Exposure to nicotine-derived nitrosamine ketone and arecoline synergistically facilitates tumor aggressiveness via overexpression of epidermal growth factor receptor and its downstream signaling in head and neck squamous cell carcinoma

Abstract

Long-term nicotine-derived nitrosamine ketone (NNK) and arecoline exposure promotes carcinogenesis and head and neck squamous cell carcinoma (HNSCC) progression, although most associated data on the two were analyzed individually. The molecular mechanisms underlying tumor progression associated with the synergistic effects of NNK and arecoline remain unclear. We treated SCC-25 and FaDu cells with NNK and arecoline (separately or in combination) for 3 months. Comparative analysis was performed to investigate the mechanism underlying the acquisition of properties related to tumor promotion, including stemness, anti-apoptosis, and resistance to HNSCC therapeutics. Long-term exposure to NNK and arecoline resulted in an increase in cancer stem cell properties, anti-apoptosis, and the resistance to cisplatin in HNSCC. We detected abundant epidermal growth factor receptor (EGFR) expression in HNSCC cells after combined treatment with NNK and arecoline. EGFR was pivotal in inducing tumor promotion and anti-apoptosis in cancer cells by inducing pAKT and NFκB. Combined treatment with NNK and arecoline synergistically facilitated tumor aggressiveness via EGFR-AKT signaling. Targeting EGFR-AKT signaling may be a feasible strategy for treating HNSCC.

Fig 1. Promotion of proliferation, enhancement of migration and invasion characteristics of the EMT phenomenon, and morphological alterations following long-term exposure to NNK and arecoline.

A. Proliferation assays showed that treatment with NNK or arecoline increased the growth rate in a short-term dependent manner in both SCC25 and FaDu cells; however, long-term treatment with NNK and arecoline caused sluggish cell proliferation until growth equilibrium was reached. B. As indicated by the arrows, long-term treatment with NNK, arecoline, or both NNK and arecoline induced EMT with a characteristic cell morphology observed for both cell lines. C. Long-term exposure to NNK, arecoline, or the combination of NNK and arecoline significantly increased the migration and invasion of HNSCC cells (p < 0.05). D. Regarding the EMT-like properties in HNSCC cells, western blot analysis revealed that long-term exposure to NNK, arecoline, and both in combination also induced alterations in the expression of representative EMT markers showing decreased expression of E-cadherin, increased expression of fibronectin, and increased expression of EMT regulators (Snail, Twist, and NFκB) in both cell lines. A synergistic effect was found in the group treated with NNK and arecoline in combination, showing more sensitivity than the other two experimental groups (p < 0.05).

Fig 2. Characterization of the sphere-forming ability and expression of CSC markers following long-term exposure to NNK and arecoline.

A. Morphological transformations under each condition through the stimulation of sphere formation by long-term alkaloid treatment were observed using a non-adhesive culture system.[26] B. C. Flow cytometric analysis showed significant overexpression (p < 0.05) of CSC representative markers, including CD24, CD44, and CD133. D. Western blot analysis also showed upregulation of Nanog, OCT4, and ALDH-1 in the three experimental groups compared with that in control cells. A prominent synergistic effect was observed in the group treated with NNK and arecoline in combination in terms of the upregulation of representative CSC markers.

Fig 3. Comparison of drug resistance and drug resistance-related gene products following exposure to NNK and arecoline.

A. Drug sensitivity after treatment with different doses of Cis for 24 h revealed marked differences between long-term alkaloid-treated cells and parental cells. Drug resistance was higher in long-term alkaloid-treated cells than in the parental cells (p < 0.05). B. Western blot analysis showed upregulation of the drug-resistance-related proteins ABCG2 and MDR-1. Alkaloid treatment upregulated both the MDR1 and ABCG2 gene products, suggesting that long-term exposure to NNK and arecoline caused greater resistance to Cis than that in untreated, parental cells. A synergistic effect was also found in the group exposed to combination treatment with NNK and arecoline, as this group exhibited more sensitivity than the other two experimental groups (p < 0.05).

Fig 4. Characterization of the anti-apoptotic ability following long-term exposure to NNK and arecoline.

A. Apoptosis analysis via annexin V staining showed that long-term alkaloid treatment attenuated apoptotic activity in both cell lines (p < 0.05). B. Western blot analysis demonstrated that treated cells displayed anti-apoptotic properties, such as increased Bcl-2 expression and downregulation of apoptotic-related proteins, including Bax, Cl-caspase-3, and Cl-PARP.

Fig 5. Induction and activation of EGFR effector molecules (other than α7-nAChR and β-AR) following long-term exposure to NNK and arecoline. EGFR was critical for the proliferation of HNSCC cells induced by long-term NNK and arecoline treatment.

A. The alkaloid receptors EGFR, α7-nAChR, and β-AR were detected by western blotting. However, following long-term exposure to NNK and arecoline, HNSCC cells strongly expressed EGFR, but not α7-nAChR and β-AR. B. Treatment with an anti-EGFR antibody downregulated EGFR signaling after long-term NNK, arecoline, and combined NNK and arecoline treatments. Western blot analysis showed that NNK and arecoline induced p-AkT and NFκB in SCC25 and FaDu cells, which was diminished in the presence of the anti-EGFR antibody.

Fig 6. Proposed model to illustrate NNK- and arecoline-mediated EGFR/AKT/NFκB signal activation resulting in enhanced anti-apoptosis, EMT, and tumor progression in HNSCC cells.

A. Long-term exposure to NNK and arecoline synergistically increased anti-apoptotic activity via the EGFR/AKT pathway. B. Such exposure also synchronized the induction of the EMT phenomenon with morphological alterations via NFκB/Snail signaling, ultimately leading to tumor progression, drug resistance, and stemness.