Human Castration-resistant Prostate Cancer Cells Research Articles

Co-targeting SKP2 and KDM5B inhibits prostate cancer progression by abrogating AKT signaling with induction of senescence and apoptosis.

Prostate cancer (PCa) is the second-leading cause of cancer mortalities in the United States and is the most commonly diagnosed malignancy in men. While androgen deprivation therapy (ADT) is the first-line treatment option to initial responses, most PCa patients invariably develop castration-resistant PCa (CRPC). Therefore, novel and effective treatment strategies are needed. The goal of this study was to evaluate the anticancer effects of the combination of two small molecule inhibitors, SZL-P1-41 (SKP2 inhibitor) and PBIT (KDM5B inhibitor), on PCa suppression and to delineate the underlying molecular mechanisms. Human CRPC cell lines, C4-2B and PC3 cells, were treated with small molecular inhibitors alone or in combination, to assess effects on cell proliferation, migration, senescence, and apoptosis. SKP2 and KDM5B showed an inverse regulation at the translational level in PCa cells. Cells deficient in SKP2 showed an increase in KDM5B protein level, compared to that in cells expressing SKP2. By contrast, cells deficient in KDM5B showed an increase in SKP2 protein level, compared to that in cells with KDM5B intact. The stability of SKP2 protein was prolonged in KDM5B depleted cells as measured by cycloheximide chase assay. Cells deficient in KDM5B were more vulnerable to SKP2 inhibition, showing a twofold greater reduction in proliferation compared to cells with KDM5B intact (p < 0.05). More importantly, combined inhibition of KDM5B and SKP2 significantly decreased proliferation and migration of PCa cells as compared to untreated controls (p < 0.005). Mechanistically, combined inhibition of KDM5B and SKP2 in PCa cells abrogated AKT activation, resulting in an induction of both cellular senescence and apoptosis, which was measured via Western blot analysis and senescence-associated β-galactosidase (SA-β-Gal) staining. Combined inhibition of KDM5B and SKP2 was more effective at inhibiting proliferation and migration of CRPC cells, and this regimen would be an ideal therapeutic approach of controlling CRPC malignancy.

Hypoxia-inducible factor 1A inhibition overcomes castration resistance of prostate tumors.

Androgen deprivation therapy (ADT) is a cornerstone of prostate cancer (PCa) management. Although tumors initially regress, many progress to a hormone-independent state termed castration-resistant PCa (CRPC), for which treatment options are limited. We here report that the major luminal cell population in tumors of Pten(i)pe-/- mice, generated by luminal epithelial cell-specific deletion of the tumor suppressor PTEN after puberty, is castration-resistant and that the expression of inflammation and stemness markers is enhanced in persistent luminal cells. In addition, hypoxia-inducible factor 1 (HIF1) signaling, which we have previously demonstrated to be induced in luminal cells of Pten(i)pe-/- mice and to promote malignant progression, is further activated. Importantly, we show that genetic and pharmacological inhibition of HIF1A sensitizes Pten-deficient prostatic tumors to castration and provides durable therapeutic responses. Furthermore, HIF1A inhibition induces apoptotic signaling in human CRPC cell lines. Therefore, our data demonstrate that HIF1A in prostatic tumor cells is a critical factor that enables their survival after ADT, and identify it as a target for CRPC management.

PD16-01 SYNERGISTIC EFFECTS OF CURCUMIN AND LACTOFERRIN TO INHIBIT THE PROSTATIC CANCER CELLS GROWTH AND MIGRATION ABILITY

