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Table of Contents
ORIGINAL ARTICLE
Year : 2022  |  Volume : 65  |  Issue : 5  |  Page : 266-275

Forkhead Box S1 inhibits the progression of lung squamous cell carcinoma cells by mediating Wnt/β-catenin pathway


1 Department of Pulmonary and Critical Care Medicine, Edong Healthcare Huangshi Central Hospital, Huangshi, Hubei, China
2 Department of Head and Neck Oncology, Edong Healthcare Huangshi Central Hospital, Huangshi, Hubei, China

Date of Submission18-May-2022
Date of Decision06-Jul-2022
Date of Acceptance19-Jul-2022
Date of Web Publication27-Oct-2022

Correspondence Address:
Dr. Song Li
Department of Head and Neck Oncology, Edong Healthcare Huangshi Central Hospital, No. 293 Hospital Street, Xisaishan District, Huangshi, Hubei Province
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0304-4920.359799

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  Abstract 


Lung squamous cell carcinoma (SCC) is considered the frequent subtype of non-small cell lung cancer (NSCLC) and results in high mortality worldwide every year. Forkhead box S1 (FOXS1) is correlated to multiple cancers, but the role and the mechanism of FOXS1 in lung SCC are unclear. This study revealed that FOXS1 was low expressed in the lung SCC tissues by utilizing UALCAN and TIMER databases. Western blotting analysis was introduced to estimate the FOXS1 expression in the lung SCC cells. Functionally, overexpression of FOXS1 dramatically inhibited cell proliferation, migration, invasion, and epithelial–mesenchymal transition in the lung SCC cells. However, knockdown of FOXS1 exerted diverse effects on lung SCC cell progression. Moreover, FOXS1 overexpression suppressed tumor growth in nude mice remarkably. Furthermore, FOXS1 overexpression reduced the activity of Wnt/β-catenin signal, while FOXS1 silence reversed the roles notably. In conclusion, our present study proved that FOXS1 inhibited lung SCC development in vitro and in vivo might by modulating Wnt/β-catenin signaling pathway.

Keywords: Epithelial–mesenchymal transition, forkhead box S1, lung squamous cell carcinoma, migration, Wnt/β-catenin signaling pathway


How to cite this article:
Wang F, Li S. Forkhead Box S1 inhibits the progression of lung squamous cell carcinoma cells by mediating Wnt/β-catenin pathway. Chin J Physiol 2022;65:266-75

How to cite this URL:
Wang F, Li S. Forkhead Box S1 inhibits the progression of lung squamous cell carcinoma cells by mediating Wnt/β-catenin pathway. Chin J Physiol [serial online] 2022 [cited 2022 Nov 26];65:266-75. Available from: https://www.cjphysiology.org/text.asp?2022/65/5/266/359799




  Introduction Top


Lung cancer is the second most frequent malignancy in the world after breast cancer, and it has the highest mortality among all cancers.[1],[2],[3] Non-small cell lung cancer (NSCLC) is the most common subtype of lung cancer, accounting for approximately 85%. Patients with NSCLC are usually diagnosed at an advanced stage, leaving a 5-year survival time of 4%–17%.[1],[4] In addition, lung squamous cell carcinoma (SCC), an important subtype of NSCLC, accounts for about 35% of patients with NSCLC and is responsible for no less than 400,000 deaths worldwide each year.[5] Previous studies discovered that the aberrant expression of various tumor-related genes is tightly associated with the occurrence and development of lung SCC.[6],[7],[8],[9] Thus, it is crucial to elucidate the molecular mechanism of lung SCC to optimize the therapeutic schedule.

