Aggressive CaP Research Articles

Myeloid differentiation factor-2/LY96, a potential predictive biomarker of metastasis and poor outcomes in prostate cancer: clinical implications as a potential therapeutic target

Prostate cancer (CaP) is the most diagnosed cancer in males and the second leading cause of cancer deaths. Patients with localized tumors are generally curable. However, no curative treatment exists for patients with advanced and metastatic disease. Therefore, identifying critical proteins involved in the metastatic process would help to develop new therapeutic options for patients with advanced and aggressive CaP. We provide strong evidence that Myeloid differentiation factor-2 (MD2) plays a critical role in metastasis and CaP progression. Analysis of tumor genomic data showed that amplifications of MD2 and increased expression are associated with poor outcomes in patients. Immunohistochemistry analysis of tumor tissues showed a correlation between the expression of MD2 and cancer progression. The Decipher-genomic test validated the potential of MD2 in predicting metastasis. In vitro studies demonstrated that MD2 confers invasiveness by activating MAPK and NF-kB signaling pathways and inducing epithelial–mesenchymal transition. Furthermore, we show that metastatic cells release MD2 (sMD2). We measured serum-sMD2 in patients and found that the level is correlated to disease extent. We determined the significance of MD2 in metastasis in vivo and as a therapeutic target, showing that the molecular and pharmacological targeting of MD2 significantly inhibited metastasis in murine models. We conclude that MD2 predicts metastatic behavior, and serum-MD2 could be studied as a potential non-invasive biomarker for metastasis, whereas MD2 presence on prostate biopsy predicts adverse disease outcome. We suggest MD2-targeted therapies could be developed as potential treatments for aggressive metastatic disease.

Deregulated kinase action in prostate cancer: molecular basis and therapeutic implications.

Prostate cancer (CaP) remains the second leading cause of cancer-related mortality in American men. Systemic treatments for metastatic CaP, which causes the majority of deaths, include androgen deprivation therapy and chemotherapy. These treatments induce remissions but do not cure CaP. Novel and functionally diverse therapeutic targets that control the cell biology that drives aggressive CaP progression are needed to overcome treatment resistance. Because signal transduction that mediates CaP cell behavior is tightly regulated by phosphorylation, kinases have attracted interest as alternative targets for CaP treatments. Here, we examine emerging evidence from recent NextGen sequencing and (phospho) proteomics analyses on clinical CaP specimens that were obtained during lethal disease progression to determine the role of deregulated kinase action in CaP growth, treatment resistance, and recurrence. We provide an overview of kinases that are impacted by gene amplification, gene deletion or somatic mutations during the progression from localized treatment-naïve CaP to metastatic castration-resistant CaP or neuroendocrine CaP, and the potential impact of such alterations on aggressive CaP behavior and treatment efficacy. Furthermore, we review knowledge on alterations in the phosphoproteome that occur during the progression to treatment-resistant CaP, the molecular mechanisms in the control of these changes, and the signal transduction associated with them. Finally, we discuss kinase inhibitors under evaluation in CaP clinical trials and the potential, challenges, and limitations to moving knowledge on the CaP kinome forward to new therapeutic strategies.

Oncologic outcomes of organ-confined Gleason grade group 4-5 prostate cancer after radical prostatectomy.

Organ-confined prostate cancer (CaP) at radical prostatectomy (RP) is associated with good long-term outcomes. However, information for aggressive Gleason organ-confined CaP is scant. To investigate the impact of Gleason grade group (GG) 4-5 on long-term oncologic outcomes after RP. Within a high-volume center database we identified patients who harbored organ-confined CaP (pT2) at RP between 1992 and 2017. Only patients with negative surgical margins, without lymph node invasion and without neo- and/or adjuvant androgen deprivation therapy and/or adjuvant radiotherapy were included. Patients with GG1 were excluded. Kaplan-Meier analyses and Cox regression models tested the effect of GG4 and GG5 on biochemical recurrence-free (BFS), metastasis-free (MFS), overall survival (OS) and cancer-specific mortality (CSM) free survival. Of 10,855 identified pT2 patients, 0.1% (n=81) and 0.1% (n=114) harbored GG4 and GG5, respectively. At 10-years after RP, BFS, MFS, OS and CSM-free rates were 80.3 vs. 68.6 vs. 55.4% (P<0.001), 96.7 vs. 89.9. vs. 83.4% (P<0.001), 93.2 vs. 78.3 vs. 72.6% (P<0.001) and 99.3 vs. 98.0 vs. 82.7% (P<0.001) for GG2 and GG3 vs. GG4 vs. GG5, respectively. In multivariable Cox regression models, GG5 represented an independent predictor for biochemical recurrence (Hazard ratio [HR] 3.00, P<0.001), metastasis (HR 5.01, P<0.001), death (HR 2.72, P<0.01) and cancer-specific death (HR 30.1, P<0.001). Conversely, GG4 represented an independent predictor for death (HR 2.10, P=0.04) and cancer-specific death (HR 6.09, P=0.01) but not for biochemical recurrence and metastasis. GG4/5 in organ-confined CaP is rare. But its associated with worse oncologic outcomes after RP, namely biochemical recurrence, metastasis, death and cancer-specific death. Patients with organ-confined GG4/5 and negative margins should be closely followed and may be candidates for risk stratification by genomic markers.

RNA-based biomarkers for the diagnosis, prognosis, and therapeutic response monitoring of prostate cancer.