You have accessJournal of UrologyCME1 Apr 2023PD16-01 SYNERGISTIC EFFECTS OF CURCUMIN AND LACTOFERRIN TO INHIBIT THE PROSTATIC CANCER CELLS GROWTH AND MIGRATION ABILITY Erica Costantini, Michele Marchioni, Lisa Aielli, Simone Ferretti, Antonio De Palma, Davide Ciavarella, Giulio Litterio, Marcella Reale, Marta Di Nicola, and Luigi Schips Erica CostantiniErica Costantini More articles by this author , Michele MarchioniMichele Marchioni More articles by this author , Lisa AielliLisa Aielli More articles by this author , Simone FerrettiSimone Ferretti More articles by this author , Antonio De PalmaAntonio De Palma More articles by this author , Davide CiavarellaDavide Ciavarella More articles by this author , Giulio LitterioGiulio Litterio More articles by this author , Marcella RealeMarcella Reale More articles by this author , Marta Di NicolaMarta Di Nicola More articles by this author , and Luigi SchipsLuigi Schips More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003271.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Castration resistant prostate cancer (CRPC) represents a challenge in urologic oncology. Several treatments are available for this tumor, but their efficacy tends to reduce in time. In recent years, several natural compounds have been tested as possible adjuvant treatment in CRPC patients. Among the other curcumin (CU) showed interesting bioactive properties, but low penetration within cells. We hypothesized that the combination with lactoferrin (LF) might exert a synergistic effect on prostatic cancer cells as anti-cancer. Their effects have been evaluated in PC3 and DU145 prostate cancer cells, that are in vitro models of CRPC. METHODS: Human CRPC cell lines (DU-145, PC3) were cultured in medium added with or without LF (175 µM) ± CU (2.5 µg/ml and 5 µg/ml). Cell viability was determined using a standard colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) reduction assay. To test ability of cell migration observation at 6 and 24 h after scratch wounds were made. Quantitative Real-time PCR was performed to evaluate gene expression of death receptors (DR) and integrins (α3/β1). The human annexin V ELISA assay was used for the quantitative detection of annexin V, involved in apoptosis regulation. RESULTS: In PC3 cells, the proliferation rate is reduced by treatments alone or in association, with a significant reduction in presence of CU, both at 2.5 (p=0.007) and 5 ug/ml (p=0.001), in a dose-dependent manner. In DU145 cells, the inhibitory effect of CU is more significant in presence of LF. In PC3 migration, LF significantly weakened invasion capacity with an increase of the cell-free area of 24.9%, 31.8%, and 24% respectively in presence of LF alone, CU2.5+LF and CU5+LF, after 6 h, with similar results in DU145 cells. In accord the integrins α3/β1, showed a significant reduction in both prostate cell lines, in presence of CU2.5, CU5, and LF175 and their combination (p<0.005). Moreover, in PC3 and DU145 an upregulation of pro-apoptotic DR4 and DR5 gene expression have been observed. Finally, in both PC3 and DU145 cells line an increased concentration of human annexin V, a marker of apoptosis, have been recorded (p<0.005). CONCLUSIONS: Our results showed that the association of CU and LF might led to beneficial effect on CRPC prostate cancer reducing the proliferative activity of cancerous cells and their ability to migrate and produce metastases. In addition, we can suggest a potential pro-apoptotic effect of CU and LF leading to a potential application as an adjuvant treatment for the treatment of CRPC. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e489 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Erica Costantini More articles by this author Michele Marchioni More articles by this author Lisa Aielli More articles by this author Simone Ferretti More articles by this author Antonio De Palma More articles by this author Davide Ciavarella More articles by this author Giulio Litterio More articles by this author Marcella Reale More articles by this author Marta Di Nicola More articles by this author Luigi Schips More articles by this author Expand All Advertisement PDF downloadLoading ...

Role of HIKESHI on Hyperthermia for Castration-Resistant Prostate Cancer and Application of a Novel Magnetic Nanoparticle with Carbon Nanohorn for Magnetic Hyperthermia.

The prognosis of castration-resistant prostate cancer (CRPC) is technically scarce; therefore, a novel treatment for CRPC remains warranted. To this end, hyperthermia (HT) was investigated as an alternative therapy. In this study, the analysis focused on the association between CRPC and heat shock protein nuclear import factor "hikeshi (HIKESHI)", a factor of heat tolerance. Silencing the HIKESHI expression of 22Rv1 cells (human CRPC cell line) treated with siRNAs inhibited the translocation of heat shock protein 70 from the cytoplasm to the nucleus under heat shock and enhanced the effect of hyperthermia. Moreover, a novel magnetic nanoparticle was developed via binding carbon nanohorn (CNH) and iron oxide nanoparticle (IONP) with 3-aminopropylsilyl (APS). Tumor-bearing model mice implanted with 22 Rv1 cells were examined to determine the effect of magnetic HT (mHT). We locally injected CNH-APS-IONP into the tumor, which was set under an alternative magnetic field and showed that tumor growth in the treatment group was significantly suppressed compared with other groups. This study suggests that HIKESHI silencing enhances the sensitivity of 22Rv1 cells to HT, and CNH-APTES-IONP deserves consideration for mHT.

Immunoproteasome inhibition prevents progression of castration-resistant prostate cancer.

Castration-resistant prostate cancer (CRPC) is refractory to hormone treatment. This study aims to explore the effect and underlying mechanisms of immunoproteasome inhibition,a novel immunotherapy, on the progression of CRPC. The immunoproteasome subunit LMP7 was silenced by using gene knockout or inhibited by theepoxyketone inhibitor ONX 0914 in a mouse CRPC tumour graft model and in interferon-γ-pretreated human CRPC cell lines in vitro. CRPC tissues reveal a significant "tumour-elicited" Th17-type inflammatory response which induces immunoproteasome subunit expression. LMP7 deficiency in host mice or in CRPC tumour grafts had no effect on the "tumour-elicited" Th17-type inflammatory response and tumour progression. However, the selective LMP7 inhibitor ONX 0914 strongly suppressed the "tumour-elicited" Th17-type inflammatory response and CRPC tumour progression. Treatment of wild-type mice receiving LMP7-deficient CRPC tumour grafts with ONX 0914 further suggested that immunoproteasome inhibition prevents CRPC progression through suppressing IL-17-induced angiogenesis and epithelial-mesenchymal transition via inactivation of COX-2/VEGF-A signalling and β-catenin/Snail signalling. Treatment of LMP7-deficient mice receiving wild-type CRPC tumour grafts with ONX 0914 and inhibition of LMP7 in PC3 and 22Rv.1 cells with ONX 0914 showed that immunoproteasome inhibition also prevents CRPC progression through inducing CRPC cell apoptosis via activation of the unfolded protein response. We define a critical role of the immunoproteasome in CRPC and propose immunoproteasome inhibition as a promising therapeutic approach to suppress CRPC progression.

