Introduction
Increasing number of studies on the molecular pathology of cancer has led to discovery of various gene mutations in non‒ small cell lung cancer (NSCLC), and those genes further being studied as important components in the development of targeted therapy. In NSCLCs, epidermal growth factor receptor (EGFR) exon 19 deletion and exon 21 L858R point mutations are most commonly observed [1]. In particular, genetic mutations in the EGFR tyrosine kinase domain mediate the sustained activity of EGFR kinase and induce cell proliferation [2]. Gefitinib and erlotinib are the first-generation EGFR tyrosine kinase inhibitors (TKIs) that reversibly bind to the ATP-binding site in the EGFR tyrosine kinase domain. These inhibitors are currently being used in clinical practice in some instances. Compared to the first-generation anti-cancer drugs, second-generation drugs such as afatinib and dacomitinib, irreversibly bind to the tyrosine kinase domain and inhibit EGFR (ErbB1), HER2 (ErbB2), and HER4 (ErbB4) receptor functions [3,4]. Moreover, the irreversible binding of afatinib to receptors can effectively inhibit cell proliferation in EGFR mutated cell lines resistant to erlotinib or gefitinib [5].
Calcium ions (Ca2+) are an essential secondary messenger in most cells that mediates various physiological cellular activities [6], and abnormal Ca2+ signaling are closely related to the development of diseases, including cancer [7]. Sarco/endoplasmic Reticulum ATPase (SERCA) regulates the basal level of cytosolic free Ca2+ through transportation of Ca2+ into the endoplasmic reticulum (ER), and its pharmacological inhibition of SERCA induces depletion of Ca2+ in the ER [8]. Depletion of Ca2+ in the ER, which is intracellular Ca2+ storage, mediates extracellular Ca2+ influx into the cell, also known as store-operated Ca2+ entry (SOCE). During this process, stromal interaction molecule 1 (STIM1) acts as a sensor of ER Ca2+ store contents [9]. In breast cancer cells, ORAI calcium release-activated calcium modulator 1 (ORAI1) induces the influx of intracellular Ca2+ by interacting with the plasma membrane protein SPCA2, which leads to cell proliferation. Additionally, ORAI1 regulates cancer cell metastasis, which shows the correlation between Ca2+ channel proteins and proliferation and metastasis of cancer cells [10]. STIM1 binds to the plasma membrane Ca2+ channel, ORAI1, and promotes extracellular Ca2+ influx into cells [11]. Extracellular Ca2+ 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 Ca2+ signal-mediated proteins and that the absence of extracellular Ca2+ enhances afatinib efficacy against NSCLC cells. However, there is a lack of understanding of afatinib-mediated changes in the extracellular Ca2+ influx and cytotoxicity according to changes in extracellular Ca2+ influx in cells with T790M gene mutation in EGFR.
In this study, autophagic flux and changes in the expression of ORAI1, STIM1, and sarco/endoplasmic reticulum Ca2+ ATPase (SERCA2) after afatinib treatment were assessed in PC-9 and gefitinib-resistant PC-9 (PC-9/GR) cells to understand cell responses in the presence and absence of extracellular Ca2+. Further, the effects of extracellular Ca2+ absence on afatinib-mediated inhibition of cell proliferation were evaluated.
Materials and Methods
1. Cell culture and reagents
PC-9 and PC-9/GR cell lines were provided by Dr. Jin Kyung Roh (Asan Medical Center, Asan Medical Institute of Convergence Science and Technology). Cells were cultured in RPMI-1640 medium (Hyclone, Logan, UT, USA) with 10% fetal bovine serum and 100 U/mL penicillin-streptomycin in a 5% CO2 incubator at 37°C. Anti-Orai1, ‒proliferating cell nuclear antigen (PCNA), goat anti-rabbit horseradish peroxidase (HRP) secondary antibody, and goat anti-mouse HRP secondary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-LC3B, -STIM1, -SERCA2, ‒poly(ADP-ribose) polymerase (PARP), ‒β-actin antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Afatinib was purchased from Selleckchem (Houston, TX, USA), and cyclopiazonic acid (CPA) was purchased from Enzo Life Sciences (Farmingdale, NY, USA). Bafilomycin A and 3-methyladenine (3-MA) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
2. Western blot assay
Cells were collected after centrifuging at 4,000 rpm and 4°C for 4 minutes. RIPA buffer (Thermo Fisher Scientific, Foster City, CA, USA) containing Protease & Phosphatase Inhibitor (Thermo Fisher Scientific) was added to lyse the cells. Proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a PVDF membrane (GE Healthcare, Little Chalfont, Notts, UK). The membrane was then blocked using TBS-T mixed with 5% (w/v) skim milk and incubated for 2 hours at 4°C in a rocker, and then washed four times for 10 minutes each time using TBS containing 0.1% Tween-20. The membrane was incubated with primary antibody diluted at a ratio of 1:1,000 in 5% skim milk solution overnight at 4°C in a rocker. The following day, the membrane was washed four times for 10 minutes each time using TBS containing 0.1% tween-20. Then it was incubated with HRP-conjugated secondary antibody diluted at a ratio of 1:2,000 in a rocker for 2 hours at 4°C, and washed again four times for 10 minutes each time using TBS containing 0.1% Tween-20. The membrane was then treated with a 1:1 mix of SuperSignal West Pico PLUS Luminol/Enhancer solution and SuperSignal West Pico PLUS Stable Peroxide (Thermo Fisher Scientific). Protein bands were detected using AzureSpot 2.0 (AzureBiosystems, Dublin, CA, USA).