Forkhead box (FOX) superfamily is a vital group of transcription factors that regulate the expression of a wide variety of genes. Abnormal function of members in the FOX superfamily is involved in various cancers as oncogenes or anti-oncogenes.[10] For example, FOXO3a is considered a suppressor in the development of breast cancer stem cells,[11] whereas FOXM1 accelerates the epithelial–mesenchymal transition (EMT) and metastasis in pancreatic cancer.[12] FOXS1, the member of FOX superfamily, has been found to exert essential effects on diverse malignancies.[13] For example, Lei et al. revealed that FOXS1 suppresses cell proliferation, colony formation, and EMT progress in hepatocellular carcinoma.[13] Diao et al. verified that knockdown of FOXS1 facilitates the viability of rhabdomyosarcoma Rh36 cells and medulloblastoma Daoy cells.[14] Lu et al. revealed that FOXS1 overexpression suppresses the proliferation and metastasis of gastric cancer in vitro and in vivo.[15] However, the role and the underlying mechanism of FOXS1 in lung SCC are still uncovered.

It has been reported that Wnt/β-catenin signaling pathway is tightly correlated to the tumorigenesis of multiple cancers such as ovarian cancer,[16] endometrial carcinoma,[17] and cervical cancer.[18] Besides, increasing evidence proved that overactivation of Wnt/β-catenin pathway leads to lung cancer.[19],[20] Previous investigations confirmed that Wnt/β-catenin signal is related to NSCLC cell progression including cell proliferation, apoptosis, differentiation, and metastasis.[21] EMT as vital progress contributes to the lack of cellular polarity and adhesion, thereby resulting in cell metastasis and tumorigenesis. In addition, it is reported that Wnt/β-catenin signaling pathway can induce EMT progress.[22],[23] Moreover, Lu et al. revealed that FOXS1 exerts its role by mediating Wnt/β-catenin pathway.[15] Nevertheless, whether FOXS1 regulates Wnt/β-catenin signaling pathway in lung SCC is still unclear.

This work, for the first time, revealed that FOXS1 expression was decreased in lung SCC cells and promotes lung SCC cell progression in vivo and in vitro at least partly by modulating Wnt/β-catenin signaling pathway.


  Materials and Methods Top


Database to analyze forkhead box S1 expression in lung squamous cell carcinoma

The UALCAN database (http://ualcan.path.uab.edu/) and TIMER database (https://cistrome.shinyapps.io/timer/) were utilized to predict the content of FOXS1 in the lung SCC tissues and paracarcinoma tissues.

Cell culture

Four human lung SCC cell lines (NCI-H520, NCI-H2170, SK-MES-1, and LTEP-s) and epithelial cell line BEAS-2B were obtained from MINGZHOUBIO, China. NCI-H520, NCI-H2170, and LTEP-s cell lines were maintained in Roswell Park Memorial Institute-1640 medium (GNM31800-S-5, GENOM, China) containing penicillin as well as streptomycin and 10% fetal bovine serum (FBS, SV30087.03, HyClone, USA). SK-MES-1 and BEAS-2B cell lines were kept in Dulbecco's modified Eagle medium (GNM12800-S-5, GENOM, China) containing penicillin as well as streptomycin and 10% FBS (SV30087.03, HyClone, USA). All cells were cultured in an incubator containing CO2 (BPN-40CRH, Yiheng Instruments, China) at 37°C.

Cell transfection

FOXS1 short-interfering RNA (si-FOXS1) and blank siRNA (si-NC) were obtained from GenePharma, China. PcDNA-FOXS1 and blank plasmid pcDNA were purchased from FENGHUI, China. The vectors were transfected into lung SCC cells when the cell density reached 50% by utilizing Lipofectamine 2000 reagent (11668-019, Invitrogen, USA) abiding by the kit's protocol. The targeted sequences of si-FOXS1 are listed here: forward - 5'-ACUCAAAGAAGAACAUUCCUG-3', reverse - 5'-GGAAUGUUCUUCUUUGAGUGA-3'.

