Circ_0098823 binding with IGF2BP3 regulates DNM1L stability to promote metastasis of hepatocellular carcinoma via mitochondrial fission.

Hepatocellular carcinoma (HCC) is highly metastatic and invasive. CircRNA participates in gene regulation of multiple tumor metastases, but little is known whether it is a bystander or an actual player in HCC metastasis. We aim to explore the molecular mechanisms of novel circRNAs in HCC metastasis. RT-qPCR was used to detect the expression of 13 circRNAs derived by the ERBB3 gene. The function of circ_0098823 and DNM1L in HCC cells were estimated by CCK-8, transwell assays, flow cytometry, electron microscope, and in vivo experiments. RNA binding protein of circ_0098823 was confirmed by RNA pull-down, mass spectrometry, and RNA immunoprecipitation. The expression of DNM1L after IGF2BP3 knockdown was detected by RT-qPCR and western blot. Circ_0098823 was significantly up-regulated both in HCC tissues and HGF induced cell lines. Circ_0098823 overexpression significantly enhanced proliferation, migration, and invasion but decreased apoptosis of HCC cells, particularly promoted mitochondrial fission. Compared with the control group, the tumors in the circ_0098823 knockdown mice were significantly smaller and lighter. Circ_0098823 silencing suppressed DNM1L expression, a key molecule for fission, which enhanced proliferation, migration and invasion, and inhibited apoptosis of HCC cell. IGF2BP3 was a binding protein of circ_0098823. The expression and mRNA stability of DNM1L were down-regulated by IGF2BP3 knockdown. IGF2BP3 knockdown significantly alleviated the excessive migration, invasion and apoptosis of HCC cells caused by circ_0098823 overexpression. This study uncovered a novel circ_0098823 with tumor-promoting effect, and the mechanism by which circ_0098823 participates in HCC progression through IGF2BP3-guided DNM1L. Our study broadens molecular understanding of HCC progression.

CRKL dictates anti-PD-1 resistance by mediating tumor-associated neutrophil infiltration in hepatocellular carcinoma.

BACKGROUND & AIMS: Immune checkpoint inhibitors (ICIs) resistance limits immunotherapy success in hepatocellular carcinoma (HCC). However, the mechanisms underlying immunotherapy resistance remain elusive. We aimed to identify the role of CT10 regulator of kinase-like (CRKL) in HCC against anti-PD-1 therapy. METHODS: Gene expression in HCC specimens from 10 patients accepted anti-PD-1 therapy was identified by RNA-sequencing. A total of 342 and 62 HCC samples from TMA1 and TMA2, respectively, were analyzed by immunohistochemistry. Transgenic mice (Alb-Cre/Trp53fl/fl) were given hydrodynamic tail vein injections (HDTVi) of adeno-associated virus serotype 8 (AAV8) vector to overexpress CRKL. Mass cytometry by time of flight (CyTOF) was used to profile the proportion and status of different immune cell lineages in the mouse tumor tissues. RESULTS: CRKL was identified as a candidate anti-PD-1 resistant gene using a pooled genetic screen. CRKL overexpression nullifies anti-PD-1 therapy efficacy by mobilizing tumor-associated neutrophils (TANs) to block infiltration and function of CD8+ T cells. PD-L1+ TANs were found to be an essential subset of TANs that were regulated by CRKL expression and display an immunosuppressive phenotype. Mechanistically, CRKL inhibits adenomatous polyposis coli (APC)-mediated proteasomal degradation of ß-catenin by competitively decreasing Axin1 binding, and thus fosters VEGFα and CXCL1 expression. Using human HCC samples, we verified the positive correlations of CRKL/ß-catenin/VEGFα and CXCL1. Targeting CRKL using CRISPR-Cas9 gene editing (CRKL knockout) or its downstream regulators effectively restored the efficacy of anti-PD-1 therapy in an orthotopic mouse model and a patient-derived organotypic tumor spheroid (PDOTS) model. CONCLUSIONS: Activation of the CRKL/ß-catenin/VEGFα and CXCL1 axis is a critical obstacle to successful anti-PD-1 therapy. Therefore, CRKL inhibitors combined with anti-PD-1 may be developed for the treatment of HCC. IMPACT AND IMPLICATIONS: Here, we found CRKL was overexpressed in anti-PD-1 resistant HCC and CRKL upregulation promotes anti-PD-1 resistance in HCC. We identified that CRKL/ß-catenin/VEGFα and CXCL1 axis upregulation contributes to anti-PD-1 tolerance through promoting tumor-associated neutrophils (TANs) infiltration. These findings support the strategy of bevacizumab-based ICIs combination therapy, and CRKL inhibitors combined with anti-PD-1 therapy may be developed for the treatment of HCC.

