Resistant Prostate Cancer Cell Lines Research Articles

Orally Bioavailable Androgen Receptor Degrader, Potential Next-Generation Therapeutic for Enzalutamide-Resistant Prostate Cancer.

Androgen receptor (AR)-targeting prostate cancer drugs, which are predominantly competitive ligand-binding domain (LBD)-binding antagonists, are inactivated by common resistance mechanisms. It is important to develop next-generation mechanistically distinct drugs to treat castration- and drug-resistant prostate cancers. Second-generation AR pan antagonist UT-34 was selected from a library of compounds and tested in competitive AR binding and transactivation assays. UT-34 was tested using biophysical methods for binding to the AR activation function-1 (AF-1) domain. Western blot, gene expression, and proliferation assays were performed in various AR-positive enzalutamide-sensitive and -resistant prostate cancer cell lines. Pharmacokinetic and xenograft studies were performed in immunocompromised rats and mice. UT-34 inhibits the wild-type and LBD-mutant ARs comparably and inhibits the in vitro proliferation and in vivo growth of enzalutamide-sensitive and -resistant prostate cancer xenografts. In preclinical models, UT-34 induced the regression of enzalutamide-resistant tumors at doses when the AR is degraded; but, at lower doses, when the AR is just antagonized, it inhibits, without shrinking, the tumors. This indicates that degradation might be a prerequisite for tumor regression. Mechanistically, UT-34 promotes a conformation that is distinct from the LBD-binding competitive antagonist enzalutamide and degrades the AR through the ubiquitin proteasome mechanism. UT-34 has a broad safety margin and exhibits no cross-reactivity with G-protein-coupled receptor kinase and nuclear receptor family members. Collectively, UT-34 exhibits the properties necessary for a next-generation prostate cancer drug.

Abstract 3826: CCS1477, a potent and selective p300/CBP bromodomain inhibitor, is targeted & differentiated from BET inhibitors in prostate cancer cell lines in vitro

Abstract Background: CCS1477 is a potent and selective p300/CBP bromodomain inhibitor, currently in a Ph1 trial for patients with metastatic castration resistant prostate cancer (mCRPC). CCS1477 works by inhibiting the expression and function of the androgen receptor (AR), as well as inhibiting c-Myc. Bromodomain and extraterminal domain (BET) protein inhibitors are also being developed in mCRPC. We have established a BET inhibitor (BETi) resistant 22Rv1 prostate cancer cell line and used this, alongside parental 22Rv1 cells, to characterise the differential effects of p300/CBP vs BET bromodomain inhibition. Methods: 22Rv1 cells which express both the wild-type and splice variant forms of AR were cultured in the presence of increasing concentrations (30-500nM) of JQ1 for several months. A parallel set of 22Rv1 cells were cultured in the presence of vehicle (0.1% DMSO). The anti-proliferative effects of JQ1, iBet762, OTX-015 and CCS1477 (10 nm-10 µM dose range) was determined in resistant and parental 22Rv1 cells in a 5d CellTitre Glo assay. The effects of combining CCS1477 with JQ1 was measured in parental 22Rv1 cells. Protein biomarker (AR, AR-splice variant, c-Myc) responses were measured by Western blot and qPCR was used to determine changes in the expression of selected genes (AR, AR-V7, c-Myc, KLK3, TMPRSS2). Gene expression microarrays (Clariom D) were used to assess global gene expression changes in cells treated for 24h with 500nM CCS1477 or JQ1. Results: JQ1 resistant 22Rv1 cells were significantly less sensitive to JQ1 compared with parental cells. (IC50; Res, 7.3 µM vs parental, 0.06 µM). There was also cross-resistance to other chemically distinct BET inhibitors, iBET762 and OTX-015. JQ1 potently inhibited c-Myc protein and gene expression in parental cells, a response that was abrogated in the JQ1 resistant line. The inhibitory effects of JQ1 on AR gene and protein expression were reduced in the resistant line. In contrast potent anti-proliferative effects of CCS1477 were retained in JQ1 resistant cells, as was the inhibitory effect on c-Myc and AR. Combination of CCS1477 & JQ1 resulted in a highly synergistic inhibitory effect on proliferation in normal 22Rv1 cells. Global gene expression analysis revealed significantly fewer altered genes after CCS1477 (27 up, 119 down) compared to JQ1 (196 up, 655 down). Conclusions: These studies provide three lines of evidence for a differentiated mode of action of CCS1477 vs BETi. First, CCS1477 continues to inhibit proliferation and relevant response biomarkers in a cell line that is resistant to BETi. Second, there is a synergistic, rather than additive effect of combining CCS1477 with JQ1. Third, there are significantly fewer genes and a distinct pattern of gene change after CCS1477 vs. JQ1. Collectively, these data point to a differentiated and more selective profile after p300/CBP inhibition with CCS1477. Citation Format: Nigel Brooks, Amy Prosser, Barbara Young, Luke Gaughan, Paul Elvin, Neil Pegg. CCS1477, a potent and selective p300/CBP bromodomain inhibitor, is targeted & differentiated from BET inhibitors in prostate cancer cell lines in vitro [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 3826.

