Clinical Prostate Cancer Samples Research Articles

FOXS1 acts as an oncogene and induces EMT through FAK/PI3K/AKT pathway by upregulating HILPDA in prostate cancer.

Prostate cancer (PCa) is a widespread global health concern characterized by elevated rates of occurrence, and there is a need for novel therapeutic targets to enhance patient outcomes. FOXS1 is closely linked to different cancers, but its function in PCa is still unknown. The expression of FOXS1, its prognostic role, clinical significance in PCa, and the potential mechanism by which FOXS1 affects PCa progression were investigated through bioinformatics analysis utilizing public data. The levels of FOXS1 and HILPDA were evaluated in clinical PCa samples using various methods, such as western blotting, immunohistochemistry, and qRT-PCR. To examine the function and molecular mechanisms of FOXS1 in PCa, a combination of experimental techniques including CCK-8 assay, flow cytometry, wound-healing assay, Transwell assay, and Co-IP assay were employed. The FOXS1 expression levels were significantly raised in PCa, correlating strongly with tumor aggressiveness and an unfavorable prognosis. Regulating FOXS1 expression, whether upregulating or downregulating it, correspondingly enhanced or inhibited the growth, migration, and invasion capabilities of PCa cells. Mechanistically, we detected a direct interaction between FOXS1 and HILPDA, resulting in the pathway activation of FAK/PI3K/AKT and facilitation EMT in PCa cells. FOXS1 collaborates with HILPDA to initiate EMT, thereby facilitating the PCa progression through the FAK/PI3K/AKT pathway activation.

Abstract 7505: Detection validation of androgen-receptor splice variant 7 in circulating tumor cells of prostate cancer by double-check assays

Abstract Introduction: Androgen-receptor splice variant 7 (AR-V7) is constitutively activated isoform of AR and has been associated with resistance towards AR targeting therapies and development into mCRPC. Confirmation of AR-V7 expression is important step in prostate cancer diagnosis and determining the drug, but the detection of AR-V7 with tissues has limitations such as clinical challenge to poorly accessible tumor tissues before and after treatment. Here, we developed the method of AR-V7 detection in circulating tumor cells (CTC) after isolation with CytoGen’s Smart BiopsyTM CTC isolator by in vitro assays of qPCR and immunofluorescence (IF) with high sensitivity and specificity, overcoming the limitations of tissue biopsy. Method: qPCR and IF methods were developed for high sensitivity and specificity of AR-V7 detection. Sensitivity and specificity were tested using AR-V7 positive cells (VCaP and 22Rv1) and negative cells (PC-3 and Raji). Limit of detection (LOD) test of AR-V7 expression was performed with AR-V7 positive cells (200 cells to 50 cells, 3 points) mixed with Raji cells (1 × 10^5cells). Then, AR-V7 positive cells (200 cells to 50 cells, 3 points) were spiked into peripheral blood mononuclear cells (PBMC) of blood sample (5mL, healthy donor) to mimic CTC after isolation by CytoGen’s Smart BiopsyTM CTC isolator, subsequently AR-V7 expression was tested with qPCR and IF assay. Results: AR-V7 expression was clearly detected by qPCR and IF in the LOD test of AR-V7 positive cells mixed with Raji cells and was not detected at all in the negative controls. The similar clear results were also observed in the spiking test of AR-V7 positive cells into PBMC isolated by CytoGen’s Smart BiopsyTM CTC isolator, suggesting that clinical samples of prostate cancer could be applied with excellent sensitivity and specificity for AR-V7. Conclusions: AR-V7 expresses only a part of cancer tissue, so it is difficult to accurately diagnose with traditional histological assays. Detection of AR-V7 in intact live CTC isolated by CytoGen’s Smart BiopsyTM CTC isolator might overcome limitations of tissue biopsy. In this study, we developed the method to detect AR-V7 in CTC with high sensitivity and specificity with qPCR and IF assay, and presented the results of verifying AR-V7 expression by double check using two kinds of assays. We plan to conduct clinical validation of the developed method through additional study using patient samples. Citation Format: Hoin Kang, Jihyun Lee, Min Hyung Kim, Soee Kim, Hyoyong Kim, Jubi Lee, Hae Ung Lee, Hai-Chon Lee, Jung Won Kim. Detection validation of androgen-receptor splice variant 7 in circulating tumor cells of prostate cancer by double-check assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7505.

