Store-Operated Calcium Entry in Breast Cancer Cells Is Insensitive to Orai1 and STIM1 N-Linked Glycosylation

SIMPLE SUMMARY: Breast cancer cells exhibit several differences in store-operated Ca(2+) entry (SOCE) as compared to non-tumoral breast epithelial cells due to altered expression and post-translational modification of STIM proteins and Orai channels, as well as their modulators. The aim of this study was to analyze Orai1 and STIM1 N-linked glycosylation in SOCE in breast cancer cells and to ascertain the potential functional relevance of this post-translational modification in the development of cancer hallmarks. Using glycosylation-deficient STIM1 and Orai1 mutants we have found SOCE in breast cancer cells is insensitive to N-linked glycosylation of these proteins, a mechanism that might be relevant to evade apoptosis. ABSTRACT: N-linked glycosylation is a post-translational modification that affects protein function, structure, and interaction with other proteins. The store-operated Ca(2+) entry (SOCE) core proteins, Orai1 and STIM1, exhibit N-glycosylation consensus motifs. Abnormal SOCE has been associated to a number of disorders, including cancer, and alterations in Orai1 glycosylation have been related to cancer invasiveness and metastasis. Here we show that treatment of non-tumoral breast epithelial cells with tunicamycin attenuates SOCE. Meanwhile, tunicamycin was without effect on SOCE in luminal MCF7 and triple negative breast cancer (TNBC) MDA-MB-231 cells. Ca(2+) imaging experiments revealed that expression of the glycosylation-deficient Orai1 mutant (Orai1N223A) did not alter SOCE in MCF10A, MCF7 and MDA-MB-231 cells. However, expression of the non-glycosylable STIM1 mutant (STIM1N131/171Q) significantly attenuated SOCE in MCF10A cells but was without effect in SOCE in MCF7 and MDA-MB-231 cells. In non-tumoral cells impairment of STIM1 N-linked glycosylation attenuated thapsigargin (TG)-induced caspase-3 activation while in breast cancer cells, which exhibit a smaller caspase-3 activity in response to TG, expression of the non-glycosylable STIM1 mutant (STIM1N131/171Q) was without effect on TG-evoked caspase-3 activation. Summarizing, STIM1 N-linked glycosylation is essential for full SOCE activation in non-tumoral breast epithelial cells; by contrast, SOCE in breast cancer MCF7 and MDA-MB-231 cells is insensitive to Orai1 and STIM1 N-linked glycosylation, and this event might participate in the development of apoptosis resistance.