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. Author manuscript; available in PMC: 2022 Oct 1.
Published in final edited form as: J Oral Pathol Med. 2021 Aug 23;50(9):919–926. doi: 10.1111/jop.13235

Fatty acid synthase mediates high glucose-induced EGFR activation in oral dysplastic keratinocytes

David J Wisniewski 1, Tao Ma 1, Abraham Schneider 1,2
PMCID: PMC8530891  NIHMSID: NIHMS1733791  PMID: 34402100

Abstract

Background:

Recent studies point to the epidermal growth factor receptor (EGFR) as a critical mediator of type 2 diabetes mellitus (T2DM)-induced renal, cardiac and ocular complications. T2DM is considered a systemic contributing factor in oral carcinogenesis. Similarly, increased EGFR gene copy number and protein expression strongly predict tumor progression. Yet, the impact of hyperglycemia on EGFR activity in oral potentially malignant disorders remains unclear. We recently reported that fatty acid synthase (FASN), a key de novo lipogenic enzyme, mediates EGFR activation in nicotine-treated oral dysplastic keratinocytes. While in non-malignant tissues FASN expression is extremely low, it is frequently upregulated in several cancers, including oral squamous cell carcinoma. The present study was carried out to investigate whether high glucose conditions trigger pro-oncogenic responses in oral dysplastic keratinocytes via FASN-mediated EGFR activation.

Methods:

Cell viability and migration of oral dysplastic keratinocytes were evaluated when exposed to normal (5 mM) or high (20 mM) glucose conditions in the presence of FASN and EGFR inhibitors. Western blotting was also performed to assess changes in FASN protein expression and EGFR activation.

Results:

Oral dysplastic keratinocytes exposed to high glucose led to EGFR activation in a FASN-dependent manner. Likewise, high glucose significantly enhanced cell viability and migration in a FASN/EGFR-mediated fashion. Notably, EGFR inhibition by the anti-EGFR monoclonal antibody cetuximab significantly reduced the proliferation of FASN-overexpressing oral dysplastic keratinocytes.

Conclusion:

These novel findings suggest that FASN may act as a key targetable metabolic regulator of glucose-induced EGFR oncogenic signaling in oral potentially malignant disorders.

Keywords: FASN, EGFR, diabetes mellitus, hyperglycemia, oral potentially malignant disorders

1. INTRODUCTION

Type 2 diabetes mellitus (T2DM) is an emergent contributing systemic factor in oral carcinogenesis1. In the United States, the prevalence of diabetes has considerably increased in recent years, currently affecting more than 34 million individuals2. Although the independent role of T2DM-associated hyperglycemia in cancer remains unclear, it cannot be ruled out1,3,4. Hyperglycemia stimulates cell proliferation, growth factor signaling and chemoresistance in various cancer types5–8. However, studies into the effects of hyperglycemia in oral potentially malignant disorders are limited1,9. A recent study involving 133 oral leukoplakia patients with malignant transformation, and 266 patients with untransformed oral leukoplakia, reported hyperglycemia as one of the most significant factors promoting neoplastic transformation9.

Fatty acid synthase (FASN), the key enzyme responsible for endogenous fatty acid biosynthesis via de novo lipogenesis is overexpressed in multiple cancer types and correlates with increasing tumor burden and poor prognosis10. In particular, high FASN protein expression impacts oral squamous cell carcinoma (OSCC) grade and recurrence rate and contributes to tumor cell proliferation and migration11,12. FASN is also linked to Epidermal Growth Factor Receptor (EGFR) activation, a pro-oncogenic mechanism strongly associated with OSCC progression13–15. Intriguingly, T2DM systemic complications including kidney failure, cardiac dysfunction and diabetic retinopathy appear to be mediated by EGFR-activated mechanisms16–19. This implies that in potentially malignant oral lesions, hyperglycemia might be a systemic risk factor promoting EGFR-dependent pro-oncogenic signaling.

