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CANCER & TREATMENTS SUPPORT & SURVIVORSHIP PREVENTION & RISK ASSESSMENT CLINICAL TRIALS & RESEARCH LIVING WITH CANCER | |
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Home > Research, Discovery & Progress > Program Overview Research ProjectsProject 1: Osteoclastogenesis as a Target for Prostate Cancer osseous Metastases TherapyPrincipal Investigator: Evan Keller, D.V.M., Ph.D. Prostate cancer skeletal metastases are considered osteoblastic; however, histopathological examination usually reveals underlying osteoclastic activity. A key molecule required for induction of osteoclastic activity is receptor activator of NFkB ligand (RANKL). We have determined that prostate cancer cells express increasing levels of RANKL and induce osteoclastogenesis as they progress to bone metastases. Furthermore, in a murine model, we have demonstrated the ability to inhibit establishment of prostate cancer in bone by blocking RANKL-induced osteoclastic activity. Thus, our hypothesis is that RANKL contributes to the development of prostate cancer skeletal metastases, which we will test in the following specific aims: Aim 1. Aim 2. Aim 3. April 2005 Update:We have identified that transforming growth factor-beta (TGF-b), a protein at high levels in the bone microenvironment, increased RANKL expression in vivo in bone. This work has now been published:
We are now exploring if prostate cancer-derived RANKL mediates its effects on osteoclast precursors through the NFkB pathway. This may identify NFkB as an important mediator of prostate-cancer-mediated osteoclastogenesis and provide viable clinical targets. To attack this issue, we transfected murine osteoclast precursors (RAW cells) with a dominant negative (DN) NFkB-inducing kinase (NIK) that inhibits activation of NFkB upstream of the IkB Kinases. Raw cells, either transfected with control empty vector (EV) or DN-NIK were incubated in prostate cancer cell C4-2B conditioned media (CM) and osteoclastogenesis was measured. The presence of DN-NIK inhibited C4-2B CM-induced by approximately 50%. The presence of DN-NIK also diminished C42B CM-induced NFkB nuclear translocation. We have demonstrated that inhibiting RANKL with osteoprotegerin (OPG) blocks establishment of prostate of CaP growth in bone. Published in:
We have also demonstrated that blocking RANKL with sRANK-Fc diminishes progression of CaP growth in bone through inhibition of bone remodeling. This work was published (and was a feature on the journal cover) in:
We are currently evaluating the efficacy of OPG-mediated inhibition of RANKL as an adjunct therapy to taxotere in prostate cancer growth in bone in a murine model. Briefly, C4-2B or LuCaP 35 tumors are injected intra-tibially into mice and then the animals are treated with sub-effective levels of taxotere in the absence or presence of OPG. We are currently evaluating several other inhibitors of osteoclastogenic activity including a Cathepsin K inhibitor and an oral inhibitor of IkB kinase, which inhibits NFkB activation, in murine models. This clinical trial is ongoing. We have recruited a total of 29 patients.
We have performed an interim analysis of bone remodeling markers to ensure we could measure them in these samples. We confirmed that we could measure serum bone alkaline phosphatase, plasma osteocalcin, serum tartrate acid phosphatase, deoxypyridinoline, serum interleukin-6, serum RANKL, and serum OPG (data not shown). Understanding the regulation of RANKL may help lead to novel targets to down-regulate RANKL expression. Down-regulation of RANKL expression would result in decreased osteoclastogenesis and bone remodeling/turnover which could ultimately could help prevent establishment or progression of CaP tumor in bone. Furthermore, our identification of TGF-b as a modulator of RANKL provides support to the idea that targeting the bone microenvironment itself may help diminish prostate cancer growth in bone. Demonstrating that prostate cancer mediates osteoclastogenesis through activation of NFkB provides the rationale to explore inhibitors of NFkB activation for targeting prostate cancer bone metastases. This is now being done in Aim 2 with the IkB kinase inhibitor. Our demonstration that sRANK-Fc is an effective inhibitor of RANKL that diminishes progression of CaP growth in bone through inhibition of bone remodeling provides support to the idea of targeting RANKL in clinical CaP. We are approaching our recruitment goal. We can measure bone remodeling markers in the samples. Publications:
Project 2: Role of Protease Activated Receptor-1 (PAR1) in Prostate Cancer MetastasisPrincipal Investigator: Kenneth J. Pienta, M.D. The hypothesis of this application is that activation of PAR1 is a critical step in prostate cancer tumorigenesis and inhibition of this step can lead to an effective treatment for prostate cancer. Effective treatment of prostate cancer will come from understanding the biology of this disease and applying this knowledge to the application of specific therapies. The development of new interventions for this disorder requires identification of specific targets to attack in men with prostate cancer. There are three major problems in attempting to treat prostate cancer. The first is the establishment of relevant models to test therapeutic agents in the pre-clinical setting before moving agents to the clinical setting. The second is the identification of therapeutic agents for intervention. The third problem is the optimal design of clinical trials that adequately test the agents identified in the pre-clinical setting. In the previous grant period, we have established in vitro and in vivo models of prostate cancer and used these models to identify potential targets and agents for intervention. This application focuses on PAR1 (protease activated receptor 1, the thrombin receptor) as a new target for therapeutic intervention in prostate tumorigenesis. In our preliminary data / progress report, we demonstrate PAR1 is overexpressed on prostate cancer cells as compared to normal prostate tissue by cDNA and tissue microarray. The Specific Aims of this proposal are as follows: Aim 1 Aim 2 Aim 3 These proposed studies will characterize the role of PAR1 in prostate tumorigenesis and begin to define therapeutic strategies to treat prostate cancer by inhibition of PAR1 activation. April, 2005 UpdateWe studied the effects of PAR-1 activation on RhoA, Rac, and cdc42 on prostate cancer cell cytoskeletal reorganization and adhesion. We found that PAR1-mediated RhoA Aactivation fascilitates cytoskeletal reorganization and decreased adhesion in PC-3 prostate cancer cells. Activation of PAR1 induced dynamic cytoskeletal reorganization and reduced PC-3 cell binding to collagen I, collagen IV and laminin (p<0.01). Expression of the cell surface integrin receptors did not change as assessed by flow cytometry. Immunofluorescence microscopy revealed that PAR1 stimulation caused reorganization of the focal adhesions, suggesting that PAR1 activation in PC-3 cells may be modulating cell adhesion through integrin function, but not expression. Furthermore, RhoA, but not Rac, was hyperactivated upon stimulation with thrombin with subsequent cell contraction, decreased cell adhesion, and reduced cell migration. It appears that thrombin stimulation may play a role in prostate cancer metastasis by decreasing cell adhesion to the extracellular matrix. In addition, our studies of how PAR-1 activation increased prostate cancer cell survival led us to discover a link to the NF?B signaling pathway. We demonstrated that stimulation of PAR-1 resulted in increased DNA binding activity of the NF?B p65 subunit. IL-6 and IL-8 levels were also elevated in conditioned media by at least 2-fold within 4 to 6 hours of PAR1 activation. This induction of cytokine production was abrogated by pretreatment of cells with the NF?B inhibitor caffeic acid phorbol ester. In addition, the p38 and ERK1/2 MAPK signaling cascades were also activated by PAR1 stimulation, wherease the SAPK/JNK pathway was unaffected. Inhibition of p38 and ERK1/2 by SB-203589 and PD-098059, respectively, did not abrogate NF?B activity, suggesting an independent induction of NF?B by PAR1 stimulation. Furthermore, TUNEL assay showed that activation of PAR1 attenuated taxotere-induced apoptosis through the upregulation of the Bcl-2 family protein Bcl-xL. However, Akt activation was not observed, suggesting that drug resistance induced by PAR1 is independent of PI3K signaling pathway. Because thrombin and PAR1 are overexpressed in prostate cancer patients, targeting the inhibition of their interaction may attenuate NF?B signaling transduction thereby reducing drug resistance of prostate cancer. We utilized the thrombin inhibitor hirudin and the PAR1 inhibitor thrombostatin in our preclinical mouse PC-3lux model to determine if we could inhibit prostate cancer cell metastasis. Neither drug was effective at inhibiting seeding of the prostate cancer cells. Attempts to inhibit prostate cancer growth through combinations of hirudin and docetaxel proved too toxic. Further work in this aim, specifically “to establish benchmarks by which specific thrombin inhibitors will be chosen for future clinical trials in prostate cancer metastasis prevention and treatment” does not appear to be feasible. While we were able to quantify circulating cells in prostate cancer patients, measuring the effects of PAR1 inhibitors is not feasible at this time. As outlined in last years progress report, we are currently doing a trial utilizing Cilengitide in hormone refractory prostate cancer patients. In this trial we are measuring circulating cells. The hypothesis of this project is that we can target the PAR1 pathway to treat prostate cancer. In the first year of the grant, we identified a target within this pathway, the avß3/ avß5 integrins [vitronectin / osteopontin receptors], which are modulated by PAR1 activation. These integrins modulate both angiogenesis as well as prostate cancer cell metastasis. We have identified an agent that inhibits the binding of vitronectin to its receptors, EMD 121974 and have received NCI approval to treat patients with advanced prostate cancer in a Phase II clinical trial. This trial contains correlative studies that will measure circulating cells serially in patients while receiving EMD 