Assessment of stromal SCD-induced drug resistance of PDAC using 3D-printed zPDX model chips.

Lipid metabolism is extensively reprogrammed in pancreatic ductal adenocarcinoma (PDAC). Stearoyl-coenzyme A desaturase (SCD) is a critical lipid regulator that was unexplored in PDAC. Here, we characterized the existence of cancer-associated fibroblasts (CAFs) with high SCD expression, and revealed them as an unfavorable prognostic factor. Therefore, primary CAFs and pancreatic cancer cells were harvested and genetically labeled. The mixture of CAFs and cancer cells were co-injected into scd-/-; prkdc-/-, or hIGF1/INS-expressing zebrafish to generate patient-derived xenograft models (zPDX). The models were aligned in 3D-printed chips for semi-automatic drug administration and high-throughput scanning. The results showed that chaperoning of the SCD-high CAFs significantly improved the drug resistance of pancreatic cancer cells against gemcitabine and cisplatin, while the administration of SCD inhibitors neutralized the protective effect. Our studies revealed the prognostic and therapeutic value of stromal SCD in PDAC, and proposed the application of zPDX model chips for drug testing.

Complement C5 is a novel biomarker for liver metastasis of colorectal cancer.

Background: Colorectal cancer (CRC) is one of the most prominent malignant diseases, with a high incidence and a dismal prognosis. Metastasis to the liver is the leading cause of death in CRC patients. This study aimed to identify accurate metastatic biomarkers of CRC and investigate the potential molecular mechanisms of liver metastasis of colorectal cancer (LMCRC). Methods: Three independent datasets were screened and downloaded from the Gene Expression Omnibus (GEO) database. The GEO2R tool was used to identify differentially expressed genes (DEGs) in CRC tissues and liver metastases. Next, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted using the Database for Annotation, Visualization, and Integrated Discovery (DAVID). Furthermore, the protein-protein interactions (PPIs) of the DEGs were analyzed using the Search Tool for the Retrieval of Interacting Genes (STRING) database, Cytoscape, and Molecular Complex Detection (MCODE). Next, the expression levels and Kaplan-Meier survival analysis of the target gene between normal colon and CRC tissues were performed by UALCAN. The expression of the target gene in tissues and cell lines was verified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blot, and immunohistochemistry (IHC) assay. The impact of the target gene on the proliferation, invasion, and migration ability of COAD cells was explored in vitro. Results: A total of 92 common DEGs were found in the three independent datasets. GO/KEGG enrichment analysis showed that the DEGs were mainly involved in 14 different pathways. The protein-protein interaction (PPI) network revealed that complement 5 (C5), the upstream gene of C8A in the complement system, was associated with C8 and other key hub genes. Meanwhile, the online UALCAN resource showed that C5 was up-regulated and facilitated malignant progression in COAD samples. Next, we confirmed that C5 remarkably increased and promoted cell proliferation, migration, and invasion in CRC cell lines, SW620 and SW480. The IHC assay showed C5 was also highly expressed in a majority of LMCRC tissues compared with paired CRC tissues. Conclusions: The findings of our integrated bioinformatics study suggest that complement C5 might serve as a potential therapeutic target in patients with CRC.

Evolutionary Trajectories of Primary and Metastatic Pancreatic Neuroendocrine Tumors Based on Genomic Variations.

Liver metastases are common in pancreatic neuroendocrine tumors (PanNETs) patients and they are considered a poor prognostic marker. This study aims to analyze the spatiotemporal patterns of genomic variations between primary and metastatic tumors, and to identify the key related biomolecular pathways. We performed next-generation sequencing on paired tissue specimens of primary PanNETs (n = 11) and liver metastases (n = 12). Low genomic heterogeneity between primary PanNETs and liver metastases was observed. Genomic analysis provided evidence that polyclonal seeding is a prevalent event during metastatic progression, and may be associated with the progression-free survival. Besides this, copy number variations of BRCA1/BRCA2 seem to be associated with better prognosis. Pathways analysis showed that pathways in cancer, DNA repair, and cell cycle regulation-related pathways were significantly enriched in primary PanNETs and liver metastases. The study has shown a high concordance of gene mutations between the primary tumor and its metastases and the shared gene mutations may occur during oncogenesis and predates liver metastasis, suggesting an earlier onset of metastasis in patients with PanNETs, providing novel insight into genetic changes in metastatic tumors of PanNETs.

