Primary Human Prostate Tumors Research Articles

Extensive androgen receptor enhancer heterogeneity in primary prostate cancers underlies transcriptional diversity and metastatic potential

Androgen receptor (AR) drives prostate cancer (PCa) development and progression. AR chromatin binding profiles are highly plastic and form recurrent programmatic changes that differentiate disease stages, subtypes and patient outcomes. While prior studies focused on concordance between patient subgroups, inter-tumor heterogeneity of AR enhancer selectivity remains unexplored. Here we report high levels of AR chromatin binding heterogeneity in human primary prostate tumors, that overlap with heterogeneity observed in healthy prostate epithelium. Such heterogeneity has functional consequences, as somatic mutations converge on commonly-shared AR sites in primary over metastatic tissues. In contrast, less-frequently shared AR sites associate strongly with AR-driven gene expression, while such heterogeneous AR enhancer usage also distinguishes patients’ outcome. These findings indicate that epigenetic heterogeneity in primary disease is directly informative for risk of biochemical relapse. Cumulatively, our results illustrate a high level of AR enhancer heterogeneity in primary PCa driving differential expression and clinical impact.

PROTEIN KINASE C ALPHA IS A CENTRAL NODE FOR TUMORIGENIC TRANSCRIPTIONAL NETWORKS IN HUMAN PROSTATE CANCER.

Aberrant expression of protein kinase C (PKC) isozymes is a hallmark of cancer. The different members of the PKC family control cellular events associated with cancer development and progression. Whereas the classical/conventional PKCα isozyme has been linked to tumor suppression in most cancer types, here we demonstrate that this kinase is required for the mitogenic activity of aggressive human prostate cancer cells displaying aberrantly high PKCα expression. Immunohistochemical analysis showed abnormal up-regulation of PKCα in human primary prostate tumors. Interestingly, silencing PKCα expression from aggressive prostate cancer cells impairs cell cycle progression, proliferation and invasion, as well as their tumorigenic activity in a mouse xenograft model. Mechanistic analysis revealed that PKCα exerts a profound control of gene expression, particularly over genes and transcriptional networks associated with cell cycle progression and E2F transcription factors. PKCα RNAi depletion from PC3 prostate cancer cells led to a reduction in the expression of pro-inflammatory cytokine and epithelial-to-mesenchymal transition (EMT) genes, as well as a prominent down-regulation of the immune checkpoint ligand PD-L1. This PKCα-dependent gene expression profile was corroborated in silico using human prostate cancer databases. Our studies established PKCα as a multifunctional kinase that plays pleiotropic roles in prostate cancer, particularly by controlling genetic networks associated with tumor growth and progression. The identification of PKCα as a pro-tumorigenic kinase in human prostate cancer provides strong rationale for the development of therapeutic approaches towards targeting PKCα or its effectors.

Acetylated HOXB13 Regulated Super Enhancer Genes Define Therapeutic Vulnerabilities of Castration-Resistant Prostate Cancer.

Androgen receptor (AR) antagonism is exacerbated by HOXB13 in castration-resistant prostate cancers (CRPC). However, it is unclear when and how HOXB13 primes CRPCs for AR antagonism. By mass-spectrometry analysis of CRPC extract, we uncovered a novel lysine 13 (K13) acetylation in HOXB13 mediated by CBP/p300. To determine whether acetylated K13-HOXB13 is a clinical biomarker of CRPC development, we characterized its role in prostate cancer biology. We identified tumor-specific acK13-HOXB13 signal enriched super enhancer (SE)-regulated targets. We analyzed the effect of loss of HOXB13K13-acetylation on chromatin binding, SE proximal target gene expression, self-renewal, enzalutamide sensitivity, and CRPC tumor growth by employing isogenic parental and HOXB13K13A mutants. Finally, using primary human prostate organoids, we evaluated whether inhibiting an acK13-HOXB13 target, ACK1, with a selective inhibitor (R)-9b is superior to AR antagonists in inhibiting CRPC growth. acK13-HOXB13 promotes increased expression of lineage (AR, HOXB13), prostate cancer diagnostic (FOLH1), CRPC-promoting (ACK1), and angiogenesis (VEGFA, Angiopoietins) genes early in prostate cancer development by establishing tumor-specific SEs. acK13-HOXB13 recruitment to key SE-regulated targets is insensitive to enzalutamide. ACK1 expression is significantly reduced in the loss of function HOXB13K13A mutant CRPCs. Consequently, HOXB13K13A mutants display reduced self-renewal, increased sensitivity to enzalutamide, and impaired xenograft tumor growth. Primary human prostate tumor organoids expressing HOXB13 are significantly resistant to AR antagonists but sensitive to (R)-9b. In summary, acetylated HOXB13 is a biomarker of clinically significant prostate cancer. Importantly, PSMA-targeting agents and (R)-9b could be new therapeutic modalities to target HOXB13-ACK1 axis regulated prostate cancers.

