IL-1RA promotes oral squamous cell carcinoma malignancy through mitochondrial metabolism-mediated EGFR/JNK/SOX2 pathway

doi:10.21203/rs.3.rs-2995954/v1

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Research Article

IL-1RA promotes oral squamous cell carcinoma malignancy through mitochondrial metabolism-mediated EGFR/JNK/SOX2 pathway

https://doi.org/10.21203/rs.3.rs-2995954/v1

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Journal Publication

published 17 Jul, 2023

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Background

Interleukin-1 receptor antagonist (IL-1RA), a member of the IL-1 family, has diverse roles in cancer development. However, the role of IL-1RA in oral squamous cell carcinoma (OSCC), in particular the underlying mechanisms, remains to be elucidated.

Methods

Tumor tissues from OSCC patients were assessed for protein expression by immunohistochemistry. Patient survival was evaluated by Kaplan-Meier curve analysis. Impact of differential IL-1RA expression on cultured OSCC cell lines was assessed in vitro by clonogenic survival, tumorsphere formation, soft agar colony formation, and transwell cell migration and invasion assays. Oxygen consumption rate was measured by Seahorse analyzer or multi-mode plate reader. PCR array was applied to screen human cancer stem cell-related genes, proteome array for phosphorylation status of kinases, and Western blot for protein expression in cultured cells. In vivo tumor growth was investigated by orthotopic xenograft in mice, and protein expression in xenograft tumors assessed by immunohistochemistry.

Results

Clinical analysis revealed that elevated IL-1RA expression in OSCC tumor tissues was associated with increased tumor size and cancer stage, and reduced survival in the patient group receiving adjuvant radiotherapy compared to the patient group without adjuvant radiotherapy. In vitro data supported these observations, showing that overexpression of IL-1RA increased OSCC cell growth, migration/invasion abilities, and resistance to ionizing radiation, whereas knockdown of IL-1RA had largely the opposite effects. Additionally, we identified that EGFR/JNK activation and SOX2 expression were modulated by differential IL-1RA expression downstream of mitochondrial metabolism, with application of mitochondrial complex inhibitors suppressing these pathways. Furthermore, in vivo data revealed that treatment with cisplatin or metformin - a mitochondrial complex inhibitor and conventional therapy for type 2 diabetes - reduced IL-1RA-associated xenograft tumor growth as well as EGFR/JNK activation and SOX2 expression. This inhibitory effect was further augmented by combination treatment with cisplatin and metformin.

Conclusions

The current study suggests that IL-1RA promoted OSCC malignancy through mitochondrial metabolism-mediated EGFR/JNK activation and SOX2 expression. Inhibition of this mitochondrial metabolic pathway may present a potential therapeutic strategy in OSCC.

IL-1RA

Mitochondrial metabolism

EGFR

JNK

SOX2

Cancer stemness

Tumor growth

Metastasis

OSCC

Oral cancer is one of the most prevalent cancers and causes of cancer-related deaths worldwide, and oral squamous cell carcinoma (OSCC) is the most frequently diagnosed form of oral cancer [19, 42, 48]. Various risk factors promote oral cancer development, including tobacco use, alcohol consumption, and betel quid [14]. However, the management of oral cancer remains a challenge owing to its tendency for frequent relapse, metastasis, and treatment resistance [12, 14, 50]. An important potential mechanism underlying some of these oncological challenges is the property of cancer stemness, which is associated with the enhanced self-proliferation and metastatic ability of cancer cells. Recent research has suggested that altered mitochondrial metabolism underpins some of these malignant cell characteristics [32, 33, 37, 47].

Interleukin-1 receptor antagonist (IL-1RA), the gene product of IL1RN which shares approximately 30% protein sequence homology with interleukin-1 (IL-1), competes with IL-1 for binding to the IL-1 receptor [18, 20]. As a member of the IL-1 family with multifaceted functions, IL-1RA plays diverse roles in a number of pathological conditions, including cancer. For example, lower levels of IL-1RA were found in several cancer types, such as leukemia, colorectal cancer, and prostate cancer [27, 30, 40], and it was negatively associated with the development of premalignant oral dysplasia [44]. However, other reports indicate higher levels of IL-1RA in cervical and gastric cancers [23, 26], suggesting that IL-1RA may function in a cancer type-specific manner. However, its clinical role and biological function in oral cancer development remain unclear.

In this study, we evaluated the potential of IL-1RA as a clinical marker for OSCC progression and patient outcomes. In addition, the cellular mechanisms of IL-1RA in OSCC malignancy were explored both in vitro and in vivo, providing insights into the development of novel therapeutic strategies for OSCC that target vulnerabilities in the IL-1RA-associated mitochondrial metabolic pathway.

Patient samples

Oral tumor tissues were obtained from patients diagnosed with OSCC at the Department of Oral and Maxillofacial Surgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan, and confirmed using clinical and histological data from the Cancer Registry. Overall survival was defined as the interval between the date of diagnosis and date of death. This study was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (approval no. KMUH-IRB-20130300 and KMUHIRB-F(I)-20220016). Patient informed consent was waived by the Institutional Review Board due to the retrospective nature of the study.

Cell culture

Human oral squamous cell carcinoma cell lines HSC-3, Ca9-22, and OECM-1 were obtained from the Bioresource Collection and Research Center, Hsinchu, Taiwan (https://www.bcrc.firdi.org.tw). HSC-3 and Ca9-22 cells were cultured in DMEM/F12 medium (Thermo Fisher Scientific, Waltham, MA, USA), and OECM-1 cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA). All cell culture media were supplemented with 10% fetal bovine serum (FBS; Biological Industries, Beit Haemek, Israel) and 1% penicillin-streptomycin-amphotericin B (Thermo Fisher Scientific, Waltham, MA, USA). Cells were maintained at 37°C in a humidified incubator with 5% CO₂ incubator.

Gene knockdown and overexpression

Knockdown of IL-1RA was conducted by lentiviral infection using a pLKO.1-puro vector carrying shRNA sequences (shIL-1RA#1, 5’-GCCTTCAGAATCTGGGATGTT–3’; shIL-1RA#2, 5’-CGAGAACAGAAAGCAGGACAA-3’; shIL-1RA#3, 5’-GCAAGGACCAAATGTCAATTT-3’; shIL-1RA#4, 5’-CGTCATGGTCACCAAATTCTA-3’) that target consensus regions of human IL1RN (Accession: NM_000577), which were obtained from the National RNAi Core Facility, Academia Sinica, Taipei, Taiwan. Another pLKO.1-puro vector carrying shRNA sequences targeting firefly luciferase (shLuc, 5’-GCGGTTGCCAAGAGGTTCCAT-3’) was used as a control (National RNAi Core Facility, Academia Sinica, Taipei, Taiwan). For gene overexpression of IL-1RA, prepackaged lentiviral particles carrying pReceiver-Lv105 vector expressing full-length human IL1RN (IL-1RA-OE; Accession: NM_173841.2), or empty pReceiver-Lv105 vector (EV) as a control, were purchased from GeneCopoeia (Rockville, MD, USA). The infection was performed by adding lentiviral particles to the corresponding cells in the cell culture medium containing 8 µg/mL polybrene (Sigma-Aldrich, St. Louis, MO, USA). After infection for 48 h, 2 µg/mL puromycin was added for selection, and the surviving cells were maintained in 2 µg/mL puromycin until further experiments.

