RESUMO
Bacterial sensing by intestinal tumor cells contributes to tumor growth through cell-intrinsic activation of the calcineurin-NFAT axis, but the role of this pathway in other intestinal cells remains unclear. Here, we found that myeloid-specific deletion of calcineurin in mice activated protective CD8+ T cell responses and inhibited colorectal cancer (CRC) growth. Microbial sensing by myeloid cells promoted calcineurin- and NFAT-dependent interleukin 6 (IL-6) release, expression of the co-inhibitory molecules B7H3 and B7H4 by tumor cells, and inhibition of CD8+ T cell-dependent anti-tumor immunity. Accordingly, targeting members of this pathway activated protective CD8+ T cell responses and inhibited primary and metastatic CRC growth. B7H3 and B7H4 were expressed by the majority of human primary CRCs and metastases, which was associated with low numbers of tumor-infiltrating CD8+ T cells and poor survival. Therefore, a microbiota-, calcineurin-, and B7H3/B7H4-dependent pathway controls anti-tumor immunity, revealing additional targets for immune checkpoint inhibition in microsatellite-stable CRC.
Assuntos
Neoplasias Colorretais , Microbiota , Animais , Antígenos B7 , Linfócitos T CD8-Positivos , Calcineurina/metabolismo , Neoplasias Colorretais/metabolismo , Camundongos , Fatores de Transcrição NFATC/metabolismo , Inibidor 1 da Ativação de Células T com Domínio V-SetRESUMO
Around 30-40% of patients with colorectal cancer (CRC) undergoing curative resection of the primary tumour will develop metastases in the subsequent years1. Therapies to prevent disease relapse remain an unmet medical need. Here we uncover the identity and features of the residual tumour cells responsible for CRC relapse. An analysis of single-cell transcriptomes of samples from patients with CRC revealed that the majority of genes associated with a poor prognosis are expressed by a unique tumour cell population that we named high-relapse cells (HRCs). We established a human-like mouse model of microsatellite-stable CRC that undergoes metastatic relapse after surgical resection of the primary tumour. Residual HRCs occult in mouse livers after primary CRC surgery gave rise to multiple cell types over time, including LGR5+ stem-like tumour cells2-4, and caused overt metastatic disease. Using Emp1 (encoding epithelial membrane protein 1) as a marker gene for HRCs, we tracked and selectively eliminated this cell population. Genetic ablation of EMP1high cells prevented metastatic recurrence and mice remained disease-free after surgery. We also found that HRC-rich micrometastases were infiltrated with T cells, yet became progressively immune-excluded during outgrowth. Treatment with neoadjuvant immunotherapy eliminated residual metastatic cells and prevented mice from relapsing after surgery. Together, our findings reveal the cell-state dynamics of residual disease in CRC and anticipate that therapies targeting HRCs may help to avoid metastatic relapse.
Assuntos
Neoplasias Colorretais , Metástase Neoplásica , Proteínas de Neoplasias , Recidiva Local de Neoplasia , Neoplasia Residual , Receptores de Superfície Celular , Animais , Humanos , Camundongos , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Progressão da Doença , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/prevenção & controle , Recidiva Local de Neoplasia/terapia , Neoplasia Residual/genética , Neoplasia Residual/patologia , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Metástase Neoplásica/prevenção & controle , Metástase Neoplásica/terapia , Modelos Animais de Doenças , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos do Interstício Tumoral/citologia , Linfócitos do Interstício Tumoral/imunologia , Terapia Neoadjuvante , ImunoterapiaRESUMO
Serrated adenocarcinoma, an alternative pathway for colorectal cancer (CRC) development, accounts for 15%-30% of all CRCs and is aggressive and treatment resistant. We show that the expression of atypical protein kinase C ζ (PKCζ) and PKCλ/ι was reduced in human serrated tumors. Simultaneous inactivation of the encoding genes in the mouse intestinal epithelium resulted in spontaneous serrated tumorigenesis that progressed to advanced cancer with a strongly reactive and immunosuppressive stroma. Whereas epithelial PKCλ/ι deficiency led to immunogenic cell death and the infiltration of CD8+ T cells, which repressed tumor initiation, PKCζ loss impaired interferon and CD8+ T cell responses, which resulted in tumorigenesis. Combined treatment with a TGF-ß receptor inhibitor plus anti-PD-L1 checkpoint blockade showed synergistic curative activity. Analysis of human samples supported the relevance of these kinases in the immunosurveillance defects of human serrated CRC. These findings provide insight into avenues for the detection and treatment of this poor-prognosis subtype of CRC.
