Glycoprotein M6A Suppresses Lung Adenocarcinoma Progression via Inhibition of the PI3K/AKT Pathway

Lung adenocarcinoma is the most common subtype of lung cancer and has high morbidity and mortality. Glycoprotein M6A (GPM6A) is a neuronal membrane glycoprotein reported to be related with cancer. However, studies on GPM6A in lung adenocarcinoma are rare. This study aimed to investigate the role of GPM6A in lung adenocarcinoma and its potential mechanism. GPM6A mRNA expression was analysed in 33 types of cancers using The Cancer Genome Atlas (TCGA) datasets. It was compared among normal lung tissues, lung adenocarcinoma tissues, and adjacent tissues using the Oncomine database. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to detect GPM6A expression in human lung adenocarcinoma cell lines (A549 and H1299) and normal pulmonary epithelial cells (BEAS-2B). When GPM6A was inhibited, cell proliferative capacity was detected by Cell Counting Kit 8 (CCK8), EdU, and colony formation assays. Cell migration ability was detected by wound healing and transwell assays. The expression of epithelial-mesenchymal transition (EMT) markers was detected by Western blotting (WB) and RT-qPCR. When GPM6A was overexpressed, cell proliferation and migration were detected again. Ten nude mice were subcutaneously injected with cells overexpressing GPM6A or empty vector, and the tumor size was recorded on day 14 and then measured every 3 days thereafter. The final tumor weight was measured on day 36. Furthermore, the expressions of phosphoinositide 3-kinase (PI3K), phosphorylated PI3K, AKT, and phosphorylated AKT were detected by WB. Results showed that GPM6A mRNA expression decreased in 15 types of tumors in TCGA dataset. GPM6A expression was lower in lung adenocarcinoma than in normal lung tissues or adjacent tissues in the Oncomine dataset. Similar results were found in lung adenocarcinoma cells. The function study showed that GPM6A downregulation enhanced the proliferation, migration, and EMT of lung adenocarcinoma cells, while GPM6A upregulation inhibited their development. The xenograft results suggested that GPM6A upregulation delayed tumor growth and reduced tumor weight. Moreover, WB showed that GPM6A knockdown activated the PI3K/AKT pathway, while GPM6A upregulation inhibited the activation of the PI3K/AKT pathway. In conclusion, GPM6A suppresses lung adenocarcinoma progression via inhibition of the PI3K/AKT pathway. Thus, GPM6A could be a possible treatment target for lung cancer therapy.


