Paul C. Marker - US grants

DESCRIPTION (provided by applicant): Interest in understanding the biology of the prostate gland is motivated by the high incidence in humans of prostatic diseases including prostatic adenocarcinoma and benign prostatic hyperplasia. One crucial but poorly understood aspect of prostate biology is the molecular basis for region specific identity within the prostate. Nevertheless, this aspect of prostatic biology is important because prostatic diseases occur in a highly region-specific manner with prostatic adenocarcinoma predominantly a disease of the peripheral zone and benign prostatic hyperplasia predominantly a disease of the transition zone. It is also clear that both epithelial region-specific identity and the progression of prostatic adenocarcinoma are influenced by paracrine signals from the prostatic stroma. In recent years, the mouse has become an increasingly important model system for understanding prostatic biology and modeling prostatic diseases. Prostate-specific promoters and enhancers have facilitated this area of research. However, the characterized prostate-specific promoters and enhancers are not adequate to explore all aspects of prostatic development and disease. Our preliminary studies have established a new and efficient method to screen for promoter and enhancer elements that direct gene expression in a region-specific manner in the prostate gland. Work described in Aim 1 of this proposal will use this new approach to identify and characterize promoter and enhancer elements associated with two genes that are expressed in prostatic cell types not accessible with currently available promoter or enhancer elements. One gene is expressed specifically in epithelial cells of the anterior and dorsal prostate. The second gene is expressed in prostatic stromal cells. Work described in Aim 2 of this proposal will use the newly defined regulatory elements to create lines of transgenic mice expressing CRE recombinase in prostatic cell types not covered by currently available CRE transgenic lines.

[unreadable] DESCRIPTION (provided by applicant): Our published and preliminary studies have shown that the mouse seminal vesicle shape (svs) mutation causes prostatic branching morphogenesis and size defects as well as increased sensitivity to hormone- induced lesions that resemble early stage prostate cancer. These data suggest that the svs mutation can be used as a unique entry-point to identify a genetic pathway that regulates branching morphogenesis and tumor suppression in the prostate. We have identified a candidate svs mutation in the Fgfr2 gene that is associated with changes in alternative splicing of the Fgfr2 transcript, and changes in the activation of downstream signal transduction pathways. Fgfr2 has previously been shown to have tumor suppressor activity prostate cancer cell lines, but studies of human prostate cancer have reported conflicting results for Fgfr2. Our investigation of svs mutant mice suggests a possible explanation for the conflicts among previous studies. We have shown that alternative splicing of Fgfr2 in the prostate and seminal vesicles is much more complex than previously recognized, and changes in the spectrum of alternatively spliced Fgfr2 isoforms without changes in protein levels (as in svs mice) can have a profound impact on prostate development and cancer susceptibility. Experiments outlined in this proposal will explore the role of Fgfr2 alternative splicing in prostatic development and cancer progression. Experiments in Aim 1 will test whether the svs mutation is allelic with Fgfr2. Experiments in Aims 2 and 3 will test the hypothesis that changes in Fgfr2 alternative splicing can contribute to prostate cancer progression in mice and humans. Experiments in Aim 4 will identify gene expression changes associated with the presence/absence and alternative splicing of Fgfr2 in the prostate. These experiments will define the types of Fgfr2 mutation (isoform switching vs. loss) that promote prostate cancer progression in mice and humans. They will also define the mechanisms used by Fgfr2 to coordinate branching morphogenesis and tumor suppression in the prostate. [unreadable] [unreadable] [unreadable]

Abstract We have conducted a two-species pilot screen to discover new oncogenes and tumor suppressor genes that can drive prostate cancer initiation and/or progression in mice and humans. The primary screen utilized transposon-based insertional mutagenesis in mice to model genetic heterogeneity in prostate cancer and conduct a genome-wide screen for new oncogenes and tumor suppressor genes. The secondary screen used human prostate cancer patient samples to validate the relevance of novel candidate cancer genes by examining the human orthologs of the genes for altered expression in human prostate cancers. This screen identified several novel candidate prostate cancer oncogenes and tumor suppressor genes. One of the first genes identified in the mouse screen, PDE4D, was also over-expressed in human prostate cancer patient samples. Furthermore, knockdown of PDE4D reduced the proliferation of human prostate cancer cells in vitro and in vivo. This proposal will investigate the novel candidate prostate cancer oncogenes and tumor suppressor genes that have been identified in our screen with a particular emphasis on evaluating the roles of PDE4D as a candidate prostate cancer oncogene and potential drug target in prostate cancer. Experiments in Aims 1 and 2 of the proposal will model PDE4D over-expression in the normal prostate and investigate the mechanism of PDE4D action in the prostate. Experiments in Aim 3 will test NVP- ABE171, a PDE4D-selective small molecule inhibitor, as a potential anti-prostate cancer drug in the context of pre-clinical models. Experiments in Aim 4 will evaluate the expression of PDE4D and other novel candidate prostate cancer genes identified in our preliminary studies for expression changes associated with different pathologic grades of human prostate cancer and/or expression that is predictive of long-term patient outcomes. Collectively, these studies will lead to a better understanding of the genetically diverse pathways that drive prostate cancer progression, and they will determine the suitability of PDE4D as a new drug target in prostate cancer.

