Murine Prostatic Intraepithelial Neoplasia Research Articles

In vivo CRISPR inactivation of Fos promotes prostate cancer progression by altering the associated AP-1 subunit Jun.

Prostate cancer is a major global health concern with limited treatment options for advanced disease. Its heterogeneity challenges the identification of crucial driver genes implicated in disease progression. Activating protein-1 (AP-1) transcription factor is associated with cancer since the first identification of its subunits, the proto-oncogenes JUN and FOS. Whereas both JUN and FOS, have been implicated in prostate cancer, this study provides the first functional evidence that FOS acts as a tumor suppressor during prostate cancer progression and invasion. Data mining revealed decreased FOS expression in prostate cancer and a further downregulation in metastatic disease, consistent with FOS expression in cell lines derived from different prostate cancer stages. FOS deficiency in prostate cancer cell lines increases cell proliferation and induces oncogenic pathway alterations. Importantly, in vivo CRISPR/Cas9-mediated Fos and Pten double mutation in murine prostate epithelium results in increased proliferation and invasiveness compared to the abrogation of Pten alone. Interestingly, enhanced Jun expression is observed in the murine prostatic intraepithelial neoplasia lacking Fos. CRISPR/Cas9-mediated knockout of Jun combined with Fos and Pten deficiency diminishes the increased proliferation rate in vivo, but not the ability to form invasive disease. Overall, we demonstrate that loss of Fos promotes disease progression from clinical latent prostate cancer to advanced disease through accelerated proliferation and invasiveness, partly through Jun.

Genetic ablation of FASN attenuates the invasive potential of prostate cancer driven by Pten loss.

Loss of the tumor suppressor gene Pten in murine prostate recapitulates human carcinogenesis and causes stromal proliferation surrounding murine prostate intraepithelial neoplasia (mPIN), which is reactive to microinvasion. In turn, invasion has been shown to be regulated in part by de novo fatty acid synthesis in prostate cancer. We therefore investigated the effects of genetic ablation of Fasn on invasive potential in prostate‐specific Pten knockout mice. Combined genetic ablation of Fasn and Pten reduced the weight and volume of all the prostate lobes when compared to single knockouts. The stromal reaction to microinvasion and the cell proliferation that typically occurs in Pten knockout were largely abolished by Fasn knockout. To verify that Fasn knockout indeed results in decreased invasive potential, we show that genetic ablation and pharmacologic inhibition of FASN in prostate cancer cells significantly inhibit cellular motility and invasion. Finally, combined loss of PTEN with FASN overexpression was associated with lethality as assessed in 660 prostate cancer patients with 14.2 years of median follow‐up. Taken together, these findings show that de novo lipogenesis contributes to the aggressive phenotype induced by Pten loss in murine prostate and targeting Fasn may reduce the invasive potential of prostate cancer driven by Pten loss. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

ELL2 Is Required for the Growth and Survival of AR-Negative Prostate Cancer Cells.

BackgroundElongation factor for RNA polymerase II 2 (ELL2) was reported as a putative tumor suppressor in the prostate. ELL2 is frequently down-regulated in prostatic adenocarcinoma specimens, and loss of ELL2 induced murine prostatic intraepithelial neoplasia and enhanced AR-positive prostate cancer cell proliferation. However, the ELL2 gene appears to be amplified in AR-negative neuroendocrine prostate tumors, suggesting a potential oncogenic role for ELL2 in AR-negative prostate cancer cells. In this study, we explored the potential function of ELL2 in PC-3 and DU145, two AR-negative prostate cancer cell lines.Materials and MethodsThe role of ELL2 in PC-3 and DU145 cells was studied using siRNA-mediated ELL2 knockdown. Genes regulated by ELL2 knockdown in PC-3 cells were identified and analyzed using RNA-Seq and bioinformatics. The expression of representative genes was confirmed by Western blot and/or quantitative PCR. Cell growth was determined by BrdU, MTT and colony formation assays. Cell death was analyzed by 7-AAD/Annexin V staining and trypan blue exclusion staining. Cell cycle was determined by PI staining and flow cytometry.ResultsELL2 knockdown inhibited the proliferation of PC-3 and DU145 cells. RNA-Seq analysis showed an enrichment in genes associated with cell death and survival following ELL2 knockdown. The interferon-γ pathway was identified as the top canonical pathway comprising of 55.6% of the genes regulated by ELL2. ELL2 knockdown induced an increase in STAT1 and IRF1 mRNA and an induction of total STAT1 and phosphorylated STAT1 protein. Inhibition of cell proliferation by ELL2 knockdown was partly abrogated by STAT1 knockdown. ELL2 knockdown inhibited colony formation and induced apoptosis in both PC-3 and DU145 cells. Furthermore, knockdown of ELL2 caused S-phase cell cycle arrest, inhibition of CDK2 phosphorylation and cyclin D1 expression, and increased expression of cyclin E.ConclusionELL2 knockdown in PC-3 and DU145 cells induced S-phase cell cycle arrest and profound apoptosis, which was accompanied by the induction of genes associated with cell death and survival pathways. These observations suggest that ELL2 is a potential oncogenic protein required for survival and proliferation in AR-negative prostate cancer cells.

