Resistant Prostate Cancer Cells Research Articles

MicroRNA-122 regulates docetaxel resistance of prostate cancer cells by regulating PKM2.

Prostate cancer (PCa), an epithelial malignancy that occurs in the prostate, is the second leading cause of cancer death worldwide. MicroRNAs (miRs/miRNAs) are reported to have important applications in the field of cancer diagnosis and treatment. The present study aimed to investigate the function of miRNA-122 in the chemoresistance of PCa cells and the underlying mechanism. Significantly decreased miR-122 and increased pyruvate kinase (PKM2) levels were observed in docetaxel-resistant PCa cells, and PKM2 was negatively correlated with miR-122. MiR-122 mimic transfection in docetaxel-resistant LNCaP cells significantly inhibited cell proliferation, promoted apoptosis and decreased glucose uptake and lactate production, which was counteracted by PKM2 overexpression. Inhibition of miR-122 in LNCaP cells had an opposite effect to miR-122 mimic transfection. In addition, miR-122 mimic transfection significantly increased the sensitivity of docetaxel-resistant LNCaP cells to docetaxel, while inhibition of miR-122 significantly decreased the sensitivity of LNCaP cells to docetaxel. Luciferase reporter assays showed that miR-122 regulated PKM2 expression by binding to the 3'-untranslated region of PKM2. The results suggest that upregulation of miR-122 could enhance docetaxel sensitivity, inhibit cell proliferation and promote apoptosis in PCa cells,possibly through the downregulation of its target protein PKM2.

Single-Cell Transcriptomics Analysis Identifies Nuclear Protein 1 as a Regulator of Docetaxel Resistance in Prostate Cancer Cells.

The majority of patients with prostate cancer treated with docetaxel develop resistance to it. To better understand the mechanism behind the acquisition of resistance, we conducted single-cell RNA-sequencing (scRNA-seq) of docetaxel-sensitive and -resistant variants of DU145 and PC3 prostate cancer cell lines. Overall, sensitive and resistant cells clustered separately. Differential gene expression analysis between resistant and sensitive cells revealed 182 differentially expressed genes common to both prostate cancer cell lines. A subset of these genes gave a gene expression profile in the resistant transcriptome-like-sensitive cells similar to the resistant cells. Exploration for functional gene pathways identified 218 common pathways between the two cell lines. Protein ubiquitination was the most differentially regulated pathway and was enriched in the resistant cells. Transcriptional regulator analysis identified 321 potential regulators across both cell lines. One of the top regulators identified was nuclear protein 1 (NUPR1). In contrast to the single-cell analysis, bulk analysis of the cells did not reveal NUPR1 as a promising candidate. Knockdown and overexpression of NUPR1 in the prostate cancer cells demonstrated that NUPR1 confers docetaxel resistance in both cell lines. Collectively, these data demonstrate the utility of scRNA-seq to identify regulators of drug resistance. Furthermore, NUPR1 was identified as a mediator of prostate cancer drug resistance, which provides the rationale to explore NUPR1 and its target genes for reversal of docetaxel resistance. IMPLICATIONS: Using single-cell sequencing of prostate cancer, we show that NUPR1 plays a role in docetaxel resistance.

Bubble Bubble, Senescent Cells Are a Cauldron of Tumor Trouble.

Senescent cells release a mélange of factors that drive multiple forms of pathology, including cancer aggressiveness. In this issue of Cancer Research, Han and colleagues show that small extracellular vesicles (sEV), membrane-enclosed bubbles that carry signaling molecules, from senescent stromal cells can promote tumorigenesis and multidrug resistance in prostate or breast cancer cells. They find that loss of SIRT1 activity drives senescence-associated sEV release, and treatment with a SIRT1 agonist prevented this effect. This adds another mechanism by which senescent cells can promote tumorigenesis and offers another activity of senescent cells that might be targeted to limit the spread of cancer.See related article by Han et al., p. 3383.

Abstract 5287: TGF-β-acetylated KLF5-Bcl-2 signaling mediates docetaxel resistance in prostate cancer cells

Abstract Purpose: In this study, we explored the role of KLF5 in TGF-β-induced drug resistance in prostate cancer cells to identify novel therapeutic targets. Procedures: We used DU 145 and PC-3 prostate cancer cell lines in in vitro cell survival assay, colony survival assay, and xenograft mouse model to measure docetaxel (DTX) resistance. TGF-β1, the SB505124 TGF-β receptor I inhibitor, and the ABT-199 Bcl-2 inhibitor were used to determine if Bcl-2 mediates TGFβ induced docetaxel resistance. To measure the function of KLF5 and its acetylation at lysine 369 in drug resistance, stable cell lines were constructed with different forms of KLF5 after the endogenous KLF5 was deleted with the CRISPR-Cas9 system. Results: We found that TGF-β induced DTX resistance depends on KLF5 expression. As a downstream molecule of TGF-β signaling, acetylated KLF5 at lysine 369 mediated TGF-β-induced DTX resistance. Moreover, acetylated KLF5 induced DTX resistance in xenograft mouse model. Mechanistically, we found that TGF-β and acetylated KLF5 upregulated Bcl-2 transcription, and inhibition of Bcl-2 sensitized cells to DTX. In addition, DTX promoted Bcl-2 degradation via a proteasome dependent pathway, and TGF-β signaling stabilized Bcl-2 via inhibition of Bcl-2 ubiquitination. Lastly, the TGF-β/acetylated KLF5/Bcl-2 signaling axis induced survival disadvantage in prostate cancer patients. Conclusions: TGF-β induces transcriptional upregulation of Bcl-2 through acetylated KLF5 at lysine 369, and TGF-β stabilizes Bcl-2 via inhibition of DTX-induced Bcl-2 ubiquitination. The TGF-β-acetylated KLF5-Bcl-2 signaling axis is thus a novel mechanism mediating DTX resistance in prostate cancer. Citation Format: Yixiang Li, Baotong Zhang, Jin-Tang Dong. TGF-β-acetylated KLF5-Bcl-2 signaling mediates docetaxel resistance in 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 5287.