Clonogenic survival assay

OSCC cells were seeded in 6-well plates (1 × 10³ cells/well; Corning, Tewksbury, MA, USA) and cultured for 14 days prior to staining with 0.5% crystal violet (Sigma-Aldrich, St. Louis, MO, USA) for 15 min at room temperature. For ionizing radiation treatment, cells were irradiated at a dose of 5 Gy using a 6-MV linear accelerator (Elekta, Stockholm, Sweden) at the Department of Radiation Oncology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan. Images were captured using a light microscope (Nikon, Tokyo, Japan) and analyzed using ImageJ software (https://imagej.nih.gov/ij).

Tumorsphere formation assay

OSCC cells were seeded in ultra-low attachment 96-well plates (5 × 10² cells/well; Corning, Tewksbury, MA, USA) in serum-free cell culture medium supplemented with 20 ng/ml epidermal growth factor (EGF; PeproTech, Rehovot, Israel), 20 ng/ml fibroblast growth factor-basic (FGF-basic; PeproTech), 10 µg/mL insulin (Sigma-Aldrich, St. Louis, MO, USA), and 1X B27 (Thermo Fisher Scientific, Waltham, MA, USA). After 14 days of cell culture, images of tumorspheres larger than 50 µm in diameter were captured using a light microscope (Nikon, Tokyo, Japan) and analyzed with ImageJ (https://imagej.nih.gov/ij).

Soft agar colony formation assay

OSCC cells were suspended in cell culture medium mixed with 0.4% low-melting agarose (Sigma-Aldrich, St. Louis, MO, USA) and then seeded in 6-cm dishes (1 × 10⁴ cells/dish; Corning, Tewksbury, MA, USA) pre-coated with a layer of 0.5% low-melting agarose (Sigma-Aldrich, St. Louis, MO, USA). After 14 days of cell culture, colonies larger than 100 µm in diameter were stained with 0.5% crystal violet for 15 min at room temperature, and images were captured using a stereo microscope (Olympus, Tokyo, Japan) and analyzed with ImageJ (https://imagej.nih.gov/ij).

XTT cell viability assay

OSCC cells were seeded in 96-well plates (1 × 10⁴ cells/well; Corning, Tewksbury, MA, USA) for 24 h, prior to the addition of XTT reagent from Cell Proliferation Kit II (Sigma-Aldrich, St. Louis, MO, USA) for 2 h at 37°C, followed by measurement of the optical density (OD) at 470 nm by subtracting the background at 660 nm.

Transwell cell migration and invasion assays

OSCC cells were re-suspended in serum-free cell culture medium and plated onto inserts (2 × 10⁴ cells/insert; 8 µm pores) in 24-well transwell plates (Corning, Tewksbury, MA, USA). The inserts were pre-coated with or without Matrigel (Corning, Tewksbury, MA, USA) for cell invasion and migration assays, respectively, and normal cell culture medium was added to the bottom wells. After 24 h, cells that remained on the upper side of the inserts were removed using cotton swabs, and cells that appeared on the lower side of the inserts were fixed with 4% formaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 15 min and stained with 0.05% crystal violet (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at room temperature. Images were captured using a light microscope (Nikon, Tokyo, Japan) and analyzed using ImageJ software (https://imagej.nih.gov/ij/).

Measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)

The basal OCR and ECAR of OSCC cells were measured using the Extracellular Oxygen Consumption Assay and Glycolysis Assay kits (Abcam, Waltham, MA, USA), respectively, according to the manufacturer’s instructions. The fluorescent signal was measured using a CLARIOstar Plus multi-mode plate reader (BMG Labtech, Ortenberg, Germany) at 1.5 min intervals with excitation/emission wavelengths of 360/650 nm for OCR and 380/615 nm for ECAR. To measure multiple statuses of OCR in mitochondria, an Agilent Seahorse XFe24 Analyzer (Agilent Technologies, Santa Clara, CA, USA) was used in accordance with the procedures from the manufacturer and our previous report [59]. The mitochondrial modulators were obtained from Seahorse XF Cell Mito Stress Test Kit (Agilent Technologies, Santa Clara, CA, USA) and injected sequentially for specific measurements as follows: first, 1 µM oligomycin was injected to measure ATP production; second, 0.5 µM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) was injected to measure maximal respiration; finally, 0.5 µM rotenone and 0.5 µM antimycin A were injected to measure spare respiratory capacity [63].

Measurement of reactive oxygen species (ROS) formation

OSCC cells were seeded in 6-well plates (3 × 10⁵ cells/well) overnight, followed by labeling with 10 µM 2’,7’-dichlorofluorescin diacetate (DCFDA; Sigma-Aldrich, St. Louis, MO, USA) for 20 min at room temperature. The cells were then analyzed using a Cytomics FC 500 Flow Cytometer (Beckman Coulter, Indianapolis, IN, USA) in accordance with the manufacturer’s instructions and our previous report [59].

Polymerase chain reaction (PCR) array

Total RNA extracted from OSCC cells was reverse transcribed and analyzed by real-time PCR using the Human Stem Cell Signaling RT² Profiler PCR Array (Qiagen, Redwood City, CA, USA) according to the manufacturer’s instructions and our previous report [53].

Proteome array

Total protein extracted from OSCC cells was analyzed using the Human Phospho Kinase Array (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions and our previous reports [52, 54].

Western blot

Total protein extracted from OSCC cells was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transferred onto nitrocellulose membranes (Merck, Darmstadt, Germany). The membranes were blocked with 2% skimmed milk (Anchor, Auckland, New Zealand) and incubated with primary antibodies overnight, followed by further incubation with species-matched horseradish peroxidase-conjugated secondary antibodies (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature. Immunoreactive protein bands were detected by incubation with Immobilon Western chemiluminescent reagents (Merck, Darmstadt, Germany), and the signals were captured by ChemiDoc XRS+ (Bio-Rad, Hercules, CA, USA) and analyzed with Image Lab (Bio-Rad, Hercules, CA, USA). The following primary antibodies were used for Western blot: Goat anti-human IL-1RA (R&D Systems, Minneapolis, MN, USA), rabbit anti-human SOX2 (GeneTex, Hsinchu, Taiwan), rabbit anti-human EGF (GeneTex, Hsinchu, Taiwan), rabbit anti-human THY1 (Abcam, Waltham, MA, USA), rabbit anti-human c-KIT (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-human p21 (GeneTex, Hsinchu, Taiwan), rabbit anti-human phospho-EGFR at Tyr1086 (GeneTex, Hsinchu, Taiwan), rabbit anti-human EGFR (GeneTex, Hsinchu, Taiwan), rabbit anti-human phospho-p44/42 MAPK (ERK1/2) at Thr202/Tyr204 (Cell Signaling Technology, Danvers, MA, USA), mouse anti-human ERK1/2 (GeneTex, Hsinchu, Taiwan), rabbit anti-human phospho-JNK1/2/3 at Thr183/Thr183/Thr221 (Abcam, Waltham, MA, USA), rabbit anti-human JNK1/2/3 (Abcam, Waltham, MA, USA), rabbit anti-human GAPDH (GeneTex, Hsinchu, Taiwan), and mouse anti-human β-actin (Sigma-Aldrich, St. Louis, MO, USA).

