Introduction
Lung cancer is one of the most predominant and fatal cancers worldwide, and non-small cell lung cancer (NSCLC) represents approximately 85% of all lung cancer cases [
1]. An important milestone in the treatment of NSCLC was the discovery of epidermal growth factor receptor (EGFR)-activating mutations as an effective therapeutic target and the successful development of third-generation EGFR tyrosine kinase inhibitors (EGFR-TKIs; Gefitinib, erlotinib, afatinib, and osimertinib). EGFR-TKIs act as competitive reversible inhibitors and provide significant clinical benefit to patients. However, the development of acquired resistance to these agents limits their long-term efficacy [
2,
3], thereby necessitating the use of new therapeutic approaches or alternative strategies. More and more resistant mechanisms have been revealed in recent years, and lipid metabolic reprogramming maybe one of them. Alterations in lipid metabolism are now gaining recognition as a hallmark of cancer [
4,
5]. The reprogramming of lipid metabolic pathways in cancer cells has been shown to play an important role in supporting cancer cell proliferation and survival [
6,
7]. The dependence of tumor cells on deregulated metabolism/biosynthesis indicates that proteins involved in these processes may be attractive chemotherapeutic targets [
8,
9].
As a key enzyme in lipid metabolism, Stearoyl-CoA Desaturase 1 (SCD1) synthesized saturated fatty acids (SFAs) into monounsaturated fatty acids (MUFAs), which can be further synthesized to neutral lipids such as triglycerides (TG) and stored in organelles termed ‘lipid droplets’ (LDs), and several studies have shown that cancer tissues display a higher rate of lipid droplets than normal tissues in several cancers including NSCLC [
10‐
12]. Previous studies have demonstrated SCD1 is highly expressed and highlighted the involvement of SCD1 in the survival of NSCLC [
13,
14].
Pisanu ME et al. reported that the use of combination therapy with SCD1 inhibitors reverts resistance to cisplatin in lung cancer stem cells, which highlight the role of SCD1 in cisplatin resistant [
15]. It was also proved that in H460 lung cancer cells, the suppression of Stearoyl-CoA Desaturase (SCD) activity, impairs the ligand-induced phosphorylation of EGFR. Recently, evaluation of non-small cell lung cancer samples reveals a positive correlation among EGFR activation, SCD1 Y55 phosphorylation and SCD1 protein expression [
16].
Even though a growing number of studies have demonstrated that EGFR-stimulated cancer growth depends on SCD1 activity especially in lung adenocarcinoma, no investigations have been carried out to identify the relevance of SCD1 expression linked to EGFR-TKI resistance in lung adenocarcinoma. Furthermore, the potential synergy between TKI therapy and SCD1 inhibition in lung adenocarcinoma has not yet been addressed. In this study, we investigated the relationship between SCD1 induced lipid synthesis and the mechanism of resistance to EGFR-TKIs in NSCLC. Our results confirmed an accumulation of intracellular lipid droplets (LDs) and higher SCD1 expression after resistance to EGFR-TKIs. Our study also proved that in EGFR-TKI-sensitive cell lines, OA (the SCD1 enzymatic product) abrogates the effect of TKIs (both first and third generation TKIs). (20S)-Protopanaxatriol (g-PPT) is an aglycone of ginsenosides isolated from
Panax ginseng that has several interesting activities, including anticancer and antimetabolic effects [
17,
18]. Given that our previous study confirm the lipid metabolism inhibition effect. As an extension of our previous research. Pharmacological treatment with g-PPT and Gefitinib triggers cell death in Gefitinib-resistant NSCLC cell lines. Furthermore, two xenografts were employed to confirm a hyposensitization to Gefitinib by OA and the reversal of Gefitinib resistance by the combination of g-PPT and Gefitinib Altogether, these results demonstrate that abnormal LD accumulation, SCD1 and lipid metabolism are candidate therapeutic targets for the treatment of TKI-resistant EGFR-mutant NSCLC and highlight the importance of detecting lipid metabolism in tumors to predict the emergence of EGFR-TKI resistance.