Introduction
Lung cancer is a common cancer that is the leading cause of death globally, with approximately 2.2 million new cases and 1.8 million deaths reported in 2020 [1]. Further, it is the second most commonly diagnosed malignancy (11.4% of total cancer cases) and the leading cause of cancer death (18% of total cancer-related deaths) [1]. Lung adenocarcinoma is a common subtype of lung cancer. It causes frst and second-order cancer morbidity to men and women in China [2]. Advanced lung adenocarcinoma is primarily treated with targeted therapies for specifc gene mutations; however, drug resistance is an inevitable issue [3,4]. Checkpoint immunotherapy has been approved as frst-line therapy for patients with Non-small cell lung cancer (NSCLC) expressing programmed cell death-ligand 1 (PD-L1) (tumor proportion score (TPS) ≥ 1%) [5,6]. However, it can also cause a spectrum of immune-related adverse events including colitis, hypophysitis, pneumonitis, thyroiditis, and infammatory arthritis [7].
M6 is a neuronal membrane glycoprotein that belongs to the proteolipid protein gene family. It is divided into two subtypes: glycoprotein M6A (GPM6A) and M6B (GPM6B). It was frst found in the developing murine embryonic central nervous system (CNS), and it mainly expressed in both neonatal and adult CNS regions [8], participating in the pathological process of CNS. Fuchsova et al. found low GPM6A expression in the hippocampus of depressed suicide victims [9]. Ma et al. reported that GPM6A showed a trend of dysregulation in schizophrenia [10]. Gregor et al. also revealed that high expression of GPM6A was associated with learning disability and behavioral anomalies [11]. Recent studies have shown the association of GPM6A with cancer. However, the function of GPM6A in cancers is controversial. Charf et al. indicated that GPM6A was highly expressed in lymphoid leukemia and promoted the transformation and proliferation of fbroblast cell (NIH/3T3), which may act as a candidate biomarker for human B-cell malignancies [12]. Ye et al. also revealed that GPM6A expression in the protein level was higher in undiferentiated or minimally diferentiated colorectal carcinoma tissues than in highly diferentiated colorectal carcinoma tissues, which suggested that GPM6A was related to poor outcomes in colorectal cancer [13]. However, Liu et al. showed that GPM6A expression was lower in hepatocellular carcinoma (HCC) than para-carcinoma tissues. Tey reported that circCCNB1 silencing which acted as a miR-106b-5p sponge inhibited GPM6A expression to promote HCC progression and activation of the AKT/ ERK signaling pathway in vivo and in vitro [14]. Jiang et al. also showed that miR-22 overexpression could inhibit the migration of small cell lung cancer cells, and elevated GPM6A was observed in cells with miR-22 overexpression [15]. Chen et al. analysed the gene expression of 100 normal specimens and 94 lung cancer samples from the Gene Expression Omnibus database, and GPM6A was identifed as a diferentially expressed gene. GPM6A expression was signifcantly lower in lung cancer samples than in normal specimens [16]. Tese results suggested that GPM6A was suppressed in cancers. However, evidence on the role of GPM6A in lung adenocarcinoma is limited.
In this study, we analysed GPM6A in 33 types of tumors in TCGA dataset and also the GPM6A expression between lung adenocarcinoma and adjacent tissues in the Oncomine database. Ten, we explored the function of GPM6A in the development of lung adenocarcinoma in vitro and in vivo and the potential mechanism of GPM6A in lung adenocarcinoma cells. Importantly, we demonstrated that GPM6A suppressed lung adenocarcinoma progression via inhibition of the PI3K/AKT pathway.

Biological Information
Data. GPM6A gene expression was compared between tumor and corresponding nontumor tissues in 33 types of cancers from Te Cancer Genome Atlas (TCGA) database using the GEPIA web tool (http://gepia. cancer-pku.cn). Hou Lung and Su Lung data were downloaded from the Oncomine database (https://www. oncomine.org) (the website was taken ofine 17 January 2022) to analyse GPM6A expression. Hou Lung data were used to compare GPM6A expression between normal lung and lung adenocarcinoma tissues, while Su Lung data were used to compare between lung adenocarcinoma and adjacent normal tissues. A survival analysis was performed between lung adenocarcinoma patients with high and with low GPM6A expression using the Kaplan-Meier plotter (http://kmplot.com/analysis/) (ID: 209469).

Cell
Culture. Human lung adenocarcinoma cell lines (A549, H1299) and normal pulmonary epithelial cells (BEAS-2B) were acquired from the Chinese Academy of Science (Shanghai, China) cell bank. Te cells were maintained in Dulbecco's modifed Eagle's medium (DMEM; Hyclone, Camarillo, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) at 37°C in a 5% CO 2 incubator [17]. Te DMEM was replaced two to three times per week.

RNA Isolation and Real-Time Quantitative Polymerase
Chain Reaction. Total RNA (1 μg) was extracted from the two cell lines using an EZ-press RNA purifcation kit (Epizyme Biomedical Technology, Shanghai, China). After mRNA was reverse transcribed to cDNA, we performed realtime quantitative polymerase chain reaction (RT-qPCR) using the SYBR Green mix (Termo, Waltham, MA, USA). Te 2 -ΔΔCT method was used to analyse gene expression. Te primer sequences were obtained from Sangon (Shanghai, China) and listed in Table 1 [17].

