- Email: [email protected]
MP62-03 NOVEL SELECTIVE LYSINE-SPECIFIC DEMETHYLASE 1 INHIBITORS AND AUTOPHAGY INHIBITORS EFFECTIVELY IMPAIR CASTRATION-RESISTANT PROSTATE CANCER GROWTH
THE JOURNAL OF UROLOGYâ
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Vol. 195, No. 4S, Supplement, Monday, May 9, 2016
Prostate Cancer: Basic Research & Pathophysiology I Moderated Poster Monday, May 9, 2016
8:00 AM-10:00 AM
MP62-01 TARGETING THE DNA BINDING DOMAIN OF THE ANDROGEN RECEPTOR: EFFICACY OF PROSTATE CANCER COMPOUND WITH NOVEL MECHANISM OF ACTION Hendrik Borgmann*, Kush Dalal, Eliana Beraldi, Artem Cherkasov, Paul Rennie, Martin Gleave, Vancouver, Canada INTRODUCTION AND OBJECTIVES: In castration-resistant prostate cancer, resistance to conventional treatments targeting the ligand binding domain of the androgen receptor (AR) occur inevitably, prompting the need to develop small molecules with a different binding location on the AR and novel mechanism of action. Recently, we identified a pocket on the AR-DNA binding domain (DBD) protein surface with potential as an alternative drug-target site. Here, we present preclinical data on an AR-DBD specific compound and its inhibitory effects across multiple prostate cancer cell lines in vitro and in vivo. METHODS: We tested the effect of VPC-14449 on cell growth of the hormone sensitive cell line LNCaP, the Enzalutamide resistant cell line MR49C and the AR Variant driven cell line 22Rv1 by MTS assay and compared to enzalutamide. Moreover, we assessed the effect of VPC-14449 on AR transcriptional activity of these cell lines using luciferase reporter assay. In castrated mice, change in volume of LNCaP tumour xenografts and serum PSA levels was determined after compound treatment over 4 weeks. RESULTS: Treatment with VPC-14449 led to dose-dependent cell growth inhibition in all three cell lines. Enzalutamide inhibited cell growth of LNCaP, but not 22Rv1 and MR49C cells for which an agonistic effect of Enzalutamide was observed at high compound concentrations. VPC-14449 blocked transcriptional activity of the AR in LNCaP, MR49C and 22Rv1 cell lines. Finally, the compound suppressed the growth of LNCaP tumor xenografts by 68% (p<0.01) and serum PSA levels by 81% (p<0.01) in mice at week 4. CONCLUSIONS: The developed AR-DBD inhibiting compound showed excellent efficacy in preclinical studies across multiple prostate cancer cell lines in vitro and in vivo. AR-DBD inhibiting compounds may complement the armamentarium of drugs as effective non-cross-resistant treatment of castration-resistant prostate cancer. Source of Funding: none
MP62-02 ERG ONCOGENE SPECIFIC INHIBITORS FOR PROSTATE CANCER Ahmed Mohamed*, Rockville, MD; Gauthaman Sukumar, Bethesda, MD; Charles Xavier, Shilpa Katta, Lakshmi Ravindranath, Muhammad Jamal, Taduru Sreenath, Gyorgy Petrovics, Albert Dobi, Rockville, MD; Meera Srivastava, Clifton Dalgard, Bethesda, MD; Shiv Srivastava, Rockville, MD INTRODUCTION AND OBJECTIVES: Treatment for advanced prostate cancer (CaP) remains challenging due to numerous genomic alterations or treatment-related genomic mutations during disease continuum. Therefore, targeting early CaP driver genes may introduce new approaches in complementing current therapeutic paradigms. ERG oncogenic activation by recurrent gene fusions is the most validated driver gene alteration in CaP and represents a high priority therapeutic target. However, the transcription factor nature of ERG poses challenges. Current ERG-targeted strategies include direct or indirect
