Clinical Outcome In Prostate Cancer Patients Research Articles

Serum ACSL4 levels in prostate cancer patients and its relationship between patient prognosis: A prospective observational study.

In this prospective observational study, our objective was to investigate the serum levels of Acyl-CoA synthetase long-chain family member 4 (ACSL4) in prostate cancer (PCa) patients and examine its association with other serum biomarkers, and the clinical outcomes of PCa patients. This prospective observational study was conducted from January 2019 to October 2021, including 103 cases of PCa patients and 101 cases of benign prostate hyperplasia (BPH) patients who received treatment at our hospital. All patients had their serum ACSL4 levels measured using enzyme-linked immunosorbent assay before treatment. The clinical outcomes included age, body mass index, gender, systolic blood pressure, diastolic blood pressure, tumor node metastasis stage, Gleason scores, and prostate volume and serum biomarkers were collected. All patients were followed up for 36 months, the overall survival and disease-free survival were recorded for all patients. All data used SPSS 26.0 for analysis. The phosphorus (P) and serum low-density lipoprotein cholesterol levels were significantly higher in PCa patients compared to BPH patients. Furthermore, compared to the BPH patients, the serum ACSL4 and free prostate-specific antigen levels were significantly decreased while serum total prostate-specific antigen (tPSA) levels were significantly elevated in PCa patients. Pearson correlation analysis showed a positive correlation between ACSL4 levels and free prostate-specific antigen levels, while a negative correlation was observed with P and tPSA levels. ACSL4 might serve as a biomarker for diagnosing PCa with the AUC was 0.747, cutoff value of 33.68 ng/mL, sensitivity of 70.3%, and specificity of 60.2%. Finally, we found that ACSL4, tPSA, and P were identified as risk factors associated with PCa patients. Our findings indicated that the serum levels of ACSL4 were significantly decreased in PCa patients compared to BPH patients. Serum ACSL4 could be used as a potential biomarker for early PCa diagnosis and prognosis.

Evaluation of glucocorticoid-related genes reveals GPD1 as a therapeutic target and regulator of sphingosine 1-phosphate metabolism in CRPC

Prostate cancer (PCa) is an androgen-dependent disease, with castration-resistant prostate cancer (CRPC) being an advanced stage that no longer responds to androgen deprivation therapy (ADT). Mounting evidence suggests that glucocorticoid receptors (GR) confer resistance to ADT in CRPC patients by bypassing androgen receptor (AR) blockade. GR, as a novel therapeutic target in CRPC, has attracted substantial attention worldwide. This study utilized bioinformatic analysis of publicly available CRPC single-cell data to develop a consensus glucocorticoid-related signature (Glu-sig) that can serve as an independent predictor for relapse-free survival. Our results revealed that the signature demonstrated consistent and robust performance across seven publicly accessible datasets and an internal cohort. Furthermore, our findings demonstrated that glycerol-3-phosphate dehydrogenase 1 (GPD1) in Glu-sig can significantly promote CRPC progression by mediating the cell cycle pathway. Additionally, GPD1 was shown to be regulated by GR, with the GR antagonist mifepristone enhancing the anti-tumorigenic effects of GPD1 in CRPC cells. Mechanistically, targeting GPD1 induced the production of sphingosine 1-phosphate (S1P) and enhanced histone acetylation, thereby inducing the transcription of p21 that involved in cell cycle regulation. In conclusion, Glu-sig could serve as a robust and promising tool to improve the clinical outcomes of PCa patients, and modulating the GR/GPD1 axis that promotes tumor growth may be a promising approach for delaying CRPC progression.

Prevalence and outcomes of atrial fibrillation in patients suffering prostate cancer: a national analysis in the United States.

Although the adverse effects of atrial fibrillation (AF) on cancers have been well reported, the relationship between the AF and the adverse outcomes in prostate cancer (PC) remains inconclusive. This study aimed to explore the prevalence of AF and evaluate the relationship between AF and clinical outcomes in PC patients. Patients diagnosed with PC between 2008 and 2017 were identified from the National Inpatient Sample database. The trends in AF prevalence were compared among PC patients and their subgroups. Multivariable regression models were used to assess the associations between AF and in-hospital mortality, length of hospital stay, total cost, and other clinical outcomes. 256,239 PC hospitalizations were identified; 41,356 (83.8%) had no AF and 214,883 (16.2%) had AF. AF prevalence increased from 14.0% in 2008 to 20.1% in 2017 (P < .001). In-hospital mortality in PC inpatients with AF increased from 5.1% in 2008 to 8.1% in 2017 (P < .001). AF was associated with adverse clinical outcomes, such as in-hospital mortality, congestive heart failure, pulmonary circulation disorders, renal failure, fluid and electrolyte disorders, cardiogenic shock, higher total cost, and longer length of hospital stay. The prevalence of AF among inpatients with PC increased from 2008 to 2017. AF was associated with poor prognosis and higher health resource utilization. Better management strategies for patients with comorbid PC and AF, particularly in older individuals, are required.

