MDA Prostate Cancer Research Articles

Deciphering the Nuclear Role of Heme Oxygenase-1 in Prostate Cancer: Transcriptional Reprogramming and Neuroendocrine Differentiation

PURPOSE This study probes the role of Heme Oxygenase-1 (HO-1) and its nuclear interactors in prostate cancer (PCa). Previous results, demonstrated the anti-tumoral role of HO-1 in PCa and its nuclear translocation, hinting at its potential regulatory roles within the nucleus. Thus, we postulate that HO-1, alongside its interactors, can reprogram PCa cells to a less aggressive phenotype. METHODS From the 11 HO-1 nuclear interacting proteins previously identified, 4 were transcription factors Using ChIP Atlas we evaluated the binding affinity of these factors to their downstream gene targets. Gene ontology and KEGG pathway analyses were then performed to associate these targets with specific biological processes and pathologies. Additionally, RNA-seq data from patient-derived xenografts (PDXs) at MD Anderson Cancer Center (MDA PCa PDXs) were analyzed to evaluate the clinical relevance of these genes in the context of PCa heterogeneity. These PDXs offer a clinically relevant model due to their representation of the disease's heterogeneity. We scrutinized the expression of the gene clusters of interest in PDXs, including their association with neuroendocrine (NE) phenotypes. RESULTS The transcription factors among the HO-1 interactors previously described, were found to bind genes enriched in processes related to histone modification, acetylation, and transcription termination, implicating their role in gene expression regulation. KEGG pathways linked these genes to neurodegenerative diseases (including Parkinson's, Huntington's, and Alzheimer's disease), mirroring the neuroendocrine (NE) phenotype in advanced PCa, which typically emerges in advanced stages and is characterized by the transdifferentiating of epithelial cells into NE cells lacking androgen receptor expression. The unsupervised clustering of gene expression from PDXs showed that high expression of these genes is associated with an AR-negative NE histopathology, suggesting a potential for these interactors in prostate cancer subtyping. CONCLUSION HO-1 nuclear interactors, especially transcription factors, appear to influence gene transcription associated with NE differentiation and cancer aggressiveness. The therapeutic relevance of modulating HO-1's interaction with its nuclear partners warrants further investigation to ascertain its role in mitigating the NE of PCa.

Abstract 3064: Unmasking the deadly tango: PKA and lipid genes in prostate cancer's bone invasion

Abstract About 88% of metastatic prostate cancer (PCa) cases involve bone metastasis. When PCa cells spread to the bone, a metabolic adaptation to the new environment takes place, partly influenced by factors secreted by the homing organ. Thus, we hypothesize that metabolic reprogramming, both in the tumor and bone niche drive PCa bone metastasis. In this work, we carried out an integrative in vitro, in vivo, and bioinformatics approach to identify key regulators governing the metabolic behavior of PCa cells. We performed an indirect co-culture between PCa (PC3) and bone progenitor (MC3T3 or Raw264.7) cells. PCa cells displayed a strong transcriptional (RNA-seq) activation of lipid metabolism, including PPAR-signaling, fat digestion, and PI3K-Akt pathway. Accordingly, treatment with the conditioned media (CM) of the co-culture promoted lipid accumulation in PC3 cells (Bodipy 493/503 staining). To assess the clinical relevance of the in vitro model, we performed an unsupervised clustering analysis using transcriptomics data from human PCa and bone metastatic samples (GSE74685). Results showed that the metabolic genes deregulated in PC3 by the co-culture, could accurately cluster samples in primary tumor or bone metastasis. We next performed a multivariable Cox regression analysis and built a prognostic model, identifying a novel signature of 5 lipid-associated genes, PPARA, VDR, SLC16A1, PAPSS2 and GPX1, associated with a 23-fold higher risk of death in PCa patients (SU2C-PCF dataset). The expression of this signature was validated in a PDX pre-clinical model (MDA PCa PDX series) when comparing MDA-PCa-183 growing intrafemorally vs. subcutaneously. In order to identify the central hubs regulating this transcriptional phenotype, we performed an Ingenuity Pathway Analysis (QIAGEN), showcasing that the lipid-related signature deregulated by the bone niche is indeed modulated by the Protein Kinase A (PKA). Accordingly, the expression of these signature was downregulated by PKA inhibition (H89; PKA inhibitor). Moreover, secretome analyses (ESI MS/MS) revealed that bone secreted type I collagen (Col1a1) and fibronectin (Fn1) regulate PKA activity in PCa cells. Further, we investigated whether PKA could modulate critical mechanisms for bone progression, focusing on osteopontin (SPP1), a secreted protein involved in the adhesion and invasion of PCa cells, whose regulatory mechanism in tumor cells is yet to be elucidated. Strikingly, we observed not only that the activation of PKA using Forskolin in PCa cells activated SPP1 expression, but also that PKA inhibition led to a significant impairment of the SPP1 induction triggered by bone soluble factors secreted during the PC3/MC3T3 co-culture. Altogether, we showcase PKA as a central hub of PCa energetic metabolism during bone progression and outline a lipid-associated gene signature that can be targeted to halt metastatic PCa. Citation Format: Pablo Sanchis, Nicolas Anselmino, Estefania Labanca, Agustina Sabater, Juan Bizzotto, Gaston Pascual, Rocio Seniuk, Sofia Lage-Vickers, Antonina Mitrofanova, Pia Valacco, Ayelen Toro, Javier Cotignola, Nora Navone, Elba Vazquez, Geraldine Gueron. Unmasking the deadly tango: PKA and lipid genes in prostate cancer's bone invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3064.

