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. / Thesis / Master of Health Sciences (MSc)

<p>Loss of chromosome 17p is frequently observed in various cancers. One of the most commonly mutated targets p53 is on chromosome 17p13.1. However, studies have also reported loss of the 17p13.2 region in breast and medulloblastoma, thereby suggesting the residence of potential tumour suppressors in 17p13.2. Cytochrome b5 domain containing 2 (CYB5D2) is located on 17p13.2 implying CYB5D2 being a candidate tumour suppressor. CYB5D2 (neuferricin) belongs to the family of membrane associated progesterone receptors (MAPR). The archetypal member of the family, progesterone receptor membrane component 1 (PGRMC1), has been shown to play a role in domains independently of its function in mediating progesterone signalling. Consistent with this, CYB5D2 was reported to promote neurogenesis and inhibit the proliferation of Neuro2a cells. However, its role in tumorigenesis remains unknown.</p> <p>To investigate the role of CYB5D2 in tumorigenesis, western blot analysis was performed on 20 matched clear cell renal cell carcinomas (ccRCC) and the adjacent non-tumour kidney (ANK) tissues; significant down-regulation of CYB5D2 was demonstrated in ccRCC in comparison to ANK tissues, an observation that was confirmed by immunohistochemistry (IHC) analysis of 9 pairs of ccRCC-ANK tissues. Ectopic expression of CYB5D2 inhibited the proliferation and the invasion of A498 ccRCC along with the inhibition of AKT activation. Collectively, the above results support the possibility of CYB5D2 being a potential tumour suppressor.</p> <p>In support of the results obtained in ccRCC, we were able to show a significant reduction of CYB5D2 in cervical squamous carcinoma compared to normal cervical tissues in our analysis of CYB5D2 expression in 35 cervical squamous tumours. In vitro, overexpression and knockdown of CYB5D2 inhibited and enhanced the invasion of HeLa cells, respectively. As a member of the MAPR family, CYB5D2 contains the signature motif of the family, the cytochrome b5 (cyt-b5) like heme/steroid binding domain. This domain is known for heme binding and research in our laboratory has shown the residue D86 being critical for heme association. Ssubstitution of D86 with G (D86G) abolished not only CYB5D2's ability to bind heme but also its capacity of inhibiting HeLa cell invasion. Taken together, we provide evidence that CYB5D2 possesses activities in suppressing tumorigenesis, at least for the tumorigenesis of ccRCC and cervical squamous carcinoma.</p> / Master of Science (MSc)