Development of a Bayesian Joint Logistic Model to Better Study the Association between Haplotypes and Disease

In 2011, there will be an estimated 1,596,670 new cancer cases and 571,950 cancer-related deaths in the US. With the ever-increasing applications of cancer genetics in epidemiology, there is great potential to identify genetic risk factors that would help identify individuals with increased genetic susceptibility to cancer, which could be used to develop interventions or targeted therapies that could hopefully reduce cancer risk and mortality. In this dissertation, I propose to develop a new statistical method to evaluate the role of haplotypes in cancer susceptibility and development. This model will be flexible enough to handle not only haplotypes of any size, but also a variety of covariates. I will then apply this method to three cancer-related data sets (Hodgkin Disease, Glioma, and Lung Cancer). I hypothesize that there is substantial improvement in the estimation of association between haplotypes and disease, with the use of a Bayesian mathematical method to infer haplotypes that uses prior information from known genetics sources. Analysis based on haplotypes using information from publically available genetic sources generally show increased odds ratios and smaller p-values in both the Hodgkin, Glioma, and Lung data sets. For instance, the Bayesian Joint Logistic Model (BJLM) inferred haplotype TC had a substantially higher estimated effect size (OR=12.16, 95% CI = 2.47-90.1 vs. 9.24, 95% CI = 1.81-47.2) and more significant p-value (0.00044 vs. 0.008) for Hodgkin Disease compared to a traditional logistic regression approach. Also, the effect sizes of haplotypes modeled with recessive genetic effects were higher (and had more significant p-values) when analyzed with the BJLM. Full genetic models with haplotype information developed with the BJLM resulted in significantly higher discriminatory power and a significantly higher Net Reclassification Index compared to those developed with haplo.stats for lung cancer. Future analysis for this work could be to incorporate the 1000 Genomes project, which offers a larger selection of SNPs can be incorporated into the information from known genetic sources as well. Other future analysis include testing non-binary outcomes, like the levels of biomarkers that are present in lung cancer (NNK), and extending this analysis to full GWAS studies.

Regulation of survivin gene expression in the human endometrium and endometrial cancer

In the United States, endometrial cancer is the leading cancer of the female reproductive tract. There are 40,100 new cases and 7,470 deaths from endometrial cancer estimated for 2008 (47). The average five year survival rate for endometrial cancer is 84% however, this figure is substantially lower in patients diagnosed with late stage, advanced disease and much higher for patients diagnosed in early stage disease (47). Endometrial cancer (EC) has been associated with several risk factors including obesity, diabetes, hypertension, previously documented occurrence of hereditary non-polyposis colorectal cancer (HNPCC), and heightened exposure to estrogen (25). As of yet, there has not been a dependable molecular predictor of endometrial cancer occurrence in women with these predisposing factors. The goal of our lab is to identify genes that are aberrantly expressed in EC and may serve as molecular biomarkers of EC progression. One candidate protein that we are exploring as a biomarker of EC progression is the cell survival protein survivin.

Factors Associated with Early Versus Late Development of Breast and Ovarian Cancer in BRCA1 and BRCA2 Positive Women

Hereditary breast and ovarian cancer (HBOC) is caused by a mutation in the BRCA1 or BRCA2 genes. Women with a BRCA1/2 mutation are at increased risks for breast and ovarian cancer and often develop cancer at an earlier age than the general population. However, some women with a BRCA1/2 mutation do not develop breast or ovarian cancer under the age of 50 years. There have been no specific studies on BRCA positive women with no cancer prior to age 50, therefore this study sought to investigate factors within these women with no cancer under age 50 with respect to reproductive risk factors, BMI, tumor pathology, screening history, risk-reducing surgeries, and family history. 241 women were diagnosed with cancer prior to age 50, 92 with cancer at age 50 or older, and 20 women were over age 50 with no cancer. Data were stratified based on BRCA1 and BRCA2 mutation status. Within the cohorts we investigated differences between women who developed cancer prior to age 50 and those who developed cancer at age 50 or older. We also investigated the differences between women who developed cancer at age 50 or older and those who were age 50 or older with no cancer. Of the 92 women with a BRCA1/2 mutation who developed cancer at age 50 or older, 46 developed ovarian cancer first, 45 developed breast cancer, and one had breast and ovarian cancer diagnosed synchronously. BRCA2 carriers diagnosed age 50 or older were more likely to have ER/PR negative breast tumors when compared to BRCA2 carriers who were diagnosed before age 50. This is consistent with one other study that has been performed. Ashkenazi Jewish women with a BRCA1 mutation were more likely to be diagnosed age 50 or older than other ethnicities. Hispanic women with a BRCA2 mutation were more likely to be diagnosed prior to age 50 when compared to other ethnicities. No differences in reproductive factors or BMI were observed. Further characterization of BRCA positive women with no cancer prior to age 50 may aid in finding factors important in the development of breast or ovarian cancer.

