PCa Cell Lines Research Articles

Lncap-AI prostate cancer cell line establishment by Flutamide and androgen-free environment to promote cell adherent

BackgroundTo establish castration-resistant prostate cancer (CRPC) - Lncap androgen-independent (AI) cell line from Lncap androgen-dependent (AD) cell line, and explore the different molecular biological between these two cell lines.MethodsThe Lncap-AD cell line was cultured and passaged 60 times over 16 months. The morphology of the Lncap-AI cell line was observed. AR levels identification were detected in qRT-PCR and Western Blot assay. CCK-8, EdU assay, wound healing assay and cell adhesion assays were used to observe the ability of proliferation, migration, and adhesion. SEM and TEM were used to observe microculture structure. At last, the PSA secrete ability was evaluated by Elisa assay.ResultsThe Lncap-AD cell line was cultured and passaged 60 times over 16 months. The Lncap-AI cell line showed a morphologic change at the end stage of culture, the cells turned slender and cell space turned separated compared to the Lncap-AD cell line. The relative levels of AR-related genes in the Lncap-AI cell line were up-regulation compared to the Lncap-AD cell line both in mRNA and protein levels. The expression of AR and HK2 proteins were influenced and down-regulation by Enzalutamide in the Lncap-AD cell line, but no obvious difference in Lncap-AI cell lines. Lncap-AI cell line showed strong viability of proliferation, migration, and adhesion by CCK-8, EdU assay, wound healing assay, and adhesion assay. The microstructure of Scanning Electron Microscopy (SEM) showed many synapses in the Lncap-AI cell line and PC3 cell line, but not in the Lncap-AD cell line. At last, the PSA secrete ability was evaluated by Elisa assay, and PCa cell lines showed no significant difference.ConclusionSimulation of CRPC progression, Lncap-AD cell line turned to Lncap-AI cell line with androgen deprivation therapy.

Targeting Castration-Resistant Prostate Cancer Using Mesenchymal Stem Cell Exosomes for Therapeutic MicroRNA-let-7c Delivery.

Castration-resistant prostate cancer (PCa; CRPC) has a poor response to androgen deprivation therapy and is considered an incurable disease. MicroRNA (miR)-lethal 7c (let-7c) was implied to be a tumor suppressor in PCa, and treatment with exogenous let-7c targets both cancer cells and their associated mesenchymal stem cells (MSCs) to prevent CRPC progression and metastasis. Exosomes are nanometer-sized membrane-bound vesicles which have an absolute predominance in biocompatibility for drug delivery and gene therapy by mediating cell-to-cell communication. By utilizing the intrinsic tumor-targeting property of MSCs, this study aimed to investigate the feasibility of MSC-derived exosomes as an exogenous miR delivery system to target CRPC, using miR let-7c as an example. Bioinformatics analysis was performed to observe miR-let-7c expression in clinical samples by utilizing the GEO database. MSC-derived exosomes were collected from a human bone marrow-derived MSC cell line after cell transfection with either a pre-miR negative control or pre-miR-let-7c, and further characterized through nanoparticle tracking analysis and Western blotting. miR-let-7c expression was determined using RT-qPCR, and the phenotypic effects of both naked and MSC-exosome-encapsulated let-7c on CRPC cells (PC3 and CWR22Rv1) were determined by WST-1 cell proliferation assay and wound healing migration assay. miR-let-7c was downregulated in metastatic PCa and high grade group patients. miR-let-7c expression was confirmed to be downregulated in PCa cell lines, with massively decreased in most metastatic CRPC-like cells. Exogenous miR-let-7c can be successfully packaged into MSC exosomes. Treatment with either naked or MSC-exosome-encapsulated miR-let-7c resulted in significant reductions in cell proliferation and migration in CRPC-like PC3 and CWR22Rv1 cells. MSC-derived exosomes could serve as a therapeutic let-7c delivery system to target CRPC.

Bone morphogenetic protein pathway responses and alterations of osteogenesis in metastatic prostate cancers.

Prostate cancer is a common cancer in men that annually results in more than 33 000 US deaths. Mortality from prostate cancer is largely from metastatic disease, reflecting on the great strides in the last century of treatments in care for the localized disease. Metastatic castrate resistant prostate cancer (mCRPC) will commonly travel to the bone, creating unique bone pathology that requires nuanced treatments in those sites with surgical, radio and chemotherapeutic interventions. The bone morphogenetic protein (BMP) pathway has been historically studied in the capacity to regulate the osteogenic nature of new bone. New mineralized bone generation is a frequent and common observation in mCRPC and referred to as blastic bone lesions. Less common are bone destructive lesions that are termed lytic. We queried the cancer genome atlas (TCGA) prostate cancer databases for the expression of the BMP pathway and found that distinct gene expression of the ligands, soluble antagonists, receptors, and intracellular mediators were altered in localized versus metastatic disease. Human prostate cancer cell lines have an innate ability to promote blastic- or lytic-like bone lesions and we hypothesized that inhibiting BMP signaling in these cell lines would result in a distinct change in osteogenesis gene expression with BMP inhibition. We found unique and common changes by comparing these cell lines response and unique BMP pathway alterations. We treated human PCa cell lines with distinct bone pathologic phenotypes with the BMP inhibitor DMH1 and found distinct osteogenesis responses. We analyzed distinct sites of metastatic PCa in the TCGA and found that BMP signaling was selectively altered in commons sites such as lymph node, bone and liver compared to primary tumors. Overall we conclude that BMPs in metastatic prostate cancer are important signals and functional mediators of diverse processes that have potential for individualized precision oncology in mCRPC.

A novel prognostic model based on three clinic-related miRNAs for prostate cancer.

