Prostate Cancer Phenotype Research Articles

Identification of a high-risk immunogenic prostate cancer patient subset as candidates for T-cell engager immunotherapy and the introduction of a novel albumin-fused anti-CD3 × anti-PSMA bispecific design

BackgroundCancer immunotherapies such as bispecific T-cell engagers have seen limited adoption in prostate cancer (PC), possibly due to differing levels of cancer receptor expression and effector T-cell infiltration between patients and inherent defects in T-cell engager design.MethodsCD8+ T-cell infiltration and PSMA expression were determined by RNA sequencing of primary PC tissue samples from 126 patients with localised PC and 17 patients with metastatic PC. Prognostic value was assessed through clinical parameters, including CAPRA-S risk score. A panel of albumin-fused anti-CD3 × anti-PSMA T-cell engagers with different neonatal Fc receptor (FcRn) affinity were characterised by flow cytometry, Bio-Layer Interferometry and functional cellular assays.ResultsA subset of patients with localised (30/126 = 24%) and metastatic (10/17 = 59%) PC showed both high PSMA expression and high CD8+ T-cell enrichment. The High/High phenotype in localised PC associated with a clinically high-risk cancer subtype, confirmed in an external patient cohort (n = 550, PRAD/TCGA). The T-cell engagers exhibited tunable FcRn-driven cellular recycling, CD3 and PSMA cellular engagement, T-cell activation and PSMA level-dependent cellular cytotoxicity.ConclusionThis work presents an albumin-fused bispecific T-cell engager with programmable FcRn engagement and identifies a high-risk PC patient subset as candidates for treatment with the T-cell engager class of immuno-oncology biologics.

Loss of miR-936 leads to acquisition of androgen-independent metastatic phenotype in prostate cancer

Prostate cancer (PCa) progresses from a hormone-sensitive, androgen-dependent to a hormone-refractory, androgen-independent metastatic phenotype. Among the many genes implicated, ANXA2, a calcium-dependent phospholipid binding protein, has been found to have a critical role in the progression of PCa into more invasive metastatic phenotype. However, the molecular mechanisms underlying the absence of ANXA2 in early PCa and its recurrence in advanced stage are yet unknown. Moreover, recent studies have observed the deregulation of microRNAs (miRNAs) are involved in the development and progression of PCa. In this study, we found the down-regulation of miR-936 in metastatic PCa wherein its target ANXA2 was overexpressed. Subsequently, it has been shown that the downregulation of miRNA biogenesis by siRNA treatment in ANXA2-null LNCaP cells could induce the expression of ANXA2, indicating the miRNA mediated regulation of ANXA2 expression. Additionally, we demonstrate that miR-936 regulates ANXA2 expression by direct interaction at coding as well as 3′UTR region of ANXA2 mRNA by luciferase reporter assay. Furthermore, the overexpression of miR-936 suppresses the cell proliferation, cell cycle progression, cell migration, and invasion abilities of metastatic PCa PC-3 cells in vitro and tumor forming ability in vivo. These results indicate that miR-936 have tumor suppressor properties by regulating the over expression of ANXA2 in hormone-independent metastatic PCa. Moreover, our results suggest that this tumor suppressor miR-936 could be developed as a targeted therapeutic molecule for metastatic PCa control and to improve the prognosis in PCa patients.

ZFP36 Inhibits Tumor Progression of Human Prostate Cancer by Targeting CDK6 and Oxidative Stress.

Background The expression of ZFP36 in previous study was reduced in prostate cancer (PCa) tissues as compared to benign prostate tissues, indicating the potential of ZFP36 as an auxiliary marker for PCa. Further evaluation was conducted in clinical samples for in vitro and in vivo experiments, to prove the potential possibility that ZFP36 dysregulation participated in the malignant phenotype of PCa, to determine its potential mechanism for tumor regulation, and to provide a new theoretical basis for gene therapy of PCa. Methods First, the expression of ZFP36 in prostate tissue and PCa tissue was explored, and the relationship between ZFP36 and clinical features of PCa patients was illustrated. Subsequently, the impact of ZFP36 on the biology of PCa cells and relevant downstream pathways of ZFP36's biological impact on PCa were elucidated. Finally, whether oxidative stress mediated the regulation of ZFP36 in PCa was verified by the determination of oxidative stress-related indicators and bioinformatics analysis. Results The downregulation of ZFP36 in PCa tissue had a positive correlation with high Gleason scores, advanced pathological stage, and biochemical recurrence. ZFP36 was identified as an independent prognostic factor for PCa patients' BCR-free survival (P = 0.022) by survival analysis. Following a subsequent experiment of function gain and loss, ZFP36 inhibited the proliferation, invasion, and migration in DU145 and 22RV1 cells and inhibits tumor growth in the mouse model. Additionally, high-throughput sequencing screened out CDK6 as the downstream target gene of ZFP36. Western blot/Q-PCR demonstrated that overexpression of ZFP36 could reduce the expression of CDK6 at both cellular and animal levels, and the dual-luciferase experiment and RIP experiment proved that CDK6 was the downstream target of ZFP36, indicating that CDK6 was a downstream target of ZFP36, which mediated tumor cell growth by blocking cell cycle at the G1 stage. Furthermore, ZFP36 inhibited oxidative stress in PCa cells. Conclusions In PCa, ZFP36 might be a tumor suppressor that regulated growth, invasion, and migration of PCa cells. The lately discovered ZFP36-CDK6 axis demonstrated the molecular mechanism of PCa progression to a certain extent which might act as a new possible therapeutic target of PCa therapy.

