Patent Application
20090162361
The invention relates to methods of diagnosing pancreatic cancer.
Pancreatic cancer has one of the highest mortality rates of any malignancy, and the 5-year-survival rate of patients is 4%. 28000 patients with pancreatic cancer are diagnosed each year, and nearly all patients will die of their disease (1). The poor prognosis of this malignancy is a result of the difficulty of early diagnosis and poor response to current therapeutic methods (1, 2). In particular currently no tumor markers are identified that allow reliable screening at an early, potentially curative stage of the disease.
cDNA microarray technologies have enabled to obtain comprehensive profiles of gene expression in normal and malignant cells, and compare the gene expression in malignant and corresponding normal cells (Okabe et al., Cancer Res 61:2129-37 (2001); Kitahara et al., Cancer Res 61: 3544-9 (2001); Lin et al., Oncogene 21:4120-8 (2002); Hasegawa et al., Cancer Res 62:7012-7 (2002)). This approach enables to disclose the complex nature of cancer cells, and helps to understand the mechanism of carcinogenesis. Identification of genes that are deregulated in tumors can lead to more precise and accurate diagnosis of individual cancers, and to develop novel therapeutic targets (Bienz and Clevers, Cell 103:311-20 (2000)). To disclose mechanisms underlying tumors from a genome-wide point of view, and discover target molecules for diagnosis and development of novel therapeutic drugs, the present inventors have been analyzing the expression profiles of tumor cells using a cDNA microarray of 23040 genes (Okabe et al., Cancer Res 61:2129-37 (2001); Kitahara et al., Cancer Res 61:3544-9 (2001); Lin et al., Oncogene 21:4120-8 (2002); Hasegawa et al., Cancer Res 62:7012-7 (2002)).
Studies designed to reveal mechanisms of carcinogenesis have already facilitated identification of molecular targets for anti-tumor agents. For example, inhibitors of farnesyltransferase (FTIs) which were originally developed to inhibit the growth-signaling pathway related to Ras, whose activation depends on posttranslational farnesylation, has been effective in treating Ras-dependent tumors in animal models (He et al., Cell 99:335-45 (1999)). Clinical trials on human using a combination or anti-cancer drugs and anti-HER2 monoclonal antibody, trastuzumab, have been conducted to antagonize the proto-oncogene receptor HER2/neu; and have been achieving improved clinical response and overall survival of breast-cancer patients (Lin et al., Cancer Res 61:6345-9 (2001)). A tyrosine kinase inhibitor, STI-571, which selectively inactivates bcr-abl fusion proteins, has been developed to treat chronic myelogenous leukemias wherein constitutive activation of bcr-abl tyrosine kinase plays a crucial role in the transformation of leukocytes. Agents of these kinds are designed to suppress oncogenic activity of specific gene products (Fujita et al., Cancer Res 61:7722-6 (2001)). Therefore, gene products commonly up-regulated in cancerous cells may serve as potential targets for developing novel anti-cancer agents.
It has been demonstrated that CD8+ cytotoxic T lymphocytes (CTLs) recognize epitope peptides derived from tumor-associated antigens (TAAs) presented on MHC Class I molecule, and lyse tumor cells. Since the discovery of MAGE family as the first example of TAAs, many other TAAs have been discovered using immunological approaches (Boon, Int J Cancer 54: 177-80 (1993); Boon and van der Bruggen, J Exp Med 183: 725-9 (1996); van der Bruggen et al., Science 254: 1643-7 (1991); Brichard et al., J Exp Med 178: 489-95 (1993); Kawakami et al., J Exp Med 180: 347-52 (1994)). Some of the discovered TAAs are now in the stage of clinical development as targets of immunotherapy. TAAs discovered so far include MAGE (van derBruggen et al., Science 254: 1643-7 (1991)), gp100 (Kawakami et al., J Exp Med 180: 347-52 (1994)), SART (Shichijo et al., J Exp Med 187: 277-88 (1998)), and NY-ESO-1 (Chen et al., Proc Nati Acad Sci USA 94: 1914-8 (1997)). On the other hand, gene products which had been demonstrated to be specifically over-expressed in tumor cells, have been shown to be recognized as targets inducing cellular immune responses. Such gene products include p53 (Umano et al., Brit J Cancer 84: 1052-7 (2001)), HER2/neu (Tanaka et al., Brit J Cancer 84: 94-9 (2001)), CEA (Nukaya et al., Int J Cancer 80: 92-7 (1999)), and so on.
In spite of significant progress in basic and clinical research concerning TAAs (Rosenbeg et al., Nature Med 4: 321-7 (1998); Mukherji et al., Proc Natl Acad Sci USA 92: 8078-82 (1995); Hu et al., Cancer Res 56: 2479-83 (1996)), only limited number of candidate TAAs for the treatment of adenocarcinomas, including colorectal cancer, are available. TAAs abundantly expressed in cancer cells, and at the same time which expression is restricted to cancer cells would be promising candidates as immunotherapeutic targets. Further, identification of new TAAs inducing potent and specific antitumor immune responses is expected to encourage clinical use of peptide vaccination strategy in various types of cancer (Boon and can der Bruggen, J Exp Med 183: 725-9 (1996); van der Bruggen et al., Science 254: 1643-7 (1991); Brichard et al., J Exp Med 178: 489-95 (1993); Kawakami et al., J Exp Med 180: 347-52 (1994); Shichijo et al., J Exp Med 187: 277-88 (1998); Chen et al., Proc Natl Acad Sci USA 94: 1914-8 (1997); Harris, J Natl Cancer Inst 88: 1442-5 (1996); Butterfield et al., Cancer Res 59: 3134-42 (1999); Vissers et al., Cancer Res 59: 5554-9 (1999); van der Burg et al., J Immunol 156: 3308-14 (1996); Tanaka et al., Cancer Res 57: 4465-8 (1997); Fujie et al., Int J Cancer 80: 169-72 (1999); Kikuchi et al., Int J Cancer 81: 459-66 (1999); Oiso et al., Int J Cancer 81: 387-94 (1999)).
It has been repeatedly reported that peptide-stimulated peripheral blood mononuclear cells (PBMCs) from certain healthy donors produce significant levels of IFN-γ in response to the peptide, but rarely exert cytotoxicity against tumor cells in an HLA-A24 or -A0201 restricted manner in 51Cr-release assays (Kawano et al., Cance Res 60: 3550-8 (2000); Nishizaka et al., Cancer Res 60: 4830-7 (2000); Tamura et al., Jpn J Cancer Res 92: 762-7 (2001)). However, both of HLA-A24 and HLA-A0201 are one of the popular HLA alleles in Japanese, as well as Caucasian (Date et al., Tissue Antigens 47: 93-101 (1996); Kondo et al., J Immunol 155: 4307-12 (1995); Kubo et al., J Immunol 152: 3913-24 (1994); Imanishi et al., Proceeding of the eleventh International Hictocompatibility Workshop and Conference Oxford University Press, Oxford, 1065 (1992); Williams et al., Tissue Antigen 49: 129 (1997)). Thus, antigenic peptides of carcinomas presented by these HLAs may be especially useful for the treatment of carcinomas among Japanese and Caucasian. Further, it is known that the induction of low-affinity CTL in vitro usually results from the use of peptide at a high concentration, generating a high level of specific peptide/MHC complexes on antigen presenting cells (APCs), which will effectively activate these CTL (Alexander-Miller et al., Proc Natl Acad Sci USA 93: 4102-7 (1996)).
The invention is based on the discovery of a pattern of gene expression correlated with pancreatic cancer (PNC). The genes that are differentially expressed in pancreatic cancer are collectively referred to herein as “PNC nucleic acids” or “PNC polynucleotides” and the corresponding encoded polypeptides are referred to as “PNC polypeptides” or “PNC proteins.”
Accordingly, the invention features a method of diagnosing or determining a predisposition to pancreatic cancer in a subject by determining an expression level of a PNC-associated gene in a patient derived biological sample, such as tissue sample. By PNC-associated gene is meant a gene that is characterized by an expression level which differs in a cell obtained from a PNC cell compared to a normal cell. A normal cell is one obtained from pancreas tissue. A PNC-associated gene is one or more of PNC 1-605. An alteration, e.g., increase or decrease of the level of expression of the gene compared to a normal control level of the gene indicates that the subject suffers from or is at risk of developing PNC.
By normal control level is meant a level of gene expression detected in a normal, healthy individual or in a population of individuals known not to be suffering from pancreatic cancer. A control level is a single expression pattern derived from a single reference population or from a plurality of expression patterns. For example, the control level can be a database of expression patterns from previously tested cells. A normal individual is one with no clinical symptoms of pancreatic cancer.
An increase in the level of PNC 1-259 detected in a test sample compared to a normal control level indicates the subject (from which the sample was obtained) suffers from or is at risk of developing PNC. In contrast, a decrease in the level of PNC 260-605 detected in a test sample compared to a normal control level indicates said subject suffers from or is at risk of developing PNC.
Alternatively, expression of a panel of PNC-associated genes in the sample is compared to a PNC control level of the same panel of genes. By PNC control level is meant the expression profile of the PNC-associated genes found in a population suffering from PNC.
Gene expression is increased or decreased 10%, 25%, 50% compared to the control level. Alternately, gene expression is increased or decreased 1, 2, 5 or more fold compared to the control level. Expression is determined by detecting hybridization, e.g., on an array, of a PNC-associated gene probe to a gene transcript of the patient-derived tissue sample.
The patient derived tissue sample is any tissue from a test subject, e.g., a patient known to or suspected of having PNC. For example, the tissue contains an epithelial cell. For example, the tissue is an epithelial cell from a pancreatic ductal adenocarcinoma.
The invention also provides a PNC reference expression profile of a gene expression level of two or more of PNC 1-605. Alternatively, the invention provides a PNC reference expression profile of the levels of expression two or more of PNC 1-259 or PNC 260-605.
The invention further provides methods of identifying an agent that inhibits or enhances the expression or activity of a PNC-associated gene, e.g. PNC 1-605 by contacting a test cell expressing a PNC-associated gene with a test agent and determining the expression level of the PNC associated gene. The test cell is an epithelial cell such as an epithelial cell from a pancreatic adenocarcinoma. A decrease of the level compared to a normal control level of the gene indicates that the test agent is an inhibitor of the PNC-associated gene and reduces a symptom of PNC, e.g. PNC 1-259. Alternatively, an increase of the level or activity compared to a normal control level or activity of the gene indicates that said test agent is an enhancer of expression or function of the PNC-associated gene and reduces a symptom of PNC, e.g, PNC 260-605.
The invention also provides a kit with a detection reagent which binds to one or more PNC nucleic acids or which binds to a gene product encoded by the nucleic acid sequences. Also provided is an array of nucleic acids that binds to one or more PNC nucleic acids.
Therapeutic methods include a method of treating or preventing pancreatic cancer in a subject by administering to the subject an antisense composition. The antisense composition reduces the expression of a specific target gene, e.g., the antisense composition contains a nucleotide, which is complementary to a sequence selected from the group consisting of PNC 1-259. Another method includes the steps of administering to a subject a short interfering RNA (siRNA) composition. The siRNA composition reduces the expression of a nucleic acid selected from the group consisting of PNC 1-259, PCDH1, CDH3 and GPR107. In yet another method, treatment or prevention of PNC in a subject is carried out by administering to a subject a ribozyme composition. The nucleic acid-specific ribozyme composition reduces the expression of a nucleic acid selected from the group consisting of PNC 1-259. Other therapeutic methods include those in which a subject is administered a compound that increases the expression of PNC 260-605 or activity of a polypeptide encoded by PNC 260-605.
The invention also includes vaccines and vaccination methods. For example, a method of treating or preventing PNC in a subject is carried out by administering to the subject a vaccine containing a polypeptide encoded by a nucleic acid selected from the group consisting of PNC 1-259 or an immunologically active fragment such a polypeptide. An immunologically active fragment is a polypeptide that is shorter in length than the full-length naturally-occurring protein and which induces an immune response. For example, an immunologically active fragment at least 8 residues in length and stimulates an immune cell such as a T cell or a B cell. Immune cell stimulation is measured by detecting cell proliferation, elaboration of cytokines (e.g., IL-2), or production of an antibody.
Alternatively, the present invention provides target molecules for treating or preventing malignant pancreatic cancer. According to the present invention, 76 (PNC 606-681), 168 (PNC 682-849) and 84 (850-933) genes were identified as genes that showed unique altered expression patterns in pancreatic cancer cells with lymph-node metastasis, liver metastasis and early recurrence, respectively. Thus, malignant pancreatic cancer can be treated or prevented via the suppression of the expression or activity of up-regulated genes selected from the group consisting of PNC 606-640 and PNC 682-741. Furthermore, recurrence of pancreatic cancer can be treated or prevented via the suppression of the expression or activity of up-regulated genes selected from the group consisting of PNC 850-893. Moreover, malignant pancreatic cancer can also be treated or prevented through enhancing the expression or activity of down-regulating genes in cancerous cells.
The present invention also provides methods for predicting recurrence of pancreatic cancer. The method comprises the step of measuring the expression level of marker genes selected from the group consisting of PNC 850-879. The marker genes were identified as genes that show unique altered expression patterns in pancreatic cancer cells of patients with recurrence within 12 month after surgery. Therefore, recurrence of the pancreatic cancer in a subject can be predicted by determining whether the expression level detected in a sample derived from the subject is closer to the mean expression level of early-recurrent cases or late-recurrent cases in reference samples.
The present invention is also based on the surprising discovery that inhibiting expression of PCDH1, CDH3 or GPR107 is effective in inhibiting the cellular growth of various cancer cells, including those involved in pancreatic ductal adenocarcinoma (PDACa). The inventions described in this application are based in part on this discovery.
The invention provides methods for inhibiting cell growth. Among the methods provided are those comprising contacting a cell with a composition comprising a small interfering RNA (siRNA) that inhibits expression of PCDH1, CDH3 or GPR107. The invention also provides methods for inhibiting tumor cell growth in a subject. Such methods include administering to a subject a composition comprising a small interfering RNA (siRNA) that hybridizes specifically to a sequence from PCDH1, CDH3 or GPR107. Another aspect of the invention provides methods for inhibiting the expression of the PCDH1, CDH3 or GPR107 gene in a cell of a biological sample. Expression of the gene may be inhibited by introduction of a double stranded ribonucleic acid (RNA) molecule into the cell in an amount sufficient to inhibit expression of the PCDH1, CDH3 or GPR107 gene. Another aspect of the invention relates to products including nucleic acid sequences and vectors as well as to compositions comprising them, useful, for example, in the provided methods. Among the products provided are siRNA molecules having the property to inhibit expression of the PCDH1, CDH3 or GPR107 gene when introduced into a cell expressing said gene. Among such molecules are those that comprise a sense strand and an antisense strand, wherein the sense strand comprises a ribonucleotide sequence corresponding to a PCDH1, CDH3 or GPR107 target sequence, and wherein the antisense strand comprises a ribonucleotide sequence which is complementary to said sense strand. The sense and the antisense strands of the molecule hybridize to each other to form a double-stranded molecule.
