Identificação e expressão gênica de células tronco tumorais no câncer de cabeça e pescoço e a resposta à quimioterápicos

Abstract

Head and neck cancer (HNC) is the fifth most common cancer and presents low survival rate. The treatment has showed unfavorable results due to the tumor resistance. This is due a small subpopulation of cells, called tumor stem cells (CSCs) that have self-innovation and tumor initiation capabilities, identified by surface biomarkers such as CD44, CD117, CD133 and ALDH. In addition, the high expression of tumor progression genes such as Epidermal Growth Factor Receptor (EGFR) and tyrosine kinase receptor (TrkB) that activate the signaling cascade through the homologous Kirsten Ras Oncogene (KRAS) gene has been evidenced in cancer development and treatment. Objectives: To identify and separate tumor stem cells by molecular biomarkers; To evaluate the tumorigenic potential of CSCs as well as the efficacy of HNC treatment in CSCs and to analyze the expression of CD44, TrkB, EGFR and KRAS genes in HNC CSCs. Materials and Methods: For identification and separation of CSCs, FACSAria Fusion equipment was used. After separation, the cells were tested for migration, invasion and colony formation assays. Cells were treated with 0.37mg/mL of 5-fluorouracil, 2.0 mg/mL of cisplatin, 0.06 mg/mL of Cetuximab and 0.05 mg/mL of Paclitaxel for 24h and the cell viability assay was analyzed by MTS. The gene expression of the CD44, TrkB, EGFR and KRAS genes was used the quantitative PCR method, using non-CSCs as relative reaction control. Results: CSCs were identified and separated by the CD44, CD117 and CD133 biomarkers in combination and ALDH1 alone. The evaluation of tumorigenic potential of CSCs showed higher migration, invasion potential and colony formation when compared to non-CSCs (p <0.0001, p = 0.0324 and p = 0.0013). The efficacy of the evaluated treatments did not show statistical differences between CSCs and non-CSCs. The subpopulation of CSCs showed high CD44 gene expression (RQ = 102.775) and low EGFR gene expression (RQ = 0.741) in the oral cancer cell line and low CD44 gene expression (RQ = 0.658), high EGFR gene expression ( RQ = 7.559) and KRAS (RQ = 1.482) and there was no expression of the TrkB gene in the laryngeal cancer cell line. In primary tumors, EGFR (mean RQ = 7.081) and KRAS (mean RQ = 1.568) genes were found to be highly expressed in CSCs, but there was no statistical difference between the two subpopulations (p = 0.5625 and p = 0.5296, respectively). Conclusion: CD44/CD117/CD133 labeling in combination and ALDH alone are efficient for separating subpopulations of CSCs in HNC. CSCs present a more aggressive tumorigenic potential and relatively more resistant to the studied treatments. In oral cancer lineage the EGFR gene was found underexpressed and CD44 gene overexpressed, however, in laryngeal cancer cell line the opposite was found in addition to KRAS overexpression and did not express the TrkB gene. The results corroborate withfindings in primary tumors expressing highly the EGFR and KRAS genes in CSCs. The present study contributes to the understanding of the mechanisms of cell proliferation, resistance and recidive to HNC treatment through the characterization of CSCs.