121974. In the second year of the grant, we did identify that PAR1 does stimulate the NF?B survival pathway. Preclinical in vivo testing of thrombin / PAR1 inhibitors was unsuccessful. PAR1 itself does not appear to be a viable target for cancer therapy. In other work, we will pursue downstream targets. Based on Dr. Pienta’s discovery that inhibitors of PAR1 did not inhibit metastasis in vivo in preclinical models which eliminated the translational potential of this project, the Clinical Applications Committee and Operating Committee elected to terminate this project in May 2005. The UM prostate spore solicited applications to replace project 2 and chose Shaomeng Wang, PhD, with his project, “Evaluation and development of non-peptide small-molecule mdm2 inhibitors as a new therapy for advanced prostate cancer.” see project 6 below. Publications:
Project 3: Signature Lethal Biomarkers of Prostate CancerPrincipal Investigator: Arul Chinnaiyan, M.D., Ph.D Afflicting one out of 9 men over age 65, prostate cancer (PCA) is a leading cause of male cancer-related death, second only to lung cancer. The American Cancer Society estimates that 198,100 American men will be diagnosed with PCA and 31,500 will die this year .While effective surgical and radiation treatments exist for clinically localized PCA, metastatic PCA remains essentially, incurable and most men diagnosed with metastatic disease will succumb over a period of months to years. The molecular differences between metastatic prostate cancer and localized prostate cancer are not well established. Before prognostic markers and rational therapies can be developed to target this lethal form of prostate cancer, the molecular alterations associated with it need to be unmasked. Our overall hypothesis is that metastatic (advanced) prostate cancer expresses genes that can be used to predict the aggressive potential of clinically localized prostate cancer. These "signature" lethal genes have potential as prognostic biomarkers, therapeutic targets, and may play a role in the progression from localized disease. The advent of high-throughput genomic and proteomic techniques raises new hopes for identifying novel molecular targets for therapy. In this proposal, we will implement novel bioinformatic approaches to identify candidate lethal biomarkers from the gene expression profiles of metastatic prostate cancer (Aim 1 ). These lethal genes will be evaluated on clinically stratified prostate cancer tissue microarrays (Aim 2). The most promising candidates will be tested in a cohort of prostate needle biopsies for their ability to predict clinical aggressiveness (Aim 3). By pursuing these studies we hope to identify a set of lethal biomarkers that will be useful for guiding treatment decisions for individual patients and that may also have potential as therapeutic targets. Specific Aim 1: Identify candidate “lethal” biomarkers of prostate cancer using the gene expression signature of advanced (metastatic) prostate cancer. Specific Aim 2: Characterize the potential of a cohort of “lethal” biomarkers to predict clinical outcome and aggressiveness of prostate cancer. Specific Aim 3: Evaluate the most promising “lethal” biomarkers on prostate needle biopsies. April 2005 Update:Specific Aim 1:
Specific Aim 2:
Specific Aim 3:
The advent of prostate specific antigen (PSA) screening has led to earlier detection of PCA. Coincident with increased serum PSA testing, there has been a dramatic increase in the number of prostate needle biopsies performed. This has resulted in a surge of equivocal prostate needle biopsies and men with the looming threat of PCA. However, the stage-shift associated with the advent of PSA screening may also be associated with diagnosis of a substantial number of prostate cancer cases that may have non-aggressive clinical natural history, or so-called ‘indolent’ prostate cancers. Even before the advent of PSA screening, it was noted that up to 70-80% of Gleason score 6 cancers, and as many as 20% of Gleason 7 cancers, may have a non-aggressive course without cancer death if observed without intervention for more than 15 years. With the population of males 65 years and older expected to increase from 14 million in year 2000 to 31 million by 2030, it will be increasingly important to discern such indolent prostate cancer from aggressive cancers that warrant intervention. Tissue biomarkers can play a vital role in such clinical decision-making, particularly as it has been shown that standard clinical parameters, such as PSA, Gleason score, and clinical stage have extremely limited utility in discerning indolent from aggressive prostate caners. As an outcome of the proposed investigation, we expect to identify lethal biomarkers that can help distinguish clinically latent prostate cancer from aggressive prostate cancer. Conversely, we also expect that some markers will also discern aggressive cancers amenable to local-regional therapy from those in which standard, regionally-targeted therapy (prostatectomy or radiation for example) would be inadequate, and wherein systemic therapy may be indicated despite ostensible lack of metastases at presentation). To identify biomarkers of