The invention features methods of inhibiting cell growth. Cell growth is inhibited by contacting a cell with a composition of a small interfering RNA (siRNA) of PCDH1, CDH3 or GPR107. The cell is further contacted with a transfection-enhancing agent. The cell is provided in vitro, in vivo or ex vivo. The subject is a mammal, e.g., a human, non-human primate, mouse, rat, dog, cat, horse, or cow. The cell is a pancreatic ductal cell. Alternatively, the cell is a tumor cell (i.e., cancer cell) such as a carcinoma cell or an adenocarcinoma cell. For example, the cell is a pancreatic ductal adenocarcinoma cell. By inhibiting cell growth is meant that the treated cell proliferates at a lower rate or has decreased viability than an untreated cell. Cell growth is measured by proliferation assays known in the art.
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. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
One advantage of the methods described herein is that the disease is identified prior to detection of overt clinical symptoms of pancreatic cancer. Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Generally pancreatic ductal adenocarcinoma has a characteristic of highly desmoplastic stromal reaction, only a low percentage (about 30%) of cancer cells are contained in the tumor mass. Furthermore, normal pancreatic ductal epithelial cells, which recently considered to be the normal counterpart of the pancreatic adenocarcinoma, occupied only less than 5% of the total population of cells composing the organ ‘pancreas’ (7, 8). Hence, the gene-expression analysis of PNC compared to normal pancreas by using whole tissue is distorted by the contamination of needless cells such as fibroblast, inflammatory cells, acinar cells, etc., and results in “noisy data”. Therefore Laser capture microdissection (LCM), or Laser microbeam microdissection (LMM), a method for isolating pure cell populations, was used to obtain specific cancer cells and normal epithelial cells (9,10).
The present invention is based in part on the discovery of changes in expression patterns of multiple nucleic acids in epithelial cells from adenocarcinomas of patients with PNC. The differences in gene expression were identified by using a comprehensive cDNA microarray system.
The gene-expression profiles of cancer cells from 18 PNCs were analyzed using cDNA microarray representing 23,040 genes couples with laser microdissection. By comparing expression patterns between cancer cells from diagnostic PNC patients and normal ductal epithelial cells purely selected with Laser Microdisection, 259 genes were identified as commonly up-regulated in PNC cells, and 346 genes were identified as being commonly down-regulated in PNC cells. In addition, selection was made of candidate molecular markers with the potential of detecting cancer-related proteins in serum or sputum of patients, and discovered some potential targets for development of signal-suppressing strategies in human PNC.
The differentially expressed genes identified herein are used for diagnostic purposes as markers of PNC and as gene targets, the expression of which is altered to treat or alleviate a symptom of PNC.
The genes whose expression levels are modulated (i.e., increased or decreased) in PNC patients are summarized in Tables 3-4 and are collectively referred to herein as “PNC-associated genes”, “PNC nucleic acids” or “PNC polynucleotides” and the corresponding encoded polypeptides are referred to as “PNC polypeptides” or “PNC proteins.” Unless indicated otherwise, “PNC” is meant to refer to any of the sequences disclosed herein. (e.g., PNC 1-605). The genes have been previously described and are presented along with a database accession number.
By measuring expression of the various genes in a sample of cells, PNC is diagnosed. Similarly, measuring the expression of these genes in response to various agents can identify agents for treating PNC.
The invention involves determining (e.g., measuring) the expression of at least one, and up to all the PNC sequences listed in Tables 3-4. Using sequence information provided by the GeneBank™ database entries for the known sequences the PNC-associated genes are detected and measured using techniques well known to one of ordinary skill in the art. For example, sequences within the sequence database entries corresponding to PNC sequences, are used to construct probes for detecting PNC RNA sequences in, e.g., Northern blot hybridization analysis. Probes include at least 10, 20, 50, 100, 200 nucleotides of a reference sequence. As another example, the sequences can be used to construct primers for specifically amplifying the PNC nucleic acid in, e.g, amplification-based detection methods such as reverse-transcription based polymerase chain reaction.
Expression level of one or more of the PNC-associated genes in the test cell population, e.g., a patient derived tissues sample, is then compared to expression levels of the some genes in a reference population. The reference cell population includes one or more cells for which the compared parameter is known, i.e., pancreatic ductal adenocarcinoma cells or normal pancreatic ductal epithelial cells.
Whether or not a pattern of gene expression levels in the test cell population compared to the reference cell population indicates PNC or predisposition thereto depends upon the composition of the reference cell population. For example, if the reference cell population is composed of non-PNC cells, a similar gene expression pattern in the test cell population and reference cell population indicates the test cell population is non-PNC. Conversely, if the reference cell population is made up of PNC cells, a similar gene expression profile between the test cell population and the reference cell population indicates that the test cell population includes PNC cells.
A level of expression of a PNC marker gene in a test cell population is considered altered in levels of expression if its expression level varies from the reference cell population by more than 1.0, 1.5, 2.0, 5.0, 10.0 or more fold from the expression level of the corresponding PNC marker gene in the reference cell population.
Differential gene expression between a test cell population and a reference cell population is normalized to a control nucleic acid, e.g. a housekeeping gene. For example, a control nucleic acid is one which is known not to differ depending on the cancerous or non-cancerous state of the cell. Expression levels of the control nucleic acid in the test and reference nucleic acid can be used to normalize signal levels in the compared populations. Control genes include, e.g., β-actin, glyceraldehyde 3-phosphate dehydrogenase or ribosomal protein P1.
The test cell population is compared to multiple reference cell populations. Each of the multiple reference populations may differ in the known parameter. Thus, a test cell population may be compared to a second reference cell population known to contain, e.g., PNC cells, as well as a second reference population known to contain, e.g., non-PNC cells (normal cells). The test cell is included in a tissue type or cell sample from a subject known to contain, or to be suspected of containing, PNC cells.
The test cell is obtained from a bodily tissue or a bodily fluid, e.g., biological fluid (such as blood or sputum). For example, the test cell is purified from pancreas tissue. Preferably, the test cell population comprises an epithelial cell. The epithelial cell is from tissue known to be or suspected to be a pancreatic ductal adenocarcinoma.
Cells in the reference cell population are derived from a tissue type as similar to test cell. Optionally, the reference cell population is a cell line, e.g. a PNC cell line (positive control) or a normal non-PNC cell line (negative control). Alternatively, the control cell population is derived from a database of molecular information derived from cells for which the assayed parameter or condition is known.
The subject is preferably a mammal. The mammal can be, e.g., a human, non-human primate, mouse, rat, dog, cat, horse, or cow.
Expression of the genes disclosed herein is determined at the protein or nucleic acid level using methods known in the art. For example, Northern hybridization analysis using probes which specifically recognize one or more of these nucleic acid sequences can be used to determine gene expression. Alternatively, expression is measured using reverse-transcription-based PCR assays, e.g., using primers specific for the differentially expressed gene sequences. Expression is also determined at the protein level, i.e., by measuring the levels of polypeptides encoded by the gene products described herein, or biological activity thereof. Such methods are well known in the art and include, e.g., immunoassays based on antibodies to proteins encoded by the genes. The biological activities of the proteins encoded by the genes are also well known.
As used herein, the term “organism” refers to any living entity comprised of at least one cell. A living organism can be as simple as, for example, a single eukaryotic cell or as complex as a mammal, including a human being.
As used herein, the term “biological sample” refers to a whole organism or a subset of its tissues, cells or component parts (e.g. bodily fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). “Biological sample” further refers to a homogenate, lysate, extract, cell culture or tissue culture prepared from a whole organism or a subset of its cells, tissues or component parts, or a fraction or portion thereof. Lastly, “biological sample” refers to a medium, such as a nutrient broth or gel in which an organism has been propagated, which contains cellular components, such as proteins or nucleic acid molecules.
PNC is diagnosed by measuring the level of expression of one or more PNC nucleic acid sequences from a test population of cells, (i.e., a patient derived biological sample). Preferably, the test cell population contains an epithelial cell, e.g., a cell obtained from pancreas tissue. Gene expression is also measured from blood or other bodily fluids such as urine. Other biological samples can be used for measuring the protein level. For example, the protein level in the blood, serum, or pancreatic juice derived from subject to be diagnosed can be measured by immunoassay or biological assay.
Expression of one or more PNC-associated genes, e.g., PNC 1-605 is determined in the test cell or biological sample and compared to the expression of the normal control level. A normal control level is an expression profile of a PNC-associated gene typically found in a population known not to be suffering from PNC. An increase or a decrease of the level of expression in the patient derived tissue sample of the PNC-associated genes indicates that the subject is suffering from or is at risk of developing PNC. For example, an increase in expression of PNC 1-259 in the test population compared to the normal control level indicates that the subject is suffering from or is at risk of developing PNC. Conversely, a decrease in expression of PNC 260-605 in the test population compared to the normal control level indicates that the subject is suffering from or is at risk of developing PNC.
When one or more of the PNC-associated genes are altered in the test population compared to the normal control level indicates that the subject suffers from or is at risk of developing PNC. For example, at least 1%, 5%, 25%, 50%, 60%, 80%, 90% or more of the panel of PNC-associated genes (PNC 1-259, PNC 260-605, or PNC 1-605) are altered.
The present invention provides a method for predicting prognosis of PNC in a subject, the method comprising the steps of:
In the present invention, marker gene(s) for prediction of prognosis of PNC may be at least one gene selected from the group consisting of PNC 850-933; 84 genes shown in Table 8. The nucleotide sequences of the genes and amino acid sequences encoded thereby are known in the art. See Table 8 for the Accession Numbers of the genes.
According to the present invention, prediction of prognosis comprises prediction of probability for recurrence of PNC. When recurrence of PNC is observed within 12 month after surgery, the subject is determined to have poor prognosis. In one embodiment, the expression levels of multiple marker genes selected from the group of PNC 850-866, 894-906 can be measured for the prediction. Preferably, the 30 genes consisting of top 17 genes (ARGBP2, CBARA1, EEF1G, LCAT, RPL23A, RPL17, ATP1A1, QARS, BZRP, TUFM, SERPINA4, SCAP, HK1, RPS11, SYNGR2, FLOT2, PSMB4) of up-regulated in late recurrence cases genes and top 13 genes of up-regulated in early recurrence cases genes (MTMR1, HT010, NPD002, YME1L1, CCT6A, HSPD1, TIMM9, GRB14, FLJ10803, LAMP1, MLLT4, CTSB, RALY) of Table 8 are useful for the prediction. In the present method, the specimen is collected from a subject. Preferable specimen includes pancreatic tissue derived from patient of pancreatic cancer. Methods for measuring the expression level of marker genes are well-known in the art. For example, DNA array is useful for measuring the expression level of multiple marker genes. According to the present invention, first, the expression level of each marker genes in a specimen is measured and then compared to that of early recurrence cases and late recurrence cases. The expression level of the marker genes of each of the cases can be measured prior to the comparison of the expression level. Then, based on the above comparison, when the expression level of one or marker genes is close to that of the early recurrence case, determining the subject to be at a risk of having recurrence of PNC and when the expression level of one or marker genes is close to that of the late recurrence case, determining the risk of the subject of having recurrence of PNC to be low. In the present invention, the recurrence of PNC can be predicted using prediction score that may be calculated by statistical methods. Methods for calculating prediction score is well-known in the art (T. R. Golub et al., Science 286, 531-7, 1999; T. J. MacDonald et al., Nat. Genet, 29, 143-52, 2001). Furthermore, prediction of recurrence using prediction score in the present invention may be also performed according to the method disclosed in the Example.
Identifying Agents that Inhibit or Enhance PNC-Associated Gene Expression
An agent that inhibits the expression or activity of a PNC-associated gene is identified by contacting a test cell population expressing a PNC-associated up-regulated gene with a test agent and determining the expression level of the PNC-associated gene. A decrease in expression in the presence of the agent compared to the normal control level (or compared to the level in the absence of the test agent) indicates the agent is an inhibitor of a PNC-associated up-regulated gene and useful to inhibit PNC.
Alternatively, an agent that enhances the expression or activity of a PNC-associated down-regulated gene is identified by contacting a test cell population expressing a PNC-associated gene with a test agent and determining the expression level or activity of the PNC-associated down-regulated gene. An increase of expression or activity compared to a normal control expression level or activity of the PNC-associated gene indicates that the test agent augments expression or activity of the PNC-associated down-regulated gene.
The test cell population is any cell expressing the PNC-associated genes. For example, the test cell population contains an epithelial cell, such as a cell is or derived from pancreas tissue. For example, the test cell is an immortalized cell line derived from an adenocarcinoma cell. Alternatively, the test cell is a cell, which has been transfected with a PNC-associated gene or which has been transfected with a regulatory sequence (e.g. promoter sequence) from a PNC-associated gene operably linked to a reporter gene.
The differentially expressed PNC-associated gene identified herein also allow for the course of treatment of PNC to be monitored. In this method, a test cell population is provided from a subject undergoing treatment for PNC. If desired, test cell populations are obtained from the subject at various time points before, during, or after treatment. Expression of one or more of the PNC-associated gene, in the cell population is then determined and compared to a reference cell population which includes cells whose PNC state is known. The reference cells have not been exposed to the treatment.
If the reference cell population contains no PNC cells, a similarity in expression between PNC-associated gene in the test cell population and the reference cell population indicates that the treatment is efficacious. However, a difference in expression between PNC-associated gene in the test population and a normal control reference cell population indicates a less favorable clinical outcome or prognosis.
By “efficacious” is meant that the treatment leads to a reduction in expression of a pathologically up-regulated gene, increase in expression of a pathologically down-regulated gene or a decrease in size, prevalence, or metastatic potential of pancreatic ductal adenocarcinoma in a subject. When treatment is applied prophylactically, “efficacious” means that the treatment retards or prevents a pancreatic tumor from forming or retards, prevents, or alleviates a symptom of clinical PNC. Assessment of pancreatic tumors is made using standard clinical protocols.
Efficaciousness is determined in association with any known method for diagnosing or treating PNC. PNC is diagnosed for example, by identifying symptomatic anomalies, e.g., weight loss, abdominal pain, back pain, anorexia, nausea, vomiting and generalized malaise, weakness, and jaundice.
Selecting a Therapeutic Agent for Treating PNC that is Appropriate for a Particular Individual
Differences in the genetic makeup of individuals can result in differences in their relative abilities to metabolize various drugs. An agent that is metabolized in a subject to act as an anti-PNC agent can manifest itself by inducing a change in gene expression pattern in the subject's cells from that characteristic of a cancerous state to a gene expression pattern characteristic of a non-cancerous state. Accordingly, the differentially expressed PNC-associated gene disclosed herein allow for a putative therapeutic or prophylactic inhibitor of PNC to be tested in a test cell population from a selected subject in order to determine if the agent is a suitable inhibitor of PNC in the subject.
To identify an inhibitor of PNC, that is appropriate for a specific subject, a test cell population from the subject is exposed to a therapeutic agent, and the expression of one or more of PNC 1-605 genes is determined.