Stable cell line construction

Stable FOXS1-overexpression cells were constructed as described in previous study.[24] Briefly, Lenti-NC or Lenti-FOXS1 brought from HANBIO (China) was transfected into HEK-293T cells for 48 h followed by harvesting the medium. Then, the supernatant medium was utilized to infect NCI-H520 or SK-MES-1 cells (1 × 105 cells/well) for another 72 h. Next, 4 μg/mL puromycin (ST551, Beyotime, China) was employed to screen the positive cells.

Western blotting analysis

NCI-H520 or SK-MES-1 cell lysates were harvested in 150 μL sodium dodecyl sulfate (SDS) lysis buffer (AR0095, Boster Biological Technology Co., Ltd., China). The protein concentration was measured by employing BCA Protein Assay Kit (P0012S, Beyotime, China). Next, total proteins were isolated using SDS polyacrylamide gel electrophoresis and were transferred to 0.45 μm polyvinylidene fluoride membrane (WJ002S, Shanghai Epizyme Biomedical Technology Co., Ltd., China). Subsequently, the membranes were blocked with 5% bovine serum albumin (BSA) Blocking Buffer (SW3015, Solarbio, China). Primary antibody targeted specific protein FOXS1 (HPA042475, MilliporeSigma, USA, 1:1000), E-cadherin (20874-1-AP, Proteintech, USA, 1:1000), N-cadherin (22018-1-AP, Proteintech, USA, 1:1000), vimentin (10366-1-AP, Proteintech, USA, 1:1000), alpha-smooth muscle actin (α-SMA) (55135-1-AP, Proteintech, USA, 1:1000), β-catenin (51067-2-AP, Proteintech, USA, 1:1000), APC (2504, Cell Signaling Technology, USA, 1:1000), c-Myc (sc-40, Santa Cruz Biotechnology, USA, 1:1000), MMP-7 (10374-2-AP, Proteintech, USA, 1:1000), or GAPDH (60004-1-Ig, Proteintech, USA, 1:1000) was employed for probing at 4°C overnight. Finally, the corresponding secondary antibody and ECL Western Blotting Substrate (PE0010, Solarbio, China) were introduced to visualize the protein bands. Besides, the relative expression level of specific proteins was determined by utilizing ImageJ software (National Institutes of Health, Bethesda, MD, USA), and the level of GAPDH serves as the internal control.

Cell counting kit-8 assay

Cell counting kit-8 (CCK-8) was employed to estimate cell growth following the manufacturer's instructions. In brief, NCI-H520 and SK-MES-1 cells were seeded in a 96-well plate (1000 cells/well) overnight. Next, the medium was replaced with 90 μL medium with 10 μL CCK-8 reagent and the cells were cultured at 37°C for another 1 h. Finally, a microplate reader was employed to determine the cell viability by recording the value of optical density at 450 nm.

Colony formation assay

600 NCI-H520 or SK-MES-1 cells were seeded in six-well plates for 2 weeks with renewing medium every 3 days. Then, the medium was removed and the cells were fixed and stained using Image-iT (R37814, Invitrogen, USA) and Image-iT (R37814, Invitrogen, USA), respectively. Finally, cell numbers were counted by utilizing an inverted fluorescence microscope (YKDZ-80, Shanghai Yongke Optical Instrument Co., Ltd., China).

Transwell assay

Transwell chamber precoating Matrigel (CLS3493, Corning, USA) was utilized to detect the effect of FOXS1 on cell invasive capacities. NCI-H520 or SK-MES-1 cells in 600 μL serum-free medium were seeded in the upper chamber, while 600 μL complete medium was added into the lower chamber for 24–48 h. Next, the cells invading the lower chamber were fixed with Image-iT (R37814, Invitrogen, USA) and stained with Image-iT (R37814, Invitrogen, USA). Finally, the positive cells were counted by utilizing an inverted fluorescence microscope (YKDZ-80, Shanghai Yongke Optical Instrument Co., Ltd., China).

