Epithelial to mesenchymal transition pathway in pituitary endocrine tumours

Detalhes bibliográficos
Autor(a) principal: Reis, Fábio Domingues Carril
Data de Publicação: 2023
Tipo de documento: Dissertação
Idioma: eng
Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
Texto Completo: http://hdl.handle.net/10362/162614
Resumo: Abstract Adenohypophyseal tumors, which were recently renamed pituitary neuroendocrine tumors (PitNET), are mostly benign tumours arising from adenohypophysis cells. The interation between elements within the microenvironment and tumor cells PitNETs, with a special emphasis on cytokines, has a substantial impact on various aspects of cancer-related processes. This includes the aggressiveness of tumors and how they respond to treatments. Among these cytokines, CCL2 has emerged as a central figure in PitNETs, although its precise biological role remains somewhat elusive. One possible explanation for its function is its influence on a process known as the epithelial-to-mesenchymal transition (EMT). EMT is a biological phenomenon where epithelial cells undergo significant transformations, including the loss of their characteristic cell polarity and the acquisition of heightened migratory capabilities. This transformation leads to increased invasiveness, aggressiveness, and resistance to treatment. A hallmark of EMT is the reduction in the expression of epithelial markers like E-cadherin, accompanied by the overexpression of mesenchymal markers like ZEB-1. In the context of pituitary neuroendocrine tumors (PitNETs), EMT remains a relatively unexplored area of study. We hypothesize that CCL2 contributes to shaping the characteristics and prognostic outcomes of PitNETs. So, with this work, we aimed to explore how CCL2 influences the epithelial-to-mesenchymal transition (EMT) pathway, with a strong focus on understanding its impact on the levels of E-cadherin and ZEB-1 expression. Additionally, we sought to determine the relationship between these molecular changes and the clinical features and outcomes observed in PitNET patients. To carry out our objectives we analyze the expression of CDH1 (which encodes E-cadherin, an epithelial marker) and ZEB1 (mesenchymal marker) and CCL2 by RT- qPCR on 86 PitNETs from patients who underwent surgery at our center between 2014-2020: 62 nonfunctioning-PitNETs (NF-PitNETs), 18 somatotropinomas and 6 corticotropinomas. CHD1, ZEB-1 and CCL2 expression was then correlated with clinico-pathological, outcome data, and CCL2 expression was also correlated with E-cadherin expression. In vitro experiments were also performed, these included proliferation assays, western blots, RT-PCR, immunofluorescence assays and morphology measurements. In our cohort, 47.7% were males, age at diagnosis was 56±15yr (mean±SD), and mean follow-up was 6±4yr. CCL2 mRNA expression did not differ among PitNET types, in the whole cohort, higher CCL2 mRNA expression was seen in males, patients who had hypopituitarism at diagnosis, and in patients who more often required multimodal therapy, needed more treatments and had active disease at last-follow-up. . CCL2 mRNA expression levels negatively correlated with CDH1 expression levels. Regarding the in vitro data, CCL2 appeared to have an anti-proliferative effect on GH3 cells. In addition, we did not observe a significant gain in the migration ability of GH3 cells in the presence of CCL2, in the transwell migration assay. Also, CCL2 did not induce significant morphological changes on GH3 cells. Nevertheless, our preliminary immunofluorescence experiments allowed us to detect the formation of filopodia in cells conditioned with rCCL2. Also, ZEB-1 mRNA expression levels were increased in cells conditioned with 5ng/ml rCCL2, consistent with a migratory phenotype, seemingly impacting EMT. Considering our results collectively, incorporating the assessment of CCL2 expression in human Pituitary Neuroendocrine Tumors (PitNETs) as a routine clinical practice, such as including it in pathology reports, may offer valuable prognostic insights, particularly in predicting the presence of more aggressive and challenging-to-treat PitNETs, working as a biomarker. However, the absence of a clear connection between CCL2 and the behavior of pituitary tumors in our in vitro experiments suggests that a prominent independent role in determining the aggressiveness of pituitary tumor cells by CCL2 alone, cannot yet be confirmed. Consequently, it does not currently justify the development or implementation of immunotherapy specifically designed to block or inhibit CCL2.