BackgroundProstate cancer (PCa) is the second most common malignant tumor in men worldwide. MiRNAs have been reported to play significant roles in prognosis prediction for patients with malignant tumors.MethodsThe survival-related miRNAs (sDMIRs) were identified by Cox regression analysis. A risk score model (RSM) was established based on three sDMIRs. The expression levels of sDMIRs in cell lines and clinical samples were detected via quantitative polymerase chain reaction. The correlations between sDMIRs and clinicopathological characteristics of PCa patients were evaluated using the chi-square test and Fisher's exact probability method.ResultsFour sDMIRs were remarkably related to the prognosis of PCa patients based on univariate Cox analysis, of which miR-10a-5p, miR-20a-5p, and miR-508-3p were used to establish the RSM. The OS in the low-risk group was better than that in the high-risk group. In the verification of various prostate cell lines and clinical samples from 162 PCa patients, the prominently higher expression of miR-10a-5p and miR-20a-5p and lower expression of miR-508-3p were detected in PCa cell lines and tumor tissues, especially the more advanced T-stage. Besides, the higher expression of miR-20a-5p and miR-10a-5p was significantly correlated to the higher level of PSA, Gleason score, more advanced T-stage, and distant metastasis status.ConclusionWe identify and validate the clinical significance of three sDMIRs and establish a verified RSM to evaluate the prognosis for PCa patients. The findings not only provide a reliable tool for clinical decision-makers to evaluate patients' prognosis but also offer a novel perspective into the field of biomarker identification.

PP1 catalytic isoforms are differentially expressed and regulated in human prostate cancer

The Ser/Thr-protein phosphatase PP1 (PP1) is a positive regulator of the androgen receptor (AR), which suggests major roles for PP1 in prostate carcinogenesis. However, studies dedicated to the characterization of PP1 in PCa are currently scarce. Here we analyzed the expression and localization of the PP1 catalytic (PP1c) isoforms in formalin-fixed, paraffin-embedded prostate tissue samples, as well as in PCa cell lines. We also analyzed well-characterized PCa cohorts to determine their transcript levels, identify genetic alterations, and assess promoter methylation of PP1c-coding genes. We found that PP-1A was upregulated and relocalized towards the nucleus in PCa and that PPP1CA was frequently amplified in PCa, particularly in advanced stages. PP-1B was downregulated in PCa but upregulated in a subset of tumors with AR amplification. PP-1G transcript levels were found to be associated with Gleason score. PP1c-coding genes were rarely mutated in PCa and were not prone to regulation by promoter methylation. Protein phosphorylation, on the other hand, might be an important regulatory mechanism of PP1c isoforms' activity. Altogether, our results suggest differential expression, localization, and regulation of PP1c isoforms in PCa and support the need for investigating isoform-specific roles in prostate carcinogenesis in future studies.

LINC00893 inhibits the progression of prostate cancer through miR-3173-5p/SOCS3/JAK2/STAT3 pathway

BackgroundProstate cancer (PCa) is one of the most common malignant tumors in the male urinary system. In recent years, the morbidity and mortality of PCa have been increasing due to the limited effects of existing treatment strategies. Long non-coding RNA (lncRNA) LINC00893 was reported to inhibit the proliferation and metastasis of papillary thyroid cancer cells, but its role in PCa has not been reported. This study aims to investigate the role and underlying mechanism of LINC00893 in regulating the progression of PCa cells.MethodsWe first compared LINC00893 expression levels between PCa tissues and normal prostate tissues through TCGA database. The relative LINC00893 expression levels were further validated in 66 pairs of PCa tissues and para-cancerous normal tissues, as well as in PCa cell lines. Gain-of-function experiment was performed by transfecting PCa cell with LINC00893 expression vector, and CCK (Cell count kit)-8, 5-Ethynyl-2′-deoxyuridine (EdU) incorporation, colony information and transwell assays were conducted to assess the functional phenotypes. Dual-luciferase reporter, RNA-binding protein immunoprecipitation (RIP) and RNA pull-down assays were performed to evaluate the molecular interactions.ResultsLINC00893 was downregulated in PCa tissues and cell lines, and patients with low expression of LINC00893 were associated with a poorer overall survival rate. LINC00893 overexpression hindered the proliferation, epithelial-mesenchymal transition (EMT) as well as the migratory ability of PCa cells, and suppressed the tumorigenesis of PCa cells in nude mice. We further demonstrated that LINC00893 acted as a sponge for miR-3173-5p and inhibited its activity, which in turn regulated the suppressor of cytokine signaling 3 (SOCS3)/Janus Kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling axis.ConclusionsOur study demonstrated that LINC00893 suppresses the progression of PCa cells through targeting miR-3173-5p/SOCS3/JAK2/STAT3 axis. Our data uncovers a novel tumor-suppressor role of LINC00893 in PCa, which may serve as a potential strategy for targeted therapy in PCa.Grapical

Abstract 5822: Leptin modulated microRNA-628-5p targets Jagged1 and inhibits prostate cancer hallmarks

