Patent Application
20120252028
1. Field of the Invention
The invention relates to the discovery of new targets for cancer chemotherapy and to the discovery of new small molecule cancer chemotherapeutics effective against such targets.
2. Summary of the Related Art
There has been much interest in the identification of genes that are essential for cancer cell growth. Such genes can be used as targets for the treatment of cancer. One approach to identifying such genes utilizes expression selection of Transdominant Genetic Inhibitors (TGIs) that inhibit the growth of carcinoma cells in vitro. TGIs are represented by Genetic Suppressor Elements (GSEs) and small hairpin RNA (shRNA) templates. GSEs are biologically active cDNA fragments that interfere with the function of the gene from which they are derived. GSEs may encode antisense RNA molecules that inhibit gene expression or peptides that interfere with the function of the target protein as dominant inhibitors (Holzmayer et al., 1992; Roninson et al., 1995). shRNA templates are small (19-21 bp) cDNA fragments, cloned into an expression vector in the form of inverted repeats and giving rise upon transcription to shRNAs, which are processed by cellular enzymes into double-stranded RNA duplexes, short interfering RNA (siRNA) that cause degradation of their cDNA target via RNA interference (RNAi) (Boutros and Ahringer, 2008). General strategies for the isolation of biologically active TGIs involves the use of expression libraries that express GSEs or shRNAs derived from either a single gene, or several genes, or all the genes expressed in a cell. These libraries are then introduced into recipient cells, followed by selection for the desired phenotype and the recovery of biologically active GSEs, which should be enriched in the selected cells.
Genes that are required for the growth of the recipient cells are expected to give rise to TGIs that would inhibit cell proliferation. Such TGIs can be isolated through negative selection techniques, such as bromodeoxyuridine (BrdU) suicide selection (Stetten et al., 1977). The applicability of this approach to the isolation of growth-inhibitory GSEs was demonstrated by Pestov and Lau (Pestov and Lau, 1994) and Primiano et al. (Primiano et al., 2003). Pestov et al. used an isopropyl-β-thio-galactoside (IPTG)-inducible plasmid expression vector to isolate cytostatic GSEs from a mixture of 19 cDNA clones of murine genes associated with the G0/G1 transition, using the BrdU suicide selection protocol. Through this approach, Pestov and Lau found that three of the genes in their mixture gave rise to growth-inhibitory GSEs. Primiano et al. (2003) used a GSE library derived from normalized (reduced-redundance) cDNA of human MCF7 breast carcinoma cells and cloned into an IPTG-inducible retroviral vector to isolate GSEs that allow MDA-MB-231 human breast carcinoma cells to survive BrdU suicide selection. That study yielded biologically active GSEs from 57 human genes, potential targets for breast cancer therapy.
There remains a need for the identification of new gene targets for cancer therapy.
The invention relates to the discovery of new gene targets for cancer chemotherapy and to the discovery of new small molecule cancer chemotherapeutics effective against such targets. The invention provides new gene targets for cancer chemotherapy, their use in assays for identifying new small molecule cancer chemotherapeutic agents, methods for inhibiting cancer cell growth comprising contacting a cell with a gene expression blocking agent that inhibits the expression of such genes and methods for therapeutic treatment of cancer in a mammal, comprising administering to the mammal such a gene expression blocking agent.
In a first aspect, the invention provides a method for identifying a small molecule anti-cancer compound, the method comprising (a) culturing a mammalian cell in the presence of a test compound; (b) culturing the mammalian cell in the absence of the test compound; (c) assaying the cells from (a) and (b) for the expression or activity of a nucleic acid or its encoded protein selected from the group of nucleic acids identified in Table 1; and (d) identifying the test compound as an anti-cancer compound if the expression or activity of the nucleic acid or its encoded protein is greater in cells cultured as in (b) than in cells cultured as in (a). In certain preferred embodiments, the nucleic acid is selected from the nucleic acids identified in Tables 2, 4, 5 and 6. In particularly preferred embodiments, the nucleic acid is selected from the nucleic acids identified in Tables 2 and 6.
More generally, in a second aspect, the method provides the use, in an assay for identifying a cancer chemotherapeutic small molecule compound, of a recombinant nucleic acid comprising a nucleic acid selected from the nucleic acids identified in Tables 2 and 6.
In a third aspect, the invention provides a method for inhibiting cancer cell growth, comprising inhibiting the expression of a nucleic acid selected from the nucleic acids identified in Tables 2 and 6.
In a fourth aspect, the invention provides a method for therapeutically treating a mammal having cancer, comprising administering to the mammal a gene expression blocking agent that inhibits the expression of a nucleic acid selected from the nucleic acids identified in Tables 2 and 6.
In a fifth aspect, the invention provides a method for selectively inhibiting the growth of cancer cells comprising selectively inhibiting expression or function of coatomer protein zeta-1 subunit gene (COPZ1) or its encoded CopI-ζ1 protein, respectively.
