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Prostate cancer (PCa) that progresses after androgen deprivation therapy (ADT) remains incurable. The underlying mechanisms that account for the ultimate emergence of resistance to ADT, progressing to castrate-resistant prostate cancer (CRPC), include those that reactivate androgen receptor (AR), or those that are entirely independent or cooperate with androgen signaling to underlie PCa progression. The intricacy of metabolic pathways associated with PCa progression spurred us to develop a metabolism-centric analysis to assess the metabolic shift occurring in PCa that progresses with low AR expression. We used PCa patient-derived xenografts (PDXs) to assess the metabolic changes after castration of tumor-bearing mice and subsequently confirmed main findings in human donor tumor that progressed after ADT. We found that relapsed tumors had a significant increase in fatty acids and ketone body (KB) content compared with baseline. We confirmed that critical ketolytic enzymes (ACAT1, OXCT1, BDH1) were dysregulated after castrate-resistant progression. Further, these enzymes are increased in the human donor tissue after progressing to ADT. In an in silico approach, increased ACAT1, OXCT1, BDH1 expression was also observed for a subset of PCa patients that relapsed with low AR and ERG (ETS-related gene) expression. Further, expression of these factors was also associated with decreased time to biochemical relapse and decreased progression-free survival. Our studies reveal the key metabolites fueling castration resistant progression in the context of a partial or complete loss of AR dependence.

Background African American (AA) patients have higher cancer mortality rates and shorter survival times compared to European American (EA) patients. Despite a significant focus on socioeconomic factors, recent findings strongly argue the existence of biological factors driving this disparity. Most of these factors have been described in a cancer-type specific context rather than a pan-cancer setting. Methods A novel in silico approach based on Gene Set Enrichment Analysis (GSEA) coupled to Transcription Factor enrichment was carried out to identify common biological drivers of pan-cancer racial disparity using The Cancer Genome Atlas (TCGA) dataset. Mitochondrial content in patient tissues was examined using a multi-cancer tissue microarray approach (TMA). Results Mitochondrial oxidative phosphorylation was uniquely enriched in AA tumors compared to EA tumors across various cancer types. AA tumors also showed strong enrichment for the ERR1-PGC1α-mediated transcriptional program, which has been implicated in mitochondrial biogenesis. TMA analysis revealed that AA cancers harbor significantly more mitochondria compared to their EA counterparts. Conclusions These findings highlight changes in mitochondria as a common distinguishing feature between AA and EA tumors in a pan-cancer setting, and provide the rationale for the repurposing of mitochondrial inhibitors to treat AA cancers.

Epigenetic changes that cause dysregulated gene expression during progression of androgen-independent prostate cancer (PCa) and metastatic skeletal lesions remain elusive. Here, we explored the role of histone demethylase NO66 in the pathogenesis of PCa and bone metastasis-related skeletal lesions. Tissue and cDNA microarrays of PCa were analyzed for NO66 mRNA and protein levels. We examined the effects of gain and loss of NO66 function on cell viability, colony formation, migration, invasion, and tumor-induced skeletal lesions in femoral bone. RNAseq and ChIPseq were performed to elucidate NO66-target genes in PCa. We report that NO66 levels were upregulated in advanced primary prostate tumors compared to normal tissue or tumors with low Gleason scores. Forced expression of NO66 promoted cell survival and invasion of PCa cells; whereas, knockdown of NO66 resulted in decreased cell survival and increased sensitivity to docetaxel. NO66-overexpressing PC3 cells implanted into the femoral bone of male SCID mice caused massive bone loss and stimulation of mouse osteoclast-promoting genes, including Dickkopf1, Cathepsin K, Nf-kβ,; and Calcr, suggesting a role for NO66 in tumor growth in bone and osteoclast activity. Combined RNAseq and ChIP-seq revealed that NO66 activates the survival gene MCL1, the invasion-associated genes IGFBP5 and MMP3, the pro-oncogenic genes CTNNB1 and CCND1, and the epigenetic modifier gene KMT2A in androgen-independent PCa. Our findings uncover the role of NO66 as a key oncogenic driver in PCa, causing osteolytic lesions through upstream epigenetic regulation of key genes for survival, invasion and metastasis, and pro-osteoclastic factors.

Unsupervised clustering and deconvolution analysis identifies three intrinsic subtypes of Metastatic Castration-Resistant Prostate Cancer (M-CRPC): AR pathway-positive Prostate Cancer (ARPC), Neuro Endocrine Prostate Cancer (NEPC), and a novel subtype endowed with hybrid epithelial/mesenchymal (E/M) and luminal progenitor-like traits (Mesenchymal and Stem-like PC, MSPC). Analysis of large patient datasets and in vitro studies support the notion that MSPC originates from ARPC as a consequence of therapy-induced lineage plasticity. Intriguingly, AR blockade instigates two separate and complementary processes: 1) transcriptional silencing of TP53 and hence acquisition of hybrid E/M and stem-like traits; and 2) inhibition of the BMP-SMAD pathway, which promotes resistance to the pro-apoptotic and anti-proliferative effects of AR inhibition. MSPC cell lines and prostate adenocarcinoma cells reprogrammed in vitro to MSPC exhibit a marked dependency on HER2/3 signaling. Moreover, combinations of the panHER inhibitor neratinib with enzalutamide or the mTORC1 inhibitor MLN0128 exhibit efficacy in preclinical models of mixed ARPC/MSPC or MSPC, respectively. Collectively, these results identify a novel subtype of M-CRPC, trace its origin to therapy induced lineage plasticity, and reveal its dependency on HER2/3 signaling.

Purpose: Advances in prostate cancer lag behind other tumor types partly due to the paucity of models reflecting key milestones in prostate cancer progression. Therefore, we develop clinically relevant prostate cancer models. Experimental Design: Since 1996, we have generated clinically annotated patient-derived xenografts (PDXs; the MDA PCa PDX series) linked to specific phenotypes reflecting all aspects of clinical prostate cancer. Results: We studied two cell line–derived xenografts and the first 80 PDXs derived from 47 human prostate cancer donors. Of these, 47 PDXs derived from 22 donors are working models and can be expanded either as cell lines (MDA PCa 2a and 2b) or PDXs. The histopathologic, genomic, and molecular characteristics (androgen receptor, ERG, and PTEN loss) maintain fidelity with the human tumor and correlate with published findings. PDX growth response to mouse castration and targeted therapy illustrate their clinical utility. Comparative genomic hybridization and sequencing show significant differences in oncogenic pathways in pairs of PDXs derived from different areas of the same tumor. We also identified a recurrent focal deletion in an area that includes the speckle-type POZ protein-like (SPOPL) gene in PDXs derived from seven human donors of 28 studied (25%). SPOPL is a SPOP paralog, and SPOP mutations define a molecular subclass of prostate cancer. SPOPLdeletions are found in 7% of The Cancer Genome Atlas prostate cancers, which suggests that our cohort is a reliable platform for targeted drug development. Conclusions: The MDA PCa PDX series is a dynamic resource that captures the molecular landscape of prostate cancers progressing under novel treatments and enables optimization of prostate cancer–specific, marker-driven therapy.

Ataxia telangiectasia mutated and RAD3 related (ATR) protein kinase plays critical roles in ensuring DNA replication, DNA repair, and cell cycle control in response to replication stress, making ATR inhibition a promising therapeutic strategy for cancer treatment. To identify genes whose loss makes tumor cells hypersensitive to ATR inhibition, we performed CRISPR/Cas9-based whole-genome screens in 3 independent cell lines treated with a highly selective ATR inhibitor, AZD6738. These screens uncovered a comprehensive genome-wide profile of ATR inhibitor sensitivity. From the candidate genes, we demonstrated that RNASEH2 deficiency is synthetic lethal with ATR inhibition both in vitro and in vivo. RNASEH2-deficient cells exhibited elevated levels of DNA damage and, when treated with AZD6738, underwent apoptosis (short-time treated) or senescence (long-time treated). Notably, RNASEH2 deficiency is frequently found in prostate adenocarcinoma; we found decreased RNASEH2B protein levels in prostate adenocarcinoma patient-derived xenograft (PDX) samples. Our findings suggest that ATR inhibition may be beneficial for cancer patients with reduced levels of RNASEH2 and that RNASEH2 merits further exploration as a potential biomarker for ATR inhibitor-based therapy.

An abrupt increase in metastatic growth as a consequence of the removal of primary tumors suggests that the concomitant resistance (CR) phenomenon might occur in human cancer. CR occurs in murine tumors and ROS-damaged phenylalanine, meta-tyrosine (m-Tyr), was proposed as the serum anti-tumor factor primarily responsible for CR. Herein, we demonstrate for the first time that CR happens in different experimental human solid tumors (prostate, lung anaplastic, and nasopharyngeal carcinoma). Moreover, m-Tyr was detected in the serum of mice bearing prostate cancer (PCa) xenografts. Primary tumor growth was inhibited in animals injected with m-Tyr. Further, the CR phenomenon was reversed when secondary implants were injected into mice with phenylalanine (Phe), a protective amino acid highly present in primary tumors. PCa cells exposed to m-Tyr in vitro showed reduced cell viability, downregulated NFκB/STAT3/Notch axis, and induced autophagy; effects reversed by Phe. Strikingly, m-Tyr administration also impaired both, spontaneous metastasis derived from murine mammary carcinomas (4T1, C7HI, and LMM3) and PCa experimental metastases. Altogether, our findings propose m-Tyr delivery as a novel approach to boost the therapeutic efficacy of the current treatment for metastasis preventing the escape from tumor dormancy. Fil: Gueron, Geraldine. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Anselmino, Nicolás. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Chiarella, Paula. Academia Nacional de Medicina de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Ortiz, Emiliano Germán. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Lage Vickers, Sofia. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Paez, Alejandra. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Giudice, Jimena. University Of North Carolina School Of Medicine; . Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Contin, Mario Daniel. Universidad de Buenos Aires; Argentina Fil: Leonardi, Daiana Beatriz. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Jaworski, Felipe Martín. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Manzano, Veronica Elena. University Of North Carolina School Of Medicine; Fil: Strazza, Ariel Ramiro. Academia Nacional de Medicina de Buenos Aires; Argentina Fil: Montagna, Daniela Romina. Academia Nacional de Medicina de Buenos Aires; Argentina Fil: Labanca, Estefania. Roswell Park Cancer Institute; Fil: Cotignola, Javier Hernan. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Daccorso, Norma. Universidad de Buenos Aires; Argentina Fil: Woloszynska-Read, Anna. University Of Texas Md Anderson Cancer Center; Fil: Navone, Nora. Roswell Park Cancer Institute; Fil: Meissl, Roberto Jose. Academia Nacional de Medicina de Buenos Aires; Argentina Fil: Ruggiero, Raul Alejandro. Academia Nacional de Medicina de Buenos Aires; Argentina Fil: Vazquez, Elba Susana. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina

BACKGROUND: While it has been challenging to establish prostate cancer patient-derived xenografts (PDXs), with a take rate of 10-40% and long latency time, multiple groups throughout the world have developed methods for the successful establishment of serially transplantable human prostate cancer PDXs using a variety of immune deficient mice. In 2014, the Movember Foundation launched a Global Action Plan 1 (GAP1) project to support an international collaborative prostate cancer PDX program involving eleven groups. Between these Movember consortium members, a total of 98 authenticated human prostate cancer PDXs were available for characterization. Eighty three of these were derived directly from patient material, and 15 were derived as variants of patient-derived material via serial passage in androgen deprived hosts. A major goal of the Movember GAP1 PDX project was to provide the prostate cancer research community with a summary of both the basic characteristics of the 98 available authenticated serially transplantable human prostate cancer PDX models and the appropriate contact information for collaborations. Herein, we report a summary of these PDX models. METHODS: PDX models were established in immunocompromised mice via subcutaneous or subrenal-capsule implantation. Dual-label species (ie, human vs mouse) specific centromere and telomere Fluorescence In Situ Hybridization (FISH) and immuno-histochemical (IHC) staining of tissue microarrays (TMAs) containing replicates of the PDX models were used for characterization of expression of a number of phenotypic markers important for prostate cancer including AR (assessed by IHC and FISH), Ki67, vimentin, RB1, P-Akt, chromogranin A (CgA), p53, ERG, PTEN, PSMA, and epithelial cytokeratins. RESULTS: Within this series of PDX models, the full spectrum of clinical disease stages is represented, including androgen-sensitive and castration-resistant primary and metastatic prostate adenocarcinomas as well as prostate carcinomas with neuroendocrine differentiation. The annotated clinical characteristics of these PDXs were correlated with their marker expression profile. CONCLUSION: Our results demonstrate the clinical relevance of this series of PDXs as a platform for both basic science studies and therapeutic discovery/drug development. The present report provides the prostate cancer community with a summary of the basic characteristics and a contact information for collaborations using these models.

Prostate cancer (PCa) incidence and mortality is nearly twice that in Black men than any other race (Non-Hispanic White, Asian/Pacific Islander, American Indian/Alaska Native, Hispanic (any race)). Characterization of the underlying biological factors that contribute to this cancer health disparity (CHD) is required to close the gap and improve patient outcome for Black men. In vitro PCa research tools which reflect the racial/ethnic diversity of this patient population are urgently needed, as most PCa cell lines are of Non-Hispanic White-origin. PCa patient-derived xenografts (PDXs) retain many of the characteristics of patient tumors and can be isolated from specific racial/ethnic groups to address this concern. Rodent PDX models provide a valuable resource for studying human cancer, however recent trends in reducing animal usage have instituted a search for alternative methods for studying PDXs that also maintain their complexity. While 2D in vitro culture of PCa PDXs has been largely unsuccessful, recent evidence suggests 3D in vitro culture of PCa PDXs may enable better long-term culture of the tumor cells. Here, we present a racially/ethnically diverse PCa PDX library of specimens (Black, Non-Hispanic White, Hispanic) developed at MD Anderson Cancer Center (the MDA PCa PDX series) compatible with 3D in vitro culture. In order to confirm self-reported race/ethnicity, each PCa PDX was characterized by whole-exome sequencing and compared to reference populations for a genetic ancestry estimation. For 3D in vitro culture, we utilized a high throughput microfluidic culture platform with 96 chips, the MIMETAS OrganoPlate® 2-lane. This platform suits both 3D tissue/cell model development and throughput needs required for drug discovery. MDA-PCa PDX cell clusters were suspended in hyaluronic acid-based hydrogel solutions, seeded into the OrganoPlate, and cultured under continuous perfusion. Genetic ancestry estimation studies revealed that patient-reported race/ethnicity often aligned with the same racial/ethnic population genetic signatures. For example, two self-reported Black PDXs were primarily of West African origin (74.6-84.6%). When cultured in 3D, PCa PDX cultures were stable and viable for at least 7 days, as determined by high content fluorescence imagining coupled with cell viability dyes. By immunofluorescent staining, PCa PDX cultures exhibited appropriate expression of phenotypic prostate-specific antigen (PSA) and androgen receptor (AR) which was maintained over the life of the culture. Studies are ongoing to screen a panel of chemotherapy drugs and determine the predictivity of the platform. This well characterized racially/ethnically diverse PCa PDX library, together with the 3D in vitro platform and methods is a valuable resource for evaluating population-based tumor response. Citation Format: Peter Shepherd, Andrei Bonteanu, Stanley Hooker, Kristin Bircsak, Divya Iyer, Dwayne Dexter, Daniel A. Harrington, Rick Kittles, Nora M. Navone. 3D in vitro prostate cancer PDX resource for studying cancer health disparities [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 2625.

Cell division cycle 6 (CDC6), an androgen receptor (AR) target gene, is implicated in regulating DNA replication and checkpoint mechanisms. CDC6 expression is increased during prostate cancer (PCa) progression and positively correlates with AR in PCa tissues. AR or CDC6 knockdown, together with AZD7762, a Chk1/2 inhibitor, results in decreased TopBP1-ATR-Chk1 signaling and markedly increased ataxia-telangiectasia-mutated (ATM) phosphorylation, a biomarker of DNA damage, and synergistically increases treatment efficacy. Combination treatment with the AR signaling inhibitor enzalutamide (ENZ) and the Chk1/2 inhibitor AZD7762 demonstrates synergy with regard to inhibition of AR-CDC6-ATR-Chk1 signaling, ATM phosphorylation induction, and apoptosis in VCaP (mutant p53) and LNCaP-C4-2b (wild-type p53) cells. CDC6 overexpression significantly reduced ENZ- and AZD7762-induced apoptosis. Additive or synergistic therapeutic activities are demonstrated in AR-positive animal xenograft models. These findings have important clinical implications, since they introduce a therapeutic strategy for AR-positive, metastatic, castration-resistant PCa, regardless of p53 status, through targeting AR-CDC6-ATR-Chk1 signaling.

Analysis of next generation transcriptome sequencing data of prostate cancer identified a novel gene fusion formed by the fusion of a protein coding gene (KLK4) with a non-coding pseudogene (KLKP1) and expression of its cognate protein. Screening of 659 prostate cancer TMA showed about 32% of positive cases predominantly expressed in higher Gleason grade tumors. Concomitant expression with ERG but not with SPINK1 and other ETS fusion positive tumors. Fusion gene expression potentially regulated by AR and ERG. Antibody specific to the KLK4-KLKP1 fusion protein was validated by immunohistochemistry and western blot methods. Oncogenic properties were validated by in vitro and in vivo functional studies. Clinical data analysis shows significant association with prostate cancer in young men and overall survival analysis indicate favorable prognosis. Non-invasive detection in urine samples has been confirmed. Taken together, we present a novel biomarker for routine screening of high Gleason grade prostate cancer at diagnosis.SIGNIFICANCEWe discovered and validated a novel prostate cancer (PCa) specific fusion gene involving a protein coding (KLK4) and a pseudogene (KLKP1) and its cognate protein. The unique feature of this fusion gene is the conversion of the noncoding pseudogene into a protein coding gene and its unique expression only in about 30% of high Gleason grade PCa. Expression of this gene is found to be concomitant in ERG fusion positive prostate cancer but mutually exclusive with SPINK1, ETV1, ETV4 and ETV5 positive tumors. Like other ETS family gene fusions, KLK4-KLKP1 can be detected in the urine samples of patients with prostate cancer enabling non-invasive detection of high Gleason grade prostate cancer. Given the unique feature of this fusion oncogenic potential, high Gleason grade specific expression and noninvasive detection, this novel gene fusion has a potential to be used as a biomarker for early detection of high-grade prostate cancer and a therapeutic target.

Next-generation antiandrogen therapies, such as enzalutamide and abiraterone, have had a profound impact on the management of metastatic castration-resistant prostate cancer (mCRPC). However, mCRPC patients invariably develop resistance to these agents. Here, a series of clonal cell lines were developed from enzalutamide-resistant prostate tumor xenografts to study the molecular mechanism of resistance and test their oncogenic potential under various treatment conditions. Androgen receptor (AR) signaling was maintained in these cell lines, which acquired potential resistance mechanisms, including expression of AR-variant 7 (AR-v7) and glucocorticoid receptor. BET bromodomain inhibitors were shown previously to attenuate AR signaling in mCRPC; here, we demonstrate the efficacy of bromodomain and extraterminal (BET) inhibitors in enzalutamide-resistant prostate cancer models. AR antagonists, enzalutamide, and ARN509 exhibit enhanced prostate tumor growth inhibition when combined with BET inhibitors, JQ1 and OTX015, respectively. Taken together, these data provide a compelling preclinical rationale to combine BET inhibitors with AR antagonists to subvert resistance mechanisms. Implications: Therapeutic combinations of BET inhibitors and AR antagonists may enhance the clinical efficacy in the treatment of mCRPC. Visual Overview: http://mcr.aacrjournals.org/content/14/4/324/F1.large.jpg. Mol Cancer Res; 14(4); 324–31. ©2016 AACR.