The test cell population contains a PNC cell expressing a PNC-associated gene. Preferably, the test cell is an epithelial cell. For example a test cell population is incubated in the presence of a candidate agent and the pattern of gene expression of the test sample is measured and compared to one or more reference profiles, e.g., a PNC reference expression profile or a non-PNC reference expression profile.
A decrease in expression of one or more of PNC 1-259 or an increase in expression of one or more of PNC 260-605 in a test cell population relative to a reference cell population containing PNC is indicative that the agent is therapeutic.
The test agent can be any compound or composition. For example, the test agents are immunomodulatory agents.
The differentially expressed genes disclosed herein can also be used to identify candidate therapeutic agents for treating PNC. The method is based on screening a candidate therapeutic agent to determine if it converts an expression profile of PNC 1-605 characteristic of a PNC state to a pattern indicative of a non-PNC state.
In the method, a cell is exposed to a test agent or a combination of test agents (sequentially or consequentially) and the expression of one or more PNC 1-605 in the cell is measured. The expression profile of the PNC-associated gene in the test population is compared to expression level of the PNC-associated gene in a reference cell population that is not exposed to the test agent.
An agent effective in stimulating expression of under-expressed genes, or in suppressing expression of over-expressed genes is deemed to lead to a clinical benefit such compounds are further tested for the ability to prevent pancreatic ductal adenocarcinomal growth in animals or test subjects.
In a further embodiment, the present invention provides methods for screening candidate agents which are potential targets in the treatment of PNC. As discussed in detail above, by controlling the expression levels or activities of marker genes, one can control the onset and progression of PNC. Thus, candidate agents, which are potential targets in the treatment of PNC, can be identified through screenings that use the expression levels and activities of marker genes as indices. In the context of the present invention, such screening may comprise, for example, the following steps:
Alternatively, the screening method of the present invention may comprise the following steps:
Alternatively, the screening method of the present invention may comprise the following steps:
Alternatively, the screening method of the present invention may comprise the following steps:
The compound isolated by the screening is a candidate for drugs that inhibit the activity of the protein encoded by marker genes and can be applied to the treatment or prevention of pancreatic cancer.
Moreover, compound in which a part of the structure of the compound inhibiting the activity of proteins encoded by marker genes is converted by addition, deletion and/or replacement are also included in the compounds obtainable by the screening method of the present invention.
When administrating the compound isolated by the method of the 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 can be formulated into a dosage form using known 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 compounds 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 tablets and capsules are, 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-mannnose, 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™ and HCO-50.
Sesame oil or Soy-bean oil can be used as a oleaginous liquid and may be used in conjunction with benzyl benzoate or benzyl alcohol as a solubilizer and may be formulated with a buffer, such as phosphate buffer and sodium acetate buffer; a pain-killer, such as procaine hydrochloride; a stabilizer, such as benzyl alcohol andphenol; and an anti-oxidant. The prepared injection may be filled into a suitable ampule.
Methods well known to one skilled in the art may be used to administer the pharmaceutical composition of the present invention to patients, for example as intraarterial, intravenous, or percutaneous injections and also as intranasal, transbronchial, intramuscular or oral administrations. The dosage and method of administration vary according to the body-weight and age of a patient and the administration method; however, one skilled in the art can routinely select a suitable method of administration. If said compound is encodable by a DNA, the DNA can be inserted into a vector for gene therapy and the vector administered to a patient to perform the therapy. The dosage and method of administration vary according to the body-weight, age, and symptoms of the patient but one skilled in the art can suitably select them.
For example, although the dose of a compound that binds to the protein of the present invention and regulates its activity depends on the symptoms, the dose is 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 kgs of body-weight.
The present invention provides target molecules for treating or preventing malignant pancreatic cancer. In the present invention, malignant cancer includes cancers having properties such as follows:
local invasion;
aggressive proliferation; and
metastasis.
Therefore, according to the present invention, malignant pancreatic cancer includes pancreatic cancer with metastasis. Screening assay for malignant PNC of the present invention can be performed according to the method for PNC described above using marker genes for malignant pancreatic cancer.
In the present invention, marker genes selected from the group consisting of PNC 606-681, and 682-849 are useful for the screening. 76 genes shown in Table 6 (PNC 606-681) were associated with lymph node metastasis. Among the genes, 35 genes (PNC 606-640) were relatively up-regulated and 41 genes (PNC 641-681) were down-regulated in node-positive tumors (
In a preferred embodiment, the present invention provides a method of screening for a compound for treating or preventing malignant pancreatic cancer, said method comprising the steps of:
In a further embodiment, the present invention provides a method of screening for a compound for treating or preventing malignant pancreatic cancer, said method comprising the steps of:
In the method of the invention, the cell for contacting with the candidate is malignant pancreatic cancer cell.
Furthermore, in other embodiment, the present invention provides a method of screening for a compound for treating or preventing malignant pancreatic cancer, said method comprising the steps of:
In addition, in one embodiment, the present invention also provides a method of screening for compound for treating or preventing malignant pancreatic cancer, said method comprising the steps of:
Furthermore, the present invention provides target molecules for treating or preventing recurrence of pancreatic cancer. Herein, recurrence of pancreatic cancer indicates recurrence of cancer in pancreas after surgery. For example, the recurrence of cancer within 12 month after surgery can be predicted by the invention. According to the present invention, early recurrence includes the recurrence within 12 month after surgery, and when no recurrence can be observed within 12 month after surgery in a case, the case is considered to be a pancreatic cancer with “late recurrence”. 84 genes (PNC 850-933) shown in Table 8 are useful as the marker genes for the screening of the present invention. Among them, the genes shown in
Accordingly, in a preferred embodiment, the present invention provides a method of screening for a compound for treating or preventing recurrence of pancreatic cancer, said method comprising the steps of:
Alternatively, in further embodiment, the present invention provides a method of screening for a compound for treating or preventing recurrence of pancreatic cancer, said method comprising the steps of:
In the present invention, the cell may comprise a recurrent pancreatic cancer cell.
Furthermore, in other embodiment, the present invention provides a method of screening for a compound for treating or preventing recurrence of pancreatic cancer, said method comprising the steps of:
In addition, in one embodiment, the preesnt invention also provides a method of screening for a compound for treating or preventing recurrence of pancreatic cancer, said method comprising the steps of:
Also provided is a method of assessing the prognosis of a subject with PNC by comparing the expression of one or more PNC-associated gene in a test cell population to the expression of the genes in a reference cell population derived from patients over a spectrum of disease stages. By comparing gene expression of one or more PNC-associated gene in the test cell population and the reference cell population(s), or by comparing the pattern of gene expression over time in test cell populations derived from the subject, the prognosis of the subject can be assessed.
A decrease in expression of one or more of PNC 260-605 compared to a normal control or an increase of expression of one or more of PNC 1-259 compared to a normal control indicates less favorable prognosis. A similar expression of one or more of PNC 1-605 indicates a more favorable prognosis compared to nomal control indicates a more favorable prognosis for the subject. Preferably, the prognosis of a subject can be assessed by comparing the expression profile of PNC 1-605. The classification score (CS) may be use for the comparing the expression profile.
The invention also includes a PNC-detection reagent, e.g., a nucleic acid that specifically binds to or identifies one or more PNC nucleic acids such as oligonucleotide sequences, which are complementary to a portion of a PNC nucleic acid or antibodies which bind to proteins encoded by a PNC nucleic acid. The reagents are packaged together in the form of a kit. The reagents are packaged in separate containers, e.g., a nucleic acid or antibody (either bound to a solid matrix or packaged separately with reagents for binding them to the matrix), 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 included in the kit. The assay format of the kit is a Northern hybridization or a sandwich ELISA known in the art.
For example, PNC detection reagent is immobilized on a solid matrix such as a porous strip to form at least one PNC detection site. The measurement or detection region of the porous strip may include a plurality of sites containing a nucleic acid. A test strip may also contain sites for negative and/or positive controls. Alternatively, control sites are located on a separate strip from the test strip. Optionally, the different detection sites may contain different amounts of immobilized nucleic acids, i.e., a higher amount in the first detection site and lesser amounts in subsequent sites. Upon the addition of test sample, the number of sites displaying a detectable signal provides a quantitative indication of the amount of PNC present in the sample. The detection sites may be configured in any suitably detectable shape and are typically in the shape of a bar or dot spanning the width of a teststrip.
Alternatively, the kit contains a nucleic acid substrate array comprising one or more nucleic acids. The nucleic acids on the array specifically identify one or more nucleic acid sequences represented by PNC 1-605. The expression of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 40 or 50 or more of the nucleic acids represented by PNC 1-605 are identified by virtue of the level of binding to an array test strip or chip. The substrate array can be on, e.g., a solid substrate, e.g., a “chip” as described in U.S. Pat. No. 5,744,305.
The invention also includes a nucleic acid substrate array comprising one or more nucleic acids. The nucleic acids on the array specifically correspond to one or more nucleic acid sequences represented by PNC 1-605. The level of expression of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 40 or 50 or more of the nucleic acids represented by PNC 1-605 are identified by detecting nucleic acid binding to the array.
The invention also includes an isolated plurality (i.e., a mixture if two or more nucleic acids) of nucleic acids. The nucleic acids are in a liquid phase or a solid phase, e.g., immobilized on a solid support such as a nitrocellulose membrane. The plurality includes one or more of the nucleic acids represented by PNC 1-605. In various embodiments, the plurality includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 40 or 50 or more of the nucleic acids represented by PNC 1-605.
The invention provides a method for treating or alleviating a symptom of PNC in a subject by decreasing expression or activity of PNC 1-259 or increasing expression or activity of PNC 260-605. Therapeutic compounds are administered prophylactically or therapeutically to subject suffering from or at risk of (or susceptible to) developing PNC. Such subjects are identified using standard clinical methods or by detecting an aberrant level of expression or activity of PNC 1-605. Therapeutic agents include inhibitors of cell cycle regulation, cell proliferation, and protein kinase activity.
The therapeutic method includes increasing the expression, or function, or both of one or more gene products of genes whose expression is decreased (“under-expressed genes”) in a PNC cell relative to normal cells of the same tissue type from which the PNC cells are derived. In these methods, the subject is treated with an effective amount of a compound, which increases the amount of one or more of the under-expressed genes in the subject. Administration can be systemic or local. Therapeutic compounds include a polypeptide product of an under-expressed gene, or a biologically active fragment thereof a nucleic acid encoding an under-expressed gene and having expression control elements permitting expression in the PNC cells; for example an agent which increases the level of expression of such gene endogenous to the PNC cells (i.e., which up-regulates expression of the under-expressed gene or genes). Administration of such compounds counters the effects of aberrantly-under expressed of the gene or genes in the subject's pancreas cells and improves the clinical condition of the subject.
The method also includes decreasing the expression, or function, or both, of one or more gene products of genes whose expression is aberrantly increased (“over-expressed gene”) in pancreas cells. Expression is inhibited in any of several ways known in the art. For example, expression is inhibited by administering to the subject a nucleic acid that inhibits, or antagonizes, the expression of the over-expressed gene or genes, e.g., an antisense oligonucleotide or small interfering RNA which disrupts expression of the over-expressed gene or genes.
As noted above, antisense nucleic acids corresponding to the nucleotide sequence of PNC 1-259 can be used to reduce the expression level of the PNC 1-259. Antisense nucleic acids corresponding to PNC 1-259 that are up-regulated in pancreatic cancer are useful for the treatment of pancreatic cancer. Specifically, the antisense nucleic acids of the present invention may act by binding to the PNC 1-259 or mRNAs corresponding thereto, thereby inhibiting the transcription or translation of the genes, promoting the degradation of the mRNAs, and/or inhibiting the expression of proteins encoded by the PNC 1-259, finally inhibiting the function of the proteins. The term “antisense nucleic acids” as used herein encompasses both nucleotides that are entirely complementary to the target sequence and those having a mismatch of one or more nucleotides, so long as the antisense nucleic acids can specifically hybridize to the target sequences. For example, the antisense nucleic acids of the present invention include polynucleotides that have a homology of at least 70% or higher, preferably at 80% or higher, more preferably 90% or higher, even more preferably 95% or higher over a span of at least 15 continuous nucleotides. Algorithms known in the art can be used to determine the homology.
The antisense nucleic acid derivatives of the present invention act on cells producing the proteins encoded by marker genes by binding to the DNAs or mRNAs encoding the proteins, inhibiting their transcription or translation, promoting the degradation of the mRNAs, and inhibiting the expression of the proteins, thereby resulting in the inhibition of the protein function.
An antisense nucleic acid derivative of the present invention can be made into an external preparation, such as a liniment or a poultice, by mixing with a suitable base material which is inactive against the derivative.
Also, as needed, the derivatives can be formulated into tablets, powders, granules, capsules, liposome capsules, injections, solutions, nose-drops and freeze-drying agents by adding excipients, isotonic agents, solubilizers, stabilizers, preservatives, pain-killers, and such. These can be prepared by following known methods.
The antisense nucleic acids derivative is given to the patient by directly applying onto the ailing site or by injecting into a blood vessel so that it will reach the site of ailment. An antisense-mounting medium can also be used to increase durability and membrane-permeability. Examples are, liposomes, poly-L-lysine, lipids, cholesterol, lipofectin or derivatives of these.
The dosage of the antisense nucleic acid derivative of the present invention can be adjusted suitably according to the patient's condition and used in desired amounts. For example, a dose range of 0.1 to 100 mg/kg, preferably 0.1 to 50 mg/kg can be administered.
The antisense nucleic acids of the invention inhibit the expression of the protein of the invention and are thereby useful for suppressing the biological activity of a protein of the invention. Also, expression-inhibitors, comprising the antisense nucleic acids of the invention, are useful since they can inhibit the biological activity of a protein of the invention.
The antisense nucleic acids of present invention include modified oligonucleotides. For example, thioated nucleotides may be used to confer nuclease resistance to an oligonucleotide.
By the term “siRNA” is meant a double stranded RNA molecule which prevents translation of a target mRNA. Standard techniques of introducing siRNA into the cell are used, including those in which DNA is a template from which RNA is transcribed. The siRNA includes a sense PNC 1-259, PCDH1, CDH3 or GPR107 nucleic acid sequence, an anti-sense PNC 1-259, PCDH1, CDH3 or GPR107 nucleic acid sequence or both. The siRNA may comprise two complementary molecules or may be constructed such that a single transcript has both the sense and complementary antisense sequences from the target gene, e.g., a hairpin, which, in some embodiments, leads to production of microRNA (miRNA).
The method is used to alter the expression in a cell of an up-regulated, e.g., as a result of malignant transformation of the cells. Binding of the siRNA to a transcript corresponding to one of the PNC 1-259 in the target cell results in a reduction in the protein production by the cell. The length of the oligonucleotide is at least 10 nucleotides and may be as long as the naturally-occurring transcript. Preferably, the oligonucleotide is 19-25 nucleotides in length. Most preferably, the oligonucleotide is less than 75, 50, 25 nucleotides in length.
