Study of molecular and cellular mechanisms involved in anaplastic thyroid carcinoma aggressiveness and response to therapy

Detalhes bibliográficos
Autor(a) principal: Rodrigues, Ricardo Miguel dos Santos
Data de Publicação: 2021
Tipo de documento: Dissertação
Idioma: eng
Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
Texto Completo: http://hdl.handle.net/10451/52108
Resumo: Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2021
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spelling Study of molecular and cellular mechanisms involved in anaplastic thyroid carcinoma aggressiveness and response to therapyCarcinoma anaplásico da tiróideTerapêuticas dirigidasResistência a fármacosMicroambiente tumoralMacrófagos associados ao tumorTeses de mestrado - 2021Departamento de Biologia VegetalTese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2021Thyroid cancer (TC) is the most common endocrine neoplasia. Thyroid carcinomas derived from follicular cells or thyrocytes are designated as nonmedullary thyroid carcinomas (NMTCs). The most undifferentiated subset of NMTC are anaplastic thyroid carcinomas (ATCs) which, although rare, are the most aggressive and lethal type of TC. ATCs are highly invasive, and lost the intrinsic functions of normal thyrocytes, presenting a downregulation of the sodium-iodide symporter (NIS), which makes the usual treatments for differentiated thyroid cancer, such as surgery and radioactive iodine, much less effective. Therefore, it is necessary to search for new therapeutic options, such as targeted therapies. The most promising targeted therapy for ATCs is the combination of dabrafenib (BRAF inhibitor) plus trametinib (MEK inhibitor) which, in a clinical trial, showed a significant response rate in ATC patients with the BRAF p.V600E mutation. This combination (DT) was recently approved only by the Food and Drug Administration (FDA) for this indication. Since some patients may develop resistance to DT, it is important to investigate the mechanisms involved and also new molecular therapeutic targets, in order to improve their overall survival. More than 50% of ATC’s tumour microenvironment (TME) is comprised by tumour associated macrophages (TAMs), which are associated with ATC aggressiveness, and may represent a possible novel target for ATC treatment. In previous proteomic studies developed by the Molecular Endocrinology group, on ATC-TAM crosstalk, Sprouty RTK Signaling Antagonist 4 (SPRY4) was identified as being a potential modulator of these interactions. The main aim of this work was to investigate the molecular mechanisms involved in ATC aggressiveness and response to DT therapy. To achieve this goal, two major approaches were used: (1) characterization of the molecular profiles of nine ATC BRAF p.V600E-positive patients, through the analysis of a multigene panel by Next Generation Sequencing (NGS) and Sanger sequencing, in order to investigate the alterations that could be associated with DT response and resistance, and to define treatment options in these cases; (2) in vitro experimental procedures, using 2D transwell cultures of ATC cell lines C3948 and T235, and a monocyte cell line (THP-1), in monocultures (without THP-1) and in co-cultures (with THP-1), to further study the role of SPRY4 in the ATC-TAM crosstalk. In the first task, we identified telomerase promotor (TERTp) mutations in all patients, which in concomitance with BRAF p.V600E, were likely contributing to the excellent initial DT therapy response. In one patient, a NRAS activating mutation was identified, representing an acquired off-target resistance mechanism, as previously described in ATCs. PIK3CA mutations were also detected in three patients and could be related with the selection of resistant clones to DT, as described in melanoma. A novel likely pathogenic mutation in MAP2K1, which encodes for MEK1, (p.E120Q), was identified in one patient, so far responding to DT. A MTOR mutation was identified in one case and may represent a novel therapeutic target in ATCs. TP53 mutations were detected, in five patients and, although known to be related with ATC development, did not appear to be directly associated with DT efficacy. Although ATC is a rare disease, the characterization of additional cases and molecular targets, may further support and extend the present findings. In the second task, western blot analysis showed that SPRY4 was downregulated in T235 co-cultures and upregulated in C3948 co-cultures, confirming previous proteomic data. Using SPRY4 siRNA-mediated silencing (> 50% of silencing), it was possible to observe that, in both cells lines, the invasion capabilities significantly increased in