Prostate cancer (CaP) is the most common malignant neoplasm of the urinary tract. The current recommendations for CaP diagnosis rely on the prostate-specific antigen levels and a digital rectal examination for anatomical abnormalities. However, these diagnostic tools are not highly sensitive. In particular, prostate-specific antigen has a low positive predictive value (approximately 30%). Thus, there is a need to develop biomarkers to improve the early clinical detection of CaP. Several novel technologies enable the identification of biomarkers from diverse sources, including the urine, serum, and prostate tissues. Furthermore, advances in genomic techniques have enabled the analysis of novel biomarkers, such as deoxyribonucleic acids (DNAs), ribonucleic acids (RNAs), proteins, and circulating tumor cells. Previous studies have demonstrated that RNAs are potential diagnostic biomarkers for various cancers using high-throughput sequencing analysis. The sensitivity and specificity of RNA biomarkers are higher than those of protein biomarkers. Polymerase chain reaction enables the amplification of trace levels of RNAs with high sensitivity and specificity. RNA biomarkers provide dynamic insights into cellular states and regulatory processes when compared with DNA biomarkers. Additionally, multiple copies of various RNAs in a cell provide more information than DNA. The levels of specific RNAs in CaP tissues are upregulated when compared with those in non-cancerous tissues. Additionally, RNAs can be easily isolated from various body fluids. Thus, RNAs are potential non-invasive biomarkers for CaP. Moreover, the analysis of RNA levels adjusted for each stage of CaP enables the determination of prognostic individualized therapy for aggressive or progressive CaP. This review focused on the diagnostic and prognostic values of RNAs for CaP.

MiCheck prostate blood test for aggressive prostate cancer designed for the clinical lab setting.

229 Background: Clinicians want a more accurate diagnostic test to identify patients for prostate biopsy. We have previously described the development of a Luminex based test for aggressive prostate cancer (MiCheck). To facilitate widespread deployment of MiCheck on standard clinical chemistry analysers such as Roche Cobas and Abbott ARCHITECT/Alinity systems, we have performed additional analytical validation and model development using the MiCheck-01 clinical samples. A novel algorithm, termed MiCheck Prostate, was developed to differentiate aggressive prostate cancer (Gleason 3+4 or above) from non-aggressive (GS3+3) or no cancer patients. Methods: Serum protein biomarkers were measured in 317 samples from the MiCheck-01 clinical trial using either Luminex Multiplex kits, Abbott ARCHITECT or Beckmann Coulter systems. Logistic regression models were used to select best analytes, then Monte Carlo cross-validation was applied to avoid overfitting. A final model was selected from the cross-validation models with best test specificity at 95% sensitivity. The MiCheck-01 samples were later all measured on a standard Abbott ARCHITECT system using commercial ARCHITECT IVD tests. The best cross-validated model was developed and the results compared to those obtained from the mixed analyte platforms using ROC curve analysis. Results: The MiCheck Prostate algorithm was developed to maximise discrimination between aggressive prostate cancer and no cancer or non-aggressive cancer. The MiCheck Prostate algorithm reports a percentage risk of aggressive prostate cancer on biopsy. MiCheck Prostate is a logistic regression algorithm that combines three serum protein markers and one clinical factor and was derived using data from the MiCheck-01 trial. The algorithm gave an AUC of 0.82, with a 48% specificity at a 95% sensitivity cutpoint, with a negative predictive value of 94% for GS3+4 and higher cancers. The samples were later re-measured using all ARCHITECT immunoassays. Good correlations of each analyte across different measurement platforms was observed (Pearson’s R &gt; 0.9). The algorithm was revalidated using the new set of analyte values. AUC of 0.82 for the detection of aggressive CaP, and sensitivity of 95%, specificity of 47% were obtained for the best model, showing no statistical difference to the previous best model derived from the mixed platforms. NPV of 94% for GS3+4 or higher cancers was maintained. Conclusions: The MiCheck Prostate test shows high sensitivity, specificity and NPV for the detection of aggressive prostate cancer. It uses analytes widely available on standard clinical chemistry analysers such as Abbott Architect and Roche Cobas, hence is simple to deploy in the laboratory setting. Studies are ongoing to confirm the MiCheck Prostate algorithm performance using additional clinical sample cohorts.

Radical prostatectomy for localized prostate cancer: 20-year oncological outcomes from a German high-volume center

IntroductionLong-term outcomes of prostate cancer (CaP) patients treated with radical prostatectomy (RP) from European cohorts are under-reported. We report on 22,843 RP patients from the Martini-Klinik Prostate Cancer Centre treated between 1992 and 2017. Patients and MethodsBiochemical recurrence (BCR) free survival, metastasis free survival (MFS), and cancer specific survival (CSS) were stratified according to National Comprehensive Cancer Network (NCCN) risk categories, pT, and pN stages, RP Gleason Grade Groups (GGG), and surgical margin status (R0/R1). For time to event analyses, uni- and multivariable Cox's proportional hazards models and univariable Kaplan-Meier analyses were applied. ResultsMedian follow up was 68 months. Most favourable 20-year survival rates were exhibited in NCCN low risk (78.7% BCR-free, 96.8% MFS, 90.1% CSS) and pT2, GGG 1 to 2, R0 patients (83.1% BCR-free, 96.7% MFS, 92.6% CSS). 20-year follow up was not constantly reached in patients with aggressive CaP features. For example, NCCN very high-risk patients exhibited 15-year BCR-free survival of 30.5%, while 20-year MFS and CSS in these individuals was reached (64.1% and 60.8%, respectively). Lowest 10-year BCR-free survival (35.6%) was exhibited in pT3b, GGG 4 to 5, R0. Lowest 10-year MFS (49.5%) was exhibited in pT2, GGG 4 to 5, R1. Lowest 10-year CSS (69.8%) was exhibited in pT3b, GGG 4 to 5, R1 patients. In separate pN1 analyses, lowest 10-year BCR-free survival (14.5%), MFS (56.9%), and CSS (71.9%) were exhibited in patients with 3 or more positive lymph nodes. ConclusionOncological outcomes after RP can be excellent for individuals with favorable CaP characteristics, also after 20 years of follow up.

