Breast cancer is highly ranked as one of the most common cause of cancer death. Although Triple Negative Breast Cancer (TNBC) is not the major subtype of breast cancer in population, it is responsible for a disproportionate share of mortality with higher grade tumors and higher rates of distant recurrence. Because most of the TNBC patients represent resistance to conventional chemotherapies, new approaches are urgently needed to improve therapeutic outcome in TNBC treatment. Calpain, a family of calcium-dependent cysteine proteases, has a wide range of cellular functions related to cancer, including functions in apoptosis, hypoxia, proliferation, migration and invasion. In this study, we aimed to identify the role of m-calpain in TNBC and to evaluate m-calpain as a novel target for TNBC therapeutics.
The correlation between m-calpain expression and clinical outcome in TNBC was investigated using database analysis. m-Calpain knock-down cellular model was constructed using MDA-MB-231 cell line. Proliferation rate and migration capacity of m-calpain KD cells were compared to those of empty vector transduced cells using WST assay and wound healing assay. EMT related gene expression was investigated using immunoblot, immunofluorescence, and quantitative RT-PCR. Subcellular localization of HIF-1α was confirmed using immunofluorescence assay. ChIP assay was conducted to analyze the interaction of HIF-1α with Twist promoter. Anoikis sensitivity in m-calpain KD cells was investigated using WST assay, Annexin V/PI double staining, and immunoblotting analysis.
m-Calpain expression was closely correlated with survival of TNBC patients in database analysis. m-Calpain silenced TNBC cells showed phenotypical changes representing inhibition of EMT signaling. Among the EMT related transcription factors, only twist expression was inhibited by m-calpain downregulation. The reduction of twist expression was due to the alteration of subcellular localization of HIF-1α.
The results indicate that m-calpain could play an important role in EMT process via HIF-1α translocation, followed by Twist expression. m-Calpain might serve as a novel target for therapeutic strategies of TNBC patients.
Clinical trial identification
Legal entity responsible for the study
Ewha Womans University
Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education (NRF-2017R1D1A1B03027846) Brain Korea 21 plus funded by the Ministry of Education
All authors have declared no conflicts of interest.