Abstract 3960: Low doses of Docetaxel in combination with anti-prostate cancer immunotherapy lead to complete tumor-free outcome through Galectin-3 silencing

Abstract Based on animal model results and the analysis of clinical data from metastatic and castration resistant prostate cancer (mCRPC) patients, we were able to demonstrate the advantages and the mechanisms by which Docetaxel(DTX)-based chemotherapy induces the correct pre-conditioning of the tumour microenvironment to allow high cytotoxic T cell activity to avoid prostate tumour growth and recurrence. First, analysis of the gene expression of human PCa cells showed that treatment with DTX decreases the expression of Galectin-3 (Gal-3) in either chemo-sensitive or -resistant prostate cancer (PCa) cell lines. In addition, using a murine PCa model (derived from TRAMP mice), treatment of TRAMP-C1 (TC1) cells or tumors with a low dose of DTX lowered the expression of Gal-3 but not Gal-1 (among others) in this cell line and tumors as well (reversible silencing of about 98% (in vitro) and 55% (in vivo)). More importantly, Gal-3 downregulation was confirmed in clinical samples of mCRPC patients treated by taxane-based chemotherapy. To evaluate whether this DTX-dependent Gal-3 downregulation in PCa tumors could be one of the principal molecular factors responsible for chemotherapy enhancement of immunotherapy, we decide to inoculate mice with an autologous BM-DC vaccine loaded with lysates from TC1 cells pretreated by DTX in vitro or expressing an anti-Gal-3 shRNA. This protocol completely protects the animals to the growth of Gal-3 deficient-PCa tumors. We then decided to go further in the understanding of the mechanisms that promote this effective anti-PCa immune response, and analysed the proliferation ability of lymphocytes in the presence or absence of TC1 tumor cells, expressing or not Gal-3. The results were encouraging as we demonstrated that Gal-3 down-regulation in tumors (through the use of TC1-shGal-3 cells or TC1-treated with low doses of DTX) was sufficient to restore the capability of CD8+ T cells to be activated and to proliferate as well as in the absence of tumor cells. Interestingly, we showed by ex vivo cytotoxicity assays that these activated and proliferating CD8+ T lymphocytes after vaccination of mice with TC1-lysate loaded BM-DC are capable of killing Gal-3 downregulated TC1 cells and protect prostate cancer recurrence and metastasis development in mice after primary tumor-resection. In conclusion, our results strongly suggest that Gal-3 is one of the principal immunosuppressor in prostate cancer that limits the success of immunotherapy. We then show that DTX-based chemotherapy would promote a decrease of Gal-3 expression by the tumors to create a permissive tumor microenvironment for successful immunotherapy. Finally, we propose a rational protocol combining no toxic/low doses of Docetaxel with simplified immunotherapy to completely control tumor recurrence for all PCa patients and chemoresistant as well. Citation Format: Daniel Compagno, Carolina Tiraboschi, Lucas Gentilini, Enrique Corapi, Felipe M. Jaworski, Anne Chauchereau, Diego J. Laderach. Low doses of Docetaxel in combination with anti-prostate cancer immunotherapy lead to complete tumor-free outcome through Galectin-3 silencing [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 3960.

Histone demethylase KDM7A controls androgen receptor activity and tumor growth in prostate cancer.