P1091 - Radical prostatectomy for localized prostate cancer: 20-year oncological outcomes from a German high-volume center

Abstract Introduction Long-term outcomes of prostate cancer (CaP) patients treated with radical prostatectomy (RP) from European cohorts are under-reported. We report on 22,843 RP patients from the Martini-Klinik Prostate Cancer Centre treated between 1992 and 2017. Patients and Methods Biochemical recurrence (BCR) free survival, metastasis free survival (MFS), and cancer specific survival (CSS) were stratified according to National Comprehensive Cancer Network (NCCN) risk categories, pT, and pN stages, RP Gleason Grade Groups (GGG), and surgical margin status (R0/R1). For time to event analyses, uni- and multivariable Cox's proportional hazards models and univariable Kaplan-Meier analyses were applied. Results Median follow up was 68 months. Most favourable 20-year survival rates were exhibited in NCCN low risk (78.7% BCR-free, 96.8% MFS, 90.1% CSS) and pT2, GGG 1 to 2, R0 patients (83.1% BCR-free, 96.7% MFS, 92.6% CSS). 20-year follow up was not constantly reached in patients with aggressive CaP features. For example, NCCN very high-risk patients exhibited 15-year BCR-free survival of 30.5%, while 20-year MFS and CSS in these individuals was reached (64.1% and 60.8%, respectively). Lowest 10-year BCR-free survival (35.6%) was exhibited in pT3b, GGG 4 to 5, R0. Lowest 10-year MFS (49.5%) was exhibited in pT2, GGG 4 to 5, R1. Lowest 10-year CSS (69.8%) was exhibited in pT3b, GGG 4 to 5, R1 patients. In separate pN1 analyses, lowest 10-year BCR-free survival (14.5%), MFS (56.9%), and CSS (71.9%) were exhibited in patients with 3 or more positive lymph nodes. Conclusion Oncological outcomes after RP can be excellent for individuals with favorable CaP characteristics, also after 20 years of follow up.

Prostate cancer in young men represents a distinct clinical phenotype: gene expression signature to predict early metastases.

Aim:Several genomic signatures are available to predict Prostate Cancer (CaP) outcomes based on gene expression in prostate tissue. However, no signature was tailored to predict aggressive CaP in younger men. We attempted to develop a gene signature to predict the development of metastatic CaP in young men.Methods:We measured genome-wide gene expression for 119 tumor and matched benign tissues from prostatectomies of men diagnosed at ≤ 50 years and > 70 years and identified age-related differentially expressed genes (DEGs) for tissue type and Gleason score. Age-related DEGs were selected using the improved Prediction Analysis of Microarray method (iPAM) to construct and validate a classifier to predict metastasis using gene expression data from 1,232 prostatectomies. Accuracy in predicting early metastasis was quantified by the area under the curve (AUC) of receiver operating characteristic (ROC), and abundance of immune cells in the tissue microenvironment was estimated using gene expression data.Results:Thirty-six age-related DEGs were selected for the iPAM classifier. The AUC of five-year survival ROC for the iPAM classifier was 0.87 (95%CI: 0.78–0.94) in young (≤ 55 years), 0.82 (95%CI: 0.76–0.88) in middle-aged (56–70 years), and 0.69 (95%CI: 0.55–0.69) in old (> 70 years) patients. Metastasis-associated immune responses in the tumor microenvironment were more pronounced in young and middle-aged patients than in old ones, potentially explaining the difference in accuracy of prediction among the groups.Conclusion:We developed a genomic classifier with high precision to predict early metastasis for younger CaP patients and identified age-related differences in immune response to metastasis development.

Abstract 5014: Identifying and treating robo1−ve/dock1+ve prostate cancer: An aggressive cancer type highly prevalent in African-American patients

Abstract Grant Funding: R01CA193739 Metastasis is the principal cause of death in prostate cancer (CaP) patients. The prognosis is very poor for metastatic CaP. On comparison, African-American patients exhibit high mortality rates than Caucasian counterparts. The failure of therapies for metastatic CaP is observed very high in patients belonging to African-American race. There is a need to identify new target-based therapies which could be either (i) beneficial to patients regardless of race or (ii) treat high-risk patients such as African-Americans who fail standard therapies compared to Caucasians. This requires identification of molecular pathways which are either (i) preferentially active in African-Americans or (ii) highly active in both races. We posit that the loss of ROBO1 in advanced stage CaP contributes to the disparity observed between African-Americans and Caucasian. We conducted a comprehensive analysis of race-based primary and metastatic tumors for ROBO1 and its downstream targets (RAC1, DOCK1, E-Cadherin, and β-catenin). We show that ROBO1 expression is progressively lost with the increasing stage of CaP in African-American patients. Next, in order to explore the underlying mechanism that leads to ROBO1 loss, we analyzed matching tumor DNA from patients and found that the ROBO1 promoter is methylated in the majority of African-American cases in agreement with the CaP stage. In addition, we found that ROBO1 loss is associated to increasing RAC1 activation in tumors and representative cell models. We show that loss of ROBO1 results in increased DOCK1 activity that in turn induces Rac1 signaling in CaP cells. We hypothesized that targeting DOCK1/Rac1 network could be an ideal strategy to treat metastatic CaP, particularly in ROBO1−/- cases. To test the hypothesis, we tested the therapeutic efficacy of a selective inhibitor of DOCK1/Rac1 interaction in ROBO1+ve and ROBO1−ve CaP cells. We observed that DOCK1-inhibitor treatment significantly decreased the RAC1 activation and β-catenin activity in metastatic CaP cells. Notably, inhibition of DOCK1/Rac1 was observed to significantly decrease the motility and invasiveness of aggressive metastatic CaP cells. Furthermore, DOCK1-inhibitor significantly blocked the potential of metastatic CaP cells to extravasate (cross-over) through the endothelial lining (constituted of HUVEC cells). These data suggested the significance of DOCK1/Rac1 pathway in a metastasis of CaP particularly in African-Americans. We conclude that the ROBO1/RAC1 ratio in tumor biopsies could be developed as an early biomarker of metastasis in African-American patients. We suggest that DOCK1-inhibitor therapy would be more beneficial to patients who exhibit ROBO1− tumor, and African-American patients are such as group who likely to benefit this therapy. Citation Format: Marina Graciela Ferrari, Arsheed Ganaie, Adrian P. Mansini. Identifying and treating robo1−ve/dock1+ve prostate cancer: An aggressive cancer type highly prevalent in African-American patients [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 5014.

