Expressão diferencial dos genes e proteínas NTRK2-TrkB, KRASe HIF1Aem célula-tronco tumoral nas linhagens celulares de câncer de cabeça e pescoço SCC-28 e FADU

Abstract

Head and neck cancer (HNC) is the sixth most prevalent tumor type worldwide with approximately 600.000 new cases annually. Even with advances in treatment, it has a low survival rate, which can be explained by the presence of cancer stem cells (CSCs), a cell subpopulation resistant to chemo and radiotherapy, favoring tumor progression. Another important structure involved in this process is Tropomyosin Related Kinase B (TrkB), able of activating proliferation pathways such as Kirsten Rat Sarcoma (KRAS). TrkB-activated KRAS leads to cell survival, angiogenesis and activation of the Hypoxia-Induced Factor (HIF1A), a subunit capable of stimulating gene transcription. Objectives: To identify and sorting CSCs from two CCP cell lines (SCC-28- laryngeal cancer and FADU-pharyngeal cancer); to evaluate the expression of NTRK2-TrkB, KRAS and HIF1A genes and proteins in CSCs and non- CSCs of these both cell lines. Material and Methods: The cell lines were cultured in supplemented DMEM and later separated by flow cytometry using the biomarker Aldehyde Dehydrogenase (ALDH) by the FacsAria equipment. The invasion, migration and formation spheres assays were performed to confirm the effectiveness of sorting. Gene expression was performed using the Real-Time PCR technique and protein expression using the Western Blotting technique. Results: The ALDH biomarker was efficient to identify and isolate CSCs from the two cell lines evaluated. CSCs showed greater capacity to invade (SCC-28 p=0.0097, FADU p=0.0029) and to migrate (SCC-28 p = 0.0253, FADU p <0.0001) when compared with non-CSCs . It was also possible to observe the greater capacity of tumor spheres formation for CTTs of the SCC-28 cell line (p=0.0030) compared to non-CTTs, however there was no statistically significant difference for FADU (p=0.5810). In the SCC-28 cell line, increased KRAS gene expression (p=0.0002) was shown in CSCs when compared to non- CSCs, however this statistically significant difference was not evident for the HIF1A gene (p=0.0730) and it was not possible to detect NTRK2 gene expression. In the FADU cell line, CSCs showed increased gene expression of NTRK2, KRAS and HIF1A (p<0.0001, p=0.0005 and p<0.0001 respectively) compared to non-CSCs. In protein expression analysis, increased expression of TrkB, KRAS HIF1A was observed in CSCs of SCC-28 cell line (p=0.0001, p=0.0004 and p=0.0007, respectively) and FADU (p=0.0064, p=0.0156 and p=0.0103, respectively) when compared with non-CSCs. Conclusion: The biomarcardor ALDH can be used to identify and isolate CSCs from the HNC and this sub-population has a greater capacity to invade, migrate and form tumor spheres. The NTRK2, KRAS and HIF1A genes show increased expression in CSCs when compared to non-CSCs, and can be promising targets for the development of new therapies against cancer.