RNA methyltransferase METTL3 induces intrinsic resistance to gefitinib by combining with MET to regulate PI3K/AKT pathway in lung adenocarcinoma

Abstract Clinical research data show that gefitinib greatly improves the progression‐free survival of patients, so it is used in advanced non‐small cell lung cancer patients with EGFR mutation. However, some patients with EGFR sensitive mutations do not have good effects on initial gefitinib treatment, and this mechanism is rarely studied. METTL3, a part of N6‐adenosine‐methyltransferase, has been reported to play an important role in a variety of tumours. In this study, we found that METTL3 is up‐regulated in gefitinib‐resistant tissues compared to gefitinib‐sensitive tissues. Cell function experiments have proved that under the treatment of gefitinib, METTL3 knockdown promotes apoptosis and inhibits proliferation of lung cancer cells. Mechanistic studies have shown that METTL3 combines with MET and causes the PI3K/AKT signalling pathway to be manipulated, which affects the sensitivity of lung cancer cells to gefitinib. Therefore, our research shows that METTL3 can be used as a molecular marker to predict the efficacy of EGFR‐TKI therapy in patients, and METTL3 may be a potential therapeutic target.

making EGFR-TKI its standard first choice. [3][4][5][6] However, there are still about 25% of patients with EGFR sensitive mutations who are not sensitive to initial gefitinib treatment, which deserves further study. 3,7 In the past few decades, research has found that epigenetics is closely related to tumours. [8][9][10][11] Epigenetics refers to changes in gene expression levels based on non-gene sequence changes, such as DNA methylation and chromatin conformation changes, which have been studied in depth. RNA methylation, as a newly discovered post-transcription epigenetic modification, has received attention in recent years. [12][13][14] As an important RNA methylation modification, N6-methyladenosine (m 6 A) is one of the most abundant chemical modifications on eukaryotic mRNA, which can regulate RNA splicing, stability, translation and nucleation. [15][16][17] There are three major types of enzymes involved in m 6 A methylation of RNA: writers (ie METTL3, METTL14, WTAP, KIAA1429 and METTL16), erasers (ie FTO and ALKBH5) and readers (ie YTHDF1, YTHDF2 and YTHDF3). 18 m 6 A modification is closely related to the occurrence and development of various tumours, such as breast cancer, glioblastoma and lung cancer. [19][20][21] As the most important component of the writers complex, METTL3 plays a very important role in the regulation of gene expression. 22,23 Studies have shown that epigenetics is related to drug resistance, and the relationship between DNA methylation and drug resistance has been widely studied. [24][25][26] However, there are few reports on RNA methylation and drug resistance. 27,28 And little is known about the important role of METTL3 in EGFR-TKI resistance. Therefore, there is an urgent need to study the role of METTL3 in EGFR-TKI resistance and describe its function and mechanism.
In this study, we demonstrated that the expression of METTL3 is up-regulated in LUAD tissues resistant to gefitinib. We found that METTL3 influences the expression of MET by integrating with MET to regulate PI3K/AKT pathway, which affect the response of lung cancer cells to gefitinib. Meanwhile, the level of m 6 A decreased in lung cancer cells that METTL3 was knocked down, suggesting that METTL3 may affect the expression of MET through the m 6 A pathway. It suggested that METTL3 could be a potential target to reverse resistance.

| Tissue samples
A total of 18 LUAD patients were enrolled in the study. Their genetic test reflected that they had either EGFR exon 19 deletion (19DEL) or L858R. The pathological tissues of both groups without medical treatment were divided into sensitivity (n = 9) and resistance (n = 9) by primary efficacy of gefitinib according to follow-up results. The project was approved by the Research Ethics Committee of Nanjing Medical University (Nanjing, Jiangsu, China), and we obtained written informed consent from all patients.

Human lung adenocarcinoma cell lines (PC9 cells and H3255 cells)
were obtained from the Institute of Biochemistry and Cell Biology at the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI 1640 medium supplemented with 10% foetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA) at 37℃ with 5% CO2 in incubators.
The target sequences for sh-METTL3 and sh-NC were described in Table S1. PC9 cells and H3255 cells were cultured into 6-well plates and transfected with 10 µL lentivirus targeting METTL3 or NC. After transfection, cells were selected for 14 days for further studies.

