Endothelial DGKG promotes tumor angiogenesis and immune evasion in hepatocellular carcinoma.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is among the most prevalent and lethal cancers worldwide. The tumor microenvironment (TME) contributes to the poor response of patients with HCC to current therapies, while tumor vascular endothelial cells (ECs) are fundamental TME components that significantly contribute to tumor progression. However, the specific functions and mechanisms of tumor vascular ECs in HCC remain unclear. METHODS: We screened and validated diacylglycerol kinase gamma (DGKG) hyper-expression specifically in HCC tumor vascular ECs. Single-cell RNA-sequencing, cytometry by time-of-flight, and in vitro and in vivo studies were performed to investigate the functions of endothelial DGKG. Multiplexed immunohistochemistry staining and flow cytometry were used to evaluate changes in the TME. RESULTS: Functionally, endothelial DGKG promotes tumor angiogenesis and immunosuppressive regulatory T-cell differentiation in HCC. Of significance, we found that HIF-1α activates DGKG transcription by directly binding to its promoter region under hypoxia. Upregulated DGKG promotes HCC progression by recruiting ubiquitin specific peptidase 16 to facilitate ZEB2 deubiquitination, which increases TGF-ß1 secretion, thus inducing tumor angiogenesis and regulatory T-cell differentiation. Importantly, targeting endothelial DGKG potentiated the efficiency of dual blockade of PD-1 and VEGFR-2. CONCLUSION: Hypoxia-induced EC-specific DGKG hyper-expression promotes tumor angiogenesis and immune evasion via the ZEB2/TGF-ß1 axis, suggesting EC-specific DGKG as a potential therapeutic target for HCC. IMPACT AND IMPLICATIONS: Here, we reported that hypoxia-induced endothelial cell-specific DGKG hyper-expression promotes angiogenesis and immune evasion in HCC by recruiting USP16 for K48-linked deubiquitination and inducing the subsequent stabilization of ZEB2, leading to increased TGF-ß1 secretion. Most importantly, endothelial DGKG inhibition greatly improved the efficacy of the dual combination of anti-VEGFR2 and anti-PD-1 treatment in a mouse HCC model, significantly inhibiting the malignant progression of HCC and improving survival. This preclinical study supports the targeting of endothelial DGKG as a potential strategy for precision HCC treatment.

TRAF6 directs FOXP3 localization and facilitates regulatory T-cell function through K63-linked ubiquitination.

Regulatory T cells (Tregs) are crucial mediators of immune control. The characteristic gene expression and suppressive functions of Tregs depend considerably on the stable expression and activity of the transcription factor FOXP3. Transcriptional regulation of the Foxp3 gene has been studied in depth, but both the expression and function of this factor are also modulated at the protein level. However, the molecular players involved in posttranslational FOXP3 regulation are just beginning to be elucidated. Here, we found that TRAF6-deficient Tregs were dysfunctional in vivo; mice with Treg-restricted deletion of TRAF6 were resistant to implanted tumors and displayed enhanced anti-tumor immunity. We further determined that FOXP3 undergoes K63-linked ubiquitination at lysine 262 mediated by the E3 ligase TRAF6. In the absence of TRAF6 activity or upon mutation of the ubiquitination site, FOXP3 displayed aberrant, perinuclear accumulation and disrupted regulatory function. Thus, K63-linked ubiquitination by TRAF6 ensures proper localization of FOXP3 and facilitates the transcription factor's gene-regulating activity in Tregs. These results implicate TRAF6 as a key posttranslational, Treg-stabilizing regulator that may be targeted in novel tolerance-breaking therapies.

Macrophage nuclear factor erythroid 2-related factor 2 deficiency promotes innate immune activation by tissue inhibitor of metalloproteinase 3-mediated RhoA/ROCK pathway in the ischemic liver.