Prognostic Significance of the Cribriform Pattern in Prostate Cancer: Clinical Outcomes and Genomic Alterations.

Given the diverse clinical progression of prostate cancer (PC) and the evolving significance of histopathological factors in its management, this study aimed to explore the impact of cribriform pattern 4 (CP4) on clinical outcomes in PC patients and examine its molecular characteristics. This retrospective study analyzed data from The Cancer Genome Atlas (TCGA) database and included PC patients who underwent radical prostatectomy (RP) and had pathology slides available for the assessment of CP4. A multivariable competing risk regression analysis was used to assess the association between CP4 and progression-free survival (PFS) while adjusting for established PC prognostic factors. The frequency of genomic alterations was compared between patients with and without CP4 using the Fisher's exact test. Among the 394 patients analyzed, 129 (32.74%) had CP4. After a median follow-up of 40.50 months (IQR: 23.90, 65.60), the presence of CP4 was significantly associated with lower PFS (AHR, 1.84; 95% CI, 1.08 to 3.114; p = 0.023) after adjusting for covariates. Seven hub genes-KRT13, KRT5, KRT15, COL17A1, KRT14, KRT16, and TP63-had significantly lower mRNA expression levels in patients with CP4 compared to those without. PC patients with CP4 have distinct genomic alterations and are at a high risk of disease progression following RP. Therefore, these patients may benefit from additional post-RP treatments and should be the subject of a prospective randomized clinical trial.

Stromal-derived MAOB promotes prostate cancer growth and progression.

Prostate cancer (PC) develops in a microenvironment where the stromal cells modulate adjacent tumor growth and progression. Here, we demonstrated elevated levels of monoamine oxidase B (MAOB), a mitochondrial enzyme that degrades biogenic and dietary monoamines, in human PC stroma, which was associated with poor clinical outcomes of PC patients. Knockdown or overexpression of MAOB in human prostate stromal fibroblasts indicated that MAOB promotes cocultured PC cell proliferation, migration, and invasion and co-inoculated prostate tumor growth in mice. Mechanistically, MAOB induces a reactive stroma with activated marker expression, increased extracellular matrix remodeling, and acquisition of a protumorigenic phenotype through enhanced production of reactive oxygen species. Moreover, MAOB transcriptionally activates CXCL12 through Twist1 synergizing with TGFβ1-dependent Smads in prostate stroma, which stimulates tumor-expressed CXCR4-Src/JNK signaling in a paracrine manner. Pharmacological inhibition of stromal MAOB restricted PC xenograft growth in mice. Collectively, these findings characterize the contribution of MAOB to PC and suggest MAOB as a potential stroma-based therapeutic target.

A Novel Signature Based on Anoikis Associated with BCR-Free Survival for Prostate Cancer.

This study aimed to elucidate the role of anoikis in the progression of prostate cancer (PCa) and to develop a prognostic signature based on anoikis-related genes (ARGs). To achieve this, PCa cases were subjected to nonnegative matrix factorization (NMF) analysis, which allowed for the identification of distinct patterns of anoikis modification. Additionally, immune infiltration was evaluated using single-sample gene-set enrichment analysis (ssGSEA). Survival analysis was performed using the Kaplan-Meier method, and a risk score was generated based on the expression levels of ARGs to quantitatively assess the modification of anoikis in PCa. Using the Least Absolute Shrinkage and Selection Operator (LASSO) method, four hub-genes were identified, and patients were classified into different risk groups based on their individual scores. Importantly, the low-risk subtype was characterized by a significantly improved biochemical recurrence-free survival, underscoring the clinical relevance of the ARG-based prognostic signature. To further improve the prognostic accuracy of the signature, patient age, pathological T stage, Gleason score, and prostate-specific antigen level were incorporated into the analysis, yielding a comprehensive prognostic signature. The clinical relevance of this signature was illustrated through a nomogram, providing a visual representation of the prognostic implications of the ARG-based signature. Taken together, these findings highlight the potential of ARGs in predicting the clinical outcomes of PCa patients and provide a novel and clinically relevant prognostic signature based on the modification of anoikis in PCa.

Comprehensive Landscape of HOXA2, HOXA9, and HOXA10 as Potential Biomarkers for Predicting Progression and Prognosis in Prostate Cancer.