Abstract 2011: MAOA Drives Anti Androgen Therapy Resistance in Advanced Prostate Cancer by Conferring Neuroendocrine Plasticity via a Twist1 BRN2 Signaling Pathway

Abstract Background: Therapy resistance in cancer is often linked to cancer cell state or lineage changes. In prostate cancer (PCa), a subset of castration-resistant prostate cancer (CRPC) loses reliance on the androgen receptor (AR) signaling and gains neuroendocrine (NE) traits through a lineage switch along disease progression. However, the molecular mechanisms driving such cellular lineage plasticity remain unclear. This study investigated the functional and mechanistic role of monoamine oxidase A (MAOA) in driving adenocarcinoma (adeno)-to-NE lineage conversion in CRPC cells upon enzalutamide (ENZ), a FDA-approved newer-generation antiandrogen drug, and evaluated the efficacy of MAOA inhibitors for treating CRPC and overcoming ENZ resistance.Methods: ENZ-resistant C4-2B and CWR-R1 cells (C4-2BENZR and CWR-R1ENZR) CRPC cells were transfected with a PSA promoter-driven GFP expression construct for isolating a PSAlo/− cell population distinct from PSAhi cells, which was used for manipulation of MAOA expression levels and subsequent functional assays in vitro and in vivo. The MAOA expression levels were determined and correlated with NE parameters in PCa clinical samples by immunohistochemistry and data mining of multiple publicly available clinical datasets. Results: MAOA protein expression was significantly upregulated in the PSAlo/− ENZ-resistant cell population, accompanied by concomitant activation of NE markers and downregulation of luminal markers including AR and AR targets, compared to the PSAhi counterpart. Elevated MAOA expression was also found in PCa patient samples associated with NE features from multiple independent cohorts. Silencing MAOA suppressed the proliferation, migration, invasion, stem-like properties and NE plasticity of both PSAlo/- C4-2BENZR and CWR-R1ENZR cells. Moreover, co-culturing adeno LNCaP or C4-2B cells with MAOA-silenced PSAlo/- cells exhibiting a repressed secretory phenotype attenuated acquired induction of NE markers and proliferation of adeno cells as compared to controls. Mechanistically, MAOA induces Twist1, which directly activates the transcription and expression of BRN2, a master neural transcription factor known to confer NE differentiation and anti-AR therapy resistance in CRPC. Further, pharmacological inhibition of MAOA greatly enhanced the growth-inhibitory efficacy of ENZ with ENZ sensitivity restored in ENZ-resistant LuCaP PC PDX-derived organoids as well as a PSAlo/− CWR-R1ENZR xenograft mouse model.Conclusion: Our results suggest MAOA as a potential therapeutic target for reversing ENZ resistance in CRPC treatment by blockade of NE transdifferentiation.Funding Acknowledgements: This work was supported by the NIH/NCI grants R37CA233658 and R01CA258634 (to B. Wu) and the DOD Department of Defense EIRA award W81XWH-21-1-0218 (to J. Wei) Citation Format: Jing Wei, Jing Wang, Chia-hui Chen, Wen Guan, Boyang Wu. MAOA Drives Anti Androgen Therapy Resistance in Advanced Prostate Cancer by Conferring Neuroendocrine Plasticity via a Twist1 BRN2 Signaling Pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2011.

PCMT1 regulates the migration, invasion, and apoptosis of prostate cancer through modulating the PI3K/AKT/GSK-3β pathway.

Protein L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) is a repair enzyme that catalyzes the conversion of isomerized aspartic acid (iso-Asp) residues into their normal structure, thereby restoring the configuration and function of proteins. Studies have shown that PCMT1 is overexpressed in several tumors and affects patients' prognosis. However, there are few reports on the role of PCMT1 in prostate cancer (PCa). In the present research, with the assistance of The Cancer Genome Atlas Program (TCGA) database, we found that PCMT1 was overexpressed in PCa tissues. The results of quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blot and immunohistochemistry staining also showed that PCMT1 expression was significantly increased in PCa tissues and cell lines. In PCa clinical samples, PCMT1 expression was closely related to Gleason score, clinical stage, lymph node metastasis and bone metastasis. The experiments of overexpression and knockdown of PCMT1 in vitro or in vivo showed that PCMT1 can significantly promote the proliferation, migration and invasion of PCa cells, inhibit cell apoptosis, and promote the growth of PCa. We furthermore confirmed that PCMT1 regulated the migration, invasion and apoptosis of PCa cells by modulating the phosphatidylinositol 3-kinase/AKT kinase/glycogen-synthase kinase-3β (PI3K/AKT/GSK-3β) signaling pathway. Collectively, PCMT1 plays a cancer-facilitative role in PCa by promoting the proliferation, migration and invasion of PCa cells, and inhibiting apoptosis. Therefore, PCMT1 is considered to represent a novel target for treating PCa.

SAT022 A Mechanism Of Resistance To Novel Inhibitors Of The N-Terminal Domain Of Androgen Receptor

