Human Prostate Cancer Patients Research Articles

Abstract 1798: Glyoxalase 1 (GLO1) as an oncometabolic enabler of prostate cancer progression: CRISPR/Cas9-based GLO1-deletion from human DU145-Luc2 cells blocks EMT and bone metastasis in SCID mice

Abstract Glyoxalase 1 (encoded by GLO1) is a glutathione-dependent enzyme detoxifying the glycolytic byproduct methylglyoxal (MG), an oncometabolite involved in metabolic reprogramming. Recently, we have demonstrated that GLO1 is overexpressed in human prostate cancer cells and patient tumors. In order to inform the ongoing debate on the role of GLO1 as an oncometabolic enabler of tumor glucose metabolism and malignant progression, we performed CRISPR/Cas9-based GLO1-deletion in human DU145-Luc2 malignant prostate cancer cells. NanoString nCounterTM ('PanCancer-Progression-Panel') comparative gene expression profiling (GLO1_KO versus GLO1_wt) revealed a significant downregulation of EMT-related pathways in GLO1_KO cells; concordantly, phenotypical screening indicated a pronounced attenuation of matrigel invasiveness observable in GLO1_KO cells. Likewise, inclusion of MG or a small molecule GLO1 inhibitor (TLSC-702) blocked invasiveness of DU145 GLO1_wt cells. Downregulation of EMT-related genes (including MMP3, SPP1, CXCL8) was accompanied by increased expression of TXNIP (thioredoxin-interacting protein), a master regulator of cellular energy metabolism and redox homeostasis. In a bioluminescent SCID mouse bone metastasis model (intracardial injection of DU145-Luc2 GLO1_wt and GLO1_KO cells), GLO1 expression was necessary to cause mandibular bone metastases (as evidenced by the complete absence of bone metastases after GLO1 deletion). Given the availability of drug-like small molecule inhibitors of GLO1 enzymatic activity these data suggest that GLO1 represents a novel molecular target for the pharmacological suppression of prostate cancer bone metastasis. Citation Format: Jana Jandova, Anne E. Cress, Georg T. Wondrak. Glyoxalase 1 (GLO1) as an oncometabolic enabler of prostate cancer progression: CRISPR/Cas9-based GLO1-deletion from human DU145-Luc2 cells blocks EMT and bone metastasis in SCID mice [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 1798.

Hybridized Chain Reaction-Amplified Alkaline Phosphatase-Induced Ag-Shell Nanostructure for the Sensitive and Rapid Surface-Enhanced Raman Scattering Immunoassay of Exosomes.

Exosomes are small extracellular vesicles that can be utilized as noninvasive biomarkers for diagnosis and treatment of cancer and other diseases. This study reports on a strategy involving a hybridized chain reaction-amplified chain coupled with an alkaline phosphatase-induced Ag-shell nanostructure for the ultrasensitive and rapid surface-enhanced Raman scattering immunoassay of exosomes. Exosomes from prostate cancer were captured using prostate-specific membrane antigen aptamer-modified magnetic beads; then, the hybridized chain reaction-amplified chain was released, incorporating a large number of functional moieties with signal amplification effects. Moreover, the steps of traditional immunoassay were simplified using magnetic materials, and the rapid, sensitive, and accurate detection of exosomes was achieved. Results could be obtained within 40 min with a detection limit of 19 particles/μL. Furthermore, the sera of human prostate cancer patients could be easily distinguished from those of healthy controls, highlighting the potential use of exosome analysis in clinical diagnostics.

Abstract 1452: Big data analysis revealed signalling activity and key regulators in human prostate cancer cell lines

Abstract Background: Human prostate cancer (PCa) cell lines are the most used models for research. The commonly used PCa cell lines can be roughly categorized into 3 groups: androgen receptor (AR)+, neuroendocrine (NE)+ and double negative (AR- NE-) cell lines. However, although these cell lines have been extensively used in PCa research since their establishment decades ago, a global molecular characterization of these cell lines is still lacking, especially in the aspect of cell signaling activity and their clinical relevance, which requires a large scale of data mining. Methods: The RNA-seq, DNA-seq and proteomic datasets were gathered from public cohort and our lab’s collection. From these data, a PCa cell line multi-omics database (PCMD) was established. Single sample gene set enrichment analysis (ssGSEA) was used to reveal the signaling pathways enriched in each PCa cell line. Additionally, human PCa patient single cell RNAseq data-based deconvolution and human PCa patient bulk RNAseq data-based nearest-neighbor (NN) graph analysis were used to evaluate the clinical relevance of each PCa cell line. The DNA-seq, ATAC-seq, DNase HS-seq and Bisulfite-seq datasets were used to annotate the key genes. Results: The PCMD database was established by compiling more than 1000 RNASeq datasets derived from various PCa cell lines. The important signaling pathways and transcription factors were revealed for each PCa cell line. Further, the cell lines were annotated with relative clinical stages. Additionally, an interactive webApp was established for scientists to explore and visualize PCMD data. Conclusion: our research characterized PCa cell lines using unbiased strategies and large sample number. It is our hope that this study would aid researchers to develop hypothesis and choose appropriate cell line to use. Funding: This study is supported by: Feist-Weiller Cancer Center, LSU Health Shreveport. Citation Format: Siyuan Cheng, Lin Li, Xiuping Yu. Big data analysis revealed signalling activity and key regulators in human prostate cancer cell lines [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 1452.