Abstract 3352: Tumor dependent co-stimulation of CD137/4-1BB in PSMA positive tumors: Preclinical characterization of CB307, a half-life extended PSMAxCD137 bispecific Humabody therapeutic

Abstract Introduction: Agonistic monoclonal antibodies targeting CD137/4-1BB have shown much preclinical promise but their clinical development has been slow due to treatment associated toxicities, in particular transaminitis and liver toxicity. We have developed CB307, a half-life extended bispecific Humabody VH co-targeting CD137 and the tumor associated antigen prostate specific membrane antigen (PSMA). PSMA is highly expressed on tumor cells in a number of cancer indications including metastatic castrate resistant prostate cancer (mCRPC) and non-small cell lung cancer, additionally PSMA expression is increased on the neovasculature of these tumors and in other solid tumors including renal cell carcinoma. Methods and Results: CD137 is a co-stimulatory receptor expressed on the surface of activated CD4+ and CD8+ T cells and NK cells. Using the Crescendo Mouse™, a transgenic platform which develops fully human VH domains in a background devoid of light chains, we have generated a half-life extended bispecific therapeutic (CB307) which has been specifically designed to enable agonism of CD137 positive cells in the tumor microenvironment of PSMA positive tumors and not in tissues where PSMA is absent. We hypothesize that the PSMA-dependent activation of CD137 signalling will enable durable anti-tumor immune responses with a more favorable therapeutic index compared to first generation monospecific CD137 antibodies. CB307 demonstrates PSMA-dependent T-cell activation in vitro as demonstrated by stimulation of T cell cytokine release, proliferation, cytotoxicity and increased expression of the pro-survival signalling molecule Bcl-XL. Cytokine production is further enhanced by combination with an anti-PD-1 antagonist antibody. In vivo, a surrogate bispecific increases CD8 T cell numbers in a prostate cancer tumor cell line model and mediates significant tumor growth delay. The serum half-life of CB307 has been extended by the inclusion of a third VH domain with specificity for human serum albumin. This has resulted in a serum half-life in cynomolgus monkey of 1.5 days (following a single i.v. dose of 3 mg/kg). In a four week, non-GLP, repeat dose toxicity study where CB307 was administered to cynomolgus monkeys at either 50 mg/kg or 160 mg/kg CB307 was well tolerated with no clinical signs, no effect on body weight or food consumption and no injection site observations. Importantly, no significant increases in ALT or AST were noted in the animals and no findings were observed on histopathology. Conclusions: There is a high unmet medical need for well tolerated and efficacious immunotherapy approaches in PSMA positive tumors such as mCRPC. We have shown that CB307, a half-life extended PSMAxCD137 bispecific delivers potent PSMA-targeted T cell activation. Non-clinical development activities for CB307 are ongoing in preparation for first in human studies. Citation Format: James William Legg. Tumor dependent co-stimulation of CD137/4-1BB in PSMA positive tumors: Preclinical characterization of CB307, a half-life extended PSMAxCD137 bispecific Humabody therapeutic [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 3352.

Abstract 2015: A transcriptionally enhanced biosensor to detect and monitor biopsy-dissociated primary prostate cancer single cell drug responses by bioluminescence microscopy