In a sixth aspect, the invention provides a method for identifying a selective small molecule inhibitor or peptide inhibitor of COPZ1 expression comprising: (a) culturing a mammalian cell comprising a recombinant DNA construct comprising a first reporter gene operatively associated with a COPZ1 promoter and a second reporter gene operatively associated with a COPZ2 promoter in the presence of a test compound; (b) culturing the mammalian cell in the absence of the test compound; (c) assaying the cells from (a) and (b) for the expression or activity of the first reporter gene and the second reporter gene, or their encoded proteins; and (d) identifying the test compound as a selective small molecule inhibitor of COPZ1 expression if the expression or activity of the first reporter gene or its encoded protein is inhibited to a greater extent than the expression or activity of the second reporter gene or its encoded protein in cells cultured as in (a), but not in cells cultured as in (b).
In a seventh aspect, the invention provides a method for identifying a selective small molecule inhibitor or peptide inhibitor of CopI-ζ1 protein comprising: (a) providing purified CopI-ζ1 protein and purified CopI-ζ1 protein in the presence of a test compound to allow an interaction of an assayable magnitude between the purified CopI-ζ1 protein and the purified CopI-γ protein; (b) providing purified CopI-ζ2 protein and purified CopI-γ protein in the absence of the test compound to allow an interaction of an assayable magnitude between the purified CopI-ζ1 protein and the purified CopI-γ protein; (c) providing purified CopI-ζ2 protein and purified CopI-ζ1 protein in the presence of the test compound to allow an interaction of an assayable magnitude between the purified CopI-ζ2 protein and the purified CopI-γ protein; (d) providing purified CopI-ζ2 protein and purified CopI-γ protein in the absence of the test compound to allow an interaction of an assayable magnitude between the purified CopI-ζ2 protein and the purified CopI-γ protein; (e) assaying the magnitude of the interaction between purified CopI-ζ1 protein and purified CopI-γ protein in steps (a) and (b); (0 assaying the magnitude of the interaction between purified CopI-ζ2 protein and purified CopI-γ protein in steps (c) and (d); and (g) identifying the test compound as a selective inhibitor of CopI-ζ1 protein if the magnitude of the interaction is lesser in step (a) than in step (c), but the magnitude of the interaction in step (b) is not lesser than the magnitude of the interaction in step (d).
In an eighth aspect, the invention provides a method for identifying a selective small molecule inhibitor or peptide inhibitor of cancer cell growth, the method comprising providing a computer model in the form of three-dimensional structural coordinates of CopI-ζ1 protein, providing three dimensional structural coordinates of a candidate compound, using a docking program to compare the three dimensional structural coordinates of the CopI-ζ1 protein with the three dimensional structural coordinates of the compound and calculate an energy-minimized conformation of the candidate compound in the CopI-ζ1 protein, and evaluating an interaction between the candidate compound and the CopI-ζ1 protein to determine binding affinity of the compound for the CopI-ζ1 protein, wherein the candidate compound is identified as a compound that selectively inhibits cancer cell growth if it has a binding affinity for the CopI-ζ1 protein site of at least 10 μM.
In a ninth aspect, the invention provides a method for determining whether a cancer in an individual is responsive to treatment by selectively inhibiting expression or function of COPZ1 or CopI-ζ1 protein, respectively, comprising obtaining cancer cells from the individual, assaying the expression of COPZ2 and/or mIR-152 in the cancer cells, and determining that the cancer in an individual is responsive to treatment by selectively inhibiting expression or function of COPZ1 or CopI-ζ1 protein, respectively, if the expression of COPZ2 and/or mIR-152 in the cancer cells is lower than in normal cells.
The invention relates to the discovery of new gene targets for cancer chemotherapy and to the discovery of new small molecule cancer chemotherapeutics effective against such targets. The invention provides new gene targets for cancer chemotherapy, their use in assays for identifying new small molecule cancer chemotherapeutic agents, methods for inhibiting cancer cell growth comprising contacting a cell with a gene expression blocking agent that inhibits the expression of such genes and methods for therapeutic treatment of cancer in a mammal, comprising administering to the mammal such a gene expression blocking agent.
The references cited herein reflect the level of knowledge in the art and are hereby incorporated by reference in their entirety. Any conflicts between the teachings of the cited references and the present specification shall be resolved in favor of the latter.
The present inventors have used both GSE and shRNA libraries constructed in tetracycline/doxycline-inducible lentiviral vectors, to select for growth-inhibitory TGIs in several types of human tumor cells, using BrdU suicide selection. As described below, this approach has enabled the inventors to select TGIs that are enriched through BrdU suicide selection. Subsequent testing of synthetic siRNAs against a set of genes enriched by this selection confirmed that the majority of these genes are required for cell growth. Some of the selected TGIs are derived from known oncogenes or known positive regulators of cell growth. Other TGIs are derived from known genes that had not been previously implicated in cell growth regulation. Genes that give rise to the isolated TGIs are identified as positive growth regulators of tumor cells. Such genes may therefore be considered as targets for the development of new anticancer drugs.