The method is also used to alter gene expression in a cell in which expression of PCDH1, CDH3 or GPR107 is up-regulated, e.g., as a result of malignant transformation of the cells. Binding of the siRNA to a PCDH1, CDH3 or GPR107 transcript in the target cell results in a reduction in PCDH1, CDH3 or GPR107 production by the cell. The length of the oligonucleotide is at least about 10 nucleotides and may be as long as the naturally-occurring PCDH1, CDH3 or GPR107 transcript. Preferably, the oligonucleotide is about 19 to about 25 nucleotides in length. Most preferably, the oligonucleotide is less than about 75, about 50, or about 25 nucleotides in length. Examples of siRNA oligonucleotides of PCDH1, CDH3 or GPR107 which inhibit PCDH1, CDH3 or GPR107 expression in mammalian cells include oligonucleotides containing target sequences, for example, nucleotides of SEQ ID NOs: 22, 23 or 24, respectively.
Methods for designing double stranded RNA having the ability to inhibit gene expression in a target cell are known. (See for example, U.S. Pat. No. 6,506,559, herein incorporated by reference in its entirety). For example, a computer program for designing siRNAs is available from the Ambion website (http://www.ambion.com/techlib/misc/siRNA_finder.html). The computer program available from Ambion, Inc. selects nucleotide sequences for siRNA synthesis based on the following protocol.
Selection of siRNA Target Sites
Also included in the invention are isolated nucleic acid molecules that include the nucleic acid sequence of target sequences, for example, nucleotides of SEQ ID NOs: 140, 141 and 142 or a nucleic acid molecule that is complementary to the nucleic acid sequence of nucleotides of SEQ ID NOs: 140, 141 and 142. As used herein, an “isolated nucleic acid” is a nucleic acid removed from its original environment (e.g., the natural environment if naturally occurring) and thus, synthetically altered from its natural state. In the present invention, isolated nucleic acid includes DNA, RNA, and derivatives thereof. When the isolated nucleic acid is RNA or derivatives thereof, base “t” should be replaced with “u” in the nucleotide sequences. As used herein, the term “complementary” refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a nucleic acid molecule, and the term “binding” means the physical or chemical interaction between two nucleic acids or compounds or associated nucleic acids or compounds or combinations thereof. Complementary nucleic acid sequences hybridize under appropriate conditions to form stable duplexes containing few or no mismatches. For the purposes of this invention, two sequences having 5 or fewer mismatches are considered to be complementary. Furthermore, the sense strand and antisense strand of the isolated nucleotide of the present invention, can form double stranded nucleotide or hairpin loop structure by the hybridization. In a preferred embodiment, such duplexes contain no more than 1 mismatch for every 10 matches. In an especially preferred embodiment, where the strands of the duplex are fully complementary, such duplexes contain no mismatches. The nucleic acid molecule is less than 3581, 3205, or 6840 nucleotides in length for PCDH1, CDH3 or GPR107, respectively. For example, the nucleic acid molecule is less than about 500, about 200, or about 75 nucleotides in length. Also included in the invention is a vector containing one or more of the nucleic acids described herein, and a cell containing the vectors. The isolated nucleic acids of the present invention are useful for siRNA against PCDH1, CDH3 or GPR107, or DNA encoding the siRNA. When the nucleic acids are used for siRNA or coding DNA thereof, the sense strand is preferably longer than about 19 nucleotides, and more preferably longer than 21 nucleotides.
The invention is based in part on the discovery that the gene encoding PCDH1, CDH3 or GPR107 is over-expressed in pancreatic ductal adenocarcinoma (PDACa) compared to non-cancerous pancreatic tissue. The cDNA of PCDH1, CDH3 or GPR107 is 3581, 3205 or 6840 nucleotides in length. The nucleic acid and polypeptide sequences of PCDH1, CDH3 or GPR107 are shown in SEQ ID NO: 119 and 120, 121 and 122 or 123 and 124, respectively. The sequence data are also available via following accession numbers.
PCDH1 (CFUPC): L11370, NM—002587
CDH3: X63629, NM—001793
GPR107: NM—032925, (KIAA1624: R39794) AB046844
Transfection of siRNAs comprising SEQ ID NOs: 140, 141 and 142 resulted in a growth inhibition of PDACa cell lines. PCDH1 (CFUPC) belongs to the protocadherin family, the largest subgroup of cadherin superfamily of calcium-dependent cell-cell adhesion molecules. Many of the protocadherin are highly expressed in the central nervous system and they are likely to play roles in neuronal circuit development and the modulation of synaptic transmission (Sano K, Tanihara H, Heimark R L, Obata S, Davidson M, St John T, Taketani S, Suzuki S. Protocadherins: a large family of cadherin-related molecules in central nervous system. EMBO J., 12:2249-56, 1993. Frank M, and Kemler R. Protocadherins. Curr Opin Cell Biol., 14:557-62, 2002). However, PCDH1 is abundant in pancreatic cancer cells, but not in central nervous system (
CDH3 is also a classical member of the cadherin family (Shimoyama Y, Yoshida T, Terada M, Shimosato Y, Abe O, Hirohashi S. Molecular cloning of a human Ca2+-dependent cell-cell adhesion molecule homologous to mouse placental cadherin: its low expression in human placental tissues. J Cell Biol., 109:1787-94. 1989) and they link to catenins and cytoskeletons through its conserved intracellular domain, mediating signal-transduction that control cell polarity, differentiation, motility and cell growth (Christofori G. Changing neighbors, changing behavior: cell adhesion molecules-mediated signaling during tumor progression. EMBO J., 22, 2318-2323, 2003). However, different form E-cadherin or N-cadherin, the function of CDH3 still remains unclear. Its expression is observed in mammary glands and ovary, and loss of expression was reported in breast cancer and prostate cancer, although the expression of P-cadherin in breast cancer correlates with poor prognosis (Peralta Soler A, Knudsen K A, Salazar H, Han A C, Keshgegian A A. P-cadherin expression in breast carcinoma indicates poor survival. Cancer, 86:1263-1272. 1999).
GPR107 (KIAA1624) is one of the G protein-coupled receptors (GPCR) with seven transmembranes. A large percentage of today's prescription drugs target one or more GPCRs with most major therapeutic area being served to some extent by several GPCR-based drugs. Clearly, GPCRs are in the highest rank in the terms of drug discovery potential. GPR107 is expressed without restriction in normal heart, placenta, skeletal muscle, prostate, testis, ovary, spinal cord as shown in Northern blot analysis (
Structure of siRNA Composition
The present invention relates to inhibiting cell growth, i.e, cancer cell growth by inhibiting expression of PCDH1, CDH3 or GPR107. Expression of PCDH1, CDH3 or GPR107 is inhibited, for example, by small interfering RNA (siRNA) that specifically target the PCDH1, CDH3 or GPR107 gene. PCDH1, CDH3 or GPR107 targets include, for example, nucleotides of SEQ ID NOs: 140, 141 and 142.
In non-mammalian cells, double-stranded RNA (dsRNA) has been shown to exert a strong and specific silencing effect on gene expression, which is referred as RNA interference (RNAi) (Sharp P A. RNAi and double-strand RNA. Genes Dev. 1999 Jan. 15; 13(2):139-41.). dsRNA is processed into 20-23 nucleotides dsRNA called small interfering RNA (siRNA) by an enzyme containing RNase III motif. The siRNA specifically targets complementary mRNA with a multicomponent nuclease complex (Hammond S M, Bernstein E, Beach D, Hannon G J. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature. 2000 Mar. 16; 404(6775):293-6; Hannon G J. RNA interference. Nature. 2002 Jul. 11; 418(6894):244-51.). In mammalian cells, siRNA composed of 20 or 21-mer dsRNA with 19 complementary nucleotides and 3′ terminal noncomplementary dimmers of thymidine or uridine, have been shown to have a gene specific knock-down effect without inducing global changes in gene expression (Elbashir S M, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001 May 24; 411(6836):494-8.). In addition, plasmids containing small nuclear RNA (snRNA) U6 or polymerase III H1-RNA promoter effectively produce such short RNA recruiting type III class of RNA polymerase III and thus can constitutively suppress its target mRNA Miyagishi M, Taira K. U6 promoter-driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells. Nat Biotechnol. 2002 May; 20(5):497-500; Brummelkamp T R, Bernards R, Agami R. A System for Stable Expression of Short Interfering RNAs in Mammalian Cells Science. 296(5567):550-553, Apr. 19, 2002.).
The growth of cells is inhibited by contacting a cell, with a composition containing a siRNA of PCDH1, CDH3 or GPR107. The cell is further contacted with a transfection agent. Suitable transfection agents are known in the art. By inhibition of cell growth is meant the cell proliferates at a lower rate or has decreased viability compared to a cell not exposed to the composition. Cell growth is measured by methods known in the art such as, the MTT cell proliferation assay.
The siRNA of PCDH1, CDH3 or GPR107 is directed to a single target of PCDH1, CDH3 or GPR107 gene sequence. Alternatively, the siRNA is directed to multiple target of PCDH1, CDH3 or GPR107 gene sequences. For example, the composition contains siRNA of PCDH1, CDH3 or GPR107 directed to two, three, four, or five or more target sequences of PCDH1, CDH3 or GPR107. By PCDH1, CDH3 or GPR107 target sequence is meant a nucleotide sequence that is identical to a portion of the PCDH1, CDH3 or GPR107 gene. The target sequence can include the 5′ untranslated (UT) region, the open reading frame (ORF) or the 3′ untranslated region of the human PCDH1, CDH3 or GPR107 gene. Alternatively, the siRNA is a nucleic acid sequence complementary to an upstream or downstream modulator of PCDH1, CDH3 or GPR107 gene expression. Examples of upstream and downstream modulators include, a transcription factor that binds the PCDH1, CDH3 or GPR107 gene promoter, a kinase or phosphatase that interacts with the PCDH1, CDH3 or GPR107 polypeptide, a PCDH1, CDH3 or GPR107 promoter or enhancer. siRNA of PCDH1, CDH3 or GPR107 which hybridize to target mRNA decrease or inhibit production of the PCDH1, CDH3 or GPR107 polypeptide product encoded by the PCDH1, CDH3 or GPR107 gene by associating with the normally single-stranded mRNA transcript, thereby interfering with translation and thus, expression of the protein. Thus, siRNA molecules of the invention can be defined by their ability to hybridize specifically to mRNA or cDNA from a PCDH1, CDH3 or GPR107 gene under stringent conditions. For the purposes of this invention the terms “hybridize” or “hybridize specifically” are used to refer the ability of two nucleic acid molecules to hybridize under “stringent hybridization conditions.” The phrase “stringent hybridization conditions” refers to conditions under which a nucleic acid molecule will hybridize to its target sequence, typically in a complex mixture of nucleic acids, but not detectably to other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 50° C.
The siRNA of the invention is less than about 500, about 200, about 100, about 50, or about 25 nucleotides in length. Preferably the siRNA is about 19 to about 25 nucleotides in length. Exemplary nucleic acid sequence for the production of PCDH1, CDH3 or GPR107 siRNA include the sequences of nucleotides of SEQ ID NOs: 140, 141 or 142 as the target sequence, respectively. Furthermore, in order to enhance the inhibition activity of the siRNA, nucleotide “u” 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 about 2, generally about 2 to about 10, preferably about 2 to about 5. The added “u”s form single strand at the 3′end of the antisense strand of the siRNA.
The cell is any cell that expresses or over-expresses PCDH1, CDH3 or GPR107. The cell is an epithelial cell such as a pancreatic ductal cell. Alternatively, the cell is a tumor cell such as a carcinoma, adenocarcinoma, blastoma, leukemia, myeloma, or sarcoma. The cell is a pancreatic ductal adenocarcinoma.
An siRNA of PCDH1, CDH3 or GPR107 is directly introduced into the cells in a form that is capable of binding to the mRNA transcripts. Alternatively, the DNA encoding the siRNA of PCDH1, CDH3 or GPR107 is in a vector.
Vectors are produced for example by cloning a PCDH1, CDH3 or GPR107 target sequence into an expression vector operatively-linked regulatory sequences flanking the PCDH1, CDH3 or GPR107 sequence in a manner that allows for expression (by transcription of the DNA molecule) of both strands (Lee, N. S., Dohjima, T., Bauer, G., Li, H., Li, M.-J., Ehsani, A., Salvaterra, P., and Rossi, J. (2002) Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nature Biotechnology 20: 500-505.). An RNA molecule that is antisense to PCDH1, CDH3 or GPR107 mRNA 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 PCDH1, CDH3 or GPR107 mRNA 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 PCDH1, CDH3 or GPR107 gene. Alternatively, two constructs are utilized to create the sense and anti-sense strands of a siRNA construct. Cloned PCDH1, CDH3 or GPR107 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 that specifically hybridizes to an mRNA or a cDNA from PCDH1, CDH3 or GPR107. In preferred embodiments, [A] is a ribonucleotide sequence corresponding to a sequence selected from the group consisting of nucleotides of SEQ ID NOs: 140, 141 and 142,
[B] is a ribonucleotide sequence consisting of 3 to 23 nucleotides, and
[A′] is a ribonucleotide sequence consisting of the complementary sequence of [A]
The region [A] hybridizes to [A′], and then a loop consisting of region [B] is formed. The loop sequence may be preferably about 3 to about 23 nucleotides in length. The loop sequence, for example, can be selected from group consisting of following sequences (http://www.ambion.com/techlib/tb/tb—506.html). Furthermore, loop sequence consisting of 23 nucleotides also provides active siRNA (Jacque, J.-M., Triques, K., and Stevenson, M. (2002) Modulation of HIV-1 replication by RNA interference. Nature 418: 435-438.).
CCC, CCACC or CCACACC: Jacque, J. M., Triques, K., and Stevenson, M (2002) Modulation of HIV-1 replication by RNA interference. Nature, Vol. 418: 435-438.
UUCG: Lee, N. S., Dohjima, T., Bauer, G., Li, H., Li, M.-J., Ehsani, A., Salvaterra, P., and Rossi, J. (2002) Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nature Biotechnology 20: 500-505. Fruscoloni, P., Zamboni, M., and Tocchini-Valentini, G. P. (2003) Exonucleolytic degradation of double-stranded RNA by an activity in Xenopus laevis germinal vesicles. Proc. Natl. Acad. Sci. USA 100(4): 1639-1644.
UUCAAGAGA: Dykxhoorn, D. M., Novina, C. D., and Sharp, P. A. (2002) Killing the messenger: Short RNAs that silence gene expression. Nature Reviews Molecular Cell Biology 4: 457-467.
For example, preferable siRNAs having hairpin loop structure of the present invention are shown below. In the following structure, the loop sequence can be selected from group consisting of CCC, UUCG, CCACC, CCACACC, and UUCAAGAGA. Preferable loop sequence is UUCAAGAGA (“ttcaagaga” in DNA (SEQ ID NO: 153)).
The regulatory sequences flanking the PCDH1, CDH3 or GPR107 sequence are identical or are different, such that their expression can be modulated independently, or in a temporal or spatial manner. siRNAs are transcribed intracellularly by cloning the PCDH1, CDH3 or GPR107 gene templates into a vector containing, e.g., a RNA polymerase III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter. For introducing the vector into the cell, transfection-enhancing agent can be used. FuGENE (Roche Diagnostices), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen), and Nucleofector (Wako pure Chemical) are useful as the transfection-enhancing agent.
