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The role of immunogenic cell death as a mechanism of tumorigenesis
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As elevadas taxas de morbilidade e mortalidade do carcinoma hepatocelular (CHC) exigem uma maior compreensão dos processos fisiopatológicos do cancro hepático e do modo como os mesmos podem comprometer a eficácia terapêutica. O CHC surge maioritariamente num contexto de doença hepática crónica, na qual os processos de morte celular imunogénica promovem reações inflamatórias crónicas e um ciclo contínuo de destruição celular que estimulam a regeneração hepática e a seleção das linhagens celulares mais capacitadas para proliferar, ou seja, com uma maior tendência carcinogénica. No entanto, a contribuição exata de cada interveniente da resposta imunológica para a esteatose, esteato-hepatite não alcoólica (EHNA) e o desenvolvimento de CHC ainda não é clara. A cinase receptor-interacting protein 3 (RIPK3) é um mediador essencial da necroptose, uma forma de morte celular lítica regulada com elevada imunogenicidade. Estudos recentes mostraram que a RIPK3 está ativada em doentes com doença hepática crónica e que a sua inibição é suficiente para impedir a progressão da doença in vivo. Por sua vez, a ausência de RIPK3 nos roedores reduziu a infiltração de células inflamatórias do fígado, o estado fibrótico e os nódulos pré-neoplásicos em modelos experimentais diéticos de EHNA. Desse modo, a RIPK3 pode exercer um papel determinante na interação entre a necrose regulada, a imunidade e a hepatocarcinogénese. Estes estudos pretenderam avaliar o impacto da deficiência em Ripk3 na carcinogénese hepática. Em primeiro lugar, foram analisadas as alterações fenotípicas decorrentes da inibição da RIPK3 no desenvolvimento tumoral, utilizando um conjunto de modelos experimentais de fígado gordo não alcoólico (FGNA) e CHC baseados numa dieta rica em gordura e deficiente em colina, na utilização de um indutor de carcinogénese, DEN, assim como, através da combinação de ambos os estímulos. De seguida, foram exploradas as subpopulações de células imunes inatas e adaptativas presentes no microambiente tumoral recorrendo a marcadores específicos (Arg1, Cd206, F4/80, Infγ, Inos, Klrb1, Nlrp3, Pd-l1, Tgfβ e Tnf-α). Os resultados comprovaram que o ambiente inflamatório que vigora durante a fase de EHNA é modulado pela RIPK3 e é determinante para o processo de carcinogénese hepática. Por outro lado, os nódulos tumorais apresentaram macrófagos pró-inflamatórios infiltrados e células natural killer, mesmo após a depleção da RIPK3, o que evidencia a intercomunicação entre os vários componentes do microambiente tumoral, os quais podem proporcionar efeitos complementares no desenvolvimento do CHC. Por fim, as funções antitumorais das células imunes adaptativas podem ser resgatadas pela deleção de Ripk3. No seu todo, os nossos estudos reforçam que a inibição dos mecanismos dependentes de RIPK3 nos doentes com EHNA poderá ser uma abordagem terapêutica para prevenir o desenvolvimento de CHC associado a FGNA.