Prostate cancer cell-platelet bidirectional signaling promotes calcium mobilization, invasion and apoptotic resistance via distinct receptor-ligand pairs

Platelets play a crucial role in cancer and thrombosis. However, the receptor-ligand repertoire mediating prostate cancer (PCa) cell-platelet interactions and ensuing consequences have not been fully elucidated. Microvilli emanating from the plasma membrane of PCa cell lines (RC77 T/E, MDA PCa 2b) directly contacted individual platelets and platelet aggregates. PCa cell-platelet interactions were associated with calcium mobilization in platelets, and translocation of P-selectin and integrin αIIbβ3 onto the platelet surface. PCa cell-platelet interactions reciprocally promoted PCa cell invasion and apoptotic resistance, and these events were insensitive to androgen receptor blockade by bicalutamide. PCa cells were exceedingly sensitive to activation by platelets in vitro, occurring at a PCa cell:platelet coculture ratio as low as 1:10 (whereas PCa patient blood contains 1:2,000,000 per ml). Conditioned medium from cocultures stimulated PCa cell invasion but not apoptotic resistance nor platelet aggregation. Candidate transmembrane signaling proteins responsible for PCa cell-platelet oncogenic events were identified by RNA-Seq and broadly divided into 4 major categories: (1) integrin-ligand, (2) EPH receptor-ephrin, (3) immune checkpoint receptor-ligand, and (4) miscellaneous receptor-ligand interactions. Based on antibody neutralization and small molecule inhibitor assays, PCa cell-stimulated calcium mobilization in platelets was found to be mediated by a fibronectin1 (FN1)-αIIbβ3 signaling axis. Platelet-stimulated PCa cell invasion was facilitated by a CD55-adhesion G protein coupled receptor E5 (ADGRE5) axis, with contribution from platelet cytokines CCL3L1 and IL32. Platelet-stimulated PCa cell apoptotic resistance relied on ephrin-EPH receptor and lysophosphatidic acid (LPA)-LPA receptor (LPAR) signaling. Of participating signaling partners, FN1 and LPAR3 overexpression was observed in PCa specimens compared to normal prostate, while high expression of CCR1 (CCL3L1 receptor), EPHA1 and LPAR5 in PCa was associated with poor patient survival. These findings emphasize that non-overlapping receptor-ligand pairs participate in oncogenesis and thrombosis, highlighting the complexity of any contemplated clinical intervention strategy.

Opposite effects of CXCR1 and CXCR2 overexpression in prostate tumorigenesis

Abstract Chemokines and their receptors play important role in tumor progression and metastasis. In prostate cancer (PCa), some members of the CXC chemokine receptor have been shown to enhance angiogenesis, proliferation and metastasis. In this study we assess the roles of the interleukin-8 (IL-8/CXCL8) receptors CXCR1 and CXCR2 in prostate tumorigenesis, using MDA PCa 2b and LNCaP cell lines. Our results show that overexpression of CXCR2 enhanced in-vitro cell proliferation, soft agar growth and in-vivo tumor xenograft in nude mice, whereas overexpression of CXCR1 inhibited cell proliferation and tumor growth, relative to control cells. CXCR1 cells showed decrease prostate specific antigen (PSA) expression whereas CXCR2 exhibited decrease androgen receptor (AR) expression. Interestingly, tumor lysates from CXCR1 cells displayed significant increase in ERK and AKT activation as well as chemokines production relative to control and CXCR2 tumors. CXCR1 tumors also exhibited a more mesenchymal phenotype, characterized by reduced E-Cadherin but increased N-Cadherin and Vimentin expression relative to CXCR2 or control tumors. Altogether, these results indicate that CXCR1 and CXCR2 may couple to distinct pathways to modulate prostate tumorigenesis. Supported by CA156735, MD012392

Abstract 2364: MicroRNA-1205 directly targets ONECUT2 in neuroendocrine prostate cancer cells

Abstract One of six deaths globally is due to cancer. In 2018, prostate cancer (PCa) represented 1.28 million cases worldwide, the second most frequent cancer diagnosis and fifth leading cause of death in men. Currently, the mortality rate of PCa has experienced a steady increase due to the progression of metastatic castration-resistant prostate cancer (mCRPC). As an androgen driven disease, androgen deprivation therapies (ADTs) have been employed for treatment. However, despite ADTs, mCRPC develops by maintaining AR signaling, which can eventually lead to progression of AR-independent neuroendocrine PCa (NEPC). NEPC is observable in 1 in 5 men with mCRPC and associated with poor outcome. Therefore, further understanding the mechanisms of progression to mCRPC and NEPC is required. The 8q24 human chromosomal region associated with Increased PCa incidence and aggressiveness contains the PVT1 (Plasmocytoma Variant Translocation 1) locus. The PVT1 locus encodes six microRNAs, including microRNA-1205 (miR-1205) in PCa. Our laboratory previously confirmed that miR-1205 is underexpressed in PCa tissue in comparison to normal prostatic tissue and in CRPC cells in comparison to non-CRPC cells. To understand the molecular mechanism of miR-1205, we performed RNA pull down and RNA sequencing analysis to identify miR-1205 molecular targets in non-tumorigenic RWPE-1 prostate epithelial cells and PCa cells (MDA PCa 2b (metastatic PCa), PC-3 (NEPC), and C4-2B (CRPC)). We identified one cut homeobox 2 (ONECUT2 or OC2) as a direct molecular target of miR-1205. ONECUT2 is a master regulator of transcriptional factors that drives NEPC by through regulating hypoxia signaling or inducing neuroendocrine markers. ONECUT2 enrichment was approximately 3-fold higher in RWPE-1 (P=0.26), 2-fold in PC-3 (P=0.06), 1-fold in C4-2B (P=0.05) and MDA PCa 2b (P=0.02) cells. The significant binding of miR-1205 to ONECUT-2 in the PC-3 NEPC cell line suggests that miR-1205 regulation of ONECUT2 may be an important molecular mechanism in NEPC. Consequently, further study of miR-1205 and ONECUT2 in NEPC is required. Citation Format: Bachelard Dieujuste, Michelle Naidoo, Olorunseun Ogunwobi. MicroRNA-1205 directly targets ONECUT2 in neuroendocrine prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2364.