Oligonucleotides and oligonucleotides complementary to various portions of PCDH1, CDH3 or GPR107 mRNA were tested in vitro for their ability to decrease production of PCDH1, CDH3 or GPR107 in tumor cells (e.g., using the pancreatic cell line such as pancreatic ductal adenocarcinoma (PDACa) cell line) according to standard methods. A reduction in PCDH1, CDH3 or GPR107 gene product in cells contacted with the candidate siRNA composition compared to cells cultured in the absence of the candidate composition is detected using specific antibodies of PCDH1, CDH3 or GPR107 or other detection strategies. Sequences which decrease production of PCDH1, CDH3 or GPR107 in in vitro cell-based or cell-free assays are then tested for there inhibitory effects on cell growth. Sequences which inhibit cell growth in vitro cell-based assay are test in vivo in rats or mice to confirm decreased PCDH1, CDH3 or GPR107 production and decreased tumor cell growth in animals with malignant neoplasms.
Patients with tumors characterized as over-expressing PCDH1, CDH3 or GPR107 are treated by administering siRNA of PCDH1, CDH3 or GPR107. siRNA therapy is used to inhibit expression of PCDH1, CDH3 or GPR107 in patients suffering from or at risk of developing, for example, pancreatic ductal adenocarcinoma (PDACa). Such patients are identified by standard methods of the particular tumor type. Pancreatic ductal adenocarcinoma (PDACa) 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 PCDH1, CDH3 or GPR107, 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 symptom of 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 a 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 PCDH1, CDH3 or GPR107 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 PCDH1, CDH3 or GPR107. For treatment of pancreatic tumors, direct infusion the celiac artery, splenic artery, or common hepatic artery, 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 promoters.
The antisense oligonucleotide or siRNA of the invention inhibit the expression of the polypeptide of the invention and is thereby useful for suppressing the biological activity of the polypeptide of the invention. Also, expression-inhibitors, comprising the antisense oligonucleotide or 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 comprising the antisense oligonucleotide or siRNA of the present invention is useful in treating a pancreatic cancer.
Alternatively, function of one or more gene products of the over-expressed genes is inhibited by administering a compound that binds to or otherwise inhibits the function of the gene products. For example, the compound is an antibody which binds to the over-expressed gene product or gene products.
The present invention refers to the use of antibodies, particularly antibodies against a protein encoded by an up-regulated marker gene, or a fragment of the antibody. As used herein, the term “antibody” refers to an immunoglobulin molecule having a specific structure, that interacts (i.e., binds) only with the antigen that was used for synthesizing the antibody (i.e., the up-regulated marker gene product) or with an antigen closely related to it. Furthermore, an antibody may be a fragment of an antibody or a modified antibody, so long as it binds to one or more of the proteins encoded by the marker genes. For instance, the antibody fragment may be Fab, F(ab′)2, Fv, or single chain Fv (scFv), in which Fv fragments from H and L chains are ligated by an appropriate linker (Huston J. S. et al. Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883 (1988)). More specifically, an antibody fragment may be generated by treating an antibody with an enzyme, such as papain or pepsin. Alternatively, a gene encoding the antibody fragment may be constructed, inserted into an expression vector, and expressed in an appropriate host cell (see, for example, Co M. S. et al. J. Immunol. 152:2968-2976 (1994); Better M. and Horwitz A. H. Methods Enzymol. 178:476-496 (1989); Pluckthun A. and Skerra A. Methods Enzymol. 178:497-515 (1989); Lamoyi E. Methods Enzymol. 121:652-663 (1986); Rousseaux J. et al. Methods Enzymol. 121:663-669 (1986); Bird R. E. and Walker B. W. Trends Biotechnol. 9:132-137 (1991)).
An antibody may be modified by conjugation with a variety of molecules, such as polyethylene glycol (PEG). The present invention provides such modified antibodies. The modified antibody can be obtained by chemically modifying an antibody. These modification methods are conventional in the field.
Alternatively, an antibody may be obtained as a chimeric antibody, between a variable region derived from a nonhuman antibody and a constant region derived from a human antibody, or as a humanized antibody, comprising the complementarity determining region (CDR) derived from a nonhuman antibody, the frame work region (FR) derived from a human antibody, and the constant region. Such antibodies can be prepared by using known technologies.
Cancer therapies directed at specific molecular alterations that occur in cancer cells have been validated through clinical development and regulatory approval of anti-cancer drugs such as trastuzumab (Herceptin) for the treatment of advanced breast cancer, imatinib methylate (Gleevec) for chronic myeloid leukemia, gefitinib (Iressa) for non-small cell lung cancer (NSCLC), and rituximab (anti-CD20 mAb) for B-cell lymphoma and mantle cell lymphoma (Ciardiello F, Tortora G. A novel approach in the treatment of cancer: targeting the epidermal growth factor receptor. Clin Cancer Res. 2001 October; 7(10):2958-70. Review; Slamon D J, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, Fleming T, Eiermann W, Wolter J, Pegram M, Baselga J, Norton L. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med. 2001 Mar. 15; 344(11):783-92; Rehwald U, Schulz H, Reiser M, Sieber M, Staak J O, Morschhauser F, Driessen C, Rudiger T, Muller-Hermelink K, Diehl V, Engert A. Treatment of relapsed CD20+ Hodgkin lymphoma with the monoclonal antibody rituximab is effective and well tolerated: results of a phase 2 trial of the German Hodgkin Lymphoma Study Group. Blood. 2003 Jan. 15; 101(2):420-424; Fang G, Kim C N, Perkins C L, Ramadevi N, Winton E, Wittmann S and Bhalla K N. (2000). Blood, 96, 2246-2253.). These drugs are clinically effective and better tolerated than traditional anti-cancer agents because they target only transformed cells. Hence, such drugs not only improve survival and quality of life for cancer patients, but also validate the concept of molecularly targeted cancer therapy. Furthermore, targeted drugs can enhance the efficacy of standard chemotherapy when used in combination with it (Gianni L. (2002). Oncology, 63 Suppl 1, 47-56; Klejman A, Rushen L, Morrione A, Slupianek A and Skorski T. (2002). Oncogene, 21, 5868-5876.). Therefore, future cancer treatments will probably involve combining conventional drugs with target-specific agents aimed at different characteristics of tumor cells such as angiogenesis and invasiveness.
These modulatory methods are performed ex vivo or in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). The method involves administering a protein or combination of proteins or a nucleic acid molecule or combination of nucleic acid, molecules as therapy to counteract aberrant expression or activity of the differentially expressed genes.
Diseases and disorders that are characterized by increased (relative to a subject not suffering from the disease or disorder) levels or biological activity of the genes may be treated with therapeutics that antagonize (i.e., reduce or inhibit) activity of the over-expressed gene or genes. Therapeutics that antagonized activity are administered therapeutically or prophylactically.
Therapeutics that may be utilized include, e.g., (i) a polypeptide, or analogs, derivatives, fragments or homologs thereof of the over-expressed or under-expressed gene or genes; (ii) antibodies to the over-expressed gene or genes; (iii) nucleic acids encoding the over-expressed or under-expressed gene or genes; (iv) antisense nucleic acids or nucleic acids that are “dysfunctional” (i.e., due to a heterologous insertion within the nucleic acids of one or more over-expressed gene or genes); (v) small interfering RNA (siRNA); or (vi) modulators (i.e., inhibitors, agonists and antagonists that alter the interaction between an over/under-expressed polypeptide and its binding partner). The dysfunctional antisense molecules are utilized to “knockout” endogenous function of a polypeptide by homologous recombination (see, e.g., Capecchi, Science 244: 1288-1292 1989). 259
Diseases and disorders that are characterized by decreased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with therapeutics that increase (i.e., are agonists to) activity. Therapeutics that up-regulate activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to, a polypeptide (or analogs, derivatives, fragments or homologs thereof) or an agonist that increases bioavailability.
Increased or decreased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or mRNAs of a gene whose expression is altered). Methods that are well-known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, etc.).
Prophylactic administration occurs prior to the manifestation of overt clinical symptoms of disease, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
Therapeutic methods include contacting a cell with an agent that modulates one or more of the activities of the gene products of the differentially expressed genes. An agent that modulates protein activity includes a nucleic acid or a protein, a naturally-occurring cognate ligand of these proteins, a peptide, a peptidomimetic, or other small molecule. For example, the agent stimulates one or more protein activities of one or more of a differentially under-expressed gene.
The present invention also relates to a method of treating or preventing pancreatic cancer in a subject comprising administering to said subject a vaccine comprising a polypeptide encoded by a nucleic acid selected from the group consisting of PNC 1-259 or an immunologically active fragment of said polypeptide, or a polynucleotide encoding the polypeptide or the fragment thereof. An administration of the polypeptide induces an anti-tumor immunity in a subject. To inducing anti-tumor immunity, a polypeptide encoded by a nucleic acid selected from the group consisting of PNC 1-259 or an immunologically active fragment of said polypeptide, or a polynucleotide encoding the polypeptide is administered. The polypeptide or the immunologically active fragments thereof are useful as vaccines against PNC. In some cases the proteins or fragments thereof may be administered in a form bound to the T cell recepor (TCR) or presented by an antigen presenting cell (APC), such as macrophage, dendritic cell (DC), or B-cells. Due to the strong antigen presenting ability of DC, the use of DC is most preferable among the APCs.
In the present invention, vaccine against PNC refers to a substance that has the function to induce anti-tumor immunity upon inoculation into animals. According to the present invention, polypeptides encoded by PNC 1-259 or fragments thereof were suggested to be HLA-A24 or HLA-A*0201 restricted epitopes peptides that may induce potent and specific immune response against PNC cells expressing PNC 1-259. Thus, the present invention also encompasses method of inducing anti-tumor immunity using the polypeptides. In general, anti-tumor immunity includes immune responses such as follows:
induction of cytotoxic lymphocytes against tumors,
induction of antibodies that recognize tumors, and
induction of anti-tumor cytokine production.
Therefore, when a certain protein induces any one of these immune responses upon inoculation into an animal, the protein is decided to have anti-tumor immunity inducing effect. The induction of the anti-tumor immunity by a protein can be detected by observing in vivo or in vitro the response of the immune system in the host against the protein.
For example, a method for detecting the induction of cytotoxic T lymphocytes is well known. A foreign substance that enters the living body is presented to T cells and B cells by the action of antigen presenting cells (APCs). T cells that respond to the antigen presented by APC in antigen specific manner differentiate into cytotoxic T cells (or cytotoxic T lymphocytes; CTLS) due to stimulation by the antigen, and then proliferate (this is referred to as activation of T cells). Therefore, CTL induction by a certain peptide can be evaluated by presenting the peptide to T cell by APC, and detecting the induction of CTL. Furthermore, APC has the effect of activating CD4+ T cells, CD8+ T cells, macrophages, eosinophils, and NK cells. Since CD4+ T cells and CD8+ T cells are also important in anti-tumor immunity, the anti-tumor immunity inducing action of the peptide can be evaluated using the activation effect of these cells as indicators.
A method for evaluating the inducing action of CTL using dendritic cells (DCs) as APC is well known in the art. DC is a representative APC having the strongest CTL inducing action among APCs. In this method, the test polypeptide is initially contacted with DC, and then this DC is contacted with T cells. Detection of T cells having cytotoxic effects against the cells of interest after the contact with DC shows that the test polypeptide has an activity of inducing the cytotoxic T cells. Activity of CTL against tumors can be detected, for example, using the lysis of 51Cr-labeled tumor cells as the indicator. Alternatively, the method of evaluating the degree of tumor cell damage using 3H-thymidine uptake activity or LDH (lactose dehydrogenase)-release as the indicator is also well known.
Apart from DC, peripheral blood mononuclear cells (PBMCs) may also be used as the APC. The induction of CTL is reported that it can be enhanced by culturing PBMC in the presence of GM-CSF and IL-4. Similarly, CTL has been shown to be induced by culturing PBMC in the presence of keyhole limpet hemocyanin (KLH) and IL-7.
The test polypeptides confirmed to possess CTL inducing activity by these methods are polypeptides having DC activation effect and subsequent CTL inducing activity. Therefore, polypeptides that induce CTL against tumor cells are useful as vaccines against tumors. Furthermore, APC that acquired the ability to induce CTL against tumors by contacting with the polypeptides are useful as vaccines against tumors. Furthermore, CTL that acquired cytotoxicity due to presentation of the polypeptide antigens by APC can be also used as vaccines against tumors. Such therapeutic methods for tumors using anti-tumor immunity due to APC and CTL are referred to as cellular immunotherapy.
Generally, when using a polypeptide for cellular immunotherapy, efficiency of the CTL-induction is known to increase by combining a plurality of polypeptides having different structures and contacting them with DC. Therefore, when stimulating DC with protein fragments, it is advantageous to use a mixture of multiple types of fragments.
Alternatively, the induction of anti-tumor immunity by a polypeptide can be confirmed by observing the induction of antibody production against tumors. For example, when antibodies against a polypeptide are induced in a laboratory animal immunized with the polypeptide, and when growth of tumor cells is suppressed by those antibodies, the polypeptide can be determined to have an ability to induce anti-tumor immunity.
Anti-tumor immunity is induced by administering the vaccine of this invention, and the induction of anti-tumor immunity enables treatment and prevention of PNC. Therapy against cancer or prevention of the onset of cancer includes any of the steps, such as inhibition of the growth of cancerous cells, involution of cancer, and suppression of occurrence of cancer. Decrease in mortality of individuals having cancer, decrease 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 vaccine against cell proliferative diseases is compared to a control without vaccine administration. For example, Student's t-test, the Mann-Whitney U-test, or ANOVA may be used for statistical analyses.
The above-mentioned protein having immunological activity or a vector encoding the protein may be combined with an adjuvant. An adjuvant refers to a compound that enhances the immune response against the protein when administered together (or successively) with the protein having immunological activity. Examples of adjuvants include cholera toxin, salmonella toxin, alum, and such, but are not limited thereto. Furthermore, the vaccine of this invention may be combined appropriately with a pharmaceutically acceptable carrier. Examples of such carriers are sterilized water, physiological saline, phosphate buffer, culture fluid, and such. Furthermore, the vaccine may contain as necessary, stabilizers, suspensions, preservatives, surfactants, and such. The vaccine is administered systemically or locally. Vaccine administration may be performed by single administration, or boosted by multiple administrations.
When using APC or CTL as the vaccine of this invention, tumors can be treated or prevented, for example, by the ex vivo method. More specifically, PBMCs of the subject receiving treatment or prevention are collected, the cells are contacted with the polypeptide ex vivo, and following the induction of APC or CTL, the cells may be administered to the subject. APC can be also induced by introducing a vector encoding the polypeptide into PBMCs ex vivo. APC or CTL induced in vitro can be cloned prior to administration. By cloning and growing cells having high activity of damaging target cells, cellular immunotherapy can be performed more effectively. Furthermore, APC and CTL isolated in this manner may be used for cellular immunotherapy not only against individuals from whom the cells are derived, but also against similar types of tumors from other individuals.
