Nicotine induces oral dysplastic keratinocyte migration via fatty acid synthase-dependent epidermal growth factor receptor activation

Abstract

Despite advances in diagnostic and therapeutic management, oral squamous cell carcinoma (OSCC) patient survival rates have remained relatively unchanged. Thus, identifying early triggers of malignant progression is critical to prevent OSCC development. Traditionally, OSCC initiation is elicited by the frequent and direct exposure to multiple tobacco-derived carcinogens, and not by the nicotine contained in tobacco products. However, other nicotine-containing products, especially the increasingly popular electronic cigarettes (e-cigs), have unknown effects on the progression of undiagnosed tobacco-induced oral premalignant lesions, specifically in regard to the effects of nicotine. Overexpression of fatty acid synthase (FASN), a key hepatic de novo lipogenic enzyme, is linked to poor OSCC patient survival. Nicotine upregulates hepatic FASN, but whether this response occurs in oral dysplastic keratinocytes is unknown. We hypothesized that in oral dysplastic keratinocytes, nicotine triggers a migratory phenotype through FASN-dependent epidermal growth factor receptor (EGFR) activation, a common pro-oncogenic event supporting oral carcinogenesis. We report that in oral dysplastic cells, nicotine markedly upregulates FASN leading to FASN-dependent EGFR activation and increased cell migration. These results raise potential concerns about e-cig safety, especially when used by former tobacco smokers with occult oral premalignant lesions where nicotine could trigger oncogenic signals commonly associated with malignant progression.

Keywords: Cell migration; EGFR; FASN; Nicotine; Oral cancer; Oral premalignant lesions.

Figure 1. Nicotine induces oral dysplastic keratinocyte migration

(A) Leuk-1 and (B) DOK cells were plated to confluency overnight. The next day cell monolayers were scratched with a pipet tip, followed by treatment with 10 μM nicotine or vehicle control for the indicated times. Quantification of percentage acellular gap closure is shown for Leuk-1 (C) and DOK (D) cells. Data represent mean ± S.E.M. **p<0.01, when compared to untreated control.

Figure 2. Nicotine induces FASN-dependent oral dysplastic keratinocyte migration

(A) Leuk-1 and (B) DOK cells were treated with nicotine for the indicated times. Following treatment, whole cell lysates were collected and subjected to western blotting for FASN. β-Actin served as loading control. (C) Leuk-1 and (E) DOK cells were plated to confluency overnight, followed by pre-treatment for 2 hours with the FASN inhibitor TVB-3166 (10 μM). The cell monolayers were next scratched with a pipet tip. Following scratching, cells were left untreated or treated with 10 μM nicotine for 24 hours. Quantification of percentage acellular gap closure is shown for Leuk-1 (D) and DOK (F) cells. Data represent mean ± S.E.M. *p<0.05, **p<0.01, and ***p<0.001.

Figure 2. Nicotine induces FASN-dependent oral dysplastic keratinocyte migration

(A) Leuk-1 and (B) DOK cells were treated with nicotine for the indicated times. Following treatment, whole cell lysates were collected and subjected to western blotting for FASN. β-Actin served as loading control. (C) Leuk-1 and (E) DOK cells were plated to confluency overnight, followed by pre-treatment for 2 hours with the FASN inhibitor TVB-3166 (10 μM). The cell monolayers were next scratched with a pipet tip. Following scratching, cells were left untreated or treated with 10 μM nicotine for 24 hours. Quantification of percentage acellular gap closure is shown for Leuk-1 (D) and DOK (F) cells. Data represent mean ± S.E.M. *p<0.05, **p<0.01, and ***p<0.001.

Figure 3. Nicotine upregulates FASN in an EGFR/PI3K/mTOR independent manner

(A) Leuk-1 and (B) DOK cells were pre-treated for 2 hours with 10 μg/mL cetuximab (CTX), 10 μM AG1478 (AG), 10 μM LY294002 (LY), or 10 nM rapamycin (Rapa) prior to treatment with nicotine for an additional 2 hours. Then, whole cell lysates were collected and subjected to western blotting for FASN, p-EGFR (Y1173), total EGFR, p-AKT (S473), total AKT, p-S6 (S240/244), total S6 and β-Actin (as loading control).