DESCRIPTION (provided by applicant): Lower urinary tract symptoms (LUTS), including bladder voiding symptoms, are a common problem in aging men. Voiding symptoms occur in many cases due to bladder outlet obstruction (BOO). While the underlying causes of BOO are unclear and may be multi-factorial, BOO is often found in association with benign prostatic hyperplasia (BPH), another highly prevalent condition in aging men. While the etiology of BPH and BOO remain largely unclear, available data are consistent with the hypothesis that changing hormone levels in aging men and/or the reactivation of developmental growth-regulatory pathways are underlying causes of BPH and associated BOO. In support of the potential role of steroid hormones in BPH and BOO, our preliminary studies showed that male mice treated with testosterone + estradiol (T+E2) at doses that mimic the hormonal milieu in aging human males developed benign enlargement of the prostate that was associated with the appearance of proliferating foci along the urethra that resembled developmental prostatic buds, changes in the morphology of the prostatic peri- urethral region, and a high incidence of urinary retention indicative of BOO. Strikingly, the severity of hormone-induced urinary retention was more than 4-fold greater in 18-month-old mice compared to 2-month-old mice indicating that this model recapitulates the age-dependent susceptibility to BOO that is also seen in humans. The hormone-induced mouse model also exhibited gene expression changes in the prostate that have been reported to occur in human BPH associated with BOO, including the up-regulation of Secreted frizzled related protein 1 (Sfrp1). Using several approaches including the creation and study of Sfrp1 knockout and transgenic mice as well as in vitro experiments, our published and preliminary studies have implicated Sfrp1 signaling via the non-canonical WNT/JNK pathway as a pro-proliferative signal during prostatic development that can be co-opted to trigger abnormal proliferation in the adult prostate. These data suggest that Sfrp1 is likely to be a mediator of BPH pathology rather than merely a biomarker for BPH. We hypothesize that a sub-type of BPH associated with BOO is caused by the reactivation of developmental growth-regulatory pathways including SFRP1/JNK signaling. This hypothesis will be tested using a multi-disciplinary approach that will include an evaluation of mouse models and a collaboration with a surgical pathologist, Wei Huang M.D., who will assist us in evaluating developmental growth factor pathways and JNK signaling in human patient samples.

? DESCRIPTION (provided by applicant): Most prostate cancers initially respond to androgen deprivation therapy but eventually progress to castration-resistant disease. Once castration-resistant prostate cancer (CRPC) arises, it cannot be effectively treated. This fact creates a need to better understand mechanisms of CRPC to enable new therapies for patients with advanced disease. In preliminary studies, we used transposon-based mutagenesis of androgen-sensitive prostate epithelial cells to derive sub-lines that were resistant to absence of androgens or the presence of bicalutamide. Molecular analysis of transposon insertions in the castration-resistant sub-lines identified MAGI2 as a potential driver of the castration resistant phenotype. Previous whole genome sequencing of human prostate cancers also found genomic rearrangements in MAGI2. Both the transposon insertions observed in our study and the rearrangements observed in human prostate cancers could potentially cause both loss of full-length MAGI2 expression and gain of expression for a truncated N-terminal fragment of MAGI2. Data mining the results of published RNA-seq studies of CRPC also identified candidate fusion transcripts involving the MAGI2 N-terminus. These data fit with additional preliminary studies of protein expression for the N-terminus of MAGI2 on human prostate cancer tissue microarrays. In these studies we observed that high expression of the MAGI2 N-terminus was associated with an increased risk of death from prostate cancer. In light of these findings, we hypothesize that over-expression of the N-terminus of MAGI2 drives progression to CRPC. We will test this hypothesis via gain- and loss-of-function approaches in cell line and mouse models (Aim 1) and by further investigating expression of the MAGI2 protein and candidate MAGI2-regulated pathways in human prostate cancer (Aim 2). This project will have high-impact outcomes for the effort to combat prostate cancer by furthering the goal of understanding mechanisms of resistance for men with prostate cancer. This project will also further the goal of distinguishing aggressive from indolent disease because our preliminary studies support the concept that MAGI2 and MAGI2-regulated pathways as potential biomarkers to distinguish aggressive from indolent disease.

