Interferon-induced transmembrane protein 1 (IFITM1) is essential for progression of laryngeal squamous cell carcinoma in an Osteopontin/NF- κ B-dependent manner

Abstract

OBJECTIVE:

To assess the expression levels of IFITM1 in human tissue samples and laryngeal squamous cell carcinoma (LSCC) cells, and to explore the potential mechanisms of IFITM1 in LSCC progression.

METHODS:

Quantitative PCR and immunohistochemical (IHC) assays were performed to detect IFITM1 expression in 62 LSCC tissues and corresponding normal tissues. We further detected the effects of IFITM1 on the proliferation, migration and invasion of LSCC cells and NF- $\kappa$ B signaling pathway through colony formation assay, wound healing assay and transwell assay, respectively.

RESULTS:

We demonstrated the possible involvement of IFITM1 in the progression of LSCC. We found the upregulated expression of IFITM1 in human LSCC tissues and cells, and analyzed the correlations between IFITM1 expression and osteopontin. Our data further confirmed that IFITM1 affected cell proliferation, migration, and invasion of LSCC cells via the regulation of NF- $\kappa$ B signaling pathway.

CONCLUSIONS:

We investigated the potential involvement of IFITM1 in the progression of LSCC, and therefore confirmed that IFITM1 was a potential therapeutic target for LSCC.

1. Introduction

Laryngeal squamous cell carcinoma (LSCC) is one of the most common tumor types with aggressive clinical characteristics [1, 2, 3]. Patients with advanced LSCC have poor prognosis and high mortality [4, 5]. LSCC patients usually lack obvious early symptoms, and are often accompanied by cervical lymph node metastases and carotid sinus syndrome at advanced stages [6, 7]. The treatment of LSCC usually adopts surgical excision, chemoradiotherapy and combination therapy [8]. Recently, targeted therapy for LSCC has shown broad prospect [9]. However, there is still an urgent need to explore more promising therapeutic targets for treatment of LSCC.

IFN-induced transmembrane protein 1 (IFITM1), a17-kDa membrane protein, is known as a cell surface protein and is involved in the inhibition of virus entry into host cell cytoplasm [10, 11]. In lymphocytes, IFITM1 is also a part of membrane complexes that transduces homotypic adhesion signals [12]. For decades, IFITM1 has been widely known as a regulator in multiple cellular processes such as cell proliferation [13]. The essential role of IFITM1 in immunity, osteogenesis and angiogenesis has also been reported in previous studies [14, 15].

In the past decade, the key role of IFITM1 in the progression of multiple types of cancers has been documented [16, 17]. IFITM1 shows abnormally high expression in several cancers, such as colorectal cancer, breast cancer and lung cancer [16, 18, 19]. A previous study indicated that depletion of IFITM1 suppressed the proliferation, migration, and invasion of lung cancer cells in vitro [19]. In addition, the combination of IFITM1 depletion and radiotherapy suppresses the progression of oral cancer [20]. Similarly, IFITM1 also affects the growth of cervical squamous cell carcinoma [21]. So far, IFITM1 has been shown to be involved in the progression and metastasis of multiple types of cancers However, the possible role of IFITM1 in the progression and development of LSCC is still unclear.

In this study, we demonstrated the abnormally high expression of IFITM1 in human LSCC tissues and cell lines, and further found that IFITM1 could affect the proliferation, migration, and invasion of LSCC cells via p65-I $\kappa$ B- $\alpha$ axis. Our findings investigated the possible involvement of IFITM1 in the progression of LSCC, and therefore identified IFITM1 as a novel LSCC therapeutic target.

2. Materials and methods

2.1 Antibodies, plasmids, and primers

Rabbit anti-IFITM1 (1:200 dilution for IHC; 1:2000 dilution for Immunoblot, ab11259, abcam, Cambridge, UK), mouse anti-GAPDH (1:2000 dilution, ab8227, abcam, Cambridge, UK), rabbit anti-cyclin A (1:2000 dilution, ab16667, abcam, Cambridge, UK), rabbit anti-cyclin D1 (1:1000 dilution, ab37150, abcam, Cambridge, UK), rabbit anti-CDK2 (1:2000 dilution, ab38898, abcam, Cambridge, UK), rabbit anti-OPN antibody (1:2000 dilution, ab184276, abcam, USA), rabbit anti-p-I $\kappa$ B- $\alpha$ antibody (1:2000 dilution, ab4461, abcam, USA), rabbit anti-I $\kappa$ B- $\alpha$ antibody (1:1500 dilution, ab92552, abcam, USA), rabbit anti-p-p65 antibody (1:1000 dilution, ab32124, abcam, USA), and rabbit anti-p65 antibody (1:1000 dilution, ab2302, abcam, USA) were all purchased from Abcam.

