Interleukin-21 receptor signaling promotes metabolic dysfunction-associated steatohepatitis-driven hepatocellular carcinoma by inducing immunosuppressive IgA+ B cells.

BACKGROUND: Dysregulation of immune surveillance is tightly linked to the development of metabolic dysfunction-associated steatohepatitis (MASH)-driven hepatocellular carcinoma (HCC); however, its underlying mechanisms remain unclear. Herein, we aimed to determine the role of interleukin-21 receptor (IL-21R) in MASH-driven HCC. METHODS: The clinical significance of IL-21R was assessed in human HCC specimens using immunohistochemistry staining. Furthermore, the expression of IL-21R in mice was assessed in the STAM model. Thereafter, two different MASH-driven HCC mouse models were applied between IL-21R-deficient mice and wild type controls to explore the role of IL-21R in MASH-driven HCC. To further elucidate the potential mechanisms by which IL-21R affected MASH-driven HCC, whole transcriptome sequencing, flow cytometry and adoptive lymphocyte transfer were performed. Finally, flow cytometry, enzyme-linked immunosorbent assay, immunofluorescent staining, chromatin immunoprecipitation assay and western blotting were conducted to explore the mechanism by which IL-21R induced IgA+ B cells. RESULTS: HCC patients with high IL-21R expression exhibited poor relapse-free survival, advanced TNM stage and severe steatosis. Additionally, IL-21R was demonstrated to be upregulated in mouse liver tumors. Particularly, ablation of IL-21R impeded MASH-driven hepatocarcinogenesis with dramatically reduction of lipid accumulation. Moreover, cytotoxic CD8+ T lymphocyte activation was enhanced in the absence of IL-21R due to the reduction of immunosuppressive IgA+ B cells. Mechanistically, the IL-21R-STAT1-c-Jun/c-Fos regulatory axis was activated in MASH-driven HCC and thus promoted the transcription of Igha, resulting in the induction of IgA+ B cells. CONCLUSIONS: IL-21R plays a cancer-promoting role by inducing IgA+ B cells in MASH-driven hepatocarcinogenesis. Targeting IL-21R signaling represents a potential therapeutic strategy for cancer therapy.

New mitochondrial DNA synthesis enables NLRP3 inflammasome activation.

Dysregulated NLRP3 inflammasome activity results in uncontrolled inflammation, which underlies many chronic diseases. Although mitochondrial damage is needed for the assembly and activation of the NLRP3 inflammasome, it is unclear how macrophages are able to respond to structurally diverse inflammasome-activating stimuli. Here we show that the synthesis of mitochondrial DNA (mtDNA), induced after the engagement of Toll-like receptors, is crucial for NLRP3 signalling. Toll-like receptors signal via the MyD88 and TRIF adaptors to trigger IRF1-dependent transcription of CMPK2, a rate-limiting enzyme that supplies deoxyribonucleotides for mtDNA synthesis. CMPK2-dependent mtDNA synthesis is necessary for the production of oxidized mtDNA fragments after exposure to NLRP3 activators. Cytosolic oxidized mtDNA associates with the NLRP3 inflammasome complex and is required for its activation. The dependence on CMPK2 catalytic activity provides opportunities for more effective control of NLRP3 inflammasome-associated diseases.

Author Correction: Inflammation-induced IgA+ cells dismantle anti-liver cancer immunity.

In this Article, the sentence: "After 7 months of HFD, MUP-uPA mice developed HCC15, which contained numerous (usually 50-100 per tumour) non-recurrent coding mutations in pathways that are mutated in human HCC (Fig. 2d and Extended Data Fig. 6a).", should have read: "After 7 months of HFD, MUP-uPA mice developed HCC15, which contained numerous (usually 50-100 per tumour) non-recurrent mutations in pathways that are mutated in human HCC (Fig. 2d and Extended Data Fig. 6a).". This has been corrected online. In Extended Data Fig. 6a and b, which show the number of point mutations identified per sample and the mutational signatures, all sequence variants (including non-coding mutations) are shown. Fig. 2d also presents all variants compared to human mutations. In the Supplementary Information to this Amendment, we now provide the comparisons of all variants and coding variants to human mutations.

