Hypoxia-mediated YTHDF2 expression and activation of the mTOR/AKT axis in lung squamous cell carcinoma

N6-methyladenosine (m6A) is a dynamic and reversible internal RNA structure of eukaryotic mRNA. YTH domain family 2 (YTHDF2), an m6A-specic reader YTH domain family, plays fundamental roles in several types of cancer. However, the function of YTHDF2 in lung squamous cell carcinoma (LUSC) remains elusive. Functionally, NCI-H226 and SK-MES-1 cells were exposed to hypoxia to detect the protein levels of hypoxia-inducible factor-1α (HIF-1α), endogenous YTHDF2, and phospho-AKT (Ser473) analyzed by western blotting and then the association of these proteins with LUSC was analyzed with a bioinformatics database. Next, we established stable YTHDF2 upregulation models in NCI-H226 and SK-MES-1 cells to explore the function of YTHDF2 in LUSC cells by performing in vitro and in vivo assays. Finally, we armed that YTHDF2 overexpression was involved in activating the mTOR/AKT signaling and inducing the EMT process in LUSC using western blotting. Clinically, immunohistochemical staining revealed the relationship between YTHDF2 expression levels and the clinicopathological characteristics of lung squamous cell carcinoma patients. The results showed that hypoxia-mediated YTHDF2, a tumor promoter, promoted cell proliferation and invasion by activating the mTOR/AKT axis and inducing the EMT process in LUSC. Moreover, YTHDF2 was closely associated with pN (pN– 37.0%, pN + 73.9%; P = 0.002) and pTNM stage (pI 50.0%, PII 43.3%, pIIIa 80.6%; P = 0.007), ultimately resulting in poor survival for LUSC patients. Hypoxia specically induces YTHDF2 expression in LUSC cells. (A) and (B) NCI-H226 and SK-MES-1 cells were exposed with or without 24h hypoxia (1% O2, 5%CO2, 94% N2). The protein levels of HIF-1α, YTHDF2, and P-AKT (be phosphorylated at serine 473) were analyzed by western blot. Data are represented by the mean ± SD of three independent experiments. *P<0.05 vs. the vector group. Using the Pearson correlation statistics, we examine the pairwise gene correlation analysis between HIF-1α and AKT1 (C), HIF-1α and YTHDF2 (D), YTHDF2 and AKT1 (E) by TCGA and GTEx expression data of GEPIA.


Introduction
Worldwide, malignant tumors of the lung are the primary cause of cancer incidence and death, ranking as the highest tumor-related mortality with more than 1.8 million deaths in 2018 and accounting for almost 1 in 5 cancer deaths [1]. Lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) are the most common histological subtypes of non-small cell lung cancer (NSCLC), which accounts for almost 80%-85% of all human lung cancers. With the development of targeted drugs for speci c gene mutations, this treatment has recently greatly improved the clinical prognosis of advanced LUAD patients recently. In contrast, LUSC patients have a poor clinical prognosis and lack targeted agents compared to LUAD patients [2][3][4]. Therefore, there is an urgent need to search for new oncogenic drivers to inhibit the development and progression of LUSC patients.
The cellular response to hypoxia, followed by activation of hypoxia-inducible factor 1 (HIF-1), has been reported to be emerging as an important mechanism promoting tumor aggressiveness, metastasis, and poor prognosis [5]. N6-methyadenosine(m6A), the most prevalent modi cation of mRNA, is not only induced by hypoxia and promotes cancer progression, angiogenesis, and metastasis in several cancers [6,7], but is also widely involved in many biological processes, such as splicing and stability of mRNA, RNA nucleation, the interaction between RNA and protein, and protein translation [8][9][10]. To date, hypoxia, as an attractive therapeutic target, has not been successfully exploited in the lung [11].
Consequently, it is critical to further investigate the molecular mechanism of hypoxia exposure and the prognosis of lung squamous cell carcinoma.
More than 90% of cancer-related mortality is associated with cancer cell metastasis [25]. Epithelialmesenchymal transition (EMT) plays an important function in cell migration, invasion, and cancer progression, endowing cells with stem cell properties and contributing to immunosuppression [26].
Furthermore, Snail superfamily members, the prominent inducers in EMT, are very strongly implicated in tumor grade, recurrence, metastasis, and poor prognosis in various tumors types [27,28]. Recently, it has been reported that RNA epigenetic factors mediate EMT progression and the development of cancer [29].
For example, adverse prognosis in liver patients was associated with the coregulation of METTL3 and YTHDF1. Moreover, studies highlight the key role of m6A in EMT progression, cancer metastasis, and YTHDF1-mediated Snail translation [30]. The roles of m6A in the EMT process and Snail expression need to be further investigated in other cancers. Certainly, our ndings indicated that upregulation of YTHDF2 induces the EMT process, promotes cancer metastasis, and predicts a worse prognosis in LUSC patients.
It has been reported that YTHDF2 expression is promoted in multiple tumors including lung cancer patients [31][32][33]. In this study, we further explored the expression level and biological role of YTHDF2 in lung squamous cell cancer. Our data showed that YTHDF2 was mediated by hypoxia exposure and orchestrated proliferation and invasion in lung squamous cell cancer. Mechanistically, hypoxia-stimulated HIF-dependent upregulation of YTHDF2 resulted in the promotion of the EMT process and activation of the mTOR/AKT signaling pathway. Thus, we hope that this study can further explore the molecular mechanism of hypoxia exposure and provide potential therapeutic targets for LUSC.