A copy number gain on 18q present in primary prostate tumors is associated with metastatic outcome

BackgroundMost prostate cancers (CaPs) grow slowly and remain indolent, yet some become aggressive and metastasize. Clinical decision-making requires prognostic markers that can be utilized at the time of diagnosis to identify aggressive tumors. Previous studies have shown a correlation between genomic alterations on the long arm of chromosome 18 (18q) and metastatic CaP. ObjectiveThe goal of this study was to comprehensively profile copy number alterations found on 18q in prostate tumors with varying outcomes to identify putative biomarkers associated with more aggressive disease MethodsA custom comparative genomic hybridization array was created composed of high-density tiling of 18q sequences. Primary prostate tumor tissues were gathered from men who underwent radical prostatectomy and were categorized based on the patient's long-term clinical outcome as either metastatic disease (MET) or no evidence of disease (NED). DNA was isolated from formalin-fixed, paraffin-embedded prostatectomy tumor tissues, and analyzed for copy number variations (CNVs). Protein levels of genes found within the region of CNVs were analyzed using immunohistochemistry. ResultsThirty-Four primary prostate tumors were analyzed: 17 NEDs and 17 METs. Two significant regions of copy number gains were found on 18q associated with outcome. One gain located at 18q11.2 was found exclusively in NED outcome tumors while another gain, located at 18q21.31, was found exclusively in MET outcome tumors (P -value< 0.0076). Immunohistochemistry analysis of protein levels showed more protein associated with copy number gain in the MET samples vs. those without the gain as indicated by H-scores of 184.7 and 121.0 respectively. ConclusionsThe latter of these CNVs represent a putative biomarker for aggressive disease and highlights a putative metastasis promoting gene. Further study of known connections to CaP suggests that the paracaspase MALT1 is the most likely target of the copy number gain and represents a potential therapeutic target. Future studies would be of interest to determine MALT1’s role in aggressive CaP and the ability of this CNV region to differentiate CaP that will eventually metastasize.

Abstract C023: Annexin A2 expression level correlates with adverse pathology and disease progression in prostate cancer

Abstract Background: Annexin A2 (ANXA2) is absent or expressed focally in the prostate epithelium of moderately differentiated tumors but is highly expressed in a subset of poorly differentiated tumors. We observed earlier that ANXA2 transcription is negatively regulated by ERG in prostate cancer (CaP) cells. Here we examined whether Annexin A2 expression in prostate tumors correlates with aggressive CaP or disease progression, whether prostate tumors of Caucasian American (CA) and African American (AA) patients have distinct Annexin A2 expression profiles, and if the levels of Annexin A2 in sera is correlated to its expression in tumor tissues. We also speculated on the underlying mechanism likely to regulate ANXA2 expression in the absence of ERG. Methods: We evaluated Annexin A2 and ERG expression in index tumors on whole-mounted prostate sections and tissue microarrays derived from radical prostatectomies of 176 patients by immunohistochemistry. Both cohorts were matched for post RP follow-up, pathologic stage, and race. ERG and Annexin A2 expression status in tumors were correlated to Gleason score, biochemical recurrence and metastasis. We measured, by enzyme-linked immunosorbent assay (ELISA), Annexin A2 proteins levels in sera of 94 controls and 222 patients, matched for race and Gleason sum. Correlation between promoter methylation and gene expression profiles of ANXA2 from TCGA studies were also examined. Results: A significant association was found between ANXA2(+) index tumors and higher Gleason Grade Group (GG) 4 and 5, and worse pathologic stage (pT 3-4). Moreover, ERG(-)/ANXA2(+) tumors were found to be associated with Gleason GG 4 and 5, and ERG(+)/ANXA2(-) tumors with Gleason GG 1 and 2. However, unlike ERG expression, which is more prevalent in prostate tumors of CA men, Annexin A2 expression was not associated with race. Trends toward earlier occurrence of BCR and worse metastasis-free survival were observed among ANXA2(+) cases. Significantly higher levels of secreted Annexin A2 were detected in the sera healthy controls than in CaP patients. Analysis of TCGA prostate cancer dataset revealed an inverse association between methylation of the ANXA2 promoter region and ANXA2 expression in prostate tumor tissues. Conclusion: The association between high Annexin A2 expression in prostate tumors with higher grade and stage suggests a potential for its use as a biomarker for aggressive CaP and to stratify patients after radical prostatectomy. Citation Format: Shyh-Han Tan, Denise Young, Wei Huang, Amina Ali, Lakshmi Ravindranath, Kurt Christen, Kevin Babcock, Huai-Ching Kuo, Yongmei Chen, Jacob Kagan, Sudhir Srivastava, Albert Dobi, Srinivasan Alagarsamy, Gyorgy Petrovics, David G. McLeod, Jennifer Cullen, Inger L. Rosner, Isabell A. Sesterhenn, Shiv Srivastava. Annexin A2 expression level correlates with adverse pathology and disease progression in prostate cancer [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C023.

