Prostate-specific Deletion Of Pten Research Articles

THU479 FOXA1 Loss Remodels The Tumor Immune Microenvironment In Late-stage Prostate Cancer

Abstract Disclosure: L.T. Brea: None. H. Shi: None. V. Keo: None. W. Xie: None. X. Lu: None. G. Gritsina: None. X. Wang: None. S.H. Park: None. J. Zhao: None. J. Yu: None. Background: Castration resistant prostate cancer (CRPC) has shown a poor response to immune checkpoint inhibitors due to its immunosuppressive nature. CRPC is infiltrated by tumor-associated macrophages (TAMs) and T regulatory cells (Tregs), which induce an immunosuppressive tumor immune microenvironment (TIME) and promote tumor progression. A deeper understanding of tumor intrinsic mechanisms shaping the TIME in prostate cancer (PCa) is needed to harness immunotherapies for PCa patients. Previous studies have found that epithelial transcription factor FOXA1 is downregulated in CRPC. In accordance, we have reported that FOXA1 loss induces tumor cell invasion and macrophage recruitment in vitro through HIF1A-CCL2 signaling. However, the extent to which FOXA1 loss regulates the TIME in PCa remains unclear, largely due to the lack of immune-proficient mouse models for the study of FOXA1 function in the context of PCa. Methods: To examine how FOXA1 loss regulates the PCa TIME, we generated a novel genetically engineered mouse model (GEMM) with prostate-specific deletion of Pten and Foxa1 (PbCre4:Ptenf/fFoxa1f/f) to compare with the established Pten-null PCa mouse model (PbCre4:Ptenf/f). We performed immunohistochemistry analyses on the GEMM prostate tumor tissues to characterize histopathology changes and assess tumor immune infiltration. We also performed spatial transcriptomics and scRNA-seq analyses of the GEMMs to comprehensively evaluate the effect of Foxa1 loss on downstream pathways and the surrounding TIME. Finally, we performed bioinformatics analysis of PCa patient datasets to validate the clinical relevance. Results: We found PbCre4:Ptenf/fFoxa1f/f mouse prostate tumors exhibited a more aggressive histopathologic tumor phenotype compared to age-matched PbCre4:Ptenf/f. Notably, immunohistochemistry staining and spatial transcriptomic analysis of the GEMMs revealed striking remodeling of the TIME upon Foxa1 loss, with marked increases in tumor infiltration by Tregs and TAMs. Moreover, CIBERSORTx analysis of patient datasets revealed FOXA1 expression negatively correlates with Treg and M2-macrophage infiltration. We also found pro-tumorigenic inflammatory cytokine signaling genes, linked to the recruitment of immunosuppressive cells, were upregulated in PbCre4:Ptenf/fFoxa1f/f prostate tumors. Furthermore, we confirmed inflammatory pathways were upregulated among FOXA1-low patient samples. Additional studies are ongoing to further elucidate the mechanisms by which FOXA1 regulates inflammatory cytokine signaling and the TIME in PCa.Conclusion: This study presents a novel GEMM for the study of FOXA1 function in an immunocompetent setting. Our data supports FOXA1 as a critical tumor intrinsic regulator of the TIME. This will have important implications on the design of immunotherapeutic approaches for late-stage PCa often with FOXA1 loss. Presentation: Thursday, June 15, 2023

Abstract B029: Foxf2 activates antitumor immunity to repress the progression of prostate cancer by repressing Fgl1

Abstract Targeting immune suppressive mechanisms in the tumor microenvironment has revolutionized cancer treatment. Although immune checkpoint inhibition (ICI) has yielded meaningful responses across many cancer types, the ICI efficacy is still limited for prostate cancer. We generated a mouse model with prostate epithelial specific expression of Foxf2, a Forkhead box transcription factor. Foxf2 overexpression suppressed tumor growth in two mouse models for prostate cancer with prostate specific Pten deletion or TRAMP. Foxf2 expression strongly induced tumor cell-intrinsic immune responses by recruiting and activating CD8+ T cells and switching the polarization of macrophages from an immunosuppressive M2-like phenotype to a more inflammatory M1-like state in both syngeneic and autochthonous mouse prostate cancer models in an immunocompetent context. Furthermore, we showed that overexpression of Foxf2 in mouse and human prostate cells repressed the expression of FGL1, a major ligand of LAG-3 that mediates T cell suppression. In this study, we take a combination of bioinformatic, molecular, cellular, and genetic approaches and reveal that Foxf2 overexpression in prostate cancer suppresses tumor progression by activating antitumor immunity. Citation Format: Deyong Jia, Zhicheng Zhou, OhJoon Kwon, Li Xin. Foxf2 activates antitumor immunity to repress the progression of prostate cancer by repressing Fgl1 [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B029.

Identification of Androgen Receptor Metabolic Correlome Reveals the Repression of Ceramide Kinase by Androgens.