CD44 potentiates hepatocellular carcinoma migration and extrahepatic metastases via the AKT/ERK signaling CXCR4 axis.

Background: Cell adhesion molecule cluster of differentiation 44 (CD44) plays a significant role in cancer cell local invasion, intravasation, migration, and the establishment of metastatic lesions. However, little is known about the underlying mechanism of how CD44 regulates hepatocellular carcinoma (HCC) extrahepatic metastasis (EHM). Methods: The expression of CD44 in HCC tissues and cell lines was detected through western blot and immunohistochemistry (IHC). Through gain- and loss-of-function assays, we examined the oncogenic roles of CD44 in regulating HCC cell growth and metastasis in vitro and in vivo. To identify the potential mechanism, we employed quantitative real-time polymerase chain reaction, and western blot. Results: In this study, CD44 was highly expressed in HCC cells and HCC-patient specimens that exhibited high malignancy potential. The overall survival (OS) was worse and the cumulative recurrence rate was higher in HCC patients with CD44 overexpression than those with low levels of CD44 expression. Our in-vitro and in-vivo experiments showed that CD44 downregulation reduced HCC cell colony formation, migration, and invasion, and HCC tumor growth and metastasis, and that the pro-metastasis effect of CD44 was mediated by the protein kinase B (AKT)/extracellular signal-regulated kinase (ERK) signaling-chemokine receptor C-X-C chemokine receptor type 4 (CXCR4) axis. The reported capacity of CD44 to induce CXCR4 expression and increase the propensity of tumors to invade and metastasize to distant organs is consistent with the aggressive clinical characteristics of HCCs. Conclusions: CD44 could represent a future therapeutic target for EHM.

Subpopulations of cancer-associated fibroblasts link the prognosis and metabolic features of pancreatic ductal adenocarcinoma.

Background: Cancer-associated fibroblasts (CAFs) are a vital constituent of the tumor microenvironment (TME) and have several activities, but the effect of CAF heterogeneity on the molecular features and clinical outcomes of pancreatic ductal adenocarcinoma (PDAC) remains unknown. Methods: An algorithm "scFrac" based on single-cell sequencing data from the Gene Expression Omnibus was introduced to emulate the enrichment of CAF subtypes in a TCGA-PDAC cohort and their prognostic influence, and confirmed by an external validation group (66 patients with PDAC) with multiplex immunohistochemistry staining. A comprehensive analysis including metabolic profile and transcription factor regulon activity was carried out among CAF subtypes. Results: Three distinct CAF populations were confirmed: myofibroblast (myCAF), inflammatory CAF (iCAF), and antigen-presenting CAF (apCAF). These subtypes expressed distinct metabolic profiles and transcriptional regulon activity. KEGG pathway annotation demonstrated that complement and coagulation cascades, as well as cytokine-cytokine receptor interaction were dominant in iCAFs, and pathways related to focal adhesion, and ECM-receptor interaction showed dominance in myCAFs, while antigen processing and presentation were the top enriched pathways in apCAFs. iCAFs trended to glycolysis with CREB3L1, EGR2 and SOX4 activation, whereas myCAFs depend on the tricarboxylic acid cycle and its derivatives with NRF2, CEBPD and YBX1 activation. iCAF is a protective factor associated with an inflammatory phenotype, but myCAF is an important factor in the poor prognosis of PDAC. Conclusions: We identified distinct molecular characteristics of 3 CAF subtypes in PDAC and plotted their metabolism profile. We introduced a novel algorism, scFrac, for exploring how CAF subgroups dysregulate cancer biology, and also shed a new therapeutic light on targeting the CAF subtype in TME.

Tumor-derived PD1 and PD-L1 could promote hepatocellular carcinoma growth through autophagy induction in vitro.