Cell culture
In a humidi ed incubator (37℃, 5% CO2), NCI-H226 and SK-MES-1 cells from the American Tissue Culture Collection (ATCC), were cultured in DMEM medium (KeyGEN BioTECH, Nanjing, China), and 10% fetal bovine serum (Gibco, United Kingdom) and 1% penicillin-streptomycin solution (KeyGEN BioTECH, Nanjing, China) were added. Using shRNA lentiviral particles (GENECHEM, Shanghai, China) containing YTHDF2, two cell lines were infected according to the infection instructions to achieve the stable upregulation of exogenous YTHDF2. The upregulation of exogenous protein expression was detected by western blotting.

Cell proliferation assay
Cell Counting Kit 8 (CCK-8) was used to detect cell proliferation. A total of 2000 cells per well were plated in 96-well plates (Costar; USA). At the indicated time points, 10 μL of CCK-8 reagent (Dojindo) was added to the cells, and the cells were cultured in a humidi ed incubator (37℃, 5% CO2) for another 1 h. Then the optical density at A490 nm was measured by an enzyme-linked immunosorbent assay (ELISA) reader.

Migration and invasion assays
The wound-healing assay measured cell migration activity. NCI-H226 and SK-MES-1 cells (4 × 10 6 ) were inoculated into six-well plates. After 80% of the cells were fused, 1.3-mm-wide scratched wounds were washed with PBS, and then photographed at 0 and 24 hours. The transfected cells were plated in the upper chamber of Transwell Matrigel chambers (Collaborative Biomedical Products, USA) at 1.2 × 10 4 per chamber. We added serum-free medium and 10% serum-containing medium to the upper and lower layers of the chamber respectively. After 24 hours, the invasive cells were xed, stained, photographed, and quanti ed. The stained cells were counted in ve random elds at ×100 magni cation, and the average number was taken.

Animal experiments
The animal experiment was approved by the Animal Research Committee of Jinan Central Hospital A liated with Shandong University.
Thirty-ve-day-old male nude mice (athymic BALB/c-nu) were obtained from Shandong University (Jinan, China). The mice were randomly divided into two groups which were inoculated with stable YTHDF2expressing LUSC cells and the vector LUSC cells. A total of 5 × 10 6 of the cells were suspended in 0.1 ml of PBS and then injected subcutaneously into the anks of mice. Tumor size was measured twice a week.
The volume formula was: width 2 × length ×π/6. After 5 weeks, well-trained individuals performed physical methods of euthanasia: cervical dislocation on nude mice in a familiar and safe environment, then the xenograft tumor load was isolated, photographed, weighed, and xed in formalin to perform immunohistochemical staining of YTHDF2.