Abstract Background: In the past decade, the therapeutic landscape of metastatic castration resistant prostate cancer (mCRPC) has been transformed with the introduction of novel pharmaceutical agents including novel antiandrogens (nAA) such as darolutamide, enzalutamide or apalutamide. Currently, the management of mCRPC is mainly based on biomarkers obtained from the patient's blood or bulk tumor samples, which do not take into account for prostate cancer (PCa) cell heterogeneity. Objectives: To develop a bioluminescence imaging technology enabling: 1) the detection of PCa single cells from dissociated biopsies and 2) single cell nAA sensitivity assessment. Methods: The PCA3 promoter (PCa specific) and PSEBC promoter (androgen receptor-driven to monitor response to nAA therapy) have been incorporated in the non-replicating adenoviral multi-promoter integrated two-step transcriptional amplification system (MP-ITSTA) that allows imaging of complementary activities of two different promoters by a single output reporter gene. Thus, PCA3/PSEBC-ITSTA system was designed to monitor androgen receptor (AR) activity specifically within each single PCa cell by bioluminescence microscopy. Prostatic biopsies were dissociated in the presence of collagenase II and DNase for 18h. PCa cell lines or fresh biopsies-dissociated cancer cells were transduced for 72h and then covered with an extracellular matrix gel. Single cell bioluminescence microscopy images were done before and after exposure to DHT or DHT+AA to specifically monitoring therapy response. Changes in cell number and luminescence intensity after AA were normalized to the DHT group to exclude non-specific cell-death due to infection or time spent in culture. Results: PCA3/PSEBC-ITSTA was specific to PCa cell lines. By determining the ratio of AR active cells before and after DHT or DHT+AA treatment in culture, PCA3-Cre-PSEBC-ITSTA was able to determine the AA sensitivity of LNCaP (sensitive), LAPC4 (moderately resistant) and 22Rv1 (resistant) PCa cell lines. Also, our method could detect and monitor AA sensitivity of primary PCa cells harvested from eight radical prostatectomy specimens. PCA3/PSEBC-ITSTA could dynamically quantify AR transcriptional activity during enzalutamide or bicalutamide treatments, in a dose dependent manner, and could unveil tumor AA response heterogeneity. As expected, inhibition of AR activity was stronger with enzalutamide than with bicalutamide. Conclusions: Our bioluminescence microscopy-based biosensor could detect primary PCa cells in culture and it allows dynamic evaluation of AA sensitivity at a single-cell level in a heterogeneous cell population harvested from radical prostatectomy biopsies. We believe our approach opens to a novel field of treatment response predictive tools for cancer treatment decision-making. Citation Format: Audrey Champagne, Pallavi Jain, Bertrand Neveu, Frédéric Pouliot. A transcriptionally enhanced biosensor to detect and monitor biopsy-dissociated primary prostate cancer single cell drug responses by bioluminescence microscopy [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 2015.

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.

Abstract 6204: Prostate cancer exosomes alter extracellular matrix composition in bone marrow and enhance metastasis

Abstract Introduction: Exosomes produced by neoplastic cells can promote the development of a pro-tumorigenic microenvironment via intercellular communication, and in some cases promote metastasis. The role of exosomes in the spread of prostate cancer, however, remains poorly understood. Here, we show that exosomes derived from enzalutamide resistant prostate cancer cells (EnzR exos) mediate intercellular communication with bone marrow-resident cells, alter the expression of extracellular matrix (ECM) proteins, and enhance metastasis in a clinically relevant mouse model of metastatic prostate cancer. Methods: Exosomes were isolated via ultracentrifugation of conditioned media from enzalutamide resistant CWR-R1 cells. Mice were primed with exosomes via three systemic injections of 10 ug exosomal protein. Exosome-mediated changes in bone marrow ECM composition were assessed via RNA sequencing of CD11b+ bone marrow cells and immunohistochemistry of formalin-fixed, decalcified, paraffin-embedded mouse femurs and tibias. For metastasis studies, intracardiac injection of luciferase-expressing enzalutamide resistant CWR-R1 cells into C.B.-17 SCID mice was used to establish metastatic tumor burden, and animals were monitored via bioluminescence imaging. Exosomal RNA sequencing was performed on EnzR exos and clinical specimens, with exosomes from normal prostate epithelial cells (PNT2) used as a control. Results: Mice primed with EnzR exos developed significantly enhanced metastatic tumor burden compared to unprimed controls. EnzR exo priming also led to decreased expression of thrombospondin-1 in the bone marrow compartment, and increased expression of versican, as determined by RNA sequencing and immunohistochemistry. Exosomal RNA sequencing revealed that five miRNAs were significantly enriched in EnzR exos compared to PNT2 exosome controls. One of these, miR-4443, was found to target thrombospondin-1. Prostate cancer patient serum samples were also enriched in miR-4443 over healthy controls, and exosomal miR-4443 correlated with disease progression. Conclusions: These data indicate that exosomes derived from enzalutamide resistant prostate cancer cells can alter ECM composition in the bone marrow compartment in a pro-tumorigenic manner. Evidence for the clinical relevance of these data was observed in both serum samples and bone biopsies from prostate cancer patients, demonstrating abundant serum exosomal miR-4443 and similarly altered bone marrow ECM composition in advanced prostate cancer patients. Exosomal miR-4443 may be a promising diagnostic and prognostic marker, as well as a potential therapeutic target. Citation Format: Stephen E. Henrich, Kaylin McMahon, Michael Plebanek, Fabio Tavora, Andre De Souza, Anthony Mega, Benedito Carneiro, C. Shad Thaxton. Prostate cancer exosomes alter extracellular matrix composition in bone marrow and enhance 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 6204.