Transfection.
Small interfering RNAs (si-NC, si-GPM6A-1, and -2) were purchased from GenePharma (Shanghai, China). Te siRNA sequences are listed in Table 1. Te two cell lines were seeded in 6-well plates at 70% confuence and transfected with siRNA using Lipofectamine 3000 ™ (Termo, Waltham, MA, USA). Te transfection efciency of siRNAs was detected after 48 h of transfection [17]. Te overexpression plasmid packaged into lentivirus (HBLV-h-GPM6A-3xfag-ZsGreen-PURO) (Lv-GPM6A), and also, the control plasmids (HBLV-ZsGreen-PURO) (vector) were synthesized by Hanheng Biotechnology (Shanghai) Co., Ltd. Te lentivirus was added to the cells after they reached 50% confuence in the 6well plates. Puromycin (3-4ug/ml) was added to the two cell lines several times until the cells successfully transfected with the lentivirus. 2.6. EdU Assay. Te cells were seeded in a 48-well plate for 24 h and were incubated with diluted EdU for 2 h. Ten, 4% polyoxymethylene was used to fx cells for 30 min, and 0.5% TritonX-100 was added to permeate cells for 10 min. Finally, the cells were stained with Apollo and Hoechst. Images of the stained cells were taken under a fuorescence microscope. Te ratio of EdU-positive cells was calculated as EdU expressed cells/DAPI stained cells in the same feld×100%.

Colony Formation Assay.
After transfection, the cells (500 per well) were seeded in 35-mm plates and cultured for 10 days. Giemsa was used to stain the cloned cells. More than 50 cells were clustered as a clone. Te rate of colony formation was calculated as the number of cloned cells number/ 500 × 100%.

Wound
Healing Assay. When the two cell lines reached 95% confuence in the 6-well plates, a 200-μL tip was used to scratch a line. Te cells were imaged at 0 and 24 h after the wound gap was formed. Te relative migration area was measured using ImageJ software [17].

Transwell
Assay. When GPM6A expression of cells was upregulated or downregulated, A549 (2.5 × 10^4) and BEAS-2B (2 × 10^4) cells in serum-free DMEM were inoculated evenly in the upper chambers of transwell plates, while DMEM containing 10% FBS was placed in the lower chambers for 24 h. Te upper chambers were placed in 4% cold paraformaldehyde to fx the cells penetrating the membranes. Te cells were then stained with 0.1% crystal violet. Photographs were taken using a light microscope, and the number of migrated cells was calculated using ImageJ.

Nude Mice Xenograft Tumor
Assays. Ten male nude mice aged 5-6 weeks were obtained from Shanghai Jie Si Jie Laboratory Animal Co. Ltd. (Shanghai, China). Te mice were randomly divided into the control group and the GPM6A overexpression group. Mice were subcutaneously injected with 1 × 10 6 cells (empty vector) or the same number of cells (Lv-GPM6A). Te tumor size was frst recorded on day 14 and then every 3 days thereafter. Te mice were subsequently sacrifced on day 36, and the subcutaneous tumor was resected and weighed. Ten, the tumor tissues were stored at −80°C.
Tis study was approved by the Ethics Review Board of Shanghai East Hospital at Tongji University (Shanghai, China). All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health.

Western Blot Analysis.
A mixed solution of SDS lysis bufer, phenylmethylsulfonyl fuoride, protease, and phosphatase inhibitor was added in the two cell lines for complete proteolysis, and the superior solution was collected after centrifugation. After the protein concentrations were measured, the collected supernatants were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difuoride membrane. After incubation with primary antibodies at 4°C overnight, the membrane was incubated with secondary antibodies (1 : 10000; goat anti-rabbit IgG; ab6721; Abcam) and exposed using an ECL assay kit [17].

Statistical
Analysis. Data were presented as the mean ± standard deviation (SD). Each experiment was conducted three times independently. Data were compared between two groups using Student's t-test and among three groups using one-way analysis of variance. All statistical analyses were performed using GraphPad Prism 6.0 software. A P value of <0.05 was considered statistically signifcant.

GPM6A Is Downregulated in Lung Adenocarcinoma.
GPM6A mRNA expression was lower in 15 types of cancers including lung adenocarcinoma (Figure 1(a)) in TCGA dataset. In the Oncomine dataset, GPM6A expression was signifcantly lower in lung adenocarcinoma tissues (42 samples) than in normal lung tissues (63 samples) (Hou lung) (Figure 1(b)). Similar results were obtained for comparison between 23 pairs of lung adenocarcinomas and adjacent lung tissues (Su lung) (Figure 1(c)). Furthermore, the Kaplan-Meier plot revealed that lung adenocarcinoma patients with lower GPM6A expression had worse prognosis (Figure 1(d)). RT-qPCR results showed lower GPM6A expression in A549 and H1299 than in BEAS-2B (Figure 1(e)).