Furthermore, a pharmaceutical composition for treating or preventing a cell proliferative disease, such as cancer, comprising a pharmaceutically effective amount of the polypeptide of the present invention is provided. The pharmaceutical composition may be used for raising anti tumor immunity.
The present invention provides a method for treating or preventing malignant pancreatic cancer, or recurrence of pancreatic cancer by increasing or decreasing the expression or activity of marker genes. According to the present invention, the marker genes that can be used for the treatment or prevention of malignant pancreatic cancer are PNC 606-681 (Table 6) and PNC 682-849 (Table 7). Alternatively, the marker genes for treating or preventing the recurrence are PNC 850-933 (Table 8). 35 genes of the PNC 606-640 (
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 the active ingredient. Formulations also include powders, granules or solutions, suspensions or emulsions. The active ingredient is optionally administered as a bolus electuary or paste. 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. 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 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 include suppositories with standard carriers such as cocoa butter or polyethylene glycol. Formulations for topical administration in the mouth, for example buccally or sublingually, include lozenges, which contain 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 or 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 or suspending agents.
For administration by inhalation the compounds are 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, dichiorotetrafluoroethane, 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, in for example, 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 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 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, e.g., polypeptides and organic compounds are administered orally or via injection at a dose of 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 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. Also the route of administration may vary depending upon the condition and its severity.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims. The following examples illustrate the identification and characterization of genes differentially expressed in PNC cells.
Genome-Wide cDNA Microarray Analysis of Gene-Expression Profiles of Pancreatic Cancer Using Cancer and Normal Ductal Epithelial Cells Purely Selected by Laser Microdissection
Tumor markers and targets for therapeutic intervention were identified by analyzing gene-expression profiles using a cDNA microarray representing 23,040 genes. Pancreatic ductal adenocarcinoma that has a characteristic of highly desmoplastic stromal reaction contained a low proportion of cancer cells in the tumor mass. Furthermore, normal duct epithelial cells from which the pancreatic carcinoma originates correspond to a few percent of the pancreas tissue. Therefore, cancer cells were purified from 18 pancreatic cancers by means of laser microbeam microdissection (LMM). Gene expression profiles were examined and compared with those of normal purified pancreatic ductal epithelial cells. These cell populations had been rendered homogenous (more than 95% purified cells). As a result, 259 genes were identified to be commonly up-regulated in pancreatic cancer cells; among them, the disease correlation and/or function of 64 (including 30 ESTs) genes were not known prior to the invention. The up-regulated genes included ones that were previously reported to be over-expressed in pancreatic cancer, such as interferon-induced transmembrane protein 1 (IFITM1), plasminogen activator, urokinase (PLAU), prostate stem cell antigen (PSCA), S100 calcium binding protein P (S100P), and baculoviral IAP repeat-containing 5 (BIRC5). 346 genes were identified as being commonly down-regulated in pancreatic cancer cells. Of them, 211 genes were functionally characterized and included some tumor suppressor genes such as AXIN1 up-regulated 1 (AXUD1), deleted in liver cancer 1 (DLC1), growth arrest and DNA-damage-inducible, beta (GADD45B), p53-inducible p53DNP1 (p53DINP1).
The present gene expression profile represents a highly accurate cancer reference, because a number of limitations of earlier methods were overcome. First, a microarray analysis using clinical samples has been difficult, because of various cellular components are present in the normal as well as cancer tissues. In particular, pancreatic ductal adenocarcinoma that has a characteristic of highly desmoplastic stromal reaction contained a low proportion of cancer cells in the tumor mass. Furthermore, the normal pancreas is mostly constituted from acinar cells and islets that accounted for more than 95% of whole pancreas, and normal duct epithelial cells from which the pancreatic carcinoma originates correspond to a few % of the pancreas. Therefore, the analysis of gene-expression profiles using bulk pancreatic cancer and normal whole pancreatic tissues is significantly influenced by the proportions of cells mixed in the tissues examined; proportional differences of acinar cells, islet cells, fibroblasts, and inflammatory cells may mask the significant increase or decrease of genes that are involved in pancreatic carcinogenesis. Hence, in this study, LMM systems were used to purify cancer and normal epithelial cells from surgical specimens to a high degree of purity (95% or higher). Because it is possible to microdissect even a single cell with LMM, this technology is critical for an accurate microarray analysis of pancreatic cancer specimens. To evaluate the purify of micordissected pancreatic cancer and normal ductal cells, the expression profile of AMY1A gene which is known to be expressed specifically in acinar cells were analyzed. As a result, the proportion of contaminating acinar cells in the dissected normal pancreatic ductal epithelial cells was estimated to be smaller than 0.29%. In addition to AMY1A, expression levels of other genes that were highly expressed in acinar cells like elastase 1, trypsin 1, and pancreatic lipase were examined. Similar results were obtained, indicating that the purify of cell populations by the LMM technique was as high as 99.2%-99.7%.
Second, the quality of extracted RNA from clinical tissue, particularly from pancreas, is one of the most important factors. Pancreas is known to be RNase-rich organ and degradation of RNA occurs very rapidly. In this study, the quality of the extracted RNA from the specimen was examined by visualization of 28S and 18S ribosomal RNAs using denaturing agarose gel electrophoresis. Following electrophoretic analysis, samples in which bands corresponding to two ribosomal RNAs were clearly observed were selected. For example, 18 cases (32%) were selected from the 56 surgically-resected cases, i.e., many were not included in the analysis due to the poor quality of RNA.
Careful purification of cancer cells as well as normal epitherial ductal cells, subsequent RNA isolation, and cDNA microarray analysis identified 259 genes whose expression was commonly up-regulated (genes which were able to obtain expression data in more than 50% cancer cases and whose expression ratio (Cy5/Cy3 intensity ratio) was more than 5.0 and the genes which were able to calculate in 33 to 50% cases and which expressed the expression ratio of more than 5.0 in all of that cases were also evaluated).
Over 90% of the gene expression profile of pancreatic cancer was different from previous pancreatic cancer expression profiles, because the expression data was obtained by testing highly purified cell populations obtained from patient tissues using laser dissection techniques.
The profiles obtained and described herein represent an improvement over earlier profiles, because they were obtained by analyzing highly purified populations of cancerous cells (pancreatic ductal adenocarcinoma) and compared to a highly purified population of the most relevant normal control, i.e., normal duct epithelial cells. Earlier methods and profiles were hampered by a high percentage of contaminating cells, which reduced the accuracy and reliability of earlier profiles. This present profile is the first one of precise and genome-wide gene expression profiles in large-scale pancreatic cancer. These data identify molecular targets for therapeutic modulation for the treatment of pancreatic cancer and specific novel tumor markers for early and accurate diagnosis of the cancer or a precancerous condition.
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. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
Tissue obtained from diseased tissue (e.g., epithelial cells from PNC) and normal tissues was evaluated to identify genes which are differently expressed or a disease state, e.g., PNC. The assays were carried out as follows.
Patients, Tissue Samples, and Laser Microdissection Tissue samples of pancreatic cancer (n=18) and normal pancreas (n=7) were obtained from surgical specimens from patients with informed consent. All pancreatic cancer tissues had histologically confirmed invasive ductal carcinoma. Clinicopathological features of the patients we used in this study are summarized in Table 1. Since almost all pancreatic ductal cells from corresponding normal tissue blocks showed dysplastic changes mostly because of downstream ductal obstruction, ductal cells for only 4 of the 18 pancreatic cancer tissues were suitable to use as normal controls. Hence, additional control ductal cells were obtained from 3 normal pancreas tissues from patients who were operated by cholangiocarcinoma, duodenal leiomyosarcoma, or ampullary tumor. In each case, the specimens were harvested immediately after surgical resection and were embedded in TissueTek OCT medium (Sakura, Tokyo, Japan) before storage at −80° C. The frozen tissues were cut to 8-μm sections using a cryostat (Sakura, Tokyo, Japan) and then stained with Hematoxylin and Eosin, and check the histological state. Pancreatic carcinoma cells and normal pancreatic ductal epithelial cells were isolated selectively using the EZ cut system with pulsed ultraviolet narrow beam focus laser (SL Microtest GmbH, Germany) in accordance with the manufacturer's protocols. After microdissection, 7 normal cases were mixed to make a “universal control of normal pancreatic ductal epithelial cells”, that was used as a control for all 118 cancer samples.
To obtain precise expression profiles of pancreatic cancer cells, LMM was used to purify cancer cells and avoid contamination of non-cancerous cells. In addition, since pancreatic cancer originates from pancreatic ductal cells, pancreatic ductal epithelial cells were used as controls. The great majority of cells in pancreas are acinar cells, it was determined that the use of the entire pancreas was inappropriate for screening genes associated with pancreatic carcinogenesis. As shown in
The proportion of acinar cells contaminated was examined in the microdissected normal pancreatic ductal epithelial cells which used as universal control (
(Ratio A) the AMY1A/ACTB intensity ratio in whole pancreas (most of the cells correspond to acinar cells)=96.74
(Ratio B) the AMY1A/ACTB intensity ratio in microdissected normal ductal epithelial cells=0.28
Contamination percentage (%); (Ratio B)/(Ratio A)×100=0.29%
Extraction of RNA and T7-Based RNA Amplification
Total RNAs were extracted from each sample of laser-microdissected cells into 350 μl of RLT lysis buffer (QIAGEN, Hilden, Germany). The extracted RNAs were treated for 30 minutes at room temperature with 30 units of DNase I (Roche, Basel, Switzerland) in the presence of 1 unit of RNase inhibitor (TOYOBO, Osaka, Japan) to remove any contaminating genomic DNA. After inactivation at 70° C. for 10 min, the RNAs were purified with an RNeasy Mini Kit (QIAGEN) according to the manufacturer's recommendations. All of the DNase I-treated RNAs were subjected to T7-based RNA amplification as described previously. Two rounds of amplification yielded 50-100 μg of aRNA from each sample. A 2.5 μg aliquot of aRNA from cancer and normal pancreatic duct epithelial cells was labeled with Cy5-dCTP or Cy3-dCTP, respectively, by a protocol described elsewhere. The hybridization, washing, and scanning were carried out according to the methods described previously (11).
Preparation of the cDNA Microarray
A genome-wide cDNA microarray with 23,040 cDNAs selected from the UniGene database (build # 131) of the National Center for Biotechnology Information (NCBI) was constructed. Briefly, the cDNAs were amplified by RT-PCR using poly(A)+ RNA isolated from various human organs as templates; the lengths of the amplicons ranged from 200 to 1,100 bp that did not contain repetitive or poly(A) sequences. The cDNA microarray system was constructed essentially as described previously (11).
Acquisition of Data
Signal intensities of Cy3 and Cy5 from the 23,040 spots were quantified and analyzed by substituting backgrounds, using ArrayVision software (Imaging Research, Inc., St. Catharines, Ontario, Canada). Subsequently, the fluorescent intensities of Cy5 (tumor) and Cy3 (control) for each target spot were adjusted so that the mean Cy3/Cy5 ratio of the 52 housekeeping genes was equal to one. Because the data derived from low signal intensities are less reliable, a cut-off value for signal intensities was determined on each slide and excluded genes from further analysis when both Cy3 and Cy5 dyes provided signal intensities lower than the cut-off as described previously (12). For other genes we calculated the Cy5/Cy3 ratio using raw data of each sample.
Semi-Quantitative RT-PCR
The 12 highly up-regulated genes were selected and examined their expression levels by applying the semi-quantitative RT-PCR experiments. A 3-μg aliquot of aRNA from each sample was reversely-transcribed for single-stranded cDNAs using random primer (Roche) and Superscript II (Life Technologies, Inc.). Each cDNA mixture was diluted for subsequent PCR amplification with the same primer sets that were prepared for the target DNA or tubulin, alpha-specific reactions. The primer sequences are listed in Table 2. Expression of tubulin-alpha served as an internal control. PCR reactions were optimized for the number of cycles to ensure product intensity within the linear phase of amplification.
The up- or down-regulated genes were identified common to pancreatic cancer using following criteria; 1) genes which were able to obtain expression data in more than 50% cancer cases, and 2) genes whose expression ratio was more than 5.0 in pancreatic cancer cells (defined as up-regulated genes) or genes whose expression ratio was under 0.2 (defined as down-regulated genes) in more than 50% of informative cases. Moreover, 3) the genes which were able to calculate in 33 to 50% cases and which expressed the expression ratio of more than 5.0 in all of that cases were also evaluated as up-regulated genes.
Identification of Genes with Clinically Relevant Expression Patterns in PNC Cells
The expression of approximately 23,000 genes in 18 pancreatic cancer patients was examined using cDNA microarray. Individual data were excluded when both Cy5 and Cy3 signals were under cut-off values. Two hundred fifty-nine up-regulated genes were identified whose expression ratio was more than 5.0 in PNC cells (see Table 3). 167 of them were expressed greater than 10-fold comparing to the normal ductal cells. Three hundred forty-six down-regulated genes whose expression ratio was less than 0.2 were identified (see Table 4).
Among the up-regulated genes, interferon induced transmembrane protein 1 (IFITM1), plasminogen activator, urokinase (PLAU), prostate stem cell antigen (PSCA), S100 calcium binding protein P (S100P), RNA binding-motif single-stranded interacting protein 1 (RBMS1), and baculoviral IAP repeat-containing 5 (BIRC5), have been reported to be over-expressed in pancreatic cancer (5, 6). Furthermore, these up-regulated genes included ones encoding proteins involved in the signal transduction pathway, transcriptional factors, cell cycle, and cell adhesion (Table 5).
Significantly over-expressed genes have diagnostic potential, and of them which were critical for tumor growth have also therapeutic potential. Specifically, genes such as regenerating gene type IV (REGIV), ephrin type-A receptor 4 precursor (EphA4), and vang (van gogh, Drosophila)-like 1 (VANGL1), are useful as a potential molecular target for new therapeutic agents.
REGIV was over-expressed in all informative pancreatic cancer cases, and the overexpression was confirmed in 7 of the 12 pancreatic cancer cases by semi-quantitative RT-PCR. Since REGIV protein was thought to be a secreted protein from the amino-acid sequences and in fact its secretion was detected in the culture medium of HT29-5M12 cells (22), it is a candidate as tumor marker.
EphA4 was indicated to be over-expressed in 12 of the 14 informative pancreatic cancer cases in the microarray, and confirmed in 9 of the 12 cases were examined by semi-quantitative RT-PCR. EphA4 is known to be a membrane receptor belonging to the ephrin family, which contains an intracellular tyrosine kinase catalytic domain (23). Involvement of EphA4 in any human cancer has not been reported. However, its nature of the cytoplasmic membrane receptor protein with possible tyrosine kinase activity as well as high level expression in cancer cells suggest that EphA4 is a candidate gene for therapeutic agents.