Since hyperglycemia upregulates FASN expression in hepatocytes20, we were prompted to explore whether high glucose could induce pro-oncogenic responses in oral dysplastic keratinocytes by activating EGFR in a FASN-dependent manner, as we recently reported in similar cells treated with nicotine15. We found that high glucose significantly increased cell viability and migration via FASN-mediated EGFR activation. These results suggest that under hyperglycemic conditions, FASN could act as key metabolic regulator of EGFR-dependent oncogenic signaling to promote the progression of potentially malignant oral lesions.

2. MATERIALS AND METHODS

2.1. Cell culture and reagents.

Human-derived dysplastic oral keratinocytes (DOK; Millipore Sigma, St. Louis, MO) were grown and maintained in high glucose Dulbecco’s modified Eagle’s media (DMEM, Millipore Sigma) supplemented with 10% fetal bovine serum (FBS, Millipore Sigma), 1% L-glutamine (Gibco, Grand Island, NY), 0.05% hydrocortisone (Millipore Sigma), 1% antibiotic/antimycotic (AA) (Millipore Sigma). Human-derived Leuk-1 cells (provided by Dr. Hening Ren, University of Maryland, Baltimore) and the NOKSI immortalized normal oral keratinocyte cell line (provided by Dr. J. Silvio Gutkind, University of California, San Diego) were cultured and maintained in Keratinocyte Serum Free medium with growth factor supplement (Gibco) and 1% AA. Cells were cultured at 37°C and 5% CO2. Before experiments, cells were serum starved overnight in low-glucose, serum-free DMEM (Gibco). FASN inhibitors cerulenin and TVB-3166 (Millipore Sigma), or the anti-EGFR monoclonal antibody cetuximab (University of Maryland Greenebaum Comprehensive Cancer Center Pharmacy) were used to pre-treat cells for 2 hours. Then, cells were continuously treated with either normal glucose (5 mM) or high glucose (20 mM) DMEM supplemented with 1% FBS, along with inhibitor treatment. We prepared 5 mM and 20 mM glucose DMEM, respectively, by adding the appropriate concentration of D-(+)-glucose (Sigma) to glucose-free, serum-free DMEM (Gibco). These experimental conditions were selected since 5 mM is within the normal range of fasting blood glucose levels in healthy adults. Also, uncontrolled hyperglycemia in humans rarely rises above 15–20 mM as well as in the obese spontaneous mutation (ob/ob) mouse model5–7,21,22.

2.2. Western blotting.

M-PER mammalian protein extraction reagent supplemented with protease and phosphatase inhibitor cocktail was used for cell lysis (Thermo Scientific, Rockford, IL). Western blotting was conducted as previously described15. The following primary antibodies were used: rabbit monoclonal against FASN (#3180, 1:1000) and rabbit monoclonal against phospho-EGFR Y1173 (#4407, 1:500) (Cell Signaling, Danvers, MA). Rabbit polyclonal against total EGFR (#sc-03, 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA) and mouse monoclonal against β-Actin (#A5441, 1:20000, Millipore Sigma) were also used as primary antibodies. Secondary antibodies included peroxidase labeled goat anti-rabbit IgG (#5220-0458, 1:10000) and goat anti-mouse IgG (#5450-0011, 1:20000 (Seracare, Milford, MA).

2.3. Wound closure migration assay.

Cells were plated to confluency in complete media, serum deprived overnight, and the indicated inhibitors were added for a 2-hour pre-treatment. Cell monolayer was then scratched with a pipet tip, followed by PBS washing. Treatments were initiated for the indicated times with normal or high glucose with 1% FBS and/or specific inhibitors. Magnification images (10X) were captured with a digital camera attached to an Axiovert 100 A microscope. The acellular gap was measured by area with ImageJ software (National Institutes of Health, Bethesda, MD) at the time of scratching (time 0) and at the final time point. Percentage of gap closure was calculated as ([Initial Area-Final Area]/Initial Area) x10015.