The quantitative PCR primer sequences for the analysis of IFITM1 were as follows: forward, 5’-ATAGTT GCCGTTCGCATG-3’ and reverse, 5’-CTGACCGACT GATATCAGAG-3’; The quantitative PCR primer sequences for the analysis of GAPDH are as follows: 5’-CGACCACTTCGTCAAGCTCA-3’ and reverse, 5’-GGTGGAGCACAGGGTACCTTATT-3’. shRNA plasmids (Ready-to-package AAV) for the knockdown of IFITM1 were bought from the Addgene.

2.2 Immunohistochemistry

Tumor tissues were obtained from Affiliated Hospital of Nantong University. The experiments was the approved by the Ethics Committee at/of the Affiliated Hospital of Nantong University (Approval no. 2019-K047). To explore the expression levels of IFITM1 in human LSCC tissues, we performed immunohistochemistry (IHC) assays. Briefly, sections were fixed with 4% paraformaldehyde (PFA) for 30 minutes at room temperature and subsequently blocked with 2% BSA for 20 minutes. Slides were subsequently incubated with IFITM1 or GAPDH antibodies at room temperature for 2 hours. Then the sections were incubated with biotinylated secondary antibody for 1.5 hours, and diaminobenzidine was used as a chromogen substrate.

2.3 Cell culture and transfection

Three types of human LSCC cell lines, including AMC-HN-8 (BNCC33877, Beijing beina technology co. LTD, China), TU177 (YS578C, Shanghai Yaji Biology, China) and TU686 (BNCC100479, Beijing beina technology co. LTD, China), which were bought from the indicated company, were maintained in DMEM culture medium supplemented with 10% of fetal bovine serum (FBS), at 37 ${}^{\circ }$ C in a 5% CO2 incubator.

The shRNA plasmids were transfected into LSCC cells using lipofectamine 2000 (11668019, Invitrogen, USA). In a 6-well plate, we diluted 1 $\mu$ m of plasmid with 5 $\mu$ L of lipofectamine 2000 transfection reagent in 1 mL of serum-free medium, left it for 20 minutes, added it to the cells, and replaced it with a complete medium 4 hours later. LSCC cells were co-transfected with 100 ng/ml exogenous OPN and/or 5 $\mu$ M TPCK and shRNA plasmids. Stably IFITM1 knockdown cellss were screened through lentivirus infection and used for the in vivo assays.

2.4 Quantitative PCR assay

Trizol (15596026, Invitrogen, USA) was used to extract total mRNA from human LSCC cells. Then the mRNAs from each type of cells were reverse-transcribed to cDNA by reverse transcriptase (M1701, Promega, Wisconsin, USA). Quantitative PCR was performed using SYBR Ex Taq kit (638319, Takara, Japan), and the expression levels of IFITM1 were normalized to the expression of GAPDH.

2.5 Immunoblot assays

Cells were lysed by RIPA lysis buffer (Beyotime, China) containing of protease inhibitors (Beyotime, China). Then the protein concentration was measured by BCA kit (Beyotime, China) and total proteins were were separated through SDS-PAGE (8% gels). After transfered onto polyvinylidene fluoride (PVDF) membranes, the membranes were blocked with 5% skim milk in TBST and incubated with primary antibodies for the detection of IFITM1, cyclin A, cyclin D1, CDK2, OPN (Osteopontin), p-I $\kappa$ B- $\alpha$ , I $\kappa$ B- $\alpha$ , p65, p-p65 and GAPHD at room temperature for 2 hours. Then, PVDF membranes were washed with TBST for four times and then incubated with HRP-conjugated secondary antibodies for 45 minutes. After washing, signals were detected using an BeyoECL Plus kit (Beyotime, China). The signals were analyzed by chemiluminescent analyzer (Beckman, USA).