Tissue samples and immunohistology
From October 2008 to May 2013, 73 LUSC patients were included in this study at the Department of Jinan Central Hospital A liated with Shandong University. We included patients with LUSC diagnosed after complete surgical resection and postsurgical pathology. TNM staging was performed according to the 8th edition of the IASLC Lung Cancer Staging Project [34]. We have obtained informed consent to conduct experiments on human subjects. This study was approved by the Ethics Committee of Jinan Central Hospital A liated with Shandong University.
All the LUSC specimens and the adjacent normal lung tissue came from 73 patients. Tissue samples were xed with a 10% neutral formalin and treated routinely. After dewaxing, inactivation of endogenous peroxidase, and antigen repair, 5% BSA blocking solution was added for 30 minutes at 37 °C. Then, the sections were incubated with YTHDF2 antibody (1:200, Catalog #A02621-1, BOSTER, Wuhan, China) overnight at 4°C and secondary antibody (Ready-to-use SABC-POD (rabbit IgG) Kit, Catalog # SA1022, BOSTER, Wuhan, China) at 37°C for 30 minutes. The specimens were developed with diaminobenzidine (DAB) and stained with Mayor's hematoxylin at 37°C for 1 minute. Finally, the specimens were analyzed by ImageScope software (Leica) and histochemistry scores were obtained. The median histochemistry score was used to divided patients into two groups.

Statistical analysis
The experimental data were analyzed by Student's t-test, the chi-squared test, or Fisher's exact probability test. GraphPad Prism 8.3.0 and the IBM SPSS Statistics 25 were used to perform statistical analyses.
Kaplan-Meier survival curves were used in univariate analysis and a Cox predictive risk model was used in multivariate analysis. All the data came from three independent experiments, in triplicate. Moreover, P values less than 0.05 were considered statistically signi cant.