In a first aspect, the invention provides a method for identifying a small molecule anti-cancer compound, the method comprising (a) culturing a mammalian cell in the presence of a test compound; (b) culturing the mammalian cell in the absence of the test compound; (c) assaying the cells from (a) and (b) for the expression or activity of a nucleic acid or its encoded protein selected from the group of nucleic acids identified in Table 1; and (d) identifying the test compound as an anti-cancer compound if the expression or activity of the nucleic acid or its encoded protein is greater in cells cultured as in (b) than in cells cultured as in (a). In certain preferred embodiments, the nucleic acid is selected from the nucleic acids identified in Tables 2, 4, 5 and 6. In particularly preferred embodiments, the nucleic acid is selected from the nucleic acids identified in Tables 2 and 6. In some embodiments the expression or activity of more than one nucleic acid or its encoded protein from the tables is assayed in step (c).
More generally, in a second aspect, the method provides the use, in an assay for identifying a cancer chemotherapeutic small molecule compound, of a recombinant nucleic acid comprising a nucleic acid selected from the nucleic acids identified in Tables 2 and 6. For purposes of this aspect of the invention, “a recombinant nucleic acid comprising a nucleic acid selected from” is intended to mean the selected nucleic acid covalently linked to other nucleic acid elements that do not occur in the normal chromosomal locus of the gene. Such other nucleic acid elements may include gene expression elements, such as heterologous promoters and/or enhancers, selectable markers, reporter genes and the like. Preferably, the other nucleic acid elements allow the selected nucleic acid to be expressed in mammalian cells. Such recombinant nucleic acids may frequently be incorporated into a chromosome of the mammalian cell.
In a third aspect, the invention provides a method for inhibiting cancer cell growth, comprising inhibiting the expression of a nucleic acid selected from the nucleic acids identified in Tables 2 and 6. In preferred embodiments of this aspect of the invention, such inhibition of expression of the nucleic acid is achieved by contacting the cell with a gene expression blocking agent. For purposes of the invention, “a gene expression blocking agent” is an agent that prevents an RNA transcribed from the nucleic acid from carrying out its normal cellular function, such function being either regulatory, or being translated into a functional protein. Such prevention may be either steric, e.g., by the agent simply binding to the RNA, or may be through the destruction of the bound RNA by cellular enzymes. Representative gene expression blocking agents include, without limitation, antisense oligonucleotides, ribozymes, short interfering RNAs (siRNA), short hairpin RNAs (shRNA), microRNAs (miRNA) and the like.
In a fourth aspect, the invention provides a method for therapeutically treating a mammal having cancer, comprising administering to the mammal a gene expression blocking agent that inhibits the expression of a nucleic acid selected from the nucleic acids identified in Tables 2 and 6. Such gene expression blocking agent is administered in a therapeutically effective amount. A therapeutically effective amount is an amount sufficient to reduce or ameliorate signs and symptoms of the cancer, such as cell proliferation or metastasis.
The inventors have surprisingly discovered that COPZ1 knockdown selectively kills tumor cells relative to normal cells and the mechanism of this selectivity, which warrants the development of COPZ1-targeting drugs. Such drugs should inhibit the expression or function of COPZ1 but not COPZ2, since the inhibition of both COPZ1 and COPZ2 kills not only tumor but also normal cells. There are several approaches to selective inhibition of COPZ1 preferentially to COPZ2.
In a fifth aspect, the invention provides a method for selectively inhibiting the growth of cancer cells comprising selectively inhibiting expression or function of coatomer protein zeta-1 subunit gene (COPZ1) or its encoded CopI-ζ1 protein, respectively. “Selective inhibition of cancer cell growth” means killing or inhibiting the growth of cancer cells without killing or inhibiting the growth of normal cells.
In some embodiments, the expression of COPZ1 is inhibited by an agent selected from an siRNA, an antisense oligonucleotide, and a ribozyme, wherein the agent selectively targets mRNA encoding CopI-ζ1 protein. siRNAs and their chemically modified variants are being actively developed for therapeutic applications (Ashihara et al., 2010; Vaishnaw et al., 2010). Related approaches targeting RNA sequences that distinguish COPZ1 from COPZ2 include the use of antisense oligonucleotides (Bennett and Swayze, 2010) and ribozymes (Freelove and Zheng, 2002; Asif-Ullah et al., 2007). In some embodiments, the expression of COPZ1 is inhibited by a small molecule that selectively inhibits COPZ1 expression. The terms “selectively targets” and selectively inhibits” mean that expression of the COPZ1 gene is inhibited, but expression of the COPZ2 gene is not inhibited.
In some embodiments, the function of CopI-ζ1 protein is inhibited by a small molecule or peptide that selectively inhibits CopI-ζ1 protein. The term “selectively inhibits CopI-ζ1 protein” means that the small molecule prevents CopI-ζ1 protein from forming CopI-ζ1 protein/CopI-γ protein dimers, to a greater extent than it prevents CopI-ζ2 protein from forming CopI-ζ2 protein/CopI-γ protein dimers.
The term “small molecule” means a molecule having a molecular weight of less than about 1500 daltons. The greater extent includes at least 10-fold, at least 20-fold, at least 50-fold and at least 100-fold. A “peptide” is an oligomer of from about 3 to about 50 naturally occurring or modified amino acids, and thus also includes peptidomimetics. Such peptides may be further modified, e.g., by pegylation.
In some embodiments, the cancer cells are in the body of an individual. Thus, the invention provides a method for treating an individual having cancer, comprising selectively inhibiting in the individual expression or function of expression or function of COPZ1 gene or its encoded CopI-ζ1 protein, respectively. The method comprises administering to the individual any of the agents discussed above in an effective amount. The term “an effective amount” means an amount sufficient to inhibit cancer cell growth in vivo.