Abstract Prostate cancer (PCa) is the most commonly diagnosed non-cutaneous cancer and the second leading cause of cancer deaths in men. Obesity is a major risk factor for prostate cancer. Leptin is an obesity hormone that controls body weight by regulating energy intake and expenditure, and clinical studies have demonstrated a correlation between blood leptin levels and PCa development. MicroRNAs (miRs) are master regulators of cancer cell signaling, including in advanced PCa. We previously identified miR-628-5p as a potential biomarker in PCa patients that was downregulated in serum samples of men with PCa (Tumor Biol., 2014, 35: 4867). In this study, we investigated the link between the obesity hormone leptin and the functional role of miR-628-5p in PCa cell lines. We demonstrate that leptin downregulates the expression of miR-628-5p and increases cell proliferation and migration in PCa cells. Enforced expression of miR-628-5p inhibited cell proliferation in PCa cells. Further, miR-628-5p reduced PCa cell survival/migration/invasion/spheroids formation and cell stemness. Mechanistically, miR-628-5p binds with JAG1-3’UTR and inhibits the expression of Jagged-1 (JAG1). JAG1 inhibition by miR-628-5p also downregulated Notch signaling, decreased the expression of snail/slug, and modulated epithelial-mesenchymal transition and invasiveness in PC3 cells. Furthermore, miR-628-5p increased sensitivity towards enzalutamide and docetaxel by inducing cell apoptosis. Collectively our data suggested that miR-628-5p is a key regulator of PCa carcinogenesis modulated by adipokine leptin and offers a novel therapeutic opportunity to inhibit the growth of advanced PCa. Citation Format: Leslimar Rios Colon, Juliet Chijioke, Suryakant Niture, Zainab Afzal, Qi Qi, Anvesha Srivastava, Malathi Ramalinga, Habib Kedir, Patrice Cagle, Elena Arthur, Gagan Deep, Simeng Suy, Sean Collins, Deepak Kumar. Leptin modulated microRNA-628-5p targets Jagged1 and inhibits prostate cancer hallmarks [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 5822.

Abstract 3675: Epigenetic variations associated with therapy resistance among prostate tumors from Puerto Rican men

Abstract Prostate cancer (PCa) is the leading cause of cancer-related death in Puerto Rican (PR) men, a population at higher risk of developing the disease than other Hispanic/Latino groups. But the relationship between this phenomenon and epigenetics, and how this relationship explains PCa racial and ethnic health disparities are controversial. The purpose of this study was to expand on the knowledge regarding epigenetic differences in terms of DNA methylation to enable risk stratification and treatment selection in this genetically admixed population. In addition, we aim to assess differential DNA methylation patterns associated with drug resistance in PCa in PR Hispanic/Latino men. Since epigenetic changes and modifications represent critical components of carcinogenesis and progression, we are expecting differential methylation levels between aggressive vs. indolent, and resistant vs. non-resistant cases. Prostate tumors from PR men were stratified based on Gleason score. Methylation levels were measured by Illumina Infinium Methylation EPIC BeadChip DNA methylation platform, and bioinformatic analyses. In parallel, DNA methylation profiles were also analyzed using genomic DNA from PCa cells selected for resistance to androgen depletion, enzalutamide resistance, or docetaxel resistance. We identified 1,225 differentially methylated genes in prostate tumors compared with normal adjacent tissues, and 141 differentially methylated genes between aggressive vs. indolent tumors. Additionally, 723 differentially methylated genes were found in therapy resistant cell lines compared to parental controls. Analysis of the methylated genes obtained from aggressive prostate tumors from Puerto Rican patients and therapy-resistant PCa cell lines revealed four overlapping methylated genes: RNF220, FAM65B, PRDM16, and DGKH. Further analysis of PCa cells resistant to androgen depletion, enzalutamide, or docetaxel demonstrated impaired cellular migration of resistant phenotypes upon DNA methyltransferase (DNMT) inhibition using 5-azacytidine or zebularine compared to parental cell lines. DNMT1 was associated with resistance in PCa cell lines, and future studies will include silencing of methyltransferases will evaluation of drug re-sensitization. These findings provide critical insight into the epigenetic landscape of aggressive prostate tumors among PR men and highlight several candidate methylation biomarkers for assessing risk in this population. Citation Format: Carlos J. Diaz Osterman, Anders Berglund, Shannalee Martinez, Camila Luis, Julie Dutil, Jaime Matta, Jarline Encarnacion-Medina, Carmen Ortiz-Sanchéz, Liang Wang, Jasreman Dhillon, Youngchul Kim, Hyun Y. Park, Gilbert Ruiz-Deya, Kosj Yamoah, Jong Y. Park. Epigenetic variations associated with therapy resistance among prostate tumors from Puerto Rican men [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 3675.

Abstract 400: Functional, structural and binding studies of the atypical ER-resident protein FKBP7, a potential target in chemoresistant prostate cancer

