Human Prostate Cancer Datasets Research Articles

27-hydroxycholesterol and DNA damage repair: implication in prostate cancer.

We previously reported that cholesterol homeostasis in prostate cancer (PC) is regulated by 27-hydroxycholesterol (27HC) and that CYP27A1, the enzyme that converts cholesterol to 27HC, is frequently lost in PCs. We observed that restoring the CYP27A1/27HC axis inhibited PC growth. In this study, we investigated the mechanism of 27HC-mediated anti-PC effects. We employed in vitro models and human transcriptomics data to investigate 27HC mechanism of action in PC. LNCaP (AR+) and DU145 (AR-) cells were treated with 27HC or vehicle. Transcriptome profiling was performed using the Affymetrix GeneChip™ microarray system. Differential expression was determined, and gene set enrichment analysis was done using the GSEA software with hallmark gene sets from MSigDB. Key changes were validated at mRNA and protein levels. Human PC transcriptomes from six datasets were analyzed to determine the correlation between CYP27A1 and DNA repair gene expression signatures. DNA damage was assessed via comet assays. Transcriptome analysis revealed 27HC treatment downregulated Hallmark pathways related to DNA damage repair, decreased expression of FEN1 and RAD51, and induced "BRCAness" by downregulating genes involved in homologous recombination regulation in LNCaP cells. Consistently, we found a correlation between higher CYP27A1 expression (i.e., higher intracellular 27HC) and decreased expression of DNA repair gene signatures in castration-sensitive PC (CSPC) in human PC datasets. However, such correlation was less clear in metastatic castration-resistant PC (mCRPC). 27HC increased expression of DNA damage repair markers in PC cells, notably in AR+ cells, but no consistent effects in AR- cells and decreased expression in non-neoplastic prostate epithelial cells. While testing the clinical implications of this, we noted that 27HC treatment increased DNA damage in LNCaP cells via comet assays. Effects were reversible by adding back cholesterol, but not androgens. Finally, in combination with olaparib, a PARP inhibitor, we showed additive DNA damage effects. These results suggest 27HC induces "BRCAness", a functional state thought to increase sensitivity to PARP inhibitors, and leads to increased DNA damage, especially in CSPC. Given the emerging appreciation that defective DNA damage repair can drive PC growth, future studies are needed to test whether 27HC creates a synthetic lethality to PARP inhibitors and DNA damaging agents in CSPC.

Abstract A060: Genetic alterations induce distinct histone post-translational modifications during the transition to castration-resistant prostate cancer

Abstract Background: Epigenetic alterations can impact gene expression and have been implicated in tumorigenesis. One major process is the post-translational modification (PTM) of histone proteins governed by histone-modifying enzyme (HME) activity. There is growing evidence that the dysregulation of HMEs plays an essential role in cancer initiation and progression. In this study, we hypothesized that alterations in histone PTMs and their associated enzymes drive the development of castration-resistant prostate cancer (CRPC) from hormone-sensitive prostate cancer (HSPC). To address this, we performed an unbiased screen of histone PTMs in a series of prostate cancer (PC) PDXs before and after the transition to CRPC. Methods: Histone PTM levels were analyzed in 10 paired HSPC and CRPC PDXs using mass spectrometry. Histone PTM patterns were subcategorized based on common genetic alterations found in PC. RNA-sequencing data for the PDXs and publicly available human PC datasets were used to further characterize HMEs. Co-expression of CHD1, NSD2, and other HMEs was examined in human PC tissue microarrays (TMAs). CHD1 KO clones were generated using CRISPR and levels of histone PTMs and HMEs were assessed and quantified. Gene set enrichment analysis (GSEA) was performed to determine the pathways altered with CHD1 in CRPC PDXs. Results: A comparison of all PDX tumors revealed increased H3K14 and H4 acetylation in CRPC tumors relative to paired HSPC derivatives. We also observed distinct histone PTM patterns associated with common genetic alterations. When compared to tumors with CHD1 WT, tumors with CHD1 loss develop reduced histone PTM H3.3K36me2. CHD1 is a chromatin remodeler commonly deleted in ~ 15% of primary PCs. Screening the differential expression and activity of HMEs in these PDXs reveals that HME NSD2 correlates with CHD1. The human CRPC SU2C/PCF dataset shows a strong positive correlation between CHD1 and NSD2 mRNA (r=0.44). Human CRPC TMAs show decreased protein expression of NSD2 and its histone mark H3K36me2 is associated with reduced CHD1 levels. CHD1 KO in DU145 results in a loss of H3.3K36me2 and a 0.5-fold decrease in NSD2 expression. GSEA revealed that a reduction in the DNA repair pathway is associated with CHD1 loss. The CRPC PDXs (n=24) and human dataset SU2C/PCF 2019 showed a strong positive correlation between CHD1, NSD2, and DNA damage repair genes. Conclusions: We have identified for the first time that genetic alterations alter histone marks during the transition to castration-resistant cancer. Reduced histone H3.3K36 methylation is associated with the genetic loss of CHD1 during the transition to CRPC. The expression of NSD2 is altered in the CRPC state in a CHD1 status-dependent manner. The CHD1-NSD2 phenotype may be the target of novel therapeutic drug combinations – the subject of ongoing research. Citation Format: Tanaya A. Purohit, Joseph Gawdzik, Bing Yang, Eric A. Armstrong, Peter W. Lewis, John M. Denu, David F. Jarrard. Genetic alterations induce distinct histone post-translational modifications during the transition to castration-resistant prostate cancer [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 A060.