Effect of modulation of CD3 binding in a PSMAxCD3 T-cell engaging bispecific antibody on maintenance of efficient tumor cell kill and cytokine release.

e17583 Background: Castration resistant prostate cancer (CRPC) is an incurable disease and represents a significant unmet need. Prostate specific membrane antigen (PSMA) is a protein highly expressed on the surface of prostate cancer cells; expression has been shown to increase with disease progression. Therapies targeting PSMA, such as anti-PSMA radioligand conjugates, have shown promise in clinical trials, validating this target for CRPC. T-cell recruiting bispecific antibodies (T-BsAbs) have demonstrated potent tumor killing activity against multiple tumor types, but immune-mediated toxicities have hampered T-cell redirecting therapies to date. Using Teneobio’s unique antibody discovery platform, we have developed a CD3xPSMA bispecific antibody (TNB-585) that retains the potent cytotoxicity of other T-BsAbs but with significantly reduced cytokine release. Methods: Antibodies targeting CD3 and PSMA were generated via immunization of our proprietary transgenic animals. Candidate antibodies were selected by repertoire deep sequencing of B-cells from draining lymph nodes, followed by high-throughput gene assembly and recombinant expression. Multiple bispecific antibodies targeting CD3 and PSMA were assembled and evaluated for their ability to selectively activate primary human T-cells and mediate killing of PSMA+ tumor cells in vitro, ex vivo, and in vivo. T-cell activation surface markers, cytokine production, and tumor cell cytotoxicity were measured. Results: In co-culture experiments, primary human T-cells were activated only in the presence of both the bispecifics and PSMA+ cells. These bispecifics mediated potent and selective cytotoxicity against PSMA-positive tumor cells, prostate tumor cell lines, or primary human prostate tumor cells isolated from patients. From among these we identified TNB-585, which showed attenuated binding to CD3. TNB-585 mediated comparable tumor cell cytotoxicity to CD3xPSMA T-BsAbs containing a high affinity anti-CD3 domain but with significantly reduced cytokine production. TNB-585 also showed tumor growth inhibition in xenograft models of prostate cancer in vivo. Conclusions: We have developed a novel CD3xPSMA T-BsAb that mediates T-cell killing of PSMA+ tumor cells with minimal production of cytokines. This molecule may improve safety, efficacy, and offer opportunities for combination therapy to treat CRPC. A Phase 1 clinical trial of this compound in CRPC is scheduled to begin in Q1 2021.

Human prostate cancer immune phenotypes after androgen deprivation therapy.

5083 Background: The prostate tumor microenvironment (TME) is generally non-inflamed. However, we previously showed in pre-clinical models that androgen deprivation therapy (ADT) induces a complex immune cell infiltrate. Whether ADT similarly promotes inflammation in patients remains unclear. Therefore, we collected specimens from patients with localized prostate cancer treated with neoadjuvant degarelix and hypothesized that we could decipher key changes in the immune TME after ADT utilizing a novel cancer systems biology approach. Methods: We identified a cohort of patients treated with neoadjuvant degarelix (240mg SQ) as part of two clinical trials (NCT01696877, NCT01542021). Patients were treated with degarelix either 4 days (N = 13), 7 days (N = 17) or 14 days (N = 8) prior to radical prostatectomy. RNA was extracted from FFPE tissue and analyzed by RNAseq. To deconvolute fractional contributions of different immune cell subsets we performed CIBERSORT and Uniform Manifold Approximation and Projection (UMAP) analysis. Treatment groups were compared to a cohort of untreated matched controls (N = 37). Results: Degarelix induced a complex immune cell infiltrate in human primary prostate tumors with an increase in both pro- and anti-inflammatory cells subsets and changes in expression of immune checkpoints compared to untreated matched controls. Degarelix therapy also significant changed associated gene signatures within the lymphoid and myeloid compartments over time. Conclusions: ADT leads to profound immune remodeling within the prostate TME. Our analysis of human primary prostate tumors supports the hypothesis that the optimal time for immunologic intervention is the peri-castration period. These data also suggest that combinatorial immunotherapy strategies that target particular immune cell subsets will likely be required to successfully promote robust anti-tumor immune responses in prostate cancer.