MP16-06 PUTATIVE TUMOR SUPPRESSOR ELL2 IS REQUIRED FOR PROLIFERATION AND SURVIVAL OF AR-NEGATIVE PROSTATE CANCER CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology I (MP16)1 Apr 2020MP16-06 PUTATIVE TUMOR SUPPRESSOR ELL2 IS REQUIRED FOR PROLIFERATION AND SURVIVAL OF AR-NEGATIVE PROSTATE CANCER CELLS Zhi Wang*, Laura E. Pascal, Uma R Chandran, Srilakshmi Chaparala, Shidong Lyu, Ding Hui, Lin Qi, and Zhou Wang Zhi Wang*Zhi Wang* More articles by this author , Laura E. PascalLaura E. Pascal More articles by this author , Uma R ChandranUma R Chandran More articles by this author , Srilakshmi ChaparalaSrilakshmi Chaparala More articles by this author , Shidong LyuShidong Lyu More articles by this author , Ding HuiDing Hui More articles by this author , Lin QiLin Qi More articles by this author , and Zhou WangZhou Wang More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000841.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Elongation Factor For RNA Polymerase II, 2 (ELL2) was reported as a putative tumor suppressor in the prostate cancer. ELL2 is frequently down-regulated in prostatic adenocarcinoma specimens. Loss of ELL2 induced murine prostatic intraepithelial neoplasia and enhanced AR-positive prostate cancer cell proliferation. However, ELL2 gene was amplified or overexpressed in neuroendocrine prostate tumors, suggesting a potential oncogenic role for ELL2 in AR-negative neuroendocrine prostate cancer cells. In this study, we explored potential function of ELL2 in PC3 and DU145, two AR-negative prostate cancer cells with some neuroendocrine phenotype. METHODS: The role of ELL2 in PC3 and DU145 was studied by determining the impact of ELL2 knockdown. Genes regulated by ELL2 knockdown in PC3 cells was identified using RNA-Seq and bioinformatics. Expression of various genes were confirmed by western blot and quantitative real-time PCR. Cell growth were determined by BrdU, MTT and colony formation assays. The cell death was analyzed by Annexin V/7-AAD staining. Cell cycle was determined by flow cytometry. RESULTS: ELL2 knockdown inhibited proliferation in PC3 and DU145 cells, in contrast to the enhancement of proliferation in AR-positive LNCaP and C4-2 prostate cancer cells by ELL2 knockdown. RNA-Seq analysis showed an enrichment in genes associated with cell death and survival following ELL2 knockdown. The interferon-γ pathway was identified as the top canonical pathway comprising of 55.6% of the differentially expressed genes. ELL2 knockdown induced an increase in STAT1 and IRF1 mRNA and an induction of total STAT1 and phosphorylated STAT1 protein. Inhibition of cell proliferation by ELL2 knockdown was partly abrogated by siSTAT1 knockdown. Colony formation and MTT assay suggested that ELL2 knockdown could induce profound cell death in both PC3 and DU145 cells. Flow cytometry analysis showed that knockdown ELL2 indeed induced apoptosis and also caused S-phase arrest. CONCLUSIONS: ELL2 knockdown inhibited cell proliferation, induced S-phase arrest, promoted apoptosis in AR-negative PC3 and DU145 prostate cancer cells, which is accompanied by the induction of genes associated with cell death and survival as well as the interferon-γ pathway. This suggests a potential oncogenic role for ELL2 in AR-negative prostate cancer cells. The mechanisms by which ELL2 promotes AR-negative prostate cancer cell proliferation and survival may represent potential targets for the treatment of AR-negative prostate cancer. Source of Funding: This project was supported in part by NIH grant R01 CA186780 and R50 CA211242. © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e216-e216 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Zhi Wang* More articles by this author Laura E. Pascal More articles by this author Uma R Chandran More articles by this author Srilakshmi Chaparala More articles by this author Shidong Lyu More articles by this author Ding Hui More articles by this author Lin Qi More articles by this author Zhou Wang More articles by this author Expand All Advertisement PDF downloadLoading ...

