Abstract 3187: Role of exosome secretion in the survival of enzalutamide-resistant prostate cancer cells: Syntaxin 6 as a novel therapeutic target

Abstract

Prostate cancer (PCa) is the most commonly diagnosed malignancy in the United States and remains the second leading cause of cancer deaths among American men. PCa deaths are typically the result of metastatic castration-resistant PCa (mCRPC). Recently, enzalutamide (Enz), an oral AR inhibitor, was approved for treating patients with mCRPC, and has significantly improved patient survival. But, invariably all PCa patients eventually develop resistance against Enz and die from mCRPC. Therefore, novel strategies aimed at overcoming Enz-resistance are urgently needed to improve the survival of PCa patients. Exosomes (extracellular vesicle, 40-150 nm in size) have previously been shown to communicate oncogenic signals and to promote growth and metastasis; however, their role in drug resistance has not been fully elucidated. Therefore, we set out to better understand exosomes role in the mechanism underlying Enz-resistance in PCa cells. We isolated exosomes secreted by Enz-sensitive (S) and -resistant (R) PCa cells by ultracentrifugation. Complete characterization of these exosomes followed using nanoparticle tracking analysis (NTA), transmission electron microscopy, proteomics, and western blotting. MTT and trypan blue exclusion assays were used to evaluate cell viability. Results showed that Enz-resistant C4-2B-R cells secreted significantly higher amount (2.67 fold) of exosomes compared to sensitive C4-2B-S cells, but without any significant change in exosome size. Proteomics analysis revealed significantly lesser number and amounts of proteins loaded in exosomes secreted by C4-2B-R cells compared to C4-2B-S cells. Western blotting validated these results, and in general less loading was observed for several exosomal biomarker proteins (e.g. CD63, Lox, TSG101, and MMP9). Of note, AR-V7, a variant of the androgen receptor, was upregulated in C4-2B-R cells as well in their exosomes. In order to better understand the biological significance of increased exosome production with concomitant decreased protein expression, we administered C4-2B-R-derived exosomes to C4-2B-S cells. Interestingly, exosomes from resistant cells significantly reduced the survival of C4-2B-S cells . This suggested not only that the resistant cells were utilizing exosomes to secrete Enz, but that these exosomes were being taken up by the sensitive PCa cells, thus leading to enhanced sensitive cell death. The inhibition of exosome biogenesis in C4-2B-R cells using GW4869 (20 µM) and dimethyl amiloride (1 µg/mL) strongly decreased their cell viability, and also increased their sensitivity towards Enz treatment. We also observed strong upregulation of syntaxin 6 in Enz-resistant C4-2B-R cells. Together, these results identified a unique mechanism underlying drug-resistance, and also offer a novel target to treat Enz-resistant CRPC.

Citation Format: Taylor C. Peak, Nicole P. Kasica, Gati K. Panigrahi, Sierra L. Patterson, Ravi Singh, Ashok K. Hemal, Rhonda L. Bitting, Gagan Deep. Role of exosome secretion in the survival of enzalutamide-resistant prostate cancer cells: Syntaxin 6 as a novel therapeutic target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3187. doi:10.1158/1538-7445.AM2017-3187