G protein‐coupled oestrogen receptor promotes cell growth of non‐small cell lung cancer cells via YAP1/QKI/circNOTCH1/m6A methylated NOTCH1 signalling

Results from various studies reveal that the role of G protein‐coupled oestrogen receptor (GPER) is cancer‐context dependent, and the function of GPER in non–small‐cell lung cancer (NSCLC) is still unclear. The present study demonstrated that neoplasm lung tissues expressed higher level of GPER compared with the normal lung tissues. The clinical data also showed that GPER expression level was positively correlated with the tumour stage of NSCLC. Our experimental data confirmed that GPER played an oncogenic role to promote cell growth of NSCLC cells. Mechanistic dissection revealed that GPER could modulate the NOTCH1 pathway to regulate cell growth in NSCLC cells. Further exploration of the mechanism demonstrated that GPER could up‐regulate circNOTCH1, which could compete with NOTCH1 mRNA for METTL14 binding. Because of the lack of m6A modification by METTL14 on the NOTCH1 mRNA, NOTCH1 mRNA was more stable and much easier to undergo protein translation. Subsequently, we found that GPER could prevent YAP1 phosphorylation and promote YAP1‐TEAD's transcriptional regulation on QKI, a transacting RNA‐binding factor involved in circRNA biogenesis, to facilitate circNOTCH1 generation. Supportively, data from preclinical mice model with implantation of H1299 cells also demonstrated that knock‐down of circNOTCH1 could block GPER‐induced NOTCH1 to suppress NSCLC tumour growth. Together, our data showed that GPER could promote NSCLC cell growth via regulating the YAP1/QKI/circNOTCH1/m6A methylated NOTCH1 pathway, and targeting our identified molecules may be a potentially therapeutic approach to suppress NSCLC development.

overall survival of NSCLC patients. 4 Even though current therapies benefit patients a lot, the 5-year overall survival (OS) rate of NSCLC patients in advanced stage was still less than 5%, 5 suggesting there is urgent need to develop novel therapeutic strategies to battle against this type of disease.
The seven-transmembrane G protein-coupled oestrogen receptor (GPER, also known as GPR30) is one member of the G protein-coupled receptor (GPCR) family. The discovery of GPER suggests an additional mechanism through which oestrogen (E2) could exert its effects. Increasing evidence has demonstrated that E2 exerts multiple biological effects through GPER but not the classical oestrogen receptors ERα and ERβ. 6 G1, the first GPER-specific agonist, which was screened out from a library of 10,000 molecules, manifested high affinity towards GPER. G15 was first identified as a GPER-selective antagonist in 2009. 7 Recent study has reported that GPER antagonist G15 could prevent oestrogen-induced cancer development of NSCLC. 8 In breast cancer, several studies have revealed that GPER could suppress YAP1 phosphorylation by turning off the Hippo pathway. 9,10 Dephosphorylated YAP1 acted as a co-transcriptional factor through binding the transcriptional enhancer-associated domain (TEAD) transcription factors to regulate downstream genes, which were involved in tumour initiation and progression. 11,12 However, the mechanism of how GPER regulates NSCLC cell growth is still unclear.
It has been validated that NOTCH1 can influence proliferation, apoptosis and differentiation of various cancers. 13,14 Activation of NOTCH1 signalling expands the population of cancer stem-like cells (CSC) in breast cancer 15 as well as ablate chemosensitivity during palliative therapy. 16 Accumulating literature has reported that oestrogen could up-regulate NOTCH1 to promote cancer cell proliferation. 17,18 Mechanistic dissection demonstrated that oestrogen activated oestrogen receptors (ERs) pathways to regulate NOTCH1 expression. 17 However, Notch signalling has also been activated in triple-negative breast cancer, 19 implying GPER, instead of ERs, might activate the Notch pathway.
Circular RNAs (circRNAs) were first identified as by-products of mRNA splicing in the early 1990s. 20 Attributing to the novel sequencing technologies and bioinformatics, more and more circRNAs have been identified and proved to be functional in physiological and pathological cellular events. 21 Encouragingly, circRNAs can be developed either as diagnostic/prognostic biomarkers or therapeutic targets in the treatments of various cancers. [22][23][24] Studies displayed that circRNA can regulate gene expression as competitive endogenous RNAs (ceRNAs) or protein sponges. [25][26][27][28][29][30] Particularly, circRNA could regulate its host gene expression and was involved in tumorigenesis and tumour progression. 31 For example, circRNA-ENO1 up-regulated ENO1 expression to promote lung adenocarcinoma progression. 32 However, so far, the relationship between circNOTCH1 and NOTCH1 underlying NSCLC progression is still unclear.
CircRNAs competitively combine with RNA-binding proteins (RBP) and decrease the binding capacity of RBP with mRNA, which determines the mRNA fate. 33,34 Among these RBPs, N6-Methyladenosine (m6A) methyltransferase is particularly important and fashionable. 35 Emerging studies have revealed that the methylated adenosine on RNA could decrease RNA stability through recruiting YTH N6-methyladenosine RNA-binding protein 2 (YTHDF2). 36 In this study, we showed that circNOTCH1 competed with NOTCH1 for the binding of methyltransferase like 14 (METTL14), decreasing NOTCH1 mRNA m6A methylation and increasing NOTCH1 mRNA stability.