Remodeling Tumor Immune Microenvironment by Using Polymer-Lipid-Manganese Dioxide Nanoparticles with Radiation Therapy to Boost Immune Response of Castration-Resistant Prostate Cancer.

Despite substantial progress in the treatment of castration-resistant prostate cancer (CRPC), including radiation therapy and immunotherapy alone or in combination, the response to treatment remains poor due to the hypoxic and immunosuppressive nature of the tumor microenvironment. Herein, we exploited the bioreactivity of novel polymer-lipid manganese dioxide nanoparticles (PLMDs) to remodel the tumor immune microenvironment (TIME) by increasing the local oxygen levels and extracellular pH and enhancing radiation-induced immunogenic cell death. This study demonstrated that PLMD treatment sensitized hypoxic human and murine CRPC cells to radiation, significantly increasing radiation-induced DNA double-strand breaks and ultimately cell death, which enhanced the secretion of damage-associated molecular patterns, attributable to the induction of autophagy and endoplasmic reticulum stress. Reoxygenation via PLMDs also polarized hypoxic murine RAW264.7 macrophages toward the M1 phenotype, enhancing tumor necrosis factor alpha release, and thus reducing the viability of murine CRPC TRAMP-C2 cells. In a syngeneic TRAMP-C2 tumor model, intravenous injection of PLMDs suppressed, while radiation alone enhanced recruitment of regulatory T cells and myeloid-derived suppressor cells. Pretreatment with PLMDs followed by radiation down-regulated programmed death-ligand 1 and promoted the infiltration of antitumor CD8+ T cells and M1 macrophages to tumor sites. Taken together, TIME modulation by PLMDs plus radiation profoundly delayed tumor growth and prolonged median survival compared with radiation alone. These results suggest that PLMDs plus radiation is a promising treatment modality for improving therapeutic efficacy in radioresistant and immunosuppressive solid tumors.

Redox-responsive nanoparticles enhance radiation therapy by altering multifaceted radio-resistance mechanisms in human castration-resistant prostate cancer cells and xenografts

IntroductionRadiation therapy (RT) is a major modality for the treatment of prostate cancer (PCa), especially castration-resistant PCa (CRPC). However, hypoxia, often seen in PCa tumors, leads to radiation-resistance. This work investigates the effect of a novel oxygen-generating polymer-lipid manganese dioxide nanoparticle (PLMDs) on improving RT outcomes in CRPC xenograft models by modulating the tumor microenvironment (TME) both before and after RT. Materials and methodsHuman PC3 and DU145 PCa cells were used to investigate clonogenic inhibition and DNA repair pathways in vitro. Tumor hypoxia and post-RT angiogenesis were evaluated in a PC3-bearing SCID mouse model. PC3 and DU145 xenografts were used to study the efficacy of PLMD in combination with single or fractionated RT. ResultsPLMD plus RT significantly inhibited clonogenic potential, increased DNA double-strand breaks, and reduced DNA damage repair in hypoxic PC3 and DU145 cells as compared to RT alone. PLMD significantly reduced hypoxia-positive areas, hypoxia induced factor 1α (HIF-1α) expression, and protein carbonyl levels (a measure of oxidative stress). Application of PLMD with RT decreased RT-induced angiogenic biomarkers by up to 3-fold. Treatment of the human CRPC xenografts with PLMD plus RT (single or fractionated doses) significantly prolonged median survival of the host compared to RT alone resulting in up to a 40% curative rate. ConclusionPLMD treatment modulated TME and sensitized hypoxic human CRPC cells to RT thus enhancing the efficacy of RT. These results confirmed the potential of PLMD as an adjuvant to RT for the treatment of hypoxic CRPC.

Ratio of the expression levels of androgen receptor splice variant 7 to androgen receptor in castration refractory prostate cancer.