BACKGROUND AND AIMS: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulator of reactive oxygen species (ROS) and inflammation and has been implicated in both human and murine inflammatory disease models. We aimed to characterize the roles of macrophage-specific Nrf2 in liver ischemia/reperfusion injury (IRI). APPROACH AND RESULTS: First, macrophage Nrf2 expression and liver injury in patients undergoing OLT or ischemia-related hepatectomy were analyzed. Subsequently, we created a myeloid-specific Nrf2-knockout (Nrf2M-KO ) strain to study the function and mechanism of macrophage Nrf2 in a murine liver IRI model. In human specimens, macrophage Nrf2 expression was significantly increased in liver tissues after transplantation or hepatectomy. Interestingly, lower Nrf2 expressions correlated with more severe liver injury postoperatively. In a mouse model, we found Nrf2M-KO mice showed worse hepatocellular damage than Nrf2-proficient controls based on serum biochemistry, pathology, ROS, and inflammation. In vitro, Nrf2 deficiency promoted innate immune activation and migration in macrophages on toll-like receptor (TLR) 4 stimulation. Microarray profiling showed Nrf2 deletion caused markedly lower transcriptional levels of tissue inhibitor of metalloproteinase 3 (Timp3). ChIP-seq, PCR, and luciferase reporter assay further demonstrated Nrf2 bound to the promoter region of Timp3. Moreover, a disintegrin and metalloproteinase (ADAM) 10/ROCK1 was specifically increased in Nrf2-deficient macrophages. Increasing Timp3 expression effectively inhibited ADAM10/ROCK1 expression and rescued the Nrf2M-KO -mediated inflammatory response on TLR4 stimulation in vitro. Importantly, Timp3 overexpression, recombinant Timp3 protein, or ROCK1 knockdown rescued Nrf2M-KO -related liver IRI by inhibiting macrophage activation. CONCLUSIONS: In conclusion, macrophage Nrf2 mediates innate proinflammatory responses, attenuates liver IRI by binding to Timp3, and inhibits the RhoA/ROCK pathway, which provides a therapeutic target for clinical organ IRI.

M2 macrophage-secreted exosomes promote metastasis and increase vascular permeability in hepatocellular carcinoma.

BACKGROUND: Metastasis is a key feature of malignant tumors and significantly contributes to their high mortality, particularly in hepatocellular carcinoma (HCC). Therefore, it is imperative to explore the mechanism of tumor metastasis. Recently, tumor-associated macrophages (TAMs) have been demonstrated to promote tumor progression, while TAM-derived molecules involved in HCC metastasis warrant further investigation. METHODS: THP-1 was treated with IL-4 (Interleukin-4) and IL-13 (Interleukin-13) for M2 polarized macrophages. Exosomes derived from M2 macrophages were characterized. Then, HCC cells or human umbilical vein endothelial cells (HUVECs) were co-cultured with M2 macrophages or treated with M2 macrophage-secreted exosomes. Next, Transwell®, Scratch assay, tube formation, and endothelial permeability assays were performed. Moreover, RT-PCR, western blotting, immunofluorescence, and ELISA were used to assess mRNA and protein expression levels. Finally, the miRNA expression profiles of exosomes derived from M2 and M0 macrophages were analyzed. RESULTS: M2 macrophage infiltration was correlated with metastasis and a poor prognosis in HCC patients. M2-derived exosomes were absorbed by HCC and HUVEC cells and promoted the epithelial-mesenchymal transition (EMT), vascular permeability, and angiogenesis. Notably, MiR-23a-3p levels were significantly higher in M2-derived exosomes and hnRNPA1 mediated miR-23a-3p packaging into exosomes. Phosphatase and tensin homolog (PTEN) and tight junction protein 1 (TJP1) were the targets of miR-23a-3p, as confirmed by luciferase reporter assays. Lastly, HCC cells co-cultured with M2-derived exosomes secreted more GM-CSF, VEGF, G-CSF, MCP-1, and IL-4, which in turn further recruited M2 macrophages. CONCLUSIONS: Our findings suggest that M2 macrophage-derived miR-23a-3p enhances HCC metastasis by promoting EMT and angiogenesis, as well as increasing vascular permeability. Video Abstract.

Apolipoprotein A-1 protected hepatic ischaemia-reperfusion injury through suppressing macrophage pyroptosis via TLR4-NF-κB pathway.