Results
Hypoxia speci cally induces YTHDF2 expression in LUSC cells.
Hypoxia induces hypoxia-inducible factor-1α (HIF-1α), which is mediated by a proline hydroxylase and has emerged as a crucial factor. Moreover, hypoxia is associated with poor prognosis and resistance to radiation and chemotherapy [35].
First, we investigated whether hypoxic exposure affected YTHDF2 expression in LUSC cells. NCI-H226 and SK-MES-1 cells were exposed to 1% oxygen for 24 hours. The expression of HIF-1α was used to validate the hypoxic response. We found that endogenous YTHDF2 expression was increased approximately 2-or 3-fold at 24 h after hypoxia exposure, respectively. Interestingly, the level of AKT that was phosphorylated at serine 473 was increased approximately 3-or 11-fold at 24 h following hypoxia ( Fig. 1A and 1B). Meanwhile, we employed bioinformatics-based screening to explore the association of HIF-1α, YTHDF2, and AKT1 in human lungs. LUSC data mining of the GEPIA http://gepia.cancer-pku.cn/ that HIF-1α expression was correlated with AKT1 and YTHDF2 ( Fig. 1C and D). As expected, the results presented a statistically positive correlation between the expression of YTHDF2 and AKT1 (Fig. 1E). In summary, the protein level of YTHDF2 in LUSC cells was speci cally increased under hypoxia, and YTHDF2 mediated activation of the mTOR/AKT signaling pathway.
We established stable YTHDF2 upregulation models in NCI-H226 and SK-MES-1 cells to explore the biological function of YTHDF2 in LUSC. Successful overexpression of YTHDF2 was con rmed at the protein level ( Fig. 2A and 2B). The CCK-8 assay was assessed cellular viability. As shown in Fig. 2C and Fig. 2D, cellular viability dramatically increased in the YTHDF2 group compared with that of the cells carrying the vector only. Moreover, to explore the effects of YTHDF2 upregulation on cell motility, we examined the invasion potential of NCI-H226 and SK-MES-1 cells by using a Transwell assay. As expected, YTHDF2 overexpression dramatically promoted LUSC cell invasion abilities (Fig. 2E and Fig.  2F). Next, the wound-healing assay showed that overexpression of YTHDF2 dramatically strengthened the migratory capabilities of LUSC cell lines (Fig. 2G and Fig. 2H). Moreover, a subcutaneous implantation experiment in nude mice was performed to investigate the oncogenic function of YTHDF2 in LUSC. Compared to those bearing the vector only, we showed that stable upregulation of YTHDF2 markedly promoted tumor growth in nude mice, as demonstrated by the signi cant increase in tumor size and weight (Fig. 3A, 3B, 3C, 3D, 3E, and 3F). In the immunohistochemical results, the YTHDF2 expression levels of the xenograft tumors using the indicated stable cells were higher than those carrying the vector only ( Fig. 3G and Fig. 3H). Hence, our data suggested that YTHDF2 plays a critical role in promoting LUSC proliferation and invasion.
YTHDF2 facilitates the mTOR/AKT signaling cascades and induces EMT in LUSC cells.
We investigated whether YTHDF2 overexpression activates those pivotal signaling pathways in LUSC cells, such as the ERK/MAPK and mTOR/AKT signaling pathways which are known to function in tumor proliferation and survival. The results showed that the phosphorylation of both AKT and mTOR was markedly increased in the YTHDF2 group compared with that of the cells carrying the vector only, but not of ERK (Fig. 4A). Furthermore, YTHDF2 expression was positively correlated with AKT and mTOR in LUSC according to the GEPIA http://gepia.cancer-pku.cn/ (Fig. 1E and 4C). Taken together, YTHDF2 upregulation promotes cell proliferation by activating the mTOR/AKT pathway rather than the ERK/MAPK pathway in LUSC.
Meanwhile, phosphorylation of mTOR and AKT by YTHDF2 promotes its activity, stabilizing Snail1 to repress the expression of E-cadherin. As a tumor suppressor, the E-cadherin change elicited by YTHDF2 upregulation was in line with the activation of the mTOR/AKT pathway. Speci cally, the downregulation of E-cadherin was accompanied by the upregulation of N-cadherin in the EMT process, which altered cell adhesion (Fig. 4B). Therefore, our study indicated that the upregulation of YTHDF2 induced the EMT process, and promoted cancer metastasis in LUSC cells.
YTHDF2, as a tumor promoter, may lead to a poor prognosis for LUSC patients.
Recently, an increasing number of studies have examined the correlation analysis between writer proteins and reader proteins [36,37]. Our data suggested that the overexpression of YTHDF2 directly affected the expression level of METTL14 at the protein level (Fig. 5A). Using the GEPIA http://gepia.cancer-pku.cn/ , we also found a marked correlation between the expression of YTHDF2 and METTL14, but not METTL3 in LUSC (Fig. 5B and 5C). Therefore, we speculated that YTHDF2 cooperating with METTL14 may be involved in LUSC.
Using the GEPIA http://gepia.cancer-pku.cn/ to detect the overall survival (OS) and the disease-free survival (DFS) of genes (YTHDF2, Snail, METTL14), the results showed that LUSC patients at the mRNA level with increased expression of Snail had poor DFS and OS, but all not of YTHDF2, while the upregulation of METTL14 only lessened DFS (Fig. 5D). In the immunohistochemical staining results, YTHDF2 protein expression levels in lung squamous cell carcinoma tissues were markedly higher than those in the corresponding normal lung tissues (Table 1 and Fig. S1). We further explored the correlation between YTHDF2 expression and clinicopathological characteristics. The 5-year survival rate of the 73 LUSC patients accounted for 39.7%. Table 2 shows that the YTHDF2 upregulation was markedly correlated with pN (pN-37.0%, pN+ 73.9%; P=0.002) and pTNM stage (pI 50.0%, pII 43.3%, pIIIa 80.6%; P=0.007). According to the log-rank test with univariate analysis, the 5-year survival rate of LUSC patients was closely related to pN (P=0.001), pTNM stage (P=0.01), and high expression of YTHDF2 (P=0.002, Table 3). Ultimately, Cox regression with multiple analyses showed that pN and YTHDF2 expression acted as independent factors affecting the 5-year survival rate (Table 4). Consistently, these data showed that YTHDF2, which induced the high expression of METTL14 and Snail, may lead to a worse prognosis for LUSC patients.