2.4. Oris™ cell migration assay.

This assay was carried out following the manufacturer’s instructions (Platypus Technologies, Madison, WI). Briefly, cells were seeded, serum starved, and pre-treated with 10 μg/mL cetuximab for 2 hours. Then, cells were cultured under normal or high glucose conditions and inhibitor treatment for 18 hours once the cell stopper was removed. Images were acquired and quantified similarly to the aforementioned scratch assay.

2.5. Cell viability assay.

DOK (1,500 cells per well) and Leuk-1 (5,000 cells per well) were plated in a 96-well plate in complete media and then serum deprived overnight. Cells were pre-treated for 2 hours with inhibitors as indicated, and then treated with normal or high glucose DMEM with 1% FBS for 5 days. CellTiter 96® AQueous One Solution (MTS) reagent (Promega, Madison, WI) was added to each well and incubated at 37°C for 1–4 hours. Cell viability was determined with a Biotek Epoch spectrophotometer by reading absorbance at 490 nm.

2.6. FASN overexpression.

We recently reported the generation of a stable FASN-overexpressing DOK cell line15. Briefly, cells were co-transfected by nucleofection according to the protocol provided by the manufacturer (Amaxa Biosystems, Köln, Germany) with plasmids encoding human FASN (pCMV-SPORT6 vector containing FASN cDNA, #MHS6278-202759913, Dharmacon, Lafayette, CO) and a puromycin resistance vector (pPUR vector, #631601, Takara, Mountain View, CA). Western blotting confirmed high FASN expression. pPUR-transfected DOK cells were used as negative controls.

2.7. Imaging-based cell counting.

DOK pPUR- or FASN-overexpressing cells (5,000 cells per well) were plated in a 96- well plate in complete media and then serum starved overnight. Cells were pre-treated the next day with 10 μg/mL cetuximab for 2 hours and incubated in normal glucose DMEM with 1% FBS for 5 days. Images of individual wells were taken with the Biotek Cytation 5 imaging system (Bioteck, Winooski, VT) at the beginning (day 0) and at the end (day 5) of treatment with a high-contrast bright field 4x objective. Cell counting analysis was performed by using a protocol for label-free cell counting provided by Biotek. Change in cell number for each individual well between 0 and 5 days was calculated and normalized relative to vehicle control in pPUR-transfected DOK cells.

2.8. Statistics.

All data are expressed as the mean value ± standard error of the mean (S.E.M.). Results are considered statistically significant when *p<0.05, **p<0.01 or ***p<0.001 by one-way analysis of variance (ANOVA) followed by Tukey multiple comparison test. In experiments containing only two data sets, we conducted analysis by unpaired Student’s t test. Statistical analyses were performed on experiments in triplicate by using the Prism 6.0 biostatistics program (GraphPad Software).

3. RESULTS

3.1. FASN is upregulated in oral dysplastic keratinocytes exposed to high glucose.

First, we determined whether protein expression levels of FASN were affected in cells cultured under high glucose conditions. Initially, Leuk-1 and DOK cells were exposed to normal (5 mM) or high glucose (20 mM) for 24 hours (Figures 1A and 1B). When compared to cells cultured in normal glucose, high glucose conditions markedly increased FASN expression. As shown in Figures 1C and 1D, a time-dependent upregulation in high glucose-induced FASN expression was also evident in both cell lines.

Figure 1. High glucose upregulates FASN protein expression in oral dysplastic keratinocytes.

Figure 1.

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(A,B) Cells were exposed to normal or high glucose-containing media for 24 hours, or for the indicated time points (C, D). Western blotting was conducted for FASN and β-Actin, which served as loading control.