Abstract Background: Metastatic Castration Resistant Prostate Cancer (mCRPC) is the latest stage of Prostate Cancer (PCa). Despite the use of taxane-based chemotherapies Docetaxel and Cabazitaxel, mCRPC remains lethal due to chemoresistance. We recently identified FKBP7 as a potential therapeutic target of interest in PCa. FKBP7 is overexpressed in taxane-resistant PCa cells and impacts both cell proliferation and Docetaxel efficacy in chemoresistant models. We demonstrated FKBP7 affects translation and binds to the translation initiation complex eIF4F. FKBP7 is an ER-resident FKBP, but its properties remain poorly described. Methods: FKBP7 expression upon chemotherapy treatment was assessed by Western Blot and correlation between FKBP7 expression and clinical prognosis was examined on public cancer databases. Subcellular localization of FKBP7 was addressed by digitonin-based fractionation. Finally, FKBP7-predicted structure was obtained with structural modeling servers and sequence alignments were performed on AliView. We then produced and purified the recombinant catalytic domain of FKBP7 and collected high quality 15N HSQC NMR spectra. Afterwards, we examined its interaction with well-known FKBP ligands. Results: Increased FKBP7 expression was observed upon treatment with several cytotoxic chemotherapies in PCa-cell lines, and TCGA data underlined FKBP7 impacts survival in other cancers than PCa. Together, this suggests an enlarged potential involvement of FKBP7 in adaptive resistance. Mechanistically, in taxane-resistant models, FKBP7 overexpression did not result from higher protein stability but rather from a transcriptional regulation. Preliminary results of subcellular fractionation showed FKBP7 is present in the Endoplasmic Reticulum (ER) and seems to localize in the cytosol, independently of proteasomal degradation. We also confirmed FKBP7 is N45-glycosylated and observed that cytosolic-FKBP7 appears mainly glycosylated. This suggests that the ER-resident FKBP7 could be refluxed in the cytosol depending on its glycosylation state to regulate the activity of the cytosolic eIF4F complex. We are currently validating this hypothesis. At last, structural modeling predicted a FKBP-type fold with a potential FKBP7 substrate specificity as its catalytic pocket presents distinct composition for charge and bulkiness compared to other FKBPs. Accordingly, FKBP7 strongly binds Rapamycin and Everolimus, but surprisingly not FK506, confirming a selective binding profile. Conclusion: Our work depicts the recently identified FKBP7/eIF4F pathway in PCa-resistance and points atypical properties for the ER-resident FKBP7. It also indicates FKBP7 could be a druggable target in adaptive resistance of other solid cancers. Besides, we gathered original data on its structure and specificity for future drug-targeting strategies. Citation Format: Luce Dreno, Carolina Tiraboschi, Sofian Lacoste, Sofia Calpe Gomez, Marine F. Garrido, Matthieu Bertrand, Mariana Tannoury, Eric Jacquet, Naima Nhiri, Yohann Loriot, Karim Fizazi, Daniel Compagno, Nadine Assrir, Ewen Lescop, Anne Chauchereau. Functional, structural and binding studies of the atypical ER-resident protein FKBP7, a potential target in chemoresistant 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 400.

Abstract LB036: Molecular insight into ERG transcriptional activity to unravel novel therapeutic options for ERG-positive castration resistant prostate cancer

Abstract Gene fusions of the AR-regulated gene TMPRSS2 with the ETS family transcription factor ERG characterize about 40% of castration resistant prostate cancer (CRPC) - an advanced and lethal stage of prostate cancer. AR-mediated overexpression of ERG hijacks AR inhibiting lineage-specific differentiation and increases cell invasion and migration. In vivo work revealed a causal role of ERG in initiating prostate cancer. Notably, in vitro and in vivo models of CRPC endogenously bearing T2ERG depend on ERG activity for proliferation and survival. Thus, pursuing the development of ERG-based therapeutic strategies represents a promising route for CRPC treatment, although major challenges in targeting transcription factors such as ERG have been reported. Yet, synthetic lethality approaches including genome-wide loss-of-function screens can be employed to avoid direct ERG targeting. Here, we used metastatic PCa cell lines (LNCaP, 22Rv1 and DU145) engineered with inducible systems to overexpress ERG to show that high levels of ERG expression lead to an apparent reduction in the fitness of AR-positive and negative cells. Prolonged overexpression of a mutant form of ERG, which is not able to bind DNA, does not affect cell viability indicating that ERG DNA binding and transcriptional activity are necessary for the induction of this phenotype. The ERG-mediated reduction in cellular fitness is influenced by cell adhesion and is characterized by cell cycle arrest mediated by several cell cycle inhibitors induced by ERG. As revealed by gene expression analyses and functional assays, ERG induces a less proliferative, more dedifferentiated and invasive phenotype, which lead to an apparent reduction of cellular fitness in vitro, but is characteristic of more aggressive tumor cells in vivo. Altogether, these results underscore the reliability of the developed in vitro models for ERG activity, which will be used for screening to identify therapeutic vulnerabilities unlocked by ERG in CRPC. Importantly, we also uncovered confounding factors (i.e. loss of ERG-positive cells not due to screen selection) that could impinge on the results arising from in vitro screens. Citation Format: Francesca Lorenzin, Giulia Fracassi, Yari Ciani, Paola Gasperini, David Rickman, Francesca Demichelis. Molecular insight into ERG transcriptional activity to unravel novel therapeutic options for ERG-positive 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 LB036.

EPA Modulates KLK Genes via miR-378: A Potential Therapy in Prostate Cancer.

Simple SummaryCastration resistant prostate cancer (CRPC) is one of the most pestilent form of prostate cancer (PCa), accounting for approximately 10–20% of all PCas, which carry poor mortality and morbidity rates. The focus in this study is on EPA, a natural supplement commonly found in fish oils. EPA induces the expression of PGC-1β gene and co-expresses miR-378 in cells, a nucleic acid sequence that inhibits PCa cell proliferation. This significance is highlighted in this study as a potential adjunctive therapy for all stages of PCa including CRPC.It is known that miRNA-378a-3p (miR-378) could be induced by eicosapentaenoic acid (EPA), an omega-3 fatty acid. Herein, we first demonstrated how miR-378 exerts anti-prostate cancer (PCa) actions by influencing multiple target genes, including KLK2, KLK4, KLK6, and KLK14, which are implicated in PCa development, cell proliferation, and cell survival. Furthermore, these genes also correlate with androgen and mTOR signaling transduction, and are considered pivotal pathways for the onset and progression of PCa. In total, four PCa cell lines and eight pairing tissues (tumor vs. normal) from clinical PCa patients were included in the current study. The results showed high significance after EPA induced tumor cells containing higher expression levels of miR-378, and led the PCa cells having low cell viabilities, and they progressed to apoptosis when compared with normal prostate cells (p < 0.001). The findings indicated that EPA might become a potential therapy for PCa, especially because it is derived from the components of natural fish oil; it may prove to be a great help for solving the problem of castration-resistant prostate cancer (CRPC).