Immunohistochemistry (IHC)

Formalin-fixed and paraffin-embedded normal and tumor tissue sections were immunostained using a Bond-Max automated IHC stainer (Leica Microsystems, Wetzlar, Germany) in accordance with the manufacturer’s instructions and our previous reports [53, 60]. The following primary antibodies were used for IHC: goat anti-human IL-1RA (R&D Systems, Minneapolis, MN, USA), rabbit anti-human SOX2 (GeneTex, Hsinchu, Taiwan), rabbit anti-human Ki67 (GeneTex, Hsinchu, Taiwan), rabbit anti-human phospho-EGFR at Tyr1086 (GeneTex, Hsinchu, Taiwan), and rabbit anti-human phospho-JNK1/2/3 at Thr183/Thr183/Thr221 (Abcam, Waltham, MA, USA). IHC images were captured using an Eclipse E600 microscope (Nikon, Tokyo, Japan), and total immunostaining scores for each tissue section were determined as the product of the percentage of positively stained cells (0, 0–4%; 1, 5–24%; 2, 25–49%; 3, 50–74%; 4, 75–100%) multiplied by the intensity of staining (0, negative; 1, weak; 2, moderate; 3, strong). To further analyze the association between IL-1RA expression and patient survival, the expression level of IL-1RA by IHC scoring was categorized into low versus high expression with cut-off values according to ROC curves [53, 60].

Animal study

Experiments involving animals were approved by and in accordance with the guidelines and regulations of the Institutional Animal Care and Use Committee of Kaohsiung Medical University, Kaohsiung, Taiwan (Approval no. 108127). Three-week-old male NOD.CB17-Prkdc^scid/JNarl mice were obtained from the National Laboratory Animal Center of Taiwan (https://www.nlac.narl.org.tw). For orthotopic xenografts, OECM-1 cells expressing luciferase (OECM-1-luc; 5 × 10⁵ cells per mouse) resuspended in 100 µL of phosphate-buffered saline were injected into mice through the intrabuccal route. At one week post-xenograft, the mice were randomly assigned to eight groups (five mice per group), including empty vector (EV) versus IL-1RA-OE from each of the untreated, metformin-treated, cisplatin-treated, and metformin combined with cisplatin-treated groups. The untreated groups were intraperitoneally injected with normal saline thrice per week. The metformin-treated group (200 mg/kg) was injected intraperitoneally three times per week. The cisplatin-treated group (2 mg/kg) was injected intraperitoneally once per week. In the metformin combined with cisplatin-treated groups, metformin (200 mg/kg) was administered three times per week and cisplatin (2 mg/kg) was administered once per week. All mice were monitored weekly using an IVIS Spectrum in vivo imaging system (PerkinElmer, Santa Clara, CA, USA), according to the manufacturer’s instructions and our previous report [53]. After treatment for 5 weeks, the mice were sacrificed, and orthotopic xenograft tumors were collected for tumor weight and volume measurements, followed by immunohistochemical analysis.

Statistical analysis

Statistical analyses were performed using JMP version 10.0.1 for Windows (SAS Institute, Cary, NC, USA). The log-rank test was used to compare patient survival using Kaplan-Meier curve analysis. The Student’s t-test was used to compare the two groups. One-way analysis of variance (ANOVA) with post-hoc Tukey’s test was used for multiple group comparisons. Pearson correlation (r) was used to evaluate the correlation between the two protein expression levels by IHC. The data are presented as the mean ± SD from three independent experiments where applicable or as otherwise specified in individual figures. P values less than 0.05 were considered statistically significant.

Clinical association of IL-1RA expression with OSCC progression and patient outcomes

To evaluate the association between IL-1RA expression and OSCC progression, immunohistochemical analysis was performed on OSCC tumor tissues and normal oral mucosal tissues adjacent to the oral fibroma tissues for comparison. The results indicated an elevated level of IL-1RA expression in OSCC tumor tissues compared to that in the normal oral mucosa (p = 0.001; Fig. 1A). Further analysis of the clinicopathological characteristics revealed that elevated IL-1RA expression in OSCC tumor tissues was associated with tumor size (p = 0.003; Fig. 1B) and cancer stage (p = 0.012; Fig. 1C). Moreover, there was an association between the expression of IL-1RA and the overall survival of patients with OSCC in response to treatment, in which high IL-1RA expression was associated with poorer overall survival probability in patients receiving adjuvant radiotherapy, but this association was not observed in patients who did not receive adjuvant radiotherapy (p = 0.043 and 0.709, respectively; Fig. 1D). These clinical data suggest that elevated IL-1RA expression is associated with adverse OSCC progression and poor treatment outcomes. Therefore, we performed gene knockdown and overexpression of IL-1RA in OSCC cells to investigate the impact of differential IL-1RA expression on cancer cell malignancy.

IL-1RA promoted in vitro cancer cell growth and migration/invasion abilities via the mitochondrial metabolic pathway

Different levels of endogenous IL-1RA expression were detected in four human OSCC cell lines, including HSC-3, SAS, Ca9-22, and OECM-1, with HSC-3 and Ca9-22 cells showing higher IL-1RA expression than SAS and OECM-1 cells (Supplementary Fig. S1A). A screen for knockdown clones of IL-1RA revealed better efficacy of shIL-1RA#1 and #2 (Supplementary Fig. S1B). Therefore, in subsequent studies, HSC-3 and Ca9-22 cells were selected for gene knockdown of IL-1RA by shIL-1RA#1 and #2 and OECM-1 cells for gene overexpression of full-length IL-1RA (Supplementary Fig. S1C).