Prostate cancer can be controlled by androgen-hormone treatment until the cancer becomes refractory. It is believed that hormone sensitivity is largely dependent on androgen receptor (AR) activity. Here, we found the histone demethylase KDM7A which demethylates histone H3K27 to be overexpressed in enzalutamide resistant castration-resistant prostate cancer cell line C4-2b, and investigated the molecular mechanism whereby androgen receptor activity is regulated by KDM7A. We engineered AR-positive LNCaP cells to stably express a short-hairpin RNA against KDM7A mRNA from a lentiviral vector. By measuring AR downstream gene expression after androgen stimulation, we found that a KDM7A-deficient cell line showed lower AR downstream gene expression compared to a control cell. KDM7A knock-down in LNCaP cell line caused decreased cell proliferation. Western blot analysis with modified-histone antibody revealed that the KDM7A-knock-down LNCaP cell line had increased H3K27 di-methylation. We confirmed KDM7A binding on AR target-gene promoters after hormone stimulation in chromatin-immunoprecipitation experiments. And increased H3K27 di-methylation was observed in KDM7A knock-down LNCaP stable cell. Treatment with KDM7A inhibitor, TC-E 5002, reduced proliferation and induced apoptosis of prostate cancer cells. Finally, we observed that the KDM7A protein was significantly upregulated in prostate cancer tissue, and that this difference correlated with the Gleason score. These data suggested that KDM7A is potentially a good therapeutic target for prostate cancer drugs and can be used as potentially a good prognostic indicator for prostate cancer and related treatment strategies.

Abstract SY45-02: 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 that 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. 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 an SSO can be developed to modulate androgen receptor signaling, inhibiting constitutive signaling and restoring ligand dependency. Such a 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: 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 American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr SY45-02.

Abstract PR04: 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 that 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. 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. Such a 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 Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr PR04.

Proteotranscriptomic Measurements of E6-Associated Protein (E6AP) Targets in DU145 Prostate Cancer Cells

Prostate cancer is a common cause of cancer-related death in men. E6AP (E6-Associated Protein), an E3 ubiquitin ligase and a transcription cofactor, is elevated in a subset of prostate cancer patients. Genetic manipulations of E6AP in prostate cancer cells expose a role of E6AP in promoting growth and survival of prostate cancer cells in vitro and in vivo However, the effect of E6AP on prostate cancer cells is broad and it cannot be explained fully by previously identified tumor suppressor targets of E6AP, promyelocytic leukemia protein and p27. To explore additional players that are regulated downstream of E6AP, we combined a transcriptomic and proteomic approach. We identified and quantified 16,130 transcripts and 7,209 proteins in castration resistant prostate cancer cell line, DU145. A total of 2,763 transcripts and 308 proteins were significantly altered on knockdown of E6AP. Pathway analyses supported the known phenotypic effects of E6AP knockdown in prostate cancer cells and in parallel exposed novel potential links of E6AP with cancer metabolism, DNA damage repair and immune response. Changes in expression of the top candidates were confirmed using real-time polymerase chain reaction. Of these, clusterin, a stress-induced chaperone protein, commonly deregulated in prostate cancer, was pursued further. Knockdown of E6AP resulted in increased clusterin transcript and protein levels in vitro and in vivo Concomitant knockdown of E6AP and clusterin supported the contribution of clusterin to the phenotype induced by E6AP. Overall, results from this study provide insight into the potential biological pathways controlled by E6AP in prostate cancer cells and identifies clusterin as a novel target of E6AP.