Abstract B068: Methionine aminopeptidase II (MetAP2) activated in situ self-assembly of small-molecule probes for imaging prostate cancer

Abstract Prostate cancer is the most common noncutaneous malignancy among men in the United States and a leading cause of cancer death worldwide. The major clinical challenges in prostate cancer diagnosis include accurate staging, localizing tumors within the prostate, and developing imaging prognostic biomarkers. PET is routinely used in the clinical management of many cancers dues to its high sensitivity (10-15 M), spatial resolution (2-4 mm3), rapid whole-body scan times (<20 min), noninvasiveness, and its ability to provide both anatomic and molecular information when combined with computed tomography (PET/CT). Several PET tracers (e.g., 18F-FDG, 18F-NaF, 11C-choline, 11C-acetate, 18F-fluciclovine, etc.) have been studied for early stage prostate cancer, bone metastases, and recurrent disease; nevertheless, all of these tracers rely on cell uptake and their applications are often limited by the low metabolic activity and a low growth rate of prostate cancer. In this project, we aim to develop a novel enzymatic activity-based PET tracer for in vivo imaging of prostate cancer by targeting methionine aminopeptidase II (MetAP2), a cytosolic metalloprotease that catalyzes the cotranslational removal of the N-terminus initiator methionine residue from nascent proteins. We hypothesize that MetAP2 can serve as a novel diagnostic biomarker of prostate cancer. A MetAP2 activatable molecular imaging probe that self-aggregates in situ can be utilized to image and define this disease. To test our hypothesis, we first measured the expression level of MetAP2 in primary benign human prostate epithelial cells (BS403) and prostate tumor cell lines by Western blot. BS403 cells express significantly lower levels of MetAP2 than PC3 (by 4-fold), DU145 (3-fold), and 22Rv1 (3-fold). The expression of MetAP2 in DU145 tumor xenografts is 12-fold higher and for 22Rv1 14-fold higher, compared to BS403 cells. Immunohistologic analysis of tissues derived from DU145 and 22Rv1 xenografts confirms strong staining of MetAP2. We performed immunohistochemistry on a prostate cancer tissue microarray containing 225 independent patient samples represented in quadruplicate. Positive staining for MetAP2 was found in nearly all of the cancer samples. Encouraged by these data, we designed and synthesized a nonradioactive MetAP2 sensitive nano-aggregation probe called M-SNAP. Incubation of M-SNAP with recombinant MetAP2 enzyme confirmed the formation of the cyclized and aggregated product M-SNAP-cycl by HPLC, TEM (transmission electron microscopy), and DLS (dynamic light scattering). Treatment of PC3 and DU145 cells with or without M-SNAP and the MetAP2 specific inhibitor TNP470 demonstrated MetAP2 dependent intracellular probe aggregation/signal retention using post fluorescent labeling of the cyclized and aggregated products. In future work, we will prepare and study an MetAP2 activatable radioisotope-labeled probe in vitro for sensing and imaging and will evaluate the PET probe in different human prostate xenograft models. MetAP2 sensing probes could provide a highly sensitive and noninvasive PET imaging approach for prostate cancer detection that could be useful in prostate cancer detection, staging, and monitoring of therapeutic response. Citation Format: Jinghang Xie, Meghan Rice, Yunfeng Cheng, Guosheng Song, Christian Kunder, James D. Brooks, Tanya Stoyanova, Jianghong Rao. Methionine aminopeptidase II (MetAP2) activated in situ self-assembly of small-molecule probes for imaging prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B068.

Differential Expression of OATP1B3 Mediates Unconjugated Testosterone Influx.

Castration-resistant prostate cancer (CRPC) has greater intratumoral testosterone concentrations than similar tumors from eugonadal men; simple diffusion does not account for this observation. This study was undertaken to ascertain the androgen uptake kinetics, functional, and clinical relevance of de novo expression of the steroid hormone transporter OATP1B3 (SLCO1B3). Experiments testing the cellular uptake of androgens suggest that testosterone is an excellent substrate of OATP1B3 (Km = 23.2 μmol/L; Vmax = 321.6 pmol/mg/minute), and cells expressing a doxycycline-inducible SLCO1B3 construct had greater uptake of a clinically relevant concentration of 3H-testosterone (50 nmol/L; 1.6-fold, P = 0.0027). When compared with Slco1b2 (-/-) mice, Slco1b2 (-/-)/hSLCO1B3 knockins had greater hepatic uptake (15% greater AUC, P = 0.0040) and lower plasma exposure to 3H-testosterone (17% lower AUC, P = 0.0030). Of 82 transporters genes, SLCO1B3 is the second-most differentially expressed transporter in CRPC cell lines (116-fold vs. androgen-sensitive cells), with a differentially spliced cancer-type ct-SLCO1B3 making up the majority of SLCO1B3 expression. Overexpression of SLCO1B3 in androgen-responsive cells results in 1.5- to 2-fold greater testosterone uptake, whereas siRNA knockdown of SLCO1B3 in CRPC cells did not change intracellular testosterone concentration. Primary human prostate tumors express SLCO1B3 to a greater extent than ct-SLCO1B3 (26% of total SLCO1B3 expression vs. 0.08%), suggesting that androgen uptake in these tumor cells also is greater. Non-liver tumors do not differentially express SLCO1B3.Implications: This study suggests that de novo OATP1B3 expression in prostate cancer drives greater androgen uptake and is consistent with previous observations that greater OATP1B3 activity results in the development of androgen deprivation therapy resistance and shorter overall survival. Mol Cancer Res; 15(8); 1096-105. ©2017 AACR.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