Using a clonogenic survival assay, we observed that knockdown of IL-1RA in HSC-3 and Ca9-22 cells reduced cancer cell growth (Fig. 2A), whereas overexpression of IL-1RA in OECM-1 cells increased cancer cell growth (Fig. 2B). Furthermore, knockdown of IL-1RA in HSC-3 cells reduced tumorsphere formation (Fig. 2C), whereas overexpression of IL-1RA in OECM-1 cells increased tumorsphere formation (Fig. 2D). In addition, overexpression of IL-1RA in OECM-1 cells enhanced colony formation on soft agar (Fig. 2E). Analysis of the cell cycle distribution revealed that knockdown of IL-1RA in HSC-3 cells led to increased G0/G1 phase and decreased G2/M phase (Supplementary Fig. S2A), whereas overexpression of IL-1RA in OECM-1 cells had the opposite effect on cell cycle distribution (Supplementary Fig. S2B). Concordantly, the expression of p21 was increased in HSC-3 cells with knockdown IL-1RA but decreased in OECM-1 cells overexpressing IL-1RA (Supplementary Fig. S2C and D). To examine the effect of IL-1RA expression on cancer cell growth under the influence of ionizing radiation (IR), OECM-1 cells were treated with IR followed by a clonogenic survival assay. We found that while ionizing irradiation caused a significant reduction in cell growth in both IL-1RA overexpression and control EV groups, the IL-1RA overexpression group had more surviving cells after IR treatment than the EV controls (Fig. 2F). These data indicate that alteration of IL-1RA expression affects cancer cell growth in OSCC cells, including clonogenic survival and colony formation in soft agar, and tumorsphere formation, which represent the properties of cancer stemness-associated growth [1, 6, 55].

As mitochondrial metabolism has been implicated in oncogenesis, such as the acquisition of cancer stemness properties, metastatic ability, and radioresistance [17, 32, 37], we further examined this possibility in IL-1RA-associated oral cancer cell malignancy. As shown in Fig. 3A, overexpression of IL-1RA in OECM-1 cells increased the oxygen consumption rate (OCR), an indicator of mitochondrial oxidative phosphorylation [35, 43]), as indicated by the upregulated basal OCR, ATP production, and maximal respiration using the Seahorse Analyzer. In concordance with this result, altered basal OCR was observed in HSC-3 cells with knockdown of IL-1RA and in OECM-1 cells overexpressing IL-1RA using a different apparatus (Fig. 3B and C). In contrast, Supplementary Fig. S3A and B show that the effect of differential IL-1RA expression on the extracellular acidification rate (ECAR; an indicator of glycolytic level [35, 43]) was the opposite compared to the results of OCR in Fig. 3B and C. Levels of reactive oxygen species (ROS) were increased by overexpression of IL-1RA in OECM-1 cells and were reduced in the presence of the mitochondrial complex inhibitor rotenone (Fig. 3D), metformin, and phenformin (Fig. 3E) [28, 38]. Furthermore, application of mitochondrial complex inhibitors to OECM-1 cells overexpressing IL-1RA effectively reduced IL-1RA-promoted tumorsphere formation (Fig. 4A and B). IL-1RA-promoted cell migration (Fig. 4C, D, and Supplementary Fig. S4A) and invasion (Fig. 4E) in OECM-1 cells were also reduced by mitochondrial complex inhibitors under circumstances where these inhibitors did not affect the viability of OECM-1 cells within the same time window of treatment (Supplementary Fig. S4B-D). Altogether, these data suggest that IL-1RA-promoted OSCC malignant behaviors, such as tumorspheric growth and cell migration/invasion abilities, are mediated through the mitochondrial metabolic pathway.

Involvement of mitochondrial metabolism in IL-1RA-associated SOX2 expression

To investigate the potential involvement of cancer stemness-related genes in IL-1RA-promoted OSCC malignancy, a screening approach using a PCR array was applied, which showed that four stemness markers, including SOX2, THY1, EGF, and KIT, were most downregulated in HSC-3 cells with IL-1RA knockdown (Fig. 5A and Supplementary Fig. S5). The protein expression levels of these four candidates were further examined by western blotting, which showed that SOX2 was consistently downregulated in HSC-3 and Ca9-22 cells with IL-1RA knockdown (Fig. 5B) and upregulated in OECM-1 cells overexpressing IL-1RA (Fig. 5C). Moreover, IL-1RA-promoted SOX2 expression in OECM-1 cells was reduced in the presence of mitochondrial complex inhibitors (Fig. 5D), suggesting that the effect of IL-1RA on SOX2 expression is mediated through the mitochondrial metabolic pathway.

IL-1RA promoted SOX2 expression via phosphorylation of EGFR and JNK downstream of mitochondrial metabolic pathway

To further investigate the signaling molecules involved in IL-1RA-associated OSCC malignancy, an antibody array was used to screen for the phosphorylation status of kinases affected by IL-1RA. Antibody array screening showed that the expression levels of phosphorylated EGFR (p-EGFR) and phosphorylated JNK (p-JNK) were increased in OECM-1 cells overexpressing IL-1RA (Fig. 6A), which was confirmed by western blotting (Fig. 6B). The increase in p-EGFR and p-JNK induced by IL-1RA overexpression in OECM-1 cells was reduced in the presence of metformin, along with a reduction in EGFR and JNK expression (Fig. 6C). We also found that inhibition of EGFR by gefitinib (an EGFR inhibitor [39]) reduced the expression of p-JNK and SOX2 (Fig. 6D). In addition, a correlation between IL-1RA expression and the expression of p-EGFR (Fig. 6E) and p-JNK (Fig. 6F) was observed in oral tumor tissues from patients with OSCC. Taken together, these data suggest that IL-1RA-associated OSCC malignancy is regulated via EGFR-mediated JNK signaling and SOX2 expression, which lie downstream of the mitochondrial metabolic pathway.

IL-1RA-promoted in vivo tumor growth and p-EGFR/p-JNK/SOX2 expression were suppressed by metformin and cisplatin

The effect of IL-1RA on in vivo tumor growth was evaluated in mice via orthotopic xenografts of OECM-1-luc cells with IL-1RA overexpression or empty vector as a control (Supplementary Fig. S6A), and the effect of treatment with metformin, cisplatin (also known as CDDP; a chemotherapeutic agent [22]), or a combination of both, was further evaluated (Supplementary Fig. S6B). Tumor formation was measured weekly using an in vivo imaging system (IVIS). At the end of the experiment, a higher level of bioluminescence resulting from luciferase activity in the orthotopic xenograft area was observed in mice treated with IL-1RA-overexpressing OECM-1-luc cells (Fig. 7A). Furthermore, the mice treated with metformin, CDDP, or a combination of both showed reduced levels of bioluminescence (Fig. 7A), as well as reduced tumor weight (Fig. 7B) and tumor volume (Supplementary Fig. S6C and D), in which the combined metformin and CDDP treatment exhibited the greatest effect on the inhibition of tumor formation. Immunohistochemical analysis for the expression of IL-1RA, Ki-67 (a cancer proliferation marker [2]), p-EGFR, p-JNK, and SOX2 revealed that IL-1RA expression remained high in the xenograft tumors collected from the mice groups with IL-1RA-overexpressing OECM-1-luc cells (Fig. 7C), whereas the elevated expression of IL-1RA-associated Ki-67, p-EGFR, p-JNK, and SOX2 was reduced in the groups treated with metformin, CDDP, or a combination of both (Fig. 7D-G). These data suggested that IL-1RA promoted in vivo tumor growth with increased p-EGFR, p-JNK, and SOX2 expression, which was suppressed by treatment with metformin or CDDP. In addition, combined treatment with metformin and CDDP may have a greater biological effect than treatment with metformin or CDDP.

