Role of calpain in breast cancer and regulation of therapeutic response to targeted treatment

The calpain system is a group of intracellular cysteine proteases. Dysregulated calpain activity, or mutation in calpain isoforms, as well as its endogenous inhibitor calpastatin, has been found to play an important role in tumorigenesis. The main aim of the current study is to explore the differential role of calpain family members in different breast cancer molecular subtypes; validate calpain-1 as a biomarker of trastuzumab response in HER2+ breast cancer patients; and explore the mechanisms by which calpain family regulates trastuzumab response in HER2+ cells. Three representative cell lines for different molecular subtypes were used: MDA-MB-231 (basal-like), MCF-7 (luminal) and SKBR3 (HER2+); and two additional HER2+ cell lines were used in the HER2+ study: acquired trastuzumab-resistant SKBR3 and inherent trastuzumab-resistant JIMT-1. The role of calpain in proliferation, signal transduction and apoptotic response was assessed using growth curves, phosphokinase arrays and Annexin V-FITC apoptosis assays, respectively. The effect of calpastatin knockdown, via shRNA, on cell migration was examined using Haptotaxis assay. The combined effect of calpeptin and trastuzumab on colony formation and cell cycle progression were examined using clonogenic survival and flow cytometry, respectively. Biomarker studies were conducted using standard immunohistochemistry. The results suggested that inhibition of calpain activity showed anti-proliferative effect on breast cancer cells across different subtypes. Knockdown of calpastatin in both MDA-MB-231 and MCF-7 cells did not have significant effects on migratory ability, either with or without calcium ionophore A23187. The study also showed that combining calpeptin and trastuzumab enhanced trastuzumab-induced anti-proliferative effects on SKBR3 and SKBR3/TR cells, but not in JIMT-1 cells. Combined treatment did not further reduce clonogenic survival either in SKBR3 or SKBR3/TR cells, compared with single agent alone. In all three HER2+ cells, combined treatment had no significant effect on trastuzumab-induced G0/G1 cell cycle arrest. Results from the immunohistochemical study suggested that high calpain-1 expression was significantly associated with adverse relapse-free survival in breast cancer patients who received adjuvant trastuzumab. Findings were validated in the expanded Nottingham and independent Newcastle patient cohort. Based on the previous in vitro study, suggesting a role of calpain in regulation of phospho-MSK1/2 expression; and because there is close link between calpain and caspase family. It was decided to explore the correlation between calpain system protein expression with MSK1 and two representative caspases (caspase-3 & -8), as well as their prognostic significance, in a large cohort of invasive breast cancer patients. Results demonstrated significant correlations between calpain-1 vs MSK1, and vs caspase-3; calpastatin vs MSK1, and vs caspase-8, however with low correlation coefficients. High MSK1 expression was significantly associated with improved breast cancer-specific survival. High caspase-3, but not caspase-8, was significantly associated with adverse breast cancer-specific survival. And combinatorial calpain-1 and caspase-3 expression provided additional prognostic values, especially in basal-like subtype. In conclusion, calpain system protein has been found to have roles in different breast cancer molecular subtypes. Calpain-1 is a potential biomarker for trastuzumab response in HER2+ breast cancer patients, and this study suggested MSK1 and caspase-3 could be potential biomarkers in breast cancer