Calpain regulates sensitivity to trastuzumab and survival in HER2-positive breast cancer.
Authors: S Kulkarni, KB Reddy, FJ Esteva, GT Budd, HC Moore, RR Tubbs
Journal: Cancer Research (2009)

Abstract Abstract #2018 Background: Overexpression of HER2 is associated with an aggressive cancer and poor prognosis for patients. HER2-positive patients are treated with trastuzumab; however resistance is common. Mechanisms that regulate activity of HER2 or downstream kinase AKT are involved in regulation of survival. Calpains are cysteine proteases involved in transmission of signals during growth, motility and transformation in cancer. Mechanisms by which calpain regulates transmission of signals in HER2-positive breast cancer are not known. Here we investigate possibility that calpain regulates survival in HER2-positive breast cancer.&amp;#x2028; Material and Methods: Activity of calpain in parental or resistant skbr3 cells was measured alone or in presence of inhibitors under conditions of adhesion or stimulation with trastuzumab using a fluorescently labeled substrate. To investigate regulation of HER2, assays were performed by mixing purified proteins or cell lysates with calpain1. Activity of HER2 was analyzed using phosphotyrosine-specific antibodies in Western blots while cleavage using antibody specific to the C-terminus. HER2-positive cancer tissue or lysates of cell lines were analyzed for presence of C-terminal fragments. Cells were transfected with the plasmid expressing catalytically inactive mutant of calpain1, selected for resistance to G418 and used in experiments to analyze the activity of HER2 and AKT. Sensitivity to trastuzumab was determined by measuring survival in presence of trastuzumab in a 96 well plate assay.&amp;#x2028; Results: In skbr3 cells, activity of calpain increased following adhesion or stimulation with trastuzumab and was inhibited by inhibitors. Calpain induced cleavage of C-terminal domains and reduced activity in full length HER2. C-terminal fragments were detectable in the lysates of HER2-positive breast cancer tissue, cell lines and also overproduced by overexpression of calpain1. Conversely, in cells expressing catalytically inactive calpain1 the activity of HER2 and AKT was increased correlating with increased survival on fibronectin in the presence of trastuzumab. As another model for calpain-induced regulation of HER2 in mechanisms of resistance, we analyzed regulation in trastuzumab-resistant skbr3 cells. Compared to parental in resistant cells adhesion- or trastuzumab-dependent activity of HER2 was increased whereas cleavage was reduced suggesting mechanisms that regulate generation of C-terminal fragments during transmission of signals were altered. Consistenly, adhesion-dependent activity of calpain was reduced whereas trastuzumab failed to stimulate activation.&amp;#x2028; Conclusion: These data are consistent with possibilities 1) HER2 is a direct target of calpain, 2) calpain induces cleavage of the cytoplasmic region during transmission of signals to regulate activity and integrity of HER2, 3) calpain-dependent mechanisms are involved in coupling HER2 to survival and 4) inhibition of calpain during adhesion-dependent transmission of signal or in the presence of trastuzumab allows increased activity of HER2 contributing to resistance. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2018.