Abstract NG01: Dendritic cell intrinsic androgen receptor signaling reduces dendritic cell function and anti-tumor immunity

Abstract NG01: Dendritic cell intrinsic androgen receptor signaling reduces dendritic cell function and anti-tumor immunity

MP20-07 NPTX1 IS A NOVEL SERUM BIOMARKER INDUCED BY ARPI TREATED PROSTATE CANCER AND ENRICHED IN LINEAGE PLASTICITY WITH BRN2 HIGH

You have accessJournal of UrologyCME1 Apr 2023MP20-07 NPTX1 IS A NOVEL SERUM BIOMARKER INDUCED BY ARPI TREATED PROSTATE CANCER AND ENRICHED IN LINEAGE PLASTICITY WITH BRN2 HIGH Takeshi Namekawa, Joshua Scurll, Maxim Kobelev, Dwaipayan Ganguli, Olena Sivak, Martin Gleave, Vancouver, Canada, Eva Corey, Colm Morrissey, and Amina Zoubeidi Takeshi NamekawaTakeshi Namekawa More articles by this author , Joshua ScurllJoshua Scurll More articles by this author , Maxim KobelevMaxim Kobelev More articles by this author , Dwaipayan GanguliDwaipayan Ganguli More articles by this author , Olena SivakOlena Sivak More articles by this author , Martin GleaveMartin Gleave More articles by this author , Vancouver Vancouver More articles by this author , Canada Canada More articles by this author , Eva CoreyEva Corey More articles by this author , Colm MorrisseyColm Morrissey More articles by this author , and Amina ZoubeidiAmina Zoubeidi More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003245.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Androgen receptor (AR) pathway inhibition is the first-line therapy for metastatic prostate cancer (PCa). However, tumors invariably develop resistance to ARPI. We previously identified a transcription factor Brain-2 (BRN2), encoded by the gene POU3F2, as a master driver of NEPC which is an aggressive form of AR-independent phenotypes treatment resistant PCa. In this study, we identified a surrogate biomarker of BRN2 activity to monitor the progression of treatment resistant PCa. METHODS: To identify BRN2 serum marker, we analyzed BRN2 ChIPseq data from 2 NEPC and 2 neural-type lung squamous cell carcinoma (nLUSC) cell lines with a focus on shared BRN2 peaks. Annotated peaks from enhancers (EnhancerAtlas) and promoters (genes within 20kb) were selected for secreted proteins and were ranked by correlation with BRN2 expression in PCa patients. Validation of protein expression was performed by western blot. R-PLEX Assay kit was used to measure concentrations in culture media from NCI-H660 cells and in serum from mice with NCI-H660 xenograft or PDX tumors and from human PCa patients. RESULTS: We identified 51 candidate serum biomarkers for BRN2 activity (or expression?) using the methods described above. These biomarkers were ranked based on their expression and correlation with BRN2 expression in PCa patient samples across four public data sets. NPTX1 was identified as the top biomarker of BRN2 expression. We further validated association of NPTX1 and BRN2 expression by BRN2 overexpression in MR42D and 22Rv1, indicating that BRN2 positively regulates NPTX1 expression. Next, we showed that NPTX1 is present in culture media from NCI-H660 cells, and that NPTX1 serum concentration positively correlated with tumor volume in mice with NCI-H660 xenografts and with NPTX1 mRNA expression in LuCaP PDX models. Importantly, evaluation of patients’ samples showed that NPTX1 levels were high in 26/35 CRPC serum samples vs levels in serum of healthy volunteers, and that the NPTX1 serum levels were highest in 4/6 patients with high POU3F2 tumor expression. CONCLUSIONS: We identified and validated NPTX1 as a serum marker that can be readily detected in serum from NEPC patients with BRN2high tumours. Source of Funding: U.S. Army Medical Research and Development Command(W81XWH1810689) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e277 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takeshi Namekawa More articles by this author Joshua Scurll More articles by this author Maxim Kobelev More articles by this author Dwaipayan Ganguli More articles by this author Olena Sivak More articles by this author Martin Gleave More articles by this author Vancouver More articles by this author Canada More articles by this author Eva Corey More articles by this author Colm Morrissey More articles by this author Amina Zoubeidi More articles by this author Expand All Advertisement PDF downloadLoading ...

Prostate tumor RON receptor signaling mediates macrophage recruitment to drive androgen deprivation therapy resistance through Gas6-mediated Axl and RON signaling.