In clinical samples, the expression of androgen receptor (AR) and of AR splice variant 7 (AR-V7) is higher in castration-resistant prostate cancer (CRPC) compared with that in hormone-sensitive prostate cancer (PCa). However, there are only a few reports on the ratio of the expression levels of AR-V7 to AR (AR-V7/AR) in prostate tissue. The present study evaluated AR-V7/AR expression in various types of human prostate tissues and CRPC cells. Pretreatment prostate tissue samples from patients with benign prostatic hyperplasia (BPH; n=18), Gleason score 7 (n=17), and Gleason score 8–10 (n=26) were collected at the time of prostate biopsy, and tissue samples from CRPC patients (n=10) were collected at the time of transurethral resection of the prostate. Furthermore, androgen-independent LNCaP cells were established. The mRNA expression levels of AR and AR-V7, cell proliferation and prostate-specific antigen (PSA) production were evaluated by reverse transcription quantitative PCR, MTS assay and chemiluminescent enzyme immunoassay, respectively. There was a significant difference in AR-V7/AR expression ratios between the CRPC group and the BPH and pre-treatment PCa groups (CRPC, 7%; BPH and pre-treatment PCa, 1%). Subsequently, we compared the AR and AR-V7 expression levels in CRPC samples with those in the pretreatment prostate tissues from the same patients. The results demonstrated that the AR-V7/AR ratio increased from 3 to 9% after CRPC onset. Furthermore, in vitro experiment demonstrated that AR-V7 expression in LNCaP cells was increased after transforming into CRPC cells. The AR-V7/AR ratio also increased from 0.05 to 0.3%. In addition, small interfering (si)-RNA-mediated knockdown of AR inhibited the proliferation of and PSA production from androgen-independent LNCaP cells; however, AR-V7 knockdown had no effect. Conversely, siRNA-mediated knockdown of both AR and AR-V7 inhibited the proliferation of VCAP cells. In summary, the findings from the present study demonstrated that AR-V7 expression and AR-V7/AR ratio were increased after the onset of CRPC, which had a limited role in CRPC cell proliferation. Further investigation is required to clarify the roles of AR other splice variants and AR-V7 in CRPC.

Role of Epigenetic Therapy in the Modulation of Tumor Growth and Migration in Human Castration-Resistant Prostate Cancer Cells with Neuroendocrine Differentiation

Introduction: Neuroendocrine transdifferentiation (NED) of prostate cancer (PC) cells is associated with the development of resistance to antiandrogen therapy and poor prognosis in patients with castration-resistant PC (CRPC). Many of the molecular events, involved in NED, appear to be mediated by epigenetic mechanisms. In this study, we evaluated the antitumor activity and epigenetic modulation of 2 epigenetic drugs, such as the demethylating agent 5-aza-2′-deoxycytidine (AZA) and the methyl donor S-adenosylmethionine (SAM), in 2 human CRPC cell lines with NED (DU-145 and PC-3). Methods: The effects of AZA and SAM on cell viability, cell cycle, apoptosis, migration, and genome-wide DNA methylation profiling have been evaluated. Results: Both drugs showed a prominent antitumor activity in DU-145 and PC-3 cells, through perturbation of cell cycle progression, induction of apoptosis, and inhibition of cell migration. AZA and SAM reversed NED in DU-145 and PC-3, respectively. Moreover, AZA treatment modified DNA methylation pattern in DU-145 cells, sustaining a pervasive hypomethylation of the genome, with a relevant effect on several pathways involved in the regulation of cell proliferation, apoptosis, and cell migration, in particular Wnt/β-catenin. Conclusions: A relevant antitumor activity of these epigenetic drugs on CRPC cell lines with NED opens a new scenario in the therapy of this lethal variant of PC.

Molecular Mechanism Mediating Cytotoxic Activity of Cabazitaxel in Docetaxel-resistant Human Prostate Cancer Cells.

Cabazitaxel is known to be effective in patients with castration-resistant prostate cancer (CRPC) showing resistance to docetaxel. The objective of this study was to investigate the molecular mechanism mediating cytotoxic activity of cabazitaxel in docetaxel-resistant human CRPC cells. Parental human CRPC cell line PC3 (PC3/P) was continuously exposed to increasing doses of docetaxel, and a cell line resistant to docetaxel, PC3/R, was developed. Phenotypic differences between these cell lines were investigated. There were no significant differences in sensitivity to cabazitaxel between PC3/P and PC3/R. In PC3/P, both docetaxel and cabazitaxel markedly inhibited the phosphorylation of AKT serine/threonine kinase 1 (AKT) and p44/42 mitogen-activated protein kinase (MAPK). In PC3/R, however, phosphorylation of AKT and p44/42 MAPK were maintained following treatment with docetaxel, whereas treatment with cabazitaxel resulted in the marked down-regulation of phosphorylation of AKT but not that of p44/42 MAPK. Furthermore, additional treatment of PC3/R with a specific inhibitor of AKT significantly enhanced the cytotoxic activity of docetaxel but not that of cabazitaxel. Growth of PC3/R in nude mice after treatment with cabazitaxel was significantly inhibited compared with that after treatment with docetaxel. Antitumor activity of cabazitaxel in docetaxel-resistant CRPC cells was explained, at least in part, by the inactivation of persistently phosphorylated AKT even after treatment with docetaxel.