Activation of NF-κB and p300/CBP potentiates cancer chemoimmunotherapy through induction of MHC-I antigen presentation.

Many cancers evade immune rejection by suppressing major histocompatibility class I (MHC-I) antigen processing and presentation (AgPP). Such cancers do not respond to immune checkpoint inhibitor therapies (ICIT) such as PD-1/PD-L1 [PD-(L)1] blockade. Certain chemotherapeutic drugs augment tumor control by PD-(L)1 inhibitors through potentiation of T-cell priming but whether and how chemotherapy enhances MHC-I-dependent cancer cell recognition by cytotoxic T cells (CTLs) is not entirely clear. We now show that the lysine acetyl transferases p300/CREB binding protein (CBP) control MHC-I AgPPM expression and neoantigen amounts in human cancers. Moreover, we found that two distinct DNA damaging drugs, the platinoid oxaliplatin and the topoisomerase inhibitor mitoxantrone, strongly up-regulate MHC-I AgPP in a manner dependent on activation of nuclear factor kappa B (NF-κB), p300/CBP, and other transcription factors, but independently of autocrine IFNγ signaling. Accordingly, NF-κB and p300 ablations prevent chemotherapy-induced MHC-I AgPP and abrogate rejection of low MHC-I-expressing tumors by reinvigorated CD8+ CTLs. Drugs like oxaliplatin and mitoxantrone may be used to overcome resistance to PD-(L)1 inhibitors in tumors that had "epigenetically down-regulated," but had not permanently lost MHC-I AgPP activity.

Inflammation-induced IgA+ cells dismantle anti-liver cancer immunity.

The role of adaptive immunity in early cancer development is controversial. Here we show that chronic inflammation and fibrosis in humans and mice with non-alcoholic fatty liver disease is accompanied by accumulation of liver-resident immunoglobulin-A-producing (IgA+) cells. These cells also express programmed death ligand 1 (PD-L1) and interleukin-10, and directly suppress liver cytotoxic CD8+ T lymphocytes, which prevent emergence of hepatocellular carcinoma and express a limited repertoire of T-cell receptors against tumour-associated antigens. Whereas CD8+ T-cell ablation accelerates hepatocellular carcinoma, genetic or pharmacological interference with IgA+ cell generation attenuates liver carcinogenesis and induces cytotoxic T-lymphocyte-mediated regression of established hepatocellular carcinoma. These findings establish the importance of inflammation-induced suppression of cytotoxic CD8+ T-lymphocyte activation as a tumour-promoting mechanism.

Erratum: Inflammation-induced IgA+ cells dismantle anti-liver cancer immunity.

This corrects the article DOI: 10.1038/nature24302.

A novel AP-1/miR-101 regulatory feedback loop and its implication in the migration and invasion of hepatoma cells.

MicroRNA-101 (miR-101) is frequently downregulated in various cancers. To date, the regulatory networks of miR-101 remain obscure. In this study, we demonstrated that miR-101 was mainly transcribed from human miR-101-2 and mouse miR-101bgene loci. Subsequent analyses revealed that activator protein-1 (AP-1) directly binded to the -17.4 to -16.4 k region upstream of pre-miR-101-2 and activated the expression of miR-101. On the other hand, miR-101 could inhibit the expression of ERK2 and c-Fos, two key factors of the AP-1 pathway, by binding to their 3'-UTRs. Furthermore, reintroduction of miR-101 efficiently suppressed the AP-1 activity and pri-miR-101-2 transcription. These data thus suggest a novel AP-1/miR-101 regulatory circuitry, that is, AP-1 promotes the transcription of miR-101, whereas the expression of miR-101 reduces the level of ERK2 and c-Fos and thereby attenuates the AP-1 signaling. Further investigation disclosed that the AP-1 activator TPA-induced MMP9 activity and the TPA-promoted migration and invasion of hepatoma cells were significantly attenuated by miR-101 but were enhanced by miR-101 inhibitor. Our results suggest that the AP-1/miR-101 feedback loop may prevent the excessive activation of metastatic signals imposed by ERK2/AP-1 and highlight the biological significance of miR-101 downregulation in cancer metastasis.

