Src Inhibition Can Synergize with Gemcitabine and Reverse Resistance in Triple Negative Breast Cancer Cells via the AKT/c-Jun Pathway.

Zhen-Hua Wu,Chen Lin,Ming-Ming Liu,Jian Zhang,Zhong-Hua Tao,Xi-Chun Hu,Ming Tan

doi:10.1371/journal.pone.0169230

Abstract

PurposeGemcitabine-based chemotherapy remains one of the standards in management of metastatic breast cancer. However, intrinsic and acquired resistance to gemcitabine inevitably occurs. The aims of this study were to assess the efficacy of the combination of src inhibition and gemcitabine in gemcitabine-resistant breast cancer cells.Methods and ResultsBy using colony formation, sphere forming, flow cytometry, cell counting kit-8 and transwell assays, 231/GEM-res (gemcitabine-resistant) cell line, which was 10 times more resistant, was shown to have elevated drug tolerance, enhanced proliferative and self-renewal abilities, compared with its parental cells. Inhibition of src by both saracatinib (AZD0530) and siRNA could partially reverse gemcitabine resistance and attenuate resistance-associated anti-apoptosis, migration and stem cell capacities. In addition, the combination of src inhibition and gemcitabine had synergistic antitumor effects. Western blot analysis revealed up-regulation of pro-apoptotic protein BAX, along with the down-regulation of anti-apoptotic proteins (BCL-XL, Survivin), migration associated proteins (p-FAK, MMP-3) and cancer stem cell (CSC) markers (CD44, Oct-4), which was probably mediated by AKT/c-Jun pathway.ConclusionIn highly gemcitabine-resistant 231 cells, src inhibition can synergize with gemcitabine, reverse drug resistance, inhibit tumor growth/metastasis/stemness of cancer stem cells, possibly via the AKT/c-Jun pathway.

Highlights

Triple-negative breast cancer (TNBC) accounts for approximately 15% of breast cancers, which is associated with aggressive behavior, high risk of recurrence and worse prognosis [1, 2]
Western blot analysis revealed upregulation of pro-apoptotic protein BAX, along with the down-regulation of anti-apoptotic proteins (BCL-XL, Survivin), migration associated proteins (p-FAK, MMP-3) and cancer stem cell (CSC) markers (CD44, Oct-4), which was probably mediated by AKT/c-Jun pathway
In highly gemcitabine-resistant 231 cells, src inhibition can synergize with gemcitabine, reverse drug resistance, inhibit tumor growth/metastasis/stemness of cancer stem cells, possibly via the AKT/c-Jun pathway

Summary

Introduction

Triple-negative breast cancer (TNBC) accounts for approximately 15% of breast cancers, which is associated with aggressive behavior, high risk of recurrence and worse prognosis [1, 2]. Chemo-resistance to gemcitabine is almost inevitable for these patients, and the underlying molecular mechanisms remain obscure. Src, a membrane-associated non-receptor tyrosine kinase, is the protein product of the proto-oncogene c-src. It participates in the activation of various downstream pathways involved in cell survival, angiogenesis, proliferation and motility [5]. Aberrant activation or overexpression of src and src-family kinases (SFK) has been observed in various tumors, including breast cancer, which is associated with metastatic progression and poor outcome [6, 7]. MDA-MB-231, a ER/PR/Her-2 negative cell line and its gemcitabine resistant subline (231/GEM) were used. Src kinase activity was significantly elevated in gemcitabine-resistant breast cancer cells. We hypothesized that src inhibition may help to overcome gemcitabine resistance, and assessed the effects of different src expression status on development and reversal of chemo-resistance of TNBC

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