inhibition of ERG/ETS transcription factor activity. Due to high homology of ERG to a large number of ETS-related proteins the specificity of these strategies remains to be established. In order to screen for ERGspecific inhibitors, we developed a new approach for screening of small molecules by assessing inhibition of ERG oncoprotein expression using an In-cell-Western based assay employing a specific anti-ERG monoclonal antibody (9FY). Here we report the characterization of a highly selective small molecule ERGi-USU. METHODS: A small molecule library from the National Cancer Institute was screened for ERG protein inhibition in TMPRSS2-ERG harboring CaP cells (VCaP) using an In-Cell-Western assay employing ERG-MAb (9FY). Among the promising candidates NSC139021 (ERGiUSU) was selected for further characterization in cell culture-based (VCaP, COLO320, MOLT4, KG1, LNCaP, LAPC4, MDAPCa2b, BPH1, RWPE and HUVEC) and VCaP nude mouse xenograft assays. Effect of ERGi-USU was determined using cell growth, cell cycle, apoptosis assays and whole transcriptome sequencing. RESULTS: Among the cell lines evaluated, ERGi-USU exhibited exclusive inhibitory effects on ERG protein as well as cell growth including TMPRSS2-ERG harboring CaP (VCaP) and ERG positive colon cancer and leukemia cells (COLO320, MOLT4, KG1) (IC50, 250400 nM). There was no inhibitory effect on cell growth of ERG negative CaP cells or normal prostate-derived epithelial cells. Importantly, ERGiUSU did not inhibit the growth of primary endothelial cells with endogenous ERG. Molecular and cellular mechanisms of ERGi-USU included inhibition of G2 phase of cell cycle, induction of apoptosis. RNA-Seq analyses revealed activation of the ER stress pathway. Importantly, significant inhibitory effect of ERGi-USU was shown on VCaP xenograft growth in nude mice without any major toxicity. CONCLUSIONS: ERG inhibition through ERGi-USU showed high therapeutic index in experimental models and has the potential in further developing ERG-targeted therapeutic strategies for CaP. Source of Funding: This work was supported in part by NIH Grants RO1 DK065977 to S.S., CPDR Program HU0001-102-0002 to D.G.M., and HJF JOTT FY15 to S.S, A.M.
MP62-03 NOVEL SELECTIVE LYSINE-SPECIFIC DEMETHYLASE 1 INHIBITORS AND AUTOPHAGY INHIBITORS EFFECTIVELY IMPAIR CASTRATION-RESISTANT PROSTATE CANCER GROWTH Toshiki Etani*, Komono, Japan; Taku Naiki, Nagoya, Japan; Takayoshi Suzuki, Kyoto, Japan; Takashi Nagai, Keitaro Iida, Ryosuke Ando, Noriyasu Kawai, Keiichi Tozawa, Nagoya, Japan; Tohru Mogami, Komono, Japan; Kenjiro Kohri, Takahiro Yasui, Nagoya, Japan INTRODUCTION AND OBJECTIVES: Lysine-specific demethylase 1 (LSD1), the first histone demethylase discovered, is a novel target for prostate cancer therapy. NCL1 and NCD38, selective inhibitors of LSD1, were first discovered at our university. We examined the anticancer effects of NCL1 and NCD38. METHODS: Various tests using LNCaP (a hormone-sensitive prostate cancer cell line) and PCai1 cells (a castration-resistant prostate cancer [CRPC] cell line established in our institute) were used to confirm the anticancer effects of NCL1 and NCD38. Cell viability was assessed by performing a WST assay in presence of a standard vehicle, NCL1, or NCD38. Chromatin immunoprecipitation (ChIP) and real-time PCR were used to confirm the methylation status. For autophagy analysis, LNCaP and PCai1 cells were incubated with and without chloroquine (CQ) and in the presence or absence of NCL1. We subsequently performed transmission electron microscopy (TEM) and a WST assay on these treated and non-treated cells. A combination index analysis was performed to assess the effects of NCL1 and CQ. Lastly, the effect on xenograft tumors in CRPC mice models was measured by subcutaneously injecting castrated nude mice with PCai1 cells. Mice were injected intraperitoneally with vehicle, NCL1, or NCD38, and subsequent growth was recorded.