Prostate cancer (PCa) is recognized as a common malignancy in male patients. The homeobox A cluster (HOXA) family members have been confirmed to be implicated in the development of several types of tumors. However, the expression pattern and prognostic values of HOXA genes in PCa have not been investigated. In this study, we analyzed TCGA datasets and identified six HOXA family members which showed a dysregulated expression in PCa specimens compared with nontumor specimens. We also explored the potential mechanisms involved in the dysregulation of HOXA family members in PCa, and the results of Pearson's correlation revealed that most HOXA members were negatively related to the methylation degree. Moreover, we explored the prognostic values of HOXA family members and identified six survival-related HOXA members. Importantly, HOXA2, HOXA9, and HOXA10 were identified as critical PCa-related genes which were abnormally expressed in PCa and associated with clinical outcomes of PCa patients. Then, we explored the association between the above three genes and immune cell infiltration. We observed that the levels of HOXA2, HOXA9, and HOXA10 were associated with the levels of immune infiltration of several kinds of immune cells. Overall, our findings identified the potential values of the HOXA family for outcome prediction in PCa, which might facilitate personalized counselling and treatment in PCa.

Downregulation of Snail by DUSP1 Impairs Cell Migration and Invasion through the Inactivation of JNK and ERK and Is Useful as a Predictive Factor in the Prognosis of Prostate Cancer.

Dual specificity phosphatase 1 (DUSP1) is crucial in prostate cancer (PC), since its expression is downregulated in advanced carcinomas. Here, we investigated DUSP1 effects on the expression of mesenchymal marker Snail, cell migration and invasion, analyzing the underlying mechanisms mediated by mitogen-activated protein kinases (MAPKs) inhibition. To this purpose, we used different PC cells overexpressing or lacking DUSP1 or incubated with MAPKs inhibitors. Moreover, we addressed the correlation of DUSP1 expression with Snail and activated MAPKs levels in samples from patients diagnosed with benign hyperplasia or prostate carcinoma, studying its implication in tumor prognosis and survival. We found that DUSP1 downregulates Snail expression and impairs migration and invasion in PC cells. Similar results were obtained following the inhibition of c-Jun N-terminal kinase (JNK) and extracellular-signal-regulated kinase (ERK). In clinical samples, we evidenced an inverse correlation between DUSP1 expression and Snail levels, which are further associated with JNK and ERK activation. Consequently, the pattern DUSP1high/activated JNKlow/activated ERKlow/Snaillow is associated with an overall extended survival of PC patients. In summary, the ratio between DUSP1 and Snail expression, with additional JNK and ERK activity measurement, may serve as a potential biomarker to predict the clinical outcome of PC patients. Furthermore, DUSP1 induction or inhibition of JNK and ERK pathways could be useful to treat PC.

Pharmacological inhibition of noncanonical EED-EZH2 signaling overcomes chemoresistance in prostate cancer.

Rationale: Chemoresistance is a major obstacle in prostate cancer (PCa) treatment. We sought to understand the underlying mechanism of PCa chemoresistance and discover new treatments to overcome docetaxel resistance.Methods: We developed a novel phenotypic screening platform for the discovery of specific inhibitors of chemoresistant PCa cells. The mechanism of action of the lead compound was investigated using computational, molecular and cellular approaches. The in vivo toxicity and efficacy of the lead compound were evaluated in clinically-relevant animal models.Results: We identified LG1980 as a lead compound that demonstrates high selectivity and potency against chemoresistant PCa cells. Mechanistically, LG1980 binds embryonic ectoderm development (EED), disrupts the interaction between EED and enhancer of zeste homolog 2 (EZH2), thereby inducing the protein degradation of EZH2 and inhibiting the phosphorylation and activity of EZH2. Consequently, LG1980 targets a survival signaling cascade consisting of signal transducer and activator of transcription 3 (Stat3), S-phase kinase-associated protein 2 (SKP2), ATP binding cassette B 1 (ABCB1) and survivin. As a lead compound, LG1980 is well tolerated in mice and effectively suppresses the in vivo growth of chemoresistant PCa and synergistically enhances the efficacy of docetaxel in xenograft models.Conclusions: These results indicate that pharmacological inhibition of EED-EZH2 interaction is a novel strategy for the treatment of chemoresistant PCa. LG1980 and its analogues have the potential to be integrated into standard of care to improve clinical outcomes in PCa patients.