In a sixth aspect, the invention provides a method for identifying a selective small molecule inhibitor or peptide inhibitor of COPZ1 expression comprising: (a) culturing a mammalian cell comprising a recombinant DNA construct comprising a first reporter gene operatively associated with a COPZ1 promoter and a second reporter gene operatively associated with a COPZ2 promoter in the presence of a test compound; (b) culturing the mammalian cell in the absence of the test compound; (c) assaying the cells from (a) and (b) for the expression or activity of the first reporter gene and the second reporter gene, or their encoded proteins; and (d) identifying the test compound as a selective small molecule inhibitor of COPZ1 expression if the expression or activity of the first reporter gene or its encoded protein is inhibited to a greater extent than the expression or activity of the second reporter gene or its encoded protein in cells cultured as in (a), but not in cells cultured as in (b). The use of reporter gene/heterologous promoter systems to identify compounds that inhibit specific gene expression has been described previously, for example, in U.S. Pat. No. 7,235,403. A selective small molecule inhibitor of COPZ1 expression is a compound having a molecular weight of less than about 1500 daltons and which inhibits expression of the COPZ1 gene, but not the COPZ2 gene. A peptide is as described previously. A test compound can be a small molecule or a peptide. The term “inhibited to a greater extent” includes extents of at least 10-fold, at least 20-fold, at least 50-fold and at least 100-fold.
The selective small molecule inhibitors or peptide inhibitor of COPZ1 expression selectively inhibit cancer cell growth. Thus, this method is also a method for identifying a selective small molecule or peptide inhibitor of cancer cell growth. “Selective inhibition of cancer cell growth” means that the compound kills or inhibits the growth of cancer cells without killing or inhibiting the growth of normal cells.
In a seventh aspect, the invention provides a method for identifying a selective small molecule inhibitor or peptide inhibitor of CopI-ζ1 protein comprising: (a) providing purified CopI-ζ1 protein and purified CopI-γ protein in the presence of a test compound to allow an interaction of an assayable magnitude between the purified CopI-ζ1 protein and the purified CopI-γ protein; (b) providing purified CopI-ζ1 protein and purified CopI-γ protein in the absence of the test compound to allow an interaction of an assayable magnitude between the purified CopI-ζ1 protein and the purified CopI-γ protein; (c) providing purified CopI-ζ2 protein and purified CopI-γ protein in the presence of the test compound to allow an interaction of an assayable magnitude between the purified CopI-ζ2 protein and the purified CopI-γ protein; (d) providing purified CopI-ζ2 protein and purified CopI-γ protein in the absence of the test compound to allow an interaction of an assayable magnitude between the purified CopI-ζ2 protein and the purified CopI-γ protein; (e) assaying the magnitude of the interaction between purified CopI-ζ1 protein and purified CopI-γ protein in steps (a) and (b); (f) assaying the magnitude of the interaction between purified CopI-ζ2 protein and purified CopI-γ protein in steps (c) and (d); and (g) identifying the test compound as a selective inhibitor of CopI-ζ1 protein if the magnitude of the interaction is lesser in step (a) than in step (c), but the magnitude of the interaction in step (b) is not lesser than the magnitude of the interaction in step (d).
An interaction between CopI-ζ1 protein and CopI-γ protein, or between CopI-ζ1 protein and CopI-γ protein, can involve either CopI-γ1 protein or CopI-γ2 protein. The interaction results in formation of an active coatomer protein complex.
In some embodiments, the purified CopI-ζ1 protein or the purified CopI-γ protein are labeled with a fluorophore suitable for fluorescence resonance energy transfer (FRET), the CopI-ζ2 protein or the purified CopI-γ protein are labeled with a fluorophore suitable for FRET, and the magnitude of the interactions are assayed by FRET. In some embodiments, the CopI-ζ1 protein and the CopI-ζ2 protein are labeled with a different fluorophore, thereby allowing the assays to take place simultaneously in the same vessel. The use of FRET to assay protein-protein interactions has been described, for example, in Boute et al., 2002; Degorce et al., 2009.
A “selective small molecule inhibitor or peptide inhibitor of CopI-ζ1 protein” is a molecule that prevents CopI-ζ1 protein from forming CopI-ζ1 protein/CopI-γ protein dimers, to a greater extent than it prevents CopI-ζ2 protein from forming CopI-ζ2 protein/CopI-γ protein dimers. The term “small molecule” means a molecule having a molecular weight of less than about 1500 daltons. A peptide is as described previously. The greater extent includes at least 10-fold, at least 20-fold, at least 50-fold and at least 100-fold.
The selective small molecule inhibitors or peptide inhibitors of CopI-ζ1 protein selectively inhibit cancer cell growth. Thus, this method is also a method for identifying a selective small molecule inhibitor or peptide inhibitor of cancer cell growth. “Selective inhibition of cancer cell growth” means that the compound kills or inhibits the growth of cancer cells without killing or inhibiting the growth of normal cells.