THE JOURNAL OF UROLOGYâ
Vol. 195, No. 4S, Supplement, Monday, May 9, 2016
RESULTS: The WST assay revealed that the number of viable cells was reduced after NCL1 and NCD38 treatment. ChIP showed NCL1-induced H3K9me2 accumulation at the ELK4 and KLK2 promoters. Flow cytometry showed that NCL1 dose-dependently induced apoptosis. Using TEM analysis, autophagosomes were observed in LNCaP cells treated with NCL1. The combination index analysis further revealed that a combination of NCL1 and CQ significantly decreased cell growth synergistically. When NCL1 and NCD38-treated mice were compared with controls, the xenograft tumor volume was reduced with no adverse effects. CONCLUSIONS: Castration resistant prostate cancer growth was effectively suppressed with NCL1 and NCD38 both in vitro and in vivo without adverse events via regulation of apoptosis and autophagy, indicating the potential use of LSD1 inhibitors as therapeutic agents for prostate cancer.
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significant difference in CD31 expression in PC3/P tumors following treatment with either docetaxel or cabazitaxel, cabazitaxel markedly inhibited the expression of CD31 in PC3/R tumors compared with docetaxel. Findings similar to those between the PC3 sublines both in vitro and in vivo were achieved between DU145/P and DU145/R. CONCLUSIONS: Collectively, these findings suggest that antitumor activity of cabazitaxel in prostate cancer cells even after the acquisition of resistance to docetaxel could be explained, at least in part, by the inactivation of Akt, which was persistently phosphorylated following treatment with docetaxel. Source of Funding: None
MP62-05 DRUG, DISEASE, GENE INTERACTION: SIMVASTATIN, PROSTATE CANCER AND THE MITOCHONDRIAL GENOME. Rebecca Arnold*, Qian Sun, John Petros, Atlanta, GA
Source of Funding: Grants-in-aid for Scientific Research, Ministry of Education, Culture, Sports, Science and Technology
MP62-04 MOLECULAR MECHANISM MEDIATING CYTOTOXIC ACTIVITY OF CABAZITAXEL IN DOCETAXEL-RESISTANT HUMAN PROSTATE CANCER CELLS Akira Miyazaki*, Kobe, Japan; Hideaki Miyake, Hamamatsu, Japan; Masato Fujisawa, Kobe, Japan INTRODUCTION AND OBJECTIVES: It has been well documented that cabazitaxel remains active in patients with castrationresistant prostate cancer (CRPC) progressing after treatment with docetaxel. The objective of this study was to evaluate molecular mechanism mediating cytotoxic activity of cabazitaxel in docataxelresistant human prostate cancer cells. METHODS: Two parental human prostate cancer cell lines, PC3 (PC3/P) and DU145 (DU145/P), which were shown to grow through androgen-independent manner, were continuously exposed to increasing doses of docetaxel, and cell lines resistant to docetaxel, PC3/R and DU145/R, showing approximately 5-fold higher IC50 than those of PC3/P and DU145/P, respectively, were developed. Differences in molecular phenotypes following treatment with docetaxel or cabazitaxel between these cell lines were comparatively investigated. RESULTS: There were no significant differences in sensitivities to cabazitaxel, between PC3/P and PC3/R. In PC3/P, both docetaxel and cabazitaxel markedly inhibited the expression levels of phosphorylated Akt and p44/42 mitogen activated protein kinase (MAPK). In PC3/R, however, phosphorylation of Akt and p44/42 MAPK were maintained following treatment with docetaxel, whereas treatment with cabazitaxel resulted in the marked down-regulation of phosphorylated Akt, but not that of p44/42 MAPK. Furthermore, additional treatment of PC3/R with a specific inhibitor of Akt significantly enhanced the cytotoxic activity of docetaxel, but not that of cabazitaxel. In vivo growth of PC3/R in nude mice after treatment with cabazitaxel was significantly inhibited compared with that following treatment with docetaxel. Despite the absence of a