Genetics and epidemiology of gallbladder disease in New World native peoples.

Native peoples of the New World, including Amerindians and admixed Latin Americans such as Mexican-Americans, are highly susceptible to diseases of the gallbladder. These include cholesterol cholelithiasis (gallstones) and its complications, as well as cancer of the gallbladder. Although there is clearly some necessary dietary or other environmental risk factor involved, the pattern of disease prevalence is geographically associated with the distribution of genes of aboriginal Amerindian origin, and levels of risk generally correspond to the degree of Amerindian admixture. This pattern differs from that generally associated with Westernization, which suggests a gene-environment interaction, and that within an admixed population there is a subset whose risk is underestimated when admixture is ignored. The risk that an individual of a susceptible New World genotype will undergo a cholecystectomy by age 85 can approach 40% in Mexican-American females, and their risk of gallbladder cancer can reach several percent. These are heretofore unrecognized levels of risk, especially of the latter, because previous studies have not accounted for admixture or for the loss of at-risk individuals due to cholecystectomy. A genetic susceptibility may, thus, be as "carcinogenic" in New World peoples as any known major environmental exposure; yet, while the risk has a genetic basis, its expression as gallbladder cancer is so delayed as to lead only very rarely to multiply-affected families. Estimates in this paper are derived in part from two studies of Mexican-Americans in Starr County and Laredo, Texas.

THE ROLE AND MECHANISM OF THE HOMEOBOX GENE DLX4 IN TRANSFORMING GROWTH FACTOR-B RESISTANCE IN CANCER

Transforming growth factor-b (TGF-b) is a cytokine that plays essential roles in regulating embryonic development and tissue homeostasis. In normal cells, TGF-b exerts an anti-proliferative effect. TGF-b inhibits cell growth by controlling a cytostatic program that includes activation of the cyclin-dependent kinase inhibitors p15Ink4B and p21WAF1/Cip1 and repression of c-myc. In contrast to normal cells, many tumors are resistant to the anti-proliferative effect of TGF-b. In several types of tumors, particularly those of gastrointestinal origin, resistance to the anti-proliferative effect of TGF-b has been attributed to TGF-b receptor or Smad mutations. However, these mutations are absent from many other types of tumors that are resistant to TGF-b-mediated growth inhibition. The transcription factor encoded by the homeobox patterning gene DLX4 is overexpressed in a wide range of malignancies. In this study, I demonstrated that DLX4 blocks the anti-proliferative effect of TGF-b by disabling key transcriptional control mechanisms of the TGF-b cytostatic program. Specifically, DLX4 blocked the ability of TGF-b to induce expression of p15Ink4B and p21WAF1/Cip1 by directly binding to Smad4 and to Sp1. Binding of DLX4 to Smad4 prevented Smad4 from forming transcriptional complexes with Smad2 and Smad3, whereas binding of DLX4 to Sp1 inhibited DNA-binding activity of Sp1. In addition, DLX4 induced expression of c-myc, a repressor of p15Ink4B and p21WAF1/Cip1 transcription, independently of TGF-b signaling. The ability of DLX4 to counteract key transcriptional control mechanisms of the TGF-b cytostatic program could explain in part the resistance of tumors to the anti-proliferative effect of TGF-b. This study provides a molecular explanation as to why tumors are resistant to the anti-proliferative effect of TGF-b in the absence of mutations in the TGF-b signaling pathway. Furthermore, this study also provides insights into how aberrant activation of a developmental patterning gene promotes tumor pathogenesis.