βArrestin1 regulates glucocorticoid receptor mitogenic signaling in castration-resistant prostate cancer.

Prostate cancer (PC) is the most commonly diagnosed malignancy and the second leading cause of cancer-related deaths in males. The disease is initially treated with methods that inhibit androgen receptor (AR) signal transduction. Laboratory-based and clinical studies have identified alternative pathways that cause the failure of AR signal inhibition and consequent development of castration-resistant prostate cancer (CRPC). Glucocorticoid receptor (GR) signaling is activated in certain PC patients and promotes the emergence of CRPC, althoughby as yet incompletely understood mechanisms. We have previously demonstrated that ubiquitous βarrestin1 (βArr1) expression levels are linked to PC progression. Here, we consider the possibility that βArr1 interacts with and activates GR in model CRPC cells. Bioinformatic analysis of tumor xenograft and human PC datasets was used to correlate the expression of βArr1 and GR. Western blot, immunohistochemistryand immunofluorescence microscopy, and subcellular fractionation were used to determine protein expression level and localization. Immunoprecipitation was applied to detect protein-protein interactions. RNA expression levels were determined using quantitative reverse transcription-polymerase chain reaction. Prostate sphere analysis was used to assess the rate of growth and invasion. The xenograft tumor implantation method was used to determine the tumor growth rate, local invasion, and metastasis. Elevated expression of βArr1 positively correlated with increased GR expression and function in CRPC xenograft and in human PC patients. βArr1 is expressed in the cell cytosol and nucleus, and it formed a complex with GR in the nucleus and not cytosol. Depletion of βArr1 in AR-null CRPC cells inhibited GR function and CRPC growth and invasion in both in vitro and in vivo settings. βArr1 binds GR that initiates mitogenic signaling cascades involved in the progression of PC to CRPC. The targeting of the βArr1-GR axis may provide a new opportunity to better manage the CRPC disease.

Anti-CCR4 treatment depletes regulatory T cells and leads to clinical activity in a canine model of advanced prostate cancer

BackgroundTargeting regulatory T cell (Treg) infiltration is an emerging strategy for cancer immunotherapy. However, its efficacy in advanced prostate cancer remains unclear. Here, we showed the therapeutic efficacy of anti-Treg...

Targeting AP-1 transcription factors by CRISPR in the prostate.

Prostate cancer is the second most diagnosed cancer in men. It is a slow progressing cancer, but when the disease reaches an advanced stage, treatment options are limited. Sequencing analyses of cancer samples have identified genes that can potentially drive disease progression. We implemented the CRISPR/Cas9 technology to simultaneously manipulate multiple genes in the murine prostate and thus to functionally test putative cancer driver genes in vivo. The activating protein-1 (AP-1) transcription factor is associated with many different cancer types, with the proto-oncogenes JUN and FOS being the two most intensely studied subunits. We analyzed expression of FOS and JUNB in human prostate cancer datasets and observed decreased expression in advanced stages. By applying CRISPR/Cas9 technology, the role of these two transcription factors in prostate cancer progression was functionally tested. Our data revealed that loss of either JunB or Fos in the context of Pten loss drives prostate cancer progression to invasive disease. Furthermore, loss of Fos increases Jun expression, and CRISPR inactivation of Jun in this context decreases cell proliferation. Overall, these in vivo studies reveal that JunB and Fos exhibit a tumor suppressor function by repressing invasive disease, whereas Jun is oncogenic and increases cell proliferation. This demonstrates that AP-1 factors are implicated in prostate cancer progression at different stages and display a dual function as tumor suppressor and as an oncogene in cancer progression.

Activated ALK Cooperates with N-Myc via Wnt/β-Catenin Signaling to Induce Neuroendocrine Prostate Cancer.

Neuroendocrine prostate cancer (NEPC) is an aggressive subtype of prostate cancer with poor prognosis, and there is a critical need for novel therapeutic approaches. NEPC is associated with molecular perturbation of several pathways, including amplification of MYCN. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase involved in the pathogenesis of neuroblastoma and other malignancies where it cooperates with N-Myc. We previously identified the first case of ALK F1174C-activating mutation in a patient with de novo NEPC who responded to the ALK inhibitor, alectinib. Here, we show that coactivation of ALK and N-Myc (ALK F1174C/N-Myc) is sufficient to transform mouse prostate basal stem cells into aggressive prostate cancer with neuroendocrine differentiation in a tissue recombination model. A novel gene signature from the ALK F1174C/N-Myc tumors was associated with poor outcome in multiple human prostate cancer datasets. ALK F1174C and ALK F1174C/N-Myc tumors displayed activation of the Wnt/β-catenin signaling pathway. Chemical and genetic ALK inhibition suppressed Wnt/β-catenin signaling and tumor growth in vitro in NEPC and neuroblastoma cells. ALK inhibition cooperated with Wnt inhibition to suppress NEPC and neuroblastoma proliferation in vitro and tumor growth and metastasis in vivo. These findings point to a role for ALK signaling in NEPC and the potential of cotargeting the ALK and Wnt/β-catenin pathways in ALK-driven tumors. Activated ALK and N-Myc are well known drivers in neuroblastoma development, suggesting potential similarities and opportunities to elucidate mechanisms and therapeutic targets in NEPC and vice versa. SIGNIFICANCE: These findings demonstrate that coactivation of ALK and N-Myc induces NEPC by stimulating the Wnt/β-catenin pathway, which can be targeted therapeutically.