Scutellaria barbata D.Don (SBD) extracts suppressed tumor growth, metastasis and angiogenesis in Prostate cancer via PI3K/Akt pathway

BackgroundScutellaria barbata D.Don (SBD) is derived from the dried whole plant of Labiate which has been widely used to treat patients with multiple cancer. It was previously reported that the ethanol extract of SBD is able to promote apoptosis, and inhibit cell proliferation and angiogenesis in cancer.Materials and methodsCCK8, Edu assays and colony formation assay were performed to assess the effect of SBD on PCa cell growth. Effect of SBD on apoptosis and cell cycle was detected by flow cytometry. Transwell and wounding healing assay were conducted to detect the invasion and migration activities of PCa cells. Western blot was employed to detect the protein expression. 2RRV1 mouse xenograft model was established to detect the effect of SBD on prostate cancer. Angiogenesis was analysed by coculturing PCa cell lines and HUVECs.ResultsThe results showed that SBD induced a significant decrease in cell viability and clonogenic growth in a dose-dependent manner. SBD induced cell apoptosis and cell cycle G2/M phase arrest by inactivating PI3K/AKT signalling pathway. Treatment with SBD also significantly decreased the cell migration and invasion via phenotypic inversion of EMT that was characterized by the increased expression of E-cadherin and Vimentin, and decreased expression of N-cadherin, which could be partially attributed to inhibiting PI3K/AKT signalling pathway. Subsequently, using AKT inhibitor MK2206, we concluded that PI3K/AKT are also involved in cell apoptosis and metastasis of PCa cells stimulated by SBD. Apart from its direct effects on PCa cells, SBD also exhibited anti-angiogenic properties. SBD alone or conditioned media from SBD-treated PCa cells reduced HUVEC tube formation on Matrigel without affecting HUVEC viability. Furthermore, 22RV1 xenograft C57BL/6 mice treated with SBD in vivo showed a significant inhibitory in tumour size and tumour weight without toxicity. In addition, administration with medium- or high-dose of SBD significantly inhibited the cell proliferation and enhanced the damage to tumour tissues.ConclusionsCollectively, our in vitro and in vivo findings suggest that SBD has the potential to develop into a safe and potent alternative therapy for PCa patients.

PD07-04 PRE-CLINICAL STUDIES FOR ADVANCING CIRMTUZUMAB-BASED ANTI-ROR1 THERAPIES IN METASTATIC PROSTATE CANCER

You have accessJournal of UrologyCME1 May 2022PD07-04 PRE-CLINICAL STUDIES FOR ADVANCING CIRMTUZUMAB-BASED ANTI-ROR1 THERAPIES IN METASTATIC PROSTATE CANCER Michelle Muldong, Sanghee Lee, Theresa Mendoza, Danielle Burner, Christina Wu, Jamillah Murtadha, Evodie Koutouan, Naomi Pineda, Gabriel Pineda, Kathleen Lennon, Hao Pham, Karl Willert, Nicholas Cacalano, Catriona Jamieson, Terry Gaasterland, Christopher Kane, Anna Kulidjian, and Christina Jamieson Michelle MuldongMichelle Muldong More articles by this author , Sanghee LeeSanghee Lee More articles by this author , Theresa MendozaTheresa Mendoza More articles by this author , Danielle BurnerDanielle Burner More articles by this author , Christina WuChristina Wu More articles by this author , Jamillah MurtadhaJamillah Murtadha More articles by this author , Evodie KoutouanEvodie Koutouan More articles by this author , Naomi PinedaNaomi Pineda More articles by this author , Gabriel PinedaGabriel Pineda More articles by this author , Kathleen LennonKathleen Lennon More articles by this author , Hao PhamHao Pham More articles by this author , Karl WillertKarl Willert More articles by this author , Nicholas CacalanoNicholas Cacalano More articles by this author , Catriona JamiesonCatriona Jamieson More articles by this author , Terry GaasterlandTerry Gaasterland More articles by this author , Christopher KaneChristopher Kane More articles by this author , Anna KulidjianAnna Kulidjian More articles by this author , and Christina JamiesonChristina Jamieson More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002526.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Increased levels of the Wnt ligand, WNT5A, are correlated with bone metastatic, castrate resistant PCa (CRPC). Wnt signaling is comprised of canonical (β-catenin dependent) and noncanonical pathways which are critical for normal development and physiology. Alterations are frequently associated with tumorigenesis and metastasis in many cancers including prostate cancer. Noncanonical Wnt5A signaling is mediated in part through ROR1, a fetal oncoprotein for which a therapeutic ROR1 inhibitory antibody, Cirmtuzumab, has been developed. Its safety has been clinically proven in trials for chronic lymphocytic leukemia (CLL) and metastatic breast cancer.Hypothesis: WNT5A may activate a stem-cell-like program via ROR1 that leads to castration resistance in bone metastatic prostate cancer. The anti-ROR1 biologic, Cirmtuzumab, may inhibit this mechanism of CRPC and re-sensitize bone metastatic prostate cancer to standard of care therapies. METHODS: We used patient-derived xenograft (PDX) bone metastatic prostate cancer models and PCa cell lines to test mechanism of action of a new therapeutic target, the WNT5A/ROR1 signaling pathway in prostate cancer for which a therapeutic ROR1 inhibitor antibody, Cirmtuzumab, has been developed and clinically tested in CLL and metastatic breast cancer patients. RESULTS: Preclinical studies using RNASeq, qRT-PCR, FACS and Westerns showed increased expression of ROR1 in the neuroendocrine prostate cancer cell lines, PC3 and DU145, and a patient derived xenograft (PDX) model of small cell bone metastatic prostate cancer, PCSD13. Pretreatment of the PCSD13 PDX with Cirmtuzumab increased the efficacy of the taxane docetaxel at inhibiting tumor growth. Intravenous infusion of anti-ROR1 CAR-T cells into mice bearing subcutaneous, ROR1-expressing, PC3 xenografts, showed marked reduction in tumor growth. ROR1 Knock out using CRISPR-Cas9 in PC3 and DU145 cells showed specificity of Cirmtuzumab-based immunotherapies. We have developed immunohistochemistry (IHC) assays to detect ROR1 in patient biopsy samples which will allow us to define the prostate cancer patient population that expressed ROR1 and to be able to evaluate the treatment in patients for a future clinical trial. CONCLUSIONS: ROR1 was expressed at high levels on castration resistant small cell PCa and neuroendocrine PCa models in PCa cell lines and PDX models. Cirmtuzumab has demonstrated efficacy in our patient derived xenograft organoid cultures. These studies have formed the rationale for development of a Phase 1b clinical trial of Cirmtuzumab in combination with docetaxel in metastatic CRPC patients. Source of Funding: Dept of Defense CDMRP PCRP Impact Award, JM Foundation © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e99 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Michelle Muldong More articles by this author Sanghee Lee More articles by this author Theresa Mendoza More articles by this author Danielle Burner More articles by this author Christina Wu More articles by this author Jamillah Murtadha More articles by this author Evodie Koutouan More articles by this author Naomi Pineda More articles by this author Gabriel Pineda More articles by this author Kathleen Lennon More articles by this author Hao Pham More articles by this author Karl Willert More articles by this author Nicholas Cacalano More articles by this author Catriona Jamieson More articles by this author Terry Gaasterland More articles by this author Christopher Kane More articles by this author Anna Kulidjian More articles by this author Christina Jamieson More articles by this author Expand All Advertisement PDF DownloadLoading ...

