Abstract 4518: Impact of hypoxia on SFK activities in prostate cancer cells

Abstract

Abstract Background: Metastasis is the major reason of disease progression and poor prognosis in prostate cancer. Src family kinases (SFKs), including c-Src, Lyn and Fyn, are non-receptor tyrosine kinases that have been shown to play an essential role in local invasion, castration resistance, and metastasis in prostate cancer. Hypoxia is able to promote metastasis-associated functions by activating various signaling molecules, including SFKs. However, whether all SFK members are upregulated by hypoxia is unclear. In the current study, the effects of hypoxia on cell functions and SFK activities are determined. Further, genetic approaches are applied to identify the typical SFK member that is mostly activated by hypoxia. Methods: For hypoxic exposure, prostate cancer cells were exposed to low oxygen tensions (1% O2) for varied durations (0, 2, 6, 24 h). For drug treatment, cells were treated with saracatinib (0, 100, 500, 1000 nM) for indicated time periods. To test cell migration, “wound-healing” assay was used by making a scratch on confluent monolayers. Cells that have migrated to the denuded area were imaged. Cell invasion was detected by seeding cells into Matrigel-coated transwell chamber and cells on the bottom membranes were counted after treatment. Clonogenic assay was performed to test cell survival. At the molecular level, SFKs phosphorylation was detected by Western blotting, and gene knockdown was accomplished by siRNA transfection. To investigate expression of p-SFK and HIF-1α in patients, tumor tissues from prostate cancer patients were probed with desired antibody by immunohistochemical staining. Results: Both p-SFK and HIF-1α were highly expressed in patient tissues with advanced stage, with HIF-1 expression significantly associated with tumor grade and Gleason score. At the cellular level, while short term hypoxic exposure (2-6 h) induced greater cell migration, invasion and survival than prolonged hypoxia (24 h) in PC-3ML and C4-2B, these behaviors were increased under hypoxia with a period of 24 h in TRAMP-C1 cells. Further, hypoxia enhanced SFK phosphorylation in the pattern that was consistent with cell functions. Knockdown SRC, but not LYN, abolished hypoxia-induced invasion and p-SFK expression. Lastly, SFK inhibitor saracatinib showed stronger inhibition on functional behaviors facilitated under hypoxia than normal conditions. Conclusions: Our data show that hypoxia, particularly short-term exposure, is able to enhance metastatic phenotypes by activating SFKs, predominantly c-Src, in prostate cancer cells, suggesting c-Src may be the most important signaling molecule of hypoxia-mediated behaviors. These findings have clarified the exact SFK member that is typically activated under hypoxia at least in prostate cancer model in vitro. More importantly, SFK inhibitors are able to impair cell functions driven by hypoxia, suggesting their therapeutic potential by suppressing tumor metastasis that is driven by hypoxia in prostate cancer. Citation Format: Yao Dai, Dietmar W. Siemann. Impact of hypoxia on SFK activities in prostate cancer cells [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 4518. doi:10.1158/1538-7445.AM2017-4518