BACKGROUND AND AIMS: Apolipoprotein A-1 (ApoA-1), the major apolipoprotein of high-density lipoprotein, plays anti-atherogenic role in cardiovascular diseases and exerts anti-inflammation effect in various inflammatory and infectious diseases. However, the role and mechanism of ApoA-1 in hepatic ischaemia-reperfusion (I/R) injury is unknown. METHODS: In this study, we measured ApoA-1 expression in human liver grafts after transplantation. Mice partial hepatic I/R injury model was made in ApoA-1 knockout mice, ApoA-1 mimetic peptide D-4F treatment mice and corresponding control mice to examine the effect of ApoA-1 on liver damage, inflammation response and cell death. Primary hepatocytes and macrophages were isolated for in vitro study. RESULTS: The results showed that ApoA-1 expression was down-regulated in human liver grafts after transplantation and mice livers subjected to hepatic I/R injury. ApoA-1 deficiency aggravated liver damage and inflammation response induced by hepatic I/R injury. Interestingly, we found that ApoA-1 deficiency increased pyroptosis instead of apoptosis during acute phase of hepatic I/R injury, which mainly occurred in macrophages rather than hepatocytes. The inhibition of pyroptosis compensated for the adverse impact of ApoA-1 deficiency. Furthermore, the up-regulated pyroptosis process was testified to be mediated by ApoA-1 through TLR4-NF-κB pathway and TLR4 inhibition significantly improved hepatic I/R injury. In addition, we confirmed that D-4F ameliorated hepatic I/R injury. CONCLUSIONS: Our study has identified the protective role of ApoA-1 in hepatic I/R injury through inhibiting pyroptosis in macrophages via TLR4-NF-κB pathway. The effect of ApoA-1 may provide a novel therapeutic approach for hepatic I/R injury.

FSTL1 promotes liver fibrosis by reprogramming macrophage function through modulating the intracellular function of PKM2.

OBJECTIVE: Follistatin-like protein 1 (FSTL1) is widely recognised as a secreted glycoprotein, but its role in modulating macrophage-related inflammation during liver fibrosis has not been documented. Herein, we aimed to characterise the roles of macrophage FSTL1 in the development of liver fibrosis. DESIGN: Expression analysis was conducted with human liver samples obtained from 33 patients with liver fibrosis and 18 individuals without fibrosis serving as controls. Myeloid-specific FSTL1-knockout (FSTL1M-KO) mice were constructed to explore the function and mechanism of macrophage FSTL1 in 3 murine models of liver fibrosis induced by carbon tetrachloride injection, bile duct ligation or a methionine-deficient and choline-deficient diet. RESULTS: FSTL1 expression was significantly elevated in macrophages from fibrotic livers of both humans and mice. Myeloid-specific FSTL1 deficiency effectively attenuated the progression of liver fibrosis. In FSTL1M-KO mice, the microenvironment that developed during liver fibrosis showed relatively less inflammation, as demonstrated by attenuated infiltration of monocytes/macrophages and neutrophils and decreased expression of proinflammatory factors. FSTL1M-KO macrophages exhibited suppressed proinflammatory M1 polarisation and nuclear factor kappa B pathway activation in vivo and in vitro. Furthermore, this study showed that, through its FK domain, FSTL1 bound directly to the pyruvate kinase M2 (PKM2). Interestingly, FSTL1 promoted PKM2 phosphorylation and nuclear translocation, reduced PKM2 ubiquitination to enhance PKM2-dependent glycolysis and increased M1 polarisation. Pharmacological activation of PKM2 (DASA-58) partially countered FSTL1-mediated glycolysis and inflammation. CONCLUSION: Macrophage FSTL1 promotes the progression of liver fibrosis by inducing M1 polarisation and inflammation based on the intracellular PKM2 reprogramming function of macrophages.

Hypoxia-Inducible Exosomes Facilitate Liver-Tropic Premetastatic Niche in Colorectal Cancer.

BACKGROUND AND AIMS: Liver metastasis is a frequent occurrence in patients with colorectal cancer (CRC), with 15%-25% of CRC patients having liver metastases at the time of initial diagnosis. Specifically, some regional-stage patients with mild symptoms (stage 1 or 2) will also advance to liver metastases rapidly, even if the CRC lesion in situ is resected in time. Nevertheless, the precise mechanism of liver metastasis is still unclear. APPROACH AND RESULTS: Fresh tumor tissues from patients with CRC, adjacent noncancerous tissues, and colorectal adenoma tissues were subjected to microarray analysis to identify differentially expressed microRNA. Exosomes from human serum and cell culture medium were separated, quantitated, and verified by transmission electronic microscopy and Zetasizer Nano. Luciferase reporter assay, real-time quantitative PCR, western blot, immunoprecipitation, chromatin and re-chromatin immunoprecipitation, migration and invasion assay, PDX mouse model, flow cytometry, immunohistochemistry, and immunofluorescence staining were employed to explore the regulation among CRC liver metastases, immunosuppression, and cell adhesion. In this study, we demonstrated that the hypoxic microenvironment in primary CRC lesions boosted exosome release, selectively initiated favorable premetastatic niche formation in the liver but not in other organs. Mechanistically, Kupffer cells (KCs) can phagocytose exosomes containing highly expressed miR-135a-5p from the blood circulation into the liver. Exosomal miR-135a-5p initiated the large tumor suppressor kinase 2-yes-associated protein-matrix metalloproteinase 7 axis to promote the occurrence of CRC liver metastasis, and cluster of differentiation 30-TNF receptor-associated factor 2-p65-mediated immunosuppression signaling also contributed to this process. CONCLUSIONS: Hypoxia-induced exosomal miR-135a-5p correlates with the development, clinical severity, and prognosis of CRC liver metastases through the premetastatic niche; and our findings revealed that miR-135a-5p might be a promising target in halting CRC liver metastases.