VANGL1 was over-expressed if all of the informative pancreatic cancer cases in the microarray data, and its high expression was also confirmed in 9 of the 12 cases by semi-quantitative RT-PCR. VANGL1, which contained four putative transmembrane domains, was expressed specifically in testis and ovary among 29 normal tissues examined (4). This gene was also highly and frequency transactivated in hepatocellular carcinoma. Since the enforced reduction of this gene expression in hepatocellular carcinomas induced apoptosis (4), this gene product is a good candidate for development of novel anti-cancer drugs. Among the genes that were functionally highly over-expressed in pancreatic cancer such as the above mentioned genes, those whose products are putative membranous or secreted are of interest for potential as novel anti-cancer drugs or as serological diagnostic markers for early detection.
To confirm the reliability of the expression profiles indicated by microarray analysis, semi-quantitative RT-PCR experiments were performed. Other 55 genes whose cancer/normal ratios were highest among the informative genes, APP, ARHGDIB, ATDC, ATP1B3, BIRC5, BUB1B, CELSR3, CKS1, CKS2, CYP2S1, E2-EPF, ELF4, ENC1, Evi-1, FOXM1, GW112, GYS1, HDGF, HOXB7, hPAD-colony 10, KNSL6, KPNB2, MMP11, MYBL2, OAS1, ORP150, PCOLN3, PPM1B, PRC1, PSCA, PYCR1, RBMS1, S100P, SFN, SLC12A2, SLC2A1, SRD5A1, TCEA1, TK1, UBCH10, WHSC1, FLJ10134, FLJ10540, FLJ20225, KIAA0101, KIAA1624, KIAA1808, FLJ21504, FXYD3, and 6 ESTs (Accession No. AI349804, AA806114, AA419568, AA570186, AA830326, AI027791) were PCR-amplified and compared with the microarray data. As shown in
APP was confirmed whose over-expression in 10 of the 12 cases,
ARHGDIB was confirmed whose over-expression in 12 cases,
ATDC was confirmed whose over-expression in 10 of the 12 cases,
ATP1B3 was confirmed whose over-expression in 12 cases,
BIRC5 was confirmed whose over-expression in 12 cases,
BUB1B was confirmed whose over-expression in 12 cases,
CELSR3 was confirmed whose over-expression in 9 of the 12 cases,
CKS1 was confirmed whose over-expression in 7 of the 12 cases,
CKS2 was confirmed whose over-expression in 11 of the 12 cases,
CYP2S1 was confirmed whose over-expression in 8 of the 12 cases,
E2-EPF was confirmed whose over-expression in 8 of the 12 cases,
ELF4 was confirmed whose over-expression in 11 of the 12 cases,
ENC1 was confirmed whose over-expression in 7 of the 12 cases,
Evi-1 was confirmed whose over-expression in 11 of the 12 cases,
FOXM1 was confirmed whose over-expression in 11 of the 12 cases,
GW112 was confirmed whose over-expression in 7 of the 12 cases,
GYS1 was confirmed whose over-expression in 10 of the 12 cases,
HDGF was confirmed whose over-expression in 10 of the 12 cases,
HOXB7 was confirmed whose over-expression in 6 of the 12 cases,
hPAD-colony 10 was confirmed whose over-expression in 6 of the 12 cases,
KNSL6 was confirmed whose over-expression in 12 cases,
KPNB2 was confirmed whose over-expression in 10 of the 12 cases,
MMP11 was confirmed whose over-expression in 10 of the 12 cases,
MYBL2 was confirmed whose over-expression in 11 of the 12 cases,
OAS1 was confirmed whose over-expression in 10 of the 12 cases,
ORP150 was confirmed whose over-expression in 8 of the 12 cases,
PCOLN3 was confirmed whose over-expression in 4 of the 12 cases,
PPM1B was confirmed whose over-expression in 3 of the 12 cases,
PRC1 was confirmed whose over-expression in 12 cases,
PSCA was confirmed whose over-expression in 6 of the 12 cases,
PYCR1 was confirmed whose over-expression in 9 of the 12 cases,
RBMS1 was confirmed whose over-expression in 12 cases,
S100P was confirmed whose over-expression in 10 of the 12 cases,
SFN was confirmed whose over-expression in 9 of the 12 cases,
SLC12A2 was confirmed whose over-expression in 5 of the 12 cases,
SLC2A1 was confirmed whose over-expression in 11 of the 12 cases,
SRD5A1 was confirmed whose over-expression in 8 of the 12 cases,
TCEA1 was confirmed whose over-expression in 8 of the 12 cases,
TK1 was confirmed whose over-expression in 10 of the 12 cases,
UBCH10 was confirmed whose over-expression in 10 of the 12 cases,
WHSC1 was confirmed whose over-expression in 8 of the 12 cases,
FLJ10134 was confirmed whose over-expression in 8 of the 12 cases,
FLJ10540 was confirmed whose over-expression in 11 of the 12 cases,
FLJ20225 was confirmed whose over-expression in 5 of the 12 cases,
KIAA0101 was confirmed whose over-expression in 12 cases,
KIAA1624 was confirmed whose over-expression in 9 of the 12 cases,
KIAA1808 was confirmed whose over-expression in 8 of the 12 cases,
FLJ21504 was confirmed whose over-expression in 11 of the 12 cases,
FXYD3 was confirmed whose over-expression in 9 of the 12 cases, and
Accession No. AI349804 was confirmed whose over-expression in 11 of the 12 cases,
AA806114 was confirmed whose over-expression in 8 of the 12 cases,
AA419568 was confirmed whose over-expression in 9 of the 12 cases,
AA570186 was confirmed whose over-expression in 6 of the 12 cases,
AA830326 was confirmed whose over-expression in 12 cases,
AI027791 was confirmed whose over-expression in 6 of the 12 cases.
These data verified the reliability of our strategy to identify commonly up-regulated genes in PNC cells.
Among the 346 down-regulated genes in pancreatic cancer cells, functions of 211 genes are characterized. These included genes that have been reported to be involved in growth suppression (24, 27, 28, 29), such as AXIN1 up-regulated 1 (AXUD1), Deleted in liver cancer 1 (DLC1), growth arrest and DNA-damage-inducible, beta (GADD45B), and P53-inducible p53DINP1 (p53DINP1).
The down-regulated genes are likely to have a tumor suppressive function. Although the representative tumor suppressor genes for pancreatic cancer such as SMAD4, TP53, INK4A, and BRCA2 (24, 25) were not observed in down-regulated gene list, other genes that were reported to be involved in tumor suppression or apoptosis, such as, AXIN1 up-regulated 1 (AXUD1), deleted in liver cancer 1 (DLC1), growth arrest and DNA-damage-inducible, beta (GADD45B), p53-inducible p53DINP1 (p53DINP1) were included in these data.
AXUD1, a nuclear protein, is induced in response to elevation of axin that is a key mediator of the Wnt-signalling pathway and is important in axis formation in early development. Dysfunction or down-regulation of the Wnt-signaling pathway is observed in human tumors, suggesting that this gene product has a tumor suppressor function (26, 27). Hence, these data imply that down-regulation of AXUD1 might lead to down-regulation of this signaling pathway and then lead to pancreatic carcinogenesis. Deleted in liver cancer 1 (DLC1) was suggested to be a candidate tumor suppressor gene for human liver cancer, as well as for prostate, lung, colorectal, and breast cancers. DLC1 shares high sequence similarity with the rat p122 RhoGap that negatively regulates the Rho GTPases. Hence, down-regulation of DLC1 is considered to result in the constitutive activation of the Rho-Rho-kinase pathway and subsequent oncogenic malignant transformation (28, 29).
Homo sapiens ribosomal protein L39 mRNA, complete cds
Homo sapiens cDNA FLJ13996 fis, clone Y79AA1002211
H. sapiens gene from PAC 106H8
Homo sapiens adlican mRNA, complete cds
Homo sapiens cDNA FLJ12150 fis, clone MAMMA1000422
Homo sapiens cDNA FLJ13154 fis, clone NT2RP3003427
Homo sapiens cDNA: FLJ21504 fis, clone COL05662
Homo sapiens cDNA: FLJ22277 fis, clone HRC03740
Homo sapiens clone 23664 and 23905 mRNA sequence
Homo sapiens HSPC285 mRNA, partial cds
Homo sapiens mRNA; cDNA DKFZp547K204 (from clone
Homo sapiens mRNA; cDNA DKFZp586A0424 (from clone
Homo sapiens cDNA FLJ11410 fis, clone HEMBA1000852
Homo sapiens cDNA FLJ12900 fis, clone NT2RP2004321
Homo sapiens cDNA FLJ13005 fis, clone NT2RP3000441
Homo sapiens cDNA FLJ13322 fis, clone OVARC1001713
Homo sapiens cDNA FLJ14115 fis, clone MAMMA1001760
Homo sapiens cDNA: FLJ20925 fis, clone ADSE00963
Homo sapiens cDNA: FLJ21545 fis, clone COL06195
Homo sapiens cDNA: FLJ22253 fis, clone HRC02763
Homo sapiens cDNA: FLJ22288 fis, clone HRC04157
Homo sapiens cDNA: FLJ22316 fis, clone HRC05262
Homo sapiens cDNA: FLJ22386 fis, clone HRC07619
Homo sapiens cDNA: FLJ22418 fis, clone HRC08590
Homo sapiens cDNA: FLJ22479 fis, clone HRC10831
Homo sapiens cDNA: FLJ22648 fis, clone HSI07329
Homo sapiens cDNA: FLJ22807 fis, clone KAIA2887
Homo sapiens cDNA: FLJ22811 fis, clone KAIA2944
Homo sapiens cDNA: FLJ23091 fis, clone LNG07220
Homo sapiens cDNA: FLJ23091 fis, clone LNG07220
Homo sapiens cDNA: FLJ23093 fis, clone LNG07264
Homo sapiens cDNA: FLJ23270 fis, clone COL10309
Homo sapiens cDNA: FLJ23527 fis, clone LNG05966
Homo sapiens clone TUA8 Cri-du-chat region mRNA
Homo sapiens GKAP42 (FKSG21) mRNA, complete cds
Homo sapiens mRNA full length insert cDNA clone
Homo sapiens mRNA; cDNA DKFZp434M229 (from clone
Homo sapiens mRNA; cDNA DKFZp564A026 (from clone
Homo sapiens mRNA; cDNA DKFZp564N1116 (from clone
Homo sapiens mRNA; cDNA DKFZp761K2024 (from clone
Comparison of clinicopathological parameters with the expression profiles indicated that altered expression of 76 genes was associated with lymph-node metastasis and that of 168 genes with liver metastasis. In addition, expression levels of 84 genes were related to the recurrence of disease. These genome-wide expression profiles should provide useful information for finding candidate genes whose products might serve as specific tumor markers and/or as molecular targets for treatment of patients with pancreatic cancer.
Materials and Methods
Genes associated with clinicopathological features, such as lymph-node-positive (r) and -negative (n), liver metastasis-positive (r) and -negative (n), and early-recurrence (r) and late-recurrence (n), were chosen according to the these two criteria; (i) signal intensities are higher than the cut-off value in at least 80% of the cases; (ii) |Medr−Medn|>=0.5, where Med indicates the median derived from log-transformed relative expression ratios in two groups. Genes were selected as candidates when they met the criteria with a permutation p-value of smaller than 0.05 in each clinicopathological status.
First, we applied a random permutation test to identify genes that were expressed differently in following two groups. The mean (μ) and standard deviation (σ) were calculated from the log-transformed relative expression ratios of each gene in node-positive (r) and node-negative (n) cases, liver-metastasis-positive (r) and -negative (n), and early-recurrence (r) and late-recurrence (n), respectively. A discrimination score (DS) for each gene was defined as follows:
DS=(μr−μn)/(σr+σn)
We carried out permutation tests to estimate the ability of individual genes to distinguish with two groups; samples were randomly permutated between the two classes 10,000 times. Since the DS dataset of each gene showed a normal distribution, we calculated a P value for the user-defined grouping (Golub et al., 1999). For this analysis, we applied the expression data of 13 cases consisting of 4 lymph-node-positive and 9 negative cases, those of 11 cases consisting of 5 liver metastasis-positive and 6 negative cases, and those of 13 cases consisting of 7 early-recurrent cases and 6 late-recurrent cases. For these analyses were performed by using only StageIV cases according to UICC TNM classification.
We further calculated the prediction score of recurrence according to procedures described previously (Golub et al., 1999). Each gene (gi) votes for either early-recurrent cases or late-recurrent cases depending on whether the expression level (xi) in the sample is closer to the mean expression level of early-recurrent cases or late-recurrent cases in reference samples. The magnitude of the vote (vi) reflects the deviation of the expression level in the sample from the average of the two classes:
V
i
=|x
i−(μr+μn)/2|
We summed the votes to obtain total votes for the early-recurrent cases (Vr) and late-recurrent cases (Vn), and calculated PS values as follows:
PS=((Vr−Vn)/(Vr+Vn))×100
reflecting the margin of victory in the direction of either early-recurrent cases or late-recurrent cases. PS values range from −100 to 100; a higher absolute value of PS reflects a stronger prediction.
We calculated the classification score (CS) by using the prediction score of early-recurrent (PSr) and late-recurrent cases (PSn) in each gene set, as follows:
CS=(μPSr−μPSn)/(σPSr+σPSn)
A larger value of CS indicates better separation of the two groups by the predictive-scoring system. For the leave-one-out test, one sample is withheld, the permutation p-value and mean expression levels are calculated using remaining samples, and the class of the withheld sample is subsequently evaluated by calculating its prediction score. We repeated this procedure for each of the 13 samples.
Identification of Genes Correlated with Clinicopathological Features Lymph-Node Metastasis and Liver Metastasis
In order to investigate relations between gene expression profiles and clinicopathological parameters, we searched genes that were possibly associated with lymph-node metastasis and liver metastasis that are important determining factors of patients' prognosis. We first examined the expression profiles and the status of lymph-node metastasis using nine lymph-node-positive and four node-negative cases, and identified 76 genes that were associated with lymph node status by a random permutation (p-value <0.05) (Table 6). Of those, 35 genes were relatively up-regulated, and 41 genes were down-regulated in node-positive tumors (
To further investigate genes that might be associated with prognosis, we compared expression profiles of 7 cases who had recurrence within 12 months after surgery (disease free interval <12 months; median 6.4 months) with those of 6 cases who had >12 months of disease free interval (median 17.0 months). As shown in
In attempt on establishment of a predictive scoring system using gene expression pattern for recurrence after surgery, we rank-ordered above prognostic 84 candidate genes on the basis of the magnitude of their permutation p-values (Table 8) and calculated the prediction score by the leave-one-out test for cross-validation using top 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 and 84 genes on the rank-ordered list. To determine the number of discriminating genes giving the best separation of the two groups, we calculated a classification score (CS) for each gene set (
Discussion
Pancreatic cancer is characterized by very aggressive progression and rapid recurrence after surgical treatment. It has been reported that the cumulative 1-, 3-, and 5-year disease free survival rate were 66%, 7%, and 3% respectively, and median disease-free survival time was the only 8 months (Sperti et al., 1997). Most common recurrent sites are the local region and the liver, and distant metastases appear in the peritoneal cavity. However, since the relationships between tumor characteristics and the recurrence patterns are still little understood, we compared the expression profiles to lymph-node status or liver metastasis. We identified 76 genes that might be associated with lymph-node status, and 168 genes with liver metastasis. These genes included some key molecules whose possible roles in tumor progression had been reported previously; ITGB4 and BSG were up-regulated in lymph-node positive cases, and KNSL6 and KRT8 were relatively up-regulated in liver metastasis cases. ITGB4 was reported to promote carcinoma invasion through a preferential and localized targeting of phosphoinositide-3 OH kinase activity (Shaw et al., 1997), supporting the possible involvement of ITGB4 in lymph-node metastasis. KNSL6, a member of the kinesin family of motor proteins, is known to be involved in chromosome segregation during mitosis (Maney T et al., 1998). The transcript of KNSL6 was highly expressed in colon cancer, and was identified as cancer antigens associated with a cancer-related serum IgG response (Scanlan M J et al., 2002). Thus, this antigen could be a biological marker for diagnosis and for monitoring of recurrence site.
