Heterogeneidade molecular entre diferentes linhagens celulares estabelecidas de câncer de mama humano triplo-negativo
Data
2022-10-28
Autores
Ferreira, Ariana Carolina 
Orientadores
Pinhal, Maria Aparecida da Silva
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Introdução: O câncer de mama triplo negativo (TNBC) representa de 15% a 20% dos casos de neoplasia maligna mamária, sendo constituído por tumores com características heterogêneas, com maior incidência de metástases, menor sobrevida dos pacientes e opções de tratamento limitadas, em que a terapia hormonal e os medicamentos direcionados ao receptor HER2 são ineficazes. Portanto, é crucial a identificação de biomarcadores moleculares que auxiliem no desenvolvimento de estratégias de tratamento mais efetivas e possíveis combinações utilizando terapias direcionadas. Objetivos: Caracterizar as linhagens celulares estabelecidas de câncer de mama triplo-negativo, MDA-MB-453, MDA-MB-231, HCC38 e BT-549, por análise do perfil de proliferação e migração celular, capacidade de formação de colônias, e determinar o padrão de expressão gênica de moléculas envolvidas com a carcinogênese (sindecans, heparanases, metaloproteases, glicosaminoglicanos, CD44 e HER2). Comparar a resposta das diferentes linhagens celulares triplo-negativas ao tratamento com Paclitaxel, bem como, avaliar possíveis alterações moleculares após tratamento. Métodos: A expressão gênica foi determinada por PCR quantitativo, microscopia confocal e citometria de fluxo. O perfil de glicosaminoglicanos foi obtido por eletroforese após incorporação de [35S]-sulfato. A resposta ao tratamento com Paclitaxel foi avaliada por ensaios de viabilidade celular (MTT) e morte celular (Anexina V/ PI). A resistência ao Paclitaxel foi obtida após tratamento contínuo das células MDA-MB-231 com tal quimioterápico. A compreensão do papel biológico do sindecam-4 (SDC4) foi feita por análises in silico e silenciamento utilizando short hairpin RNAs (shRNA). Resultados: A análise da expressão gênica revelou que as linhagens celulares triplo-negativas MDA-MB-453 e HCC38 expressam mRNA para HER2, sendo que tal receptor está ativado na linhagem HCC38. A expressão de CD44 foi significantemente menor nas células MDA-MB-453, sendo que tal linhagem não exibiu isoformas variantes do receptor (CD44v). As linhagens de origem metastática, MDA-MB-453 e MDA-MB-231, não exibiram as isoformas CD44v3I e CD44v5. As células triplo-negativas estudadas também exibiram diferentes perfis de glicosaminoglicanos sulfatados. As células HCC38 e BT-549, apresentaram maior expressão gênica de sindecam-4 e resposta mais efetiva ao Paclitaxel em relação às células de origem metastática, MDA-MB-453 e MDA-MB-231. Análises in silico evidenciaram que a baixa expressão de sindecam-4 (SDC4) está associada à presença de linfonodos metastáticos e baixa sobrevida dos pacientes acometidos por câncer de mama. O silenciamento gênico de SDC4 acarretou menor resposta ao tratamento com Paclitaxel. As células MDA-MB-231 resistentes ao Paclitaxel apresentaram alterações morfológicas e diminuição da expressão de SDC4. O tratamento quimioterápico foi capaz de induzir alterações significativas na expressão gênica das diferentes linhagens de câncer triplo-negativo. Conclusões: As linhagens celulares, embora pertencentes ao subtipo molecular triplo-negativo de câncer de mama, exibiram características moleculares e resposta ao tratamento quimioterápico distintos, evidenciando possíveis alvos para o desenvolvimento de abordagens terapêuticas mais efetivas. Os dados in silico e in vitro mostraram a importância do SDC4 na tumorigênese do câncer de mama, sugerindo que tal molécula possa servir como potencial biomarcador para o prognóstico do câncer de mama e, desta forma, auxiliar na decisão de estratégias terapêuticas mais assertivas.
