Impact of hormonal therapy on survival and racial disparities in a large community-based cohort of older men with local/regional prostate cancer

Prostate cancer (CaP) is the most diagnosed non-cutaneous malignancy and the second leading cause of cancer mortality among United States males. Major racial disparities in incidence, survival, as well as treatment persist. The mortality is three times higher among African Americans (AAs) compared with Caucasians. Androgen carcinogenesis has been persistently implicated but results are inconsistent; and hormone manipulation has been the main stay of treatment for metastatic disease, supportive of the androgen carcinogenesis. The survival disadvantage of AAs has been attributed to the differences in socioeconomic factors (SES), tumor stage, and treatment. We hypostasized that HT prolongs survival in CaP and that the racial disparities in survival is influenced by variation in HT and primary therapies as well as SES. To address these overall hypothesis, we first utilized a random-effect meta-analytic design to examine evidence from randomized trials on the efficacy of androgen deprivation therapy in localized and metastatic disease, and assessed, using Cox proportional hazards models, the effectiveness of HT in prolonging survival in a large community-based cohort of older males diagnosed with local/regional CaP. Further we examined the role of HT and primary therapies on the racial disparities in CaP survival. The results indicated that adjuvant HT compared with standard care alone is efficacious in improving overall survival, whereas HT has no significant benefit in the real world experience in increasing the overall survival of older males in the community treated for local/regional disease. Further, racial differences in survival persist and were explained to some extent by the differences in the primary therapies (radical prostatectomy, radiation and watchful waiting) and largely by SES. Therefore, given the increased used of hormonal therapy and the cost-effectiveness today, more RCTs are needed to assess whether or not survival prolongation translates to improved quality of life, and to answer the research question on whether or not the decreased use of radical prostatectomy by AAs is driven by the Clinicians bias or AAs's preference of conservative therapy and to encourage AAs to seek curative therapies, thus narrowing to some degree the persistent mortality disparities between AAs and Caucasians. ^

PROSTATE CANCER STEM CELLS: DRUG TOLERANCE, EXPERIMENTAL RECONSTITUTION AND CASTRATION RESISTANCE

Prostate cancer (PCa) is one of the leading malignancies affecting men in the Western world. Although tremendous effort has been made towards understanding PCa development and developing clinical treatments in the past decades, the exact mechanisms of PCa are still not clearly understood. Emerging evidence has postulated that a population of stem cell-like cells inside a tumor, termed ‘cancer stem cells (CSCs)’, may be the cells responsible for tumor initiation, progression, recurrence, metastasis and therapy resistance. Like CSC studies in other cancer types, it has been reported that PCa also contains CSCs. However, there remain several unresolved questions that need to be clarified. First, the relationship between prostate CSCs (PCSCs) and therapy resistance (chemo- and radio-) is not known. Herein, we have found that not all CSCs are drug-tolerant, and not all drug-tolerant cells are CSCs. Second, whether primary human PCa (HPCa) actually contain PCSCs remains unclear, due to the well-known fact that we have yet to establish a reliable assay system that can reproducibly and faithfully reconstitute tumor regeneration from single HPCa cells. Herein, after utilizing more than 114 HPCa samples we have provided evidence that immortalized bone marrow-derived stromal cells (Hs5) can help dissociated HPCa cells generate undifferentiated tumors in immunodeficient NOD/SCID-IL2Rγ-/- mice, and the undifferentiated PCa cells seem to have a survival advantage to generate tumors. Third, the evolution of PCa from androgen dependent to the lethally castration resistant (CRPC) stage remains enigmatic, and the cells responsible for CRPC development have not been identified. Herein, we have found a putative cell population, ALDH+CD44+α2β1+ PCa cells that may represent a cell-of-origin for CRPC. Taken together, our work has improved our understanding of PCSC properties, possibly highlighting a potential therapeutic target for CRPC.