Materials and methods
Patients and samples
A total of 20 formalin-fixed paraffin-embedded tissue samples and frozen tissue samples were included in this study. These samples were obtained from 13 lung cancer patients (shown in Table
1). Case number 01–07 patients were diagnosed with primary NSCLC with cTNM stages of IIIB or IV and were unfit for surgery. Biopsy and EGFR mutational testing verified the presence of EGFR-TKI-sensitive mutations (ADx-ARMS, AmoyDx, China). After at least 2 months, first-generation EGFR-TKI (Gefitinib, AstraZeneca, UK) treatment (Patient’s medication time is up to 12 months and the shortest is 3 months) and clinical assessment according to the Response Evaluation Criteria In Solid Tumors (RECIST) confirmed cTNM downstaging to IIIA. The patients underwent initial surgery at the Department of Thoracic Surgery, Affiliated Tongji Hospital of Huazhong University of Science and Technology Tongji Medical College (Wuhan, China) from 2016 to 2018. Those patients harbor paired tissue of pre- and post- treatment. Case number 07–10 patients were underwent initial surgery after downstaging post-TKI treatment. For they initially subjected to EGFR mutational testing using peripheral blood, tissue samples were collected only after TKI treatment. Case number 11–13 underwent initial surgery at the Department of Thoracic Surgery during the same period and were confirmed to possess sensitive EGFR mutations.
Table 1
The baseline characteristics of the patients
01 | Female | IV | cT2aN2M1 | L858R | 12 | IIIA | pT2N2M0 | T790 M/L858R |
02 | Female | IV | cT2aN2M1 | 19-Del | 9 | IB | pT2N0M0 | T790 M |
03 | Female | IV | cT4N2M1 | 19-Del | 7 | IIIA | pT2N2M0 | 19-Del |
04 | Male | IV | cT1cN2M1 | L858R | 7 | IA | pT1bN0M0 | L858R |
05 | Male | IIIB | cT4N2M0 | 19-Del | 3 | IA | pT1N0M0 | 19-Del |
06 | Male | IIIC | cT3N3M0 | 19-Del | 3 | IB | pT2aN0M0 | 19-Del |
07 | Female | IIIA | cT2bN3M0 | 19-Del | 2 | IA | pT1N0M0 | 19-Del |
08 | Female | IIIB | cT3N3M0 | 19-Del | 5 | IB | pT2bN1M0 | 19-Del |
09 | Male | IIIA | cT2aN2M0 | 19-Del | 3 | IB | pT2aN0M0 | 19-Del |
10 | Male | IIIB | cT4N2M0 | 19-Del | 3 | IA | pT1N0M0 | 19-Del |
11 | Male | IB | pT2BN0M0 | 19-Del | N | | | |
12 | Male | IIIA | pT2N2M0 | 19-Del | N | | | |
13 | Female | IIB | pT3N0M0 | L858R | N | | | |
All tissue samples including both paraffin-embedded tissue and frozen tissue were obtained with informed consent from patients. Each specimen has a corresponding tumor and adjacent tissues. None of the patients had any prior history of chemotherapy, radiation, or hormonal therapy except TKI treatment before the surgery. Clinical information of the patients was recorded in detail, and the diagnoses were confirmed by at least two pathologists. pTNM stage and tumor differentiation grade were obtained from the Tongji Hospital records. The baseline characteristics of the patients are shown in Table
1 and Additional file
1: Table S1. This study was approved by the Research Ethics Committee, Tongji Medical College, Huazhong University of Science and Technology. (The IRB ID number is TJ-IRB20180403).
Cell lines and cell culture
The EGFR-mutant NSCLC cell lines HCC827, PC-9, and H1975 were originally obtained from ATCC and the Chinese Academy of Sciences (Shanghai, China) prior to 2014. Gefitinib-resistant HCC827GR (T790 M) cells were provided by Dr. J Yang (Guangxi Medical University, Guangxi, China) in 2016. All cell lines were cultured in RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin). All cell lines were cultured at 37 °C in a humidified atmosphere with 5% CO2 and 95% air.
