Abstract 4150: Exploration of the role of Hsps in exosome derived from prostate cancer cells

Abstract

Abstract Introduction: Prostate cancer (PCa) is the leading type of cancer diagnosed in men. In 2007 approximately 218, 890 new cases of prostate cancer were reported in the United States (American Cancer Society). Initially the disease is locally confined to the prostate and is hormone or androgen-dependent. It is curable at this stage upon surgical or radiation treatment however, with time, which varies from months to years, many prostate cancers metastasize and even with aggressive hormone ablation treatment approaches, progress to castration resistance (CRPC) and lethality. During early metastasis a response to androgen deprivation therapy (ADT) is usually observed however despite a reduction in androgen levels after ADT, androgen receptor remains active contributing to CRPC progression. Hsp27 is a stress-activated cytoprotective chaperone, up-regulated in CRPC, which predicts a poor prognosis and confers hormone and chemo-resistance in animal models. Targeting Hsp27 using antisense delays time post castration and enhances taxane activity. Recent studies linked cancer with exosome formation; exosomes are 30-100 nm membrane-bound compartment secreted form normal and tumor cells. The mechanisms underlying exosome formation and secretion are not fully understood. Factors such as stage of cancer, cell type and cell cycle could affect the amount and composition of exosomes formed and secreted. Since Hsp27 is up-regulated in CRPC, we hypothesize that Hsp27 can influence exosome formation in androgen independent PCa. Methods-Results: PC3 cells were seeded and grown in Dulbecco's modified Eagle's medium (DMEM) with 5% fetal bovine serum until 70% confluency. Media was changed to 5% charcoal strip serum DMEM and finally to a serum free media for 72 hours before exosome isolation. Exosomes were purified from the conditioned media using differential centrifugation after remove cell debris and protein aggregates. Further analysis by transmission electron microscopy detected the integrity of purified exosomes. Western blot analysis was also used to probe for different exosome markers including Actin, Tubulin, TSG 101, HSP70 and CD36. In this study, we functionally analyzed the role of Hsp27 in relation to exosome formation from androgen independent prostate cancer cells. Hsp27 was knocked down in PC3 cells using siRNA, and confirmed with western blot analysis. The relative content of Hsp27 in exosomes mirrors and is therefore reflective of that found in cells. Upon Hsp27 knockdown in PC3 cells we can also observe that Hsp27 does not influence exosome formation. Conclusion: Hsp27 chaperone protein is expressed within exosomes, perhaps serving as a novel exosome marker in prostate cancer cell, while Hsp27 knockdown does not affect exosome formation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4150.