3.2. High glucose supports oral dysplastic keratinocyte viability in a FASN-dependent manner.

Next, we evaluated the effects of high glucose on cell viability, a measurement reflective of increased cell number (Figures 2A and 2B). Cell viability was significantly enhanced over a 5-day period in response to high glucose (p<0.001). Further, in both cell lines, FASN inhibition by cerulenin or TVB-3166 abrogated the apparent cell growth-stimulating effects of high glucose (p<0.001). It is well-known that FASN inhibitors alter fatty acid biosynthesis by blocking FASN enzymatic activity, but not its protein expression10,23. Accordingly, we observed no major alterations in FASN expression when cells were cultured in the presence of cerulenin or TVB-3166 (Figures 2C and 2D). These observations imply that a functional FASN activity is required to support high glucose-induced oral dysplastic keratinocyte proliferation.

Figure 2. FASN supports high glucose-induced oral dysplastic keratinocyte viability.

Figure 2.

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(A,B) Cells were pre-treated with 10 μM cerulenin (Cer) or TVB-3166 (TVB) for 2 hours, then cultured in normal or high glucose-containing media for 5 days followed by the addition of MTS reagent. Data represent mean ± S.E.M. ***p<0.001 when compared to indicated treatment. (C, D) FASN immunoblotting following exposure to high glucose for 24 hours in the absence (-) or presence of 10 μM cerulenin (Cer) or TVB-3166 (TVB). β-actin, loading control.

3.3. FASN mediates high glucose-induced oral dysplastic keratinocyte migration.

Next, we evaluated the effects of high glucose-induced FASN on cell motility. We analyzed Leuk-1 and DOK migration when cultured under normal and high glucose concentrations over a 20-hour period in the presence of FASN inhibitors TVB-3166 and cerulenin (Figure 3). High glucose significantly increased cell migration on Leuk-1 (p<0.05) and DOK (p<0.01). Furthermore, FASN inhibitors markedly diminished these responses.

Figure 3. FASN mediates high glucose-induced oral dysplastic keratinocyte migration.

Figure 3.

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(A,B) Cells were pre-treated with TVB-3166 (TVB) or cerulenin (Cer) for 2 hours. Following pre-treatment, cells were cultured in normal or high glucose-containing media. Gap closure was quantified as the change in acellular area for (B) Leuk-1 and (E) DOK cells. Leuk-1 (C) and DOK (F) cells were treated with normal or high glucose-containing media followed by incubation with MTS reagent. Fold change relative to day 0 was calculated to determine total change in viability over 24 hours. Data represent mean ± S.E.M. *p<0.05 and **p<0.01 when comparing 3 or more groups. Not statistically significant (n.s) indicates p>0.05.

Altogether, these data suggest that under high glucose conditions, cell viability and motility are dependent on the proper function of FASN. Interestingly, high glucose treatment for 24 hours did not enhance the relative number of Leuk-1 (Figure 3C) or DOK (Figure 3F) cells, implying the observed 20-hour cell migratory responses were independent of cell doubling and proliferation. In addition, no significant differences were found in cell migration when normal oral keratinocytes were cultured under normal versus high glucose conditions (Supplemental Figure 1).

3.4. FASN inhibition downregulates high glucose-induced EGFR phosphorylation.

Based on our recent findings linking FASN to EGFR activation in nicotine-stimulated oral dysplastic keratinocytes15, we then explored whether high glucose-induced FASN affected EGFR activation. Exposure of Leuk-1 and DOK cells to high glucose for 24 hours led to EGFR activation as evidenced by phosphorylation (Figure 4). In addition, FASN inhibition strongly reduced the expression levels of high glucose-induced phosphorylated EGFR. These findings underscore the need of a functionally active FASN to mediate EGFR phosphorylation in response to high glucose.

Figure 4. FASN inhibition downregulates high glucose-induced EGFR phosphorylation.

Figure 4.

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(A,B) Cells were pre-treated with TVB-3166 (TVB) or cerulenin (Cer), and then exposed to normal or high glucose-containing media for 24 hours. Western blotting for pEGFR (Y1173) and total EGFR is shown. β-Actin, loading control.