Cell treatment and cell morphological observation
Gefitinib (ZD1839) and osimertinib (AZD9291) were either supplied by AstraZeneca (Cambridge, UK) or purchased from MedChemExpress (MCE) (HY-50895/HY-15772, USA). g-PPT was purchased from MCE (HY-N0835, USA). OA was purchased from Sigma Chemical Co. (O1008, Sigma-Aldrich, USA). Cell culture experiments were performed using reagents formulated in 100% dimethyl sulfoxide (DMSO).
Equal amount of cells planted in a six-well plate and treated for 48 h. Cell were cultured at 37 °C in a humidified atmosphere with 5% CO2 and 95% air. After 48 h treatment the cell morphology and cell death were visualized with Inverted phase contrast microscope (Olympus, Tokyo, Japan).
Oil red O staining
To detect the difference in basal LD content between the two groups of patients who received EGFR-TKI treatment or not, we performed Oil Red O staining. Frozen cancer tissues were embedded in OCT compound (Sakura, Tokyo, Japan) and cut into 10 μm sections. The sections washed several times with distilled water, followed by preincubation in 60% isopropanol prior to the final staining with filtered Oil Red O working solution (60% Oil Red O stock solution (BA-4081, Baso, Zhuhai, China) and 40% deionized water). After a series of washing steps in 60% isopropanol, the nuclei were counterstained with hematoxylin prior to differentiation in 1% hydrochloric acid in alcohol. Finally, after washing several times with distilled water, the slides were sealed with glycerin-gelatin. Representative images were captured using an inverted microscope (Olympus, Tokyo, Japan).
Nile red staining
Live cells seeded on cover glasses were fixed in 4% paraformaldehyde (PFA) for 20 min at room temperature (RT) and then incubated with Nile red (HY-D0718, MCE, USA) at 1:2000 in phosphate-buffered saline (PBS) for 10 min. The slides were counterstained with Hoechst 33342 (H1399, Thermo Fisher, USA) at 1 μg/ml in PBS for 5 min at RT before imaging. The cells were visualized with a fluorescence microscope (Olympus, Tokyo, Japan). A representative image is shown from three independent experiments.
Fluorescence emitted by cells stained by Nile red was measured at 595 nm by flow cytometry (BD Biosciences, USA), during the assay 10,000 cells per sample were collect and count. The result analyzed with Cell Quest software (BD Biosciences, USA) and expressed as mean of fluorescence intensity (MFI).
Immunohistochemistry (IHC)
Formalin-fixed paraffin-embedded tissue blocks were retrieved from the archive and analyzed by IHC as previously described [
13,
19]. In short, the Avidin-Biotin Complex (ABC) Vectastain Kit (ZSGB-Bio, Beijing, China) was used, and anti-SCD1 (ab19862), anti-p-EGFR1068 (#3777), anti-c-Caspase3 (#9664), anti-KI67 (ab15580), and anti-perilipin (#9349) were used as primary antibodies to incubate the tissue sections (4-μm thick) after heat-induced epitope retrieval (in 10 mM sodium citrate buffer of pH 6.0), followed by incubation with a secondary antibody conjugated to peroxidase (1:100; Dako). Detection was performed using diaminobenzidine for 3 min, and the slides were counterstained with hematoxylin. The scoring system used incorporated both the intensity of scoring (0 = absent, −, 1 = weak, +, 2 = moderate, ++, and 3 = strong, +++) and the percentage of positive tumor cells (0 = 0%, 1 = 1–25%, 2 = 26–50%, 3 = 51–75%, and 4 = 76–100%). Points for the intensity and percentage of staining were added to calculate the overall score according to the method described before [
20]. Investigators scored all slides for SCD1, perilipin, KI67, c-Caspase3 and p-EGFR expression and followed the criteria of double-blind trials. Based on the overall score, SCD1, perilipin, KI67, c-Caspase3 and p-EGFR expression was classified as negative (≤ 4) and positive (> 4).