Simple SummaryProstate cancer cells require androgens to survive and grow. In turn, targeting androgen signaling has become a predominant therapeutic strategy in this disease. These hormones regulate a plethora of biological processes, which identification could aid the refinement of future anticancer treatments. Our aim was to uncover metabolic processes under the control of androgens, taking advantage of bioinformatics analyses using publicly accessible data in prostate cancer. We found that these hormones control the abundance of an enzyme, ceramide kinase (CERK). CERK produces ceramide-1-phosphate, a metabolite with prosurvival and migration properties. This finding suggests that antiandrogen therapies could be limited by the reactivation of this metabolic process. Prostate cancer (PCa) is one of the most prevalent cancers in men. Androgen receptor signaling plays a major role in this disease, and androgen deprivation therapy is a common therapeutic strategy in recurrent disease. Sphingolipid metabolism plays a central role in cell death, survival, and therapy resistance in cancer. Ceramide kinase (CERK) catalyzes the phosphorylation of ceramide to ceramide 1-phosphate, which regulates various cellular functions including cell growth and migration. Here we show that activated androgen receptor (AR) is a repressor of CERK expression. We undertook a bioinformatics strategy using PCa transcriptomics datasets to ascertain the metabolic alterations associated with AR activity. CERK was among the most prominent negatively correlated genes in our analysis. Interestingly, we demonstrated through various experimental approaches that activated AR reduces the mRNA expression of CERK: (i) expression of CERK is predominant in cell lines with low or negative AR activity; (ii) AR agonist and antagonist repress and induce CERK mRNA expression, respectively; (iii) orchiectomy in wildtype mice or mice with PCa (harboring prostate-specific Pten deletion) results in elevated Cerk mRNA levels in prostate tissue. Mechanistically, we found that AR represses CERK through interaction with its regulatory elements and that the transcriptional repressor EZH2 contributes to this process. In summary, we identify a repressive mode of AR that influences the expression of CERK in PCa.

Anti-androgen therapy induces transcriptomic reprogramming in metastatic castration-resistant prostate cancer in a murine model

Despite recent development of next-generation androgen receptor (AR) antagonists, metastatic castration-resistant prostate cancer (CRPC) remains incurable and requires deeper understanding through studies in suitable animal models. Prostate-specific deletion of Pten and Smad4 in mice recapitulated the disease progression of human prostate adenocarcinoma, including metastasis to lymph nodes and lung. Moreover, Pten/Smad4 tumors fostered an immunosuppressive microenvironment dominated by myeloid-derived suppressor cells (MDSCs). However, the response of Pten/Smad4 tumors to androgen deprivation and anti-androgen therapies has not been described. Here, we report that the combination of surgical castration and enzalutamide treatment in Pten/Smad4 mice slowed down the tumor growth and prolonged the median survival of the mice for 8 weeks. Treatment-naïve and castration-resistant primary tumors exhibited comparable levels of immune infiltrations with the exception of reduced monocytic MDSCs in CRPC. RNA profiling of treatment-naïve and castration-resistant primary tumors revealed largely preserved transcriptome with modest expressional alterations of collagen-related and immune-related genes, among which CC chemokine receptor type 2 (Ccr2) downregulation and predicted negative activation in CRPC was consistent with reduced monocytic MDSC infiltration. Importantly, significant transcriptomic reprograming was observed in lung metastatic CRPC compared with primary CRPC and enriched for immune-related and coagulation-related pathways. At the individual gene level, we validated the expression changes of some of the most upregulated (Cd36, Bmp5, Bmp6, Etv5, Prex2, Ptprb, Egfl6, Itga8 and Cxcl12) and downregulated genes (Cxcl9 and Adamts5). Together, this study uncovers the inherent activity of Pten/Smad4 tumors to progress to CRPC and highlights potentially targetable transcriptomic signatures associated with CRPC metastasis.

Locally invasive, castrate-resistant prostate cancer in a Pten/Trp53 double knockout mouse model of prostate cancer monitored with non-invasive bioluminescent imaging.

Here we have improved an existing mouse model of prostate cancer based on prostate-specific deletion of Pten and Trp53 by incorporating a Cre-activatable luciferase reporter. By coupling the deletion of those genes to the activation of a luciferase reporter, we were able to monitor tumor burden non-invasively over time. We show that, consistent with previous reports, deletion of both Pten and Trp53 on a C57BL/6 background accelerates tumor growth and results in both the loss of androgen receptor expression and castrate resistant tumors as compared with loss of Pten alone. Loss of Trp53 results in the development of sarcomatoid histology and the expression of markers of epithelial-to-mesenchymal transition Zeb1 and vimentin, with kinetics and penetrance dependent on whether one or both alleles of Trp53 were deleted. Homozygous deletion of Trp53 and Pten resulted in uniformly lethal disease by 25 weeks. While we were able to detect locally invasive disease in the peritoneal cavity in aggressive tumors from the double knockout mice, we were unable to detect lymphatic or hematogenous metastatic disease in lymph nodes or at distant sites.

Early intervention exercise training does not delay prostate cancer progression in Pten−/− mice

There is convincing evidence that men with advanced prostate cancer experience improved quality of life as a result of exercise therapy, although there is limited preclinical, and no clinical, data to directly support the notion that exercise training improves prostate cancer prognosis or outcome. The aim of this study was to investigate the effect of regular exercise training on the early stages of prostate cancer progression, as well as assessing whether alterations to prostate cancer metabolism are induced by exercise. Mice with prostate-specific deletion of Pten (Pten-/- ) remained sedentary or underwent 6 weeks of endurance exercise training or high-intensity exercise training involving treadmill running. At the conclusion of the training period, the prostate lobes were excised. A portion of fresh tissue was used to assess glucose, glutamine, and fatty acid metabolism by radiometric techniques and a second portion was fixed for histopathology. Despite the implementation of an effective exercise regime, as confirmed by improvements in running capacity, neither prostate mass, cell proliferation or the incidence of high-grade prostate intraepithelial hyperplasia or noninvasive carcinoma in situ were significantly different between groups. Similarly, neither glucose uptake, oxidation and de novo lipogenesis, glutamine oxidation, or fatty acid uptake, oxidation and storage into various lipids were significantly different in prostate tissue obtained from untrained and exercise trained mice. These results show that 6 weeks of moderate or high-intensity exercise training does not alter substrate metabolism in the prostate or slow the progression of Pten-null prostate cancer. These results question whether exercise is a useful therapy to prevent or delay prostate cancer progression.

