Glucose upregulates amphiregulin in oral dysplastic keratinocytes: A potential role in diabetes-associated oral carcinogenesis

Abstract

Background: Compelling evidence implicates diabetes-associated hyperglycemia as a promoter of tumor progression in oral potentially malignant disorders (OPMD). Yet, information on hyperglycemia-induced cell signaling networks in oral oncology remains limited. Our group recently reported that glucose-rich conditions significantly enhance oral dysplastic keratinocyte viability and migration through epidermal growth factor receptor (EGFR) activation, a pathway strongly linked to oral carcinogenesis. Here, we investigated the basal metabolic phenotype in these cells and whether specific glucose-responsive EGFR ligands mediate these responses.

Methods: Cell energy phenotype and lactate concentration were evaluated via commercially available assays. EGFR ligands in response to normal (5 mM) or high (20 mM) glucose were analyzed by quantitative real-time PCR, ELISA, and western blotting. Cell viability and migration assays were performed in the presence of pharmacological inhibitors or RNA interference.

Results: When compared to normal keratinocytes, basal glycolysis in oral dysplastic keratinocytes was significantly elevated. In highly glycolytic cells, high glucose-activated EGFR increasing viability and migration. Notably, we identified amphiregulin (AREG) as the predominant glucose-induced EGFR ligand. Indeed, enhanced cell migration in response to high glucose was blunted by EGFR inhibitor cetuximab and AREG siRNA. Conversely, AREG treatment under normal glucose conditions significantly increased cell viability, migration, lactate levels, and expression of glycolytic marker pyruvate kinase M2.

Conclusion: These novel findings point to AREG as a potential high glucose-induced EGFR activating ligand in highly glycolytic oral dysplastic keratinocytes. Future studies are warranted to gain more insight into the role of AREG in hyperglycemia-associated OPMD tumor progression.

Keywords: EGFR; Oral potentially malignant disorders; amphiregulin; hyperglycemia; oral carcinogenesis.

Figure 1.. Elevated glucose concentration induces viability and migration of highly glycolytic oral dysplastic keratinocytes.

(A) Metabolic potential of indicated cells tested with the Agilent Seahorse XFp Cell Energy Phenotype assay. Open square-baseline; closed square-stress condition. OCR: oxygen consumption rate; ECAR: extracellular acidification. (B) Basal glycolysis analyzed with Seahorse glycolytic rate assay in indicated cells (glycoPER: glycolytic proton efflux rate). Data represent mean ± S.E.M., *p<0.001. (C) Whole cell extracts from PGK, LEUK1 and DOK were subjected to western blotting for GLUT1 and HKII. β-Actin served as loading control. (D) DOK were exposed to normal (5 mM) or high glucose (20 mM)-containing media for 72 hours. Then, MTS reagent was added to evaluate cell viability. Data represent mean ± S.E.M. *p<0.0001 vs. 5 mM glucose. (E) Representative image showing DOK migration following exposure to normal or high glucose. Acellular gap closure was quantified relative to time 0 as depicted by the dotted white line (F). Data represent mean ± S.E.M. *p<0.01 vs. 5 mM glucose.

Figure 2.. High glucose markedly enhances cell migration in an EGFR-dependent manner.

(A) DOK were exposed to normal or high glucose-containing media for 48 hours. Western blotting for pEGFR (Y1173) and total EGFR was performed in whole cell lysates. β-actin served as loading control. (B) DOK pre-treated with 10 μg/ml cetuximab were exposed to normal or high glucose-containing media for 72 hours. MTS reagent was then added to evaluate cell viability. Data represent mean ± S.E.M. *p<0.0001, **p<0.05. (C) Representative image showing cell migration following exposure to high glucose for 20 hours in the presence or absence of cetuximab and (D) quantification of acellular gap closure relative to time 0 as depicted by the white dotted line in C. Data represent mean ± S.E.M. *p<0.001 vs. vehicle-treated control.

Figure 3.. Amphiregulin: a predominant high glucose-induced EGFR ligand.