Cell viability assay
Eight thousand cells per well were transferred to 96-well flat-bottomed plates and cultured overnight before exposure to various concentrations of Gefitinib, osimertinib, and OA in medium containing 10% FBS for 48 h. After incubation for the indicated times, cell proliferation was measured. In brief, 10 μl commercial Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Inc., Japan) reagent was added to each well and incubated at 37 °C for 1 h. Absorbance was measured at 450 nm using a spectrophotometer. Each experiment was performed in triplicate and repeated at least 3 times.
Western blotting of cultured cells
Cells were harvested and lysed using ice-cold RIPA lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM sodium chloride, 1% Nonidet P-40, and 0.5% sodium deoxycholate) supplemented with a protease inhibitor cocktail (Roche). Following centrifugation at 10,000×g for 15 min at 4 °C, proteins in the supernatants were quantified by the Bradford method and separated using 10% SDS-PAGE gels and electrotransferred from the gel to nitrocellulose membranes (Merck & Co., Inc., Whitehouse Station, NJ, USA). Following blocking with 5% skimmed milk in PBS, the membranes cut into strips according to the different molecular weights. The membranes were immunoblotted with the primary antibodies against p-ERK (#4370), (#4695), AKT (#4691), p-AKT (#4060), c-PARP (#5625), c-Caspase3 (#9664), p-EGFR1068 (#3777) and p-Stat3 (#9145), BCL-XL (10783–1-AP), GAPDH (10494–1-AP), KI67 (ab15580), c-MET (ab51067), and SCD1 (ab19862) at 4 °C overnight. Subsequently, HRP-conjugated secondary antibodies bound to the primary antibodies were detected using an ECL detection system (ChemiDocTM XRS+ machine, Bio-Rad Laboratories). GAPDH protein levels were employed as loading controls. Densitometric analyses were performed using ImageJ software. Relative quantification was carried out after normalization against the band intensities of GAPDH. A Mann-Whitney test was performed to assess the difference in protein expression between groups. A representative blot is shown from three independent experiments. The expression of target protein by image lab (Bio-Rad Laboratories).
Apoptosis analysis using flow cytometry
An Annexin V-FITC apoptosis kit (BD Biosciences, NJ, USA) was used to determine the number of apoptotic cells according to the manufacturer’s instructions. Different cell groups were harvested with 0.25% trypsin and washed with PBS. After centrifugation, the cells were re-suspended in 100 μl buffer and then stained with Annexin V (3 μl) and propidium iodide (PI; 5 μl), and the mixture was incubated in the dark at 4 °C for 15 min. The cells were sorted using a FACS Calibur flow cytometer (BD Biosciences, USA), during the assay 10,000 cells per sample were collect and count. The result were analyzed using Cell Quest software (BD Biosciences, USA). The experiments were repeated three times.
BODIPY 493/503 staining
Cells were fixed in 4% PFA for 20 min at RT and incubated with BODIPY 493/503 (D3299, Thermo Fisher, USA) at 1:2000 and Hoechst 33342 (H1399, Thermo Fisher, USA) in PBS for 15 min at RT. Finally, the cells were visualized with a fluorescence microscope (Olympus, Tokyo, Japan). A representative image is shown from three independent experiments.
For LD staining, live cells were washed twice in PBS and incubated in 2 μg/mL BODIPY in PBS for 15 min at 37 °C. After staining, cells were washed twice in PBS and fixed in 2% paraformaldehyde for 15 min. Fixed cells were washed and re-suspended in PBS, passed through a cell strainer, and analyzed on an FACS Calibur flow cytometer (BD Biosciences, USA) under FL-1.