Introduction: Triple-negative breast cancer (TNBC) represents 15% to 20% of malignant breast cancer cases, consisting tumors with very heterogeneous characteristics, with a higher incidence of metastases, lower patient survival, and limited treatment options, since hormone therapy and drugs targeting the HER2 receptor are ineffective. Therefore, identifying molecular biomarkers that help develop more effective treatment strategies and possible combinations using targeted therapies is critical. Objectives: To characterize established triple-negative breast cancer cell lines, MDA-MB-453, MDA-MB-231, HCC38, and BT-549, by analyzing the cell proliferation and migration profile, colony formation capacity, and determining the pattern of gene expression of molecules involved in carcinogenesis (syndecans, heparanases, metalloproteases, glycosaminoglycans, CD44 and HER2). Compare the response of different triple-negative cell lines to treatment with Paclitaxel and evaluate possible molecular changes after treatment. Methods: Gene expression was determined by quantitative PCR, confocal microscopy, and flow cytometry. The glycosaminoglycan profile was obtained by electrophoresis after incorporation of [35S]-sulfate. Response to Paclitaxel treatment was assessed by cell viability (MTT) and cell death (Annexin V/PI) assays. Paclitaxel resistance was obtained after continuous treatment of MDA-MB-231 cells. The biological role of syndecan-4 (SDC4) was evaluated by in silico analysis and silencing assay using short hairpin RNAs (shRNA). Results: The analysis of gene expression revealed that the triple-negative cell lines MDA-MB-453 and HCC38 express mRNA for HER2, and this receptor is activated in the HCC38 lineage. CD44 expression was significantly lower in MDA-MB-453 cells, and this strain did not exhibit variant isoforms of that receptor (CD44v). The lines of metastatic origin, MDA-MB-453 and MDA-MB-231 did not exhibit the CD44v3I and CD44v5 isoforms. The triple-negative cells studied also exhibited different profiles of sulfated glycosaminoglycans. The HCC38 and BT-549 cells showed greater gene expression of syndecan-4 and a more effective response to Paclitaxel concerning cells of metastatic origin, MDA-MB-453 and MDA-MB-231. In silico analyses showed that the low expression of syndecan-4 (SDC4) is associated with metastatic lymph nodes and poor survival of patients with breast cancer. SDC4 gene silencing resulted in a lower response to treatment with Paclitaxel. Paclitaxel-resistant MDA-MB-231 cells showed morphological changes and decreased SDC4 expression. The chemotherapy treatment induced significant changes in the gene expression of different triple-negative cancer lines. Conclusions: The cell lines, although belonging to the triple-negative molecular subtype of breast cancer, exhibited distinct molecular characteristics and responses to chemotherapy treatment, highlighting possible targets for developing more effective therapeutic approaches. The in silico and in vitro data showed the importance of SDC4 in the tumorigenesis of breast cancer, suggesting that this molecule can serve as a potential biomarker for the prognosis of breast cancer and thus help in the decision of more assertive therapeutic strategies.
Introduction: Triple-negative breast cancer (TNBC) represents 15% to 20% of malignant breast cancer cases, consisting tumors with very heterogeneous characteristics, with a higher incidence of metastases, lower patient survival, and limited treatment options, since hormone therapy and drugs targeting the HER2 receptor are ineffective. Therefore, identifying molecular biomarkers that help develop more effective treatment strategies and possible combinations using targeted therapies is critical. Objectives: To characterize established triple-negative breast cancer cell lines, MDA-MB-453, MDA-MB-231, HCC38, and BT-549, by analyzing the cell proliferation and migration profile, colony formation capacity, and determining the pattern of gene expression of molecules involved in carcinogenesis (syndecans, heparanases, metalloproteases, glycosaminoglycans, CD44 and HER2). Compare the response of different triple-negative cell lines to treatment with Paclitaxel and evaluate possible molecular changes after treatment. Methods: Gene expression was determined by quantitative PCR, confocal microscopy, and flow cytometry. The glycosaminoglycan profile was obtained by electrophoresis after incorporation of [35S]-sulfate. Response to Paclitaxel treatment was assessed by cell viability (MTT) and cell death (Annexin V/PI) assays. Paclitaxel resistance was obtained after continuous treatment of MDA-MB-231 cells. The biological role of syndecan-4 (SDC4) was evaluated by in silico analysis and silencing assay using short hairpin RNAs (shRNA). Results: The analysis of gene expression revealed that the triple-negative cell lines MDA-MB-453 and HCC38 express mRNA for HER2, and this receptor is activated in the HCC38 lineage. CD44 expression was significantly lower in MDA-MB-453 cells, and this strain did not exhibit variant isoforms of that receptor (CD44v). The lines of metastatic origin, MDA-MB-453 and MDA-MB-231 did not exhibit the CD44v3I and CD44v5 isoforms. The triple-negative cells studied also exhibited different profiles of sulfated glycosaminoglycans. The HCC38 and BT-549 cells showed greater gene expression of syndecan-4 and a more effective response to Paclitaxel concerning cells of metastatic origin, MDA-MB-453 and MDA-MB-231. In silico analyses showed that the low expression of syndecan-4 (SDC4) is associated with metastatic lymph nodes and poor survival of patients with breast cancer. SDC4 gene silencing resulted in a lower response to treatment with Paclitaxel. Paclitaxel-resistant MDA-MB-231 cells showed morphological changes and decreased SDC4 expression. The chemotherapy treatment induced significant changes in the gene expression of different triple-negative cancer lines. Conclusions: The cell lines, although belonging to the triple-negative molecular subtype of breast cancer, exhibited distinct molecular characteristics and responses to chemotherapy treatment, highlighting possible targets for developing more effective therapeutic approaches. The in silico and in vitro data showed the importance of SDC4 in the tumorigenesis of breast cancer, suggesting that this molecule can serve as a potential biomarker for the prognosis of breast cancer and thus help in the decision of more assertive therapeutic strategies.