DOWNREGULATION OF PAX2 SUPPRESSES OVARIAN CANCER CELL GROWTH

PAX2 is one of nine PAX genes regulating tissue development and cellular differentiation in embryos. PAX2 promotes cell proliferation, oncogenic transformation, cell-lineage specification, migration, and survival. Unattenuated PAX2 has been found in several cancer types. We therefore sought to elucidate the role of PAX2 in ovarian carcinomas. We found that PAX2 was expressed in low-grade serous, clear cell, endometrioid and mucinous cell ovarian carcinomas, which are relatively chemoresistant compared to high grade serous ovarian carcinomas. Four ovarian cancer cell lines, RMUGL (mucinous), TOV21G (clear cell), MDAH-2774 (endometrioid) and IGROV1 (endometrioid), which express high-levels of PAX2, were used to study the function of PAX2. Lentiviral shRNAs targeting PAX2 were used to knock down PAX2 expression in these cell lines. Cellular proliferation and motility assays subsequently showed that PAX2 stable knockdown had slower growth and migration rates. Microarray gene expression profile analysis further identified genes that were affected by PAX2 including the tumor suppressor gene G0S2. Reverse phase protein array (RPPA) data showed that PAX2 knockdown affected several genes that are involved in apoptosis, which supports the fact that downregulation of PAX2 in PAX2-expressing ovarian cancer cells inhibits cell growth. We hypothesize that this growth inhibition is due to upregulation of the tumor suppressor gene G0S2 via induction of apoptosis. PAX2 represents a potential therapeutic target for chemoresistant PAX2-expressing ovarian carcinomas.

Role of germ line p53 mutations in aneuploidy and immortalization of dermal fibroblasts from Li-Fraumeni cancer patients

Pedigree analysis of certain families with a high incidence of tumors suggests a genetic predisposition to cancer. Li and Fraumeni described a familial cancer syndrome that is characterized by multiple primary tumors, early age of onset, and marked variation in tumor type. Williams and Strong (1) demonstrated that at least 7% of childhood soft tissue sarcoma patients had family histories that is readily explained by a highly penetrant autosomal dominant gene. To characterize the mechanism for genetic predisposition to many tumor types in these families, we have studied genetic alterations in fibroblasts, a target tissue from patients with the Li-Fraumeni Syndrome (LFS).^ We have observed spontaneous changes in initially normal dermal fibroblasts from LFS patients as they are cultured in vitro. The cells acquire an altered morphology, chromosomal anomalies, and anchorage-independent growth. This aberrant behavior of fibroblasts from LFS patients had never been observed in fibroblasts from normal donors. In addition to these phenotypic alterations, patient fibroblasts spontaneously immortalize by 50 population doublings (pd) in culture; unlike controls that remain normal and senesce by 30-35 (2). At 50 pd, immortal fibroblasts from two patients were found to be susceptible to tumorigenic transformation by an activated T24 H-ras oncogene (3). Approximately 80% of the oncogene expressing transfectants were capable of forming tumors in nude mice within 2-3 weeks. p53 has been previously associated with immortalization of cells in culture and cooperation with ras in transfection assays. Therefore, patients' fibroblast and lymphocyte derived DNA was tested for point mutations in p53. It was shown that LFS patients inherited certain point mutations in one of the two p53 alleles (4). Further studies on the above LFS immortal fibroblasts have demonstrated loss of the remaining p53 allele concomitant with escape from senescence. While the loss of the second allele correlates with immortalization it is not sufficient to transformation by an activated H-ras or N-ras oncogene. These immortal fibroblasts are resistant to tumorigenic transformation by v-abl, v-src, c-neu or v-mos oncogene; implying that additional steps are required in the tumorigenic progression of LFS patients' fibroblasts.^ References. (1) Williams et al., J. Natl. Cancer Inst. 79:1213, 1987. (2) Bischoff et al., Cancer Res. 50:7979, 1990. (3) Bischoff et al., Oncogene 6:183, 1991. (4) Malkin et al., Science 250:1233, 1990. ^