Prostac: A New Composite Score With Potential Predictive Value in Prostate Cancer.

Prostate cancer (PCa) is the most commonly diagnosed solid organ cancer in men worldwide. Current diagnosis of PCa includes use of initial prostate specific antigen assay which has a high false positive rate, low specificity, and low sensitivity. The side effects of unnecessary prostate biopsies that healthy men are subjected to, often result in unintended health complications. New PCa biomarkers are being discovered to address this unmet need. Here, we report on the creation of a composite score (Prostac) based on three recently discovered PCa biomarkers, Plasmacytoma Variant Translocation 1 (PVT1) exons 4A, 4B, and 9. Statistical analysis of copy numbers derived from a real-time quantitative polymerase chain (qPCR) reaction - based assay, showed these PCa biomarkers to be linearly separable and significantly over expressed in PCa epithelial cells. We train a supervised learning algorithm using support vector machines to generate a classification hyperplane from which a user-friendly composite score is developed. Cross validation of Prostac using data from prostate epithelial cells (RWPE1) and PCa cells (MDA PCa 2b) accurately classified 100% of PCa cells. Creation of the Prostac score lays the groundwork for clinical trial of its use in PCa diagnosis.

Host versus cell-dependent effects of β-arrestin 1 expression in prostate tumorigenesis.

Prostate cancer (PCa) constitutes a serious health challenge and remains one of the main causes of cancer-related death among men. The more aggressive form of the disease has been attributed to androgen independence, resulting in a lack of response to androgen deprivation therapy and sustained activation of other growth pathways. The scaffold proteins β-arrestin 1 and 2 (βarr1 and βarr2), which are known to mediate G protein-coupled receptor desensitization and internalization, were also shown to modulate prostate tumorigenesis. βarr1 is significantly overexpressed (>4-fold) in PCa cells relative to βarr2. In this study, we investigated the effect of βarr1 overexpression in PCa development and progression using the mouse and human PCa cell xenografts, and autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP) models deficient in β-arrestin depletion of βarr1 in TRAMP mice (TRAMP/βarr1-/-) increased PCa growth and decreased overall survival relative to control TRAMP or TRAMP/βarr2-/- animals. Prostate tissues from TRAMP/βarr1-/- tumors displayed an increase in androgen receptor (AR) expression, whereas overexpression of βarr1 in TRAMP-C1 (TRAMP-C1-βarr1-GFP) which derived from TRAMP decreased AR expression, cell proliferation and tumor growth in nude mice xenografts, relative to control TRAMP-C1-GFP. Knockdown of βarr1 expression in human MDA PCa 2b cells (MDA PCa 2b-βarr1-/-) also decreased AR expression cell proliferation and tumor growth relative to control (MDA PCa 2b-Sham) cells. Interestingly, both TRAMP-C1-βarr1-GFP and MDA PCa 2b-βarr1-/- xenografts showed a decrease in AKT phosphorylation but an increase in MAPK activation. Altogether, the data indicate that the effect of βarr1 in modulating AR signaling to regulate PCa aggressiveness is cell and host autonomous.

Abstract D064: Race-related RNA splicing dysregulation of PI3Kd signaling: A therapeutic target for aggressive prostate cancer

Abstract Background: Age-adjusted incidence and mortality rates for prostate cancer (PCa) among African Americans (AAs) are greater than among whites. The more aggressive characteristics of PCa in AAs contribute to the disparity, in addition to social determinants of health. Prior work has shown race-related differential RNA splicing of PI3Kd in PCa tissue and a novel short RNA splice variant of PI3Kd enriched in PCa from AAs. This variant drives PCa aggressiveness and associates with poorer survival for PCa. PCa cells engineered to overexpress the PI3Kd variant enriched in PCa from AAs are resistant to CAL-101, a PI3Kd inhibitor, and those overexpressing the PI3Kd variant enriched in PCa from whites are sensitive to CAL-101. Available inhibitors that target race-related RNA splicing dysregulated pathways in PCa represent potential novel therapeutic strategies for aggressive PCa. Methods: We identified a panel of PI3Kd inhibitors of varying subunit specificity, which are available and/or currently being tested in other diseases. A panel of non-engineered PCa cell lines derived from AAs or whites were treated with the inhibitors and resulting alterations in proliferation were assessed using an Incucyte Live-Cell Imaging System. The cell lines were tested for baseline RNA and protein levels of total PI3K and levels of each PI3K subunit. After treatment, cells were tested for RNA levels of targets in pathways that cross talk with the PI3K pathway and downstream targets of PI3K signaling. Results: The seven non-engineered PCa cell lines derived from AAs or whites varied in PI3Kd subunit expression across a 60-fold range. A panel of seven PI3Kd inhibitors varied in subunit specificity for PI3Kd, with PI3Kg (lowly expressed) being the most common secondary target. Specificity for all other subunits ranged from 5-19,000x relative to PI3Kd IC50. LNCaP and LN95 cell lines had the highest baseline expression of PI3Kd and the largest inhibition of proliferation in response to the highly PI3Kd-specific inhibitors, parsaclisib and idelalisib, whereas 22RV1, MDA PCa 2b and VCaP cell lines had the lowest baseline expression of PI3Kd and the smallest response to the highly PI3Kd-specific inhibitors. However, none of the cell lines exhibited sensitivity to umbralisib, a highly PI3Kd-specific inhibitor, suggesting that subunit baseline expression is not the only factor determining sensitivity. All cell lines exhibited marked proliferation inhibition in response to copanlisib and LY3023414, which have less subunit specificity and/or known off-target effects. Further studies of effects of these inhibitors on oncogenic signaling and PCa cell biology are underway, including evaluation of inhibitor activity in PCa patient-derived xenografts derived from AAs or whites. Conclusions: PI3Kd inhibitors have a potential therapeutic role in PCa dependent on the PI3K subunit status of the tumor. PI3Kd inhibition has potential as a novel therapeutic strategy to improve outcomes for men with PCa driven by this mechanism. Citation Format: Bonnie L LaCroix, Daniel J George, Jennifer A Freedman, Steven R Patierno. Race-related RNA splicing dysregulation of PI3Kd signaling: A therapeutic target for aggressive prostate cancer [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr D064.

