RESUMO
A hallmark of macroautophagy is the covalent lipidation of LC3 and insertion into the double-membrane phagophore, which is driven by the ATG16L1/ATG5-ATG12 complex. In contrast, non-canonical autophagy is a pathway through which LC3 is lipidated and inserted into single membranes, particularly endolysosomal vacuoles during cell engulfment events such as LC3-associated phagocytosis. Factors controlling the targeting of ATG16L1 to phagophores are dispensable for non-canonical autophagy, for which the mechanism of ATG16L1 recruitment is unknown. Here we show that the WD repeat-containing C-terminal domain (WD40 CTD) of ATG16L1 is essential for LC3 recruitment to endolysosomal membranes during non-canonical autophagy, but dispensable for canonical autophagy. Using this strategy to inhibit non-canonical autophagy specifically, we show a reduction of MHC class II antigen presentation in dendritic cells from mice lacking the WD40 CTD Further, we demonstrate activation of non-canonical autophagy dependent on the WD40 CTD during influenza A virus infection. This suggests dependence on WD40 CTD distinguishes between macroautophagy and non-canonical use of autophagy machinery.
Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Proteínas de Transporte/fisiologia , Membranas Intracelulares/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Repetições WD40 , Animais , Apresentação de Antígeno , Proteínas Relacionadas à Autofagia/genética , Células Cultivadas , Classe III de Fosfatidilinositol 3-Quinases/genética , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Células Dendríticas/metabolismo , Endossomos/metabolismo , Feminino , Humanos , Vírus da Influenza A/isolamento & purificação , Influenza Humana/metabolismo , Influenza Humana/patologia , Influenza Humana/virologia , Lisossomos/metabolismo , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genéticaRESUMO
BACKGROUND & AIMS: The increasing incidence of anal canal carcinomas requires better knowledge on anal human papillomavirus (HPV) infection. We aimed to assess anal canal HPV infection prevalence and risk factors among patients seen at a gastroenterology department in France. METHODS: We analyzed anal tissue samples collected from 469 consecutive patients (median age 54 years, 52% women), including 112 who received immunosuppressant therapies and 101 with inflammatory bowel disease (70 with Crohn's disease), who underwent colonoscopy examinations from April 1, 2012 to April 30, 2015. HPV was detected and genotyped using the INNO-LiPA assay, and we collected medical and demographic data from all subjects. Risk factors for any HPV, high-risk HPV (HR-HPV) and HPV16 infection were assessed by bivariate and multivariate analysis. The primary outcomes association of HR-HPV or HPV16 with medical and demographic features. RESULTS: We detected HPV DNA in anal tissues from 34% of the subjects and HR-HPV in 18%. HPV16 was the most prevalent genotype (detected in 7%), followed by HPV51, HPV52, and HPV39. HR-HPV was detected in a significantly higher proportion of samples from women (23.1%) than men (12.8%) (P = .0035); HR-HPV and HPV16 were detected in a significantly higher proportion of patients with Crohn's disease (30.0%) than without (18.1%) (P = .005). Female sex, history of sexually transmitted disease, lifetime and past year-number of sexual partners, active smoking, and immunosuppressive therapies were independent risk factors for anal HR-HPV infection in multivariate analysis. CONCLUSION: One third of patients who underwent colonoscopy at a gastroenterology department were found to have anal canal HPV infection. We detected HR-HPV infection in almost 20% of patients and in a significantly higher proportion of patients with Crohn's disease than without. Increasing our knowledge of HPV infection of anal tissues could help physicians identify populations at risk and promote prophylaxis with vaccination and adequate screening.