| Oncomine analysis
Oncomine (www.oncom ine.org), a cancer microarray database and web-based data mining platform, aims at integrating discovery from genome-wide expression analyses. GPER expression data were extracted from array results of 96 samples, which were conducted by Beer DG, et al. 37 Differential analysis of GPER was conducted between lung adenocarcinoma (LUAD) and normal tissues. Meanwhile, all the tumour samples were subtyped into high tumour stage and low tumour stage. Subsequently, GPER expression differentiation was analysed between the two groups.

| MTT assay
Cells were cultured in phenol red-free medium which was supple-

| Colony formation assay
Cells were plated at 500 per well in 6-well plates and incubated in RPMI 1640 with 10% FBS at 37°C. Two weeks later, the cells were fixed and stained with 0.1% crystal violet. The number of visible colonies was counted manually.

| RNA extraction and quantitative real-time PCR (qRT-PCR) analysis
Total RNAs were extracted using Trizol reagent (Invitrogen). 1 µg of total RNA was reverse transcribed using Superscript III transcriptase (Invitrogen). Quantitative real-time PCR (qRT-PCR) was conducted using a Bio-Rad CFX96 system. SYBR green was used to determine the mRNA expression level of a gene of interest. Expression levels were normalized to the GAPDH mRNA level using the 2−ΔΔCt method.

| Western blot
Cells were washed twice with cold PBS and lysed in RIPA buffer.
The signals of the probes were detected by FISH Kit (K2191050, BioChain) following the manufacturer's instructions.

| RNA immunoprecipitation (RIP)
Cells were lysed in ice-cold lysis buffer supplemented with RNase inhibitor after different treatments.

| Chromatin immunoprecipitation assay (ChIP)
Cell lysates were pre-cleared with normal mouse IgG and protein A-agarose. We then added 2.0 µg anti-YAP1 antibody to the cell lysates and incubated overnight at 4°C. IgG was used as the negative control. Specific primer sets were designed to amplify a target sequence within QKI promoter, and agarose gel electrophoresis was used to identify PCR products. The specific primers were listed as follows: the forward 5′-TCTCAGCCGCGAATTACTCT-3′; the reverse 5′-TCAGCTGCACCAATGAAGTC-3′.

| Luciferase reporter assay
The 3kb length of QKI's promoter was constructed into pGL3-basic luciferase reporter vector. A549 cells w/wo YAP1 shRNAs were seeded in 24-well plates, and plasmids were transfected using lipofectamine 3000 (Invitrogen). After 36 hours transfection, luciferase activity was measured by Dual-Luciferase Assay (Promega) according to the manufacturer's manual.

| mRNA stability assay
H1299 cells with/without circNOTCH1 shRNAs were cultured, then actinomycin D (5 µg/mL, Apexbio) was used to block de novo RNA synthesis. Subsequently, total RNA was harvested at 0, 40, 80 and 120 minutes time points and qRT-PCR was conducted to detect the NOTCH1 mRNA.

