Abstract P3-07-05: Potential of breast tumour kinase (Brk) as a therapeutic target: Brk modulates drug responses in breast cancer cell lines

Abstract

Abstract Background: Breast tumour kinase (Brk/PTK6) is over-expressed in up to 86% of all breast cancers and has shown to be involved in the processes regulating tumour development and progression. A few Brk inhibitors are in development and our studies indicate potential for a Brk inhibitor to be used in combination with current breast cancer therapies. Furthermore the effect of common breast cancer therapies and Brk inhibition on the expression of ptk6 as well as it's alternatively spliced isoform (ALT-PTK6) have not been previously been shown. Using qPCR we have determined gene expression of both genes before and after treatment. Methods: Breast cancer cell lines T47D, GI101, BT474, SKBR3, MDA-MB 231, and MDA-MB 436 were treated with Brk inhibitor, Compound 4f (Mahmoud et al, 2012) and common breast cancer therapies (Taxol, Doxorubicin, Lapatinib and Tamoxifen) at concentrations ranging from 0µM to 10µM. Western blotting was carried out to determine levels of Brk and activation of Brk substrate; STAT3 after treatment with Brk inhibitor. RNA was extracted using Qiagen Mini RNeasy prep kit, cDNA synthesized using Invitrogen Superscript II Reverse Transcriptase and qPCR carried out using primers specific for PTK6 or primers that recognized both transcripts (PTK6 and ALT-PTK6) for total expression. Results: In all cell lines tested there was a moderate reduction in cell proliferation following treatment with the Brk inhibitor (4f). However a greater effect was observed in combination therapy. Triple negative breast cancer cell lines MDA-MB 231 and MDA-MB 436 were treated with Compound 4f and Taxol or Doxorubicin (n=3) resulting in modest but statistically significant reduction in cell numbers. Cell responses to Taxol in both cell lines were significantly greater in the presence of 4f over a range of doses (P between 0.05 and 0.007). Responses to Doxorubicin were also significantly improved in the presence of 4f (P<0.03 for MDA-MB-231 and P=0.03 for MDA-MB-436). Co-treatment of HER2 positive breast cancer cell lines BT474 and Sk-Br-3 with Lapatinib and 4f showed significant increase in responses over a range of doses between 0.31 and 5µM (n=3, P<0.05 for BT474 and P between 0.03 and 0.0004 for Sk-Br-3). In comparison to untreated cells, there was statistically significant reduction in ptk6 and Total gene expression observed at various time points within multiple breast cancer cell lines in response to Compound 4f treatment. Significant differences between untreated and treated cells for T47D cell line were at 8 hours post treatment (p=0.02), 2 and 4 hours post treatment in GI101 cell line (p=0.04 and p=0.02 respectively), 8 and 24 hours post treatment in Sk-Br-3 (p=0.001 and p=0.017 respectively) and 8 hours post treatment in MDA-MB 231 cell lines (p=0.03). Conclusion: Inhibition of Brk led to a decrease in ptk6 gene expression. Expression ratios of ptk6 and the short isoform ALT-PTK6 were determined and a reduction of ptk6 gene correlated with elevation of ALT-PTK6 and vice versa. Inhibition of Brk also indicated an increase in breast cancer cell sensitivity to current breast cancer therapies. Our studies thus indicate potential for inhibition of Brk kinase activity in combination with current breast cancer treatments. Citation Format: Hussain H, Harvey A. Potential of breast tumour kinase (Brk) as a therapeutic target: Brk modulates drug responses in breast cancer cell lines [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 P3-07-05.