IL-1RA, a critical regulator of the IL-1 signaling network, has emerged as a key player in cancer development [5, 34], yet its role in oral cancer malignancy remains to be elucidated. In this study, we highlighted the significance of IL-1RA as a clinical marker of OSCC and explored its biological mechanisms and therapeutic potential. Specifically, our data suggest that elevated IL-1RA levels are associated with adverse OSCC progression and poor patient outcomes. In addition, IL-1RA-promoted OSCC malignancy involved enhanced properties of cancer stemness via a mitochondrial metabolism-associated EGFR/JNK pathway, which may be suppressed by application of mitochondrial complex inhibitors both in vitro and in vivo (Fig. 8).

A novel IL-1RA/EGFR/JNK pathway in oral cancer

EGFR is a transmembrane protein that can transduce signals via translocation to intracellular organelles upon stimulation of various cancer cells, in addition to its canonical receptor tyrosine kinase activity [45, 49, 51]. For example, mitochondrial translocation of EGFR occurs in breast cancer cells in response to EGF stimulation, which subsequently induces the phosphorylation of cytochrome c oxidase subunit II (Cox II) in the mitochondria and enhances cancer cell survival [4, 15]. Mitochondrial translocation of EGFR following EGF stimulation was also observed in non-small cell lung cancer (NSCLC), where increased mitochondrial EGFR upregulates ATP production and enhances the metastatic ability of NSCLC cells [9]. Another study revealed that nuclear translocation of EGFR is regulated by AKT, which is associated with gefitinib resistance in breast cancer cells through enhanced gene expression of the breast cancer-resistant protein (BCRP; also known as ATP-binding cassette subfamily G member 2, ABCG2) [25]. Furthermore, attenuation of estrogen receptor β expression leads to increased EGFR nuclear translocation, which is associated with poor patient outcomes in prostate cancer [57]. Our current study revealed that the activation of EGFR by IL-1RA induced the activation of the JNK pathway and was associated with enhanced malignant behavior of OSCC cells. Clinicopathological analysis supported these findings by revealing a positive correlation between the expression of IL-1RA and phosphorylated EGFR and JNK in OSCC tumor tissues. This newly identified pathway involving IL-1RA/EGFR/JNK indicates that targeting the EGFR pathway may be a potential therapeutic strategy for IL-1RA-associated OSCC malignancy.

OSCC cancer stemness is enhanced by IL-1RA1

Cancer stemness, the enhanced ability for self-proliferation and cancer cell motility, has been shown to correlate with tumor recurrence and metastasis and is a critical area in oral cancer research [33, 46, 56]. Our current study showed that IL-1RA enhanced cancer stemness-associated properties of OSCC cells, such as self-proliferation, anchorage-independent growth, and cell migration/invasion abilities. In addition, IL-1RA promoted the expression of SOX2 in OSCC cells. As SOX2 is a pleiotropic transcription factor and an important hallmark of cancer stemness, with functional roles in regulating tumor growth and metastasis [36, 41], our current findings suggest that increased SOX2 expression may play a role in IL-1RA-promoted OSCC malignancy.

Inhibition of mitochondrial metabolism may abrogate IL-1RA-mediated malignant behaviors

Alteration of cellular energy and redox status via mitochondrial metabolism, a process known as mitochondrial metabolic reprogramming, has been shown to be associated with cancer progression [13, 64]. As a result, targeting mitochondria by inhibiting mitochondrial complexes presents a novel therapeutic strategy for cancer [3, 58]. Our in vitro data showed that IL-1RA overexpression increased the rate of mitochondrial oxidative metabolism and inhibition of mitochondrial complexes, including the use of conventional compound inhibitors (e.g., metformin, phenformin, rotenone, or antimycin A [21, 38]) and novel peptide inhibitors (e.g., CT20 [31] or MITOx20 [24]), suppressed IL-1RA-promoted malignant behaviors of OSCC cells. In particular, the widely used diabetic medication metformin has been found to possess antitumor effects, with cancer risk reduction noted in epidemiological studies [7, 29]. The current study explored the potential of metformin as a therapeutic drug for IL-1RA-associated OSCC malignancy in an orthotopic xenograft mouse model, showing that IL-1RA-promoted tumor growth and activation of EGFR/JNK and SOX2 expression in tumor tissues was reduced by metformin or cisplatin treatment, and the inhibitory effect was further augmented by combination therapy. Together, these data not only suggest a crucial involvement of mitochondrial metabolism in IL-1RA-promoted OSCC growth with participation of the EGFR/JNK/SOX2 pathway but also provide pre-clinical evidence for drug repurposing of metformin as an adjunctive treatment in combination with existing cancer therapies to enhance the therapeutic response [16, 62]. Furthermore, our data may help identify those who would benefit most from such adjunctive treatments, suggesting risk stratification of patients with high IL 1RA tumor tissue levels requiring radiotherapy to additional metformin treatment.

Limitations and future work

While this study has unveiled a novel mechanism underlying IL-1RA-promoted OSCC malignancy through mitochondrial metabolism-associated EGFR/JNK activation and SOX2 expression, there are some limitations inherent in our current data, as well as areas that warrant further investigation. For example, whether IL-1RA-activated EGFR transduces signals via its receptor tyrosine kinase activity or via translocation to intracellular organelles remains to be determined. The genetic control of SOX2 expression induced by IL-1RA in OSCC cells also requires further investigation, such as the potential for transcriptional regulation by c-Jun, which can bind to the promoter region of SOX2 [8, 10]. Furthermore, apart from the effect of metformin on in vivo tumor growth examined in this study, the therapeutic potential of metformin on metastasis in a pre-clinical in vivo model merits further investigation.

The current study demonstrates a critical role for IL-1RA in the pathogenesis of OSCC, and its elevated expression in OSCC tumor tissues may be considered an adverse clinical marker for cancer progression and treatment response. In addition, the newly identified biological mechanisms linking IL-1RA and OSCC malignancy suggest that mitochondrial metabolism plays a crucial role in regulating the malignant behavior of OSCC cells. In particular, the administration of metformin, which has been proposed as a repurposed drug for cancer treatment [7, 16, 29, 62], alleviates IL-1RA-promoted OSCC malignancy through EGFR/JNK signaling and SOX2 expression. These findings not only reveal the impact of IL-1RA and its associated mechanisms on OSCC malignancy but also provide insights for further development of targeted therapies, such as the potential for targeted delivery of metformin to mitochondria [11] or cancer cell surfaces [61], as a treatment strategy for OSCC.

