently of EGF stimulation, and led to the activation of PLCgamma and phosphorylation of MAPK. In addition, point mutations were identified in the tyrosine kinase domain of the TTK gene in two patients with metastatic brain tumors derived from primary lung adenocarcinoma and in a lung-cancer cell line RERF-LC-AI. In vitro, the TTK mutant increased the invasive ability of mammalian cells. Together, these data imply that TTK functions as oncogene and its activation is likely to play an important role in the intracellular stimulation of EGFR-MAPK signaling in cancer cells, and that a novel intracellular signaling pathway between TTK kinase and EGFR, independent from the presence of EGF, plays a significant role in pulmonary carcinogenesis. Thus, the present invention suggests that targeting the TTK enzymatic activity will be a promising therapeutic strategy for treatment of lung-cancer patients.
In the course of screening for novel molecular targets for diagnosis, treatment and prevention of human cancers, genome-wide expression profile analyses of 101 lung cancers was performed on cDNA microarray containing 27,648 genes, coupled with laser microdissection (Kikuchi T, et al. Oncogene. 2003 Apr. 10; 22(14):2192-205.; Kikuchi T, et al. Int J. Oncol. 2006 April; 28(4):799-805.; Kakiuchi S, et al., Mol Cancer Res. 2003 May; 1(7):485-99.; Kakiuchi S, et al., Hum Mol. Genet. 2004 Dec. 15; 13(24):3029-43. Epub 2004 Oct. 20.; Taniwaki M, et al., Int J. Oncol. 2006 September; 29(3):567-75.). The results demonstrate that the gene encoding the TTK protein kinase (alias hMps1) is frequently over-expressed in the great majority of primary lung cancers.
Epidermal growth factor receptor (EGFR) has been recognized as an important mediator of growth signaling pathways (Carpenter G. Annu Rev Biochem. 1987; 56:881-914.; Wells C. Int J Biochem Cell Biol. 1999 June; 31(6):637-43.). Aberrant EGFR activity, arising from genetic and epigenetic changes, has been shown to enhance cell proliferation and cause tumor progression in many tumors (Salomon D S, et al., Crit. Rev Oncol Hematol. 1995 July; 19(3):183-232.; Mendelson J. Clin Cancer Res. 2000 March; 6(3):747-53.). Therefore, agents that selectively block EGFR signaling have been under development; to that end, anti-EGFR monoclonal antibody, cetuximab (Erbitux), and small-molecule inhibitors of EGFR tyrosine kinase such as gefitinib (Iressa) and erlotinib (Tarceva), have been used in clinical practice (Dowell J, et al., Nat Rev Drug Discov. 2005 January; 4(1):13-4.; Herbst R S, et al., Nat Rev Cancer. 2004 December; 4(12):956-65.). Stimulation of its ligands, such as EGF, causes EGFR to undergo a conformational change and autophosphorylation that activates the EGFR signaling pathways includes the MAPK (mitogen activated protein kinase) cascade and the c-Src (cellular Src) cascade (Yarden Y. Eur J. Cancer. 2001 September; 37 Suppl 4:S3-8.; Pal SK & Pegram M. Anticancer Drugs. 2005 June; 16(5):483-94.; Tice D A, et al., Proc Natl Acad Sci USA. 1999 Feb. 16; 96(4):1415-20.). c-Src phosphorylates the cytoplasmic tail of EGFR in the presence of EGF and activates the EGFR signals (Yarden Y. Eur J. Cancer. 2001 September; 37 Suppl 4:S3-8.; Pal SK & Pegram M. Anticancer Drugs. 2005 June; 16(5):483-94.; Tice D A, et al., Proc Natl Acad Sci USA. 1999 Feb. 16; 96(4):1415-20.). However, no kinase that phosphorylates EGFR and consequently activates the EGFR pathways in an EGF-independent manner has yet been reported.
The evidence disclosed herein demonstrates that TTK plays a significant role in pulmonary carcinogenesis through EGF-independent phosphorylation of EGFR Tyr-992 and Ser-967, and subsequent activation of downstream MAPK signals that are considered to be indispensable for tumor growth/survival. These data suggest that novel signaling between TTK and EGFR, independent of the presence of EGF, represents a potential target for development of novel therapeutic drugs for lung cancer.
Also disclosed herein are variant TTK proteins composed of various amino acid substitutions, for example, an exchange from leucine to proline at codon 72 (L72P), serine to threonine at codon 76 (S76T), tyrosine to cysteine at codon 574 (Y574C), proline to Glutamine at codon 789 (P789Q) and lysine to Isoleusine at codon 856 (K856I) (Table 4). A missense mutation at codon 574 (Y574C) on the TTK kinase domain found in a RERF-LC-AI cell line was not present in the SNP databases (JSNP: http://snp.ims.u-tokyo.ac.jp/index_ja.html; DBSNP: http://www.ncbi.nlm.nih.gov/projects/SNP/). In addition, two missense mutations were identified in the TTK kinase domain in clinical samples of two metastatic brain tumors derived from primary lung adenocarcinoma. The mutations resulted in two amino acid substitution, namely the substitution of Valine to Phenylalanine at codon 610 (V610F) and Glutamine to Histidine at codon 753 (Q753H). Matched normal brain tissue was available for these two patients and showed only the wild-type DNA sequence, indicating that the mutations had arisen somatically during tumor formation or progression.
The mutant-TTK (Y574C) transfected cells showed a high-autophosphorylation level as compared to non-transfected cells, indicating that the mutation could promote the TTK kinase activity. Furthermore, it was confirmed that the invasive ability of mutant-TTK (Y574C) transfected cells was significantly enhanced by matrigel invasion assay using the mutant-TTK construct. These results doubtlessly indicate that the TTK mutation originated from RERF-LC-AI cell could be an activating mutation involved in lung carcinogenesis.
#location in kinase domain
Thus, the present invention is based, in part, on the discovery of the EGF-independent phosphorylation of EGFR Tyr-992 and Ser-967 by TTK, and subsequent activation of downstream MAPK signals that are considered to be indispensable for tumor growth and/or survival.
Accordingly, the present invention provides a method of diagnosing lung cancer or a predisposition for developing lung cancer in a subject, including the step of determining TTK expression level and a level of kinase activity of TTK for EGFR in a biological sample derived from the subject, wherein an increase in said level as compared to a normal control level indicates that the subject suffers from or is at risk of developing lung cancer. In particular, the kinase activity of TTK for EGFR is EGF-independent and one of the phosphorylation sites of EGFR is Tyr-992 or Ser-967.
The present invention also provides methods of assessing or determining a lung cancer prognosis. In some embodiments, the method includes the steps of:
In particular, the kinase activity of TTK for EGFR is EGF-independent and the phosphorylation site of EGFR is Tyr-992 or Ser-967.
In a further embodiment, the present invention features a method of measuring TTK kinase activity, the method involving the incubation of polypeptides under conditions suitable for a phosphorylation of EGFR by TTK. Suitable polypeptides include a TTK polypeptide or functional equivalent thereof and an EGFR polypeptide or functional equivalent thereof.
For example, the TTK polypeptide may possess the amino acid sequence of SEQ ID NO: 2. Alternatively, the TTK polypeptide may possess an amino acid sequence of SEQ ID NO: 2, where one or more amino acids are modified by substitution, deletion or insertion, so long as the resulting polypeptide retains the biological activity of the polypeptide of SEQ ID NO: 2. Biological activities of the polypeptide of SEQ ID NO: 2 include, for example, the promotion of cell proliferation and the kinase activity of TTK for EGFR. Additionally, the polypeptide may take the form of an 857-amino acid protein encoded by the open reading frame of SEQ. ID. NO. 1, or a polynucleotide that hybridizes under stringent conditions, e.g., low or high, to the nucleotide sequence of SEQ ID NO: 1, so long as the resulting polynucleotide encodes a protein that retains the biological activity of the polypeptide of SEQ ID NO: 2, e.g. the region including Asp-647 of SEQ ID NO: 2.
The EGFR polypeptide may possess the amino acid sequence of SEQ ID NO; 4 (GenBank Accession No. NP—005219). In the cells, EGFR is cleavage at the N-terminal domain and forms an 1186 residue protein (SEQ ID NO: 42). The EGFR polypeptide may possess an amino acid sequence of SEQ ID NO: 4, wherein one or more amino acids are modified by substitution, deletion or insertion, so long as the resulting polypeptide retains the target region of TTK kinase on SEQ ID NO: 4, e.g. the region including Tyr-992 and Ser-967 at cleavage type of EGFR: Additionally, the EGFR polypeptide may take the form of a 1210-amino acid protein encoded by the open reading frame of SEQ. ID. NO. 3 (GenBank Accession No. NM—005228), or a polynucleotide that hybridizes under stringent conditions, e.g. low or high, to the nucleotide sequence of SEQ ID NO: 3, so long as the resulting polynucleotide encodes a protein that retains the target site of TTK kinase on SEQ ID NO: 4.
In the context of the present invention, the kinase activity of TTK for EGFR can be defined by the detection of the phospho-EGFR, especially phosphorylated at Tyr-992 (
The present invention further provides methods of identifying an agent that modulates (e.g., increases or decreases) kinase activity of TTK for EGFR is detected by incubating a TTK polypeptide, or functional equivalent thereof and EGFR polypeptide, or functional equivalent thereof in the presence of ATP as a phosphate donor and determining the phospho-EGFR level. A decrease in the phospho-EGFR level as compared to a normal control level indicates that the test agent is an inhibitor of TTK kinase. Compounds that inhibit (e.g., decreases) kinase activity of TTK for EGFR are useful for treating, preventing or alleviating a symptom of lung cancer. For example, such compounds may inhibit the proliferation of lung cancer cells. Alternatively, an increase in the level or activity as compared to a normal control level indicates that the test agent is an enhancer of kinase activity of TTK for EGFR. Herein, the phrase normal control level refers to a level of kinase activity of TTK for EGFR detected in the absence of the test compound. For example, phosphorylation of EGFR by TTK is EGF-independent and examples of the phosphorylated sites of EGFR are Tyr-992 and Ser-967, and so on.
The present invention also encompasses compositions and methods for treating or preventing of lung cancer by contacting a lung cancer cell with a compound identified as described above. In a further embodiment, the present invention provides for the use of a compound identified as described above, for manufacturing a pharmaceutical composition suitable for treating or preventing lung cancer. For example, a method of treating lung cancer may involve the step of administering to a mammal, e.g. a human patient having been diagnosed with such a disease state, with a composition containing a pharmaceutically effective amount of a compound identified as described above and a pharmaceutical carrier.
The present invention also provides a kit for the detecting the kinase activity of TTK for EGFR. The reagents are preferably packaged together in the form of a kit. The reagents may be packaged in separate containers and may include, for example, a TTK polypeptide, an EGFR polypeptide, reagent for detecting a phospho-EGFR, e.g. phospho-EGFR (Tyr992) or phospho-EGFR (Ser967), a control reagent (positive and/or negative), and/or a detectable label. Instructions (e.g., written, tape, VCR, CD-ROM, etc.) for carrying out the assay are preferably included in the kit. The assay format of the kit may include any kinase assay known in the art.
The present invention is based in part on the discovery of the various amino acid substitutions of TTK at kinase domain, especially the mutations resulted in the amino acid substitution; Valine to Phenylalanine at codon 610 (V610F) and Glutamine to Histidine at codon 753 (Q753H), which are available for these two patients respectively, and a missense mutation Tyrosine to Cysteine at codon 574 (Y574C) on the TTK kinase domain found in a RERF-LC-AI cell line, which was not present in the SNP databases. Because the mutation of TTK (Y574C or Q753H) could promote the TTK kinase activity and the invasive ability, the mutation of TTK (Y574C or Q753H) originated from RERF-LC-AI cell could be an activating mutation involved in lung carcinogenesis. Accordingly, the present invention provides the method of predicting a metastasis of lung cancer, especially a brain metastasis of lung cancer, by using the mutations of kinase domain of TTK, e.g. Y574C or Q753H as index.
In other embodiment, the present invention also provides A method for treating or preventing lung cancer comprising administering to subject a composition comprising a double-stranded molecule which reduces TTK (SEQ ID NO: 1) or EGFR (SEQ ID NO: 3) gene expression, wherein the double-stranded molecule comprises a sense nucleic acid and an anti-sense nucleic acid, wherein the sense nucleic acid comprises a ribonucleotide sequence corresponding to a sequence of SEQ ID NO: 62 or 63 as the target sequence.
Alternatively, the present invention also provides a composition for treating or preventing lung cancer comprising a pharmaceutically effective amount of a double-stranded molecule which reduces TTK (SEQ ID NO: 1) or EGFR (SEQ ID NO: 3) gene expression, and a pharmaceutically acceptable carrier.
In addition, the present invention provides inhibitory polypeptides that are selected from group of ISSILEKGERLPQPPICTI (SEQ ID NO: 44), DVYMIMVKCWMIDADSRPK (SEQ ID NO. 45) and FRELIIEFSKMARDPQRYL (SEQ ID NO. 46). The present invention further provides pharmaceuticals or methods using these inhibitory polypeptides for prevention and/or treatment of lung cancer.
The present invention also relates to methods for treatment and/or prevention of lung cancer comprising the step of administering an inhibitory polypeptide that is selected from group of ISSILEKGERLPQPPICTI (SEQ ID NO: 44), DVYMIMVKCWMIDADSRPK (SEQ ID NO. 45) and FRELIIEFSKMARDPQRYL (SEQ ID NO. 46), or a polynucleotide encoding the same.
These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying figures and examples. However, it is to be understood that both the foregoing summary of the invention and the following detailed description are of a preferred embodiment, and not restrictive of the invention or other alternate embodiments of the invention. In particular, while the invention is described herein with reference to a number of specific embodiments, it will be appreciated that the description is illustrative of the invention and is not constructed as limiting of the invention. Various modifications and applications may occur to those who are skilled in the art, without departing from the spirit and the scope of the invention, as described by the appended claims. Likewise, other objects, features, benefits and advantages of the present invention will be apparent from this summary and certain embodiments described below, and will be readily apparent to those skilled in the art. Such objects, features, benefits and advantages will be apparent from the above in conjunction with the accompanying examples, data, figures and all reasonable inferences to be drawn therefrom, alone or with consideration of the references incorporated herein.
Regarding the specific aims and objectives recited above, it will be understood by those skilled in the art that one or more aspects of this invention can meet certain objectives, while one or more other aspects can meet certain other objectives. Each objective may not apply equally, in all its respects, to every aspect of this invention. As such, the objects herein can be viewed in the alternative with respect to any one aspect of this invention.
Various aspects and applications of the present invention will become apparent to the skilled artisan upon consideration of the brief description of the figures and the detailed description of the present invention and its preferred embodiments which follows:
Part a depicts the expression of TTK in clinical samples of NSCLC (T) and corresponding normal lung tissues (N), examined by semiquantitative RT-PCR.
Part b depicts the expression of TTK in lung-cancer cell lines by semiquantitative RT-PCR.
Part c depicts the expression of TTK protein in 7 lung-cancer cell lines, normal airway epithelial cells (SAEC), and two normal lung fibroblast cells (CCD19Lu and MRC-5) detected by western-blot analysis.
Part d provides representative images of immunohistochemical analysis of TTK protein in lung adenocarcinomas (ADC), squamous-cell carcinomas (SCC), and small cell lung cancer (SCLC) tissues. Magnification, ×200.
Part e depicts the expression of TTK in A549 (left panels) and LC319 cells (right panels) by immunocytochemical analyses. Cells were fixed and stained using anti-TTK antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG as secondary antibody. TTK was visualized in green and the cell nuclei in blue (DAPI).
Part f depicts the results of western blot analyses with anti-TTK antibody, confirming that TTK localizes in the cytosolic and nuclear fraction of A549 (left panels) and LC319 cells (right panels).
Part a depicts the results of immunohistochemical evaluation of TTK protein expression on tissue microarrays. Examples are shown for strong, weak, or absent TTK expression in lung SCCs, and for no expression in normal lung. Magnification, ×100.
Part b depicts the results of Kaplan-Meier analysis of tumor-specific survival in patients with non-small cell lung cancer (NSCLC) according to TTK expression (P<0.0001 by the Log-rank test).
Part a depicts the expression of TTK in response to si-TTKs (si-TTK-1, -2) or control siRNAs (luciferase (LUC), or scramble (SCR)) in LC319 cells, analyzed by semiquantitative RT-PCR (left upper panels). Viability of LC319 cells evaluated by MTT assay in response to si-TTKs, -LUC, or -SCR (left lower panels). Colony-formation assays of LC319 cells transfected with specific siRNAs or control plasmids (right lower panels).
Part b depicts the expression of TTK protein in TTK-stable transfectants of HEK293 cells on western-blot analysis.
Part c depicts the transfectants expressing low levels (clone 1) or high levels (clone 2) of TTK, or controls cells transfected with mock vector were each cultured in triplicate; at each time point, the cell viability was evaluated by MTT assay.
Part d depicts the growth curves of TTK-stable transfectants of HEK293 cells or mock-HEK293 cells transplanted to subcutaneous of nude mice (5×106 TTK-transfected HEK29J cells/mouse).
Part e depicts the results of Matrigel invasion assay demonstrating the increased invasive ability of NIH-3T3 cells transfected with TTK-, the catalytically inactive TTK-KD (kinase dead)-, or mock-vector. The number of invading cells through Matrigel-coated filters was shown.
Part a depicts results of Tyr-992 phosphorylation of EGFR in COS-7 cells that transiently over-expressed TTK. COS-7 cells that scarcely expressed endogenous TTK were transfected with the TTK-expression vector, the catalytically inactive TTK-KD (D647A)-expression vector, or mock vector. Whole cell extracts from these cells were used for western-blot analysis using a total of 31 antibodies against various phospho-proteins involved in cancer-cell signaling (see Table 2). Blots were stripped and re-probed for total EGFR or ACTB to verify equal loading; all bands of EGFR are about 175 kDa.
Part b depicts the results of interaction of endogenous TTK with EGFR in lung cancer cells. Immunoprecipitations were performed using anti-TTK antibodies and extracts from A549 cells in the absence or presence of EGF (100 nM). Immunoprecipitates were subjected to western blot analysis to detect endogenous EGFR. IP, immunoprecipitation; IB, immunoblot.
Part c depicts the results of phase contrast images of A549 cells treated with AG1478, or transfected with siRNA (oligo) against TTK, or siRNA (oligo) against EGFR. Non-treated A549 cells were served as controls.
