Breast Tumour Kinase (Brk/PTK6) Contributes to Breast Tumour Xenograft Growth and Modulates Chemotherapeutic Responses In Vitro

Abstract

Breast tumour kinase (Brk/PTK6) is overexpressed in up to 86% of breast cancers and is associated with poorer patient outcomes. It is considered a potential therapeutic target in breast cancer, even though the full spectrum of its kinase activity is not known. This study investigated the role of the kinase domain in promoting tumour growth and its potential in sensitising triple negative breast cancer cells to standard of care chemotherapy. Triple negative human xenograft models revealed that both kinase-inactive and wild-type Brk promoted xenograft growth. Suppression of Brk activity in cells subsequently co-treated with the chemotherapy agents doxorubicin or paclitaxel resulted in an increased cell sensitivity to these agents. In triple negative breast cancer cell lines, the inhibition of Brk kinase activity augmented the effects of doxorubicin or paclitaxel. High expression of the alternatively spliced isoform, ALT-PTK6, resulted in improved patient outcomes. Our study is the first to show a role for kinase-inactive Brk in human breast tumour xenograft growth; therefore, it is unlikely that kinase inhibition of Brk, in isolation, would halt tumour growth in vivo. Breast cancer cell responses to chemotherapy in vitro were kinase-dependent, indicating that treatment with kinase inhibitors could be a fruitful avenue for combinatorial treatment. Of particular prognostic value is the ratio of ALT-PTK6:Brk expression in predicating patient outcomes.

Keywords: ALT-PTK6; Brk/PTK6; breast cancer; chemotherapy; kinase; prognosis.

Figure 1

Brk promotes breast tumour xenograft growth (A) MDA-MB-157 cells were stably transfected with either the pRc/CMV vector (Vector), or a vector encoding wild-type (WT Brk) or kinase-inactive Brk (KM Brk), and the expression was confirmed by Western blotting with β-Actin expression as a loading control. (B) Cells were injected into the mammary fat pad of female CrTac:NCr-Fox-1(nu) athymic mice and tumour volume was calculated from the measurement of two perpendicular diameters over 118 days. Mean tumour volume (n = 10) +/− SEM is plotted. * indicates p < 0.05 (student’s t-test, WT-Brk compared to the vector control).

Figure 2

Brk moderates cell responses to chemotherapeutic agents. Brk levels were suppressed in T47D and GI101 cells by RNAi (Brk KD) and suppression was confirmed by Western blotting (A); the cells were seeded and allowed to adhere overnight in 96-well plates. Cells were treated with either doxorubicin (B,D) or Paclitaxel (C,E) for 72 h. Cell viability was determined by MTT assay and expressed as a percentage of the control (vehicle only) wells. The mean % survival (+/− SD) of a representative experiment is plotted with the control (transfected with no-target siRNA) in open diamonds and Brk-suppressed cells in closed squares; * indicates p < 0.05.

Figure 3

Brk’s effects on drug responses are dependent on kinase function and not proliferation. MDA-MB-468 cells, stably transfected with empty vector (V) or constructs expressing either wild-type (WT) or kinase-inactive Brk (KM), were seeded and allowed to adhere overnight in 96-well plates. Cells were treated with either (A) doxorubicin (upper panel) or (B) paclitaxel (lower panel) for 72 h. Cell viability was determined by MTT assay and expressed as a percentage of the control (vehicle only) wells. The mean % survival (+/− SD) of a representative experiment is plotted; * indicates p < 0.05. (C) Whole cells lysates were analysed for Brk expression by Western blotting with β-Actin expression as a loading control. (D) Cells were seeded into 24 well plates and replicate wells counted over 10 days.

Figure 4

Inhibition of Brk’s kinase function increases cell sensitivity to chemotherapeutic agents. Triple negative MDA-MB-231 and MDA-MB-436 cells were seeded and allowed to adhere overnight in 96 wells plates. They were then treated with the Brk inhibitor Compound 4f alone (A,D) or in combination with paclitaxel (B,E) or doxrubicin (C,F) over a range of concentrations (0–5 µm). In B–F, open squares represent cells treated with doxorubicin or paclitaxel only; closed diamonds represent cells treated with both 4f and the chemotherapeutic agent. Cell viability was determined by MTT assay and expressed as a percentage of the control (vehicle only) wells. The mean % survival (+/− SD) of n = 3 experiments is plotted; * indicates p < 0.05.

Figure 5

Effect of ptk6 transcripts on patient outcomes. Ptk6 transcript expression was determined in breast cancer tissue (n = 127) in relation to the normal background tissue (n = 33) using real-time qPCR and correlated with conventional clinic-pathological parameters and clinical outcomes. The Kaplan–Meier curves show high ptk6 expression (green) and low ptk6 (blue) levels in relation to (A) overall and (B) disease-free survival over time, as well as ALT-TPK6 in relation to (C) overall and (D) disease-free survival over time, where high expression is again in green and low expression is in blue. The statistical analysis is shown in the Supplemental Tables S1–S4.

Figure 6

High ALT-PTK6: ptk6 ratio results in favourable overall survival. ptk6 transcript expression was determined in breast cancer tissue (n = 127) in relation to the normal background tissue (n = 33) using real-time qPCR and correlated with conventional clinic-pathological parameters and clinical outcomes. The Kaplan–Meier curves show high ALT-PTK: ptk6 ratios (green) and low ALT-PTK: ptk6 ratios (blue) levels in relation to overall survival. The statistical analysis is shown in the Supplemental Table S5.