Background
Oral squamous cell carcinoma (OSCC) is a familiar malignant tumor, accounting for about 95% [
1]. There are more than 300,000 new cases of OSCC yearly, and the age of onset displays a decreasing trend year by year [
2]. Currently, the pathogenesis of OSCC remains unclear. Bad habits, such as smoking and drinking alcohol, are the risk factors for OSCC, and other risk factors also contain HPV infection, chewing areca, and immune deficiency [
3]. At present, the therapy of OSCC is mainly radical surgical resection, combined with comprehensive remedy [
4]. Although the therapy for OSCC has improved, there has been no significant reduction in mortality, and the 5-year survival rate is only 50%-60%. OSCC, as an aggressive epithelial tumor, is associated with aberrant expression of multiple genes. Therefore, it is particularly critical to verify the mechanism of OSCC to improve OSCC remedy.
Interleukin-6 (IL-6) can act on cell receptors through autocrine or paracrine pathways to induce intercellular signal transduction and exchange, thus completing the relevant biological functions of cells [
5,
6]. There are two pathways of IL-6 transduction: classical pathway and trans-pathway. In the classical pathway, IL-6 can directly bind to IL-6R on cell membrane to mediate cell signal transduction [
6,
7]. In the trans-pathway, IL-6 can bind to sIL-6R and interacts with gpl30 to activate the downstream pathways including Janus protein tyrosine kinase (JAK), STAT3, MAPK, P13K, etc. [
8]. Researches testified that IL-6 is relevant to tumor growth, differentiation, apoptosis, drug resistance, and immune regulation, etc. IL-6 was also reported to be notably elevated in patients with advanced tumors [
9,
10]. And IL-6, as a diagnostic and therapeutic target, plays crucial roles in the OSCC progression, such as radio resistance, proliferation, and metastasis, etc. [
11‐
14]. JAK/STAT pathway is one of the most crucial cytokine signal transduction pathways and extensively involved in the regulation of multiple pathophysiological processes, especially inflammatory responses [
15,
16]. Activation of STAT3 can bind to target genes to change gene expression, and further participate in cell renewal, survival, division, metastasis, and other processes [
17,
18]. Study certified that JAK/STAT pathway could participate in the migration and apoptosis of OSCC cells [
19]. Besides, IL-6/JAK/STAT3 axis has also been discovered to have pleiotropic effects in cancers, including gastric cancer [
20], colorectal cancer [
21], and breast cancer [
22], etc. However, it is not clear whether IL-6 can influence OSCC progression through JAK/STAT3 pathway. And the detailed downstream regulatory mechanism of IL-6/JAK/STAT3 axis is not elucidated in OSCC.
In this research, we first confirmed the expressions of IL-6, IL-1β, and IL-18 in OSCC tissues. Meanwhile, we also identified the impacts of IL-6 on proliferation capacity in OSCC cells and xenograft tumor of model mice. Besides, we verified that whether JAK/STAT3 is a key regulatory pathway in OSCC proliferation mediated by IL-6. Moreover, we further investigated the possible mechanisms by which the IL-6/JAK/STAT3/Sox4 axis could affect OSCC progression. Therefore, our study preliminarily revealed the importance of IL-6/JAK/STAT3/Sox4 axis in the progression of OSCC and its potential regulatory mechanism, which might be effective approaches for the therapy of OSCC.
Materials and methods
Patients and samples
Twenty normal oral mucosa tissues, forty oral lichen planus (OLP) tissues and 108 cases of OSCC tissues were harvested from patients or volunteers in the Sichuan Provincial People’s Hospital from 2010 to 2015 and stored at -80 °C in the refrigerator for further analysis. Among the 108 patients, 9 patients were progressed from OLP to OSCC. At the same time, we collected peripheral venous blood (5 mL) from these participants, and the supernatant was collected by centrifugation (4 °C, 3 000 × g/min, 20 min). The supernatant was divided into sterile EP tubes and stored at -80 °C in the refrigerator. Inclusion criteria: The diagnosis of OSCC was confirmed by pathological sections and clinical diagnosis; all were first-time cancer patients with complete pathological data; no radiotherapy, chemotherapy and drug treatment; patients and their relatives agreed and signed the informed consent. Exclusion criteria: combination of tumors from other sites; combination of hypertension, diabetes mellitus; patients during pregnancy or lactation. In addition, clinical findings and follow-up data pertaining to these patients were recorded. The study was approved by Ethics Committee of Sichuan Provincial People’s Hospital [Sichuan, P.R. China; approval no. 2015NSF(7)], all patients signed informed consent.