| In vivo studies
Thirty-two 6 week-old female nude mice were purchased from the National Cancer Institute (NCI) and divided into four groups week. The mice were killed after 8 weeks. Tumours were removed for study.

| Statistics
Experiments were repeated at least three times with triplicate data points. The continuous data were analysed by using the Mann-Whitney U test. Results of laboratory experiments are expressed as mean ± SD. Statistical significance for in vitro or in vivo experiments was determined using the independent-sample t test.
P < .05 was considered statistically significant.
F I G U R E 1 GPER promoted NSCLC cell growth. (A) GPER expression in lung cancer tissues vs normal cancerous tissues from the Oncomine data set. (B) The GPER expression level was highly expressed in T 3+4 NSCLC samples compared with T 3+4 ones. (C) In A549 cells pre-treated with G1 (10 nmol/L) or G15 (1 µmol/L), MTT assay was conducted to examine cell growth.
(D) MTT was used to detect cell growth of A549 cells after GPER knock-down. (E) Colony formation assay was conducted to examine cell growth of A549 cells treated with/without shGPER. Quantification is at the right. (F) Cell growth was detected by MTT assay in H1299 cells treated with/ without oeGPER. (G) Colony formation assay was conducted to examine cell growth of H1299 cells treated with/ without oeGPER. Quantification was made at the right. Quantitation was presented as mean ± SD, and P values were calculated by t test, *P < .05, **P < .01

| GPER promoted cell growth of NSCLC cells
To explore the role of GPER in lung cancer, we first examined its expression level in different human lung specimens by analysing the Oncomine database of lung adenocarcinomas (www.oncom ine.org).
Data revealed that lung adenocarcinomas exhibited a high GPER expression profile compared with normal lung tissues ( Figure 1A). The expression level of GPER was much higher in large size tumours (T 3-4 ) compared with small size ones (T 1-2 ), suggesting GPER may play an oncogenic role in the development of lung cancer ( Figure 1B). To test our speculation, we treated A549 cells with GPER-selective agonist G1 (10 nmol/L) or GPER antagonist G15 (1 µmol/L). The result of MTT assay revealed that G1 promoted cell growth while G15 suppressed cell growth of A549 cells ( Figure 1C). And G1-induced cell growth of A549 cells could be reversed by G15 treatment ( Figure 1C). We se-

lected A549 cells to conduct loss-of-function experiments and H1299
to perform gain-of-function experiments due to the relatively high level of GPER in A549 cells compared with H1299 cells ( Figure S1A). First, we applied the lentivirus system to knock down GPER with shRNA (shGPER) in A549 cell line ( Figure S1B) and to overexpress GPER with GPER-CDS (oeGPER) in H1299 cell line ( Figure S1C). Subsequently, MTT and colony formation assays were performed to examine cell  Together, the results from Figure 1A-G and Figure S1A-C suggested that GPER increased NSCLC cell growth.

| GPER promoted NSCLC cell growth via upregulating the expression level of NOTCH1
To dissect the mechanism by which GPER altered NSCLC cell growth, we then examined the expression levels of some selective oncogenes related to cell growth/proliferation and found that knockdown of GPER in A549 cells led to decreasing the expression levels of NOTCH1, Hif-1α, β-catenin, CXCR4, CENPE and C-MYC at mRNA level ( Figure 2A). In contrast, overexpression of GPER in H1299 cells led to increasing the expression of NOTCH1, Hif-1α, IGF2BP3 and CXCR4 ( Figure 2B). Western blot was conducted to detect the expression levels of three potential oncogenes in A549 cells transfected with/without (w/wo) shGPER. The result showed that only NOTCH1 protein was markedly decreased when GPER was depleted ( Figure 2C).
Consistently, induction of GPER increased NOTCH1 at protein level in H1299 cells ( Figure 2D). To further confirm the impact on NOTCH1 expression level upon the alteration of GPER signalling, we treated A549 cells with G1, G15 and G1 + G15, respectively. The Western blotting analysis demonstrated that G1 had capacity to increase NOTCH1 level, which was blocked by G15 treatment, while G15 alone could reduce NOTCH1 expression level in A549 cells ( Figure 2E). Consistently, a strongly positive correlation between GPER and NOTCH1 (R = 0.3716, P < .01) was observed in TCGA data set ( Figure 2F). To verify that GPER-mediated NSCLC cell growth is dependent of NOTCH1, we conducted interruption assays and the data revealed that knock-down of NOTCH1 ( Figure S1D) could partially block GPER-induced cell growth of H1299 cells, monitored by MTT ( Figure 2G) and colony formation approach ( Figure 2H).
Together, the results from Figure 2A-H and Figure S1D suggested that GPER increased NSCLC cell growth relied on NOTCH1 induction.