| RNA extraction, reverse transcription and qPCR
Total RNA was extracted from tissues or cells with TRIzol reagent (Thermo Fisher Scientific, USA). Then, 1.0 μg of isolated RNA was reverse-transcribed to cDNA by using PrimeScript™ RT reagent (Takara, Japan) under manufacturer's instructions. Real-time PCR analysis was performed with SYBR Green (Takara, Japan). According to the manufacturer's instructions, qPCR and data collection were carried out on StepOnePlus RT-PCR system (Applied Biosystems, USA). β-ACTIN was used as a housekeeping gene. The primers used in this study were described in Table S1.

| Western blot analysis and antibodies
RIPA buffer (Sigma, USA) was used to lyse total cellular proteins.
Cells treated with gefitinib were extracted after 6 hours. After ultrasonic cracking, the lysates were quantified using a BCA Protein Assay kit (Pierce, USA). Total protein was separated on 8% SDS-PAGE gel and transferred to a PVDF membrane (Millipore, USA). anti-rabbit (150 µg/mL, 1:3000 dilution) and anti-mouse (688 µg/ mL, 1:3000 dilution) secondary antibodies were purchased from Cell Signaling Technology. β-ACTIN was used to as an internal control.

| CCK8 assay
The survival of cells with gefitinib treatment was assessed by CCK8 assay (Selleck, Shanghai, China). PC9 cells and H3255 cells transfected with sh-NC or sh-METTL3 were seeded into 96-well plates with 3000 cells/well. The next day, the cells were exposed to different concentrations of gefitinib (MedChemExpress, China) for 72 hours. Then, 10 µL of CCK8 was added into each well and incubated at 37°C for 1 hour. The absorbance was measured at 450 nm by an enzyme-labelled instrument. Set triplicate and repeated three times independently.

| Colony-formation assay
The PC9 cells and H3255 cells transfected with sh-METTL3 or sh-NC were placed in 6-well plates with 500 cells/well. Then, the cells were treated with different concentrations of gefitinib. Change fresh medium with gefitinib every 3 days. Two weeks later, the colonies were fixed in paraformaldehyde, washed with PBS and stained with 0.1% crystal violet (Sigma, St. Louis, MO, USA). The above experiment was performed in triplicate.

| Flow cytometric analysis of apoptosis
The PC9 cells and H3255 cells transfected with sh-METTL3 or sh-NC were treated with gefitinib and cultured for 72 hours. Then, trypsin without EDTA was used to harvest the cells. The cells were

| m 6 A dot blot assay
Total RNA was extracted from PC9 cells and H3255 cells transfected with sh-METTL3 or sh-NC with TRIzol reagent (Thermo Fisher Scientific, USA). NanoDrop was used to test the concentration of RNA and dilute RNA to 100 ng/µL with RNase-free water.

| Statistical analysis
The data were performed with SPSS software 22.0 (IBM, SPSS, Chicago, IL, USA) and GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA). Evaluate the correlation between METTL3 and MET using the linear correlation analysis. Student's t test was used to compare the difference between groups. P value < .05 was considered to indicate the result statistically significant.

| METTL3 is up-regulated in LUAD tissues resistant to gefitinib
In order to study the relationship between METTL3 and gefitinib resistance, we first examined METTL3 mRNA expression in 18 LUAD tissues by quantitative real time (qPCR). The tissues without medical treatment harboured EGFR sensitive mutation: EGFR exon 19 deletion or exon 21 mutation. After following up cases, we divided the tissues into two groups: those who were sensitive to gefitinib (n = 9) and resistant to gefitinib (n = 9). Compared with gefitinib-sensitive tissues, METTL3 expression was significantly higher (P < .01) in gefitinib-resistant tissues ( Figure 1A). Then, we analysed TCGA database and found that METTL3 expression of LUAD tissues exhibited a significant increase compared to normal tissues ( Figure 1B). So, the expression of METTL3 is higher in LUAD tissues compared to normal tissues and higher in gefitinibresistant LUAD tissues compared to gefitinib-sensitive LUAD tissues.

| Knockdown of METTL3 sensitizes LUAD cells to gefitinib
To study the biological significance of METTL3 in LUAD, PC9 (EGFR  Figure 2C). According to the above result, we found that METTL3 reduce the sensitivity of LUAD to gefitinib.

| Knockdown of METTL3 reduced the m 6 A level of total RNAs in PC9 cells and H3255 cells significantly
To test the change of m 6 A level of total RNAs, we conducted m 6 A dot blot assays. The result reflected that the m 6 A level of total RNAs reduced significantly in PC9 cells and H3255 cells transfected with sh-METTL3 compared to control groups ( Figure 3). Therefore, we speculated that the sensitivity of PC9 cells and H3255 cells transfected with sh-METTL3 to gefitinib may be related to the changes in m 6 A levels of total RNA.