A serum microRNA classifier for early detection of hepatocellular carcinoma: a multicentre, retrospective, longitudinal biomarker identification study with a nested case-control study.

BACKGROUND: The ability of circulating microRNAs (miRNAs) to detect preclinical hepatocellular carcinoma has not yet been reported. We aimed to identify and assess a serum miRNA combination that could detect the presence of clinical and preclinical hepatocellular carcinoma in at-risk patients. METHODS: We did a three-stage study that included healthy controls, inactive HBsAg carriers, individuals with chronic hepatitis B, individuals with hepatitis B-induced liver cirrhosis, and patients with diagnosed hepatocellular carcinoma from four hospitals in China. We used array analysis and quantitative PCR to identify 19 candidate serum miRNAs that were increased in six patients with hepatocellular carcinoma compared with eight control patients with chronic hepatitis B. Using a training cohort of patients with hepatocellular carcinoma and controls, we built a serum miRNA classifier to detect hepatocellular carcinoma. We then validated the classifiers' ability in two independent cohorts of patients and controls. We also established the classifiers' ability to predict preclinical hepatocellular carcinoma in a nested case-control study with sera prospectively collected from patients with hepatocellular carcinoma before clinical diagnosis and from matched individuals with hepatitis B who did not develop cancer from the same surveillance programme. We used the sensitivity, specificity, and area under the receiver operating characteristic curve (AUC) to evaluate diagnostic performance, and compared the miRNA classifier with α-fetoprotein at a cutoff of 20 ng/mL (AFP20). FINDINGS: Between Aug 1, 2009, and Aug 31, 2013, we recruited 257 participants to the training cohort, and 352 and 139 participants to the two independent validation cohorts. In the third validation cohort, 27 patients with hepatocellular carcinoma and 135 matched controls were included in the nested case-control study, which ran from Aug 1, 2009, to Aug 31, 2014. We identified a miRNA classifier (Cmi) containing seven differentially expressed miRNAs (miR-29a, miR-29c, miR-133a, miR-143, miR-145, miR-192, and miR-505) that could detect hepatocellular carcinoma. Cmi showed higher accuracy than AFP20 to distinguish individuals with hepatocellular carcinoma from controls in the validation cohorts, but not in the training cohort (AUC 0·826 [95% CI 0·771-0·880] vs 0·814 [0·756-0·872], p=0·72 in the training cohort; 0·817 [0·769-0·865] vs 0·709 [0·653-0·765], p=0·00076 in validation cohort 1; and 0·884 [0·818-0·951] vs 0·796 [0·706-0·886], p=0·042 for validation cohort 2). In all four cohorts, Cmi had higher sensitivity (range 70·4-85·7%) than did AFP20 (40·7-69·4%) to detect hepatocellular carcinoma at the time of diagnosis, whereas its specificity (80·0-91·1%) was similar to that of AFP20 (84·9-100%). In the nested case-control study, sensitivity of Cmi to detect hepatocellular carcinoma was 29·6% (eight of 27 cases) 12 months before clinical diagnosis, 48·1% (n=13) 9 months before clinical diagnosis, 48·1% (n=13) 6 months before clinical diagnosis, and 55·6% (n=15) 3 months before clinical diagnosis, whereas sensitivity of AFP20 was only 7·4% (n=2), 11·1% (n=3), 18·5% (n=5), and 22·2% (n=6) at the corresponding timepoints (p=0·036, p=0·0030, p=0·021, p=0·012, respectively). Cmi had a larger AUC than did AFP20 to identify small-size (AUC 0·833 [0·782-0·883] vs 0·727 [0·664-0·792], p=0·0018) and early-stage (AUC 0·824 [0·781-0·868] vs 0·754 [0·702-0·806], p=0·015) hepatocellular carcinoma and could also detect α-fetoprotein-negative (AUC 0·825 [0·779-0·871]) hepatocellular carcinoma. INTERPRETATION: Cmi is a potential biomarker for hepatocellular carcinoma, and can identify small-size, early-stage, and α-fetoprotein-negative hepatocellular carcinoma in patients at risk. The miRNA classifier could be valuable to detect preclinical hepatocellular carcinoma, providing patients with a chance of curative resection and longer survival. FUNDING: National Key Basic Research Program, National Science and Technology Major Project, National Natural Science Foundation of China.

