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The Role and Mechanism of Action of BRK in Tamoxifen-resistant Breast Cancer

Date

2022-09-29

Journal Title

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ORCID

Type

Thesis

Degree Level

Doctoral

Abstract

The anti-Estrogen Receptor (ER) therapy Tamoxifen has historically been used as a first -line treatment against ER-positive breast cancer. However, 30% of Tamoxifen-treated tumours develop resistance against the drug (TamR). Breast Tumour Kinase (BRK), a tyrosine kinase, presents itself as a possible target to combat TamR resistance as it drives tumourigenesis in breast cancer cells. Previous research has shown that BRK knockdown re-sensitizes TamR cells to the drug, though the mechanisms behind BRK’s functioning in TamR have yet to be elucidated. To address this, I used a global phosphoproteomics approach to compare MCF7 cell lines, that differed in their sensitivity to Tamoxifen, and TamR T47D cells, that differed in BRK expression, and found a total of 1048 differentially expressed phosphopeptides. Pathway analysis revealed overrepresentation of the IGFR and insulin receptor signaling in both MCF7 and T47D TamR cells as well as when BRK was knocked down in T47D TamR cells. Specifically, BRK knockdown resulted in the inhibition of Insulin Receptor Substrate-1 (IRS1) through the hyperphosphorylation of the S1101 site and the hypophosphorylation of the Y896. Subsequent RT-PCR and ChIP-qPCR analyses revealed that both BRK knockdown and inhibition reduced downstream changes in cyclin D1 gene expression mediated by IRS1. To further identify BRK-specific targets, phosphotyrosine-enriched phosphoproteomics analysis was also conducted, comparing T47D Parental, T47D TamR and T47D TamR BRK knockdown cells. Out of 6492 phosphosites identified, 118 high -confidence phosphotyrosine sites were analyzed for significant changes in phosphorylation levels to identify differentially regulated pathways in TamR versus Parental cells and changes in these pathways when BRK is knocked down in TamR. Total proteomics analysis was then used to calculate the phosphorylation levels of these peptides relative to their total levels. Through this, I identified potential BRK-specific targets involved in TamR such as CDK1, GSK3-beta and catenin delta-1. Of these targets, I was able to validate that both the knockdown and inhibition of BRK in TamR cells resulted in the hypophosphorylation of both CDK1 and catenin delta-1 at the Y15 and Y904 phosphosites respectively. Overall, these findings indicate that BRK helps regulate TamR through its interaction with signaling intermediaries in the IGFR/insulin receptor signaling pathway.

Description

Keywords

Phosphoproteomics, proteomics, breast cancer, drug resistance, tamoxifen, breast tumour kinase, non-receptor tyrosine kinases, insulin receptor signaling, pathway analysis, mass spectrometry, beta catenin signaling pathway, cyclin dependent kinases,

Citation

Degree

Doctor of Philosophy (Ph.D.)

Department

Biochemistry

Program

Biochemistry

Advisor

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DOI

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