FOXA1 regulates alternative splicing in prostate cancer.

SummaryDysregulation of alternative splicing in prostate cancer is linked to transcriptional programs activated by AR, ERG, FOXA1, and MYC. Here, we show that FOXA1 functions as the primary orchestrator of alternative splicing dysregulation across 500 primary and metastatic prostate cancer transcriptomes. We demonstrate that FOXA1 binds to the regulatory regions of splicing-related genes, including HNRNPK and SRSF1. By controlling trans-acting factor expression, FOXA1 exploits an “exon definition” mechanism calibrating alternative splicing toward dominant isoform production. This regulation especially impacts splicing factors themselves and leads to a reduction of nonsense-mediated decay (NMD)-targeted isoforms. Inclusion of the NMD-determinant FLNA exon 30 by FOXA1-controlled oncogene SRSF1 promotes cell growth in vitro and predicts disease recurrence. Overall, we report a role for FOXA1 in rewiring the alternative splicing landscape in prostate cancer through a cascade of events from chromatin access, to splicing factor regulation, and, finally, to alternative splicing of exons influencing patient survival.

Histone lysine demethylase inhibition reprograms prostate cancer metabolism and mechanics

ObjectiveAberrant activity of androgen receptor (AR) is the primary cause underlying development and progression of prostate cancer (PCa) and castration-resistant PCa (CRPC). Androgen signaling regulates gene transcription and lipid metabolism, facilitating tumor growth and therapy resistance in early and advanced PCa. Although direct AR signaling inhibitors exist, AR expression and function can also be epigenetically regulated. Specifically, lysine (K)-specific demethylases (KDMs), which are often overexpressed in PCa and CRPC phenotypes, regulate the AR transcriptional program. MethodsWe investigated LSD1/UTX inhibition, two KDMs, in PCa and CRPC using a multi-omics approach. We first performed a mitochondrial stress test to evaluate respiratory capacity after treatment with MC3324, a dual KDM-inhibitor, and then carried out lipidomic, proteomic, and metabolic analyses. We also investigated mechanical cellular properties with acoustic force spectroscopy. ResultsMC3324 induced a global increase in H3K4me2 and H3K27me3 accompanied by significant growth arrest and apoptosis in androgen-responsive and -unresponsive PCa systems. LSD1/UTX inhibition downregulated AR at both transcriptional and non-transcriptional level, showing cancer selectivity, indicating its potential use in resistance to androgen deprivation therapy. Since MC3324 impaired metabolic activity, by modifying the protein and lipid content in PCa and CRPC cell lines. Epigenetic inhibition of LSD1/UTX disrupted mitochondrial ATP production and mediated lipid plasticity, which affected the phosphocholine class, an important structural element for the cell membrane in PCa and CRPC associated with changes in physical and mechanical properties of cancer cells. ConclusionsOur data suggest a network in which epigenetics, hormone signaling, metabolite availability, lipid content, and mechano-metabolic process are closely related. This network may be able to identify additional hotspots for pharmacological intervention and underscores the key role of KDM-mediated epigenetic modulation in PCa and CRPC.

Identification and Validation of the Prognostic Impact of Metastatic Prostate Cancer Phenotypes.

Castration-sensitive metastatic prostate cancer is heterogeneous. Our objective is to identify metastatic prostate cancer phenotypes and their prognostic impact on survival. The National Cancer Database was queried. The Surveillance, Epidemiology, and End Results database was used for validation. Patterns were split into: nonregional lymph node, bone only, and visceral (any brain/liver/lung). Hazard ratios (HR) with 95% confidence intervals (CI) were calculated for the univariate and multivariate Cox proportional hazards regression models, odds ratios were calculated, Kaplan-Meier curves were generated, and a nomogram of the multivariate regression model was created. The training set included 13,818 men; bone only was most common (n=11,632, 84.2%), then nonregional lymph node (n=1388, 10.0%), and any visceral (brain/liver/lung; n=798, 5.8%). Risk of death was increased by metastases to a visceral organ versus nonregional lymph node (HR=2.26; 95% CI [2.00, 2.56]), bone only metastases versus nonregional lymph node (HR=1.57; 95% CI [1.43, 1.72]), T-stage 4 versus 1 (HR=1.27; 95% CI [1.17, 1.36]), Grade Group 5 versus 1 (HR=1.93; 95% CI [1.61, 2.31]), PSA > 20 ng/mL versus < 10 ng/mL (HR=1.32; 95% CI [1.23, 1.42]), and age ≥ 80 versus < 50 (HR=1.96; 95% CI [1.69, 2.29]). On internal validation, the model had C-indices 20.5%, 22.7%, and 14.6% higher than the current staging system for overall survival, 1-year, and 5-year survival, respectively. We developed and validated prognostic metastatic prostate cancer phenotypes that can assist risk stratification to potentially personalize therapy. Our nomogram (https://tinyurl.com/prostate-met) may be used to predict survival.