SLC38A5 aggravates DC-mediated psoriasiform skin inflammation via potentiating lysosomal acidification.

Amino acid (aa) metabolism is closely correlated with the pathogenesis of psoriasis; however, details on aa transportation during this process are barely known. Here, we find that SLC38A5, a sodium-dependent neutral aa transporter that counter-transports protons, is markedly upregulated in the psoriatic skin of both human patients and mouse models. SLC38A5 deficiency significantly ameliorates the pathogenesis of psoriasis, indicating a pathogenic role of SLC38A5. Surprisingly, SLC38A5 is almost exclusively expressed in dendritic cells (DCs) when analyzing the psoriatic lesion and mainly locates on the lysosome. Mechanistically, SLC38A5 potentiates lysosomal acidification, which dictates the cleavage and activation of TLR7 with ensuing production of pro-inflammatory cytokines such as interleukin-23 (IL-23) and IL-1ß from DCs and eventually aggravates psoriatic inflammation. In summary, this work uncovers an auxiliary mechanism in driving lysosomal acidification, provides inspiring insights for DC biology and psoriasis etiology, and reveals SLC38A5 as a promising therapeutic target for treating psoriasis.

An enhanced genetic mutation-based model for predicting the efficacy of immune checkpoint inhibitors in patients with melanoma.

Background: Programmed death ligand 1 (PD-L1) and tumor mutation burden (TMB) have been developed as biomarkers for the treatment of immune checkpoint inhibitors (ICIs). However, some patients who are TMB-high or PD-L1-high remained resistant to ICIs therapy. Therefore, a more clinically applicable and effective model for predicting the efficacy of ICIs is urgently needed. Methods: In this study, genomic data for 466 patients with melanoma treated with ICIs from seven independent cohorts were collected and used as training and validation cohorts (training cohort n = 300, validation cohort1 n = 61, validation cohort2 n = 105). Ten machine learning classifiers, including Random Forest classifier, Stochastic Gradient Descent (SGD) classifier and Linear Support Vector Classifier (SVC), were subsequently evaluated. Results: The Linear SVC with a 186-gene mutation-based set was screened to construct the durable clinical benefit (DCB) model. Patients predicted to have DCB (pDCB) were associated with a better response to the treatment of ICIs in the validation cohort1 (AUC=0.838) and cohort2 (AUC=0.993). Compared with TMB and other reported genetic mutation-based signatures, the DCB model showed greater predictive power. Furthermore, we explored the genomic features in determining the benefits of ICIs treatment and found that patients with pDCB were associated with higher tumor immunogenicity. Conclusion: The DCB model constructed in this study can effectively predict the efficacy of ICIs treatment in patients with melanoma, which will be helpful for clinical decision-making.

c-Myc-activated intronic miR-210 and lncRNA MIR210HG synergistically promote the metastasis of gastric cancer.

The relationship between microRNA (miRNA) and hosting long non-coding RNA (lncRNA) remains unclear. Here, the expression levels of microRNA-210 (miR-210) and hosting lncRNA MIR210HG are significantly increased and positively correlated in gastric cancer (GC). Gain- and loss-of-function studies demonstrate that miR-210 and MIR210HG synergistically promote the migration and invasion of GC cells in vitro. Furthermore, GC sublines simultaneously expressing miR-210 and MIR210HG display synergistic promotion of lung metastasis in vivo. Mechanistically, MIR210HG interacts with DExH-box helicase 9 (DHX9) to increase DHX9/c-Jun complex's occupancy on the promoter of matrix metallopeptidases (MMPs), and thus promotes migration and invasion of GC cells. Additionally, miR-210 directly suppresses the expression of dopamine receptor D5 (DRD5), serine/threonine kinase 24 (STK24) and MAX network transcriptional repressor (MNT), resulting in enhanced migration and invasion. Finally, MYC proto-oncogene (c-Myc) transactivates miR-210 and MIR210HG. Overexpression of miR-210 or/and MIR210HG can rescue the inhibitory effect on the migration and invasion by silencing c-Myc. Moreover, c-Myc inhibitor significantly decreases lung metastasis of GC in vivo. Collectively, our findings identify a novel mechanism, by which c-Myc-activated miR-210 and MIR210HG synergistically promote the metastasis of GC.