Genetic predisposition to prostate cancer

Prostate cancer (PC) is a significant economic and health burden in the U.S. and Europe but its causes are largely unknown. The most significant risk factors (after gender) are age and family history of the disease. A gene with high penetrance but low frequency on chromosome 1q, HPC 1, has been suggested to cause a proportion of the familial aggregation of PC but other more common genes, conferring less risk, are also thought to contribute to disease predisposition. We have pursued a strategy to study both types of genetic risk in PC. To identify high penetrance genes, affected men from thirteen families have been genotyped for genetic linkage analysis at six microsatellite markers spanning 45 cM of 1q24-25. Both LOD score and non-parametric statistics provide no significant support for HPC1 in this genomic region, although 3 of the families did combine to produce a LOD score of 0.9. These families will be included in a genome wide search for other PC predisposition genes as part of a multinational collaboration.^ For study of common genetic factors in PC development, leukocyte DNA samples from an unselected series of 55 patients and 67 controls have been examined for genetic differences in two other candidate genes, the androgen receptor gene, hAR, at Xq11-12, and the vitamin D receptor gene, hVDR, at 12q12-14. hAR was typed for two trinucleotide repeat length polymorphisms, (CAG)$\rm\sb{n}$ and (GGC)$\rm\sb{n},$ encoding polyglutamine and polyglycine tracts, respectively, which have been implicated in PC susceptibility. These data, combined with similarly processed patients and controls from the U.K. show no consistent association of allele length with PC risk. A novel finding, however, has been a significant association between the number of GGC repeats and the length of time between diagnosis and relapse in stage T1-T4 Caucasian patients irrespective of therapy and age of the patient. Of 49 patients who relapsed out of 108 entering the study, those with 16 or fewer GGC repeats had an average relapse-free-period of 101 (+/$-$7.7) months while for those with more than 16 repeats the period averaged 48 (+/$-$2.9) months, a difference of 2.1 fold or 4.4 years.^ The second gene, hVDR, was genotyped at two polymorphisms, a synonymous C/T substitution in exon 9 identified by differential TaqI enzymatic digestion and a variable length polyA tract in the 3$\sp\prime$ UTR. Although these polymorphisms are in strong linkage disequilibrium only the polyA region showed a possible association with PC risk. Men homozygous for alleles with fewer than 18 A's had an increased risk (OR = 3.0, p = 0.0578) compared to controls. This result is opposite to the findings of others and may either indicate off-setting random errors which together balance out to no significant overall effect or reflect more complex genetic and/or environmental associations.^ Overall, this research suggests that single gene familial predisposition may be less prominent in PC than in other cancers and that the characteristics of PC pathology may be useful in identifying the effects of common genetic factors. ^

Effect of cancer chemotherapy on the frequency of minisatellite repeat number changes in human sperm

The objective of this study was to determine whether cancer chemotherapy induces detectable mutations in DNA of the human germline and whether minisatellite repeat number changes can be used as a sensitive indicator of genetic damage in human sperm caused by mutagens. We compared the mutation frequencies in sperm of the same cancer patients pre- and post-, pre- and during, or during and post-treatment. Small pool polymerase chain reaction (SP-PCR) (DNA equivalent to approximately 100 sperm) and Southern blotting techniques were used to detect mutations and quantify the frequency of repeat number changes at the minisatellite MS205 locus. One pre- and one post-treatment semen sample was obtained from each Hodgkin's disease patient treated with either: (1) a regimen without alkylating agents, Novantrone, Oncovin, Vinblastine, and Prednisone (NOVP), 4 patients; (2) a regimen containing alkylating agents, Cytoxan, Vinblastine, Procarbazine, and Prednisone (CVPP)/Adriamycin, Bleomycin, DTIC, CCNU, and Prednisone (ABDIC), 2 patients; and (3) a regimen containing alkylating agents, Mechlorethamine, Oncovin, Procarbazine, and Prednisone (MOPP), 1 patient. One pre- and one during treatment semen sample from each of two Hodgkin's disease patients treated with Adriamycin, Bleomycin, Vinblastine, and Dacarbazine (ABVD) were obtained. One during and one post-treatment semen sample from a Hodgkin's disease patient treated with NOVP were also obtained. At least 7900 sperm in each sample were screened for the repeat number changes at the MS205 locus by multi-aliquots of SP-PCR. The mutation frequencies of pre- and post-treatment for the four patients treated with NOVP were 0.22 and 0.18%; 0.24 and 0.16%; 0.35 and 0.28%; and 0.19 and 0.18%. With CVPP/ABDIC, they were 0.22 and 0.23%; and 0.94 and 0.98% for the two patients and with MOPP they were 0.79 and 1.14%. The mutation frequencies of pre- and during treatment with ABVD were 0.09 and 0.07%; and 0.34 and 0.27% for the two patients. The mutation frequencies of during and post-treatment with NOVP for one patient were 0.31 and 0.25%. A statistically significant increase in mutation frequency was only found in the patient treated with MOPP. According to the time of samples collected after or during treatment and the above results, we conclude that there is no effect of NOVP and CVPP/ABDIC regimens on the mutation frequency in spermatogonia. The spermatocytes are not highly sensitive to chemotherapy agents compared to spermatogonia at the minisatellite MS205 locus. MOPP treatment may increase the mutation frequency at the MS205 locus in spermatogonia. ^

