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SORAFENIB) ENHANCES SENSITIVITY IN RENAL CELL CARCINOMA
THE JOURNAL OF UROLOGYâ
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be solved. We show that the autocrine secretion of IL-6 induced by TKIs-stimulation causes activation of AKT- mTOR pathway, and also show that combination therapy with IL-6 receptor antibody and TKI reduces angiogenesis and GLUT1 expression. METHODS: Human renal cell carcinoma cell line 786-O was used for this study. Sorafenib, sunitinib and pazopanib were used as TKIs. GLUT1 expreiion and VEGF secretions from 786-O cells cultured with TKIs were measured by real time PCR and ELISA. Western blot analyses were applied for detection of GLUT1 as well as activated Akt, mTOR proteins. The growth of tumors in athymic mice receiving combination therapy with tocilizumab and sorafenib were compared with those in the sorafenib treated mice. The effects of the combination therapy were evaluated by FDG-PET and immunohistochemical examinations for CD31 and GLUT1. RESULTS: The 786-O RCC cell line secreted IL-6 and VEGF when they were cultured with TKIs. Western blot analysis revealed that Akt and mTOR were phosphorylated by TKI treatment. Tocilizumab treatment in combination with TKIs reduced activation of IL-6 signaling pathway and also suppressed cell proliferation and GLUT1 expression. The mean SUV max was decreased on day 3 in athymic mice receiving combination therapy with tocilizumab and sorafenib in comparison with those in the sorafenib alone (9.81.6 vs. 11.50.8 respectively. p-0.04). No histopathological differences were found on day 3 despite decreased CD31 positive cells. On the day 21, the CD31 positive cells increased again in the tumors of sorafenib monotherapy. In contrast, when tocilizumab was given with sorafenib the tumor showed extensive central necrosis with absence of CD31 positive cells. CONCLUSIONS: Our results indicate that TKI treatment induces GLUT1 expression on RCC cells. IL-6 antibody in combination with TIK would restrain angiogenesis and GLUT1 expression on RCC.
Vol. 195, No. 4S, Supplement, Tuesday, May 10, 2016
CONCLUSIONS: Anti-androgen therapy suppresses tumor growth in AR-positive RCC in a xenograft model suggesting AR as a new potential target of intervention in the treatment of AR-positive RCC.
Source of Funding: Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No.22591773).
MP92-12 ANTI-ANDROGEN THERAPY SUPPRESSES TUMOR GROWTH IN ANDROGEN RECEPTOR-POSITIVE RENAL CELL CARCINOMA: A XENOGRAFT STUDY Christopher Han*, Geun Taek Lee, Rutveej Patel, Parth Modi, Seok Joo Kwon, Izak Faiena, Neal Patel, Eric Singer, Isaac Kim, New Brunswick, NJ INTRODUCTION AND OBJECTIVES: The presence of androgen receptor (AR) in renal cell carcinoma (RCC) has been shown to be associated with higher tumor stage irrespective of gender. Our previous study with androgen treatment in AR-positive RCC cell lines has resulted in tumor proliferation while enzalutamide (Enz) treatment caused reduction in cell viability. We have, therefore, performed a xenograft study to study the in vivo effect of anti-androgen therapy in AR-positive RCC. METHODS: Male nude mice were randomly assigned to the castration and non-castration groups following the subcutaneous injection of an AR-positive RCC cell line, CAKI2. After surgical castration of the castration group, each group was further divided into six experimental arms: the main control arm without any treatment nor vehicle administration, the treatment arms with Enz, abiraterone acetate (AA), or the combination of Enz and AA treatments, and the respective vehicle-only arms for the Enz and AA arms. Both groups were treated for three weeks. RESULTS: The castration group showed almost 40% reduction in the average tumor size compared to the non-castration group at three weeks (p<0.01). Compared to the respective vehicle-only arm, Enz arm showed 4- and 20-fold reductions in tumor sizes in the castration and non-castration groups, respectively (p<0.01). AA arm also showed 2.5and 9-fold size reductions in each group, respectively (p<0.01). Furthermore, the combination therapy with Enz and AA showed 12- and 23-fold reductions, respectively, in tumor sizes compared to the main control arm (p<0.01).