β-elemonic acid inhibits growth and triggers apoptosis in human castration-resistant prostate cancer cells through the suppression of JAK2/STAT3/MCL-1 and NF-ĸB signal pathways

Castration-resistant prostate cancer (CRPC) has become a significant problem in the current treatment of prostate cancer (PCa) with the characteristics of high metastatic potential, resistance and easy recurrence. The abnormal activation of JAK2/STAT3/MCL-1 and NF-κB has been confirmed as the main reason for the development of CRPC. We previously found that β-elemonic acid (β-EA) as a natural triterpene has potential anti-inflammatory and anti-osteosarcoma effects with lower toxicity. But it remains unknown whether it had effects on CRPC. The present research in vitro and in vivo systematically investigates anti-cancer effects and mechanisms of β-EA on human CRPC. β-EA treatment resulted in apoptotic cell death in human PCa cells by mitochondrial apoptotic pathways (including up-regulation of cleaved caspase-3, cleaved PARP, and Bax or down-regulation of Bcl-2). Besides, β-EA at relatively lower levels inhibited colony-forming, the migration and invasion potential of PCa cells, indicating its anti-proliferation and anti-metastasis activities. After exploring the potential mechanism, our results suggested that it subsequently inhibited the activation of JAK2/STAT3/MCL-1 and NF-κB signaling pathway by the administration of β-EA. The silencing of NF-κB/p65, JAK2 and STAT3, respectively, increased the sensitivity of the PCa cells to β-EA induced apoptosis. Moreover, β-EA exhibited a strong affinity with its essential proteins JAK2, RELA/p65, NF-κBIα/IκBα by molecular docking analysis. Importantly, β-EA retards tumor growth in a murine xenograft model, consistent with our study in vitro. Taken together, findings from this study reveal for the first time the potential role and mechanisms of β-EA on CRPC.

Identification of marker genes and cell subtypes in castration-resistant prostate cancer cells

The diverse tumor cell populations may be the critical roles in relapse and resistance to treatment in prostate cancer patients. This study aimed to identify new marker genes and cell subtypes among castration-resistant prostate cancer (CRPC) cells. We downloaded single-cell RNA seq profiles (GSE67980) from the Gene Expression Omnibus (GEO) database. Principal component (PC) analysis and t-Distributed Stochastic Neighbor Embedding (TSNE) analysis were performed to identify marker genes. CRPC cells were clustered and annotated. GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses among marker genes were performed. A total of 1500 genes with larger standardized variance were obtained. The top 20 genes were demonstrated in each identified 20 PCs. PC with P-value < 0.05 was selected, including PC1, PC7, PC8, and PC14. The TSNE analysis classified cells as two clusters. The top 6 genes in cluster 0 included HBB, CCL5, SLITRK4, GZMB, BBIP1, and PF4V1. Plus, the top 6 genes in cluster 1 included MLEC, CCT8, CCT3, EPCAM, TMPRSS2, EIF4G2. The GO analysis revealed that these marker genes were mainly enriched in RNA catabolic process, translational initiation, mitochondrial inner membrane, cytosolic part, ribosome, cell adhesion molecule binding, cadherin binding, and structural constituent of ribosome. The KEGG analysis showed that these marker genes mainly enriched in metabolism associated pathways, including carbon metabolism, cysteine and methionine metabolism, propanoate metabolism, pyruvate metabolism, and citrate cycle pathways. To conclude, our results provide essential insights into the spectrum of cellular heterogeneity within human CRPC cells. These marker genes, GO terms and pathways may be critical in the development and progression of human CRPC.

Abstract 4097: Enhanced synergistic efficacy of simvastatin and metformin on enzalutamide resistant prostate cancer cells