Part d depicts the results of in vitro kinase assay by incubating purified recombinant TTK protein with whole cell lysates isolated from COS-7 cells. After in vitro kination reaction, the samples were subjected to western-blot analysis with anti-phospho-EGFR antibodies (Tyr-845, Tyr-992, Tyr-1045, Tyr-1068, Tyr-1148, and Tyr-1173). Blots were stripped and re-probed for total EGFR or ACTB to verify equal loading (left panels). COS-7 cells maintained in serum-free medium for 24 hours were exposed to EGF (100 nM) for 5 or 15 min at 37° C. COS-7 cells without exposure to the EGF treatment were served as a control. Whole cell extracts from these cells were used for western-blot analysis with these anti-phospho-EGFR antibodies (right panels); all bands shown are around 175 kDa.
Part e is a schematic representation of the EGFR-deletion mutants (DELs). GST fusion proteins with three partial EGFR sequences at cytoplasmic region were constructed (deletion mutants). Individual mutants are shown as a bar with amino acid residue number at both ends. Locations of the extracellular domain, transmembrane region (TM), tyrosine kinase domain, Src phosphorylation site (Tyr-845), and tyrosine autophosphorylation sites (Tyr-992, Tyr-1045, Tyr-1068, Tyr-1148, and Tyr-1173) are indicated. All three EGFR deletion mutants (EGFR DELs) are kinase-deficient constructs.
Part f depicts the results of three recombinant EGFR-DELs loaded on SDS-PAGE were visualized by Coomassie Brilliant Blue staining (upper panel). In vitro kinase assay by incubating purified recombinant TTK with three deletion mutants of EGFR as substrates (lower panels). After the kinase reaction, samples were subjected to western-blot analysis with anti-phospho-EGFR antibodies. Blots were stripped and re-probed for GST to verify equal loading.
Part g depicts the results of in vitro kinase assays by incubating the recombinant TTK (as kinase) and catalytically active recombinant GST-tagged EGFR (active-rhEGFR as substrates; Upstate). After the kinase reaction, samples were subjected to western-blot analysis with anti-phospho-tyrosine antibodies. The kinase activity of active-rhEGFR was detected in the presence of ATP (lane 2, # indicates the autophosphorylation of active-rhEGFR). The phosphorylation of active-rhEGFR pre-treated with EGFR tyrosine kinase inhibitor (AG1378) was not detected in the absence of recombinant TTK (lane 3), while it was detected in the presence of recombinant TTK (lane 4). Autophosphorylated form of recombinant TTK (arrow) and phosphorylated form of active-rhEGFR by recombinant TTK were indicated.
Part h depicts the results of immunohistochemical staining of representative surgically-resected samples including NSCLC (lung-ADC and —SCC) and SCLC as well as normal lung, using anti-phospho-EGFR (Tyr-992) antibody on tissue microarrays (×200).
Part i depicts the results of Kaplan-Meier analysis of tumor-specific survival in patients with NSCLC according to phospho-EGFR (Tyr-992) expression (P<0.0001 by the Log-rank test).
Part j depicts the results of association of co-activation of TTK and phospho-EGFR (Tyr-992) with poor prognosis of NSCLC patients. The 366 NSCLC cases were divided into three groups; group-1 for cases with strong-positive staining for both TTK and phospho-EGFR (Tyr-992) (63 patients), group-2 for cases with negative staining for both markers (74 patients), group-3 for any other cases (229 patients, shown as others).
Part k and l depict the results of immunofluorescence analysis of phospho-EGFR (Tyr-992) in COS-7 cells that were transiently over-expressed TTK. COS-7 cells that scarcely expressed endogenous TTK, were transfected with TTK-expressing vector or with empty-vector (mock), and were maintained in serum-free medium for 12 hours, and subsequently washed and fixed; The TTK-Alexa488, phospho-EGFR (Tyr-992)-Alexa594, or cell nuclei (DAPI) were visualized in green, red, or blue, respectively. Internalization of phospho-EGFR (Tyr-992) was observed in cells transfected with TTK-expressing plasmids (k). Arrows indicate localization of phospho-EGFR (Tyr-992) (1).
Part m depicts the levels of phospho-EGFR (Tyr-992) detected by immunofluorescence analysis in A549 cells transfected with the RNAi (oligo) against TTK (si-TTK). RNAi mediated suppression of TTK reduced the phosphorylation of EGFR at Tyr-992.
Part a depicts the expression levels of TTK and the phosphorylation levels of EGFR (Tyr-992) in A549 cells that had been arrested at mitosis with colcemid treatment (0, 100, 200 nM) (WAKO) for 24 hours, was detected by western-blot analysis using anti-TTK or pEGFR (Tyr-992) antibody (top and third panels). To assess the mobility-shift of TTK or EGFR band by phosphorylation, the cell lysate was treated or untreated with Lambda Protein Phosphatase (λ-PPase; New England Biolabs) in phosphatase buffer or buffer alone for 1 hour at 37° C. The treatment abolished the mobility-shift of TTK and EGFR bands detected by western-blotting (second and forth panels).
Part b depicts the expression levels of TTK, phospho-EGFR (Tyr-992), total EGFR, phospho-PLCγ1 (Tyr-771), total PLCγ1, phospho-p44/42 MAPK (Thr202/Tyr204), and total p44/42 MAPK, detected by western-blot analysis in A549 cells transfected with the RNAi (oligo) against TTK. RNAi mediated suppression of TTK reduced the phosphorylation of both EGFR (Tyr-992), PLCγ1 (Tyr-771) and p44/42 MAPK (Thr202/Tyr204).
Part c depicts results of immunofluorescence analysis of phospho-p44/42 MAPK in COS-7 cells transiently over-expressing TTK. Transfected cells were maintained in serum-free medium for 12 hours, and subsequently washed and fixed; The Flag-TTK-Alexa488, phospho-p44/42 MAPK (Thr202/Tyr204)-Alexa594, or cell nuclei (DAPI) were visualized in green (upper panel), red (middle panel), or blue, respectively. Phosphorylation of p44/42 MAPK was observed only in TTK-transfected cells, but it was not detected in TTK-non-transfected cells (lower panel).
Part d depicts the results of co-immunoprecipitaion of PLCγ1 with EGFR in COS-7 cells that were transfected with the TTK-, TTK-KD- or empty-vector (mock). The EGFR was immunoprecipitated from whole cell extracts of these cells by using anti-EGFR antibody. Immunoprecipitates were analyzed by western-blotting.
Part a depicts the results of in vitro kinase assay performed by incubating recombinant EGFR-DEL-2 (wild type) or EGFR-DEL-2 mutant whose Tyr-992 residue was replaced with an alanine (Y992A), with recombinant TTK in the presence of [γ-32P] ATP. The products were separated on SDS-PAGE and phosphorylation was visualized by autoradiography.
Part b depicts the levels of TTK, phospho-EGFR (Ser-967), and total EGFR proteins in lung-cancer cell lines, detected by western-blot analysis.
Part c depicts the results of phospho-EGFR (Ser-967) in COS-7 cells that transiently over-expressed TTK, detected by immunofluorescence analysis. COS-7 cells that scarcely expressed endogenous TTK were transfected with the TTK-expression vector. The TTK-Alexa594, phospho-EGFR (Ser-967)-Alexa488, or cell nuclei (DAPI) were visualized in red (right panels), green (middle panels), or blue (left panels), respectively. TTK over-expression induced the phosphorylation of EGFR (Ser-967).
Part d depicts the phosphorylation level of EGFR (Ser-967) detected by immunofluorescence analysis of A549 cells transfected with the RNAi (oligo) against TTK. RNAi mediated suppression of TTK reduced the phosphorylation of EGFR (Ser-967).
Part e depicts the results of immunohistochemical evaluation of phospho-EGFR (Ser-967) expression on tissue microarrays. Examples are shown for strong, weak, or absent phospho-EGFR (Ser-967) expression. Magnification, ×100.
Part f depicts the results of Kaplan-Meier analysis of tumor-specific survival in patients with NSCLC according to phospho-EGFR (Ser-967) expression (P<0.0001 by the Log-rank test).
Part a depicts the results of MTT assay demonstrating the increased growth promoting effect of TTK-stable transfectants of HEK293. TTK- or mock-stable transfectants of HEK293 cells were each cultured in triplicate; at each time point, the cell viability was evaluated by MTT assay. These stable transfectants were transfected with the RNAi (oligo) against EGFR (si-EGFR).
Part b depicts the results of Matrigel invasion assay demonstrating the increased invasive ability of TTK- or mock-stable transfectants of HEK293. These stable transfectants were transfected with si-EGFR.
Part c depicts the results of inhibition of growth of lung cancer cells by cell-permeable EGFR peptides (11R-EGFR) detected by MTT assay. Peptides that were introduced into TTK-over-expressing A549 cells. Growth-suppressive effect of 11R-EGFR899-917, 11R-EGFR918-936, or 11R-EGFR937-955 that was derived from TTK-binding region in EGFR (left panel). The treatment with scramble peptides derived from the most effective 11R-EGFR937-955 peptides resulted in no growth-suppressive effect in cell viability as measured by MTT assay (right panel). Columns, relative absorbance of triplicate assays; bars, SD.
Part a depicts the results of Sequence analyses of TTK mutations in lung cancer. Left panels, a point mutation (Y574C; arrow) resulting in amino acid substitution within the tyrosine kinase domain of TTK found in a lung cancer cell line, RERF-LC-AI. Representative wild type sequence is shown as a reference. Middle and right panels, Point mutations (arrows) resulting in amino acid substitution within the tyrosine kinase domain of TTK in two metastatic brain tumors derived from primary lung adenocarcinoma (V610F in Case 2 and Q753H in Case 8). The corresponding part of wild type DNA sequence from paired normal brain tissues is also shown.
Part b depicts the results of western-blot analysis using anti-Flag antibody detecting exogenously expressed mutant TTKs (Y574C) and (Q753H) in NIH-3T3 cells. Phospho-TTK was indicated by arrowhead.
Part c and d depict the results of Matrigel invasion assay demonstrating the increased invasive ability of NIH-3T3 cells transfected wt-TTK-, TTK-KD-, mutant TTK (Y574C)—, or mutant TTK (Q753H)—, or mock-vector. Invading cells through Matrigel-coated filters evaluated by Giemsa staining (×200) (c) and the cell numbers counted (d).
Expression of TTK in clinical samples of early primary NSCLC (stage I-III A), advanced primary NSCLC (stage III B-IV), and metastatic brain tumor from ADC (T) and normal lung tissues (N), examined by semiquantitative RT-PCR. (upper panel). Densitmetric intensity of PCR product was quantified by image analysis software (lower panel).
It is to be understood that the present invention is not limited to the specific methodologies and protocols herein described, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to a “cell” is a reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, will control. Accordingly, in the context of the present invention, the following definitions apply:
In the context of the present invention, the TTK polypeptide may be a polypeptide having the amino acid sequence of SEQ ID NO: 2 or a polypeptide having the amino acid sequence of SEQ ID NO: 2 in which one or more amino acids are modified by substitution, deletion or insertion, provided the resulting polypeptide is functionally equivalent to the polypeptide of SEQ ID NO: 2. Additionally, the TTK polypeptide may take the form of an 857-amino acid protein encoded by the open reading frame of SEQ. ID. NO. 1, or a polynucleotide that hybridizes under stringent conditions, e.g., low or high, to the nucleotide sequence of SEQ ID NO: 1, provided the resulting polynucleotide encodes a protein that is functionally equivalent to the polypeptide of SEQ ID NO: 2.
In the context of the present invention, the term “functionally equivalent” means that the subject protein retains a biological activity of the original protein. Biological activities of the polypeptide of SEQ ID NO: 2 include, for example, the promotion of cell proliferation and the kinase activity of TTK for EGFR. In the context of the present invention, a protein that is functionally equivalent to TTK preferably has kinase activity for EGFR. Whether or not a subject protein has the target activity can be determined in accordance with the present invention. For example, kinase activity for EGFR can be determined by incubating a polypeptide under conditions suitable for phosphorylation of EGFR and detecting the phosphor-EGFR level. For example, phosphorylation site of EGFR by TTK is Tyr992 or Ser967.
Proteins that are functionally equivalent to the human TTK protein, encoded by the DNA isolated through the above hybridization techniques or gene amplification techniques, normally have a high homology to the amino acid sequence of the human TTK protein. In the context of the present invention, the term “high homology” refers to a homology of 40% or higher, preferably 60% or higher, more preferably 80% or higher, even more preferably 95% or higher. The homology of a protein can be determined by following the algorithm in “Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad. Sci. USA 80, 726-30”.
In the context of the present invention, the term “stringency” in the context of hybridization refers to the relative rigor of hybridization standards utilized. Examples of suitable low stringency conditions include, for example, 42° C., 2×SSC, 0.1% SDS, or preferably 51° C., 2×SSC, 0.1% SDS. Preferably, a high stringency condition is used. An example of a suitable high stringency condition includes, for example, washing 3 times in 2×SSC, 0.01% SDS at room temperature for 20 min, then washing 3 times in 1×SSC, 0.1% SDS at 37° C. for 20 min, and washing twice in 1×SSC, 0.1% SDS at 50° C. for 20 min. However, several factors, such as temperature and salt concentration, can influence the stringency of hybridization and one skilled in the art can suitably select the factors to achieve the requisite stringency.
In the context of the present invention, the phrase “control level” refers to an mRNA or protein expression level detected in a control sample and may include any of (a) a normal control level or (b) a lung cancer specific control level. A control level can be a single expression pattern from a single reference population or composed from a plurality of expression patterns. For example, in the context of the present invention, the control level can be a database of expression patterns from previously tested cells. The phrase “normal control level” refers to a level of gene expression detected in a normal, healthy individual or in a population of individuals known not to be suffering from cancer, such as lung cancer. A normal individual is one with no clinical symptoms of cancer, particularly lung cancer. On the other hand, a “lung cancer control level” refers to a level of gene expression found in a population suffering from lung cancer.
In the context of the present invention, an expression level of a particular gene is deemed “increased” when the expression of the gene or the activity of its gene product is increased by at least 0.1, at least 0.2, at least 1, at least 2, at least 5, or at least 10 or more fold as compared to a control level. TTK gene expression can be determined by detecting mRNA of TTK from a tissue sample from a patient, e.g., by RT-PCR or Northern blot analysis, or detecting a protein encoded by TTK, e.g., by immunohistochemical analysis of a tissue sample from a patient.
In the context of the present invention, the specimen obtained from a subject may be any biological sample for example, a solid tissue or bodily fluid sample, obtained from a test subject, e.g., a patient known to or suspected of having cancer, more particularly lung cancer. For example, in the context of tissue specimen, the tissue can contain epithelial cells. More particularly, the tissue can be epithelial cells from lung cancer cells, e.g. non-small cell lung cancer or small cell lung cancer. Alternatively, the specimen may be a bodily fluid, such a blood, serum, or plasma.
The present invention relates to cancer therapy and prevention. In the context of the present invention, therapy against cancer or prevention of the onset of cancer includes any of the following steps, including inhibition of the growth of cancerous cells, involution of cancer, and suppression of the occurrence of cancer. A decrease in mortality and morbidity of individuals having cancer, decrease in the levels of tumor markers in the blood, alleviation of detectable symptoms accompanying cancer, and such are also included in the therapy or prevention of cancer. Such therapeutic and preventive effects are preferably statistically significant. For example, in observation, at a significance level of 5% or less, wherein the therapeutic or preventive effect of a pharmaceutical composition against cell proliferative diseases is compared to a control without administration. For example, Student's t-test, the Mann-Whitney U-test, or ANOVA can be used for statistical analysis.
Furthermore, in the context of the present invention, the term “prevention” encompasses any activity which reduces the burden of mortality or morbidity from disease. Prevention can occur at primary, secondary and tertiary prevention levels. While primary prevention avoids the development of a disease, secondary and tertiary levels of prevention encompass activities aimed at preventing the progression of a disease and the emergence of symptoms as well as reducing the negative impact of an already established disease by restoring function and reducing disease-related complications.
In the context of the present invention, an “efficacious” treatment is one that leads to a reduction in the level of TTK or the phosphorylation levels of EGFR or a decrease in size, prevalence, or metastatic potential of lung cancer in a subject. When a treatment is applied prophylactically, “efficacious” means that the treatment retards or prevents occurrence of lung cancer or alleviates a clinical symptom of lung cancer. The assessment of lung cancer can be made using standard clinical protocols. Furthermore, the efficaciousness of a treatment can be determined in association with any known method for diagnosing or treating lung cancer. For example, lung cancer is routinely diagnosed histopathologically or by identifying symptomatic anomalies.
Additional definitions are interspersed in the subsequent text, where applicable.
Although advances have been made in development of molecular-targeting drugs for cancer therapy, the ranges of tumor types that respond as well as the effectiveness of the treatments remain very limited (Ranson, M., et al. (2002) J Clin Oncol, 20: 2240-50.; Blackledge, G. and Averbuch, S. (2004) Br J Cancer, 90: 566-72.). Hence, there is an urgent need to develop new anti-cancer agents that are highly specific to malignant cells with minimal or no adverse reactions. A powerful strategy toward these ends would combine the screening of up-regulated genes in cancer cells, identified on the basis of genetic information obtained on cDNA microarrays with high-throughput screening of their effect on cell growth, by inducing loss-of-function phenotypes with RNAi systems, with validation of the potential drug targets by analyzing hundreds of clinical samples on tissue microarray (Sauter, G., et al. (2003) Nat Rev Drug Discov, 2: 962-72.; Kononen, J., et al. (1998) Nat Med, 4: 844-7.). Following such a strategy, it is herein demonstrated that TTK is not only frequently co-over-expressed in clinical NSCLC samples and cell lines, but also that the high levels of expression of the gene products are indispensable for the disease progression as well as the growth of NSCLC cells.