3. Intracellular Ca2+ measurement
Fura-2/AM (Sigma-Aldrich), which is a fluorescent dye used to detect Ca2+, was used to measure intracellular Ca2+ ([Ca2+]i). A 1 μM Fura-2/AM solution was added to the cell culture medium in a 5% CO2 incubator at 37°C. The cell surface was washed briefly using HEPES buffer with a pH of 7.4 and osmotic pressure of 310 mOsm. [Ca2+]i was measured using Fura-2/AM at 340 and 380 nm. Fluorescence at 510 nm was captured using a CCD (charge-coupled device) camera. Captured images were analyzed using MetaFluor software (Molecular Devices, San Jose, CA, USA) and expressed as F340/F380 ratio.
4. Flow cytometry
FITC Annexin V Apoptosis Detection Kit with propidium iodide (PI) (BioLegend, San Diego, CA, USA) was used to assess cell apoptosis. In summary, cells were detached using trypsin-EDTA and washed twice with cold phosphate buffered saline. After cells were washed with cell staining buffer, they were then incubated with cell staining buffer containing Annexin V-FITC and PI for 15 minutes in the dark. The stained cells were analyzed using a FACScan analyzer (Becton Dickinson Frankin Lakes, NJ, USA), and a minimum of 20,000 events were analyzed.
5. Cell proliferation assay
Cell proliferation was assessed using EZ-CYTOX (Daeil Lab Service Co. Ltd., Seoul, Korea) according to the manufacturer’s protocol. Cells were plated in a 96-well plate and treated according to the experiment design. The cells were then incubated in a 5% CO2 incubator at 37°C. After 72 hours, EZ-Cytox (10 μL) was added to each well, and cells were incubated at 37°C and with 5% CO2 for 30 minutes. Optical density was measured at 450 nm using iMAX Microplate Reader (Bio-Rad, Hercules, CA, USA).
6. Statistical analysis
Origin 2020 software (OriginLab Corporation, Northampton, MA, USA) was used to compare the statistical significance between the groups. One-way analysis of variance and t-test were conducted to assess significance, and Tukey’s post hoc test was conducted for post hoc analysis. p<0.05 was considered statistically significant. All results were obtained from three or more independent experiments and expressed as mean±SD.
Results
1. Afatinib-mediated alterations in the expression of ORAI1, STIM1, and SERCA2 proteins in the presence and absence of extracellular Ca2+
Alterations in the expression of Ca2+ channels and Ca2+- binding proteins in lung cancer cells regulate cell proliferation and apoptosis signaling pathways15. In particular, expression of STIM1, 2 and ORAI1, 2, and 3, which mediate the extracellular Ca2+ influx in non-excitable cells, were altered in tissues of various cancers, including breast and colorectal cancers [16]. We examined the levels of ORAI1, STIM1, and SERCA2, which are directly related to SOCE, after treating PC-9 and PC-9/GR cells with afatinib. PC-9 and PC-9/GR cells were cultured in a normal culture medium (1 mM CaCl2) and Ca2+-free culture medium (0 mM CaCl2), and cells were treated with 0.1 μM afatinib for different durations of time (0, 24, and 48 hours). The whole-cell lysates were used to assess the intracellular levels of ORAI1, STIM1, and SERCA2 proteins through western blot assay. In PC-9 cells that were cultured in a normal medium (1 mM CaCl2), intracellular expression of ORAI1, STIM1, and SERCA2 proteins were significantly decreased after 48 hours of treatment with afatinib. In contrast, the levels of ORAI1, STIM1, and SERCA2 proteins in PC-9/GR cells were not decreased after afatinib treatment (Figure 1A). In PC-9 and PC-9/GR cells cultured in Ca2+-free medium (0 mM CaCl2), expression of ORAI1, STIM1, and SERCA2 proteins were significantly reduced after afatinib treatment (Figure 1B). These results suggested that a decreased expression of SOCE-mediating proteins by afatinib depends on extracellular Ca2+.
