AGK::BRAF no carcinoma papilífero da tiroide pediátrico: investigação do mecanismo associado à agressividade tumoral e análise funcional
Arquivos
Data
2023-03-31
Autores
Sisdelli, Luiza de Mello Oliveira 
Orientadores
Cerutti, Janete Maria
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Introdução: O carcinoma papilífero da tireoide (CPT), apesar de ser raro em crianças e adolescentes, geralmente têm uma apresentação clínica mais agressiva nesta população do que o CPT em adultos, apresentando multifocalidade, subtipos histológicos mais agressivos, presença de invasão tumoral, e maior prevalência de metástase loco-regional e à distância. Apesar disso, a mortalidade pelo câncer na faixa etária pediátrica é inferior àquela reportada em adultos. Tem sido sugerido que estas características clínico patológicas observadas em pacientes pediátricos (≤ 18 anos) deve-se, principalmente, às diferenças observadas quanto ao tipo de alteração observada nos genes “drivers” que levam à ativação constitutiva da via MAPK/ERK neste grupo de pacientes, bem como, maior responsividade ao tratamento convencional como iodo.
Objetivos: Esta tese teve como objetivo analisar alterações genéticas associadas a ativação das vias RAS/MAPK/ERK e PI3K/AKT em 79 casos de CPT pediátricos e avaliar o efeito da presença da fusão AGK::BRAF na ativação da via MAPK, na expressão do gene NIS (do inglês, sodium/iodide symporter) e na atividade de NIS, bem como, em processos biológicos associados à progressão tumoral.
Metodologia: As fusões gênicas (RET::PTC1, RET::PTC3, AGK::BRAF, ETV6::NTRK3 e STRN::ALK) foram avaliadas por RT-PCR e as variantes patogênicas do tipo SNVs (do inglês, Single Nucleotide Variants) (BRAF V600E, e TERT C228T/C250T) avaliadas por sequenciamento de Sanger. A análise funcional foi realizada na linhagem celular derivada de células foliculares de tiroide normal de rato (PCCL3) permanentemente e independentemente transfectadas com plasmídeo contendo cDNA de AGK::BRAF, ETV6::NTRK3, RET::PTC1, RET::PTC3, BRAF V600E, e BRAF WT (do inglês, wild type). Para examinar o efeito destes eventos genéticos nas vias de sinalização RAS/MAPK/ERK e PIK3/AKT e nas taxas da expressão de NIS, utilizamos a técnica de Western-blot. A atividade de NIS foi analisada por um ensaio de captação de iodeto não radioativo. A instabilidade genômica foi investigada por ensaio de micronúcleos com DAPI e microscopia fluorescente. A organização dos telômeros foi avaliada por hibridização in situ fluorescente quantitativa (Q-FISH) seguida por análise da organização tridimensional (3D) dos telômeros utilizando o software TeloView®. A organização da cromatina, por microscopia de super-resolução de iluminação estruturada tridimensional (3D-SIM), seguido por granulometria.
Resultados: Na análise do perfil molecular, identificamos alterações genéticas em 53/79 (67%) dos casos de CPT, sendo que 59% (47/79) apresentavam fusões gênicas. RET::PTC1 foi a alteração genética mais frequente (22/79, 28%), seguida de AGK::BRAF (15/79, 19%), ETV6::NTRK3 (14/79, 18%), RET::PTC3 (12/79, 15%), BRAF V600E (11/79, 14%) e STRN::ALK (3/79, 4%). Entre as amostras analisadas para mutações do promotor de TERT (C228T e C250T) (n = 42), apenas uma (1/42, 2,4%) carregava uma variante patogênica (C250T). Além disso, identificamos a coexistência de mais de uma alteração genética associada à via MAPK em cerca de 1/3 desta coorte. Encontramos uma associação entre a presença de fusão RET::PTC3 e metástases linfonodais, multifocalidade e tamanho do tumor. A análise funcional demonstrou que a fusão AGK::BRAF leva a maior ativação da via MAPK e menor expressão e atividade de NIS em células PCCL3. Estes dados foram acompanhados por um maior número de micronúcleos e pontes anafásicas, alteração da organização 3D dos telômeros e da estrutura da cromatina. Demonstramos que os tumores de pacientes pediátricos têm diferentes assinaturas de organização telomérica e estrutura do DNA, em comparação com o tecido normal, sugerindo uma ruptura progressiva da organização da cromatina e dos telômeros, que pode estar relacionada à instabilidade genômica. Além disso, dados preliminares do 3D-SIM demonstram que pacientes com características clinicopatológicas de mau prognóstico (metástase à distância, multifocalidade e tamanho tumoral > 2 cm) apresentam mais espaços pobres em DNA nos núcleos dos tumores. Curiosamente, os tumores positivos para a fusão AGK::BRAF, mostraram uma tendência a ter mais espaços pobres em DNA em seus núcleos. De fato, demonstramos, em uma análise feita em um relato de caso, que a fusão AGK::BRAF resulta em uma organização diferente dos telômeros e do DNA, quando comparada a RET::PTC3.