Epidermal growth factor receptor (EGFR) has been recognized as an important mediator of various growth signaling pathways (Carpenter G. Annu Rev Biochem. 1987; 56:881-914.; Wells C. Int J Biochem Cell Biol. 1999 June; 31(6):637-43.). Aberrant EGFR activity arising from genetic and epigenetic changes, has been shown to enhance cell proliferation and drive tumor progression in many tumors (Salomon D S, et al., Crit. Rev Oncol Hematol. 1995 July; 19(3):183-232.; Mendelson J. Clin Cancer Res. 2000 March; 6(3):747-53.). Therefore, agents that selectively block EGFR signaling have been under development; examples currently in clinical use include anti-EGFR monoclonal antibody, cetuximab (Erbitux), and small-molecule inhibitors of EGFR tyrosine kinase such as gefitinib (Iressa) and erlotinib (Tarceva) (Dowell J, et al., Nat Rev Drug Discov. 2005 January; 4(1): 13-4.; Herbst R S, et al., Nat Rev Cancer. 2004 December; 4(12):956-65.). Stimulation of its ligands, such as EGF, causes EGFR to undergo a conformational change and autophosphorylation that activates the EGFR signaling pathways includes the MAPK (mitogen activated protein kinase) cascade and c-Src (cellular Src) cascade (Yarden Y. Eur J. Cancer. 2001 September; 37 Suppl 4:S3-8.; Pal SK & Pegram M. Anticancer Drugs. 2005 June; 16(5):483-94.; Tice D A, et al., Proc Natl Acad Sci USA. 1999 Feb. 16; 96(4):1415-20.). c-Src phosphorylates the cytoplasmic tail of EGFR in the presence of EGF and activates the EGFR signals (Yarden Y. Eur J. Cancer. 2001 September; 37 Suppl 4:S3-8.; Pal SK & Pegram M. Anticancer Drugs. 2005 June; 16(5):483-94.; Tice D A, et al., Proc Natl Acad Sci USA. 1999 Feb. 16; 96(4):1415-20.). However, to date, no kinase that phosphorylates EGFR and consequently activates the EGFR pathways in an EGF-independent manner has been reported.
However, evidence is provided herein that TTK plays a significant role in pulmonary carcinogenesis through EGF-independent phosphorylation of EGFR Tyr-992 or Ser-967 and subsequent activation of downstream MAPK signals that are considered to be indispensable for tumor growth/survival. Thus, the data suggest that a novel signaling between TTK and EGFR, independent of the presence of EGF, represents a potential target for development of novel therapeutic drugs for lung cancer.
As noted above, the present invention is based, in part, on the discovery of a novel intracellular target molecule of a TTK kinase, EGFR. The present invention is also based on the finding that a high expression level of TTK and/or a high level of phospho-EGFR is associated with a poor prognosis in lung cancer patients. Especially, the lung cancer is non-small cell lung cancer (NSCLC). In view of the evidence provided herein, that TTK expression and/or a kinase activity of TTK for EGFR or the phosphorylation level of EGFR is associated with poor prognosis of cancer patients, the present invention thus provides methods for assessing or determining a prognosis for lung cancer patients. For example, the pohsphorylation site of EGFR is Tyr-992 or Ser-967. An example of such a method includes the steps of:
In the context of the present method, the specimen is collected from a subject. An example of a preferred specimen for use in the context of the present invention is a lung tissue obtained by biopsy or surgical-resection from lung cancer patients. In the context of the present invention, when the TTK expression level or a phosphorylation level of EGFR detected in a test specimen is higher than a control level, then the test specimen is deemed to have an elevated level of TTK expression or a phosphorylation level of EGFR. An example of a useful control level in the context of the present invention may include a standard value of TTK expression or a phosphorylation level of EGFR level taken from a group associated with good prognosis. The standard value may be obtained by any method known in the art. For example, a range of mean±2S.D. or mean±3 S.D. may be used as the standard value. Alternatively, poor prognosis can be determined, when strong staining is observed by immunohistochemical analysis of sample tissue.
In the context of the present invention, an expression level of TTK may be detected by any one of the method selected from the group consisting of:
In the context of the present invention, the mRNA, the protein, or biological activity of the protein may be detected by any method. Methods for detecting a given protein, mRNA or biological activity thereof are well known to those skilled in the art. For example, mRNA may be detected using known PCR or hybridization based technologies. Alternatively, any immunoassay format may be applied for detection of a protein. Furthermore, the biological activity of TTK, e.g. a kinase activity of TTK for EGFR, may also detected using any suitable assay method, such as those described herein. For example, the kinase activity of TTK for EGFR may be detected at tyrosine of 992 amino acid residue or serine of 967 amino acid residue in SEQ ID NO: 42.
In the context of the present invention, a phosphorylation level of EGFR may be detected by measuring the amount of phosphorylated EGFR, e.g. Tyr-992 or Ser-967 phosphorylated EGFR. The method of detecting the phosphorylated EGFR is well known to those skilled in the art. For example, immunoassay by using a specific antibody may be useful.
In the context of the present invention, determination of a poor prognosis may be used to determine further treatment, e.g., to stop further treatments that reduce quality of life, to treat the cancer in a different manner than previously used, or to treat the cancer more aggressively. In other words, the assessment of a prognosis by TTK or phosphorylation of EGFR enables clinicians to choose, in advance, the most appropriate treatment for an individual lung cancer patient without even the information of conventional clinical staging of the disease, using only routine procedures for tissue-sampling.
Further, the methods of the present invention may be used to assess the efficacy of a course of treatment. For example, in a mammal with cancer from which a biological sample is found to contain an elevated level of TTK expression or phosphorylation of EGFR; the efficacy of an anti-cancer treatment can be assessed by monitoring the TTK expression level or the phosphorylation level of EGFR over time. For example, a decrease in TTK expression level or the phosphorylation level of EGFR in a biological sample taken from a mammal following a course of treatment, as compared to a level observed in a sample taken from the mammal before treatment onset, or earlier in, the treatment, may be indicative of efficacious treatment.
Alternatively, according to the present invention, an intermediate result may also be provided in addition to other test results for assessing or determining the prognosis of a subject. Such intermediate result may assist a doctor, nurse, or other practitioner to assess, determine, or estimate the prognosis of a subject. Additional information that may be considered, in combination with the intermediate result obtained by the present invention, to assess prognosis includes clinical symptoms and physical conditions of a subject.
As noted above, the present invention also provides kits for assessing or determining lung cancer prognosis, including any one component selected from the group consisting of:
TTK has a kinase activity for EGFR, and its expression level and phospho-EGFR level (Tyr-992 or Ser-967) revealed a shorter tumor-specific survival period. Furthermore, the phosphorylation of EGFR at Tyr-992 or Ser-967 by TTK is independent from the EGF stimulation. Thus, TTK-mediated phosphorylation of EGFR is useful as a diagnostic parameter of lung cancer, e.g. non-small cell lung cancer.
The present invention also provides kits for assessing or determining lung cancer or a predisposition for developing lung cancer in a subject, wherein the kit includes a reagent for detecting the kinase activity of TTK for EGFR. The kit is also useful as a diagnostic of lung cancer, e.g. non-small cell lung cancer. Furthermore, the kinase activity of TTK for EGFR, e.g. an EGF-independent phosphorylation of EGFR by TTK, may also detected using any suitable reagent.
The selective phosphorylation of EGFR by TTK is revealed herein. Consequently, in another aspect, the present invention provides a method of measuring a kinase activity of TTK for EGFR. Such a method may include the steps of:
In the context of present invention, the conditions suitable for the EGFR phosphorylation may be provided with an incubation of EGFR and TTK in the presence of phosphate donor. In the present invention, preferable phosphate donor is ATP. The conditions suitable for the EGFR phosphorylation by TTK also include culturing cells expressing the polypeptides. For example, the cell may be a transformant cell harboring an expression vector that contains a polynucleotide encoding the polypeptide. In another embodiment, the phosphorylation reaction against EGFR is performed by incubation of EGFR and TTK in kinase assay buffer (for example, 50 mM Tris, pH 7.4, 10 mM MgCl2, 2 mM dithiothreitol, 1 mM NaF, 0.2 mM ATP) for 60 min at 30° C. In the context of present invention, functional equivalent of EFGR is fragment of EGFR which may be comprised TTK-mediated phosphorylation site of EGFR, Tyr992 or Ser967. For example, the fragment of EGFR may be comprised amino acid sequence of SEQ ID NO: 43.
After the incubation, a phospho-EGFR level can be detected with an antibody recognizing phosphorylated EGFR. Prior to the detection of phosphorylated EGFR, EGFR may be separated from other elements, or cell lysate of EGFR expressing cells. For instance, gel electrophoresis may be used for separation of EGFR. Alternatively, EGFR may be captured by contacting EGFR with a carrier having an anti-EGFR antibody. When the labeled phosphate donor was used, phospho-EGFR level can be detected via tracing the label. For example, radio-labeled ATP (e.g. 32P-ATP) was used as phosphate donor, radio activity of the separated EGFR correlates with phospho-EGFR level.
In the context of present invention, kinase activity of TTK in biological samples may be estimated. For example, the biological sample of the present invention may include cancer tissues obtained from a patient or cancer cell lines. The kinase activity of TTK in such biological samples is useful as credible marker for indicating lung cancer, or assessing or determining prognosis. The present invention further provides a reagent for measuring a kinase activity of TTK for EGFR. Examples of such reagents include EGFR and phosphate donor. In the present invention, the kit for measuring a kinase activity of TTK for EGFR is also provided. Such a kit may include the reagent of the present invention and detecting agent for detecting phospho-EGFR level. Preferable detecting agent is an antibody specifically recognizing phosphorylated EGFR from unphosphorylated EGFR. For example, in the present invention, preferable antibody recognizes phosphorylated EGFR at Tyr992 or Ser967.
The present invention also provides a method of diagnosing lung cancer or a predisposition for developing lung cancer in a subject, such a method including the step of determining a level of the TTK expression or the phosphorylation of EGFR in a biological sample derived from the subject, wherein an increase in said level, as compared to a normal control level, indicates that said subject suffers from or is at risk of developing lung cancer. In the present invention, any sample derived from a subject to be diagnosed may be used. An example of a preferred sample for use in the context of the present invention is a lung tissue obtained by biopsy or surgical-resection. For example, the phosphorylation site of EGFR by TTK is Tyr992 or Ser967.
Alternatively, according to the present invention, an intermediate result for examining the condition of a subject may be provided. Such intermediate result may be combined with additional information to assist a doctor, nurse, or other practitioner to determine that a subject suffers from lung cancer. Further, the present invention relates to a method for screening a person who is required to be further diagnosed for lung cancer. After the screening, persons indicating positive result are recommended to be submitted further screening test, or medical treatment to confirm whether they truly suffer from lung cancer.
Alternatively, the present invention may be used to detect cancerous cells in a subject-derived tissue, and provide a doctor with useful information to determine that the subject suffers from lung cancer. Accordingly, the present invention involves determining (e.g., measuring) a level of a kinase activity of TTK for EGFR in subject derived samples. In the present invention, a method for diagnosing lung cancer also includes a method for testing or detecting lung cancer. Alternatively, in the present invention, diagnosing lung cancer also refers to showing a suspicion, risk, or possibility of lung cancer in a subject.
The diagnostic method of the present invention involves the step of determining (e.g., measuring) the expression of TTK. Using sequence of TTK gene can be detected and measured using conventional techniques well known to one of ordinary skill in the art. For example, northern blot hybridization analyses can be used for determining the expression of TTK gene. Hybridization probes typically include at least 10, at least 20, at least 50, at least 100, or at least 200 consecutive nucleotides of TTK sequence. As another example, the sequences can be used to construct primers for specifically amplifying the TTK nucleic acid in, e.g., amplification-based detection methods, for example, reverse-transcription based polymerase chain reaction (RT-PCR). As another example, an antibody against TTK, e.g., an anti-TTK polyclonal antibody or anti-TTK monoclonal antibody, can be used for immunoassay, for example, immunohistochemical analysis, western blot analysis or ELISA, etc.
Alternatively, the expression of TTK can be detected by the biological activity. For example, the biological activity is cell proliferative activity or invasion activity or kinase activity against EGFR Tyr997 or Ser967. The method of detecting the kinase activity described in above.
Also, the diagnostic method of the invention involves the step of determining the pohsphorylation level of EGFR. For example, the pohsphorylation site of EGFR is Tyr992 or Ser967. The antibody that specifically recognizes the pohsphorylation without non-phosphorylation type can be used for immunoassay, for example, immunohistochemical analysis, western blot analysis or ELISA, etc.
The level of the TTK expression or the pohsphorylation level of EGFR detected in a test cell population, e.g., a tissue sample from a subject, can then be compared to that in a reference cell population. The reference cell population may include one or more cells for which the compared parameter is known, i.e., lung cancer cells or normal lung epithelial cells (non-lung cancer cells).
Whether or not the level of the TTK expression or the pohsphorylation level of EGFR in a test cell population as compared to a reference cell population indicates the presence of lung cancer or a predisposition thereto depends upon the composition of the reference cell population. For example, if the reference cell population is composed of non-lung cancer cell, a similarity in the level between the test cell population and the reference cell population indicates the test cell population is non-lung cancer. Conversely, if the reference cell population is made up of lung cancer cells, a similarity in gene expression between the test cell population and the reference cell population indicates that the test cell population includes lung cancer cells.
The level of the TTK expression or the pohsphorylation level of EGFR in a test cell population is considered “altered” or deemed to “differ” if it varies from the level in a reference cell population by more than 1.1, more than 1.5, more than 2.0, more than 5.0, more than 10.0 or more fold.
Differential gene expression between a test cell population and a reference cell population can be normalized to a control gene, e.g. a housekeeping gene. For example, a control gene is one which is known not to differ depending on the cancerous or non-cancerous state of the cell. The expression level of a control gene can thus be used to normalize signal levels in the test and reference cell populations. Exemplary control genes include, but are not limited to, e.g., beta-actin, glyceraldehyde 3-phosphate dehydrogenase and ribosomal protein P1.
The test cell population can be compared to multiple reference cell populations. Each of the multiple reference cell populations can differ in the known parameter. Thus, a test cell population can be compared to a first reference cell population known to contain, e.g., lung cancer cells, as well as a second reference cell population known to contain, e.g., non-lung cancer cells (normal cells). The test cell population can be included in a tissue or cell sample from a subject known to contain, or suspected of containing, lung cancer cells.
The test cell population can be obtained from a bodily tissue or a bodily fluid, e.g., biological fluid (for example, blood, sputum, saliva). For example, the test cell population can be purified from lung tissue. Preferably, the test cell population comprises an epithelial cell. The epithelial cell is preferably from a tissue known to be or suspected to be lung carcinoma.
Cells in the reference cell population are preferably from a tissue type similar to that of the test cell population. Optionally, the reference cell population is a cell line, e.g. a lung cancer cell line (i.e., a positive control) or a normal non-lung cancer cell line (i.e., a negative control). Alternatively, the control cell population can be from a database of molecular information obtained from cells for which the assayed parameter or condition is known.
The subject is preferably a mammal. Exemplary mammals include, but are not limited to, e.g., a human, non-human primate, mouse, rat, dog, cat, horse, or cow.
The present invention also provides a kit for detection of kinase activity of TTK for EGFR. Examples of components contained within such kits include, EGFR, an antibody that binds to phospho-EGFR, e.g. anti-phospho-EGFR (Tyr992) antibody or anti-phospho-EGFR (Ser967) antibody, and a detectable label for detecting the antibody. An antibody recognizing phosphorylated Tyr992 or Ser967 of EGFR is commercially available. Alternatively, it is well known that such antibody can be obtained by immunization with phosphorylated EGFR at Tyr992 or Ser967, or fragment thereof that includes the Tyr992 or Ser967 residue.
The TTK cDNA consists of 2,984 nucleotides that contain an open reading frame of 2,571 nucleotides as set forth in SEQ. ID. NO.: 1 (GenBank Accession No. NM—003318). The open reading frame encodes an 857-amino acid protein having amino acid sequence as set forth in SEQ. ID. NO.: 2 (GenBank Accession No. NP—003309). Mps1 (TTK is its human homologue) was first discovered in budding yeast as a factor to be required in centrosome duplication and was subsequently shown to have a critical function in the spindle checkpoint.
The present invention also relates to the finding that TTK has the kinase activity for EGFR. For example, the phosphorylation site of EGFR by TTK is Tyr992 or Ser967 and the phosphorylation is EGF-independent. To that end, one aspect of the invention involves identifying test compounds that regulate TTK-mediated phosphorylation of EGFR. Accordingly, the present invention provides novel methods for identifying compounds that modulates a kinase activity of TTK for EGFR. For instance, the present invention provides a method of identifying an agent that modulates a kinase activity of TTK for EGFR, such a method including the steps of:
Agents identified by the present method constitute candidate compounds that may slow or arrest the progression of, e.g., lung cancer, by inhibiting TTK-mediated phosphorylation of EGFR. Accordingly, the invention thus provides a method of screening for a compound that modulates TTK kinase activity for EGFR, e.g. EGF independent phosphorylation of EGFR by TTK. For example the phosphorylation site of EGFR is Tyr992 or Ser967. The method is practiced by contacting a TTK, or a functional equivalent thereof having kinase activity for EGFR, and EGFR or functional equivalent thereof capable of phosphorylation by TTK, with one or more candidate compounds, and assaying phospho-EGFR level. For example, the functional equivalent of EGFR that is capable of phosphorylation by TTK may be comprised TTK-mediated phosphorylation site of EGFR, Tyr992 or Ser967. More preferably, the fragment of EGFR may be comprised the region from 889aa to 1045aa of SEQ ID NO: 42. A compound that modulates phosphorylation of EGFR by TTK or functional equivalent is thereby identified. Consequently, the present invention also provides a method of screening for a compound for treating and/or preventing lung cancer, such a method including the steps of:
The other respect of the invention, a kit for detecting the ability of a test compound to modulate kinase activity of TTK for EGFR also provided. Such a kit may include the components of:
A) a polypeptide selected from the group consisting of:
B) EGFR or EGFR or functional equivalent thereof.
C) a reagent for detecting a phospho-EGFR.
Further, this invention also provides a kit for detecting for the ability of a test compound to modulate kinase activity of TTK for EGFR. Such a kit may include the components of:
A) a cell expressing EGFR or functional equivalent thereof a polypeptide selected from the group consisting of:
B) a reagent for detecting a phospho-EGFR.
In the present invention, the functional equivalent of EGFR is the fragment consisting of amino acid sequence of SEQ ID NO: 43. Furthermore, the kit may further include phosphate donor. Preferable phosphate donor is ATP. The reagent for detection in the kit of the present invention may also include an antibody recognizes phosphorylated Tyr992 or Ser967 of EGFR as the reagent for detecting a phospho-EGFR.