African-American (AA) men have a 60% higher incidence of prostate cancer compared to European-American (EA) men and tend to have a more aggressive clinical outcome. An understanding of the altered biological responses and the factors that lead to health disparity in prostate cancer development and progression is unclear. Metabolism defines the physiological state of the cell and metabolic reprogramming is a hallmark of cancer. Thus, studying the metabolic landscape will be critical for understanding the biological changes that might contribute to this disparity. Analysis of 190 metabolites across prostate cancer/benign adjacent tissue pairs from ancestry typed AA and EA men performed in our laboratory, revealed altered levels of nucleosides adenosine and inosine. High inosine to adenosine ratio is observed in AA men compared to EA men. In line with this finding, the enzyme adenosine deaminase (ADA), which converts adenosine to inosine was found elevated in AA men, potentially explaining the high inosine to adenosine ratio in AA PCa. The consequences of the accumulation of these metabolites on AA PCa progression are unknown. The current study will address the knowledge gap on the consequences of elevated ADA activity in AA PCa and attempt to dissect its role in effecting an aggressive phenotype in PCa. To determine the role of elevated ADA in PCa, it was overexpressed in ancestry-typed AA (MDA-PCa-2A) and EA PCa(LNCaP) cell lines (termed ADA OE). Phenotypic examination of these cells revealed increased ADA enzyme activity decreases the cell-ECM adhesion. This adhesion decrease is facilitated by a decrease in cell adhesion protein integrin β1 (ITGB1). Elevated ADA enzyme activity decreases cAMP which mediates this adhesion decrease by disrupting the Epac-Rap1 pathway. It is postulated that high inosine upon ADA OE activates adenosine receptors A1 and A3, which causes this decrease in cAMP levels. These molecular findings suggest that ADA-mediated adhesion decrease could facilitate metastatic dissemination from the primary tumor. From a translational viewpoint, ADA enzyme activity could serve as a biomarker for metastatic prostate cancer. Therefore, we further look to analyze the inosine to adenosine levels (ADA enzyme activity) in patient samples and correlate with various clinical outcomes in PCa. Our initial analysis reveals high inosine to adenosine ratio in biopsy positive patient samples compared to biopsy negative samples. Our next steps of analysis include correlating the enzyme activity with (i) Biochemical Recurrence (ii) Time to attaining castration resistance in castration sensitive patients (iii) Time to metastasis (iv) Response to Androgen deprivation in patients after BCr. Based on our current findings, we expect inosine to adenosine ratio in plasma or urine to serve as a predictive marker for PCa incidence or progression in AA men. In addition, we expect ADA to serve as a potential therapeutic target for AA PCa. Citation Format: Christy Charles, Jie Gohlke, Stacy Lloyd, Uttam Rasaily, James Henderson, Balasubramanyam Karanam, Brian Simons, Nora Navone, Rick Kittles, Stefan Ambs, George Michailidis, Nagireddy Putluri, Arun Sreekumar. Metabolic re-wiring in African-American prostate cancer: A role of adenosine-inosine axis [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PR17.

Aggressive Variant Prostate Cancers (AVPC) are a clinically and molecularly distinct subset of prostate cancers with a poor prognosis characterized by androgen-indifference, platinum-sensitivity and combined defects in Tp53, RB1 and PTEN, for which novel therapeutic approaches are urgently needed. Defects observed in these tumor suppressor pathways have been linked to lineage plasticity in genetically engineered mouse models. We hypothesized that this plasticity can be identified in human tumor samples, and can be modulated to serve as the foundation for effective combination therapies for the AVPC subset. We previously defined a gene expression signature derived from mouse embryonic day 6.5 that is representative of extensive cellular plasticity (PMID:26123483). We determined the plasticity scores in 12 patient-derived xenograft (PDX) models representative of the spectrum of prostate cancer biology (using available RNA sequencing profiles), and in 26 macrodissected tumor biopsies obtained from patients who received platinum-based chemotherapy for aggressive prostate cancer (using ALMAC Affymetrix gene expression microarray). As expected, the AVPC PDX models exhibited high plasticity scores as did 11 (42.3%) of the patient samples, associated with patient progression free- and overall survival. We next treated a well characterized AVPC PDX model (144-4) with the anti-EMT drug (p38MAPK inhibitor) SB203580 at 10mg/kg, and observed a decrease in stem-cell and EMT factors associated with plasticity. These data confirm the association between plasticity and AVPC in human tumor samples, and suggest that changes in plasticity can be measured in tumor samples to determine the value of its modulation in clinical trials. Acknowledgments: ALMAC Diagnostics Services, UK, and UTMDACC ATGC Core Lab (CCSG-CA016672-ATGC) for RNA sequencing services; UTMDACC Prostate Cancer SPORE (RS, SAM, AA), UTMDACC IRG (RS), and the UTMDACC Prostate Cancer Moonshot Program (AA) for financial support. Citation Format: Rama Soundararajan, Petra den Hollander, Peter Shepherd, Nora Navone, Daniele Lorenzini, Caddie Laberiano-Fernandez, Wei Lu, Jianling Zhou, Jingjing Liu, Jianhua Zhang, Luisa Maren Solis-Soto, Sendurai A. Mani, Ana Aparicio. Modulating plasticity in aggressive variant prostate cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5443.

Advanced prostate cancer (PCa) is one of the leading causes of cancer death in men in the United States. The FGF/FGFR1 signaling axis has been implicated in the progression of PCa. Different isoforms of FGFR1 have been described in PCa (e.g., alpha and beta), which might account for the typical heterogeneity of PCa. The isoforms may also mediate different clinical responses to treatments targeting this pathway. Pan-FGFR tyrosine kinase inhibitor JNJ-42756493 (Janssen) has been demonstrated to have antiproliferative effects in FGFR pathway-activated cancer cell lines and in vivo mouse antitumor activity. Our goal was to study the effects of JNJ-42756493 in PCa growth and signaling in vitro and establish whether these effects differ according to the FGFR1 isoform (alpha or beta) expressed. Using transfected bone-derived PCa cell lines and patient-derived xenografts (PDXs), we measured the effects of FGFR1 expression on cell proliferation and activation of the FGFR1-pMAPK signaling pathway. We then performed drug treatment experiments on FGFR1-expressing sublines and high-FGFR1 PDXs and observed the response in FGFR1-pMAPK signaling pathway activation. Our results showed that FGFR1 isoforms alpha and beta trigger different biological effects (i.e., proliferation), possibly contributing to the heterogeneity of PCa. We found that targeting the tyrosine kinase domain of FGFR1 with JNJ-42756493 results in pMAPK inhibition in PCa sublines. These findings indicate that the presence of specific FGFR1 variants should be considered for treatment selection in advanced PCa. The results also suggest that FGFR1-pMAPK pathway blockade by JNJ-42756493 should be further studied as a strategy to guide the development of therapies to hinder PCa progression. Citation Format: Louise Medina Bengtsson, Estefania Labanca, Nora Navone. Tyrosine kinase inhibition of fibroblast growth factor receptor 1 in advanced prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3748.

Advanced prostate cancer displays conspicuous chromosomal instability and rampant copy number aberrations, yet the identity of functional drivers resident in many amplicons remain elusive. Here, we implemented a functional genomics approach to identify new oncogenes involved in prostate cancer progression. Through integrated analyses of focal amplicons in large prostate cancer genomic and transcriptomic datasets as well as genes upregulated in metastasis, 276 putative oncogenes were enlisted into an in vivo gain-of-function tumorigenesis screen. Among the top positive hits, we conducted an in-depth functional analysis on Pygopus family PHD finger 2 (PYGO2), located in the amplicon at 1q21.3. PYGO2 overexpression enhances primary tumor growth and local invasion to draining lymph nodes. Conversely, PYGO2 depletion inhibits prostate cancer cell invasion in vitro and progression of primary tumor and metastasis in vivo. In clinical samples, PYGO2 upregulation associated with higher Gleason score and metastasis to lymph nodes and bone. Silencing PYGO2 expression in patient-derived xenograft models impairs tumor progression. Finally, PYGO2 is necessary to enhance the transcriptional activation in response to ligand-induced Wnt/β-catenin signaling. Together, our results indicate that PYGO2 functions as a driver oncogene in the 1q21.3 amplicon and may serve as a potential prognostic biomarker and therapeutic target for metastatic prostate cancer. Significance: Amplification/overexpression of PYGO2 may serve as a biomarker for prostate cancer progression and metastasis. Cancer Res; 78(14); 3823–33. ©2018 AACR.

Significance Prostate cancer has an unpredictable natural history: While most tumors are clinically indolent, some patients display lethal phenotypes. Serum prostate-specific antigen is the most often used test in prostate cancer but screening is controversial. Treatment options are limited for metastatic disease, hence the need for early diagnosis. Prostate cancer antigen 3 ( PCA3 ), a long noncoding RNA, is the most specific biomarker identified and approved as a diagnostic test. However, its inherent biological function (if any) has remained elusive. We uncovered a negative transdominant oncogenic role for PCA3 that down-regulates an unrecognized tumor suppressor gene, PRUNE2 (a human homolog of the Drosophila prune gene) thereby promoting malignant cell growth. This work defines a unique biological function for PCA3 in prostate cancer.

Purpose: Conditioning strategies constitute a relatively unexplored and exciting opportunity to shape tumor fate by targeting the tumor microenvironment. In this study, we assessed how hemin, a pharmacologic inducer of heme oxygenase-1 (HO-1), has an impact on prostate cancer development in an in vivo conditioning model. Experimental Design: The stroma of C57BL/6 mice was conditioned by subcutaneous administration of hemin prior to TRAMP-C1 tumor challenge. Complementary in vitro and in vivo assays were performed to evaluate hemin effect on both angiogenesis and the immune response. To gain clinical insight, we used prostate cancer patient-derived samples in our studies to assess the expression of HO-1 and other relevant genes. Results: Conditioning resulted in increased tumor latency and decreased initial growth rate. Histologic analysis of tumors grown in conditioned mice revealed impaired vascularization. Hemin-treated human umbilical vein endothelial cells (HUVEC) exhibited decreased tubulogenesis in vitro only in the presence of TRAMP-C1-conditioned media. Subcutaneous hemin conditioning hindered tumor-associated neovascularization in an in vivo Matrigel plug assay. In addition, hemin boosted CD8+ T-cell proliferation and degranulation in vitro and antigen-specific cytotoxicity in vivo. A significant systemic increase in CD8+ T-cell frequency was observed in preconditioned tumor-bearing mice. Tumors from hemin-conditioned mice showed reduced expression of galectin-1 (Gal-1), key modulator of tumor angiogenesis and immunity, evidencing persistent remodeling of the microenvironment. We also found a subset of prostate cancer patient-derived xenografts and prostate cancer patient samples with mild HO-1 and low Gal-1 expression levels. Conclusions: These results highlight a novel function of a human-used drug as a means of boosting the antitumor response. Fil: Jaworski, Felipe Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Gentilini, Lucas Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Gueron, Geraldine. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Meiss, Roberto P.. Academia Nacional de Medicina de Buenos Aires; Argentina Fil: Ortiz, Emiliano Germán. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Berguer, Paula Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Ahmed, Asif. University of Aston; Reino Unido Fil: Navone, Nora. University of Texas; Estados Unidos Fil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Compagno, Daniel Georges. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Laderach, Diego Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Universidad Nacional de Luján. Departamento de Ciencias Básicas; Argentina Fil: Vazquez, Elba Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina

Purpose: We investigated MYCN-regulated molecular pathways in castration-resistant prostate cancer (CRPC) classified by morphologic criteria as adenocarcinoma or neuroendocrine to extend the molecular phenotype, establish driver pathways, and identify novel approaches to combination therapy for neuroendocrine prostate cancer (NEPC). Experimental Design and Results: Using comparative bioinformatics analyses of CRPC-Adeno and CRPC-Neuro RNA sequence data from public data sets and a panel of 28 PDX models, we identified a MYCN–PARP–DNA damage response (DDR) pathway that is enriched in CRPC with neuroendocrine differentiation (NED) and CRPC-Neuro. ChIP-PCR assay revealed that N-MYC transcriptionally activates PARP1, PARP2, BRCA1, RMI2, and TOPBP1 through binding to the promoters of these genes. MYCN or PARP1 gene knockdown significantly reduced the expression of MYCN–PARP–DDR pathway genes and NED markers, and inhibition with MYCNsi and/or PARPsi, BRCA1si, or RMI2si significantly suppressed malignant activities, including cell viability, colony formation, and cell migration, in C4-2b4 and NCI-H660 cells. Targeting this pathway with AURKA inhibitor PHA739358 and PARP inhibitor olaparib generated therapeutic effects similar to those of gene knockdown in vitro and significantly suppressed tumor growth in both C4-2b4 and MDACC PDX144-13C subcutaneous models in vivo. Conclusions: Our results identify a novel MYCN–PARP–DDR pathway that is driven by N-MYC in a subset of CRPC-Adeno and in NEPC. Targeting this pathway using in vitro and in vivo CRPC-Adeno and CRPC-Neuro models demonstrated a novel therapeutic strategy for NEPC. Further investigation of N-MYC–regulated DDR gene targets and the biological and clinical significance of MYCN–PARP–DDR signaling will more fully elucidate the importance of the MYCN–PARP–DDR signaling pathway in the development and maintenance of NEPC. Clin Cancer Res; 24(3); 696–707. ©2017 AACR.

Synthetic lethality and collateral lethality are two well-validated conceptual strategies for identifying therapeutic targets in cancers with tumour-suppressor gene deletions. Here, we explore an approach to identify potential synthetic-lethal interactions by screening mutually exclusive deletion patterns in cancer genomes. We sought to identify 'synthetic-essential' genes: those that are occasionally deleted in some cancers but are almost always retained in the context of a specific tumour-suppressor deficiency. We also posited that such synthetic-essential genes would be therapeutic targets in cancers that harbour specific tumour-suppressor deficiencies. In addition to known synthetic-lethal interactions, this approach uncovered the chromatin helicase DNA-binding factor CHD1 as a putative synthetic-essential gene in PTEN-deficient cancers. In PTEN-deficient prostate and breast cancers, CHD1 depletion profoundly and specifically suppressed cell proliferation, cell survival and tumorigenic potential. Mechanistically, functional PTEN stimulates the GSK3β-mediated phosphorylation of CHD1 degron domains, which promotes CHD1 degradation via the β-TrCP-mediated ubiquitination-proteasome pathway. Conversely, PTEN deficiency results in stabilization of CHD1, which in turn engages the trimethyl lysine-4 histone H3 modification to activate transcription of the pro-tumorigenic TNF-NF-κB gene network. This study identifies a novel PTEN pathway in cancer and provides a framework for the discovery of 'trackable' targets in cancers that harbour specific tumour-suppressor deficiencies.

Prostate cancer (PCa) cells display abnormal expression of cytoskeletal proteins resulting in an augmented capacity to resist chemotherapy and colonize distant organs. We have previously shown that heme oxygenase 1 (HO-1) is implicated in cell morphology regulation in PCa. Here, through a multi 'omics' approach we define the HO-1 interactome in PCa, identifying HO-1 molecular partners associated with the integrity of the cellular cytoskeleton. The bioinformatics screening for these cytoskeletal-related partners reveal that they are highly misregulated in prostate adenocarcinoma compared with normal prostate tissue. Under HO-1 induction, PCa cells present reduced frequency in migration events, trajectory and cell velocity and, a significant higher proportion of filopodia-like protrusions favoring zippering among neighboring cells. Moreover forced expression of HO-1 was also capable of altering cell protrusions in transwell co-culture systems of PCa cells with MC3T3 cells (pre-osteoblastic cell line). Accordingly, these effects were reversed under siHO. Transcriptomics profiling evidenced significant modulation of key markers related to cell adhesion and cell-cell communication under HO-1 induction. The integration from our omics-based research provides a four molecular pathway foundation (ANXA2/HMGA1/POU3F1; NFRSF13/GSN; TMOD3/RAI14/VWF; and PLAT/PLAU) behind HO-1 regulation of tumor cytoskeletal cell compartments. The complementary proteomics and transcriptomics approaches presented here promise to move us closer to unravel the molecular framework underpinning HO-1 involvement in the modulation of cytoskeleton pathways, pushing toward a less aggressive phenotype in PCa. Fil: Paez, Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Pallavicini, Carla. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: Schuster, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Valacco, Maria Pia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Giudice, Jimena. University of North Carolina; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Ortiz, Emiliano Germán. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Anselmino, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Labanca, Estefania. University of Texas; Estados Unidos Fil: Binaghi, María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Salierno, Marcelo Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Marti, Marcelo Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Cotignola, Javier Hernan. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Woloszynska Read, Anna. Roswell Park Cancer Institute; Estados Unidos Fil: Bruno, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: Levi, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Navone, Nora. University of Texas; Estados Unidos Fil: Vazquez, Elba Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Gueron, Geraldine. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina

Aggressive variant prostate cancers (AVPC) are a clinically defined group of tumors of heterogeneous morphologies, characterized by poor patient survival and for which limited diagnostic and treatment options are currently available. We show that the cell surface 78-kDa glucose-regulated protein (GRP78), a receptor that binds to phage-display-selected ligands, such as the SNTRVAP motif, is a candidate target in AVPC. We report the presence and accessibility of this receptor in clinical specimens from index patients. We also demonstrate that human AVPC cells displaying GRP78 on their surface could be effectively targeted both in vitro and in vivo by SNTRVAP, which also enabled specific delivery of siRNA species to tumor xenografts in mice. Finally, we evaluated ligand-directed strategies based on SNTRVAP-displaying adeno-associated virus/phage (AAVP) particles in mice bearing MDA-PCa-118b, a patient-derived xenograft (PDX) of castration-resistant prostate cancer bone metastasis that we exploited as a model of AVPC. For theranostic (a merging of the terms therapeutic and diagnostic) studies, GRP78-targeting AAVP particles served to deliver the human Herpes simplex virus thymidine kinase type-1 (HSVtk) gene, which has a dual function as a molecular-genetic sensor/reporter and a cell suicide-inducing transgene. We observed specific and simultaneous PET imaging and treatment of tumors in this preclinical model of AVPC. Our findings demonstrate the feasibility of GPR78-targeting, ligand-directed theranostics for translational applications in AVPC.

The arachidonic acid pathway is important in the development and progression of numerous malignant diseases, including prostate cancer. To more fully evaluate the role of individual cyclooxygenases (COXs), lipoxygenases (LOXs) and their metabolites in prostate cancer, we measured mRNA and protein levels of COXs and LOXs and their arachidonate metabolites in androgen-dependent (LNCaP) and androgen-independent (PC-3 and DU145) prostate cancer cell lines, bone metastasis-derived MDA PCa 2a and MDA PCa 2b cell lines and their corresponding xenograft models, as well as core biopsy specimens of primary prostate cancer and nonneoplastic prostate tissue taken ex vivo after prostatectomy. Relatively high levels of COX-2 mRNA and its product PGE2 were observed only in PC-3 cells and their xenografts. By contrast, levels of the exogenous 12-LOX product 12-HETE were consistently higher in MDA PCa 2b and PC-3 cells and their corresponding xenograft tissues than were those in LNCaP cells. More strikingly, the mean endogenous level of 12-HETE was significantly higher in the primary prostate cancers than in the nonneoplastic prostate tissue (0.094 vs. 0.010 ng/mg protein, respectively; p=0.019). Our results suggest that LOX metabolites such as 12-HETE are critical in prostate cancer progression and that the LOX pathway may be a target for treating and preventing prostate cancer.

The identification of new and effective therapeutic targets for the lethal, castration-resistant stage of prostate cancer (CRPC) has been challenging because of both the paucity of adequate frozen tissues and a lack of integrated molecular analysis. Therefore, in this study, we performed a genome-wide analysis of DNA copy number alterations from 34 unique surgical CRPC specimens and 5 xenografts, with matched transcriptomic profiling of 25 specimens. An integrated analysis of these data revealed that the asparagine synthetase (ASNS) gene showed a gain in copy number and was overexpressed at the transcript level. The overexpression of ASNS was validated by analyzing other public CRPC data sets. ASNS protein expression, as detected by reverse-phase protein lysate array, was tightly correlated with gene copy number. In addition, ASNS protein expression, as determined by IHC analysis, was associated with progression to a therapy-resistant disease state in TMAs that included 77 castration-resistant and 40 untreated prostate cancer patient samples. Knockdown of ASNS by small-interfering RNAs in asparagine-deprived media led to growth inhibition in both androgen-responsive (ie, LNCaP) and castration-resistant (ie, C4-2B) prostate cancer cell lines and in cells isolated from a CRPC xenograft (ie, MDA PCa 180-30). Together, our results suggest that ASNS is up-regulated in cases of CRPC and that depletion of asparagine using ASNS inhibitors will be a novel strategy for targeting CRPC cells.

