Sucheta Kulkarni

Calpain regulates sensitivity to trastuzumab and survival in HER2-positive breast cancer

Resistance to anti-HER2 (human epithelial growth factor receptor 2) trastuzumab therapy occurs commonly in HER2-positive breast cancer and involves overactivation of HER2 and/or AKT1. Using the model of trastuzumab-sensitive or trastuzumab-resistant HER2-positive cells with wild-type PTEN, negative regulator of AKT1, we explore the involvement of cysteine protease calpain in mechanisms of trastuzumab resistance. Overexpression of calpain1 or activation of endogenous calpain during adhesion or trastuzumab treatment of trastuzumab-sensitive cells induces cleavage of cytoplasmic domains of HER2/phospho-HER2; cleavage occurs in HER2-positive tumors. Expression of the catalytically inactive mutant of calpain1 reduces the cleavage to enhance the activity of HER2, inactivates PTEN to enhance the activation of AKT1, induces desensitization to trastuzumab and promotes survival of trastuzumab-sensitive cells. In the model of trastuzumab resistance, constitutive overactivation of HER2 and AKT1 correlates with reduced activation of calpain. Moreover, inhibitors of the catalytic site of calpain reduce the increase in constitutive activity of AKT1 and survival of trastuzumab-resistant cells selectively. Together, by regulating the activation of HER2 and PTEN/AKT1, calpain regulates trastuzumab sensitivity and survival, and the deregulation of the activation of calpain promotes trastuzumab resistance. Trastuzumab-resistant cells activate AKT1 in a mechanism dependent on the residual calpain activity, inhibition of which restores trastuzumab sensitivity and rescues resistance. These data identify calpain as a new therapeutic target in HER2-positive breast cancer.

Use of similar immunoglobulin VH gene segments by MALT lymphomas of the ocular adnexa

Extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue type (MALT lymphomas) develop from acquired reactive infiltrates directed against external or autoantigens. Although some European cases of ocular adnexal MALT lymphoma have been associated with Chlamydia psittaci infections, C. psittaci has not been detected in large studies of US-based cases. To evaluate whether the growth of US-based ocular adnexal MALT lymphomas may be promoted by a similar antigen, we identified and analyzed the expressed immunoglobulin VH genes in 10 cases. Interestingly, the VH genes in two cases used the same VH1 family V1-2 gene segment, and three cases used the same VH4 family V4-34 gene segment. The other five cases all used different gene segments V4-31, V5-51, V3-23, V3-30, and V3-7. All of the VH genes were mutated from germ line, with percent homologies ranging between 96.9 and 89.0%. The distribution of replacement and silent mutations within the VH genes was nonrandom consistent with the maintenance of immunoglobulin function and also strongly suggestive of antigen selection in the six VH genes with highest mutation loads. The CDR3 sequences in two of three VH-34 cases were the same size (15 amino acids) and had similar sizes in the two VH1-2 cases (18 and 16 amino acids). In conclusion, US-based MALT lymphomas of the ocular adnexa preferentially express a limited set of VH gene segments not frequently used by other MALT lymphomas and consistent with some recognizing similar antigens. Analysis of somatic mutations present within the VH genes is also consistent with antigen binding stimulating the growth of these lymphomas.

Calpain4 is required for activation of HER2 in breast cancer cells exposed to trastuzumab and its suppression decreases survival and enhances response.

Overactivation of HER2 and crosstalk of other HER family members contribute to a survival pathway of breast cancer cells exposed to trastuzumab, the therapeutic inhibitor of HER2 and thus, decrease response and promote resistance. We have explored the involvement of the intracellular cysteine protease calpain4, the common partner of isoforms calpain1 and calpain2, in the regulation of cell survival and trastuzumab‐response. Increase of calpain4 expression and isoform activities were detected in breast cancer cells and HER2‐positive tumors. Molecular analyses of parent and resistant cells suggested that perturbation of regulations, induced by calpain4 and of activities of HER2 and HER3, was associated with trastuzumab‐resistance. The suppression of calpain4 destabilized calpain1 and calpain2, however, did not prevent the activation of HER2 and HER3 or cell proliferation in the absence of trastuzumab. To understand the significance, the survival of parent and trastuzumab‐resistant cells in which calpain4 was suppressed, was assessed in the presence of trastuzumab; survival in each cell type was decreased and associated with a loss of HER2 and HER3 activity. Taken together, by contributing to the activation and the crosstalk of HER2, calpain4 promotes the survival pathway of breast cancer cells, and therefore, its suppression enhances trastuzumab‐response and decreases resistance.

Calpain regulates sensitivity to trastuzumab and survival in HER2-positive breast cancer.

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.
 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.
 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.
 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.