Tuberculosis and Respiratory Diseases

The Korean Academy of Tuberculosis and Respiratory Diseases (대한결핵및호흡기학회)
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Afatinib Mediates Autophagic Degradation of ORAI1, STIM1, and SERCA2, Which Inhibits Proliferation of Non-Small Cell Lung Cancer Cells

  • Kim, Mi Seong (Department of Oral Physiology, Institute of Biomaterial-Implant, School of Dentistry, Wonkwang University) ;
  • Kim, So Hui (Department of Carbon Convergence Engineering, College of Engineering, Wonkwang University) ;
  • Yang, Sei-Hoon (Department of Internal Medicine, School of Medicine, Wonkwang University) ;
  • Kim, Min Seuk (Department of Oral Physiology, Institute of Biomaterial-Implant, School of Dentistry, Wonkwang University)
  • Received : 2021.07.06
  • Accepted : 2021.10.24
  • Published : 2022.04.30
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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 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 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 gefitinib-resistant 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.

Keywords

Acknowledgement

We thank Dr. Jin Kyung Rho for providing us with different NSCLC cell lines. We would like to thank Editage (https://www.Editage.co.kr) for English language editing.

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