| GPER functioned to positively regulate NOTCH1 expression level through circNOTCH1 in NSCLC cells
To explore the mechanism by which GPER regulated NOTCH1 expression level, we focused on the circRNAs generated from the host gene NOTCH1. 38 We utilized the online tool circbase (http:// circb ase.org/) to predict all the circRNAs spliced and generated from the NOTCH1 transcript. Our detection from qRT-PCR demonstrated that the expression levels of has_circ_0089548 and has_circ_0089552 were consistently altered upon the manipulation of GPER in A549 and H1299 cells, indicating they may be involved in the process of GPER-induced NOTCH1 in NSCLC cells ( Figure 3A). Furthermore, we conducted an exoribonuclease RNase R degrading RNA experiment to confirm the circular property of hsa_circ_0089548 and hsa_circ_0089552. The result revealed that the two circRNAs were resistant to RNase R treatment when linear-NOTCH1 mRNA was used as a control ( Figure 3B). We then applied the lentivirus system to target specific 5′-3′ splice junctions for knocking down these two circRNAs ( Figure 3C), which successfully reduced the expression levels of these two circRNAs in H1299 cells ( Figure 3D). We found that reduction of has_circ_0089552 (shcirc_0089552) but not has_circ_0089548 (shcirc_0089548) could suppress cell growth of H1299 cells ( Figure S1E,F). Western blotting also validated that only knock-down of has_circ_0089552 (circNOTCH1) could decrease the protein level of NOTCH1 in H1299 cells ( Figure 3E). In addition, fluorescent in situ hybridization (FISH) assay clearly demonstrated that circNOTCH1 (has_circ_0089552) was predominantly localized in cell cytoplasm of A459 and H1299 cells ( Figure 3F). To further verify that circNOTCH1 was indeed involved in GPER-mediated cell growth of NSCLC, we performed interruption assays in H1299 cells, and the results displayed that knock-down of circNOTCH1 could block GPER-induced cell growth of H1299 cells, monitored by colony formation assay ( Figure 3G) and MTT assay ( Figure 3H).
Together, the results from Figure 3A-H and Figure S1E,F indicated that GPER functioned through circNOTCH1/NOTCH1 signalling pathway to increase NSCLC cell growth.