BACKGROUNDS: Autophagy in tumor was also found to influence immune microenvironment. The relation between autophagy and cancer intrinsic PD1 and PD-L1 expression was not clear. METHODS: With data from TCGA and GTEx databases, mRNA expression levels of autophagy-related genes were compared between tumor samples and normal tissues, which were also correlated with survival status. Expression of autophagy-related genes were also associated with clinical traits in datasets of GSE14520 and ICGC LIRI. Single sample gene set enrichment analysis (ssGSEA) was used to calculate autophagy scores in tumor samples, using signatures from MSigDB database. Lentivirus (PD1 and PD-L1), siRNA (ATG13) and plasmids (LC3A/B) were used to target specific genes in tumor cells; Western blot was used to examine protein expression accordingly. Co-immunoprecipitation was performed to find PD1 or PD-L1 interacting proteins; colony formation and EdU analysis were used to evaluate tumor cell growth abilities. RESULTS: mRNA levels of autophagy markers were increased in tumor and correlated with worse survival of cancer patients. In hepatocellular carcinoma (HCC), high mRNA expression of autophagy markers was related to poor clinical status; increasing LC3 expression in HCC cell lines could promote tumor growth. Tumor intrinsic PD1 or PD-L1 were related to higher autophagy levels in specific tumor types; over-expression of PD1 or PD-L1 could increase autophagy in tumor cells through ATG13 interaction. CONCLUSION: Autophagy could promote tumor growth in specific cancer types. Tumor intrinsic PD1 or PD-L1 could both increase autophagy through ATG13 interaction.

PARG inhibition limits HCC progression and potentiates the efficacy of immune checkpoint therapy.

BACKGROUND & AIMS: Although the treatment of hepatocellular carcinoma (HCC) has been revolutionized by the advent of effective systemic therapies, the prognosis of patients with HCC remains dismal. Herein, we examined the pathophysiological role of PARG and assessed the utility of targeting dePARylation for HCC therapy. METHODS: The oncogenic function of PARG was evaluated in 2 orthotopic xenograft models and a Pargflox/flox mice model. The therapeutic efficacy of PARG inhibitors in combination with an anti-PD-1 antibody were assessed in murine orthotopic models. Microarray analysis was used to evaluate the pathological relevance of the PARG/DDB1/c-Myc/MMR axis. RESULTS: High PARG expression was strongly associated with poor HCC prognosis. Hepatocyte-specific PARG deletion significantly impaired liver tumorigenesis. PARG promoted HCC growth and metastasis through DDB1-dependent modulation of c-Myc. Specifically, PARG dePARylated DDB1 and consequently promoted DDB1 autoubiquitination, thus stabilizing the c-Myc protein in HCC cells. PARG downregulation attenuated c-Myc-induced MMR expression and PARG deficiency was correlated with a favorable prognosis in patients with HCC treated with anti-PD-1-based immunotherapy. In addition, PARG inhibitors could act in synergy with anti-PD-1 antibodies in orthotopic mouse models. CONCLUSIONS: PARG can act as an oncogene in HCC by modulating PARG/DDB1/c-Myc signaling and could be used as a biomarker to identify patients with HCC who may benefit from anti-PD-1 treatment. Our findings suggest that co-inhibition of PARG and PD-1 is an effective novel combination strategy for patients with HCC. LAY SUMMARY: The increase in deaths due to hepatocellular carcinoma (HCC) is a growing concern, with the mechanisms responsible for HCC development still incompletely understood. Herein, we identify a novel mechanism by which the protein PARG contributes to HCC development. Inhibition of PARG increased the efficacy of anti-PD-1 therapy (a type of immunotherapy) in HCC. These findings support the future clinical development of PARG inhibitors, potentially in combination with anti-PD-1 inhibitors.

PNOC Expressed by B Cells in Cholangiocarcinoma Was Survival Related and LAIR2 Could Be a T Cell Exhaustion Biomarker in Tumor Microenvironment: Characterization of Immune Microenvironment Combining Single-Cell and Bulk Sequencing Technology.