PD59-02 HIGH DIMENSIONAL PROFILING OF THE IMMUNE MICROENVIRONMENT IN PROSTATE CANCER REVEALED NOVEL TUMOR-DRIVEN ALTERATIONS IN INFILTRATING SUBSETS

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology III (PD59)1 Apr 2020PD59-02 HIGH DIMENSIONAL PROFILING OF THE IMMUNE MICROENVIRONMENT IN PROSTATE CANCER REVEALED NOVEL TUMOR-DRIVEN ALTERATIONS IN INFILTRATING SUBSETS Massimo Lazzeri*, Michela Masetti, Federica Portale, Roberta Carriero, Bianca Partini, Andrea Ponzetta, Sebastien Jaillon, Piergiuseppe Colombo, Grazia Elefante, Alberto Saita, Paolo Casale, Clelia Peano, Paolo Kunderfranco, Javier Cibella, Giorgio Guazzoni, and Diletta Di Mitri Massimo Lazzeri*Massimo Lazzeri* More articles by this author , Michela MasettiMichela Masetti More articles by this author , Federica PortaleFederica Portale More articles by this author , Roberta CarrieroRoberta Carriero More articles by this author , Bianca PartiniBianca Partini More articles by this author , Andrea PonzettaAndrea Ponzetta More articles by this author , Sebastien JaillonSebastien Jaillon More articles by this author , Piergiuseppe ColomboPiergiuseppe Colombo More articles by this author , Grazia ElefanteGrazia Elefante More articles by this author , Alberto SaitaAlberto Saita More articles by this author , Paolo CasalePaolo Casale More articles by this author , Clelia PeanoClelia Peano More articles by this author , Paolo KunderfrancoPaolo Kunderfranco More articles by this author , Javier CibellaJavier Cibella More articles by this author , Giorgio GuazzoniGiorgio Guazzoni More articles by this author , and Diletta Di MitriDiletta Di Mitri More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000969.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Prostate adenocarcinoma is one of the most common cancers in the male world population and the incidence of this type of tumor has undergone a significant increase in the last decades. In recent years, research in this field has led to a greater awareness of the clinical and molecular characteristics of this tumor type. As a result, new therapeutic options have emerged to date, mainly aimed at manipulating the immune system. Here we investigated the profile of the immune composition in PCa by single cell analysis. The primary endpoint was to obtain a comprehensive profiling of the immune composition in PCa patients. Secondary endpoint was the identification of tumor-driven immune changes in the immune microenvironment that may be used in the diagnosis and prediction of progression of prostate cancer. METHODS: We conduct a single cell immune profiling strategy based on single cell RNA sequencing (scRNAseq) and multiparametric flow cytometry (FACS) in order to dissect the immune landscape of prostate cancer, both at tumor site and periphery. CD45+ immune cells infiltrating tumoral and adjacent tissues were isolated from patients with PCa and analyzed by single cell sequencing. In addition both peripheral blood and fresh tumoral tissue were collected and analyzed by FACS. RESULTS: For the scRNAseq the study included 3 patients with clinically significant prostate cancer who underwent prostatectomy (GS 4+5). Flow cytometry analysis was then extended to 45 patients bearing prostate cancer at different progression stage. Our analysis revealed a significant modulation in the abundance of several immune subsets infiltrating the tumoral tissue when compared to the non-tumoral. Furthermore we identified a subset of tumor infiltrating macrophages showing an altered gene expression profile when compared with macrophages infiltrating the non-tumoral tissue. Importantly we derived a genetic signature from this cluster of TAMs that resulted to be associated with cancer progression in human prostate cancer datasets. CONCLUSIONS: The results of our study provided a comprehensive analysis of the immune landscape in prostate cancer patients and identified tumor-driven immune changes that may be useful to develop new therapeutic strategies and for risk-stratification and decision-making process of patients. Source of Funding: AIRC foundation © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e1205-e1205 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Massimo Lazzeri* More articles by this author Michela Masetti More articles by this author Federica Portale More articles by this author Roberta Carriero More articles by this author Bianca Partini More articles by this author Andrea Ponzetta More articles by this author Sebastien Jaillon More articles by this author Piergiuseppe Colombo More articles by this author Grazia Elefante More articles by this author Alberto Saita More articles by this author Paolo Casale More articles by this author Clelia Peano More articles by this author Paolo Kunderfranco More articles by this author Javier Cibella More articles by this author Giorgio Guazzoni More articles by this author Diletta Di Mitri More articles by this author Expand All Advertisement PDF downloadLoading ...