Characterization of novel cell lines derived from a MYC-driven murine model of lethal metastatic adenocarcinoma of the prostate.

Loss or mutation of PTEN alleles at 10q23 in combination with 8q24 amplification (encompassing MYC) are common findings in aggressive, human prostate cancer. Our group recently developed a transgenic murine model of prostate cancer involving prostate-specific Pten deletion and forced expression of MYC under the control of the Hoxb13 promoter. MYC overexpression cooperated with Pten loss to recapitulate lethal, human prostate cancer. We now report on the generation of two mouse prostate cancer cell lines, BMPC1 and BMPC2, derived from a lymph node, and liver metastasis, respectively. Both cell lines demonstrate a phenotype consistent with adenocarcinoma and grew under standard tissue culture conditions. Androgen receptor (AR) protein expression is minimal (BMPC1) or absent (BMPC2) consistent with AR loss observed in the BMPC mouse model of invasive adenocarcinoma. Growth in media containing charcoal-stripped serum resulted in an increase in AR mRNA in BMPC1 cells with no effect on protein expression, unless androgens were added, in which case AR protein was stabilized, and showed nuclear localization. AR expression in BMPC2 cells was not effected by growth media or treatment with androgens. Treatment with an anti-androgen/castration or androgen supplemented media did not affect in vitro or in vivo growth of either cell line, irrespective of nuclear AR detection. These cell lines are a novel model of androgen-insensitive prostatic adenocarcinoma driven by MYC over-expression and Pten loss.

Prostate-specific PTen deletion in mice activates inflammatory microRNA expression pathways in the epithelium early in hyperplasia development

PTen loss is one of the most frequent events in prostate cancer both at the initiation stage and during late stage metastatic development. The mouse model of prostate-specific probasin-mediated Pten deletion leads to prostate intraepithelial neoplasia (PIN) leading to adenocarcinoma. Using this model, we analysed the miR and mRNA transcriptome profile of Pten−/− PIN versus wild type age-matched prostate tissues and analysed the effects of Pten loss on miR expression in the early neoplastic process. At the PIN stage, Pten loss significantly changed the expression of over 20 miRNAs and over 4000 genes. The observed miR expression indicated a strong immunological cohort, which is seen in many human and mouse cancers and is thought to derive from infiltrating B and T immune cells. However, upon in situ hybridisation, these immunologically related miRs did not correlate with immune cell location, and emanated from the prostate epithelium itself and not from the associated immune cells present. Growing Pten−/− prostate cells in culture showed that the overexpressed miRNAs seen in Pten−/− were directly in response to the overactive PI3 kinase pathway and were in part responsible in reducing target gene expression levels. Inhibition of PI3 kinase downstream regulators, or re-introducing wild type PtencDNA reduced miR overexpression resulting in increased miR target gene expression. MiR inhibitors also showed this pattern, and synergised with an mTORC1 inhibitor. Overall, Pten deletion in the prostate epithelium activated a cohort of inflammation-related miRs usually associated with immune responses from B and T cells. These oncomiRs may then accelerate carcinogenesis.

The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

Phosphoinositide-3 (PI-3) kinase signaling has a pervasive role in cancer. One of the key effectors of PI-3 kinase signaling is AKT, a kinase that promotes growth and survival in a variety of cancers. Genetically engineered mouse models of prostate cancer have shown that AKT signaling is sufficient to induce prostatic epithelial neoplasia (PIN), but insufficient for progression to adenocarcinoma. This contrasts with the phenotype of mice with prostate-specific deletion of Pten, where excessive PI-3 kinase signaling induces both PIN and locally invasive carcinoma. We reasoned that additional PI-3 kinase effector kinases promote prostate cancer progression via activities that provide biological complementarity to AKT. We focused on the PKN kinase family members, which undergo activation in response to PI-3 kinase signaling, show expression changes in prostate cancer, and contribute to cell motility pathways in cancer cells. PKN kinase activity was measured by incorporation of 32 P into protein substrates. Phosphorylation of the turn-motif (TM) in PKN proteins by mTOR was analyzed using the TORC2-specific inhibitor torin and a PKN1 phospho-TM-specific antibody. Amino acid substitutions in the TM of PKN were engineered and assayed for effects on kinase activity. Cell motility-related functions and PKN localization was analyzed by depletion approaches and immunofluorescence microscopy, respectively. The contribution of PKN proteins to prostate tumorigenesis was characterized in several mouse models that express PKN transgenes. The requirement for PKN activity in prostate cancer initiated by loss of phosphatase and tensin homolog deleted on chromosome 10 (Pten), and the potential redundancy between PKN isoforms, was analyzed by prostate-specific deletion of Pkn1, Pkn2, and Pten. PKN1 and PKN2 contribute to motility pathways in human prostate cancer cells. PKN1 and PKN2 kinase activity is regulated by TORC2-dependent phosphorylation of the TM, which together with published data indicates that PKN proteins receive multiple PI-3 kinase-dependent inputs. Transgenic expression of active AKT and PKN1 is not sufficient for progression beyond PIN. Moreover, Pkn1 is not required for tumorigenesis initiated by loss of Pten. Triple knockout of Pten, Pkn1, and Pkn2 in mouse prostate results in squamous cell carcinoma, an uncommon but therapy-resistant form of prostate cancer.