MP64-08 A NOVEL BIGUANIDE DERIVATIVE DRUG, IM176, INHIBITS PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II1 Apr 2018MP64-08 A NOVEL BIGUANIDE DERIVATIVE DRUG, IM176, INHIBITS PROSTATE CANCER Choung-Soo KIM, Wonseok Choi, YOONRIM KIM, and Bong-Min Kim Choung-Soo KIMChoung-Soo KIM More articles by this author , Wonseok ChoiWonseok Choi More articles by this author , YOONRIM KIMYOONRIM KIM More articles by this author , and Bong-Min KimBong-Min Kim More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2053AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Biguanides were originally developed for the treatment of hyperglycemia and type 2 diabetes mellitus. Recently, the biguanides metformin and phenformin have been shown to exert potential anticancer effects in prostate cancer. We evaluated the anti-cancer efficacy and mechanism of IM176, a novel biguanide derivate drug on prostate cancer using prostate cancer cell lines and patient-derived castration-resistant prostate cancer cell lines. METHODS Cell viability assay, annexin V-FITC apoptosis detection, microscopy with immunofluorescence staining, real-time quantitative reverse transcription-polymerase chain reaction, and western blotting were conducted. Efficacy of IM176 was also evaluated using 2 cell lines derived from castration-resistant prostate cancer patient. RESULTS IM176 dose-dependently inhibited cell viability in all prostate cancer cell lines at lowest IC50 concentrations (LNCaP: 18.5μM, 22Rv1: 36.8 μM) compared to those of metformin, and phenformin. IM176-mediated AMPK activation caused mTOR inhibition, and a decrement in the phosphorylation of p70S6K1 and S6. IM176 inhibited the expression of AR, AR-splice variant 7 (AR-V7) and prostate-specific antigen in LNCaP and 22Rv1. IM176 induced apoptosis with increased levels of cleavage of caspase-3, and annexin V-positive / PI-positive, respectively. Moreover, IM176 inhibited cell viability at lowest IC50 concentrations in 2 cell lines derived from castration-resistant prostate cancer patient. We evaluate the relationship between AMPK-mTOR pathway and AR signaling pathway by blocking each pathway separately. After AR knockdown, phosphorylation of AMPK was significantly increased. However, AR and AR-V7 were not increased after treating AMPK inhibitor, Compound C. CONCLUSIONS IM176 showed comparable anti-tumor effects via AMPK-mTOR pathway and AR signaling pathway with the lowest IC50 compared to other biguanide derivative drugs in prostate cancer cell lines, including patient-derived castration resistant prostate cancer cell line and may be a novel anti-cancer drug for the treatment of prostate cancer. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e853 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Choung-Soo KIM More articles by this author Wonseok Choi More articles by this author YOONRIM KIM More articles by this author Bong-Min Kim More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Mechanistic Studies of Drug‐Like Inhibitors of P‐glycoprotein using ATPase Assays, Electron Spin Resonance Spectroscopy and Cancer Cell Models

Multidrug resistance (MDR) in cancer and other diseases is frequently associated with transmembrane efflux proteins, one of which is P‐glycoprotein (P‐gp). Over‐expression of P‐gp in cancer cells increases the efflux of therapeutic drugs rendering them ineffective. In order to re‐sensitize multidrug resistant cancers to chemotherapy, we have found inhibitors of P‐gp by in silico screening methods (Brewer et al., Molecular pharmacology 86, 716–726, 2014), and several of these inhibitors were shown to successfully overcome MDR in the drug resistant DU145TXR prostate cancer cell line (Follit et al., Pharmacol. Res. Perspect. 3, e00170, 2015). In this study, we expanded our cell culture screening to evaluate 25 new compounds identified by our in silico screening method and report that over 30% of these compounds re‐sensitized DU145TXR cancer cells to chemotherapy, while not exhibiting significant toxicity to the non‐cancerous human lung fibroblast (HFL‐1) cell line. The best of the identified hit compounds were further investigated with regard to the mechanism of inhibition using ATPase activity assays and electron spin resonance spectroscopy (ESR). Several of the best compounds were found to inhibit ATPase activity. Using a spin‐labeled ATP analog and ESR spectroscopy, we assessed the effects of the identified inhibitors on ATP binding to P‐gp. Variants of one of the original hit compounds, SMU 29, that were computationally predicted to interact more strongly with P‐gp have been synthesized and were analyzed here for their effects on P‐gp ATPase activity and ATP binding characteristics.Support or Funding InformationThis work is supported by NIH NIGMS [R15GM094771‐02] to John G. Wise, SMU University Research Council, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas, as well as the Hamilton Undergraduate Research Scholars Program and the SMU Summer Research Assistantship and Undergraduate Research Assistantship Programs.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MP70-11 REVERSE OF DOCETAXEL RESISTANCE IN PROSTATE CANCER VIA NOTCH SIGNALING INHIBITION

