Increased Expression of c-Met is Associated with Chemotherapy-Resistant Breast Cancer and Poor Clinical Outcome

Abstract

BACKGROUND The relevance of c-Met expression as a prognostic or predictive clinical indicator in chemotherapy-resistant breast cancer remains unknown. The aims of this study were to investigate the expression of c-Met in breast cancer tissues and its association with expression of type II topoisomerase (TOPO II), including in patients who received neoadjuvant chemotherapy (NAC), and to investigate chemotherapy resistance in vitro in breast cancer cell lines. MATERIAL AND METHODS Tissue samples from 255 patients with breast cancer, with matched adjacent normal breast tissue, were used in tissue microarrays (TMAs). c-Met protein expression levels were determined using immunohistochemistry. Forty-five cases of breast cancer treated with NAC were studied to investigate the association between c-Met and TOPO II expression and clinical outcome. Chemotherapy resistance was evaluated in vitro in the MCF-7 and MDA-MB-231 breast cancer cell lines. RESULTS Expression of c-Met protein was increased in breast cancer tissue compared with normal breast tissue. In breast cancer tissue samples, increased c-Met expression was significantly associated with increased Ki-67 expression, tumor size, tumor stage, and TOPO II expression, and with reduced overall survival (OS) rates. Increased c-Met expression and reduced TOPO II expression were associated with chemotherapy resistance. In breast cancer cell lines, knockdown of c-Met expression induced TOPO II expression and increased tumor cell sensitivity to chemotherapy. CONCLUSIONS The findings of this study support a role for c-Met as a clinical prognostic marker and for c-Met and TOPO II as predictive markers for response to chemotherapy in patients with breast cancer.

Figure 1

Representative photomicrographs of c-Met protein expression using immunohistochemistry (IHC) in tissue microarrays (TMAs) containing sections of breast tissue. 1. Matched adjacent normal breast tissue with low levels of c-Met expression. 2. Breast tissue containing invasive ductal carcinoma shows high levels of c-Met expression; Row A shows c-Met immunostaining at a magnification of ×4 (bar=500 mm). Row B shows c-Met immunostaining at a magnification of ×40 (bar=50 mm).

Figure 2

Overall survival (OS) curves for patients with breast cancer using the Kaplan-Meier method and the log-rank test. Overall survival (OS) curves for patients with breast cancer with high c-Met expression (blue line, 1) and patients with low or no c-Met expression (green line, 2).

Figure 3

Representative photomicrographs of c-Met and TOPO II protein expression using immunohistochemistry (IHC) in tissue microarrays (TMAs) containing sections of breast tissue. Row 1 shows high levels of c-Met expression or low or negative TOPO II expression. Row 2 shows high levels of c-Met expression or high levels of TOPO II expression. Row 3 shows low or no expression of c-Met or TOPO II. Row 4 shows low or no expression of c-Met and high levels of TOPO II expression. Line 1 shows c-Met immunostaining with magnification ×20. Line 2 shows TOPO II immunostaining with magnification ×20.

Figure 4

Knockdown of c-Met induced TOPO II expression and drug resistance in breast cancer cell lines, MCF-7 and MDA-MB-231, compared with the normal breast epithelial cell line, MCF10A. (A) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot show c-Met expression in the MCF-7 and MDA-MB-231 breast cancer cell lines, compared with the normal breast epithelial cell line, MCF10A. GAPDH was used as the internal control. (B) Ingenuity Pathway Analysis identifies the molecular interaction between c-Met and topoisomerase. (C) Western blot and qRT-PCR shows that c-Met protein and mRNA levels in MCF-7 and MDA-MB-231 breast cancer cell lines were significantly reduced after transfection with c-Met short interfering RNA (siRNA) (si1 and si2), knocked down c-Met and induced TOPO II expression in MCF-7 and MDA-MB-231 cells. ^# The means compared with TOPO II expression of the siNC group, ^## P<0.001, * The means compared with c-Met expression of the siNC group, ** P<0.001. (D) si1 and si2 c-Met significantly induced the proliferation of the MCF7 and MDA-MB-231 breast cancer cells lines, which was treated by different concentrations of daunorubicin, from 0.0625–1.0 mg/ml. * P<0.05.