Jagged1 upregulation in prostate epithelial cells promotes formation of reactive stroma in the Pten null mouse model for prostate cancer

The role of Notch signaling in prostate cancer has not been defined definitively. Several large scale tissue microarray studies revealed that the expression of some Notch signaling components including the Jagged1 ligand are upregulated in advanced human prostate cancer specimens. Jagged1 expressed by tumor cells may activate Notch signaling in both adjacent tumor cells and cells in tumor microenvironment. However, it remains undetermined whether increased Jagged1 expression reflects a cause for or a consequence of tumor progression in vivo. To address this question, we generated a novel R26-LSL-JAG1 mouse model that enables spatiotemporal Jagged1 expression. Prostate specific upregulation of Jagged1 neither interferes with prostate epithelial homeostasis nor significantly accelerates tumor initiation or progression in the prostate-specific Pten deletion mouse model for prostate cancer. However, Jagged1 upregulation results in increased inflammatory foci in tumors and incidence of intracystic adenocarcinoma. In addition, Jagged1 overexpression upregulates Tgfβ signaling in prostate stromal cells and promotes progression of a reactive stromal microenvironment in the Pten null prostate cancer model. Collectively, Jagged1 overexpression does not significantly accelerate prostate cancer initiation and progression in the context of loss-of-function of Pten, but alters tumor histopathology and microenvironment. Our study also highlights an understudied role of Notch signaling in regulating prostatic stromal homeostasis.

Changes in Susceptibility to Oncolytic Vesicular Stomatitis Virus during Progression of Prostate Cancer.

A major challenge to oncolytic virus therapy is that individual cancers vary in their sensitivity to oncolytic viruses, even when these cancers arise from the same tissue type. Variability in response may arise due to differences in the initial genetic lesions leading to cancer development. Alternatively, susceptibility to viral oncolysis may change during cancer progression. These hypotheses were tested using cells from a transgenic mouse model of prostate cancer infected with vesicular stomatitis virus (VSV). Primary cultures from murine cancers derived from prostate-specific Pten deletion contained a mixture of cells that were susceptible and resistant to VSV. Castration-resistant cancers contained a higher percentage of susceptible cells than cancers from noncastrated mice. These results indicate both susceptible and resistant cells can evolve within the same tumor. The role of Pten deletion was further investigated using clonal populations of murine prostate epithelial (MPE) progenitor cells and tumor-derived Pten(-/-) cells. Deletion of Pten in MPE progenitor cells using a lentivirus vector resulted in cells that responded poorly to interferon and were susceptible to VSV infection. In contrast, tumor-derived Pten(-/-) cells expressed higher levels of the antiviral transcription factor STAT1, activated STAT1 in response to VSV, and were resistant to VSV infection. These results suggest that early in tumor development following Pten deletion, cells are primarily sensitive to VSV, but subsequent evolution in tumors leads to development of cells that are resistant to VSV infection. Further evolution in castration-resistant tumors leads to tumors in which cells are primarily sensitive to VSV. There has been a great deal of progress in the development of replication-competent viruses that kill cancer cells (oncolytic viruses). However, a major problem is that individual cancers vary in their sensitivity to oncolytic viruses, even when these cancers arise from the same tissue type. The experiments presented here were to determine whether both sensitive and resistant cells are present in prostate cancers originating from a single genetic lesion in transgenic mice, prostate-specific deletion of the gene for the tumor suppressor Pten. The results indicate that murine prostate cancers are composed of both cells that are sensitive and cells that are resistant to oncolytic vesicular stomatitis virus (VSV). Furthermore, androgen deprivation led to castration-resistant prostate cancers that were composed primarily of cells that were sensitive to VSV. These results are encouraging for the use of VSV for the treatment of prostate cancers that are resistant to androgen deprivation therapy.