3.5. Cetuximab reduces high glucose-induced oral dysplastic keratinocyte viability and motility.

To determine the contribution of EGFR to the previously observed increase in cell viability by high glucose, we treated cells with cetuximab, a clinically approved anti-EGFR monoclonal antibody for the treatment of head and neck cancer24 (Figure 5). Cetuximab pre-treatment markedly compromised high glucose-supported cell viability in a statistically significant manner (p<0.001), while having no effect on cells cultured under normal glucose. Because EGFR activity also affects cell migration, we investigated whether high glucose-induced cell motility was dependent on EGFR activation. To that end, we carried out a wound healing assay like the one presented in Figure 3. In this experiment, however, we treated Leuk-1 and DOK cells with cetuximab under both normal and high glucose conditions (Figures 5C and 5E). Leuk-1 and DOK cells both exhibited reduced migration under high glucose when pre-treated with cetuximab (Figures 5D and 5E). Collectively, these data suggest that high glucose-induced EGFR activation also acts as a major mechanism supporting oral dysplastic keratinocyte viability and migration.

Figure 5. Cetuximab reduces high glucose-induced oral dysplastic keratinocyte viability and motility.

Figure 5.

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(A,B) Cells were pre-treated with 10 μg/mL cetuximab (CTX) or vehicle control followed by exposure to normal or high glucose-containing media for 5 days. MTS reagent was then added to evaluate cell viability. Data represent mean ± S.E.M. ***p<0.001 when compared to indicated treatment. (C) Leuk-1 and (E) DOK cells were pre-treated with 10 μg/mL CTX) or vehicle. Following pre-treatment, cells were exposed to normal or high glucose-containing media for 18 hours. Gap closure was quantified as the change in acellular area for both (D) Leuk-1 and (F) DOK cells. Data represent mean ± S.E.M. *p<0.05, **p<0.01 and ***p<0.001 when compared to indicated treatment.

3.6. Cetuximab inhibits proliferation of FASN-overexpressing DOK cells.

To determine whether FASN overexpression alone, independently of glucose concentration, affected cell growth in an EGFR-dependent manner, we treated a FASN-overexpressing DOK cell line with cetuximab (Figure 6). We have reported that in DOK cells, FASN overexpression alone enhanced cell migration in an EGFR-dependent manner15. To validate the role of FASN/EGFR on increased cell proliferation, we imaged and subsequently counted total cell number in both the pPUR- and FASN-overexpressing cells following cetuximab treatment. While in a 5-day period the total number of FASN-overexpressing cells consistently increased, this response was significantly inhibited upon cetuximab treatment (p<0.001). In support of what we observed when FASN and EGFR were upregulated by high glucose, these results highlight the critical role of FASN-mediated EGFR function as driver of oral dysplastic keratinocyte proliferation.

Figure 6. Cetuximab inhibits increased proliferation of FASN-overexpressing DOK cells.

Figure 6.

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(A) pPUR-transfected and FASN-overexpressing DOK cell lysates were subjected to FASN immunoblotting. β-actin, loading control. (B) Similar cells were treated with vehicle control or 10 μg/mL cetuximab (CTX). Imaging-based cell counting was conducted after 5 days. Data represent mean ± S.E.M, with **p<0.01 and ***p<0.001 when compared to indicated treatment. n.s. indicates data not significant (p>0.05).