Androgen deprivation therapy (ADT), or chemical castration, is the first-line therapy for prostate cancer; however, resistance leaves few treatment options. Prostatic tumor-associated macrophages (TAMs) have been shown to promote prostate cancer growth and are abundant in castration-resistant prostate cancer (CRPC), suggesting a role in promoting CRPC. We recently showed a tumor cell-intrinsic mechanism by which RON promotes CRPC. Given previous reports that RON alters prostate cancer cell chemokine productionand RON-overexpressing tumors alter macrophage function, we hypothesized that a macrophage-dependent mechanism regulated by tumor cell intrinsic RON also promotes CRPC. Using RON-modulated genetically engineered mouse models (GEMMs) and GEMM-derived cell lines and co-cultures with bone marrow-derived macrophages, we show functional and molecular characteristics of signaling pathways in supporting CRPC. Further, we used an unbiased phosphokinase array to identify pathway interactions regulated by RON. Finally, using human prostate cancer cell lines and prostate cancer patient data sets, we show the relevance of our findings to human prostate cancer. Studies herein show that macrophages recruited into the prostate tumor microenvironment (TME) serve as a source for Gas6 secretion which serves to further enhance RON and Axl receptor activation in prostate tumor cells thereby driving CRPC. Further, we show targeting RON and macrophages in a murine model promotes CRPC sensitization to ADT. We discovered a novel role for the RON receptor in prostate cancer cells in promoting CRPC through the recruitment of macrophages into the prostate TME. Macrophage-targeting agents in combination with RON/Axl inhibition are likely to provide clinical benefits for patients with CRPC.

βArrestin1 regulates glucocorticoid receptor mitogenic signaling in castration-resistant prostate cancer.

Prostate cancer (PC) is the most commonly diagnosed malignancy and the second leading cause of cancer-related deaths in males. The disease is initially treated with methods that inhibit androgen receptor (AR) signal transduction. Laboratory-based and clinical studies have identified alternative pathways that cause the failure of AR signal inhibition and consequent development of castration-resistant prostate cancer (CRPC). Glucocorticoid receptor (GR) signaling is activated in certain PC patients and promotes the emergence of CRPC, althoughby as yet incompletely understood mechanisms. We have previously demonstrated that ubiquitous βarrestin1 (βArr1) expression levels are linked to PC progression. Here, we consider the possibility that βArr1 interacts with and activates GR in model CRPC cells. Bioinformatic analysis of tumor xenograft and human PC datasets was used to correlate the expression of βArr1 and GR. Western blot, immunohistochemistryand immunofluorescence microscopy, and subcellular fractionation were used to determine protein expression level and localization. Immunoprecipitation was applied to detect protein-protein interactions. RNA expression levels were determined using quantitative reverse transcription-polymerase chain reaction. Prostate sphere analysis was used to assess the rate of growth and invasion. The xenograft tumor implantation method was used to determine the tumor growth rate, local invasion, and metastasis. Elevated expression of βArr1 positively correlated with increased GR expression and function in CRPC xenograft and in human PC patients. βArr1 is expressed in the cell cytosol and nucleus, and it formed a complex with GR in the nucleus and not cytosol. Depletion of βArr1 in AR-null CRPC cells inhibited GR function and CRPC growth and invasion in both in vitro and in vivo settings. βArr1 binds GR that initiates mitogenic signaling cascades involved in the progression of PC to CRPC. The targeting of the βArr1-GR axis may provide a new opportunity to better manage the CRPC disease.

HRM method for identification of TP53 exon 5 and 8 mutations in human prostate cancer patients

BackgroundThe purpose of the present study was to perform a high-resolution melting (HRM) analysis to discover mutations in gene exons 5–8 of tumor protein p53 (TP53), as well as the relationships of these mutations to clinical parameters in prostate cancer (PC). MethodsGenomic DNA was extracted from 50 formalin-fixed paraffin-embedded (FFPE) tissues with PC. Mutations in exons 5 and 8 of TP53 were analyzed using the HRM method. Sanger sequencing was used to describe mutations. ResultsAccording to the HRM analysis results, 21 (42%) PC samples had different normalized and shifted melting curves from other samples. Mutations in TP53 exons 5 and8 were observed in 12 (24%) patients by the Sanger method. The detection sensitivity of the HRM method in exon 5 and exon 8 mutations was 66.7% and 50%, respectively. PSA levels of PC patients with TP53 mutation were found to be lower than that of patients with no mutation (p = 0.8270). However, we did not find any correlations between TP53 mutations and clinical parameters (p > 0.05). ConclusionsHRM analysis is a simple, rapid, and efficient mutation-scanning method for known/unknown mutations in TP53 exons 5and8, as well as an attractive method for detection of mutations and their analysis in FFPE tissues. Additional studies with larger patient populations are warranted to confirm the correlation between the TP53 mutations and PC risk.

Elevated expression of the colony-stimulating factor 1 (CSF1) induces prostatic intraepithelial neoplasia dependent of epithelial-Gp130.

Macrophages are increased in human benign prostatic hyperplasia and prostate cancer. We generate a Pb-Csf1 mouse model with prostate-specific overexpression of macrophage colony-stimulating factor (M-Csf/Csf1). Csf1 overexpression promotes immune cell infiltration into the prostate, modulates the macrophage polarity in a lobe-specific manner, and induces senescence and low-grade prostatic intraepithelial neoplasia (PIN). The Pb-Csf1 prostate luminal cells exhibit increased stem cell features and epithelial-to-mesenchymal transition. Human prostate cancer patients with high CSF-1 expression display similar transcriptional alterations with the Pb-Csf1 model. P53 knockout alleviates senescence but fails to progress PIN lesions. Ablating epithelial Gp130 but not Il1r1 substantially blocks PIN lesion formation. The androgen receptor (AR) is downregulated in Pb-Csf1 mice. ChIP-Seq analysis reveals altered AR binding in 2482 genes although there is no significant widespread change in global AR transcriptional activity. Collectively, our study demonstrates that increased macrophage infiltration causes PIN formation but fails to transform prostate cells.