Assuntos
Mucosa Intestinal/imunologia , Neoplasias Intestinais/imunologia , Isoenzimas/imunologia , Proteína Quinase C/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/imunologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/metabolismo , Feminino , Humanos , Vigilância Imunológica/genética , Vigilância Imunológica/imunologia , Mucosa Intestinal/enzimologia , Mucosa Intestinal/patologia , Neoplasias Intestinais/enzimologia , Neoplasias Intestinais/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Camundongos Knockout , Camundongos Transgênicos , Pessoa de Meia-Idade , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismoRESUMO
Metastatic colorectal cancer (CRC) is highly resistant to therapy and prone to recur. The tumor-induced local and systemic immunosuppression allows cancer cells to evade immunosurveillance, facilitating their proliferation and dissemination. Dendritic cells (DCs) are required for the detection, processing, and presentation of tumor antigens, and subsequently for the activation of antigen-specific T cells to orchestrate an effective antitumor response. Notably, successful tumors have evolved mechanisms to disrupt and impair DC functions, underlining the key role of tumor-induced DC dysfunction in promoting tumor growth, metastasis initiation, and treatment resistance. Conventional DC type 2 (cDC2) are highly prevalent in tumors and have been shown to present high phenotypic and functional plasticity in response to tumor-released environmental cues. This plasticity reverberates on both the development of antitumor responses and on the efficacy of immunotherapies in cancer patients. Uncovering the processes, mechanisms, and mediators by which CRC shapes and disrupts cDC2 functions is crucial to restoring their full antitumor potential. In this study, we use our recently developed 3D DC-tumor co-culture system to investigate how patient-derived primary and metastatic CRC organoids modulate cDC2 phenotype and function. We first demonstrate that our collagen-based system displays extensive interaction between cDC2 and tumor organoids. Interestingly, we show that tumor-corrupted cDC2 shift toward a CD14+ population with defective expression of maturation markers, an intermediate phenotype positioned between cDC2 and monocytes, and impaired T-cell activating abilities. This phenotype aligns with the newly defined DC3 (CD14+ CD1c+ CD163+) subset. Remarkably, a comparable population was found to be present in tumor lesions and enriched in the peripheral blood of metastatic CRC patients. Moreover, using EP2 and EP4 receptor antagonists and an anti-IL-6 neutralizing antibody, we determined that the observed phenotype shift is partially mediated by PGE2 and IL-6. Importantly, our system holds promise as a platform for testing therapies aimed at preventing or mitigating tumor-induced DC dysfunction. Overall, our study offers novel and relevant insights into cDC2 (dys)function in CRC that hold relevance for the design of therapeutic approaches.