Transforming growth factor beta 1 (TGF-β1) has been implicated in the pathogenesis of prostate cancer (PCa) bone metastasis. In this study, we tested the antitumor efficacy of a selective TGF-β receptor I kinase inhibitor, LY2109761, in preclinical models. The effect of LY2109761 on the growth of MDA PCa 2b and PC-3 human PCa cells and primary mouse osteoblasts (PMOs) was assessed in vitro by measuring radiolabeled thymidine incorporation into DNA. In vivo, the right femurs of male SCID mice were injected with PCa cells. We monitored the tumor burden in control- and LY2109761-treated mice with MRI analysis and the PCa-induced bone response with X-ray and micro-CT analyses. Histologic changes in bone were studied by performing bone histomorphometric evaluations. PCa cells and PMOs expressed TGF-β receptor I. TGF-β1 induced pathway activation (as assessed by induced expression of p-Smad2) and inhibited cell growth in PC-3 cells and PMOs but not in MDA PCa 2b cells. LY2109761 had no effect on PCa cells but induced PMO proliferation in vitro. As expected, LY2109761 reversed the TGF-β1-induced pathway activation and growth inhibition in PC-3 cells and PMOs. In vivo, LY2109761 treatment for 6. weeks resulted in increased volume in normal bone and increased osteoblast and osteoclast parameters. In addition, LY2109761 treatment significantly inhibited the growth of MDA PCa 2b and PC-3 in the bone of SCID mice (p

SummaryRecurrent fusions of ETS genes are considered driving mutations in a diverse array of cancers, including Ewing's sarcoma, acute myeloid leukemia, and prostate cancer. We investigate the mechanisms by which ETS fusions mediate their effects, and find that the product of the predominant ETS gene fusion, TMPRSS2:ERG, interacts in a DNA-independent manner with the enzyme poly (ADP-ribose) polymerase 1 (PARP1) and the catalytic subunit of DNA protein kinase (DNA-PKcs). ETS gene-mediated transcription and cell invasion require PARP1 and DNA-PKcs expression and activity. Importantly, pharmacological inhibition of PARP1 inhibits ETS-positive, but not ETS-negative, prostate cancer xenograft growth. Finally, overexpression of the TMPRSS2:ERG fusion induces DNA damage, which is potentiated by PARP1 inhibition in a manner similar to that of BRCA1/2 deficiency.

Prostate cancer (PCa) is the second leading cause of cancer-associated death in men. Energetic metabolism alterations have become a new hallmark of cancer, since variations in a single gene can orchestrate changes in metabolic pathways and confer an adaptive advantage. Heme-oxygenase 1 (HO-1) exerts an antitumoral role in PCa inhibiting cell proliferation, migration, tumor growth, and angiogenesis. The aim of this work was to assess the role of HO-1 in the metabolic signature of PCa. Through RNA-Seq we found a set of metabolic genes deregulated under pharmacologic induction (hemin treatment) or genetic induction of HO-1 in PC3 cells. STAR and ATP5L2 were upregulated, while HMGCS2, PRODH, and ACOT12 were downregulated. These genes encode for steroid hormone metabolism, ATP synthesis, ketogenesis, proline and lipid metabolism. The analysis of the deregulated genes (2-fold) by Gene Ontology revealed alterations in several metabolic pathways such as steroid, proline and lipid metabolism, and ATP synthesis. Functional analysis highlighted a decrease in oxygen consumption rate and ATP production under hemin treatment. Furthermore, HO-1 induction led to a decrease in the extracellular lactate levels. Bone is the only site of PCa progression, and bone cells are able to produce factors that favor progression. However, the molecular nature of this interaction remains elusive. Our results performed on cocultures of PC3 cells (treated or not with hemin) with Raw264.7 (preosteoclastic) or MC3T3 (preosteoblastic) cells demonstrate that HO-1 directs the metabolic fate of bone precursor cells due to the deregulation of glycolytic genes. HO-1 induction in PC3 cells downregulated PKM2 and LDHA expression in cocultured Raw264.7 and MC3T3 cells (p< 0.05). Based on our results, we propose HO-1 as a key regulator of the metabolic status of PCa cells and a powerful mediator capable of redefining the metabolic signature of bone precursor cells, thus favoring the establishment of a less aggressive phenotype. Citation Format: Valeria G. Antico Arciuch, Florencia Cascardo, Florencia Velazquez, Sofia Lage Vickers, Nicolas Anselmino, Estefania Labanca, Javier Cotignola, Geraldine Gueron, Nora Navone, Elba Vazquez. Heme-oxygenase 1 drives the metabolic fate in 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 A019.

Bone metastases typically develop in patients with advanced prostate cancer (PCa). These metastases are osteoblastic (bone-forming) and constitute the main cause of morbidity and mortality. Androgen deprivation is commonly used to treat bone metastases of PCa, but responses to such therapy are short, and eventually the disease progresses to a castration-resistant form. Bone is the primary site of castration-resistant PCa (CRPC) progression. Further development of therapies for bone metastases of PCa requires an understanding of the mechanisms underlying the growth of CRPC in bone. Clinical and laboratory-based investigations have implicated a role for PCa cell-bone cell interaction in the growth of PCa and have demonstrated that osteoblasts produce factors that favor the growth and survival of PCa cells. Collectively, these observations strongly suggest that PCa cells interact with osteoblasts and that this interaction contributes to the growth of PCa in bone. Recently, a study showed 18% of patients with metastatic CRPC harbored alterations in the Wnt canonical pathway, including hotspot mutations of β-catenin (β-cat), the molecular node of the Wnt canonical pathway, that leads to pathway activation. This report is in agreement with previous publications of β-cat nuclear accumulation, an indirect measure of Wnt-canonical pathway activation, in 20 to 40% of advanced PCas. We previously reported that a patient-derived xenograft (PDX) MDA-PCa-118b (118b) developed in our lab harbors a β-cat mutation (D32G) and has an activated Wnt canonical pathway. 118b induces an osteoblastic reaction when it is implanted in the bone of mice in vivo. In vitro, 118b induces osteoblast proliferation in coculture. In contrast, the PCa cell line, PC3, induces osteolytic lesions in vivo and in vitro inhibits osteoblast proliferation in coculture. Strikingly, expression of β-cat mutated at D32G in PC3 cells reverses the osteoblast inhibitory effect of PC3 cells, resulting in increased proliferation of osteoblasts when grown in coculture. We therefore hypothesize that β-cat/Wnt signaling in PCa cells mediates the PCa-bone interaction that favors PCa growth in bone. Our future studies are focusing on studying the importance of β-cat mutations recently identified in patients with metastatic CRPC and determining whether these mutations also result in an osteoblastic phenotype, which in turn supports growth of prostate cancer in bone. Additionally, we are screening our collection of PCa PDX lines for increased β-cat expression through immunohistochemistry and will sequence DNA from PDX lines that demonstrate increased nuclear β-cat expression to test for activating mutations in β-cat. Our aim is to gain insight into the mechanism of the paracrine interaction between PCa cells and bone that supports the growth of PCa bone lesions, with the ultimate goal of developing therapies to block these interactions and reducing the morbidity and mortality associated with metastatic disease. Citation Format: Justin M. Roberts, Estefania Labanca, Peter Shepherd, Jun Yang, Michael W. Starbuck, Nora M. Navone. The role of activated beta-catenin/Wnt signaling in the progression of castration resistance of prostate cancer in bone [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 B050.

Bone metastases typically develop in patients with advanced prostate cancer (PCa). Androgen deprivation is commonly used as treatment, but responses are short, and eventually the disease progresses to a castration-resistant form (CRPC). The fibroblast growth factor (FGF) axis is commonly altered during PCa progression. Our group and others have implicated the FGF axis in the pathogenesis of PCa progression in bone, identified it as a candidate target for therapy, and suggested that FGF receptor (FGFR)-1 mediates a positive feedback loop between PCa cells and bone cells. RNA sequencing studies of 183 human PCa samples indicated that the mean expression of FGFR-1 is the highest of all the FGFR family genes studied. Analyses of these FGFR-1 transcripts identified eight different protein-coding transcripts to be the most abundantly expressed with diverse human PCa tissue samples expressing different FGFR-1 isoforms. The overall goal of this project is to investigate implications of FGFR1/FGFR1 isoforms expression in the pathogenesis of PCa bone metastases. When we assessed the expression of the two best-characterized FGFR-1 isoforms (alpha (NM_023110.2) and beta (NM_023105.2), which represent the most abundant protein coding transcripts found in PCa, we observed that all patient-derived xenografts (PDXs) studied express significantly higher levels of FGFR-1 alpha than beta while PCa cell lines mostly express the beta isoform. Mining The Cancer Genome Atlas (TCGA) PCa data, we identified distinct patterns of gene expression associated with each FGFR-1 isoform (alpha and beta). Briefly, FGFR-1 beta (but not alpha) is significantly associated with pathways including the MAP-kinase signaling cascade, signaling by FGFR in disease and pathways in cancer. In vitro studies of FGF signaling activation in PCa cells expressing FGFR-1 isoforms alpha, beta or empty vector, confirmed these results. Male SCID mice injected intracardially with PC3-FGFR-1 alpha exhibited higher death rates compared to animals injected with either PC3-FGFR-1 beta or PC3-empty vector. Furthermore, higher number of metastases per mice as well as higher number of mice with bone metastases were observed in animals injected with PC3-FGFR-1 alpha. The fact that different prostate tumors express different FGFR-1 isoforms suggests that FGFR1 alpha and beta isoforms activate different genes or pathways in PCa cells and this may underlie, at least in part, PCa heterogeneity, pattern of progression, and differences in response to FGFR1 inhibitors. Further studies will warrant the understanding of the implications of FGFR-1 isoforms expression in the pathogenesis of PCa. Citation Format: Estefania Labanca, Jun Yang, Peter Shepherd, Justin Roberts, Michael Starbuck, Bradley Broom, Matthew Iyer, Christopher Logothetis, Arul Chinnaiyan, Nora Navone. Expression of fibroblast growth factor receptor 1 isoforms and activation of different pathways in prostate cancer progression [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 A026.