Oleocanthal Attenuates Metastatic Castration-Resistant Prostate Cancer Progression and Recurrence by Targeting SMYD2.

Simple SummaryThe Mediterranean, extra-virgin-olive-oil-rich diet ingredient S-(-)-oleocanthal (OC) has emerged as a potential inhibitor for the growth and relapse of the most aggressive prostate cancer type. This effect is mediated through suppression of important enzyme, SMYD2, that drives the activation of several downstream protein effectors. OC treatments reduced SMYD2 downstream substrates, which are critical for prostate cancer growth and relapse. OC is more advantageous than other reported SMYD2 inhibitors because it has shown potent anticancer activity in animal models. OC’s anti-prostate-cancer effect was prominent compared with some standard drugs currently used to control prostate cancer. OC is a potential, novel natural compound appropriate for immediate use by prostate cancer patients and survivors as a nutraceutical or dietary supplement product.Metastatic castration-resistant prostate cancer (mCRPC) is the most aggressive prostate cancer (PC) phenotype. Cellular lysine methylation is driven by protein lysine methyltransferases (PKMTs), such as those in the SET- and MYND-containing protein (SMYD) family, including SMYD2 methylate, and several histone and non-histone proteins. SMYD2 is dysregulated in metastatic PC patients with high Gleason score and shorter survival. The Mediterranean, extra-virgin-olive-oil-rich diet ingredient S-(-)-oleocanthal (OC) inhibited SMYD2 in biochemical assays and suppressed viability, migration, invasion, and colony formation of PC-3, CWR-R1ca, PC-3M, and DU-145 PC cell lines with IC50 range from high nM to low µM. OC’s in vitro antiproliferative effect was comparable to standard anti-PC chemotherapies or hormone therapies. A daily, oral 10 mg/kg dose of OC for 11 days effectively suppressed the progression of the mCRPC CWR-R1ca cells engrafted into male nude mice. Daily, oral OC treatment for 30 days suppressed tumor locoregional and distant recurrences after the primary tumors’ surgical excision. Collected OC-treated animal tumors showed marked SMYD2 reduction. OC-treated mice showed significant serum PSA reduction. For the first time, this study showed SMYD2 as novel molecular target in mCRPC, and OC emerged as a specific SMYD2 lead inhibitor. OC prevailed over previously reported SMYD2 inhibitors, with validated in vivo potency and high safety profile, and, therefore, is proposed as a novel nutraceutical for mCRPC progression and recurrence control.

Neuropilin-2 axis in regulating secretory phenotype of neuroendocrine-like prostate cancer cells and its implication in therapy resistance.

SUMMARYNeuroendocrine (NE)-like tumors secrete various signaling molecules to establish paracrine communication within the tumor milieu and to create a therapy-resistant environment. It is important to identify molecular mediators that regulate this secretory phenotype in NE-like cancer. The current study highlights the importance of a cell surface molecule, Neuropilin-2 (NRP2), for the secretory function of NE-like prostate cancer (PCa). Our analysis on different patient cohorts suggests that NRP2 is high in NE-like PCa. We have developed cell line models to investigate NRP2’s role in NE-like PCa. Our bioinformatics, mass spectrometry, cytokine array, and other supporting experiments reveal that NRP2 regulates robust secretory phenotype in NE-like PCa and controls the secretion of factors promoting cancer cell survival. Depletion of NRP2 reduces the secretion of these factors and makes resistant cancer cells sensitive to chemotherapy in vitro and in vivo. Therefore, targeting NRP2 can revert cellular secretion and sensitize PCa cells toward therapy.

Abstract 273: Prostate cancer metastasis induces irregular bone formation associated to specific androgen dependent phenotypes