ANOVA

One-way analysis of variance

ECAR

Extracellular acidification rate

EGF

Epidermal growth factor

EGFR

Epidermal growth factor receptor

Empty vector

FBS

Fetal bovine serum

FGF-basic

Fibroblast growth factor-basic

FCCP

Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

IHC

Immunohistochemistry

IL-1

Interleukin-1

IL-1RA

Interleukin-1 receptor antagonist

IL-1RA-OE

Overexpression of IL-1RA

Ionizing radiation

IVIS

in vivo imaging system

JNK

c-Jun N-terminal kinase

OCR

Oxygen consumption rate

OSCC

Oral squamous cell carcinoma

PCR

Polymerase chain reaction

RFU

Relative fluorescence unit

ROS

Reactive oxygen species

SDS-PAGE

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

shIL-1RA

Knockdown of IL-1RA

shLuc

knockdown of firefly luciferase

SOX2

SRY-box transcription factor 2

Ethics approval and consent to participate

This study was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (approval no. KMUH-IRB-20130300 and KMUHIRB-F(I)-20220016). Patient informed consent was waived by the Institutional Review Board due to the retrospective nature of the study. All animal experiments were approved by the Institutional Animal Care and Utilization Committee of Kaohsiung Medical University, Kaohsiung, Taiwan (approval no. 108127).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Funding

This work was supported by grants from the Ministry of Science and Technology (MOST 111-2314-B-037-011-, MOST 111-2314-B-037-020-, MOST 111-2314-B-037-046-) and the Center for Intelligent Drug Systems and Smart Bio-devices (IDS²B) from the Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education, Taiwan. This work was also supported by grants from Kaohsiung Medical University Hospital (KMUH110-0R43, KMUH110-0M65, KMUH111-1M56, KMUH111-1R37, KMUH-DK(A)112001) and Kaohsiung Medical University (KMU-DK(A)112006, NYCU-KMU-112-I005), Taiwan.

Authors’ contributions

S.S.F.Y., Y.M.W., L.P.C., A.C.H., H.D.H.N., Y.K.C., S.C.S.H., S.L., Y.Y.W. conceived and designed the study. S.S.F.Y., Y.M.W., L.P.C., A.C.H., and Y.Y.W. performed experiments. S.S.F.Y., Y.M.W., H.D.H.N., Y.K.C., and Y.Y.W. analyzed and interpreted the data. S.S.F.Y. and Y.Y.W. prepared the manuscript. Y.M.W., L.P.C., A.C.H., H.D.H.N., Y.K.C., S.C.S.H., and S.L. revised the manuscript critically. All authors have reviewed and approved the final manuscript.

Acknowledgements

Not applicable.