Abstract B70: Thymoquinone regulates cytochrome P450 genes involved in prostate cancer disparity

Abstract African American (AA) men have a higher incidence and greater mortality from prostate cancer (PCa) than Caucasian American (CA) men. The factor influencing the racial disparity is not clearly understood and is probably genetic variation attributed to the disease. Androgen and androgen receptor (AR) pathways have long been associated with prostate growth. Racial differences have also been found among variants of the genes of the enzymes involved in androgen biosynthesis and metabolism, such as CYP3A4, CYP3A7, and CYP17A1. Although several inhibitors are approved for CYPs genes, the major drawback of these inhibitors is they contain the steroid scaffold, which contributes to the undesirable side effects (dyspnea, edema, contusion, etc.) observed in patients. In this regard, using nonsteroid scaffolds such as natural compound acts as a more potent inhibitor and interacts more selectively with the cytochrome P450 family. In this study, our data showed the effect of natural compound thymoquinone (TQ), a constituent of Nigella sativa (black seed), on CYP3A4, CYP3A7, and CYP17A1 genes in PCa cells, and found that TQ-treated cells significantly downregulated the expression of CYP3A4, CYP3A7, and CYP17A1 in MDA PCa 2b and E006AA-hT (African American) compared to LNCaP (Caucasian) cell lines. These findings were further confirmed by flow cytometry, Western blots, and immunofluorescence. These studies suggested that TQ could be the potent inhibitor of the active sites of the cytochrome P450 enzymes, which are an important target in the treatment of PCa. Additionally, knowledge of the PCa susceptibility genes (CYPs) could be used to identify individuals at risk of developing PCa with poor outcomes for heightened screening or prevention modalities and to identify optimal treatment strategies for men of African descent. Citation Format: Santosh K. Singh, James W. Lillard, Jr., Rajesh Singh. Thymoquinone regulates cytochrome P450 genes involved in prostate cancer disparity [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr B70.

Abstract 539: Pro-proliferative function of the histone deacetylase SIRT3 in prostate cancer

Abstract Worldwide, prostate cancer (PCa) is the second most frequently diagnosed cancer and fifth leading cause of cancer death in males. The existing treatments, as well as surgical approaches, have not been fully effective either for prevention or treatment of PCa. This necessitates a need to intensify our efforts towards the understanding of genetics and mechanism(s) of PCa. This may lead to the identification of the novel molecular target(s) and mechanism-based approaches for the management of this neoplasm. Sirtuin-3 (SIRT3) is a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase primarily located in the mitochondria and known to play important roles in the regulation of a variety of cellular processes, including transcription and programmed cell death. The fact that SIRT3 can regulate several cellular processes those are critical in cancer cell proliferation, makes it a potential therapeutic target for cancer management. Moreover, SIRT3 is a central regulator of mitochondrial adaptive responses that relate to metabolic reprogramming of cancer cells. However, the role of SIRT3 in cancer, including PCa, is not well understood and it has been shown to act both as a tumor suppressor as well as a tumor promoter. In this study, we determined the role of SIRT3 in PCa, employing in vitro and ex vivo approaches. The first step in our efforts to understand the role of SIRT3 in PCa was to check the expression profile of SIRT3 in a panel of human PCa cell line (DU145, 22Rν1, PC3, LNCaP, C4-2, MDA PCa 2b, E006AA-Par and E006AA-hT) by RT-qPCR and immunoblot analyses. Compared to normal human prostate epithelial cells (NrPEC), PCa cells showed higher expression of SIRT3, both at mRNA and protein levels. Further, we determined the expression profile of SIRT3 by immunostaining of a tissue microarray (TMA) containing paraffin-embedded sections of 40 cases of prostate adenocarcinoma and 8 normal prostate tissues. Our data demonstrated a significant upregulation of SIRT3 in cancerous prostatic tissues compared to the normal tissues. We next determined the effect of chemical inhibition of SIRT3 using a recently described SIRT3 inhibitor viz. 4'-Bromo-Resveratrol (4BR), in human PCa cells (DU145 and 22Rν1). 4BR treatment at 10, 20 and 40 μM concentrations for 48 and 72 h resulted in a dose- and time-dependent decrease in cell growth and proliferation. Further, 4BR treatment resulted in a marked decrease in clonogenic survival of DU145 and 22Rν1 PCa cells. Furthermore, 4BR treatment resulted in a marked cleavage of PARP, an indicator of apoptosis induction; and a decrease in the level of PCNA, a marker of cellular proliferation, in human PCa cells. Overall, our data suggest a possible pro-proliferative function of SIRT3 in PCa. Further studies are underway to unravel the role and functional significance of SIRT3 during PCa development and progression. Citation Format: Chandra K. Singh, Gagan Chhabra, Minakshi Nihal, Kenneth A. Iczkowski, Nihal Ahmad. Pro-proliferative function of the histone deacetylase SIRT3 in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 539.