INTRODUCTION AND OBJECTIVES: Statins, 3-hydroxy-3 methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, are currently the most widely used cholesterol-lowering drugs. There is an epidemiologic association between statin use and decreased risk of prostate cancer progression. Both inherited and somatic mutations of the mitochondrial genome are linked to prostate cancer. We previously demonstrated that simvastatin treatment induced apoptosis in human cybrid prostate cancer cells and that the response to drug was different depending on mitochondrial genotype with a prostate cancer-derived mitochondrial DNA (mtDNA) mutation conferring resistance to simvastatin induced apoptosis (The Prostate 75: 1916, 2015) The purpose of this study was to determine the molecular mediators of this effect. METHODS: Cytoplasmic hybrid (cybrid) cell lines were constructed that contained a prostate cancer nucleus and either wild type or mutant mtDNA derived from a prostate cancer patient with the cytochrome oxidase subunit 1 gene mutation T6124C (Met74Thr). Multiple clones for each genotype were tested for mitochondrial protein expression. Both wild type and mutant cells were treated with increasing concentrations of simvastatin and reactive oxygen species (ROS) and reactive nitrogen species (RNS) levels measured. RESULTS: The prostate cancereassociated mtDNA mutation T6124C (Met74Thr) caused a dramatic loss of cytochrome oxidase peptide subunits I, II and IV and substantial alterations of reactive oxygen and nitrogen species. Specifically, the mutation was associated with a shift from superoxide anion to hydrogen peroxide by increasing the mitochondrial superoxide dismutase, an effect that was further augmented in the presence of simvastatin. The mutation also lowered the cellular nitric oxide levels by approximately 2/3, a difference that simvastatin only partially decreased. CONCLUSIONS: While we have previously demonstrated that a prostate cancer-derived mitochondrial DNA mutation confers resistance to simvastatin-induced prostate cancer cell apoptosis, the current study demonstrates that this is likely due to a more global downregulation of multiple subunits of respiratory complex IV (the cytochrome oxidase) and that this is associated with decreased reactive nitrogen species. Individual prostate cancer patients response to simvastatin may be substantially altered by mitochondrial genetics and associated with alterations in cellular ROS and RNS production. Source of Funding: none
MP62-06 COMBINED AKT AND MEK PATHWAY BLOCKADE IN PRECLINICAL MODELS OF ENZALUTAMIDE-RESISTANT PROSTATE CANCER bec, Canada; Soojin Kim, Amina Zoubeidi, Paul Toren*, Que Vancouver, Canada INTRODUCTION AND OBJECTIVES: Despite recent advances with newer androgen receptor (AR) pathway inhibitors,
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Vol. 195, No. 4S, Supplement, Monday, May 9, 2016
Prostate Cancer: Basic Research & Pathophysiology I Moderated Poster Monday, May 9, 2016
8:00 AM-10:00 AM
MP62-01 TARGETING THE DNA BINDING DOMAIN OF THE ANDROGEN RECEPTOR: EFFICACY OF PROSTATE CANCER COMPOUND WITH NOVEL MECHANISM OF ACTION Hendrik Borgmann*, Kush Dalal, Eliana Beraldi, Artem Cherkasov, Paul Rennie, Martin Gleave, Vancouver, Canada INTRODUCTION AND OBJECTIVES: In castration-resistant prostate cancer, resistance to conventional treatments targeting the ligand binding domain of the androgen receptor (AR) occur inevitably, prompting the need to develop small molecules with a different binding location on the AR and novel mechanism of action. Recently, we identified a pocket on the AR-DNA binding domain (DBD) protein surface with potential as an alternative drug-target site. Here, we present preclinical data on an AR-DBD specific compound and its inhibitory effects across multiple prostate cancer cell lines in vitro and in vivo. METHODS: We tested the effect of VPC-14449 on cell growth of the hormone sensitive cell line LNCaP, the Enzalutamide resistant cell line MR49C and the AR Variant driven cell line 22Rv1 by MTS assay and compared to enzalutamide. Moreover, we assessed the effect of VPC-14449 on AR transcriptional activity of these cell lines using luciferase reporter assay. In castrated mice, change in volume of LNCaP tumour