T-Cell Immunoglobulin and Mucin Domain-Containing Protein-4 Is Critical for Kupffer Cell Homeostatic Function in the Activation and Resolution of Liver Ischemia Reperfusion Injury.

BACKGROUND AND AIMS: Liver ischemia reperfusion injury (IRI) remains an unresolved clinical problem. This study dissected roles of liver-resident macrophage Kupffer cells (KCs), with a functional focus on efferocytosis receptor T-cell immunoglobulin and mucin domain-containing protein-4 (TIM-4), in both the activation and resolution of IRI in a murine liver partial warm ischemia model. APPROACH AND RESULTS: Fluorescence-activated cell sorting results showed that TIM-4 was expressed exclusively by KCs, but not infiltrating macrophages (iMФs), in IR livers. Anti-TIM-4 antibody depleted TIM-4+ macrophages in vivo, resulting in either alleviation or deterioration of liver IRI, which was determined by the repopulation kinetics of the KC niche with CD11b+ macrophages. To determine the KC-specific function of TIM-4, we reconstituted clodronate-liposome-treated mice with exogenous wild-type or TIM-4-deficient KCs at either 0 hour or 24 hours postreperfusion. TIM-4 deficiency in KCs resulted in not only increases in the severity of liver IRI (at 6 hours postreperfusion), but also impairment of the inflammation resolution (at 7 days postreperfusion). In vitro analysis revealed that TIM-4 promoted KC efferocytosis to regulate their Toll-like receptor response by up-regulating IL-10 and down-regulating TNF-α productions. CONCLUSIONS: TIM-4 is critical for KC homeostatic function in both the activation and resolution of liver IRI by efferocytosis.

M2 Macrophage-Derived Exosomes Facilitate HCC Metastasis by Transferring αM ß2 Integrin to Tumor Cells.

BACKGROUND AND AIMS: The development and progression of hepatocellular carcinoma (HCC) is dependent on its local microenvironment. Tumor-associated macrophages (TAMs) are deemed a key factor for the tumor microenvironment and attribute to contribute to tumor aggressiveness. However, the detailed mechanism underlying the pro-metastatic effect of TAMs on HCC remains undefined. APPROACH AND RESULTS: The present study proved that TAMs were enriched in HCC. TAMs were characterized by an M2-polarized phenotype and accelerated the migratory potential of HCC cells in vitro and in vivo. Furthermore, we found that M2-derived exosomes induced TAM-mediated pro-migratory activity. With the use of mass spectrometry, we identified that integrin, αM ß2 (CD11b/CD18), was notably specific and efficient in M2 macrophage-derived exosomes (M2 exos). Blocking either CD11b and/or CD18 elicited a significant decrease in M2 exos-mediated HCC cell metastasis. Mechanistically, M2 exos mediated an intercellular transfer of the CD11b/CD18, activating the matrix metalloproteinase-9 signaling pathway in recipient HCC cells to support tumor migration. CONCLUSIONS: Collectively, the exosome-mediated transfer of functional CD11b/CD18 protein from TAMs to tumor cells may have the potency to boost the migratory potential of HCC cells, thus providing insights into the mechanism of tumor metastasis.

Development and validation of a gradient boosting machine to predict prognosis after liver resection for intrahepatic cholangiocarcinoma.