TWIST1-WDR5-Hottip Regulates Hoxa9 Chromatin to Facilitate Prostate Cancer Metastasis.

TWIST1 is a transcription factor critical for development that can promote prostate cancer metastasis. During embryonic development, TWIST1 and HOXA9 are coexpressed in mouse prostate and then silenced postnatally. Here we report that TWIST1 and HOXA9 coexpression are reactivated in mouse and human primary prostate tumors and are further enriched in human metastases, correlating with survival. TWIST1 formed a complex with WDR5 and the lncRNA Hottip/HOTTIP, members of the MLL/COMPASS-like H3K4 methylases, which regulate chromatin in the Hox/HOX cluster during development. TWIST1 overexpression led to coenrichment of TWIST1 and WDR5 as well as increased H3K4me3 chromatin at the Hoxa9/HOXA9 promoter, which was dependent on WDR5. Expression of WDR5 and Hottip/HOTTIP was also required for TWIST1-induced upregulation of HOXA9 and aggressive cellular phenotypes such as invasion and migration. Pharmacologic inhibition of HOXA9 prevented TWIST1-induced aggressive prostate cancer cellular phenotypes in vitro and metastasis in vivo This study demonstrates a novel mechanism by which TWIST1 regulates chromatin and gene expression by cooperating with the COMPASS-like complex to increase H3K4 trimethylation at target gene promoters. Our findings highlight a TWIST1-HOXA9 embryonic prostate developmental program that is reactivated during prostate cancer metastasis and is therapeutically targetable. Cancer Res; 77(12); 3181-93. ©2017 AACR.

Abstract A25: Sensing stress in cancer: a novel therapy targeting protein synthesis through the unfolded protein response in prostate cancer development

Abstract Distinct oncogenic lesions within components of the Myc or PI3K pathways hijack the translation machinery, to establish an aberrant “cancer proteome”, that drives cancer cell growth and tumor development. Invariably, however, increases in the protein synthesis capacity of cancer cells impose a cellular stress that requires an adaptive or evasive response in order for cancer cells to survive and continue proliferating. One of the key cytoprotective stress response pathways is known as the unfolded protein response (UPR), which is engaged when an excess of unfolded/misfolded proteins accumulate within the lumen of the endoplasmic reticulum. How the specific arms of UPR are coupled to increased protein synthesis in cancer etiology and drug response is still poorly understood. In this study we established a new genetic mouse model to address the role of protein synthesis-dependent activation of the UPR in prostate cancer initiation and maintenance promoted by loss of the PTEN tumor suppressor and overexpression of the Myc oncogene. We show that in this epithelial cancer model, Myc hyperactivation and PTEN loss synergize to dramatically stimulate PERK and IRE-1α activity, two major signaling arms of the UPR pathway. Distinct mechanisms transduce adaptive signals under conditions of ER stress: PERK control of protein synthesis through phosphorylation of the key translation initiation factor eIF2α and IRE1α-mediated splicing of the XBP-1 mRNA to produce an active and stable XBP1 transcription factor (XBP1s). Importantly, we observed that UPR activation is associated with formation of prostatic intraepithelial neoplasia (PIN) and invasive carcinoma. Strikingly, the pharmacologic inhibition of eIF2α phosphorylation leads to tumor inhibition. We have further established patient derived xenograft (PDX) models from primary and metastatic human prostate tumors to extend the role of UPR in specific steps of human tumor development. We have also developed a series of novel mouse and human prostate cancer cell culture systems to define the role of localized translation of ER-associated ribosomes during oncogenic transformation. Together this work provides critical insight into a fundamental mechanism by which the augmented protein synthesis requirement of cancer cells is coupled to an adaptation of ER cellular stress, reflecting a new cancer-specific vulnerability that is targetable. Citation Format: Crystal S. Conn, Hao G. Nguyen, Yae Kye, John T. Cunningham, Charles Truillet, Michael Evans, Tony L. Huynh, Peter Walters, Davide Ruggero. Sensing stress in cancer: a novel therapy targeting protein synthesis through the unfolded protein response in prostate cancer development. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A25.