xenografts and serum PSA levels was determined after compound treatment over 4 weeks. RESULTS: Treatment with VPC-14449 led to dose-dependent cell growth inhibition in all three cell lines. Enzalutamide inhibited cell growth of LNCaP, but not 22Rv1 and MR49C cells for which an agonistic effect of Enzalutamide was observed at high compound concentrations. VPC-14449 blocked transcriptional activity of the AR in LNCaP, MR49C and 22Rv1 cell lines. Finally, the compound suppressed the growth of LNCaP tumor xenografts by 68% (p<0.01) and serum PSA levels by 81% (p<0.01) in mice at week 4. CONCLUSIONS: The developed AR-DBD inhibiting compound showed excellent efficacy in preclinical studies across multiple prostate cancer cell lines in vitro and in vivo. AR-DBD inhibiting compounds may complement the armamentarium of drugs as effective non-cross-resistant treatment of castration-resistant prostate cancer. Source of Funding: none
MP62-02 ERG ONCOGENE SPECIFIC INHIBITORS FOR PROSTATE CANCER Ahmed Mohamed*, Rockville, MD; Gauthaman Sukumar, Bethesda, MD; Charles Xavier, Shilpa Katta, Lakshmi Ravindranath, Muhammad Jamal, Taduru Sreenath, Gyorgy Petrovics, Albert Dobi, Rockville, MD; Meera Srivastava, Clifton Dalgard, Bethesda, MD; Shiv Srivastava, Rockville, MD INTRODUCTION AND OBJECTIVES: Treatment for advanced prostate cancer (CaP) remains challenging due to numerous genomic alterations or treatment-related genomic mutations during disease continuum. Therefore, targeting early CaP driver genes may introduce new approaches in complementing current therapeutic paradigms. ERG oncogenic activation by recurrent gene fusions is the most validated driver gene alteration in CaP and represents a high priority therapeutic target. However, the transcription factor nature of ERG poses challenges. Current ERG-targeted strategies include direct or indirect
inhibition of ERG/ETS transcription factor activity. Due to high homology of ERG to a large number of ETS-related proteins the specificity of these strategies remains to be established. In order to screen for ERGspecific inhibitors, we developed a new approach for screening of small molecules by assessing inhibition of ERG oncoprotein expression using an In-cell-Western based assay employing a specific anti-ERG monoclonal antibody (9FY). Here we report the characterization of a highly selective small molecule ERGi-USU. METHODS: A small molecule library from the National Cancer Institute was screened for ERG protein inhibition in TMPRSS2-ERG harboring CaP cells (VCaP) using an In-Cell-Western assay employing ERG-MAb (9FY). Among the promising candidates NSC139021 (ERGiUSU) was selected for further characterization in cell culture-based (VCaP, COLO320, MOLT4, KG1, LNCaP, LAPC4, MDAPCa2b, BPH1, RWPE and HUVEC) and VCaP nude mouse xenograft assays. Effect of ERGi-USU was determined using cell growth, cell cycle, apoptosis assays and whole transcriptome sequencing. RESULTS: Among the cell lines evaluated, ERGi-USU exhibited exclusive inhibitory effects on ERG protein as well as cell growth including TMPRSS2-ERG harboring CaP (VCaP) and ERG positive colon cancer and leukemia cells (COLO320, MOLT4, KG1) (IC50, 250400 nM). There was no inhibitory effect on cell growth of ERG negative CaP cells or normal prostate-derived epithelial cells. Importantly, ERGiUSU did not inhibit the growth of primary endothelial cells with endogenous ERG. Molecular and cellular mechanisms of ERGi-USU included inhibition of G2 phase of cell cycle, induction of apoptosis. RNA-Seq analyses revealed activation of the ER stress pathway. Importantly, significant inhibitory effect of ERGi-USU was shown on VCaP xenograft growth in nude mice without any major toxicity. CONCLUSIONS: ERG inhibition through ERGi-USU showed high therapeutic index in experimental models and has the potential in further developing ERG-targeted therapeutic strategies for CaP. Source of Funding: This work was supported in part by NIH Grants RO1 DK065977 to S.S., CPDR Program HU0001-102-0002 to D.G.M., and HJF JOTT FY15 to S.S, A.M.