Conditional Deletion of Eaf1 Induces Murine Prostatic Intraepithelial Neoplasia in Mice

ELL-associated factor 1 is a transcription elongation factor that shares significant homology and functional similarity to the androgen-responsive prostate tumor suppressor ELL-associated factor 2. EAF2 is frequently down-regulated in advanced prostate cancer and Eaf2 deletion in the mouse induced the development of murine prostatic intraepithelial neoplasia. Here we show that similar to EAF2, EAF1 is frequently down-regulated in advanced prostate cancer. Co-downregulation of EAF1 and EAF2 occurred in 40% of clinical specimens with Gleason score >7. We developed and characterized a murine model of prostate-epithelial specific deletion of Eaf1 in the prostate and crossed it with our previously generated mouse with conventional deletion of Eaf2. The prostates of Eaf1 deletion mice displayed murine prostatic intraepithelial neoplasia lesions with increased proliferation and inflammation. Combined deletion of Eaf1 and Eaf2 in the murine model induced an increased incidence in mPIN lesions characterized by increased proliferation and CD3+ T cells and CD19+ B cells infiltration compared to individual deletion of either Eaf1 or Eaf2 in the murine prostate. These results suggest that EAF1 may play a tumor suppressive role in the prostate. Cooperation between EAF1 and EAF2 may be important for prostate maintaining prostate epithelial homeostasis, and concurrent loss of these two tumor suppressors may promote prostate tumorigenesis and progression.

Conditional deletion of ELL2 induces murine prostate intraepithelial neoplasia

Conditional deletion of ELL2 induces murine prostate intraepithelial neoplasia

Interleukin-30/IL27p28 Shapes Prostate Cancer Stem-like Cell Behavior and Is Critical for Tumor Onset and Metastasization.

Prostate cancer stem-like cells (PCSLC) are believed to be responsible for prostate cancer onset and metastasis. Autocrine and microenvironmental signals dictate PCSLC behavior and patient outcome. In prostate cancer patients, IL30/IL27p28 has been linked with tumor progression, but the mechanisms underlying this link remain mostly elusive. Here, we asked whether IL30 may favor prostate cancer progression by conditioning PCSLCs and assessed the value of blocking IL30 to suppress tumor growth. IL30 was produced by PCSLCs in human and murine prostatic intraepithelial neoplasia and displayed significant autocrine and paracrine effects. PCSLC-derived IL30 supported PCSLC viability, self-renewal and tumorigenicity, expression of inflammatory mediators and growth factors, tumor immune evasion, and regulated chemokine and chemokine receptor genes, primarily via STAT1/STAT3 signaling. IL30 overproduction by PCSLCs promoted tumor onset and development associated with increased proliferation, vascularization, and myeloid cell recruitment. Furthermore, it promoted PCSLC dissemination to lymph nodes and bone marrow by upregulating the CXCR5/CXCL13 axis, and drove metastasis to lungs through the CXCR4/CXCL12 axis. These mechanisms were drastically hindered by IL30 knockdown or knockout in PCSLCs. Collectively, these results mark IL30 as a key driver of PCSLC behavior. Targeting IL30 signaling may be a potential therapeutic strategy against prostate cancer progression and recurrence.Significance: IL30 plays an important role in regulating prostate cancer stem-like cell behavior and metastatic potential, therefore targeting this cytokine could hamper prostate cancer progression or recurrence. Cancer Res; 78(10); 2654-68. ©2018 AACR.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

ELL2 regulates DNA non-homologous end joining (NHEJ) repair in prostate cancer cells