Abstract 4688: Interaction of LEDGF/p75 with oncogenic transcription factors in docetaxel resistant prostate cancer cells

Abstract Prostate Cancer (PCa) progression leads to an advanced stage called metastatic castration PCa (mCRPC), for which currently there is no cure in spite of advances in treatment with new generation anti-androgen drugs and chemotherapy with taxanes such as docetaxel (DTX). Our group demonstrated previously that the stress oncoprotein Lens Epithelium Derived Growth Factor of 75 kD (LEDGF/p75) is upregulated in clinical prostate tumors and contributes to DTX-resistance in mCRPC cells. However, little is known about the molecular mechanisms by which LEDGF/p75 promotes taxane resistance. The C-terminus of LEDGF/p75 contains a domain called the Integrase Binding Domain (IBD), which in T cells is responsible for tethering the HIV-integrase complex to transcriptionally active chromatin. In non-PCa cells, the LEDGF/p75 IBD interacts with transcription complexes, such as Menin-MLL and the c-MYC binding protein JPO2, to promote cell survival. However, the relevance of these and others protein-protein interactions (PPIs) in the context of PCa chemoresistance has never been explored. This study was designed to test the hypothesis that interactions between LEDGF/p75, the c-MYC binding protein JPO2, Menin and MLL in DTX-resistant mCRPC cells contribute to chemoresistance. We demonstrated by immunoblotting a significant co-upregulation of JPO2, c-MYC, Menin, MLL, and LEDGF/p75 in the DTX-resistant PC3-DR and DU145-DR cell lines, compared to the parental, drug sensitive cells. We also observed nuclear co-localization of these proteins by immunofluorescence microscopy and confocal microscopy. Using an immunoprecipitation approach, we confirmed that endogenous JPO2, c-MYC, Menin, and MLL co-immunoprecipitate with LEDGF/p75 in the DTX-resistant PCa cell lines. We are extending these studies to other known interacting partners of LEDGF/p75 to determine if they are also upregulated in DTX-resistant PCa cells and interact with this protein in the chemoresistance context. In addition, we have initiated studies to evaluate the effects of pharmacologic and genetic inhibition of LEDGF/p75 and its interacting partners on sensitization of DR cells to DTX. We predict that targeting LEDGF/p75 PPIs will disrupt transcriptional activity contributing to DTX resistance. Our goals are to establish the contribution of these PPIs to LEDGF/p75-mediated transactivation of DTX resistance-associated genes, and target these interactions to overcome PCa chemoresistance. Citation Format: Greisha L. Ortiz Hernandez, Evelyn S. Sanchez-Hernández, Carlos A. Casiano. Interaction of LEDGF/p75 with oncogenic transcription factors in docetaxel 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 4688.

Abstract 4850: Targeting prostate cancer with the CDK4 and CDK6 inhibitor abemaciclib

Abstract Background: Prostate cancer (PCa) is a leading cause of cancer death in men and represents a substantial public health burden [1]. Most PCa are primarily dependent on androgen receptor (AR) activity and castration is an effective approach to treat PCa patients. Despite the recent significant treatment advances, PCa inevitably becomes androgen-independent and progresses to the castration-resistant disease state (CRPC), the deadliest form of the disease [2]. Progression of the disease to castration-resistance is often mediated by a reactivation of AR signaling pathway [3]. Upon androgen stimulation, expression of D-type cyclin is up-regulated which results in an increased cyclin-dependent kinase 4 and 6 (CDK4/6) activity and stimulation of the cell cycle. [4]; Thus, inhibition of CDK4/6 may represent an effective strategy to delay or overcome primary androgen resistance. Abemacicilb is a CDK4 and CDK6 inhibitor with a clinical safety profile allowing continuous dosing to achieve sustained target inhibition [5]. Abemaciclib is FDA-approved for the treatment of patients with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer [6,7]. Methods: The anti-proliferative activity of the abemaciclib was evaluated using iodide staining in a panel of 15 PCa cell lines. In order to get new insights on abemaciclib effects, deeper in vitro analysis was carried out in LNCaP, PC-3 and 22RV1, as ADT responding and resistant PCa cell models, respectively. Cell cycle analysis was done by FACS and High Content Imaging; cellular signaling was assessed by Western blotting. Apoptosis was measured by detection of caspase 3 and Tunnel assay. 22RV1 xenograft mouse model was used to evaluate abemaciclib efficacy in vivo. Results: Anti-proliferative activity of abemaciclib was observed across a panel of PCa cell lines, mainly in hormone receptor positive (AR+) cell lines. Overall, abemaciclib efficiently inhibited CDK4 and CDK6 which prevented the phosphorylation of Rb with the consequent effect in cell cycle and induced a G1 cell cycle arrest. Prolonged treatment promoted a marked senescence phenotype indicated by an increased b-galactosidase staining and morphological changes to result ultimately in apoptosis. In 22RV1 xenograft models, abemaciclib significantly reduced tumor growth. Taken together these data provide insights on sensitivity of PCa models to abemaciclib and its mode of action, demonstrating the potential of this drug for the treatment of prostate cancer patients. Conclusions: Abemaciclib inhibits proliferation of AR positive prostate cancer cell lines by inducing cell cycle arrest mediated by inhibition of Rb phosphorylation. Abemaciclib is a CDK4/6 inhibitor with potential to treat prostate cancer by blocking cell proliferation leading to induction of senescence and apoptosis. Citation Format: Raquel Torres-Guzmán, Carmen Baquero, Maria Patricia Ganado, Carlos Marugán, Huimin Bian, Yi Zeng, Ramón Rama, Jian Du, Maria José Lallena. Targeting prostate cancer with the CDK4 and CDK6 inhibitor abemaciclib [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 4850.