BACKGROUND: Accurate prognosis assessment is essential for surgically resected intrahepatic cholangiocarcinoma (ICC) while published prognostic tools are limited by modest performance. We therefore aimed to establish a novel model to predict survival in resected ICC based on readily-available clinical parameters using machine learning technique. METHODS: A gradient boosting machine (GBM) was trained and validated to predict the likelihood of cancer-specific survival (CSS) on data from a Chinese hospital-based database using nested cross-validation, and then tested on the Surveillance, Epidemiology, and End Results (SEER) database. The performance of GBM model was compared with that of proposed prognostic score and staging system. RESULTS: A total of 1050 ICC patients (401 from China and 649 from SEER) treated with resection were included. Seven covariates were identified and entered into the GBM model: age, tumor size, tumor number, vascular invasion, number of regional lymph node metastasis, histological grade, and type of surgery. The GBM model predicted CSS with C-Statistics ≥ 0.72 and outperformed proposed prognostic score or system across study cohorts, even in sub-cohort with missing data. Calibration plots of predicted probabilities against observed survival rates indicated excellent concordance. Decision curve analysis demonstrated that the model had high clinical utility. The GBM model was able to stratify 5-year CSS ranging from over 54% in low-risk subset to 0% in high-risk subset. CONCLUSIONS: We trained and validated a GBM model that allows a more accurate estimation of patient survival after resection compared with other prognostic indices. Such a model is readily integrated into a decision-support electronic health record system, and may improve therapeutic strategies for patients with resected ICC.

Isoform- and Cell Type-Specific Roles of Glycogen Synthase Kinase 3 N-Terminal Serine Phosphorylation in Liver Ischemia Reperfusion Injury.

Glycogen synthase kinase 3 (Gsk3) α and ß are both constitutively active and inhibited upon stimulation by N-terminal serine phosphorylation. Although roles of active Gsk3 in liver ischemia reperfusion injury (IRI) have been well appreciated, whether Gsk3 N-terminal serine phosphorylation has any functional significance in the disease process remains unclear. In a murine liver partial warm ischemia model, we studied Gsk3 N-terminal serine mutant knock-in (KI) mice and showed that liver IRI was decreased in Gsk3αS21A but increased in Gsk3ßS9A mutant KI mice. Bone marrow chimeric experiments revealed that the Gsk3α, but not ß, mutation in liver parenchyma protected from IRI, and both mutations in bone marrow-derived cells exacerbated liver injuries. Mechanistically, mutant Gsk3α protected hepatocytes from inflammatory (TNF-α) cell death by the activation of HIV-1 TAT-interactive protein 60 (TIP60)-mediated autophagy pathway. The pharmacological inhibition of TIP60 or autophagy diminished the protection of the Gsk3α mutant hepatocytes from inflammatory cell death in vitro and the Gsk3α mutant KI mice from liver IRI in vivo. Thus, Gsk3 N-terminal serine phosphorylation inhibits liver innate immune activation but suppresses hepatocyte autophagy in response to inflammation. Gsk3 αS21, but not ßS9, mutation is sufficient to sustain Gsk4 activities in hepatocytes and protect livers from IRI via TIP60 activation.

Mutational landscape of paired primary and synchronous metastatic lymph node in chemotherapy naive gallbladder cancer.

BACKGROUND: Comprehensive genomic analysis of paired primary tumors and their metastatic lesions may provide new insights into the biology of metastatic processes and therefore guide the development of novel strategies for intervention. To date, our knowledge of the genetic divergence and phylogenetic relationships in gallbladder cancer (GBC) is limited. METHODS: We performed whole exome sequencing for 5 patients with primary tumor, metastatic lymph node (LNM) and corresponding normal tissue. Mutations, mutation signatures and copy number variations were analyzed with state-of-art bioinformatics methods. Phylogenetic tree was also generated to infer metastatic pattern. RESULTS: Five driver mutations were detected in these patients. Among which, TP53 was the only shared mutation between primary tumor and LNM. Although tumor mutational burden was comparable between primary tumor and LNM, higher mutation burden was observed in LNM of one patient. Copy number variations (CNVs) burden was higher in LNM than their primary tumor. Phylogenetic analysis indicated both linear and parallel progression of metastasis exist in these patients. TP53 mutation and CNVs were homogenously between primary tumor and LNM. CONCLUSIONS: High consistence of genetic landscape were shown between primary tumor and LNM in GBC. However, heterogenicity still exist between primary tumor and LNM in particular patients in term of driver mutation, TMB and CNV burden. Phylogenetic analysis indicated both Linear and parallel progression of metastasis were exist among these patients.

Integrating Machine Learning and Tumor Immune Signature to Predict Oncologic Outcomes in Resected Biliary Tract Cancer.