ELL2 is an androgen-responsive gene that is expressed by prostate epithelial cells and is frequently down-regulated in prostate cancer. Deletion of Ell2 in the murine prostate induced murine prostatic intraepithelial neoplasia and ELL2 knockdown enhanced proliferation and migration in C4-2 prostate cancer cells. Here, knockdown of ELL2 sensitized prostate cancer cells to DNA damage and overexpression of ELL2 protected prostate cancer cells from DNA damage. Knockdown of ELL2 impaired non-homologous end joining repair but not homologous recombination repair. Transfected ELL2 co-immunoprecipitated with both Ku70 and Ku80 proteins. ELL2 could bind to and co-accumulate with Ku70/Ku80 proteins at sites of DNA damage. Knockdown of ELL2 dramatically inhibited Ku70 and Ku80 recruitment and retention at DNA double-strand break sites in prostate cancer cells. The impaired recruitment of Ku70 and Ku80 proteins to DNA damage sites upon ELL2 knockdown was rescued by re-expression of an ELL2 transgene insensitive to siELL2. This study suggests that ELL2 is required for efficient NHEJ repair via Ku70/Ku80 in prostate cancer cells.

Conditional deletion of ELL2 induces murine prostate intraepithelial neoplasia.

Elongation factor, RNA polymerase II, 2 (ELL2) is an RNA Pol II elongation factor with functional properties similar to ELL that can interact with the prostate tumor suppressor EAF2. In the prostate, ELL2 is an androgen response gene that is upregulated in benign prostatic hyperplasia (BPH). We recently showed that ELL2 loss could enhance prostate cancer cell proliferation and migration, and that ELL2 gene expression was downregulated in high Gleason score prostate cancer specimens. Here, prostate-specific deletion of ELL2 in a mouse model revealed a potential role for ELL2 as a prostate tumor suppressor in vivoEll2-knockout mice exhibited prostatic defects including increased epithelial proliferation, vascularity and PIN lesions similar to the previously determined prostate phenotype in Eaf2-knockout mice. Microarray analysis of prostates from Ell2-knockout and wild-type mice on a C57BL/6J background at age 3 months and qPCR validation at 17 months of age revealed a number of differentially expressed genes associated with proliferation, cellular motility and epithelial and neural differentiation. OncoPrint analysis identified combined downregulation or deletion in prostate adenocarcinoma cases from the Cancer Genome Atlas (TCGA) data portal. These results suggest that ELL2 and its pathway genes likely play an important role in the development and progression of prostate cancer.

Redox-activatable hyaluronic acid-SS-mertansine prodrug: superior drug loading, enhanced toleration, and targeted breast tumor therapy

ELL2 is an androgen-responsive gene that is expressed by prostate epithelial cells and is frequently down-regulated in prostate cancer. Deletion of Ell2 in the murine prostate induced murine prostatic intraepithelial neoplasia and ELL2 knockdown enhanced proliferation and migration in C4-2 prostate cancer cells. Here, knockdown of ELL2 sensitized prostate cancer cells to DNA damage and overexpression of ELL2 protected prostate cancer cells from DNA damage. Knockdown of ELL2 impaired non-homologous end joining repair but not homologous recombination repair. Transfected ELL2 co-immunoprecipitated with both Ku70 and Ku80 proteins. ELL2 could bind to and co-accumulate with Ku70/Ku80 proteins at sites of DNA damage. Knockdown of ELL2 dramatically inhibited Ku70 and Ku80 recruitment and retention at DNA double-strand break sites in prostate cancer cells. The impaired recruitment of Ku70 and Ku80 proteins to DNA damage sites upon ELL2 knockdown was rescued by re-expression of an ELL2 transgene insensitive to siELL2. This study suggests that ELL2 is required for efficient NHEJ repair via Ku70/Ku80 in prostate cancer cells.

Abstract 2674: High fat diet accelerates MYC-driven prostate cancer through metabolic and epigenomic rewiring

