Afatinib Mediates Autophagic Degradation of ORAI1, STIM1, and SERCA2, Which Inhibits Proliferation of Non–Small Cell Lung Cancer Cells

BACKGROUND: The expression of calcium signaling pathway molecules is altered in various carcinomas, which are related to the proliferation and altered characteristics of cancer cells. However, changes in calcium signaling in anti-cancer drug-resistant cells (bearing a T790M mutation in epidermal growth factor receptor [EGFR]) remain unclear. METHODS: Afatinib-mediated changes in the level of store-operated Ca(2+) entry (SOCE)-related proteins and intracellular Ca(2+) level in non–small cell lung cancer cells with T790M mutation in the EGFR gene were analyzed using western blot and ratiometric assays, respectively. Afatinib-mediated autophagic flux was evaluated by measuring the cleavage of LC3B-II. Flow cytometry and cell proliferation assays were conducted to assess cell apoptosis and proliferation. RESULTS: The levels of SOCE-mediating proteins (ORAI calcium release-activated calcium modulator 1 [ORAI1], stromal interaction molecule 1 [STIM1], and sarco/endoplasmic reticulum Ca(2+) ATPase [SERCA2]) decreased after afatinib treatment in non–small cell lung cancer cells, whereas the levels of SOCE-related proteins did not change in gefitinibresistant non–small cell lung cancer cells (PC-9/GR; bearing a T790M mutation in EGFR). Notably, the expression level of SOCE-related proteins in PC-9/GR cells was reduced also responding to afatinib in the absence of extracellular Ca(2+). Moreover, extracellular Ca(2+) influx through the SOCE was significantly reduced in PC-9 cells pre-treated with afatinib than in the control group. Additionally, afatinib was found to decrease the level of SOCE-related proteins through autophagic degradation, and the proliferation of PC-9GR cells was significantly inhibited by a lack of extracellular Ca(2+). CONCLUSION: Extracellular Ca(2+) plays important role in afatinib-mediated autophagic degradation of SOCE-related proteins in cells with T790M mutation in the EGFR gene and extracellular Ca(2+) is essential for determining anti-cancer drug efficacy.