Abstract B02: Genomic and transcriptomic profiling of urine in prostate cancer

Abstract Prostate cancer (CaP) affects one in 8 men in the US and Canada, but many of these men could be spared aggressive treatment, often associated with significant morbidity, if diagnostic tools could more accurately assess the risk that the cancer will metastasize. Low pathologic/Gleason grade (Gleason Grade 3+3=6, equivalent to WHO Grade Group 1) is the main defining feature of nonlethal low-risk CaP, and active surveillance (AS) is the standard of care for such men. However, current diagnostic methods cannot accurately separate low and higher risk CaP based on core biopsies, presenting a major dilemma as repeated biopsies during AS carry significant risks. At present no diagnostic tools, either imaging-based or biomarker-based, can safely supplant repeated invasive biopsies. Genomic profiling of liquid biospecimens (“liquid biopsies”) is quickly becoming translated as part of contemporary clinical trials and is being translated for use in the diagnostic setting. Less invasive than traditional solid tissue biopsy approaches, liquid biopsy specimens have the potential to be used not only for early detection but also to monitor therapeutic response and progression. Leveraging the advantage of the anatomic relationship between the prostate and urinary tract, we obtained 11 post-digital rectal exam (DRE) urine samples from patients who presented at the Kingston Health Sciences Centre (Kingston, Ontario) to be evaluated for prostate cancer. Through a pan-Canadian research consortium called PRONTO, we developed a tissue-based classifier (PRONTO-T) differentiating between low- and high-risk cancer with an independently validated AUC of 0.85. This proof-of-concept study tests post-digital rectal examination (DRE) urine samples for genomic features associated with aggressive CaP, including features included in the PRONTO-T risk classifier. DNA and RNA were extracted from 11 post-DRE urine samples. Profiling with a customized NGS-based targeted-methylation panel by Thermo Fisher demonstrated robust signals from urine-derived DNA. Similarly, urine samples profiled with the pan-cancer Oncomine Comprehensive Assay (v3) detected potentially actionable mutations in MET and PIK3CA. RNA obtained from post-DRE urines met quality control standards for transcriptional profiling using the NanoString nCounter System and revealed patterns of gene expression changes consistent with prostate cancer biology. These encouraging preliminary results demonstrate that the profiling of urine can provide sufficient diagnostic and potentially prognostic information to significantly impact the clinical management of prostate cancer. Citation Format: Jane Bayani, Palak G. Patel, Dan Dion, Vanessa Bratti, Ania Ahmadian-Namin, Tamara Jamaspishvili, Jason Izard, D. Robert Siemens, David M. Berman, John M. S. Bartlett. Genomic and transcriptomic profiling of urine in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B02.

Associations of cell cycle genetic variants with aggressive prostate cancer in the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial.

175 Background: The need to differentiate patients at risk for developing aggressive prostate cancer (CaP) from those at risk for less aggressive disease has led to efforts to identify genetic markers to predict disease course and personalize treatment. A study with participants from Johns Hopkins Medical Institutions (JHMI) and Washington University (WU) found single nucleotide polymorphisms (SNPs) in cell cycle genes were associated with risk of aggressive CaP. We sought to replicate those results in the European-American population of the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial. Methods: We focused on variants associated with aggressive CaP in the JHMI and WU cohorts, which included 13 SNPs in 12 genes ( CCNC, CCND3, CCNG1, CCNT2, CDK2, CDK6, MDM2, SKP2, TERF2, WEE1, YWHAB, YWHAH). Variants were genotyped using the Pyrosequencing assay. Patients were classified into high risk (Gleason≥8, pT3b, N+, M+), low risk (Gleason≤7, ≤pT3a, N0, M0) or non-cancer control groups based on clinicopathologic characteristics. Logistic regression analysis was used to compare genotype frequencies of each variant between groups using the dominant model. Results: There were 108 aggressive and 1080 non-aggressive CaP patients, and 1155 controls . CDK6 ( rs8) was associated with increased risk of any CaP (OR 1.2, 95% CI 1.02-1.42; p = 0.032) and high risk disease (OR 1.63, 95% CI 1.09-2.42; p = 0.017) vs. controls. In contrast, the JHMI and WU cohort found CDK6 variants to be protective against aggressive CaP. CCNG1 ( rs11541970) approached significance (p = 0.052) between high risk and control groups, and CCNC conferred a protective effect consistent with the prior study, but did not reach significance (p = 0.101). No associations with any cell cycle gene variants were detected when comparing high and low risk patients. Conclusions: Our study did not replicate the results from the JHMI and WU cohorts. CDK6 predicted an increased risk of developing any CaP and high risk CaP. However, directionality was opposite to the prior study, indicating that this variant is unlikely to be a true predictor of increased risk of or protection from aggressive CaP.

Protein Kinase N1 control of androgen-responsive serum response factor action provides rationale for novel prostate cancer treatment strategy.

Sustained reliance on androgen receptor (AR) after failure of AR-targeting androgen deprivation therapy (ADT) prevents effective treatment of castration-recurrent (CR) prostate cancer (CaP). Interfering with the molecular machinery by which AR drives CaP progression may be an alternative therapeutic strategy but its feasibility remains to be tested. Here, we explore targeting the mechanism by which AR, via RhoA, conveys androgen-responsiveness to serum response factor (SRF), which controls aggressive CaP behavior and is maintained in CR-CaP. Following a siRNA screen and candidate gene approach, RNA-Seq studies confirmed that the RhoA effector Protein Kinase N1 (PKN1) transduces androgen-responsiveness to SRF. Androgen treatment induced SRF-PKN1 interaction, and PKN1 knockdown or overexpression severely impaired or stimulated, respectively, androgen regulation of SRF target genes. PKN1 overexpression occurred during clinical CR-CaP progression, and hastened CaP growth and shortened CR-CaP survival in orthotopic CaP xenografts. PKN1's effects on SRF relied on its kinase domain. The multikinase inhibitor lestaurtinib inhibited PKN1 action and preferentially affected androgen regulation of SRF over direct AR target genes. In a CR-CaP patient-derived xenograft, expression of SRF target genes was maintained while AR target gene expression declined and proliferative gene expression increased. PKN1 inhibition decreased viability of CaP cells before and after ADT. In patient-derived CaP explants, lestaurtinib increased AR target gene expression but did not significantly alter SRF target gene or proliferative gene expression. These results provide proof-of-principle for selective forms of ADT that preferentially target different fractions of AR's transcriptional output to inhibit CaP growth.

