Adaptive Resistance to EGFR-Targeted Therapy by Calcium Signaling in NSCLC Cells

Abstract

Targeted therapies against oncogenic receptor tyrosine kinases (RTK) show promising results in the clinic. Unfortunately, despite the initial positive response, most patients develop therapeutic resistance. Most research has focused on acquired resistance occurring after an extensive time of treatment; however, the question remains as to how cells can survive an initial treatment, as early resistance to apoptosis will enable cells to develop any growth-stimulating mechanism. Here, the non-small cell lung cancer (NSCLC) PC9 cell line was used to systematically profile, by mass spectrometry, changes in the proteome, kinome, and phosphoproteome during early treatment with the EGFR inhibitor afatinib. Regardless of the response, initial drug-sensitive cells rapidly adapt to targeted therapy, and within days, cells regained the capacity to proliferate, despite persisting target inhibition. These data reveal a rapid reactivation of mTOR and MAPK signaling pathways after initial inhibition and an increase in abundance and activity of cytoskeleton and calcium signaling-related proteins. Pharmacologic inhibition of reactivated pathways resulted in increased afatinib efficacy. However more strikingly, cells that were restricted from accessing extracellular calcium were extremely sensitive to afatinib treatment. These findings were validated using three additional inhibitors tested in four different NSCLC cell lines, and the data clearly indicated a role for Ca signaling during the development of adaptive resistance. From a therapeutic point of view, the increased inhibitor efficacy could limit or even prevent further resistance development. Combined targeting of calcium signaling and RTKs may limit drug resistance and improve treatment efficacy. .

Role of Calcium Homeostasis in Modulating EMT in Cancer

“…They can also remain in a state of dormancy for extended periods of time, which, in conjunction with a decelerated cell cycle, enables them to acquire mutations that allow for cellular progression [ 73 ]. Research has substantiated a consistent trait of these persister cells as having increased calcium signaling in parallel with cytoskeleton remodeling [ 74 ]. Understanding the vast array of mechanisms of resistance remains crucial for future drug discovery and optimizing combination therapy.…”

“…Epidermal growth factor (EGF), the ligand for EGFR, was found to play a role in the calcium oscillations essential for EMT [ 87 ]. The evidence suggests that the blockage of cells from extracellular calcium via a calcium chelator known as EGTA increases the efficacy of EGFR inhibitors such as afatinib [ 74 ]. Immunotherapies are also on the rise in cancer treatment.…”

Restricting extracellular Ca2+ on gefitinib-resistant non-small cell lung cancer cells reverses altered epidermal growth factor-mediated Ca2+ response, which consequently enhances gefitinib sensitivity

“…We observed that low [Ca 2+ ]e-mediated [Ca 2+ ]i oscillations were abolished by gefitinib and U73122 in PC-9/GR and PC-9 cells, suggesting that the absence of [Ca 2+ ]e led to enhanced gefitinib sensitivity and reversed gefitinib-mediated cytotoxicity and apoptosis. Mulder et al demonstrated that initial targeted therapy on PC-9 cells increases the activity of Ca 2+ signaling-related proteins and deprives the cell of extracellular Ca 2+ , which results in the marked enhancement of afatinib efficacy [12]. Our data indicated that increased [Ca 2+ ]e influx in PC-9/GR cells contributed to gefitinib resistance; therefore, restricting [Ca 2+ ]e could be key in enhancing drug sensitivity.…”

“…Notably, treatment using EGFR-targeting afatinib causes Ca 2+ signaling-related gene expression in PC-9 cells. Reduced extracellular Ca 2+ ([Ca 2+ ]e) levels increase the sensitivity of PC-9 cells to afatinib [12]. These reports strongly suggest that Ca 2+ signaling in cancer cells is significantly associated with the development of acquired drug resistance.…”

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

“…Extracellular Ca 2+ influx plays important roles in cell survival, cell proliferation, invasiveness [ 12 ], and the regulation of the early G1 phase of the cell cycle [ 13 ]. Mulder et al [ 14 ] reported that afatinib treatment in PC-9 cells regulates the expression of various Ca 2+ signal-mediated proteins and that the absence of extracellular Ca 2+ enhances afatinib efficacy against NSCLC cells. However, there is a lack of understanding of afatinib-mediated changes in the extracellular Ca 2+ influx and cytotoxicity according to changes in extracellular Ca 2+ influx in cells with T790M gene mutation in EGFR.…”