SLC4A4 promotes prostate cancer progression in vivo and in vitro via AKT-mediated signalling pathway

BackgroundProstate cancer (PCa) is the second leading cause of cancer-related male deaths worldwide. The purpose of this study was to investigate the effects of homo sapiens solute carrier family 4 member 4 (SLC4A4), which encodes the electrogenic Na+/HCO3− cotransporter isoform 1 (NBCe1), in the development and progression of PCa.MethodsThe expression levels of SLC4A4 in PCa and normal prostate tissues were evaluated by immunohistochemistry. The SLC4A4 knockdown cell model was structured by lentiviral infection, and the knockdown efficiency was validated by RT-qPCR and Western blotting. The effects of SLC4A4 knockdown on cell proliferation, apoptosis and cycle, migration, and invasion were detected by Celigo cell counting assay and CCK-8 assay, flow cytometry analysis, wound-healing, and Transwell assay, respectively. Tumor growth in nude mice was surveyed by in vivo imaging and Ki-67 staining. Furthermore, underlying mechanism of SLC4A4 silence induced inhibition of PCa progression was explored by human phospho-kinase array.ResultsOur results revealed that SLC4A4 expression was up-regulated in PCa tissues and human PCa cell lines. High expression of SLC4A4 in tumor specimens was significantly correlated with disease progression. SLC4A4 knockdown inhibited cell proliferation, migration and invasion, while facilitated apoptosis, which was also confirmed in vivo. Moreover, SLC4A4 promoted PCa progression through the AKT-mediated signalling pathway.ConclusionThe results of this study indicated that SLC4A4 overexpression was closely associated with the progression of PCa; SLC4A4 knockdown suppressed PCa development in vitro and in vivo. SLC4A4 acts as a tumor promotor in PCa by regulating key components of the AKT pathway and may therefore act as a potential therapeutic target for PCa treatment.

MiR-363-3p promotes prostate cancer tumor progression by targeting Dickkopf 3.

BackgroundProstate cancer (PCa) is a frequent malignant tumor worldwide with high morbidity along with mortality. MicroRNAs (miRNAs) have been identified as key posttranscriptional modulators in diverse cancers. Herein, we purposed to explore the impacts of miR‐363‐3p on PCa growth, migration, infiltration along with apoptosis.MethodsThe expressions of miR‐363‐3p along with Dickkopf 3 (DKK3) were assessed in clinical PCa specimens. We adopted the PCa cell line PC3 and transfected it using miR‐363‐3p repressors or mimic. The relationship of miR‐363‐3p with DKK3 was verified by a luciferase enzyme reporter assay. Cell viability along with apoptosis were determined by MTT assay coupled with flow cytometry analysis. Cell migration along infiltration were detected via wound healing, as well as Transwell assays. The contents of DKK3, E‐cadherin, vimentin along with N‐cadherin were analyzed via Western blotting accompanied with qRT–PCR.ResultsMiR‐363‐3p was found to be inversely associated with the content of DKK3 in clinical PCa specimens. Further investigations revealed that DKK3 was miR‐363‐3p's direct target. Besides, overexpression of miR‐363‐3p decreased the contents of DKK3, promoted cell viability, migration coupled with infiltration, and reduced cell apoptosis, while silencing of miR‐363‐3p resulted in opposite influence. Upregulation of miR‐363‐3p diminished E‐cadherin contents but increased vimentin along with N‐cadherin protein contents in PC3 cells; in contrast, miR‐363‐3p downregulation produced the opposite result.ConclusionOur study indicates that miR‐363‐3p promotes PCa growth, migration coupled with invasion while dampening apoptosis by targeting DKK3.