Abstract LB-211: The cardiac transcription factor Nkx2-5 contributes to SRF-dependent AR action in prostate cancer

Abstract Ligand-activated androgen receptor (AR) drives prostate cancer (CaP) growth. Heterogeneity in AR action exists and AR often remains active when therapies targeting its ligand-activation have failed. Understanding the mechanisms by which AR mediates CaP progression may lead to new treatments. Previously, we identified a mechanism in which AR imparts androgen-responsiveness to the transcription factor Serum Response Factor (SRF). SRF-dependent AR action is enriched in CaP, correlates with CaP recurrence and is maintained in late-stage CaP. SRF binds constitutively at its target genes and becomes transcriptionally active via upstream signaling cascades or SRF cofactors. Our prior work showed that RhoA signaling transduces androgen regulation to a significant fraction, but not all, SRF target genes. Here, we isolate the homeodomain protein Nkx2-5 as a novel regulator of androgen-responsive SRF action. Nkx2-5 co-operates with SRF in transcriptional control of heart development. In CaP, Nkx2-5 promoter hypermethylation is a biomarker that distinguishes CaP from benign prostate, yet consequences of differential Nkx2-5 expression on CaP are unknown. In the CaP cell models LNCaP, C4-2 and VCaP, siRNA-mediated loss of Nkx2-5 increased basal and androgen-dependent expression of exogenously and endogenously expressed SRF target genes, and these effects were reversed after silencing of SRF. Conversely, overexpression of Nkx2-5 decreased androgen-responsiveness of SRF target genes. Co-IP and ChIP assays indicated androgen-stimulated SRF-Nkx2-5 interaction in CaP cell nuclei. Loss of Nkx2-5 resulted in distinct morphological changes in CaP cells, which depended entirely on SRF. In rhodamine phalloidin staining assays, loss of Nkx2-5 increased significantly (p&amp;lt;0.01) cell length, width and circumference, and the number of cell protrusions in cultures with and without androgen stimulation. Changes in cell shape were accompanied by modest decreases in CaP cell proliferation and migration, but significant (p&amp;lt;0.05) decreases in CaP cell-matrix adhesion. Nkx2-5 silencing increased the size of the cell nucleus and led to multilobed and grooved nuclei and enlarged nucleoli. These nuclear shape changes were not associated with alterations in DNA ploidy, but were associated with changed heterochromatin organization as reflected in a reduction in heterochromatin protein 1 immunostaining and nuclear content. Such Nkx2-5-dependent changes in nuclear shape and size and heterochromatin organization occurred also in non-malignant prostate epithelial RWPE2 cells. This work uncovered a novel role for Nkx2-5 in the transition from benign prostate to CaP, showing that Nkx2-5 regulates SRF transcriptional activity in CaP cells and demonstrating that differential Nkx2-5 expression alters cell-matrix adhesion and nuclear morphology, both processes that are associated with aggressive CaP progression. Citation Format: Salma Ben-Salem, Dhirodatta Senapati, Giridhar Mudduluru, Yixue Su, Varadha Balaji Venkadakrishnan, Hannelore V. Heemers. The cardiac transcription factor Nkx2-5 contributes to SRF-dependent AR action in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-211.

Abstract 3745: Inhibition of Protein Kinase N1 prevents control of AR over SRF action in advanced prostate cancer

Abstract A major limitation in the management of castration-recurrent prostate cancer (CR-CaP) is the lack of treatments to inhibit androgen receptor (AR) action that is driving CaP growth when androgen-deprivation therapy (ADT) has failed. AR action breaks down in fractions that are regulated differently at the molecular level, control diverse aspects of CaP biology and contribute differentially to CaP progression. Blocking a specific fraction(s) of AR action that mediates CaP progression may serve as alternative treatment strategy, yet evidence for such an approach and drugs that render this approach feasible are not known. Our laboratory has identified a mechanism of AR action in which AR conveys androgen-responsiveness to Serum Response Factor (SRF) target genes via androgen activation of RhoA. Androgen-responsive SRF action mediates cell migration and is associated with aggressive CaP behavior and recurrence. Importantly, control of AR over RhoA-SRF action is maintained in CR-CaP, which renders this signaling a potentially attractive novel therapeutic target. Using an siRNA screen, we identified Protein Kinase N1 (PKN1) as the Rho effector that transduces androgen-responsiveness from RhoA to SRF. In promoter-reporter, qRT-PCR and oligoarray assays, knockdown of PKN1 severely impaired the androgen-regulation of SRF target genes, but affected androgen response of only a few (12 of 452) direct AR target genes. In contrast, transient overexpression of PKN1 preferentially stimulated androgen-responsive SRF target gene expression. Stable overexpression of PKN1 hastened growth and shortened survival in CaP orthotopic xenografts. Site-directed mutagenesis showed that the effect of PKN1 on SRF target genes relied on its kinase activity. Treatment with lestaurtinib, a multikinase inhibitor that is also a potent inhibitor of PKN1, mimicked the effects of PKN1 knockdown on expression of SRF and AR target genes in several CaP cell lines and ex vivo CaP explants. Lestaurtinib also inhibited the proliferation of CaP cells that are either ADT-naïve or -resistant, express only the constitutively active AR variant ARv567es, or are growth-stimulated by the AR-activating ADT drug metabolite 5α-abiraterone. RNA-Seq followed by MSigDB analyses confirmed that lestaurtinib impairs androgen-dependent PKN1 activity and revealed that 100 of the 127 MgSigDB gene sets isolated after PKN1 silencing overlapped with 123 gene sets derived after lestaurtinib treatment. Overlapping gene sets included several related to hormonal carcinogenesis and cancer progression, prostate development, signaling cascades that have been implicated in SRF and RhoA function and/or are relevant to CaP. PKN1 inhibition via lestaurtinib, which is used already to treat human hematologic malignancies and is well tolerated by carcinoma patients, may thus serve as novel alternative treatment strategy to target AR-dependent SRF action in CR-CaP. Citation Format: Varadha Balaji Venkadarkrishnan, Adam DePriest, Yixue Su, Giridhar Mudduluru, Salma Ben-Salem, Sangeeta Kumari, Qiang Hu, Eduardo Cortes, Scott Dehm, Cristina Magi-Galluzzi, Eric Klein, Nima Sharifi, Song Liu, Hannelore Heemers. Inhibition of Protein Kinase N1 prevents control of AR over SRF action in advanced 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 3745.