Overexpression and knock-down studies highlight that a disintegrin and metalloproteinase 28 controls proliferation and migration in human prostate cancer.

Prostate cancer is one of the most prevalent cancers in men. It is critical to identify and characterize oncogenes that drive the pathogenesis of human prostate cancer. The current study builds upon previous research showing that a disintegrin and metallproteinase (ADAM)28 is involved in the pathogenesis of numerous cancers. Our novel study used overexpression, pharmacological, and molecular approaches to investigate the biological function of ADAM28 in human prostate cancer cells, with a focus on cell proliferation and migration. The results of this study provide important insights into the role of metalloproteinases in human prostate cancer.The expression of ADAM28 protein levels was assessed within human prostate tumors and normal adjacent tissue by immunohistochemistry. Immunocytochemistry and western blotting were used to assess ADAM28 protein expression in human prostate cancer cell lines. Functional assays were conducted to assess proliferation and migration in human prostate cancer cells in which ADAM28 protein expression or activity had been altered by overexpression, pharmacological inhibition, or by siRNA gene knockdown.The membrane bound ADAM28 was increased in human tumor biopsies and prostate cancer cell lines. Pharmacological inhibition of ADAM28 activity and/or knockdown of ADAM28 significantly reduced proliferation and migration of human prostate cancer cells, while overexpression of ADAM28 significantly increased proliferation and migration.ADAM28 is overexpressed in primary human prostate tumor biopsies, and it promotes human prostate cancer cell proliferation and migration. This study supports the notion that inhibition of ADAM28 may be a potential novel therapeutic strategy for human prostate cancer.

A Novel Class of Hsp90 C-Terminal Modulators Have Pre-Clinical Efficacy in Prostate Tumor Cells Without Induction of a Heat Shock Response.

While there is compelling rationale to use heat shock protein 90 (Hsp90) inhibitors for treatment of advanced prostate cancer, agents that target the N-terminal ATP-binding site of Hsp90 have shown little clinical benefit. These N-terminal binding agents induce a heat shock response that activates compensatory heat shock proteins, which is believed to contribute in part to the agents' lack of efficacy. Here, we describe the functional characterization of two novel agents, SM253 and SM258, that bind the N-middle linker region of Hsp90, resulting in reduced client protein activation and preventing C-terminal co-chaperones and client proteins from binding to Hsp90. Inhibition of Hsp90 activity in prostate cancer cells by SM253 and SM 258 was assessed by pull-down assays. Cell viability, proliferation and apoptosis were assayed in prostate cancer cell lines (LNCaP, 22Rv1, PC-3) cultured with N-terminal Hsp90 inhibitors (AUY922, 17-AAG), SM253 or SM258. Expression of HSR heat shock proteins, Hsp90 client proteins and co-chaperones was assessed by immunoblotting. Efficacy of the SM compounds was evaluated in human primary prostate tumors cultured ex vivo by immunohistochemical detection of Hsp70 and Ki67. SM253 and SM258 exhibit antiproliferative and pro-apoptotic activity in multiple prostate cancer cell lines (LNCaP, 22Rv1, and PC-3) at low micromolar concentrations. Unlike the N-terminal inhibitors AUY922 and 17-AAG, these SM agents do not induce expression of Hsp27, Hsp40, or Hsp70, proteins that are characteristic of the heat shock response, in any of the prostate cell lines analyzed. Notably, SM258 significantly reduced proliferation within 2 days in human primary prostate tumors cultured ex vivo, without the significant induction of Hsp70 that was caused by AUY922 in the tissues. Our findings provide the first evidence of efficacy of this class of C-terminal modulators of Hsp90 in human prostate tumors, and indicate that further evaluation of these promising new agents is warranted. Prostate 76:1546-1559, 2016. © 2016 Wiley Periodicals, Inc.

Inducible expression of antigen processing and antigen presentation molecules and cancer testis antigens in human prostate cancer