Trpm8 Expression in Human and Mouse Castration Resistant Prostate Adenocarcinoma Paves the Way for the Preclinical Development of TRPM8-Based Targeted Therapies.

Metastatic prostate cancer (mPCa) is one of the leading causes of cancer-related mortality in both the US and Europe. Androgen deprivation is the first-line therapy for mPCa; however, resistance to therapy inevitably occurs and the disease progresses to the castration resistant stage, which is uncurable. A definition of novel targeted therapies is necessary for the establishment of innovative and more effective protocols of personalized oncology. We employed genetically engineered mouse models of PCa and human samples to characterize the expression of the TRPM8 cation channel in both hormone naïve and castration resistant tumors. We show that Trpm8 expression marks both indolent (Pten-null) and aggressive (Pten/Trp53 double-null and TRAMP) mouse prostate adenocarcinomas. Importantly, both mouse and human castration-resistant PCa preserve TRPM8 protein expression. Finally, we tested the effect of TRPM8 agonist D-3263 administration in combination with enzalutamide or docetaxel on the viability of aggressive mouse PCa cell lines. Our data demonstrate that D-3263 substantially enhances the pro-apoptotic activity of enzalutamide and docetaxel in TRAMP-C1 e TRAMP-C2 PCa cell lines. To conclude, this study provides the basis for pre-clinical in vivo testing of TRPM8 targeting as a novel strategy to implement the efficacy of standard-of-care treatments for advanced PCa.

Abstract PO-152: Metabolic rewiring in African-American prostate cancer: A role for adenosine-inosine axis

Abstract African-American (AA) men have a 60% higher incidence of prostate cancer compared to European-American (EA) men and tend to have a more aggressive clinical outcome. An understanding of the altered biological responses and the factors that lead to health disparity in prostate cancer development and progression is unclear. Metabolism defines the physiological state of the cell and metabolic reprogramming is a hallmark of cancer. Thus, studying the metabolic landscape will be critical for understanding the biological changes that might contribute to this disparity. Analysis of 190 metabolites across prostate cancer/benign adjacent tissue pairs from ancestry typed AA and EA men performed in our laboratory, revealed altered levels of adenosine and inosine. High inosine to adenosine ratio is observed in AA men compared to EA men. In line with this finding, the enzyme adenosine deaminase (ADA), which converts adenosine to inosine was found elevated in AA men, potentially explaining the high inosine to adenosine ratio in AA PCa. The effects of these metabolic alterations on AA PCa progression are unknown. The current study will address the knowledge gap on the consequences of elevated ADA activity in AA PCa and attempt to dissect its role in effecting an aggressive phenotype in PCa. To determine the role of elevated ADA in PCa, it was overexpressed in ancestry-typed AA (MDA-PCa-2A) and EA PCa(LNCaP) cell lines (termed ADA OE). Phenotypic examination of these cells revealed increased ADA enzyme activity decreases the cell-ECM adhesion. Molecular analyses revealed a significant reduction in Tenascin C(TNC), a matrix protein known for its anti-adhesive properties. TNC is critical for modulating cell motility and adhesion. TNC is also a marker for the reactive stromal microenvironment. Therefore, we evaluated the effects of altered adenosine-inosine levels on stromal cells. TNC induction was observed in stromal cells upon treatment with ADA OE conditioned media and inosine addition. These molecular findings suggest that high ADA activity could facilitate adhesion decrease and modulate the stromal microenvironment to aid in the metastatic dissemination from the primary tumor. From a translational viewpoint, ADA enzyme activity could serve as a biomarker for metastatic prostate cancer. Therefore, we further look to analyze the inosine to adenosine levels in patient samples and correlate with various clinical outcomes in PCa. Our initial analysis reveals high inosine to adenosine ratio in biopsy positive patient samples compared to biopsy negative samples. Our next steps of analysis include correlating the enzyme activity with (i)Biochemical Recurrence(BCr) (ii)Time to attaining castration resistance in castration-sensitive patients (iii)Time to metastasis (iv)Response to Androgen deprivation in patients after BCr. Based on our current findings, we expect the inosine to adenosine ratio in plasma or urine to serve as a predictive marker for PCa incidence or progression in AA men. In addition, we expect ADA to serve as a potential therapeutic target for AA PCa. Citation Format: Christy Charles. Metabolic rewiring in African-American prostate cancer: A role for adenosine-inosine axis [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-152.

Integrative Analysis of MALT1 as a Potential Therapeutic Target for Prostate Cancer and its Immunological Role in Pan-Cancer.

Background: Mucosa-associated lymphoma antigen 1 (MALT1) is an oncogene in subsets of diffuse large B cell lymphoma (DLBCL) and mucosa-associated lymphoid tissue type (MALT) lymphoma. However, the role of MALT1 across cancers, especially in prostate cancer is still poorly understood. Methods: Here, we used several public datasets to evaluate MALT1 expression. Then, PCa cell lines and nude mice were used to investigate the cellular functions in vitro and in vivo. Microarray data were downloaded from The Cancer Genome Atlas and MALT1 was subjected to gene set enrichment analysis (GSEA) and Gene Ontology (GO) analysis to identify the biological functions and relevant pathways. Additionally, the correlations between MALT1 expression and mismatch repair (MMR) gene mutation, immune checkpoint gene expression, tumor mutational burden (TMB), and microsatellite instability (MSI) were investigated by Pearson correlation analysis. Moreover, the correlation between MALT1 expression and tumor immune infiltration was analyzed by the Tumor Immune Evaluation Resource (TIMER) database. Results: MALT1 overexpression was significantly correlated with MMR gene mutation levels and crucially promoted proliferation and colony genesis while reducing PCa cell apoptosis levels in vivo and in vitro. MALT1 expression showed strong correlations with immune checkpoint genes, TMB, and MSI in most cancers. The GO analysis indicated that MALT1-coexpressed genes were involved in heterotypic cell-cell adhesion, actin filament-based movement regulation, and action potential regulation. GSEA revealed that MALT1 expression was associated with several signaling pathways, including the NF-κB signaling, Wnt/β-catenin and TGF-β signaling pathways, in PCa. Additionally, MALT1 expression was significantly correlated with the infiltration of immune cells, including B cells, CD8+ T cells, dendritic cells and macrophages, and negatively correlated with CD4+ cell infiltration in PCa. Conclusion: MALT1 expression is higher in pancancer samples than in normal tissues. MALT1 promoted proliferation and colony genesis while reducing PCa cell apoptosis levels, and MALT1 suppression could inhibit xenograft tumor establishment in nude mice. Furthermore, MALT1 expression is closely related to the occurrence and development of multiple tumors in multiple ways. Therefore, MALT1 may be an emerging therapeutic target for a variety of cancers especially PCa.