Immunofluorescence (IF) staining of cultured cells and mouse lung cancer sections
Cells cultured in cover slides were pretreated with drugs for 48 h. The cells were fixed with 4% PFA for 20 min, permeabilized with 0.1% Triton™ X-100 (30–5140 SAJ, Sigma-Aldrich, USA) for 10 min, blocked with 5% bovine serum albumin (BSA) for 1 h and incubated with antibodies against p-EGFR 1068 and SCD1 (1:100, rabbit) at 4 °C overnight. Highly cross-adsorbed donkey anti-rabbit IgG (H + L) secondary antibody was used at a concentration of 2 μg/ml in PBS containing 0.2% BSA for 1 h at RT to label the cells, and then the cells were counterstained with 1 μg/ml Hoechst 33342 for 5 min at RT. The cells were visualized with a fluorescence microscope (Olympus, Tokyo, Japan).
Mouse lung tumors were prepared and subjected to IHC staining as described above. The Avidin-Biotin Complex (ABC) Vectastain Kit (ZSGB-Bio, Beijing, China) was used, and anti-p-EGFR 1068 and anti-SCD1 (1:100, rabbit) were used as primary antibodies to incubate the tissue sections (4-μm thick) after heat-induced epitope retrieval (with 10 mM sodium citrate buffer of pH 6.0) and blockage with 5% BSA for 1 h. Highly cross-adsorbed donkey anti-rabbit IgG (H + L) secondary antibody was used at a concentration of 2 μg/ml in PBS containing 0.2% BSA for 1 h at RT to label the cells, and then the cells were counterstained with Hoechst 33342 (H1399, Thermo Fisher, USA). The tissues were visualized with a fluorescence microscope (Olympus, Tokyo, Japan). The fluorescence intensity Quantified by image J (Rawak Software, Inc. Germany).
Cell proliferation assay and plate clone formation assay
After 48 h of treatment, cell proliferation was quantified based on the incorporation of 5-ethynyl-2′-deoxyuridine (Edu) into DNA using a Cell-Light™ Edu Apollo®567 In Vitro Imaging Kit (Rio-Bio, Guangzhou, China). Before fixation, the cells were incubated with Edu for 2 h, permeabilized in 1.0% Triton X-100 for 15 min, and then subjected to Edu staining. Cell nuclei were stained with Hoechst 33342 (H1399, Thermo Fisher, USA) for 15 min. Fluorescence microscopy (Olympus, Tokyo, Japan) was used to determine the proportion of nucleated cells that had incorporated Edu. The cell proliferation rate was calculated as a percentage of Edu-positive nuclei to total nuclei in five high-power fields per well. The assay was performed in triplicate and repeated three times in independent experiments.
The plate clone formation assay was performed using the indicated cells. For each group, 800 surviving cells per well were incubated in 6-well plates containing 2 ml complete medium per well, followed by incubation for 10 days. Then, treatment was commenced when a mass of cells was visible to the naked eye for 48 h. At the indicated time point, the cells were washed twice with PBS, treated with crystal violet for 10 min, washed again, counted, and assessed. All experiments were performed at least 3 times. Colonies with > 40 cells were counted under phase contrast microscopy at × 40 magnification.
Xenograft mouse models
Four-week-old female Kunming mice (Animal Purchase No. 11401300077528, No. 32002100004219) were obtained from the Experimental Animal Center of Hubei Province (Animal Study Permit No. SCXK 2010–0009) and maintained in an environment with a standardized barrier system (System Barrier Environment No. 00021082) in the Experimental Animal Center of Tongji Hospital of Huazhong University of Science and Technology. These manipulations were approved by the Animal Care and Use Committee in Tongji Hospital of Huazhong University of Science and Technology. To establish the H1975-luc xenograft model, which is resistant to Gefitinib, and the HCC827-luc xenograft model, which is sensitive to Gefitinib, we transfected H1975 and HCC827 cells with luciferase and confirmed the efficiency of transfection using the IVIS Spectrum system (Caliper Life Sciences Inc., Xenogen Corporation).
Approximately 1 × 107 H1975-luc cells were subcutaneously injected into the right hind limbs of mice. Treatment began 1 week following injection. The mice were randomized into four groups (n = 4 per group) and intraperitoneally injected with vehicle (PBS, NT), g-PPT (50 mg/kg/day), Gefitinib (50 mg/kg/day) or g-PPT (50 mg/kg/day) + Gefitinib (50 mg/kg/day).