Defects in immune surveillance have been correlated with tumorigenesis and poor clinical outcomes in cancer patients. Given recent advances in vaccine therapies and checkpoint inhibition, therapeutic targeting of defective antigen processing may play a critical role in improving the broad utility of immunotherapies. Epigenetic alterations have been found to alter expression of genes involved in antigen processing and presentation. We evaluated the ability of epigenetic modifying agents to increase expression of antigen presentation machinery (APM) molecules ex vivo drug culture using primary human prostate tumor tissue biopsies. Prostate tissue biopsies were collected from patients with advanced, localized prostate cancer undergoing radical prostatectomy. These tumor biopsies sectioned into 200uM slices and cultured with the hypomethylating agent decitabine (5AZA) and/or histidine-deacetylase inhibitor panobinostate (LBH589). After 72 hours of treatment, tissue slices were harvested and subjected to qRT-PCR analysis. 5AZA alone or in combination with LBH589 have increased expression of MHC Class I molecules (HLA-ABC, B2M) and APM elements including TAP2, Tapasin and LMP7. Increased expression of MHC class I molecules was identified in human prostate cancer treated with 5AZA and the combination of 5AZA and LBH589. Significant inter-patient heterogeneity was also observed, suggesting other mechanisms by which prostate cancer downregulates MHC class I expression. We further identify increased expression of Cancer-Testis Antigens in these samples, indicating multiple mechanisms by which epigenetic modifying agents may improve immune recognition of human prostate cancer. These include NY-ESO, SSX2 and NYSAR35. These results suggest novel therapeutic strategies that can be employed with other immune based therapies for men with localized and advanced prostate cancer.

Transient induction of ING4 by Myc drives prostate epithelial cell differentiation and its disruption drives prostate tumorigenesis.

The mechanisms by which Myc overexpression or Pten loss promotes prostate cancer development are poorly understood. We identified the chromatin remodeling protein, ING4, as a crucial switch downstream of Myc and Pten that is required for human prostate epithelial differentiation. Myc-induced transient expression of ING4 is required for the differentiation of basal epithelial cells into luminal cells, while sustained ING4 expression induces apoptosis. ING4 expression is lost in >60% of human primary prostate tumors. ING4 or Pten loss prevents epithelial cell differentiation, which was necessary for tumorigenesis. Pten loss prevents differentiation by blocking ING4 expression, which is rescued by ING4 re-expression. Pten or ING4 loss generates tumor cells that co-express basal and luminal markers, indicating prostate oncogenesis occurs through disruption of an intermediate step in the prostate epithelial differentiation program. Thus, we identified a new epithelial cell differentiation switch involving Myc, Pten, and ING4, which when disrupted leads to prostate tumorigenesis. Myc overexpression and Pten loss are common genetic abnormalities in prostate cancer, whereas loss of the tumor suppressor ING4 has not been reported. This is the first demonstration that transient ING4 expression is absolutely required for epithelial differentiation, its expression is dependent on Myc and Pten, and it is lost in the majority of human prostate cancers. This is the first demonstration that loss of ING4, either directly or indirectly through loss of Pten, promotes Myc-driven oncogenesis by deregulating differentiation. The clinical implication is that Pten/ING4 negative and ING4-only negative tumors may reflect two distinct subtypes of prostate cancer.

Abstract 3016: Implication of delta-catenin-promoted E-cadherin processing on human prostate cancer progression.

Abstract Although δ-catenin overexpression is frequently observed in some human cancers, the mechanistic implications of δ-catenin expression in epithelial cells and carcinomas remain elusive. In this study, we found that prostate cancer cells overexpressing δ-catenin show an increase in multi-layer growth in culture. In these cells, δ-catenin colocalizes with E-cadherin at the plasma membrane, and the E-cadherin processing is noticeably elevated. A deletion mutant of δ-catenin totally abolishes the δ-catenin-induced E-cadherin processing and the multi-layer growth of the cells. In addition, prostate cancer cells overexpressing δ-catenin display an elevated total β-catenin level and increase nuclear distribution, resulting in the activation of β-catenin/LEF-1-mediated transcription. Indeed, human prostate tumor xenograft in nude mice, which is derived from cells overexpressing δ-catenin, shows increased β-catenin nuclear localization and more rapid growth rates. Moreover, the metastatic xenograft tumor weights positively correlate with the level of 29 kD E-cadherin fragment, and primary human prostate tumor tissues also show elevated levels of δ-catenin expression and the E-cadherin processing. Taken together, these results suggest that δ-catenin plays an important role in prostate cancer progression through inducing E-cadherin processing and thereby activating β-catenin-mediated oncogenic signals. Citation Format: Hangun Kim, Somy Yoon, So-Yeon Park, Jun Eul Hwang, Kwonseop Kim, Kyung Keun Kim. Implication of delta-catenin-promoted E-cadherin processing on human prostate cancer progression. [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 3016. doi:10.1158/1538-7445.AM2013-3016 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 2793: Development of an androgen-dependent prostate Champions TumorGraft™ cancer model.