Wound healing assay

NCI-H520 or SK-MES-1 cells were seeded in a six-well plate (1 × 105 cells/well) and were transfected with indicated vectors. Then, the cell monolayer was treated with 200 μL sterile tip to produce a horizontal scratch. Next, cells cultured at 37°C in an incubator were photographed (0 h and 24 h) with an inverted fluorescence microscope (YKDZ-80, Shanghai Yongke Optical Instrument Co., Ltd., China) to analyze the wound healing of the lung SCC cells.

Tumor growth

All BALB/c nude mice (6 weeks old) were obtained from Trophic Animal Feed High-Tech Co., Ltd., China, and were raised under sterile conditions. Then, the mice were randomly divided into two groups of five and were inoculated subcutaneously with SK-MES-1 cells (1 × 107) carrying Lenti-NC or Lenti-FOXS1. Tumor size (length and width) was recorded every 7 days. After 35 days, all tumors were isolated from nude mice and were weighted and measured. In addition, tumor volume = l (length) × w (width)2/2. This experiment was permitted by the Ethical Committee of Edong Healthcare Huangshi Central Hospital (Approval Number 2021-012).

Immunohistochemistry staining

Immunohistochemistry (IHC) staining was conducted as introduced in previous investigation.[25] In brief, the isolated tumor tissues were fixed using a formalin-free tissue fixator (A5472, Sigma-Aldrich, USA) for no less than 24 h, and then the tissues were embedded into Sakura Tissue-Tek® OCT Compound (4583, Sakura, USA). Next, the tissues were cut into 10-μm slices and were treated in a microwave oven to extract the antigen. Then, the slices were incubated with 10% H2O2 (323381, Sigma-Aldrich, USA) for 5 min and were treated with goat serum (C0265, Beyotime, China). Subsequently, the slices were treated with primary antibody FOXS1 (HPA042475, MilliporeSigma, USA, 1:1000) or ki-67 (ab15580, Abcam, UK) overnight at 4°C. Finally, the positive cells were exhibited by an enhanced DAB chromogenic kit (DA1016, Solarbio, China) according to its manufacturer's instruction and were photographed using an Olympus microscope (CX31, Olympus, Japan).

Statistics

All data were analyzed with SPSS 22.0 software (Statistical package for the social science, IBM, Chicago, IL, USA). The comparison was carried out using Student's t-test and analysis of variance followed by Tukey's test based on the data type. ***P < 0.001, **P < 0.01, *P < 0.05, &&&P < 0.001 and &&P < 0.01 were considered statistically significant. All data were obtained from no less than 3 experiments.


  Results Top


FOXS1 is downregulated in the lung SCC tissues and cell lines and inhibits lung SCC cell progression