The functional role of the tumor suppressor MMAC /PTEN in glioblastoma multiforme and prostate cancer

Investigations into the molecular basis of glioblastoma multiforme led to the identification of a putative tumor suppressor gene, MMAC/ PTEN. Initial studies implicated MMAC/PTEN in many different tumor types, and identified a protein phosphatase motif in its sequence. This project aimed to identify the biological and biochemical functions of MMAC/PTEN by transiently expressing the gene in cancer cells that lack a functional gene product. ^ Expression of MMAC/PTEN mildly suppressed the growth of U251 human glioma cells and abrogated the growth advantage mediated by overexpression of the epidermal growth factor receptor (EGFR). Immunoblotting demonstrated that MMAC/PTEN expression did not affect the phosphorylation of the EGFR itself, or the intermediates of several downstream signaling pathways. However, MMAC/PTEN expression significantly reduced the phosphorylation and catalytic activity of the proto-oncogene Akt/PKB. While Akt/PKB regulates the survival of many cell types, expression of MMAC/PTEN did not induce apoptosis in adherent U251 cells. Instead, MMAC/PTEN expression sensitized the cells to apoptosis when maintained in suspension (anoikis). As the survival of suspended cells is one of the hallmarks leading to metastasis, MMAC/PTEN expression was examined in a system in which metastasis is more clinically relevant, prostate cancer. ^ Expression of MMAC/PTEN in both LNCaP and PC3-P human prostate cancer cells specifically inhibited Akt/PKB phosphorylation. MMAC/PTEN expression in LNCaP cells resulted in a profound inhibition of growth that was significantly greater than that achieved with expression of p53. Expression of MMAC/PTEN in PC3-P cells resulted in greater growth inhibition than was observed in U251 glioma cells, but less than was observed in LNCaP cells, or upon p53 expression. To determine if MMAC/PTEN could function as a tumor suppressor in vivo, the effects of MMAC/PTEN expression on PC3-P cells implanted orthotopically in nude mice were examined. The ex-vivo expression of MMAC/PTEN did not decrease tumor incidence, but it did significantly decrease tumor size and metastasis. In-vivo expression of MMAC/PTEN in pre-established PC3-P tumors did not significantly inhibit tumor incidence or size, but did inhibit metastasis formation. ^ These studies demonstrate that MMAC/PTEN is a novel and important tumor suppressor gene, which functions to downregulate an important cell survival signaling pathway. ^

A NEW TUMOR SUPPRESSOR GENE CANDIDATE REGULATED BY THE NON-CODING RNA PCA3 IN HUMAN PROSTATE CANCER

Prostate cancer is the second leading cause of cancer-related death and the most common non-skin cancer in men in the USA. Considerable advancements in the practice of medicine have allowed a significant improvement in the diagnosis and treatment of this disease and, in recent years, both incidence and mortality rates have been slightly declining. However, it is still estimated that 1 man in 6 will be diagnosed with prostate cancer during his lifetime, and 1 man in 35 will die of the disease. In order to identify novel strategies and effective therapeutic approaches in the fight against prostate cancer, it is imperative to improve our understanding of its complex biology since many aspects of prostate cancer initiation and progression still remain elusive. The study of tumor biomarkers, due to their specific altered expression in tumor versus normal tissue, is a valid tool for elucidating key aspects of cancer biology, and may provide important insights into the molecular mechanisms underlining the tumorigenesis process of prostate cancer. PCA3, is considered the most specific prostate cancer biomarker, however its biological role, until now, remained unknown. PCA3 is a long non-coding RNA (ncRNA) expressed from chromosome 9q21 and its study led us to the discovery of a novel human gene, PC-TSGC, transcribed from the opposite strand and in an antisense orientation to PCA3. With the work presented in this thesis, we demonstrate that PCA3 exerts a negative regulatory role over PC-TSGC, and we propose PC-TSGC to be a new tumor suppressor gene that contrasts the transformation of prostate cells by inhibiting Rho-GTPases signaling pathways. Our findings provide a biological role for PCA3 in prostate cancer and suggest a new mechanism of tumor suppressor gene inactivation mediated by non-coding RNA. Also, the characterization of PCA3 and PC-TSGC led us to propose a new molecular pathway involving both genes in the transformation process of the prostate, thus providing a new piece of the jigsaw puzzle representing the complex biology of prostate cancer.