The establishment of a new tumor niche for most carcinomas is orchestrated primarily by the bidirectional engagement of cancer cells and fibroblasts. Cancer cells reprogram and activate fibroblasts into a wound healing-like state and, in turn, these cancer-associated fibroblasts (CAFs) react by remodeling the extracellular matrix (ECM) and secreting chemokines and growth factors. Macrophages are rapidly recruited to the tumor and represent the primary immune cell in the microenvironment. Tumor-associated macrophages (TAMs) enter a wound healing-like loop, displaying the full spectrum of macrophage phenotypes and behaviors as the disease progresses. Among these activities is the ability to modulate growth factor bioavailability. Deposition of the heparan sulfate (HS) proteoglycan perlecan/HSPG2 in the ECM and secretion of HS modulators are increased as result of the cancer-CAF-TAM trialogue and play critical roles in the availability and signaling of growth factors, cytokines, and chemokines. In this work, we developed a novel, biomimetic 3D hydrogel system composed of hyaluronic acid and collagen I (1:1) to study prostate cancer bone metastases. Our goal was to explore the molecular signaling between TAMs and CAFs to learn how this interaction affects the expression of the enzymes that modulate HS as a growth factor depot. Essential modulators of HS include heparanase (HPSE) and sulfatases 1 and 2 (SULF1, 2), all of which selectively release growth factors to influence signaling in the tumor microenvironment. We found that alternatively activated macrophages (M2) and TAMs (M2-like) increase levels of HPSE and SULFs in CAFs. We next examined the impact of TAM-CAF co-signaling on prostate cancer viability, growth, and dependence on HS modulators. Our data show that as a consequence of the TAM-CAF interaction, the elevated levels of HS-modifying enzymes are sufficient to trigger substantial changes in prostate cancer cell behavior, including viability, proliferation, and response to growth factors. We currently are assessing if these outcomes are seen in a series of patient-derived prostate xenograft lines representing different prostate cancer subtypes. We conclude that our 3D hydrogel model is a physiologically relevant in vitro system to discover fundamental aspects of HS signaling in the tumor microenvironment and thus uncover new ways to reduce the bioavailability of growth factors in the prostate cancer microenvironment and interfere with cancer progression. (Supported by NIH P01 and CAPES.) Citation Format: Fabio Henrique Brasil da Costa, Nora M. Navone, Mary C. Farach-Carson, Daniel D. Carson. Heparan sulfate modifiers and signaling between tumor-associated macrophages and cancer-associated fibroblasts in a biomimetic hydrogel model of bone-metastatic 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 3799.

Men with castration-resistant prostate cancer (PCa) frequently develop metastasis in bone. The reason for this association is unclear. We have previously shown that cadherin-11 (also known as OB-cadherin), a homophilic cell adhesion molecule that mediates osteoblast adhesion, plays a role in the metastasis of PCa to bone. Here, we report that androgen deprivation therapy upregulates cadherin-11 expression in PCa. In human PCa specimens, immunohistochemical staining showed that 22 of 26 (85%) primary PCa tumors from men with castration-resistant PCa expressed cadherin-11. In contrast, only 7 of 50 (14%) androgen-dependent PCa tumors expressed cadherin-11. In the MDA-PCa-2b xenograft animal model, cadherin-11 was expressed in the recurrent tumors following castration. In the PCa cell lines, there is an inverse correlation between expression of cadherin-11 and androgen receptor (AR), and cadherin-11 is expressed in very low levels or not expressed in AR positive cell lines, including LNCaP, C4-2B4, and VCaP cells. We showed that AR likely regulates cadherin-11 expression in PCa through an indirect mechanism. Although re-expression of AR in the AR-negative PC3 cells led to the inhibition of cadherin-11 expression, depletion of androgen from the culture medium or down regulation of AR by RNA interference in the C4-2B4 cells or VCaP cells only produce a modest increase of cadherin-11 expression. Promoter analysis indicated that cadherin-11 promoter does not contain a typical AR binding element, and AR elicits a modest inhibition of cadherin-11 promoter activity, suggesting that AR does not regulate cadherin-11 expression directly. Together, these results suggest that androgen deprivation upregulates cadherin-11 expression in prostate cancer and this may contribute to the metastasis of PCa to bone. Our study suggests that therapeutic strategies that block cadherin-11 expression or function may be considered when applying androgen ablation therapy.

The tendency of prostate cancer to produce osteoblastic bone metastases suggests that cancer cells and osteoblasts interact in ways that contribute to cancer progression. To identify factors that mediate these interactions, we compared gene expression patterns between two bone-derived prostate cancer cell lines that produce osteoblastic (MDA PCa 2b) or osteolytic lesions (PC-3). Both cell lines expressed Wnt ligands, including WNT7b, a canonical Wnt implicated in osteogenesis. PC-3 cells expressed 50 times more Dickkopf-1 (DKK1), an inhibitor of Wnt pathways, than did MDA PCa 2b cells. Evaluation of the functional role of these factors (in cocultures of prostate cancer cells with primary mouse osteoblasts (PMOs) or in bone organ cultures) showed that MDA PCa 2b cells activated Wnt canonical signaling in PMOs and that DKK1 blocked osteoblast proliferation and new bone formation induced by MDA PCa 2b cells. MDA PCa 2b cells did not induce bone formation in calvaria from mice lacking the Wnt co-receptor Lrp5. In human specimens, WNT7b was not expressed in normal prostate but was expressed in areas of high-grade prostate intraepithelial neoplasia, in three of nine primary prostate tumor specimens and in 16 of 38 samples of bone metastases from prostate cancer. DKK1 was not expressed in normal or cancerous tissue but was expressed in two of three specimens of osteolytic bone metastases (P=0.0119). We conclude that MDA PCa 2b induces new bone formation through Wnt canonical signaling, that LRP5 mediates this effect, and that DKK1 is involved in the balance between bone formation and resorption that determines lesion phenotype. Fil: Li, Z. G.. University of Texas; Estados Unidos Fil: Yang, J.. University of Texas; Estados Unidos Fil: Vazquez, Elba Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Rose, D.. University of Texas; Estados Unidos Fil: Vakar Lopez, F.. University of Texas; Estados Unidos Fil: Mathew, P.. University of Texas; Estados Unidos Fil: Lopez, A.. University of Texas; Estados Unidos Fil: Logothetis, C. J.. University of Texas; Estados Unidos Fil: Lin, S. H.. University of Texas; Estados Unidos Fil: Navone, N. M.. University of Texas; Estados Unidos

ErbB-3, an ErbB receptor tyrosine kinase, has been implicated in the pathogenesis of several malignancies, including prostate cancer. We found that ErbB-3 expression was up-regulated in prostate cancer cells within lymph node and bone metastases. Despite being a plasma membrane protein, ErbB-3 was also detected in the nuclei of the prostate cancer cells in the metastatic specimens. Because most metastatic specimens were from men who had undergone androgen ablation, we examined the primary tumors from patients who have undergone hormone deprivation therapy and found that a significant fraction of these specimens showed nuclear localization of ErbB3. We thus assessed the effect of androgens and the bone microenvironment on the nuclear translocation of ErbB-3 by using xenograft tumor models generated from bone-derived prostate cancer cell lines, MDA PCa 2b, and PC-3. In subcutaneous tumors, ErbB-3 was predominantly in the membrane/cytoplasm; however, it was present in the nuclei of the tumor cells in the femur. Castration of mice bearing subcutaneous MDA PCa 2b tumors induced a transient nuclear translocation of ErbB-3, with relocalization to the membrane/cytoplasm upon tumor recurrence. These findings suggest that the bone microenvironment and androgen status influence the subcellular localization of ErbB-3 in prostate cancer cells. We speculate that nuclear localization of ErbB-3 may aid prostate cancer cell survival during androgen ablation and progression of prostate cancer in bone. (Mol Cancer Res 2007;5(7):675–84)

Prostate cancer is the second leading cause of cancer-related death in men worldwide. Many factors that participate in the development of prostate cancer promote imbalance in the redox state of the cell. Accumulation of reactive oxygen species causes injury to cell structures, ultimately leading to cancer development. The antioxidant enzyme heme oxygenase 1 (HMOX1/HO-1) is responsible for the maintenance of the cellular homeostasis, playing a critical role in the oxidative stress and the regulation of prostate cancer development and progression. In the present study, the transcriptional regulation of HO-1 was investigated in prostate cancer. Interestingly, the tumor suppressor BRCA1 binds to the HO-1 promoter and modulates HO-1, inducing its protein levels through both the increment of its promoter activity and the induction of its transcriptional activation. In addition, in vitro and in vivo analyses show that BRCA1 also controls HO-1-negative targets: MMP9, uPA, and Cyclin D1. HO-1 transcriptional regulation is also modulated by oxidative and genotoxic agents. Induction of DNA damage by mitoxantrone and etoposide repressed HO-1 transcription, whereas hydrogen peroxide and doxorubicin induced its expression. Xenograft studies showed that HO-1 regulation by doxorubicin also occurs in vivo. Immunofluorescence analysis revealed that BRCA1 overexpression and/or doxorubicin exposure induced the cytoplasmic retention of HO-1. Finally, the transcription factor NRF2 cooperates with BRCA1 protein to activate HO-1 promoter activity. In summary, these results show that the activation of BRCA1-NRF2/HO-1 axis defines a new mechanism for the maintenance of the cellular homeostasis in prostate cancer. Fil: Labanca, Estefania. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: de Luca, Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Gueron, Geraldine. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Paez, Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Moiola, Cristian Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Massillo, Cintia Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Porretti, Juliana Carla. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Giudice, Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Zalazar, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Navone, Nora. University of Texas; Estados Unidos Fil: Vazquez, Elba Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: de Siervi, Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina

A major problem in patients with multiple myeloma is chemotherapy resistance, which develops in myeloma cells upon interaction with bone marrow stromal cells. However, few studies have determined the role of bone marrow adipocytes, a major component of stromal cells in the bone marrow, in myeloma chemotherapy resistance. We reveal that mature human adipocytes activate autophagy and upregulate the expression of autophagic proteins, thereby suppressing chemotherapy-induced caspase cleavage and apoptosis in myeloma cells. We found that adipocytes secreted known and novel adipokines, such as leptin and adipsin. The addition of these adipokines enhanced the expression of autophagic proteins and reduced apoptosis in myeloma cells. In vivo studies further demonstrated the importance of bone marrow-derived adipocytes in the reduced response of myeloma cells to chemotherapy. Our findings suggest that adipocytes, adipocyte-secreted adipokines, and adipocyte-activated autophagy are novel targets for combatting chemotherapy resistance and enhancing treatment efficacy in myeloma 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.