Abstract Bone metastases (BM) occur in ~80% of advanced prostate cancer (PC) patients, and are a major cause of morbidity. PCBMs are predominantly osteoblastic with mixed, and lytic regions; all of which compromise bone strength. How PCs alter bone structure is unresolved. Prolonged use of modern androgen receptor (AR) targeting agents has increased development of AR pathway-independent PC phenotypes. We hypothesize that defining the structural changes, cellular composition, and PC classification of PCBM-affected bone will help reveal mechanisms of PCBM pathology, and identify potential therapeutic opportunities. We analyze the 3D structure of 14 cadaveric PCBM lumbar vertebrae using micro-computed tomography (microCT). We performed scanning electron microscopy quantitative backscattering electron (qBSE), and energy-dispersive X-ray spectroscopy (EDX) to determine mineral morphology and composition. We analyzed sequential decalcified sections for collagen structure, fibril orientation, and extracellular matrix composition using bright field and polarized light microscopy. We determined lesion structure and cell distribution from two PCBM vertebral specimens resected during decompression surgery using histology and immunohistochemistry, and characterized PCBM cell populations using single cell RNA sequencing (sc RNA Seq) on one vertebral specimen. MicroCT imaging revealed three distinct dysmorphic bone patterns: 1) osteolytic, defined by thinned broken trabecula of well-organized mineral and collagen structures; 2) osteoblastic, with disorganized matrix deposited on pre-existing trabecula; and 3) osteoblastic, with little, if any, residual trabecula, and dominated by accumulation of disorganized mineralized matrix. qBSE and EDX revealed heterogeneous mineral composition, with higher variability in mineral content. Disorganized matrix had a higher voids or “lacunae” density, poorly organized collagen fiber alignment, and higher decorin and pan-proteoglycan content. Sc RNA Seq revealed a PCBM tumor microenvironment composed of a variety of immune cells, hematopoietic progenitors, and bone resident cells. Importantly, we identified AR-high, AR-low, and double-negative PC cells in the same lesion, with minimum neuroendocrine component. Simultaneous presence of these PC phenotypes was confirmed by immunohistochemistry. Morphologically, AR-positive cells adopt two configurations in PCBM, i) clusters within bone cavities in which cells maintain secretory polarization and resemble prostate acini, and ii) diffuse cells in intimate contact with bone-resident cells that lack polarization. PCBM lesions have abnormal bone structures lacking collagen organization, and with mineral heterogeneity consistent with bone weakness. PC cells interact with bone cells, but how distinct PC phenotypes affect bone turnover and structure need further analysis. Citation Format: Felipe Eltit, Qiong Wang, Raphaële Charest-Morin, Colm Morrissey, Eva Corey, Rizhi Wang, Michael E. Cox. Prostate cancer metastasis induces irregular bone formation associated to specific androgen dependent phenotypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 273.

Abstract 818: New targets in prostate cancer: Nuclear localization of galectin-1 in androgen receptor indifferent prostate cancer

Abstract Currently, no morphological characteristics have been described in clinical specimens to delineate an androgen receptor (AR)-active from an AR-inactive phenotype in AR-expressing aggressive variant prostate cancer (AVPC). 36 localized and 51 metastatic prostate cancer (CaP) samples were analyzed - 19 (37.3%) of the metastases were in the bone, 20 (39.2%) were in the lung, while the rest were in other areas such as brain, lymph node, bladder, etc). Expression level of Gal-1 was graded individually in the nucleus and the cytoplasm of both primary and metastatic tissues. Very low Gal-1 expression was observed in epithelial cells of benign prostate, while stromal staining was strong. Localized prostate tumors obtained from prostatectomy samples showed strong gal-1 expression in the epithelial cells, but remarkably the Gal-1 staining was confined to the cytoplasm. In contrast, in metastatic tumors many nuclei stained for Gal-1. Among localized tumors, Kaplan-Meier curves showed a significant increase in metastasis formation (p=0.022) and decreased overall survival (p=0.015) among those expressing nuclear Gal-1 (n=16) compared to those with no nuclear Gal-1 (n=20). Whereas cytoplasmic Gal-1 negatively correlated with patient age (R=-0.51, p=0.008) and PSA (R=-0.41, p=0.006), and positively correlated with Ki67 (R=0.35, p=0.014), the expression of nuclear Gal-1 correlated strongly with the expression of NE markers including chromogranin A (R=0.32, p=0.031). Significantly, nuclear Gal-1 was expressed in AVPC models but not in those derived from localized tissues. Significantly, androgen deprivation, including treatment with AR antagonists, resulted in nuclear localization of Gal-1. We adapted the powerful one-bead-two-compound (OB2C) combinatorial library method for the discovery of synthetic small molecule death ligands against cancer cells in an ultra-high throughput fashion. One of these benzimidazole-based ligands, LLS80, was active against tumor cells at low micromolar IC50. Pull-down assay followed by LC MS/MS indicated that galectin-1 is the target protein of LLS80, which induced apoptosis preferentially in AVPC cells expressing high level of Gal-1. LLS80 has all the indicators of being a good therapeutic agent: (i) LLS80 has a low in vitro IC50 in AVPC cells, (ii) it shows significant in vivo anti-tumor efficacy using xenograft models, (iii) it is a novel small molecule that can penetrate AVPC cells, (iv) it can synergize with enzalutamide in vivo and in vitro, (v) it inhibits a known therapeutic target, Gal-1, and (vi) low toxicity. Treatment with LLS80 (2 µM) prevented AR antagonist-induced Gal-1 nuclear localization, and also downregulated NE markers in AVPC models of CaP progression. We conclude that nuclear Gal-1 is a novel marker of AVPC. Citation Format: Maria Malvina Tsamouri, Salma Siddiqui, Blythe P. Durbin-Johnson, Kit S. Lam, Ruiwu Liu, Paramita M. Ghosh. New targets in prostate cancer: Nuclear localization of galectin-1 in androgen receptor indifferent prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 818.