The present invention further provides a composition for treating or preventing lung cancer, such a composition composed of a pharmaceutically effective amount of a compound that decreases a kinase activity of TTK for EGFR and a pharmaceutically acceptable carrier. As noted above, in the context of the present invention, the term “functionally equivalent” means that the subject protein retains the biological activity of the original protein, in this case the kinase activity for EGFR. Whether or not a subject protein has the target activity can be determined in accordance with the present invention. For example, kinase activity for EGFR can be determined by incubating a polypeptide under conditions suitable for phosphorylation of EGFR and detecting the phosphor-EGFR level. For example, the phosphorylation site of EGFR by TTK is Tyr992 or Ser967.
Methods for preparing proteins functionally equivalent to a given protein are well known to those skilled in the art and include conventional methods of introducing mutations into the protein. For example, one skilled in the art can prepare proteins functionally equivalent to the human TTK protein by introducing an appropriate mutation in the amino acid sequence of the human TTK protein via site-directed mutagenesis (Hashimoto-Gotoh, T. et al. (1995), Gene 152, 271-5; Zoller, M J, and Smith, M. (1983), Methods Enzymol. 100, 468-500; Kramer, W. et al. (1984), Nucleic Acids Res. 12, 9441-56; Kramer W, and Fritz H J. (1987) Methods. Enzymol. 154, 350-67; Kunkel, T A (1985), Proc. Natl. Acad. Sci. USA. 82, 488-92; Kunkel (1991), Methods Enzymol. 204, 125-39). Amino acid mutations can occur in nature, too. The proteins suitable for use in the context present invention include those proteins having the amino acid sequences of the human TTK protein in which one or more amino acids are mutated, provided the resulting mutated proteins are functionally equivalent to the human TTK protein. The number of amino acids to be mutated in such a mutant is generally 25 amino acids or less, preferably 10 to 15 amino acids or less, more preferably 5 to 6 amino acids or less, and even more preferably 2 to 3 amino acids or less. To maintain kinase activity for EGFR, it is preferable to conserve the kinase domain in the amino acid sequence of the mutated protein. For example, to maintain the kinase domain of TTK, Asp647 of TTK can not be altered.
Mutated or modified proteins, proteins having amino acid sequences modified by substitution, deletion or insertion of one or more amino acid residues of a certain amino acid sequence, are known to retain the original biological activity (Mark, D. F. et al., Proc. Natl. Acad. Sci. USA (1984) 81, 5662-6, Zoller, M. J. & Smith, M., Nucleic Acids Research (1982) 10, 6487-500, Wang, A. et al., Science (1984) 224, 1431-3, Dalbadie-McFarland, C et al., Proc. Natl. Acad. Sci. USA (1982) 79, 6409-13).
The amino acid residue to be mutated is preferably mutated into a different amino acid in which the properties of the amino acid side-chain are conserved (a process known in the art as “conservative amino acid substitution”). Examples of properties of amino acid side chains are hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side chains having the following functional groups or characteristics in common: an aliphatic side-chain (G, A, V, L, I, P); a hydroxyl group containing side-chain (S, T, Y); a sulfur atom containing side-chain (C, M); a carboxylic acid and amide containing side-chain (D, N, E, Q); a base containing side-chain (R, K, H); and an aromatic containing side-chain (H, F, Y, W). Note, the parenthetic letters indicate the one-letter codes of amino acids.
An example of a protein to which one or more amino acids residues are inserted or added to the amino acid sequence of human TTK protein (SEQ ID NO: 2) is a fusion protein containing the human TTK protein. Fusion proteins suitable for use in the context of the present invention include, for example, fusions of the human TTK protein and other peptides or proteins. Fusion proteins can be made using techniques well known to those skilled in the art, for example by linking the DNA encoding the human TTK protein of the invention with DNA encoding other peptides or proteins, so that the frames match, inserting the fusion DNA into an expression vector and expressing it in a host. There is no restriction as to the peptides or proteins to be fused to the protein of the present invention.
Known peptides that can be used as peptides that are fused to the TTK protein include, for example, FLAG (Hopp, T. P. et al., (1988) Biotechnology 6, 1204-10), 6×His containing six His (histidine) residues, 10×His, Influenza agglutinin (HA), human c-myc fragment, VSP-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag, alpha-tubulin fragment, B-tag, Protein C fragment, and the like. Examples of proteins that may be fused to a protein of the invention include GST (glutathione-S-transferase), Influenza agglutinin (HA), immunoglobulin constant region, beta-galactosidase, MBP (maltose-binding protein), and such.
Fusion proteins can be prepared by fusing commercially available DNA, encoding the fusion peptides or proteins discussed above, with the DNA encoding a protein of the present invention and expressing the fused DNA prepared.
An alternative method known in the art to isolate functional equivalent proteins is, for example, the method using a hybridization technique (Sambrook, J. et al., (1989) Molecular Cloning 2nd ed. 9.47-9.58, Cold Spring Harbor Lab. Press). One skilled in the art can readily isolate a DNA having high homology with a whole or part of the TTK DNA sequence (e.g., SEQ ID NO: 1) encoding the human TTK protein, and isolate functional equivalent proteins to the human TTK protein from the isolated DNA. The proteins used for the present invention include those that are encoded by DNA that hybridize with a whole or part of the DNA sequence encoding the human TTK protein and are functional equivalent to the human TTK protein. These proteins include mammal homologues corresponding to the protein derived from human or rat (for example, a protein encoded by a monkey, mouse, rabbit and bovine gene). In isolating a cDNA highly homologous to the DNA encoding the human TTK protein from animals, it is particularly preferable to use tissues from testis or lung cancer.
The conditions of hybridization for isolating a DNA encoding a protein functionally equivalent to the human TTK protein can be routinely selected by a person skilled in the art. For example, hybridization may be performed by conducting pre-hybridization at 68° C. for 30 min or longer using “Rapid-hyb buffer” (Amersham LIFE SCIENCE), adding a labeled probe, and warming at 68° C. for 1 hour or longer. The following washing step can be conducted, for example, for a low stringency condition. Exemplary low stringency conditions include, for example, 42° C., 2×SSC, 0.1% SDS, or preferably 50° C., 2×SSC, 0.1% SDS. More preferably, high stringency conditions are selected. Exemplary high stringency conditions include, for example, washing 3 times in 2×SSC, 0.01% SDS at room temperature for 20 min, then washing 3 times in 1×SSC, 0.1% SDS at 37° C. for 20 min, and washing twice in 1×SSC, 0.1% SDS at 50° C. for 20 min. However, several factors, such as temperature and salt concentration, can influence the stringency of hybridization. Selection of the factors necessary to achieve a requisite level of stringency constitutes routine optimization that is well within the purview of one skilled in the art.
In place of hybridization, a gene amplification method, for example, the polymerase chain reaction (PCR) method, can be utilized to isolate a DNA encoding a protein functionally equivalent to the human TTK protein, using a primer synthesized based on the sequence information of the DNA (SEQ ID NO: 1) encoding the human TTK protein (SEQ ID NO: 2).
As noted above, proteins that are functionally equivalent to the human TTK protein, encoded by the DNA isolated through the above hybridization techniques or gene amplification techniques, normally have a high homology to the amino acid sequence of the human TTK protein. In the context of the present invention, the term “high homology” refers to a homology of 40% or higher, preferably 60% or higher, more preferably 80% or higher, even more preferably 95% or higher. The homology of a protein can be determined by following the algorithm in “Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad. Sci. USA 80, 726-30”.
A protein useful in the context of the present invention may have variations in amino acid sequence, molecular weight, isoelectric point, the presence or absence of sugar chains, or form, depending on the cell or host used to produce it or the purification method utilized. Nevertheless, so long as it is functionally equivalent to a human TTK protein (SEQ ID NO: 2), it is useful in the context of the present invention.
The proteins useful in the context of the present invention can be prepared as recombinant proteins or natural proteins, by methods well known to those skilled in the art. A recombinant protein can be prepared, for example, by inserting a DNA encoding a protein of the present invention (for example, the DNA having the nucleotide sequence of SEQ ID NO: 1) into an appropriate expression vector, introducing the vector into an appropriate host cell, obtaining the extract, and purifying the protein by subjecting the extract to chromatography, for example, ion exchange chromatography, reverse phase chromatography, gel filtration, or affinity chromatography utilizing a column to which antibodies against the protein of the present invention are fixed, or by combining more than one of aforementioned columns.
In addition, when a protein useful in the context of the present invention is expressed within host cells (for example, animal cells and E. coli) as a fusion protein with glutathione-S-transferase protein or as a recombinant protein supplemented with multiple histidines, the expressed recombinant protein can be purified using a glutathione column or nickel column.
After purifying the fusion protein, it is also possible to exclude regions, other than the objective protein, by cutting with thrombin or factor-Xa as required.
A natural protein can be isolated by methods known to those skilled in the art, for example, by contacting an affinity column, in which antibodies binding to the TTK protein described below are bound, with the extract of tissues or cells expressing a protein of the present invention. The antibodies can be polyclonal antibodies or monoclonal antibodies.
In the present invention, a kinase activity of TTK or its functional equivalent can be determined by methods known in the art. For example, TTK and EGFR can be incubated with an ATP, under suitable assay conditions for a phosphorylation of EGFR by TTK. In the present invention, exemplary conditions for the phosphorylation of EGFR include the steps of contacting TTK with EGFR or cell extracts, and incubating them. In the present invention, conditions for the phosphorylation of EGFR may be provided by the incubation of TTK with EGFR in the presence of phosphate donor. An ATP is an example of suitable phosphate donor. For example, a radio labeled ATP can be the phosphate donor. The increased radio-labeled-EGFR may be detected by any suitable method. For example, in a hot assay, the radio-labeled-EGFR is detected by scintillation counter. On the other hand, in a cold assay, the phospho-EGFR is detected using an antibody binding to phospho-EGFR, e.g. western blot assay or ELISA. For example, suitable conditions for phosphorylation of EGFR by TTK are set forth below:
Reaction mixture:
50 mM Tris-HCl (pH 7.4),
10 mM MgCl2,
2 mM dithiothreitol,
1 mM NaF,
0.2 mM ATP
The reaction mixture is mixed with a sample containing TTK to be determined, and incubated for 60 min. at 30° C. The reactions were stopped by addition of Laemmli sample buffer and heating at 95° C. for 5 min. Proteins were resolved by SDS-PAGE and then western-blot using an antibody binding to phospho-EGFR.
Alternatively, a kinase activity of TTK for EGFR can be estimated based on a radio-labeled-EGFR by scintillation counter. A phospho-EGFR can be detected by an antibody based detection system, e.g. ELISA or western blot assay. Alternatively, a phospho-EGFR can be detected with mass spectrometry, e.g. MALDI-TOF-MS. For example, the phosphorylation site of EGFR by TTK is Tyr992 or Ser967. The kinase activity of TTK for EGFR at Tyr992 or Ser967 may be detected by using an antibody specific for phospho-EGFR (Tyr992 or Ser967).
Various low-throughput and high-throughput enzyme assay formats are known in the art and can be readily adapted for detection or measuring of the phosphorylation level of EGFR by TTK. For high-throughput assays, the EGFR is preferably immobilized on a solid support, such as a multi-well plate, slide or chip. Following the reaction, the phospho-EGFR can be detected on the solid support. In order to detect phospho-EGFR, for example, an antibody binding to phospho-EGFR can be used. For example, the phosphorylation site of EGFR by TTK is Tyr992 or Ser967. The phosphorylation of EGFR at Tyr992 or Ser967 by TTK may be detected by using an antibody specific for phosphor-EGFR (Tyr992 or Ser967). Alternatively, P32 labeled ATP may be used as a phosphate donor. The phospho-EGFR can be traced with radioactive P32 To facilitate such assays, the solid support may be coated with streptavidin and the EGFR labeled with biotin. The skilled person can determine suitable assay formats depending on the desired throughput capacity of the screen.
Any test compound, including, but not limited to, cell extracts, cell culture supernatant, products of fermenting microorganisms, extracts from marine organisms, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic micromolecular compounds and natural compounds, can be used in the screening methods of the present invention. The test compound of the present invention can be also obtained using any of the numerous approaches in combinatorial library methods known in the art, including (1) biological libraries, (2) spatially addressable parallel solid phase or solution phase libraries, (3) synthetic library methods requiring deconvolution, (4) the “one-bead, one-compound” library method and (5) synthetic library methods using affinity chromatography selection. The biological library methods using affinity chromatography selection are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12: 145-67). Examples of methods for the synthesis of molecular libraries can be found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6909-13; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11422-6; Zuckermann et al. (1994) J. Med. Chem. 37: 2678-85; Cho et al. (1993) Science 261: 1303-5; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2061; Gallop et al. (1994) J. Med. Chem. 37: 1233-51.). Libraries of compounds may be presented in solution (see Houghten (1992) Bio/Techniques 13: 412-21.) or on beads (Lam (1991) Nature 354: 82-4.), chips (Fodor (1993) Nature 364: 555-6.), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484, and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1865-9.) or phage (Scott and Smith (1990) Science 249: 386-90.; Devlin (1990) Science 249: 404-6.; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87: 6378-78.; Felici (1991) J. Mol. Biol. 222: 301-10.; US Pat. Application 2002103360).
A compound isolated by the screening method of the present invention is a candidate for the development of drugs that inhibit a kinase activity of TTK for EGFR and can be applied to the treatment or prevention of lung cancer. Especially, the kinase activity of TTK for EGFR is. EGF-independent., Alternatively, the phosphorylation site of EGFR is Tyr-992 or Ser-967.
Moreover, a compound in which a part of the structure of the compound inhibiting the kinase activity of TTK for EGFR is converted by addition, deletion and/or replacement are also included in the compounds obtainable by the screening method of the present invention.
The present invention provides compositions for treating or preventing lung cancer containing any of the compounds selected by the screening methods of the present invention.
When administrating a compound isolated by a method of the present invention as a pharmaceutical for humans and other mammals, such as mice, rats, guinea-pigs, rabbits, cats, dogs, sheep, pigs, cattle, monkeys, baboons, and chimpanzees, the isolated compound can be directly administered or, alternatively, can be formulated into a dosage form using conventional pharmaceutical preparation methods. For example, according to the need, the drugs can be taken orally, as sugar-coated tablets, capsules, elixirs and microcapsules, or non-orally, in the form of injections of sterile solutions or suspensions with water or any other pharmaceutically acceptable liquid. For example, the compound can be mixed with pharmaceutically acceptable carriers or media, specifically, sterilized water, physiological saline, plant-oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, and such, in a unit dose form required for generally accepted drug implementation. The amount of active ingredients in these preparations makes a suitable dosage within the indicated range acquirable.
Examples of additives that can be mixed to form tablets and capsules include, for example, binders, such as gelatin, corn starch, tragacanth gum and arabic gum; excipients, such as crystalline cellulose; swelling agents, such as corn starch, gelatin and alginic acid; lubricants, such as magnesium stearate; sweeteners, such as sucrose, lactose or saccharin; and flavoring agents, such as peppermint, Gaultheria adenothrix oil and cherry. When the unit-dose form is a capsule, a liquid carrier, such as an oil, can also be further included in the above ingredients. Sterile composites for injections can be formulated following normal drug implementations using vehicles such as distilled water used for injections.
Physiological saline, glucose, and other isotonic liquids, including adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, can be used as aqueous solutions for injections. These can be used in conjunction with suitable solubilizers, such as alcohol, specifically ethanol, polyalcohols such as propylene glycol and polyethylene glycol, non-ionic surfactants, such as Polysorbate 80 (TM) and HCO-50.
Sesame oil and soy-bean oil are examples of suitable oleaginous liquids and may be used in conjunction with benzyl benzoate or benzyl alcohol as solubilizers. They may be further formulated with a buffer, such as phosphate buffer or sodium acetate buffer; a pain-killer, such as procaine hydrochloride; a stabilizer, such as benzyl alcohol or phenol; and an anti-oxidant. The prepared injection may be filled into a suitable ampule.
Methods well known to those skilled in the art may be used to administer a pharmaceutical composition of the present invention to patients, for example, as intra-arterial, intravenous, or percutaneous injections and also as intranasal, transbronchial, intramuscular or oral administrations. The dosage and method of administration may vary according to the body-weight and age of the patient and the selected administration method; however, one skilled in the art can routinely select a suitable method of administration and dosage. If said compound is encodable by a DNA, the DNA can be inserted into a vector for gene therapy and the vector can be administered to a patient to perform the therapy. The dosage and method of administration may again vary according to the body-weight, age, and symptoms of the patient; however, one skilled in the art can suitably select them.
For example, although the dose of a compound that binds to TTK and regulates its activity depends on the symptoms, a suitable dose is generally about 0.1 mg to about 100 mg per day, preferably about 1.0 mg to about 50 mg per day and more preferably about 1.0 mg to about 20 mg per day, when administered orally to a normal adult (weight 60 kg).
When administering parenterally, in the form of an injection to a normal adult (weight 60 kg), although there are some differences according to the patient, target organ, symptoms and method of administration, it is convenient to intravenously inject a dose of about 0.01 mg to about 30 mg per day, preferably about 0.1 to about 20 mg per day and more preferably about 0.1 to about 10 mg per day. Also, in the case of other animals too, it is possible to administer an amount converted to 60 kg of body-weight.
In another aspect, the present invention includes pharmaceutical, or therapeutic, compositions containing one or more therapeutic compounds described herein. Pharmaceutical formulations may include those suitable for oral, rectal, nasal, topical (including buccal and sub-lingual), vaginal or parenteral (including intramuscular, subcutaneous and intravenous) administration, or for administration by inhalation or insufflation. The formulations may, where appropriate, be conveniently presented in discrete dosage units and may be prepared by any of the methods conventional in the art of pharmacy. All such Pharmaceutical methods herein include the steps of bringing into association the active compound with liquid carriers or finely divided solid carriers or both as needed and then, if necessary, shaping the product into the desired formulation.
Pharmaceutical formulations suitable for oral administration may conveniently be presented as discrete units, such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; or as a solution, a suspension or as an emulsion. The active ingredient may also be presented as a bolus electuary or paste, and be in a pure form, i.e., without a carrier. Tablets and capsules for oral administration may contain conventional excipients, such as binding agents, fillers, lubricants, disintegrant or wetting agents. A tablet may be made by compression or molding, optionally with one or more formulational ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredients in a free-flowing form, such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may be coated according to methods well known in the art. Oral fluid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives, such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), or preservatives. Furthermore, the tablets may optionally be formulated so as to provide slow or controlled release of the active ingredient therein.
Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline, water-for-injection, immediately prior to use. Alternatively, the formulations may be presented for continuous infusion. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
Formulations for rectal administration may be presented as a suppository with the usual carriers, such as cocoa butter or polyethylene glycol. Formulations for topical administration in the mouth, for example, buccally or sublingually, include lozenges, containing the active ingredient in a flavored base, such as sucrose and acacia or tragacanth, and pastilles containing the active ingredient in a base, such as gelatin and glycerin or sucrose and acacia. For intra-nasal administration, the compounds of the present invention may be used as a liquid spray or dispersible powder or in the form of drops. Drops may be formulated with an aqueous or non-aqueous base also including one or more dispersing agents, solubilizing agents or suspending agents. Liquid sprays are conveniently delivered from pressurized packs.
For administration by inhalation, the compounds of the present invention are conveniently delivered from an insufflator, nebulizer, pressurized pack or other convenient aerosol spray delivery means. Pressurized packs may include a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.
Alternatively, for administration by inhalation or insufflation, the compounds of the present invention may take the form of a dry powder composition, for example a powder mix of the compound and a suitable powder base, such as lactose or starch. The powder composition may be presented in a unit dosage form, in for example, capsules, cartridges, gelatin or blister packs from which the powder may be administered with the aid of an inhalator or insufflators.
When desired, the above-described formulations, adapted to give sustained release of the active ingredient, may be employed. The pharmaceutical compositions of the present invention may also contain other active ingredients, such as antimicrobial agents, immunosuppressants or preservatives.
It should be understood that in addition to the ingredients particularly mentioned above, the formulations of the present invention may include other agents conventional in the art, having regard to the type of formulation in question; for example, those suitable for oral administration may include flavoring agents.
Preferred unit dosage formulations are those containing an effective dose, as recited below, or an appropriate fraction thereof, of the active ingredient.
For each of the aforementioned conditions, the compositions may be administered orally or via injection at a dose ranging from about 0.1 to about 250 mg/kg per day. The dose range for adult humans is generally from about 5 mg to about 17.5 g/day, preferably about 5 mg to about 10 g/day, and most preferably about 100 mg to about 3 g/day. Tablets or other unit dosage forms of presentation provided in discrete units may conveniently contain an amount which is effective at such dosage or as a multiple of the same, for instance, units containing about 5 mg to about 500 mg, usually from about 100 mg to about 500 mg.
The pharmaceutical composition preferably is administered orally or by injection (intravenous or subcutaneous), and the precise amount administered to a subject will be the responsibility of the attendant physician. However, the dose employed will depend upon a number of factors, including the age and sex of the subject, the precise disorder being treated, and its severity. In addition, the route of administration may vary depending upon the condition and its severity.
The present invention is based in part on the discovery of the various amino acid substitutions of TTK at kinase domain, especially the mutations resulted in promoting the TTK kinase activity and the invasive ability. In view of the evidence provided herein, that one or more amino acid substitutions of TTK at kinase domain are associated with a metastasis of lung cancer, the present invention thus provides methods for predicting metastasis of lung cancer. An example of such a method includes the steps of: detecting one or more mutations of TTK at kinase domain, and then indicating a high risk of metastasis of lung cancer when a mutation is detected. In the context of the present invention, one or more amino acid substitutions of TTK at kinase domain are Valine to Phenylalanine at codon 610 (V610F), Glutamine to Histidine at codon 753 (Q753H) and Tyrosine to Cysteine at codon 574 (Y574C) of SEQ ID NO: 2. The detection can be accomplished by sequencing, mini-sequencing, hybridization, restriction fragment analysis, oligonucleotide ligation assay or allele specific PCR.
It is revealed herein that the presence of a previously unknown TTK mutant correlates with a high risk of metastasis of lung cancer. Based on this finding, the present invention also provides a method and reagent for detecting such TTK mutant. For instance, the present invention provides a method for detecting one or more mutation of TTK, wherein the mutation is at least one mutation selected from the group consisted of Valine to Phenylalanine at codon 610 (V610F), Glutamine to Histidine at codon 753 (Q753H) and Tyrosine to Cysteine at codon 574 (Y574C) of SEQ ID NO: 2. The method of the present invention includes steps of:
In the present invention, a binding agent recognizing any one of the mutation of the polypeptide is preferably an antibody that binds to polypeptide having at least one mutation selected from the group consisted of Valine to Phenylalanine at codon 610 (V610F), Glutamine to Histidine at codon 753 (Q753H) and Tyrosine to Cysteine at codon 574 (Y574C) of SEQ ID NO: 2, and substantially not binds to the polypeptide of SEQ ID NO: 2. Further, the antibody may be monoclonal antibody or polyclonal antibody, or fragment thereof that contains the antigen-binding region. In the present invention, such antibodies may be obtained by conventional methods of immunization of TTK polypeptide or immunological-active fragment containing the mutation. Alternatively, a screening of antibody variable region recognizing the TTK mutant from antibody library may be performed using TTK mutant as antigen.
In the present invention, a preferred antibody will specifically recognize the TTK mutant. Such antibody is referred as TTK mutant specific antibody. Alternatively, in the present invention, a preferred TTK mutant specific antibody will substantially not bind to the polypeptide of SEQ ID NO: 2. In the context, antibody which substantially does not bind with wild type of TTK having the amino acid sequence of SEQ ID NO: 2 shows generally 30% or less, preferably 20% or less, more preferably 10% or less of reactivity with wild type of TTK, comparing with that of TTK mutant.
In the present invention, the TTK mutant includes at least one mutation selected from the group consisted of Valine to Phenylalanine at codon 610 (V610F), Glutamine to Histidine at codon 753 (Q753H) and Tyrosine to Cysteine at codon 574 (Y574C) of SEQ ID NO: 2. Such mutant may be prepared by conventional method of introducing site specific mutations into the protein. For example, one skilled in the art can prepare proteins having the mutation by introducing site specific mutation in the amino acid sequence of the human TTK protein via site-directed mutagenesis (Hashimoto-Gotoh, T. et al. (1995), Gene 152, 271-5; Zoller, M J, and Smith, M. (1983), Methods Enzymol. 100, 468-500; Kramer, W. et al. (1984), Nucleic Acids Res. 12, 9441-56; Kramer W, and Fritz H J. (1987) Methods. Enzymol. 154, 350-67; Kunkel, T A (1985), Proc. Natl. Acad. Sci. USA. 82, 488-92; Kunkel (1991), Methods Enzymol. 204, 125-39).
In the present invention, TTK mutant can also be detected by determining of the nucleotide sequence of cDNA encoding TTK mutant. For example, primers or probes annealing with the cDNA (or mRNA) at region may include any one codon of Valine to Phenylalanine at codon 610 (V610F), Glutamine to Histidine at codon 753 (Q753H) and Tyrosine to Cysteine at codon 574 (Y574C) of SEQ ID NO: 2.
Further, the present invention also provides a reagent for detecting one or more mutation of TTK, wherein the mutation is at least one mutation selected from the group consisted of Valine to Phenylalanine at codon 610 (V610F), Glutamine to Histidine at codon 753 (Q753H) and Tyrosine to Cysteine at codon 574 (Y574C) of SEQ ID NO: 2, wherein the reagent includes a binding agent recognizing any one of the mutation of the polypeptide or cDNA encoding thereto.
In addition, polynucleotides that encode a TTK mutant or fragment thereof that includes the mutated position are useful as control samples for detecting the mutation. Accordingly, the present invention provides an isolated polynucleotide having the nucleotide sequence of SEQ ID NO: 1, in which one or more mutations selected from group consisting of A1870G (for Y574C), G1977T (for V610F), and G2408C (for Q753H) are included, or fragment thereof containing the one or more mutations. For example, in the detection of the mutation using a hybridization technique, polynucleotide of TTK mutant can be used as positive control. In the present invention, any fragment of the polynucleotide of TTK mutant may be used as such control, unless at least one mutation is included. Preferable length of the fragments of the polynucleotide of TTK mutant is generally 25 or more, 50 or more, 100 or more, and more preferably 200 or more nucleotides.
A polypeptide encoding a TTK mutant or fragment thereof containing the mutated position is also useful as control sample for detecting the mutation. Accordingly, the present invention provides an isolated polypeptide having the amino acid sequence of SEQ ID NO: 2, in which one or more mutations selected from group consisting of V610F, Q753H and for Y574C are included, or fragment thereof containing the one or more mutations. For example, in the detection of the mutation using an immunoassay technique, polypeptide of TTK mutant can be used as positive control. In the present invention, any fragment of the polypeptide of TTK mutant may be used as such control, unless at least one mutation is included. The mutation of TTK polypeptide can also be detected by molecular mass analysis of whole peptide or fragment thereof. For instance, preferable technique for the molecular mass analysis is MALDI-TOF MS. Length of the fragments of the polypeptide of TTK mutant is generally 10 or more, preferably 20 or more, or 50 or more, and more preferably 100 or more amino acid residues.
Dominant Negative Protein that Inhibits TTK Kinase Activity for EGFR
The present invention relates to inhibitory polypeptides that contain ISSILEKGERLPQPPICTI (SEQ ID NO: 44) or DVYMIMVKCWMIDADSRPK (SEQ ID NO: 45) or FRELIIEFSKMARDPQRYL (SEQ ID NO: 46). In some preferred embodiments, the inhibitory polypeptide comprises ISSILEKGERLPQPPICTI (SEQ ID NO: 44) or DVYMIMVKCWMIDADSRPK (SEQ ID NO: 45) or FRELIIEFSKMARDPQRYL (SEQ ID NO: 46); a polypeptide functionally equivalent to the polypeptide; or polynucleotide encoding those polypeptides, wherein the polypeptide lacks the kinase activity of TTK for EGFR. It is a novel finding proved by the present invention that that EGFR fragment inhibits the lung cancer cell proliferation.
The polypeptides comprising the selected amino acid sequence of the present invention, can be of any length, so long as the polypeptide contain the amino acid sequence of ISSILEKGERLPQPPICTI (SEQ ID NO: 44) or DVYMIMVKCWMIDADSRPK (SEQ ID NO: 45) or FRELIIEFSKMARDPQRYL (SEQ ID NO: 46) and inhibits cancer cell proliferation. For example, the length of the amino acid sequence may range from 19 to 76 residues preferably from 19 to 57, more preferably from 19 to 38.
The polypeptides of the present invention may contain two or more “selected amino acid sequences”. The two or more “selected amino acid sequences” may be the same or different amino acid sequences. Furthermore, the “selected amino acid sequences” can be linked directly. Alternatively, they may be disposed with any intervening sequences among them.
Furthermore, the present invention relates to polypeptides homologous (i.e., share sequence identity) to the ISSILEKGERLPQPPICTI (SEQ ID NO: 44) or DVYMIMVKCWMIDADSRPK (SEQ ID NO: 45) or FRELIIEFSKMARDPQRYL (SEQ ID NO: 46) polypeptide specifically disclosed here. In the present invention, polypeptides homologous to the ISSILEKGERLPQPPICTI (SEQ ID NO: 44) or DVYMIMVKCWMIDADSRPK (SEQ ID NO: 45) or FRELIIEFSKMARDPQRYL (SEQ ID NO: 46) polypeptide are those which contain any mutations selected from addition, deletion, substitution and insertion of one or several amino acid residues and are functionally equivalent. The phrase “functionally equivalent” refers to having the function to inhibit the kinase activity of TTK for EGFR and inhibit the cell proliferation. Therefore, polypeptides functionally equivalent to the ISSILEKGERLPQPPICTI (SEQ ID NO: 44) or DVYMIMVKCWMIDADSRPK (SEQ ID NO: 45) or FRELIIEFSKMARDPQRYL (SEQ ID NO: 46) peptide in the present invention preferably have amino acid mutations in sites other than the ISSILEKGERLPQPPICTI (SEQ ID NO: 44) or DVYMIMVKCWMIDADSRPK (SEQ ID NO: 45) or FRELIIEFSKMARDPQRYL (SEQ ID NO: 46) sequence. Amino acid sequences of polypeptides functionally equivalent to the ISSILEKGERLPQPPICTI (SEQ ID NO: 44) or DVYMIMVKCWMIDADSRPK (SEQ ID NO: 45) or FRELIIEFSKMARDPQRYL (SEQ ID NO: 46) peptide in the present invention conserve the ISSILEKGERLPQPPICTI (SEQ ID NO: 44) or DVYMIMVKCWMIDADSRPK (SEQ ID NO: 45) or FRELIIEFSKMARDPQRYL (SEQ ID NO: 46) sequence, and have 60% or higher, usually 70% or higher, preferably 80% or higher, more preferably 90% or higher, or 95% or higher, and further more preferably 98% or higher homology to a “selected amino acid sequence”. Amino acid sequence homology can be determined using algorithms well known in the art, for example, BLAST or ALIGN set to their default settings.
The polypeptides of the present invention can be chemically synthesized from any position based on selected amino acid sequences. Methods used in the ordinary peptide chemistry can be used for the method of synthesizing polypeptides. Specifically, the methods include those described in the following documents and Japanese Patent publications:
The polypeptides of the present invention can be also synthesized by known genetic engineering techniques. An example of genetic engineering techniques is as follows. Specifically, DNA encoding a desired peptide is introduced into an appropriate host cell to prepare a transformed cell. The polypeptides of the present invention can be obtained by recovering polypeptides produced by this transformed cell. Alternatively, a desired polypeptide can be synthesized with an in vitro translation system, in which necessary elements for protein synthesis are reconstituted in vitro.
When genetic engineering techniques are used, the polypeptide of the present invention can be expressed as a fused protein with a peptide having a different amino acid sequence. A vector expressing a desired fusion protein can be obtained by linking a polynucleotide encoding the polypeptide of the present invention to a polynucleotide encoding a different peptide so that they are in the same reading frame, and then introducing the resulting nucleotide into an expression vector. The fusion protein is expressed by transforming an appropriate host with the resulting vector. Different peptides to be used in forming fusion proteins include the following peptides:
FLAG (Hopp et al., (1988) BioTechnology 6, 1204-10),
6×His consisting of six H is (histidine) residues, 10×His,
Influenza hemagglutinin (HA),
Human c-myc fragment,
VSV-GP fragment,
p18 HIV fragment,
T7-tag,
HSV-tag,
E-tag,
SV40T antigen fragment,
lck tag,
α-tubulin fragment,
B-tag,
Protein C fragment,
GST (glutathione-S-transferase),
HA (Influenza hemagglutinin),
Immunoglobulin constant region,
β-galactosidase, and
MBP (maltose-binding protein).
The polypeptide of the present invention can be obtained by treating the fusion protein thus produced with an appropriate protease, and then recovering the desired polypeptide. To purify the polypeptide, the fusion protein is captured in advance with affinity chromatography that binds with the fusion protein, and then the captured fusion protein can be treated with a protease. With the protease treatment, the desired polypeptide is separated from affinity chromatography, and the desired polypeptide with high purity is recovered.
The polypeptides of the present invention include modified polypeptides. In the present invention, the term “modified” refers, for example, to binding with other substances. Accordingly, in the present invention, the polypeptides of the present invention may further comprise other substances such as cell-membrane permeable substance. The other substances include organic compounds such as peptides, lipids, saccharides, and various naturally-occurring or synthetic polymers. The polypeptides of the present invention may have any modifications so long as the polypeptides retain the desired activity of inhibiting the kinase activity of TTK for EGFR. In some embodiments, the inhibitory polypeptides can directly compete with EGFR binding to TTK. Modifications can also confer additive functions on the polypeptides of the invention. Examples of the additive functions include targetability, deliverability, and stabilization.
Preferred examples of modifications in the present invention include, for example, the introduction of a cell-membrane permeable substance. Usually, the intracellular structure is cut off from the outside by the cell membrane. Therefore, it is difficult to efficiently introduce an extracellular substance into cells. Cell membrane permeability can be conferred on the polypeptides of the present invention by modifying the polypeptides with a cell-membrane permeable substance. As a result, by contacting the polypeptide of the present invention with a cell, the polypeptide can be delivered into the cell to act thereon.
The “cell-membrane permeable substance” refers to a substance capable of penetrating the mammalian cell membrane to enter the cytoplasm. For example, a certain liposome fuses with the cell membrane to release the content into the cell. Meanwhile, a certain type of polypeptide penetrates the cytoplasmic membrane of mammalian cell to enter the inside of the cell. For polypeptides having such a cell-entering activity, cytoplasmic membranes and such in the present invention are preferable as the substance. Specifically, the present invention includes polypeptides having the following general formula.
[R]−[D];
wherein,
[R] represents a cell-membrane permeable substance; [D] represents a fragment sequence containing ISSILEKGERLPQPPICTI (SEQ ID NO: 44) or DVYMIMVKCWMIDADSRPK (SEQ ID NO: 45) or FRELIIEFSKMARDPQRYL (SEQ ID NO: 46). In the above-described general formula, [R] and [D] can be linked directly or indirectly through a linker. Peptides, compounds having multiple functional groups, or such can be used as a linker. Specifically, amino acid sequences containing -GGG- can be used as a linker. Alternatively, a cell-membrane permeable substance and a polypeptide containing a selected sequence can be bound to the surface of a minute particle. [R] can be linked to any positions of [D]. Specifically, [R] can be linked to the N terminal or C terminal of [D], or to a side chain of amino acids constituting [D]. Furthermore, more than one [R] molecule can be linked to one molecule of [D]. The [R] molecules can be introduced to different positions on the [D] molecule. Alternatively, [D] can be modified with a number of [R]s linked together.
For example, there have been reported a variety of naturally-occurring or artificially synthesized polypeptides having cell-membrane permeability (Joliot A. & Prochiantz A., Nat Cell Biol. 2004; 6: 189-96). All of these known cell-membrane permeable substances can be used for modifying polypeptides in the present invention. In the present invention, for example, any substance selected from the following group can be used as the above-described cell-permeable substance:
In the present invention, the poly-arginine, which is listed above as an example of cell-membrane permeable substances, is constituted by any number of arginine residues. Specifically, for example, it is constituted by consecutive 5-20 arginine residues. The preferable number of arginine residues is 11 (SEQ ID NO: 60).