Assuntos
Neoplasias Colorretais , Células Dendríticas , Dinoprostona , Interleucina-6 , Organoides , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Humanos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Organoides/imunologia , Organoides/metabolismo , Dinoprostona/metabolismo , Interleucina-6/metabolismo , Interleucina-6/imunologia , Técnicas de Cocultura , Fenótipo , Plasticidade CelularRESUMO
BACKGROUND & AIMS: The liver is one of the organs most commonly affected by metastasis. The presence of liver metastases has been reported to be responsible for an immunosuppressive microenvironment and diminished immunotherapy efficacy. Herein, we aimed to investigate the role of IL-10 in liver metastasis and to determine how its modulation could affect the efficacy of immunotherapy in vivo. METHODS: To induce spontaneous or forced liver metastasis in mice, murine cancer cells (MC38) or colon tumor organoids were injected into the cecum or the spleen, respectively. Mice with complete and cell type-specific deletion of IL-10 and IL-10 receptor alpha were used to identify the source and the target of IL-10 during metastasis formation. Programmed death ligand 1 (PD-L1)-deficient mice were used to test the role of this checkpoint. Flow cytometry was applied to characterize the regulation of PD-L1 by IL-10. RESULTS: We found that Il10-deficient mice and mice treated with IL-10 receptor alpha antibodies were protected against liver metastasis formation. Furthermore, by using IL-10 reporter mice, we demonstrated that Foxp3+ regulatory T cells (Tregs) were the major cellular source of IL-10 in liver metastatic sites. Accordingly, deletion of IL-10 in Tregs, but not in myeloid cells, led to reduced liver metastasis. Mechanistically, IL-10 acted on Tregs in an autocrine manner, thereby further amplifying IL-10 production. Furthermore, IL-10 acted on myeloid cells, i.e. monocytes, and induced the upregulation of the immune checkpoint protein PD-L1. Finally, the PD-L1/PD-1 axis attenuated CD8-dependent cytotoxicity against metastatic lesions. CONCLUSIONS: Treg-derived IL-10 upregulates PD-L1 expression in monocytes, which in turn reduces CD8+ T-cell infiltration and related antitumor immunity in the context of colorectal cancer-derived liver metastases. These findings provide the basis for future monitoring and targeting of IL-10 in colorectal cancer-derived liver metastases. IMPACT AND IMPLICATIONS: Liver metastasis diminishes the effectiveness of immunotherapy and increases the mortality rate in patients with colorectal cancer. We investigated the role of IL-10 in liver metastasis formation and assessed its impact on the effectiveness of immunotherapy. Our data show that IL-10 is a pro-metastatic factor involved in liver metastasis formation and that it acts as a regulator of PD-L1. This provides the basis for future monitoring and targeting of IL-10 in colorectal cancer-derived liver metastasis.
Assuntos
Neoplasias Colorretais , Neoplasias Hepáticas , Animais , Humanos , Camundongos , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Linfócitos T CD8-Positivos , Linhagem Celular Tumoral , Interleucina-10 , Neoplasias Hepáticas/patologia , Receptores de Interleucina-10 , Microambiente TumoralRESUMO
Most patients with colorectal cancer die as a result of the disease spreading to other organs. However, no prevalent mutations have been associated with metastatic colorectal cancers. Instead, particular features of the tumour microenvironment, such as lack of T-cell infiltration, low type 1 T-helper cell (TH1) activity and reduced immune cytotoxicity or increased TGFß levels predict adverse outcomes in patients with colorectal cancer. Here we analyse the interplay between genetic alterations and the tumour microenvironment by crossing mice bearing conditional alleles of four main colorectal cancer mutations in intestinal stem cells. Quadruple-mutant mice developed metastatic intestinal tumours that display key hallmarks of human microsatellite-stable colorectal cancers, including low mutational burden, T-cell exclusion and TGFß-activated stroma. Inhibition of the PD-1-PD-L1 immune checkpoint provoked a limited response in this model system. By contrast, inhibition of TGFß unleashed a potent and enduring cytotoxic T-cell response against tumour cells that prevented metastasis. In mice with progressive liver metastatic disease, blockade of TGFß signalling rendered tumours susceptible to anti-PD-1-PD-L1 therapy. Our data show that increased TGFß in the tumour microenvironment represents a primary mechanism of immune evasion that promotes T-cell exclusion and blocks acquisition of the TH1-effector phenotype. Immunotherapies directed against TGFß signalling may therefore have broad applications in treating patients with advanced colorectal cancer.