Sulfiredoxin as a Potential Therapeutic Target for Advanced and Metastatic Prostate Cancer.

The incidence of prostate cancer (PCa) is increasing, and it is currently the second most frequent cause of death by cancer in men. Despite advancements in cancer therapies, new therapeutic approaches are still needed for treatment-refractory advanced metastatic PCa. Cross-species analysis presents a robust strategy for the discovery of new potential therapeutic targets. This strategy involves the integration of genomic data from genetically engineered mouse models (GEMMs) and human PCa datasets. Considering the role of antioxidant pathways in tumor initiation and progression, we searched oxidative stress-related genes for a potential therapeutic target for PCa. First, we analyzed RNA-sequencing data from Pb-Cre4; Ptenf/f mice and discovered an increase in sulfiredoxin (Srxn1) mRNA expression in high-grade prostatic intraepithelial neoplasia (PIN), well-differentiated adenocarcinoma (medium-stage tumors), and poor-differentiated adenocarcinoma (advanced-stage prostate tumors). The increase of SRXN1 protein expression was confirmed by immunohistochemistry in mouse prostate tumor paraffin samples. Analyses of human databases and prostate tissue microarrays demonstrated that SRXN1 is overexpressed in a subset of high-grade prostate tumors and correlates with aggressive PCa with worse prognosis and decreased survival. Analyses in vitro showed that SRXN1 expression is also higher in most PCa cell lines compared to normal cell lines. Furthermore, siRNA-mediated downregulation of SRXN1 led to decreased viability of PCa cells LNCaP. In conclusion, we identified the antioxidant enzyme SRXN1 as a potential therapeutic target for PCa. Our results suggest that the use of specific SRXN1 inhibitors may be an effective strategy for the adjuvant treatment of castration-resistant PCa with SRXN1 overexpression.

Multifocal Signal Modulation Therapy by Celecoxib: A Strategy for Managing Castration-Resistant Prostate Cancer.

Background: Prostate cancer (PCa) is a significant health concern throughout the world. Standard therapy for advanced disease consists of anti-androgens, however, almost all prostate tumors become castration resistant (CRPC). Progression from androgen-sensitive PCa to CRPC is promoted by inflammatory signaling through cyclooxygenase-2 (COX-2) expression and ErbB family receptors/AKT activation, compensating androgen receptor inactivity. Methods: Making use of CRPC cell lines, we investigated the effects of the anti-inflammatory drug celecoxib. Biochemical data obtained using immunoblotting, enzyme-linked immunosorbent assay (ELISA), invasion, and xenografts were further integrated by bioinformatic analyses. Results: Celecoxib reduced cell growth and induced apoptosis through AKT blockade, cleavage of poly (ADP-ribose) polymerase-1 (PARP-1), and proteasomal degradation of the anti-apoptotic protein Mcl-1. Epidermal growth factor receptor (EGFR), ErbB2, and ErbB3 degradation, and heterogeneous nuclear ribonucleoprotein K (hnRNP K) downregulation, further amplified the inhibition of androgen signaling. Celecoxib reduced the invasive phenotype of CRPC cells by modulating NF-κB activity and reduced tumor growth in mice xenografts when administered in association with the anti-EGFR receptor antibody cetuximab. Bioinformatic analyses on human prostate cancer datasets support the relevance of these pathways in PCa progression. Conclusions: Signaling nodes at the intersection of pathways implicated in PCa progression are simultaneously modulated by celecoxib treatment. In combination therapies with cetuximab, celecoxib could represent a novel therapeutic strategy to curb signal transduction during CRPC progression.

Abstract 3017: Role of long noncoding RNA H19 in driving enzalutamide resistant neuroendocrine prostate cancer