The roles of Y-box-binding protein (YB)-1 and C-X-C motif chemokine ligand 14 (CXCL14) in the progression of prostate cancer via extracellular-signal-regulated kinase (ERK) signaling

ABSTRACT The cold-shock protein Y-box-binding protein (YB)-1 regulates the expression of various chemokines and their receptors at the transcriptional level. Expression of the orphan chemokine CXCL14 is repressed by EGF induced signaling. The possible links between EGF-mediated YB-1 and CXCL14 as well as the functions of critical kinase pathways in the progression of prostate cancer have remained unexplored. Here we examined the correlation between YB-1 and CXCL14, and the ERK/AKT/mTOR pathways in prostate cancer. Knockdown of YB-1 decreased cyclinD1 expression with an upregulation of cleaved-PARP in human prostate cancer cells. EGF treatment upregulated phospho-YB-1 expression in a time-dependent manner, while treatment with an ERK inhibitor completely silenced its expression in prostate cancer cells. EGF treatment stimulates CyclinD1 and YB-1 phosphorylation in an ERK-dependent pathway. Positive and negative regulation of YB-1 and CXCL14 was observed after EGF treatment in prostate cancer cells, respectively. EGF rescues cell cycle and apoptosis via the AKT and ERK pathways. Furthermore, YB-1 silencing induces G1 arrest and apoptosis, while knockdown of CXCL14 facilitates cell growth and inhibits apoptosis in prostate cancer cells. YB-1 and CXCL14 were inversely correlated in prostate cancer cells and tissues. A significant association between poor overall survival and High YB-1 expression was observed in human prostate cancer patients. In conclusion, our data reveal the functional relationship between YB-1 and CXCL14 in EGF mediated ERK signaling, and YB-1 expression is a significant prognostic marker to predict prostate cancer.

Alternative splicing of NF-YA promotes prostate cancer aggressiveness and represents a new molecular marker for clinical stratification of patients

BackgroundApproaches based on expression signatures of prostate cancer (PCa) have been proposed to predict patient outcomes and response to treatments. The transcription factor NF-Y participates to the progression from benign epithelium to both localized and metastatic PCa and is associated with aggressive transcriptional profile. The gene encoding for NF-YA, the DNA-binding subunit of NF-Y, produces two alternatively spliced transcripts, NF-YAs and NF-YAl. Bioinformatic analyses pointed at NF-YA splicing as a key transcriptional signature to discriminate between different tumor molecular subtypes. In this study, we aimed to determine the pathophysiological role of NF-YA splice variants in PCa and their association with aggressive subtypes.MethodsData on the expression of NF-YA isoforms were extracted from the TCGA (The Cancer Genome Atlas) database of tumor prostate tissues and validated in prostate cell lines. Lentiviral transduction and CRISPR-Cas9 technology allowed the modulation of the expression of NF-YA splice variants in PCa cells. We characterized 3D cell cultures through in vitro assays and RNA-seq profilings. We used the rank-rank hypergeometric overlap approach to identify concordant/discordant gene expression signatures of NF-YAs/NF-YAl-overexpressing cells and human PCa patients. We performed in vivo studies in SHO-SCID mice to determine pathological and molecular phenotypes of NF-YAs/NF-YAl xenograft tumors.ResultsNF-YA depletion affects the tumorigenic potential of PCa cells in vitro and in vivo. Elevated NF-YAs levels are associated to aggressive PCa specimens, defined by Gleason Score and TNM classification. NF-YAl overexpression increases cell motility, while NF-YAs enhances cell proliferation in PCa 3D spheroids and xenograft tumors. The transcriptome of NF-YAs-spheroids has an extensive overlap with localized and metastatic human PCa signatures. According to PCa PAM50 classification, NF-YAs transcript levels are higher in LumB, characterized by poor prognosis compared to LumA and basal subtypes. A significant decrease in NF-YAs/NF-YAl ratio distinguishes PCa circulating tumor cells from cancer cells in metastatic sites, consistently with pro-migratory function of NF-YAl. Stratification of patients based on NF-YAs expression is predictive of clinical outcome.ConclusionsAltogether, our results indicate that the modulation of NF-YA isoforms affects prostate pathophysiological processes and contributes to cancer-relevant phenotype, in vitro and in vivo. Evaluation of NF-YA splicing may represent a new molecular strategy for risk assessment of PCa patients.

A Novel Splice Variant of the Inhibitor of Growth 3 Lacks the Plant Homeodomain and Regulates Epithelial-Mesenchymal Transition in Prostate Cancer Cells.