Assuntos
Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Evasão da Resposta Imune , Imunoterapia , Metástase Neoplásica/genética , Metástase Neoplásica/imunologia , Fator de Crescimento Transformador beta/imunologia , Alelos , Animais , Diferenciação Celular/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/imunologia , Modelos Animais de Doenças , Sinergismo Farmacológico , Feminino , Humanos , Evasão da Resposta Imune/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Intestinos/patologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/secundário , Masculino , Camundongos , Mutação , Metástase Neoplásica/tratamento farmacológico , Metástase Neoplásica/patologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Células-Tronco/patologia , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologiaRESUMO
The development of metastasis severely reduces the life expectancy of patients with colorectal cancer (CRC). Although loss of SMAD4 is a key event in CRC progression, the resulting changes in biological processes in advanced disease and metastasis are not fully understood. Here, we applied a multiomics approach to a CRC organoid model that faithfully reflects the metastasis-supporting effects of SMAD4 inactivation. We show that loss of SMAD4 results in decreased differentiation and activation of pro-migratory and cell proliferation processes, which is accompanied by the disruption of several key oncogenic pathways, including the TGFß, WNT, and VEGF pathways. In addition, SMAD4 inactivation leads to increased secretion of proteins that are known to be involved in a variety of pro-metastatic processes. Finally, we show that one of the factors that is specifically secreted by SMAD4-mutant organoidsâDKK3âreduces the antitumor effects of natural killer cells (NK cells). Altogether, our data provide new insights into the role of SMAD4 perturbation in advanced CRC.
Assuntos
Neoplasias Colorretais , Multiômica , Humanos , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Fator de Crescimento Transformador beta/metabolismo , Proliferação de Células/genética , Proteína Smad4/genéticaRESUMO
Mammalian Wnt proteins are believed to act as short-range signals, yet have not been previously visualized in vivo. Self-renewal, proliferation and differentiation are coordinated along a putative Wnt gradient in the intestinal crypt. Wnt3 is produced specifically by Paneth cells. Here we have generated an epitope-tagged, functional Wnt3 knock-in allele. Wnt3 covers basolateral membranes of neighbouring stem cells. In intestinal organoids, Wnt3-transfer involves direct contact between Paneth cells and stem cells. Plasma membrane localization requires surface expression of Frizzled receptors, which in turn is regulated by the transmembrane E3 ligases Rnf43/Znrf3 and their antagonists Lgr4-5/R-spondin. By manipulating Wnt3 secretion and by arresting stem-cell proliferation, we demonstrate that Wnt3 mainly travels away from its source in a cell-bound manner through cell division, and not through diffusion. We conclude that stem-cell membranes constitute a reservoir for Wnt proteins, while Frizzled receptor turnover and 'plasma membrane dilution' through cell division shape the epithelial Wnt3 gradient.
Assuntos
Membrana Celular/metabolismo , Mucosa Intestinal/citologia , Nicho de Células-Tronco , Células-Tronco/citologia , Células-Tronco/metabolismo , Via de Sinalização Wnt , Proteína Wnt3/metabolismo , Alelos , Animais , Adesão Celular , Divisão Celular , Difusão , Feminino , Receptores Frizzled/metabolismo , Técnicas de Introdução de Genes , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Camundongos , Organoides/citologia , Organoides/metabolismo , Celulas de Paneth/citologia , Celulas de Paneth/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína Wnt3/genéticaRESUMO
Wnt/ß-catenin signaling controls development and adult tissue homeostasis by regulating cell proliferation and cell fate decisions. Wnt binding to its receptors Frizzled (FZD) and low-density lipoprotein-related 6 (LRP6) at the cell surface initiates a signaling cascade that leads to the transcription of Wnt target genes. Upon Wnt binding, the receptors assemble into large complexes called signalosomes that provide a platform for interactions with downstream effector proteins. The molecular basis of signalosome formation and regulation remains elusive, largely due to the lack of tools to analyze its endogenous components. Here, we use internally tagged Wnt3a proteins to isolate and characterize activated, endogenous Wnt receptor complexes by mass spectrometry-based proteomics. We identify the single-span membrane protein TMEM59 as an interactor of FZD and LRP6 and a positive regulator of Wnt signaling. Mechanistically, TMEM59 promotes the formation of multimeric Wnt-FZD assemblies via intramembrane interactions. Subsequently, these Wnt-FZD-TMEM59 clusters merge with LRP6 to form mature Wnt signalosomes. We conclude that the assembly of multiprotein Wnt signalosomes proceeds along well-ordered steps that involve regulated intramembrane interactions.