In a parallel experiment, 1 × 107 HCC827-luc cells were subcutaneously injected into the left upper limbs of mice. Treatment began 1 week following injection. The mice were randomized into three groups and intraperitoneally injected with vehicle (PBS, NT), Gefitinib (50 mg/kg/day) or Gefitinib (50 mg/kg/day) + OA (50 mg/kg/day) (n = 4 per group).
Tumor growth was monitored using caliper measurements twice every week, and tumor volume was calculated using the formula length x width2 × 0.52. Body weight was assessed twice weekly. Approximately 4 weeks later, the mice were analyzed using the IVIS Spectrum system (Caliper Life Sciences Inc., Xenogen Corporation). The total flux (photons/s) of the xenografts was calculated using Living Image software version 4.3.1. Then, the xenografts from each group were collected for further IHC and IF analyses.
Gene expression data (GSE83666, GSE38310 profiling data) were downloaded as raw signals from the Gene Expression Omnibus (
http://www.ncbi.nlm.nih.gov/geo), interpreted, normalized and log2-scaled using the online analysis tool GCBI website (
https://www.gcbi.com.cn). The gene expression of SCD1, perilipin (PLIN) and FASN in NSCLC cell lines was obtained from published gene expression profiles included in the Cancer Genome Atlas (TCGA) dataset using cBioPortal tools (
http://cbioportal.org).
Statistics
The results are expressed as the mean ± standard deviation (SD) from at least three independent experiments. Single comparisons between two groups were performed by Student’s t-tests. Comparisons between multiple groups were performed by one-way ANOVA followed by Tukey’s post-test. All statistical analyses were performed in SPSS 18.0.0 (SPSS Inc., Chicago, IL). p values < 0.05 were considered significant.
Discussion
The efficacy of conventional and molecular-targeted cancer therapies has been hindered by the emergence of drug resistance to therapy. Although EGFR-TKIs are the standard of care for patients with EGFR-mutant NSCLC, TKIs are not curative, mainly due to rapid progression after drug resistance [
19,
25,
26]. Currently, several major resistance mechanisms to TKIs have been identified and characterized, including T790 M and C797S mutations and Met amplification [
27‐
29]. The mechanism underlying the emergence of resistance is still unclear, and the origin of the resistant mutations remains controversial [
30‐
32]. Hence, there is an unmet need to identify the mechanism underlying resistance and novel targets in NSCLC for therapeutic intervention.
Lipid metabolic reprogramming is widely known as a hallmark of cancer that enables cancer cells to adapt and sustain survival signals [
33‐
35]. Previous joint drug trials have confirmed that blocking abnormal lipid metabolism can solve chemotherapy resistance or endocrine therapy resistance [
15,
34]. Accumulating evidence suggests that EGFR mutations can drive alterations in metabolism [
16,
36,
37], indicating that metabolic reprogramming plays a vital role in the emergence of EGFR-TKI resistance. To this end, our initial approach involved assessing the content of LDs and the expression of SCD1. SCD1 is known as a key enzyme in lipid metabolism that is involved in the introduction of a double bond into palmitic and stearic acids, giving rise to palmitoleic acid and oleic acid, respectively [
38]. Paired pre- and post-TKI treatment tumor tissues were collected, and we observed LD accumulation and SCD1 expression upregulated in post-TKI treatment tissues. Cell lines with different sensitivities to TKIs also confirmed that the extent of LDs was much higher in the cell lines with resistant EGFR mutations (including both cell line with acquired resistance (HCC827GR) and cell line with primary resistance (H1975)) than in the cell lines with sensitive EGFR mutations. Analysis of paired TKI resistance cell line PC9GR and parental PC9 data from public databases also supported our findings and indicated a correlation between abnormal lipid metabolism and TKI resistance. In our study the post-TKI treatment tissue expression higher LD. Additionally resistance cell lines show higher LD expression and database analysis the resistance cell line PC9GR and parental PC9 cell line, PC9GR show higher lipid metabolic associated gene expression. Taken together we thought this may indicated lipid metabolism correlation to resistance. A recent report also suggested that FASN mediates resistance in NSCLC [
39]. We also found that although the cell lines with different sensitivities to TKIs had variable LD content, when treated with TKI for a short time, the LD content decreased in all cell lines. We think that this is due to the adaptation of cells to sustain survival signals, and LDs are mobilized and metabolized into small molecule phospholipids and fatty acids to maintain the survival of cells. SCD1 can regulate the ratio between SFAs and MUFAs and affect membrane fluidity and cell function. Previous studies have confirmed the effect of altered plasma membrane fluidity on cell proliferation [
40‐
42]. This led us to hypothesize that SCD1 may induce resistance in NSCLC by increasing membrane fluidity. Nevertheless, the definite mechanism underlying sustained NSCLC cell survival by increasing membrane fluidity requires further study.