Source of Funding: Cancer Institute of New Jersey
MP92-13 NOVEL COMBINATION TREATMENT (OGX427/ SORAFENIB) ENHANCES SENSITIVITY IN RENAL CELL CARCINOMA Sebastian Frees*, Claudia Chavez-Munoz, Peter Raven, Betty Zhou, Martin Gleave, Kim Chi, Alan So, Vancouver, Canada INTRODUCTION AND OBJECTIVES: Novel targeted therapies, tyrosine kinase inhibitors (TKI) have led to significant improvement
THE JOURNAL OF UROLOGYâ
Vol. 195, No. 4S, Supplement, Tuesday, May 10, 2016
in outcome of patients with metastatic renal cell carcinoma (mCCRCC). However, eventual drug resistance to TKI universally occurs due to upregulation of non-VEGF genes that allows for tumor progression. Heat shock protein 27 (Hsp27), a chaperone molecule involved in cell stress response, has been shown to be involved in treatment resistance in many malignancies. In this study, we assess the expression of Hsp27 in CCRCC and the effects of knocking down Hsp27 in cCCRCC with or without TKIs. METHODS: Immunhistochemistry of a tissue microarray from 15 patients treated with Sorafenib was stained for expression of Hsp27. Several renal cell lines (CAKI-1, CAKI-2, 7680 and ACHN) were treated with Sorafenib and analysed for Hsp27 expression. After specific knockdown of Hsp27 with siRNA and antisense oligonucleotide (OGX427) cell viability was measured using Presto Blue. RESULTS: Staining of the tissue microarray revealed a significant upregulation in the protein expression of Hsp27 after treatment compared to a historical untreated cohort. In human renal cancer cell lines, we were able to observe similar results. Targeting Hsp27 with siRNA and antisense oligonucleotide revealed a decrease in cell viability in combination with Sorafenib treatment. CONCLUSIONS: Treatment of renal cell carcinoma with Sorafenib leads to an upregulation of the Hsp27 in-vivo and in vitro. Targeting Hsp27 in combination with Sorafenib treatment might lead to better response rates in the treatment of advanced renal cancer. Source of Funding: none
MP92-14 INHIBITION OF MTOR/AKT PATHWAY ENHANCES CELL DEATH VIA AUTOPHAGY IN RENAL CELL CARCINOMA Hua Chen, Kyle Potts, Allan Murray, Ronald Moore*, Edmonton, Canada INTRODUCTION AND OBJECTIVES: mTOR (mammalian target of rapamycin) kinase, the catalytic subunit of two multiprotein complexes (mTORC1 and mTORC2), lies at a critical signaling node of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway and plays a key role in cell proliferation, survival and autophagy. Although mTORC1 is a validated therapeutic target for renal cell carcinoma (RCC), the mechanisms by which allosteric inhibitors of mTORC1, temsirolimus and everolimus, retard tumor growth are not fully understood. In this study, we tested the impact of inhibitors of the PI3K/ AKT/mTOR pathway on cell proliferation, apoptosis and autophagy in a panel of RCC cell lines and explored molecular mechanisms of the anticancer activities. METHODS: Five RCC cell lines (786-O, Caki-1, Caki-2, A498 and ACHN) were used in this study. Cell viability and clonogenicity were evaluated by XTT and colony formation assays. We analyzed cell cycle distribution and apoptosis by flow cytometry and Western blotting. The efficacy of PI3K/AKT/mTOR pathway inhibition by the mTORC1 inhibitor RAD001, the AKT inhibitor MK2206, the PI3K/mTOR inhibitor PI103 and the ATP-competitive dual mTORC1 and mTORC2 inhibitor AZD8055 were evaluated by Western blotting for changes in phosphorylated AKT, S6K, N-myc downstreamregulated gene 1 (NDRG1) expression and autophagy marker expression. Autophagic vacuole formation was evaluated by immunocytochemistry. RESULTS: We found that mTORC2 was activated in RCC cell lines. AZD8055 induced cell cycle arrest in G0/G1 phases and increased clonogenic cell death and apoptosis better than MK2206, PI103 and RAD001. In exploring the underlying mechanisms of cytotoxicity, we found that autophagic flux was induced in Caki-1 cells treated with AZD8055, MK2206 and PI103 but not RAD001, resulting in significantly increased expression of LC3-II, decreased levels of p62 and the formation of autophagic vacuoles, all markers of autophagic flux. Pharmacologic inhibition or knockdown of autophagy prevented Caki-1 cells from AZD8055-induced cell death. Interestingly, defective autophagy marked by the presence of
e1167
sustained p62 was evidenced in several RCC cell lines and appeared to contribute to tumor growth via p62 accumulation. Mechanistically, we identified that NDRG1 was involved in the regulation of AZD8055-induced autophagy. CONCLUSIONS: The results illustrate the importance of mTORC2 as a cancer target and suggest that inactivation of p62 accumulation (a selective receptor in autophagy defective cells) could be a novel predictive marker for mRCC. Source of Funding: Mr. Lube Foundation, Canadian Cancer Research Society (CCRS) and Alberta Innovates Health Solutions (AIHS).