Abstract Androgen deprivation therapy (ADT) continues to be the mainstay therapy for advance-stage prostate cancer. Although malignant cells respond to ADT initially, subsequently colonize and re-emerge as castration-resistant prostate cancer (CRPC). Enzalutamide (ENZU), a second-generation AR antagonist exhibits survival advantage in CRPC patients, however, ~30% of patients develop resistance after ENZU treatment, activating AR in these tumors. These ENZU-resistant tumors poorly respond to chemotherapy and other treatment modalities. Therefore, identification of an effective low-cost therapeutic alternative with fewer side effects may lead to increased survival and greatly benefit the quality-of-life of these patients. Activation of aerobic glycolysis and biomass production is implicated, in part, to be responsible for CRPC cells to acquire androgen resistance. Previous studies from our laboratory have demonstrated that synergistic combination of simvastatin (SIM), a drug for the treatment of hypercholesterolemia and metformin (MET), a glucose-lowering drug inhibits CRPC growth, invasiveness and migration with minimal effect on normal prostate epithelial cells (Mol Cancer Ther. 13(10):2288-2302, 2014). Here we examine whether the combination of SIM and MET could be effective in the treatment of ENZU-resistant prostate cancer cells. Human CRPC cells viz. C4-2B and its variant C4-2B-ENZU (C4-2B enzalutamide resistant) and 22Rv1 were used for the experiments. C4-2B-ENZU cells were generated by growing C4-2B cells in 20µM ENZU over 6 months and maintained in 5µM ENZU in the cell culture medium. 22Rv1 cells were inherently resistant to ENZU treatment. Both cells were treated with SIM (4µM) and MET (2mM) individually and in combination, followed by the assessment of cell viability, crystal violet assay, cell cycle analysis, migration, invasion, and expression of various target genes by Western blotting. C4-2B-ENZU and 22Rv1 cells were treated with a combination of SIM and MET at pharmacological dose range (500nM-4µM SIM and 250µM-2mM MET). Combination treatment with 4μM SIM and 2mM MET (SIM+MET) led to significant and synergistic inhibition of cell viability, migration, invasion and cell cycle blockade in both cancer cell lines. The individual treatments of SIM and MET exhibited little or no effect on these cells. Furthermore, SIM+MET combination decreased the expression of AR, AR-V7, p-Akt (Ser473), p-AMPKα1/α2 (Ser-485/491) and simultaneously increased p-AMPKα1 (Thr-172) and AMPKα kinase activity in these cells. We demonstrate, for the first time, that synergistic combination of SIM and MET may be an effective regimen for treatment ENZU-resistant prostate cancer cells. Citation Format: Eswar Shankar, Pingfu Fu, Gregory T. MacLennan, Sanjay Gupta. Enhanced synergistic efficacy of simvastatin and metformin on enzalutamide resistant prostate cancer cells [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 4097.

Small molecule DTDQ exerts anti-metastatic effects in DU145 human castration-resistant prostate cancer cells via modulations of ERK, JNK, p38 and c-Myc signaling pathways

Small molecule is an organic compound with low molecular mass and can be used as a drug to treat cancer cells. 6,7-dimethyl-4-(3,4,5-trimethoxyphenyl)-3,4-dihydroquinolin-2(1H)-one (DTDQ) is a small molecule known to have potential anti-metastatic effects in human non-small cell lung cancer cells. Prostate cancer is one of the most common cancers in men and can progress to metastatic castration-resistant prostate cancer (CRPC) which possesses resistance to androgen deprivation therapy. In this study, we investigated the anti-metastatic effects of DTDQ in DU145 human CRPC cells. The results showed that DTDQ inhibited proliferation, migration and invasion of DU145 human CRPC cells. DTDQ suppressed activities of MMP-2 and MMP-9 of DU145 human CRPC cells via transcriptional regulation. DTDQ modulates the three major mitogen-activated protein kinases (MAPKs), ERK, JNK and p38 that are intimately associated with cancer cell metastasis. DTDQ also down-regulates c-Myc transcription factor of DU145 CRPC cells. Finally, we observed anti-metastatic effects of DTDQ in PC3 human CRPC cells, indicating that repressive effects of DTDQ are not limited to DU145 human CRPC cells. These results suggest that DTDQ may be a potential candidate of an anti-metastatic drug to treat human CRPC.

Aspalathin-rich green Aspalathus linearis extract suppresses migration and invasion of human castration-resistant prostate cancer cells via inhibition of YAP signaling

BackgroundMore than 80% of advanced prostate cancer (PCa) cases have bone metastasis, with a 5-year survival rate of 25%. Previously, we reported that GRT, a standardized, pharmaceutical-grade aspalathin-rich extract (12.78 g aspalathin/100 g extract), prepared from green rooibos produced from the leaves and fine stems of Aspalathus linearis, inhibits the proliferation of PCa cells, meriting this investigation to determine if GRT can suppress the migration and invasion of castration-resistant prostate cancer (CRPC) cells. PurposeIn the present study, we investigated whether GRT extract can interfere with the migration and invasion of human CRPC cells. MethodsTranswell assays were used to explore the effects of GRT on the migration and invasion of CRPC cells. Micro-Western Array (MWA) and Western blot analysis were carried out to unravel the underlying molecular mechanism(s). ResultsTreatment with 25–100 μg/ml GRT suppressed the migration and invasion of LNCaP C4-2B and 22Rv1 CRPC cells. MWA and Western blot analysis indicated that GRT treatment suppressed the protein level of yes-associated protein (YAP), macrophage stimulating 1 protein (MST1), phospho-MST1/phospho-MST2 T183/T180, and paxillin, but increased the abundance of E-cadherin. Over-expression of YAP rescued the suppressive effects of GRT on migration and invasion of CRPC cells. Treatment with the major flavonoid of GRT — the C-glucosyl dihydrochalcone, aspalathin — at a concentration of 75–100 μg/ml also reduced the migration and invasion of CRPC cells, and the inhibition was partially rescued by YAP over-expression. ConclusionsGRT treatment suppresses the migration and invasion of CRPC cells via inhibition of YAP signaling and paxillin.