co-culture, similar to that observed in T235 using the non-target control (siNT) but contrasting with the decrease observed for C3948 cells with siNT. Also, the analysis of ATC cells’ cytoskeletal alterations and morphology, through immunofluorescence with phalloidin to detect actin filaments, confirmed the spreading behavior under siSPRY4 conditions in both cell lines, consistent with invasion assays results. Taken together, these results unveiled a tumour suppressor gene role for SPRY4 in ATC, in the presence of TAMs, with an influence in ATC-TAMs interactions and ATC aggressiveness. Additional studies are warranted to further elucidate SPRY4-related signaling mechanisms in this context. In conclusion, this study contributed to improve the present knowledge on the cellular and molecular mechanisms involved ATC response to therapy, and TME/TAM interactions, that are associated with ATC aggressiveness, which may eventually lead to new therapeutic approaches capable of improving the prognosis and overall survival of ATC patients.Cavaco, Branca Maria Prudêncio Limón, 1969-Dias, Deodália Maria Antunes, 1952-Repositório da Universidade de LisboaRodrigues, Ricardo Miguel dos Santos202120212024-12-30T00:00:00Z2021-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10451/52108TID:202994538enginfo:eu-repo/semantics/embargoedAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-11-08T16:57:11Zoai:repositorio.ul.pt:10451/52108Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T22:03:15.531Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse
dc.title.none.fl_str_mv Study of molecular and cellular mechanisms involved in anaplastic thyroid carcinoma aggressiveness and response to therapy
title Study of molecular and cellular mechanisms involved in anaplastic thyroid carcinoma aggressiveness and response to therapy
spellingShingle Study of molecular and cellular mechanisms involved in anaplastic thyroid carcinoma aggressiveness and response to therapy
Rodrigues, Ricardo Miguel dos Santos
Carcinoma anaplásico da tiróide
Terapêuticas dirigidas
Resistência a fármacos
Microambiente tumoral
Macrófagos associados ao tumor
Teses de mestrado - 2021
Departamento de Biologia Vegetal
title_short Study of molecular and cellular mechanisms involved in anaplastic thyroid carcinoma aggressiveness and response to therapy
title_full Study of molecular and cellular mechanisms involved in anaplastic thyroid carcinoma aggressiveness and response to therapy
title_fullStr Study of molecular and cellular mechanisms involved in anaplastic thyroid carcinoma aggressiveness and response to therapy
title_full_unstemmed Study of molecular and cellular mechanisms involved in anaplastic thyroid carcinoma aggressiveness and response to therapy
title_sort Study of molecular and cellular mechanisms involved in anaplastic thyroid carcinoma aggressiveness and response to therapy
author Rodrigues, Ricardo Miguel dos Santos
author_facet Rodrigues, Ricardo Miguel dos Santos
author_role author
dc.contributor.none.fl_str_mv Cavaco, Branca Maria Prudêncio Limón, 1969-
Dias, Deodália Maria Antunes, 1952-
Repositório da Universidade de Lisboa
dc.contributor.author.fl_str_mv Rodrigues, Ricardo Miguel dos Santos
dc.subject.por.fl_str_mv Carcinoma anaplásico da tiróide
Terapêuticas dirigidas
Resistência a fármacos
Microambiente tumoral
Macrófagos associados ao tumor
Teses de mestrado - 2021
Departamento de Biologia Vegetal
topic Carcinoma anaplásico da tiróide
Terapêuticas dirigidas
Resistência a fármacos
Microambiente tumoral
Macrófagos associados ao tumor
Teses de mestrado - 2021
Departamento de Biologia Vegetal
description Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2021
publishDate 2021
dc.date.none.fl_str_mv 2021
2021
2021-01-01T00:00:00Z
2024-12-30T00:00:00Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/masterThesis
format masterThesis
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://hdl.handle.net/10451/52108
TID:202994538
url http://hdl.handle.net/10451/52108
identifier_str_mv TID:202994538
dc.language.iso.fl_str_mv eng
language eng
dc.rights.driver.fl_str_mv info:eu-repo/semantics/embargoedAccess
eu_rights_str_mv embargoedAccess
dc.format.none.fl_str_mv application/pdf
dc.source.none.fl_str_mv reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
instacron:RCAAP
instname_str Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
instacron_str RCAAP
institution RCAAP
reponame_str Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
collection Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
repository.name.fl_str_mv Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
repository.mail.fl_str_mv
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