Abstract 1338: Exosome secretion promotes proliferation of African American prostate cancer cells under hypoxia: Role of HIF2A and RAB signaling
Authors: Gatikrushna Panigrahi, Taylor C. Peak, Sierra L. Patterson, Nicole Kasica, Ravi Singh, Ashok Hemal, Gagan Deep
Journal: Cancer Research (2017)

Abstract Background and Objectives: African American men experience worse prostate cancer (PCa) outcomes compared with those of Caucasian men not only in incidence and mortality rates but also harbor higher grade tumors compared to Caucasians. While the disparity is likely multifactorial, there may be biological basis for such phenomenon. Hypoxia in the tumor microenvironment is the key determinant of disease aggressiveness regulating primary growth, angiogenesis, metastasis and treatment outcomes. But role of hypoxia in the aggressiveness of PCa in African-American men has not been characterized yet. Here, we focused on hypoxia signaling, exosome biogenesis and characterization in African-American PCa cells compared to Caucasian PCa cells. Methods: We employed several techniques (cell viability assays, ultracentrifugation, nanoparticle tracking analyses [NTA], electron microscopy, and western blotting) to compare exosomes biogenesis and underlying molecular mechanisms in African-American and Caucasian PCa cells under normoxic and hypoxic conditions. Results: Compared to normoxic condition, African-American PCa cells (E006AA-hT and RC77T) either survived better or proliferated under hypoxia (1% O2), while growth of Caucasian PCa cells (PC3 and LNCaP) was strongly inhibited under hypoxia. NTA analyses showed that all PCa cells secreted more exosomes under hypoxia compared to normoxia, but more prominently in African-American PCa cells (E006AA-hT: 4.4 fold, RC77T: 35 fold, PC3: 3 fold and LNCaP: 2 fold). Exosomes secreted by E006AA-hT cells under hypoxia were loaded with higher amount of HSP70, HSP90, IL6 and Annexin II, and lower TGFβ. These exosomes also showed significantly higher MMP9 loading compared to E006AA-hT cell lysates. More importantly, under hypoxia, exosome biogenesis inhibition by GW4869 (20 µM) and 5-(N,N-dimethyl) amiloride (0.1-5 µg/ml) significantly reduced the E006AA-hT cell viability by 30% (p=0.0001) and 20-60% (p&amp;lt; 0.005 - 0.0001), respectively in 48 hrs. Further studies on hypoxia signaling revealed that E006AA-hT cells have significantly higher HIF2α and HIF1β but low HIF1α expression compared to LNCaP cells. Interestingly, HIF2α was present largely in nuclear fraction and its expression was similar under both normoxic and hypoxic conditions in E006AA-hT cells. Studies on the RAB and its effector proteins indicated that RabGAP5 expression was strongly increased under hypoxia in both E006AA-hT and LNCaP cells. Further, compared to LNCaP cells, E006AA-hT cells showed higher expression of Rab5 and Rabex5 but lower Rab4, and these molecules did not change significantly under hypoxia. Conclusion: African-American PCa cells have growth advantage under hypoxia mainly through higher exosome secretion; and higher expression/activity of HIF2α and RAB-signaling molecules could be associated with PCa aggressiveness in African-Americans. Citation Format: Gatikrushna Panigrahi, Taylor C. Peak, Sierra L. Patterson, Nicole Kasica, Ravi Singh, Ashok Hemal, Gagan Deep. Exosome secretion promotes proliferation of African American prostate cancer cells under hypoxia: Role of HIF2A and RAB signaling [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 1338. doi:10.1158/1538-7445.AM2017-1338