RANGE ADAPTIVE PROTON THERAPY FOR PROSTATE CANCER

Purpose: The rapid distal falloff of a proton beam allows for sparing of normal tissues distal to the target. However proton beams that aim directly towards critical structures are avoided due to concerns of range uncertainties, such as CT number conversion and anatomy variations. We propose to eliminate range uncertainty and enable prostate treatment with a single anterior beam by detecting the proton’s range at the prostate-rectal interface and adaptively adjusting the range in vivo and in real-time. Materials and Methods: A prototype device, consisting of an endorectal liquid scintillation detector and dual-inverted Lucite wedges for range compensation, was designed to test the feasibility and accuracy of the technique. Liquid scintillation filled volume was fitted with optical fiber and placed inside the rectum of an anthropomorphic pelvic phantom. Photodiode-generated current signal was generated as a function of proton beam distal depth, and the spatial resolution of this technique was calculated by relating the variance in detecting proton spills to its maximum penetration depth. The relative water-equivalent thickness of the wedges was measured in a water phantom and prospectively tested to determine the accuracy of range corrections. Treatment simulation studies were performed to test the potential dosimetric benefit in sparing the rectum. Results: The spatial resolution of the detector in phantom measurement was 0.5 mm. The precision of the range correction was 0.04 mm. The residual margin to ensure CTV coverage was 1.1 mm. The composite distal margin for 95% treatment confidence was 2.4 mm. Planning studies based on a previously estimated 2mm margin (90% treatment confidence) for 27 patients showed a rectal sparing up to 51% at 70 Gy and 57% at 40 Gy relative to IMRT and bilateral proton treatment. Conclusion: We demonstrated the feasibility of our design. Use of this technique allows for proton treatment using a single anterior beam, significantly reducing the rectal dose.

A spatial-temporal approach to surveillance of prostate cancer disparities in population subgroups

BACKGROUND: Prostate cancer mortality disparities exist among racial/ethnic groups in the United States, yet few studies have explored the spatiotemporal trend of the disease burden. To better understand mortality disparities by geographic regions over time, the present study analyzed the geographic variations of prostate cancer mortality by three Texas racial/ethnic groups over a 22-year period. METHODS: The Spatial Scan Statistic developed by Kulldorff et al was used. Excess mortality was detected using scan windows of 50% and 90% of the study period and a spatial cluster size of 50% of the population at risk. Time trend was analyzed to examine the potential temporal effects of clustering. Spatial queries were used to identify regions with multiple racial/ethnic groups having excess mortality. RESULTS: The most likely area of excess mortality for blacks occurred in Dallas-Metroplex and upper east Texas areas between 1990 and 1999; for Hispanics, in central Texas between 1992 and 1996: and for non-Hispanic whites, in the upper south and west to central Texas areas between 1990 and 1996. Excess mortality persisted among all racial/ethnic groups in the identified counties. The second scan revealed that three counties in west Texas presented an excess mortality for Hispanics from 1980-2001. Many counties bore an excess mortality burden for multiple groups. There is no time trend decline in prostate cancer mortality for blacks and non-Hispanic whites in Texas. CONCLUSION: Disparities in prostate cancer mortality among racial/ethnic groups existed in Texas. Central Texas counties with excess mortality in multiple subgroups warrant further investigation.

Risk of Second Malignant Neoplasms Following Proton Arc Therapy and Volumetric Modulated Arc Therapy for Prostate Cancer

The risk of second malignant neoplasms (SMNs) following prostate radiotherapy is a concern due to the large population of survivors and decreasing age at diagnosis. It is known that parallel-opposed beam proton therapy carries a lower risk than photon IMRT. However, a comparison of SMN risk following proton and photon arc therapies has not previously been reported. The purpose of this study was to predict the ratio of excess relative risk (RRR) of SMN incidence following proton arc therapy to that after volumetric modulated arc therapy (VMAT). Additionally, we investigated the impact of margin size and the effect of risk-minimized proton beam weighting on predicted RRR. Physician-approved treatment plans were created for both modalities for three patients. Therapeutic dose was obtained with differential dose-volume histograms from the treatment planning system, and stray dose was estimated from the literature or calculated with Monte Carlo simulations. Then, various risk models were applied to the total dose. Additional treatment plans were also investigated with varying margin size and risk-minimized proton beam weighting. The mean RRR ranged from 0.74 to 0.99, depending on risk model. The additional treatment plans revealed that the RRR remained approximately constant with varying margin size, and that the predicted RRR was reduced by 12% using a risk-minimized proton arc therapy planning technique. In conclusion, proton arc therapy was found to provide an advantage over VMAT in regard to predicted risk of SMN following prostate radiotherapy. This advantage was independent of margin size and was amplified with risk-optimized proton beam weighting.

