RESUMO
Tumor necrosis factor receptor 1 (TNFR1) activates NF-κB-dependent pro-inflammatory gene expression, but also induces cell death by triggering apoptosis and necroptosis. Inhibition of inhibitor of NF-κB kinase (IKK)/NF-κB signaling in keratinocytes paradoxically unleashed spontaneous TNFR1-mediated skin inflammation in mice, but the underlying mechanisms remain poorly understood. Here, we show that TNFR1 causes skin inflammation in mice with epidermis-specific knockout of IKK2 by inducing receptor interacting protein kinase 1 (RIPK1)-dependent necroptosis, and to a lesser extent also apoptosis, of keratinocytes. Combined epidermis-specific ablation of the NF-κB subunits RelA and c-Rel also caused skin inflammation by inducing TNFR1-mediated keratinocyte necroptosis. Contrary to the currently established model that inhibition of NF-κB-dependent gene transcription causes RIPK1-independent cell death, keratinocyte necroptosis, and skin inflammation in mice with epidermis-specific RelA and c-Rel deficiency also depended on RIPK1 kinase activity. These results advance our understanding of the mechanisms regulating TNFR1-induced cell death and identify RIPK1-mediated necroptosis as a potent driver of skin inflammation.
Assuntos
Queratinócitos/metabolismo , Necroptose/fisiologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Apoptose/fisiologia , Feminino , Quinase I-kappa B/metabolismo , Inflamação/metabolismo , Queratinócitos/patologia , Masculino , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , NF-kappa B/fisiologia , Necroptose/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais , Pele/metabolismo , Pele/patologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
BACKGROUND: The profound disparity in response to immune checkpoint blockade (ICB) by cutaneous melanoma (CM) and uveal melanoma (UM) patients is not well understood. Therefore, we characterized metastases of CM and UM from the same metastatic site (liver), in order to dissect the potential underlying mechanism in differential response on ICB. METHODS: Tumor liver samples from CM (n=38) and UM (n=28) patients were analyzed at the genomic (whole exome sequencing), transcriptional (RNA sequencing) and protein (immunohistochemistry and GeoMx Digital Spatial Profiling) level. RESULTS: Comparison of CM and UM metastases from the same metastatic site revealed that, although originating from the same melanocyte lineage, CM and UM differed in somatic mutation profile, copy number profile, tumor mutational burden (TMB) and consequently predicted neoantigens. A higher melanin content and higher expression of the melanoma differentiation antigen MelanA was observed in liver metastases of UM patients. No difference in B2M and human leukocyte antigen-DR (HLA-DR) expression was observed. A higher expression of programmed cell death ligand 1 (PD-L1) was found in CM compared with UM liver metastases, although the majority of CM and UM liver metastases lacked PD-L1 expression. There was no difference in the extent of immune infiltration observed between CM and UM metastases, with the exception of a higher expression of CD163 (p<0.0001) in CM liver samples. While the extent of immune infiltration was similar for CM and UM metastases, the ratio of exhausted CD8 T cells to cytotoxic T cells, to total CD8 T cells and to Th1 cells, was significantly higher in UM metastases. CONCLUSIONS: While TMB was different between CM and UM metastases, tumor immune infiltration was similar. The greater dependency on PD-L1 as an immune checkpoint in CM and the identification of higher exhaustion ratios in UM may both serve as explanations for the difference in response to ICB. Consequently, in order to improve current treatment for metastatic UM, reversal of T cell exhaustion beyond programmed cell death 1 blockade should be considered.
Assuntos
Melanoma/complicações , Neoplasias Cutâneas/complicações , Neoplasias Uveais/complicações , Feminino , Humanos , Neoplasias Hepáticas/secundário , Masculino , Melanoma/patologia , Estudos Retrospectivos , Neoplasias Cutâneas/patologia , Neoplasias Uveais/patologia , Melanoma Maligno CutâneoRESUMO
Characterization of spatial protein expression for multiple targets from a single tissue is difficult to perform, especially due to the limitations of multiplex immunohistochemistry and tissue heterogeneity. Therefore, a new technology is required that permits detailed and simultaneous expression profiling of proteins within a defined region of interest (ROI). To address this unmet need, NanoString Technologies developed a new technology, GeoMxTM digital spatial profiling (DSP), which currently enables simultaneous and guided detection of up to 40 antibodies (probes) from a single formalin-fixed paraffin-embedded (FFPE) tissue. DSP probes are tagged with unique photocleavable DNA oligos that are released after guided ultraviolet exposure in specific ROIs. Digital quantification of the released oligos by NanoString's nCounter® system provides a detailed expression profile of proteins within these discrete ROIs. In this article, we will describe our experience with the GeoMx DSP platform using cancer FFPE tissues. These expression profiles will provide better characterization and understanding of tumor heterogeneity and the tumor micro-environment, enabling the improvement of patient therapy and the identification of potential biomarker signatures. The purpose of this article is to offer potential future users an independent insight into the DSP platform and a comprehensive idea of usability, including advantages and current limitations of the technology based on our current experience with the beta version of NanoString's DSP platform as part of the DSP beta-testing program. The GeoMxTM Digital Spatial Profiling (DSP) platform is a non-destructive technique for regional in-depth protein expression profiling. Using oligonucleotide detection technologies, the GeoMxTM DSP enables simultaneous high-level multiplexing on a single FFPE tissue. Here, we focus on our current experience derived from our biomarker research using the beta version of the DSP instrument.