Discussion
In this study, we suggested that YTHDF2 upregulation was signi cantly induced by hypoxia in LUSC cells. Overexpression of YTHDF2 positively activated the mTOR/AKT pathway and regulated the progression of EMT which may act as a tumor promoter to induce LUSC cell proliferation and invasion.
Generally, it has been reported that m6A modi cation is associated with tumorigenesis. A better explanation of the molecular mechanisms of a complete m6A modi cation process requires the cooperation of m6A writer genes, erasers genes, and readers genes, rather than a single isolated gene. For example, m6A modi cation mediated by the cooperation of METTL14, ALKBH5, and YTHDF3 was reported to in uence the cell cycle, induce the progression of EMT, and contribute to angiogenesis of cancer cells in breast cancer [37]. YTHDF1-mediated the translation of Snail was veri ed, as a portion of EMT was altered by deletion of METTL3 in liver patients [30]. In bladder cancer, the mutual interaction between METTL3 and YTHDF2 induced the degradation of SETD7 and KLF4 mRNA in the proliferation and metastasis process [32]. Moreover, the methyltransferase METTL3 was discovered to regulate the degradation of SOCS2 mRNA, enhancing the progression of liver cancer in a YTHDF2-mediated m6Adependent manner [36]. Here, we also demonstrated that YTHDF2 was positively related to METTL14 and cooperated with METTL14 in LUSC. However, how this mutual collaboration between METTL14 and YTHDF2 activates the oncogenic signaling pathway in LUSC is largely unknown. We will address the unknown mechanism in our next study.
In our study, the YTHDF2 protein expression level was markedly higher in human LUSC tissues than in normal tissues. Cell proliferation was signi cantly enhanced in YTHDF2-overexpression cells compared with control cells. Moreover, YTHDF2 upregulation promoted tumor growth and increased tumor volume in vivo compared with control cells. The mechanism underlying YTHDF2-mediated LUSC tumorigenesis was also investigated. The results indicated that compared with control cells, AKT and mTOR phosphorylation was signi cantly increased following YTHDF2 overexpression, which is crucial for tumor progression. Based on the results, it was hypothesized that YTHDF2 may promote LUSC cell proliferation by activating the AKT/mTOR signaling pathway which is known to play a pivotal role in multiple types of cancer, such as breast cancer and ovarian carcinoma [38,39]. To the best of our knowledge, the present study is the rst study to demonstrate the role of YTHDF2 in LUSC cell proliferation. However, our present study failed to analyze AKT and mTOR expression levels by immunohistochemistry.
Our study was not without limits: for instance, there are reports that YTHDF2 regulates m6A-modi ed mRNA degradation, which is seemingly contrary to our study. One possible explanation is that YTHDF2 decays the m6A-modi ed mRNA of tumor suppressor genes, thus promoting cell growth. Another possible explanation is that YTHDF2-mediated mRNA decay might not be the only mechanism underlying m6A function in cancer progression. In addition, one report suggests that PI3K-AKT signaling is involved in promoting the EMT process via the mTOR or MAPK cascade [40], and another report indicates that the m6A modi cation is associated with the EMT progression and cancer metastasis that is induced by YTHDF1-mediated Snail translation in liver patients [38]. Therefore, the existence of these two signaling axes remains to be veri ed in LUSC, and whether m6A mediates E-cadherin expression via other factors/pathways also deserves further exploration. In future studies, we will contribute to addressing these unresolved limitations.
In summary, the present study indicated that YTHDF2 was involved in mediating LUSC cell proliferation and invasion. These results may improve the current understanding of the mechanism underlying the biological role of YTHDF2 during tumor development and might provide a potential therapeutic target for LUSC. All data are included in this article.

Con icts of interest
All authors declare no con ict of interest.

Funding
The present study was funded by Shandong provincial Natural Science Foundation (grant no. ZR2020MH201).
Authors' contributions Nan Zhang and Zhi-Gang Sun designed this work. Peng Xu wrote the manuscript and prepared the gures and tables. Kang Hu drafted and revised the manuscript. All the authors contributed to manuscript revision, read and approved the submitted version.

Acknowledgments
None Tables    Figure 1 Hypoxia speci cally induces YTHDF2 expression in LUSC cells. (A) and (B) NCI-H226 and SK-MES-1 cells were exposed with or without 24h hypoxia (1% O2, 5%CO2, 94% N2). The protein levels of HIF-1α, YTHDF2, and P-AKT (be phosphorylated at serine 473) were analyzed by western blot. Data are represented by the mean ± SD of three independent experiments. *P<0.05 vs. the vector group. Using the Pearson correlation statistics, we examine the pairwise gene correlation analysis between HIF-1α and AKT1 (C), HIF-1α and YTHDF2 (D), YTHDF2 and AKT1 (E) by TCGA and GTEx expression data of GEPIA.