Cell culture
Oral epithelial cells (HOEC) and OSCC cell line Cal27 were purchase from Procell (Wuhan, China), OSCC cell lines (SCC-4, SCC-15, SCC-9, SCC-25, and Tca83) were from ATCC (Manassas, VA). All cells were hatched in DMEM (Gibco) with L-glutamine, sodium pyruvate, 10% fetal bovine serum (FBS, Sigma) at 37 °C and 5% CO2.
Cell treatment and transfection
SCC-15 and SCC-25 cells were first addressed with 0, 0.1, 1, 5, 10, 25, 50 ng/mL IL-6 for 24 h, respectively. SCC-15 and SCC-25 cells were also processed with 25 ng/ml IL-6, 5 μmol/L JAK2 inhibitor (Fedratinib) [
23], and 25 μmol/L STAT3 inhibitor (Protosappanin A) for 24 h [
24]. Empty vector (pcDNA4.0), Sox4 overexpression plasmid (pcDNA4.0-Sox4), Sox4 shRNAs (shSox4, 5’-GCGACAAGATCCCTTTCATTC-3’), NLRP3 overexpression plasmid (pcDNA4.0-NLRP3), NLRP3 shRNAs (shNLRP3, 5’-GCTTCATCCACATGACTTTCC-3’), and negative control (NC) shRNAs were gained from HanBio Biotechnology (HanBio, Shanghai, China). SCC-15 and SCC-25 cells (1 × 10
5 cells/well) in 6-well plates were transfected with shSox4, pcDNA4.0-Sox4, shNLRP3, or pcDNA4.0-NLRP3 for 48 h using lipofectamine 3000 (Invitrogen) in accordance with the specification.
RT-qPCR
The processed OSCC cells were harvested or the tumors in each group were ground, and total RNAs were isolated applying TRIzol reagent (Invitrogen, MA, USA). Subsequently, cDNAs were synthesized with the RNAs (as template) and PrimeScript™ RT reagent Kit (TaKaRa). Then PCR amplification was then conducted with SYBR Green qPCR Master Mix (DBI Bioscience) after reverse transcription. The data in this experiment were calculated with 2−△△CT method, and GAPDH was the internal control. The primers for different genes are given as followed: IL-6: 5’-ACTCACCTCTTCAGAACGAATTG-3’ (forward) and 5’-CCATCTTTGGAAGGTTCAGGTTG-3’ (reverse); IL-6R: 5’-CCCCTCAGCAATGTTGTTTGT-3’ (forward) and 5’-CTCCGGGACTGCTAACTGG-3’ (reverse); Sox4: 5’-AGCGACAAGATCCCTTTCATTC-3’ (forward) and 5’-CGTTGCCGGACTTCACCTT-3’ (reverse); NLRP3: 5’-GATCTTCGCTGCGATCAACAG-3’ (forward) and 5’-CGTGCATTATCTGAACCCCAC-3’ (reverse); GAPDH: 5’-CTGGGCTACACTGAGCACC-3’ (forward) and 5’-AAGTGGTCGTTGAGGGCAATG-3’ (reverse).