Abstract 5591: Differential profiles of pro-inflammatory chemokines identified in patients with prostate cancer and benign prostatic hyperplasia

Abstract Background: Prostate cancer (PCa) is the most frequently diagnosed malignant disease and the second leading cause of cancer-related deaths of men in the United States. The development of PCa has been linked to inflammation; thus, systemic inflammatory biomarkers may be useful to differentiate PCa from other prostatic disorders, such as benign prostatic hyperplasia (BPH). Proinflammatory chemokines are among the factors that regulate inflammatory processes through the recruitment of leukocytes to the sites of infection or injury. The aim of the present study was to compare the serum profiles of pro-inflammatory chemokines of patients newly diagnosed either with PCa or BPH. Methods: Serum was prepared from blood samples collected at the time of diagnosis from a cohort of patients newly diagnosed with PCa or BPH. Protein concentrations of chemokines were measured in each group of samples with a multiplex ELISA kit specific for the quantification of 12 proinflammatory chemokines: CCL2, CCL3, CCL4, CCL5, CCL11, CCL17, CCL22, CXCL1, CXCL8, CXCL9, CXCL10 and CXCL11. Results were further validated for selected chemokines using single analyte ELISA kits. This work was approved by the Institutional Review Board of the University of Cartagena. Results: Our study showed that PCa patients had a significantly different proinflammatory chemokine profile compared to BPH patients. Serum samples in the PCa group had significant higher levels of CXCL8, CCL2, CCL3, CCL4, CXCL11 and CXCL1 compared with the BPH group. Conclusion: Our study showed that PCa patients had a high proinflammatory chemokine profile. Additional studies are required to determine whether proinflammatory chemokine profiles can serve as a diagnostic signature to differentiate between PCa and BPH, and whether proinflammatory chemokine profiles are correlated with clinical outcomes of PCa patients. Keywords: Prostate cancer, chemokine, inflammation, Benign Prostatic Hyperplasia Citation Format: Niradiz Reyes, Raj Tiwari, Jan Geliebter. Differential profiles of pro-inflammatory chemokines identified in patients with prostate cancer and benign prostatic hyperplasia [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 5591.

Abstract C029: Exosomes-associated miR-5001, miR-3692 and miR-4529 are novel biomarkers for aggressive prostate cancer and associated with poor prognosis in African American patients

Abstract Background: Although microRNA (miR) profiling has been widely used to predict clinical outcomes, differential miR expressions that can segregate prostate cancer (PCa) patients based on their races and tumor aggressiveness have not been fully investigated. We aimed to determine the diagnostic and prognostic abilities of exosomal miRs to identify the aggressive phenotypes of PCa in African American (AA) men. Methods: Exosomes were isolated from blood of twenty AA and European Americans (EuA) PCa patients at low and high Gleason scores and their aged-matched healthy subjects (n=20) as well as AA and EuA normal and PCa cells. miR profiling was performed on PCa exosomes derived from blood and PCa cells. The expression level was correlated with clinical outcomes of PCa patients. The sensitivity and specificity of exosomal miRs were assessed using receiver operating characteristic (ROC) curve. Results: Results from miR profiling showed a number of exosomal miRs that were able to differentiate normal from PCa, low from high Gleason scores and AA from EuA PCa patients. These dysregulated miRs were validated in another cohort of forty PCa patients in addition to a large panel of PCa cell lines. In the validation cohort, miR-5001, miR-3692 and miR-4529 were upregulated in the exosomes derived from blood of AA compared to EuA men. These miRs were correlated with age, T-stage, residual tumor, involvement of lymph nodes, Gleason score, and overall survival of AA patients. The combination of these miRs showed high discriminatory power (AUC=0.91) for segregation of PCa patients according to their clinical outcomes. Conclusion: miR profiling identified a new set of miRs that can differentiate PCa specimens based on their race and Gleason score. The differential expression of these miRs demonstrates their potential role as biomarkers in the context of racial disparity. Further studies are warranted to determine their role in PCa at advanced stages. Citation Format: Rofaida Gaballa, Mohamed Gaballah, Hamdy E.A. Ali, Andrew S. Sholl, Hamed I. Ali, Zakaria Y. Abd Elmageed. Exosomes-associated miR-5001, miR-3692 and miR-4529 are novel biomarkers for aggressive prostate cancer and associated with poor prognosis in African American patients [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 C029.

Significance of distinct specifically enriched missense TP53 mutations in prostate cancer.