Assuntos
Doenças do Ânus/epidemiologia , Doenças do Ânus/virologia , Doença de Crohn/complicações , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/epidemiologia , Infecções por Papillomavirus/virologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , França/epidemiologia , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Papillomaviridae/classificação , Papillomaviridae/genética , Prevalência , Estudos Prospectivos , Medição de Risco , Adulto JovemRESUMO
BACKGROUND INFORMATION: Efficient clearance of apoptotic cells, named efferocytosis, is a fundamental physiological process for tissue development and homeostasis. The contribution of non-professional phagocytes like fibroblasts to efferocytosis has been established, although the underlying mechanisms are not well understood. We recently demonstrated that horizontal DNA transfer can occur through the uptake of apoptotic human papillomavirus-positive cancer cells by human primary fibroblasts leading to their transformation. The aim of this present study was to analyse the cellular and molecular mechanisms that drive the phagocytic activity of human primary fibroblasts in the context of apoptotic cervical cancer cell removal. RESULTS: Here we provide evidence that human primary fibroblasts engulf late more efficiently than early apoptotic cells, but their phagocytic ability remains limited compared to professional phagocytes such as human monocyte-derived macrophages. The engulfment occurs in a time-, temperature- and calcium-dependent manner. Remodelling of actin-fibers contributes to the biogenesis of apoptotic cell containing macroendocytic vacuoles. Both morphological analyses and pharmacological approaches confirmed the involvement of actin-driven phagocytosis and likely macropinocytotic mechanisms in apoptotic target internalization. The uptake of apoptotic cells requires phosphatidylserine recognition, which is mainly mediated by phosphatidylserine-receptor brain-specific angiogenesis inhibitor 1. Confocal microscopy analyses with organelle-specific markers revealed that internalised apoptotic material traffics into late phagolysosomes and specific features of microtubule-associated protein 1 light chain 3-associated phagocytosis were observed. CONCLUSIONS: Our in vitro data show that fibroblasts contribute to apoptotic tumour cell removal by phagocytosis and likely macropinocytotic mechanisms. Efferocytosis by fibroblasts involves phosphatidylserine receptor brain-specific angiogenesis inhibitor 1, which participates in subsequent uptake orchestration via actin cytoskeleton remodelling. SIGNIFICANCE: Our results highlight the cellular and molecular mechanisms of fibroblast-mediated clearance of apoptotic tumour cells. Consequences regarding alternative mechanism of carcinogenesis or tumour progression should be addressed.
Assuntos
Apoptose , Fibroblastos/metabolismo , Papillomaviridae , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/virologia , Adulto , Feminino , Fibroblastos/patologia , Células HeLa , Humanos , Pessoa de Meia-Idade , Neoplasias do Colo do Útero/patologiaRESUMO
High-risk human Papillomaviruses (HR-HPV) - the most carcinogenic infectious agents - are responsible for the development of cervical cancer. The knowledge of HPV infection natural history and viral carcinogenesis led to the investigation of viral biomarkers (genotype, viral load, integration, E6/E7 mRNA expression, viral DNA methylation) from clinical samples representative of the evolution of cervical lesions. Mostly concerning HPV16, the literature data agree on an increase of viral load, proportion of samples harboring integrated HPV genomes and methylation of CpG located in the L1 gene with the lesion severity. Viral load and L1 CpG methylation are interesting for clinical practice since appropriate cutoff values allow the identification of precancerous lesions with a high specificity. Although HPV E6/E7 transcript detection is more specific than HPV DNA detection to identify precancerous cervical lesions, viral transcript quantitation and cutoff value determination are unlikely feasible in clinical practice. Taken together, data highlight promising biomarkers that could be integrated to screening algorithms.
RESUMO
Although survival from breast cancer has dramatically increased, many will develop recurrent, metastatic disease. Unfortunately, survival for this stage of disease remains very low. Activating the immune system has incredible promise since it has the potential to be curative. However, immune checkpoint blockade (ICB) which works through T cells has been largely disappointing for metastatic breast cancer. One reason for this is a suppressive myeloid immune compartment that is unaffected by ICB. Cholesterol metabolism and proteins involved in cholesterol homeostasis play important regulatory roles in myeloid cells. Here, we demonstrate that NR0B2, a nuclear receptor involved in negative feedback of cholesterol metabolism, works in several myeloid cell types to impair subsequent expansion of regulatory T cells (Tregs); Tregs being a subset known to be highly immune suppressive and associated with poor therapeutic response. Within myeloid cells, NR0B2 serves to decrease many aspects of the inflammasome, ultimately resulting in decreased IL1ß; IL1ß driving Treg expansion. Importantly, mice lacking NR0B2 exhibit accelerated tumor growth. Thus, NR0B2 represents an important node in myeloid cells dictating ensuing Treg expansion and tumor growth, thereby representing a novel therapeutic target to re-educate these cells, having impact across different solid tumor types. Indeed, a paper co-published in this issue demonstrates the therapeutic utility of targeting NR0B2.