Abstract INTRODUCTION: Approximately one-fourth of the prostate cancer (PCa) patients relapsing androgen deprivation therapy (ADT) have been shown to develop neuroendocrine prostate cancer (NEPC). One of the primary mechanisms attributed to this resistance is the lineage switching of epithelial cells to neuroendocrine (NE) phenotype making them independent of androgen receptor (AR) pathways for survival. Molecular basis essential to this lineage switch requires further elucidation. Thus, we aim to understand key mechanistic pathways that drive the development and maintenance of NEPC, and to investigate if the inhibition of pathway can re-sensitize to ADT in NEPC. METHODS: Bioinformatics analysis was performed with existing databases to compare prostate adenocarcinoma (PCA) vs NEPC. Molecular features of NEPC have been shown to depict RB1 loss in 70-90% and TP53 loss in 56-67% of the cases. Thus, we established prostate organoid cultures from P53fl/fl/Rbfl/fl mouse and infected them with Cre-lentivirus for combined loss of TP53/Rb1. Various PCA and NEPC cell lines and patient derived organoids were used to study the role of the long non-coding RNA H19 in NEPC. Organoid growth were assessed with Incucyte and invasive capacity was measured by Matrigel invasion assay. RESULTS: Bioinformatic analysis of exisiting human PCa data sets demonstrate that H19 is one of the most highly expressed genes in NEPC and parallels the expression of NE markers. Indeed, we confirmed that H19 expression was high in various NEPC cell lines and NE patient derived organoids. H19 was also found to be markedly induced in mouse prostate organoids and LNCaP cells after TP53/Rb1 loss. Importantly, overexpression of H19 induced NE genes while suppressing the genes involved in AR signaling in LNCaP and primary PCa patient derived organoids and inversely, knockdown (KD) of H19 in LNCaP and mouse prostate organoids with TP53/Rb1 loss reduced the expression of NE gene. Interestingly, the KD of H19 in these cells re-induced the luminal phenotype, consequently restoring their sensitivity to ADT. These findings strongly implicate H19 in driving the NE lineage switch. The KD of H19 in these organoids led to a regression of tumor growth and invasiveness, suggesting H19 is required in malignant states. H19 KD decreases stem cell genes (SOX2, OCT4) via methylation of their promoters, and levels of EZH2, which methylates 'Lys-9' (H3K9me) and 'Lys-27' (H3K27me) of histone H3 and represses transcription of the target genes. H3K27me3 levels, which are high in NEPC, was reduced in the H19 KD, suggesting H19 works in an epigenetic regulation to induce NEPC phenotype. CONCLUSION: These data highlight H19 as a novel regulator in controlling the lineage switch to NEPC, tumor growth, and the sensitivity to hormone blockade, suggesting that H19 regulation will be important for developing treatments specifically aimed for this highly aggressive form of prostate cancer. Citation Format: Neha Singh, Virginie Olive, Ritu Pandey, Jin Song, Jeremiah Bearrs, Sathish K. Padi, Koichi Okumura, Andrew S. Kraft. Role of long noncoding RNA H19 in driving enzalutamide resistant neuroendocrine prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3017.

Abstract 4486: Androgen-deprivation therapy promotes immune suppression in a murine model of prostate cancer

Abstract Most prostate cancer (PCa) deaths are due to castration resistant PCa (CRPC), following failure of androgen deprivation therapy (ADT). ADT is the standard of care for patients with advanced PCa but nearly universal progression to CRPC occurs 2-3 years after ADT is initiated. Immunotherapy with checkpoint inhibitors has not been effective in most prostate cancers, perhaps because such cancers lack functional CD8 T-cells. This may be caused by infiltration of myeloid cell populations into the tumor immune cell microenvironment (TIME). Recently, we found that in a PTEN-deficient mouse PCa model, castration induces an immunosuppressive state within the tumor that is concurrent with tumor recurrence. The response to castration/ADT is tri-phasic: a pro-apoptotic regression phase when tumor shrinks, followed by selection for a residual population of resistant tumor cells and finally recurrent growth as CRPC. Using PCa cell lines to model the first two phases of the response to ADT, we have shown that ADT induces apoptosis, thereby enriching for an ADT-resistant stem/progenitor population that we propose is the in vivo source of TNF. Mechanistically, in our model system the response to ADT is driven by the soluble mediators TNF and CCL2, which facilitate communication within the TIME. Specifically, a TNF-CCL2-CCR2 paracrine loop is induced between prostate cancer cells and non-tumor cells in the microenvironment: TNF produced by tumor cells acts on myofibroblasts to induce CCL2 production, which in turn recruits CCR2+ tumor-associated macrophages (TAMs). To investigate the ADT response within the TIME in an in vivo model of prostate cancer, we employed a prostate-specific PTEN-deficient mouse model (PbCre4 x PTENf/f). Castration caused the tumors to regress, consistent with initial phase of the response that is seen in the human disease. At late times post-castration (5-6 weeks), corresponding to the selection phase, we observed a coordinate increase in the stem/progenitor tumor cell population, as well as TNF and CCL2, within the TIME. Immunohistochemical staining of tumors 5 weeks post-castration revealed an increase in TAMs, and a decrease in CD8 T cells, consistent with an immuno-suppressive or immuno-evasive state. This phenotype was reversed by a soluble receptor that binds TNF (etanercept). Thus, following ADT, TNF derived from an ADT-resistant stem/progenitor epithelial tumor cell population promotes an immunosuppressive state via CCL2 in the TIME. Analysis of public human PCa data sets show the transcripts for TNF, as well as gene signatures for stem/progenitor tumor cells and M2 TAMs are increased in CRPC, consistent with our hypothesis that ADT drives the development of myeloid immuno-suppressive state via a TNF-CCL2-CCR2 axis. Our results set the stage for the future development of immunotherapies that could improve the efficacy of ADT. Citation Format: John J. Krolewski, Kai Sha, Kent L. Nastiuk. Androgen-deprivation therapy promotes immune suppression in a murine model of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4486.