Delineation of a novel genetic region at 5q11 harboring tumor suppressor gene(s) and identification of the telomeric DNA as a marker for human prostate cancer

Prostate cancer is the most commonly diagnosed cancer and the second leading cause of cancer mortality in American men. The distinction between those cases of prostate cancer destined to progress rapidly to lethal metastatic disease and those with little likelihood of causing morbidity and mortality is a major goal of current research. Some type of diagnostic method is urgently needed to identify which histological prostate cancers have completed the progression to a stage that will produce a life-threatening disease, thus requiring immediate therapeutic intervention. The objectives of this dissertation are to delineate a novel genetic region harboring tumor suppressor gene(s) and to identify a marker for prostate tumorigenesis. I first established an in vitro cell model system from a human prostate epithelial cells derived from tissue fragments surrounding a prostate tumor in a patient with prostatic adenocarcinoma. Since chromosome 5 abnormality was present in early, middle and late passages of this cell model system, I examined long-term established prostate cancer cell lines for this chromosome abnormality. The results implicated the region surrounding marker D5S2068 as the locus of interest for further experimentation and location of a tumor suppressor gene in human prostate cancer. ^ Cancer is a group of complex genetic diseases with uncontrolled cell; division and prostate cancer is no exception. I determined if telomeric DNA, and telomerase activity, alone or together, could serve as biomarkers of prostate tumorigenesis. I studied three newly established human prostate cancer cell lines and three fibroblast cell cultures derived from prostate tissues. In conclusion, my data reveal that in the presence of telomerase activity, telomeric repeats are maintained at a certain optimal length, and analysis of telomeric DNA variations might serve as early diagnostic and prognostic biomarkers for prostate cancer. (Abstract shortened by UMI.)^

The localization and identification of candidate tumor suppressor genes involved in prostate cancer

Frequent loss of heterozygosity (LOH) at specific chromosomal regions are highly associated with the inactivation of tumor suppressor genes (TSGs) (Weinberg, 1991; Bishop, 1989). Chromosome 8p is the most frequently reported site of LOH (∼60%) in prostate cancer (PC), suggesting that there may be inactivated TSG(s) involved in PC on chromosome 8p. (Bergerheim et. al., 1991; Kagan et. al., 1995). In order to identify the smallest common regions of frequent LOH (SCLs) on chromosome 8, we screened 52 PC patient/tumor samples with 39 polymorphic markers in successive screenings. In the course of refining the SCLs, we identified 3 tumors with >6 Mb homozygous deletions (HZDs) at 8p22 and 8p21, suggesting the presence of candidate TSGs at both loci. These HZDs spanned the two SCLs at 8p22 (46%) and 8p21 (45%). The SCLs were narrowed to 3.2 cM at 8p22 and less than 3 cM at 8p21. ^ In order to identify candidate TSGs within the SCLs on 8p, two approaches were used. In the candidate gene approach, thirty genes that mapped to the SCLs were evaluated for expression in normal prostate and in PC cell lines. One of the candidate genes, Clusterin, showed decreased expression in 4/7 (57%) prostate cancer cell lines by Northern blot analysis. Clusterin will be further examined as a candidate TSG. ^ The second approach involved utilizing subtractive hybridization and hybrid affinity capture to generate pools of expressed sequence tags (ESTs) enriched for genes that are downregulated or deleted in PC and that map to specific regions of interest. We took advantage of a prostate cancer cell line (PC3) with a known HZD of a candidate TSG, CTNNA1 on 5q31, to develop and validate a model system. We then developed subtracted libraries enriched for 8p22 and 8p21 ESTs by this method, using two cell lines, MDAPCa-2b and PC3. The ESTs were cloned, and 40 were sequenced and evaluated for expression in normal prostate and PC cell lines. Three ESTs from the subtracted libraries, C2, C17 and F12, showed decreased expression in 29–57% of the prostate tumor cell lines studied, and will be further examined as candidate TSGs. ^

