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Title: STAG3 gene expression in breast cancer cells
Author: Lafta, Inam Jasim
ISNI:       0000 0004 5991 6638
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2016
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The expression of cohesin genes has been found to be disregulated in a number of cancers including prostate, breast and squamous cell carcinoma; and mutations in genes that encode cohesin components have been noticed in colorectal cancer and myeloid malignancies (reviewed by Rhodes et al., 2011). It has been suggested that members of the cohesin complex might be considered as a subgroup of cancer biomarkers (Xu et al., 2011a). Therefore, this study focused on studying the expression of STAG genes in breast cancer cell lines and primary breast tumours. More attention was paid for STAG3, because in addition of being the meiotic component of cohesin is also a member of Cancer Testis (CT) antigens. CT antigens have been used successfully as cancer biomarkers as well as therapeutic targets for various malignancies. Our findings show that the STAG1, STAG2 and STAG3 genes are highly expressed at the mRNA level in the breast cancer cell lines including: MCF-7, T-47D, MDA-MB-231 and MDA-MB-468 and primary breast tumours as well compared to normal breast tissue or normal breast cell line, MCF-10A, using qRT-PCR. Interestingly, a tendency for increasing STAG3 mRNA expression was recorded from stage I through stage IV of breast tumour implying that there might be increased expression as the tumour develops. Therefore, STAG3 expression was confirmed at the protein level by immunoblotting, where STAG3 protein bands were produced by all of the studied cancer cells when compared with the normal breast. Jurkat cells were used as a positive control as STAG3 expression in these had been previously established. Further confirmation of STAG3 protein signal was achieved in primary breast tumour tissue sections compared with the normal tissue using immunohistochemistry. Overall, these data suggest that STAG3 may be a suitable novel biomarker for breast cancer detection. Because STAG3 is a potential therapeutic target for breast cancer, RNA interference was successfully used to deplete STAG3 in MCF-7 cells. Analysis of the cell cycle profile by FACS revealed an accumulation of cells in G1 phase, and simultaneous reduction in the number of cells in both S and G2/M phases of the cell cycle. However, when depleting STAG3 using other si-RNAs specific for STAG3, more breast cancer dead cells were reported in MTT toxicity assay compared to MCF-10A. Finally, we studied STAG3 regulation by the transcription factors, E2F4/E2F6, no correlation was found between STAG3 expression and either of E2Fs as depleting any of them did not affect STAG3 expression. Interestingly, we found that RNAi-mediated E2F6 silencing, but not E2F4, in cancer cells caused cell death. On the other hand, MCF-10A cells depleted of E2F6 showed higher survival fraction in MTT. This finding suggests E2F6 as another potential therapeutic target for breast cancer.
Supervisor: Goldman, Alastair ; Bryant, Helen Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available