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spelling Epithelial to mesenchymal transition pathway in pituitary endocrine tumourspituitary endocrine tumoursCiências MédicasAbstract Adenohypophyseal tumors, which were recently renamed pituitary neuroendocrine tumors (PitNET), are mostly benign tumours arising from adenohypophysis cells. The interation between elements within the microenvironment and tumor cells PitNETs, with a special emphasis on cytokines, has a substantial impact on various aspects of cancer-related processes. This includes the aggressiveness of tumors and how they respond to treatments. Among these cytokines, CCL2 has emerged as a central figure in PitNETs, although its precise biological role remains somewhat elusive. One possible explanation for its function is its influence on a process known as the epithelial-to-mesenchymal transition (EMT). EMT is a biological phenomenon where epithelial cells undergo significant transformations, including the loss of their characteristic cell polarity and the acquisition of heightened migratory capabilities. This transformation leads to increased invasiveness, aggressiveness, and resistance to treatment. A hallmark of EMT is the reduction in the expression of epithelial markers like E-cadherin, accompanied by the overexpression of mesenchymal markers like ZEB-1. In the context of pituitary neuroendocrine tumors (PitNETs), EMT remains a relatively unexplored area of study. We hypothesize that CCL2 contributes to shaping the characteristics and prognostic outcomes of PitNETs. So, with this work, we aimed to explore how CCL2 influences the epithelial-to-mesenchymal transition (EMT) pathway, with a strong focus on understanding its impact on the levels of E-cadherin and ZEB-1 expression. Additionally, we sought to determine the relationship between these molecular changes and the clinical features and outcomes observed in PitNET patients. To carry out our objectives we analyze the expression of CDH1 (which encodes E-cadherin, an epithelial marker) and ZEB1 (mesenchymal marker) and CCL2 by RT- qPCR on 86 PitNETs from patients who underwent surgery at our center between 2014-2020: 62 nonfunctioning-PitNETs (NF-PitNETs), 18 somatotropinomas and 6 corticotropinomas. CHD1, ZEB-1 and CCL2 expression was then correlated with clinico-pathological, outcome data, and CCL2 expression was also correlated with E-cadherin expression. In vitro experiments were also performed, these included proliferation assays, western blots, RT-PCR, immunofluorescence assays and morphology measurements. In our cohort, 47.7% were males, age at diagnosis was 56±15yr (mean±SD), and mean follow-up was 6±4yr. CCL2 mRNA expression did not differ among PitNET types, in the whole cohort, higher CCL2 mRNA expression was seen in males, patients who had hypopituitarism at diagnosis, and in patients who more often required multimodal therapy, needed more treatments and had active disease at last-follow-up. . CCL2 mRNA expression levels negatively correlated with CDH1 expression levels. Regarding the in vitro data, CCL2 appeared to have an anti-proliferative effect on GH3 cells. In addition, we did not observe a significant gain in the migration ability of GH3 cells in the presence of CCL2, in the transwell migration assay. Also, CCL2 did not induce significant morphological changes on GH3 cells. Nevertheless, our preliminary immunofluorescence experiments allowed us to detect the formation of filopodia in cells conditioned with rCCL2. Also, ZEB-1 mRNA expression levels were increased in cells conditioned with 5ng/ml rCCL2, consistent with a migratory phenotype, seemingly impacting EMT. Considering our results collectively, incorporating the assessment of CCL2 expression in human Pituitary Neuroendocrine Tumors (PitNETs) as a routine clinical practice, such as including it in pathology reports, may offer valuable prognostic insights, particularly in predicting the presence of more aggressive and challenging-to-treat PitNETs, working as a biomarker. However, the absence of a clear connection between CCL2 and the behavior of pituitary tumors in our in vitro experiments suggests that a prominent independent role in determining the aggressiveness of pituitary tumor cells by CCL2 alone, cannot yet be confirmed. Consequently, it does not currently justify the development or implementation of immunotherapy specifically designed to block or inhibit CCL2.Resumo Os tumores adeno-hipofisários, que foram recentemente rebatizados de tumores neuroendócrinos da hipófise (PitNET), são na sua maioria tumores benignos que surgem das células adeno-hipofisárias. A interação entre os elementos do microambiente e as células tumorais destes tumores, com especial destaque para as citocinas, tem um impacto substancial em vários aspetos dos processos relacionados com o cancro, incluindo a agressividade dos tumores e a forma como respondem aos tratamentos. Entre estas citocinas, a CCL2 emergiu como uma figura central nos tumores neuroendócrinos da hipófise, embora o seu papel biológico exato