Background: Cholangiocarcinoma was a highly malignant liver cancer with poor prognosis, and immune infiltration status was considered an important factor in response to immunotherapy. In this investigation, we tried to locate immune infiltration related genes of cholangiocarcinoma through combination of bulk-sequencing and single-cell sequencing technology. Methods: Single sample gene set enrichment analysis was used to annotate immune infiltration status in datasets of TCGA CHOL, GSE32225, and GSE26566. Differentially expressed genes between high- and low-infiltrated groups in TCGA dataset were yielded and further compressed in other two datasets through backward stepwise regression in R environment. Single-cell sequencing data of GSE138709 was loaded by Seurat software and was used to examined the expression of infiltration-related gene set. Pathway changes in malignant cell populations were analyzed through scTPA web tool. Results: There were 43 genes differentially expressed between high- and low-immune infiltrated patients, and after further compression, PNOC and LAIR2 were significantly correlated with high immune infiltration status in cholangiocarcinoma. Through analysis of single-cell sequencing data, PNOC was mainly expressed by infiltrated B cells in tumor microenvironment, while LAIR2 was expressed by Treg cells and partial GZMB+ CD8 T cells, which were survival related and increased in tumor tissues. High B cell infiltration levels were related to better overall survival. Also, malignant cell populations demonstrated functionally different roles in tumor progression. Conclusion: PNOC and LAIR2 were biomarkers for immune infiltration evaluation in cholangiocarcinoma. PNOC, expressed by B cells, could predict better survival of patients, while LAIR2 was a potential marker for exhaustive T cell populations, correlating with worse survival of patients.

High serum gamma-glutamyl transpeptidase concentration associates with poor postoperative prognosis of patients with hepatitis B virus-associated intrahepatic cholangiocarcinoma.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) caused by chronic hepatitis B virus (HBV) infection has become prominent. Prospectively stratifying postoperative risk factors is a challenging task. METHODS: We retrospectively assessed the relationship between serum gamma-glutamyl transpeptidase (GGT) concentration and postoperative outcomes in 107 subjects with HBV-associated ICC. Cox proportionate hazard models and subgroup analyses were used to test the hypothesis with adjustment for potential confounders. RESULTS: Serum GGT concentration was negatively correlated with postoperative outcomes. For a 1-standard deviation (per-SD) (117 µ/L) increase of serum GGT concentration, the relative risk (RR) for overall survival (OS) and time to recurrence (TTR) were 1.72 [95% confidence interval (CI), 1.37 to 2.16] and 1.53 (95% CI, 1.22 to 1.91), respectively. In addition, the RRs of middle and top tertiles of GGT for death were 1.81 (95% CI, 0.98 to 3.32) and 3.56 (95% CI, 1.97 to 6.42), respectively (P for trend <0.001). Similarly, the RRs for recurrence of the corresponding tertiles were 1.70 (95% CI, 0.93 to 3.10) and 3.27 (95% CI, 1.77 to 6.06), respectively (P for trend =0.002). In our study, the negative correlation between serum GGT levels and OS did not differ significantly between groups stratified by age, sex, HBV DNA level, carbohydrate antigen 19-9 (CA19-9) level and liver resection type (all P for interaction >0.05); however, there was a significant interactive effect of serum GGT and adjuvant chemotherapy on OS (RR =0.64 vs. 1.77, P for interaction =0.04). CONCLUSIONS: High serum GGT concentration is associated with an increased risk of postoperative death and tumor recurrence in patients with HBV-associated ICC. However, this relationship became less significant with the implementation of adjuvant chemotherapy.

GOLM1 upregulates expression of PD-L1 through EGFR/STAT3 pathway in hepatocellular carcinoma.

GOLM1, a type II transmembrane protein, is associated with tumor progression, metastasis and immunosuppression. However, the relationship between GOLM1 and the immunosuppressive molecule PD-L1 in HCC remains largely unclear. Here, we revealed that GOLM1 acts as a novel positive regulator of PD-L1, whose abnormal expression plays a crucial role in cancer immune evasion and progression. We found that GOLM1 is overexpressed and positively correlated with PD-L1 expression in HCC. Mechanistically, we found that GOLM1 promotes the phosphorylation of STAT3 by enhancing the level of EGFR, which in turn upregulates the transcriptional expression of PD-L1. Taken together, we demonstrated that GOLM1 acts as a positive regulator of PD-L1 expression via the EGFR/STAT3 signaling pathway in human HCC cells. This study provides a new insight into the regulatory mechanism of PD-L1 expression in HCC, which may provide a novel therapeutic target for HCC immunotherapy.

Tumor Derived SIGLEC Family Genes May Play Roles in Tumor Genesis, Progression, and Immune Microenvironment Regulation.