MP92-15 SUPPRESSION OF CHAPERONE-MEDIATED AUTOPHAGY: A NOVEL MECHANISM OF ACTION OF SILIBININ AGAINST BLADDER AND RENAL CANCER Jin Zeng*, Wei Liu, Feng Li, Yi Sun, Lei Li, Xinyang Wang, Dalin He, Xi’an, China, People’s Republic of INTRODUCTION AND OBJECTIVES: Chaperone-mediated autophagy (CMA) contributes to cellular quality control through the selective degradation of cytosolic proteins in lysosomes. Dysregulation of CMA activity occurs in age-related disorders and cancers. Chemical regulation of CMA is not currently possible, owing to the lack of information on the signaling mechanisms. A bulk of evidence from others and us suggests that silibinin, a natural flavonoid from milk thistle, exerts strong pleiotropic anticancer capabilities in various cancers. However, underlying mechanisms are poorly understood. The aim of this study was to investigate the role of CMA in silibinininduced cell death in bladder cancer (BCa) and renal cell carcinoma (RCC). METHODS: Human BCa 5637 and T24 cell lines and RCC ACHN and 769-P cell lines served as the model system in vitro and in vivo. Cell viability was determined by MTS assay. Lysosomes were isolated from cells by iodixanol-based density gradient centrifugation. The expression of two key CMA components heat shockcognate chaperone of 70 kDa (hsc70) and lysosome-associated membrane protein type 2a (LAMP2a) on lysosomes were examined by western blot after silibinin treatment. Co-immunoprecipitation was used to determine the interaction between hsc70 and LAMP2a. CMA activity was monitored by in vitro lysosome binding and uptake assay. RNase and GAPDH, two well-characterized CMA substrates, were used in this functional assay allow tracking CMA activity. Immunohistochemistry analysis was performed for evaluating hsc70 and LAMP2a expression in BCa and RCC xenografts after oral silibinin in vivo. RESULTS: Exposure of BCa and RCC cells to silibinin resulted in significant inhibition of cell growth. Downregulation of hsc70 and LAMP2a on lysosomes were observed after silibinin treatment. Overexpression of exogenous hsc70 or LAMP2a attenuated silibinin-induced cell death, while knocking down hsc70 or LAMP2a sensitized cells to death by silibinin. Furthermore, silibinin inhibited the interaction between hsc70 and LAMP2a on lysosome in both BCa and RCC cells. Additionally, silibinin treatment decreased the ability of substrates to be taken up by the lysosomes and inhibited the lysosomal degradation of substrates, suggesting the suppression of CMA activity. Oral silibinin suppressed the growth of BCa and RCC xenografts in vivo, which were accompanied with downregulation of hsc70 and LAMP2a. CONCLUSIONS: This study reveals suppression of CMA as a novel mechanism for silibinin0 s anti-proliferative effect, and suggests targeting CMA pathway may have antitumor potential against BCa and RCC. Source of Funding: Grants from the National Natural Science Foundation of China (NSFC 81101936 to JZ; NSFC 81172436 to YS)
e1166
be solved. We show that the autocrine secretion of IL-6 induced by TKIs-stimulation causes activation of AKT- mTOR pathway, and also show that combination therapy with IL-6 receptor antibody and TKI reduces angiogenesis and GLUT1 expression. METHODS: Human renal cell carcinoma cell line 786-O was used for this study. Sorafenib, sunitinib and pazopanib were used as TKIs. GLUT1 expreiion and VEGF secretions from 786-O cells cultured with TKIs were measured by real time PCR and ELISA. Western blot analyses were applied for detection of GLUT1 as well