The data obtained from UALCAN database (http://ualcan.path.uab.edu/) and TIMER database (https://cistrome.shinyapps.io/timer/) illustrated that FOXS1 expression was decreased in the lung SCC tissues compared with paracarcinoma tissues [Figure 1]a. In addition, FOXS1 expression in the human lung SCC cell lines (NCI-H520, NCI-H2170, SK-MES-1, and LTEP-s) was dramatically lower than that in the pulmonary epithelial cell line BEAS-2B [Figure 1]b. Then, we overexpressed or knocked-down FOXS1 in NCI-H520 or SK-MES-1 cell lines to perform subsequent assays. Western blotting analysis demonstrated that FOXS1 expression was increased in pcDNA-FOXS1 group compared to pcDNA group and was reduced in si-FOXS1 group compared to si-NC group [Figure 1]c and [Figure 1]d. CCK-8 assay indicated that FOXS1 overexpression decreased cell viability, while FOXS1 knockdown enhanced cell viability in the NCI-H520 and SK-MES-1 cells remarkably [Figure 1]e. Besides, colony formation assay further proved that cell colony-forming abilities were notably weakened by overexpression of FOXS1 and increased by knockdown of FOXS1 in NCI-H520 and SK-MES-1 cells [Figure 1]f. Collectively, these findings suggested that FOXS1 suppresses lung SCC cell proliferation.
Figure 1: FOXS1 is downregulated in the lung SCC tissues and cell lines and inhibits lung SCC cells progression. (a) The mRNA level of FOXS1 analyzed by UALCAN and TIMER databases. **P < 0.01 versus normal group. (b) The protein level of FOXS1 was low expressed in human lung SCC cell lines (NCI-H520, NCI-H2170, SK-MES-1, and LTEP-s) compared with pulmonary epithelial cell line (BEAS-2B) detected via western blotting analysis. ***P < 0.001, **P < 0.01, *P < 0.05 versus BEAS-2B group. (c) The protein level of FOXS1 was increased by pcDNA-FOXS1 while was decreased by si-FOXS1 in NCI-H520 and SK-MES-1 cell lines detected via western blotting analysis. (d) The quantitative analysis of FOXS1 protein expression level in NCI-H520 and SK-MES-1 cell lines. **P < 0.01 versus pcDNA group. &&P < 0.01 versus si-NC group. (e) Overexpression of FOXS1 inhibited cell growth while knockdown of FOXS1 increased cell viability at 24, 48, and 72 h in NCI-H520 and SK-MES-1 cell lines according to CCK-8 assay. **P < 0.01 versus pcDNA group. &&P < 0.01 versus si-NC group. (f) Overexpression of FOXS1 suppressed cell cloning ability while knockdown of FOXS1 enhanced that in NCI-H520 and SK-MES-1 cell lines detected via colony formation assay. **P < 0.01 versus pcDNA group. &&P < 0.01 versus si-NC group. All data were obtained from no less than 3 experiments, FOXS1: Forkhead box S1, CCK-8: Cell counting kit-8, SCC: Squamous cell carcinoma.

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Forkhead box S1 inhibits lung squamous cell carcinoma cell migration and invasion

Transwell in addition to wound healing assays was employed to explore the roles of FOXS1 in migration and invasion of the lung SCC cells. As presented in [Figure 2]a, the invasive capacity of NCI-H520 and SK-MES-1 cell lines was impaired in pcDNA-FOXS1 group compared with pcDNA group while was enhanced in si-FOXS1 group compared with si-NC group [Figure 2]b. Similar, FOXS1 overexpression inhibited cell migration, whereas FOXS1 silence promoted cell migration in NCI-H520 and SK-MES-1 cell lines, as exhibited in [Figure 2]b. Taken together, these results proved that FOXS1 inhibited the migration and invasion of the lung SCC cells.
Figure 2: FOXS1 inhibits lung SCC cell migration and invasion. (a) FOXS1 overexpression inhibited cell invasion while FOXS1 knockdown promoted that in NCI-H520 and SK-MES-1 cell lines measured through transwell assay. **P < 0.01 versus pcDNA group. &&P < 0.01 versus si-NC group. (b) FOXS1 overexpression inhibited cell migration while FOXS1 knockdown promoted that in NCI-H520 and SK-MES-1 cell lines measured through wound healing assay. **P < 0.01 versus pcDNA group. &&P < 0.01 versus si-NC group. All data were obtained from no less than 3 experiments. FOXS1: Forkhead box S1, CCK-8: Cell counting kit-8, SCC: Squamous cell carcinoma.

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Forkhead box S1 inhibits epithelial–mesenchymal transition progress in lung squamous cell carcinoma cells

Given that EMT exerts a vital role in the metastasis of cancer cells,[26] the expression level of EMT-related proteins (E-cadherin, N-cadherin, vimentin, and α-SMA) was detected to verify the effects of FOXS1 on EMT progress in the lung SCC cells. The data showed that the overexpression of FOXS1 increased the protein level of E-cadherin but reduced the expression of N-cadherin, vimentin, and α-SMA in the NCI-H520 and SK-MES-1 cell lines remarkably. However, knockdown of FOXS1 exerted opposite effects obviously [Figure 3]. These findings concluded that FOXS1 suppressed EMT in the lung SCC cells.
Figure 3: FOXS1 inhibits EMT progress in lung SCC cells. FOXS1 overexpression increased E-cadherin expression and decreased the expression level of N-cadherin, vimentin, and α-SMA, whereas FOXS1 knockdown exerted opposite effects on NCI-H520 and SK-MES-1 cell lines detected via western blotting analysis. **P < 0.01, *P < 0.05 versus pcDNA group. &&P < 0.01 versus si-NC group. All data were obtained from no less than 3 experiments, FOXS1: Forkhead box S1, EMT: Epithelial-mesenchymal transition, α-SMA: Alpha-smooth muscle actin.