377 Background: The most common TP53 alterations are missense mutations occurring in the DNA binding domain. The majority of missense p53 mutants (mut-p53) demonstrate oncogenic gain-of-function (GOF) abilities, irrespective of wild-type p53 presence, and thus contribute to a more aggressive disease. In prostate cancer (PCa), characterized by comparatively low overall mutational burden, TP53 is frequently mutated in both primary and advanced disease. Despite significant progress made in the field, detailed mechanisms of GOF in PCa remain undefined due to differing features of p53 mutants. Methods: Analysis of available datasets was performed to assess TP53 mutational status in PCa patient samples and its correlation with the clinical outcome. Using hormone therapy sensitive and CRPC cells, a panel of cell lines was generated to model the two most frequently occurring mutations in the presence or absence of wild-type TP53, as occurs clinically. CHIP-seq, gene expression arrays, and in vitro and in vivo biological assays were performed to interrogate the significance of mut-p53 in PCa. Results: In PCa, missense mutations are significantly associated with decreased progression-free and overall survival. In PCa patient samples these mutations most commonly occur at the R273 residue, demonstrating specific enrichment when compared to other cancers, with R273C alteration being the most frequent. Using our cell panel, CHIP-seq data revealed an expansion of the p53 cistrome upon expression of R273C and R273H mutants in a manner distinct from p53 stabilization in the presence of wt-p53. Moreover, analysis of the TP53 missense mutant-sensitive transcriptomes demonstrated differential gene expression between these mutants, related to the expression of wild-type TP53 in those cells. Finally, R273C and R273H p53 mutants elicited context dependent effects on canonical p53 functions, thereby modulating distinct downstream biological outcomes. Conclusions: These data expand our knowledge of the underlying mechanisms by which distinct gain-of-function p53 mutants affect prostate cancer, and can lead to identification of novel therapeutic targets to improve clinical outcomes in PCa patients harboring these mutations.

Effect of DNA damage response mutations on prostate cancer prognosis: a systematic review.

The prognosis of men with prostate cancer (PC) with mutations in DNA damage response (DDR) genes undergoing different treatments is unclear. This systematic review compared clinical outcomes in PC patients with DDR mutations (DDR+) versus no mutations (DDR-). 14resources plus gray literature were searched for studies in PC and subgroups (castration-resistant PC, metastatic PC and metastatic castration-resistant PC) by DDR gene (ATM, ATR, BRCA1, BRCA2, CHEK2, FANCA, MLH1, MRE11A, NBN, PALB2, RAD51C) mutation status. From 11,648 records, 26 studies were included. For mCRPC, six studies reported comparative efficacy for key outcomes. Improvements in several clinical outcomes were observed for DDR+ (vs DDR-) after PARP inhibitor therapy or immunotherapy. DDR+ PC patients may have improved outcomes depending on the treatment they undergo.

Cytokines expression levels from tissue, plasma or serum as promising clinical biomarkers in adenocarcinoma of the prostate: a systematic review of recent findings.

Prostate cancer (PC) is a common cancer (excluding non-melanoma skin cancer) in men in many parts of the world, although incidence and mortality rates vary significantly by population. In current medical practice, prognostic markers for PC include the presenting serum prostate-specific antigen (PSA) level, tumour Gleason score (GS) and clinical tumour stage. However, existing pre-treatment factors cannot be used to predict acute radiotherapy (RT)-induced toxicity. Therefore, new protein biomarkers are required in RT oncology to improve decision-making, treatment and therapy monitoring for PC patients. The aim of this systematic review is to the update potential research to address the difference in cytokine expression and their association with RT-induced toxicity and clinical outcomes. Studies were collected after searching three electronic databases: PubMed, Medline, and Google Scholar. An additional search was carried out through cross-check on a bibliography of selected articles. After the selection process made by two of the authors, 19 articles met the inclusion criteria and were included in the systematic review. Results from previous studies identified elevated levels of cytokines have been reported in several types of cancers and have sometimes correlated with disease progression or prognosis. Elevated levels of cytokine were noticed after immediate exposure to RT and their association with RT-induced acute/late toxicity of PC patients. Moreover, above studies also identified overexpression of cytokines on tumour biopsies and correlation with shortening cancer-specific survival and biochemical recurrence. Thus, altered levels of cytokine might be predictive biomarkers for RT-induced and clinical outcomes of PC patients.