Abstract Disclosure: J. Setiawan: None. N.R. Mawji: None. A.H. Tien: None. R.J. Andersen: Stock Owner; Self; ESSA Pharma. M.D. Sadar: Stock Owner; Self; ESSA Pharma. Background: Androgen receptor (AR) is a steroid hormone receptor and a major therapeutic target in prostate cancer. Antiandrogen therapies directed at the AR C-terminal ligand-binding domain (LBD) provide initial benefit to patients with advanced prostate cancer, but they eventually progress to lethal metastatic castration-resistant prostate cancer (CRPC). AR N-terminal domain (NTD) inhibitors are a novel class of antagonists that were developed to overcome resistance mechanisms related to the AR-LBD, including splice variants lacking the LBD (AR-V7). While mutations in the AR-LBD have been well-characterized, the impact of NTD mutations on AR transcriptional activity and drug efficacy remains unclear. The mutation W435L was discovered from clinical samples of prostate cancer from patients treated with antiandrogens. This mutation stabilizes interaction between the AR’s NTD and LBD (N/C interaction) to increase AR transcriptional activity. Tryptophan residues W435 and W397 in AR NTD are essential in the binding site for the AR-NTD inhibitor EPI-7386 that is currently in clinical trials. We hypothesize that tryptophan mutations within the EPI-7386 binding site of AR are a potential mechanism of resistance. Methods: Site-directed mutagenesis was used to create expression vectors with W435 and W397 point mutations in AR. Plasmids were transfected into AR-negative CV-1 cells to compare the transcriptional activity of wild-type (WT) versus mutant ARs. IC50s for each inhibitor were generated for androgen-induced reporter gene constructs. A mammalian two-hybrid system assay was used to assess the impact of inhibitors and AR mutations on N/C interaction. Results: The AR-W435L mutation led to higher IC50s to inhibit androgen-induced MMTV-luciferase activity by NTD inhibitors and antiandrogens. Differences in N/C interaction were observed between WT and mutant ARs treated with EPI-7386. Importantly, the ability of another EPI analog, EPI-7170, to decrease AR-N/C interaction was not significantly impacted by W435L. These data support that loss of efficacy with EPI-7386 is not across the entire class of EPI analogs and may be specific to EPI-7386. Conclusion: Tryptophan mutations in the EPI-7386 binding site are predictive of resistance to this specific antagonist and do not broadly apply to the entire class of EPI analogs. Key differences were revealed between AR-LBD and AR-NTD inhibitors that support the recruitment of different tau regions in the NTD. Presentation: Saturday, June 17, 2023

CDC20 Is Regulated by the Histone Methyltransferase, KMT5A, in Castration-Resistant Prostate Cancer.

The methyltransferase KMT5A has been proposed as an oncogene in prostate cancer and therefore represents a putative therapeutic target. To confirm this hypothesis, we have performed a microarray study on a prostate cancer cell line model of androgen independence following KMT5A knockdown in the presence of the transcriptionally active androgen receptor (AR) to understand which genes and cellular processes are regulated by KMT5A in the presence of an active AR. We observed that 301 genes were down-regulated whilst 408 were up-regulated when KMT5A expression was reduced. KEGG pathway and gene ontology analysis revealed that apoptosis and DNA damage signalling were up-regulated in response to KMT5A knockdown whilst protein folding and RNA splicing were down-regulated. Under these conditions, the top non-AR regulated gene was found to be CDC20, a key regulator of the spindle assembly checkpoint with an oncogenic role in several cancer types. Further investigation revealed that KMT5A regulates CDC20 in a methyltransferase-dependent manner to modulate histone H4K20 methylation within its promoter region and indirectly via the p53 signalling pathway. A positive correlation between KMT5A and CDC20 expression was also observed in clinical prostate cancer samples, further supporting this association. Therefore, we conclude that KMT5A is a valid therapeutic target for the treatment of prostate cancer and CDC20 could potentially be utilised as a biomarker for effective therapeutic targeting.

Aspartoacylase suppresses prostate cancer progression by blocking LYN activation

BackgroundGlobally, despite prostate cancer (PCa) representing second most prevalent malignancy in male, the precise molecular mechanisms implicated in its pathogenesis remain unclear. Consequently, elucidating the key molecular regulators that govern disease progression could substantially contribute to the establishment of novel therapeutic strategies, ultimately advancing the management of PCa.MethodsA total of 49 PCa tissues and 43 adjacent normal tissues were collected from January 2017 to December 2021 at Zhongnan Hospital of Wuhan University. The advanced transcriptomic methodologies were employed to identify differentially expressed mRNAs in PCa. The expression of aspartoacylase (ASPA) in PCa was thoroughly evaluated using quantitative real-time PCR and Western blotting techniques. To elucidate the inhibitory role of ASPA in PCa cell proliferation and metastasis, a comprehensive set of in vitro and in vivo assays were conducted, including orthotopic and tumor-bearing mouse models (n = 8 for each group). A combination of experimental approaches, such as Western blotting, luciferase assays, immunoprecipitation assays, mass spectrometry, glutathione S-transferase pull-down experiments, and rescue studies, were employed to investigate the underlying molecular mechanisms of ASPA’s action in PCa. The Student’s t-test was employed to assess the statistical significance between two distinct groups, while one-way analysis of variance was utilized for comparisons involving more than two groups. A two-sided P value of less than 0.05 was deemed to indicate statistical significance.ResultsASPA was identified as a novel inhibitor of PCa progression. The expression of ASPA was found to be significantly down-regulated in PCa tissue samples, and its decreased expression was independently associated with patients’ prognosis (HR = 0.60, 95% CI 0.40–0.92, P = 0.018). Our experiments demonstrated that modulation of ASPA activity, either through gain- or loss-of-function, led to the suppression or enhancement of PCa cell proliferation, migration, and invasion, respectively. The inhibitory role of ASPA in PCa was further confirmed using orthotopic and tumor-bearing mouse models. Mechanistically, ASPA was shown to directly interact with the LYN and inhibit the phosphorylation of LYN as well as its downstream targets, JNK1/2 and C-Jun, in both PCa cells and mouse models, in an enzyme-independent manner. Importantly, the inhibition of LYN activation by bafetinib abrogated the promoting effect of ASPA knockdown on PCa progression in both in vitro and in vivo models. Moreover, we observed an inverse relationship between ASPA expression and LYN activity in clinical PCa samples, suggesting a potential regulatory role of ASPA in modulating LYN signaling.ConclusionOur findings provide novel insights into the tumor-suppressive function of ASPA in PCa and highlight its potential as a prognostic biomarker and therapeutic target for the management of this malignancy.