γ-Klotho is correlated with resistance to docetaxel in castration-resistant prostate cancer.

The Klotho (KL) gene was first identified as a potent aging suppressor. The KL family currently comprises of three proteins: α-Klotho (KLA), β-Klotho (KLB), and γ-Klotho (KLG). Many studies have shown that KLA and KLB participate in tumor progression or suppression, depending on the type of cancer; however, the relationship between KLG and prostate cancer has not yet been studied. Some studies have claimed that KL is correlated to sensitivity to chemotherapy. Here, we investigated the oncogenic potential of KLG in castration-resistant prostate cancer (CRPC). Immunohistochemical analysis using prostate biopsy specimens revealed that patients with high KLG expression in primary prostate cancer tissue had a significantly poor prognosis for overall survival. In addition, the prostate-specific antigen response rate after docetaxel (DTX) therapy in patients with high KLG expression was lower than that in patients with low KLG expression. To evaluate the potential of KLG as a therapeutic target in human prostate cancer, we generated a xenograft model of human CRPC cell line (PC-3) in male athymic mice. The animals were randomly divided into four groups as follows: i) control group (vehicle only); ii) DTX group (intraperitoneal administration); iii) small interfering RNA targeting KLG (KLG siRNA) group (intratumoral administration); and iv) a combination group (DTX plus KLG siRNA). After 3 weeks of treatment, the tumor weight and tumor Ki-67 labeling index were significantly lower in the KLG siRNA group and the combination group than in the control group. Sensitivity to DTX was increased upon treatment with KLG siRNA. These findings suggest that KLG expression in primary prostate cancer lesions is associated with resistance to DTX in CRPC and has potential as a diagnostic and therapeutic target for patients with CRPC.

Recruitment of miR-8080 by luteolin inhibits androgen receptor splice variant 7 expression in castration-resistant prostate cancer.

A need exists for seeking effective treatments for castration-resistant prostate cancer (CRPC) in response to its emergence following androgen deprivation therapy as a major clinical problem. In the present study, we investigated the chemopreventive and chemotherapeutic potential of luteolin, a flavonoid with antioxidative properties, on prostate cancer, including CRPC. Luteolin inhibited the progression of rat prostate carcinogenesis by induction of apoptosis in a transgenic rat for adenocarcinoma of prostate (TRAP) model. Luteolin decreased cell proliferation in a dose-dependent manner and induced apoptosis with the activation of caspases 3 and 7 in both rat (PCai1, established from a TRAP prostate tumor) and human (22Rv1) CRPC cells. Dietary luteolin also suppressed tumor growth via an increase in apoptosis and inhibition of angiogenesis in PCai1 and 22Rv1 xenografts implanted in castrated nude mice. We also focused on androgen receptor splice variant 7 (AR-V7), which contributes to cell proliferation and therapeutic resistance in CRPC. Luteolin dramatically suppressed AR-V7 protein expression in 22Rv1 cells in vitro and ex vivo. Microarray analysis identified MiR-8080, which contains a possible target sequence for AR-V7 3′-UTR, as a gene upregulated by luteolin. MiR-8080 transfection decreased the AR-V7 expression level and the induction of apoptosis in 22Rv1 cells. Furthermore, miR-8080 knockdown canceled luteolin decreasing AR-V7 and the cell growth of 22Rv1. MiR-8080 induced by luteolin intake enhanced the therapeutic effect of enzalutamide on 22Rv1 xenografts under castration conditions. These results indicate luteolin inhibits CRPC by AR-V7 suppression through miR-8080, highlighting luteolin and miR-8080 as promising therapeutic agents for this disease.

Successful Targeting of the Warburg Effect in Prostate Cancer by Glucose-Conjugated 1,4-Naphthoquinones.

Treatment of castration-resistant prostate cancer (CRPC) remains challenging due to the development of drug resistance. The Warburg effect describes the ability of cancer cells to consume larger amounts of glucose compared to normal tissues. We identified derivatives of natural 1,4-naphthoquinones to be active in CRPC and further synthetically modified them via glucose conjugation to increase selectivity by Warburg effect targeting. Mechanisms of action were examined by quantitative proteomics followed by bioinformatical analysis and target validation. Four synthesized molecules revealed the highest selectivity towards human CRPC cells, which correlated with higher GLUT-1 activity and expression. The compounds were able to induce pro-apoptotic signs and to inhibit the pro-survival processes and mechanisms of drug resistance (i.e., AR-signaling and autophagy). Proteome analysis suggested a disruption of the mitochondria/oxidative phosphorylation, which was validated by further functional analysis: thus, mitochondria depolarization, elevated levels of cytotoxic ROS, an increase of Bax/Bcl-2 ratio as well as release of mitochondrial AIF and cytochrome C to cytoplasm were observed. In conclusion, glucose-conjugated 1,4-naphthoquinones show potent activity and selectivity in human CRPC exerted via mitochondrial targeting. The compounds can overcome drug resistance against current standard therapies and suppress pro-survival mechanisms. This unique combination of properties makes them new promising candidates for the treatment of CRPC.