MP62-03 NOVEL SELECTIVE LYSINE-SPECIFIC DEMETHYLASE 1 INHIBITORS AND AUTOPHAGY INHIBITORS EFFECTIVELY IMPAIR CASTRATION-RESISTANT PROSTATE CANCER GROWTH Toshiki Etani*, Komono, Japan; Taku Naiki, Nagoya, Japan; Takayoshi Suzuki, Kyoto, Japan; Takashi Nagai, Keitaro Iida, Ryosuke Ando, Noriyasu Kawai, Keiichi Tozawa, Nagoya, Japan; Tohru Mogami, Komono, Japan; Kenjiro Kohri, Takahiro Yasui, Nagoya, Japan INTRODUCTION AND OBJECTIVES: Lysine-specific demethylase 1 (LSD1), the first histone demethylase discovered, is a novel target for prostate cancer therapy. NCL1 and NCD38, selective inhibitors of LSD1, were first discovered at our university. We examined the anticancer effects of NCL1 and NCD38. METHODS: Various tests using LNCaP (a hormone-sensitive prostate cancer cell line) and PCai1 cells (a castration-resistant prostate cancer [CRPC] cell line established in our institute) were used to confirm the anticancer effects of NCL1 and NCD38. Cell viability was assessed by performing a WST assay in presence of a standard vehicle, NCL1, or NCD38. Chromatin immunoprecipitation (ChIP) and real-time PCR were used to confirm the methylation status. For autophagy analysis, LNCaP and PCai1 cells were incubated with and without chloroquine (CQ) and in the presence or absence of NCL1. We subsequently performed transmission electron microscopy (TEM) and a WST assay on these treated and non-treated cells. A combination index analysis was performed to assess the effects of NCL1 and CQ. Lastly, the effect on xenograft tumors in CRPC mice models was measured by subcutaneously injecting castrated nude mice with PCai1 cells. Mice were injected intraperitoneally with vehicle, NCL1, or NCD38, and subsequent growth was recorded.
THE JOURNAL OF UROLOGYâ
Vol. 195, No. 4S, Supplement, Monday, May 9, 2016
RESULTS: The WST assay revealed that the number of viable cells was reduced after NCL1 and NCD38 treatment. ChIP showed NCL1-induced H3K9me2 accumulation at the ELK4 and KLK2 promoters. Flow cytometry showed that NCL1 dose-dependently induced apoptosis. Using TEM analysis, autophagosomes were observed in LNCaP cells treated with NCL1. The combination index analysis further revealed that a combination of NCL1 and CQ significantly decreased cell growth synergistically. When NCL1 and NCD38-treated mice were compared with controls, the xenograft tumor volume was reduced with no adverse effects. CONCLUSIONS: Castration resistant prostate cancer growth was effectively suppressed with NCL1 and NCD38 both in vitro and in vivo without adverse events via regulation of apoptosis and autophagy, indicating the potential use of LSD1 inhibitors as therapeutic agents for prostate cancer.
e813
significant difference in CD31 expression in PC3/P tumors following treatment with either docetaxel or cabazitaxel, cabazitaxel markedly inhibited the expression of CD31 in PC3/R tumors compared with docetaxel. Findings similar to those between the PC3 sublines both in vitro and in vivo were achieved between DU145/P and DU145/R. CONCLUSIONS: Collectively, these findings suggest that antitumor activity of cabazitaxel in prostate cancer cells even after the acquisition of resistance to docetaxel could be explained, at least in part, by the inactivation of Akt, which was persistently phosphorylated following treatment with docetaxel. Source of Funding: None
MP62-05 DRUG, DISEASE, GENE INTERACTION: SIMVASTATIN, PROSTATE CANCER AND THE MITOCHONDRIAL GENOME. Rebecca Arnold*, Qian Sun, John Petros, Atlanta, GA
Source of Funding: Grants-in-aid for Scientific Research, Ministry of Education, Culture, Sports, Science and Technology