The polypeptides of the present invention inhibit proliferation of lung cancer cells. Therefore, the present invention provides therapeutic and/or preventive agents for cancer which comprise as an active ingredient a polypeptide which comprises ISSILEKGERLPQPPICTI (SEQ ID NO: 44) or DVYMIMVKCWMIDADSRPK (SEQ ID NO: 45) or FRELIIEFSKMARDPQRYL (SEQ ID NO: 46); or a polynucleotide encoding the same. Alternatively, the present invention relates to methods for treating and/or preventing lung cancer comprising the step of administering a polypeptide of the present invention. Furthermore, the present invention relates to the use of the polypeptides of the present invention in manufacturing pharmaceutical compositions for treating and/or preventing lung cancer. Furthermore, the present invention also relates to a polypeptide selected from peptides comprising ISSILEKGERLPQPPICTI (SEQ ID NO: 44) or DVYMIMVKCWMIDADSRPK (SEQ ID NO: 45) or FRELIIEFSKMARDPQRYL (SEQ ID NO: 46) for treating and/or preventing lung cancer.
Alternatively, the inhibitory polypeptides of the present invention can be used to induce apoptosis of cancer cells. Therefore, the present invention provides apoptosis inducing agents for cells, which comprise as an active ingredient a polypeptide which comprises ISSILEKGERLPQPPICTI (SEQ ID NO: 44) or DVYMIMVKCWMIDADSRPK (SEQ ID NO: 45) or FRELIIEFSKMARDPQRYL (SEQ ID NO: 46); or a polynucleotide encoding the same. The apoptosis inducing agents of the present invention may be used for treating cell proliferative diseases such as cancer. Alternatively, the present invention relates to methods for inducing apoptosis of cells which comprise the step of administering the polypeptides of the present invention. Furthermore, the present invention relates to the use of polypeptides of the present invention in manufacturing pharmaceutical compositions for inducing apoptosis in cells.
The inhibitory polypeptides of the present invention induce apoptosis in TTK-expressing cells such as lung cancer. In the meantime, TTK expression has not been observed in most of normal organs. In some normal organs, the expression level of TTK is relatively low as compared with lung cancer tissues. Accordingly, the polypeptides of the present invention may induce apoptosis specifically in lung cancer cells.
When the polypeptides of the present invention are administered, as a prepared pharmaceutical, to human and other mammals such as mouse, rat, guinea pig, rabbit, cat, dog, sheep, pig, cattle, monkey, baboon and chimpanzee for treating lung cancer or inducing apoptosis in cells, isolated compounds can be administered directly, or formulated into an appropriate dosage form using known methods for preparing pharmaceuticals. For example, if necessary, the pharmaceuticals can be orally administered as a sugar-coated tablet, capsule, elixir, and microcapsule, or alternatively parenterally administered in the injection form that is a sterilized solution or suspension with water or any other pharmaceutically acceptable liquid. For example, the compounds can be mixed with pharmacologically acceptable carriers or media, specifically sterilized water, physiological saline, plant oil, emulsifier, suspending agent, surfactant, stabilizer, corrigent, excipient, vehicle, preservative, and binder, in a unit dosage form necessary for producing a generally accepted pharmaceutical. Depending on the amount of active ingredient in these formulations, a suitable dose within the specified range can be determined.
Examples of additives that can be mixed in tablets and capsules are binders such as gelatin, corn starch, tragacanth gum, and gum arabic; media such as crystalline cellulose; swelling agents such as corn starch, gelatin, and alginic acid; lubricants such as magnesium stearate; sweetening agents such as sucrose, lactose or saccharine; and corrigents such as peppermint, wintergreen oil and cherry. When the unit dosage from is capsule, liquid carriers such as oil can be further included in the above-described ingredients. Sterilized mixture for injection can be formulated using media such as distilled water for injection according to the realization of usual pharmaceuticals.
Physiological saline, glucose, and other isotonic solutions containing, adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride can be used as an aqueous solution for injection. They can be used in combination with a suitable solubilizer, for example, alcohol, specifically ethanol and polyalcohols such as propylene glycol and polyethylene glycol, non-ionic surfactants such as Polysorbate 80™ and HCO-50.
Sesame oil or soybean oil can be used as an oleaginous liquid, and also used in combination with benzyl benzoate or benzyl alcohol as a solubilizer. Furthermore, they can be further formulated with buffers such as phosphate buffer and sodium acetate buffer; analgesics such as procaine hydrochloride; stabilizers such as benzyl alcohol and phenol; and antioxidants. Injections thus prepared can be loaded into appropriate ampoules.
Methods well-known to those skilled in the art can be used for administering pharmaceutical compounds of the present invention to patients, for example, by intraarterial, intravenous, or subcutaneous injection, and similarly, by intranasal, transtracheal, intramuscular, or oral administration. Doses and administration methods are varied depending on the body weight and age of patients as well as administration methods. However, those skilled in the art can routinely select them. DNA encoding a polypeptide of the present invention can be inserted into a vector for the gene therapy, and the vector can be administered for treatment. Although doses and administration methods are varied depending on the body weight, age, and symptoms of patients, those skilled in the art can appropriately select them. For example, a dose of the compound which bind to the polypeptides of the present invention so as to regulate their activity is, when orally administered to a normal adult (body weight 60 kg), about 0.1 mg to about 100 mg/day, preferably about 1.0 mg to about 50 mg/day, more preferably about 1.0 mg to about 20 mg/day, although it is slightly varied depending on symptoms.
When the compound is parenterally administered to a normal adult (body weight 60 kg) in the injection form, it is convenient to intravenously inject a dose of about 0.01 mg to about 30 mg/day, preferably about 0.1 mg to about 20 mg/day, more preferably about 0.1 mg to about 10 mg/day, although it is slightly varied depending on patients, target organs, symptoms, and administration methods. Similarly, the compound can be administered to other animals in an amount converted from the dose for the body weight of 60 kg.
siRNA of TTK or EGFR.
siRNA of TTK or EGFR gene can be used to reduce the expression level of the TTK or EGFR gene. For example, siRNA of TTK or EGFR gene is useful for the treatment of lung cancer. Specifically, siRNA of the present invention can act by binding to mRNAs corresponding thereto, thereby promoting the degradation of the mRNA, and/or inhibiting the expression of protein encoded by the TTK or EGFR gene, thereby, inhibiting the function of the protein.
The term “polynucleotide” and “oligonucleotide” are used interchangeably herein unless otherwise specifically indicated and are referred to by their commonly accepted single-letter codes. The terms apply to nucleic acid (nucleotide) polymers in which one or more nucleic acids are linked by ester bonding. The polynucleotide or oligonucleotide may be composed of DNA, RNA or a combination thereof.
As use herein, the term “double-stranded molecule” refers to a nucleic acid molecule that inhibits expression of a target gene including, for example, short interfering RNA (siRNA; e.g., double-stranded ribonucleic acid (dsRNA) or small hairpin RNA (shRNA)) and short interfering DNA/RNA (siD/R-NA; e.g. double-stranded chimera of DNA and RNA (dsD/R-NA) or small hairpin chimera of DNA and RNA (shD/R-NA)).
Also, an siRNA against the TTK or EGFR gene can be used to reduce the expression level of the TTK or EGFR gene. Herein, term “siRNA” refers to a double stranded RNA molecule which prevents translation of a target mRNA. Standard techniques for introducing siRNA into the cell can be used, including those in which DNA is a template from which RNA is transcribed. In the context of the present invention, the siRNA is composed of a sense nucleic acid sequence and an anti-sense nucleic acid sequence against SEQ ID NO: 62 or 63. The siRNA is constructed such that a single transcript has both the sense and complementary antisense sequences from the target gene, e.g., a hairpin. The siRNA may either be a dsRNA or shRNA.
As used herein, the term “dsRNA” refers to a construct of two RNA molecules having sequences complementary to one another annealed together via the complementary sequences to form a double-stranded RNA molecule. The nucleotide sequence of two strands may include not only the “sense” or “antisense” RNAs selected from a protein coding sequence of target gene sequence, but also RNA molecule having a nucleotide sequence selected from non-coding region of the target gene.
The term “shRNA”, as used herein, refers to an siRNA having a stem-loop structure, composed of first and second regions complementary to one another, i.e., sense and antisense strands. The degree of complementarity and orientation of the regions being sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The loop region of an shRNA is a single-stranded region intervening between the sense and antisense strands and may also be referred to as “intervening single-strand”.
As use herein, the term “siD/R-NA” refers to a double-stranded polynucleotide molecule which is composed of both RNA and DNA, and includes hybrids and chimeras of RNA and DNA and prevents translation of a target mRNA. Herein, a hybrid indicates a molecule wherein a polynucleotide composed of DNA and a polynucleotide composed of RNA hybridize to each other to form the double-stranded molecule; whereas a chimera indicates that one or both of the strands composing the double stranded molecule may contain RNA and DNA. Standard techniques of introducing siD/R-NA into the cell are used. The siD/R-NA includes a TTK or EGFR sense nucleic acid sequence (also referred to as “sense strand”), a TTK or EGFR antisense nucleic acid sequence (also referred to as “antisense strand”) or both. The siD/R-NA may be constructed such that a single transcript has both the sense and complementary antisense nucleic acid sequences from the target gene, e.g., a hairpin. The siD/R-NA may either be a dsD/R-NA or shD/R-NA.
As used herein, the term “dsD/R-NA” refers to a construct of two molecules having sequences complementary to one another annealed together via the complementary sequences to form a double-stranded polynucleotide molecule. The nucleotide sequence of two strands may include not only the “sense” or “antisense” polynucleotides sequence selected from a protein coding sequence of target gene sequence, but also polynucleotide having a nucleotide sequence selected from non-coding region of the target gene. One or both of the two molecules constructing the dsD/R-NA are composed of both RNA and DNA (chimeric molecule), or alternatively, one of the molecules is composed of RNA and the other is composed of DNA (hybrid double-strand).
The term “shD/R-NA”, as used herein, refers to an siD/R-NA having a stem-loop structure, including first and second regions complementary to one another, i.e., sense and antisense strands. The degree of complementarity and orientation of the regions being sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The loop region of an shD/R-NA is a single-stranded region intervening between the sense and antisense strands and may also be referred to as “intervening single-strand”
The double-stranded molecules of the invention may contain one or more modified nucleotides and/or non-phosphodiester linkages. Chemical modifications well known in the art are capable of increasing stability, availability, and/or cell uptake of the double-stranded molecule. The skilled person will be aware of other types of chemical modification which may be incorporated into the present molecules (WO03/070744; WO2005/045037). In one embodiment, modifications can be used to provide improved resistance to degradation or improved uptake. Examples of such modifications include phosphorothioate linkages, 2′-O-methyl-4′ linked ribonucleotides, 2′-O-methyl ribonucleotides (especially on the sense strand of a double-stranded molecule), 2′-deoxy-fluoro ribonucleotides, 2′-deoxy ribonucleotides, “universal base” nucleotides, 5′-C— methyl nucleotides, and inverted deoxyabasic residue incorporation (US20060122137).
In another embodiment, modifications can be used to enhance the stability or to increase targeting efficiency of the double-stranded molecule. Modifications include chemical cross linking between the two complementary strands of a double-stranded molecule, chemical modification of a 3′ or 5′ terminus of a strand of a double-stranded molecule, sugar modifications, nucleobase modifications and/or backbone modifications, 2-fluoro modified ribonucleotides and 2′-deoxy ribonucleotides (WO2004/029212). In another embodiment, modifications can be used to increased or decreased affinity for the complementary nucleotides in the target mRNA and/or in the complementary double-stranded molecule strand (WO2005/044976). For example, an unmodified pyrimidine nucleotide can be substituted for a 2-thio, 5-alkynyl, 5-methyl, or 5-propynyl pyrimidine. Additionally, an unmodified purine can be substituted with a 7-deza, 7-alkyl, or 7-alkenyi purine. In another embodiment, when the double-stranded molecule is a double-stranded molecule with a 3′ overhang, the 3′-terminal nucleotide overhanging nucleotides may be replaced by deoxyribonucleotides (Elbashir S M et al., Genes Dev 2001 Jan. 15, 15(2): 188-200). For further details, published documents such as US20060234970 are available. The present invention is not limited to these examples and any known chemical modifications may be employed for the double-stranded molecules of the present invention so long as the resulting molecule retains the ability to inhibit the expression of the target gene.
Furthermore, the double-stranded molecules of the invention may include both DNA and RNA, e.g., dsD/R-NA or shD/R-NA. Specifically, a hybrid polynucleotide of a DNA strand and an RNA strand or a DNA-RNA chimera polynucleotide shows increased stability. Mixing of DNA and RNA, i.e., a hybrid type double-stranded molecule consisting of a DNA strand (polynucleotide) and an RNA strand (polynucleotide), a chimera type double-stranded molecule including both DNA and RNA on any or both of the single strands (polynucleotides), or the like may be formed for enhancing stability of the double-stranded molecule. The hybrid of a DNA strand and an RNA strand may be the hybrid in which either the sense strand is DNA and the antisense strand is RNA, or the opposite so long as it has an activity to inhibit expression of the target gene when introduced into a cell expressing the gene. Preferably, the sense strand polynucleotide is DNA and the antisense strand polynucleotide is RNA. Also, the chimera type double-stranded molecule may be either where both of the sense and antisense strands are composed of DNA and RNA, or where any one of the sense and antisense strands is composed of DNA and RNA so long as it has an activity to inhibit expression of the target gene when introduced into a cell expressing the gene.
In order to enhance stability of the double-stranded molecule, the molecule preferably contains as much DNA as possible, whereas to induce inhibition of the target gene expression, the molecule is required to be RNA within a range to induce sufficient inhibition of the expression. As a preferred example of the chimera type double-stranded molecule, an upstream partial region (i.e., a region flanking to the target sequence or complementary sequence thereof within the sense or antisense strands) of the double-stranded molecule is RNA. Preferably, the upstream partial region indicates the 5′ side (5′-end) of the sense strand and the 3′ side (3′-end) of the antisense strand. The upstream partial region preferably is a domain consisting of 9 to 13 nucleotides counted from the terminus of the target sequence or complementary sequence thereto within the sense or antisense strands of the double-stranded molecules. Moreover, preferred examples of such chimera type double-stranded molecules include those having a strand length of 19 to 21 nucleotides in which at least the upstream half region (5′ side region for the sense strand and 3′ side region for the antisense strand) of the polynucleotide is RNA and the other half is DNA. In such a chimera type double-stranded molecule, the effect to inhibit expression of the target gene is much higher when the entire antisense strand is RNA (US20050004064).
In the present invention, the double-stranded molecule may form a hairpin, such as a short hairpin RNA (shRNA) and short hairpin consisting of DNA and RNA (shD/R-NA). The shRNA or shD/R-NA is a sequence of RNA or mixture of RNA and DNA making a tight hairpin turn that can be used to silence gene expression via RNA interference. The shRNA or shD/R-NA preferably includes the sense target sequence and the antisense target sequence on a single strand wherein the sequences are separated by a loop sequence. Generally, the hairpin structure is cleaved by the cellular machinery into dsRNA or dsD/R-NA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs which match the target sequence of the dsRNA or dsD/R-NA.
In another embodiment, halogenated RNAs, RNAs partially replaced with DNAs, or methylated RNAs can be used to confer RNAase resistance to the siRNA. Such nucleic acid derivatives that confer RNAase resistance are also included in the double-stranded RNA. In the present invention, the double stranded molecule may include a double stranded RNA constructed from ribonucleotides, modified ribonucleotides, or ribonucleotide derivatives.
An siRNA of the TTK or EGFR gene hybridizes to target mRNA and thereby decreases or inhibits production of the polypeptides encoded by the TTK or EGFR gene by associating with the normally single-stranded mRNA transcript, thereby interfering with translation and thus, expression of the protein. In the context of the present invention, an siRNA is preferably less than 500, 200, 100, 50, or 25 nucleotides in length. More preferably an siRNA is 19-25 nucleotides in length. Exemplary nucleic acid sequence for the production of TTK or EGFR siRNA includes the sequences of nucleotides of SEQ ID NOs: 63 or 64 as the target sequence. In order to enhance the inhibition activity of the siRNA, one or more uridine (“u”) nucleotides can be added to 3′ end of the antisense strand of the target sequence. The number of “u's” to be added is at least 2, generally 2 to 10, preferably 2 to 5. The added “u's” form a single strand at the 3′ end of the antisense strand of the siRNA.
An siRNA of the TTK or EGFR gene can be directly introduced into the cells in a form that is capable of binding to the mRNA transcripts. Alternatively, a DNA encoding the siRNA can be carried in a vector.
Vectors can be produced, for example, by cloning an TTK or EGFR gene target sequence into an expression vector having operatively-linked regulatory sequences flanking the sequence in a manner that allows for expression (by transcription of the DNA molecule) of both strands (Lee, N. S., et al., (2002) Nature Biotechnology 20: 500-5). An RNA molecule that is antisense to mRNA of the TTK or EGFR gene is transcribed by a first promoter (e.g., a promoter sequence 3′ of the cloned DNA) and an RNA molecule that is the sense strand for the mRNA of the TTK or EGFR gene is transcribed by a second promoter (e.g., a promoter sequence 5′ of the cloned DNA). The sense and antisense strands hybridize in vivo to generate siRNA constructs for silencing of the TTK or EGFR gene. Alternatively, the two constructs can be utilized to create the sense and anti-sense strands of a siRNA construct. Cloned TTK or EGFR gene can encode a construct having secondary structure, e.g., hairpins, wherein a single transcript has both the sense and complementary antisense sequences from the target gene.
A loop sequence consisting of an arbitrary nucleotide sequence can be located between the sense and antisense sequence in order to form the hairpin loop structure. Thus, the present invention also provides siRNA having the general formula 5′-[A]-[B]-[A′]-3′,
wherein [A] is a ribonucleotide sequence corresponding to a sequence of the TTK or EGFR gene,
[B] is a ribonucleotide sequence composed of 3 to 23 nucleotides, and
[A′] is a ribonucleotide sequence having the complementary sequence of [A].
The region [A] hybridizes to [A′], and then a loop composed of region [B] is formed. The loop sequence can be 3 to 23 nucleotides in length. The loop sequence, for example, can be selected from the following sequences (found on the worldwide web at ambion.com/techlib/tb/tb—506.html). Furthermore, a loop sequence consisting of 23 nucleotides also provides active siRNA (Jacque, J. M., et al., (2002) Nature 418: 435-8.).
CCC, CCACC or CCACACC: Jacque, J. M, et al., (2002) Nature, Vol. 418: 435-8.