Abstract 2437: Novel metabolic adaptations support proliferation of African American prostate cancer cells under hypoxia

Abstract Hypoxia in prostate cancer (PCa) selects for aggressive clones and causes treatment failure, metastatic progression and mortality. Despite the key role of hypoxia in prostate carcinogenesis, little is known about the specific effect of hypoxia in African Americans, a race that faces a serious PCa-related health disparity with the highest incidence and mortality rate. We hypothesize that metabolic adaptation to hypoxia is one mechanism that contributes to PCa aggressiveness in African Americans. Therefore, in the present work we focused on the metabolic adaptations, both in vitro and in vivo, contributing to proliferation of African American PCa cells under hypoxic conditions. PCa cells from men of African American ancestry (E006AA-hT, MDA PCa 2b, WFCB17 and 22Rv1) showed proliferation and significantly higher clonogenicity under hypoxia (1% O2) compared to normoxia. In contrast, Caucasian PCa cells (LNCaP and PC3) showed significant growth inhibition under similar hypoxic condition. The proliferation under hypoxia in African American PCa cells was associated with higher beta-oxidation of lipids as measured by malate response in Oroboros Oxygraph system. In addition, [18F]-fluoro-4-thia-oleate ([18F]-FTO) uptake in African American PCa cells under hypoxia was more than 2-fold higher (p <0.001) compared to normoxic cells, confirming higher beta-oxidation in hypoxic PCa cells. Furthermore, African American PCa cells showed more than 2-fold (p <0.001) higher uptake of [11C]-acetate under hypoxia, suggesting increased fatty acid synthesis during hypoxia in African American PCa cells. This suggests that African American PCa cells may increase fatty acid synthesis under hypoxia to provide metabolic fuel. Fatty acid oxidation is associated with mitochondrial metabolism; accordingly, we observed higher oxidative phosphorylation, complex II activity and expression under hypoxia in African American PCa cells. Importantly, treatment with complex II inhibitor Atpenin A5 (1-2 microM) strongly reduced the clonogenicity (51%-79%, p <0.0001) of African American PCa cells under hypoxia. In vivo, mPET/CT imaging using [18F]-FTO and [18F]-fluoromisonidazole as PET tracers showed greater tumor uptake of both tracers in 22Rv1 tumors in male athymic nude mice, suggesting that hypoxia and fatty acid oxidation are associated with African American PCa growth. Furthermore, analyses of 22Rv1 tumor tissues by confocal microscopy showed higher level of carnitine palmitoyltransferase 1 (a key regulator of β-oxidation) and succinate dehydrogenase B (a subunit of complex II) in hypoxic areas (pimonidazole+) of the tumor. Taken together, these data demonstrate that proliferation of African American PCa cells under hypoxia is supported by high lipid beta-oxidation, lipid synthesis and complex II activity, providing a novel mechanism to promote disease aggressiveness. Citation Format: Gati Panigrahi, Prakash Praharaj, Kiran Sai, Gargi Mahapatra, Taylor Peak, Sierra Patterson, Hakeem Oufkir, Anthony Molina, Steven Kridel, Gagan Deep. Novel metabolic adaptations support proliferation of African American prostate cancer cells under hypoxia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2437.

Exosomal miR-141-3p regulates osteoblast activity to promote the osteoblastic metastasis of prostate cancer.

Exosomes from cancer cells, which contain microRNA and reach metastasis loci prior to cancer cells, stimulate the formation of a metastatic microenvironment. Previous studies have shown that exosomal miR-141-3p is associated with metastatic prostate cancer (PCa). However, the role and regulatory mechanism of miR-141-3p in the microenvironment of bone metastases require further study. In this study, we performed a series of experiments in vivo and in vitro to determine whether exosomal miR-141-3p from MDA PCa 2b cells regulates osteoblast activity to promote osteoblastic metastasis. We demonstrate that extracts obtained from cell culture supernatants contained exosomes and that miR-141-3p levels were significantly higher in MDA PCa 2b cell exosomes. Via confocal imaging, numerous MDA PCa 2b exosomes were observed to enter osteoblasts, and miR-141-3p was transferred to osteoblasts through MDA PCa 2b exosomes in vitro. Exosomal miR-141-3p from MDA PCa 2b promoted osteoblast activity and increased osteoprotegerin OPG expression. miR-141-3p suppressed the protein levels of the target gene DLC1, indicating its functional significance in activating the p38MAPK pathway. In animal experiments, exosomal miR-141-3p had bone-target specificity and promoted osteoblast activity. Mice injected with miR-141-3p-mimics exosomes developed apparent osteoblastic bone metastasis. Exosomal miR-141-3p from MDA PCa 2b cells promoted osteoblast activity and regulated the microenvironment of bone metastases, which plays an important role in the formation of bone metastases and osteogenesis damage in PCa. Clarifying the specific mechanism of bone metastasis will help generate new possibilities for the treatment of PCa.

Abstract 4139: Role of zinc transporters in prostate cancer and a potential association with racial disparity