Abstract 4098: Topotecan-mediated inhibition of HIF1α/ARV7 heterodimers in 22RV1 prostate cancer cells prevents ARV7 nuclear entry, reversing enzalutamide resistance

Abstract Background: Although enzalutamide (ENZ) is an effective treatment for prostate cancer (PC), development of ENZ resistance is inevitable. One mechanism for resistance is upregulation of ARV7, a splice variant of the full-length androgen receptor (FLAR) that lacks the androgen binding and hinge region domains. Nuclear translocation of FLAR requires the hinge region for coupling to tubulin or importin. ARV7 gains nuclear entry independent of the canonical pathway. FLAR has been reported to bind to Hypoxia Inducible Factor-1α (HIF1α) under normoxic conditions in the presence of dihydrotestosterone (DHT). We hypothesized that ARV7 may also heterodimerize with HIF1α in the cytoplasm, enabling nuclear localization and transcriptional activity of both transcription factors. We reported that topotecan (TPT) blocks HIF1α mRNA translation by targeting topoisomerase I (TOPO1) resident on HIF1α mRNA. We determined if ARV7 was bound to HIF1α in ENZ resistant cells and if inhibition of HIF1α translation by topotecan (TPT) could block ARV7 nuclear localization, thereby resensitizing ARV7 expressing cells to ENZ. We also evaluated if TOPO1 resided on FLAR/ARV7 mRNA, providing an additional target for TPT. Methods: 22Rv1 ENZ resistant PC cells that express FLAR and ARV7 protein were treated with the hypoxia mimetic cobalt chloride to stabilize HIF1α. Cytoplasmic and nuclear extracts from 22Rv1 cells were used for co-immunoprecipitation (Co-IP) using anit-HIF1α antibody. The effect of DHT, ENZ, and TPT on HIF1α and AR/ARV7 expression, and interactions between Topo-1 and HIF1α and AR mRNA were determined by western blot and RNA-IP. The effects of ENZ and TPT on 22Rv1 cell viability were evaluated using the XTT viability assay. Results: Topo-I RNA IP demonstrated its linkage to HIF-1α, FLAR and ARV7 mRNA. Co-IP of 22Rv1 whole cell lysates and nuclear extracts with HIF1α-specific antibodies yielded both full length AR and ARV7 protein bands on western blots under normoxic conditions in the presence of DHT and under hypoxic conditions. TPT and ENZ treatment of 22Rv1 cells under hypoxic conditions resulted in significantly reduced HIF1α, FLAR and ARV7 nuclear localization. TPT had an IC50 of 20 nM in normoxia and 91 nM in hypoxia. ENZ had an IC50 of 24.4 uM in normoxia and 388.7 uM in hypoxia. Dose-response curves for TPT / ENZ combinations at concentrations achieved clinically demonstrated synergistic activity, with combination index values of 0.54 under normoxic and 0.19 under hypoxic conditions. Conclusions: Our data indicate that Topo I resides on HIF-1α, FLAR and ARV7 mRNA, providing a potential target for TPT-mediated inhibition of their translation. Clinically achievable TPT concentrations caused HIF1α knockdown in hypoxic 22Rv1 cells in association with diminished nuclear localization of ARV7. 22Rv1 cells showed a synergistic dose-response to the combination of TPT and enzalutamide. These findings support exploration of TPT in combination with ENZ for treatment of PC. Citation Format: John P. Fruehauf, Diana C. Gomez, Leslie Spitalny, Jai-Hyun Kim. Topotecan-mediated inhibition of HIF1α/ARV7 heterodimers in 22RV1 prostate cancer cells prevents ARV7 nuclear entry, reversing enzalutamide resistance [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 4098.

Sea buckthorn (Hippophae rhamnoides L.) inhibits cellular proliferation, wound healing and decreases expression of prostate specific antigen in prostate cancer cells in vitro

Abstract Androgen receptor (AR) is a ligand-dependent transcription factor that governs the expression of androgen-responsive genes. If somehow AR is retained in the cytoplasm and its shuttling into the nucleus is inhibited it cannot activate the target androgen responsive genes and prevent prostate cancer progression. The primary goal of our study was to evaluate plant based functional food extract from sea buckthorn (Hippophae rhamnoides L.) that can target AR nuclear localization in prostate cancer cells and inhibit prostate cancer growth in vitro. Differential extraction using various solvents based on polarity revealed that the end phase aqueous cocktail extracted from leaves of Hippophae rhamnoides L. (SKICDDL-3) can effectively target AR and downregulate androgen responsive genes, PSA, ELL2, EAF2 and CALR significantly in vitro. Colony formation Unit assay and Wound healing assay further shows that SKICDDL-3 can effectively inhibit proliferation and migration of castration resistant C4-2 prostate cancer cells in vitro.