(A) Gene expression analysis of seven cognate EGFR ligands by RT-qPCR in DOK following a 24-hour exposure to normal or high glucose. Data represent mean ± S.E.M. *p<0.0003 when compared to 5 mM glucose-induced gene expression; X: undetected; EGF: epidermal growth factor; TGFα: transforming growth factor alpha; HB-EGF: heparin binding-EGF; AREG: amphiregulin; EPGN: epigen; BTC: betacellulin; EREG: epiregulin. (B) TGFα and AREG mRNA levels relative to β-actin in DOK exposed to normal or high glucose for 24 hours. Data represent mean ± S.E.M. *p<0.0003. (C) Soluble AREG was quantified by ELISA in conditioned medium from DOK exposed for 24 hours to normal or high glucose in the presence or absence of 10 μg/ml cetuximab. Data represent mean ± S.E.M. *p<0.01; **p<0.05; ***p<0.0001; n.s.: no statistical significance.

Figure 4.. AREG inhibition significantly decreases high glucose-induced cell migration and aerobic glycolysis.

(A) Western blotting for AREG, phosphorylated EGFR (pEGFR) and total EGFR in whole cell lysates from DOK untransfected or transfected with scrambled control (Sc) or AREG siRNA (50 nM and 100 nM). Cells were exposed to normal or high glucose for 24 hours. β-actin served as loading control. (B) DOK were either left untransfected (−) or transfected with scrambled control (Sc) or AREG siRNA (50 nM). Cells were exposed the next day to normal or high glucose for 48 hours. CCK-8 reagent was added to evaluate cell viability. Data represent mean ± S.E.M. *p<0.0001; **p<0.001; n.s.: no statistical significance among 20 mM glucose groups. (C) Representative image of wound closure migration assay in DOK untransfected or transfected with Sc or AREG siRNA (50 nM) that were exposed to normal or high glucose for 20 hours, and (D) quantification of acellular gap closure relative to time 0 as depicted by the white dotted line in B. Data represent mean ± S.E.M. *p<0.0001; **p<0.05; n.s.: no statistical significance. (E) Lactate concentration in conditioned media from DOK untransfected or transfected with Sc or AREG siRNA (50 nM) exposed to normal or high glucose for 48 hours. Data represent mean ± S.E.M. *p<0.0001; n.s.: no statistical significance. (F) Western blotting for AREG and PKM2 in whole cell lysates from DOK untransfected or transfected with Sc or AREG siRNA (50 nM and 100 nM) and exposed to normal or high glucose for 24 hours. β-actin served as loading control.

Figure 5.. Recombinant human AREG stimulates cell viability, cell migration, and aerobic glycolysis under normal glucose conditions.

(A) DOK were treated for 72 hours with increasing concentrations of recombinant human AREG under normal glucose culture conditions. MTS reagent was added to evaluate cell viability. Data represent mean ± S.E.M. When compared to vehicle control, *p<0.05 and **p<0.001. (B) Representative image showing significant DOK migration following treatment with AREG for 20 hours. (C) Quantification of gap closure relative to time 0. Data represent mean ± S.E.M.; *p<0.0001. (D) Lactate concentration in conditioned media following AREG treatment. When compared within same time point, *p<0.003, **p<0.0001, n.s.: no statistical significance. (E) Cells were treated with vehicle control or AREG (2.5, 5 and 10 ng/ml) for 24 hours. Western blotting for pEGFR, total EGFR and PKM2 with β-Actin as loading control.

Figure 6.. Proposed model for the effects of high glucose-induced AREG/EGFR activation on promoting aerobic glycolysis and increasing cell migration in oral dysplastic keratinocytes.

High glucose uptake by GLUT1 transporters upregulates AREG expression and secretion leading to EGFR activation. AREG/EGFR signaling sustains a glycolytic phenotype as evidenced by increased PKM2 expression and lactate production to enhance cell migration in a yet to be elucidated mechanisms.