We performed combinatorial peptide library screening in vivo on a novel human prostate cancer xenograft that is androgen-independent and induces a robust osteoblastic reaction in bonelike matrix and soft tissue. We found two peptides, PKRGFQD and SNTRVAP, which were enriched in the tumors, targeted the cell surface of androgen-independent prostate cancer cells in vitro, and homed to androgen receptor-null prostate cancer in vivo. Purification of tumor homogenates by affinity chromatography on these peptides and subsequent mass spectrometry revealed a receptor for the peptide PKRGFQD, α-2-macroglobulin, and for SNTRVAP, 78-kDa glucose-regulated protein (GRP78). These results indicate that GRP78 and α-2-macroglobulin are highly active in osteoblastic, androgen-independent prostate cancer in vivo. These previously unidentified ligand–receptor systems should be considered for targeted drug development against human metastatic androgen-independent prostate cancer.

DOC-2/DAB2 is a member of the disable gene family with tumor-inhibitory activity. Its down-regulation is associated with several neoplasms, and serine phosphorylation of its N terminus modulates DOC-2/DAB2's inhibitory effect on AP-1 transcriptional activity. We describe the cloning of DIP1/2, a novel gene that interacts with the N-terminal domain of DOC-2/DAB2. DIP1/2 is a novel GTPase-activating protein containing a Ras GTPase-activating protein homology domain (N terminus) and two other unique domains (i.e. 10 proline repeats and leucine zipper). Interaction between DOC-2/DAB2 and DIP1/2 is detected in normal tissues such as the brain and prostate. Altered expression of these two proteins is often detected in prostate cancer cells. Indeed, the presence of DIP1/2 effectively blocks mitogen-induced gene expression and inhibits the growth of prostate cancer. Thus, DOC-2/DAB2 and DIP1/2 appear to represent a unique negative regulatory complex that maintains cell homeostasis.

Androgen deprivation is the standard treatment for advanced prostate cancer (PCa), but most patients ultimately develop resistance and tumor recurrence. We found that MYB is transcriptionally activated by androgen deprivation therapy or genetic silencing of the androgen receptor (AR). MYB silencing inhibited PCa growth in culture and xenografts in mice. Microarray data revealed that c-Myb and AR shared a subset of target genes that encode DNA damage response (DDR) proteins, suggesting that c-Myb may supplant AR as the dominant regulator of their common DDR target genes in AR inhibition-resistant or AR-negative PCa. Gene signatures including AR, MYB, and their common DDR-associated target genes positively correlated with metastasis, castration resistance, tumor recurrence, and decreased survival in PCa patients. In culture and in xenograft-bearing mice, a combination strategy involving the knockdown of MYB, BRCA1, or TOPBP1 or the abrogation of cell cycle checkpoint arrest with AZD7762, an inhibitor of the checkpoint kinase Chk1, increased the cytotoxicity of the poly[adenosine 5'-diphosphate (ADP)-ribose] polymerase (PARP) inhibitor olaparib in PCa cells. Our results reveal new mechanism-based therapeutic approaches for PCa by targeting PARP and the DDR pathway involving c-Myb, TopBP1, ataxia telangiectasia mutated- and Rad3-related (ATR), and Chk1.

Impairment of double-stranded DNA break (DSB) repair is essential to many cancers. However, although mutations in DSB repair proteins are common in hereditary cancers, mechanisms of impaired DSB repair in sporadic cancers remain incompletely understood. Here, we describe the first role for a long noncoding RNA (lncRNA) in DSB repair in prostate cancer. We identify PCAT-1, a prostate cancer outlier lncRNA, which regulates cell response to genotoxic stress. PCAT-1 expression produces a functional deficiency in homologous recombination through its repression of the BRCA2 tumor suppressor, which, in turn, imparts a high sensitivity to small-molecule inhibitors of PARP1. These effects reflected a posttranscriptional repression of the BRCA2 3′UTR by PCAT-1. Our observations thus offer a novel mechanism of “BRCAness” in sporadic cancers. Cancer Res; 74(6); 1651–60. ©2014 AACR.

The androgen receptor (AR) is involved in the development, growth and progression of prostate cancer (CaP). CaP often progresses from an androgen-dependent to an androgen-independent tumor, making androgen ablation therapy ineffective. However, the mechanisms for the development of androgen-independent CaP are unclear. More than 80% of clinically androgen-independent prostate tumors show high levels of AR expression. In some CaPs, AR levels are increased because of gene amplification and/or overexpression, whereas in others, the AR is mutated. Nonetheless, the involvement of the AR in the transition of CaP to androgen-independent growth and the subsequent failure of endocrine therapy are not fully understood. Here we show that in CaP cells from a patient who failed androgen ablation therapy, a doubly mutated AR functioned as a high-affinity cortisol/cortisone receptor (ARccr). Cortisol, the main circulating glucocorticoid, and its metabolite, cortisone, both equally stimulate the growth of these CaP cells and increase the secretion of prostate-specific antigen in the absence of androgens. The physiological concentrations of free cortisol and total cortisone in men greatly exceed the binding affinity of the ARccr and would activate the receptor, promoting CaP cell proliferation. Our data demonstrate a previously unknown mechanism for the androgen-independent growth of advanced CaP. Understanding this mechanism and recognizing the presence of glucocorticoid-responsive AR mutants are important for the development of new forms of therapy for the treatment of this subset of CaP.

Purpose: This study was undertaken to establish the pattern of specific p53 gene mutations in prostate cancer within primary tumors and distant metastases.Materials and Methods: We performed polymerase chain reaction-single-strand conformation polymorphism and sequencing analyses of p53 exons 5-8 in DNA extracted from 22 formalin-fixed, paraffin-embedded tissues from 17 patients. Samples from three patients included specimens from primary and metastatic sites (paired specimens).Results: G:C-to-A:T transitions were the most common point mutations (64%). Six (55%) of 11 G:C-to-A:T transitions occurred at CpG dinucleotides in five hot-spot codons (175, 245, 248, 273, and 282). Sequencing analysis of the paired samples revealed that two of the three pairs had the same mutation in both. Sequencing analysis of DNA from a different area of one of the primary tumors revealed a different mutation in the p53 gene.Conclusions: Our results suggest that specific p53 mutations participate in the progression of ...

1. The effect of long-term griseofulvin (GRIS) topical administration on some indicators of liver damage was examined. 2. Liver porphyrin accumulation was significant; however, no porhyrin crystals were observed under light microscopy. 3. An earlier onset of hepatopathy was established (3-fold) increase of direct bilirubin values after 7 days of treatment; hepatic injury was confirmed by measuring a 6-fold increase of free bilirubin. 4. Enhanced values of alkaline phosphatase and glutamic oxalacetic transaminase (GOT) confirmed the onset of cholestasis. 5. Topical application of GRIS induced measurable hepatopathy. Nevertheless, we cannot discard the possibility that this hepatopathy could also be attributed in part to a direct reaction to xenobiotics.

Prostate cancer (PCa) is a leading cause of death among males. It is currently estimated that inflammatory responses are linked to 15-20% of all deaths from cancer worldwide. PCa is dominated by complications arising from metastasis to the bone where the tumor cells interact with the bone microenvironment impairing the balance between bone formation and degradation. However, the molecular nature of this interaction is not completely understood. Heme oxygenase-1 (HO-1) counteracts oxidative damage and inflammation. Previous studies from our laboratory showed that HO-1 is implicated in PCa, demonstrating that endogenous HO-1 inhibits bone derived-prostate cancer cells proliferation, invasion and migration and decreases tumor growth and angiogenesis in vivo. The aim of this work was to analyze the impact of HO-1 modulated PCa cells on osteoblasts proliferation in vitro and on bone remodeling in vivo. Using a co-culture system of PC3 cells with primary mice osteoblasts (PMOs), we demonstrated that HO-1 pharmacological induction (hemin treatment) abrogated the diminution of PMOs proliferation induced by PCa cells and decreased the expression of osteoclast-modulating factors in osteoblasts. No changes were detected in the expression of genes involved in osteoblasts differentiation. However, co-culture of hemin pre-treated PC3 cells (PC3 Hem) with PMOs provoked an oxidative status and activated FoxO signaling in osteoblasts. The percentage of active osteoblasts positive for HO-1 increased in calvarias explants co-cultured with PC3 Hem cells. Nuclear HO-1 expression was detected in tumors generated by in vivo bone injection of HO-1 stable transfected PC3 (PC3HO-1) cells in the femur of SCID mice. These results suggest that HO-1 has the potential to modify the bone microenvironment impacting on PCa bone metastasis. Fil: Ferrando, María Mercedes Catalina. DTO.DE QUIMICA BIOLOGICA; Fil: Wan, Xinahi. Fil: Meissl, Roberto Jose. Academia Nacional de Medicina de Buenos Aires; Fil: Yang, Yung. Fil: de Siervi, Adriana. Consejo Nacional de Invest.cientif.y Tecnicas. Instituto de Biologia y Medicina Experimental (i); Fil: Navone, Nora. Fil: Vazquez, Elba Susana. Universidad de Buenos Aires. Facultad de Cs.exactas y Naturales. Departamento de Quimica Biologica

Advanced prostate cancer carries a poor prognosis and novel therapies are needed. Research has focused on identifying mechanisms that promote angiogenesis and cellular proliferation during prostate cancer progression from the primary tumor to bone—the principal site of prostate cancer metastases. One candidate pathway is the fibroblast growth factor (FGF) axis. Aberrant expression of FGF ligands and FGF receptors leads to constitutive activation of multiple downstream pathways involved in prostate cancer progression including mitogen-activated protein kinase, phosphoinositide 3-kinase, and phospholipase Cγ. The involvement of FGF pathways in multiple mechanisms relevant to prostate tumorigenesis provides a rationale for the therapeutic blockade of this pathway, and two small-molecule tyrosine kinase inhibitors—dovitinib and nintedanib—are currently in phase II clinical development for advanced prostate cancer. Preliminary results from these trials suggest that FGF pathway inhibition represents a promising new strategy to treat castrate-resistant disease. Clin Cancer Res; 19(21); 5856–66. ©2013 AACR.