Abstract Introduction: The mechanisms underlying the association between high dietary fat intake and prostate cancer (PCa) are unknown. Using a MYC-driven PCa mouse model, we sought to identify metabolic and epigenomic alterations driven by high fat diet (HFD) that facilitate PCa progression. Additionally, we investigated whether these alterations were relevant to PCa progression and lethality in humans. Material and Methods: Wild-type (WT) and transgenic Hi-MYC (MYC) mice were assigned either a HFD or control diet and were sacrificed at 12, 24, and 36 weeks of age for histologic and phenotypic characterization. Metabolic and epigenomic analyses were carried on the ventral prostates of 12-week old mice. Human PCa gene expression profiling data were obtained from 319 men with PCa and well-annotated post-diagnostic saturated fat intake (SFI) data from the Physicians’ Health Study and Health Professionals Follow-up Study prospective cohorts. Results: HFD does not affect the incidence of MYC-induced murine prostate intraepithelial neoplasia (mPIN) at 12 weeks, but increases mPIN proliferative index (Ki-67) at 24 weeks and tumor burden at 36 weeks. MYC overexpression, as expected, induces a significant metabolic reprogramming and HFD further enhances this rewiring to provide additional anabolic metabolites to sustain the increased proliferation of MYC prostate while having little effect on the WT prostate. Moreover, MYC altered key metabolites of the methionine cycle in a direction suggestive of a global hypomethylation, again amplified by HFD. Targeted quantitative histone mass spectrometry revealed a robust MYC-driven signature, including a global demethylation of H3K27 and H4K20 marks, the latter enhanced by HFD. Moreover, ChIP-seq revealed an intricate crosstalk between MYC and the H4K20me1 demethylase PHF8, resulting in enhanced genomic instability in the context of HFD. Finally, RNA-seq and ATAC-seq analyses showed that HFD rewires MYC-driven PCa through the alteration of genes implicated in chromatin function and remodeling. In humans, SFI was associated with enrichment in genes associated with increased MYC transcriptional activity in the prostate. Furthermore, this MYC transcriptional signature was associated with PCa lethality overall (OR = 3.21; 95% CI = 1.47, 7.35 comparing extreme score tertiles), and the association was stronger among men with high post-diagnostic SFI (OR = 1.32; 95%CI = 1.11, 1.66) than those with low SFI (OR = 1.05; 95%CI = 0.98, 1.12). Conclusions: HFD supports a coordinated metabolomic and epigenomic rewiring to increase epigenomic plasticity and MYC transcriptional activity prior to the appearance of phenotypic alterations in the prostate. Importantly, HFD requires MYC-mediated transformation to trigger its deleterious effects. In humans, SFI also enhances MYC transcriptional activity, which is associated with increased PCa lethality. Citation Format: David P. Labbe’, Giorgia Zadra, Meng Yang, Charles Y. Lin, Jaime M. Reyes, Stefano Cacciatore, Ericka M. Ebot, Maura B. Cotter, Amanda L. Creech, Jacob D. Jaffe, Philip W. Kantoff, James E. Bradner, Lorelei A. Mucci, Jorge E. Chavarro, Massimo Loda, Myles Brown. High fat diet accelerates MYC-driven prostate cancer through metabolic and epigenomic rewiring. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2674.

Abstract 4190: A mouse model of pRb2/p130 in prostate cancer

Abstract INTRODUCTION Prostate Cancer (PCa) is the most commonly diagnosed cancer in men and it is responsible for the second highest number of cancer-related deaths in the United States. There is an urgent need for new treatments and better experimental animal models to study PCa development, progression, and metastasis. Prostate cancer is a heterogeneous and multifocal disease that arises from several genetic and epigenetic aberrations. Among different altered pathways, the Retinoblastoma is inactivated in more than fifty percent of PCa. Despite a plentiful literature, the activity of pRb members (pRb, pRb2/p130 and p107) is not fully elucidated. From a previously published mouse model, we know that the inactivation of all the pRb members induces aberrant proliferation and it is sufficient for tumor initiation. Here, we describe a novel double ko (Rb, pRb2/p130) mouse model, which offers a new opportunity to dissect prostate tumor initiation and development. MATERIALS AND METHODS To study the effect of pRb2/p130 inactivation on prostate tumorigenesis and progression, we knocked out pRb and/or pRb2/p130 in the prostate tissue (Probasin-CRE). The genotypes of each gene were determined by PCR. To analyze the effects of pRb2/p130 loss, we collected prostate tissues at different time points (3, 6, 9, 12 and 18 months). A portion of each prostate sample was fixed in formalin and embedded in paraffin to perform immunohistochemistry analyses and another portion was flash-frozen for molecular studies. The histological classification of each prostate samples was made on H&E-stained sections by A.K.S., a veterinary pathologist with extensive experience in murine prostate pathology. RESULTS As reported in literature, pRb family disruption in the prostate epithelium induces prostatic intraepithelial neoplasia. The prostate of triple ko mice showed extensive abnormal gland architecture beginning as early as 2 months of age with a focal hyperplasia. By 3 months of age, all the triple ko mice displayed characteristic traits of murine prostatic intraepithelial neoplasia (mPIN). Double ko (pRb and pRb2/p130) mice developed hyperplasia after 6 months and focally invasive well-differentiated adenocarcinomas by 12-18 months. CONCLUSIONS Our preliminary findings suggest that the inactivation of pRb and pRb2/p130 leads to the development of a prostate tumor phenotype and highlight the importance of the function of pRb2/p130 in the prostatic epithelium. This model will be useful to clarify the molecular mechanism of pRb2/p130 function and represents a new opportunity for cancer therapy. Citation Format: Silvia Boffo, Andres J. Klein-Szanto, Flavio Rizzolio, Antonio Giordano. A mouse model of pRb2/p130 in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4190. doi:10.1158/1538-7445.AM2015-4190