Expression cloning of a candidate prostate cancer oncogene

Genetic analysis, both karyotyping and comparative genomic hybridization, of prostate cancer cell lines and specimens have revealed multiple areas of concordant increases in DNA content. An increase of DNA in specific regions of the genome in cancer is often associated with the amplification of oncogenes. Based on these observations we have hypothesized that oncogenes are involved in the initiation or progression of prostate cancer. An expression cloning approach was utilized to identify candidate oncogenes in prostate cancer. ^ A full-length, unidirectional cDNA expression library was constructed from DU145 prostate cancer cells. The cDNA library was screened using CP12, a rat prostate epithelial cell line. In soft agarose assays, CP12 (parental or vector transfected) do not form colonies. However, upon the introduction of a number of known oncogenes CP12 becomes anchorage independent in soft agarose. Based on this in-vitro phenotypic shift, a DU145 cDNA library was stably transfected into CP12, and selected for anchorage independence. Two hundred fifty nine anchorage independent clones were isolated. Some colonies contained more than one insert, bringing the candidate oncogene pool to approximately 400. Seven inserts were sequenced at random. Using the sequences obtained, GenBank was screened, and matches were found with p53, PARG1, a mitochondrial ATPase, RNF6, and three unknown genes that mapped to Unigene clusters. As the pool of cDNA inserts appeared promising, overexpressed genes were further selected. From 259 clones, 17 clones were overexpressed more than 6-fold in DU145 compared to Normal Prostate. From the 17 clones, 12 cDNA inserts were strongly expressed in DU145 and were isolated for sequencing. ^ Two of the sequences, 1G6 and 3E9, were identical. Expression of 1G6/2G9/3E9 was tested by RT-PCR. 1G6/2G9/3E9 was not expressed in normal prostate, but was expressed in all prostate cancer cell lines tested as well as six prostate cancer samples. When retransfected into CP12, 1G6/2G9/3E9 induced the formation of foci and anchorage independent colonies. Thus, functional and expression data suggest that 1G6/2G9/3E9 may be a prostate cancer oncogene. ^

Genetic alterations associated with prostate cancer progression

Prostate cancer is the second most commonly diagnosed cancer among men in the United States. In this study, evidence is presented to support the hypothesis that specific chromosomal aberrations (involving one or more chromosomal regions) are associated with prostate cancer progression from organ-confined to locally advanced tumors and that some aberrations seen in high frequency in metastatic tumors may also be present in a subset of primary tumors. To determine the appropriate approach to address this hypothesis, I have established a modified CGH protocol by microdissection and DOP-PCR for use in detecting chromosomal changes in clinical prostate tumor specimens that is more sensitive and accurate than conventional CGH methods. I have successfully performed the improved CGH protocol to screen for genetic changes of 24 organ confined (pT2) and 21 locally advanced (pT3b) clinical prostate cancer specimens without metastases (N0M0). Comparisons of tumors by stage or Gleason scores following contingency table analysis showed that seven regions of the genome differed significantly between pT2 and pT3b tumors or between low and high Gleason tumors suggesting that these regions may be important in local prostate cancer progression. These included losses on 6p21–25, 6q24–27, 8p, 10q25–26, 15q22–26, and 18cen–q12 as well as gain of 3p13–q13. Multivariate analyses showed that loss of 8p (step1) and loss of 6q25–26 (or 6p21–25 or 10q25–26) (step 2) were predictive of pathologic stage or Gleason groups with 80% accuracy. Additional 5–7 steps in the multivariate model increased the predictive value to 91–95%. Comparison of the CGH data from the primary prostate tumors of this study with those obtained from published literature on metastases and recurrent tumors showed that the clinically more aggressive stage pT3b tumors shared more abnormalities in high frequency with metastases and recurrent tumors than less aggressive stage pT2 tumors. Furthermore, loss of 11cen–q22 was shared only between the primary tumors and metastases while gain of Xcen–q13 and loss of 18cen–q12 were in common between primary and recurrent tumors. These analyses suggest that the multistage model of prostate cancer progression is not linear and that some early primary tumors may be predisposed to metastasize or evolve into recurrent tumors due to the presence of specific genetic alterations. ^

