CYB5D2 PRODUCES TUMOR SUPPRESSING EFFECTS IN BREAST CANCER

Abstract

Breast Cancer (BC) is the leading cause of cancer related deaths among women worldwide. Its etiology includes inactivation of tumor suppressors. In line with this notion, our laboratory has recently reported that CYB5D2 possesses tumor suppressing activities in cervical cancer; evidence also suggests CYB5D2 as a novel tumor suppressor of BC. I thus hypothesize that CYB5D2 mitigates cell propagation in vitro. To examine this possibility, I transiently expressed CYB5D2 in two typical BC subtype cell lines, HCC 1954 (HER2+) and MCF7 (Luminal A). Remarkably, cell population was diminished over a period of at least five days post-transfection when compared to empty vector (EV). To characterize CYB5D2-derived inhibition of BC cell proliferation, I generated a Tet-On inducible system in both cell lines in which CYB5D2 expression is induced upon addition of doxycycline. As expected, induction of CYB5D2 led to a decline of cell propagation and colony formation based on cell proliferation and colony formation assays respectively. Moreover, knockdown of CYB5D2 via lentivirus-based shCYB5D2 transfection in HCC 1954 and MCF7 cells resulted in an incline of cell propagation and colony formation when compared to the shCTRL (control) line. To examine possible apoptotic mechanism of tumor suppressor, we performed TUNEL assay analysis in cell lines expressing CYB5D2. Both HCC 1954 and MCF7 obtained similar results exhibiting evidence of apoptotic cells when compared to their respective controls. Interestingly, upon CYB5D2 expression HCC 1954 also displayed evidence of halting G1 phase progression when performing cell cycle analysis, suggesting a promising alternate tumor suppressing mechanism. Lastly, to corroborate with previous observations of an existing molecular interaction between CYB5D2 and tumor suppressor Phosphatase and tensin homolog (PTEN) we performed a Co-Immunoprecipitation (Co-Ip) assay. As expected, endogenous CYB5D2 and PTEN lysate from HCC 1954 and MCF7 were demonstrated to interact on a molecular level. These observations support the notion that CYB5D2 elicits tumor-suppressing activities in both Her2+ and Luminal A breast cancer cell lines. It is important to note that we detected a greater tumor suppressing impact of CYB5D2 in HCC 1954 when compared to MCF7 suggesting a potential mechanism to favour HER2+ status. Additional research in determining potential tumor-suppressing pathway of CYB5D2 in BC is encouraged as we established promising insight of novel tumor suppressor.