Abstract Optimal intracellular zinc concentration is essential for many cellular functions as it serves as a catalytic and/or structural cofactor for a variety of proteins. Although, a number of proteins are tangled in regulating cellular zinc homeostasis, the most important are two protein families of zinc transporters, 14 members of solute carrier family 39 (SLC39A) and 10 members of solute carrier family 30 (SLC30A). These two families are known to transport zinc into- and out of- the cytoplasm, respectively. Prostate cells accumulate a high amount of zinc to sustain a metabolic condition unique to the prostate which is characterized by a truncation of the Krebs cycle and production of high amounts of citrate. Zinc depletion has frequently been noted in the progression of prostate cancer (PCa). Whether zinc transporters are a steering cause of zinc depletion in PCa development and progression and/or are key determinants in the racial disparity in PCa is not well studied. In this study, we determined the connection of zinc transporters (SLC39A 1-14 and SLC30A 1-10) in PCa, in the perspective of racial health disparity in human PCa samples taken from African-American (AA) and European-American (EA) patients and compared them with respective adjacent benign samples. We also assessed the mRNA level of zinc transporters in normal prostate epithelial cells (NrPEC and RWPE1) and among PCa cells derived from AA (MDA PCa 2b, E006AA-PAR, E006AA-HT) and EA patients (DU145, PC3, 22Rν1, LNCaP, C4-2B). In addition, we performed a dataset analysis of the Oncomine database for differential expression profile of zinc transporters in PCa versus normal prostate. We found that SLC39As mRNA levels were differentially expressed in PCa with a significant downregulation of SLC39A1, SLC39A10, SLC39A11, SLC39A13 and SLC39A14, and upregulation of SLC39A3, SLC39A5, SLC39A6 and SLC39A8. Further, SLC30As showed a significant downregulation of SLC30A5 and SLC30A6 and upregulation of SLC30A1, SLC30A9 and SLC30A10, in PCa. Further, compared to EA samples, the AA PCa showed an increasing trend of SLC39A5, SLC39A6 and SLC30A9. In addition, compared to AA samples, the EA PCa showed a trend of increasing SLC30A1 and SLC30A9 and decreasing SLC39A10. These data provide evidence that the zinc transporters may be linked to racial disparity of PCa in AA versus EA. Moreover, Ingenuity Pathway Analysis revealed regulatory interactions between zinc transporters and tumor suppressor/promoter genes proven to be modulated in PCa. These genes are HOXB13, ELAVL1, DIRAS3, ALPP, CSF2, CCL4, INSR, AKT, IL6, TGFBP1, CHI3L1, EGFR and HNF4A. Overall, our study provides interesting data about the expression profiles of zinc transporters and their interaction with tumor suppressor and promoter genes in PCa, which may offer novel strategies for the management of PCa by pharmacologically modulating zinc transporters. Citation Format: Chandra K. Singh, Kareem M. Malas, Caitlin Tydrick, Kenneth A. Iczkowski, Nihal Ahmad. Role of zinc transporters in prostate cancer and a potential association with racial disparity [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 4139. doi:10.1158/1538-7445.AM2017-4139

The AR/NCOA1 axis regulates prostate cancer migration by involvement of PRKD1.

Due to the urgent need for new prostate cancer (PCa) therapies, the role of androgen receptor (AR)-interacting proteins should be investigated. In this study we aimed to address whether the AR coactivator nuclear receptor coactivator 1 (NCOA1) is involved in PCa progression. Therefore, we tested the effect of long-term NCOA1 knockdown on processes relevant to metastasis formation. [(3)H]-thymidine incorporation assays revealed a reduced proliferation rate in AR-positive MDA PCa 2b and LNCaP cells upon knockdown of NCOA1, whereas AR-negative PC3 cells were not affected. Furthermore, Boyden chamber assays showed a strong decrease in migration and invasion upon NCOA1 knockdown, independently of the cell line's AR status. In order to understand the mechanistic reasons for these changes, transcriptome analysis using cDNA microarrays was performed. Protein kinase D1 (PRKD1) was found to be prominently up-regulated by NCOA1 knockdown in MDA PCa 2b, but not in PC3 cells. Inhibition of PRKD1 reverted the reduced migratory potential caused by NCOA1 knockdown. Furthermore, PRKD1 was negatively regulated by AR. Immunohistochemical staining of PCa patient samples revealed a strong increase in NCOA1 expression in primary tumors compared with normal prostate tissue, while no final conclusion could be drawn for PRKD1 expression in tumor specimens. Thus, our findings directly associate the AR/NCOA1 complex with PRKD1 regulation and cellular migration and support the concept of therapeutic inhibition of NCOA1 in PCa.

Abstract 3913: Fibroblast growth factor receptor 1 isoforms in prostate cancer bone metastases

Abstract Bone metastases dominate the clinical picture of men with advanced prostate cancer (PCa). The fibroblast growth factor (FGF)/FGF receptor (FGFR) complex is commonly altered during PCa progression. Four highly conserved genes (FGFR1, FGFR2, FGFR3, and FGFR4) encode a repertoire of alternatively spliced variants of FGFRs that vary in the extracellular ligand-binding and intracellular kinase domains. A previous study from our group implicated FGFR1 as a therapy target for PCa bone metastases (Sci Transl Med 2014; 6:252ra122). One of the most important mechanisms by which FGFRs determine specificity for different FGFs is by alternative exon usage of the immunoglobulin-like (Ig-like) motif of the extracellular domain. We analyzed FGFR1 transcripts in 183 human PCa samples and in PCa patient-derived xenografts (PDXs) assessed by RNA sequencing. We identified eight different protein coding transcript to be the most abundantly expressed, namely ENST00000326324; ENST00000356207; ENST00000397103 (with a predicted protein length of 731 to 733 aa) and ENST00000397091; ENST00000397108; ENST00000397113; ENST00000425967; ENST00000532791 (with a predicted protein length of 820 to 853aa). Probably reflecting FGFR1alpha/beta isoforms: FGFR1alpha, containing three Ig-like domains and FGFR1beta, containing only two Ig-like domains. Interestingly, different PCa tissue samples expressed different isoforms. We subsequently assessed, by real-time reverse transcription polymerase chain reaction, the expression of FGFR1alpha and beta in three PCa cell lines (PC3, DU145 and C4-2B) and seven PCa PDXs (MDA PCa 2b, MDA PCa 118b, MDA PCa 155-12; MDA PCa 146-10; MDA PCa 146-12; MDA PCa 150-3 and MDA PCa 183) derived from primary PCa, bone metastases and brain metastases and reflecting the typical adenocarcinoma as well as, adenocarcinomas with neuroendrocrine differentiation and small cell carcinomas of PCa. We found that all PDXs express primarily FGFR1alpha isoform while PCa cell lines express FGFR1beta. We stably transfected PC3 cells with FGFR1alpha (NM_023110.2) and FGFR1beta (NM_023105.2) isoforms. After clones were established, we placed the cells for 10 days in culture and found that tissue culture plates containing PC3 expressing FGFR1alpha had significantly more cells compared with dishes containing PC3 expressing FGF1beta isoform as assessed by direct cell counting in a phase contrast microscope. Furthermore, PC3 cells transfected with FGFR1 alpha display a more adhesive phenotype than cells transfected with FGFR1beta or empty vector as assessed by cell attachment in a 24-well plate. We conclude that FGFR1 isoforms are involved in the pathogenesis of PCa and can be used to develop markers of response to FGFR1 blockade. These results warrant further studies to fully understand the biological implications of FGFR1 isoforms in the pathogenesis of PCa. Citation Format: Estefania Labanca, Xinhai Wan, Jun Yang, Matthew Iyer, Christopher Logothetis, Arul Chinnaiyan, Nora Navone. Fibroblast growth factor receptor 1 isoforms in prostate cancer bone metastases. [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 3913. doi:10.1158/1538-7445.AM2015-3913