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology III1 Apr 2018MP70-11 REVERSE OF DOCETAXEL RESISTANCE IN PROSTATE CANCER VIA NOTCH SIGNALING INHIBITION Qiu Shi, Xiang Tu, Qiang Wei, and Lu Yang Qiu ShiQiu Shi More articles by this author , Xiang TuXiang Tu More articles by this author , Qiang WeiQiang Wei More articles by this author , and Lu YangLu Yang More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2255AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Chemotherapeutic Docetaxel (Doc) is the standard of care for the hormone resistant prostate cancer (HRPC). However, many HRPC patients do not respond to Doc chemotherapy in a long-term treatment because the cancerous tissue acquires resistance to Doc treatment. The molecular mechanism underlying the development of Doc resistance in HRPC remains unclear. METHODS Human prostate cancer cell line DU145 were purchased and Doc resistant human prostate cancer cell line DU145/adr were generated by culturing cells with Docetaxel in a dose-escalation manner. Cell viability was determined by MTT assay kit. After enumeration of tumor stem-like cells in 3D fibrin gels, CD133 positive cells were selected by fluorescence-activated cell sorting. A Notch signaling pathway inhibitor was applied to combine with Doc to overcome the Doc resistance in HRPC. The ability to release doxorubicin from DU145 and DU145/adr cells was detected by HPLC. Quantitative real-time PCR and western blotting were performed for the detection of the target genes and proteins. RESULTS A subpopulation of prostate cancer cells, owing enhanced stem-like properties, was found in both prostate cancer cell line and HRPC patient tumor tissues, showed high resistance to Doc. The subpopulation of Doc resistance cells has up-regulation of Notch2 and activation of Notch signaling pathway, leading to the high tumor initiating ability and colony formation capability. Most importantly, the Notch signaling pathway up-regulates the expression of drug resistant associated gene ABCB1, which encodes P-GP, resulting in the Doc resistance. CONCLUSIONS Our results illustrated the role of a stem cell-like subpopulation of prostate cancer cells in Doc-resistance that is dependent on Notch signaling pathway activation. Importantly, regulation of Notch signaling was evident in samples derived from the tumors of HRPC patients indicating the clinical relevance of Notch as a potential Doc resistance biomarker. Furthermore, inhibition of Notch signaling pathway in Doc treatment regimens can re-sensitize cancer cells, revealing a potential therapeutic strategy in cases of HRPC. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e938 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Qiu Shi More articles by this author Xiang Tu More articles by this author Qiang Wei More articles by this author Lu Yang More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Association of NRP2 and EGFR co-expression with cancer-specific survival in prostate cancer and the influence of NRP2-depletion combined with EGFR-inhibition on cell functioning in cisplatin-sensitive and –resistant DU145 prostate cancer cell lines

Association of NRP2 and EGFR co-expression with cancer-specific survival in prostate cancer and the influence of NRP2-depletion combined with EGFR-inhibition on cell functioning in cisplatin-sensitive and –resistant DU145 prostate cancer cell lines

Targeted inhibitors of P-glycoprotein increase chemotherapeutic-induced mortality of multidrug resistant tumor cells

Overexpression of ATP-binding cassette (ABC) transporters is often linked to multidrug resistance (MDR) in cancer chemotherapies. P-glycoprotein (P-gp) is one of the best studied drug transporters associated with MDR. There are currently no approved drugs available for clinical use in cancer chemotherapies to reverse MDR by inhibiting P-glycoprotein. Using computational studies, we previously identified several compounds that inhibit P-gp by targeting its nucleotide binding domain and avoiding its drug binding domains. Several of these compounds showed successful MDR reversal when tested on a drug resistant prostate cancer cell line. Using conventional two-dimensional cell culture of MDR ovarian and prostate cancer cells and three dimensional prostate cancer microtumor spheroids, we demonstrated here that co-administration with chemotherapeutics significantly decreased cell viability and survival as well as cell motility. The P-gp inhibitors were not observed to be toxic on their own. The inhibitors increased cellular retention of chemotherapeutics and reporter compounds known to be transport substrates of P-gp. We also showed that these compounds are not transport substrates of P-gp and that two of the three inhibit P-gp, but not the closely related ABC transporter, ABCG2/BCRP. The results presented suggest that these P-gp inhibitors may be promising leads for future drug development.

Integrative molecular network analysis identifies emergent enzalutamide resistance mechanisms in prostate cancer.