Western blot
The processed OSCC cells and the ground tumors were increased with the lysates including RIPA (Beyotime, China) and protease inhibitors (Beyotime, China). The extracted proteins were quantified through BCA method, mixed with appropriate loading, and heated at 100℃ for denaturation. Then same amount (50 μg) of proteins were added to 10% SDS-PAGE, separated by electrophoresis at constant pressure, and transferred to PVDF membrane (Millipore). Next, the membrane with protein was sealed with 5% skim milk for 2 h, exposed to primary antibodies, including ASC (Abcam, ab283684, 1:1000), IL-1β (Abcam, ab254360, 1:1000), IL-18 (Abcam, ab207324, 1:1000), Pro-IL-18 (Proteintech, 10,663–1-AP, 1:1000), NLRP3 (Abcam, ab263899, 1:1000), IL-6 (Abcam, ab9324, 1 µg/ml), Sox4 (Abcam, ab70598, 1:500), JAK2 (Abcam, ab108596, 1:1000), pJAK2 (Abcam, ab32101, 1:1000), STAT3 (Abcam, ab68153, 1:1000), pSTAT3 (Abcam, ab267373, 1:1000) at 4℃ overnight, and secondary antibodies (Abcam) for 1 h. Finally, western blotting was developed after processing with ECL kit (Thermo scientific), and the brightness of each strip can be controlled by adjusting the exposure time.
ELISA assay
In line with the instruction, IL-1β ELISA kit (Abcam, ab214025 [for human] and ab197742 [for mouse]) and IL-18 ELISA kit (Abcam, ab215539 [for human] and ab216165 [for mouse]) was utilized to test IL-1β and IL-18 activities.
CCK-8
The treated OSCC cells were evenly increased into 96-well plates (100 μL, 1 × 103 cells/well). Then cells were growth in a 37 ℃ incubator and each well was supplemented with 10 μL CCK-8 (Dojindo, Tokyo, Japan) at 0, 24, 48, and 72 h. After additional 2 h of incubation, the OD was monitored by applying a microplate reader (Bio-TekEpoch) at 450 nm.
The processed OSCC cells (1 × 103 cells) were routinely hatched in a 6-well plate at 37 ℃ for 14 days. Then the adherent clones were fixed and dyed using 0.1% crystal violet, and clones were counted.
EdU staining
The processed OSCC cells (1 × 105 cells/well) were incubated in 6-well plates until they adhere to the wall. Then cells were processed with 100 μL EdU regent (50 μM, Life Technologies) for 2 h at 37 °C. Then cells were fixed using 4% formaldehyde (30 min), disposed of 50 μL 2 mg/mL glycine and 100 μL 0.5 Triton X-100. The Edu-positive cells were photographed using a fluorescence microscope (Olympus, Tokyo, Japan).
Chromatin immunoprecipitation (ChIP) assay
SCC-15 cells (1 × 106 cells/dish) were uniformly placed in 10 cm cell culture dishes and fixed using 37% formaldehyde at 37 ℃ for 10 min. Then cells were disposed of 0.125 M glycine and SDS Lysis Buffers, and DNA was interrupted into 200–1000 bp through ultrasound. Next, the mixture was addressed with 8 μL NaCl (5 M) at 65 ℃ and cross-linked for 4 h. Subsequently, GenClean Agarose Gel DNA Recovery Kit was applied to extract DNA, and co-precipitation was conducted with the CHIP kit (Millipore). The Sox4 primers for ChIP as followed: forward 5’-GCACCAGAGGCTGATTCT-3’ and reverse 5’-CTGCTTAAAAGCCAAGTG-3’.
Luciferase reporter assay
The downstream target genes for transcription factor STAT3 were identified by JASPAR 2022 (
https://jaspar.genereg.net/). We first constructed the wild type (WT) and mutant (Mut) Sox4 promoter plasmids (pGL4.1-Sox4-promoter-WT or pGL4.1-Sox4-promoter-MUT) by referring to the binding sites between STAT3 and Sox4 promoter region. 293 T cells were then co-transfected with the corresponding plasmids and internal plasmids (pTK-RL) containing the Renilla luciferase gene by applying Lipofectamine 3000 (Invitrogen). After 48 h of transfection, luciferase (Firefly/Renilla) activity was measured using the Dual luciferase assay kit (Promega).