BACKGROUND: Improved methods are needed to predict outcomes in biliary tract cancers (BTCs). We aimed to build an immune-related signature and establish holistic models using machine learning. METHODS: Samples were from 305 BTC patients treated with curative-intent resection, divided into derivation and validation cohorts in a two-to-one ratio. Spatial resolution of T cell infiltration and PD-1/PD-L1 expression was assessed by immunohistochemistry. An immune signature was constructed using classification and regression tree. Machine learning was applied to develop prediction models for disease-specific survival (DSS) and recurrence-free survival (RFS). RESULTS: The immune signature composed of CD3+, CD8+, and PD-1+ cell densities and PD-L1 expression within tumor epithelium significantly stratified patients into three clusters, with median DSS varying from 11.7 to 80.8 months and median RFS varying from 6.2 to 62.0 months. Gradient boosting machines (GBM) outperformed rival machine-learning algorithms and selected the same 11 covariates for DSS and RFS prediction: immune signature, tumor site, age, bilirubin, albumin, carcinoembryonic antigen, cancer antigen 19-9, tumor size, tumor differentiation, resection margin, and nodal metastasis. The clinical-immune GBM models accurately predicted DSS and RFS, with respective concordance index of 0.776-0.816 and 0.741-0.781. GBM models showed significantly improved performance compared with tumor-node-metastasis staging system. CONCLUSIONS: The immune signature promises to stratify prognosis and allocate treatment in resected BTC. The clinical-immune GBM models accurately predict recurrence and death from BTC following surgery.

Deficiency of TGR5 exacerbates immune-mediated cholestatic hepatic injury by stabilizing the ß-catenin destruction complex.

Intrahepatic cholestasis induced by drug toxicity may cause cholestatic hepatic injury (CHI) leading to liver fibrosis and cirrhosis. The G protein-coupled bile acid receptor 1 (TGR5) is a membrane receptor with well-known roles in the regulation of glucose metabolism and energy homeostasis. However, the role and mechanism of TGR5 in the context of inflammation during CHI remains unclear. Wild-type (WT) and TGR5 knockout (TGR5-/-) mice with CHI induced by bile duct ligation (BDL) were involved in vivo, and WT and TGR5-/- bone marrow-derived macrophages (BMDMs) were used in vitro. TGR5 deficiency significantly exacerbated BDL-induced liver injury, inflammatory responses and hepatic fibrosis compared with WT mice in vivo. TGR5-/- macrophages were more susceptible to lipopolysaccharide (LPS) stimulation than WT macrophages. TGR5 activation by its ligand suppressed LPS-induced pro-inflammatory responses in WT but not TGR5-/- BMDMs. Notably, expression of ß-catenin was effectively inhibited by TGR5 deficiency. Furthermore, TGR5 directly interacted with Gsk3ß to repress the interaction between Gsk3ß and ß-catenin, thus disrupting the ß-catenin destruction complex. The pro-inflammatory nature of TGR5-knockout was almost abolished by lentivirus-mediated ß-catenin overexpression in BMDMs. BMDM migration in vitro was accelerated under TGR5-deficient conditions or supernatant from LPS-stimulated TGR5-/- BMDMs. From a therapeutic perspective, TGR5-/- BMDM administration aggravated BDL-induced CHI, which was effectively rescued by ß-catenin overexpression. Our findings reveal that TGR5 plays a crucial role as a novel regulator of immune-mediated CHI by destabilizing the ß-catenin destruction complex, with therapeutic implications for the management of human CHI.

Radiomic Features at Contrast-enhanced CT Predict Recurrence in Early Stage Hepatocellular Carcinoma: A Multi-Institutional Study.

