Cancer stem cells and fibroblast niche cross talk in an in-vitro oral dysplasia model.

Understanding the cellular interactions during oral carcinogenesis has the potential to identify novel prognostic and therapeutic targets. This study aimed at investigating the cancer stem cell (CSC)-fibroblast niche interactions using in-vitro dysplastic cell line models developed from different stages of 4NQO-induced oral carcinogenic mice model. The spontaneously transformed epithelial cells (DysMSCTR6, 14 and 16) were developed from three time points (mild/moderate/severe), while two fibroblast cell lines (FibroMSCTR12, 16) were developed from moderate and severe dysplastic tissue. The epithelial (Epcam+/Ck+) and the fibroblast cell lines (Vimentin+/α-SMA+/Ck-) were authenticated and assessment of cells representing progressive grades of dysplastic severity indicated a significant increase in dysplastic marker profile (P < 0.05). Evaluation of the CSC characteristics showed that an increase in expression of Cd133, Cd44, Aldh1a1, Notch1, and Sox2 was accompanied by an increase in migratory (P > 0.05) and colony formation capacity (P > 0.005). Targeting Notch1 (GSI inhibitor PZ0187; 30 µM), showed a significant reduction in cell proliferation capacity (P < 0.05) and in the dysplastic marker profile. Further, Notch1 inhibition resulted in down regulation of Cd133 and Aldh1a 1 (P < 0.05) and a complete abrogation of colony formation ability (P < 0.0001). The effect of niche interactions evaluated using FibroMSCTR12-conditioned media studies, revealed an enrichment of ALDH1A1+ cells (P < 0.05), induction of spheroid formation ability (P < 0.0001) and increased proliferation capacity (3.7 fold; P < 0.005). Although PZ0187 reduced cell viability by ∼40%, was unable to abrogate the conditioned-media induced increase in proliferation capacity completely. This study reports a Notch-1 dependent enrichment of CSC properties during dysplastic progression and a Notch-1 independent dysplastic cell-fibroblast interaction during oral carcinogenesis.

Curcumin and metformin-mediated chemoprevention of oral cancer is associated with inhibition of cancer stem cells.

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 mm3 ; 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.

Cancer stem cell mediated acquired chemoresistance in head and neck cancer can be abrogated by aldehyde dehydrogenase 1 A1 inhibition.

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.

Establishment and characterization of triple drug resistant head and neck squamous cell carcinoma cell lines.

Resistance to chemotherapy leading to poor outcome and survival remains a challenge for developing strategies for therapeutic interventions in all types of cancer, including head and neck cancer. In vitro chemoresistant cell line models are an indispensable resource towards delineating the mechanisms involved in drug resistance/response and for the development of novel drugs. Current treatment for head and neck cancer includes chemotherapy with cisplatin, docetaxel and 5-fluorouracil (5-FU) and the response rates to these drugs in patients is 60-80%. The present study aimed to generate head and neck cancer triple drug-resistant cell lines in an effort towards elucidating the mechanisms underlying chemoresistance and providing a resourceful tool for drug design. Using two head and neck squamous cell carcinoma cell lines, Hep-2 (larynx) and CAL-27 (oral cavity), the present study sequentially exposed these cells to increasing concentrations of the combination of docetaxel, cisplatin and 5-FU (TPF) to generate triple drug-resistant cells, termed Hep-2 TPF resistant (TPFR) and CAL-27 TPFR. The effect of the drug treatments on the cell viability, apoptosis, cell cycle and the expression of genes associated with multidrug resistance were analyzed in the parental cells and drug-resistant counterparts.