In an eighth aspect, the invention provides a method for identifying a selective small molecule inhibitor or peptide inhibitor of cancer cell growth, the method comprising providing a computer model in the form of three-dimensional structural coordinates of CopI-ζ1 protein, providing three dimensional structural coordinates of a candidate compound, using a docking program to compare the three dimensional structural coordinates of the CopI-ζ1 protein with the three dimensional structural coordinates of the compound and calculate an energy-minimized conformation of the candidate compound in the CopI-ζ1 protein, and evaluating an interaction between the candidate compound and the CopI-ζ1 protein to determine binding affinity of the compound for the CopI-ζ1 protein, wherein the candidate compound is identified as a compound that selectively inhibits cancer cell growth if it has a binding affinity for the CopI-ζ1 protein site of at least 10 μM. The solution structure of CopI-ζ1 protein has been described by Yu et al., 2009.
siRNAs or other RNA-targeting drugs, inhibitors of COPZ1 expression, and molecules identified in cell-free assays (such as FRET) or predicted by computer modeling to be selective inhibitors of CopI-ζ1 function can be further tested for the expected biological effects in tumor cells. These effects include inhibition of cell proliferation, induction of cell death, disruption of Golgi and inhibition of autophagy. COPZ1-specific inhibitors inducing such biological effects in tumor cells can be considered as therapeutic candidates for further development.
In a ninth aspect, the invention provides a method for determining whether a cancer in an individual is responsive to treatment by selectively inhibiting expression or function of COPZ1 or CopI-ζ1 protein, respectively, comprising, obtaining cancer cells from the individual, assaying the expression of COPZ2 and/or mIR-152 in the cancer cells, and determining that the cancer in an individual is responsive to treatment by selectively inhibiting expression or function of COPZ1 or CopI-ζ1 protein, respectively, if the expression of COPZ2 and/or mIR-152 in the cancer cells is lower than in normal cells. The expression level in normal cells may be measured from any normal cell, meaning a cell that is not neoplastically transformed. Alternatively, a standardized signal may be provided as a surrogate for normal cell expression. Such expression may be at least 10-fold greater, at least 20-fold greater, at least 50-fold greater or at least 100-fold greater.
In the methods for treatment according to of the invention, the gene expression blocking agent may be formulated with a physiologically acceptable carrier, excipient, or diluent. Such physiologically acceptable carriers, excipients and diluents are known in the art and include any agents that are not physiologically toxic and that do not interfere with the function of the gene expression blocking agent. Representative carriers, excipients and diluents include, without limitation, lipids, salts, hydrates, buffers and the like.
Administration of the gene expression blocking agents or formulations thereof may be by any suitable route, including, without limitation, parenteral, mucosal, transdermal and oral administration.
Homo sapiens v-Ki-ras2 Kirsten rat sarcoma viral
norvegicus]
Homo sapiens actin, beta (ACTB), mRNA
Homo sapiens heat shock protein 90 kDa alpha
Homo sapiens CD9 molecule (CD9)
Homo sapiens immature colon carcinoma transcript
Homo sapiens tripartite motif-containing 29
Homo sapiens mannosidase, endo-alpha-like
Homo sapiens casein kinase 2, alpha 1 polypeptide
sapiens]
Homo sapiens TSC22 domain family, member 2,
Homo sapiens ATP synthase, H+ transporting,
Homo sapiens farnesyl diphosphate synthase
Homo sapiens heat shock 60 kDa protein 1
Homo sapiens integrin, beta 1 (fibronectin receptor,
sapiens (human)
Homo sapiens neurofibromin 1 (neurofibromatosis,
Homo sapiens HIG1 domain family, member 2A
Homo sapiens ribosomal protein L23 (RPL23)
norvegicus]
Homo sapiens SH3KBP1 binding protein 1
Homo sapiens v-maf musculoaponeurotic
Homo sapiens calpain 2, (m/II) large subunit
Homo sapiens iduronidase, alpha-L-(IDUA),
Homo sapiens pregnancy specific beta-1-
Homo sapiens brain protein 13 (BR13)
tropicalis]
norvegicus]
Homo sapiens actin related protein ⅔ complex,
Homo sapiens keratin 8 (KRT8), mRNA
Homo sapiens DNA directed RNA polymerase II
Homo sapiens Rho GDP dissociation inhibitor (GDI)
troglodytes]
troglodytes]
Homo sapiens, clone IMAGE: 6016214, mRNA
norvegicus]
troglodytes]
Homo sapiens, clone IMAGE: 5211852, mRNA
sapiens]
sapiens]
sapiens
Arabidopsis)
troglodytes]
pombe]
Homo sapiens,
norvegicus]
norvegicus]
The following examples are intended to further illustrate the invention and are not intended to be construed to limit the scope of the invention.