Abstract 5999: TBX2 promotes prostate cancer bone-metastatic phenotype through exosomal microRNA-375-3p

Abstract Introduction: Bone is the preferred site of metastasis in about 80% of advanced prostate cancer (PCa) patients. The molecular mechanisms that drive bone metastatic PCa are not well understood. Recent reports have increasingly shed light on the role of cancer-cell-derived extracellular vesicles (exosomes) in driving the metastatic cascade including priming the secondary site for metastatic manifestation. We have previously reported that TBX2 promotes multiple facets of the PCa bone metastatic cascade via a cell-intrinsic manner. In this study, we sought to determine if the bone metastasis promoted by TBX2 is driven by microRNAs (miRs) in an exosome-mediated paracrine manner. Methods: Two converse approaches were utilized for the genetic modulation of TBX2: a) TBX2 was blocked in the metastatic PC3 and C4-2B human PCa cells using a dominant negative (DN) approach, and b) TBX2 was over-expressed in the low-TBX2 expressing LNCaP human PCa cells. Exosomes isolated from TBX2-modulated human PCa cells were added to MC3T3 pre-osteoblastic cells or mouse calvaria bone cells to examine the differential effects of TBX2 modulation on osteoblast or osteoclast differentiation. Next, unbiased next generation sequencing (NGS) was performed to identify the differential miR expression in exosomes derived from the TBX2-blocked PC3 cells. Results: Our results demonstrate that compared with control exosomes, exosomes derived from TBX2-blocked PC3 cells slowed the proliferation and migration/wound healing when added to 22Rv1 cells. Further, compared with control exosomes, exosomes derived from TBX2-blocked C4-2B cells resulted in reduced osteoblast differentiation when added to: a) MC3T3 pre-osteoblastic cells, or b) mouse calvaria bone cells. Along similar lines, when compared with the control exosomes, exosomes derived from TBX2-blocked PC3 and C4-2B cells resulted in reduced osteoclast differentiation when added to bone-marrow derived cells. These results were confirmed by q RT-PCR analysis of the osteoblastic/osteolytic bone markers. In order to identify exosomal miRs downstream of TBX2 signaling, NGS analysis of exosomal miRs obtained from TBX2-blocked PC3 human PCa cells identified miR-375-3p as the top-most differentially-expressed (over-expressed miR) when compared with control exosomes. In silico analysis revealed that miR-375-3p could potentially bind and target the 3’UTR of RBPJ, an established driver of PCa bone metastasis. In agreement, q RT-PCR analysis revealed that rescue of miR-375-3p in TBX2 modulated human PCa cells rescued RBPJ expression. Conclusions: Our studies have identified the exosome-mediated TBX2/miR-375-3p/RBPJ signaling axis as a promoter of the bone-metastatic phenotype in human PCa. Citation Format: Girijesh Kumar Patel, Sayanika Dutta, Hamed Khedmatgozar, Manisha Tripathi, Srinivas Nandana. TBX2 promotes prostate cancer bone-metastatic phenotype through exosomal microRNA-375-3p [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5999.

Abstract 1507: Tumor only analysis of whole exome sequencing from a multi-institutional Nigerian prostate cancer cohort reveals DNA repair genes associated with African ancestry

Abstract Men of African ancestry (MAA) have the highest global incidence and mortality of prostate cancer (PCa); however, the biology underlying this harsh disease presentation remains poorly understand, largely due to Africans and people within the African diaspora being under-represented in genomics research. MAA are younger at diagnosis, have higher tumor volume at diagnosis and have higher tumor aggression compared to European American men. Additionally, genomic profiling continues to show that PCa etiology and phenotype are influenced by higher amounts of African ancestry and that West African ancestry is associated with unique genomic alterations. Herein we utilize whole exome sequencing of a unique cohort of 45 advanced stage, treatment naïve Nigerian primary PCa tumors and 11 unmatched non-tumor tissue to compare genomic variants with African (AA) and European (EA) American TCGA PCa tumors. Nigerian samples were collected from 6 sites in central and southwest Nigeria. After whole exome sequencing, samples were processed using GATK best practices. Four genes [BRCA1 (100%), BARD1 (45%), BRCA2 (27%) and PMS2 (18%)] had germline variants in at least two Nigerian non-tumor samples. Across 111 germline variants, the AA cohort reflected a pattern [BRCA1 (68%), BARD1 (34%), BRCA2 (28%) and PMS2 (16%)] similar to Nigerian samples. Of the most frequently mutated genes, BRCA1 showed a statistically (p ≤ 0.05) higher mutation frequency in MAA. Disaggregating gene level mutation frequencies by variant revealed both ancestry linked and Nigerian specific germline variant patterns. Driven by rs799917, BRCA1 showed increasing mutation frequency as African admixture increased. BRCA2_rs11571831 was only present in MAA and BRCA2_rs766173 was increased in Nigerian men. 133 somatic variants were present in 26 PCa associated genes within the Nigerian tumor cohort. Nine genes [BRCA2 (27%), APC (20%), ATM (20%), BRCA1 (13%), DNAJC6 (13%), EGFR (13%), MAD1L1 (13%), MLH1 (11%) and PMS2 (11%)] showed mutation frequencies above 10%. Compared to TCGA cohorts, BRCA2, APC and BRCA1 showed statistically significant increases in Nigerian tumors. The Nigerian cohort variant pattern shared similarities (cosign similarities ≥ 0.734) with COSMIC signatures 5 and 6 and mutated genes showed significant (q &amp;lt; 0.001) GO and functional enrichment in mismatch repair and non-homologous repair deficiency (HRD) pathways. Here, we show that variants in DDR genes are increased in Nigerian PCa and that a portion of those variants correlate with increased African ancestry. Moreover, we identify variants of unknown significance that may contribute to population specific routes of tumorigenesis and treatment. These results present the most comprehensive characterization of the Nigerian PCa exome to date and further highlight the need to increase study population diversity. Citation Format: Jason A. White, Ernest Kaninjing, Kayode A. Adeniji, Paul Jibrin, John O. Obafunwa, Chidiebere N. Ogo, Mohammed Faruk, Solomon Rotimi, Ademola Popoola, Omolara A. Fatiregun, Olabode P. Oluwole, Balasubramanyam Karanam, Wei Tang, Stefan Ambs, Folake T. Odedina, Damali N. Martin, Clayton Yates. Tumor only analysis of whole exome sequencing from a multi-institutional Nigerian prostate cancer cohort reveals DNA repair genes associated with African ancestry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1507.