Discussion
Te role of GPM6A in lung cancer is yet to be clarifed. In the current study, GPM6A mRNA expression was lower in 15 types of tumors including lung adenocarcinoma in TCGA dataset.
GPM6A expression was also lower in lung adenocarcinoma than in adjacent tissues in the Oncomine database. GPM6A downregulation enhanced the proliferation and migration of lung adenocarcinoma cells, while GPM6A upregulation suppressed the proliferation and metastasis of lung adenocarcinoma in vitro and in vivo. Furthermore, GPM6A knockdown activated the PI3K/AKT pathway, whereas GPM6A upregulation inactivated the PI3K/AKT pathway.  BRCA  CHOL  DLBC  ESCA  GBM  HNSC  LAML  LGG  ACC  BLCA  CESC  COAD  KICH  KIRC  KIRP  LIHC  LUAD  LUSC  MESO  PAAD  PCPG  PRAD  READ  SARC  SKCM  STAD  TGCT  THCA  THYM  UCEC     GPM6A is a stress-responsive gene belonging to the proteolipid protein family. It is expressed on neuronal membrane proteins in the CNS. It binds to the μ-opioid receptor and regulates immune response and stress [18]. Alvarez Juliá et al. reported that GPM6A is involved in neurite extension, flopodium and spine formation, and synaptogenesis under stress [19]. Michibata et al. also showed that GPM6A regulates the opiate drug receptor during treatment of chronic stress and pain [20]. Recent studies reported that GPM6A is associated with various diseases. In our study, TCGA dataset results showed that GPM6A mRNA expression was obviously decreased in 15 types of tumors and increased in 18 types of tumors. Terefore, its role in tumor is ambiguous.
Liu et al. found low GPM6A expression in HCC, and GPM6A downregulation regulated the cell cycle and promoted the development of HCC by activating the AKT/ERK pathway [14]. Cai et al. analysed TCGA dataset and found that rectal cancer patients with low GPM6A expression had longer overall survival time [21]. However, studies on the relevance of GPM6A in lung cancer are limited. Jiang et al. reported that GPM6A was     Journal of Oncology 9 inhibited in small-cell lung cancer cells, which was regulated by miR-22 [15]. In the current study, GPM6A expression at the mRNA level was lower in lung adenocarcinoma than in normal or adjacent lung tissues.
Further function study showed that GPM6A inhibits the propagation and migration of lung adenocarcinoma cells. Tese fndings indicated that GPM6A acts as a tumor suppressor.  Te PI3Ks are a large family of lipid enzymes that phosphorylate the 3′-OH group of phosphatidylinositols on the plasma membrane [22]. AKT is one of the major downstream efectors of PI3K. Te signaling pathway participates in many aspects of cell growth and survival in physiological and pathological conditions [23]. Te PI3K/ AKT pathway is a key regulator of survival during cellular stress. As tumors exist in a stressful environment, this pathway plays a crucial role in the development of cancer [23]. Given that GPM6A is associated with stress, we detected PI3K, p-PI3K, AKT, and p-AKT expression at the protein level and found that GPM6A downregulation promoted the progression of lung adenocarcinoma cells. In contrast, GPM6A upregulation inhibited the progression of lung cancer. Although we detected the other typical tumor signaling pathways including MAPK, JAK/STAT3, and Wnt/β-catenin signaling pathways, it had no diferential expression between Lv-GPM6A and the empty vector. Terefore, GPM6A might suppress lung adenocarcinoma progression via inhibition of the PI3K/AKT pathway.
Tis study has some limitations. We did not investigate the function of GPM6A in PI3K inhibitor treatment. Te role of GPM6A inhibition in lung adenocarcinoma was also not observed in vivo. Tese will be addressed in our future research.

Conclusions
In conclusion, GPM6A acts as a tumor suppressor and inhibits the proliferation and migration of lung adenocarcinoma via inhibition of the PI3K/AKT pathway. Terefore, GPM6A could be a possible treatment target for lung cancer therapy.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that there are no conficts of interest regarding the publication of this paper.