Is a 3-mm intrafractional margin sufficient for daily image-guided intensity-modulated radiation therapy of prostate cancer?

PURPOSE: To determine whether a 3-mm isotropic target margin adequately covers the prostate and seminal vesicles (SVs) during administration of an intensity-modulated radiation therapy (IMRT) treatment fraction, assuming that daily image-guided setup is performed just before each fraction. MATERIALS AND METHODS: In-room computed tomographic (CT) scans were acquired immediately before and after a daily treatment fraction in 46 patients with prostate cancer. An eight-field IMRT plan was designed using the pre-fraction CT with a 3-mm margin and subsequently recalculated on the post-fraction CT. For convenience of comparison, dose plans were scaled to full course of treatment (75.6 Gy). Dose coverage was assessed on the post-treatment CT image set. RESULTS: During one treatment fraction (21.4+/-5.5 min), there were reductions in the volumes of the prostate and SVs receiving the prescribed dose (median reduction 0.1% and 1.0%, respectively, p<0.001) and in the minimum dose to 0.1 cm(3) of their volumes (median reduction 0.5 and 1.5 Gy, p<0.001). Of the 46 patients, three patients' prostates and eight patients' SVs did not maintain dose coverage above 70 Gy. Rectal filling correlated with decreased percentage-volume of SV receiving 75.6, 70, and 60 Gy (p<0.02). CONCLUSIONS: The 3-mm intrafractional margin was adequate for prostate dose coverage. However, a significant subset of patients lost SV dose coverage. The rectal volume change significantly affected SV dose coverage. For advanced-stage prostate cancers, we recommend to use larger margins or improve organ immobilization (such as with a rectal balloon) to ensure SV coverage.

CLINICAL TRIAL ENROLLMENT IN A MULTIDISCIPLINARY PROSTATE CANCER

Purpose: Clinical oncology trials are hampered by low accrual rates. Less than 5% of adult cancer patients are treated on a clinical trial. We aimed to evaluate clinical trial enrollment in our Multidisciplinary Prostate Cancer Clinic and to assess if a clinical trial initiative, introduced in 2006, increased our trial enrollment.Methods: Prostate cancer patients with non-metastatic disease who were seen in the clinic from 2004 to 2008 were included in the analysis. Men were categorized by whether they were seen before or after the clinical trial enrollment initiative started in 2006. The initiative included posting trial details in the clinic, educating patients about appropriate clinical trial options during the treatment recommendation discussion, and providing patients with documentation of trials offered to them. Univariate and multivariate (MVA) logistic regression analysis evaluated the impact of patient characteristics and the clinical trial initiative on clinical trial enrollment.Results: The majority of the 1,370 men were white (83%), and lived within the surrounding counties or state (69.4%). Median age was 64.2 years. Seventy-three point five percent enrolled in at least one trial and 28.5% enrolled in more than one trial. Sixty-seven percent enrolled in laboratory studies, 18% quality of life studies, 13% novel studies, and 3.7% procedural studies. On MVA, men seen in later years (p < 0.0001) were more likely to enroll in trials. The proportion of men enrolling increased from 38.9% to 84.3% (p<0.0001) after the clinical trial initiative. On MVA, older men (p < 0.0001) were less likely to enroll in clinical trials. There was a trend toward men in the high-risk group being more likely to participate in clinical trials (p = 0.056). There was a second trend for men of Hispanic, Asian, Native American and Indian decent being less likely to participate in clinical trials (p = 0.054).Conclusion: Clinical trial enrollment in the multidisciplinary clinic increased after introduction of a clinical trial initiative. Older men were less likely to enroll in trials. We speculate we achieved high enrollment rates because 1) specific trials are discussed at time of treatment recommendations, 2) we provide a letter documenting offered trials and 3) we introduce patients to the research team at the same clinic visit if they are interested in trial participation.

