Chemoresistance leading to disease relapse is one of the major challenges to improve outcome in head and neck cancers. Cancer Stem Cells (CSCs) are increasingly being implicated in chemotherapy resistance, this study investigates the correlation between CSC behavior and acquired drug resistance in in vitro cell line models. Cell lines resistant to Cisplatin (Cal‐27 CisR, Hep‐2 CisR) and 5FU (Cal‐27 5FUR) with high Resistance Indices (RI) were generated (RI ≥ 3) by short‐term treatment of head and neck squamous cell carcinoma (HNSCC) cell lines with chemotherapeutic drugs (Cisplatin, Docetaxel, 5FU), using a dose‐incremental strategy. The cell lines (Cal‐27 DoxR, Hep‐2 DoxR, Hep‐2 5FUR) that showed low RI, nevertheless had a high cross resistance to Cisplatin/5FU (P < 0.05). Cal‐27 CisR and DoxR showed 12–14% enrichment of CD44+ cells, while CisR/5FUR showed 4–6% increase in ALDH1A1+ cells as compared to parental cells (P < 0.05). Increased expression of stem cell markers (CD44, CD133, NOTCH1, ALDH1A1, OCT4, SOX2) in these cell lines, correlated with enhanced spheroid/colony formation, migratory potential, and increased in vivo tumor burden (P < 0.05). Inhibition of ALDH1A1 in Cal‐27 CisR led to down regulation of the CSC markers, reduction in migratory, self‐renewal and tumorigenic potential (P < 0.05) accompanied by an induction of sensitivity to Cisplatin (P < 0.05). Further, ex vivo treatment of explants (n = 4) from HNSCC patients with the inhibitor (NCT‐501) in combination with Cisplatin showed a significant decrease in proliferating cells as compared to individual treatment (P = 0.001). This study hence suggests an ALDH1A1‐driven, CSC‐mediated mechanism in acquired drug resistance of HNSCC, which may have therapeutic implications. © 2016 Wiley Periodicals, Inc.

Effective chemoprevention is critical for improving outcomes of oral cancer. As single agents, curcumin and metformin are reported to exhibit chemopreventive properties, in vitro as well as in patients with oral cancer. In this study, the chemopreventive efficacy of this drug combination was tested in a 4-nitro quinoline-1-oxide (4NQO) induced mice oral carcinogenesis model. Molecular analysis revealed a cancer stem cell (CSC)-driven oral carcinogenic progression in this model, wherein a progressive increase in the expression of CSC-specific markers (CD44 and CD133) was observed from 8th to 25th week, at transcript (40-100-fold) and protein levels (P ≤ 0.0001). Chemopreventive treatment of the animals at 17th week with curcumin and metformin indicated that the combination regimen decreased tumor volume when compared to the control arm (0.69+0.03 vs 6.66+2.4 mm ; P = 0.04) and improved overall survival of the animals (P = 0.03). Assessment of the molecular status showed an overall downregulation of CSC markers in the treatment arms as compared to the untreated control. Further, in vitro assessment of the treatment on the primary cells generated from progressive stages of 4NQO-induced mice tissue showed a concordant and consistent downregulation of the CSC markers following combination treatment (P < 0.05). The treatment also inhibited the migratory and self-renewal properties of these cells; the effect of which was prominent in the cultures of early dysplastic tissue (P < 0.002). Collectively, our observations suggest that the combination of curcumin and metformin may improve chemopreventive efficacy against oral squamous cell carcinoma through a CSC-associated mechanism.

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