Conclusão: Aqui confirmamos que rearranjos são o evento genético mais comum no CPT pediátrico, e ainda demonstramos que co-ocorrência de mais de uma alteração genética que levam à ativação da via RAS/MAPK/ERK são prevalentes nos tumores desta população. Importante, observamos que o rearranjo RET::PTC3 está associado a características de mau prognóstico no CPT pediátrico. Os dados da análise funcional ajudaram a elucidar os mecanismos pelos quais a fusão AGK::BRAF causa a desdiferenciação das células tiroidianas e um comportamento mais agressivo das células tumorais em casos pediátricos de CPT com metástase à distância. Também demonstramos como o estudo da organização tridimensional nuclear e da estrutura do DNA do CPT pediátrico pode ser de extrema importância para o melhor entendimento desses tumores.
Introduction: The papillary thyroid carcinoma (PTC), despite being rare in children and adolescents, generally has a more aggressive clinical presentation in this population than in adult PTC, with multifocality, more aggressive histological subtypes, presence of tumor invasion, and higher prevalence of locoregional and distant metastasis. Despite this, cancer mortality in the pediatric age group is lower than that reported in adults. It has been suggested that these clinical and pathological characteristics observed in pediatric patients (≤ 18 years old) are mainly due to the differences observed in the type of alteration observed in the ―driver‖ genes that lead to the constitutive activation of the MAPK/ERK pathway in this group of patients, as well as greater responsiveness to conventional treatment such as iodine. Objectives: This thesis aimed to analyze the genetic alterations associated with the activation of the RAS/MAPK/ERK and PI3K/AKT pathway in 79 cases of pediatric PTC and to evaluate the effect of the presence of the AGK::BRAF fusion on the activation of the MAPK pathway, on the expression of the NIS protein (sodium/iodide symporter) and NIS activity, as well as biological processes associated with tumor progression. Methodology: Gene fusions (RET::PTC1, RET::PTC3, AGK::BRAF, ETV6::NTRK3 and STRN::ALK) were evaluated by RT-PCR and point genetic alterations (BRAF V600E, and TERT C228T/C250T) were screened by sequencing from Sanger. Functional analysis was performed on a cell line derived from rat normal thyroid follicular cells (PCCL3) permanently and independently transfected with plasmid containing cDNA of AGK::BRAF, ETV6::NTRK3, RET::PTC1, RET::PTC3, BRAF V600E, and BRAF WT (wild type). To examine the effect of these genetic events on RAS/MAPK/ERK and PIK3/AKT signaling pathways and on NIS expression rates, we used the Western-blot technique. NIS activity was analyzed by a non-radioactive iodide uptake assay. Genomic instability was investigated by DAPI micronucleus assay and fluorescent microscopy. Telomere organization was assessed by xiv quantitative fluorescent in situ hybridization (Q-FISH) followed by three-dimensional (3D) telomere organization analysis using the TeloView® software. Chromatin organization was analyzed by super-resolution three-dimensional structured illumination microscopy (3D-SIM), followed by granulometry. Results: In the molecular profile analysis, we identified genetic alterations in 53/79 (67%) of PTC cases, with 59% (47/79) having gene fusions. RET::PTC1 was the most frequent genetic alteration (22/79, 28%), followed by AGK::BRAF (15/79, 19%), ETV6::NTRK3 (14/79, 18%), RET::PTC3 (12/79, 15%), BRAF V600E (11/79, 14%), and STRN::ALK (3/79, 4%). Among the samples analyzed for TERT promoter mutations (C228T and C250T) (n = 42), only one (1/42, 2.4%) carried a mutation (C250T). Furthermore, we identified the coexistence of more than one genetic alteration associated with the RAS/MAPK/ERK pathway in approximately 1/3 of our cohort. We found an association between the presence of RET::PTC3 fusion and lymph node metastases, multifocality and tumor size. Functional analysis demonstrated that the AGK::BRAF fusion leads to greater activation of the RAS/MAPK/ERK pathway and lower expression and activity of NIS in PCCL3 cells. These data were accompanied by a greater number of micronuclei and anaphase bridges, changes in the 3D organization of telomeres and chromatin structure. We demonstrated that tumors from pediatric patients have different signatures of telomere organization and DNA structure compared to normal tissue, suggesting a progressive disruption of chromatin and telomere organization, which may be related to genomic instability. Furthermore, preliminary data from 3D-SIM demonstrate that patients with clinicopathological features of poor prognosis (distant metastasis, multifocality, and tumor size > 2 cm) have more DNA-poor spaces in tumor nuclei. Interestingly, tumors positive for the AGK::BRAF fusion showed a tendency to have more DNA-poor spaces in their nuclei. In fact, we demonstrated, in an analysis made in a case report, that the AGK::BRAF fusion results in a different organization of telomeres and DNA, when compared to RET::PTC3. Conclusion: Here we confirm that rearrangements are the most common genetic event in pediatric PTC, and we also demonstrated that the co-occurrence of more xv than one genetic alteration that led to the activation of the RAS/MAPK/ERK pathway are prevalent in tumors in this population. Importantly, we observed that the RET::PTC3 rearrangement is associated with poor prognostic features in pediatric PTC. Functional analysis data helped to elucidate the mechanisms by which the AGK::BRAF fusion causes thyroid cell dedifferentiation and more aggressive tumor cell behavior in pediatric cases of PTC with distant metastasis. We also demonstrate how the study of the three-dimensional nuclear organization and DNA structure of pediatric PTC can be extremely important for a better understanding of these tumors.