A novel GSK-3 beta-C/EBP alpha-miR-122-insulin-like growth factor 1 receptor regulatory circuitry in human hepatocellular carcinoma.

UNLABELLED: miR-122 is a highly abundant, hepatocyte-specific microRNA. The biomedical significance and regulatory mechanisms of miR-122 remain obscure. We explored the role of miR-122 in tumorigenesis in the context of gene regulatory network. The miR-122 promoter and its transactivator were identified by way of luciferase reporter system, electrophoretic mobility shift, and chromatin immunoprecipitation assays. The miR-122 regulatory circuitry and its implication in hepatocarcinogenesis were identified using livers of different development stages, human hepatocellular carcinoma (HCC) tissues and cell lines, and aflatoxin B1 (AFB1)-transformed cells. We characterized the -5.3 to -4.8 kb region upstream of miR-122 precursor as miR-122 promoter. Further investigation revealed that deletion of predicted CCAAT/enhancer-binding protein alpha (C/EBPα) binding sites C/EBPα knockdown significantly reduced miR-122 promoter activity and endogenous miR-122 expression; and C/EBPα directly interacted with the miR-122 promoter in vitro and in vivo. These data suggest that C/EBPα is a transactivator for miR-122 transcription. We further demonstrated that miR-122 suppressed insulin-like growth factor 1 receptor (IGF-1R) translation and sustained glycogen synthase kinase-3 beta (GSK-3ß) activity. The activated GSK-3ß not only repressed cell proliferation, but also activated C/EBPα, which maintained miR-122 levels and thereby enforced IGF-1R suppression. Interestingly, down-regulation of miR-122 and C/EBPα, and up-regulation of IGF-1R were frequently observed in HCC tissues, and decreased miR-122 levels were associated with worse survival of HCC patients. Moreover, AFB1 exposure resulted in decreased activity in GSK-3ß, C/EBPα, and miR-122 and increased levels of IGF-1R, whereas restoration of miR-122 suppressed the tumorigenicity of HCC and AFB1-transformed cells. CONCLUSION: We have identified a novel GSK-3ß-C/EBPα-miR-122-IGF-1R regulatory circuitry whose dysfunction may contribute to the development of HCC. Our findings provide new insight into miR-122's function and the mechanisms of hepatocarcinogenesis.

Hepatocellular Carcinoma Cell-Secreted Exosomal MicroRNA-210 Promotes Angiogenesis In Vitro and In Vivo.

We previously found that 19 microRNAs (miRNAs) significantly increased in the sera of hepatocellular carcinoma (HCC) patients. Here, we evaluated whether these miRNAs were secreted by HCC cells and contributed to tumor angiogenesis. High level of miR-210-3p (miR-210) was detected in the exosomes isolated from the sera of HCC patients and the conditioned media of hepatoma cells. Higher miR-210 level in serum was correlated with higher microvessel density in HCC tissues. Moreover, the HCC cell-secreted exosomes promoted in vitro tubulogenesis of endothelial cells, which was strengthened by overexpressing miR-210 in HCC cells but was attenuated by repressing miR-210 or DROSHA in HCC cells. This pro-tubulogenesis effect by HCC exosomes was also abrogated by antagonizing miR-210 in endothelial cells. Subsequent in vivo studies revealed that Matrigel plug and subcutaneous tumor xenografts treated with HCC cell-derived exosomal miR-210 displayed much more vessels. Furthermore, exosomal miR-210 could be delivered into endothelial cells and directly inhibited the expression of SMAD4 and STAT6, resulting in enhanced angiogenesis. Collectively, HCC cell-secreted exosomal miR-210 may be transferred into endothelial cells and thereby promotes tumor angiogenesis by targeting SMAD4 and STAT6. Our findings identify a novel mechanism of HCC angiogenesis and highlight the biological importance of exosomal miR-210.