In addition, we identified 84 genes possibly associated with tumor recurrence of pancreatic cancers. Expression levels of a subset of 30 genes selected from these 84 genes would be useful for predicting the disease free interval after surgical operation (
Homo sapiens mRNA; cDNA DKFZp566L203 (from clone
Homo sapiens PAC clone RP4-751H13 from 7q35-qter
Homo sapiens mRNA full length insert cDNA clone
Homo sapiens clone 25194 mRNA sequence
Homo sapiens clone 24711 mRNA sequence
Homo sapiens cDNA: FLJ23091 fis, clone LNG07220
Homo sapiens cDNA FLJ20532 fis, clone KAT10877
Homo sapiens cDNA FLJ12750 fis, clone NT2RP2001168,
Homo sapiens cDNA: FLJ23591 fis, clone LNG14729
Human Pancreatic cell lines PK45P, KLM1 and MIA-PaCa2 (ATCC Number: CRL-1420) were obtained from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University. All these cells are publicly available.
Isolation of Over-Expressing Genes in PDACa Cells by Using cDNA Microarray
Fabrication of the cDNA microarray slides has been described (Ono K, Tanaka T, Tsunoda T, Kitahara O, Kihara C, Okamoto A, Ochiai K, Takagi T, and Nakamura Y. Cancer Res., 60: 5007-5011, 2000). For each analysis of expression profiles it was prepared duplicate sets of cDNA microarray slides containing approximately 23,040 DNA spots, to reduce experimental fluctuation. Briefly, total RNA was purified from PDACa cells and normal pancreatic duct epithelium microdissected from 18 pancreatic cancer tissues. T7-based RNA amplification was carried out to obtain adequate RNA for microarray experiments. Aliquots of amplified RNA from PDACa cells and normal duct epithelium were labeled by reverse transcription with Cy5-dCTP and Cy3-dCTP, respectively (Amersham Biosciences). Hybridization, washing, and detection were carried out as described previously (Ono K, Tanaka T, Tsunoda T, Kitahara O, Kihara C, Okamoto A, Ochiai K, Takagi T, and Nakamura Y. Cancer Res., 60: 5007-5011, 2000). Subsequently, among the up-regulated genes, it was focused three genes, PCDH1, CDH3 and GPR107 because its expression ratio was greater than 5.0 in more than 50% of informative cancers and their expression level in normal vital major organs was relatively low according to the our previous data of gene expression in 29 normal human tissues (Saito-Hisaminato A, Katagiri T, Kakiuchi S, Nakamura T, Tsunoda T, Nakamura Y. Genome-wide profiling of gene expression in 29 normal human tissues with a cDNA microarray. DNA Res., 9: 35-45, 2002).
RNA from the microdissected PDACa cells and normal pancreatic ductal epithelial cells were subject to two-round amplification by T7-based in vitro transcription (Epicentre Technologies) and synthesized to single-strand cDNA. It was prepared appropriate dilutions of each single-stranded cDNA for subsequent PCR amplification by monitoring β-actin (ACTB) as a quantitative control. The primer sequences the present inventors used were 5′-AGAAGGAGACCAAGGACCTGTAT-3′ (SEQ.ID.NO. 125) and 5′-AGAACTTTATTGTCAGGGTCAAGG-3′ (SEQ.ID.NO. 126) for PCDH1, 5′-CTGAAGGCGGCTAACACAGAC-3′ (SEQ.ID.NO127) and 5′-TACACGATTGTCCTCACCCTTC-3′ (SEQ.ID.NO.128) for CDH3, and 5′-CATCCACGAAACTACCTTCAACT-3′ (SEQ.ID.NO.129) and 5′-TCTCCTTAGAGAGAAGTGGGGTG-3′ (SEQ.ID.NO.130) for ACTB. All reactions involved initial denaturation at 94° C. for 2 min followed by 21 cycles (for ACTB) or 28-32 cycles (for PCDH1 and CDH3) at 94° C. for 30 s, 58° C. for 30 s, and 72° C. for 1 min, on a GeneAmp PCR system 9700 (PE Applied Biosystems).
Formalin-fixed and paraffin-embedded PDACa sections were immunostained using a mouse anti-CDH3 monoclonal antibody (BD Transduction Laboratories) for CDH3 expression. Deparaffinized tissue sections were placed in 10 mM citrate buffer, pH 6.0, and heated to 108° C. in an autoclave for 15 minutes for antigen retrieval. Sections were incubated with a 1:10 dilution or a 1:100 dilution of primary antibody for CDH3, respectively, in a humidity chamber for an hour at room temperature, and developed with peroxidase labeled-dextran polymer followed by diaminobenzidine (DAKO Envision Plus System; DAKO Corporation, Carpinteria, Calif.). Sections were counterstained with hematoxylin. For negative controls, primary antibody was omitted.
Human multiple-tissue Northern blots (Clontech) were hybridized with a [α32P] dCTP-labeled PCR product amplified by the primers described above. Pre-hybridization, hybridization and washing were performed according to the supplier's recommendations. The blots were auto-radiographed with intensifying screens at −80° C. for 5 days.
Construction of psiU6BX Plasmid
The DNA flagment encoding siRNA was inserted into the GAP at nucleotide 485-490 as indicated (−) in the following plasmid sequence (SEQ ID No: 144).
snRNA U6 gene is reported to be transcribed by RNA polymerase III, which produce short transcripts with uridines at the 3′ end. The genomic fragment of the snRNA U6 gene containing the promoter region was amplified by PCR using a set of primers,
5′-GGGGATCAGCGTTTGAGTAA-3′ (SEQ ID No: 145), and
5′-TAGGCCCCACCTCCTTCTAT-3′ (SEQ ID No: 146) and human placental DNA as a template. The product was purified and cloned into pCR plasmid vector using a TA cloning kit according to the supplier's protocol (Invitrogen). The BamHI, XhoI fragment containing the snRNA U6 gene was purified and cloned into nucleotide 1257 to 56 fragment of pcDNA3.1(+) plasmid, which was amplified by PCR with a set of primer, 5′-TGCGGATCCAGAGCAGATTGTACTGAGAGT-3′ (SEQ ID No: 147) and 5′-CTCTATCTCGAGTGAGGCGGAAAGAACCA-3′ (SEQ ID No: 148). The ligated DNA was used for a template of PCR with primers, 5′-TTTAAGCTTGAAGACTATTTTACATCAGGTTGTTTTTCT-3′ (SEQ ID No: 149) and 5′-TTTAAGCTTGAAGACACGGTGTTTGTCCTTTCCACA-3′ (SEQ ID No: 150). The product was digested with HindIII, which was subsequently self-ligated to produce psiU6BX vector plasmid. For the control, psiU6BX-EGFP was prepared by cloning double-stranded oligonucleotides of
5′-CACCGAAGCAGCACGACTTCTTCTTCAAGAGAGAAGAAGTCGTGCTGC TTC-3′ (SEQ ID No: 151) and
5′-AAAAGAAGCAGCACGACTTCTTCTCTCTTGAAGAAGAAGTCGTGCTGC TTC-3′ (SEQ ID No: 152) into the BbsI site in the psiU6BX vector.
sIRNA-Expressing Constructs
The nucleotide sequences of the siRNAs were designed using an siRNA design computer program available from the Ambion website. (http://www.ambion.com/techlib/misc/siRNA_finder.html). Briefly, nucleotide sequences for siRNA synthesis are selected using the following protocol.
Selection of siRNA Target Sites:
1. Starting with the AUG start codon of the each gene transcript, scan downstream for an AA dinucleotide sequences. The occurrence of each AA and the 3′ adjacent 19 nucleotides are recorded as potential siRNA target sites. Tuschl et al. don't recommend against designing siRNA to the 5′ and 3′ untranslated regions (UTRs) and regions near the start codon (within 75 bases) as these may be richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex.
2. The potential target sites are compared to the appropriate genome database (human, mouse, rat, etc.) to eliminate target sequences with significant homology to other coding sequences.
3. Qualifying target sequences are selected for synthesis. Several target sequences along the length of the gene are selected for evaluation. The oligonucleotides used for siRNAs of PCDH1, CDH3 or GPR107 are shown below. Each oligionucleotide is a combination of a sense nucleotide sequence and an antisense nucleotide sequence of the target sequence. The nucleotide sequences of the hairpin loop structure and target sequence are shown in SEQ ID NO:137 to SEQ ID NO:139 and SEQ ID NO: 140 to SEQ ID NO:142, respectively (endonuclease recognition cites are eliminated from each hairpin loop structure sequence).
Insert sequence of siRNA for PCDH1
Insert sequence of siRNA for CDH3
Insert sequence of siRNA for GPR107
Insert sequence of siRNA for control
Sequence ID NO of each sequences are listed in Table 9.
Human PDACa cell lines among PK45P, KLM1 and MIA-PaCa2, were plated onto 10-cm dishes (5×105 cells/dish) and transfected with psiU6BX containing EGFP target sequence (EGFP) and psiU6BX containing target sequence using Lipofectamine 2000 (Invitrogen) or FuGENE6 (Roche), according to manufacture's instruction. Cells were selected by 500 mg/ml Geneticin for one week, and preliminary cells were harvested 48 hours after transfection and analyzed by RT-PCR to validate knockdown effect on PCDH1, CDH3 and GPR107. The primers of RT-PCR were the same ones described above. These cells were also stained by Giemsa solution and performed MTT assay to evaluate the colony formation and the cell number, respectively.
In previous study, it was generated precise expression profiles of PDACa by combining laser microdissection with genome-wide cDNA microarrays with 27,000 genes spotted. The present inventors identified more than 200 genes as up-regulated genes in PDACa cells comparing with the expression pattern of normal pancreatic ductal epithelium that was thought to be the origin of PDACa (Nakamura T, Furukawa Y, Nakagawa H, Tsunoda T, Ohigashi H, Murata K, Ishikawa O, Ohgaki, Kashimura N, Miyamoto M, Hirano S, Kondo S, Katoh H, Nakamura Y, and Katagiri T. Genome-wide cDNA microarray analysis of gene-expression profiles in pancreatic cancers using populations of tumor cells and normal ductal epithelium cells selected for purity by laser microdissection. Oncogene, 2004 Feb. 9, Epub ahead of print). Based on these expression profile of PDACa cells, the present inventors selected three over-expressing genes, and PCDH1 and CDH3 were validated their overexpression in PDACa by RT-PCR using the cDNA from microdissected PDACa cells to (FIG. 6A,B) or immunohistochemistry (
To investigate the growth or survival effect of PCDH1 on PDACa cells, the present inventors knocked down their endogenous expression of PCDH1 specifically by mammalian vector-based RNA interference (RNAi) technique in PDACa cell line. PCDH1 is expressed inrestrictedly in normal heart, placenta, prostate as shown in Northern blot analysis (
The transfection of the siRNA-producing vectors clearly resulted in reduction of the endogenous expression in one designed siRNA, 410si, for PCDH1 (
(2) CDH3 (P-Cadherin) (Genbank Accession No. NM—001793; SEQ ID No.121, 122)
The present inventors validated CDH3 overexpression in PDACa cells by RT-PCR (
To investigate the growth or survival effect of CDH3 on PDACa cells, the present inventors knocked down their endogenous expression of CDH3 specifically by mammalian vector-based RNA interference (RNAi) technique in PDACa cell line. The transfection of the siRNA-producing vectors clearly resulted in reduction of the endogenous expression in one designed siRNA, si24, for CDH3 (
(3) GPR107 (G Protein-Coupled Receptor 107) (Genbank Accession No. AB046844; SEQ ID No.123, 124)
The present inventors identified this orphan GPCR as a target for pancreas cancer, which function and ligands are unknown. GPR107 is expressed inrestrictedly in normal heart, placenta, skeltal muscle, prostate, testis, ovary, spinal cord as shown in Northern blot analysis (
In conclusion, the present inventors identified three membrane-type molecules over-expressed in PDACa cells and all of them are likely to be associated with cancer cell growth, suggested these membrane-type molecules are ideal molecular targets for deadly pancreatic cancer treatment and antibodies against these membrane molecules are promising therapeutic approach.
The gene-expression analysis of pancreatic cancer described herein, obtained through a combination of laser-capture dissection and genome-wide cDNA microarray, has identified specific genes as targets for cancer prevention and therapy. Based on the expression of a subset of these differentially expressed genes, the present invention provides molecular diagnostic markers for identifying or detecting pancreatic cancer.
The methods described herein are also useful in the identification of additional molecular targets for prevention, diagnosis and treatment of pancreatic cancer. The data reported herein add to a comprehensive understanding of pancreatic cancer, facilitate development of novel diagnostic strategies, and provide clues for identification of molecular targets for therapeutic drugs and preventative agents. Such information contributes to a more profound understanding of pancreatic tumorigenesis, and provide indicators for developing novel strategies for diagnosis, treatment, and ultimately prevention of pancreatic cancer.
The present inventors have also shown that the cell growth is suppressed by small interfering RNA (siRNA) that specifically target the PCDH1, CDH3 or GPR107 gene. Thus, this novel siRNAs are useful target for the development of anti-cancer pharmaceuticals. For example, agents that block the expression of PCDH1, CDH3 or GPR107 or prevent its activity may find therapeutic utility as anti-cancer agents, particularly anti-cancer agents for the treatment of pancreatic cancer, such as pancreatic ductal adenocarcinoma (PDACa).
All patents, patent applications, and publications cited herein are incorporated by reference in their entirety. Furthermore, while the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention.
This application is a continuation-in-part of PCT/JP2003/011817 (WO 2004/031412), which claims priority to U.S. Provisional Application Ser. No. 60/414,872, filed Sep. 30, 2002 and Ser. No. 60/450,889, filed Feb. 28, 2003. This application also claims the benefit of Ser. No. 60/555,809 filed Mar. 24, 2004. All of these applications are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60555809 | Mar 2004 | US | |
| 60414872 | Sep 2002 | US | |
| 60450889 | Feb 2003 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11090739 | Mar 2005 | US |
| Child | 12291912 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/JP2003/011817 | Sep 2003 | US |
| Child | 11090739 | US |
