The Role of Hypoxia-Regulated RhoB Expression in Breast Cancer Tumorigenesis and Metastasis
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Although Rho GTPases RhoA, RhoB, and RhoC share more than 85% amino acid sequence identity, they may play very distinct roles in tumor progression. RhoA and RhoC have been suggested in many studies to contribute positively to tumor progression, but the role of RhoB in cancer still remains unclear. RhoB contains a unique C-terminal region that undergoes specific post-translational modifications affecting its localization and function. In contrast to RhoA and RhoC, RhoB not only localizes at the plasma membrane, but also on endosomes, multivesicular bodies and has even been reported in the nucleus. Recent studies have shown that small GTPases such as RhoA, Rac1 and Cdc42 are induced in vitro during hypoxia. Hypoxia within a solid tumor arises from the increase in oxygen consumption of rapidly proliferating cancer cells, and a decrease in oxygen availability due to structurally and functionally abnormal blood vessels that form within these tumors. Although RhoA and RhoC are well characterized and have been extensively studied under hypoxia, whether and how hypoxia regulates RhoB remains elusive. We investigated the effect of hypoxia on the expression of RhoB and the mechanism and significance of RhoB expression in breast cancer. We found that hypoxia significantly upregulated the mRNA and protein expression of RhoB in a HIF-1 independent manner in SUM159 and MDA-MB-231 cell lines. CRISPR-mediated reduction in RhoB expression caused a marked decrease in HIF-1α expression in an AKT-dependent manner, but did not show the reverse for RhoB overexpression. Therefore, we conclude that RhoB is necessary, but not sufficient for HIF-1 induction under hypoxia. Additionally, we observed that loss of RhoB in 3D cellular environments led to a small, but significant, decrease in cell motility and proliferation. Furthermore, orthotopic implantation of RhoB knockdown cells into immune-deficient mice contributed to a slower tumor growth and decreased metastasis compared to the wild type and non-targeted controls. Our work suggests that RhoB may play an oncogenic role in breast cancer potentially by increasing HIF-1α. This work warrants additional studies to determine whether targeting RhoB could have implications in the treatment of breast cancer.