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Forkhead box S1 inhibits Wnt/β-catenin signaling pathway in lung squamous cell carcinoma cells

To explore whether FOXS1 regulates Wnt/β-catenin signaling pathway in the lung SCC cells, we measured the content of important proteins related to Wnt/β-catenin pathway (β-catenin, APC, c-Myc, and MMP-7). The results illustrated that the overexpression of FOXS1 reduced the content of β-catenin, c-Myc, and MMP-7 while increased APC expression. However, knockdown of FOXS1 exhibited adverse results [Figure 4]. These results indicated that FOXS1 suppressed Wnt/β-catenin signaling pathway in the lung SCC cells.
Figure 4: FOXS1 inhibits Wnt/β-catenin signaling pathway in lung SCC cells. FOXS1 overexpression increased APC expression and decreased the expression level of β-catenin, c-Myc, and MMP-7, whereas FOXS1 knockdown exerted opposite effects on NCI-H520 and SK-MES-1 cell lines detected via western blotting analysis. ***P < 0.001, **P < 0.01 versus pcDNA group. &&&P < 0.001, &&P < 0.01 versus si-NC group. All data were obtained from no less than 3 experiments. FOXS1: Forkhead box S1, SCC: Squamous cell carcinoma.

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Forkhead box S1 inhibits tumor growth in vivo

To further verify the suppressive role of FOXS1 in lung SCC in vivo, we injected SK-MES-1 cells carrying Lenti-FOXS1 or Lenti-NC into nude mice. As exhibited in [Figure 5]a, Lenti-FOXS1 reduced the size, volume, and weight of tumor compared with Lenti-NC group. IHC assay demonstrated that the higher level of FOXS1 contributed to the less positive cells of ki-67 notably [Figure 5]b. Therefore, these results confirmed that FOXS1 suppressed tumor growth in vivo.
Figure 5: FOXS1 inhibits tumor growth in vivo. (a) The size, volume, and weight of each tumor were reduced by overexpression of FOXS1. ***P < 0.001, **P < 0.01 versus Lenti-NC group. (b) Ki-67 positive cells were decreased by overexpression of FOXS1 detected via IHC assay. All data were obtained from no less than 3 experiments. FOXS1: Forkhead box S1.

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  Discussion Top


This study uncovered that FOXS1 was low expressed in the lung SCC tissues and cell lines compared with paracarcinoma tissues and pulmonary epithelial cell. Besides, the overexpression of FOXS1 inhibited cell proliferation, colony formation, migration, invasion, and EMT progress in NCI-H520 and SK-MES-1 cell lines. However, FOXS1 silence exerted opposite effects on the development of the lung SCC cells. In addition, FOXS1 overexpression blocked Wnt/β-catenin signaling pathway, whereas FOXS1 knockdown activated this pathway effectively. Furthermore, increased FOXS1 notably facilitated lung SCC cell growth in mice.

Although antineoplastic treatments have improved as time goes by, the mortality rates of lung SCC remained high.[27] Early diagnosis of lung SCC patients is a challenge due to the absence of clinical symptoms. Besides, few effective therapeutic targeted genes were identified and the markers regarding prognosis were lacking in lung SCC.[28],[29] Hence, we aimed to explore the effective prognostic biomarkers and therapeutic targets of lung SCC for improving clinical outcomes.