PROJECT SUMMARY - PROJECT 3 Lower urinary tract symptoms (LUTS) are a costly and potentially critical medical problem for millions of aging men. This spectrum disorder encompasses symptoms such as weak stream, nocturia, incomplete emptying and intermittent urination, all of which are indicative of lower urinary tract dysfunction (LUTD). Surgical ablation of prostate tissue and medical approaches may improve urinary flow, but such treatments are not effective for all men, can produce adverse effects that result in discontinuation of the therapeutic regimen, and do not abrogate the risk for disease progression. If left untreated or treated ineffectively, LUTD can progress to bladder dysfunction, which can lead to urinary retention, recurrent UTI, bladder calculi, and, eventually, renal impairment. Work accomplished by the Macoska laboratory and this Center have shown that collagen accumulation around the prostatic urethra consistent with tissue fibrosis is an untreated pathobiology contributing to LUTD. The overarching goal of the O?Brien Center for Benign Urology Research is to identify mechanisms that result in lower urinary tract dysfunction and prostate-related lower urinary tract symptoms (LUTS). New evidence (presented in this application) indicates heterogeneity among peri-urethral collagen-producing cells as well as inflammatory cells within the prostatic microenvironment. Inflammatory cells secrete a medley of pro-fibrotic proteins into the prostatic microenvironment. Among these proteins, IL-4 and IL-13 are of particular interest because they share a common signaling axis which, as shown here for the first time, establishes and perpetuates an autocrine loop that activates JAK/STAT signaling to promote the expression and maintenance of IL-4, IL-13, their cognate receptors, regulatory transcription factors, and ECM components, even in the absence of inflammatory cells. Based on preliminary data presented here we hypothesize that some peri-urethral stromal cell populations establish an IL-4/IL-13 axis that self-perpetuates, induces myofibroblast phenoconversion and survival, and upregulates collagen accumulation, thereby promoting consequent urinary voiding dysfunction. To test this hypothesis, Project 3 will: 1) Determine whether signaling through the IL-4 receptor creates a STAT6- and GATA-3-mediated positive feedback loop; 2) Elucidate the mechanisms through which the IL-4/IL-13 axis promotes myofibroblast phenoconversion and concordant collagen expression; 3) Determine whether IL-4 pathologically represses myofibroblast apoptosis and thereby promotes myofibroblast survival, and 4) Test whether the IL-4/IL-13 signaling axis promotes lower urinary tract fibrosis and urinary voiding dysfunction in vivo. The results of these preclinical studies will elucidate previously unknown interleukin-mediated molecular and cellular mechanisms that promote lower urinary tract fibrosis and dysfunction. Using JAK/STAT inhibitors to therapeutically target this potentially self-perpetuating mechanism may ?break the cycle? and successfully treat recalcitrant peri-urethral prostatic fibrosis contributing to LUTD development and progression.

Abstract The growth hormone/insulin-like growth factor-1 (GH/IGF-1) axis has been implicated in age related diseases including prostate cancer. Humans with Laron syndrome lack functional growth hormone receptors (GHRs) and have reduced rates of cancer and diabetes. Mice with homozygous null mutations in Ghr are also protected from cancer in the C3(1)/TAg prostate cancer model. Similarly, rats lacking a functional GH gene are protected prostate cancers in the Probasin/TAg model. GHRs are expressed in multiple tissues including the liver and prostate. Activation of GHRs in the liver by GH is the main mechanism that stimulates the release of IGF-1 into circulation. Many GH actions are mediated by the subsequent actions of IGF-1 in target tissues including the prostate. Furthermore, evidence from humans indicates that elevated circulating IGF-1 confers an increased risk for the development of several cancers including prostate cancer. The expression of GHRs in the normal prostate and by prostate cancer cells suggests that local signaling by GHRs may also be important in prostate cancer cells. Recent studies demonstrating that up-regulation of local GH synthesis by prostate cancer cells is a common feature of cancer progression further supports the potential importance of local GH/GHR actions in prostate cancer. However, several important aspects of the role of GH/IGF-1 axis in prostate cancer remain uncertain. Available studies have not addressed the potential ongoing requirement for GH signaling at different stages of prostate cancer progression, the role of local GHR signaling in prostate cancer cells, or the signal transduction mechanisms downstream of GHR activation important in prostate cancer. This project will use innovative genetic approaches in mice to address these limitations of current knowledge. Previous rodent studies were also limited because they focused only on tumors driven by disruption the function of the TP53 and RB1 tumor suppressors. This project will further determine if the requirement for intact GHR signaling extends to mouse models for prostate cancers driven by activation of the PI3K/AKT pathway that is also commonly observed in human prostate cancers. Completion of these studies will be an important step toward identifying the best ways to employ pegvisomant or other agents targeting the GH/IGF-1 axis for the treatment and/or prevention of prostate cancer.