Abstract 5637: Transformation of the gut microbiome in response to androgen deprivation and the transition to castration-resistant prostate cancer

Abstract Androgen deprivation therapy (ADT) remains the standard care for men with advanced prostate cancer. Although initially effective, many patients fail to maintain durable responses and progress to lethal castration-resistant prostate cancer. Commensal bacteria play important role in maintaining homeostasis and proper immune function. However, ADT alters gut bacterial compositions and the ramifications being investigated. In this study, we used a mouse model of Pten-deficient prostate cancer to characterize differences in bacterial composition of gut bacteria in mice with castration naïve-prostate cancer (CNPC) and gut microbiota from mice three and six weeks after treatment ADT via surgical castration representing castration-sensitive prostate cancer (CSPC) and CRPC, respectively. Fecal samples were collected and processed for 16s rRNA amplicon sequencing. ADT resulted in significant changes to biological diversity and local diversity was highest in CSPC (Shannon index, P=0.04). Community composition also differed significantly different between the groups (Adonis OTU Bray-Curtis, P=0.001, NMDS stress=0.07). Despite dissimilarities between bacterial communities among the groups, CNPC and CRPC shared a greater similarity. Taxa associated with CNPC included Unclassified S247, Prevotella L. reuteri, unclassified Rikenellaceae and unclassified Bacteroidales; taxa associated with CSPC included R. gnavus, Dorea, unclassified Desulfovibrionaceae, Dehalobacterium, A. muciniphilia, Bifidobacterium, and B. producta; and taxa associated with CRPC included Turicibacter, R. flavefaciens, L. garvieae, unclassified Peptostreptococcaceae and unclassified Bacilli. Mice with CSPC and CRPC had an increased abundance genes associated with of catabolic metabolic pathways associated with aromatic compound, steroid Benzoate, and toluene degradation as well as genes associated to pathways corresponding to methane metabolism, phosphonate and phosphinate metabolism, and nitrogen metabolism. Genes enriched in fecal taxa from CNPC and CRPC mice were associated with folate biosynthesis, galactose metabolism, amino sugar and nucleotide sugar metabolism, and carbon fixation pathways in prokaryotes. This study provides preclinical evidence to support the association between the gut microbiome and prostate cancer phenotypes associated with sensitivity to ADT. Citation Format: Chisato Wakamori, Marco A. De Velasco, Kazuko Sakai, Yurie Kura, Naomi Ando, Kazutoshi Fujita, Eri Banno, Kazuhiro Yoshimura, Masahiro Nozawa, Kazuto Nishio, Hirotsugu Uemura. Transformation of the gut microbiome in response to androgen deprivation and the transition to castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5637.

CircSCAF8 promotes growth and metastasis of prostate cancer through the circSCAF8-miR-140-3p/miR-335-LIF pathway

Circular RNAs (circRNAs) have been increasingly linked to cancer progression. However, the detailed biological functions of circRNAs in prostate cancer (PCa) remain unclear. Using high-throughput circRNA sequencing, we previously identified 18 urine extracellular vesicle circRNAs that were increased in patients with PCa compared with those with benign prostatic hyperplasia. Spearman correlation analysis of the expression levels of the 18 circRNAs between the tumor tissue and matched urine extracellular vesicles in 30 PCa patients showed that circSCAF8 had the highest R2 (R2 = 0.635, P < 0.001). The Cox proportional hazards regression model was used to estimate the effect of circSCAF8 on progression-free survival. The in vitro and in vivo functional experiments were implemented to investigate the effects of circSCAF8 on the phenotype of PCa. We found that the knockdown of circSCAF8 in PCa cells suppressed the proliferation, migration, and invasion ability, while overexpression of circSCAF8 had the opposite effects. Similar results were observed in vivo. In a cohort of 85 patients who had undergone radical prostatectomy, circSCAF8 expression in PCa tissues was a powerful predictor of progression-free survival (HR = 2.14, P = 0.022). Mechanistically, circSCAF8 can function by binding to both miR-140-3p and miR-335 to regulate LIF expression and activate the LIF-STAT3 pathway that leads to the growth and metastasis of PCa. Collectively, our findings demonstrate that circSCAF8 contributes to PCa progression through the circSCAF8-miR-140-3p/miR-335-LIF pathway.