Hyperactivated FRS2α-mediated signaling in prostate cancer cells promotes tumor angiogenesis and predicts poor clinical outcome of patients

Metastasis of tumors requires angiogenesis, which is comprised of multiple biological processes that are regulated by angiogenic factors. The fibroblast growth factor (FGF) is a potent angiogenic factor and aberrant FGF signaling is a common property of tumors. Yet, how the aberration in cancer cells contributes to angiogenesis in the tumor is not well understood. Most studies of its angiogenic signaling mechanisms have been in endothelial cells. FGF receptor substrate 2α (FRS2α) is an FGF receptor-associated protein required for activation of downstream signaling molecules that include those in the mitogen-activated protein and AKT kinase pathways. Herein, we demonstrated that overactivation and hyperactivity of FRS2α, as well as overexpression of cJUN and HIF1α, were positively correlated with vessel density and progression of human prostate cancer (PCa) toward malignancy. We also demonstrate that FGF upregulated the production of vascular endothelial growth factor A mainly by increasing expression of cJUN and HIF1α. This then promoted recruitment of endothelial cells and vessel formation for the tumor. Tumor angiogenesis in mouse PCa tissues was compromised by tissue-specific ablation of Frs2α in prostate epithelial cells. Depletion of Frs2α expression in human PCa cells and in a preclinical xenograft model, MDA PCa 118b, also significantly suppressed tumor angiogenesis accompanied with decreased tumor growth in the bone. The results underscore the angiogenic role of FRS2α-mediated signaling in tumor epithelial cells in angiogenesis. They provide a rationale for treating PCa with inhibitors of FGF signaling. They also demonstrate the potential of overexpressed FRS2α as a biomarker for PCa diagnosis, prognosis and response to therapies.

Phase 1 study of the PSMA-tubulysin small-molecule drug conjugate EC1169 in pts with metastatic castrate-resistant prostate cancer (mCRPC).

2586Background: EC1169 is a small molecule drug conjugate designed to bind to prostate cancer (PCa) cells via prostate specific membrane antigen (PSMA) and, following endocytosis, to then release t...

P1.05 - A ph 1 study of 2 different schedules of the PSMA-tubulysin small-molecule drug conjugate EC1169 in pts with rec Met Cast-Resist PC (MCRPC)

ABSTRACT Background: EC1169 is a conjugate of a prostate-specific membrane antigen (PSMA) targeting moiety linked to tubulysin B hydrazide (TubBH) that specifically binds to PSMA-expressing tumor cells to exert its anti-tumor effect in vivo. The PSMA “target” that binds EC1169 is overexpressed on a majority of prostate cancer (PCa) cells. TubBH, a member of the tubulysin class of anti-neoplastic agents, exerts an anti-tumor effect by inhibiting the polymerization of tubulin into microtubules, thus blocking spindle formation and arresting cells in metaphase. Xenograft studies demonstrated good EC1169's antitumor activity with complete remissions and cures observed in mice bearing PSMA-positive LNCaP and MDA PCa 2b tumors. 99mTc-EC0652 has a 99mTc binding moiety linked to a DUPA moiety, which binds to PSMA on the surface of target cells, and has displayed more sustained retention in PCa masses than any previously described PCa imaging agent. Materials and Methods: Key inclusion criteria are age ≥18 years, ECOG performance status 0–1, and adequate organ function. Patients must have failed prior or ongoing androgen-deprivation therapy, progressed on abiraterone or enzalutamide, and previously treated with a taxane unless contraindicated. All patients undergo a 99mTc-EC0652 scan, and EC1169 is IV bolus administered TIW or QW on wks 1, 2 of a 3-week cycle, with the continuous reassessment method (CRM) used for dose escalations. The primary study objective is to determine EC1169 MTD and recommended phase 2 dose. Secondary objectives include evaluating EC1169 safety, pharmacokinetic profiles, antitumor activity, and its correlation with PSMA expression as measured by 99mTc-EC0652. Results: Five patients had been enrolled into 5 dose levels, 3 in the TIW, and 2 in the QIW schedule. The number of cycles administered ranged from 1 to 4, with no dose delay or omission due to toxicity. No DLT was observed and only TEAEs of grade 1, were reported (e.g. dysgeusia, arthralgia and anorexia). One pt had SD for 4 cycles, and four pts are still on study. Conclusions: Dose escalation is ongoing, currently at 0.45 mg/m2 for both the TIW and QIW schedules. Updated data will be presented at the conference.