prostate cancer aggressiveness, we will focus on those differentially expressed genes that are most highly associated with metastatic prostate cancer or post-prostatectomy recurrence. These genes will be designated as harbingers of lethal disease and be examined in clinically localized disease. The research proposed in this application is significant because we expect to develop tissue-based tests (and in the future serum-based tests) to predict the aggressiveness of prostate cancer and what treatment approach should be implemented. The plans for this project have not changed significantly since funding of this proposal. Additionally we are developing a cohort of frozen needle biopsies to determine if we can predict recurrence based on gene expression obtained from amplified RNA derived from laser-captured cells. Publications:
Project 4: Defining Genetic Risk Factors for Brothers of Men with Prostate CancerPrincipal Investigator: Kathleen A. Cooney, M.D. Men with a family history of prostate cancer have a two to four-fold excess risk of developing prostate cancer compared to those with no family history. The degree of risk elevation associated with familial prostate cancer may depend on several factors including the age at diagnosis of the affected family members and the total number of affected first and/or second-degree relatives. Many multiplex prostate cancer families have been studied over the last decade with the goal of identifying highly penetrant prostate cancer genes using linkage approaches, however, many of these prostate cancer genes remain elusive. An alternative strategy for the identification of prostate cancer susceptibility genes is the use of association studies, which have generally used case:control datasets to study low penetrance genes. The University of Michigan Prostate Cancer Genetics Project (PCGP) is a family-based study with the goal of characterizing the molecular basis for the inherited predisposition to prostate cancer. We hypothesize that prostate cancer susceptibility loci with modest penetrance can also be identified and characterized using family-based association studies. Since prostate cancer is a late-onset disease, and parental genotype information from parents is typically unavailable, we will focus primarily on understanding the genetic differences between men with prostate cancer and their unaffected male siblings. Therefore, to characterize prostate cancer susceptibility genes using prostate cancer families, the following three Specific Aims are proposed:
The translational goal of our project is to identify genes that can be used to determine risk of prostate cancer as well as clinically aggressive prostate cancer in unaffected men with a family history of prostate cancer. April, 2005 updateIn this second year of funding, we have made significant progress towards both Aims 1 and 2. DSP recruitment. At the present time, we are recruiting ~10 unaffected brothers every month to create new DSPs. In addition to providing prostate cancer screening information and a blood sample for DNA extraction, men are asked to complete a 15-30 minute computer-assisted telephone interview. Men are asked a number of questions regarding their general health, use of physician-prescribed as well as complementary and alternative medication use (CAMs), and risk perception and concern about a potential prostate cancer diagnosis (see below).For these studies, we generally recruit only the oldest unaffected brother from a given sibship but we also have families with multiple affected and unaffected brothers. As of March 1. 2005, we have 812 siblings (increased from 728 last year) from 311 families. This number includes only those families in which we have collected DNA samples from both an affected and unaffected brother and received all of the relevant medical records/family history information. Over the past year, we have focused our recruitment to include unaffected brothers from families already participating in the PCGP. This strategy results in a more efficient use of time and study personnel. Thus, there are an additional 110 new DSP families in which recruitment is underway but not completed. There are an additional 60 families identified that are eligible but recruitment has not been initiated. Over the past year, we also successfully renewed the R01 grant that supports continued recruitment of hereditary prostate cancer families. Thus, we do not anticipate difficulties in reaching our recruitment goal of 600 families with at least one DSP by year 5 of funding. 1. Collection of clinical data. This data is being entered to our Microsoft Access database and will be available for use in the analyses proposed in Specific Aim 2b. Based in our preliminary studies, we estimate that one-third of our affected siblings will have clinically aggressive prostate cancer using strict clinical criteria:
2. New genotype data. 3. Follow up studies for BRCA1 SNP. Furthermore, the association between BRCA1 and prostate cancer risk persisted among the much smaller subset of families with a strong history of prostate cancer (4 or more men with confirmed prostate cancer) (p=0.039) and clinically aggressive prostate cancer (0.011). To follow up on this observation, we have selected a series of 6 SNPs over a region spanning approximately 100 kB on either side of the BRCA1 SNP. These 7 SNPs should tag over 90% of the potential haplotypes in this region of 17q21. In addition, we are genotyping all of our PCGP families with three or more confirmed cases of prostate cancer with the initial BRCA1 SNP. Our research team as well as other collaborative groups has observed evidence of prostate cancer linkage to 17q21-22 markers. We have also screened the entire BRCA1 gene for mutations using a set of probands from 93 PCGP families with evidence of 17q21 linkage. Our failure to identify a significant number of deleterious BRCA1 mutations in linked families suggests that there is a prostate cancer susceptibility gene other than BRCA1 in this chromosomal region. The proposed experiments will contribute to the search for this putative prostate cancer gene on chromosome 17q21-22. 4. SNP discovery. For each of the 3 genes, primers were designed to sequence all exons, introns as well as upstream regulatory and 3’ untranslated regions. Twenty affected men (representing both low and high stage disease) and 20 unaffected men from the DSP dataset were sequenced. The data is currently being analyzed to characterize the haplotype structures for each of the 3 genes and to identify haplotype-tagged SNPs for use in analysis of the entire DSP dataset. Given the overall prevalence of prostate cancer in the general population, many American men will report a family history of the disease. Men who have a brother with prostate cancer have an approximately 3–fold excess risk of prostate cancer. These men represent an appropriate population for targeted screening and chemoprevention given their elevated risk of prostate cancer. Our goal is to identify genetic markers that can be used to stratify these unaffected brothers into high and low risk categories. Over the next 12 months, we will continue to recruit unaffected brothers of men with prostate cancer to expand our set of DSPs. We will continue our strategy of selecting SNPs in candidate susceptibility genes to test for association with prostate cancer using family-based methods. In the coming year, we will perform SNP discovery on one or more candidate genes to complement our knowledge of SNPs in the available public databases. Publications:
Project 5: Multiple Mechanisms of Androgen Resistance in Prostate Cancer ProgressionPrincipal Investigator: Diane Robins, Ph.D. Prostate cancer initially depends on androgens for growth and initially responds to androgen ablation therapy. Nevertheless, the cancer ultimately becomes resistant to anti-androgens and disease progression occurs. However, the tumors retain androgen receptor (AR) and its downstream signaling pathway. Resistance may be due to several factors. First, anti-androgens may display partial agonist behavior, the basis of which may vary with the compound. Second, mutant ARs may arise during treatment, with reduced ligand specificity or ligand-independent activity. These mutant ARs may show differential resistance to particular antagonists. An overall hypothesis of this proposal is that mutant ARs are selected during treatment of prostate cancer with anti-androgens and allow progression of androgen-independent disease. Our specific hypotheses are that:
Aim 1. Aim 2. Aim 3. April, 2005 UpdateIn this project, we are working on Aims I and II concurrently, and will initiate Aim III as results from the mouse models provide support for the hypothesis. Aim I is based on our finding that AR displayed differential interactions with coactivator (SRC-1) and corepressor (NCoR), dependent on antagonist. In particular, hydroxyflutamide (OHF)-AR showed greater interaction with GST-fused fragments of SRC-1 than did bicalutamide (BIC)-AR. In contrast, BIC-AR interacted more strongly with corepressor fragments than did OHF-AR. These results agree with transfection data from our and other laboratories, and may account for the greater partial agonism of OHF than BIC. We compared these interactions for wild type AR and the LNCaP AR, T877A (a mutant receptor that is resistant to OHF but sensitive to BIC), but saw little difference in binding of wild type versus T877A AR. Overall, the GST pulldowns did not adequately distinguish quantitative differences that are demonstrable in functional transfection assays. We are therefore developing more sensitive approaches using mammalian one- and two-hybrid assays to reveal protein-protein interactions that may vary with ligand. We have found that the interaction domains of both SRC-1 and NCoR interact well with DHT-AR tethered to an androgen responsive DNA element (HRE-3). With flutamide, neither coregulator showed significant interaction in this assay. However, with BIC, there is detectable albeit modest interaction between AR and NCoR, in agreement with the preliminary GST and transfection data. In the complementary interaction test, where coregulator interaction domains are tethered to DNA via the Gal4 DNA binding domain, AR interacts with the SRC-1 interaction domain fusion but not with the NCoR fusion. However, in this context, interaction