Background Early stage hepatocellular carcinoma (HCC) is the ideal candidate for resection in patients with preserved liver function; however, cancer will recur in half of these patients and no reliable prognostic tool has been established. Purpose To investigate the effectiveness of radiomic features in predicting tumor recurrence after resection of early stage HCC. Materials and Methods In total, 295 patients (median age, 58 years; interquartile range, 50-65 years; 221 men) who underwent contrast material-enhanced CT and curative resection for early stage HCC that met the Milan criteria between February 2009 and December 2016 were retrospectively recruited from three independent institutions. Follow-up consisted of serum α-fetoprotein level, liver function tests, and dynamic imaging examinations every 3 months during the first 2 years and then every 6 months thereafter. In the development cohort of 177 patients from institution 1, recurrence-related radiomic features were computationally extracted from the tumor and its periphery and a radiomics signature was built with least absolute shrinkage and selection operator regression. Two models, one integrating preoperative and one integrating pre- and postoperative variables, were created by using multivariable Cox regression analysis. An independent external cohort of 118 patients from institutions 2 and 3 was used to validate the proposed models. Results The preoperative model integrated radiomics signature with serum α-fetoprotein level and tumor number; the postoperative model incorporated microvascular invasion and satellite nodules into the above-mentioned predictors. In both study cohorts, two radiomics-based models provided better predictive performance (concordance index ≥0.77, P < .05 for all), lower prediction error (integrated Brier score ≤0.14), and larger net benefits, as determined by means of decision curve analysis, than rival models without radiomics and widely adopted staging systems. The radiomics-based models gave three risk strata with high, intermediate, or low risk of recurrence and distinct profiles of recurrent tumor number. Conclusion The proposed radiomics models with pre- and postresection features helped predict tumor recurrence for early stage hepatocellular carcinoma. © RSNA, 2020 Online supplemental material is available for this article.

Circular RNA MAT2B Promotes Glycolysis and Malignancy of Hepatocellular Carcinoma Through the miR-338-3p/PKM2 Axis Under Hypoxic Stress.

Glucose metabolism reprogramming, which is a well-established characteristic of multiple cancers, demands a higher rate of glycolysis to meet the increasing demands for macromolecular synthesis and to maintain rapid proliferation in a hypoxic environment. However, the mechanism underlying this switch remains to be elucidated. In this study, we investigated the function of circular RNA MAT2B (circMAT2B) in hepatocellular carcinoma (HCC) glucose metabolism reprogramming and malignancy. CircMAT2B was identified by bioinformatics analysis of Gene Expression Omnibus data sets. CircMAT2B expression was up-regulated in HCC tissues and cell lines. HCC patients with high circMAT2B expression had shortened overall survival. We analyzed the positive correlation between glycolysis and circMAT2B expression in HCC using a maximum standardized uptake value determined by preoperative positron emission tomography/computed tomography scanning combined with high-performance liquid chromatography assessment of the metabolites of glycolysis and the citric acid cycle. The effect of circMAT2B on glycolysis was validated in vitro and in vivo under hypoxic (1% O2 ) conditions. Functional assays were performed in HCC cells, HCC organoids, and nude mice to explore the tumor-promoting roles of circMAT2B in HCC. Biotin-coupled probe pull-down assays, biotin-coupled microRNA capture, luciferase reporter assays, fluorescence in situ hybridization, and RNA immunoprecipitation assays were performed to confirm the interaction among different RNAs. Mechanistically, we demonstrated that circMAT2B up-regulated expression levels of the microRNA (miR)-338-3p target gene PKM2, which encodes a key enzyme in the process of glycolysis, through "sponging" miR-338-3p; thus, glycolysis and HCC progression are promoted through this mechanism. Conclusion: CircMAT2B promoted HCC progression by enhanced glycolysis by activating the circMAT2B/miR-338-3p/PKM2 axis under hypoxia, which may provide a therapeutic target for HCC.

5-Hydroxytryptamine Receptor 1D Aggravates Hepatocellular Carcinoma Progression Through FoxO6 in AKT-Dependent and Independent Manners.

Serotonin and its receptors have been shown to play critical regulatory roles in cancer biology. Nevertheless, the contributions of 5-hydroxytryptamine 1D (5-HT1D), an indispensable member of the serotonergic system, to hepatocellular carcinoma (HCC) remain unknown. The present study demonstrated that the 5-HT1D expression level was significantly up-regulated in HCC tissues and cell lines. The 5-HT1D expression level was closely correlated with unfavorable clinicopathological characteristics. Survival analyses show that elevated 5-HT1D expression level predicts poor overall survival and high recurrence probability in HCC patients. Functional studies revealed that 5-HT1D significantly promoted HCC proliferation, epithelial-mesenchymal transition, and metastasis in vitro and in vivo. Mechanistically, 5-HT1D could stabilize PIK3R1 by inhibiting its ubiquitin-mediated degradation. The interaction between 5-HT1D and phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) enhanced the expression of FoxO6 through the PI3K/Akt signaling pathway; FoxO6 could also be directly transcriptionally activated by 5-HT1D in an Akt-independent manner. MicroRNA-599 was found to be an upstream suppressive modulator of 5-HT1D. Additionally, 5-HT1D could attenuate tryptophan hydroxylase 1 expression through the PI3K/Akt/cut-like homeobox 1 axis in HCC. Conclusion: Herein, we uncovered the potent oncogenic effect of 5-HT1D on HCC by interacting with PIK3R1 to activate the PI3K/Akt/FoxO6 pathway, and provided a potential therapeutic target for HCC.