Abstract A084: Towards combining androgen deprivation and immunotherapy to prevent progression to castration-resistant prostate cancer

Abstract Most prostate cancer (PCa) deaths are due to castration-resistant PCa (CRPC), following failure of androgen-deprivation therapy (ADT). ADT is the standard of care for patients with advanced PCa. However, nearly universal progression to castration-resistant prostate cancer (CRPC) occurs 2-3 years after ADT is initiated. Although there have been recent improvements in the treatment of CRPC, even the most promising therapies are still not curative. One approach to this problem is to improve the initial treatment of advanced prostate cancers, by combining complementary therapies with ADT, to prevent progression of such advanced cancers to CRPC. Immunotherapy with checkpoint inhibitors (CPIs) has not been effective in prostate cancers, perhaps because such cancers are “cold” (lacking cytolytic CD8 T-cells). Some cold tumors may be caused by infiltration of myeloid cell populations (tumor associated macrophages and myeloid-derived suppressor cells) into the tumor immune cell microenvironment (TIME). Recently, we found that in a PTEN-deficient mouse PCa model, castration induces an immunosuppressive state within the tumor that is concurrent with tumor recurrence. The response to castration/ADT is tri-phasic: a pro-apoptotic regression phase when tumor shrinks, followed by selection for a residual population of resistant tumor cells and finally recurrent growth as CRPC. Using PCa cell lines to model the first two phases of the response to ADT, we have shown that ADT induces apoptosis, thereby enriching for an ADT-resistant stem/progenitor population that we propose is the in vivo source of TNF. Mechanistically, in our model system the response to ADT is driven by the soluble mediators TNF and CCL2, which facilitate communication within the TIME. Specifically, a TNF-CCL2-CCR2 paracrine loop is induced between prostate cancer cells and non-tumor cells in the microenvironment: TNF produced by tumor cells acts on myofibroblasts to induce CCL2 production, which in turn recruits CCR2+ tumor-associated macrophages (TAMs). To investigate the ADT response within the TIME in an in vivo model of prostate cancer, we employed a prostate-specific PTEN-deficient mouse model (PbCre4 x PTENf/f). Castration caused the tumors to regress, consistent with initial phase of the response that is seen in the human disease. At late times post-castration (5-6 weeks), corresponding to the selection phase, we observed a coordinate increase in the stem/progenitor tumor cell population, as well as TNF and CCL2, within the TIME. Immunohistochemical staining of tumors 5 weeks post-castration revealed an increase in TAMs, and a decrease in CD8 T-cells, consistent with an immunosuppressive or immunoevasive state. This phenotype was reversed by a soluble receptor that binds TNF (etanercept). We also observed increased myeloid-derived suppressor cells (MDSC). Thus, following ADT, TNF derived from an ADT-resistant stem/progenitor epithelial tumor cell population promotes an immunosuppressive state via CCL2 in the TIME. Analysis of public human PCa data sets shows TNF and stem/progenitor marker expression are both increased in CRPC, consistent with our hypothesis that ADT drives the development of an immunosuppressive state via a TNF-CCL2-CCR2 axis. Our results set the stage for the future development of immunotherapies that could improve the efficacy of ADT. Citation Format: John J. Krolewski, Kai Sha, Michalis Mastri, Dean Tang, Kevin Eng, Kent L. Nastiuk. Towards combining androgen deprivation and immunotherapy to prevent progression to castration-resistant prostate cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A084.

Modifier locus mapping of a transgenic F2 mouse population identifies CCDC115 as a novel aggressive prostate cancer modifier gene in humans

BackgroundIt is well known that development of prostate cancer (PC) can be attributed to somatic mutations of the genome, acquired within proto-oncogenes or tumor-suppressor genes. What is less well understood is how germline variation contributes to disease aggressiveness in PC patients. To map germline modifiers of aggressive neuroendocrine PC, we generated a genetically diverse F2 intercross population using the transgenic TRAMP mouse model and the wild-derived WSB/EiJ (WSB) strain. The relevance of germline modifiers of aggressive PC identified in these mice was extensively correlated in human PC datasets and functionally validated in cell lines.ResultsAggressive PC traits were quantified in a population of 30 week old (TRAMP x WSB) F2 mice (n = 307). Correlation of germline genotype with aggressive disease phenotype revealed seven modifier loci that were significantly associated with aggressive disease. RNA-seq were analyzed using cis-eQTL and trait correlation analyses to identify candidate genes within each of these loci. Analysis of 92 (TRAMP x WSB) F2 prostates revealed 25 candidate genes that harbored both a significant cis-eQTL and mRNA expression correlations with an aggressive PC trait. We further delineated these candidate genes based on their clinical relevance, by interrogating human PC GWAS and PC tumor gene expression datasets. We identified four genes (CCDC115, DNAJC10, RNF149, and STYXL1), which encompassed all of the following characteristics: 1) one or more germline variants associated with aggressive PC traits; 2) differential mRNA levels associated with aggressive PC traits; and 3) differential mRNA expression between normal and tumor tissue. Functional validation studies of these four genes using the human LNCaP prostate adenocarcinoma cell line revealed ectopic overexpression of CCDC115 can significantly impede cell growth in vitro and tumor growth in vivo. Furthermore, CCDC115 human prostate tumor expression was associated with better survival outcomes.ConclusionWe have demonstrated how modifier locus mapping in mouse models of PC, coupled with in silico analyses of human PC datasets, can reveal novel germline modifier genes of aggressive PC. We have also characterized CCDC115 as being associated with less aggressive PC in humans, placing it as a potential prognostic marker of aggressive PC.