The shRNA library was prepared as follows. The strategy for shRNA library construction is depicted in
Lung (A549, H69), colon (HCT116, SW480), breast (MCF-7, MDA-MB321), prostate (LNCaP, PC3), cervical (HeLa), ovarian (A2780), renal (ACHN) carcinomas cell lines, fibrosarcoma (HT1080), osteosarcoma (Saos-2) cell lines, melanoma (MALME-3M), glioblastoma (U251), chronic myelogenous leukemia (K562), promyelocytic leukemia (HL60), and acute lymphoblastic leukemia (CCRF-CEM) cell lines were obtained from ATCC. mRNA from these cell lines was used to prepare normalized cDNA, through duplex-specific nuclease (DSN) normalization (Zhulidov et al., 2004); the normalization was carried by Evrogen (Moscow, Russia) as a service. Normalization efficacy was tested by Q-PCR analysis of representation of cDNAs of seven transcripts with high (β-actin, GAPDH, EF1-α), medium (L32, PPMM) and low (Ubch5b, c-Yes) expression levels in parental cells. The representation of highly expressed transcripts decreased up to 70-fold in the normalized mixture, while the level of rare cDNAs increased up to 30-fold after normalization. Normalized cDNA mixture was fragmented by DNAse I digestion to obtain 100-500 by fragments, followed by end repair by treatment with T4 DNA polymerase and Klenow fragment as described (Gudkov and Roninson, 1997). cDNA fragments were amplified by ligation-mediated PCR. For amplification, adaptors containing translation start sites with Age I and Sph I restriction sites were used. cDNA fragments were digested with Age I and Sph I and ligated into a modified tetracycline/doxycycline-inducible vector, pLLCEm (Wiznerowicz and Trono, 2003), under the control of the CMV promoter. The ligation produced a library of approximately 260 million clones. The percent recombination in this library was assessed by direct sequencing of 192 clones. The number of clones containing an insert was >90%. The average length of the inserts was 135 bp.
As the recipient cell lines for TGI selection, we have chosen four human cancer cell lines and human immortalized fibroblasts. The tumor cell lines are MDA-MB-231 breast carcinoma, PC3 prostate carcinoma, HT108 fibrosarcoma and T24 bladder carcinoma. The immortalized fibroblasts are BJ-hTERT. To obtain tetracycline/doxycycline-inducible cells, tTR-KRAB, a tetracycline/doxycycline-sensitive repressor was overexpressed in all the cell lines, by infecting them with a lentiviral vector expressing tTR-KRAB and dsRED fluorescent protein (Wiznerowicz and Trono, 2003), followed by two rounds of FACS selection for dsRed positive cells. To analyze the tetracycline/doxycycline-dependent regulation, tTR-KRAB expressing cell lines were infected with an EGFP-expressing tetracycline/doxycycline-inducible lentiviral vector. The level of activation of GFP expression by treatment with 100 ng/ml of doxycycline ranged from about 30-fold to 300-fold in different cell lines.
The shRNA library in pLLCE-TU6-LX vector described in above was transduced into MDA-MB-231 breast carcinoma cells expressing ttR-KRAB. The GSE library in pLLCEm lentiviral vector, described above, was transduced into all five cell lines. Lentiviral transduction was carried out using a pseudotype packaging system, by co-transfecting plasmid library DNA with Δ8.91 lentiviral packaging plasmid and VSV-G (pantropic receptor) plasmid into 293FT cells in DMEM with 10% FC2 using TransFectin reagent. 2.5×107 recipient cells were infected with the shRNA library, and 1×108 cells of each recipient cell line were infected with the GSE library. The infection rate (as determined by Q-PCR analysis of integrated provirus) was 95%. 25% of the infected cells were subjected to DNA purification, and the rest were plated at a density of 1×106 cells per P150, to a total of 100 million cells. These cells were subjected to selection for Doxycycline-dependent resistance to BrdU suicide, as follows. Cells were treated with 0.1 μg/ml of doxycycline for 18 hrs, then with 0.1 μg/ml of doxycycline and 50 μM BrdU for 48 hrs. Cells were then incubated with 10 μM Hoechst 33258 for 3 hrs and illuminated with fluorescent white light for 15 min on a light box, to destroy the cells that replicated their DNA and incorporated BrdU in the presence of doxycycline. Cells were then washed twice with phosphate-buffered saline and allowed to recover in normal medium (DMEM, 10% FBS) for 7-10 days. The surviving cells were collected, followed by DNA purification. The cDNA fragments were amplified by PCR from genomic DNA extracted from the infected unselected and BrdU-selected cells using vector specific primers and subjected to ultra high throughput sequencing by 454 Life Science Inc (http://www.454.com/enabling-technology/the-process.asp).
High-throughput sequencing of shRNA sequences recovered by PCR from the genomic DNA of MDA-MB-231 cells before and after BrdU selection, followed by BLAST analysis, yielded 53201 sequences with homology to Unigene database entries before selection and 53803 sequences after selection. These sequences matched 14699 and 3316 Unigene clusters respectively. Among the genes found in the selected subset, 741 were targeted by four or more shRNA sequences (Table 1). The genes in Table 1 are sorted by the “enrichment factor” (EF), a value defined by multiplying the number of different shRNA sequences found to be enriched for each gene after BrdU suicide selection by the fold enrichment in the frequency of any shRNA sequences derived from the corresponding gene. By this criterion, which takes into account both the likelihood that the shRNA target gene has been correctly identified by being targeted by multiple shRNA sequences and the degree of enrichment, one of the most enriched genes was KRAS, a well-known oncogene that has undergone an activating, mutation in MDA-MB-231 cells (Kozma et al., 1987). This result validates the selection system as capable of identifying oncogenes, potential targets for anticancer drugs.