as activated Akt, mTOR proteins. The growth of tumors in athymic mice receiving combination therapy with tocilizumab and sorafenib were compared with those in the sorafenib treated mice. The effects of the combination therapy were evaluated by FDG-PET and immunohistochemical examinations for CD31 and GLUT1. RESULTS: The 786-O RCC cell line secreted IL-6 and VEGF when they were cultured with TKIs. Western blot analysis revealed that Akt and mTOR were phosphorylated by TKI treatment. Tocilizumab treatment in combination with TKIs reduced activation of IL-6 signaling pathway and also suppressed cell proliferation and GLUT1 expression. The mean SUV max was decreased on day 3 in athymic mice receiving combination therapy with tocilizumab and sorafenib in comparison with those in the sorafenib alone (9.81.6 vs. 11.50.8 respectively. p-0.04). No histopathological differences were found on day 3 despite decreased CD31 positive cells. On the day 21, the CD31 positive cells increased again in the tumors of sorafenib monotherapy. In contrast, when tocilizumab was given with sorafenib the tumor showed extensive central necrosis with absence of CD31 positive cells. CONCLUSIONS: Our results indicate that TKI treatment induces GLUT1 expression on RCC cells. IL-6 antibody in combination with TIK would restrain angiogenesis and GLUT1 expression on RCC.
Vol. 195, No. 4S, Supplement, Tuesday, May 10, 2016
CONCLUSIONS: Anti-androgen therapy suppresses tumor growth in AR-positive RCC in a xenograft model suggesting AR as a new potential target of intervention in the treatment of AR-positive RCC.
Source of Funding: Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No.22591773).
MP92-12 ANTI-ANDROGEN THERAPY SUPPRESSES TUMOR GROWTH IN ANDROGEN RECEPTOR-POSITIVE RENAL CELL CARCINOMA: A XENOGRAFT STUDY Christopher Han*, Geun Taek Lee, Rutveej Patel, Parth Modi, Seok Joo Kwon, Izak Faiena, Neal Patel, Eric Singer, Isaac Kim, New Brunswick, NJ INTRODUCTION AND OBJECTIVES: The presence of androgen receptor (AR) in renal cell carcinoma (RCC) has been shown to be associated with higher tumor stage irrespective of gender. Our previous study with androgen treatment in AR-positive RCC cell lines has resulted in tumor proliferation while enzalutamide (Enz) treatment caused reduction in cell viability. We have, therefore, performed a xenograft study to study the in vivo effect of anti-androgen therapy in AR-positive RCC. METHODS: Male nude mice were randomly assigned to the castration and non-castration groups following the subcutaneous injection of an AR-positive RCC cell line, CAKI2. After surgical castration of the castration group, each group was further divided into six experimental arms: the main control arm without any treatment nor vehicle administration, the treatment arms with Enz, abiraterone acetate (AA), or the combination of Enz and AA treatments, and the respective vehicle-only arms for the Enz and AA arms. Both groups were treated for three weeks. RESULTS: The castration group showed almost 40% reduction in the average tumor size compared to the non-castration group at three weeks (p<0.01). Compared to the respective vehicle-only arm, Enz arm showed 4- and 20-fold reductions in tumor sizes in the castration and non-castration groups, respectively (p<0.01). AA arm also showed 2.5and 9-fold size reductions in each group, respectively (p<0.01). Furthermore, the combination therapy with Enz and AA showed 12- and 23-fold reductions, respectively, in tumor sizes compared to the main control arm (p<0.01).