RESUMO
The mechanisms that regulate cell death and inflammation play an important role in liver disease and cancer. Receptor-interacting protein kinase 1 (RIPK1) induces apoptosis and necroptosis via kinase-dependent mechanisms and exhibits kinase-independent prosurvival and proinflammatory functions. Here, we have used genetic mouse models to study the role of RIPK1 in liver homeostasis, injury, and cancer. While ablating either RIPK1 or RelA in liver parenchymal cells (LPCs) did not cause spontaneous liver pathology, mice with combined deficiency of RIPK1 and RelA in LPCs showed increased hepatocyte apoptosis and developed spontaneous chronic liver disease and cancer that were independent of TNF receptor 1 (TNFR1) signaling. In contrast, mice with LPC-specific knockout of Ripk1 showed reduced diethylnitrosamine-induced (DEN-induced) liver tumorigenesis that correlated with increased DEN-induced hepatocyte apoptosis. Lack of RIPK1 kinase activity did not inhibit DEN-induced liver tumor formation, showing that kinase-independent functions of RIPK1 promote DEN-induced hepatocarcinogenesis. Moreover, mice lacking both RIPK1 and TNFR1 in LPCs displayed normal tumor formation in response to DEN, demonstrating that RIPK1 deficiency decreases DEN-induced liver tumor formation in a TNFR1-dependent manner. Therefore, these findings indicate that RIPK1 cooperates with NF-κB signaling to prevent TNFR1-independent hepatocyte apoptosis and the development of chronic liver disease and cancer, but acts downstream of TNFR1 signaling to promote DEN-induced liver tumorigenesis.
Assuntos
Transformação Celular Neoplásica/metabolismo , Hepatócitos/enzimologia , Neoplasias Hepáticas Experimentais/enzimologia , Proteínas de Neoplasias/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/patologia , Dietilnitrosamina/toxicidade , Hepatócitos/patologia , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/patologia , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismoRESUMO
IκB kinase/nuclear [corrected] factor κB (IKK/NF-κB) signaling exhibits important yet opposing functions in hepatocarcinogenesis. Mice lacking NEMO in liver parenchymal cells (LPC) spontaneously develop steatohepatitis and hepatocellular carcinoma (HCC) suggesting that NF-κB prevents liver disease and cancer. Here, we show that complete NF-κB inhibition by combined LPC-specific ablation of RelA, c-Rel, and RelB did not phenocopy NEMO deficiency, but constitutively active IKK2-mediated NF-κB activation prevented hepatocellular damage and HCC in NEMO(LPC-KO) mice. Knock-in expression of kinase inactive receptor-interacting protein kinase 1 (RIPK1) prevented hepatocyte apoptosis and HCC, while RIPK1 ablation induced TNFR1-associated death domain protein (TRADD)-dependent hepatocyte apoptosis and liver tumors in NEMO(LPC-KO) mice, revealing distinct kinase-dependent and scaffolding functions of RIPK1. Collectively, these results show that NEMO prevents hepatocarcinogenesis by inhibiting RIPK1 kinase activity-driven hepatocyte apoptosis through NF-κB-dependent and -independent functions.
Assuntos
Carcinoma Hepatocelular/metabolismo , Fígado Gorduroso/metabolismo , Hepatócitos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Hepáticas/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Apoptose/genética , Carcinoma Hepatocelular/genética , Células Cultivadas , Fígado Gorduroso/genética , Expressão Gênica , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Immunoblotting , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genéticaRESUMO
The serine/threonine kinase RIPK1 is recruited to TNFR1 to mediate proinflammatory signaling and to regulate TNF-induced cell death. A RIPK1 deficiency results in perinatal lethality, impaired NFκB and MAPK signaling, and sensitivity to TNF-induced apoptosis. Chemical inhibitor and in vitro-reconstitution studies suggested that RIPK1 displays distinct kinase activity-dependent and -independent functions. To determine the contribution of RIPK1 kinase to inflammation in vivo, we generated knock-in mice endogenously expressing catalytically inactive RIPK1 D138N. Unlike Ripk1(-/-) mice, which die shortly after birth, Ripk1(D138N/D138N) mice are viable. Cells expressing RIPK1 D138N are resistant to TNF- and polyinosinic-polycytidylic acid-induced necroptosis in vitro, and Ripk1(D138N/D138N) mice are protected from TNF-induced shock in vivo. Moreover, Ripk1(D138N/D138N) mice fail to control vaccinia virus replication in vivo. This study provides genetic evidence that the kinase activity of RIPK1 is not required for survival but is essential for TNF-, TRIF-, and viral-initiated necroptosis.