LncRNA MIR99AHG mediated by FOXA1 modulates NOTCH2/Notch signaling pathway to accelerate pancreatic cancer through sponging miR-3129-5p and recruiting ELAVL1

BackgroundPancreatic cancer (PCa) is a fatal malignancy with poor prognosis, high recurrence and mortality. Substantial reports have suggested long non-coding RNAs (lncRNAs) are implicated in development of numerous malignant tumors, and PCa is included. However, the correlation between novel lncRNA mir-99a-let-7c cluster host gene (MIR99AHG) and PCa remains elusive and needs to be deeply investigated.MethodsIn this study, we firstly used RT-qPCR to examine MIR99AHG expression. Functional assays were implemented for determination of the role of MIR99AHG in PCa cells. Mechanism experiments were designed and carried out for exploring the regulatory mechanism involving MIR99AHG.ResultsMIR99AHG was distinctly overexpressed in PCa cell lines. MIR99AHG deficiency abrogated PCa cell proliferation, migration and invasion. Moreover, MIR99AHG up-regulation was induced by transcription factor forkhead box A1 (FOXA1). Furthermore, MIR99AHG modulated notch receptor 2 (NOTCH2) expression and stimulated Notch signaling pathway through sequestering microRNA-3129-5p (miR-3129-5p) and recruiting ELAV like RNA binding protein 1 (ELAVL1).ConclusionsAltogether, the exploration of FOXA1/MIR99AHG/miR-3129-5p/ELAVL1/NOTCH2 axis in the progression of PCa might provide a meaningful revelation for PCa diagnosis and treatment.

Co-administration of tyrosine kinase inhibitors with rottlerin in metastatic prostate cancer cells.

After prostatectomy due to prostate carcinoma, patients often develop metastases. Although prostate cancer is susceptible to hormonal manipulation, many patients become castration-resistant. Therefore, new therapies are the focus of investigations. We analyzed the effect of the tyrosine kinase inhibitors (TKIs), sorafenib and sunitinib, in combination with rottlerin, a PKCδ inhibitor, on metastatic mechanisms in prostate carcinoma cells. LNCaP and PC-3 prostate carcinoma cells were treated with sorafenib or sunitinib alone at various concentrations (1-20 µM) or in combination with rottlerin (10 µM) for 24 h. Then, cell toxicity (MTT test) and cell proliferation (BrdU incorporation assay) were quantified. The study demonstrated a dose-dependent inhibitory effect of sorafenib and sunitinib on PC-3 and LNCaP cell activity and proliferation. Both agents showed significantly stronger cytotoxic effects in LNCaP cells. At the highest concentrations, sorafenib and sunitinib inhibited the viability of LNCaP cells up to 2 % and 31 %, respectively, and the viability of PC-3 cell line up to 20 % and 43 %, respectively. The proliferation of both cell lines was significantly stronger inhibited by sorafenib than by sunitinib. In LNCaP cells, sorafenib and sunitinib at the highest concentrations inhibited cell proliferation up to 46 % and 49 %, respectively, and the proliferation of PC-3 line up to 40 % and 47 %, respectively. Rottlerin reduced the viability and proliferation of PC3 cells to 81 % and 42 %, whereas the viability and proliferation of LNCaP cells were reduced to 25 % and 57 %, respectively. Sorafenib and sunitinib at low concentrations partly neutralized the inhibitory effect of rottlerin on cell viability and proliferation. On the other hand, in PC-3 cells, rottlerin reduced the inhibitory effects of sorafenib and sunitinib at the highest concentrations on cell viability from 20 % to 30 % and from 43 % to 61 %, respectively. An additive effect on cell activity was observed after treating LNCaP cells with both sunitinib at high concentrations and rottlerin. This combination increased the cytotoxic effect from 31 % to 13 % at the highest sunitinib concentration. Our results showed that monotherapy with sorafenib was the most efficient in both PCa cell lines. A marginally additive effect of rottlerin was only observed in LNCaP cells treated with sunitinib at a high concentration. Sorafenib and sunitinib reduced cell migration in PC-3 cells to 10 % and 32 % of untreated cells, respectively. Co-treatment with sorafenib/sunitinib and rottlerin did not result in a significantly stronger anti-migratory effect than the treatment with each TKI alone. Given the strong cytotoxic effect of TKIs, especially sorafenib, on LNCaP cells, the results of the migration assay in this line were severely biased and not considered in the analysis. Unlike in other malignancies, combination therapy with TKI and rottlerin seems not beneficial in prostate cancer. More promising seems to be monotherapy with rottlerin, but further studies are needed to confirm this observation.