2.6 MTT assays

Approximately 500 LSCC cells were seeded into 96-well plates, transfected with control or shIFITM1 plasmids and maintained for 48 hours. LSCC cells were then washed with PBS twice and incubated with MTT agent for 4 hours. Then dissolved in 150 $\mu$ L DMSO Then the OD value was measured with a microplate reader (Thermo Fisher, USA) at 562 nm wavelength.

2.7 Colony formation assay

One thousand cells were seeded into a 6-well culture plate and transfected with shRNA plasmids and maintained in a 37 ${}^{\circ }$ C, 5% CO2 incubator. and refreshed the medium every 3 days. After 2 weeks, cells were fixed with 4% PFA for 20 minutes at room temperature, stained with 0.2% crystal violet buffer for 30 minutes, and washed with PBS twice for 5 minutes. Then, the colony numbers were manually counted under microscope.

2.8 Wound healing assay

LSCC cells were transfected with shRNA plasmids, maintained in serum-free culture medium for 48 hours and the wound was made mechanically with a 20- $\mu$ L pipette tip. Subsequently, cells were washed with 1X PBS to remove debris, and the 2% serum containing culture medium was added. Photographs were taken using a microscope at 0 hour and 24 hours, and the relative extent of wound closure was calculated. Additionally, the agent of exogenous OPN (100 ng/mL) or TPCK (5 $\mu$ M) was treated in LSCC cells after the transfection of IFITM1 shRNA plasmids and maintained for 24 hours, then the wound closure assays were performed.

2.9 Transwell assay

LSCC cells were transfected with control or shIFITM1 plasmids for 48 hours and then re-suspended in serum-free RPMI1640 culture medium. The upper chambers of filters (8.0 $\mu$ m membrane pores) were containing 20% matrigel and the filters were incubated at 37 ${}^{\circ }$ C for 30 minutes. Five thousand LSCC cells in 200 $\mu$ L of serum free medium were then plated into the upper chambers of the inserts and induced to migrate toward the bottom chambers containing complete medium. After 24 hours, cells in the top chamber were removed with cotton swabs, and remaining cells were fixed in 4% PFA for 20 minutes, stained with 0.2% crystal violet buffer for 30 minutes. Then the migrated cell numbers was manually counted.

2.10 Reporter gene assay

LSCC cells were co-transfected with 0.8 $\mu$ g NF- $\kappa$ B luciferase reporter plasmid (Beyotime, China), 0.4 $\mu$ g pSV- $\beta$ -Galactosidase Control Vector (Addgene, USA). AMC-HN-8 cells were cultured and transfected with control or shIFITM1 plasmids overnight. After 24 hr, cells were washed and subjected to lysis. An equal volume of luciferase substrate was then added to all samples, and luminescence was measured in a microplate luminometer. The value of luciferase activity was normalized to transfection efficiency monitored by the co-transfected $\beta$ -galactosidase expression vector.

2.11 Statistical analysis

GraphPad 6.0 was used for statistical analysis in this study. All data were represented as mean $\pm$ SEM. Additionally, the correlation analysis between clinical pathological features and IFITM1 expression were performed through $\chi$ 2 analysis. K-M analysis were performed to evaluate the prognosis. Student’s $t$ -test was used for statistical comparisons. $P<$ 0.05 was considered as statistically significant.

Figure 1.

IFITM1 was upregulared in human laryngeal squamous cell carcinoma (LSCC) tissues and cells. Quantitative PCR and immunohistochemical assays were performed to analyze the expression of IFITM1 in LSCC tissues and cells. A. Quantitative PCR assay showed an upregulation ofin IFITM1 in 62 human LSCC tissues samples. B. Immunohistochemical analysis of IFITM1 expression levels in LSCC tissues and the corresponding normal tissues (100 $\times$ and 200 $\times$ magnification, respectively). C. Quantitative PCR assays indicate the increased mRNA expression levels of IFITM1 in LSCC cell lines, including AMC-HN-8, TU177 and TU686. Human normal bronchial epithelial cells (HBE) were used as control.