Abstract 2547: YAP1 associates with the EPHA3 receptor tyrosine kinase to promote prostate cancer progression

Abstract Metastatic castration-resistant prostate cancer is lethal, and the mechanisms contributing to this deadly disease are largely unknown. The disparity in our knowledge poses a significant barrier to developing effective therapeutics against advanced prostate cancer in humans. Evidence suggests that the ephrin family of receptor tyrosine kinase signaling may play a critical role in the pathobiology of prostate cancer progression. However, the mechanism is elusive. Our initial study showed that YAP1, in concert with the TEAD1 transcription factor, regulated EPHA3 expression. However, the significance of EPHA3 signaling in prostate cancer remains elusive. Here we investigated the role of EPHA3 signaling in prostate cancer cells. We showed that the expression of EPHA3 correlated with YAP1 in prostate cancer clinical samples. We also showed that EPHA3 knockout reduced cell growth in 2D and 3D cultures. In addition, EPHA3 loss sensitized castration-resistant prostate cancer cells to enzalutamide. Moreover, EPHA3 knockout reduced the GTPase activity of RHOA compared to cells with EPHA3-WT, which coincided with reduced phospho-ERK1/2 levels. Consistently, EPHA3 knockout reduces phosphor-ERK1/2, similar to the inhibition of phospho-ERK1/2 by an inhibitor of ROCK1 (Rho-associated coiled-coil-containing protein kinase 1). Besides, EPHA3 knockout reduced the expression of EMT markers ZEB1/2 and the cancer stem cell marker CD44 compared to the wild type. Furthermore, the levels of EPAH3 transcripts were higher in high-grade PC of AA patients compared to other races. Finally, AA and Caucasian Americans (CA) PC cell lines differentially express the member of ephrin-A and ephrin-B receptors. These observations suggest that the YAP1-EPHA3 signaling is a viable therapeutic target for patients with advanced prostate cancer. Citation Format: Marwah M. Al-Mathkour, Abdulrahman M. Dwead, Kezhan Khazaw, Bekir Cinar. YAP1 associates with the EPHA3 receptor tyrosine kinase to promote prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2547.

HMGB1 promotes the development of castration‑resistant prostate cancer by regulating androgen receptor activation.

AR signalling pathway reactivation plays a key role in the development of castration-resistant prostate cancer (CRPC). High-mobility group protein B1 (HMGB1) is an important factor involved in the occurrence and development of a variety of tumours by regulating gene transcription. In the present study, the association between HMGB1 and prostate cancer (PCa) and the effects of HMGB1 on androgen receptor (AR) transcription and signalling pathway reactivation in PCa cells in vitro and in vivo were evaluated. A bioinformatics method was used to determine the mRNA expression level of HMGB1 in PCa specimens and its correlation with the mRNA expression of AR. Immunohistochemical staining was used to detect the expression of these proteins in clinical PCa samples. Reporter gene and ChIP assays were performed to determine the activity of AR and the effect of HMGB1 on the ability of AR to bind to the promoters of prostate specific antigen and transmembrane protease, serine 2. A bioluminescence resonance energy transfer assay was employed to observe the direct interaction between HMGB1 and AR protein. Additionally, a castrated nude mouse xenograft tumour model was established to verify the effect of HMGB1 on PCa. The results revealed that HMGB1 expression was significantly increased in PCa specimens, which may have a strong correlation with AR expression. Moreover, HMGB1 could reactivate the AR signalling pathway, directly interact with AR, and promote the development of CRPC in an androgen-independent manner. The results of the present study indicated that HMGB1 promoted the development of CRPC by interacting with AR, which inferred that decreasing the expression of HMGB1 may be a potential effective method for CRPC prevention and treatment.