Abstract 2896: STAT1 dependent interferon-related DNA damage resistance signature (IRDS) as a survival mechanism in castrate resistant prostate cancer (CRPC)

Abstract Using the organoid culture system and aggressive Pten/Tp53-null mouse model (GEMM), we have previously shown that the prostate cancer cell population harbors two classes of self-renewing luminal progenitors which are resistant to in vivo castration and to androgen receptor (AR) inhibitors (enzalutamide) ex vivo. Understanding signaling pathways governing intrinsic survival/self-renewal ability of luminal progenitors in castrate conditions can highlight pathways that play a role in acquired drug resistance. To identify mechanisms of castration resistance in luminal progenitors, we performed RNAseq analysis of luminal progenitor organoids derived from wild-type(WT) and Pten/Tp53-null mice (intact and castrated (two weeks), n=5; each). Gene enrichment analysis identified key signaling pathways altered in luminal tumor organoids (AR signaling, lipid metabolism, protein secretion, inflammation etc.) that have also been described for FACS-purified human prostate luminal (CD49flo) fraction. Interestingly, we found no difference in transcriptional profiles of intact and castrated tumor organoids, suggesting intrinsic survival ability of luminal progenitors upon castration. Of note, we observed most significant enrichment of STAT1-dependent IRDS in luminal progenitor tumor organoids relative to wild type luminal organoids. IRDS comprises of a subset of STAT1-driven genes that have been previously associated with survival of cancer cells and with breast cancer therapy resistance. Our analysis of human prostate cancer datasets revealed IRDS as a prognostic marker for progression in the TCGA primary prostate cancer cohort (p<0.05). Further, high IRDS-expressing CRPC samples (SU2C dataset) were enriched for low AR signaling (r= -0.33, p< 0.05). CRPC patients in IRDS-hi cohort showed enrichment for cancer stem cell phenotype and for genes associated with drug resistance, Consistent with the bioinformatics analysis real time PCR, immunofluorescence and western blot analysis of ex-vivo organoid cultures of castration-resistant Pten/Tp53-null tumor organoids showed higher protein expression of STAT1 and IRDS genes in luminal tumor organoids relative to luminal WT organoids. In vivo, castrated prostate tumors showed higher STAT1 levels than the intact tumors. Treatment with enzalutamide of luminal tumor organoids resulted in time dependent increase in STAT1 expression. STAT1 KD in tumor organoids decreased number of progeny organoids in subsequent generations suggesting either a direct or indirect effect upon self-renewal. Overall, our initial findings suggest STAT1 dependent signaling as a potential mechanism of androgen-independent survival in prostate cancer. Citation Format: Supreet Agarwal, Kerry McGowen, Keith Jansson, Mike Beshiri, Fathi Elloumi, Maggie Cam, Kathy Kelly. STAT1 dependent interferon-related DNA damage resistance signature (IRDS) as a survival mechanism in castrate resistant prostate cancer (CRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2896. doi:10.1158/1538-7445.AM2017-2896

Mining Human Prostate Cancer Datasets: The “camcAPP” Shiny App

Core CRUK funding: MD, AGL, ADL. Academy of Medical Sciences Clinical Lecturer Starter Grant SGCL11 (prinicipal funder of this work): ADL.

Notch promotes tumor metastasis in a prostate-specific Pten-null mouse model.

Although Notch signaling is deregulated in prostate cancer, the role of this pathway in disease development and progression is not fully understood. Here, we analyzed 2 human prostate cancer data sets and found that higher Notch signaling correlates with increased metastatic potential and worse disease survival rates. We used the Pten-null mouse prostate cancer model to investigate the function of Notch signaling in the initiation and progression of prostate cancer. Disruption of the transcription factor RBPJ in Pten-null mice revealed that endogenous canonical Notch signaling is not required for disease initiation and progression. However, augmentation of Notch activity in this model promoted both proliferation and apoptosis of prostate epithelial cells, which collectively reduced the primary tumor burden. The increase in cellular apoptosis was linked to DNA damage-induced p53 activation. Despite a reduced primary tumor burden, Notch activation in Pten-null mice promoted epithelial-mesenchymal transition and FOXC2-dependent tumor metastases but did not confer resistance to androgen deprivation. Notch activation also resulted in transformation of seminal vesicle epithelial cells in Pten-null mice. Our study highlights a multifaceted role for Notch signaling in distinct aspects of prostate cancer biology and supports Notch as a potential therapeutic target for metastatic prostate cancer.