For NLRP3, the wild type promoter of NLRP3 was constructed (pGL4.1-NLRP3-promoter-WT). SCC-15 and SCC-25 cells were then co-transfected with pGL4.1-NLRP3-promoter-WT plasmids, different concentration of Sox4 overexpression plasmids (0, 0.1, 0.5, 1, 5, 10, 20, 50 ng) and internal plasmids (pTK-RL) containing the Renilla luciferase gene by applying Lipofectamine 3000 (Invitrogen). After 48 h of transfection, luciferase (Firefly/Renilla) activity was measured using the Dual luciferase assay kit (Promega).
DNA Pull-down assay
The interaction between STAT3 and Sox4 promoter was confirmed by applying a DNA pull-down test kit (Gzscbio, Guangzhou, China). Briefly, cells were lysed after centrifugation. And then we mixed the streptavidin magnetic beads (125 μL) and probes targeting Sox4 promoter (25 μL, 8 μmol/L). And the mixture continued to mix with cell lysis for 12 h on ice. After elution, the conjunct protein was collected and Western blot was utilized to test STAT3 expression.
Tumor xenograft model
BALB/c male nude mice (SPF, 4 weeks, 20 ± 2 g) were obtained from Shanghai slack laboratory. And the experimental mice were fed through separate cages under the conditions of humidity 45%-55%, temperature 22–25 ℃, light for 12 h, adequate standard feed, and purified water. After one week, SCC15 cells were harvested and counted, and the right anterior axilla of each mouse was subcutaneously injected with 2 × 105 cells in 0.2 mL PBS. To evaluate the role of IL-6 on tumor growth, when the tumor volume reached 150 mm3, the tumor xenograft was directly injected with 100 μl PBS (Sigma-Aldrich, P2272, pH = 7.2), 10 mg/kg IL-6 recombinant protein (Fully biologically active, Abcam, ab259381) in 100 μl PBS (Sigma-Aldrich, P2272, pH = 7.2), or 10 mg/kg IL-6 antibody (Abcam, ab259341) in 100 μl PBS (Sigma-Aldrich, P2272, pH = 7.2) once a week, and the mice were divided into control, PBS, IL-6 group and IL-6 antibody groups. To evaluate the role of Sox4 on tumor growth, SCC15 cells were transfected with shSox4 and a Sox4 knockdown stable cell line was constructed. Each mouse was subcutaneously injected with 2 × 105 SCC15 cells with Sox4 knockdown in 0.2 mL PBS. when the tumor volume reached 150 mm3, the tumor xenograft was directly injected with 10 mg/kg IL-6 recombinant protein (Fully biologically active, Abcam, ab259381) in 100 μl PBS (Sigma-Aldrich, P2272, pH = 7.2), and the mice were divided into control, shSox4, IL-6 group and shSox4 + IL-6 groups. Tumor length was tested every 7 days for 21 days. At 21 days, the BALB/c nude mice were dislocated and sacrificed, and the tumors were collected. All animal experiments were done in animal laboratory center as per the study protocol according to the NIH Guide for the Care and Use of Laboratory Animals, approved by the Animal Care and Use Committee of the Sichuan Provincial People’s Hospital.
Immunohistochemistry (IHC)
Normal oral mucosa, OLP and OSCC tissues from human and the tumors from mice were fixed in 10% formaldehyde for 24 h. All tissues were conventionally dehydrated, paraffin embedded, and cut into 4 μm thick slices. Then the slices were conventionally dewaxed, washed, and repaired using sodium citrate (pH = 6.0) for 8 min under high temperature and high pressure. After washing, the slices were added into 3% H2O2 and then heated. Subsequently, the slices were blocked using 10% BSA, placed at 4℃ overnight with anti-IL-6, anti-Sox4, anti-NLRP3, anti-Ki-67, followed by secondary antibody (Abcam) for 1 h. After washing, the slices were then subjected to multiple processing in the later stage, including DAB treatment (10 s), washing, hematoxylin redyeing (30 s), dehydration, neutral gum sealing and natural drying. The results were obtained under a light microscope.