Crosstalking between Androgen and PI3K/AKT Signaling Pathways in Prostate Cancer Cells

Both androgen action and PI3K medicated signaling pathways have been implicated in prostate tumorigenesis. Our androgen receptor (AR) conditional transgenic mice developed murine prostatic intraepithelial neoplasia (mPIN) and prostatic adenocarcinoma lesions recapitulating human prostate cancer development and progression. Role of transgenic AR contributing to malignancy was demonstrated by high degree of transgenic AR expression in atypical and tumor cells in mPIN as well as prostatic adenocarcinoma lesions of the transgenic mice, but not in adjacent normal tissue. Interestingly, reduced PI3K/Akt activation also appeared in these mouse atypical and tumor cells, suggesting an interaction between androgen and PI3K/AKT pathways. In this study, we further investigated this interaction. We showed that the androgen depletion or knockdown of AR expression results in elevated levels of active phosphorylated AKT in prostate cancer cells. Castration of conditional Pten knock-out mice showed increased Akt, phosphorylated Akt, and pS6 expression in the mouse prostate. Using a series of newly generated Ar reporter and Pten knock-out compound mice, we showed that Pten loss directly represses endogenous Ar expression in prostatic epithelial cells. Moreover, Pten loss and PI3K/Akt activation reduced Ar-mediated transcription in purified Pten-null cells. This study provides novel evidence demonstrating interplay between androgen and PI3K pathways, as well as introduces unique and relevant mouse models for further studies of PI3K and AR pathways in the context of prostate tumorigenesis.

Chronic Chlorpyrifos Exposure Does Not Promote Prostate Cancer in Prostate Specific PTEN Mutant Mice

Environmental factors are likely to interact with genetic determinants to influence prostate cancer progression. The Agricultural Health Study has identified an association between exposure to organophosphorous pesticides including chlorpyrifos, and increased prostate cancer risk in pesticide applicators with a first-degree family history of this disease. Exploration of this potential gene-environment interaction would benefit from the development of a suitable animal model. Utilizing a previously described mouse model that is genetically predisposed to prostate cancer through a prostate-specific heterozygous PTEN deletion, termed C57/Luc/Ptenp+/-, we used bioluminescence imaging and histopathological analyses to test whether chronic exposure to chlorpyrifos in a grain-based diet for 32 weeks was able to promote prostate cancer development. Chronic exposure to chlorpyrifos in the diet did not promote prostate cancer development in C57/Luc/Ptenp+/- mice despite achieving sufficient levels to inhibit acetylcholinesterase activity in plasma. We found no significant differences in numbers of murine prostatic intraepithelial neoplasia lesions or disease progression in chlorpyrifos versus control treated animals up to 32 weeks. The mechanistic basis of pesticide-induced prostate cancer may be complex and may involve other genetic variants, multiple genes, or nongenetic factors that might alter prostate cancer risk during pesticide exposure in agricultural workers.