Assuntos
Neoplasias da Mama , Progressão da Doença , Células Mieloides , Linfócitos T Reguladores , Linfócitos T Reguladores/imunologia , Animais , Feminino , Camundongos , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Humanos , Células Mieloides/imunologia , Células Mieloides/metabolismo , Camundongos Knockout , Interleucina-1beta/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Inflamassomos/metabolismo , Inflamassomos/imunologiaRESUMO
Immune checkpoint blockade (ICB) has had limited utility in several solid tumors such as breast cancer, a major cause of cancer-related mortality in women. Therefore, there is considerable interest in alternate strategies to promote an anti-cancer immune response. A paper co-published in this issue describes how NR0B2, a protein involved in cholesterol homeostasis, functions within myeloid immune cells to modulate the inflammasome and reduce the expansion of immune-suppressive regulatory T cells (Treg). Here, we develop NR0B2 as a potential therapeutic target. NR0B2 in tumors is associated with improved survival for several cancer types including breast. Importantly, NR0B2 expression is also prognostic of ICB success. Within breast tumors, NR0B2 expression is inversely associated with FOXP3, a marker of Tregs. While a described agonist (DSHN) had some efficacy, it required high doses and long treatment times. Therefore, we designed and screened several derivatives. A methyl ester derivative (DSHN-OMe) emerged as superior in terms of (1) cellular uptake, (2) ability to regulate expected expression of genes, (3) suppression of Treg expansion using in vitro co-culture systems, and (4) efficacy against the growth of primary and metastatic tumors. This work identifies NR0B2 as a target to re-educate myeloid immune cells and a novel ligand with significant anti-tumor efficacy in preclinical models.
Assuntos
Células Mieloides , Linfócitos T Reguladores , Humanos , Células Mieloides/imunologia , Células Mieloides/metabolismo , Células Mieloides/efeitos dos fármacos , Feminino , Animais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Neoplasias da Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/imunologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Camundongos , Linhagem Celular Tumoral , Microambiente Tumoral , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêuticoRESUMO
High-risk (HR) human papillomavirus (HPV)-associated carcinogenesis is driven mainly by the overexpression of E7 and E6 oncoproteins following viral DNA integration and the concomitant loss of the E2 open reading frame (ORF). However, the integration of HR-HPV DNA is not systematically observed in cervical cancers. The E2 protein acts as a transcription factor that governs viral oncogene expression. The methylation of CpGs in the E2-binding sites (E2BSs) in the viral long control region abrogates E2 binding, thus impairing the E2-mediated regulation of E7/E6 transcription. Here, high-resolution melting (HRM)-PCR was developed to quantitatively analyze the methylation statuses of E2BS1, E2BS2, and the specificity protein 1 (Sp1)-binding site in 119 HPV16-positive cervical smears. This is a rapid assay that is suitable for the analysis of cervical samples. The proportion of cancer samples with methylated E2BS1, E2BS2, and Sp1-binding site CpGs was 47%, whereas the vast majority of samples diagnosed as being within normal limits, low-grade squamous intraepithelial lesions (LSIL), or high-grade squamous intraepithelial lesions (HSIL) harbored unmethylated CpGs. Methylation levels varied widely, since some cancer samples harbored up to 60% of methylated HPV16 genomes. A pyrosequencing approach was used as a confirmation test and highlighted that quantitative measurement of methylation can be achieved by HRM-PCR. Its prognostic value deserves to be investigated alone or in association with other biomarkers. The reliability of this single-tube assay offers great opportunities for the investigation of HPV16 methylation in other HPV-related cancers, such as head and neck cancers, which are a major public health burden.
Assuntos
Metilação de DNA , DNA Viral/genética , DNA Viral/metabolismo , Proteínas de Ligação a DNA/metabolismo , Papillomavirus Humano 16/genética , Proteínas Oncogênicas Virais/metabolismo , Neoplasias do Colo do Útero/virologia , Sítios de Ligação , Feminino , Regulação Viral da Expressão Gênica , Papillomavirus Humano 16/isolamento & purificação , Humanos , Reação em Cadeia da Polimerase , Temperatura de TransiçãoRESUMO
The incidence of oropharyngeal cancers (OPC) is increasing in the world. Among OPC, those induced by human papillomaviruses have a better prognosis than non-HPV-associated OPC. The objective of this study was to highlight the relevance of HPV16 load, HPV16 DNA integration and HPV16-L1 serology on progression-free survival and overall survival of OPC patients. The PAPILLOPHAR cohort consists of 362 patients with oropharyngeal squamous cell carcinomas prospectively followed up for 5 years after treatment. Tumor biopsies and sera were collected at inclusion to investigate tumor HPV DNA/RNA characteristics and HPV16 L1 serology, respectively. Twenty-seven percent of tumor biopsies were HPV DNA- and RNA-positive and HPV16 represented 93% of HPV-positive cases. Among them, neither HPV16 viral load nor HPV16 DNA integration was associated with overall survival (OS) or progression-free survival (PFS). In contrast, high anti-HPV16 L1 antibody titers were significantly associated with a better OS and PFS. This study reveals that HPV16 load and integration are not relevant prognosis biomarkers in OPC patients.Clinical Relevance: High levels of HPV16 L1 antibodies may be useful to predict OPC patient outcome following treatment.ClinicalTrials.gov Identifier: NCT00918710, May 2017.