Introduction: The papillary thyroid carcinoma (PTC), despite being rare in children and adolescents, generally has a more aggressive clinical presentation in this population than in adult PTC, with multifocality, more aggressive histological subtypes, presence of tumor invasion, and higher prevalence of locoregional and distant metastasis. Despite this, cancer mortality in the pediatric age group is lower than that reported in adults. It has been suggested that these clinical and pathological characteristics observed in pediatric patients (≤ 18 years old) are mainly due to the differences observed in the type of alteration observed in the ―driver‖ genes that lead to the constitutive activation of the MAPK/ERK pathway in this group of patients, as well as greater responsiveness to conventional treatment such as iodine. Objectives: This thesis aimed to analyze the genetic alterations associated with the activation of the RAS/MAPK/ERK and PI3K/AKT pathway in 79 cases of pediatric PTC and to evaluate the effect of the presence of the AGK::BRAF fusion on the activation of the MAPK pathway, on the expression of the NIS protein (sodium/iodide symporter) and NIS activity, as well as biological processes associated with tumor progression. Methodology: Gene fusions (RET::PTC1, RET::PTC3, AGK::BRAF, ETV6::NTRK3 and STRN::ALK) were evaluated by RT-PCR and point genetic alterations (BRAF V600E, and TERT C228T/C250T) were screened by sequencing from Sanger. Functional analysis was performed on a cell line derived from rat normal thyroid follicular cells (PCCL3) permanently and independently transfected with plasmid containing cDNA of AGK::BRAF, ETV6::NTRK3, RET::PTC1, RET::PTC3, BRAF V600E, and BRAF WT (wild type). To examine the effect of these genetic events on RAS/MAPK/ERK and PIK3/AKT signaling pathways and on NIS expression rates, we used the Western-blot technique. NIS activity was analyzed by a non-radioactive iodide uptake assay. Genomic instability was investigated by DAPI micronucleus assay and fluorescent microscopy. Telomere organization was assessed by xiv quantitative fluorescent in situ hybridization (Q-FISH) followed by three-dimensional (3D) telomere organization analysis using the TeloView® software. Chromatin organization was analyzed by super-resolution three-dimensional structured illumination microscopy (3D-SIM), followed by granulometry. Results: In the molecular profile analysis, we identified genetic alterations in 53/79 (67%) of PTC cases, with 59% (47/79) having gene fusions. RET::PTC1 was the most frequent genetic alteration (22/79, 28%), followed by AGK::BRAF (15/79, 19%), ETV6::NTRK3 (14/79, 18%), RET::PTC3 (12/79, 15%), BRAF V600E (11/79, 14%), and STRN::ALK (3/79, 4%). Among the samples analyzed for TERT promoter mutations (C228T and C250T) (n = 42), only one (1/42, 2.4%) carried a mutation (C250T). Furthermore, we identified the coexistence of more than one genetic alteration associated with the RAS/MAPK/ERK pathway in approximately 1/3 of our cohort. We found an association between the presence of RET::PTC3 fusion and lymph node metastases, multifocality and tumor size. Functional analysis demonstrated that the AGK::BRAF fusion leads to greater activation of the RAS/MAPK/ERK pathway and lower expression and activity of NIS in PCCL3 cells. These data were accompanied by a greater number of micronuclei and anaphase bridges, changes in the 3D organization of telomeres and chromatin structure. We demonstrated that tumors from pediatric patients have different signatures of telomere organization and DNA structure compared to normal tissue, suggesting a progressive disruption of chromatin and telomere organization, which may be related to genomic instability. Furthermore, preliminary data from 3D-SIM demonstrate that patients with clinicopathological features of poor prognosis (distant metastasis, multifocality, and tumor size > 2 cm) have more DNA-poor spaces in tumor nuclei. Interestingly, tumors positive for the AGK::BRAF fusion showed a tendency to have more DNA-poor spaces in their nuclei. In fact, we demonstrated, in an analysis made in a case report, that the AGK::BRAF fusion results in a different organization of telomeres and DNA, when compared to RET::PTC3. Conclusion: Here we confirm that rearrangements are the most common genetic event in pediatric PTC, and we also demonstrated that the co-occurrence of more xv than one genetic alteration that led to the activation of the RAS/MAPK/ERK pathway are prevalent in tumors in this population. Importantly, we observed that the RET::PTC3 rearrangement is associated with poor prognostic features in pediatric PTC. Functional analysis data helped to elucidate the mechanisms by which the AGK::BRAF fusion causes thyroid cell dedifferentiation and more aggressive tumor cell behavior in pediatric cases of PTC with distant metastasis. We also demonstrate how the study of the three-dimensional nuclear organization and DNA structure of pediatric PTC can be extremely important for a better understanding of these tumors.