Statistical characterization and development of summary measures of non-normal distributions of nuclear morphometry data from prostate cancer cells for use as prognostic indicators

Nuclear morphometry (NM) uses image analysis to measure features of the cell nucleus which are classified as: bulk properties, shape or form, and DNA distribution. Studies have used these measurements as diagnostic and prognostic indicators of disease with inconclusive results. The distributional properties of these variables have not been systematically investigated although much of the medical data exhibit nonnormal distributions. Measurements are done on several hundred cells per patient so summary measurements reflecting the underlying distribution are needed.^ Distributional characteristics of 34 NM variables from prostate cancer cells were investigated using graphical and analytical techniques. Cells per sample ranged from 52 to 458. A small sample of patients with benign prostatic hyperplasia (BPH), representing non-cancer cells, was used for general comparison with the cancer cells.^ Data transformations such as log, square root and 1/x did not yield normality as measured by the Shapiro-Wilks test for normality. A modulus transformation, used for distributions having abnormal kurtosis values, also did not produce normality.^ Kernel density histograms of the 34 variables exhibited non-normality and 18 variables also exhibited bimodality. A bimodality coefficient was calculated and 3 variables: DNA concentration, shape and elongation, showed the strongest evidence of bimodality and were studied further.^ Two analytical approaches were used to obtain a summary measure for each variable for each patient: cluster analysis to determine significant clusters and a mixture model analysis using a two component model having a Gaussian distribution with equal variances. The mixture component parameters were used to bootstrap the log likelihood ratio to determine the significant number of components, 1 or 2. These summary measures were used as predictors of disease severity in several proportional odds logistic regression models. The disease severity scale had 5 levels and was constructed of 3 components: extracapsulary penetration (ECP), lymph node involvement (LN+) and seminal vesicle involvement (SV+) which represent surrogate measures of prognosis. The summary measures were not strong predictors of disease severity. There was some indication from the mixture model results that there were changes in mean levels and proportions of the components in the lower severity levels. ^

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. ^

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. ^

Collagen I modulates growth and gene expression in prostate cancer cells

Prostate cancer is the second leading cause of male cancer-related deaths in the United States. Interestingly, prostate cancer preferentially metastasizes to skeletal tissue. Once in the bone microenvironment, advanced prostate cancer becomes highly resistant to therapeutic modalities. Several factors, such as extracellular matrix (ECM) components, have been implicated in the spread and propagation of prostatic carcinoma. In these studies, we have utilized the PC3 cell line, derived from a human bone metastasis, to investigate the influence of the predominant bone ECM protein, type I collagen, on prostate cancer cell proliferation and gene expression. We have also initiated the design and production of ribozymes to specific gene targets that may influence prostate cancer bone metastasis. ^ Our results demonstrate that PC3 cells rapidly adhere and spread on collagen I to a greater degree than on fibronectin (FN) or poly-L-lysine (PLL). Flow cytometry analysis reveals the presence of the α1, α2 and α3 collagen binding integrin subunits. The use of antibody function blocking studies reveals that PC3 cells can utilize α2β 1 and α3β1 integrins to adhere to collagen I. Once plated on collagen I, the cells exhibit increased rates of proliferation compared with cells plated on FN or tissue culture plastic. Additionally, cells plated on collagen I show increased expression of proteins associated with progression through G1 phase of the cell cycle. Inhibitor studies point to a role for phosphatidylinositol 3-kinase (PI3K), MAP kinase (MAPK), and p70 S6 kinase in collagen I-mediated PC3 cell proliferation and cyclin D1 expression. To further characterize the effect of type I collagen on prostate cancer bone metastasis, we utilized a cDNA microarray strategy to monitor type I collagen-mediated changes in gene expression. Results of this analysis revealed a gene expression profile reflecting the increased proliferation occurring on type I collagen. Microarray analysis also revealed differences in the expression of specific gene targets that may impact on prostate cancer metastasis to bone. ^ As a result of our studies on the interaction of prostate cancer cells and the skeletal ECM, we sought to develop novel molecular tools for future gene therapy of functional knockdown experiments. To this end, we developed a series of ribozymes directed against the α2 integrin and at osteopontin, a protein implicated in the metastasis of various cancers, including prostate. These ribozymes should facilitate the future study of the mechanism of prostate cancer cell proliferation, and disease progression occurring at sites of skeletal metastasis where a type I collagen-based environment predominates. ^ Together these studies demonstrate the involvement of bone ECM proteins on prostate cancer cell proliferation and suggest that they may play a significant role on the growth of prostate metastases once in the bone microenvironment. ^

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. ^