4. DISCUSSION

To the best of our knowledge, we present the first evidence demonstrating that high glucose, a microenvironmental stimulus mimicking hyperglycemia, may impact the progression of oral potentially malignant lesions by enhancing cell growth and migration of oral dysplastic keratinocytes. These findings hold a high translational relevance due to the rising number of individuals diagnosed with T2DM2. In general, tumor tissues increase their lipid metabolism to adapt to the essential needs in cell membrane biogenesis, protein modification, energy storage, and signaling functions. In humans, FASN is a major lipogenic enzyme capable of synthesizing fatty acids de novo by catalyzing the biosynthesis of palmitate from acetyl-coenzyme A (CoA) and malonyl-CoA in a nicotinamide adenine dinucleotide phosphate-reduced (NADPH)-dependent reaction10. We found that high glucose upregulates FASN expression significantly increasing oral dysplastic cell growth and migration. We recently reported that in oral dysplastic keratinocytes FASN overexpression alone stimulated cell migration underscoring its potential pro-oncogenic role in oral potentially malignant disorders15. This study demonstrates that FASN upregulation in response to high glucose contributes to the malignant-like behavior in oral potentially malignant dysplastic cells.

Compelling evidence indicates that signaling pathways activated by EGFR mediates T2DM systemic complications affecting the heart, kidneys and eyes16–19. The potential implications of hyperglycemia and EGFR signaling are particularly concerning in oral oncology since EGFR overexpression and increased EGFR gene copy number results in a significant advantage for tumor progression25. Moreover, FASN can affect localization and modification of cell membrane-bound growth factor receptors including EGFR13,14. Thus, we investigated whether high glucose FASN upregulation could potentially trigger EGFR activation. Indeed, high glucose induced EGFR activation through phosphorylation. This effect was markedly prevented by FASN inhibitors cerulenin and TVB-3166. Likewise, inhibition of EGFR by cetuximab markedly affected high glucose induced oral dysplastic cell growth and migration. FASN and EGFR inhibitor dosages were based on previously published work by our group and others in non-tumor and tumor cell lines15,23,26,27. Noteworthy, we show here that neither cerulenin, TVB-3166 or cetuximab in the selected doses were detrimental to cells cultured under normal glucose conditions (controls).

Future mechanistic studies should investigate the regulation of FASN-controlled EGFR activation and possibly other receptor-mediated signaling pathways. Since FASN affects cell membrane biogenesis, it is possible that FASN modifies cell membrane dynamics by altering lipid rafts or other similar structures. Other processes regulating FASN expression should also be considered, including the role of the de-ubiquitinating enzyme USP2a, and the transcription factors sterol regulatory element binding protein 1c and carbohydrate-responsive element binding protein28,29.

Within the limitations associated with in vitro experimental conditions, this study highlights the timely need to further explore the role of hyperglycemia relative to FASN/EGFR signaling in preclinical animal models of oral carcinogenesis. These endeavors may unravel novel chemopreventive and therapeutic targetable pathways for personalized interventions in oral oncology.

Supplementary Material

fS1

Figure S1. High glucose does not increase cell migration in NOKSI cells. (A) NOKSI cells were plated overnight to confluency. Cell monolayers were scratched with a pipet tip, allowing cells to migrate while being exposed to normal or high glucose in 1% FBS containing media for 24 hours. (B) Quantification of percentage acellular gap closure for imaged NOKSI cells. Data represent mean ± S.E.M. “ns” indicates no statistically significant differences between conditions when analyzed by Student’s t test (p>0.05).

Acknowledgements

This work was funded by the National Institutes of Health/National Institute of Dental and Craniofacial Research Grant R01 DE023578 and University of Maryland School of Dentistry funds (to A.S.).

Footnotes

Conflicts of interest: None to declare

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Associated Data

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Supplementary Materials

fS1

Figure S1. High glucose does not increase cell migration in NOKSI cells. (A) NOKSI cells were plated overnight to confluency. Cell monolayers were scratched with a pipet tip, allowing cells to migrate while being exposed to normal or high glucose in 1% FBS containing media for 24 hours. (B) Quantification of percentage acellular gap closure for imaged NOKSI cells. Data represent mean ± S.E.M. “ns” indicates no statistically significant differences between conditions when analyzed by Student’s t test (p>0.05).

NIHMS1733791-supplement-fS1.pdf (2.1MB, pdf)

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