1698P Correlation between sex steroid hormone receptors expression in prostate cancer (PCa)

PCa is well known to be an androgen-dependent disease. However, estrogens and estrogen receptors alpha and beta (ERα and ERβ) also play an important role in PCa progression. All types of receptors share similar structure and can be activated by same ligands. As so, relative amounts of all receptor types may be important in predicting overall response to therapy, including androgen deprivation. We aimed to assess possible correlations between ERα, ERβ and androgen receptor (AR) expression in clinical PCa samples.

FOXA1 inhibits hypoxia programs through transcriptional repression of HIF1A.

Intratumoral hypoxia is associated with castration-resistant prostate cancer (CRPC), a lethal disease. FOXA1 is an epithelial transcription factor that is down-regulated in CRPC. We have previously reported that FOXA1 loss induces epithelial-mesenchymal transition (EMT) and cell motility through elevated TGFβ signaling. However, whether FOXA1 directly regulates hypoxia pathways of CRPC tumors has not been previously studied. Here we report that FOXA1 down-regulation induces hypoxia transcriptional programs, and FOXA1 level is negatively correlated with hypoxia markers in clinical prostate cancer (PCa) samples. Mechanistically, FOXA1 directly binds to an intragenic enhancer of HIF1A to inhibit its expression, and HIF1A, in turn, is critical in mediating FOXA1 loss-induced hypoxia gene expression. Further, we identify CCL2, a chemokine ligand that modulates tumor microenvironment and promotes cancer progression, as a crucial target of the FOXA1-HIF1A axis. We found that FOXA1 loss leads to immunosuppressive macrophage infiltration and increased cell invasion, dependent on HIF1A expression. Critically, therapeutic targeting of HIF1A-CCL2 using pharmacological inhibitors abolishes FOXA1 loss-induced macrophage infiltration and PCa cell invasion. In summary, our study reveals an essential role of FOXA1 in controlling the hypoxic tumor microenvironment and establishes the HIF1A-CCL2 axis as one mechanism of FOXA1 loss-induced CRPC progression.

Implementation of a prostate cancer-specific targeted sequencing panel for credentialing of patient-derived cell lines and genomic characterization of patient samples.

Primary and metastatic prostate cancers have low mutation rates and recurrent alterations in a small set of genes, enabling targeted sequencing of prostate cancer-associated genes as an efficient approach to characterizing patient samples (compared to whole-exome and whole-genome sequencing). For example, targeted sequencing provides a flexible, rapid, and cost-effective method for genomic assessment of patient-derived cell lines to evaluate fidelity to initial patient tumor samples. We developed a prostate cancer-specific targeted next-generation sequencing (NGS) panel to detect alterations in 62 prostate cancer-associated genes as well as recurring gene fusions with ETS family members, representing the majority of common alterations in prostate cancer. We tested this panel on primary prostate cancer tissues and blood biopsies from patients with metastatic prostate cancer. We generated patient-derived cell lines from primary prostate cancers using conditional reprogramming methods and applied targeted sequencing to evaluate the fidelity of these cell lines to the original patient tumors. The prostate cancer-specific panel identified biologically and clinically relevant alterations, including point mutations in driver oncogenes and ETS family fusion genes, in tumor tissues from 29 radical prostatectomy samples. The targeted panel also identified genomic alterations in cell-free DNA and circulating tumor cells (CTCs) from patients with metastatic prostate cancer, and in standard prostate cancer cell lines. We used the targeted panel to sequence our set of patient-derived cell lines; however, no prostate cancer-specific mutations were identified in the tumor-derived cell lines, suggesting preferential outgrowth of normal prostate epithelial cells. We evaluated a prostate cancer-specific targeted NGS panel to detect common and clinically relevant alterations (including ETS family gene fusions) in prostate cancer. The panel detected driver mutations in a diverse set of clinical samples of prostate cancer, including fresh-frozen tumors, cell-free DNA, CTCs, and cell lines. Targeted sequencing of patient-derived cell lines highlights the challenge of deriving cell lines from primary prostate cancers and the importance of genomic characterization to credential candidate cell lines. Our study supports that a prostate cancer-specific targeted sequencing panel provides an efficient, clinically feasible approach to identify genetic alterations across a spectrum of prostate cancer samples and cell lines.

Construction of a risk prediction model using m6A RNA methylation regulators in prostate cancer: comprehensive bioinformatic analysis and histological validation