MP31-07 ESTROGEN RECEPTOR α REGULATES PROLIFERATION IN PTEN NULL PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research II1 Apr 2014MP31-07 ESTROGEN RECEPTOR α REGULATES PROLIFERATION IN PTEN NULL PROSTATE CANCER Itsuhiro Takizawa, Mitchell Lawrence, Helen Pearson, John Pedersen, Normand Pouliot, Australian Prostate, Cancer BioResource, Patrick Humbert, Luc Furic, and Gail Risbridger Itsuhiro TakizawaItsuhiro Takizawa More articles by this author , Mitchell LawrenceMitchell Lawrence More articles by this author , Helen PearsonHelen Pearson More articles by this author , John PedersenJohn Pedersen More articles by this author , Normand PouliotNormand Pouliot More articles by this author , Australian ProstateAustralian Prostate More articles by this author , Cancer BioResourceCancer BioResource More articles by this author , Patrick HumbertPatrick Humbert More articles by this author , Luc FuricLuc Furic More articles by this author , and Gail RisbridgerGail Risbridger More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.916AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES High doses of estrogen, in combination with androgens, can initiate prostate cancer. This effect is attributed to the activity of the estrogen receptor α (ERα). Although it is well established that ERα stimulates proliferation via genomic and non-genomic activities in breast and ovarian cancer, relatively little is known about the functions of ERα in prostate cancer cells. We observed that ERα expression is up-regulated in high Gleason Score specimens of human prostate cancer. We also noted that the incidence and severity of tumours in PTEN conditional knockout mice with prostate-specific deletion of PTEN, correlates with the estrogen sensitivity of each lobe of the prostate. Therefore, we hypothesized that this model could be used to study the role of ERα in prostate cancer progression. METHODS Immunohistochemistry and stereology were used to quantify ERα and Ki67 expression in PTEN null mice. To assess the functional role of ERα, a cell line derived from a PTEN null tumour was treated with shRNA or TPSF, a non-competitive ERα antagonist. Rescue experiments with expression constructs for either full length ERα, capable of genomic and non-genomic actions, or membrane-only ERα, only able to trigger rapid non-genomic signalling, were used to determine the mechanism underlying ERα-regulated proliferation. RESULTS There was a dramatic increase in ERα expression in prostate tumours of PTEN null mice compared with normal prostates of control animals. Within the PTEN null prostate, there was a consistent pattern of ERα expression: low in benign glands, moderate in tumours within the dorsal, lateral and ventral lobes, and high in tumours within the anterior prostate. This pattern significantly correlated with the levels of the proliferative marker Ki67. There was also a significant correlation between ERα and Ki67 within individual malignant glands in the anterior prostate. In vitro knockdown of ERα attenuated the proliferation of PTEN null cells as did treatment with TPSF. Loss of ERα reduced the activity of both the PI3K and MAPK pathways and decreased MYC levels. This effect was reversed by re-expressing full-length or membrane-only ERα. CONCLUSIONS Collectively, these results demonstrate that ERα drives the proliferation of prostate cancer cells through classical genomic and rapid non-genomic signalling. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e324-e325 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Itsuhiro Takizawa More articles by this author Mitchell Lawrence More articles by this author Helen Pearson More articles by this author John Pedersen More articles by this author Normand Pouliot More articles by this author Australian Prostate More articles by this author Cancer BioResource More articles by this author Patrick Humbert More articles by this author Luc Furic More articles by this author Gail Risbridger More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

PTEN loss mediated Akt activation promotes prostate tumor growth and metastasis via CXCL12/CXCR4 signaling

IntroductionThe chemokine CXCL12, also known as SDF-1, and its receptor, CXCR4, are overexpressed in prostate cancers and in animal models of prostate-specific PTEN deletion, but their regulation is poorly understood. Loss of the tumor suppressor PTEN (phosphatase and tensin homolog) is frequently observed in cancer, resulting in the deregulation of cell survival, growth, and proliferation. We hypothesize that loss of PTEN and subsequent activation of Akt, frequent occurrences in prostate cancer, regulate the CXCL12/CXCR4 signaling axis in tumor growth and bone metastasis.MethodsMurine prostate epithelial cells from PTEN+/+, PTEN+/−, and PTEN−/− (prostate specific knockdown) mice as well as human prostate cancer cell lines C4-2B, PC3, and DU145 were used in gene expression and invasion studies with Akt inhibition. Additionally, HA-tagged Akt1 was overexpressed in DU145, and tumor growth in subcutaneous and intra-tibia bone metastasis models were analyzed.ResultsLoss of PTEN resulted in increased expression of CXCR4 and CXCL12 and Akt inhibition reversed expression and cellular invasion. These results suggest that loss of PTEN may play a key role in the regulation of this chemokine activity in prostate cancer. Overexpression of Akt1 in DU145 resulted in increased CXCR4 expression, as well as increased proliferation and cell cycle progression. Subcutaneous injection of these cells also resulted in increased tumor growth as compared to neo controls. Akt1 overexpression reversed the osteosclerotic phenotype associated with DU145 cells to an osteolytic phenotype and enhanced intra-osseous tumor growth.ConclusionsThese results suggest the basis for activation of CXCL12 signaling through CXCR4 in prostate cancer driven by the loss of PTEN and subsequent activation of Akt. Akt1-associated CXCL12/CXCR4 signaling promotes tumor growth, suggesting that Akt inhibitors may potentially be employed as anticancer agents to target expansion of PC bone metastases.

Abstract 315: Proteomic profiling of PTEN-deletion mouse prostate and its molecular pharmacodynamic responses to methylseleninic acid .