Statistical analysis
All experiments were conducted in thrice, and all data was displayed with mean ± SD and counted with SPSS 21.0 (SPSS, Inc.). And the statistical charts were made with GraphPad Prism 8.0. And the paired Student's t test or One-Way ANOVA were utilized for statistical calculations. The curves of Kaplan–Meier and log-rank assessments were employed to evaluate differences in survival outcomes between groups, while associations between IL-6 and Sox4, IL-1β or IL-18 were appraised through Pearson correlation assessments. Chi-square test was adopted to determine the relation between IL-6 or Sox4 expression level and clinical characteristics of OSCC patients. P < 0.05 represented the statistical significance.
Discussion
The incidence of head and neck cancer is relatively high, ranking 6th in the incidence of systemic malignant tumors, among which 90% are OSCC, and the prognosis is poor [
2,
25]. However, the pathogenesis of OSCC is not clear at present. Tumor-associated inflammation is known as the 7th biological feature of malignant tumors [
26]. On the one hand, chronic inflammation has key roles in tumor genesis, development, migration, and invasion through NF-κB-IL-6-STAT pathway; on the other hand, chronic inflammation can recruit immune cells and inflammatory cells to the tumor tissue, and with the development of tumor, the function of these cells evolved from inhibition and immune surveillance to promoting tumor cell proliferation [
27]. IL-6, as a multifunctional molecule, is involved in immune and inflammatory responses [
7]. Besides, IL-6 has been reported to be overexpressed in patients with cancers, including OSCC, which is associated with poor prognosis [
11,
28]. IL-6 also could induce tumor cell growth, metastasis, and angiogenesis through IL-6R-mediated pathways [
7]. In our study, we also testified that IL-6 was upregulated in OSCC tissues, and connected with inflammatory cytokines (IL-1β and IL-18). Besides, IL-6 also could upregulate IL-6R and accelerate proliferation of OSCC cells, which was consistent with previous research [
29]. Moreover, we also certified that IL-6 could activate JAK2 and STAT3 pathways in OSCC cells.
It is reported that JAK2/STAT3 can receive extracellular stimulation signals through inflammatory cytokine receptors, then induce JAK2 and STAT3 phosphorylation and regulate the expression of downstream genes [
30,
31]. JAK2/STAT3 has also been confirmed to be critically involved in the growth and metastasis of multiple cancers, including OCSS [
32‐
34]. In our study, we proved that JAK2 inhibitor (Fedratinib) and STAT3 inhibitor (Protosappanin A) could notably attenuate the induction of IL-6 on the proliferation and inflammation of OCSS cells. thus, we demonstrated that IL-6 could accelerate OSCC progression by JAK2 and STAT3 pathway.
To further investigate the possible downstream genes regulated by STAT3, we reviewed vast literature. It was reported that STAT3 can induce abnormal proliferation of tumor cells through regulation of BCL-2, FAS, survivin and CyclinD1 [
35,
36]. STAT3 can accelerate neovascularization by regulating VEGF and bFGF [
37]. Knockdown of STAT3 can increase NF-κB promoter activity and enhance NF-κB accumulation in the nucleus [
38]. STAT3 silencing also could reduce the expressions of cytokines IL-6, IL-1β and inflammatory mediators ICAM1 and COX2 in the tumor microenvironment [
39]. And MMP-2 and S100A4 may be the key targets of STAT3 involved in cancer metastasis [
40,
41]. Besides, recent studies have also confirmed that STAT3 can participate in the cancer process by mediating the expression of Sox4 [
42,
43]. Moreover, our previous research also revealed that Sox4 is significantly upregulated in oral lichen planus (OLP), OSCC versus OLP tissues, and Sox4 might be actively involved in the progression of OLP to OSCC, suggesting that Sox4 could induce the progression in OLP-associated OSCC [
44]. Therefore, we selected Sox4 as a downstream gene of STAT3 for the next step in OSCC. SOX family is a novel family of genes that can regulate development [
45]. SOX is characterized by a conserved HMG box, which can specifically bind to DNA sequences and is a key transcription regulatory factor [
46]. And Sox4 is a transcription factor associated with development and differentiation [
47]. Sox4 could induce tumorigenesis by endowing cancer cells with survivability, mobility, and invasiveness [
48]. Several researches verified that Sox4 is a critical oncogene that is highly expressed in cancers, including prostate cancer [
49], colorectal cancer [
50], bladder cancer [
51], and breast cancer [
52], etc. Sox4 has also been testified to be associated with differentiation, metastasis, and chemoradioresistance in OSCC [
53,
54]. Sox4 might have the potential to be a reliable prognostic factor for OSCC. While the regulatory pathways of Sox4 in OSCC progression have not been clearly elucidated. In our study, we also discovered that Sox4 was also upregulated and positively correlated with IL-6 in OSCC tissues. While it is unclear for the regulatory relationship between IL-6 and Sox4 in OSCC progression. Next, we further certified that Sox4 silencing could prevent proliferation and NLRP3 inflammasome activation mediated by IL-6 in OSCC cells. Moreover, we discovered that STAT3 could target Sox4, and STAT3 silencing could prevented OSCC progression by downregulating Sox4 in IL-6-induced OSCC cells. therefore, we demonstrated the IL-6/JAK2/STAT3/Sox4 pathway in OSCC.