2. Reduction in SOCE in PC-9 cells by afatinib
A ratiometric assay using a fluorescent Ca2+ indicator was performed to confirm the decrease in SOCE based on the reduced levels of ORAI1, STIM1, and SERCA2 proteins in PC-9 cells after afatinib treatment. PC-9 cells cultured in a normal medium (1 mM CaCl2) were treated for 48 hours with afatinib (0.1 μM) or DMSO (control). CPA (25 μM) was diluted in a Ca2+-free HEPES buffer (1 mM EGTA) and perfused to cells to induce ER Ca2+ depletion. The cells were then treated with CPA (25 μM) diluted in a normal HEPES buffer (1 mM Ca2+) to measure SOCE. SOCE significantly decreased in experimental group pre-treated with afatinib (0.31031±0.09007, n=10) compared to that in the control group (0.59053±0.11451, n=11) (Figure 2).
3. Reduced expression of ORAI1, STIM1, and SERCA2 after afatinib treatment depends on autophagy
To confirm that the decrease of ORAI1, STIM1, and SERCA2 expression after afatinib treatment was due to intracellular autophagy, LC3B-II cleavage was assessed at specific time points after treating PC-9 and PC-9/GR cells with afatinib. Afatinib (0.1 μM)-mediated LC3B-II cleavage in PC-9 cells was significantly increased after 1 and 3 hours of treatment. However, no significant change was observed in PC-9/GR cells (Figure 3A). After 3 and 6 hours of treatment with afatinib, significantly different LC3B-II cleavage levels were observed between PC-9 and PC-9/GR cells (Figure 3A). To further determine the association between decreased expression of ORAI1, STIM1, and SERCA2 and intracellular autophagy activity, bafilomycin A1 (5 nM) and 3-MA (5 mM), which are autophagy inhibitors, were added for the last 1, 3, and 6 hours of afatinib (0.1 μM) treatment (ORAI1 for 24 hours; STIM1 and SERCA2 for 48 hours). Reduced ORAI1, STIM1, and SERCA2 levels were significantly inhibited by bafilomycin A1 and 3-MA treatment (Figure 3B). These findings suggest that decreased level of SOCE-mediating proteins by afatinib depends on the intracellular autophagic degradation.
4. Analysis of afatinib-mediated apoptosis in the presence and absence of extracellular Ca2+
We demonstrated that afatinib reduces ORAI1, STIM1, and SERCA2 protein levels in PC-9/GR cells in the absence of extracellular Ca2+. Therefore, we assumed that the absence of extracellular Ca2+ would regulate apoptosis of PC-9 and PC-9/GR cells, and flow cytometry was used to test this hypothesis. PC-9 and PC-9/GR cells were cultured in experimental conditions of different calcium concentrations and afatinib treatment times. Apoptosis was then assessed by measuring the fluorescence levels of propidium iodide and FITC. In PC-9 cells, similar to the decreased expression of ORAI1, STIM1, and SERCA2 at 48 hours, apoptotic cells increased by >40% under all conditions, with and without extracellular Ca2+. However, although ORAI1, STIM1, and SERCA2 protein levels decreased in the absence of extracellular Ca2+ in PC-9/GR cells, there was no difference in the percentage of apoptotic cells (Figure 4).
5. Lack of extracellular Ca2+ enhances the inhibitory effects of afatinib on the proliferation of PC-9/GR cells
To determine inhibition level of PC-9 and PC-9/GR cell proliferation by afatinib without extracellular Ca2+, the expression of PCNA, a marker protein for cell proliferation, and cleavage of PARP, a marker protein for cytotoxicity, were assessed. In addition, cell proliferation was evaluated by MTT assay. In PC-9 cells, the expression of PCNA was decreased by afatinib regardless of extracellular Ca2+, and PARP cleavage increased in the absence of extracellular Ca2+ (Figure 5A). In PC-9/GR cells, PCNA expression was not affected by afatinib when the extracellular Ca2+ level was normal. Additionally, intracellular PCNA expression was decreased after 48 hours of treatment with afatinib without extracellular Ca2+ (Figure 5B). Notably, PARP cleavage in PC-9/GR cells after afatinib treatment was not observed regardless of extracellular Ca2+ (Figure 5A). PC-9 and PC-9/GR cells were cultured with and without extracellular Ca2+, and cell proliferation was assessed after 72 hours of treatment with afatinib (0.1 μM). The viability of PC-9 cells was reduced by >50% after afatinib (0.1 μM) treatment under all conditions, with and without extracellular Ca2+. The viability of PC-9/GR cells was reduced by 15% after afatinib (0.1 μM) treatment in the presence of extracellular Ca2+. In the absence of extracellular Ca2+, the viability of cells was reduced by >30% after afatinib (0.1 μM) treatment. These findings suggest that lack of extracellular Ca2+ further improved the inhibitory effects of afatinib on PC-9/GR cell proliferation.