Source of Funding: Cancer Institute of New Jersey
MP92-13 NOVEL COMBINATION TREATMENT (OGX427/ SORAFENIB) ENHANCES SENSITIVITY IN RENAL CELL CARCINOMA Sebastian Frees*, Claudia Chavez-Munoz, Peter Raven, Betty Zhou, Martin Gleave, Kim Chi, Alan So, Vancouver, Canada INTRODUCTION AND OBJECTIVES: Novel targeted therapies, tyrosine kinase inhibitors (TKI) have led to significant improvement
THE JOURNAL OF UROLOGYâ
Vol. 195, No. 4S, Supplement, Tuesday, May 10, 2016
in outcome of patients with metastatic renal cell carcinoma (mCCRCC). However, eventual drug resistance to TKI universally occurs due to upregulation of non-VEGF genes that allows for tumor progression. Heat shock protein 27 (Hsp27), a chaperone molecule involved in cell stress response, has been shown to be involved in treatment resistance in many malignancies. In this study, we assess the expression of Hsp27 in CCRCC and the effects of knocking down Hsp27 in cCCRCC with or without TKIs. METHODS: Immunhistochemistry of a tissue microarray from 15 patients treated with Sorafenib was stained for expression of Hsp27. Several renal cell lines (CAKI-1, CAKI-2, 7680 and ACHN) were treated with Sorafenib and analysed for Hsp27 expression. After specific knockdown of Hsp27 with siRNA and antisense oligonucleotide (OGX427) cell viability was measured using Presto Blue. RESULTS: Staining of the tissue microarray revealed a significant upregulation in the protein expression of Hsp27 after treatment compared to a historical untreated cohort. In human renal cancer cell lines, we were able to observe similar results. Targeting Hsp27 with siRNA and antisense oligonucleotide revealed a decrease in cell viability in combination with Sorafenib treatment. CONCLUSIONS: Treatment of renal cell carcinoma with Sorafenib leads to an upregulation of the Hsp27 in-vivo and in vitro. Targeting Hsp27 in combination with Sorafenib treatment might lead to better response rates in the treatment of advanced renal cancer. Source of Funding: none
MP92-14 INHIBITION OF MTOR/AKT PATHWAY ENHANCES CELL DEATH VIA AUTOPHAGY IN RENAL CELL CARCINOMA Hua Chen, Kyle Potts, Allan Murray, Ronald Moore*, Edmonton, Canada INTRODUCTION AND OBJECTIVES: mTOR (mammalian target of rapamycin) kinase, the catalytic subunit of two multiprotein complexes (mTORC1 and mTORC2), lies at a critical signaling node of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway and plays a key role in cell proliferation, survival and autophagy. Although mTORC1 is a validated therapeutic target for renal cell carcinoma (RCC), the mechanisms by which allosteric inhibitors of mTORC1, temsirolimus and everolimus, retard tumor growth are not fully understood. In this study, we tested the impact of inhibitors of the PI3K/ AKT/mTOR pathway on cell proliferation, apoptosis and autophagy in a panel of RCC cell lines and explored molecular mechanisms of the anticancer activities. METHODS: Five RCC cell lines (786-O, Caki-1, Caki-2, A498 and ACHN) were used in this study. Cell viability and clonogenicity were evaluated by XTT and colony formation assays. We analyzed cell cycle distribution and apoptosis by flow cytometry and Western blotting. The efficacy of PI3K/AKT/mTOR pathway inhibition by the mTORC1 inhibitor RAD001, the AKT inhibitor MK2206, the PI3K/mTOR inhibitor PI103 and the ATP-competitive dual mTORC1 and mTORC2 inhibitor AZD8055 were evaluated by Western blotting for changes in phosphorylated AKT, S6K, N-myc downstreamregulated gene 1 (NDRG1) expression and autophagy marker expression. Autophagic vacuole formation was evaluated by immunocytochemistry. RESULTS: We found that mTORC2 was activated in RCC cell lines. AZD8055 induced cell cycle arrest in G0/G1 phases and increased clonogenic cell death and apoptosis better than MK2206, PI103 and RAD001. In exploring the underlying mechanisms of cytotoxicity, we found that autophagic flux was induced in Caki-1 cells treated with AZD8055, MK2206 and PI103 but not RAD001, resulting in significantly increased expression of LC3-II, decreased levels of p62 and the formation of autophagic vacuoles, all markers of autophagic flux. Pharmacologic inhibition or knockdown of autophagy prevented Caki-1 cells from AZD8055-induced cell death. Interestingly, defective autophagy marked by the presence of
e1167
sustained p62 was evidenced in several RCC cell lines and appeared to contribute to tumor growth via p62 accumulation. Mechanistically, we identified that NDRG1 was involved in the regulation of AZD8055-induced autophagy. CONCLUSIONS: The results illustrate the importance of mTORC2 as a cancer target and suggest that inactivation of p62 accumulation (a selective receptor in autophagy defective cells) could be a novel predictive marker for mRCC. Source of Funding: Mr. Lube Foundation, Canadian Cancer Research Society (CCRS) and Alberta Innovates Health Solutions (AIHS).