Assuntos
Papillomavirus Humano 16 , Neoplasias Orofaríngeas , Humanos , Papillomavirus Humano 16/genética , Anticorpos Antivirais , Neoplasias Orofaríngeas/patologia , Prognóstico , DNA Viral/genéticaRESUMO
Immune checkpoint blockade (ICB) has revolutionized cancer therapy but has had limited utility in several solid tumors such as breast cancer, a major cause of cancer-related mortality in women. Therefore, there is considerable interest in alternate strategies to promote an anti-cancer immune response. We demonstrate that NR0B2, a protein involved in cholesterol homeostasis, functions within myeloid immune cells to modulate the NLRP3 inflammasome and reduce the expansion of immune-suppressive regulatory T cells (Treg). Loss of NR0B2 increased mammary tumor growth and metastasis. Small molecule agonists, including one developed here, reduced Treg expansion, reduced metastatic growth and improved the efficacy of ICB. This work identifies NR0B2 as a target to re-educate myeloid immune cells providing proof-of-principle that this cholesterol-homeostasis axis may have utility in enhancing ICB.
RESUMO
Non-canonical autophagy is a key cellular pathway in immunity, cancer, and neurodegeneration, characterized by conjugation of ATG8 to endolysosomal single membranes (CASM). CASM is activated by engulfment (endocytosis, phagocytosis), agonists (STING, TRPML1), and infection (influenza), dependent on K490 in the ATG16L1 WD40-domain. However, factors associated with non-canonical ATG16L1 recruitment and CASM induction remain unknown. Here, using pharmacological inhibitors, we investigate a role for V-ATPase during non-canonical autophagy. We report that increased V0-V1 engagement is associated with, and sufficient for, CASM activation. Upon V0-V1 binding, V-ATPase recruits ATG16L1, via K490, during LC3-associated phagocytosis (LAP), STING- and drug-induced CASM, indicating a common mechanism. Furthermore, during LAP, key molecular players, including NADPH oxidase/ROS, converge on V-ATPase. Finally, we show that LAP is sensitive to Salmonella SopF, which disrupts the V-ATPase-ATG16L1 axis and provide evidence that CASM contributes to the Salmonella host response. Together, these data identify V-ATPase as a universal regulator of CASM and indicate that SopF evolved in part to evade non-canonical autophagy.
Assuntos
Proteínas Relacionadas à Autofagia , Autofagia , Proteínas Associadas aos Microtúbulos , Fagocitose , ATPases Vacuolares Próton-Translocadoras , Proteínas Relacionadas à Autofagia/metabolismo , Linhagem Celular , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
Chemotherapy with anti PD-1/PD-L1 antibodies has become the standard of care for patients with metastatic non-small cell lung cancer (mNSCLC). Using lung tumor models, where pemetrexed and cisplatin (PEM/CDDP) chemotherapy remains unable to synergize with immune checkpoint inhibitors (ICIs), we linked the failure of this treatment with its inability to induce CXCL10 expression and CD8+ T cell recruitment. Using drug screening, we showed that combining a MEK inhibitor (MEKi) with PEM/CDDP triggers CXCL10 secretion by cancer cells and CD8+ T cell recruitment, sensitizing it to ICIs. PEM/CDDP plus a MEKi promotes optineurin (OPTN)-dependent mitophagy, resulting in CXCL10 production in a mitochondrial DNA- and TLR9-dependent manner. TLR9 or autophagy/mitophagy inhibition abolishes the anti-tumor efficacy of PEM/CDDP plus MEKi/anti-PD-L1 therapy. In human NSCLCs, high OPTN, TLR9, and CXCL10 expression is associated with a better response to ICIs. Our results underline the role of TLR9- and OPTN-dependent mitophagy in enhancing chemoimmunotherapy efficacy.