BackgroundEpigenetic reprogramming reportedly has a crucial role in prostate cancer (PCa) progression. RNA modification is a hot topic in epigenetics, and N6-methyladenosine (m6A) accounts for approximately 60% of RNA chemical modifications. The aim of this study was to evaluate the m6A modification patterns in PCa patients and construct a risk prediction model using m6A RNA regulators.Materials and methodsAnalyses were based on the levels of 25 m6A regulators in The Cancer Genome Atlas (TCGA). Differentially expressed gene (DEG) and survival analyses were performed according to TCGA-PRAD clinicopathologic and follow-up information. To detect the influences of m6A regulators and their DEGs, consensus clustering analysis was performed, and tumor mutational burden (TMB) estimation and tumor microenvironment (TME) cell infiltration were assessed. mRNA levels of representative genes were verified using clinical PCa data.ResultsDiverse expression patterns of m6A regulators between tumor and normal (TN) tissues were detected regarding Gleason score (GS), pathological T stage (pT), TP53 mutation, and survival comparisons, with HNRNPA2B1 and IGFBP3 being intersecting genes. HNRNPA2B1 was upregulated in advanced stages (GS > 7, pT3, HR > 1, and TP53 mutation), as verified using clinical PCa tissue. Three distinct m6A modification patterns were identified through consensus clustering analysis, but no significant difference was found among these groups in recurrence-free survival (RFS) analysis. Six DEGs of m6A clusters (m6Aclusters) were screened through univariate Cox regression analysis. MMAB and PAIAP2 were intersecting genes for the five clinical factors. MMAB, which was upregulated in PCa compared with TN, was verified using clinical PCa samples. Three distinct subgroups were established according to the 6 DEGs. Cluster A involved the most advanced stages and had the poorest RFS. The m6A score (m6Ascore) was calculated based on the 6 genes, and the low m6Ascore group showed poor RFS with a negative association with infiltration for 16 of 23 immune-related cells.ConclusionWe screened DEGs of m6Aclusters and identified 6 genes (BAIAP2, TEX264, MMAB, JAGN1, TIMM8AP1, and IMP3), with which we constructed a highly predictive model with prognostic value by dividing TCGA-PRAD into three distinct subgroups and performing m6Ascore analysis. This study helps to elucidate the integral effects of m6A modification patterns on PCa progression.

Concomitant High Apoptosis Inhibitor of Macrophage (AIM) and Low Prostate-Specific Antigen (PSA) Indicates Activated T Cell-Mediated Anticancer Immunity, Enhance Sensitivity to Pembrolizumab, and Elicit Good Prognosis in Prostate Cancer.

Background: Despite its widespread use, the use of prostate-specific antigen (PSA) alone as a screening biomarker for prostate cancer (PCa) leads often to unwarranted prostate biopsy, over-diagnosis, and consequently, over-treatment, because of its limited specificity. There are reports that the apoptosis inhibitor of macrophage (AIM), secreted mainly by macrophages and epithelial cells, is upregulated during inflammation and facilitates immune recognition of cancerous cells by blocking human regulator of complement activation. Objective: These controversies around the PSA utility necessitate a reexamination of its use as a screening tool. More so, despite the suggested implication of AIM in anticancer immunosurveillance, there is a dearth of information on its role in therapy response, disease progression, and clinical outcomes of patients with PCa. These inform the present study to probe the nature and role of AIM/PSA signaling in anticancer immunity and prognosis in PCa. Methods: A combination of bioinformatics-aided statistical analyses, gene function annotation, and immune infiltrate analyses, coupled with tissue staining, and function assays, namely migration, invasion, and clonogenicity assays, we employed. Results: We demonstrated that AIM and PSA expression levels are inversely correlated in PCa clinical samples and cell lines, with AIMlowPSAhigh defining PCa, compared to AIMhighPSAlow in normal samples. Concomitant aberrant PSA and significantly suppressed AIM expression levels positively correlated with high-grade disease and characterized by advanced stage prostate cancer, regardless of mutation status. We found that a high PSA/AIM ratio is associated with disease recurrence in patients with prostate cancer but is equivocal for overall survival. In addition, PSA-associated AIM suppression is implicated in the enhanced ‘metastability’ of PCa and a high AIM/PSA ratio is associated with strong castration-induced regression. CRISPR-mediated AIM knockout was associated with higher PSA expression while ectopic expression of AIM significantly attenuated the migration and invasive capability of PC3 and DU145 cells. Interestingly, compared to normal samples, we observed that AIM, biomarkers of T-cell activation and M1 phenotype markers are co-suppressed in PCa samples. Conclusion: Herein, we demonstrate that AIM/CD5L binds to PSA and that a high PSA/AIM ratio defines advanced stage PCa (regardless of mutation status), is implicated in enhanced metastability, and associated with disease recurrence, while a high AIM/PSA ratio is associated with strong castration-induced regression. More so, the ectopic expression of AIM significantly enhances the anticancer effect of Pembrolizumab and elicits an increased CD8+ T-cell count in AIMhiPSAloPDL1+ PCa cases that are respondent to Pembrolizumab treatment.