Abstract Recent genomic and exome sequencing analyses revealed that loss of one allele of tumor suppressor gene Pten (phosphatase and tensin homolog deleted on chromosome 10) occurs frequently in primary prostate tumors, and homozygous deletion of Pten plays a casual role in advanced prostate cancer. Therefore, the prostate-specific PTEN-deletion driven prostate cancer mouse model resembles many features of human prostate cancer including disease progression to metastasis. Due to its high clinical relevance, this model is gaining wider utility for prostate cancer chemoprevention and therapeutic studies. Very recently, our collaborative team found that oral administration of methylseleninic acid (MSeA), a second-generation selenium compound, significantly inhibited PTEN-loss driven prostate tumorigenesis in vivo. In particular, the efficacy was associated with a super-activation of p53-p21 senescence pathway (PTEN-loss induced cellular senescence, PICS). To gain insights into additional molecular targets, we profiled the prostate proteome of PTEN-deletion mice. Whole prostates from PTEN-deletion mice (12-15 weeks of age) and their wild type littermate were obtained from the NCI Mouse Model Repository. Homogenate from 4 prostate pairs (Pten−/− and wild type) was prepared and digested individually and the peptides were labeled with 8-plex iTRAQTM reagents. Labeled peptides were pooled and analyzed by 2D-LC-MS/MS (LTQ Orbitrap Velos, Thermo Scientific). Out of 949 proteins identified, we further analyzed 152, the expressions of which were significantly and concordantly changed in all four pairs of mice. Among them, we detected enrichment of proteins related to cellular senescence (chitinase-3 like protein3 [CH3L3], cathelin-related antimicrobial peptide [Camp]), p53 (S100-A8, sorbitol dehydrogenase [DHSO]) and molecular targets involved in AKT signaling pathway (lipocalin 2, CH3L3), in line with the well-described AKT driven p53-mediated senescence in Pten-deficient prostate cancer mouse model. We also found decreased protein expression of GRP78 (78 kDa glucose-regulated protein), endoplasmin, probasin and TGM4 (transglutaminase4), same as we reported in the TRAMP model. Some of these protein changes (over or under) were predicated by published changes of their mRNA levels in prostates (probasin, TGM4, etc) and/or protein levels in sera (probasin, endoplasmin, GRP78, etc). Moreover, the congruence on some proteins (DHSO, S100-A8, etc) between the mouse model and clinical specimens (e.g. by C. Sawyers) highlights the clinical relevance of the model. The proteomic changes induced by MSeA are being investigated. Citation Format: Jinhui Zhang, Yibin Deng, Junxuan Lü. Proteomic profiling of PTEN-deletion mouse prostate and its molecular pharmacodynamic responses to methylseleninic acid . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 315. doi:10.1158/1538-7445.AM2013-315

Abstract LB-405: Identification of master regulators driving advanced prostate cancer and treatment response through the assembly of mouse and human prostate cancer interactomes

Abstract Identification of master regulators driving advanced prostate cancer and treatment response through the assembly of mouse and human prostate cancer interactomes. Alvaro Aytes1,4, Antonina Mitrofanova2,4, Celine Lefebvre2,4, Carolyn W. Kinkade1,4, Michael M. Shen3,4, Andrea Califano2,4, and Cory Abate-Shen1,4. 1Departments of Urology, Pathology and Cell Biology, 2Biomedical Informatics and Center for Computational Biology and Bioinformatics, 3Medicine and Genetics & Development, 4Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, NY 10032 We have developed new genetically-engineered mouse models (GEM) based on inducible prostate-specific deletion of Pten alone or together with activation of oncogenic Kras. These models recapitulate all stages of prostate tumorigenesis, including metastases. We have used these GEM models to develop unbiased molecular networks of transcriptional and post-translational interactions, namely interactomes. Importantly, interactomes are predicated on the availability of large gene expression profile (GEP) datasets representative of the phenotypic variability and genetic perturbations of prostate cancer. We have generated GEPs using a collection of prostate cancer mouse models from our laboratory, as well as from the mouse modeling community. Each model has been treated with a variety of drugs against members of the AR, MAPk, Akt/mTOR, JAK/STAT, NF-KB signaling pathways and other kinase inhibitors as well as chemotherapeutic agents. In parallel, a human prostate cancer interactome has been generated using publicly available datasets. Interrogation of these interactomes represents a new paradigm for ‘human to mouse to human’ analyses. In particular, we have used these interactomes to identify molecular mechanisms and biomarkers of response to combinatorial targeting of Akt/mTOR and MAPK pathways in our metastatic prostate cancer GEM model of Kras activation and Pten loss-of-function. Molecular profiles from these tumors parallel those of human prostate cancers, having poor prognosis, concomitant activation of Akt/mTOR and MAP kinase signaling and displaying robust phosphorylation (inactivation) of FoxO3a tumor suppressor. This combined therapy profoundly reduces tumor and metastatic burden, improves survival and decreases FoxO3a phosphorylation. Computational analyses show that combination therapy in mice shifts the malignant signature toward that of normal prostate, while cross-species analyses identify a two-gene signature downstream of FoxO3a, including FOXM1 and CENP-F that is expressed in human metastatic tumors and is predictive of clinical outcome. Our findings suggest that targeting the FoxO3a regulatory network may be particularly beneficial for patients who have failed conventional chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-405. doi:1538-7445.AM2012-LB-405

Abstract A46: A new transgenic mouse model of prostate cancer that displays rapid progression from prostate intraepithelial neoplasia to invasive carcinoma.