Assuntos
Quimiocina CXCL10/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Autofagia/efeitos dos fármacos , Autofagia/genética , Antígeno B7-H1/antagonistas & inibidores , Biomarcadores Tumorais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Linhagem Celular Tumoral , Quimiocina CXCL10/metabolismo , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Proteínas de Checkpoint Imunológico/genética , Proteínas de Checkpoint Imunológico/metabolismo , Camundongos , Mitofagia/genética , Mitofagia/imunologia , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Ligação Proteica , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: While stimulator of interferon genes (STING) activation in innate immune cells of the tumor microenvironment can result in CD8 T cell-dependent antitumor immunity, whether STING signaling affects CD4 T-cell responses remains elusive. METHODS: Here, we tested whether STING activation modulated the effector functions of CD4 T cells in vivo by analyzing tumor-infiltrating CD4 T cells and evaluating the contribution of the CD4 T cell-derived cytokines in the antitumor activity of the STING ligand 2'3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) in two mouse tumor models. We performed ex vivo experiments to assess the impact of STING activation on CD4 T-cell differentiation and investigate the underlying molecular mechanisms. Finally, we tested whether STING activation enhances TH9 cell antitumor activity against mouse melanoma upon adoptive transfer. RESULTS: We found that activation of STING signaling cell-intrinsically enhances the differentiation and antitumor functions of TH1 and TH9 cells by increasing their respective production of interferon gamma (IFN-γ) and interleukin-9. IRF3 and type I interferon receptors (IFNARs) are required for the STING-driven enhancement of TH1 cell differentiation. However, STING activation favors TH9 cell differentiation independently of the IFNARs/IRF3 pathway but through mammalian target of rapamycin (mTOR) signaling, underscoring that STING activation differentially affects the fate of distinct CD4 T-cell subsets. The therapeutic effect of STING activation relies on TH1 and TH9-derived cytokines, and STING activation enhances the antitumor activity of TH9 cells upon adoptive transfer. CONCLUSION: Our results reveal the STING signaling pathway as a therapeutic target to boost CD4 T-cell effector functions and antitumor immunity.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Interleucina-9/fisiologia , Proteínas de Membrana/fisiologia , Células Th1/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Diferenciação Celular , Feminino , Fator Regulador 3 de Interferon/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Nucleotídeos Cíclicos/farmacologia , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/fisiologia , Células Th1/citologiaRESUMO
High-risk human papillomavirus (hrHPVs), particularly HPV16 and HPV18, are the etiologic factors of ano-genital cancers and some head and neck squamous cell carcinomas (HNSCCs). Viral E6 and E7 oncoproteins, controlled at both transcriptional and post-transcriptional levels, drive hrHPVs-induced carcinogenesis. In the present study, we investigated the implication of the DEAD-box helicase eukaryotic translation initiation factor 4A3 (eIF4A3,) an Exon Junction Complex factor, in the regulation of HPV16 gene expression. Our data revealed that the depletion of the factor eIF4A3 up-regulated E7 oncoprotein levels. We also showed that the inhibition of the nonsense-mediated RNA decay (NMD) pathway, resulted in the up-regulation of E7 at both RNA and protein levels. We therefore proposed that HPV16 transcripts might present different susceptibilities to NMD and that this pathway could play a key role in the levels of expression of these viral oncoproteins during the development of HPV-related cancers.