Physical and functional mapping of a novel tumor suppressor locus for prostate cancer within chromosome 10p12.31-q11

Prostate cancer remains the second leading cause of male cancer deaths in the United States, yet the molecular mechanisms underlying this disease remain largely unknown. Cytogenetic and molecular analyses of prostate tumors suggest a consistent association with the loss of chromosome 10. Previously, we have defined a novel tumor suppressor locus PAC-1 within chromosome 10pter-q11. Introduction of the short arm of chromosome 10 into a prostatic adenocarcinoma cell line PC-3H resulted in dramatic tumor suppression and restoration of a programmed cell death pathway. Using a combined approach of comparative genomic hybridization and microsatellite analysis of PC-3H, I have identified a region of hemizygosity within 10p12-p15. This region has been shown to be involved in frequent loss of heterozygosity in gliomas and melanoma. To functionally dissect the region within chromosome 10p containing PAC-1, we developed a strategy of serial microcell fusion, a technique that allows the transfer of defined fragments of chromosome 10p into PC-3H. Serial microcell fusion was used to transfer defined 10p fragments into a mouse A9 fibrosarcoma cell line. Once characterized by FISH and microsatellite analyses, the 10p fragments were subsequently transferred into PC-3H to generate a panel of microcell hybrid clones containing overlapping deletions of chromosome 10p. In vivo and microsatellite analyses of these PC hybrids identified a small chromosome 10p fragment (an estimated 31 Mb in size inclusive of the centromere) that when transferred into the PC-3H background, resulted in significant tumor suppression and limited a region of functional tumor suppressor activity to chromosome 10p12.31-q11. This region coincides with a region of LOH demonstrated in prostate cancer. These studies demonstrate the utility of this approach as a powerful tool to limit regions of functional tumor suppressor activity. Furthermore, these data used in conjunction with data generated by the Human Genome Project lent a focused approach to identify candidate tumor suppressor genes involved in prostate cancer. ^

Metastatic lung cancer in a new mouse model inheriting p53 and latent K-ras mutations

Lung cancer is a devastating disease with very poor prognosis. The design of better treatments for patients would be greatly aided by mouse models that closely resemble the human disease. The most common type of human lung cancer is adenocarcinoma with frequent metastasis. Unfortunately, current models for this tumor are inadequate due to the absence of metastasis. Based on the molecular findings in human lung cancer and metastatic potential of osteosarcomas in mutant p53 mouse models, I hypothesized that mice with both K-ras and p53 missense mutations might develop metastatic lung adenocarcinomas. Therefore, I incorporated both K-rasLA1 and p53RI72HΔg alleles into mouse lung cells to establish a more faithful model for human lung adenocarcinoma and for translational and mechanistic studies. Mice with both mutations ( K-rasLA1/+ p53R172HΔg/+) developed advanced lung adenocarcinomas with similar histopathology to human tumors. These lung adenocarcinomas were highly aggressive and metastasized to multiple intrathoracic and extrathoracic sites in a pattern similar to that seen in lung cancer patients. This mouse model also showed gender differences in cancer related death and developed pleural mesotheliomas in 23.2% of them. In a preclinical study, the new drug Erlotinib (Tarceva) decreased the number and size of lung lesions in this model. These data demonstrate that this mouse model most closely mimics human metastatic lung adenocarcinoma and provides an invaluable system for translational studies. ^ To screen for important genes for metastasis, gene expression profiles of primary lung adenocarcinomas and metastases were analyzed. Microarray data showed that these two groups were segregated in gene expression and had 79 highly differentially expressed genes (more than 2.5 fold changes and p<0.001). Microarray data of Bub1b, Vimentin and CCAM1 were validated in tumors by quantitative real-time PCR (QPCR). Bub1b , a mitotic checkpoint gene, was overexpressed in metastases and this correlated with more chromosomal abnormalities in metastatic cells. Vimentin, a marker of epithelial-mesenchymal transition (EMT), was also highly expressed in metastases. Interestingly, Twist, a key EMT inducer, was also highly upregulated in metastases by QPCR, and this significantly correlated with the overexpression of Vimentin in the same tumors. These data suggest EMT occurs in lung adenocarcinomas and is a key mechanism for the development of metastasis in K-ras LA1/+ p53R172HΔg/+ mice. Thus, this mouse model provides a unique system to further probe the molecular basis of metastatic lung cancer.^