Testosterone treatment and the risk of aggressive prostate cancer in men with low testosterone levels.

PurposeTestosterone treatment of men with low testosterone is common and, although relatively short-term, has raised concern regarding an increased risk of prostate cancer (CaP). We investigated the association between modest-duration testosterone treatment and incident aggressive CaP.Materials and methodsRetrospective inception cohort study of male Veterans aged 40 to 89 years with a laboratory-defined low testosterone measurement from 2002 to 2011 and recent prostate specific antigen (PSA) testing; excluding those with recent testosterone treatment, prostate or breast cancer, high PSA or prior prostate biopsy. Histologically-confirmed incident aggressive prostate cancer or any prostate cancer were the primary and secondary outcomes, respectively.ResultsOf the 147,593 men included, 58,617 were treated with testosterone. 313 aggressive CaPs were diagnosed, 190 among untreated men (incidence rate (IR) 0.57 per 1000 person years, 95% CI 0.49–0.65) and 123 among treated men (IR 0.58 per 1000 person years; 95% CI 0.48–0.69). After adjusting for age, race, hospitalization during year prior to cohort entry, geography, BMI, medical comorbidities, repeated testosterone and PSA testing, testosterone treatment was not associated with incident aggressive CaP (HR 0.89; 95% CI 0.70–1.13) or any CaP (HR 0.90; 95% CI 0.81–1.01). No association between cumulative testosterone dose or formulation and CaP was observed.ConclusionsAmong men with low testosterone levels and normal PSA, testosterone treatment was not associated with an increased risk of aggressive or any CaP. The clinical risks and benefits of testosterone treatment can only be fully addressed by large, longer-term randomized controlled trials.

Abstract 2830: Secreted protein acidic and rich in cysteine antigen and autoantibodies in sera of prostate cancer patients: Potential use in diagnosis /prognosis

Abstract Objectives: Improvements in blood based biomarkers for detecting clinically significant prostate cancer (CaP) or for distinguishing between indolent and aggressive CaP are critical in enhancing the management of this most common non-skin cancer of men in the US. These biomarkers include tumor associated antigens (TAAs) and autoantibodies (AAbs) against TAAs in patient sera. SPARC/Osteonectin is highly expressed in metastatic cancers (glioblastoma, melanoma, breast cancer and prostate cancer) and promotes bone metastasis and epithelial-mesemchymal transition. Our comparative transcriptome analyses of well/moderately differentiated CaP with poorly differentiated CaP, defined SPARC as central node in the network of genes with common regulatory elements (NKXH_NKXH_HOX) associating with poorly differentiated CaP. Further, we reported associations of high levels of SPARC mRNA or protein with Gleason 8-10 or poorly differentiated primary tumors and association with metastatic progression. The aims of this study are: i) Quantify SPARC antigen in the sera CaP patients; ii) Are AAbs against SPARC present in the serum of CaP patients?; iii) Is there a correlation between SPARC AAb level in patient sera and disease status? iii) Does SPARC AAb level vary according to distinct ethnic groups? Methods: Sera from CaP patients and healthy controls were evaluated for SPARC antigen using a commercial enzyme-linked immunosorbent assay (ELISA) kit. AAbs against SPARC were determined by ELISA utilizing recombinant full-length human SPARC protein as a substrate. For evaluation we used Caucasian American (CA, n=117) and African American (AA, n=111) CaP patients comprising Gleason 6-10, and healthy controls (CA, n=52; AA, n=45). Results: SPARC antigen levels in the sera showed a difference in the overall case versus control groups (p=0.0016) and this trend was observed only in AA (p=0.0015) in comparison to CA patients (p=0.1709). SPARC AAbs were detected in the sera and the values were significant in the overall case versus control (p&amp;lt; .0001) and also in CA (p&amp;lt;.0001) and AA (p&amp;lt;.0001) CaP patients with levels significantly lower in patients compared to controls. AAb reactivity for CaP patients was similar between CA and AA groups. Conclusions: This study demonstrated the presence of AAbs against SPARC in CaP patient serum for the first time. Of note, highly significant differences were noted between CaP patient (low) and controls (high) sera, across different ethnic groups. These data suggest further evaluation of SPARC AAbs as a promising serum biomarker for CaP. Source of Funding: This research was supported by the Center for Prostate Disease Research, Uniformed Services University Grant HU0001-10-2-0002. Citation Format: Anshu Rastogi, Andy Martinez, Amina Ali, Shyh-Han Tan, Jennifer Cullen, Yongmei Chen, Gyorgy Petrovics, Albert Dobi, Lakshmi Ravindranath, Denise Young, Isabell Sesterhenn, Jacob Kagan, Sudhir Srivastava, David McLeod, Inger Rosner, Shiv Srivastava, Alagarsamy Srinivasan. Secreted protein acidic and rich in cysteine antigen and autoantibodies in sera of prostate cancer patients: Potential use in diagnosis /prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2830. doi:10.1158/1538-7445.AM2017-2830