Recent work demonstrates that castration-resistant prostate cancer (CRPC) tumors harbor countless genomic aberrations that control many hallmarks of cancer. While some specific mutations in CRPC may be actionable, many others are not. We hypothesized that genomic aberrations in cancer may operate in concert to promote drug resistance and tumor progression, and that organization of these genomic aberrations into therapeutically targetable pathways may improve our ability to treat CRPC. To identify the molecular underpinnings of enzalutamide-resistant CRPC, we performed transcriptional and copy number profiling studies using paired enzalutamide-sensitive and resistant LNCaP prostate cancer cell lines. Gene networks associated with enzalutamide resistance were revealed by performing an integrative genomic analysis with the PAthway Representation and Analysis by Direct Reference on Graphical Models (PARADIGM) tool. Amongst the pathways enriched in the enzalutamide-resistant cells were those associated with MEK, EGFR, RAS, and NFKB. Functional validation studies of 64 genes identified 10 candidate genes whose suppression led to greater effects on cell viability in enzalutamide-resistant cells as compared to sensitive parental cells. Examination of a patient cohort demonstrated that several of our functionally-validated gene hits are deregulated in metastatic CRPC tumor samples, suggesting that they may be clinically relevant therapeutic targets for patients with enzalutamide-resistant CRPC. Altogether, our approach demonstrates the potential of integrative genomic analyses to clarify determinants of drug resistance and rational co-targeting strategies to overcome resistance.

RNF7 knockdown inhibits prostate cancer tumorigenesis by inactivation of ERK1/2 pathway

Development of castration resistance is a key contributor to mortality in patients with prostate cancer. High expression of RING finger protein 7 (RNF7) in cancer cells is known to play a key role in tumor progression. However, the role of RNF7 in prostate cancer progression is not well elucidated. In this study, we silenced RNF7 by shRNA interference in two castration resistant prostate cancer (CRPC) cell lines, DU145 and PC3. RNF7 knockdown attenuated proliferation and enhanced sensitivity of prostate cancer cells to cisplatin treatment. Invasive property of DU145 and PC3 cells was also attenuated by RNF7 silencing. The underlying mechanisms appear to be associated with accumulation of tumor suppressive proteins p21, p27 and NOXA, while inactivation of ERK1/2 by RNF7 knockdown. We demonstrated that RNF7 knockdown induced growth suppression of prostate cancer cells and inactivated ERK1/2 pathway, which suggested RNF7 might be a potential novel therapeutic target for CRPC.

Design, synthesis and in vitro cytotoxicity studies of novel β-carbolinium bromides

A series of novel β-carbolinium bromides has been synthesized from easily accessible β-carbolines and 1-aryl-2-bromoethanones. The newly synthesized compounds were evaluated for their in vitro anticancer activity. Among the synthesized derivatives, compounds 16l, 16o and 16s exhibited potent anticancer activity with IC50 values of <10μM against tested cancer cell lines. The most potent analogue 16l was broadly active against all the tested cancer cell lines (IC50=3.16–7.93μM). In order to test the mechanism of cell death, we exposed castration resistant prostate cancer cell line (C4-2) to compounds 16l and 16s, which resulted in increased levels of cleaved PARP1 and AO/EB staining, indicating that β-carbolinium salts induce apoptosis in these cells. Additionally, the most potent β-carbolines 16l and 16s were found to inhibit tubulin polymerization.

Amino Acid Profiling of Zinc Resistant Prostate Cancer Cell Lines: Associations With Cancer Progression.

Failure in intracellular zinc accumulation is a key process in prostate carcinogenesis. Nevertheless, epidemiological studies of zinc administration have provided contradicting results. In order to examine the impact of the artificial intracellular increase of zinc(II) ions on prostate cancer metabolism, PNT1A, 22Rv1, and PC-3 prostatic cell lines-depicting different stages of cancer progression-and their zinc-resistant counterparts were used. To determine "benign" and "malignant" metabolic profiles, amino acid patterns, gene expression, and antioxidant capacity of these cell lines were assessed. Amino acid profiles were examined using an ion-exchange liquid chromatography. Intracellular zinc content was measured by atomic absorption spectrometry. Metallothionein was quantified using differential pulse voltammetry. The content of reduced glutathione was determined using high performance liquid chromatography coupled with an electrochemical detector. Cellular antioxidant capacity was determined by the ABTS test and gene expression analysis was performed by qRT-PCR. Long-term zinc treatment was shown to reroute cell metabolism from benign to more malignant type. Long-term application of high concentration of zinc(II) significantly enhanced cisplatin resistance, invasiveness, cellular antioxidant capacity, synthesis of glutathione, and expression of treatment resistance- and stemness-associated genes (SOX2, POU5F1, BIRC5). Tumorous cell lines universally displayed high accumulation of aspartate and sarcosine and depletion of essential amino acids. Increased aspartate/threonine, aspartate/methionine, and sarcosine/serine ratios were associated with cancer phenotype with high levels of sensitivity and specificity. Prostate 77: 604-616, 2017. © 2017 Wiley Periodicals, Inc.