| GPER regulated circNOTCH1 expression: via transcriptional regulation on QKI by YAP1/ TEAD complex
To dissect the mechanism by which GPER regulated circNOTCH1, we first checked several transacting RNA-binding factors including ADAR, quaking (QKI), FUS, HNRNPL and DHX9, which were involved in circRNA biogenesis. Our data showed that only QKI expression level was increased upon GPER induction in H1299 cells ( Figure 4A). Consistently, inhibition of GPER with shRNA ( Figure 4B) or its antagonist G15 ( Figure 4C) all led to decreased QKI expression in A549 cells. We then applied the lentivirus system to attenuate QKI expression level with shRNA-QKI (shQKI #1 and shQKI #2) in A549 cells and to overexpress QKI with QKI-CDS in H1299 cells ( Figure S2A). We noticed that overexpression of QKI could increase circNOTCH1 expression in H1299 cells and knock-down of QKI had ability to decrease circNOTCH1 expression in A549 cells ( Figure 4D). Of note, we utilized the online tool (http://gepia.cance r-pku.cn) to analyse LUAD data of The Cancer Genome Atlas (TCGA), and we observed a positive correlation between GPER and QKI, which supported our study ( Figure 4E).
Next, the interruption experiment was conducted to examine whether GPER functioned through QKI to regulate circNOTCH1 expression level. The result of qRT-PCR demonstrated that knockdown of QKI could block GPER-induced circNOTCH1 expression F I G U R E 3 GPER functioned to positively regulate NOTCH1 expression level through circNOTCH1 in NSCLC cells. (A) We applied qRT-qPCR to screen all the circRNAs originated from NOTCH1 gene in H1299 cells transfected with oeGPER or pWPI vector (left) and A549 cells transfected with shGPER or pLKO.1 vector (right). (B) qRT-PCR assay was conducted to validate circRNAs expression when treated with RNase R. (C) Schematic illustration showed the principle of using shRNA to knock down the circRNAs (sh_circ_89548, sh_circ_89552). (D) qRT-PCR assay was conducted to validate the knock-down efficiency of circRNAs. (E) Western blot was performed to test NOTCH1 expression in H1299 cells after knocking down the two circRNAs. (F) FISH demonstrated that circNOTCH1 was predominantly localized in the cytoplasm of A549 and H1299 cells. DAPI was used to indicate the nucleus. Scale bar, 100 µm. (G) Colony formation assay was conducted to test cell growth using H1299 cells transfected as indicated: pLKO.1 + pWPI, pLKO.1 + oeGPER, shcircNOTCH1 + pWPI, shcircNOTCH1 + oeGPER, and quantification was at the right. Quantitation was presented as mean ± SD and P values calculated by t test. (H) MTT assay was conducted in H1299 cells according to the above groups. *P < .05, **P < .01  level in A549 cells ( Figure 4F). Similar result was gained in the shQKI A549 cells treated with G1 ( Figure 4G). Recent reports illustrated that GPER could function through the Gαq-11, PLCβ/PKC, and Rho/ROCK signalling pathways to promote YAP1 dephosphorylation, which entered the nucleus and regulated its downstream genes through the recruitment of TEADs. 9,10,39 To test this mechanism in NCLSC cells, we conducted Western blot to examine the phosphorylation level of YAP1. As expected, overexpression of GPER could decrease YAP1 phosphorylation level, which was accompanied by increased expression levels of QKI and NOTCH1 in H1299 cells (Figure 4H left). On the contrary, GPER reduction by shRNA could increase the phosphorylation level of YAP1 as well as decreasing QKI and NOTCH1 in A549 cells (Figure 4H right).
These results were consistent with previous study showing GPER could regulate YAP1 activation. 9 Next, we applied the lentivirus activates the TEAD transcription factor family to output its important roles in various biological processes and human diseases. [40][41][42] Here, we hypothesized that YAP1 acted as a co-transcriptional factor combing with TEAD to regulate QKI transcription. To test our hypothesis, we used the Ensemble website with JASPAR database to screen the potential TEAD elements on the upstream 3 kb region of QKI gene locus. We found three putative TEAD elements in vivo binding assay also confirmed that YAP1-TEAD could bind to the second TEAD element ( Figure 4K).
Together, the results from Figure 4A-K and Figure S2A,B suggested that GPER could increase circNOTCH1 expression via YAP1-TEAD/QKI signalling.

| CircNOTCH1 competitively bond with METTL14 for protecting NOTCH1 mRNA
To determine how circNOTCH1 regulated NOTCH1 expression, we referred to the competing endogenous RNAs (ceRNAs). To test this assumption, we examined the potential regulation of NOTCH1 by miRNAs through detecting the NOTCH1 mRNA in the Argonaute 2 (Ago 2) complex using RNA interaction-precipitation (RIP) assay ( Figure S2C), because numeral studies reported that showed that circNOTCH1 failed to alter NOTCH1 protein level in the METTL14-depleted H1299 cells ( Figure 5D). Of note, we also noticed that GPER had little ability to regulate METTL14 expression level in A549 cells ( Figure 5E), implying the regulatory specificity of circNOTCH1 towards NOTCH1.
Together, the results from Figure 5A-E and Figure 2C-E suggested that circNOTCH1 could increase NOTCH1 expression via competitively binding with the m6A methyltransferase, METTL14.