One important observation of this study was that OA abrogated the cytotoxic effect of TKIs in cell lines with sensitive EGFR mutations. OA is a major product of intracellular lipid metabolism (MUFA) triggered by SCD1. Accumulating evidence suggests that OA alters the activation of signaling pathways to promote cancer progression [
43,
44]. It known to all TKIs inhibit the phosphorylation of EGFR and the activation of the downstream pathway leads to cytotoxicity in EGFR sensitive mutation NSCLC cells; Zhang et al. reported that EGFR stabilizes SCD1 through Y55 phosphorylation and up-regulating MUFA synthesis to promote lung cancer growth [
16]. SCD1 can be subtly controlled by tyrosine phosphorylation and uncover a previously unknown direct linkage between oncogenic receptor tyrosine kinase and lipid metabolism in lung cancer. In our study although the effects are different in different cell lines, the trend is similar. We found co-treatment with OA lead to an accumulation of LDs, abrogates the effect of EGFR-TKIs and recovers the proliferation ability of cells, finally sustaining cell survival both in vitro and in vivo. Our results corroborate previous findings, suggesting that high OA expression triggered by SCD1 acts as a central node connecting lipid metabolism with therapeutic resistance. Taken together, our data demonstrate that abnormal lipid metabolism may be responsible for resistance to EGFR-TKIs.
Given that LDs accumulate and SCD1 upregulated in TKIs resistant cell lines, OA lead to an accumulation of LDs and abrogated the cytotoxic effect of TKIs. To detect whether inhibit abnormal lipid metabolism can reverse the resistance of NSCLC cells to TKIs, we used g-PPT to inhibit lipid metabolism. g-PPT has been proved with several interesting activities, including anticancer and antimetabolic effects [
17,
18,
45]. Whereas, a limited effect of g-PPT and TKI single used was observed in TKI-resistant NSCLC cell lines. However, when we co-treated TKI-resistant cells with g-PPT and Gefitinib, we observed a synergistic cytotoxic effect both in vitro and in vivo. Interestingly, this synergistic cytotoxic effect was not the same in TKI-resistant cells (HCC827GR/H1975) as in TKI-sensitive cells (HCC827/PC9), which may be due to differences in lipid metabolism between TKI-sensitive cells and TKI-resistant cells. There are also differences in primary resistant (the sensitive and resistant mixed) cell lines H1975 when compared with the secondary resistance cell line HCC827GR, which proves that the mutation state of the cells itself has a different effect on the combination therapy. Additionally, it is well known that ligand binding to EGFR phosphorylates EGFR, finally activating the downstream signaling pathways. Both the AKT and ERK signaling pathways are known to be downstream targets of EGFR, and EGFR activating mutations have been reported to deregulate these pathways in cancer [
46,
47]. In our study the level p-EGFR and the activation of both ERK/AKT pathways of Gefitinib-resistance cell lines was sharply decreased in the combination treatment group, meanwhile Gefitinib not. This result suggests that co-administration of EGFR-TKIs with g-PPT inhibit the expression of SCD1 and affects EGFR phosphorylation which provide a linkage between EGFR-TKI resistance and lipid metabolism in lung cancer.