Key Genes And Pathways Controlled By E2F1 In Human Castration-Resistant Prostate Cancer Cells.

BackgroundTreatment of castration-resistant prostate cancer (CRPC) is an enormous challenge. As E2F transcription factor 1 (E2F1) is an essential factor in CRPC, this study investigated the genes and pathways controlled by E2F1 and their effects on cellular behavior in CRPC.MethodsIn vitro assays were used to evaluate cellular proliferation, apoptosis, and behavior. Cellular expression was quantified by RNA sequencing (RNA-seq). Gene co-expression was assessed using the GeneMANIA database, and correlations were analyzed with the GEPIA server. Altered pathways of differentially expressed genes (DEGs) were revealed by functional annotation. Module analysis was performed using the STRING database and hub genes were filtered with the Cytoscape software. Some DEGs were validated by real-time quantitative PCR (RT-qPCR).ResultsKnockdown of E2F1 significantly inhibited proliferation and accelerated apoptosis in PC3 cells but not in DU145 cells. Invasion and migration were reduced for both cell lines. A total of 1811 DEGs were identified in PC3 cells and 27 DEGs in DU145 cells exhibiting E2F1 knockdown. Ten overlapping DEGs, including TMOD2 and AIF1L, were identified in both knockdown cell lines and were significantly enriched for association with actin filament organization pathways. TMOD2 and KREMEN2 were genes co-expressed with E2F1; six overlapping DEGs were positively correlated with transcription factor E2F1. DEGs of the PC3 and DU145 groups were associated with multiple pathways. Five DEGs that overlapped between the two cell lines and three hub DEGs from PC3 cells were validated by RT-qPCR.ConclusionThe results of this study suggest that E2F1 has a critical role in regulating actin filaments, as indicated by the change in expression level of several genes, including TMOD2 and AIF1L, in CRPC. This extends our understanding of the cellular responses affected by E2F1 in CRPC.

Abstract 339: Mechanisms of resistance to poly (ADP-ribose) polymerase inhibitors in prostate cancer

Abstract Background: Poly (ADP-ribose) polymerase inhibitors (PARPi) have demonstrated promise in treating cancers with DNA damage repair (DDR) gene abnormalities. As a result, olaparib and more recently, rucaparib have been given Breakthrough Therapy designation by the FDA for use in metastatic castration resistant prostate cancer (mCRPC) with BRCA1/2 and ATM mutations. One of the problems encountered with PARPi is drug resistance which generally limits drug efficacy. Mechanistic studies on PARPi resistance have shown one of the main mechanisms of acquired resistance to be a reversion mutation of BRCA1/2. However, this is likely more applicable to cancers in which impaired BRCA1/2 function is due to a mutation rather than in prostate cancer (PC) where BRCA2 tends to be frequently deleted. Therefore, we hypothesize that mechanisms of resistance to PARPi in PC may involve alternative molecular mechanisms rather than a reversion mutation. Methods: We used human castration-resistant PC cell lines that harbor genomic deletions of BRCA2, PC-3 and LnCaP-Abl, and performed cell viability (MTT) assays to determine the inhibitory growth (IG) concentrations of these cell lines with talazoparib and olaparib. We cultured parental PC-3 cells in sublethal concentrations (IG 50% and IG 90%) of talazoparib-supplemented media to develop talazoparib-resistant cells. RNA sequencing followed by gene-set enrichment analysis (GSEA) of hallmark gene sets was performed on the talazoparib-resistant PC-3 cells to understand the underlying molecular mechanisms. Results: We observed that the talazoparib-resistant PC-3 cells exhibited significantly enhanced cell growth compared to parental cells when cultured in the IG 90% concentration of olaparib. However, interestingly, the talazoparib-resistant cells grew much slower in 2D compared to parental PC-3 cells when cultured in the PARPi-free media. Our transcriptomic analysis showed significant enrichment of various inflammatory response pathways, including TNF-α and IFNα/γ signaling pathways, in the talazoparib-resistant cells and even in the parental PC-3 cells transiently treated with talazoparib. Conclusion: We hypothesize that resistance to PARPi in PC may be related to upregulation of inflammatory signaling. Therefore, further exploration of TNF-α and IFNα/γ and their role in PARPi resistance mechanisms may lead to the identification of targets that allow for overcoming PARPi resistance in PC. Citation Format: Mohammad Atiq, Goutam Chakraborty, Subhiksha Nandakumar, Ying Z. Mazzu, Konrad Stopsack, Yuki Yoshikawa, Nabeela Khan, Gwo-Shu Mary Lee, Philip W. Kantoff. Mechanisms of resistance to poly (ADP-ribose) polymerase inhibitors in prostate cancer [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 339.