permaneça inexplorado. Uma possível explicação para a sua função é a sua influência na transição epitélio-mesenquimal (MET). A MET é um fenómeno biológico em que as células epiteliais sofrem transformações significativas, incluindo a perda da sua polaridade celular caraterística e a aquisição de capacidades migratórias. Esta transformação leva a um aumento da capacidade de invasão, da agressividade e da resistência ao tratamento destes tumores. Uma caraterística marcante da MET é a redução da expressão de marcadores epiteliais como a E-caderina, acompanhada pela sobre-expressão de marcadores mesenquimais como o ZEB-1. No contexto dos tumores neuroendócrinos da hipófise (PitNETs), a MET continua a ser uma área de estudo relativamente inexplorada. A nossa hipótese é que a CCL2 contribui para moldar as características e os prognósticos dos tumores neuroendócrinos de hipófise. Com esta hipótese em mente, pretendemos explorar a forma como a CCL2 influencia a via da transição epitélio-mesenquimal (MET), com um foco na compreensão do seu impacto nos níveis de expressão da E-caderina e do ZEB-1. Além disso, procurámos determinar a relação entre estas alterações moleculares, as características clínicas e os resultados observados em doentes com tumores neuroendócrinos da hipófise. Para atingir os nossos objetivos, analisamos a expressão de CDH1 (que codifica E-caderina, um marcador epitelial), ZEB1 (marcador mesenquimal) e CCL2 por RT-qPCR em 86 PitNETs de pacientes submetidos a cirurgia no nosso centro entre 2014-2020: 62 PitNETs não funcionais (NF-PitNETs), 18 somatotropinomas e 6 corticotropinomas. Na nossa coorte, 47,7% eram homens, a idade ao diagnóstico foi de 56±15 anos (média ± DP) e o seguimento médio dos doentes foi de 6±4 anos. A expressão de CHD1, ZEB-1 e CCL2 foi então correlacionada com dados clínico-patológicos e a expressão de CCL2 foi também correlacionada com a expressão de E-caderina. Foram também efetuadas experiências in vitro, que incluíram ensaios de proliferação, western blots, RT-PCR, ensaios de imunofluorescência e medições morfológicas. A expressão de mRNA da CCL2 não diferiu entre os tipos de tumores neuroendócrinos hipofisários humanos, tendo sido observada uma maior expressão de mRNA da CCL2 nos homens, nos doentes que apresentavam hipopituitarismo ao diagnóstico, nos doentes que necessitavam mais frequentemente de multiterapia, que necessitavam de mais tratamentos e que tinham doença ativa no último seguimento. Os níveis de expressão de mRNA da CCL2 correlacionaram-se negativamente com os níveis de expressão de CDH1. Relativamente aos dados in vitro, a CCL2 parece ter um efeito anti-proliferativo nas células GH3, e não foi possível confirmar uma migração significativa das células GH3 com o ensaio de migração “transwell”. A CCL2 também não induziu alterações morfológicas significativas nas células GH3. No entanto, as nossas experiências preliminares de imunofluorescência permitiram-nos detetar a formação de filopodia nas células condicionadas com rCCL2. Observou-se também um aumento nos níveis de expressão de mRNA do ZEB-1 nas células condicionadas com 5 ng/ml de rCCL2, o que é consistente com um fenótipo migratório e, consequentemente, com um impacto da CCL2 na MET. Considerando estes resultados, a incorporação de uma avaliação da expressão de CCL2 nos tumores neuroendócrinos hipofisários humanos (PitNETs) como prática clínica de rotina, tal como a sua inclusão nos relatórios de patologia, pode oferecer informações prognósticas valiosas, particularmente na previsão da presença de tumores neuroendócrinos hipofisários humanos mais agressivos e difíceis de tratar, funcionando como um biomarcador. No entanto, a ausência de uma ligação clara entre o CCL2 e o comportamento dos tumores da hipófise nas nossas experiências in vitro sugere que ainda não é possível confirmar com certeza, que a CCL2 tenha um papel independente proeminente na determinação da agressividade das células tumorais. Consequentemente, atualmente não se justifica o desenvolvimento ou a implementação de imunoterapia especificamente concebida para bloquear ou inibir a CCL2.Silva, Ana Luísa Ribeiro daMarques, Pedro Miguel De SousaRUNReis, Fábio Domingues Carril2023-12-152026-12-15T00:00:00Z2023-12-15T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10362/162614TID:203474996enginfo: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:RCAAP2024-03-11T05:45:32Zoai:run.unl.pt:10362/162614Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T03:58:59.030399Repositório 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 Epithelial to mesenchymal transition pathway in pituitary endocrine tumours
title Epithelial to mesenchymal transition pathway in pituitary endocrine tumours
spellingShingle Epithelial to mesenchymal transition pathway in pituitary endocrine tumours
Reis, Fábio Domingues Carril
pituitary endocrine tumours
Ciências Médicas
title_short Epithelial to mesenchymal transition pathway in pituitary endocrine tumours
title_full Epithelial to mesenchymal transition pathway in pituitary endocrine tumours