Discussion
The concentration of intracellular Ca2+ is regulated by various intracellular molecules, known as the “calcium-signaling toolkit.” Intracellular calcium ions are precisely controlled spatially and temporally to mediate various cellular physiological activities, including cell proliferation, differentiation, migration, and secretion [6]. Moreover, abnormal Ca2+ signals are also correlated to the development of various diseases. In particular, abnormal changes in the expression of “calcium-signaling toolkit” proteins were reported in various carcinoma cells, and these changes mediate the induction of tumorigenesis in normal cells, tissue invasion and metastasis of cancer cells, and resistance to anti-cancer drugs [7]. In a recent study, Mulder et al. reported that intracellular Ca2+ signaling plays a key role in reactivating the mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase signaling pathways inhibited by afatinib in NSCLC cells over time [14]. Notably, apoptosis and inhibition of proliferation by afatinib were regulated by extracellular Ca2+ level, and the absence of exogenous calcium significantly improved afatinib efficacy [14]. Afatinib is an irreversible ErbB receptor family blocker and inhibits ATP-binding against tumors with exon 19 deletion, exon 21 L858R mutation, and exon 20 T790M mutation17. Additionally, afatinib has high inhibitory activity on cell proliferation against carcinomas resistant to first-generation EGFR-TKIs, including gefitinib and erlotinib [17]. However, changes in the “calcium-signaling toolkit” in NSCLC cells resistant to first-generation EGFR-TKIs, such as gefitinib, are not well known. In this study, we assessed the alterations in the expression of SOCE-mediating proteins by afatinib and the resulting changes in the physiological activities in a PC-9 cell line and a gefitinibresistant PC-9/GR cell line (bearing a T790M mutation in EGFR gene). The expression of ORAI1, STIM1, and SERCA2 proteins in PC-9 cells was significantly decreased after treatment with afatinib for 48 hours regardless of extracellular Ca2+. In cells resistant to gefitinib, there was no significant change in protein levels after afatinib treatment in an environment with a normal level of extracellular Ca2+ (1 mM). However, in the absence of extracellular Ca2+, afatinib treatment caused a significant decrease in protein levels (Figure 1). Notably, afatinib, which has obvious inhibitory effects on the proliferation of cells with T790M gene mutation, did not affect ORAI1, STIM1, and SERCA2 protein levels in PC-9/GR cells, unlike in PC-9 cells in the presence of extracellular Ca2+. Additionally, in the absence of extracellular Ca2+, protein levels were decreased by afatinib in both PC-9 and PC-9/GR cells. These results indicate that the cellular response to afatinib is altered by a T790M gene mutation and in PC-9/GR cells, the presence of exogenous calcium determines the levels of proteins that mediate SOCE.
In 2007, Hoyer-Hansen and Jaattela [18] reported that an increase in intracellular Ca2+ was fundamental in the early stages of LC3 aggregation and autophagosome formation. It was shown that Ca2+-dependent AMP that activated protein kinase inhibits target of rapamycin (mTOR) activity, thereby promoting the autophagic influx [18]. We observed an increase in autophagic influx triggered by afatinib presence in PC-9 cells. However, there was no significant increase in autophagic flux in PC-9/GR cells. This finding is consistent with the decrease in the expression of SOCE-mediating proteins (ORAI1, STIM1, and SERCA2) by afatinib and suggests that afatinib decreases protein levels through autophagic degradation. Further, we observed that afatinib effectively inhibited cell proliferation in PC-9/GR cells without extracellular Ca2+ more than with extracellular Ca2+. This finding shows that extracellular Ca2+ plays a crucial role in afatinib-mediated suppression of SOCE-related proteins and regulation of cell proliferation.
The findings of this study suggest that the expression of SOCE-mediating proteins (ORAI1, STIM1, and SERCA2) are decreased by afatinib-activated autophagic degradation and demonstrates that extracellular Ca2+ levels increase the efficacy of afatinib. Therefore, the concentration of extracellular Ca2+ is an important factor in enahncing the efficiency of anti-cancer treatment using afatinib.
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