Figure 4. Nicotine activates EGFR in a FASN-dependent manner

(A) Leuk-1 and (B) DOK cells were stimulated with nicotine for 24 hours. Following treatment, whole cell lysates were collected and subjected to western blotting for p-EGFR (Y1173), total EGFR and β-Actin (as loading control). (C) Leuk-1 and (D) DOK cells were pre-treated with TVB-3166, a FASN inhibitor, for 2 hours. Then, without removing the inhibitor, 10 μM nicotine was added to the media for 24 hours. Cells were then collected, lysed, and immunoblotted for p-EGFR (Y1173), total EGFR and β-Actin (loading control). (E) Leuk-1 and (F) DOK cells were transfected with either 50 nM of control scramble siRNA or 100 nM FASN siRNA followed by a 24-hour nicotine stimulation. Cells were then lysed and western blotting was performed to assess protein expression levels of FASN, p-EGFR (Y1173), total EGFR and β-Actin (loading control).

Figure 5. EGFR kinase inhibition prevents FASN-mediated oral dysplastic keratinocyte migration in response to nicotine

(A) Leuk-1 and (C) DOK cells were plated overnight to confluency and pre-treated for 2 hours with 1 μM AG1478, an EGFR tyrosine kinase inhibitor. Then, cell monolayers were scratched with a pipet tip, and cells allowed to migrate with continued AG1478 treatment for 24 hours. Quantification of percentage acellular gap closure is shown for imaged Leuk-1 (B) and DOK cells (D). Data represent mean ± S.E.M. **p<0.01 and ***p<0.001.

Figure 5. EGFR kinase inhibition prevents FASN-mediated oral dysplastic keratinocyte migration in response to nicotine

(A) Leuk-1 and (C) DOK cells were plated overnight to confluency and pre-treated for 2 hours with 1 μM AG1478, an EGFR tyrosine kinase inhibitor. Then, cell monolayers were scratched with a pipet tip, and cells allowed to migrate with continued AG1478 treatment for 24 hours. Quantification of percentage acellular gap closure is shown for imaged Leuk-1 (B) and DOK cells (D). Data represent mean ± S.E.M. **p<0.01 and ***p<0.001.

Figure 6. EGFR tyrosine kinase inhibition markedly reduces cell migration in FASN-overexpressing oral dysplastic keratinocytes

(A) Parental DOK, DOK expressing pPUR vector, and FASN-overexpressing DOK cells were all cultured in serum free media for 24 hours. Then, whole cell lysates were collected and immunoblotted for FASN, p-EGFR (Y1173), total EGFR and β-Actin (as loading control). (B) Stably FASN overexpressing DOK cells were plated to confluency overnight, along with DOK parental cells (non-transfected) and DOK transfected with a puromycin-resistance vector (pPUR) as controls. Next, cell monolayers were scratched with a pipet tip, and cell migration assessed after 24 hours. (C) Quantification of percentage acellular gap closure is shown for imaged parental, pPUR-transfected and FASN-transfected DOK cells. (D) pPUR and FASN-overexpressing DOK cells were pre-treated with 1 μM AG1478 for 2 hours, the cell monolayer was scratched, and cells were allowed to migrate for 24 hours with continuous AG1478 treatment. (E) Quantification of percentage acellular gap closure is shown for imaged cells. Data represent mean ± S.E.M. **p<0.01 and ***p<0.001.

Figure 6. EGFR tyrosine kinase inhibition markedly reduces cell migration in FASN-overexpressing oral dysplastic keratinocytes

(A) Parental DOK, DOK expressing pPUR vector, and FASN-overexpressing DOK cells were all cultured in serum free media for 24 hours. Then, whole cell lysates were collected and immunoblotted for FASN, p-EGFR (Y1173), total EGFR and β-Actin (as loading control). (B) Stably FASN overexpressing DOK cells were plated to confluency overnight, along with DOK parental cells (non-transfected) and DOK transfected with a puromycin-resistance vector (pPUR) as controls. Next, cell monolayers were scratched with a pipet tip, and cell migration assessed after 24 hours. (C) Quantification of percentage acellular gap closure is shown for imaged parental, pPUR-transfected and FASN-transfected DOK cells. (D) pPUR and FASN-overexpressing DOK cells were pre-treated with 1 μM AG1478 for 2 hours, the cell monolayer was scratched, and cells were allowed to migrate for 24 hours with continuous AG1478 treatment. (E) Quantification of percentage acellular gap closure is shown for imaged cells. Data represent mean ± S.E.M. **p<0.01 and ***p<0.001.