PT092 - SPINK1 promotes castration resistance in prostate cancer cells

PT092 - SPINK1 promotes castration resistance in prostate cancer cells

Design and Synthesis of a Trifunctional Molecular System "Programmed" to Block Epidermal Growth Factor Receptor Tyrosine Kinase, Induce High Levels of DNA Damage, and Inhibit the DNA Repair Enzyme (Poly(ADP-ribose) Polymerase) in Prostate Cancer Cells.

Resistance to chemotherapy in advanced cancers can be mediated by different factors such as epidermal growth factor receptor (EGFR) overexpression and DNA repair enzymes. Therefore, current standards of care usually involve combinations of multiple treatments. Here, to reduce the adverse effects of multiple drug combinations and improve outcome, we proposed a single drug approach to block multiple overlapping effects that characterize chemoresistance. Thus, we designed a new linker that allows assembly of multiple functions (e.g., inhibition of EGFR phosphorylation, induction of DNA lesions, and blockade of their repair) into a single molecule. This led to the successful synthesis of a novel and potent combi-molecule JS230. Here, we demonstrated that in resistant prostate cancer cells overexpressing EGFR, it was capable of (a) inhibiting EGFR in a dose-dependent manner, (b) damaging DNA, and (c) sustaining the damage by inhibiting the DNA repair protein poly(ADP-ribose) polymerase (PARP). The triple mechanism of action of JS230 cumulated into growth inhibitory potency superior to that of classical two- or three-drug combinations.

Abstract D116: Glucocorticoid receptor interacts with beta-catenin to promote stemness and therapy resistance in prostate cancer cells

Abstract Prostate cancer (PCa) is the most commonly diagnosed male cancer, with men of African ancestry showing a disproportionately high incidence and mortality. There is increasing evidence that glucocorticoid signaling through glucocorticoid receptor (GR) is amplified in African American men due to cumulative life stress, and that GR signaling is a key driver of resistance to androgen-targeted therapy, radiotherapy, and taxane chemotherapy in advanced PCa. Recent studies indicate that GR also promotes in PCa cells the formation of tumorspheres, a property of cancer stem cells (CSC); however, the mechanisms remain unclear. CSCs are intrinsically resistant to therapies due to their low-frequency, expression of transporters and efflux pumps, quiescent cell cycle and metabolic profile. Our previous studies demonstrated that docetaxel (DTX)-resistant PCa cells exhibit increased capacity for CSC-like properties, suggesting that these properties contribute to chemoresistance. Like GR, beta-catenin is overexpressed in metastatic and therapy-resistant tumors, and is considered a key regulator of cancer stemness and androgen-targeted therapy resistance. Thus, we hypothesized that GR may interact with beta-catenin in PCa cells to support stemness and chemoresistance. Using whole-cell and nuclear co-immunoprecipitation, we demonstrated the interaction between GR and beta-catenin in several DTX-sensitive and -resistant PCa cell line pairs. Pharmacological inhibition of GR using the selective inhibitor CORT-108297 with concomitant inhibition of beta-catenin using the small molecule inhibitor MSAB significantly enhanced DTX cytotoxicity in resistant PCa cells grown in both adherent and spheroid cultures. The BET bromodomain inhibitor JQ1, an antagonist of enhancer-mediated GR upregulation, also significantly reduced tumorsphere formation and stemness in DTX-resistant PCa cells. In order to gain insights into transcriptomic profiles activated by GR signaling that may contribute to stemness and therapy resistance, we performed RNA sequencing in a racially diverse panel of PCa cell lines (MDA-PCa-2b, VCaP, 22RV1, and PC3) treated with and without dexamethasone, a potent glucocorticoid. We observed that the African American cell line MDA-PCa-2b yielded the largest number of differentially regulated genes, perhaps due to the ability of dexamethasone to signal through both AR and GR in this particular cell line. RNAseq data is currently being mined and validated in cellular models treated with glucocorticoids or androgens, in the presence or absence of anti-GR or anti-androgen inhibitors, in order to identify GR-regulated genes. Our results offer novel insights into mechanisms by which GR signaling may influence tumorsphere formation and stemness, and suggest that combinatorial targeting of GR and beta-catenin could be a promising therapeutic strategy to overcome PCa therapy resistance and consequently reduce PCa mortality and its racial disparities. Citation Format: Shannalee R Martinez, Evelyn S Sanchez-Hernandez, Xin Chen, Alfonso M Duran, Charles H Wang, Carlos A Casiano. Glucocorticoid receptor interacts with beta-catenin to promote stemness and therapy resistance in prostate cancer cells [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr D116.

Loss of epithelial AR increase castration resistant stem-like prostate cancer cells and promotes cancer metastasis via TGF-β1/EMT pathway.