Abstract A36: Notch signaling promotes tumor metastasis in the Pten null mouse model for prostate cancer

Abstract The role of Notch signaling in prostate cancer remains inconclusive. We show that a higher Notch signature score is significantly correlated with higher disease grade, metastatic potential and higher probability of lethal outcome in two published human prostate cancer datasets. We employ genetic approaches to investigate the role of Notch signaling in initiation and progression of prostate cancer in a Pten null mouse model. Notch signaling is dispensable for disease initiation and progression in this model. Elevated Notch activity in the Pten null model promotes both epithelial proliferation and apoptosis, which collectively results in a reduction of primary tumor burdens. Despite reduced primary tumor burdens, Notch activation drives epithelial-mesenchymal-transition and promotes distal tumor metastases at full penetrance. Our study reveals distinct impacts of Notch signaling on cancer cell proliferation, survival and metastasis in the context of Pten loss, and highlights a multifaceted and pleiotropic role of Notch in regulating the different aspects of prostate cancer cell biology. Citation Format: Oh-Joon Kwon, Li Zhang, Wang Jianghua, Chad Creighton, Michael Ittmann, Li Xin. Notch signaling promotes tumor metastasis in the Pten null mouse model for prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A36.

Cosuppression of Sprouty and Sprouty-related negative regulators of FGF signalling in prostate cancer: a working hypothesis.

Deregulation of FGF receptor tyrosine kinase (RTK) signalling is common in prostate cancer. Normally, to moderate RTK signalling, induction of Sprouty (SPRY) and Sprouty-related (SPRED) antagonists occurs. Whilst decreased SPRY and SPRED has been described in some cancers, their role in prostate cancer is poorly understood. Therefore, we hypothesise that due to the need for tight regulation of RTK signalling, SPRY and SPRED negative regulators provide a degree of redundancy which ensures that a suppression of one or more family member does not lead to disease. Contrary to this, our analyses of prostates from 24-week-old Spry1- or Spry2-deficientmice, either hemizygous (+/−) or homozygous (−/−) for the null allele, revealed a significantly greater incidence of PIN compared to wild-type littermates. We further investigated redundancy of negative regulators in the clinical setting in a preliminary analysis of Gene Expression Omnibus and Oncomine human prostate cancer datasets. Consistent with our hypothesis, in two datasets analysed a significant cosuppression of SPRYs and SPREDs is evident. These findings demonstrate the importance of negative regulators of receptor tyrosine signalling, such as Spry, in the clinical setting, and highlight their importance for future pharmacopeia.

Identifying actionable targets through integrative analyses of GEM model and human prostate cancer genomic profiling.

Copy-number alterations (CNA) are among the most common molecular events in human prostate cancer genomes and are associated with worse prognosis. Identification of the oncogenic drivers within these CNAs is challenging due to the broad nature of these genomic gains or losses which can include large numbers of genes within a given region. Here, we profiled the genomes of four genetically engineered mouse prostate cancer models that reflect oncogenic events common in human prostate tumors, with the goal of integrating these data with human prostate cancer datasets to identify shared molecular events. Met was amplified in 67% of prostate tumors from Pten p53 prostate conditional null mice and in approximately 30% of metastatic human prostate cancer specimens, often in association with loss of PTEN and TP53. In murine tumors with Met amplification, Met copy-number gain and expression was present in some cells but not others, revealing intratumoral heterogeneity. Forced MET overexpression in non-MET-amplified prostate tumor cells activated PI3K and MAPK signaling and promoted cell proliferation and tumor growth, whereas MET kinase inhibition selectively impaired the growth of tumors with Met amplification. However, the impact of MET inhibitor therapy was compromised by the persistent growth of non-Met-amplified cells within Met-amplified tumors. These findings establish the importance of MET in prostate cancer progression but reveal potential limitations in the clinical use of MET inhibitors in late-stage prostate cancer.

Tumor-Suppressive Activity of Lunatic Fringe in Prostate through Differential Modulation of Notch Receptor Activation

Elevated Notch ligand and receptor expression has been associated with aggressive forms of prostate cancer, suggesting a role for Notch signaling in regulation of prostate tumor initiation and progression. Here, we report a critical role for Lunatic Fringe (Lfng), which encodes an O-fucosylpeptide 3-ß-N-acetylglucosaminyltransferase known to modify epidermal growth factor repeats of Notch receptor proteins, in regulation of prostate epithelial differentiation and proliferation, as well as in prostate tumor suppression. Deletion of Lfng in mice caused altered Notch activation in the prostate, associated with elevated accumulation of Notch1, Notch2, and Notch4 intracellular domains, decreased levels of the putative Notch3 intracellular fragment, as well as increased expression of Hes1, Hes5, and Hey2. Loss of Lfng resulted in expansion of the basal layer, increased proliferation of both luminal and basal cells, and ultimately, prostatic intraepithelial neoplasia. The Lfng-null prostate showed down-regulation of prostatic tumor suppressor gene NKX3.1 and increased androgen receptor expression. Interestingly, expression of LFNG and NKX3.1 were positively correlated in publically available human prostate cancer data sets. Knockdown of LFNG in DU-145 prostate cancer cells led to expansion of CD44+CD24− and CD49f+CD24− stem/progenitor-like cell population associated with enhanced prostatosphere-forming capacity. Taken together, these data revealed a tumor-suppressive role for Lfng in the prostate through differential regulation of Notch signaling.