| CircNOTCH1 depletion could block GPERinduced tumour growth in the subcutaneous xenograft mouse model
To  ( Figure 6A,B). The results showed that mice injected with cells with pLKO.1 + oeGPER had increased tumour growth compared with the control cohorts ( Figure 6A,B). However, this increased tumour growth by GPER was suppressed when circNOTCH1 was reduced ( Figure 6A,B). Importantly, Western blotting analysis on tumour samples also revealed that depletion of circNOTCH1 could block GPER-induced NOTCH1 expression ( Figure 6C).
Together, results from the in vivo studies in Figure 6A-C confirmed our in vitro studies and demonstrated that GPER could promote NSCLC progression by the modulation of YAP1-TEAD/QKI/ circNOTCH1/m6A methylated NOTCH1 signalling ( Figure 6D).

| D ISCUSS I ON
Previous global statistics showed that lung cancer was the most commonly diagnosed cancer and the leading cause of cancer-related deaths. 44 Several in vitro and in vivo studies have proved that oestrogen could promote lung cancer proliferation. 45 However, the therapeutic effect of pure antiestrogen (tamoxifen) was still unsatisfied. The utility of tamoxifen combined with GPER antagonists exhibited marked effects in blocking the progression of the primary breast tumour in the experimental animal model. 46 Mechanistic study revealed that tamoxifen acted as a GPER agonist to activate GPER, which in turn provided survival signal for breast cancer cells.
Consistent with this report, our study also exhibited that GPER can function through YAP1-TEAD/QKI/circNOTCH1 signalling to regulate NOTCH1 expression and to promote NSCLC tumour growth, supplementing the oncogenic role of GPER in lung cancer development.
Among several genes related to cell growth/proliferation, NOTCH1 was proved as the downstream gene regulated by GPER.  51 However, the m6A modification RNAs' fate is determined by a group of RNA-binding proteins that specifically recognize the methylated adenosine on RNA named m6A 'readers'. 52 For example, in breast cancer cells, METTL14 promotes cancer cell invasion and migration, but in colorectal cancer, METTL14 inhibits proliferation and metastasis. 51,53 In the present study, m6A antibody pull-down data confirmed that knock-down of circNOTCH1 increased the m6A modification on Nothch1 mRNA ( Figure 5C). However, circNOTCH1 failed to alter NOTCH1 expression when METTL14 was depleted, and knock-down GPER did not modulate METTL14 expression ( Figure 5D,E). Combined with the RNA degradation data ( Figure 5B), our data demonstrated that circ-NOTCH1 modulated NOTCH1 expression through competing for the binding of METTL14.
YAP1 has been regarded as an oncogene in tumorigenesis and progression through YAP1-TEAD transcriptional regulation. [54][55][56] Mechanistic dissection revealed that GPER functioned through YAP1-TEAD to regulate QKI expression, which in turn promoted the formation of circNOTCH1 by binding to the normal linear pre-NOTCH1. 57 Indeed, knock-down of QKI in A549 cells could block GPER-induced circNOTCH1 expression and YAP1 inhibition by shRNA also significantly decreased QKI mRNA level. The luciferase reporter assay demonstrated that the second YAP-TEAD element on the promoter of QKI was responsible for the transcriptional regulation of YAP on QKI. Together, all these data illustrated that GPER regulates circNOTCH1 expression through dephosphorylating YAP1 that promotes QKI expression.
Collectively, the findings of the present study demonstrate that GPER may promote NSCLC cell growth by regulating YAP1-TEAD/ QKI/circNOTCH1/m6A methylated NOTCH1 signalling ( Figure 6D) and targeting this signalling with small molecules may be promising therapeutic strategies to retard NSCLC progression.

CO N FLI C T O F I NTE R E S T
This study has no potential conflict of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets used and/or analysed during the current study are available from the corresponding author on reasonable request.