Abstract Understanding the molecular, biochemical, and cellular mechanisms that underpin prostate cancer progression will reveal new opportunities for targeted therapies. Our NCI-supported Program Project on “Signaling and Progression in Prostate Cancer” is a team of basic and clinician scientists that share the overall goal of defining how prostate cancer cells subvert signal transduction pathways during progression to androgen independence. Projects and cores within our program are developing new model systems intended to recapitulate human disease progression. Core B in the program (Transgenic Models and Animal Imaging) has developed a new transgenic mouse model by combining mutant alleles of genes known to have important roles in human prostate cancer. The model is based on the phosphatase and tensin homolog (Pten) tumor suppressor, which is mutated in approximately 30% of primary prostate cancers. Prostate-specific deletion of Pten in mice has been shown by multiple groups to cause prostatic intraepithelial neoplasia (PIN), which slowly progresses to invasive cancer. This suggests that Pten loss allows the initiation of cancer, but that progression is subject to restraint mechanisms. We hypothesized that a restraint mechanism might depend on signaling via the TGFβ pathway, owing to the prominent role that TGFβ plays in growth control. We show that conditional deletion of Pten and Tgfbr2, the type II receptor for TGFβ, in prostate epithelium results in large, poorly differentiated prostate tumors with 100% penetrance by 12 weeks of age. Mice with Pten and Tgfbr2 deletions acquire invasive adenocarcinoma and bladder obstruction and most die by 15 weeks of age. Androgen depletion by castration does not provide a survival benefit for these mice. Pten loss causes an increase in expression of the cyclin-dependent kinase inhibitor p27, which likely restrains progression in this setting. However, when both Pten and Tgfbr2 are deleted, p27 levels are reduced and cyclin D levels increase. Our results suggest that Tgfbr2 is an important tumor suppressor in prostate cancer and that its loss allows rapid progression when combined with loss of the Pten tumor suppressor. Disruption of the TGFβ-mediated restraint provides a rapid, penetrant, and physiologically relevant preclinical model that will be helpful for analyzing restraint- and progression-related gene expression and signal transduction changes in Pten-dependent tumorigenesis. Citation Format: Glen Bjerke, Chun-Song Yang, Henry Frierson, Bryce Paschal, David Wotton. A new transgenic mouse model of prostate cancer that displays rapid progression from prostate intraepithelial neoplasia to invasive carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A46.

Abstract C48: Regulation of expression of CXCL12 and its receptors CXCR4 and CXCR7 by PTEN in prostate cancer

Abstract Introduction: Expression of the chemokine CXCL12, also known as SDF-1, and its receptors, CXCR4 and CXCR7, are associated with prostate cancer (PC) metastasis. Loss of the tumor suppressor PTEN (phosphatase and tensin homolog) occurs in 30-80% of primary PCs, and up to 50% of PC bone metastases. In mouse models, prostate-specific deletion of PTEN leads to Akt activation and increased expression of CXCR4 and CXCL12. Moreover, the level of PTEN expression is inversely associated with prostate tumorigenesis. Additionally, HIF-1α, a hypoxia-induced transcription factor previously shown to induce expression of CXCR4, is regulated by Akt. Thus, we hypothesize that the PTEN loss-mediated activation of HIF-1α regulates the function of CXCR4 and CXCR7 receptor pathways in PC progression. Methods: Prostate-specific deletion of floxed exon 5 of PTEN was achieved by Cre recombinase expressed under the control of an androgen-responsive probasin promoter. Stable cell lines developed from PTEN+/+, PTEN+/−, and PTEN−/− murine prostate epithelial cells that were isolated from the prostates of corresponding mice at 8 weeks of age, and the PTEN status in these cell lines was confirmed by genotyping and immunoblot analyses. Subsequently, these cells were treated with inhibitors of the PI3K/Akt pathway, and gene expression was determined by real time PCR. Results: We found that loss of PTEN resulted in increased expression of CXCR4, CXCR7, and CXCL12, suggesting that loss of PTEN may play a key role in the regulation of these chemokines in prostate cancer. Additionally, expression of HIF-1α was also upregulated upon PTEN loss. Upon treatment of PTEN−/− cells with inhibitors of the PI3K/Akt pathway, the induction of CXCL12 and CXCR7 was reversed, further demonstrating the role of the PTEN regulated pathway in the expression of these genes. Conclusions: These results suggest a molecular basis for activation of CXCL12 signaling through its receptors in prostate cancer driven by the loss of PTEN through the induction of HIF-1. Citation Format: M. Katie Conley-LaComb, Yong Q. Chen, Hyeong-Reh C. Kim, Michael L. Cher, Sreenivasa R. Chinni. Regulation of expression of CXCL12 and its receptors CXCR4 and CXCR7 by PTEN in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C48.

Abstract A36: Identification of master regulators driving advanced prostate cancer and treatment response through the assembly of mouse and human prostate cancer interactomes