title_fullStr Epithelial to mesenchymal transition pathway in pituitary endocrine tumours
title_full_unstemmed Epithelial to mesenchymal transition pathway in pituitary endocrine tumours
title_sort Epithelial to mesenchymal transition pathway in pituitary endocrine tumours
author Reis, Fábio Domingues Carril
author_facet Reis, Fábio Domingues Carril
author_role author
dc.contributor.none.fl_str_mv Silva, Ana Luísa Ribeiro da
Marques, Pedro Miguel De Sousa
RUN
dc.contributor.author.fl_str_mv Reis, Fábio Domingues Carril
dc.subject.por.fl_str_mv pituitary endocrine tumours
Ciências Médicas
topic pituitary endocrine tumours
Ciências Médicas
description Abstract Adenohypophyseal tumors, which were recently renamed pituitary neuroendocrine tumors (PitNET), are mostly benign tumours arising from adenohypophysis cells. The interation between elements within the microenvironment and tumor cells PitNETs, with a special emphasis on cytokines, has a substantial impact on various aspects of cancer-related processes. This includes the aggressiveness of tumors and how they respond to treatments. Among these cytokines, CCL2 has emerged as a central figure in PitNETs, although its precise biological role remains somewhat elusive. One possible explanation for its function is its influence on a process known as the epithelial-to-mesenchymal transition (EMT). EMT is a biological phenomenon where epithelial cells undergo significant transformations, including the loss of their characteristic cell polarity and the acquisition of heightened migratory capabilities. This transformation leads to increased invasiveness, aggressiveness, and resistance to treatment. A hallmark of EMT is the reduction in the expression of epithelial markers like E-cadherin, accompanied by the overexpression of mesenchymal markers like ZEB-1. In the context of pituitary neuroendocrine tumors (PitNETs), EMT remains a relatively unexplored area of study. We hypothesize that CCL2 contributes to shaping the characteristics and prognostic outcomes of PitNETs. So, with this work, we aimed to explore how CCL2 influences the epithelial-to-mesenchymal transition (EMT) pathway, with a strong focus on understanding its impact on the levels of E-cadherin and ZEB-1 expression. Additionally, we sought to determine the relationship between these molecular changes and the clinical features and outcomes observed in PitNET patients. To carry out our objectives we analyze the expression of CDH1 (which encodes E-cadherin, an epithelial marker) and ZEB1 (mesenchymal marker) and CCL2 by RT- qPCR on 86 PitNETs from patients who underwent surgery at our center between 2014-2020: 62 nonfunctioning-PitNETs (NF-PitNETs), 18 somatotropinomas and 6 corticotropinomas. CHD1, ZEB-1 and CCL2 expression was then correlated with clinico-pathological, outcome data, and CCL2 expression was also correlated with E-cadherin expression. In vitro experiments were also performed, these included proliferation assays, western blots, RT-PCR, immunofluorescence assays and morphology measurements. In our cohort, 47.7% were males, age at diagnosis was 56±15yr (mean±SD), and mean follow-up was 6±4yr. CCL2 mRNA expression did not differ among PitNET types, in the whole cohort, higher CCL2 mRNA expression was seen in males, patients who had hypopituitarism at diagnosis, and in patients who more often required multimodal therapy, needed more treatments and had active disease at last-follow-up. . CCL2 mRNA expression levels negatively correlated with CDH1 expression levels. Regarding the in vitro data, CCL2 appeared to have an anti-proliferative effect on GH3 cells. In addition, we did not observe a significant gain in the migration ability of GH3 cells in the presence of CCL2, in the transwell migration assay. Also, CCL2 did not induce significant morphological changes on GH3 cells. Nevertheless, our preliminary immunofluorescence experiments allowed us to detect the formation of filopodia in cells conditioned with rCCL2. Also, ZEB-1 mRNA expression levels were increased in cells conditioned with 5ng/ml rCCL2, consistent with a migratory phenotype, seemingly impacting EMT. Considering our results collectively, incorporating the assessment of CCL2 expression in human Pituitary Neuroendocrine Tumors (PitNETs) as a routine clinical practice, such as including it in pathology reports, may offer valuable prognostic insights, particularly in predicting the presence of more aggressive and challenging-to-treat PitNETs, working as a biomarker. However, the absence of a clear connection between CCL2 and the behavior of pituitary tumors in our in vitro experiments suggests that a prominent independent role in determining the aggressiveness of pituitary tumor cells by CCL2 alone, cannot yet be confirmed. Consequently, it does not currently justify the development or implementation of immunotherapy specifically designed to block or inhibit CCL2.
publishDate 2023
dc.date.none.fl_str_mv 2023-12-15
2023-12-15T00:00:00Z
2026-12-15T00:00:00Z
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