Assuntos
Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Via de Sinalização Wnt/fisiologia , Proteína Wnt3A/metabolismo , Animais , Células HEK293 , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteínas de Membrana/genética , Camundongos , Complexos Multiproteicos/genética , Proteínas do Tecido Nervoso/genética , Proteína Wnt3A/genéticaRESUMO
The mechanism by which Wnt receptors transduce signals to activate downstream beta-catenin-mediated target gene transcription remains incompletely understood but involves Frizzled (Fz) receptor-mediated plasma membrane recruitment and activation of the cytoplasmic effector Dishevelled (Dvl). Here, we identify the deubiquitinating enzyme CYLD, the familial cylindromatosis tumor suppressor gene, as a negative regulator of proximal events in Wnt/beta-catenin signaling. Depletion of CYLD from cultured cells markedly enhances Wnt-induced accumulation of beta-catenin and target gene activation. Moreover, we demonstrate hyperactive Wnt signaling in human cylindroma skin tumors that arise from mutations in CYLD. At the molecular level, CYLD interacts with and regulates K63-linked ubiquitination of Dvl. Enhanced ubiquitination of the polymerization-prone DIX domain in CYLD-deficient cells positively links to the signaling activity of Dvl. Together, our results argue that loss of CYLD instigates tumor growth in human cylindromatosis through a mechanism in which hyperubiquitination of polymerized Dvl drives enhancement of Wnt responses.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinoma Adenoide Cístico/metabolismo , Carcinoma de Apêndice Cutâneo/metabolismo , Fosfoproteínas/metabolismo , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Neoplasias Cutâneas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Carcinoma Adenoide Cístico/genética , Carcinoma Adenoide Cístico/patologia , Carcinoma de Apêndice Cutâneo/genética , Carcinoma de Apêndice Cutâneo/patologia , Proliferação de Células , Enzima Desubiquitinante CYLD , Proteínas Desgrenhadas , Células HeLa , Humanos , Lisina , Camundongos , Mutação , NF-kappa B/metabolismo , Fosfoproteínas/genética , Multimerização Proteica , Estrutura Terciária de Proteína , Interferência de RNA , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Fatores de Tempo , Ativação Transcricional , Transfecção , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Supressoras de Tumor/genética , Ubiquitinação , Proteínas Wnt/genética , Proteína Wnt3 , beta Catenina/genéticaRESUMO
Wnt binding to members of the seven-span transmembrane Frizzled (Fz) receptor family controls essential cell fate decisions and tissue polarity during development and in adulthood. The Fz-mediated membrane recruitment of the cytoplasmic effector Dishevelled (Dvl) is a critical step in Wnt/ß-catenin signaling initiation, but how Fz and Dvl act together to drive downstream signaling events remains largely undefined. Here, we use an Fz peptide-based microarray to uncover a mechanistically important role of the bipartite Dvl DEP domain and C terminal region (DEP-C) in binding a three-segmented discontinuous motif in Fz. We show that cooperative use of two conserved motifs in the third intracellular loop and the classic C-terminal motif of Fz is required for DEP-C binding and Wnt-induced ß-catenin activation in cultured cells and Xenopus embryos. Within the complex, the Dvl DEP domain mainly binds the Fz C-terminal tail, whereas a short region at the Dvl C-terminal end is required to bind the Fz third loop and stabilize the Fz-Dvl interaction. We conclude that Dvl DEP-C binding to Fz is a key event in Wnt-mediated signaling relay to ß-catenin. The discontinuous nature of the Fz-Dvl interface may allow for precise regulation of the interaction in the control of Wnt-dependent cellular responses.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Receptores Frizzled/metabolismo , Fosfoproteínas/metabolismo , Transdução de Sinais , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Linhagem Celular , Proteínas Desgrenhadas , Polarização de Fluorescência , Receptores Frizzled/química , Humanos , Microscopia Confocal , Dados de Sequência Molecular , Fosfoproteínas/química , Ligação Proteica , Proteínas de XenopusRESUMO