can be obtained in the absence of AR’s LBD, suggesting AR N-C interaction may compete with NCoR interaction. Further, neither OHF nor BIC enhanced NCoR interaction with AR, but mifepristone did, supporting work of others showing this compound to be a potent AR antagonist. In more functional transfection assays, we are comparing OHF and BIC effects on diverse promoters and in different cell types. High levels of OHF, but not BIC, consistently show partial agonism in CV-1 but not PC-3 cells, and this agonism is more pronounced on the complex PSA promoter than on simple HREs. Thus the partial agonism of OHF is likely to vary with the promoter and cell environment. This is under further study. In Aim II, we are testing differential selection pressures conferred by antagonist treatment in vivo. In the first model, we are comparing xenograft growth in intact vs. castrated SCID mice, for the following 4 cell lines: VCaP, PC-3, PC-3 stably transfected with wild type AR, and PC-3 stably transfected with LNCaP AR. Tumor growth rates have been assessed and tumors harvested for comparison of molecular markers at RNA and protein levels [including AR, PSA, coactivators (SRC-1, SRC-3), corepressors, and several growth factors], and tissue fixed for histological comparison. Currently, mice bearing VCaP xenografts are being treated with flutamide (as pellet implants) and casodex (compounded in food). AR cDNAs will be sequenced and compared for mutations in the VCaP series, which is likely to be the most informative for testing the hypothesis that mutations will vary dependent on treatment. Substantial progress continues to be made with our engineered mouse model HART (Humanized Androgen Receptor Target Mouse) containing the “humanized” h/mAR. These animals are grossly normal, with respect to behavior, growth and fertility. More detailed analyses are revealing subtle differences in induced levels of expression of specific AR target genes in different tissues. These mice were crossed with TRAMP mice, and h/mAR-TRAMP males were compared to mAR-TRAMP littermates, either intact or following castration at 12 weeks. Tumor progression was tracked by palpation, noting appearance of tumor and approximate growth rate. We have also tracked tumor progression by MRI analysis, to corroborate the accuracy of palpation (performed blinded by two investigators), for several individual mice. MRI detects tumor initiation 2 weeks prior to palpation, but the cost is prohibitive compared to the information gained. We initially planned to sacrifice mice to harvest tumors at an endpoint of 29 weeks, but at this time many mice did not have substantial tumor. Thus we use 29 weeks as a somewhat arbitrary distinction, allowing division of mice into 3 classes (aggressive, moderate, or slow disease) reflecting status at 29 weeks– those mice that became moribund before 29 weeks were euthanized and these tumors are deemed “aggressive”; mice with moderate disease have a palpable tumor at 29 weeks but are not moribund, and mice with slow disease do not yet have a palpable tumor at 29 weeks. This allows us to maximize informative samples from these mice and to obtain survival data, as well as compare approximate growth rates of tumors (by time from palpation until death). All the harvested tumors are thus late-stage and histology confirms that they represent grossly undifferentiated disease. We have created a tissue microarray with these samples, using the SPORE microarray core. Immunohistochemistry shows stronger nuclear staining for AR in the h/mAR than wild type TRAMP mice, in both residual glandular epithelium and cancer cells. While all the adjacent normal epithelium shows nuclear staining, staining of cells in cancer tissue is heterogeneous. In the castrated mice, AR staining is diffuse in the wild type mice, but in the h/mAR mice numerous cells (~10-20%) still have distinct nuclear staining. In intact mice (androgen-dependent), survival curves were similar for h/mAR compared to wild type TRAMP mice, with tumors falling approximately equally into aggressive, moderate and slow disease. In experiments carried out in parallel and funded by the DOD, for which we created h/mAR mice with AR alleles varying in glutamine tract length, we are seeing notable effects of glutamine tract length in intact mice. In the castrated h/mAR mice (mimicking androgen-independent disease), significantly more mice showed early and aggressive disease with the stronger humanized AR than for the wild type mouse allele. In fact, it appears that despite genetic homogeneity within mouse groups, a strong stochastic element exists for tumor initiation. The castrated mice reveal 2 responses to androgen withdrawal – in one group early aggressive disease occurs whereas in others disease is, as might be more expected, very delayed in onset and slow-growing. We find this result exciting as it suggests that the h/mAR mouse will indeed reveal novel findings, and further, that this model may accentuate the difference between androgen-dependent and –independent prostate cancer. We are currently optimizing steps required to sequence AR cDNAs from these tumors to obtain results in a more high through-put manner. This