UUCG: Lee, N. S., et al., (2002) Nature Biotechnology 20: 500-5.; Fruscoloni, P., et al., (2003) Proc. Natl. Acad. Sci. USA 100(4): 1639-44.
UUCAAGAGA: Dykxhoorn, D. M., et al., (2003) Nature Reviews Molecular Cell Biology 4: 457-67.
Accordingly, in some embodiments, the loop sequence can be selected from group consisting of, CCC, UUCG, CCACC, CCACACC, and UUCAAGAGA. A preferable loop sequence is UUCAAGAGA (“ttcaagaga” in DNA). Exemplary hairpin siRNA suitable for use in the context of the present invention include:
The nucleotide sequence of suitable siRNAs can be designed using an siRNA design computer program available from the Ambion website on the worldwide web at ambion.com/techlib/misc/siRNA_finder.html. The computer program selects nucleotide sequences for siRNA synthesis based on the following protocol.
The regulatory sequences flanking the TTK or EGFR gene sequences can be identical or different, such that their expression can be modulated independently, or in a temporal or spatial manner. siRNAs are transcribed intracellularly by cloning the TTK or EGFR gene templates, respectively, into a vector containing, e.g., a RNA polymerase III transcription unit from the small nuclear RNA (snRNA) U6 or the human Hi RNA promoter. For introducing the vector into the cell, transfection-enhancing agent can be used. FuGENE (Roche diagnostics), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen), and Nucleofector (Wako pure Chemical) are useful as the transfection-enhancing agent.
The siRNA of the present invention inhibits the expression of a polypeptide of the present invention and is thereby useful for suppressing the biological activity of a polypeptide of the invention. Also, expression-inhibitors, including the siRNA of the invention, are useful in the point that they can inhibit the biological activity of the polypeptide of the invention. Therefore, a composition composed of one or more siRNAs of the present invention is useful for treating a lung cancer. Alternatively, the present invention provides use of inhibitory nucleic acids including the siRNAs, or vector expressing the nucleic acids for manufacturing a pharmaceutical composition for treating or preventing lung cancer. Further, the present invention also provides such inhibitory nucleic acids including the siRNAs, or vector expressing the nucleic acids for treating or preventing lung cancer.
Patients with tumors characterized as over-expressing TTK or EGFR are treated by administering siRNA of TTK or EGFR, respectively. siRNA therapy is used to inhibit expression of TTK or EGFR in patients suffering from or at risk of developing lung cancer. Such patients are identified by standard methods of the particular tumor type. Lung cancer cell is diagnosed for example, by CT, MRI, ERCP, MRCP, computer tomography, or ultrasound. Treatment is efficacious if the treatment leads to clinical benefit such as, a reduction in expression of TTK or EGFR, or a decrease in size, prevalence, or metastatic potential of the tumor in the subject. When treatment is applied prophylactically, “efficacious” means that the treatment retards or prevents tumors from forming or prevents or alleviates a clinical symptom of the tumor. Efficaciousness is determined in association with any known method for diagnosing or treating the particular tumor type.
siRNA therapy is carried out by administering to a patient an siRNA by standard vectors encoding the siRNAs of the invention and/or gene delivery systems such as by delivering the synthetic siRNA molecules. Typically, synthetic siRNA molecules are chemically stabilized to prevent nuclease degradation in vivo. Methods for preparing chemically stabilized RNA molecules are well known in the art. Typically, such molecules comprise modified backbones and nucleotides to prevent the action of ribonucleases. Other modifications are also possible, for example, cholesterol-conjugated siRNAs have shown improved pharmacological properties. (Song et al. Nature Med. 9:347-351 (2003)).
Suitable gene delivery systems may include liposomes, receptor-mediated delivery systems, or viral vectors such as herpes viruses, retroviruses, adenoviruses and adeno-associated viruses, among others. A therapeutic nucleic acid composition is formulated in a pharmaceutically acceptable carrier. The therapeutic composition may also include a gene delivery system as described above. Pharmaceutically acceptable carriers are biologically compatible vehicles which are suitable for administration to an animal, e.g., physiological saline. A therapeutically effective amount of a compound is an amount which is capable of producing a medically desirable result such as reduced production of a TTK or EGFR gene product, reduction of cell growth, e.g., proliferation, or a reduction in tumor growth in a treated animal.
Parenteral administration, such as intravenous, subcutaneous, intramuscular, and intraperitoneal delivery routes, may be used to deliver siRNA compositions of TTK or EGFR. For treatment of lung cancer, direct infusion into the tissue or near the site of cancer, is useful.
Dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular nucleic acid to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Dosage for intravenous administration of nucleic acids is from approximately 106 to 1022 copies of the nucleic acid molecule.
The polynucleotides are administered by standard methods, such as by injection into the interstitial space of tissues such as muscles or skin, introduction into the circulation or into body cavities or by inhalation or insufflation. Polynucleotides are injected or otherwise delivered to the animal with a pharmaceutically acceptable liquid carrier, e.g., a liquid carrier, which is aqueous or partly aqueous. The polynucleotides are associated with a liposome (e.g., a cationic or anionic liposome). The polynucleotide includes genetic information necessary for expression by a target cell, such as a promoter.
Pharmaceutical Compositions Comprising siRNA
Accordingly, the present invention includes medicaments and methods useful in either or both preventing and treating lung cancer. These medicaments and methods comprise a siRNA that inhibits expression of TKK (SEQ ID NO: 62) or EGFR (SEQ ID NO: 63) in an amount effective to achieve attenuation or arrest of disease cell proliferation. More specifically, in the context of the present invention, a therapeutically effective amount means an amount effective to prevent development of, or to alleviate existing symptoms of, the subject being treated.
Individuals to be treated with methods of the present invention include any individual afflicted with lung cancer. Such an individual can be, for example, a vertebrate such as a mammal, including a human, dog, cat, horse, cow, or goat; or any other animal, particularly a commercially important animal or a domesticated animal.
In the context of the present invention, suitable pharmaceutical formulations include those suitable for oral, rectal, nasal, topical (including buccal and sub-lingual), vaginal or parenteral (including intramuscular, sub-cutaneous and intravenous) administration, or for administration by inhalation or insufflation. Preferably, administration is intravenous. The formulations are optionally packaged in discrete dosage units.
Pharmaceutical formulations suitable for oral administration include capsules, cachets or tablets, each containing a predetermined amount of active ingredient. Suitable formulations also include powders, granules, solutions, suspensions and emulsions. The active ingredient is optionally administered as a bolus electuary or paste. Tablets and capsules for oral administration may contain conventional excipients, for example, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol, cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP), binding agents, lubricants, and/or wetting agents. If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
A tablet may be made by compression or molding, optionally with one or more formulational ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredients in a free-flowing form, such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active and/or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may be coated according to methods well known in the art. Oral fluid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), pH maintaining agents, and/or preservatives. The tablets may optionally be formulated so as to provide slow or controlled release of the active ingredient therein. A package of tablets may contain one tablet to be taken on each of the month.
Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, optionally contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; as well as aqueous and non-aqueous sterile suspensions including suspending agents and/or thickening agents. The formulations may be presented in unit dose or multi-dose containers, for example as sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline, water-for-injection, immediately prior to use. Alternatively, the formulations may be presented for continuous infusion. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
Formulations suitable for rectal administration include suppositories with standard carriers such as cocoa butter or polyethylene glycol. Formulations suitable for topical administration in the mouth, for example buccally or sublingually, include lozenges, containing the active ingredient in a flavored base such as sucrose and acacia or tragacanth, and pastilles comprising the active ingredient in a base such as gelatin and glycerin or sucrose and acacia. For intra-nasal administration the compounds of the invention may be used as a liquid spray, a dispersible powder or in the form of drops. Drops may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing agents and/or suspending agents.
For administration by inhalation the compounds can be conveniently delivered from an insufflator, nebulizer, pressurized packs or other convenient means of delivering an aerosol spray. Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.
Alternatively, for administration by inhalation or insufflation, the compounds may take the form of a dry powder composition, for example a powder mix of the compound and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form, for example, as capsules, cartridges, gelatin or blister packs from which the powder may be administered with the aid of an inhalator or insufflators.
Other formulations include implantable devices and adhesive patches; which release a therapeutic agent.
When desired, the above described formulations, adapted to give sustained release of the active ingredient, may be employed. The pharmaceutical compositions may also contain other active ingredients such as antimicrobial agents, immunosuppressants and/or preservatives.
It should be understood that in addition to the ingredients particularly mentioned above, the formulations of this invention may include other agents conventional in the art with regard to the type of formulation in question. For example, formulations suitable for oral administration may include flavoring agents.
It will be apparent to those persons skilled in the art that certain excipients may be more preferable depending upon, for instance, the route of administration, the concentration of test compound being administered, or whether the treatment uses a medicament that includes a protein, a nucleic acid encoding the test compound, or a cell capable of secreting a test compound as the active ingredient.
The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Proper formulation is dependent upon the route of administration chosen.
Lung cancer cell lines were grown in monolayers in appropriate medium supplemented with 10% fetal calf serum (FCS) and were maintained at 37° C. in an atmosphere of humidified air with 5% CO2. Primary lung cancer and metastatic brain tumors derived from lung adenocarcinoma samples had been obtained earlier with informed consent (Kikuchi et al., Oncogene. 2003 Apr. 10; 22(14):2192-205., Int J. Oncol. 2006 April; 28(4):799-805., Taniwaki et al., Int J. Oncol. 2006 September; 29(3):567-75.). An independent set of 366 formalin-fixed primary tumors (234 adenocarcinomas (ADC), 104 squamous-cell carcinomas (SCC), and 28 large-cell carcinomas (LCC)) and adjacent normal lung tissue samples from patients undergoing surgery at Saitama Cancer Center (Saitama, Japan) were used in this invention. A total of 12 SCLC tissue samples were also obtained at Saitama Cancer Center.
Total RNA was extracted from cultured cells and clinical tissues using Trizol reagent (Life Technologies, Inc.) according to the manufacturer's protocol. Extracted RNAs and normal human tissue poly(A) RNAs were treated with DNase I (Nippon Gene) and were reverse-transcribed using oligo(dT)20 primer and SuperScript II reverse transcriptase (Invitrogen). Semiquantitative RT-PCR experiments were carried out with the following synthesized gene-specific primers or with beta-actin (ACTB)-specific primers as an internal control: TTK,
PCR reactions were optimized for the number of cycles to ensure product intensity within the logarithmic phase of amplification.
Human multiple-tissue blots (BD Biosciences Clontech) were hybridized with a 32P-labeled PCR product of TTK. The full-length cDNA of TTK was prepared by RT-PCR using primers. Prehybridization, hybridization, and washing were performed according to the supplier's recommendations. The blots were autoradiographed with intensifying screens at room temperature for 72 hours.
Cells were lysed with RIPA buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 0.5% deoxychorate-Na, 0.1% SDS] containing Protease Inhibitor Cocktail Set III (CALBIOCHEM) and phosphatase inhibitor (1 mM sodium fluoride, 1 mM sodium orthovanadate, 2 mM imidazole, 4 mM sodium tartrate). Cytoplasmic and nuclear extractions were isolated by NE-PER Nuclear and Cytoplasmic Extraction Reagents (PIERCE). Protein samples were separated by SDS-polyacrylamide gels and electroblotted onto Hybond-ECL nitrocellulose membranes (GE Healthcare Bio-sciences). Blots were incubated with a mouse monoclonal anti-Mps1 (TTK) antibody (Upstate), rabbit polyclonal anti-phospho-EGFR antibodies (Tyr-845, Tyr-992, Tyr-1045, Tyr-1068, Tyr-1.48, and Tyr-1173; Cell Signaling Technology, Inc.), a rabbit polyclonal anti-EGFR antibody (Cell Signaling Technology, Inc.), a rabbit polyclonal anti-phospho-PLCgamma1 (Tyr-771) antibody (Cell Signaling Technology, Inc.), a rabbit polyclonal anti-PLCgamma1 antibody (Cell Signaling Technology, Inc.), a rabbit polyclonal anti-phospho-p44/42 MAPK (Thr202/Tyr204) antibody (Cell Signaling Technology, Inc.), a rabbit polyclonal anti-p44/42 MAPK antibody (Cell Signaling Technology, Inc.), a mouse monoclonal anti-Flag antibody (SIGMA) or a mouse monoclonal anti-beta-actin antibody (SIGMA). Anti-phospho-EGFR (Ser-967) antibodies were raised against Ser-967-phosphorylated synthetic peptides and purified with the phosphopeptide column. Antigen-antibody complexes were detected using secondary antibodies conjugated to horseradish peroxidase (GE Healthcare Bio-sciences). Protein bands were visualized by ECL Western Blotting Detection Reagents (GE Healthcare Bio-sciences).
Cultured cells were fixed with 4% paraformaldehyde solution for 15 min at 37° C., or 10% Trichloro acetic acid for 15 min on ice. Fixed cells were incubated in PBS(−) containing 0.1% Triton X-100 for 10 min and subsequently washed with PBS(−). Prior to the primary antibody reaction, fixed cells were covered with CAS-BLOCK (ZYMED Laboratories Inc.) for 10 min to block non-specific-antibody binding. Then the cells were incubated with a mouse monoclonal anti-Mps1 (TTK) antibody (Upstate) and a rabbit polyclonal anti-phospho-EGFR (Tyr-992) (Cell Signaling Technology, Inc.), or a rabbit polyclonal anti-phospho-p44/42 MAPK (Thr202/Tyr204) antibody (Cell Signaling Technology, Inc.), or a mouse monoclonal anti-Flag antibody (SIGMA). Antibodies were stained with an anti-mouse secondary antibody conjugated to Alexa Fluor 488 (Molecular Probes) and an anti-rabbit secondary antibody conjugated to Alexa Fluor 594 (Molecular Probes). DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were viewed and assessed using a confocal microscope at wavelengths of 488, 594 nm (TCS SP2 AOBS: Leica Microsystems).
To investigate the presence of TTK or phospho-EGFR (Tyr-992) protein in clinical samples, the sections were stained using ENVISION+ Kit/horseradish peroxidase (HRP) (DakoCytomation). Briefly, anti-human TTK antibody (NOVUS Biologicals, Inc.) or anti-phospho-EGFR (Tyr-992) antibody (Cell Signaling Technology, Inc.) or anti-phospho-EGFR (Ser-967) antibody (described above), was added after blocking endogenous peroxidase and proteins, and the sections were incubated with HRP-labeled anti-rabbit IgG as the secondary antibody. Substrate-chromogen was added and the specimens were counterstained with hematoxylin.
The tumor tissue microarrays were constructed as published previously (Chin et al., Mol. Pathol. 2003 October; 56(5):275-9.; Callagy et al., Diagn Mol. Pathol. 2003 March; 12(1):27-34., J. Pathol. 2005 February; 205(3):388-96.). The tissue area for sampling was selected based on a visual alignment with the corresponding HE-stained section on a slide. Three, four, or five tissue cores (diameter 0.6 mm; height 3-4 mm) taken from the donor tumor blocks were placed into a recipient paraffin block using a tissue microarrayer (Beecher Instruments). A core of normal tissue was punched from each case. 5-micro-m sections of the resulting microarray block were used for immunohistochemical analysis. Positivity of TTK or phospho-EGFR (Tyr-992) or phospho-EGFR (Ser-967) protein was assessed semiquantitatively by three independent investigators without prior knowledge of the clinicopathological data, each of who recorded staining intensity as absent (score3211d as 0), weak (1+) or strongly positive (2+). Cases were accepted only as strongly positive (2+) if all reviewers defined them as such.
Using contingency tables, attempts were made to correlate clinicopathological variables such as age, gender, histology, smoking history, tumor size (pT), and lymph-node metastasis (pN) with the positivity of TTK and/or phospho-EGFR (Tyr-992) and/or phospho-EGFR (Ser-967) determined by tissue-microarray analysis. Tumor-specific survival curves were calculated from the date of surgery to the time of death related to NSCLC, or to the last follow-up observation. Kaplan-Meier curves were calculated for each relevant variable and for TTK and/or phospho-EGFR (Tyr-992) and/or phospho-EGFR (Ser-967) expression; differences in survival times among patient subgroups were analyzed using the log-rank test. Univariate analyses were performed with the Cox proportional-hazard regression model to determine associations between clinicopathological variables and cancer-related mortality.
(i) vector Based Assay
A vector-based RNA interference (RNAi) system, psiH1BX3.0, had previously established to direct the synthesis of siRNAs in mammalian cells (Suzuki et al., 2003, 2005; Kato et al, 2005; Furukawa et al, 2005). 10 micro-g of siRNA-expression vector were transfected into NSCLC cell lines LC319 and A549 both of which over-expressed TTK endogenously, using 30 micro-1 of Lipofectamine 2000 (Invitrogen). The transfected cells were cultured for five days in the presence of appropriate concentrations of geneticin (G418), after which cell numbers and viability were measured by Giemsa staining and triplicate MTT assays. The target sequences of the synthetic oligonucleotides for RNAi were as follows: control 1 (Luciferase: Photinus pyralis luciferase gene), 5′-CGTACGCGGAATACTTCGA-3′ (SEQ ID NO: 9); control 2 (Scramble: chloroplast Euglena gracilis gene coding for 5S and 16S rRNAs),
To validate our RNAi system, individual control siRNAs (Luciferase and Scramble) were initially confirmed using semiquantitative RT-PCR to decrease expression of the corresponding target genes that had been transiently transfected into COS-7 cells. Down-regulation of TTK expression by si-TTK, but not by controls, was confirmed with semiquantitative RT-PCR in the cell lines used for this assay.
The oligo-siRNAs (Dharmacon, Inc., Lafayette, Colo.) (600 μM) were transfected into appropriate lung-cancer cell lines using 30 micro-1 of Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) following the manufacturer's protocol. The ribonucleotide sequences corresponding to following sequences were used as the oligo-siRNA.
The growth effect of TTK on mammalian cells was also examined using COS-7 and NIH-3T3 cells transiently transfected with plasmids expressing TTK or mock plasmids. The cells were cultured in DMEM containing 10% FCS and geneticin for 14 days, and MTT assay were performed.
To obtain mitotically arrested cells, cultured cells were treated with colcemid (WAKO) for 24 hours before extracts were prepared for western-blot analysis. For phosphatase treatment, cell extract were incubated with lambda-phosphatase (New England Biolabs) in phosphatase buffer or buffer alone for 1 hour at 37° C., then analyzed by immunoblotting.