Hypoxia-induced hsa_circ_0000826 is linked to liver metastasis of colorectal cancer.

BACKGROUND: hsa_circ_0000826 has been previously linked to CRC through the competing endogenous RNA network; however, the upstream driver of hsa_circ_0000826 elevation remains unknown. In this study, we aim to elucidate the effect of hypoxia-induced hsa_circ_0000826 on CRC tumorigenesis and metastasis. METHODS: RNA scope assay was used to evaluate the expression of hsa_circ_0000826 in CRC cells under hypoxia condition. The effects of hsa_circ_0000826 on phenotypes of CRC cells were evaluated through cell migration and invasion assay. The nude, AOM-DSS model mice and APCMin /+ mice were used to investigate the relationship between circ_0000826, hypoxia, and CRC in mice. A total of 100 CRC tissue samples, as well as the paired adjacent tissues, were collected, and qRT-PCR assay was used to detect the expression of hsa_circ_0000826 in these samples. RESULTS: Hypoxia-induced hsa_circ_0000826 overexpression can increase the malignant phenotypes, tumor formation, and metastasis capability of CRC cells in vitro. mmu_circ_0000826 levels were significantly increased in the CRC tissues from AOM-DSS and APC mice model under hypoxia conditions. Further, the hypoxia-induced upregulation of mmu_circ_0000826 can also promote CRC tumorigenesis and liver metastasis in vivo. The expression of hsa_circ_0000826 in serum was significantly increased in CRC tissues in 100-pair of CRC and according to the adjacent normal tissues by qRT-PCR assays. Moreover, the expression levels of hsa_circ_0000826 in serum of patient with liver metastasis were significantly increased than those without metastasis. CONCLUSION: Our results suggested that hsa_circ_0000826 was induced by the hypoxia in CRC, which can be a potential biomarker of CRC liver metastasis.

Current perspectives on the immunosuppressive tumor microenvironment in hepatocellular carcinoma: challenges and opportunities.

Incidence of hepatocellular carcinoma (HCC) is on the rise due to the prevalence of chronic hepatitis and cirrhosis. Although there are surgical and chemotherapy treatment avenues the mortality rate of HCC remains high. Immunotherapy is currently the new frontier of cancer treatment and the immunobiology of HCC is emerging as an area for further exploration. The tumor microenvironment coexists and interacts with various immune cells to sustain the growth of HCC. Thus, immunosuppressive cells play an important role in the anti-tumor immune response. This review will discuss the current concepts of immunosuppressive cells, including tumor-associated macrophages, marrow-derived suppressor cells, tumor-associated neutrophils, cancer-associated fibroblasts, and regulatory T cell interactions to actively promote tumorigenesis. It further elaborates on current treatment modalities and future areas of exploration.

circLARP4 induces cellular senescence through regulating miR-761/RUNX3/p53/p21 signaling in hepatocellular carcinoma.

Circular RNAs (circRNAs), a novel class of non-coding RNAs, have emerged as indispensable modulators in human malignancies. Aberrant cellular senescence is a phenotype observed in various cancers. The association of circRNAs with cellular senescence in tumors is yet to determined. Here, we investigated the role of circLARP4 in cellular senescence and cell proliferation in hepatocellular carcinoma (HCC). Downregulated circLARP4 level was observed in HCC tissues and cell lines. Low expression level of circLARP4 independently predicted poor survival outcome. Gain-of-function and loss-of-function assays demonstrated that circLARP4 suppressed HCC cell proliferation, mediated cell cycle arrest and induced senescence in vitro. Levels of p53 and p21, 2 key regulatory molecules in cellular senescence, were increased in circLARP4-overexpressed HCC cells and decreased in circLARP4-silenced HCC cells. In vivo experiments further confirmed the tumor-suppressing activity of circLARP4. Further mechanistic studies showed that circLARP4 dampened HCC progression by sponging miR-761, thereby promoting the expression level of RUNX3 and activating the downstream p53/p21 signaling. Our study revealed the role of circLARP4/miR-761/RUNX3/p53/p21 signaling in HCC progression, providing a potential survival predictor and therapeutic candidate for HCC.