Retraction Note to: Loss of PDEF, a prostate-derived Ets factor is associated with aggressive phenotype of prostate cancer: Regulation of MMP 9 by PDEF

Prostate-derived Ets factor (PDEF) is expressed in tissues of high epithelial content including prostate, although its precise function has not been fully established. Conventional therapies produce a high rate of cure for patients with localized prostate cancer, but there is, at present, no effective treatment for intervention in metastatic prostate cancer. These facts underline the need to develop new approaches for early diagnosis of aggressive prostate cancer patients, and mechanism based anti-metastasis therapies that will improve the outlook for hormone-refractory prostate cancer. In this study we evaluated role of prostate-derived Ets factor (PDEF) in prostate cancer. We observed decreased PDEF expression in prostate cancer cell lines correlated with increased aggressive phenotype, and complete loss of PDEF protein in metastatic prostate cancer cell lines. Loss of PDEF expression was confirmed in high Gleason Grade prostate cancer samples by immuno-histochemical methods. Reintroduction of PDEF profoundly affected cell behavior leading to less invasive phenotypes in three dimensional cultures. In addition, PDEF expressing cells had altered cell morphology, decreased FAK phosphorylation and decreased colony formation, cell migration, and cellular invasiveness. In contrast PDEF knockdown resulted in increased migration and invasion as well as clonogenic activity. Our results also demonstrated that PDEF downregulated MMP9 promoter activity, suppressed MMP9 mRNA expression, and resulted in loss of MMP9 activity in prostate cancer cells. These results suggested that loss of PDEF might be associated with increased MMP9 expression and activity in aggressive prostate cancer. To confirm results we investigated MMP9 expression in clinical samples of prostate cancer. Results of these studies show increased MMP9 expression correlated with advanced Gleason grade. Taken together our results demonstrate decreased PDEF expression and increased MMP9 expression during the transition to aggressive prostate cancer. These studies demonstrate for the first time negative regulation of MMP9 expression by PDEF, and that PDEF expression was lost in aggressive prostate cancer and was inversely associated with MMP9 expression in clinical samples of prostate cancer. Based on these exciting results, we propose that loss of PDEF along with increased MMP9 expression should serve as novel markers for early detection of aggressive prostate cancer.

IRE1α Expedites the Progression of Castration-Resistant Prostate Cancers via the Positive Feedback Loop of IRE1α/IL-6/AR.

Castration-resistant prostate cancer (CRPC) is the lethal form of prostate cancer (PCa), and the underlying molecular mechanism has not been fully elucidated. Inositol requiring enzyme 1 alpha (IRE1α), a key regulator of unfolded protein response (UPR), is intimately associated with PCa progression. However, whether IRE1α is implicated in CRPC development remains unknown. Here, we showed that IRE1α expression was significantly increased in CRPC tissues and high-grade PCa tissues. Overexpression of IRE1α promoted PCa cell proliferation under the androgen deficiency condition in vitro and in vivo. Mechanistically, increased IRE1α expression induced IL-6 secretion via the IRE1α/XBP-1s signal pathway. IRE1α-induced IL-6 activated androgen receptor (AR), and the activation of AR by IL-6, in turn, promoted IRE1α expression. IRE1α formed a positive feedback loop with IL-6 and AR to promote prostate cancer cell proliferation under the androgen-deficient condition. In clinical PCa samples, high IRE1α expression correlated with elevated IL-6 and increased PSA expression. Our findings demonstrated a novel mechanism of CRPC progression and suggest targeting IRE1α may be a potential target for the prevention and treatment of CRPC.

Functional mapping of androgen receptor enhancer activity

BackgroundAndrogen receptor (AR) is critical to the initiation, growth, and progression of prostate cancer. Once activated, the AR binds to cis-regulatory enhancer elements on DNA that drive gene expression. Yet, there are 10–100× more binding sites than differentially expressed genes. It is unclear how or if these excess binding sites impact gene transcription.ResultsTo characterize the regulatory logic of AR-mediated transcription, we generated a locus-specific map of enhancer activity by functionally testing all common clinical AR binding sites with Self-Transcribing Active Regulatory Regions sequencing (STARRseq). Only 7% of AR binding sites displayed androgen-dependent enhancer activity. Instead, the vast majority of AR binding sites were either inactive or constitutively active enhancers. These annotations strongly correlated with enhancer-associated features of both in vitro cell lines and clinical prostate cancer samples. Evaluating the effect of each enhancer class on transcription, we found that AR-regulated enhancers frequently interact with promoters and form central chromosomal loops that are required for transcription. Somatic mutations of these critical AR-regulated enhancers often impact enhancer activity.ConclusionsUsing a functional map of AR enhancer activity, we demonstrated that AR-regulated enhancers act as a regulatory hub that increases interactions with other AR binding sites and gene promoters.

Prostate Cancer Stimulation by a Novel Liver X Receptor (LXRa)-Estrogen Related Receptor (ERRa) Axis