Oral cavity, as a microenvironment colonized by more than 700 kinds of microorganisms, has been in the inflammatory environment with pathogenic bacteria for a long time [
55]. About 25% of malignancies are reported to be associated with chronic inflammation or infection [
56]. Chronic inflammation can cause the disappearance of cell growth inhibition, autonomic angiogenesis, apoptosis avoidance, transformation from benign to malignant, and enhancement of metastases [
57]. In the early stage of tumor formation, reactive oxygen species (ROS) and active nitrogen substances produced by immune cell infiltration can lead to epigenetic changes of oncogenes and tumor suppressor genes, thus promoting tumor genesis [
58]. During tumor metastasis, cytokines secreted by immune cells will enhance cell viability and invasion, resulting in transformation from epithelial cells to mesenchymal cells [
59]. Therefore, the molecular mechanism between tumor and inflammation are key for the prevention and therapy of cancer. Inflammasome is a key molecular structure for the body to resist pathogens and recognize its own danger signal. NLRP3 inflammasome, as the core protein of NLRs family, is a member of innate immune system [
60]. And its abnormal activation is associated with various chronic inflammation, mitochondrial diseases, and tumors [
61]. The NLRP3 inflammasome consists of NLRP3, ASC, and pro-caspase-1. NLRP3 inflammasome can activate pro-caspase to form active caspase-1, which causes the maturation and secretion of IL-1β and IL-18, leading to inflammation [
62]. IL-1β, as a class of pleiotropic pro-inflammatory cytokines, plays a key role in tumor development [
63]. IL-1β can promote tumor growth and metastasis by inducing the expression of various metastasis-related factors, such as matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF), inflammatory chemokines and growth factor genes [
64]. IL-18 has been reported to exert antitumor effects through multiple pathways. IL-18 can induce proliferation and enhance the activity of T lymphocytes and NK cells [
65]. IL-18 also promotes the production and secretion of cytokines such as IFN-γ, IL-2, GM-CSF, and TNF-α. And these cytokines are able to exert anti-tumor effects either directly by killing or by modulating immunity [
64]. The relationship between NLRP3 inflammasome and malignant tumors is complex and has some tissue or cell specificity. Related studies also testified that NLRP3 inflammasome was increased in OSCC, and its expression was relevant to tumor stage and lymph node metastasis [
66,
67]. And the activation of NLRP3 also could enhance the proliferation, migration, and invasion of OSCC cells [
66]. However, the regulatory relationship between NLRP3 and Sox4 remains unclear. In our study, we also proved that IL-6 could induce OSCC progression through activation of NLRP3 and its downstream cascade reaction (secretion of IL-1β/IL-18). And silencing of NLRP3 also could prevent proliferation and NLRP3 inflammasome activation in IL-6-mediated OSCC cells. In addition, NLRP3 pathway could participate in IL-6-mediated OSCC process as a downstream pathway of Sox4.
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