Breast cancer is unanimously considered a highly heterogeneous disease due to its diverse molecular features. Breast tumor kinase (BRK), also known as protein tyrosine kinase 6 (PTK6), is a non-receptor tyrosine kinase that is highly expressed in over 80% of breast carcinomas. The role and mechanism of action of enzymatically activated BRK in breast pathology are unclear. The objectives of this project were to reveal the effect of BRK activation on cell migration, proliferation and tumorigenesis. We also aimed to determine the mechanism of action of BRK in the promotion of cell proliferation. We used BRK-negative cells (MCF10A, MDA-MB-231 and HEK293) to generate three sets of stable cell lines that stably expressed GFP alone, GFP-BRK-WT or GFP-BRK-Y447F (constitutively active) by retroviral infections. We also stably knocked down BRK from BRK-positive cells BT20 and SKBR3 by RNA interference using shRNAs against BRK. Western blotting, immunoprecipitation and qPCR studies were conducted to evaluate protein expression, protein-protein interaction and mRNA expression, respectively. Both sets of cell lines were used to determine the effect of BRK on cell proliferation (automated cell counter), cell migration (transwell and wound healing assay), transformation (colony formation assay) and tumor formation (mouse Xenograft assay). To investigate the mechanism of action of BRK, we validated downstream of tyrosine kinases 1 (Dok1), a tumor suppressor, as a BRK substrate. Deletion or site-directed mutagenesis was performed to map BRK-targeted tyrosines in Dok1 protein. Results obtained from this research project showed that stable expression of the constitutively active mutant of BRK (BRK-Y447F) in MDA-MB-231 cells led to a significant increase in the cell proliferation, migration rate and promoted colony formation and drastically enhanced tumor formation in athymic nude mice in comparison to control cells. Additionally, depletion of BRK abrogated the migration of BT20 and SKBR3 cells. Furthermore, we showed that BRK interacts with and phosphorylates Dok1, inducing Dok1 downregulation via a ubiquitin-proteasome-mediated mechanism. Together, our results show that the activation of BRK is essential for mammary gland tumorigenesis and suggest that targeting of Dok1 for degradation is a novel mechanism of action of BRK in the promotion of cell proliferation, migration and tumor formation.
| Identifer | oai:union.ndltd.org:USASK/oai:ecommons.usask.ca:10388/ETD-2015-11-2310 |
| Date | 2015 November 1900 |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text, thesis |
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