Tuberc Respir Dis.  2022 Apr;85(2):147-154. 10.4046/trd.2021.0095.

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

• Kim MS
• Kim SH
• Yang SH
• Kim MS

• KMID: 2528418
• DOI: http://doi.org/10.4046/trd.2021.0095

Abstract

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 drugresistant cells (bearing a T790M mutation in epidermal growth factor receptor [EGFR]) remain unclear.
Methods
Afatinib-mediated changes in the level of store-operated Ca²⁺ entry (SOCE)-related proteins and intracellular Ca²⁺ 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 Ca²⁺ 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 Ca²⁺. Moreover, extracellular Ca²⁺ 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 Ca²⁺.
Conclusion
Extracellular Ca²⁺ plays important role in afatinib-mediated autophagic degradation of SOCE-related proteins in cells with T790M mutation in the EGFR gene and extracellular Ca²⁺ is essential for determining anti-cancer drug efficacy.

Keyword

Afatinib; Calcium Channels; Non–Small Cell Lung; Autophagy