Effect of Combination therapy of Desmopressin and Docetaxel on prostate cancer cell DU145 proliferation, migration and growth

Background: This study was designed to assess the efficacy of the combination of Desmopressin and Docetaxel for prostate cancer. Desmopressin has been demonstrated to inhibit tumor progression and metastasis in in vitro and in vivo models of breast cancer. Docetaxel, an anti-mitotic chemotherapeutic agent, is widely used for the treatment of castration resistant prostate cancer. However, it is associated with adverse effects and eventual drug resistance. This is the first report on the effect of combining Desmopressin and Docetaxel in prostate cancer, both in vitro and in vivo. Methods: An established castrate resistant prostate cancer cell line DU145 was used. Cellular proliferation was determined using the MTS assay. The migratory inhibition potential of Desmopressin alone and in combination with Docetaxel was accessed using the wound healing assay. In vivo evaluation was performed on a prostate cancer xenograft model using athymic nude mouse. Treatment was administered bi- weekly and tumor volume were measured throughout the treatment period. Eventually, after a six-week treatment period, tumors were excised and measured.  Results: A combination therapy of 1 µM Desmopressin with 100nM Docetaxel resulted in dramatic inhibition of proliferation of DU145 cells 72 hours post treatment compared to either agent along (p &lt; 0.05). Wound healing assay revealed inhibition of cellular migration as well (p&lt;0.05). The use of a xenograft mouse model followed by treatment with 5 mg/kg Docetaxel intraperitoneally with concomitant 2 µg/ml/kg Desmopressin administered intravenously 30 minutes before administering chemotherapy and 24 hours after, resulted in a significant decrease in tumor volume (P&lt;0.05), while not impacting body weight.Conclusions: Desmopressin significantly enhanced the anti-proliferative and inhibiting the migratory potential of Docetaxel. Combination treatment had no additional effect on mice weight or mortality. These studies could enhance the efficacy of Docetaxel- based chemotherapy treatment for castrate resistant prostate cancer.

Comparison of the effect of multidrug resistance modifiers on prostate cancer, mouse lymphoma and colon cancer cells

The reason for unsuccessful tumor chemotherapy is related to multidrug resistance. An important factor is the overexpression of efflux pumps, such as P-glycoprotein. Amino- and amide-substituted steroid compounds and phenothiazine derivatives were investigated in tumor models in vitro. The inhibition of P-glycoprotein was evaluated by flow cytometry and the interaction of these compounds with doxorubicin was investigated as well. Molecular docking was used to estimate the binding energies of the compounds to P-glycoprotein. The aminosteroids showed anticancer activity on multidrug resistant mouse T-lymphoma and prostate cancer cell lines. The combination of steroids and doxorubicin potentiated its effect in hormone resistant prostate cancer cells. Among the N-hydroxyalkyl-2-aminophenothiazines, secondary amines exhibited anticancer effects on multidrug resistant colon adenocarcinoma cells. The tested phenothiazine and steroid derivatives showed potent anticancer activity, furthermore, the stereoisomerism of thioridazine did not play a role in the antitumor properties. Neither steroids nor thioridazine influenced apoptosis in hormone resistant cells. Orv. Hetil., 2016, 157(37), 1489-1495.

Overcoming chemoresistance in prostate cancer with Chinese medicine Tripterygium wilfordii via multiple mechanisms.