MP62-04 MOLECULAR MECHANISM MEDIATING CYTOTOXIC ACTIVITY OF CABAZITAXEL IN DOCETAXEL-RESISTANT HUMAN PROSTATE CANCER CELLS Akira Miyazaki*, Kobe, Japan; Hideaki Miyake, Hamamatsu, Japan; Masato Fujisawa, Kobe, Japan INTRODUCTION AND OBJECTIVES: It has been well documented that cabazitaxel remains active in patients with castrationresistant prostate cancer (CRPC) progressing after treatment with docetaxel. The objective of this study was to evaluate molecular mechanism mediating cytotoxic activity of cabazitaxel in docataxelresistant human prostate cancer cells. METHODS: Two parental human prostate cancer cell lines, PC3 (PC3/P) and DU145 (DU145/P), which were shown to grow through androgen-independent manner, were continuously exposed to increasing doses of docetaxel, and cell lines resistant to docetaxel, PC3/R and DU145/R, showing approximately 5-fold higher IC50 than those of PC3/P and DU145/P, respectively, were developed. Differences in molecular phenotypes following treatment with docetaxel or cabazitaxel between these cell lines were comparatively investigated. RESULTS: There were no significant differences in sensitivities to cabazitaxel, between PC3/P and PC3/R. In PC3/P, both docetaxel and cabazitaxel markedly inhibited the expression levels of phosphorylated Akt and p44/42 mitogen activated protein kinase (MAPK). In PC3/R, however, phosphorylation of Akt and p44/42 MAPK were maintained following treatment with docetaxel, whereas treatment with cabazitaxel resulted in the marked down-regulation of phosphorylated Akt, but not that of p44/42 MAPK. Furthermore, additional treatment of PC3/R with a specific inhibitor of Akt significantly enhanced the cytotoxic activity of docetaxel, but not that of cabazitaxel. In vivo growth of PC3/R in nude mice after treatment with cabazitaxel was significantly inhibited compared with that following treatment with docetaxel. Despite the absence of a
INTRODUCTION AND OBJECTIVES: Statins, 3-hydroxy-3 methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, are currently the most widely used cholesterol-lowering drugs. There is an epidemiologic association between statin use and decreased risk of prostate cancer progression. Both inherited and somatic mutations of the mitochondrial genome are linked to prostate cancer. We previously demonstrated that simvastatin treatment induced apoptosis in human cybrid prostate cancer cells and that the response to drug was different depending on mitochondrial genotype with a prostate cancer-derived mitochondrial DNA (mtDNA) mutation conferring resistance to simvastatin induced apoptosis (The Prostate 75: 1916, 2015) The purpose of this study was to determine the molecular mediators of this effect. METHODS: Cytoplasmic hybrid (cybrid) cell lines were constructed that contained a prostate cancer nucleus and either wild type or mutant mtDNA derived from a prostate cancer patient with the cytochrome oxidase subunit 1 gene mutation T6124C (Met74Thr). Multiple clones for each genotype were tested for mitochondrial protein expression. Both wild type and mutant cells were treated with increasing concentrations of simvastatin and reactive oxygen species (ROS) and reactive nitrogen species (RNS) levels measured. RESULTS: The prostate cancereassociated mtDNA mutation T6124C (Met74Thr) caused a dramatic loss of cytochrome oxidase peptide subunits I, II and IV and substantial alterations of reactive oxygen and nitrogen species. Specifically, the mutation was associated with a shift from superoxide anion to hydrogen peroxide by increasing the mitochondrial superoxide dismutase, an effect that was further augmented in the presence of simvastatin. The mutation also lowered the cellular nitric oxide levels by approximately 2/3, a difference that simvastatin only partially decreased. CONCLUSIONS: While we have previously demonstrated that a prostate cancer-derived mitochondrial DNA mutation confers resistance to simvastatin-induced prostate cancer cell apoptosis, the current study demonstrates that this is likely due to a more global downregulation of multiple subunits of respiratory complex IV (the cytochrome oxidase) and that this is associated with decreased reactive nitrogen species. Individual prostate cancer patients response to simvastatin may be substantially altered by mitochondrial genetics and associated with alterations in cellular ROS and RNS production. Source of Funding: none
MP62-06 COMBINED AKT AND MEK PATHWAY BLOCKADE IN PRECLINICAL MODELS OF ENZALUTAMIDE-RESISTANT PROSTATE CANCER bec, Canada; Soojin Kim, Amina Zoubeidi, Paul Toren*, Que Vancouver, Canada INTRODUCTION AND OBJECTIVES: Despite recent advances with newer androgen receptor (AR) pathway inhibitors,