| METTL3 regulated the expression of MET positively and activate the PI3K/AKT pathway to reduce sensitivity to gefitinib
To investigate the mechanism of METTL3 in decreasing sensitivity to gefitinib in PC9 cells and H3255 cells, we checked published methylated RNA immunoprecipitation sequencing and data analysis in lung cancer.
We found that MET had m 6 A peaks identified in H1299 cell. 22 According to previous reports, MET is related to primary gefitinib resistance. 29 So, we conjectured that METTL3 regulated MET expression. qPCR demonstrated our conjecture that the expression of MET decreased significantly after METTL3 knockdown ( Figure 4A,B). Western blot assays also show that the knockdown of METTL3 caused a significant decrease in MET protein expression ( Figure 4C,D). Next, we verified in the patient tissues. We tested the expression of METTL3 and MET in the 18 tissues

| D ISCUSS I ON
Gefitinib is a small molecule that targets EGFR and has been successfully used as a first-line treatment for NSCLC patients with EGFR mutations. The use of targeted therapy has greatly improved the survival rate of NSCLC patients. However, some patients with EGFR mutations are still insensitive to gefitinib. 3 The molecular mechanism of initial gefitinib resistance in NSCLC patients remains unclear.
In recent years, m 6 A modification is closely related to the occurrence and development of lung cancer. [31][32][33] Growing evidence has revealed that m 6 A modification is associated with drug resistance. [34][35][36] It was reported that METTL3 knockdown enhances sensitivity to cisplatin. 37 However, the relationship between m 6 A modification and EGFR-TKI resistance is not studied. In this study, we found that the relative expression level of METTL3 is higher in F I G U R E 4 METTL3 is involved in MET up-regulation. A and B, qPCR analysis of MET expression in PC9 cells and H3255 cells transfected with sh-NC, sh-METTL3-1# or sh-METTL3-2#. C and D, Western blot assay was used to detect the level of MET in PC9 cells and H3255 cells after shRNA treatment targeting METTL3 or NC. E, qPCR assay was used to analyse the relationship between MET expression (rCt value) and METTL3 expression (rCt value) in the 18 LUAD tissues mentioned above. F and G, qPCR analysis of immunoprecipitation with METTL3 antibody in PC9 cells and H3255 cells to assess the expression of MET. The above results are mean ± SD of three independent experiments performed in triplicate. **, P < .01, and ***, P < .001 F I G U R E 5 METTL3 regulates PI3K/ AKT pathway through MET. A and B, Western blot assays confirmed PI3K/AKT pathway change after transfection with sh-METTL3 or sh-NC in PC9 cells and H3255 cells. β-ACTIN protein was used as an internal control. Representative results were from three independent experiments gefitinib-resistant tissues compared to gefitinib-sensitive tissues.
Knockdown of METTL3 sensitized PC9 cells and H3255 cells to gefitinib, leading to cell apoptosis. Whether the expression of METTL3 is related to the response to gefitinib and prognosis need further study. Meanwhile, we found that knockdown of METTL3 was accompanied by a decreased expression of MET. In patient tissues, we also found METTL3 and MET are positively related. Furthermore, RNA immunoprecipitation (RIP) assays showed that METTL3 could bind MET in PC9 cells and H3255 cells. m 6 A dot blot assays reflected the level of m 6 A in cells with METTL3 knockdown decreased significantly. METTL3, an important RNA methyltransferase, we conjectured that METTL3 may regulate MET through the m 6 A modification. MET was reported to be associated with resistance to gefitinib by activating PI3K/AKT signalling pathway. 7 In sensitive cells, gefitinib can inhibit EGFR phosphorylation, leading to inactivation of PI3K/AKT signalling. However, in resistant cells, even if gefitinib inhibits the phosphorylation of EGFR, the PI3K/AKT signalling pathway is still activated by MET amplification. 29 In this study, we found that knockdown of METTL3 also decreased the expression of MET and phosphorylation-AKT compared to control cells with gefitinib treatment. It suggested that METTL3 could affect the PI3K/AKT signalling pathway by regulating the expression of MET and leads to TKI resistance.
In conclusion, our experiment indicated that METTL3 may be a potential molecular marker to predict patients' efficacy on EGFR-TKI drugs and METTL3 may be a target to overcome the intrinsic resistance to EGFR-TKI.

ACK N OWLED G EM ENTS
This work was supported by grants from the National Natural Science Foundation of China (81972188) and the Medical Important Talents of Jiangsu Province (ZDRCA2016024).

CO N FLI C T O F I NTE R E S T
The authors declare no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data are free access to available upon request.