Assuntos
RNA Helicases DEAD-box/metabolismo , Fator de Iniciação 4A em Eucariotos/metabolismo , Proteínas E7 de Papillomavirus/genética , Linhagem Celular Tumoral , Interações Hospedeiro-Patógeno , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Humanos , Proteínas E7 de Papillomavirus/metabolismoRESUMO
BACKGROUND: We have previously shown that 5-fluorouracil (5-FU) selectively kills myeloid-derived suppressor cells (MDSCs) and activates NLRP3 (NOD-leucine rich repeat and pyrin containing protein 3) inflammasome. NLRP3 activation leads to caspase-1 activation and production of IL-1ß, which in turn favors secondary tumor growth. We decided to explore the effects of either a heat shock (HS) or the deficiency in heat shock protein (HSP) 70, previously shown to respectively inhibit or increase NLRP3 inflammasome activation in macrophages. METHODS: Caspase-1 activation was detected in vitro in MSC-2 cells by western blot and in vivo or ex vivo in tumor and/or splenic MDSCs by flow cytometry. The effects of HS, HSP70 deficiency and anakinra (an IL-1 inhibitor) on tumor growth and mice survival were studied in C57BL/6 WT or Hsp70-/- tumor-bearing mice. Finally, Th17 polarization was evaluated by qPCR (Il17a, Rorc) and angiogenic markers by qPCR (Pecam1, Eng) and immunohistochemistry (ERG). RESULTS: HS inhibits 5-FU-mediated caspase-1 activation in vitro and in vivo without affecting its cytotoxicity on MDSCs. Moreover, it enhances the antitumor effect of 5-FU treatment and favors mice survival. Interestingly, it is associated to a decreased Th17 and angiogenesis markers in tumors. IL-1ß injection is able to bypass HS+5-FU antitumor effects. In contrast, in Hsp70-/- MDSCs, 5-FU-mediated caspase-1 activation is increased in vivo and in vitro without effect on 5-FU cytotoxicity. In Hsp70-/- mice, the antitumor effect of 5-FU was impeded, with an increased Th17 and angiogenesis markers in tumors. Finally, the effects of 5-FU on tumor growth can be restored by inhibiting IL-1ß, using anakinra. CONCLUSION: This study provides evidence on the role of HSP70 in tuning 5-FU antitumor effect and suggests that HS can be used to improve 5-FU anticancer effect.
Assuntos
Caspase 1/metabolismo , Fluoruracila/farmacologia , Proteínas de Choque Térmico HSP70/metabolismo , Resposta ao Choque Térmico , Inflamassomos/imunologia , Linfoma/imunologia , Células Supressoras Mieloides/imunologia , Animais , Antimetabólitos Antineoplásicos/farmacologia , Ativação Enzimática , Feminino , Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Linfoma/tratamento farmacológico , Linfoma/metabolismo , Linfoma/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Células Supressoras Mieloides/efeitos dos fármacos , Células Supressoras Mieloides/metabolismo , Células Supressoras Mieloides/patologiaRESUMO
Monitoring of ATG8 proteins by western blotting and immunofluorescence microscopy are the most common methods to monitor the autophagy pathway. However, it has recently been shown that ATG8 proteins can be lipidated to non-autophagosome, single-membrane compartments through a noncanonical autophagy pathway. This is commonly found to occur during macro-endocytic processes such as phagocytosis, where it has been termed LC3-associated phagocytosis, and upon lysosomotropic drug treatment. Therefore, care is required when interpreting data based on ATG8 in order to conclude whether a signal relates to the canonical or noncanonical pathway. Here we provide methods to monitor noncanonical autophagy through fluorescence microscopy.
Assuntos
Autofagia , Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , Animais , Família da Proteína 8 Relacionada à Autofagia/análise , Células da Medula Óssea/citologia , Técnicas de Cultura de Células/métodos , Linhagem Celular , Separação Celular/métodos , Células Cultivadas , Células HCT116 , Humanos , Macrófagos/citologia , Camundongos , Microscopia Confocal/métodos , Proteínas Associadas aos Microtúbulos/análise , FagocitoseRESUMO
The catabolic process of autophagy plays important functions in inflammatory and immune responses by modulating innate immunity and adaptive immunity. Over the last decade, a cell-intrinsic role for autophagy in modulating CD4 T cell functions and differentiation was revealed. After the initial observation of autophagosomes in effector CD4 T cells, further work has shown that not only autophagy levels are modulated in CD4 T cells in response to environmental signals but also that autophagy critically affects the biology of these cells. Mouse models of autophagy deletion in CD4 T cells have indeed shown that autophagy is essential for CD4 T cell survival and homeostasis in peripheral lymphoid organs. Furthermore, autophagy is required for CD4 T cell proliferation and cytokine production in response to T cell receptor activation. Recent developments have uncovered that autophagy controls CD4 T cell differentiation and functions. While autophagy is required for the maintenance of immunosuppressive functions of regulatory T cells, it restrains the differentiation of TH9 effector cells, thus limiting their antitumor and pro-inflammatory properties. We will here discuss these findings that collectively suggest that therapeutic strategies targeting autophagy could be exploited for the treatment of cancer and inflammatory diseases.