Clinical, environmental, genetic, and genomic profile of men with early-onset aggressive prostate cancer.

e17049 Background: Although prostate cancer (PC) is heterogeneous, the rate of patients diagnosed with aggressive metastatic disease at a young age has been increasing. Prior studies defining genetic abnormalities in high-risk PC have not focused on this unique population of patients, thus the clinical and molecular features of these lethal PC phenotypes are not well described. Methods: This multi-institutional study evaluated two cohorts. Cohort 1, reported here, included early-onset (age ≤ 60) PC that was metastatic (N+ or M+) at diagnosis or PC that metastasized within 5 years post curative intent/local therapy. Cohort 2 included men with metastatic hormone sensitive PC who rapidly progressed (≤ 14 months) after systemic therapy. Data was collected to define clinical and genomic profiles, including sequencing of somatic &amp; germline DNA, circulating tumor DNA and tumor RNA. Standard descriptive statistics were used. Results: 44 patients were enrolled. Median age at diagnosis was 55 years (range 41-60); 84% were White and 14% were Black. Median prostate specific antigen at diagnosis was 20 (range 1-534 ng/mL). 54% reported a family history of PC, while breast and colorectal cancer were reported in 35% and 14%, respectively. 4.5% reported a history of biochemical agent exposure. 58.5% of patients had De Novo distant metastatic disease (56% of these were low-volume) and 59.5% had a Gleason score of 9-10. 59% had received prior local therapy. Germline and somatic genetic data are available for 36 patients (4 are pending). The most common somatic mutation was in TP53 (n=15), followed by BRAF (n=14), AR (n=7), ERBB3 &amp; MYC (n=5 each), CDKN2B, HRAS, MUC4, OBSCN &amp; SPOP (n=4 each). Additional unique mutations in over 1,000 genes were also identified. Germline mutations were detected in BRCA2, ATM, ATP7B &amp; FBN1 (n=3 each), RB1, CDH1, MYBPC3, MYH11 &amp; MYH7 (n=2 each). 11 other unique germline mutations were also identified. Germline mutations were identified in genes previously implicated in hereditary PC ( BRCA2, ATM, PALB2, BRIP1 &amp; CHEK2), with an overall germline incidence of 25%. There were also incidental germline mutations in genes related to hereditary cardiac conditions ( MYBPC, MYH11, MYH7), as well as other hereditary cancers ( RB1 and CDH1). Conclusions: This study evaluated specific criteria to define risk factors associated with the development of aggressive PC at a young age. Nearly 90% of these patients had a family history of cancer, with over 50% reporting a family history of PC. Somatic mutations were identified in genes such as TP53 that are frequently associated with aggressive disease. Additionally, there was enrichment for germline mutations associated with PC that exceeded what has previously been reported and enrichment of mutations not commonly included in PC genetic risk panels. Thus, more work is needed to define characteristics of this high-risk population and optimize management.

Retraction Note: Hypoxia-associated p38 mitogen-activated protein kinase-mediated androgen receptor activation and increased HIF-1α levels contribute to emergence of an aggressive phenotype in prostate cancer.

Retraction Note: Hypoxia-associated p38 mitogen-activated protein kinase-mediated androgen receptor activation and increased HIF-1α levels contribute to emergence of an aggressive phenotype in prostate cancer.

Fatty acid oxidation enzyme Δ3, Δ2-enoyl-CoA isomerase 1 (ECI1) drives aggressive tumor phenotype and predicts poor clinical outcome in prostate cancer patients.

Prostate cancer (PCa) metastases are highly enriched with genomic alterations including a gain at the 16p13.3 locus, recently shown to be associated with disease progression and poor clinical outcome. ECI1, residing at the 16p13.3 gain region, encodes Δ3, Δ2-Enoyl-CoA Delta Isomerase 1 (ECI1), a key mitochondrial fatty acid β-oxidation enzyme. Although deregulated mitochondrial fatty acid β-oxidation is known to drive PCa pathogenesis, the role of ECI1 in PCa is still unknown. We investigated the impacts of ECI1 on PCa phenotype in vitro and in vivo by modulating its expression in cell lines and assessed the clinical implications of its expression in human prostate tissue samples. In vitro, ECI1 overexpression increased PCa cell growth while ECI1 deficiency reduced its growth. ECI1 also enhanced colony formation, cell motility, and maximal mitochondrial respiratory capacity. In vivo, PCa cells stably overexpressing ECI1 injected orthotopically in nude mice formed larger prostate tumors with higher number of metastases. Immunohistochemistry analysis of the human tissue microarray representing 332 radical prostatectomy cases revealed a stronger ECI1 staining in prostate tumors compared to corresponding benign tissues. ECI1 expression varied amongst tumors and was higher in cases with 16p13.3 gain, high Gleason grade, and advanced tumor stage. ECI1 overexpression was a strong independent predictor of biochemical recurrence after adjusting for known clinicopathologic parameters (hazard ratio: 3.65, P < 0.001) or the established CAPRA-S score (hazard ratio: 3.95, P < 0.001). ECI1 overexpression was also associated with significant increased risk of distant metastasis and reduced overall survival. Overall, this study demonstrates the functional capacity of ECI1 in PCa progression and highlights the clinical implication of ECI1 as a potential target for the management of PCa.