Abstract Subsets of prostate tumors are highly aggressive and progress to a castration-resistant stage, which is metastatic and invariably lethal. Prostate tumors are associated with genetic perturbations such as chromosomal alterations, but rarely with mutational events. Loss of the NKX3.1 gene is an early and prevalent event while deletion of PTEN tumor suppressor, is prevalent in early disease and strongly associated with metastatic tumors. Ras is rarely mutated in prostate tumors but components of the RAS signaling pathway are frequently deregulated in primary tumors and profoundly altered in metastases. Additionally, translocations resulting in constitutive activation of Kras occur in a subset of aggressive prostate cancers. Our laboratory has developed new genetically-engineered mouse models (GEM) based on inducible prostate-specific deletion of Pten alone or together with activation of oncogenic Kras. These models recapitulate all stages of prostate tumorigenesis, including distant metastases with high penetrance. We have exploited these GEM models using reverse-engineering of regulatory programs to develop genome-wide molecular networks of transcriptional and post-translational interactions, namely interactomes. These approaches are unbiased and encompass the entire genome. We have focused on comparing regulatory networks for both mouse and human prostate cancer. Importantly, interactomes are predicated on the availability of large gene expression profile (GEP) datasets representative of the natural phenotypic variability and genetic perturbations of prostate cancer. We have generated GEP using a collection of prostate cancer mouse models from our laboratory, as well as from the mouse modeling community. Each model has been treated with a variety of targeting drugs against members of the androgen-signaling, MAPk, Akt/mTOR, JAK/STAT, NF-KB pathways and other kinase inhibitors as well as chemotherapeutic agents. These perturbations have been instrumental in increasing transcriptome variability within the same genetic background. In parallel, a human prostate cancer interactome has been generated using publicly available datasets. Interrogation of these interactomes represents a new paradigm for ‘human to mouse to human’ analyses. In particular, we have used these interactomes to identify molecular mechanisms and biomarkers of response to combinatorial targeting of Akt/mTOR and MAP kinase pathways in our metastatic prostate cancer GEM model of Kras activation and Pten loss-of-function. Molecular profiles from these tumors parallel those of human prostate cancers having poor prognosis, concomitant activation of Akt/mTOR and MAP kinase signaling and displaying robust phosphorylation (inactivation) of FoxO3a tumor suppressor. This combined therapy profoundly reduces tumor and metastatic burden, improves survival, decreases FoxO3a phosphorylation, and is significantly more effective than standard of care chemotherapy. Computational analyses show that combination therapy in mice shifts the malignant signature toward that of normal prostate, while cross-species analyses identify a two-gene signature downstream of FoxO3a, including FOXM1 and CENP-F that is expressed in human metastatic tumors and is predictive of clinical outcome. Our findings suggest that targeting the FoxO3a regulatory network may be particularly beneficial for patients who have failed conventional chemotherapy. Citation Format: Alvaro Aytes, Antonina Mitrofanova, Celine Lefebvre, Michael M. Shen, Andrea Califano, Cory Abate-Shen. Identification of master regulators driving advanced prostate cancer and treatment response through the assembly of mouse and human prostate cancer interactomes [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A36.

PTEN Regulates PDGF Ligand Switch for β-PDGFR Signaling in Prostate Cancer

Platelet-derived growth factor (PDGF) family members are potent growth factors that regulate cell proliferation, migration, and transformation. Clinical studies have shown that both PDGF receptor β (β-PDGFR) and its ligand PDGF D are up-regulated in primary prostate cancers and bone metastases, whereas PDGF B, a classic ligand for β-PDGFR, is not frequently detected in clinical samples. In this study, we examined the role of the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in the regulation of PDGF expression levels using both a prostate-specific, conditional PTEN-knockout mouse model and mouse prostate epithelial cell lines established from these mice. We found an increase in PDGF D and β-PDGFR expression levels in PTEN-null tumor cells, accompanied by a decrease in PDGF B expression. Among Akt isoforms, increased Akt3 expression was most prominent in mouse PTEN-null cells, and phosphatidylinositol 3-kinase/Akt activity was essential for the maintenance of increased PDGF D and β-PDGFR expression. In vitro deletion of PTEN resulted in a PDGF ligand switch from PDGF B to PDGF D in normal mouse prostate epithelial cells, further demonstrating that PTEN regulates this ligand switch. Similar associations between PTEN status and PDGF isoforms were noted in human prostate cancer cell lines. Taken together, these results suggest a mechanism by which loss of PTEN may promote prostate cancer progression via PDGF D/β-PDGFR signal transduction.

Slow Disease Progression in a C57BL/6 Pten-Deficient Mouse Model of Prostate Cancer

Prostate-specific deletion of Pten in mice has been reported to recapitulate histological progression of human prostate cancer. To improve on this model, we introduced the conditional ROSA26 luciferase reporter allele to monitor prostate cancer progression via bioluminescence imaging and extensively backcrossed mice onto the albino C57BL/6 genetic background to address variability in tumor kinetics and to enhance imaging sensitivity. Bioluminescence signal increased rapidly in Pten(p-/-) mice from 3 to 11 weeks, but was much slower from 11 to 52 weeks. Changes in bioluminescence signal were correlated with epithelial proliferation. Magnetic resonance imaging revealed progressive increases in prostate volume, which were attributed to excessive fluid retention in the anterior prostate and to expansion of the stroma. Development of invasive prostate cancer in 52-week-old Pten(p-/-) mice was rare, indicating that disease progression was slowed relative to that in previous reports. Tumors in these mice exhibited a spontaneous inflammatory phenotype and were rapidly infiltrated by myeloid-derived suppressor cells. Although Pten(p-/-) tumors responded to androgen withdrawal, they failed to exhibit relapsed growth for up to 1 year. Taken together, these data identify a mild prostate cancer phenotype in C57BL/6 prostate-specific Pten-deficient mice, reflecting effects of the C57BL/6 genetic background on cancer progression. This model provides a platform for noninvasive assessment of how genetic and environmental risk factors may affect disease progression.