Abstract C70: Trop2 regulates prostate tumorigenesis and stem cell self-renewal

Abstract Cancer cells share multiple characteristics with adult tissue stem cells such as self-renewal, proliferative capacity and differentiation. Several groups have identified Trop2, a type I trans-membrane glycoprotein, as a marker of stem/progenitor cells in adult tissues. Consistent with the relationship between stem cells and tumorigenesis, we have previously identified that Trop2hi cells are efficient targets for transformation in the prostate. Additionally, Trop2 is highly expressed in various cancers including advanced colorectal, pancreatic, prostate, ovarian and pulmonary carcinomas and is associated with unfavorable outcome. However the functional role of Trop2 in stem cell self-renewal, its relation to transformation, and the molecular mechanisms by which Trop2 transmits signals to regulate these processes remain unclear. We hypothesize that heightened Trop2 may promote the stem-like properties of self-renewal and proliferative activity in prostate cancer. We observe significantly elevated levels of Trop2 in human prostate cancer, lymph node metastases and in mouse models of prostate cancer. Over-expression of Trop2 induces anchorage-independent growth in vitro and murine prostatic intraepithelial neoplasia (mPIN) in vivo. In vitro sphere and in vivo regeneration assays demonstrate that Trop2 regulates stem/progenitor self-renewal in the prostate, suggesting that elevated levels of Trop2 in prostate cancer are selected for by promoting tumor cell self-renewal. Biochemical analysis reveals that Trop2 is activated by regulated intra-membrane proteolysis (RIP), resulting in shedding of the extracellular domain (ECD) and release of the intracellular domain (ICD) to the nucleus. RIP is required for Trop2 activity and cleavage mutants of Trop2 fail to enhance self-renewal in vitro. We generated expression constructs of Trop2 ICD and ECD to test their role in self-renewal and tumorigenesis. Secreted Trop2 ECD induces Trop2 cleavage and results in increased stem/progenitor cell proliferation. Trop2 ICD alone promotes self-renewal activity and transformation as measured by sphere formation and in vivo regeneration assays, respectively. Trop2 ICD is sufficient to induce mPIN lesions similar to previously reported mouse models of prostate cancer. These findings suggest that the ICD is the functional unit of Trop2. Trop2 ICD is involved in a signaling cascade that is dependent upon β-catenin to promote self-renewal. We find Trop2 ICD, but not the ECD, forms a complex with β-catenin. While β-catenin loss does not affect baseline sphere-forming activity, we find that over-expression of Trop2 requires the presence of β-catenin to enhance progenitor activity in vitro. Taken together, our study provides evidence that Trop2 is a key regulator of transformation and self-renewal in the prostate and its function depends on its activation by RIP and downstream signaling through β-catenin. Delineating the molecular mechanism of Trop2 action allows development of therapeutic strategies applicable to not only prostate cancer, but other epithelial malignancies that exhibit increased Trop2 expression. Such strategies include small molecules able to block Trop2 proteolysis, thus inhibiting Trop2 function. More specific therapies such as monoclonal antibodies that will interfere with Trop2 processing and consequent activation could also represent promising future anticancer therapies. Citation Format: Tanya Stoyanova, Andrew Goldstein, Houjian Cai, Justin M. Drake, Jiaoti Huang, Hong Zhang, Owen N. Witte. Trop2 regulates prostate tumorigenesis and stem cell self-renewal [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 C70.

Abstract B57: Oncostatin M promotes progression of Pten-deficient prostate cancer

Abstract Chronic inflammation has been proposed to play a crucial role in several cancers including gastric, pancreatic, colon and lung, with recent evidence suggesting involvement in prostate cancer. While both interleukin 6 (IL6) and the related cytokine oncostatin M (OSM) have been implicated in prostate cancer, there have been few studies using in vivo models. To address this issue, we employed a dissociated prostate tissue recombination system using epithelium derived from conditional Pten knockout mice. Loss of Pten is common in prostate cancer and generates an intermediate murine prostatic intraepithelial neoplasia (mPIN) lesion in our system, providing an ideal means to assess functional synergy. We hypothesize that increased expression of these cytokines in the prostate microenvironment will promote malignant progression of intermediate prostate cancer lesions. Paracrine expression of IL6 did not transform naïve prostate epithelium nor did it exhibit functional synergy with Pten null epithelium in our system. Grafts expressing OSM alone did not transform naïve tissue, though in contrast, presented dramatic functional synergy with loss of Pten in prostate epithelium. Loss of Pten combined with OSM exhibited progression to poorly differentiated adenocarcinoma with features of epithelial-to-mesenchymal transition (EMT). Transformed tissues displayed loss of cytokeratin expression with down-regulation and mis-localization of e-cadherin. These tumors exhibit activation of several pathways downstream of OSM signaling including STAT3 and Erk1/2 as seen by immunohistochemical analysis. Further, we interrogated the role of OSM in promoting hormone refractory prostate cancer by castrating host animals following tumor establishment. Loss of Pten alone failed to progress beyond mPIN lesions and eventually regressed at 6 months post castration. In dramatic contrast, grafts expressing the OSM ligand exhibited initial regression with castration resistant growth following a latency period, recapitulating events commonly observed in human disease. Our evidence suggests that in the context of Pten loss, IL6 does not appear to promote prostate malignancy in the murine system. Conversely, we have identified that OSM synergizes with loss of Pten to promote aggressive prostate malignancy. Paracrine expression of OSM results in activation of several pathways, providing a means to interrogate hierarchy within signaling modules in the context of prostate cancer progression. Our study further indicates that OSM could play a role in promoting castration resistance, allowing investigation of potential points for therapeutic intervention. Citation Format: Daniel A. Smith, Yang Zong, Atsushi Kiba, Owen N. Witte. Oncostatin M promotes progression of Pten-deficient 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 B57.