The full-length cDNA of TTK was subcloned into pFastBac HT vector. The recombinant shuttle vector was transformed into the baculovirus genome (bacmid DNA) in DH10Bac competent cells (Invitrogen) by using Tn7 site-specific transposition according to the minufacture's instructions for the Bac-to-Bac expression system (Invitrogen). The recombinant bacmid DNA was isolated, purified, transfected into Sf9 cells (Invitrogen) (recombinant baculovirus), and used for next infection. Log-phase Sf-9 cells were infected with recombinant baculovirus at a multiplicity of infection (MOI) of 1, and then infected Sf-9 cells were grown in suspension at 27° C. for 72 hours. Extracts of Sf-9 cells were collected and the His fusion proteins were purified using HiTrap HP column (GE Healthcare Bio-sciences) using standard protocols. To construct the catalytically inactive TTK (TTK-KD), a point mutation (Ala) was introduced at codon 647 (Asp). TTK-KD was cloned into pGEX6T vector (GE Healthcare Bio-sciences). GST fusion protein was expressed in E. coli stain BL21 and purified by Glutathione Sepharose 4B (GE Healthcare Bio-sciences) using standard protocols.
To investigate phosphorylation of proteins by TTK, purified recombinant His-tagged TTK was incubated with whole extracts prepared from cell lines or purified GST-tagged proteins containing various cytoplasmic region of EGFR in kinase assay buffer (50 mM Tris, pH 7.4, 10 mM MgCl2, 2 mM dithiothreitol, 1 mM NaF, 0.2 mM ATP) for 60 min at 30° C. The reactions were stopped by addition of Laemmli sample buffer and heating at 95° C. for 5 min. Proteins were resolved by SDS-PAGE and then western-blot or 32P incorporation analysis.
TTK transfectants were harvested, and 5×106 cells were suspended in growth factor-reduced, phenol red-free Mtrigel (BD Bioscience) and injected s.c. into the right dorsum of 6-weeks-old nu/nu BalbC female mice (Charles River Laboratories). Tumor size was measured using a ruler, and tumor volume was calculated using the formula V=(W/2)2×L, where W is the distance across and L is the measurement lengthwise of the tumor.
TTK-expressing stable transfectants were established according to a standard protocol. Using FuGENE 6 Transfection Reagent (Roche Diagnostics), we transfected HEK293 and NIH3T3 cells with plasmids expressing TTK (pCAGGS-n3FH-TTK) or mock plasmids (pCAGGS-n3FH). Transfected cells were cultured in DMEM containing 10% FCS and G418, then individual colonies were trypsinized and screened for stable transfectants by a limiting-dilution assay. Expression of TTK was determined in each clone by RT-PCR, western blotting, and immunostaining. HEK293 or NIH3T3 transfectants that stably expressed TTK were seeded onto 6-well plates (5×104 cells/well), and maintained in medium containing 10% FCS and 0.4 mg/ml G418 for 7 days. At each time point, cell proliferation was evaluated by the MTT assay using Cell Counting Kits (WAKO). All experiments were done in triplicate.
Total RNA was extracted from NSCLC cell lines using RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. Extracted RNAs were treated with DNase I (Nippon Gene) and were reverse-transcribed using oligo (dT) primer and SuperScript II reverse transcriptase (Invitrogen). Amplification of TTK was carried out with the synthesized TTK specific primers,
Then, sequencing analysis of the PCR products were performed using an ABI Prism 3700 DNA sequencer (Applied Biosystems).
Using FuGENE 6 Transfection Reagent (Roche Diagnostics) according to the manufacturer's instructions, NIH-3T3 or COS-7 cells were transfected with plasmids expressing TTK (pCAGGS-n3FH-TTK), TTK-KD (D647A), mutant TTK (originated in RERF-LC-AI cells) or mock plasmids (pCAGGS-n3FH). Transfected cells were harvested and suspended in DMEM without FCS. Before the cell suspension was prepared, the dried layer of Matrigel matrix (Becton Dickinson Labware) was rehydrated with DMEM for 2 hours at room temperature. Then, DMEM containing 10% FCS was added to each lower chamber of 24-well Matrigel invasion chambers and cell suspension was added to each insert of the upper chamber. The plates of inserts were incubated for 22 hours at 37° C. After incubation, cells invading through the Matrigel-coated inserts were fixed and stained by Giemsa.
17˜31 amino acid peptide sequences corresponding to a part of EGFR protein that contained possible TTK-interacting region were covalently linked at its NH2 terminus to a membrane transducing 11 poly-arginine sequence (Hayama, S, et al. Cancer Res. 66, 10339-48 (2006), Futaki S, et al. J Biol. Chem. 276, 5836-5840 (2001)). Three cell-permeable peptides were synthesized:
Scramble peptides derived from the most effective 11R-EGFR937-955 peptides were synthesized as a control: Scramble, RRRRRRRRRRR-GGG-EFMAELLRYFRPQSKRDII. Peptides were purified by preparative reverse-phase high-pressure liquid chromatography. A549 cells were incubated with the 11R peptides at the concentration of 2.5, 5, and 7.5 micro-mol/L for 5 days. The medium was exchanged at every 48 hours at the appropriate concentrations of each peptide and the viability of cells was evaluated by MTT assay at 5 days after the treatment.
To search for novel molecular targets for development of therapeutic agents and/or diagnostic markers for lung cancer, genes that showed 5-fold higher expression in more than 50% of lung cancers analyzed by cDNA microarray were screened first. Among 27,648 genes screened, the gene encoding TTK protein kinase was identified as frequently over-expressed in various types of lung cancers. This expression level was confirmed in 11 of 14 additional NSCLC cases (5 of 7 adenocarcinomas (ADCs) and in 6 of 7 squamous-cell carcinomas (SCCs) (
To verify the biological and clinicopathological significance of TTK, TTK protein expression was examined in clinical NSCLCs by means of tissue microarrays containing NSCLC tissues from 366 patients as well as SCLC tissues from 12 patients. Positive staining for TTK was observed in 68.6% of surgically-resected NSCLCs (251/366) and in 66.7% of SCLCs (8/12), while no staining was observed in any of normal lung tissues examined (
To assess whether TTK is essential for growth/survival of lung-cancer cells, plasmids were constructed to express siRNA against TTK (si-TTK-1 and -2) as well as two control plasmids (siRNAs for Luciferase (LUC), or Scramble (SCR)), and transfected each of them into LC319 and A549 cells (Representative data of LC319 was shown in
To determine whether TTK plays an important role in the growth of cells, HEK293-derived transfectants that stably expressed TTK were established. Growth of HEK293 cells expressing exogenous TTK was promoted at a significant degree in accordance with the expression level of TTK compared their growth with control cells transfected with mock vector (
As the immunohistochemical analysis on tissue microarray had indicated that lung-cancer patients with TTK strong-positive tumors showed shorter cancer-specific survival period than patients whose tumors were negative for TTK, the present inventers performed Matrigel invasion assays to determine whether TTK might play a role in cellular invasive ability. Invasion of in NIH-3T3 cells transfected with TTK-expression vector through Matrigel was significantly enhanced, compared to the control cells transfected with mock or TTK-KD vector, suggesting that TTK could also contribute to the highly malignant phenotype of lung-cancer cells (
To elucidate the function of TTK kinase in carcinogenesis, the present inventers attempted to identify substrate proteins that would be phosphorylated by TTK and activate cell-proliferation signaling. Immunoblot-screening of kinase substrates for TTK was performed by using cell lysates of COS-7 cells transfected with TTK-expression vector and a series of antibodies specific for phospho-proteins related to cancer-cell signaling (see Example 1). A total of 31 phosphoproteins (Table 2) were screened. It was found that Tyr-992 of EGFR was significantly phosphorylated in the cells transfected with the TTK-expression vector, compared with those with mock vector (
The endogenous association between TTK and EGFR in lung cancer cells was also investigated by immunoprecipitaion experiment using extracts from A549 cells, and the interaction between these two proteins regardless the EGF-stimulation was detected (
To further confirm specific phosphorylation of EGFR Tyr-992 by TTK, in vitro kinase assays were performed by incubating purified His-tagged TTK with whole cell extracts prepared from COS-7 cells. To investigate individual phosphorylation sites, the phospho-specific antibodies for EGFR (Tyr-845, Tyr-992, Tyr-1045, Tyr-1068, Tyr-1148, and Tyr-1173) corresponding to various phospho-Tyr residues within the cytoplasmic domain of the EGFR were applied. Western-blot analyses revealed phosphorylation of EGFR Tyr-992 by the recombinant TTK in a dose dependent manner, while phosphorylation of other Tyr residues in EGFR was not detected (
The EGFR tyrosine phosphorylation sites were further evaluated by TTK using GST-tagged proteins containing various cytoplasmic region of EGFR as substrates (EGFR-DEL1, −DEL2, and −DEL3;
Immunohistochemical analysis was further performed with anti-phospho-EGFR (Tyr-992) antibody using tissue microarrays composed of 366 NSCLC and 12 SCLC tissues. Of the 366 NSCLC cases, 123 (33.6%) revealed strong phospho-EGFR (Tyr-992) staining (score 2+), 121 (33.1%) were stained weakly (score 1+), and no staining (score 0) was observed in 122 cases (33.3%), while, no staining for phospho-EGFR (Tyr-992) was observed in any of normal lung tissues examined (
The requirement of EGF for the phosphorylation of EGFR by TTK was then examined. TTK was exogenously over-expressed in serum-starved COS-7 cells and microscopically observed that phospho-EGFR (Tyr-992) was detected as small spots around the nucleus in only TTK-expressing cells (
The phosphorylation level of EGFR Tyr-992 was then examined in the mitotic phase of A549 cells, when the expression level of TTK was remarkably enhanced (
GST-tagged EGFR-DEL2 protein phosphorylated by TTK was detected as double bands (
Immunohistochemical analysis was also performed with anti-phospho-EGFR (Ser-967) antibody using tissue microarrays consisting of 374 NSCLC, and it was found that the pattern of phospho-EGFR (Ser-967) positivity was significantly concordant with TTK positivity in these tumors (P<0.0001) independently conformed the results obtained by in vitro assays. Strong expression of phospho-EGFR (Ser-967) in NSCLCs was significantly associated with tumor-specific survival (P<0.0001 by the Log-rank test) (
+P < 0.05 (Fisher's exact test)
1ADC, adenocarcinoma
To further investigate the involvement of EGFR in TTK-induced cell proliferation/invasion, the present inventors next introduced RNAi-EGFR (oligo) to HEK293-derived transfectants that stably expressed TTK. The proliferation of HEK293 cells was scarcely reduced (P=0.2439) by RNAi-EGFR (oligo) (control cells transfected with mock vector), whereas the TTK-induced cell proliferation was significantly reduced (P=0.0032) by RNAi-EGFR (
The biological importance of the association of TTK and EGFR proteins and its potential as therapeutic targets for lung cancer were subsequently investigated. As shown in
Oncogenic protein kinase activation by somatic mutation in kinase domain is one of the common mechanisms of tumorigenesis (Herbst, R. S., et al., Nat Rev Cancer. 4, 956-965. (2004); Pal, S. K. and Pegram, M., Anticancer Drugs. 16, 483-494. (2005)). To examine the presence of activating mutations of TTK, the TTK kinase domain was directly sequenced by using mRNAs prepared from 36 lung-cancer cell lines and 60 clinical lung cancer tissue samples (30 primary NSCLCs and 30 metastatic brain tumors derived from primary NSCLCs). A missense mutation was found at codon 574 (Y574C) in a RERF-LC-AI cell line; which had not been reported in the SNP databases (JSNP: http://snp.ims.u-tokyo.ac.jp/index_ja.html; DBSNP: http://www.ncbi.nlm.nih.gov/projects/SNP/) (
To evaluate the functional properties of the mutant TTK identified by mutational analysis, two mutant-TTK proteins (Y574C or Q753H) were expressed in cultured mammalian cells. The level of autophosphorylated TTK (phospho-TTK) was significantly higher in the mutant-TTK-expressing NIH-3T3 cells than in the cells transfected with the wild type-TTK (wt-TTK)-expression vector, indicating that these mutations could promote the kinase activity of TTK protein (
Mps1 (TTK is its human homologue) was first discovered in budding yeast as a factor to be required in centrosome duplication (Winey M, et al., J. Cell Biol. 1991 August; 114(4):745-54.) and was subsequently shown to have a critical function in the spindle checkpoint (Weiss E & Winey. M. J. Cell Biol. 1996 January; 132(1-2):111-23.). In human, over-expression of TTK was found in several cancers, but their functional significance in carcinogenesis has been remained unclear (Stucke V M, et al., EMBO J. 2002 Apr. 2; 21(7):1723-32.).
It is herein demonstrated that the treatment of NSCLC cells with specific siRNA to TTK reduces its expression and caused growth suppression. The growth-promotive effect by introduction of TTK in mammalian cells also supports its oncogenic function. The results obtained by in vitro and in vivo assays suggest an important role of TTK in human cancer, and that screening of molecules targeting, the TTK pathway presents a promising therapeutic approach for treating lung cancers. EGFR whose pathway was known to be involved in carcinogenesis of various tissues is herein revealed as a novel intracellular target molecule of TTK kinase. Also revealed herein is the discovery by tissue microarray analysis that NSCLC patients showing high levels of TTK and phospho-EGFR (Tyr-992 or Ser-967) revealed a shorter tumor-specific survival period. EGFR autophosphorylation has been shown to play a critical role in the activation of the MAPK cascade following EGF stimulation. Of the phosphorylation sites in EGFR, Tyr-992 was proven to be a high binding-affinity site to PLCgamma, and is required for PLCgamma activation by EGF stimulation (Rotin D, et al., EMBO J. 1992 February; 11(2):559-67.). PLCgamma activates the Ras/MAPK cascade through inositol 1,4,5-triphosphate production and oscillations in cytosolic Ca2+ (Schmidt-Ullrich RK, Oncogene. 2003 Sep. 1; 22(37):5855-65.). The activation of the MAP kinase pathway is associated with cell division and its aberrant activity is supposed to be involved in the uncontrolled cell proliferation that occurs in tumors (Pal S K & Pegram M. Anticancer Drugs. 2005 June; 16(5):483-94.). Interestingly, the data herein indicate that phosphorylation of EGFR at Tyr-992 and Ser-967 by TTK is independent from the EGF stimulation. Phosphorylation of EGFR Ser-967 has been reported to be constitutive phosphorylation (Elisabetta et al., 2005), but any role of its function has not been implicated. Phosphorylation of EGFR at Ser-967 was mainly detected in nuclear EGFR. Nuclear EGFR was recently reported as a transcription factor or transcriptional coactivator (Lin et al., Nat Cell Biol. 3:802-8 (2001)). Our data indicated that phosphorylation of EGFR at Ser-967 may be important for nuclear localization of EGFR during lung carcinogenesis.
DNA sequences of the TTK kinase domain in 36 lung-cancer cell lines and 60 primary and metastatic NSCLCs were also examined, from which one lung squamous-cell carcinoma cell line that has an activating mutation promoting cellular invasive activity was identified. Point mutations were also found in 2 cases of brain metastatic lesion of adenocarcinoma. Interestingly, abundant expression of TTK protein kinase was observed in the great majority of lung-cancer samples of various histological types and much higher expression in advanced stage tumor and especially in brain metastasis (
In summary, the present invention strongly demonstrates for the first time that EGFR signaling is intracellularly regulated by the activated TTK kinase and that targeting of the enzymatic activity of TTK holds promise for development of a new strategy for treatment of lung-cancer patients.
As demonstrated herein, TTK has kinase activity for EGFR, and the suppression of this activity leads to the inhibition of cell proliferation of lung cancer cells. Thus, agents that inhibit the kinase activity of TTK for EGFR find therapeutic utility as anti-cancer agents for the treatment of lung cancer. For example, the phosphorylation site of EGFR by TTK is Try922 or Ser967, which is an EGF-independent phosphorylation.
In addition, the present invention provides a screening method for anti-cancer agents that inhibit the kinase activity of TTK for EGFR. EGFR has been recognized as an important mediator of growth signaling pathways. Accordingly, it is expected that candidate compounds that inhibit the critical step for cell proliferation can be isolated by the present invention.
In addition, the present invention demonstrates that treatment of lung cancer cells with siRNA against TTK suppresses its expression as well as the kinase activity of TTK for EGFR at Tyr-992 or Ser-967, and, thus, suppresses growth of cancer cells. This data implies that up-regulation of TTK function and enhancement of kinase activity of TTK for EGFR are common features of pulmonary carcinogenesis. Accordingly, the selective suppression of TTK kinase activity may be a promising therapeutic strategy for treatment of lung-cancer patients.
Alternatively, lung cancer can be detected using the kinase activity of TTK for EGFR as a diagnostic marker.
Furthermore, it was revealed herein that a high level of TTK expression and/or phospho-EGFR is significantly associated with poor prognosis for patients with lung cancer. Accordingly, a prognosis of lung cancer can be assessed or determined by measuring a TTK expression level and/or phosphor-EGFR (Tyr992 or Ser967).
In addition, in the present invention, it is revealed that TKK mutations are associated with a high risk of metastasis of lung cancer. Accordingly, risk assessment for metastasis of lung cancer can be achieved by detecting such mutations. Furthermore, method for detecting a TKK mutant is also provided by the present invention.
All patents, patent applications, and publications cited herein are incorporated by reference in their entirety. However, nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. While the invention has been described in detail and with reference to specific embodiments thereof, it is to be understood that the foregoing description is exemplary and explanatory in nature and is intended to illustrate the invention and its preferred embodiments. Through routine experimentation, one skilled in the art will readily recognize that various changes and modifications can be made therein without departing from the spirit and scope of the invention. Further advantages and features will become apparent from the claims filed hereafter, with the scope of such claims to be determined by their reasonable equivalents, as would be understood by those skilled in the art. Thus, the invention is intended to be defined not by the above description, but by the following claims and their equivalents.
The present application claims the benefit of U.S. Provisional Application No. 60/874,791, filed Dec. 13, 2006, the entire disclosure of which is hereby incorporated herein by reference for all purposes.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2007/074359 | 12/12/2007 | WO | 00 | 9/15/2009 |
| Number | Date | Country | |
|---|---|---|---|
| 60874791 | Dec 2006 | US |