Abstract 2969: Paracrine effect of the endothelium on prostate cancer cells

Abstract Prostate cancer (CaP) is the most commonly diagnosed cancer and the second leading cause of cancer deaths among males in the United States. Androgen deprivation therapy (ADT) is the standard treatment for advanced or metastatic CaP. However, during ADT, CaP progresses from an androgen-sensitive (AS-CaP) to a more aggressive, and eventually lethal, castration-resistant (CRPC) phenotype. There is evidence to suggest that the prostate tumor mass is under tight control of endothelial microvasculature due to an increase in angiogenesis by tumor cells. Nevertheless, now there is evidence to support that this influence is not one-directional and that the endothelial cells secrete a large number of active substances (angiocrine factors), which may directly or indirectly influence tumor growth and progression. However, the direct impacts of the endothelium on prostate tumor progression or the molecular mechanisms that are involved in this communication remain unclear. Here we investigated the potential influence of endothelium-derived paracrine factors on prostate cancer biology and the role of connexins in these interactions, since connexins play a major role in cell-cell communication and form a bidirectional signaling pathway to assemble gap junctions and alter cell behaviors. We measured the effect of conditioned media (CM) obtained from a primary culture of human endothelial cells isolated from umbilical vein (HUVEC) on viability, proliferation, migration and invasion of CaP cell lines (LNCaP, LNCaP-C4-2 and PC3) and in the metastatic potential by in vivo assays using co-injection of CaP cell with HUVEC or injection of CaP cells pre-incubated with CM from HUVEC in a zebrafish embryo model. Finally, we studied the expression and the role of connexins on this stimulation using pharmacological (GJIC inhibitors) approaches. All together, our results showed that CM from endothelial cell induces an increases in the viability and proliferation in all CaP cell lines (LNCaP, LNCaP-C4-2 and PC3) but only increases migration of the CRPC cell lines (LNCaP-C4-2 and PC3). We also observed in our in vivo model that endothelial cells either through cell-cell interaction or by paracrine communication increases the metastatic ability of the CaP cells. Moreover, the increase in viability and migration of CaP cells observed with CM from endothelial cells was blocked using inhibitors of gap junctions. Real-time PCR analyses detected an up-regulation of Cx43 mRNA after exposition to CM from endothelial cells. Our data suggest that angiocrine communication between endothelial cells and CaP cells increases proliferation and migration of more aggressive CaP cells which could be important for the acquisition of the aggressive phenotype of the disease, and this interaction could be mediated by Cx43. Delineation of such critical players may culminate in identifying therapeutic targets or biomarkers to counteract CaP, especially advanced CaP. Citation Format: Verónica Torres-Estay, Patricia Fuenzalida, Catalina Ascencio, Carla Cembrano, Daniela Carreño, Néstor Corro, Viviana Montecinos, Gareth Owen, Xavier Figueroa, Julio Amigo, Juan Carlos Saéz, Alejandro Godoy. Paracrine effect of the endothelium on prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2969. doi:10.1158/1538-7445.AM2017-2969

Abstract 865: Isolation of WDR77-mediated interaction between androgen receptor and p53 uncovers novel treatment strategy for prostate cancer

Abstract The ∼30,000 prostate cancer (CaP) deaths in the United States annually are due to failure of androgen deprivation therapy (ADT). ADT prevents ligand-activation of androgen receptor (AR), which is the main target for treatment of non-organ-confined CaP. ADT fails while CaP remains dependent on AR. Recent NextGen sequencing approaches on ADT-recurrent CaP specimens have confirmed AR amplification, and emergence of gain-of-function AR mutations and forms of AR that do not require ligand-activation. The same sequencing studies also identified a striking enrichment in gain-of-function p53 mutations during ADT. AR and p53 have been recognized as the 2 major genomic drivers of lethal CaP progression, do not interact directly and are not targeted efficiently for therapy in ADT-recurrent CaP. Recently, using a customized gene expression oligoarray, we defined the contribution of 18 clinically relevant coregulators to androgen regulation of 452 AR target genes. Coregulator contribution to AR action was highly gene-specific and context-dependent. Selectivity in dependence of AR target genes on individual coregulators was reflected in the composition of associated AR binding sites, as well as in CaP cell biology and clinical CaP progression that was controlled by these AR target genes. The existence of diverse AR-coregulator-transcription factor (TF) transcriptional codes was evidenced by WDR77-dependent interaction between AR and p53. In this novel AR-, p53- and WDR77-dependent transcriptional mechanism, the coregulator WDR77 physically and functionally bridged the action of AR and p53. WDR77 controlled selectively androgen regulation of 96 AR target genes that are associated with aggressive CaP and that mediate cell death, DNA replication, recombination and repair. Using Cistrome project tools, binding motifs for p53 were found close to AR binding sites in WDR77-dependent AR target genes, and ChIP validated androgen-dependent recruitment of p53 and WDR77 to those genomic regions. Genome-wide gene expression profiling confirmed significant overlap (n = 262) in p53- and WDR77-dependent androgen-responsive genes in CaP cells. Co-immunoprecipitation and mammalian-2-hybrid assays demonstrated p53 and WDR77 interaction, and delineated the p53-WDR77 interacting domains. In conclusion, WDR77-dependent interaction between AR and p53 provides the rationale for a novel CaP treatment strategy, namely disruption of functional interaction(s) between AR and TFs, such as p53, that control the CaP biology responsible for lethal disease progression. Citation Format: Sangeeta Kumari, Simon Schlanger, Dan Wang, Song Liu, Hannelore Heemers. Isolation of WDR77-mediated interaction between androgen receptor and p53 uncovers novel treatment strategy for 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 865.