Inhibitor of growth 3 (ING3) is one of five members of the ING tumour suppressor family, characterized by a highly conserved plant homeodomain (PHD) as a reader of the histone mark H3K4me3. ING3 was reported to act as a tumour suppressor in many different cancer types to regulate apoptosis. On the other hand, ING3 levels positively correlate with poor survival prognosis of prostate cancer (PCa) patients. In PCa cells, ING3 acts rather as an androgen receptor (AR) co-activator and harbours oncogenic properties in PCa. Here, we show the identification of a novel ING3 splice variant in both the human PCa cell line LNCaP and in human PCa patient specimen. The novel ING3 splice variant lacks exon 11, ING3∆ex11, which results in deletion of the PHD, providing a unique opportunity to analyse functionally the PHD of ING3 by a natural splice variant. Functionally, overexpression of ING3Δex11 induced morphological changes of LNCaP-derived 3D spheroids with generation of lumen and pore-like structures within spheroids. Since these structures are an indicator of epithelial–mesenchymal transition (EMT), key regulatory factors and markers for EMT were analysed. The data suggest that in contrast to ING3, ING3Δex11 specifically modulates the expression of key EMT-regulating upstream transcription factors and induces the expression of EMT markers, indicating that the PHD of ING3 inhibits EMT. In line with this, ING3 knockdown also induced the expression of EMT markers, confirming the impact of ING3 on EMT regulation. Further, ING3 knockdown induced cellular senescence via a pathway leading to cell cycle arrest, indicating an oncogenic role for ING3 in PCa. Thus, the data suggest that the ING3Δex11 splice variant lacking functional PHD exhibits oncogenic characteristics through triggering EMT in PCa cells.

DNA methylome, transcriptome, and prostate cancer prevention by phenethyl isothiocyanate in TRAMP mice.

Epigenetics/epigenomics has been shown to be involved in carcinogenesis. However, how the epigenome would be altered in the transgenic adenocarcinoma of the mouse prostate (TRAMP) cancer model and the effect of cancer chemopreventive phytochemical phenethyl isothiocyanate (PEITC) on the epigenome in TRAMP mice are not known. PEITC has been reported to reduce the risk of many cancers including prostate cancer (PCa). In this study, male TRAMP mice were fed a control diet or diet containing 0.05% PEITC from 8 weeks to 16 weeks. The tumor incidence was reduced in the PEITC diet (0/6) as compared with the control diet (6/7). RNA-sequencing (RNA-seq) analyses on nontumor and tumor prostatic tissues revealed several pathways like cell cycle/Cdc42 signaling, inflammation, and cancer-related signaling, were activated in prostate tissues of TRAMP mice but were reversed or attenuated in TRAMP mice fed with PEITC diet. DNA CpG methyl-seq analyses showed that global methylation patterns of prostate samples from TRAMP mice were hugely different from those of wild-type mice. Dietary PEITC partially reversed the global methylation changes during prostatic carcinogenesis. Integration of RNA-seq and DNA methyl-seq analyses identified a list of genes, including Adgrb1 and Ebf4, with an inverse regulatory relationship between their RNA expression and CpG methylation. In summary, our current study demonstrates that alteration of the global epigenome in TRAMP prostate tumor and PEITC administration suppresses PCa carcinogenesis, impacts global CpG epigenome and transcriptome, and attenuates carcinogenic pathways like cell cycle arrest and inflammation. These results may provide insights and epigenetic markers/targets for PCa prevention and treatment in human PCa patients.

Neuropeptide Y nerve paracrine regulation of prostate cancer oncogenesis and therapy resistance.

BackgroundNerves are key factors in prostate cancer (PCa) progression. Here, we propose that neuropeptide Y (NPY) nerves are key regulators of cancer–nerve interaction.MethodsWe used in vitro models for NPY inhibition studies and subsequent metabolomics, apoptotic and migration assays, and nuclear transcription factor‐κB (NF‐κB) translocation studies. Human naïve and radiated PCa tissues were used for NPY nerve density biomarker studies. Tissues derived from a Botox denervation clinical trial were used to corroborate metabolomic changes in humans.ResultsCancer cells increase NPY positive nerves in vitro and in preneoplastic human tissues. NPY‐specific inhibition resulted in increased cancer apoptosis, decreased motility, and energetic metabolic pathway changes. A comparison of metabolomic response in NPY‐inhibited cells with the transcriptome response in human PCa patients treated with Botox showed shared 13 pathways, including the tricarboxylic acid cycle. We identified that NF‐κB is a potential NPY downstream mediator. Using in vitro models and tissues derived from a previous human chemical denervation study, we show that Botox specifically, but not exclusively, inhibits NPY in cancer. Quantification of NPY nerves is independently predictive of PCa‐specific death. Finally, NPY nerves might be involved in radiation therapy (RT) resistance, as radiation‐induced apoptosis is reduced when PCa cells are cocultured with dorsal root ganglia/nerves and NPY positive nerves are increased in prostates of patients that failed RT.ConclusionThese data suggest that targeting the NPY neural microenvironment may represent a therapeutic approach for the treatment of PCa and resistance through the regulation of multiple oncogenic mechanisms.

Abstract 4706: Transcriptional repression of mitochondrial deacetylase SIRT3 promotes prostate cancer metastasis