Increasing evidence illustrated that FOX superfamily is closely associated with various biological functions including tumorigenesis.[10],[30] FOXS1, the essential member of FOX superfamily, was reported by Cederberg et al. in 1997 and presented distinct expression pattern.[31] FOXS1 has been revealed to be mainly expressed in the central nervous system, especially in the internal cerebellum particles.[32] Functionally, previous findings have demonstrated that FOXS1 is capable of regulating energy conversion and testicular vascular diseases.[32],[33] In addition, it has been reported that FOXS1 serves as a tumor suppressive gene in several malignancies such as gastric cancer[15] and liver cancer.[13] In this investigation, we discovered that FOXS1 was downregulated in the lung SCC tissues and cells obviously, implying that FOXS1 might be correlated to lung SCC progression. To our knowledge, this work is the first to elucidate the role of FOXS1 in lung SCC. Further assays verified that the overexpression of FOXS1 suppressed cell viability and colony formation capacities, while knockdown of FOXS1 exerted opposite effects, which confirmed that FOXS1 inhibited cell proliferation. Besides, consistent with recent studies,[15] FOXS1 overexpression impaired cell migratory and invasive abilities in the lung SCC cells, whereas FOXS1 knockdown reversed these results.

EMT is considered the crucial progress involved in cancer progressions including tumorigenesis and metastasis, which is usually characterized by the lack of epithelial features and the gain of mesenchymal features.[34],[35] Tumor cells employ EMT as a transitional form to adapt to the microenvironment in vivo.[36] Moreover, EMT is accompanied by the decrease of epithelioid proteins (e.g., E-cadherin) and the increase of mesenchymal proteins (e.g., N-cadherin and vimentin).[34] Besides, α-SMA is reported to be the vital protein to monitor EMT progress.[37] Interestingly, our present work proved that the overexpression of FOXS1 inhibited EMT progress by increasing E-cadherin expression and decreasing the expression of N-cadherin, vimentin, and α-SMA; however, FOXS1 silence still presented adverse effects on EMT progress.

Subsequently, we further explore the underlying signaling pathway by which FOXS1 regulated in lung SCC progression. It is reported that Wnt/β-catenin pathway is associated with lung SCC development.[38],[39] The aberrant activation Wnt/β-catenin pathway affected cell proliferation, metastasis, and EMT in various cancers.[40] In addition, previous study has illustrated that FOXS1 effectively regulated Wnt/β-catenin signal in gastric cancer.[15] Therefore, we investigated whether FOXS1 played its role in lung SCC cells by mediating Wnt/β-catenin pathway. It is well known that the abnormal transduction of Wnt/β-catenin pathway is featured in the unusual expression of downstream genes, including β-catenin, APC, c-Myc, and MMP-7.[41],[42] As expected, this investigation revealed that FOXS1 overexpression reduced the expression level of β-catenin, c-Myc, and MMP-7 and increased APC expression, whereas FOXS1 silence exerted opposite effects. These results suggested that FOXS1 significantly inhibited the activation of Wnt/β-catenin signaling cascade.

Finally, xenograft model in nude mice was conducted to confirm that increased FOXS1 effectively decelerated the tumor growth in vivo, indicating that FOXS1 inhibited lung SCC progression both in vitro and in vivo.

We only tested that FOXS1 regulated Wnt/β-catenin signaling pathway in the lung SCC cells, more rescue experiments will be performed to verify whether FOXS1 exerted its suppressive role by mediating Wnt/β-catenin pathway in the lung SCC cells. In addition, we need to conduct a series of analyses to expound how FOXS1 influences Wnt/β-catenin signal cascade in lung SCC cells.


  Conclusion Top


In conclusion, this work, for the first time, elucidated that FOXS1 inhibited lung SCC progression in vitro and in vivo at least partly by modulating Wnt/β-catenin signaling pathway.

Data availability

The authors declare that all data supporting the findings of this study are available within the paper and any raw data can be obtained from the corresponding author upon request.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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