Agrez M.V., Kovach J.S. and Lieber M.M. Cell aggregates in the soft agar "human tumour stem-cell assay". Br J Cancer 46(6):880-887, 1982.
Andres-Sanchez N., Fisher D. and Krasinska L. Physiological functions and roles in cancer of the proliferation marker Ki-67. J Cell Sci 135(11), 2022.
Bian C., Zheng Z., Su J., Wang H., Chang S., Xin Y. and Jiang X. Targeting mitochondrial metabolism to reverse radioresistance: an alternative to glucose metabolism. Antioxidants 11(11), 2022.
Boerner J.L., Demory M.L., Silva C. and Parsons S.J. Phosphorylation of Y845 on the epidermal growth factor receptor mediates binding to the mitochondrial protein cytochrome c oxidase subunit II. Mol Cell Biol 24(16):7059-7071, 2004.
Boersma B., Jiskoot W., Lowe P. and Bourquin C. The interleukin-1 cytokine family members: role in cancer pathogenesis and potential therapeutic applications in cancer immunotherapy. Cytokine Growth Factor Rev 62:1-14, 2021.
Brix N., Samaga D., Belka C., Zitzelsberger H. and Lauber K. Analysis of clonogenic growth in vitro. Nat Protoc 16(11):4963-4991, 2021.
Chae Y.K., Arya A., Malecek M.K., Shin D.S., Carneiro B., Chandra S., Kaplan J., Kalyan A., Altman J.K., Platanias L. and Giles F. Repurposing metformin for cancer treatment: current clinical studies. Oncotarget 7(26):40767-40780, 2016.
Chang C.C., Hsu W.H., Wang C.C., Chou C.H., Kuo M.Y., Lin B.R., Chen S.T., Tai S.K., Kuo M.L. and Yang M.H. Connective tissue growth factor activates pluripotency genes and mesenchymal-epithelial transition in head and neck cancer cells. Cancer Res 73(13):4147-4157, 2013.
Che T.F., Lin C.W., Wu Y.Y., Chen Y.J., Han C.L., Chang Y.L., Wu C.T., Hsiao T.H., Hong T.M. and Yang P.C. Mitochondrial translocation of EGFR regulates mitochondria dynamics and promotes metastasis in NSCLC. Oncotarget 6(35):37349-37366, 2015.
Chen H., Jin K., Song J., Zuo Q., Yang H., Zhang Y. and Li B. Functional characterization of the Sox2, c-Myc, and Oct4 promoters. J Cell Biochem 120(1):332-342, 2019.
Cheng G., Zielonka J., Ouari O., Lopez M., McAllister D., Boyle K., Barrios C.S., Weber J.J., Johnson B.D., Hardy M., Dwinell M.B. and Kalyanaraman B. Mitochondria-targeted analogues of metformin exhibit enhanced antiproliferative and radiosensitizing effects in pancreatic cancer cells. Cancer Res 76(13):3904-3915, 2016.
Cheng Y., Li S., Gao L., Zhi K. and Ren W. The molecular basis and therapeutic aspects of cisplatin resistance in oral squamous cell carcinoma. Front Oncol 11:761379, 2021.
Choudhury F.K. Mitochondrial redox metabolism: the epicenter of metabolism during cancer progression. Antioxidants 10(11), 2021.
Coletta R.D., Yeudall W.A. and Salo T. Grand challenges in oral cancers. Front Oral Health 1:3, 2020.
Demory M.L., Boerner J.L., Davidson R., Faust W., Miyake T., Lee I., Huttemann M., Douglas R., Haddad G. and Parsons S.J. Epidermal growth factor receptor translocation to the mitochondria: regulation and effect. J Biol Chem 284(52):36592-36604, 2009.
Deng J., Peng M., Wang Z., Zhou S., Xiao D., Deng J., Yang X., Peng J. and Yang X. Novel application of metformin combined with targeted drugs on anticancer treatment. Cancer Sci 110(1):23-30, 2019.
Diehn M., Cho R.W., Lobo N.A., Kalisky T., Dorie M.J., Kulp A.N., Qian D., Lam J.S., Ailles L.E., Wong M., Joshua B., Kaplan M.J., Wapnir I., Dirbas F.M., Somlo G., Garberoglio C., Paz B., Shen J., Lau S.K., Quake S.R., Brown J.M., Weissman I.L. and Clarke M.F. Association of reactive oxygen species levels and radioresistance in cancer stem cells. Nature 458(7239):780-783, 2009.
Dinarello C.A. Interleukin-1, interleukin-1 receptors and interleukin-1 receptor antagonist. Int Rev Immunol 16(5-6):457-499, 1998.
Du M., Nair R., Jamieson L., Liu Z. and Bi P. Incidence trends of lip, oral cavity, and pharyngeal cancers: global burden of disease 1990-2017. J Dent Res 99(2):143-151, 2020.
Eisenberg S.P., Evans R.J., Arend W.P., Verderber E., Brewer M.T., Hannum C.H. and Thompson R.C. Primary structure and functional expression from complementary DNA of a human interleukin-1 receptor antagonist. Nature 343(6256):341-346, 1990.
Fisher N., Meunier B. and Biagini G.A. The cytochrome bc₁ complex as an antipathogenic target. FEBS Lett 594(18):2935-2952, 2020.
Forgie B.N., Prakash R. and Telleria C.M. Revisiting the anti-cancer toxicity of clinically approved platinating derivatives. Int J Mol Sci 23(23), 2022.
Fujiwaki R., Iida K., Nakayama K., Kanasaki H., Hata K., Katabuchi H., Okamura H. and Miyazaki K. Clinical significance of interleukin-1 receptor antagonist in patients with cervical carcinoma. Gynecol Oncol 89(1):77-83, 2003.
Giddings E.L., Champagne D.P., Wu M.H., Laffin J.M., Thornton T.M., Valenca-Pereira F., Culp-Hill R., Fortner K.A., Romero N., East J., Cao P., Arias-Pulido H., Sidhu K.S., Silverstrim B., Kam Y., Kelley S., Pereira M., Bates S.E., Bunn J.Y., Fiering S.N., Matthews D.E., Robey R.W., Stich D., D'Alessandro A. and Rincon M. Mitochondrial ATP fuels ABC transporter-mediated drug efflux in cancer chemoresistance. Nat Commun 12(1):2804, 2021.
Huang W.C., Chen Y.J., Li L.Y., Wei Y.L., Hsu S.C., Tsai S.L., Chiu P.C., Huang W.P., Wang Y.N., Chen C.H., Chang W.C., Chang W.C., Chen A.J., Tsai C.H. and Hung M.C. Nuclear translocation of epidermal growth factor receptor by Akt-dependent phosphorylation enhances breast cancer-resistant protein expression in gefitinib-resistant cells. J Biol Chem 286(23):20558-20568, 2011.
Iizuka N., Hazama S., Hirose K., Abe T., Tokuda N., Fukumoto T., Tangoku A. and Oka M. Interleukin-1 receptor antagonist mRNA expression and the progression of gastric carcinoma. Cancer Lett 142(2):179-184, 1999.
Iwagaki H., Hizuta A. and Tanaka N. Interleukin-1 receptor antagonists and other markers in colorectal cancer patients. Scand J Gastroenterol 32(6):577-581, 1997.
Koopman W.J., Nijtmans L.G., Dieteren C.E., Roestenberg P., Valsecchi F., Smeitink J.A. and Willems P.H. Mammalian mitochondrial complex I: biogenesis, regulation, and reactive oxygen species generation. Antioxid Redox Signal 12(12):1431-1470, 2010.
Kourelis T.V. and Siegel R.D. Metformin and cancer: new applications for an old drug. Med Oncol 29(2):1314-1327, 2012.
Kurzrock R. Cytokine deregulation in cancer. Biomed Pharmacother 55(9-10):543-547, 2001.
Lee M.W., Bassiouni R., Sparrow N.A., Iketani A., Boohaker R.J., Moskowitz C., Vishnubhotla P., Khaled A.S., Oyer J., Copik A., Fernandez-Valle C., Perez J.M. and Khaled A.R. The CT20 peptide causes detachment and death of metastatic breast cancer cells by promoting mitochondrial aggregation and cytoskeletal disruption. Cell Death Dis 5(5):e1249, 2014.
Lee S.Y., Jeong E.K., Ju M.K., Jeon H.M., Kim M.Y., Kim C.H., Park H.G., Han S.I. and Kang H.S. Induction of metastasis, cancer stem cell phenotype, and oncogenic metabolism in cancer cells by ionizing radiation. Mol Cancer 16(1):10, 2017.
Liu Y., Yang M., Luo J. and Zhou H. Radiotherapy targeting cancer stem cells "awakens" them to induce tumour relapse and metastasis in oral cancer. Int J Oral Sci 12(1):19, 2020.
Mantovani A., Barajon I. and Garlanda C. IL-1 and IL-1 regulatory pathways in cancer progression and therapy. Immunol Rev 281(1):57-61, 2018.
Marchetti P., Fovez Q., Germain N., Khamari R. and Kluza J. Mitochondrial spare respiratory capacity: mechanisms, regulation, and significance in non-transformed and cancer cells. FASEB J 34(10):13106-13124, 2020.
Mirzaei S., Paskeh M.D.A., Entezari M., Mirmazloomi S.R., Hassanpoor A., Aboutalebi M., Rezaei S., Hejazi E.S., Kakavand A., Heidari H., Salimimoghadam S., Taheriazam A., Hashemi M. and Samarghandian S. SOX2 function in cancers: association with growth, invasion, stemness and therapy response. Biomed Pharmacother 156:113860, 2022.
Moindjie H., Rodrigues-Ferreira S. and Nahmias C. Mitochondrial metabolism in carcinogenesis and cancer therapy. Cancers 13(13), 2021.
Murai M. and Miyoshi H. Current topics on inhibitors of respiratory complex I. Biochim Biophys Acta 1857(7):884-891, 2016.
Ono M. and Kuwano M. Molecular mechanisms of epidermal growth factor receptor (EGFR) activation and response to gefitinib and other EGFR-targeting drugs. Clin Cancer Res 12(24):7242-7251, 2006.
Parekh D.J., Ankerst D.P., Baillargeon J., Higgins B., Platz E.A., Troyer D., Hernandez J., Leach R.J., Lokshin A. and Thompson I.M. Assessment of 54 biomarkers for biopsy-detectable prostate cancer. Cancer Epidemiol Biomarkers Prev 16(10):1966-1972, 2007.
Porter L. and McCaughan F. SOX2 and squamous cancers. Semin Cancer Biol 67(Pt 1):154-167, 2020.
Rivera C. Essentials of oral cancer. Int J Clin Exp Pathol 8(9):11884-11894, 2015.
Schmidt C.A., Fisher-Wellman K.H. and Neufer P.D. From OCR and ECAR to energy: perspectives on the design and interpretation of bioenergetics studies. J Biol Chem 297(4):101140, 2021.
Shiiba M., Saito K., Yamagami H., Nakashima D., Higo M., Kasamatsu A., Sakamoto Y., Ogawara K., Uzawa K., Takiguchi Y. and Tanzawa H. Interleukin-1 receptor antagonist (IL1RN) is associated with suppression of early carcinogenic events in human oral malignancies. Int J Oncol 46(5):1978-1984, 2015.
Sigismund S., Avanzato D. and Lanzetti L. Emerging functions of the EGFR in cancer. Mol Oncol 12(1):3-20, 2018.
Sinha N., Mukhopadhyay S., Das D.N., Panda P.K. and Bhutia S.K. Relevance of cancer initiating/stem cells in carcinogenesis and therapy resistance in oral cancer. Oral Oncol 49(9):854-862, 2013.
Sotgia F., Fiorillo M. and Lisanti M.P. Hallmarks of the cancer cell of origin: comparisons with "energetic" cancer stem cells (e-CSCs). Aging 11(3):1065-1068, 2019.
Sung H., Ferlay J., Siegel R.L., Laversanne M., Soerjomataram I., Jemal A. and Bray F. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 71(3):209-249, 2021.
Tomas A., Futter C.E. and Eden E.R. EGF receptor trafficking: consequences for signaling and cancer. Trends Cell Biol 24(1):26-34, 2014.
Vig N., Mackenzie I.C. and Biddle A. Phenotypic plasticity and epithelial-to-mesenchymal transition in the behaviour and therapeutic response of oral squamous cell carcinoma. J Oral Pathol Med 44(9):649-655, 2015.
Wang Y.N. and Hung M.C. Nuclear functions and subcellular trafficking mechanisms of the epidermal growth factor receptor family. Cell Biosci 2(1):13, 2012.
Wang Y.Y., Chen H.D., Lo S., Chen Y.K., Huang Y.C., Hu S.C., Hsieh Y.C., Hung A.C., Hou M.F. and Yuan S.F. Visfatin enhances breast cancer progression through CXCL1 induction in tumor-associated macrophages. Cancers 12(12), 2020.
Wang Y.Y., Chen Y.K., Lo S., Chi T.C., Chen Y.H., Hu S.C., Chen Y.W., Jiang S.S., Tsai F.Y., Liu W., Li R.N., Hsieh Y.C., Huang C.J. and Yuan S.F. MRE11 promotes oral cancer progression through RUNX2/CXCR4/AKT/FOXA2 signaling in a nuclease-independent manner. Oncogene 40(20):3510-3532, 2021.
Wang Y.Y., Hung A.C., Wu Y.C., Lo S., Chen H.D., Chen Y.K., Hsieh Y.C., Hu S.C., Hou M.F. and Yuan S.F. ADSCs stimulated by resistin promote breast cancer cell malignancy via CXCL5 in a breast cancer coculture model. Sci Rep 12(1):15437, 2022.
Weiswald L.B., Bellet D. and Dangles-Marie V. Spherical cancer models in tumor biology. Neoplasia 17(1):1-15, 2015.
Wolmarans E., Boy S.C., Nel S., Mercier A.E. and Pepper M.S. Cancer stem cells in head and neck carcinomas: identification and possible therapeutic implications. Adv Exp Med Biol 1083:89-102, 2018.
Wu W.F., Wang L., Spetsieris N., Boukovala M., Efstathiou E., Brossner C., Warner M. and Gustafsson J.A. Estrogen receptor β and treatment with a phytoestrogen are associated with inhibition of nuclear translocation of EGFR in the prostate. Proc Natl Acad Sci U S A 118(13), 2021.
Yan L., Tan Y., Chen G., Fan J. and Zhang J. Harnessing metabolic reprogramming to improve cancer immunotherapy. Int J Mol Sci 22(19), 2021.
Yang Y.F., Wang Y.Y., Hsiao M., Lo S., Chang Y.C., Jan Y.H., Lai T.C., Lee Y.C., Hsieh Y.C. and Yuan S.F. IMPAD1 functions as mitochondrial electron transport inhibitor that prevents ROS production and promotes lung cancer metastasis through the AMPK-Notch1-HEY1 pathway. Cancer Lett 485:27-37, 2020.
Yuan S.F., Hung A.C., Hsu C.W., Lan T.H., Su C.W., Chi T.C., Chang Y.C., Chen Y.K. and Wang Y.Y. CD44 mediates oral squamous cell carcinoma-promoting activity of MRE11 via AKT signaling. J Pers Med 12(5), 2022.
Zhang F., Liu W., Long Y. and Peng H. Targeted delivery of metformin against lung cancer cells via hyaluronan-modified mesoporous silica nanoparticles. Appl Biochem Biotechnol, 2023.
Zhang H.H. and Guo X.L. Combinational strategies of metformin and chemotherapy in cancers. Cancer Chemother Pharmacol 78(1):13-26, 2016.
Zhang J. and Zhang Q. Using Seahorse machine to measure OCR and ECAR in cancer cells. Methods Mol Biol 1928:353-363, 2019.
Zheng J. Energy metabolism of cancer: glycolysis versus oxidative phosphorylation. Oncol Lett 4(6):1151-1157, 2012.