Assuntos
Autofagia/imunologia , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/imunologia , Proliferação de Células , Animais , Autofagossomos , Ensaios Clínicos como Assunto , Citocinas/imunologia , Humanos , Imunidade Inata , Inflamação/terapia , Ativação Linfocitária/imunologia , Camundongos , Neoplasias/terapia , Linfócitos T Reguladores/imunologiaRESUMO
High-risk human papillomaviruses are the etiological agents of cervical cancer and HPV16 is the most oncogenic genotype. Immortalization and transformation of infected cells requires the overexpression of the two viral oncoproteins E6 and E7 following HPV DNA integration into the host cell genome. Integration often leads to the loss of the E2 open reading frame and the corresponding protein can no longer act as a transcriptional repressor on p97 promoter. Recently, it has been proposed that long control region methylation also contributes to the regulation of E6/E7 expression.To determine which epigenetic mechanism is involved in HPV16 early gene regulation, 5-aza-2'-deoxycytidine was used to demethylate Ca Ski and SiHa cell DNA. Decreased expression of E6 mRNA and protein levels was observed in both cell lines in an E2-independent manner. E6 repression was accompanied by neither a modification of the main cellular transcription factor expression involved in long control region regulation, nor by a modification of the E6 mRNA splicing pattern. In contrast, a pronounced upregulation of miR-375, known to destabilize HPV16 early viral mRNA, was observed. Finally, the use of miR-375 inhibitor definitively proved the involvement of miR-375 in E6 repression. These results highlight that cellular DNA methylation modulates HPV16 early gene expression and support a role for epigenetic events in high-risk HPV associated-carcinogenesis.
Assuntos
Azacitidina/análogos & derivados , Papillomavirus Humano 16/genética , MicroRNAs/genética , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/tratamento farmacológico , Proteínas Repressoras/genética , Neoplasias do Colo do Útero/tratamento farmacológico , Azacitidina/uso terapêutico , Linhagem Celular Tumoral , Metilação de DNA , Decitabina , Feminino , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/genética , Proteínas Repressoras/metabolismo , Regulação para Cima , Neoplasias do Colo do Útero/genéticaRESUMO
The modulation of canonical macroautophagy/autophagy for therapeutic benefit is an emerging strategy of medical and pharmaceutical interest. Many drugs act to inhibit autophagic flux by targeting lysosome function, while others were developed to activate the pathway. Here, we report the surprising finding that many therapeutically relevant autophagy modulators with lysosomotropic and ionophore properties, classified as inhibitors of canonical autophagy, are also capable of activating a parallel noncanonical autophagy pathway that drives MAP1LC3/LC3 lipidation on endolysosomal membranes. Further, we provide the first evidence supporting drug-induced noncanonical autophagy in vivo using the local anesthetic lidocaine and human skin biopsies. In addition, we find that several published inducers of autophagy and mitophagy are also potent activators of noncanonical autophagy. Together, our data raise important issues regarding the interpretation of LC3 lipidation data and the use of autophagy modulators, and highlight the need for a greater understanding of the functional consequences of noncanonical autophagy.
Assuntos
Autofagia/fisiologia , Metabolismo dos Lipídeos/fisiologia , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitofagia/fisiologia , Linhagem Celular , Endossomos/metabolismo , Humanos , LipídeosRESUMO
BACKGROUND: Colorectal cancer (CRC) is one of the most common cancers. As in other cancer locations, the involvement of human papillomaviruses (HPV) has been suggested but remains highly debated with wide differences among reported prevalence of HPV infection in CRCs. AIM: To determine the actual prevalence of high risk HPV16 and 18 in a large case-control study. METHODS: CRC specimens were used for analysis of both tumor and distant healthy tissue. As a non-malignant control group, samples from sigmoid diverticulosis resections were studied. Detection of HPV16 and HPV18 DNA was performed using a real time polymerase chain reaction (qPCR). Ten percent of tumor samples were also randomly subjected to a complete HPV genotyping using the INNO-LiPA technique. RESULTS: 467 samples were analyzed: 217 tumor samples from 210 CRCs, 210 distant healthy tissue samples, and 40 sigmoid samples. HPV18 DNA was never amplified and HPV16 was amplified only three times in tumor tissues with viral loads under or at the limit of quantification. New extraction from the same tumor blocks for these samples revealed no HPV with qPCR and INNO-Lipa assays. CONCLUSION: With adequate procedures and reliable techniques, no HPV was detected in the largest case-control study so far, bringing more evidence on the absence of involvement of HPV in CRCs.