Ack1 Mediated AKT/PKB Tyrosine 176 Phosphorylation Regulates Its Activation

The AKT/PKB kinase is a key signaling component of one of the most frequently activated pathways in cancer and is a major target of cancer drug development. Most studies have focused on its activation by Receptor Tyrosine Kinase (RTK) mediated Phosphatidylinositol-3-OH kinase (PI3K) activation or loss of Phosphatase and Tensin homolog (PTEN). We have uncovered that growth factors binding to RTKs lead to activation of a non-receptor tyrosine kinase, Ack1 (also known as ACK or TNK2), which directly phosphorylates AKT at an evolutionarily conserved tyrosine 176 in the kinase domain. Tyr176-phosphorylated AKT localizes to the plasma membrane and promotes Thr308/Ser473-phosphorylation leading to AKT activation. Mice expressing activated Ack1 specifically in the prostate exhibit AKT Tyr176-phosphorylation and develop murine prostatic intraepithelial neoplasia (mPINs). Further, expression levels of Tyr176-phosphorylated-AKT and Tyr284-phosphorylated-Ack1 were positively correlated with the severity of disease progression, and inversely correlated with the survival of breast cancer patients. Thus, RTK/Ack1/AKT pathway provides a novel target for drug discovery.

Effect of Low-Fat Diet on Development of Prostate Cancer and Akt Phosphorylation in the Hi-Myc Transgenic Mouse Model

This study evaluated the effect of dietary fat on prostate cancer development by using the Hi-Myc mouse transgenic prostate cancer model. Hi-Myc mice develop murine prostatic intraepithelial neoplasia (mPIN) as early as 2 to 4 weeks and invasive adenocarcinoma between 6 and 9 months due to the overexpression of human c-Myc in the mouse prostate. Three-week-old male Hi-Myc mice were placed on high-fat (HF; 42% Kcal) or low-fat (LF; 12% Kcal) diets, and equal caloric intake was maintained until euthanasia at 7 months. The number of mice that developed invasive adenocarcinoma at 7 months was 27% less in the LF diet group (12/28) compared with the HF diet group (23/33, P < 0.05). Epithelial cells in mPIN lesions in the LF group had a significantly lower proliferative index compared with epithelial cells in the HF group (21.7% versus 28.9%, P < 0.05). During the mPIN phase of carcinogenesis (4 months), the LF group had higher serum insulin-like growth factor (IGF) binding protein-1 levels (21.0 +/- 8.9 ng/mL versus 3.2 +/- 0.8 ng/mL, P < 0.05) relative to the HF group. Akt (Ser(473)) phosphorylation, Akt kinase activity, and phosphorylation of downstream targets of Akt in prostates were significantly reduced in the LF diet group compared with the HF group. We conclude that dietary fat reduction delays transition from mPIN to invasive cancer in this Myc-driven transgenic mouse model, possibly through suppression of the IGF-Akt pathway and decreased proliferation of mPIN epithelial cells.

Myc-driven murine prostate cancer shares molecular features with human prostate tumors

Increased Myc gene copy number is observed in human prostate cancer. To define Myc's functional role, we generated transgenic mice expressing human c-Myc in the mouse prostate. All mice developed murine prostatic intraepithelial neoplasia followed by invasive adenocarcinoma. Microarray-based expression profiling identified a Myc prostate cancer expression signature, which included the putative human tumor suppressor NXK3.1. Human prostate tumor databases revealed modules of human genes that varied in concert with the Myc prostate cancer signature. This module includes the Pim-1 kinase, a gene known to cooperate with Myc in tumorigenesis, and defines a subset of human, “ Myc-like” human cancers. This approach illustrates how genomic technologies can be applied to mouse cancer models to guide evaluation of human tumor databases.