Tumor Suppressor Role and Clinical Implication of the Fifth Ewing Variant (FEV) Gene, an ETS Family Gene, in Prostate Cancer

Background: Our previous study identified a 32-gene risk index model as a robust prognostic tool for disease recurrence of prostate cancer (PCa) after surgery. Among the 32 genes, the Fifth Ewing Variant (FEV) Gene was one of the top downregulated genes in the relapsed PCa. However, very little is known about FEV gene and its involvement in PCa. Methods: FEV mRNA expression was analyzed and correlated to clinical outcomes in PCa patients who underwent prostatectomy in Massachusetts General Hospital. FEV expression level was also detected in PCa cell lines and an immortalized prostate epithelial cell line BPH-1 by Western blot and qRT-PCR. Furthermore, we established stable cell lines (LNCaP and PC-3) transfected with either empty vector or the full-length FEV gene and conducted cell function assays in vitro. The role of FEV in tumor xenografts growth was also determined in vivo Finding: Of 191 patients who enrolled in this study, 77 (40.3%) and 24 (13.6%) developed prostate-specific antigen (PSA) relapse and metastasis respective after radical prostatectomy. FEV was significantly downregulated in PCa patients with PSA failure and metastasis. FEV expression was significantly reduced in all PCa cell lines compared to BPH-1 (all P<0.05). Functionally, the enforced expression of FEV markedly inhibited cell growth, migration and invasion efficacies in PCa cells and led to a significant repression of tumor xenografts growth in vivo. Interpretation: Our data indicate that FEV downregulation may be potentially associated with PSA relapse in PCa patients. Functionally, FEV may act as a tumor suppressor in PCa. Funding Statement: This work was supported by grants from National Key Basic Research Program of China (2015CB553706), National Natural Science Foundation of China (81874099, 81571 427, 81802555), the Fundamental Research Funds for the Central Universities(2018MS 18), Natural Science Foundation of Guangdong Province (2018A030313668, 2017A030 310149), Guangzhou Municipal Science and Technology Project (201710010081, 20180 3040001, 201707010291), Projects of Guangdong Key Laboratory of Clinical Molecular Medicine and Diagnostics, Guangzhou Medical University Initiation Project for PHD (201 6C12), General Research Projects of Guangzhou Science and Technology Department (201804010053). Declaration of Interests: No potential conflicts of interest were disclosed. Ethics Approval Statement: The study was approved by the human study ethics committees at Massachusetts General Hospital (MGH; Boston, MA). All specimens were handled and made anonymous according to the ethical and legal standards.

PO-119 Accelerated glutamine metabolism is conferring radioresistance to prostate cancer

IntroductionPathophysiological conditions within the tumour microenvironment induce metabolic adaptations of cancer cells. These metabolic features correlating with aggressive tumour growth pattern and the relapse risk after radiotherapy. Beside glucose, fast-growing cancer cells consume glutamine for energy production. Within the presented project we investigated the potential of the glutamine metabolism as putative therapeutic target and predictive biomarker for an individualised radiotherapy of prostate cancer (PC) patients.Material and methodsGenome and metabolome analysis of radioresistant PC cells identified the glutamine metabolism as regulator for intrinsic sensitivity to irradiation. Radiosensitizing effects of glutamine deprivation, molecular targeting and genetic suppression of key enzymes were determined for clonogenic survival, DNA repair and sphere-formation ability to determine the cancer stem cell (CSC) potential in PC cell lines, primary culture models and ex vivo treated primary biopsies. Analysis of tumour growth after glutamine deprivation were performed in xenograft models. The intracellular level of the glutamine metabolism and tricarboxylic acid cycle metabolites, reactive oxygen species (ROS) and glutathione (GSH) were determined. The clinical validation of the identified metabolic biomarkers was carried out with the NanoString technology.Results and discussionsRadioresistant PC cells exhibit an accelerated glutaminolysis with enhanced α-ketoglutarate to succinate ratio. The elevated α-ketoglutarate is leading to GSH consumption, increased intracellular ROS level, modulated epigenetic regulators and induction of a CSC phenotype. Metabolic, chemical and genetic targeting of glutaminolysis results in the inhibition of mTOR signalling, enhanced endoplasmic reticulum stress and reduction of DNA repair. In combination with irradiation this targeting therapy is effectively radiosensitizing PC cells in vitro and in vivo. Moreover, the c-MYC gene expression, a key regulator of glutaminolysis, significantly correlates with the PSA-free survival after radiotherapy.ConclusionThis study shows that the enhanced glutaminolysis of PC cells is conferring resistance to radiotherapy. The therapeutic targeting of the glutamine metabolism is elevating the cytotoxic effects of irradiation. In addition, metabolic enzymes involved in the glutamine metabolism can be potentially used to predict clinical outcome of PC patients after radiotherapy.