BackgroundPrevious study has reported that loss of epithelial androgen receptor (AR) may promote tumor progression and cause TRAMP mouse model die earlier. The detail mechanisms, however, remain unclear.MethodsImmunohistochemistry assay, Western blot and real-time PCR were used to detect the expression of epithelial and mesenchymal markers. RNA extraction, RT-PCR, quantitative RT-PCR, BrdU incorporation assays, flow cytometry and other experimental technics were also used in present work.ResultsDecreased expression of epithelial markers (Cytokeratin 8, NKX3.1 and E-cadherin) and increased expression of mesenchymal markers (α-SMA, Vimentin, and N-cadherin) in were found in AR knockout TRAMP tumors. Further investigation indicated that AR signal deprivation is associated with cell morphology transition, high cell mobility, high cell invasion rate and resistance to anoikis in TRAMP prostate tumor cells. Together, these findings implied knockout AR in TRAMP prostate tumor may lead to EMT, which may result in earlier metastasis, and then cause TRAMP mice die earlier. TGF-β1 is responsible for EMT in AR knockout TRAMP tumor cells.ConclusionsIn conclusion, ADT therapy induced hormone refractory prostate cancer may gain the ability of metastasis through cell’s EMT which is a phase of poor differentiation. Anti-EMT drugs should be developed to battle the tumor metastasis induced by ADT therapy.

Abstract B006: Development of a novel therapeutic splice-switching oligonucleotide targeting race-related androgen receptor signaling and aggressive prostate cancer

Abstract Background: Age-adjusted incidence and mortality rates for prostate cancer (PCa) among African American (AA) men are 1.6- and 2.4-fold greater, respectively, than among white men. The more aggressive characteristics of AA PCa account for a significant component of the PCa disparity, in addition to social determinants of health. Differences in androgenic activities in AA versus white populations and PCa patients have been observed and, clinically, AAs have a less complete response to androgen deprivation than whites do. One of the critical androgen receptor signaling targets in PCa is AR-V7, an androgen receptor variant that lacks the ligand-binding domain, is constitutively active, and associates with castration-resistant PCa, poorer clinical outcomes, and resistance to androgen ablation/androgen receptor inhibition therapies. This work addresses the urgent need to develop a novel therapeutic strategy capable of inhibiting AR-V7. Methods: We have designed and synthesized a novel chemically modified splice switching oligonucleotide (SSO) to correct aberrant splicing leading to production of AR-V7 as well as a control scrambled SSO. After transfection of PCa cell lines derived from AA and white patients with these SSOs, we have examined AR-V7 as well as androgen receptor RNA and protein levels using qPCR and Western blot analysis, respectively. Resulting alterations in proliferation have been assessed by monitoring cell growth using an Incucyte Live-Cell Imaging System and associated software. Results: Transfection of a panel of PCa cell lines derived from AA and white patients with AR-V7 SSO decreases AR-V7 RNA and protein in a dose-dependent manner while maintaining expression levels of full-length androgen receptor. Preliminary data suggest this biochemical response correlates with a biologically significant phenotype. AR-V7 SSO decreases proliferation in PCa cells, including proliferation in the presence of the androgen receptor inhibitor, enzalutamide, over and above the reduction seen in response to enzalutamide alone in both enzalutamide-sensitive and -resistant PCa cell lines. Additionally, transfection of PCa cells with SSOs designed to target nearby splicing enhancer sequences partially reduces AR-V7 RNA expression. Further studies to examine the effects of this SSO on transactivation activity, signaling and PCa cell biology, and the therapeutic efficacy of this SSO in AA and white PCa patient-derived primary cell lines and xenografts are under way. Conclusions: These studies suggest that a SSO can be developed to modulate androgen receptor signaling, inhibiting constitutive signaling and restoring ligand dependency, with interference of nearby splicing enhancer sequences contributing to its function. Such an SSO could represent a novel therapeutic strategy with the potential to reduce PCa disparities for AA men and improve outcomes for men of all races with aggressive disease driven by this mechanism. Citation Format: Bonnie L. LaCroix, Brendon M. Patierno, Bruce A. Sullenger, Daniel J. George, Steven R. Patierno, Jennifer A. Freedman. Development of a novel therapeutic splice-switching oligonucleotide targeting race-related androgen receptor signaling and aggressive prostate cancer [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr B006.

Repurposing antimuscarinics to treat lethal chemoresistant prostate and lung cancers.

