Publicação
Molecular characterisation of the poorly differentiated and undifferentiated thyroid carcinomas using genome-wide approaches
| Resumo: | Poorly differentiated (PDTC) and anaplastic thyroid carcinomas (ATC) are highly malignant tumours composed by dedifferentiated cells, for which current therapeutic options have been ineffective. In the present project, the molecular signatures and genetic alterations associated with these tumours were elucidated, by using genome-wide expression analysis as first assessment. The role of the microRNA stability in thyroid tumourigenesis was also evaluated. The comparison of the expression profiles between PDTC, well-differentiated thyroid carcinomas and normal thyroid tissues, and between ATC and normal thyroid tissues, revealed that PDTC and ATC have common deregulated signatures, indicative of cell adhesion impairment and increased cell cycle, proliferation and chromosomal instability. Additionally, ATC were specifically characterized by loss of epithelial and thyroid-related functions and activation of components from TGF-β pathway. The gene expression data suggested that follicular variants of papillary carcinomas were possible precursors of PDTC, whereas, ATC were more similar to classical papillary carcinomas. Validation by quantitative RT-PCR in independent sample sets, allowed to confirm the significant over-expression of UHRF1 (associated to proliferation) and downregulation of ITIH5 (associated to cell adhesion and invasion) in PDTC relatively to normal tissues. Moreover, CDKN3 gene, which encodes a cyclin-dependent kinase inhibitor, was shown to be significantly upregulated in PDTC and ATC relatively to normal thyroid, and in accordance with its suppressive function, CDKN3 was aberrantly spliced in ATC samples. The over-expression of the epithelial-tomesenchymal transition factor SNAI2, which is induced by TGF-β, was significant in ATC. Attending to the deregulated pathways identified, a mutational screening was undertaken in 26 ATC and 22 PDTC, which included the hot-spot regions of RAS, BRAF, TP53, CTNNB1 (β-catenin) and PIK3CA genes, and, for the first time, a comprehensive mutational analysis of cell cycle [CDKN1A (p21CIP1); CDKN1B (p27KIP1); CDKN2A (p14ARF, p16INK4A); CDKN2B (p15INK4B); CDKN2C (p18INK4C)], and cell adhesion regulators (AXIN1). Most mutations were identified in TP53 (42% of ATC; 27% of PDTC) and in RAS (31% of ATC; 18% of PDTC). The alterations in these genes were mutually exclusive (P=0.0354), and were associated with a lower patient survival (P=0.0383). CDKN2A and CDKN2B were found to be mutated, for the first time, in up to 25% of PDTC. Other known recurrent mutations in ATC (in BRAF, PIK3CA, CTNNB1 and AXIN1 genes) were rarely detected, undermining their role in ATC development. The study of the microRNA stability in thyroid tumourigenesis is still ongoing. Turnover rates were determined in a dedifferentiated thyroid carcinoma cell line (BCPAP), under standard conditions. A non-disruptive pulse-labelling method was applied and the decrease in microRNA expression along the time was quantified by two-colour arrays. These results represent the first report for an atlas of human microRNA half-lives. The determined average half-life for 249 microRNA was 2.5 days, a stability time more comparable to proteins than to mRNA. The comparison of these data with decay rates in normal thyroid cells will probably uncover miRNA for which stability is deregulated. In conclusion, this project presents several genes and molecular events that were found deregulated in PDTC and ATC. More importantly, these represent potential therapeutic targets that can be used for the treatment of these highly aggressive tumours, in the near future. |
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| Autores principais: | Pita, Jaime Miguel Gomes, 1985- |
| Assunto: | Glândula tiróide Tumores Expressão genética Mutação (Biologia) MicroRNA Teses de doutoramento - 2013 |
| Ano: | 2013 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Poorly differentiated (PDTC) and anaplastic thyroid carcinomas (ATC) are highly malignant tumours composed by dedifferentiated cells, for which current therapeutic options have been ineffective. In the present project, the molecular signatures and genetic alterations associated with these tumours were elucidated, by using genome-wide expression analysis as first assessment. The role of the microRNA stability in thyroid tumourigenesis was also evaluated. The comparison of the expression profiles between PDTC, well-differentiated thyroid carcinomas and normal thyroid tissues, and between ATC and normal thyroid tissues, revealed that PDTC and ATC have common deregulated signatures, indicative of cell adhesion impairment and increased cell cycle, proliferation and chromosomal instability. Additionally, ATC were specifically characterized by loss of epithelial and thyroid-related functions and activation of components from TGF-β pathway. The gene expression data suggested that follicular variants of papillary carcinomas were possible precursors of PDTC, whereas, ATC were more similar to classical papillary carcinomas. Validation by quantitative RT-PCR in independent sample sets, allowed to confirm the significant over-expression of UHRF1 (associated to proliferation) and downregulation of ITIH5 (associated to cell adhesion and invasion) in PDTC relatively to normal tissues. Moreover, CDKN3 gene, which encodes a cyclin-dependent kinase inhibitor, was shown to be significantly upregulated in PDTC and ATC relatively to normal thyroid, and in accordance with its suppressive function, CDKN3 was aberrantly spliced in ATC samples. The over-expression of the epithelial-tomesenchymal transition factor SNAI2, which is induced by TGF-β, was significant in ATC. Attending to the deregulated pathways identified, a mutational screening was undertaken in 26 ATC and 22 PDTC, which included the hot-spot regions of RAS, BRAF, TP53, CTNNB1 (β-catenin) and PIK3CA genes, and, for the first time, a comprehensive mutational analysis of cell cycle [CDKN1A (p21CIP1); CDKN1B (p27KIP1); CDKN2A (p14ARF, p16INK4A); CDKN2B (p15INK4B); CDKN2C (p18INK4C)], and cell adhesion regulators (AXIN1). Most mutations were identified in TP53 (42% of ATC; 27% of PDTC) and in RAS (31% of ATC; 18% of PDTC). The alterations in these genes were mutually exclusive (P=0.0354), and were associated with a lower patient survival (P=0.0383). CDKN2A and CDKN2B were found to be mutated, for the first time, in up to 25% of PDTC. Other known recurrent mutations in ATC (in BRAF, PIK3CA, CTNNB1 and AXIN1 genes) were rarely detected, undermining their role in ATC development. The study of the microRNA stability in thyroid tumourigenesis is still ongoing. Turnover rates were determined in a dedifferentiated thyroid carcinoma cell line (BCPAP), under standard conditions. A non-disruptive pulse-labelling method was applied and the decrease in microRNA expression along the time was quantified by two-colour arrays. These results represent the first report for an atlas of human microRNA half-lives. The determined average half-life for 249 microRNA was 2.5 days, a stability time more comparable to proteins than to mRNA. The comparison of these data with decay rates in normal thyroid cells will probably uncover miRNA for which stability is deregulated. In conclusion, this project presents several genes and molecular events that were found deregulated in PDTC and ATC. More importantly, these represent potential therapeutic targets that can be used for the treatment of these highly aggressive tumours, in the near future. |
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