The Rho-GTPase Rac1 promotes actin polymerization and membrane protrusion that mediate initial contact and subsequent maturation of cell-cell junctions. Here we report that Rac1 associates with the ubiquitin-protein ligase neural precursor cell expressed developmentally down-regulated 4 (Nedd4). This interaction requires the hypervariable C-terminal domain of Rac1 and the WW domains of Nedd4. Activated Rac1 colocalises with endogenous Nedd4 at epithelial cell-cell contacts. Reduction of Nedd4 expression by shRNA results in reduced transepithelial electrical resistance (TER) and concomitant changes in the distribution of adherens and tight junction markers. Conversely, expression of Nedd4 promotes TER, suggesting that Nedd4 cooperates with Rac1 in the induction of junctional maturation. We found that Nedd4, but not Nedd4-2, mediates the ubiquitylation and degradation of the adapter protein dishevelled-1 (Dvl1), the expression of which negatively regulates cell-cell contact. Nedd4-mediated ubiquitylation requires its binding to the C-terminal domain of Dvl1, comprising the DEP domain, and targets an N-terminal lysine-rich region upstream of the Dvl1 DIX domain. We found that endogenous Rac1 colocalises with endogenous Dvl1 in intracellular puncta as well as on cell-cell junctions. Finally, activated Rac1 was found to stimulate Nedd4 activity, resulting in increased ubiquitylation of Dvl1. Together, these data reveal a novel Rac1-dependent signalling pathway that, through Nedd4-mediated ubiquitylation of Dvl1, stimulates the maturation of epithelial cell-cell contacts.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adesão Celular/fisiologia , Comunicação Celular/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Fosfoproteínas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Junções Aderentes/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Proteínas Desgrenhadas , Células HeLa , Humanos , Pulmão/citologia , Ubiquitina-Proteína Ligases Nedd4 , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de Sinais , UbiquitinaçãoRESUMO
Although aberrant activation of the KRAS and PI3K pathway alongside TP53 mutations account for frequent aberrations in human gastric cancers, neither the sequence nor the individual contributions of these mutations have been clarified. Here, we establish an allelic series of mice to afford conditional expression in the glandular epithelium of KrasG12D;Pik3caH1047R or Trp53R172H and/or ablation of Pten or Trp53. We find that KrasG12D;Pik3caH1047R is sufficient to induce adenomas and that lesions progress to carcinoma when also harboring Pten deletions. An additional challenge with either Trp53 loss- or gain-of-function alleles further accelerated tumor progression and triggered metastatic disease. While tumor-intrinsic STAT3 signaling in response to gp130 family cytokines remained as a gatekeeper for all stages of tumor development, metastatic progression required a mutant Trp53-induced interleukin (IL)-11 to IL-6 dependency switch. Consistent with the poorer survival of patients with high IL-6 expression, we identify IL-6/STAT3 signaling as a therapeutic vulnerability for TP53-mutant gastric cancer.
RESUMO
Colorectal cancer (CRC) raises considerable clinical challenges, including a high mortality rate once the tumor spreads to distant sites. At this advanced stage, more accurate prediction of prognosis and treatment outcome is urgently needed. The role of cancer immunity in metastatic CRC (mCRC) is poorly understood. Here, we explore cellular immune cell status in patients with multi-organ mCRC. We analyzed T cell infiltration in primary tumor sections, surveyed the lymphocytic landscape of liver metastases, and assessed circulating mononuclear immune cells. Besides asking whether immune cells are associated with survival at this stage of the disease, we investigated correlations between the different tissue types; as this could indicate a dominant immune phenotype. Taken together, our analyses corroborate previous observations that higher levels of CD8+ T lymphocytes link to better survival outcomes. Our findings therefore extend evidence from earlier stages of CRC to indicate an important role for cancer immunity in disease control even after metastatic spreading to multiple organs. This finding may help to improve predicting outcome of patients with mCRC and suggests a future role for immunotherapeutic strategies.