MP92-15 SUPPRESSION OF CHAPERONE-MEDIATED AUTOPHAGY: A NOVEL MECHANISM OF ACTION OF SILIBININ AGAINST BLADDER AND RENAL CANCER Jin Zeng*, Wei Liu, Feng Li, Yi Sun, Lei Li, Xinyang Wang, Dalin He, Xi’an, China, People’s Republic of INTRODUCTION AND OBJECTIVES: Chaperone-mediated autophagy (CMA) contributes to cellular quality control through the selective degradation of cytosolic proteins in lysosomes. Dysregulation of CMA activity occurs in age-related disorders and cancers. Chemical regulation of CMA is not currently possible, owing to the lack of information on the signaling mechanisms. A bulk of evidence from others and us suggests that silibinin, a natural flavonoid from milk thistle, exerts strong pleiotropic anticancer capabilities in various cancers. However, underlying mechanisms are poorly understood. The aim of this study was to investigate the role of CMA in silibinininduced cell death in bladder cancer (BCa) and renal cell carcinoma (RCC). METHODS: Human BCa 5637 and T24 cell lines and RCC ACHN and 769-P cell lines served as the model system in vitro and in vivo. Cell viability was determined by MTS assay. Lysosomes were isolated from cells by iodixanol-based density gradient centrifugation. The expression of two key CMA components heat shockcognate chaperone of 70 kDa (hsc70) and lysosome-associated membrane protein type 2a (LAMP2a) on lysosomes were examined by western blot after silibinin treatment. Co-immunoprecipitation was used to determine the interaction between hsc70 and LAMP2a. CMA activity was monitored by in vitro lysosome binding and uptake assay. RNase and GAPDH, two well-characterized CMA substrates, were used in this functional assay allow tracking CMA activity. Immunohistochemistry analysis was performed for evaluating hsc70 and LAMP2a expression in BCa and RCC xenografts after oral silibinin in vivo. RESULTS: Exposure of BCa and RCC cells to silibinin resulted in significant inhibition of cell growth. Downregulation of hsc70 and LAMP2a on lysosomes were observed after silibinin treatment. Overexpression of exogenous hsc70 or LAMP2a attenuated silibinin-induced cell death, while knocking down hsc70 or LAMP2a sensitized cells to death by silibinin. Furthermore, silibinin inhibited the interaction between hsc70 and LAMP2a on lysosome in both BCa and RCC cells. Additionally, silibinin treatment decreased the ability of substrates to be taken up by the lysosomes and inhibited the lysosomal degradation of substrates, suggesting the suppression of CMA activity. Oral silibinin suppressed the growth of BCa and RCC xenografts in vivo, which were accompanied with downregulation of hsc70 and LAMP2a. CONCLUSIONS: This study reveals suppression of CMA as a novel mechanism for silibinin0 s anti-proliferative effect, and suggests targeting CMA pathway may have antitumor potential against BCa and RCC. Source of Funding: Grants from the National Natural Science Foundation of China (NSFC 81101936 to JZ; NSFC 81172436 to YS)