Abstract LB-320: Evaluating changes in circulating tumor cell count in prostate cancer patients during androgen deprivation plus chemotherapy by using AxonDx enumeration device

Abstract Background: Circulating tumor cell (CTC) counts have clinical utility as a prognostic marker in cancer prostate cancer (PCa). Using the FDA-approved CellSearch system, CTC counts &amp;gt5 CTC/7.5 ml convey a negative prognosis with respect to overall survival. The significance of sequential CTC counts during treatment is not fully understood. To determine whether longitudinal CTC counts can be used as a prognostic and/or predictive marker of clinical outcome in PCa patients undergoing androgen deprivation therapy (ADT) plus chemotherapy, we used the AxonDx nCyte non-enrichment-based CTC detection and enumeration system to evaluate CTC counts at baseline, during, and after therapy in 16 patients for up to 11 months. Methods: Whole blood was drawn from PCa patients participating in a clinical trial of ADT plus chemotherapy. CTCs were determined at multiple time points: at PSA evaluation (every 8 wks, ±radiographic reevaluation [every 16 wks]). Blood was collected into vacutainers containing K3-EDTA and processed within 6 hr. PBMC containing nucleated cells harvested from 6 ml of whole blood were fixed, permeabilized and blocked with BSA buffer, and stained using a proprietary antibody cocktail containing DAPI, an anti-cytokeratin mixture, the pan-leukocyte marker CD45, and markers selective for granulocytes and lymphocytes. Stained cells were drawn onto 1.2 µm pore filters, washed, placed on counting slides, and analyzed. CTCs are identified as cytokeratin-positive, nucleus-positive and leukocyte marker-negative. Results: CTCs were obtained from 12 patients for 2-9 months at 2-7 time points; from 2 patients for 11 months at 5-7 time points, and from 2 patients for 1 month at 1 time point. We detected time-dependent changes in CTC counts following treatment that did not necessarily correlate with patient's local prostate specific antigen (PSA) levels but were early indicators of subsequent increase in tumor burden. Conclusions: The AxonDx nCyte system can reliably detect CTCs from PCa patient samples that trend along with other signs of disease progression. CTCs can be used longitudinally for clinical monitoring of patients with PCa prior to signs of progression via tumor markers or scans during ADT plus chemotherapy. To further evaluate sequential CTC counts as a reliable biomarker for PCa, we plan additional studies to confirm our data and compare the AxonDx nCyte system with the CellSearch device in our ongoing clinical trial PCa program. Citation Format: Robert J. Amato, Reynolds Brobey, Yue Wang, Mehdi Dehghani, Zoe Tramel, Kent Murphy, Jeff Smith. Evaluating changes in circulating tumor cell count in prostate cancer patients during androgen deprivation plus chemotherapy by using AxonDx enumeration device [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-320.

Quercetin inhibits prostate cancer by attenuating cell survival and inhibiting anti-apoptotic pathways

BackgroundDespite recent advances in diagnosis and treatment, prostate cancer (PCa) remains the leading cause of cancer-related deaths in men. Current treatments offered in the clinics are often toxic and have severe side effects. Hence, to treat and manage PCa, new agents with fewer side effects or having potential to reduce side effects of conventional therapy are needed. In this study, we show anti-cancer effects of quercetin, an abundant bioflavonoid commonly used to treat prostatitis, and defined quercetin-induced cellular and molecular changes leading to PCa cell death.MethodsCell viability was assessed using MTT. Cell death mode, mitochondrial outer membrane potential, and oxidative stress levels were determined by flow cytometry using Annexin V-7 AAD dual staining kit, JC-1 dye, and ROS detection kit, respectively. Antibody microarray and western blot were used to delineate the molecular changes induced by quercetin.ResultsPCa cells treated with various concentrations of quercetin showed time- and dose-dependent decrease in cell viability compared to controls, without affecting normal prostate epithelial cells. Quercetin led to apoptotic and necrotic cell death in PCa cells by affecting the mitochondrial integrity and disturbing the ROS homeostasis depending upon the genetic makeup and oxidative status of the cells. LNCaP and PC-3 cells that have an oxidative cellular environment showed ROS quenching after quercetin treatment while DU-145 showed rise in ROS levels despite having a highly reductive environment. Opposing effects of quercetin were also observed on the pro-survival pathways of PCa cells. PCa cells with mutated p53 (DU-145) and increased ROS showed significant reduction in the activation of pro-survival Akt pathway while Raf/MEK were activated in response to quercetin. PC-3 cells lacking p53 and PTEN with reduced ROS levels showed significant activation of Akt and NF-κB pathway. Although some of these changes are commonly associated with oncogenic response, the cumulative effect of these alterations is PCa cell death.ConclusionsOur results demonstrated quercetin exerts its anti-cancer effects by modulating ROS, Akt, and NF-κB pathways. Quercetin could be used as a chemopreventive option as well as in combination with chemotherapeutic drugs to improve clinical outcomes of PCa patients.