Assuntos
Neoplasias Colorretais , Neoplasias Hepáticas , Humanos , Neoplasias Colorretais/patologia , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/mortalidade , Masculino , Feminino , Neoplasias Hepáticas/secundário , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/mortalidade , Idoso , Pessoa de Meia-Idade , Prognóstico , Linfócitos T CD8-Positivos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Metástase Neoplásica , AdultoRESUMO
The tumor microenvironment is a complex ecosystem that drives cancer progression and restrains immunity. Although immune checkpoint inhibitors have shown strong potential in a subset of patients, a better understanding of suppressive mechanisms may inspire ways to improve immunotherapeutic efficacy. A new study in this issue of Cancer Research focuses on targeting cancer-associated fibroblasts in preclinical models of gastric tumors. Aiming to rebalance anticancer immunity and enhance treatment responses to checkpoint-blocking antibodies, this work also addresses the potential for multitarget tyrosine kinase inhibitors in treating gastrointestinal cancer. See related article by Akiyama et al., p. 753.
Assuntos
Receptor alfa de Fator de Crescimento Derivado de Plaquetas , Neoplasias Gástricas , Humanos , Ecossistema , Imunoterapia , Anticorpos Bloqueadores , Receptores Proteína Tirosina Quinases , Microambiente TumoralRESUMO
Colorectal cancer (CRC) remains one of the most aggressive and lethal cancers, with metastasis accounting for most deaths. As such, there is an unmet need for improved therapies for metastatic CRC (mCRC). Currently, the research focus is shifting towards the reciprocal interactions within the tumor microenvironment (TME), which prevent tumor clearance by the immune system. Dendritic cells (DCs) play a key role in the initiation and amplification of anti-tumor immune responses and in driving the clinical success of immunotherapies. Dissecting the interactions between DCs and CRC cells may open doors to identifying key mediators in tumor progression, and possible therapeutic targets. This requires representative, robust and versatile models and tools. Currently, there is a shortage of such in vitro systems to model the CRC TME and its tumor-immune cell interactions. Here we develop and establish a dynamic organotypic 3D co-culture system to recapitulate and untangle the interactions between DCs and patient-derived mCRC tumor organoids. To our knowledge, this is the first study investigating human DCs in co-culture with tumor organoids in a 3D, organotypic setting. This system reveals how mCRC organoids modulate and shape monocyte-derived DCs (MoDCs) behavior, phenotype, and function, within a collagen matrix, using techniques such as brightfield and fluorescence microscopy, flow cytometry, and fluorescence-activated cell sorting. Our 3D co-culture model shows high viability and extensive interaction between DCs and tumor organoids, and its structure resembles patient tissue sections. Furthermore, it is possible to retrieve DCs from the co-cultures and characterize their phenotypic and functional profile. In our study, the expression of activation markers in both mature and immature DCs and their ability to activate T cells were impacted by co-culture with tumor organoids. In the future, this direct co-culture platform can be adapted and exploited to study the CRC-DC interplay in more detail, enabling novel and broader insights into CRC-driven DC (dys)function.
Assuntos
Neoplasias do Colo , Neoplasias Retais , Humanos , Técnicas de Cocultura , Neoplasias do Colo/patologia , Neoplasias Retais/patologia , Células Dendríticas , Organoides , Fenótipo , Microambiente TumoralRESUMO
Transforming growth factor-ß (TGFß) signalling controls multiple cell fate decisions during development and tissue homeostasis; hence, dysregulation of this pathway can drive several diseases, including cancer. Here we discuss the influence that TGFß exerts on the composition and behaviour of different cell populations present in the tumour immune microenvironment, and the context-dependent functions of this cytokine in suppressing or promoting cancer. During homeostasis, TGFß controls inflammatory responses triggered by exposure to the outside milieu in barrier tissues. Lack of TGFß exacerbates inflammation, leading to tissue damage and cellular transformation. In contrast, as tumours progress, they leverage TGFß to drive an unrestrained wound-healing programme in cancer-associated fibroblasts, as well as to suppress the adaptive immune system and the innate immune system. In consonance with this key role in reprogramming the tumour microenvironment, emerging data demonstrate that TGFß-inhibitory therapies can restore cancer immunity. Indeed, this approach can synergize with other immunotherapies - including immune checkpoint blockade - to unleash robust antitumour immune responses in preclinical cancer models. Despite initial challenges in clinical translation, these findings have sparked the development of multiple therapeutic strategies that inhibit the TGFß pathway, many of which are currently in clinical evaluation.