A leading cause of cancer chemotherapy failure is chemoresistance, which often involves multiple mechanisms. Chinese medicines (CM) usually contain multiple components which could potentially target many mechanisms simultaneously and may offer an advantage over single compounds that target one mechanism at a time. The purpose of this study was to investigate the chemosensitizing effect (CE) of a specific CM, Tripterygium wilfordii (TW), on prostate cancer cells resistant to docetaxel (Dtx) and identify the potential mechanisms. The CE of TW (in combination with Dtx) was evaluated in two Dtx resistant prostate cancer cell lines (PC3-TxR and DU145-TxR) and the efficacy of the combination for resistant PC3-TxR tumor was investigated using a xenograft mouse model. For mechanistic study, the inhibitory effect of TW on P-glycoprotein activity was assessed. In addition, novel gene targets of TW were identified using DNA microarray and quantitative PCR. Results showed that TW induced a CE of 8 and >38 folds in PC3-TxR and DU145-TxR cells, respectively with Dtx IC50 reversed back to that of the sensitive parent cells. An optimum dose of TW+Dtx significantly retarded tumor growth in mice compared to TW or Dtx alone. TW inhibited P-glycoprotein activity and induced a significant gene expression changes in genes related to angiogenesis, cell cycle regulation and differentiation. Our in vitro and in vivo studies demonstrate that TW in combination with Dtx was able to overcome the chemoresistance and suppress resistant prostate tumor growth via multi-mechanisms.

Abstract 4335: Nanoformulations of PARP inhibitors Olaparib and Talazoparib for targeted cancer therapy

Abstract Introduction: Poly(ADP-ribose) Polymerase (PARP) plays an important role in a number of DNA repair pathways. PARP inhibitors (PARPi) such as Olaparib and Talazoparib exploit the concept of synthetic lethality by selectively targeting cancer cells with defective DNA repair pathways. These drugs are currently only available in oral form which results in limited bioavailability, poor tumor accumulation, and systemic toxicity. Here we report the development of novel nanoformulations of Olaparib and Talazoparib to allow intravenous or intraperitoneal delivery, providing greater bioavailability and tumor accumulation, while limiting systemic toxicities. Methods: Nanoparticle formulations of Olaparib and Talazoparib were synthesized and tested in vitro and in vivo. Short-and long-term dose response with a panel of ovarian cancer cell lines were conducted. These cell lines include KURAMOCHI, SKOV3, OVSAHO, JHOS2, PA1, COV318, 403 and 404, derived from BRCA2-/-, PTEN-/-, TP53mut mice, and 4306 and 4412, developed from conditional LSL-K-rasG12D/+, PTENloxP/loxP mice. Radiosensitization with NanoOlaparib was tested in the radiation resistant prostate cancer cell line FK01, derived from Ptenpc-/-;Trp53pc-/- mice. In vivo, NanoOlaparib was tested in an IP spread model using 404 cells. Animals were treated IP with NanoOlaparib alone, and in combination with cisplatin. Radiosensitization with NanoOlaparib in vivo was tested in a xenograft model using FK01 cells to mimic castration resistant prostate cancer. Animals were treated biweekly with NanoOlaparib before and after radiation treatment. Results: The murine cell lines 403 and 404 were highly sensitive to this treatment due to the mutations in BRCA2, PTEN, and TP53. 4412 and 4306 showed comparable sensitivity, suggesting that a PTEN deletion confers similar sensitivity to PARP inhibitors as a BRCA2 deletion. PA1 demonstrated high sensitivity to NanoOlaparib which may be attributed to genetic instability. NanoTalazoparib is more potent than NanoOlaparib, resulting in a similar relationship in cell line sensitivity with overall lower IC50’s. Strong synergistic radiosensitization was observed in FK01 cells with NanoOlaparib. Bioluminescence imaging illustrated that NanoOlaparib administered IP daily resulted in a greater inhibition of tumor growth than those treated with oral Olaparib daily. The FK01 xenografts are highly radioresistant with little difference between untreated and radiation only animals. NanoOlaparib delays tumor growth, while the combination of radiation and NanoOlaparib clearly shrinks tumors. Conclusions: Robust nanoparticle formulations of NanoTalazoparib and NanoOlaparib have been successfully developed for in vitro and in vivo studies. These results show that NanoOlaparib and NanoTalazoparib amplify the therapeutic efficacy of PARP inhibition and imply a very promising role for the nanoformulation in ovarian and prostate cancers. Citation Format: Paige Baldwin, Anders Ohman, Jeremy Thong, Shifalika Tangutoori, Anne van de Ven, Rajiv Kumar, Daniela Dinulescu, Srinivas Sridhar. Nanoformulations of PARP inhibitors Olaparib and Talazoparib for targeted cancer therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4335.