3. Results

3.1 IFITM1 is upregulated in human laryngeal squamous cell carcinoma tissues (or IFITM1 over-expressed in human LSCC)

To explore the potential role of IFITM1 in the progression of laryngeal squamous cell carcinoma (LSCC), we compared its expression levels between tumor tissues and the adjacent normal tissues. A total of 62 LSCC tumor tissue were used in this study, and the mRNA levels or expression levels of IFITM1 in tumor tissues or corresponding normal tissues were detected through quantitative PCR assay and immunohistochemistry (IHC) assay, respectively. Interestingly, our results showed that the expression levels of IFITM1 mRNA was significantly upregulated in tumor tissues, compared to adjacent normal tissues (Fig. 1A). To further confirm the expression difference of IFITM1 in tumor and normal tissues, we performed IHC assays. In line with the quantitative PCR assays, the expression of IFITM1 in human LSCC tissues was profoundly increased, whereas the expression levels of IFITM1 in normal tissues were lower than in tumor tissues (Fig. 1B).

Figure 2.

IFITM1 promotes the proliferation of LSCC cells in vitro. AMC-HN-8 cells were transfected with shIFITM1 plasmid or a control plasmid. A. Quantitative PCR assays showed significantly decreased expression levels of IFITM1 mRNA in IFITM1-shRNA transfected AMC-HN-8 cells. B. Immunoblot assays confirmed the efficient silencing of IFITM1 expression in IFITM1-shRNA-transfected AMC-HN-8 cells. C. The results of MTT assays showed the inhibition of cell proliferation caused by IFITM1 silence. D. Representive images showing the results of colony formation assays of AMC-HN-8 cells transfected with control or IFITM1 shRNA plasmids. The relative proliferation levels were quantified. E. Immunoblot assays indicated the expression level of cyclin A, cyclin D1, CDK2 and GAPDH in AMC-HN-8 cells transfected with the indicated plasmids. The experiments were repeated for at least three times. Results are presented as mean $\pm$ SEM, ${}^{**}P<$ 0.01.

Figure 3.

IFITM1 contributes to the migration and invasion of LSCC cells in vitro. A. Wound healing assays were performed using AMC-HN-8 cells transfected with control or shIFITM1 plasmids, and the relative wound width was detected. B. Transwell assays using control or IFITM1 depletion AMC-HN-8 cells were performed and the extent of transwell migration was quantified by relative cell number. The experiments were repeated for at least 3 times. Results are presented as mean $\pm$ SEM, ${}^{**}P<$ 0.01.

Subsequently, we detected the mRNA levels of IFITM1 in human normal bronchial epithelial cells (HBE) and LSCC cell lines, including AMC-HN-8, TU177 and TU686. Consistent with the previous results, we found that IFITM1 was dramatically up-regulated in LSCC cells, compared with normal bronchial epithelial cells (Fig. 1C). Together, these results suggest that IFITM1 shows abnormally high expression in human LSCC cells and tissues.

3.2 Knockdown of IFITM1 inhibited proliferation of LSCC cells

To further explore the function of IFITM1, we transfected the shRNA plasmid targeted against IFITM1 into human LSCC cell line AMC-HN-8 (the highest IFITM1 expression level), to suppress the expression level of IFITM1. As excessive cell proliferation can lead to tumorigenesis, therefore, the effect of IFITM1 on LSCC cells proliferation was examined. The results of quantitative PCR assays showed that the transfection of IFITM1 shRNA effectively decreased its expression level in AMC-HN-8 cell (Fig. 2A). Consistent with quantitative PCR results, the immunoblot assays further proved the obvious decrease of IFITM1 expression level in AMC-HN-8 cell transfected with shIFITM1 plasmid (Fig. 2B).

We further detected the effects of IFITM1 on cell proliferation of LSCC by MTT assays and colony formation assay. MTT assays showed that IFITM1 silence significantly decreased cell viability (Fig. 2C). Additionally, colony formation results showed that the colony formation capacity was significantly restrained after knocking-down IFITM1, indicated by the obviously decreased colony numbers (Fig. 2D). Next, we examined the effect of IFITM1 silence on the level of cell cycle control proteins such as cyclin A, cyclin D1 and CDK2. A significantly reduced expression of cyclin A, cyclin D1 and CDK2 in IFITM1 shRNA-transfected group was found, suggesting that the decline in proliferative capacity was mainly caused by cell cycle arrest (Fig. 2E).

