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Unravelling the Mechanism of the SHON Protein in Triple Negative Breast Cancer MDA-MB-231 Cell Line

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Thesis embarged until 4th June 2027(5.16 MB)

Date

2025

Authors

Supervisor

Li, Yan

Item type

Thesis

Degree name

Master of Science (Research)

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Publisher

Auckland University of Technology

Abstract

Triple-negative breast cancer (TNBC) is the most deadly subtype of breast cancer, and accounts for approximately 10-15% of all breast cancer diagnoses. TNBC acquired its name, due to the fact that its cancer cells are negative for two crucial molecular receptors; estrogen and progesterone, and it also does not produce enough of the HER2 protein, and thus, is triple-negative for all three of these molecular markers. This makes this subtype of breast cancer incredibly difficult to treat as the absence of these molecular receptors, prevents the use of more effective treatments, such as endocrine therapy and immunotherapy. The anthracycline-based chemotherapy drug doxorubicin has been adopted as the standard preferred treatment for TNBC. However, while doxorubicin is the preferred treatment, it is unfortunately problematic and is highly associated with the formation of drug resistance in patients and dose-limiting toxicity. Recent research has identified a new potential oncogene associated with the formation of breast cancer (subsequently also TNBC) known as the SHON (secreted hominoid-specific oncogene) gene. Previous literature identified that the forced expression of this gene in breast cancer cell lines enhanced the oncogenicity of the cells, increased cell proliferation, and increased overall cancer cell survival. As well as enhancing the oncogenicity of breast cancer cells, there was also evidence to suggest that the SHON gene could be a potential biomarker for TNBC, and that it can be used as a way to predict patient response to anthracycline chemotherapy. Based on previous literature we hypothesized that by using CRISPR-Cas9 gene editing technology, we could knock out the SHON gene from MDA-MB-231 cells, to study and compare the differences between MDA-MB-231 cells with this gene and MDA-MB-231 cells without this gene. Allowing, not only a better understanding of how this gene behaves and functions as an oncogene, but also determining whether or not this gene knock out would cause MDA-MB-231 cells to be more sensitive to the anticancer effects of doxorubicin. The knock out of the SHON gene via CRISPR-Cas9 technology was confirmed using Sanger sequencing. To distinguish the difference in cell proliferation a colony formation assay was performed. To understand the differences in metabolic activity between SHON-KO MDA-MB-231 cells and SHON-WT MDA-MB-231 cells, a PrestoBlue assay was performed. Then an MTT assay was used to compare the differences in sensitivity to doxorubicin, and finally an apoptosis assay was performed to determine the doxorubicin-induced apoptosis rates between the isogenic cell pair. Knock out of SHON gene in MDA-MB-231 cells significantly decreased cell proliferation and colony formation, further confirming its oncogenicity roles. The results also suggest that the MDA-MB-231 cells without the SHON gene were more susceptible to undergoing apoptosis. For the first time, I generated experimental evidence that knock out of SHON gene in MDA-MB-231 cells is associated with decreased sensitivity to doxorubicin. This result is consistent with the clinical evidence that SHON expression is a positive prognostic biomarker for predicting the response to anthracycline-based therapy in TNBC patients.

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http://hdl.handle.net/10292/19270

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