Abstract 2030: Culturing patient-derived prostate cancer xenografts using a three-dimensional hydrogel system for drug testing and mechanistic studies

Abstract Advanced prostate cancer (PCa) typically metastasizes to bone and current treatments are mainly palliative. While a mechanistic understanding of PCa progression in bone is needed to develop new therapies, the complex phenotypes in PCa are poorly reflected by cell lines and cell line-based xenograft models, impeding progress in this endeavor. To overcome this limitation, a panel of PCa patient-derived xenograft (PDX) models was established at the University of Texas MD Anderson Cancer Center. While these PDX models better recapitulate the phenotypic signature of the original tumors as compared to cell lines, limitations exist, including the inability to manipulate these cells ex vivo before implantation in vivo and issues associated with the foreign animal physiology. Based on previous studies demonstrating the potential of using hyaluronan (HA)-based hydrogels for the 3D engineering of PCa cell line-derived ectotumors suitable for mechanistic studies, we evaluated the ability of these hydrogels to maintain viability of the PCa PDX tumors in vitro and optimize culture conditions. Using the MDA PCa 183 and 118b PDX tumors as proof-of-concept, tumors freshly harvested were digested and encapsulated within a HA-based hydrogel, comprised of thiol-modified HA crosslinked with poly(ethylene glycol)-diacrylate. Tumor cells remained as multicellular aggregates or ectotumors in 3D, and were viable even up to 2 weeks in culture. Furthermore, cells were positive for Ki-67, indicating that the hydrogel system is able to retain the proliferative capacity of the tumor cells. In probing for the androgen receptor (AR) expression, we found that while the hydrogel-encapsulated MDA PCa 183 cells stained positive for AR, it was not the case for the MDA PCa 118b, as would be expected in vivo, demonstrating that the hydrogel system is also able to preserve critical phenotypic characteristics of the original tumors. Lastly, to evaluate this novel system as a potential platform for rapid drug testing, we treated the 3D PDX ectotumors to docetaxel, and compared their response against a traditional bone metastatic PCa cell line, C4-2B. Interestingly, the 3D PDX ectotumors exhibited a significantly greater resistance to the drug than the C4-2B cells, reminiscent of chemoresistant tumors in vivo. We demonstrate for the first time that PCa PDX tumors are amenable to in vitro culture with the use of a 3D HA-based hydrogel system. The hydrogel supported the maintenance of multicellular ectotumors of PCa cells, which remained viable and phenotypically similar to the tumors of origin. Furthermore, a differential drug response to docetaxel was observed for both the 3D PCa PDX ectotumors tested as compared to the C4-2B cell line, the clinical relevance of which is currently being investigated. This model may have broad applicability for the culture of other bone metastatic cancers and enable mechanistic studies of disease biology. Citation Format: Eliza L.S. Fong, Mariane Martinez, Jun Yang, Leland M.K. Chung, Antonios G. Mikos, Nora M. Navone, Daniel A. Harrington, Mary C. Farach-Carson. Culturing patient-derived prostate cancer xenografts using a three-dimensional hydrogel system for drug testing and mechanistic studies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2030. doi:10.1158/1538-7445.AM2014-2030

Abstract 618: Implications of inhibition of steroid receptor co-activator-1 in human prostate cancer

Abstract Background: Prostate cancer (PCa) is still the most commonly diagnosed cancer and 2nd leading cause of death in the USA and the 3rd leading cause of death behind lung and colorectal cancers in Europe, respectively. Depending on the stage of the tumor different therapy options exist. Initially, those therapies show success and lead to tumor regression but eventually castration-resistant PCa (CRPC) develops. Androgen receptor (AR) is still active in CRPC through various mechanisms. One possibility of targeting the AR axis is inhibition of its co-regulators such as SRC-1, needed for efficient transcription of AR downstream genes. SRC-1, a member of the p160 coactivator family, is known to promote the transcriptional activity of steroid receptors like the AR as well as that of other non-steroidal transcription factors. Due to the fact that SRC-1 staining is increased in metastatic tissue of patients samples compared to clinically localized PCa our goal is to investigate the specific role of SRC-1 in PCa progression and metastatogenesis. Moreover, the question whether SRC-1 could be a target for a novel therapeutic approach will be addressed. Methods: Knock-down cell lines were generated using lentiviral-based RNAi technology. Western Blotting and qRT-PCR analysis were performed to verify knock-down efficiency. The proliferation of the different cell lines was addressed by 3H thymidine incorporation assays. Scratch (wound healing) and Boyden chamber assays were used to investigate the migratory and invasive behavior of the cell lines. Results: Immortalized benign and malignant prostate cell lines were screened for SRC-1 expression on mRNA and protein level, revealing that SRC-1 is expressed in all tested cell lines. Furthermore, knock down of SRC-1 in LNCaP and MDA PCa 2b (AR+) led to a markedly reduced AR transcriptional activity, which was verified by a reduced transcription of a downstream target gene prostate specific antigen. Whereas SRC-1 knockdown in MDA PCa 2b and PC3 cells did not show any effect on proliferation, depletion of SRC-1 negatively influenced cell proliferation of LNCaP cells. Moreover, we investigated the role of SRC-1 in the migratory and invasive behavior. While depletion of SRC-1 in PC3 and MDA PCa 2b cells significantly diminished migration, it had no effect on invasiveness. Conclusion: Together these findings demonstrate that SRC-1 is not only an important regulator of AR transcriptional activity but also affects other signaling pathways. Furthermore, SRC-1 seems to play a crucial role in migration but not in invasion and proliferation, depending on the cell lines used. Thus, SRC-1 may be a possible therapeutic target in metastatic prostate cancer, although its specific role has to be further investigated. Citation Format: Birgit Luef, Florian Handle, Frédéric R. Santer, Zoran Culig. Implications of inhibition of steroid receptor co-activator-1 in human prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 618. doi:10.1158/1538-7445.AM2014-618