3.3 Downregulation of IFITM1 blocks cell migration and invasion of LSCC cells in vitro

We next performed wound healing and transwell assays to evaluate the possible effects of IFITM1 on the migration and invasion of LSCC cells. As was expected, our results exhibited that IFITM1 silence remarkably inhibited the extent of wound closure in AMC-HN-8 cell (Fig. 3A). In addition, IFITM1 silence significantly blocked the invasion of AMC-HN-8 cell, with dramatically dropped cell numbers (Fig. 3B), confirmed by transwell assay. Collectively, our results indicated that IFITM1 inhibited cell migration and invasion of LSCC in vitro.

3.4 IFITM1 mediates NF- $\kappa$ B signaling pathway through the regulation of Osteopontin

LSCC is one of the most common malignancies in head and neck. In order to study the molecular mechanism of IFITM1 affecting the progression of LSCC, we focused on Osteopontin (OPN), a phosphoprotein which functions as a cell attachment protein and cytokine which regulate cancer growth and metastasis through two cell adhesion molecules. Through immunoblot assays, we found that OPN expression was significantly reduced after IFITM1 knockdown (Fig. 4A). It has been known that OPN is necessary for NF- $\kappa$ B signaling pathway activation, and therefore OPN suppression may affect the activation of NF- $\kappa$ B signaling pathway.

Figure 4.

IFITM1 mediates NF- $\kappa$ B signaling pathway through the regulation of Osteopontin (OPN). A. Immunoblot assays indicated the levels of OPN, p-I $\kappa$ B- $\alpha$ , I $\kappa$ B- $\alpha$ , p65, p-p65 and GAPDH in AMC-HN-8 cells transfected with control or shIFITM1 plasmids. B. AMC-HN-8 cells transfected with $\kappa$ B-luciferase plasmid for 24 h were either co-transfected with control or shIFITM1 plasmids. Luciferase activity was also measured. The experiments were repeated for at least 3 times. Results are presented as mean $\pm$ SEM, ${}^{**}P<$ 0.01.

Figure 5.

Exogenous OPN rescue cell migration and invasion of LSCC cells blocked by ablation of IFITM1. A. Representive photographs showed the results of colony formation assays of AMC-HN-8 cells transfected with the indicated plasmids. The relative proliferation levels were quantified. B. Wound healing assays were also performed using AMC-HN-8 cells transfected with the indicated plasmids, and the relative wound width was detected. C. Transwell assays using AMC-HN-8 cells transfected with the indicated plasmids were performed and the extent of relative transwell migration was quantified. The experiments were repeated for at least 3 times. Results are presented as mean $\pm$ SEM, ${}^{**}P<$ 0.01.

To verify this hypothesis, we evaluated the expression level of I $\kappa$ B- $\alpha$ and p65, which play an important role in the NF- $\kappa$ B signaling pathway. Interestingly, through immunoblot assays, we found that OPN overexpression induced the phosphorylation of I $\kappa$ B- $\alpha$ , whereas knockdown of IFITM1 blocked this phenomenon in AMC-HN-8 cells (Fig. 4A). Additionally, we found OPN suppression led to the significant decrease of p65 phosphorylation levels in AMC-HN-8 cells, whereas the expression of total p65 was not affected (Fig. 4A). Then we conducted the relative $\kappa$ B promoter activity assays to test the activity of the NF- $\kappa$ B signaling pathway. In line with the hypothesis, our results showed that OPN suppression caused by IFITM1 silence remarkably weakened the relative $\kappa$ B promoter activity (Fig. 4B). According to these results, we demonstrated that IFITM1 mediated NF- $\kappa$ B signaling pathway through the regulation of OPN.

3.5 Exogenous OPN rescues cell migration and invasion of LSCC cells blocked by downregulation of IFITM1

We next investigated whether adding exogenous OPN or TPCK, a protein inhibitor which inhibiting I $\kappa$ B- $\alpha$ degradation, had an effect on LSCC cells behavior. Colony formation assays showed that the addition of exogenous OPN alone resulted in a significant increase in colony formation capacity compared to IFITM1 silence group, whereas addition of exogenous OPN and TPCK simultaneously resulted in a comparable colony formation capacity (Fig. 5A). Additionally, the results of wound healing and transwell assays had the similar tendency of colony formation assay (Fig. 5B and C). Altogether, these results indicated that adding exogenous OPN promoted cell migration and invasion of LSCC cells.