Evaluating changes in circulating tumor cell count in prostate cancer patients during androgen deprivation plus chemotherapy by using AxonDx enumeration device.

266 Background: Circulating tumor cell (CTC) counts have clinical utility as a prognostic marker in cancer. For prostate cancer (PCa), CTC counts ≥5 CTC/7.5 ml using the FDA-approved CellSearch system convey a negative prognosis with respect to overall survival, but the significance of changes in CTC count during treatment is not fully understood. To determine whether longitudinal CTC counts can be used as a prognostic and/or predictive marker of clinical outcome in PCa patients undergoing androgen deprivation therapy (ADT) plus chemotherapy, we used the AxonDx nCyte non-enrichment-based CTC detection and enumeration system to evaluate CTC counts at baseline, during, and after therapy in 18 patients for up to 11 months. Methods: Whole blood was drawn from PCa patients on clinical trials for ADT plus chemotherapy at PSA evaluation (±radiographic reevaluation) into vacutainers containing K3-EDTA and processed within 6 hr. PBMC containing nucleated cells harvested from 6 ml of whole blood were fixed, permeabilized and blocked with BSA buffer, and stained using a proprietary antibody cocktail containing DAPI, an anti-cytokeratin mixture, the pan-leukocyte marker CD45, and markers selective for granulocytes and lymphocytes. Stained cells were drawn onto 1.2 µm pore filters, washed, placed on counting slides, and analyzed. CTCs are identified as cytokeratin-positive, nucleus-positive and leukocyte marker-negative. Results: CTCs were obtained from 12 patients for 2-9 months at 2-7 time points; from 2 patients for 11 months at 5-7 time points, and from 4 patients for 1 month at 1 time point. We detected time-dependent changes in CTC counts following treatment that did not necessarily correlate with patient’s local prostate specific antigen (PSA) levels but were early indicators of subsequent increase in tumor burden. Conclusions: The AxonDx nCyte system can reliably detect CTCs from PCa patient samples that trend along with other signs of disease progression. CTCs can be used longitudinally for clinical monitoring of patients with PCa prior to signs of progression via tumor markers or scans during ADT plus chemotherapy, providing a reliable biomarker for PCa.

Abstract 4952: Co-expression of pAKT and MYC is associated with aggressive disease in human prostate cancer

Abstract Introduction: AKT and MYC are amongst the most prevalent oncogenes in prostate cancer (PCa). MYC cooperates with activated AKT in pre-clinical, genetically engineered in vivo models accelerating disease progression and increasing resistance to therapy. However, the clinical relevance of phosphoAKT/MYC co-expression in human prostate tumors remains unknown. Experimental Procedures: A prospective cohort (Physicians’ Health Study and Health Professionals Follow-up Study) of 578 men with PCa with a mean 13-year follow up was used. Protein expression of MYC and phosphoAKT (Serine 473) was characterized by immunohistochemistry on tissue microarrays. pAKT and MYC expression was analyzed using the Cri Vectra/InForm multispectral analysis platform. The InForm software package was trained to segment PCa regions only (i.e., no stromal regions were included in the analysis). We used quantile normalization to adjust for the potential batch effects of protein expression staining across each of the tissue microarrays using the limma package R. The continuous score for pAKT and MYC were divided into quartiles and assigned a value of 1, 2, 3 or 4 for low (1), moderate (2 or 3) and high staining (4). Spearman correlations between pAKT, MYC and the clinico-pathological data were calculated. Results: High pAKT and High MYC were co-expressed in 10% of patients. (Quartile 4). Also 10% were negative or weakly positive for both pAKT and MYC (Quartile 1). Only 3% of the High pAKT patients had weak or no expression of MYC and 4% of High_MYC patients had Low pAKT expression. The majority of patients were distributed in the Quartiles 2 and 3 (73%). Importantly, only High pAKT and High pMYC co-expressors were associated with tumor stage (pT3 or pT4) (p = 0.03), high Gleason Score (p = 0.021) and presence of metastatic disease (p = 0.011). GEP analysis showed that the same patients exhibited up-regulation of genes involved in cell invasion/migration capacity and stromal remodeling. Conclusions: Co-expression of AKT and MYC is associated poor clinical outcome in PCa patients. Gene expression profiling suggest that concomitant activation of AKT and MYC results in the activation of pathways associated with an invasive phenotype. Citation Format: Nelma Pértega-Gomes, Svitlana Tyekucheva, Kathryn Penney, Giorgia Zadra, Lorelei A. Mucci, Massimo Loda. Co-expression of pAKT and MYC is associated with aggressive disease in human 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 4952.