Patients with metastatic prostate cancer (PCa) have effective therapy options, but none of them are curative. Thus, their mortality rates are persistently high. Essential to furthering our progress in PCa research and therapy development is a spectrum of models that reflect the heterogeneity of the disease at each tumor site as well as the different histological variants of PCa (e.g., adenocarcinoma, small cell carcinoma). To address this challenge, we developed a strategy to establish PCa patient-derived xenografts (PDXs), using PCa tissue specimens taken from PCa sites demonstrating clinical progression. This approach provided a diverse repository of PDXs that can be linked prospectively with clinical progression and led to the identification of clinically relevant therapy targets and have proven valuable for testing drugs. We studied the first 50 PDXs developed under our program to a) define the histopathological features of paired human PCa and corresponding PDXs applying the clinically defined morphological characterization groupings of human cancer to the PDX tumors; b) assess the expression of genes known to play roles in PCa pathogenesis (e.g., androgen receptor, PTEN, ETS gene fusions) in PDXs and the human tumors of origin using immunohistochemistry and fluorescence in situ hybridization and c) perform array comparative genomic hybridization to 42 PDXs. We found that the histopathological and molecular pattern of these PDXs maintain the fidelity with the human tumor of origin. Furthermore, of the 50 cases studied, 32 (64%) were adenocarcinomas, and 16 (32%) were small cell carcinomas, poorly differentiated neuroendocrine carcinomas or mixed adenocarcinoma/ small cell carcinomas. In our cohort, we also have one sarcomatoid tumor and one ductal adenocarcinoma. Of the 32 adenocarcinomas in this cohort, 26 were AR-positive (81%), and 11 of the 27 AR-positive adenocarcinomas (41%) had aberrant expression of genes frequently involved in recurrent rearrangement (e.g., ERG, ETV1, ETV5). Also, SCCs and poorly differentiated neuroendocrine carcinomas did not express AR and were negative for ERG. This distribution recapitulates that of human PCa in the general population. Comparative genomic hybridization demonstrated gains and losses previously reported in PCa with a defined cluster of genomic aberrations. Significant differences in oncogenic pathways activation in pairs of PDXs derived from different areas of the same tumor suggesting divergent cellular progression. Finally, using this platform, we identified a focal deletion of speckle-type POZ protein-like (SPOPL) gene in 7/28 PDX. SPOPL is a MATH-BTB protein that shares an overall 85% sequence identity with SPOP (a SPOPL paralog). SPOP was recently reported to be mutated in about 8% of PCa and to define a molecular subclass of PCa. No mutations were found in SPOP in our cohort. In support of our findings, deletions on SPOPL were also found in about 7% of the PCa in TCGA data suggesting that our cohort is a reliable platform for discovery. In conclusion, we have developed a dynamic repository of clinically annotated samples that can be used as a discovery platform. Furthermore, these clinically annotated samples can be linked prospectively to clinical progression/response to therapy and thus will help define therapeutic targets for subpopulations of men and to identify likely responders to previous and upcoming therapies. Citation Format: Nallasivam Palanisamy, Jun Yang, Xinhai Wan, Elsa M. li Ning Tapia, John C. Araujo, Eleni Efstathiou, Estefania Labanca, Louis Pisters, Ana Aparicio, Ritu Bhalla, Scott Tomlins, Lakshmi P. Kunju, Arul Chinnaiyan, Christopher J. Logothetis, Patricia Troncoso, Nora M. Navone. Analyses of a prostate cancer patient-derived xenografts series, a resource for translational research. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A03.

Castrate-resistant progression and bone metastases are hallmarks of advanced prostate cancer (PCa). The fibroblast growth factor (FGF)/FGF receptor (FGFR) complex mediates tumor-stromal interactions and is commonly altered in PCa. FGFR1, FGFR2, FGFR3, and FGFR4 genes encode alternatively spliced variants of FGFRs that vary in the extracellular ligand-binding and intracellular kinase domains. A published study from our group implicated FGFR1 as a therapy target for PCa bone metastases (STM 2014; 6:252ra122). Further, our studies of FGFR1 transcripts by RNA sequencing of 183 human PCas identified eight different protein coding transcripts as the most abundantly expressed with different human PCas expressing different FGFR1 isoforms (Abstract #3913 AACR 2015). These results suggest that different FGFR1 isoforms in PCa cells may partially underlie the biological heterogeneity of PCa. The studies presented here focus in two of the best-characterized isoforms: FGFR1alpha (R1alpha), with 3 Ig-like domains, and 822 aa in length; and FGFR1beta (R1beta), with only 2 Ig-like domains and 733 aa in length. We assessed whether these isoforms induce activation of the same pathways using PC3 cells transiently transfected with empty vector (EV), R1alpha (NM_023110.2) or R1beta (NM_023105.2) and treated with vehicle, FGF2 or FGF9. By Western blot analysis we found that total FGFR1 expression (relative to a loading control) was similar in cells transfected with R1beta or R1alpha. Levels of p-FGFR1 were high in untreated cells transfected with R1alpha, but no further induction was observed after treatment with FGF2 or FGF9. However, p-FGFR1 expression was almost undetectable in untreated cells expressing R1beta and was slightly induced by FGF2 but not by FGF9. p-PLCγ expression was found only in cells expressing R1alpha. We subsequently stably transfected these isoforms in PC3 cells and discovered that while no significant difference in R1alpha and R1beta transcript levels was detected, the levels of R1alpha protein were higher than those of R1beta suggesting that these isoforms may undergo different translational regulation, We also performed in vivo studies by subcutaneous injection of R1alpha or R1beta expressing PC3 cells in immunocompromised mice and weekly monitored tumor volume. We found that PC3 cells expressing R1alpha developed significantly larger tumors than PC3 cells expressing R1beta. We are now in the process of analyzing at the molecular level these tissue specimens. In conclusion, R1alpha and R1beta isoforms trigger different biological effects in PCa cells. Because different isoforms are expressed in different prostate tumors, the expression of these isoforms could be associated with the typical PCa heterogeneity and could explain differences in therapy responses 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. Alpha and beta isoforms of fibroblast growth factor receptor 1 in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1871.

Prostate Cancer (PCa) is the second leading cause of cancer in men. PCa cells display abnormal expression of cytoskeletal proteins resulting in an augmented capacity to resist chemotherapy and colonize other organs. We have previously shown that heme-oxygenase 1 (HO-1) has a strong anti-tumoral effect in prostate cancer (PCa) and regulates the adhesive properties of PCa cells. Innovative high-throughput proteomic platforms are now identifying and quantifying new specific and sensitive biomarkers for PCa detection, stratification and treatment. Towards this end, we undertook an in-depth mass spectrometry-based proteomics study to build the Heme-oxygenase 1 (HO-1) interactome in PCa, in an effort to identify HO-1 molecular partners associated to the integrity of the cellular architecture and assess actin dynamics of PCa cells under HO-1 modulation. The proteomics analysis of HO-1 interacting proteins yielded several cytoskeletal-associated proteins regulating actin filament dynamics, such as gelsolin, lasp1, SIPA1L1, testin, moesin, tropomodulin and vinculin. The bioinformatics screening across the Oncomine platform revealed that the RNA expression profiles of the cytoskeletal HO-1 interacting proteins, lie within the 10 percent of the most consistently low-expressed genes in prostate adenocarcinoma compared to normal tissue. Motility changes were assessed on fiber-like 2D migration scenarios displaying a reduced frequency in migration events and in migration speed under hemin exposure, a specific pharmacological inducer of HO-1. A significant higher proportion of filopodia-like protrusions among neighboring cells and cellular contacts were observed when HO-1 was induced; effects reversed under HO-1 silencing. Altogether, our experimental findings demonstrate that HO-1 modulation in PCa induces the remodeling of the actin filament architecture at filopodia, alters the migratory patterns and cellular morphology, showcasing the relevance of the cytoskeletal proteins as potential therapeutic targets against the aggressive and metastatic disease. Citation Format: Alejandra V. Paez, Carla Pallavicini, Federico Schuster, Jimena Giudice, Pia Valacco, Estefania Labanca, Nicolas Anselmino, Emiliano Ortiz, Maria Binaghi, Javier Cotignola, Marcelo Marti, Luciana Bruno, Valeria Levi, Nora Navone, Elba Vazquez, Geraldine Gueron. Hitting the brakes on the migratory capacity of tumoral cells: Targeting key regulators of actin dynamics in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5058.

Androgen ablation by pharmacological use or castration is a standard therapy for treating prostate cancer (PCa), however, at the later stage PCa becomes castration-resistant and is metastasized primarily to bones. Epigenetic changes that cause dysregulated gene expression during progression and metastasis of androgen-independent prostate cancer are not clearly understood. We previously reported that the jumonji C domain-containing NO66 is a histone demethylase specific for H3K4me3 and H3K36me3, which negatively regulates osteoblast differentiation and bone formation. By analyzing a multi-cancer tissue microarray, the serendipitous observation was made that NO66 is overexpressed in a variety of cancers. Our objective was to examine NO66's role in the pathogenesis of prostate cancer and bone metastasis. Our findings indicate that NO66 knockdown resulted in significant decreases in the proliferation and invasiveness of prostate cancer cells (PC3 and DU145) while overexpressing NO66 promoted their proliferation and invasion. Furthermore, NO66 depletion resulted in a two- to three fold decrease in colony formation and anchorage-independent growth of PCa cells suggesting an oncogenic function of NO66. RNAseq analysis comparing between control and NO66-depleted DU-145 cells revealed a repertoire of differentially expressed genes. NO66 up-regulates genes with proliferative activity such as chemokine CXCL5, growth factor IGFBP5, IL6, WNT and MAPK4 pathway, which are known to drive PCa proliferation. Furthermore, ChIP sequencing for NO66 and H3K9AC in NO66-overexpressing PC3 cells revealed that interaction sites of NO66 overlap with peaks of H3K9AC in majority of the genes. These data clearly indicate that NO66 activates a cohort of genes which are involved in tumor progression and metastasis. Strikingly, implanting NO66-overexpressing prostate cancer cells into the femur of SCID male mice caused massive femoral bone loss after three weeks, which suggests that NO66 causes osteolytic bone lesions similar to the clinico-pathological features of advanced PCa. Finally, analysis of tumor xenografts derived from patients by immunohistochemistry showed high expression of NO66 in high grade advanced PCa compared to low grade and hyperplasia suggesting that NO66 may be used as a biomarker to distinguish aggressive prostate cancer from indolent stage. Our data suggest that NO66 plays an important role in cell proliferation, cell invasion and bone metastasis of prostate cancer. Citation Format: Rozita Bagheri-Yarmand, Nora Navone, Xinhai Wan, Christopher Logothetis, Johnny Huard, Krishna Sinha. Oncogenic and osteolytic function of histone demethylase NO66 in castration resistant prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 693.