Downregulation of SHMT2 promotes the prostate cancer proliferation and metastasis by inducing epithelial-mesenchymal transition

Serine hydroxymethyltransferase 2 (SHMT2) is a key enzyme that regulates serine/glycine transition; however, its specific function and molecular mechanisms in tumors remain controversial. In this study, we aimed to enhance the understanding in this regard. Through in vitro and in vivo experiments, as well as data analyses using public databases, we investigated the effect of SHMT2 in prostate cancer. Our results indicated that SHMT2 acts as a prostate cancer tumor proliferation suppressor and negatively regulates the aggressive behavior of prostate cancer through activation of epithelial–mesenchymal transition. Additionally, downregulated SHMT2 expression was observed in more advanced prostate cancer phenotypes, and further analysis showed that its depletion promoted proliferation and migration in prostate cancer cell lines. Taken together, our results revealed the function of SHMT2 in prostate cancer and may potentially play a role in the exploration of new therapeutic strategies.

Hsa-miR-1290 is associated with stemness and invasiveness in prostate cancer cell lines by targeting RORA.

This work examined microRNA-1290 (miR-1290)'s effect on regulating the malignant phenotype of prostate cancer (PC) cells. We detected miR-1290 expression within PC based on open-sourced datasets as well as in cancer cells and tissues. Loss-of-function experiments by miR-1290 knockdown in PC cell lines were performed. We performed CCK-8, clone forming, Transwell, and sphere formation assays for examining PC cells' malignant phenotypes following miR-1290 knockdown. We estimated miR-1290's target genes using online resources including miRDB, miRbase, miRTarBase and TargetScan. We also performed in vivo studies for validating how miR-1290 affected tumour formation within the mouse model. According to findings in this work, miR-1290 showed overexpression within PC cells and tissues. miR-1290 was indispensable for PC cell growth, stemness and invasion as well as mesenchymal status. Further, we identified RORA (retinoic acid receptor-related orphan receptor A) as miR-1290's target gene for mediating miR-1290 within PC cells. To sum up, this work suggests that miR-1290 up-regulation enhances PC cell growth and invasion by regulating RORA expression.

Early PSA density kinetics predicts biochemical and local failure following extreme hypofractionated radiotherapy in intermediate-risk prostate cancer

PurposeThe present study explores PSA density (PSA-D) as predictor of biochemical and local failure in organ-confined prostate cancer at 3–6 months after hypofractionated stereotactic ablative radiotherapy (SABR). Methods and materialsA cohort of 219, hormone-naïve, NCCN intermediate-risk prostate cancer patients were derived from a phase 2 study of 5 × 9 Gy prostate cancer SABR. PSA-D was calculated at 3 and 6 months by dividing serum PSA by the MR-derived prostate CTV, while the slope of the 3–6 months curve was used to express the kinetics of PSA-D decay. ResultsThe median follow-up was 60.3 (range 46–76) months, and the actuarial 7-year bRFS was 98.0% for intermediate-risk favorable (FIR) patients versus 84.5% for the unfavorable (UIR) subgroup (P = 0.02). Fourteen patients developed a Phoenix-defined biochemical PSA relapse (bRFS) at a median of 34.2 months, 11 confirmed with 68Ga-PSMA PET/CT scan that revealed tracer uptake at the site of dominant intraprostatic pretreatment lesion in 8 patients. The 3-month PSA-D concertation (cut-off 0.08 ng/ml2) and 3–6 months decay slope (cut-off −0.45) values were predictive of long-term bRFS. A dual adverse PSA-D permutation was detected in 25/148 UIR patients, exhibiting 47.5% 7-year bRFS compared with 94.1% for the remaining 123 UIR patients with favorable PSA-D kinetics (P = 0.0006). Intraprostatic local relapse in patients with a 3-month PSA-D > 0.080 ng/ml2 was 11.0% vs. 1.7% for patients with PSA-D ≤ 0.080 ng/ml2 (P = 0.01) and 2.3% vs. 4.3%, respectively, for nodal progression (P = 0.68). ConclusionsEarly post-treatment PSA-D kinetics transcends pre-treatment risk stratification of tumor relapse and adds a nuance in the biological characterization of intermediate-risk prostate cancer phenotypes. The dual adverse PSA-D algorithm may serve as a tool to validate current search of classifiers of radioresistance in prostate cancer with therapeutic implications.