298 CLUSTERIN INHIBITION USING OGX-011 SYNERGISTICALLY ENHANCES ANTITUMOR ACTIVITY OF SORAFENIB IN A HUMAN RENAL CELL CARCINOMA MODEL

Vol. 187, No. 4S, Supplement, Sunday, May 20, 2012

tively. Tumor proliferation and apoptosis were measured using the markers ki67 and TUNEL, respectively. HIF-1␣ activity was determined using a firefly luciferase assay and mRNA levels quantified using qRT-PCR. PDH activity and ␣-ketoglutarate were measured using commerically available kits. Angiogenesis was assessed in-vitro by matrigel assay. RESULTS: RCC cells have more hyperpolarized ⌿⌬m (TMRM, p⬍0.001) and less mROS (Mitosox, p⬍0.001) than PT cells. Treatment with DCA reversed these changes in the RCC line without significantly altering PT ⌿⌬m or mROS. This is associated with an increase in PDH activity (p⬍0.05) and increased levels of the Krebs cycle metabolite ␣-KG (p⬍0.01). RCC cells are characterized by significantly more HIF-1␣ activity than PT cells (p⬍0.01). Treatment with DCA reduces mRNA levels of the HIF responsive genes GLUT1, GLUT4, and VEGF (p⬍0.01) as well as decreasing VEGF protein level. (p⬍0.01) RCC cells treated with DCA demonstrate decreased markers of proliferation (p⬍0.001) and increased rates of apoptosis (p⬍0.001). Supernatant, containing the angiogenic signals from RCC cells was placed on microvascular endothelial cells. The supernatant from RCC cells increased vascularity (tubular structures and total length), which was blocked by DCA treatment (p⬍0.05). CONCLUSIONS: DCA is a novel, inexpensive, oral chemotherapeutic agent that reverses the mitochondrial remodeling of RCC. This decreases proliferation and angiogenesis while increasing apoptosis in a human RCC line. Additional human tumor samples will be used to generalize these findings in RCC. Source of Funding: CIHR, AHFMR

298 CLUSTERIN INHIBITION USING OGX-011 SYNERGISTICALLY ENHANCES ANTITUMOR ACTIVITY OF SORAFENIB IN A HUMAN RENAL CELL CARCINOMA MODEL Yuji Kusuda*, Hideaki Miyake, Kobe, Japan; Martin Gleave, Vancouver, Canada; Masato Fujisawa, Kobe, Japan INTRODUCTION AND OBJECTIVES: Clusterin is expressed in a variety of malignant tumors, including renal cell carcinoma (RCC), and associated with resistance to broad-spectrum treatment. OGX-011 is a second-generation antisense oligodeoxynucleotide (ODN) currently in late-stage clinical development that potently inhibits clusterin expression and enhances the efficacy of anticancer therapies in various human cancers. The objective of this study was to investigate whether inactivation of clusterin with OGX-011 potentiates the effect of sorafenib, a novel inhibitor of multiple tyrosine kinases, in a human RCC ACHN model. METHODS: Effects of a combined treatment with OGX-011 and sorafenib on ACHN cells were examined by MTT assay, DNA fragmentation assay and Western bolt analyses of proteins involved in apoptosis and signal transduction. In vivo efficacy of this combined therapy was assessed in nude mice given subcutaneous injection of ACHN cells. RESULTS: We initially revealed the dose-dependent inhibition of clusterin expression in ACHN cells by treatment with OGX-011. Although clusterin expression was increased in a dose-dependent manner by sorafenib, additional treatment of ACHN cells with OGX-011 significantly blocked the upregulation of clusterin expression induced by sorafenib. Despite the lack of a significant effect on the growth of ACHN cells, OGX-011 treatment synergistically enhanced the sensitivity of ACHN cells to sorafenib in a dose-dependent manner, reducing the IC50 by more than 50%. Characteristic apoptotic DNA ladder formation was detected after combined treatment with OGX-011 and sublethal dose of sorafenib, but not either agent alone. Furthermore, combined treatment of ACHN cells with OGX-011 and sorafenib resulted in the marked downregulation of Mcl-1, phosphorylated Akt and p44/42 MAP kinase compared with treatment with either agent alone. In vivo systemic administration of OGX-011 and sorafenib synergistically decreased the subcutaneous ACHN tumor volume compared with that of control ODN plus sorafenib.