Abstract Prostate cancer (PCa)is the second most frequently diagnosed cancer and the sixth leading cause of cancer death in males, with the highest incidence rates recorded in developed countries such as those in North America (Jemal et al., 2011). While the five year-survival rate for PCa patients with localized disease is 100%, it is only 30.6% once the cancer metastasizes (Vishnu et al., 2010). One of the biggest challenges for finding a better treatment for metastatic PCa is the lack of predictable and accurate preclinical models that closely recapitulate different stages of the pathogenesis of human PCa. Champions Oncology focuses on the development of Champions TumorGraft™ models derived from the direct implantation of patient tumors into immunocompromised mice. Compared to traditional cell line-based xenograft models, the patient-derived TumorGraft models maintain stable gene-expression patterns and mutational status and correlate to clinical predictability. To overcome the challenge for PCa, Champions Oncology has utilized its TumorGraft technology to establish and characterize a new androgen-dependent prostate cancer TumorGraft model, CTG-0488. This involved the implantation of a primary human prostate tumor in immunocompromised mice in a manner that preserves the biological properties of the original human tumor. In this preliminary work, the characteristics of Champion TumorGraft™ model CTG-0488 are showcased, including tumor histology, mutational status, and chemosensitivity profiles to several standard of care compounds. Overall the development of an androgen-dependent prostate cancer Champions TumorGraft™ model will significantly aid in the development of oncology compounds focused on androgen-dependent prostate cancer. Citation Format: Tin Oo Khor, Keren Paz, Dhanrajan Tiruchinapalli, David Sidransky, Elizabeth M. Bruckheimer. Development of an androgen-dependent prostate Champions TumorGraft™ cancer model. [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 2793. doi:10.1158/1538-7445.AM2013-2793

Abstract 3213: Selective killing of advanced prostate cancer cells by targeting the Warburg effect and autophagy

Abstract Mutations of multiple tumor suppressor genes, such as PTEN and p53, have been proposed to play important roles in the development of prostate cancer. Loss of one allele of PTEN occurs in 70-80% of human primary prostate tumors and homozygous inactivation of PTEN is associated with advanced disease. Similarly, p53 is found completely lost or mutated almost exclusively in advanced human prostate cancer. Thus, selective killing of prostate cancer cells harboring mutations of PTEN and p53 may prove to be a promising strategy for the treatment of advanced prostate cancer. The Warburg effect of aerobic glycolysis has now been generally accepted as a key metabolic hallmark of cancer. In this study, we investigated the molecular target leading to the Warburg effect in the growth and aggressiveness of prostate cancer cells harboring inactivation of PTEN and p53 and delineated the underlying mechanism. We identified that expression of hexokinase II (HK2), an enzyme involved in the first step of glycolysis, is preferentially elevated in human prostate cancer cells bearing mutations of both PTEN and p53 (PC3). Functional studies demonstrated that HK2 expression is crucial for the Warburg effect in PC3 cells and knockdown of HK2 inhibits tumor growth in PC3 xenograft mouse model. These novel findings prompted us to test whether 2-de-oxyglucose (2-DG), an inhibitor of HK2, could potentially suppress prostate cancer growth by targeting the Warburg effect. We found that an induction of AMPK-dependent autophagy prevents cancer cells from apoptosis upon 2-DG treatment, thereby limiting therapeutic efficacy on prostate cancer in vivo. Consistent with cell survival function of autophagy, its inhibition by chloroquine (a small molecule inhibitor of autophagy) or individual knockdown of the essential genes involved in autophagy (Atg5, Atg7, Beclin1, and ULK1) induced massive cell death when combined with 2-DG. This cell death can be rescued by overexpression of anti-apoptotic protein Bcl-2 or downregulation of pro-apoptotic protein Bax in PC3. More importantly, we demonstrated that combination of chloroquine and 2-DG could specifically kill prostate cancer cells, leaving normal prostate epithelial cells untouched. This specificity is due to the preferential induction of HK2 through the activated Rictor-AKT-mTOR pathway in cancer cells. Finally, combination of chloroquine and 2-DG caused synthetic lethality in prostate cancer cells and effectively suppressed tumor growth in PC3 xenograft mouse model. Towards therapeutic translation, we have observed that expression of HK2 by staining primary human prostate tumor samples with HK2 antibodies correlated with the stages of prostate cancer. Given our findings, we therefore propose that targeting the Warburg effect and autophagy pathways may serve as an effective and selective treatment for patients with advanced prostate cancer, in particular those with PTEN and p53 mutations. 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 3213. doi:1538-7445.AM2012-3213

Abstract C23: MicroRNA-106b targets caspase-7 and is associated with recurrence in human prostate cancer

