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
