Abstract P1-03-10: Breast tumour kinase and its role in mTOR signalling

Abstract

Abstract Background: The development of distal metastases and the acquisition of resistance to chemotherapeutic agents are one of the leading causes of cancer related death in breast cancer patients. There is both a scientific and clinical need to understand the alterations in cellular signalling pathways that could contribute to chemotherapeutic resistance in breast cancer, thereby identifying novel targets for therapy. The intracellular tyrosine kinase Brk/PTK6 enhances coupling of ErbB signalling to PI3K/Akt and we hypothesise that Brk plays a role in chemo-resistance and activation of downstream effectors such as mTOR. Methods: The protein and activation levels of mTOR pathway components and expression levels of Brk, in both Taxol-sensitive and resistant breast cancer cells, were examined by western blotting and immunofluorescence. Brk transfected cells were treated with BEZ-235 for 72 hours and relative cell numbers determine by MTT assay. Immunoprecipitation studies were carried out on lysates from T47D cells, transfected to express FLAG-tagged Brk, with ANTI-FLAGM2 agarose beads. Sequence alignment was done using the NCBI BLAST Tool. Results: We found that mTOR signalling was up-regulated in the Taxol-resistant cell line compared to parental Taxol-sensitive cells. This up-regulation was also accompanied by increased Brk levels. Transfection of a Brk-negative breast cancer cell line, MDA-MB-468 with wild-type Brk resulted in increased levels of both mTOR and, to a lesser extent, the downstream signalling component GβL at the protein level compared to cells transfected with vector only. Levels of the mTOR inhibitor DEPTOR were also decreased in response to Brk expression and mTOR co-precipitated in FLAG-Brk pull downs using FLAG M2 beads. The Taxol resistant cells also showed altered responses to the mTOR inhibitor RAD-001/everolimus. Interestingly, sequence alignment revealed that there are common amino acid motifs between Brk and the mTOR regulatory molecule DEPTOR. Conclusions: These data implicate Brk in up-regulating mTOR expression and indicate that Brk may influence mTOR signalling in the development of Taxol resistance. It is possible that Brk could substitute for DEPTOR in mTOR complexes providing a mechanism for elevated mTOR signalling in many breast cancers. Citation Format: Harvey A, Foster H, Thorpe M, Karteris E. Breast tumour kinase and its role in mTOR signalling [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-03-10.