To verify that genes enriched by the selection are required for MDA-MB-231 cell growth, we have selected 22 genes represented by at least two selected shRNA sequences and showing the highest EF value. We then used synthetic short interfering RNA (siRNA) targeting these genes and designed by Qiagen, Inc. according to Qiagen's siRNA design algorithms, for transfection into MDA-MB-231 cells, to determine if such siRNAs will inhibit cell growth. Four siRNAs per gene, obtained from Qiagen, were transfected into MDA-MB231 cells in 96-well plates, in triplicates, using Silentfect® transfection reagent (Biorad) and manufacturer's instructions, and 5 nM of siRNA per well. A cytotoxic mixture of siRNA derived from several essential genes (Qiagen, All-star Cell Death Hs siRNA, #1027298), was used as a positive control, and siRNAs targeting either no known genes (Qiagen, Negative Control siRNA #1022076) or the Green Fluorescent Protein (GFP) (Qiagen, GFP-22 siRNA, #1022064) were used as negative controls. Cells were cultured in DMEM media with 10% FBS serum, and the relative cell number was determined six days after siRNA transfection by staining cellular DNA with Hoechst 33342 (Polysciences Inc; #23491-52-3). As shown in
Sequencing of GSE fragments recovered by PCR from the genomic DNA of five different cell lines before and after BrdU selection was followed by BLAST analysis. The numbers of cDNA fragment sequences with homology to Unigene database entries revealed by BLAST analysis in each PCR product and the number of sequences enriched two or more fold by BrdU selection are shown in Table 3. Among the selected genes, 178 were enriched in two or more different cell lines (Table 4), and 98 genes were enriched in tumor cell lines only but not in BJ-hTERT (Table 5). These genes represent potential targets for cancer treatment.
To verify the growth-regulatory activity of 26 genes enriched by GSE selection, we have used transfection of the corresponding siRNAs from Qiagen siRNA collection, four siRNAs per gene, as described in section 5 above. In these assays we have used HT1080 fibrosarcoma cells (3 days analysis after transfection) T24 bladder carcinoma (3 days analysis after transfection), and MDA-MB-231 breast carcinoma cells (6 day analysis after transfection). The results presented in
Among the new potential targets listed in Table 6, we have investigated in greater detail COPZ1, which was targeted by GSEs identified in BrdU-selected populations of tumor cell lines HT1080, MDA-MB-231, T24, and PC3, but not in immortalized normal BJ-hTERT fibroblasts. COPZ1 encodes CopI-ζ1, one of the two isoforms of a coatomer of COPI secretory vesicles involved in Golgi to ER and Golgi to Golgi traffic (Beck et al., 2009). The other CopI-ζ isoform, CopI-ζ2, is encoded by the COPZ2 gene; the two CopI-ζ proteins have 75% amino acid identity (Wegmann et al., 2004). CopI-1 and CopI-ζ2 are alternative components of a dimeric complex that also includes one of the two isoforms of CopI-γ, encoded by another pair of closely related genes, COPG1 and COPG2. The CopI-ζ/CopI-γ dimers interact within COPI complexes with additional CopI proteins, which are encoded by the genes COPA, COPB1, COPB2, COPD and COPE (Wegmann et al., 2004; Moelleken et al., 2007).
As shown in
The knockdown of COPZ1 by siRNA was verified by quantitative reverse transcription-PCR (QPCR), as described (VanGuilder et al., 2008). The sequences of the primers used to amplify GAPDH and RPL13A (normalization standards), COPZ1 and other COPI component genes analyzed herein are listed in Table 8. QPCR analysis showed that COPZ1 Qiagen B and COPZ1. Qiagen D siRNAs decreased COPZ1 mRNA levels in MDA-MB-231, PC3 and BJ-hTERT cells by >95% relative to cells transfected with a control siRNA targeting no known genes (Qiagen).
The knockdown of COPA or COPB was reported to cause the collapse of endoplasmic reticulum and Golgi compartments and cellular traffic arrest (Styers et al., 2008). Disruption of intracellular traffic either by inhibition of COPI complex formation or by blocking COPI assembly on Golgi membrane by inhibition of adenosine diphosphate ribosylation factor with brefeldin A (Donaldson et al., 1991; Fujiwara et al., 1988) resulted in cell death (Citterio et al., 2008; Shao et al., 1996). Additionally COPA or COPB knockdown inhibits the maturation of the autophagosome (Razi et al., 2009), an essential step in autophagy, a process involving the degradation of cell components through lysosomes. Autophagy is a physiological program that plays a role in cell growth, development, and homeostasis (Mizushima et al., 2008), and therefore interference with autophagy may result in cell death (Platini et al., 2010; Filimonenko et al., 2007). To determine if COPZ1 knockdown, like that of COPA or COPB, interferes with autophagy and causes Golgi disruption, we have transfected COPZ1 siRNA (from Thermo Scientific; Table 7), in parallel with siRNAs targeting COPA and COPZ2, into PC3 cells expressing LC3, a protein marker of autophagosomes fused with Green Fluorescent Protein (GFP-LC3) (Fung et al., 2008). The knockdown effects on autophagosome accumulation and Golgi integrity were analyzed 72 hrs later by fluorescence microscopy analysis after staining with monoclonal antibodies against a Golgi marker GM130 (Golgi membrane protein 130 kD, BD Bioscience) and GFP-LC3 localization. Fluorescent microscopy analysis (
To determine if siRNA knockdown of the other COPI components would mimic the antiproliferative effect of COPZ1 siRNA, we have compared the effects of siRNAs targeting COPA, COPB1, COPB2, COPE, COPG1, COPG2, COPZ1 and COPZ2 on the proliferation of HT1080, MDA-MB-231, T24 and PC3 tumor cell lines and immortalized normal BJ-hTERT fibroblasts. This analysis was conducted through the same experimental setup as in the experiments shown in
The differential effect of COPZ1 siRNAs on tumor and normal cells was verified using an independent set of siRNAs (from Thermo Scientific; Table 7).