4. Discussion

Laryngeal squamous cell carcinoma (LSCC) is a common head and neck malignancy with a poor overall 5-year survival rate [22]. Debulking surgery, radiation and chemotherapy have limited efficiency on LSCC treatment due to the high invasiveness [23]. Targeted therapy is a promising treatment for LSCC, however, novel and effective therapeutic targets still need to be developed [9, 24, 25]. Through quantitative PCR and IHC assays, we found the obvious high expression of IFITM1 in human LSCC tissues at mRNA and protein levels, compared to the normal tissues. Similarly, our data also confirmed IFITM1 was highly expressed in LSCC cell lines, including AMC-HN-8, TU177, and TU886 cells. We therefore proposed the possible involvement of IFITM1 in the progression of LSCC. The abnormally high expression of IFITM1 was found in multiple types of tumors, including non-small cell lung cancer, breast cancer, and esophageal adenocarcinoma, and correlated with the prognosis of patients. These findings, together with our data in this study, suggested that IFITM1 has the potential of serving as an independent prognostic factor and therapeutic target for LSCC.

We further evaluated the effects of IFITM1 on LSCC progression in vitro. By performing MTT and colony formation assays, we noticed that downregulation of IFITM1 by siRNA transfection significantly inhibited the LSCC cell proliferation. Our data further confirmed that the expression of cell cycle markers, including cyclin A, cyclin D1, and CDK2, was dramatically decreased after IFITM1 silence. Meanwhile, through wound healing and transwell assays, we further demonstrated that IFITM1 affected the migration and invasion of LSCC cells. Similarly in lung cancer cells, IFITM1 siRNA transfection also resulted in the inhibition of cell proliferation, migration, and invasion [19]. In oral cancer, IFITM1 could affect cell proliferation, DNA damage and cell apoptosis [20]. Additionally, IFITM1 silence had growth inhibitory effects in cervical squamous cell carcinoma [21]. These studies proved that IFITM1 has multiple roles in cancer, and the precise mechanisms of its action still needs to be further studied.

As a cell surface protein, IFITM1 is involved in a variety of physiological and pathological processes through different mechanisms. For example, the depletion of IFITM1 suppressed proliferation and invasion of aromatase inhibitor-resistant breast cancer cells through JAK/STAT signaling pathway [17, 26]. Overexpression of IFITM1 promoted the aggressive phenotype of breast cancer cells via activation of signal transducer and activator of transcription 2 (STAT2)-dependent manner [27]. IFITM1 also contributes to the metastasis of colorectal cancer via the regulation of CAV-1 expression [28]. Here, we showed that IFITM1 regulated the proliferation, migration, and invasion of LSCC cells via the phosphorylation of p65 and I $\kappa$ B- $\alpha$ . p65-I $\kappa$ B- $\alpha$ axis is known to be involved in the progression of multiple cancers, such as bladder cancer, ovarian cancer, and breast cancer. Our findings further confirmed the critical role of this pathway in LSCC progression. In the future, we will clarify whether the inhibitors of IFITM1 will have promising effects on LSCC treatment, making it a desirable drug target. IFITM1 is also known as a regulator in immunity, mainly through OPN and I $\kappa$ B/NF $\kappa$ B pathway. In this study, we found that the effects of IFITM1 on the proliferation, migration and invasion of LSCC was achieved through this signaling pathway. Therefore, the relationship between IFITM1 and immunity cannot be ignored. In conclusion, we investigated the potential involvement of IFITM1 in the progression of LSCC. Our findings indicated the upregulated expression of IFITM1 in human LSCC tissues and cell lines. We further found that IFITM1 silence blocked the proliferation, migration, and invasion of LSCC cells in vitro, via the phosphorylation of p65 and I $\kappa$ B- $\alpha$ . We therefore provide evidence that IFITM1 has the potential to be explored as a novel and promising therapeutic target for LSCC treatment.