Abstract Prostate cancer (PCa) is the most common cancer and the second leading cause of cancer death among men in America. Aggressive prostate tumors that rapidly metastasize to other organs such as bones account for high morbidity and mortality. Metastatic PCa is resistant to androgen depletion therapy, and increased expression of androgen receptor (AR) coregulator Steroid Receptor Coregulator-2 (SRC-2) remains a major underlying driver of lethal PCa progression. Increased expression of SRC-2 positively correlates with PCa recurrence and poor disease outcome. SRC-2 directly binds to AR and regulates its transcriptional activity even in castrate-levels of androgen. Although the role of SRC-2 as a coactivator of AR is well established, we recently discovered that SRC-2 can also function as a co-repressor of AR on certain gene promoters such as mitochondrial deacetylase Sirtuin 3 (SIRT3). Analysis of human PCa databases revealed that expression of SIRT3 is reduced in prostate tumors as the disease progresses to metastatic PCa, and there exists an inverse correlation with SRC-2 expression. To confirm that SRC-2 is functioning as a co-repressor on SIRT3 promoter we performed chromatin immunoprecipitation (ChIP) and identified strong occupancy of SRC-2 on the SIRT3 promoter along with AR, whereas SRC-2 knockdown reduced AR enrichment. Consistently with this observation SRC-2 or AR knockdown significantly increased SIRT3 expression in PCa lines C4-2 and 22RV1, validating that SRC-2 functions as a transcriptional co-repressor of AR on SIRT3 promoter. This suggests that AR/SRC-2 can transcriptionally silence potential tumor suppressors. Next, we investigated the role of SIRT3 in PCa, and adenovirus-mediated ectopic expression of SIRT3 significantly impaired cell cycle progression arresting TRAMP-C2 and C4-2 cells in G0/G1 phase with reduced cell growth and viability, suggesting SIRT3 may function as a tumor suppressor in PCa. Since SRC-2 represses SIRT3 gene expression we analyzed mRNA expression of SRC-2 and SIRT3 in metastatic lesions from human prostate cancer patients using an advanced prostate cancer genomics dataset. We found that increased levels of SRC-2 with a concomitant reduction in SIRT3 expression is prevalent in metastatic lesions of PCa patients that spread to bones and lymph nodes. We used intra-iliac artery (IIA) injection to selectively deliver C4-2 cells expressing stable knockdown of SRC-2 (shSRC-2) or NTshRNA into the hind limb tissues. After 12 weeks we found SRC-2 ablation almost completely abolished PCa colonization in the bone whereas all animals transplanted with C4-2-NTshRNA developed bone metastasis. In conclusion, our study indicates that transcriptional repression of tumor suppressor SIRT3 by SRC2 may function as an oncogenic driver of lethal PCa progression to the bone. This work is supported by DoD grant: W81XWH-16-1-0297, NCI grant: K22CA207578 and Roswell Park Alliance Foundation. Citation Format: Mayrel Palestino Dominguez, Abhisha Sawant Dessai, Eriko Katsuta, Tao Dai, Subhamoy Dasgupta. Transcriptional repression of mitochondrial deacetylase SIRT3 promotes prostate cancer metastasis [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 4706.

Immune landscape of human prostate cancer: immune evasion mechanisms and biomarkers for personalized immunotherapy

BackgroundDespite recent advances in cancer immunotherapy, the efficacy of these therapies for the treatment of human prostate cancer patients is low due to the complex immune evasion mechanisms (IEMs) of prostate cancer and the lack of predictive biomarkers for patient responses.MethodsTo understand the IEMs in prostate cancer and apply such understanding to the design of personalized immunotherapies, we analyzed the RNA-seq data for prostate adenocarcinoma from The Cancer Genome Atlas (TCGA) using a combination of biclustering, differential expression analysis, immune cell typing, and machine learning methods.ResultsThe integrative analysis identified eight clusters with different IEM combinations and predictive biomarkers for each immune evasion cluster. Prostate tumors employ different combinations of IEMs. The majority of prostate cancer patients were identified with immunological ignorance (89.8%), upregulated cytotoxic T lymphocyte-associated protein 4 (CTLA4) (58.8%), and upregulated decoy receptor 3 (DcR3) (51.6%). Among patients with immunologic ignorance, 41.4% displayed upregulated DcR3 expression, 43.26% had upregulated CTLA4, and 11.4% had a combination of all three mechanisms. Since upregulated programmed cell death 1 (PD-1) and/or CTLA4 often co-occur with other IEMs, these results provide a plausible explanation for the failure of immune checkpoint inhibitor monotherapy for prostate cancer.ConclusionThese findings indicate that human prostate cancer specimens are mostly immunologically cold tumors that do not respond well to mono-immunotherapy. With such identified biomarkers, more precise treatment strategies can be developed to improve therapeutic efficacy through a greater understanding of a patient’s immune evasion mechanisms.

MP51-09 THE NOVEL ROLE OF SPOP IN REGULATING TOPOISOMERASE 2A IN PROSTATE CANCER CELLS AS A POTENTIAL THERAPEUTIC MARKER FOR DNA REPAIR TARGETED THERAPY

