A novel association between p130Cas and resistance to the chemotherapeutic drug adriamycin in human breast cancer cells

Abstract

Resistance to chemotherapy remains a major obstacle for the treatment of breast cancer. Understanding the molecular mechanism(s) of resistance is crucial for the development of new effective therapies to treat this disease. This study examines the putative role of p130(Cas) (Cas) in resistance to the cytotoxic agent Adriamycin. High expression of Cas in primary breast tumors is associated with the failure to respond to the antiestrogen tamoxifen and poor prognosis, highlighting the potential clinical importance of this molecule. Here, we show a novel association between Cas and resistance to Adriamycin. We show that Cas overexpression renders MCF-7 breast cancer cells less sensitive to the growth inhibitory and proapoptotic effects of Adriamycin. The catalytic activity of the nonreceptor tyrosine kinase c-Src, but not the epidermal growth factor receptor, is critical for Cas-mediated protection from Adriamycin-induced death. The phosphorylation of Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) is elevated in Cas-overexpressing cells treated with Adriamycin, whereas expression of the proapoptotic protein Bak is decreased. Conversely, Cas depletion in the more resistant T47D and MDA-MB-231 cell lines increases sensitivity to Adriamycin. Based on these data, we propose that Cas activates growth and survival pathways regulated by c-Src, Akt, and ERK1/2 that lead to the inhibition of mitochondrial-mediated apoptosis in the presence of Adriamycin. Because Cas is frequently expressed at high levels in breast cancers, these findings raise the possibility of resensitizing Cas-overexpressing tumors to chemotherapy through perturbation of Cas signaling pathways.

Figure 1

Human breast cancer cell lines differ in their sensitivity to adriamycin. A. 2 × 10⁵ MCF-7, T47D, and MDA-MB-231 cells were plated in 60-mm dishes and allowed to adhere for 24 h. Cells were treated with 0-20 μM adriamycin for an additional 48 h and the mitochondrial transmembrane potential was measured by R123 incorporation and flow cytometry as described in the Materials and Methods. Lines, the mean of three independent experiments; bars, standard error (SE). B. Expression of Cas in human breast cancer cell lines. Proteins (50 μg) were isolated from MCF-7, T47D, and MDA-MB-231 cells and immunoblotted for Cas and the small adaptor molecule Crk as a loading control. Densitometric values represent the ratio of Cas to loading control (Crk) relative to the value obtained for MCF-7 cells.

Figure 2

Cas promotes resistance to adriamycin. A, Cells overexpressing Cas incorporate greater amounts of R123. Stable doxycycline-regulated MCF-7 (Cas4) cells were cultured in the presence (diamonds) or absence (squares) of 1 μg/ml dox for 48 h and then treated with 0-20 μM adriamycin for an additional 48 h in the presence or absence of dox. Mitochondrial transmembrane potential was measured by R123 incorporation and flow cytometry as described in Materials and Methods. Lines, mean of three independent experiments; bars, SE. *, significant difference from the mean at a ≥ 95% confidence interval (CI) relative to cells expressing endogenous levels of Cas under the same treatment conditions. Inset, Cas4 cells were cultured in the presence or absence of 1 μg/ml dox for 48 h. Proteins were isolated from these cells and immunoblotted for Cas and GAPDH. B, Cas overexpression renders cells resistant to the apoptosis-inducing effect of adriamycin. Cells were cultured as described for panel A. Apoptosis was measured by TUNEL positivity and flow cytometry as described in Materials and Methods. Columns, mean of four independent experiments; bars, SE. *, significant difference from the mean at a ≥ 95% CI relative to cells expressing endogenous levels of Cas under the same treatment conditions. C, Cas overexpression abrogates adriamycin-induced G0/G1 arrest and results in a greater percentage of cells in S phase. Cells were cultured as described for panel A. Cell cycle was measured by PI staining and flow cytometry as described in Materials and Methods. Data, mean of three independent experiments. D, Cas depletion sensitizes cells to adriamycin. MDA-MB-231 or T47D cells were transfected with mock, control, or Cas-targeted siRNAs and then treated with 0-2 μM adriamycin for 48 h. Mitochondrial transmembrane potential was measured by R123 incorporation and flow cytometry as described in Materials and Methods. Columns, mean of three independent experiments; bars, SE. *, significant difference from the mean at a ≥ 95% CI relative to mock-transfected cells under the same treatment conditions. In parallel, proteins were isolated from siRNA-treated cells and immunoblotted for Cas and GAPDH.

Figure 3

Cas-mediated protection from adriamycin-induced cell death requires the kinase activity of c-Src. A, PP2 reduces the amount of rhodamine 123 incorporation in Cas overexpressing cells. Dox-regulated MCF-7 Cas4 cells were grown as described for Fig. 2. Where indicated, PP2 or AG1478 was included in the media. Mitochondrial transmembrane potential was measured by R123 incorporation and flow cytometry as described in Materials and Methods. Columns, mean of three independent experiments; bars, SE. *, significant difference from the mean at a ≥ 95% CI. B, Protection from adriamycin-induced death provided by Cas overexpression requires c-Src but not EGFR catalytic activity. Dox-regulated MCF-7 Cas4 cells were grown as described in Panel A. Apoptosis was measured by TUNEL positivity and flow cytometry as described in Materials and Methods. Columns, mean of three independent experiments; bars, SE. *, significant difference from the mean at a ≥ 95% CI. C. Relative TUNEL positivity. Columns, mean of the percentage of TUNEL-positive cells for three independent experiments divided by the mean percentage measured for each condition in cells expressing endogenous levels of Cas; bars, SE. *, significant difference for the mean at ≥ 95% CI.

Figure 4

Cas overexpression correlates with increased activation of Akt and ERK1/2. A, Proteins (50μg) isolated from cells cultured in the presence or absence of adriamycin for 48 h were separated on 10% SDS-PAGE and immunoblotted with the indicated antibodies. Immunoblots shown are representative of three experiments. B, Relative protein expression levels. The relative expression levels of Cas, pAkt^S473, and pERK1/2 were determined by dividing the band intensities obtained by densitometry for the proteins of interest by the band intensities of GAPDH (Cas), Akt (pAKT ^S473), or ERK1/2 (pERK1/2). These values were then normalized to the corresponding value for vehicle-treated cells expressing 1X Cas. Columns, mean of three independent experiments; bars, SE. *, significant difference from the mean at a ≥ 95% CI.

Figure 5

Cas mediates resistance to adriamycin through modulation of the mitochondrial-mediated cell death pathway. Proteins (50μg) isolated from cells cultured in the presence or absence of adriamycin for 48 h were separated by SDS-PAGE and immunoblotted with the indicated antibodies. The relative expression levels of Cas and Bak were determined by dividing the band intensities obtained by densitometry for the proteins of interest by the band intensities of the loading control (β-tubulin or GAPDH). These values were then normalized to the corresponding value for vehicle-treated cells expressing 1X Cas (A) or vehicle-treated cells transfected with control siRNAs (B). Columns, mean of three (A) and 2 (B) independent experiments; bars, SE. *, significant difference from the mean at a ≥ 95% CI. Immunoblots in panel C are representative of three independent experiments.

Figure 6

Models for Cas-mediated resistance to adriamycin. A, Effect of Cas overexpression in “sensitive” cell lines treated with adriamycin (MCF-7 cells expressing 6X Cas). B, Effect of Cas depletion in “resistant” cell lines treated with adriamycin (MDA-MB-231 cells treated with siCas). C, Effect of “resistant” cells treated with adriamycin (MDA-MB-231 cells treated with siControl).