e15636 Background: Advanced non-small cell lung cancer (NSCLC) and prostate cancer (PCa) remain leading causes of cancer-related deaths in the United States and worldwide. The current study investigates the ability of antimuscarinics to resensitize chemotherapy resistant NSCLC and PCa to chemotherapy-induced apoptosis. Methods: The chemotherapy drug docetaxel (DTX) resistant human NSCLC and PCa cell cultures were established to recapitulate clinical emergence of chemoresistance. These cultures were used to harvest total RNA and protein for qPCR and Western blot analyses, respectively. DTX’s ability to induce apoptosis was measured with crystal violet survival assay and TUNEL assay. Results: DTX-resistant NSCLC (A549R) and PCa (DU145R) cells show increased expression of the muscarinic acetylcholine receptors 1 and 3 (CHRM1 / CHRM3). Treatment of these DTX-resistant cells with the CHRM1 antagonist dicyclomine (Dic) or the CHRM3 antagonist darifenacin (Dari) resensitize these cells to DTX-induced cell death. This treatment repurposes Dic and Dari, which are currently used in the clinic to treat irritable-bowel syndrome and overactive bladder, respectively. Decreased cell survival was observed with crystal violet survival assay, which led to increased cell death at a lower concentration of DTX compared to cells treated with DTX alone (A549R [DTX IC50]: DTX = No response, Dic + DTX = 5.2 nM, Dari + DTX = 2.2 nM / DU145R [DTX IC50]: DTX = No response, Dic + DTX = 4.2 nM, Dari + DTX = 1.4 nM). Furthermore, cells treated with Dic + DTX or Dari + DTX show increased apoptosis measured by TUNEL assay compared to cells treated with DTX alone (A549R [TUNEL positive %]: DTX = 0.9%, Dic + DTX = 22.2%, Dari + DTX = 88.2% / DU145R [TUNEL positive %]: DTX = 3.6%, Dic + DTX = 96.4%, Dari + DTX = 90.1%). Conclusions: We show that chemoresistant NSCLC and PCa have high expression of CHRM1 and CHRM3 and that antimuscarinics effectively resensitize these lethal forms of cancer to chemotherapeutics. Our data support the rationale of repurposing these therapies to treat chemoresistant NSCLC and PCa, allowing quick entry into a Phase II clinical trial and further translation into clinical practice, in a more cost-effective and less financially risky way, to benefit cancer patients.

RESTORE: A single-arm, open-label phase II trial of bipolar androgen therapy (BAT) in men with metastatic castration resistant prostate cancer (mCRPC)—A comparison of post-abiraterone (Abi) versus post-enzalutamide (Enza) patients (Pts).

5576 Background: A paradoxical inhibition of cell growth has been observed in both androgen-sensitive and castration resistant prostate cancer cell lines following the addition of high-dose testosterone.We have conducted several clinical trials investigating a mode of supraphysiologic testosterone therapy termed, BAT, in which testosterone levels are rapidly driven to the supraphysiologic range followed by a return to near-castrate levels over 28-day treatment cycles with favorable results. We previously reported the efficacy of BAT in mCRPC pts that were progressing on enza. In this study, we compared the effect of BAT in mCRPC pts whose last therapy was abi vs. enza. In addition, we examined the benefit of abi or enza rechallenge after progression on BAT. Methods: 59 mCRPC pts (n = 29 post abi; n = 30 post enza) were enrolled and received at lease one dose of BAT monotherapy, 400mg intramuscularly every 28 days. After clinical or radiographic progression on BAT, pts were rechallenged with the AR targeted therapy to which they were most recently resistant. The co-primary endpoints were a 50% decline in PSA from baseline (PSA50) for BAT and for enza/abi rechallenge. Results: 5/29 (17.2%) of post-abi pts compared to 9/30 (30%) in the post enza group achieved a PSA50 response (P = 0.36). Post BAT rechallenge with abi (n = 19) or enza (n = 22) resulted in a PSA50 response rate of 15.8% (n = 3) and 68.2% (n = 15), respectively (P = 0.001). The total duration of benefit (i.e. PFS on BAT + PFS on rechallenge = “PFS2”) was significantly longer in the post enza vs. post-abi patients (Median PFS2: 12.75 vs. 8.125 months; P = 0.04. Lastly, AR-V7 negative (n = 42) pts has a significantly longer median PFS2 compared to AR-V7 positive (n = 10) pts. (10.3 vs. 7.1 months, P = 0.005). Conclusions: Our data suggest that BAT may be more effective at resensitizing mCRPC to direct AR antagonists (i.e. enza) compared to abi. Detection of AR-V7 portended a worse outcome on BAT/rechallenge. Further clinical study is warranted. Clinical trial information: NCT02090114 . [Table: see text]

Cyclopentadienyl ruthenium(II) carbosilane metallodendrimers as a promising treatment against advanced prostate cancer

In searching for efficient and selective antitumour drugs, a new family of carbosilane metallodendrimers functionalized with [Ru(η5-C5H5)(PTA)Cl] (PTA = 1,3,5-triaza-7-phosphatricyclo-[3.3.1.1] decane) is reported. Experiments of the biophysical characterization showed an ability to interact with biological membranes, as well as with proteins (e.g. human serum albumin) without affecting their usual biological activity. These metallodendrimers possessed potent and selective anticancer activity in vitro in a panel of tumour cell lines. Importantly, the first generation metallodendrimer, bearing 4 Ru(II) complexes, was remarkably active towards resistant prostate cancer cells, inhibiting both cell proliferation and metastasis to bone tissues. Such promising antitumour activity can be further improved when given with docetaxel, with in vitro cytotoxicity being in the nanomolar range. Furthermore, its intravenous administration to an advanced prostate cancer mice model inhibited tumour growth up to 25% and 45% when given 10 mg/kg/week and 7.5 mg/kg/4–5 days, respectively.