will then be applied to patient samples for Aim III. Despite diverse genetic and environmental factors that affect risk of prostate cancer, all prostate tumors have in common an initial dependence on the steroid hormone androgen for growth. Androgen works via its nuclear receptor, which functions to regulate expression of specific genes. Successful means to slow the spread of disease include shutting off androgen synthesis by chemical or surgical castration and blocking receptor function with compounds that bind but do not activate receptor. While these treatments are initially successful, they ultimately fail, and cancer recurs. It is critical for a cure to understand the basis of resistance to hormonal ablation and antiandrogen therapy. We think that mutations in androgen receptor may underlie some cases of relapse, particularly by altering receptor interactions with accessory proteins that help to activate or repress receptor function. These mutations may be detected because they are selected by, or survive, the treatment. Furthermore, since the treatments vary, the mutant ARs that are resistant to them may vary as well. We are trying to gain evidence in support of these hypotheses in experiments both in vitro and in mice that aim to discern distinct molecular bases for flutamide versus bicalutamide action, and to determine whether mutations resistant to one are not resistant to the other. Some evidence for this already exists but has not been examined on a broader scale. Further support would be significant to clinical progress in two ways. First, confirming different mutations arising in response to different drugs might encourage testing of regimens that would alternate drugs on a short time scale or use them in conjunction, so that resistance to both could not occur. Second, the sites of mutations that are effective highlight sites on androgen receptor that interact with particular proteins to function, and these partners might themselves become novel targets for therapy. A more short-term and practical outcome is that the mice we are creating with human androgen receptors replacing those of the mouse may prove to be excellent animals for drug testing of novel agents targeting the human AR. Our plans have not altered significantly from those originally described in the proposal. Aim II has taken precedence as it is progressing well. As we identify mutant ARs in mouse xenograft and tumor samples, the mutations will be introduced into cDNA expression vectors and tested in accord with goals of Aim I. That is, the mutant ARs will be compared to wild type for ability to interact with coactivators and corepressors in physical interaction studies and in functional transfection assays. These experiments will determine whether mutations are functionally significant in androgen-independent tumor progression. Once several mutations have been identified in the mouse tumor models, we will begin to sequence ARs from the patient samples (Aim III) to determine whether antagonist-specific (or –influenced) mutations are demonstrable in human tumors. Given the complementarity and multiplicity of our models, we believe we are in a strong position to determine whether AR mutations are differentially selected by different treatment modes. This should be of translational value in devising appropriate treatment regimens, and encourage the search for more potent antiandrogens. Related Publications:
Project 6: Evaluation and Development of Non-Peptide Small-Molecule MDM2 Inhibitors as a New Therapy for Advanced Prostate CancerPrincipal Investigator: Shaomeng Wang, Ph.D. After Project 2 was terminated, this project was chosen by competition as a replacement. This project began 6/1/05. Our major objectives in this proposal are to evaluate the therapeutic potential of potent, non-peptide, drug-like, water-soluble small-molecule inhibitors of MDM2 which we have recently designed and synthesized as a new therapy for the treatment of advanced prostate cancer; and to elucidate their molecular mechanism of action in prostate cancer models. Sccessfully carriedout, this project will lead to the development of an entirely new class of molecularly targeted anti-cancer therapy for the treatment of advanced prostate cancer by stimulating the activity of p53 through blocking the p53-MDM2 interaction. Aim 1: (a) In vitro evaluation of 8-10 potent, non-peptide small-molecule MDM2 inhibitors for their anticancer activity in human prostate cancer cells, alone and in combination with chemotherapeutic drugs; and (b) elucidation of their molecular mechanism of action in prostate cancer cells. Aim 2: Determination of the therapeutic potential of the 3-5 most promising MDM2 inhibitors in animal models of human prostate cancer, alone and in combination with current chemotherapeutic agents. Aim 3: (a) Analysis of MDM2 and related proteins of prostate cancer tissue microarrays and correlations with measures of clinical outcome; and (b) isolation of cells from the circulation of patients with androgen-independent prostate cancer and determination of p53 status of circulating cancer cells. Related Publications
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