THE JOURNAL OF UROLOGY姞

e121

CONCLUSIONS: Collectively, these findings demonstrated that clusterin helps confer a resistant phenotype of human RCC cells to sorafenib through the inhibition of apoptosis and activation of major signal transduction pathways, and that combined use of OGX-011 may be useful in enhancing the cytotoxic effect of sorafenib in patients with advanced RCC. Source of Funding: None

299 ESTABLISHMENT OF SORAFENIB-RESISTANT MOUSE RENAL CELL CARCINOMA RENCA CELLS AND CHARACTERIZATION OF THEIR ACQUIRED RESISTANT MECHANISM Yuji Kusuda*, Hideaki Miyake, Iori Sakai, Masato Fujisawa, Kobe, Japan INTRODUCTION AND OBJECTIVES: Sorafenib, a novel orally available inhibitor of multiple tyrosine kinases, has been shown to have encouraging antitumor activity against advanced renal cell carcinoma (RCC); however, acquired resistance to sorafenib invariably develops in RCC patients. The objectives of this study were to establish RCC cell line resistant to sorafenib and to characterize the mechanism underlying the acquisition of this resistant mechanism. METHODS: A mouse RCC cell line, RenCa, was continuously exposed to increasing doses of sorafenib in vitro, and a sorafenibresistant cell line RenCa/R was developed. Changes in the phenotype in RenCa/R cells before and after sorafenib treatment were compared with those in parental RenCa (RenCa/P) cells. RESULTS: The IC50 of sorafenib in RenCa/R was approximately 5-fold higher than that in RenCa/P, while there was no significant difference in the growth between RenCa/P and RenCa/R cells when cultured in the standard medium without sorafenib. Furthermore, there were no significant differences in the sensitivities to several types of therapeutic agents for RCC, including interferon-␣, interleukin-2, sunitinib, temsirolimus and everolimus, between RenCa/P and RenCa/R cells. Phosphorylated patterns of p44/42 MAPK, Akt and STAT3 in RenCa/P cells were similar to those in RenCa/R cells before sorafenib treatment; however, following the administration of sorafenib, expression level of phosphorylated Akt, but not those of p44/42 MAPK and STAT3, in RenCa/P cells were significantly downregulated compared with those in RenCa/R cells. Despite the lack of a growth inhibitory effect on RenCa/P cells, additional treatment with LY294002, a specific inhibitor of the Akt signaling pathway, significantly increased the sensitivity of RenCa/R cells to sorafenib. Furthermore, receptor tyrosine kinase assay showed that treatment of RenCa/P cells with sorafenib resulted in the marked downregulation of various phosphorylated receptors compared with that of RenCa/R cells. However, there were no significant differences in the secreted levels of major angigenic factors between RenCa/P and RenCa/R cells irrespective of treatment with sorafenib. CONCLUSIONS: Maintained phosphorylation of several types of protein kinases during treatment with sorafenib may be involved in the acquisition of resistant phenotype in RCC cells to this agent; therefore, it would be worthy to further investigate the significance of agents inactivating protein kinases as possible candidates for overcoming resistance to sorafenib in RCC. Source of Funding: None

300 A COMPARISON OF KU0063794, A DUAL MTORC1 AND MTORC2 INHIBITOR, AND TEMSIROLIMUS IN PRECLINICAL RENAL CELL CARCINOMA MODELS Hao Zhang, Dror Berel, Yanping Wang, Robert Figlin, Hyung Kim*, Los Angeles, CA INTRODUCTION AND OBJECTIVES: Rapamycin analogs, temsirolimus and everolimus, are approved for the treatment of advance renal cell carcinoma (RCC). Currently approved agents inhibit