Liu, Dong-XuLi, YanLi, Jiawei2023-10-192023-10-192023http://hdl.handle.net/10292/16799The number of female breast cancer (BC) had increased to almost 2.3 million in 2020 with more two-thirds being estrogen receptor (ER) positive (ER+). Endocrine therapy, also called hormone therapy or anti-oestrogen therapy, which lowers oestrogen levels and inhibits the growth of the cancer, has been commonly used to treat ER+ BC. However, some patients are not responsive to endocrine therapy, resulting in metastasis recurrence and costing lives. SHON protein in BC tumors is a potential biomarker for anti-estrogen therapy. Emerging evidence has demonstrated that SHON protein is produced in and secreted from various cancer cells including BC (Jung et al., 2013). SHON contains a predicted open reading frame of 282 bp encoding a 93-amino acid residue protein with a predicted molecular mass of 9.7 kDa (Jung et al., 2013). It is imperative to understand the mechanism through which SHON regulates cell signaling to exert its oncogenic functions and serve as a predictive biomarker for anti-estrogen therapy. We used CRISPR-Cas9 gene editing technique to create SHON gene knockouts (KO) in somatic BC cells, coupled with the SHON knock-down and overexpression cell models and in vitro functional assays. Despite COVID19 lockdown and subsequent loss of BC SHON KO clones, the attempt to establish SHON KO cells using CRISPR-Cas9 system generates heterozygous SHON KO MCF-7 and T47D cell models, plus a potential homozygous SHON KO MDA-MB-231 cell model (Chapter 3). Our results could potentially contribute to current knowledge especially in the importance of considering BC p53 and KRAS status, target gene copy number and gene function in the future design of CRISPR/Cas system. SHON nuclear expression in breast tumors predicted the clinical outcome of patients with estrogen receptor α positive (ERα+) BC who received tamoxifen. However, the mechanism underlying has not been fully understood. The main purpose of Chapter 4 was to investigate the sensitivity of tamoxifen after depletion of endogenous SHON in two ERα+ cell lines. Our results, for the first time, show silencing SHON in MCF-7 and T47D cells significantly decreased cell proliferation and colony formation and increased their sensitivity to tamoxifen. Silencing SHON also resulted in significantly increased tamoxifen-induced apoptosis rates in MCF-7 cells compared to the scramble control. Such apoptosis alteration effects are FBS concentration dependent since decreasing FBS to 5% induced extensive but comparable apoptosis in SHON-siRNA and control MCF-7 cells. More interestingly, the FBS concentration dependence in T47D cells shows a totally opposite trend, as silencing SHON increased tamoxifen-induced apoptosis rate when T47D cells were incubated with 5% FBS but not with 10% FBS. In Chapter 5, forced expression of SHON in MCF-7 cells significantly increased cell proliferation and colony formation, enhanced the anchorage independent 3D growth of MCF7 cells, and enhanced the colony growth in the presence of Matrigel, further confirming its oncogenicity roles. For the first time, I generated experimental evidence that forced expression of SHON in MCF7 cells increased its sensitivity to tamoxifen and enhanced tamoxifen-induced apoptosis rates in ERα+ SHON-overexpressing MCF-7 cells compared with control. This result is also consistent with the clinical evidence that SHON expression in tumors has been shown to be a positive prognostic biomarker for predicting the response to anti-estrogen therapy in patients with ERα+ BC. I postulate that the constitutively active SHON protein may be also activating estrogen receptor β (ERβ), which may further activate estrogen-ERβ complex induced apoptosis especially when estrogen binding to ERα was blocked by tamoxifen in SHON-overexpressing MCF-7 cells. Given the importance of SHON as a biomarker for antiestrogen therapy, further studies are warranted for elucidating its genomic activities and proapoptotic signaling pathways.enOpenAccess