RESUMO
Telomeres are nucleoprotein structures at the end of chromosomes that maintain their integrity. Mutations in genes coding for proteins involved in telomere protection and elongation produce diseases such as dyskeratosis congenita or idiopathic pulmonary fibrosis known as telomeropathies. These diseases are characterized by premature telomere shortening, increased DNA damage and oxidative stress. Genetic diagnosis of telomeropathy patients has identified mutations in the genes TERT and TERC coding for telomerase components but the functional consequences of many of these mutations still have to be experimentally demonstrated. The activity of twelve TERT and five TERC mutants, five of them identified in Spanish patients, has been analyzed. TERT and TERC mutants were expressed in VA-13 human cells that express low telomerase levels and the activity induced was analyzed. The production of reactive oxygen species, DNA oxidation and TRF2 association at telomeres, DNA damage response and cell apoptosis were determined. Most mutations presented decreased telomerase activity, as compared to wild-type TERT and TERC. In addition, the expression of several TERT and TERC mutants induced oxidative stress, DNA oxidation, DNA damage, decreased recruitment of the shelterin component TRF2 to telomeres and increased apoptosis. These observations might indicate that the increase in DNA damage and oxidative stress observed in cells from telomeropathy patients is dependent on their TERT or TERC mutations. Therefore, analysis of the effect of TERT and TERC mutations of unknown function on DNA damage and oxidative stress could be of great utility to determine the possible pathogenicity of these variants.
Assuntos
Disceratose Congênita , Telomerase , Humanos , Apoptose/genética , DNA/metabolismo , Dano ao DNA/genética , Disceratose Congênita/genética , Disceratose Congênita/metabolismo , Disceratose Congênita/patologia , Mutação , Estresse Oxidativo/genética , RNA/genética , Telomerase/genética , Telomerase/metabolismo , Telômero/genética , Telômero/metabolismoRESUMO
Salivary gland squamous cell carcinomas (SG-SCCs) constitute a rare type of head and neck cancer which is linked to poor prognosis. Due to their low frequency, the molecular mechanisms responsible for their aggressiveness are poorly understood. In this work we studied the role of the phosphatase DUSP1, a negative regulator of MAPK activity, in controlling SG-SCC progression. We generated DUSP1 KO clones in A253 human cells. These clones showed a reduced ability to grow in 2D, self-renew in ECM matrices and to form tumors in immunodeficient mice. This was caused by an overactivation of the stress and apoptosis kinase JNK1/2 in DUSP1-/+ clones. Interestingly, RNAseq analysis revealed that the expression of SOX2, a well-known self-renewal gene was decreased at the mRNA and protein levels in DUSP1-/+ cells. Unexpectedly, CRISPR-KO of SOX2 did not recapitulate DUSP1-/+ phenotype, and SOX2-null cells had an enhanced ability to self-renew and to form tumors in mice. Gene expression analysis demonstrated that SOX2-null cells have a decreased squamous differentiation profile -losing TP63 expression- and an increased migratory phenotype, with an enhanced epithelial to mesenchymal transition signature. In summary, our data indicates that DUSP1 and SOX2 have opposite functions in SG-SCC, being DUSP1 necessary for tumor growth and SOX2 dispensable showing a tumor suppressor function. Our data suggest that the combined expression of SOX2 and DUSP1 could be a useful biomarker to predict progression in patients with SG-SCCs.