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II (MP51)1 Apr 2020MP51-09 THE NOVEL ROLE OF SPOP IN REGULATING TOPOISOMERASE 2A IN PROSTATE CANCER CELLS AS A POTENTIAL THERAPEUTIC MARKER FOR DNA REPAIR TARGETED THERAPY Ryuta Watanabe*, Masashi Maekawa, Miki Hieda, Tomohiko Taguchi, Noriyoshi Miura, Tadahiko Kikugawa, Shigeki Higashiyama, and Takashi Saika Ryuta Watanabe*Ryuta Watanabe* More articles by this author , Masashi MaekawaMasashi Maekawa More articles by this author , Miki HiedaMiki Hieda More articles by this author , Tomohiko TaguchiTomohiko Taguchi More articles by this author , Noriyoshi MiuraNoriyoshi Miura More articles by this author , Tadahiko KikugawaTadahiko Kikugawa More articles by this author , Shigeki HigashiyamaShigeki Higashiyama More articles by this author , and Takashi SaikaTakashi Saika More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000913.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: SPOP, speckle-type POZ protein, is a substrate recognizing receptor of the Cullin-3/RING ubiquitin E3 complex. Heterozygous point mutations in the substrate-binding domain of SPOP have been found in 10-15% of human prostate cancer patients. Although SPOP is essential for a DNA repair in exogenous DNA damage stresses, it still remains unclear whether SPOP is involved in the process of a DNA repair in endogenous DNA damage stresses. In this study, we focus on relationships the functions of SPOP, especially those involved in topoisomerase 1 (TOP1) and topoisomerase 2 (TOP2), to the DNA-protein crosslink repair from endogenous DNA damage in prostate cancer cell lines. METHODS: We first examined the level of γH2AX, a marker of DNA breaks in various prostate cancer cell lines; C4-2, LNCaP, PC3, DU145 upon SPOP knockdown (KD). To elucidate the site of action of SPOP during DNA repair, we treated control or SPOP-KD C4-2 cells with topoisomerase (TOP) inhibitors, which inhibit DNA repair. To investigate functions of SPOP in the regulation of TOP, we analyzed protein expression and cellular localization of TOPs by western blotting and immunofluorescence staining. We also measured the amounts of TOPs which covalently bound on DNA by cesium chloride-density gradient centrifugation. We finally detected the level of enzymes (TDP1, TDP2 and MRE11) which liberate TOPs from the TOP-DNA cleavage complex. RESULTS: We found that SPOP KD in androgen receptor (AR)-positive prostate cancer cells increased the amount of γH2AX. Because the level of γH2AX in SPOP-knockdown cells was not additive by treatment of TOP inhibitors, it was suggested that SPOP could act upstream of TOP1 and TOP2-dependent pathways. Immunofluorescence staining indicated that TOP2A, but not TOP1, was accumulated in the nuclei by SPOP knockdown. By cesium chloride-density gradient centrifugation, we found that SPOP KD increased the amount of TOP2A on genomic DNA. The amount of TDP1 and TDP2, which liberate TOP2A from the TOP2A-DNA complex was decreased by SPOP knockdown. We also confirmed that the increase of γH2AX in SPOP-KD C4-2 cells was dependent on both AR and TOP2A. CONCLUSIONS: Our results suggest that SPOP is involved in the DNA-protein crosslink repair process through the elimination of TOP2A from the TOP2A cleavage complex in prostate cancer cell lines. These results imply that TOP inhibitor might be effective to AR-positive, SPOP-wild type prostate cancer. Also, PARP inhibitor might be effective to SPOP-mutated prostate cancer. Source of Funding: JSPS KAKENHI Grant Number 19K18613 © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e767-e767 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ryuta Watanabe* More articles by this author Masashi Maekawa More articles by this author Miki Hieda More articles by this author Tomohiko Taguchi More articles by this author Noriyoshi Miura More articles by this author Tadahiko Kikugawa More articles by this author Shigeki Higashiyama More articles by this author Takashi Saika More articles by this author Expand All Advertisement PDF downloadLoading ...

Expression of TMBIM6 in Cancers: The Involvement of Sp1 and PKC.

Transmembrane Bax Inhibitor Motif-containing 6 (TMBIM6) is upregulated in several cancer types and involved in the metastasis. Specific downregulation of TMBIM6 results in cancer cell death. However, the TMBIM6 gene transcriptional regulation in normal and cancer cells is least studied. Here, we identified the core promoter region (−133/+30 bp) sufficient for promoter activity of TMBIM6 gene. Reporter gene expression with mutations at transcription factor binding sites, EMSA, supershift, and ChIP assays demonstrated that Sp1 is an essential transcription factor for basal promoter activity of TMBIM6. The TMBIM6 mRNA expression was increased with Sp1 levels in a concentration dependent manner. Ablation of Sp1 through siRNA or inhibition with mithramycin-A reduced the TMBIM6 mRNA expression. We also found that the protein kinase-C activation stimulates promoter activity and endogenous TMBIM6 mRNA by 2- to 2.5-fold. Additionally, overexpression of active mutants of PKCι, PKCε, and PKCδ increased TMBIM6 expression by enhancing nuclear translocation of Sp1. Immunohistochemistry analyses confirmed that the expression levels of PKCι, Sp1, and TMBIM6 were correlated with one another in samples from human breast, prostate, and liver cancer patients. Altogether, this study suggests the involvement of Sp1 in basal transcription and PKC in the enhanced expression of TMBIM6 in cancer.