The hepatocellular carcinoma (HCC) high morbidity and mortality call for a better understanding about liver cancer evolution and how it drives resistance to therapies. HCC arises in a background of chronic liver disease, where sustained immunogenic cell death fosters inflammation and a continuous cycle of cellular destruction that trigger liver regenerative responses, and later select cell clones better adapted to survive and proliferate. Still, the exact contribution of immune cells to steatosis, non-alcoholic steatohepatitis (NASH), and HCC development is unclear. Receptor-interacting protein kinase 3 (RIPK3) is a key executor of necroptosis, which is a lytic immunogenic form of regulated cell death. Recent evidence shows that RIPK3 is triggered under chronic liver injury in humans, while its inhibition could arrest disease progression in vivo. Further, Ripk3-deficient mice display decreased liver inflammatory cell infiltration, fibrosis and preneoplastic nodules upon dietary models of NASH. Thereby, RIPK3 exerts an intricate role in the interplay between regulated necrosis, immunity and hepatocarcinogenesis. In this thesis, we aimed to evaluate the impact of Ripk3 deficiency in hepatocarcinogenesis. First, we evaluated the phenotypic outcomes of Ripk3 ablation in tumour development in a collection of preclinical models of NAFLD and HCC, using CD-HFD-induced NASH, DEN-induced HCC, and a combination of DEN plus CD-HFD HCC models. We next explored the innate and adaptive immune cell subpopulations in the tumour microenvironment (TME) by evaluating the expression profile of selected markers (Arg1, Cd206, F4/80, Infγ, Inos, Klrb1, Nlrp3, Pd-l1, Tgfβ and Tnf-α). Indeed, our results proved that the inflammatory environment that is imposed during the NASH stage is modulated by RIPK3 and determinant for hepatocarcinogenesis. Surprisingly, tumour nodules presented infiltered pro-inflammatory macrophages and natural killer cells, even upon RIPK3 ablation, which evidences part of the crosstalk between the distinct components of the TME that may afford complementary effects on HCC progression. Finally, the anti-tumoral functions of the adaptive immune cells could be rescued by Ripk3 deletion. Taken together, our studies reinforce that inhibition of RIPK3-dependent mechanisms may be a therapeutic approach to prevent NAFLD-associated HCC.
The hepatocellular carcinoma (HCC) high morbidity and mortality call for a better understanding about liver cancer evolution and how it drives resistance to therapies. HCC arises in a background of chronic liver disease, where sustained immunogenic cell death fosters inflammation and a continuous cycle of cellular destruction that trigger liver regenerative responses, and later select cell clones better adapted to survive and proliferate. Still, the exact contribution of immune cells to steatosis, non-alcoholic steatohepatitis (NASH), and HCC development is unclear. Receptor-interacting protein kinase 3 (RIPK3) is a key executor of necroptosis, which is a lytic immunogenic form of regulated cell death. Recent evidence shows that RIPK3 is triggered under chronic liver injury in humans, while its inhibition could arrest disease progression in vivo. Further, Ripk3-deficient mice display decreased liver inflammatory cell infiltration, fibrosis and preneoplastic nodules upon dietary models of NASH. Thereby, RIPK3 exerts an intricate role in the interplay between regulated necrosis, immunity and hepatocarcinogenesis. In this thesis, we aimed to evaluate the impact of Ripk3 deficiency in hepatocarcinogenesis. First, we evaluated the phenotypic outcomes of Ripk3 ablation in tumour development in a collection of preclinical models of NAFLD and HCC, using CD-HFD-induced NASH, DEN-induced HCC, and a combination of DEN plus CD-HFD HCC models. We next explored the innate and adaptive immune cell subpopulations in the tumour microenvironment (TME) by evaluating the expression profile of selected markers (Arg1, Cd206, F4/80, Infγ, Inos, Klrb1, Nlrp3, Pd-l1, Tgfβ and Tnf-α). Indeed, our results proved that the inflammatory environment that is imposed during the NASH stage is modulated by RIPK3 and determinant for hepatocarcinogenesis. Surprisingly, tumour nodules presented infiltered pro-inflammatory macrophages and natural killer cells, even upon RIPK3 ablation, which evidences part of the crosstalk between the distinct components of the TME that may afford complementary effects on HCC progression. Finally, the anti-tumoral functions of the adaptive immune cells could be rescued by Ripk3 deletion. Taken together, our studies reinforce that inhibition of RIPK3-dependent mechanisms may be a therapeutic approach to prevent NAFLD-associated HCC.
Descrição
Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, 2021, Universidade de Lisboa, Faculdade de Farmácia.
Palavras-chave
Hepatocellular carcinoma Immunogenic cell death Non-alcoholic steatohepatitis RIPK3 Mestrado integrado - 2021