Assuntos
Neoplasias , Fator de Crescimento Transformador beta , Humanos , Evasão da Resposta Imune , Imunoterapia , Neoplasias/patologia , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/uso terapêutico , Microambiente TumoralRESUMO
Patients with advanced or metastatic colorectal cancer ((m)CRC) have limited effective treatment options resulting in high mortality rates. A better understanding of the molecular basis of this disease has led to growing interest in small molecule tyrosine kinase inhibitors (TKIs) for its treatment. However, of around 42 TKIs demonstrating preclinical anti-tumour activity, and despite numerous clinical trials, only 1 has been approved for clinical use in mCRC. Clearly, there is a huge gap in the translation of these targeted therapies to the clinic. This underlines the limitations of preclinical models to predict clinical drug efficacy and to fully characterize the mechanism of action. Moreover, several relevant topics remain poorly resolved. Do we know the actual intracellular concentrations that are required for anticancer efficacy, and what range of intra-tumoral drug concentrations is reached in clinical setting? Are the intended targeted kinases responsible for the anti-cancer activity or are other unexpected cellular targets involved? Do we have any idea of the effect of these drugs on the tumour microenvironment and does this help explain therapy success, failure or heterogeneity? In this review, we address these questions and discuss concepts that jointly complicate the clinical translation of TKIs for CRC. Finally, we will argue that an integrated approach with more sophisticated preclinical models and techniques may improve the prediction of clinical treatment efficacy.
Assuntos
Neoplasias Colorretais , Inibidores de Proteínas Quinases , Neoplasias Colorretais/tratamento farmacológico , Humanos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Microambiente TumoralRESUMO
RAS genes are the most frequently mutated oncogenes in cancer. These mutations occur in roughly half of the patients with colorectal cancer (CRC). RAS mutant tumors are resistant to therapy with anti-EGFR monoclonal antibodies. Therefore, patients with RAS mutant CRC currently have few effective therapy options. RAS mutations lead to constitutively active RAS GTPases, involved in multiple downstream signaling pathways. These alterations are associated with a tumor microenvironment (TME) that drives immune evasion and disease progression by mechanisms that remain incompletely understood. In this review, we focus on the available evidence in the literature explaining the potential effects of RAS mutations on the CRC microenvironment. Ongoing efforts to influence the TME by targeting mutant RAS and thereby sensitizing these tumors to immunotherapy will be discussed as well.
Assuntos
Neoplasias Colorretais , Microambiente Tumoral , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Receptores ErbB/genética , Genes ras , Humanos , Mutação , Microambiente Tumoral/genéticaRESUMO
Patients with advanced cancer refractory to standard treatment were treated with sunitinib at a dose of 300 mg once every week (Q1W) or 700 mg once every two weeks (Q2W). Tumor, skin and plasma concentrations were measured and immunohistochemical staining for tumor cell proliferation (TCP), microvessel density (MVD) and T-cell infiltration was performed on tumor biopsies before and after 17 days of treatment. Oral administration of 300 mg sunitinib Q1W or 700 mg Q2W resulted in 19-fold (range 5-35×) and 37-fold higher (range 10-88×) tumor drug concentrations compared to parallel maximum plasma drug concentrations, respectively. Patients with higher tumor sunitinib concentrations had favorable progression-free and overall survival than those with lower concentrations (p = 0.046 and 0.024, respectively). In addition, immunohistochemistry of tumor biopsies revealed an induction of T-cell infiltration upon treatment. These findings provide pharmacological and biological insights in the clinical benefit from high-dose intermittent sunitinib treatment. It emphasizes the potential benefit from reaching higher tumor drug concentrations and the value of measuring TKI tumor- over plasma-concentrations. The finding that reaching higher tumor drug concentrations provides most clinical benefit in patients with treatment refractory malignancies indicates that the inhibitory potency of sunitinib may be enforced by a high-dose intermittent treatment schedule. These results provide proof of concept for testing other clinically available multitargeted tyrosine kinase inhibitors in a high-dose intermittent treatment schedule.