Abstract 4523: A novel syngeneic mouse model of prostate cancer bone metastasis: Mechanisms of chemotaxis and bone colonization

Abstract Background: Bone metastasis in human prostate cancer remains a major clinical problem since no effective therapy exists. The RANKL/RANK pathway plays a predominant role in the interaction between metastasized prostate cancer cells and osteoclasts that increases the bone turnover. The current therapies, including targeting RANKL with denosumab, address the growth of prostate tumor cells that have already colonized the bone, but are largely ineffective in prolonging the survival of human prostate cancer patients with bone metastasis. Further, a major impediment to prostate cancer bone metastasis research is the lack of an animal model that spontaneously recapitulates human prostate cancer bone metastasis in the context of an intact immune system. Results: To overcome this major limitation, we have developed a novel syngeneic mouse model to study prostate cancer bone metastasis. Both the CXCL12/CXCR4 and RANKL/RANK pathways have been reported to be overexpressed / dysregulated in human prostate cancer bone metastatic samples. Data generated utilizing our immune-intact mouse model shows that the CXCL12/CXCR4 and RANKL/RANK pathways co-operate with each other to drive prostate cancer bone metastasis. Studies have shown that targeting the CXCL12/CXCR4 and RANKL/RANK pathways individually affects the immune system, thereby making our syngeneic mouse model an indispensable tool for studying the critical co-operation between these 2 pathways in the manifestation of human prostate cancer bone metastasis. Extending our earlier findings that RANKL drives PCa metastases in immune-deficient mice (Chu et al., 2014) to immune-intact C57/Bl6 mice, we found that MPC3 mouse PCa cells with RANKL overexpression (MPC3-Luc-GFP-RANKL) develop 70-80% limb and jaw within 4 weeks of intra-cardiac injection in these syngeneic mice. Control MPC3 cells had no bone metastasis. Bone lesions visualized by luciferase imaging and X-ray were confirmed by micro CT and immunohistochemistry. RANKL signaling drove bone and visceral metastases via the downstream CXCL12/CXCR4 signaling axis. MPC3-Luc-GFP-RANKL cells showed increased CXCR4 protein levels by immunohistochemistry. Metastatic bone marrow flush showed dramatically increased levels of CXCL12 mRNA compared with control mice (MPC3-Luc-GFP-EV). Conclusion: In sum, 1) circulating PCa cells induce a marked CXCL12 elevation after colonizing bone, triggering chemotaxis and recruiting CXCR4-positive PCa cells to migrate to bone; and 2) osteomimetic PCa cells with increased RANKL expression interact with osteoclasts to enhance bone resorption and turnover, releasing additional growth factors and chemokines for PCa cell growth and survival in bone. Citation Format: Srinivas Nandana, Murali Gururajan, Manisha Tripathi, Chia-Yi Chu, Haiyen Zhau, Stephen Shiao, Leland Chung. A novel syngeneic mouse model of prostate cancer bone metastasis: Mechanisms of chemotaxis and bone colonization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4523.

Abstract LB-267: Transcriptomic and epigenomic analysis of metastatic castration-resistant prostate cancer and a pan-cancer analysis of its genetic signatures

Abstract Prostate cancer (PCa) is the 2nd leading cause of non-cutaneous cancer related deaths in the United States. The 5-year survival rate for local or regional disease is high. However, many patient’s disease will progress to the aggressive metastatic castration-resistant form of prostate cancer (mCRPC) - the estimated mortality rates of mCRPC is 19.5% for 2020 (www.cancer.org). Treatment with docetaxel or cabazitaxel alone or in combination with bevacizumab, thalidomide and prednisone increases survival rate slightly, but lead to drug resistance. The use of immunotherapy has further increased overall survival by 4-5 months. Therefore, a greater understanding of the underlying molecular mechanisms responsible for disease progression is needed.To understand and identify appropriate targets for therapy, we performed mRNA and miRNA expression studies and identified potential candidate cancer pathway genes and miRNA-mRNA pairs as biomarkers for aggressive Prostate cancer (mCRPC). Gene expression signatures were identified using differential expression (DE) analysis softwares. Spearman’s rank-based correlation and TargetScan were used to assess the association between mRNA and miRNA expression. We identified disparate mRNA and miRNA expression signatures for aggressive vs non-aggressive prostate cancers. The gene signatures were validated using the human prostate cancer patient cohort from The Cancer Genome Atlas (TCGA) database. The top ten genes that were significantly (p<0.0001) associated with patient survival in PCa patients, stratified by Gleason scores, were PLAU, TGFB1, SERPINE1, MET, TIPM1, ITGA3, SERPINB5, PLAUR, CDKN1A and IGF1. Ingenuity Pathway Analysis (IPA) identified the activation of angiogenesis pathway as key factor for cancer aggressiveness. Epigenetic analysis showed distinct alterations in aggressive vs non-aggressive PCa. We also identified miRNA-mRNA pairs (including PLAU- mir181) which may influence differential gene expression in advanced-stage cancers. Furthermore, we performed a pan-cancer analysis of these top PCa survival-associated genes in other solid tumor types using patient expression profiles from the TCGA database. Our results identified significant (p<0.05) associations of the expression of the following genes with cancer survival: PLAU (testicular, skin, lung, pancreatic and brain); TGBF1 (bladder, cervical and testicular); SERPINE1 (bladder and cervical); TIPM1 (bladder); ITGA3 (endometrial and cervical); SERPINB5 (endometrial); PLAUR (cervical); CDKN1A (bladder); and IGF1 (ovarian). In conclusion, we used genomic, transcriptomic and epigenomic approaches to identify and validate genetic signatures that may be used to further understand the molecular basis of aggressive cancers (specifically mCRPC) and develop personalized treatments with the goal of improving overall survival. Citation Format: Taraswi Mitra Ghosh, Amit Kumar Mitra, Joshua Davis, Brian Cummings, Clayton Yates, Robert Arnold. Transcriptomic and epigenomic analysis of metastatic castration-resistant prostate cancer and a pan-cancer analysis of its genetic signatures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-267.