Background: Liver X Receptors (LXRα, β) are oxysterol sensing nuclear receptors that regulate lipogenesis, cholesterol homeostasis and immune cell function. While oxysterols are agonist ligands of LXRs, sulfated oxysterols, catalytically produced by the SULT2B1b sulfotransferase, are LXR-inert. Increased SULT2B1b expression leads to attenuation of LXR signaling. In a previous report, we showed that SULT2B1b is undetectable in clinical samples of castration-resistant prostate cancer (CRPC), and its level is significantly reduced in a subset of primary prostate cancer. In cell models, genetic ablation of SULT2B1b exacerbated aggressive traits of CRPC, evident from EMT-like activation, enhanced invasion, faster xenograft tumor growth and reduced cell adhesiveness and stiffness in single-cell atomic force microscopy analysis. AKR1C3, which promotes androgen biosynthesis and shows elevated expression in advanced prostate cancer, is markedly upregulated in SULT2B-depleted cells. Elevated AKR1C3 leads to activation of the ERK1/2 Map kinase survival signal in CRPC cells. Results: We report here that AKR1C3 upregulation is a consequence of enhanced LXRα signaling in SULT2B1b-deficient cells, since the upregulation was abolished in multiple cell models when LXRα was silenced by siRNAs or inactivated by the small molecule inhibitor SR9243, which is an LXRs-selective inverse agonist. Conversely, LXR agonism induced by an oxysterol, or by the synthetic ligand GW3965, resulted in elevated AKR1C3 expression. Consistent with a recent report that the nuclear receptor ERRα is a positive regulator of AKR1C3, we found that ERRα ablation prevented AKR1C3 upregulation in SULT2B1b-deficient cells. Notably, LXRα inactivation caused marked reduction of ERRα, indicating that ERRα functions downstream of LXRα to induce AKR1C3 and ERK1/2. Dependence of ERRα and AKR1C3 expression on LXRα was observed in both androgen receptor (AR)-positive and AR-negative CRPC cells. Elevated ERRα in prostate cancer is known to be associated with a poor disease outcome. This association may be in part due to ERRα activation by cholesterol, which is the endogenous agonist ligand for ERRα (Cell Metab 23: 479, 2016), and high cholesterol is a risk factor for aggressive prostate cancer. Furthermore, statins, which inhibit cholesterol biosynthesis, are beneficial to CRPC patients with elevated blood cholesterol. We identified two cholesterol-responsive ERRα-binding sites in the far upstream region of the AKR1C3 promoter. This result confirms that ERRα plays a direct role in the transcriptional upregulation of AKR1C3. Significance: Our study establishes a novel LXRα→ERRα→AKR1C3→ERK1/2 survival axis that is activated in CRPC cells under SULT2B1b deficiency. The LXRα→ERRα regulatory axis may be exploited for developing novel therapeutics against AR-positive and AR-negative CRPC.

Acetylation of interferon regulatory factor-5 suppresses androgen receptor and downregulates expression of Sox2.

Interferon regulatory factor-5 (IRF5) is a transcription factor and has essential cellular mechanisms as a tumour suppressor gene. IRF5 protein function is irregular in various human cancers, and its role in prostate cancer is also unknown. This study presents the first evidence that IRF5 expression is controlled with androgen receptor (AR) signalling interaction and stem cell factors (Nanog, Oct4, Sox2) in prostate cancer. Human prostate cancer cell lines (PC3, DU145 and LNCaP) were transfected plasmids and assessed for cellular localization of IRF5 and AR interaction with IF-staining. Co-immunoprecipitation and ChIP assay were used to detect the IRF5 and AR protein-protein interaction and IRF5 stem cell factors protein-gene interaction. The target relation between IRF5, AR, CREB, p300, ISRE, ARE and NF-кB was tested by luciferase assay. IRF5 was low expressed in androgen-dependent prostate cancer cells and tissues. The analysis of human prostate cancer clinical samples supports the interaction of IRF5 and AR in a pathological role, as IRF5 expression is down-regulated in the tumours' advanced stages. Tumour suppression mechanism of IRF5 and SOX2 levels in cells reduces and causes AR acetylation. Those affect the prostate cancer mechanism by modifying the cellular response in the signal pathway. IRF5 can be promising for treating androgen-dependent prostate cancers and is a therapeutic protein for new drug studies.

MiRNA-145-5p expression and prospective molecular mechanisms in the metastasis of prostate cancer.

The clinicopathological implication and prospective molecular mechanisms of miRNA‐145‐5p in the metastasis of prostate cancer (PCa) stand unclear. Herein, it is found that miRNA‐145‐5p expression was remarkably reduced in 131 cases of metastatic PCa than 1371 cases of localised ones, as the standardised mean differences (SMD) was −1.26 and the area under the curve (AUC) was 0.86, based on miRNA‐chip and miRNA‐sequencing datasets. The potential targets of miRNA‐145‐5p in metastatic PCa (n = 414) was achieved from the intersection of miRNA‐145‐5p transfected metastatic PCa cell line data, differential expression of metastatic PCa upregulated genes and online prediction databases. TOP2A was screened as one of the target hub genes by PPI network analysis, which was adversely related to miRNA‐145‐5p expression in both metastatic PCa (r = −0.504) and primary PCa (r = −0.281). Gene‐chip and RNA‐sequencing datasets, as well as IHC performed on clinical PCa samples, showed consistent upregulated expression of TOP2A mRNA and protein in PCa compared with non‐PCa. The expression of TOP2A mRNA was also significantly higher in metastatic than localised PCa with the SMD being 1.72 and the AUC of sROC being 0.91. In summary, miRNA‐145‐5p may participate in PCa metastasis by binding TOP2A and be useful as a biomarker for the detection of metastatic PCa.