To understand why the knockdown of COPZ1 but not of the other COPI proteins selectively inhibits the proliferation of tumor cells relative to normal fibroblasts, we have measured the expression of COPZ1, COPZ2, COPA, COPB1 and COPB2 in BJ-hTERT, HT1080, MDA-MB-231, T24, and PC3 cell lines by QPCR, using primers listed in Table 8.
We have expanded the QPCR analysis of COPZ2 and COPZ1 expression to a large set of different normal human tissues (from Ambion) (
COPZ2 downregulation in cancer cells offers an explanation for tumor-selective cytotoxicity of COPZ1-targeting siRNAs. Since COPZ1 and COPZ2 gene products are alternative components of CopI-ζ/CopI-γ dimers, it is likely that they can substitute for each other, and that COPI complexes remain functional if either COPZ1 or COPZ2 gene products are present. Therefore, COPZ1 knockdown is not toxic to normal cells that express COPZ2.
However, COPZ2 is expressed at very low levels or not at all in tumor cells, and therefore such cells become dependent on COPZ1 for normal COPI function and survival. Therefore, COPZ1 knockdown kills COPZ2-deficient tumor cells but not COPZ2-proficient normal cells. To test this explanation, we asked if the restoration of COPZ2 expression in tumor cells would protect them from killing by COPZ1 siRNA. We have cloned full-length COPZ1 and COPZ2 cDNAs from MGC cDNA collection (distributed by Open Biosystems) into a lentiviral expression vector pLenti6-bsd-FLAG constructed in our laboratory, which expresses the cloned protein with a FLAG tag at the C-terminus. These recombinant lentiviruses (as well as the insert-free vector) were then transduced into PC3 cells. The transduced cells were selected with blasticidine and tested for the expression of COPZ1 and COPZ2 by immunoblotting, using FLAG-specific antibody (M2 Anti-FLAG, Sigma-Aldrich) and antibodies specific for COPZ1 (D20 anti-COPZ antibody, Santa-Cruz Biotechnology) and COPZ2 (a gift of Dr. F. Wieland, University of Heidelberg). The results of this analysis, shown in
COPZ2 siRNA alone had no effect on the proliferation of any of the three PC3 populations (
The reason for COPZ2 downregulation in tumor cells is presently unknown. However, COPZ2 gene contains in one of its introns a gene encoding the precursor of a microRNA (miRNA) mIR-152 (Weber, 2005; Rodriguez et al., 2004). miRNAs are pleiotropic regulators of gene expression, a number of which have been identified as playing important roles in cancer, either as oncogenes or as tumor suppressors (Ryan et al., 2010). Remarkably, mIR-152 was shown to be downregulated in clinical samples of several types of cancer, including breast cancer where mIR-152 gene is hypermethylated (Lehmann et al., 2008), endometrial serous adenocarcinoma where decreased expression of miR-152 was a statistically independent risk factor for overall survival (Hiroki et al., 2010), cholangiocarcinoma (Braconi et al., 2010) and gastric and colorectal cancers, where low expression of miR-152 was correlated with increased tumor size and advanced pT stage (Chen and Carmichael, 2010). Furthermore, mIR-152 overexpression in cholangiocarcinoma cells decreased cell proliferation (Braconi et al., 2010), and mIR-132 overexpression in a placental human choriocarcinoma cell line sensitized the cells to lysis by natural killer cells (Zhu et al., 2010). Hence, mIR-152 displays expression changes and biological activities indicative of a tumor suppressor. Many miRNAs located within protein-coding genes are transcriptionally linked to the expression of their host genes (Stuart et al., 2004), and a correlation between COPZ2 and mIR-152 expression has been noted among normal tissues (Bak et al., 2008). Therefore, COPZ2 downregulation in cancers could be a corollary of the downregulation of a tumor-suppressive miRNA mIR-152. To test this hypothesis, we have measured mIR-152 expression in a series of cell lines where COPZ2 expression has been determined, using QPCR with a combination of the universal miRNA (Hurteau et al., 2006) and miR-152 specific primers (Table 8). The results of this analysis, shown in
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US10/45600 | 8/16/2010 | WO | 00 | 5/16/2012 |
| Number | Date | Country | |
|---|---|---|---|
| 61234140 | Aug 2009 | US |
