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Establishment and Characterization of Three Afatinib-resistant Lung Adenocarcinoma PC-9 Cell Lines Developed with Increasing Doses of Afatinib

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Published on: June 26, 2019

Afatinib Mediates Autophagic Degradation of ORAI1, STIM1, and SERCA2, Which Inhibits Proliferation of Non-Small Cell

Mi Seong Kim^1,2, So Hui Kim³, Sei-Hoon Yang⁴

¹Department of Oral Physiology, Institute of Biomaterial-Implant, School of Dentistry, Wonkwang University, Iksan, Republic of Korea.

Tuberculosis and Respiratory Diseases

|November 30, 2021

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.

Insights

Extracellular calcium is crucial for afatinib efficacy in EGFR-mutated lung cancer. Afatinib triggers autophagic degradation of calcium signaling proteins, inhibiting cancer cell proliferation.

Area of Science:

Oncology
Cell Biology
Pharmacology

Background:

Calcium signaling pathways are altered in carcinomas, impacting cancer cell proliferation.
The role of calcium signaling in drug-resistant cancer cells with epidermal growth factor receptor (EGFR) T790M mutations is not well understood.

Purpose of the Study:

To investigate the effect of afatinib on calcium signaling in non-small cell lung cancer (NSCLC) cells harboring the EGFR T790M mutation.
To elucidate the mechanisms underlying afatinib resistance and potential therapeutic strategies.

Main Methods:

Western blot and ratiometric assays were used to analyze store-operated Ca2+ entry (SOCE)-related proteins and intracellular Ca2+ levels.
Autophagic flux was assessed by measuring LC3B-II cleavage; apoptosis and proliferation were evaluated using flow cytometry and cell proliferation assays.

Main Results:

Afatinib decreased SOCE-related proteins (ORAI1, STIM1, SERCA2) in NSCLC cells but not in gefitinib-resistant PC-9/GR cells (EGFR T790M).
Afatinib induced autophagic degradation of SOCE proteins, and extracellular calcium depletion inhibited proliferation in PC-9/GR cells.

Conclusions:

Extracellular calcium is vital for afatinib-mediated autophagic degradation of SOCE proteins in EGFR T790M-mutated cells.
Extracellular calcium availability is essential for determining the efficacy of anti-cancer drugs like afatinib.

Read full analysis

Keywords:

Afatinib Autophagy Calcium Channels Non–Small Cell Lung

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