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IL-1RA promotes oral squamous cell carcinoma malignancy through mitochondrial metabolism-mediated EGFR/JNK/SOX2 pathway

Status:

Journal Publication

Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

Background

Methods

Patient samples

Cell culture

Gene knockdown and overexpression

Clonogenic survival assay

Tumorsphere formation assay

Soft agar colony formation assay

XTT cell viability assay

Transwell cell migration and invasion assays

Measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)

Measurement of reactive oxygen species (ROS) formation

Polymerase chain reaction (PCR) array

Proteome array

Western blot

Immunohistochemistry (IHC)

Animal study

Statistical analysis

Results

Clinical association of IL-1RA expression with OSCC progression and patient outcomes

IL-1RA promoted in vitro cancer cell growth and migration/invasion abilities via the mitochondrial metabolic pathway

Involvement of mitochondrial metabolism in IL-1RA-associated SOX2 expression

IL-1RA promoted SOX2 expression via phosphorylation of EGFR and JNK downstream of mitochondrial metabolic pathway

IL-1RA-promoted in vivo tumor growth and p-EGFR/p-JNK/SOX2 expression were suppressed by metformin and cisplatin

Discussion

A novel IL-1RA/EGFR/JNK pathway in oral cancer

OSCC cancer stemness is enhanced by IL-1RA1

Inhibition of mitochondrial metabolism may abrogate IL-1RA-mediated malignant behaviors

Limitations and future work

Conclusions

Abbreviations

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1