Abstract Previously, we found several microRNA (miR) of the polycistronic miR-106b-25 cluster to be elevated in prostate cancer. This cluster is a homolog to the prototypical oncomiR-1 (miR-17-92) and is thought to act as a super-oncogene when co-amplified with its host gene minichromosome complex maintenance component 7 (MCM7). In this study we validated our previous findings in an independent prostate cancer dataset and further found that miR-106b is elevated in metastatic tissues and associated with disease recurrence. Genome-wide analysis of miR-106b-25 correlated transcripts revealed enrichment of focal adhesion and ECM-receptor interaction genes. Further, we found miR-106b target enrichment in transcripts that were protective against recurrence, suggesting that miR-106b targets collectively have a functionally protective role in the recurrence of prostate cancer. In cell based assays, overexpression of miR-106b increased proliferation, adhesion-free survival, soft agar survival, and adhesion to ECM components (laminin, and collagen type I and IV). In addition, microarray analysis showed that miR-106b down-regulated transcripts were enriched for miR-106b targets sites and cell death pathways genes. When we compared genes that were related to recurrence, altered by miR-106b, and predicted to have miR-106b 3′UTR target sites we identified CASP7, a central executioner of apoptosis. Corroborating these findings we found that the expression of CASP7 was consistently reduced in primary human prostate tumors and metastatic lesions across multiple prostate cancer datasets, and is by itself a candidate predictor of disease recurrence. Western blot analysis in RWPE-1, 22Rv1, and LNCaP prostate cells showed that miR-106b was capable of reducing CASP7 protein levels. Direct targeting of the CASP7 3′UTR by miR-106b was confirmed using wild-type and mutant 3′UTR luciferase reporter assays. This research reveals a novel oncogenic role for the miR-106b-25 cluster in prostate cancer and, for the first time, indentifies CASP7 as a direct target of miR-106b. Future work is aimed at understanding the role of miR-106b-dependent regulation of CASP7 and the possible utility of targeting miR-106b as novel therapy for prostate cancer. Citation Format: Robert S. Hudson, Tiffany H. Dorsey, Ming Yi, Dominic Esposito, Robert M. Stephens, Carlo M. Croce, Stefan Ambs. MicroRNA-106b targets caspase-7 and is associated with recurrence in human 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 C23.

δ-Catenin promotes E-cadherin processing and activates β-catenin-mediated signaling: Implications on human prostate cancer progression

δ-Catenin binds the juxtamembrane domain of E-cadherin and is known to be overexpressed in some human tumors. However, the functions of δ-catenin in epithelial cells and carcinomas remain elusive. We found that prostate cancer cells overexpressing δ-catenin show an increase in multi-layer growth in culture. In these cells, δ-catenin colocalizes with E-cadherin at the plasma membrane, and the E-cadherin processing is noticeably elevated. E-Cadherin processing induced by δ-catenin is serum-dependent and requires MMP- and PS-1/γ-secretase-mediated activities. A deletion mutant of δ-catenin that deprives the ability of δ-catenin to bind E-cadherin or to recruit PS-1 to E-cadherin totally abolishes the δ-catenin-induced E-cadherin processing and the multi-layer growth of the cells. In addition, prostate cancer cells overexpressing δ-catenin display an elevated total β-catenin level and increase its nuclear distribution, resulting in the activation of β-catenin/LEF-1-mediated transcription and their downstream target genes as well as androgen receptor-mediated transcription. Indeed, human prostate tumor xenograft in nude mice, which is derived from cells overexpressing δ-catenin, shows increased β-catenin nuclear localization and more rapid growth rates. Moreover, the metastatic xenograft tumor weights positively correlate with the level of 29kD E-cadherin fragment, and primary human prostate tumor tissues also show elevated levels of δ-catenin expression and the E-cadherin processing. Taken together, these results suggest that δ-catenin plays an important role in prostate cancer progression through inducing E-cadherin processing and thereby activating β-catenin-mediated oncogenic signals.

Prostate cancer stem cells: The case for model systems

Advanced prostate cancers are treated with androgen deprivation therapy, which usually leads to a rapid and significant reduction in tumor burden but subsequent development of castration-resistant and metastatic disease almost always occurs. The source of tumor heterogeneity and the accompanying mechanisms leading to treatment resistance are major areas of prostate cancer research. Although our understanding of tumor heterogeneity is evolving, the functional isolation of tumor propagating populations, also known as cancer stem cells (CSCs), is fundamental to the identification and molecular characterization of castration-resistant prostate cancer cells. Of clinical importance, knowledge of prostate CSCs has implications for design of next generation-targeted therapies aimed at both eradicating primary tumor mass and preventing castration-resistant disease. The inability to routinely transplant fractionated primary human prostate tumors has prevented progress in analyzing the source of heterogeneous and treatment-resistant populations in prostate cancer. Here, we briefly overview the mechanisms of castration resistance, including the hypothesis for the existence of androgen-independent prostate CSCs. Finally, we discuss the interpretation of preclinical models and their utility for characterizing prostate CSCs in androgen-replete and androgen-deprived conditions.