BACKGROUND: SIGLEC family genes can also be expressed on tumor cells in different cancer types, and though it has been found that SIGLEC genes expressed by immune cells can be exploited by tumors to escape immune surveillance, functions of tumor derived SIGLEC expression in tumor microenvironment (TME) were barely investigated, which could play roles in cancer patients' survival. METHODS: Using bioinformatic analysis, mutation status of SIGLEC family genes was explored through the cBioPortal database, and expression of them in different tumors was explored through the UALCAN database. The GEPIA database was used to compare SIGLEC family genes' mRNA between cancers and to generate a highly correlated gene list in tumors. A KM-plotter database was used to find the association between SIGLEC genes and survival of patients. The associations between SIGLEC family genes' expression, immune infiltration, and immune regulators' expression in TME were generated and examined by the TIMER 2.0 database; the differential fold changes of SIGLEC family genes in specific oncogenic mutation groups of different cancer types were also yielded by TIMER 2.0. The networks of SIGLEC family genes and highly correlated genes were constructed by the STRING database, and gene ontology and pathway annotation of SIGLEC family highly correlated genes were performed through the DAVID database. RESULTS: SIGLEC family genes were highly mutated and amplified in melanoma, endometrial carcinoma, non-small cell lung cancer, bladder urothelial carcinoma, and esophagogastric adenocarcinoma, while deep deletion of SIGLEC family genes was common in diffuse glioma. Alteration of SIGLEC family genes demonstrated different levels in specific tumors, and oncogenic mutation in different cancer types could influence SIGLEC family genes' expression. Most SIGLEC family genes were related to patients' overall survival and progression free survival. Also, tumor derived SIGLEC family genes were related to tumor immune cell infiltration and may regulate TME by influencing chemokine axis. CONCLUSION: Our computational analysis showed SIGLEC family genes expressed by tumor cells were associated with tumor behaviors, and they may also influence TME through chemokine axis, playing vital roles in patients' survival. Further experiments targeting tumor derived SIGLEC family genes are needed to confirm their influences on tumor growth, metastasis, and immune environment regulation.

Gamma-Glutamyltransferase Elevation Is Frequent in Patients With COVID-19: A Clinical Epidemiologic Study.

A newly identified coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes the infectious coronavirus disease 2019 (COVID-19), emerged in December 2019 in Wuhan, Hubei Province, China, and now poses a major threat to global public health. Previous studies have observed highly variable alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in patients with COVID-19. However, circulating levels of the cholangiocyte injury biomarker gamma-glutamyltransferase (GGT) have yet to be reported in the existing COVID-19 case studies. Herein, we describe the relationship between GGT levels and clinical and biochemical characteristics of patients with COVID-19. Our study is a retrospective case series of 98 consecutive hospitalized patients with confirmed COVID-19 at Wenzhou Central Hospital in Wenzhou, China, from January 17 to February 5, 2020. Clinical data were collected using a standardized case report form. Diagnosis of COVID-19 was assessed by symptomatology, reverse-transcription polymerase chain reaction (RT-PCR), and computed tomography scan. The medical records of patients were analyzed by the research team. Of the 98 patients evaluated, elevated GGT levels were observed in 32.7%; increased C-reactive protein (CRP) and elevated ALT and AST levels were observed in 22.5%, 13.3%, and 20.4%, respectively; and elevated alkaline phosphatase (ALP) and triglycerides (TGs) were found in 2% and 21.4%, respectively. Initially, in the 82 patients without chronic liver disease and alcohol history, age older than 40 years (P = 0.027); male sex (P = 0.0145); elevated CRP (P = 0.0366), ALT (P < 0.0001), and ALP (P = 0.0003); and increased TGs (P = 0.0002) were found to be associated with elevated GGT levels. Elevated GGT (P = 0.0086) and CRP (P = 0.0162) levels had a longer length of hospital stay. Conclusion: A sizable number of patients with COVID-19 infection have elevated serum GGT levels. This elevation supports involvement of the liver in persons with COVID-19.

Recent advances of GOLM1 in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common liver malignancies and is a leading cause of cancer-related deaths. Most HCC patients are diagnosed at an advanced stage and current treatments show poor therapeutic efficacy. It is particularly urgent to explore early diagnosis methods and effective treatments of HCC. There are a growing number of studies that show GOLM1 is one of the most promising markers for early diagnosis and prognosis of HCC. It is also involved in immune regulation, activation and degradation of intracellular signaling factors and promotion of epithelial-mesenchymal transition. GOLM1 can promote HCC progression and metastasis. The understanding of the GOLM1 regulation mechanism may provide new ideas for the diagnosis, monitoring and treatment of HCC.

Hepatoma cell-intrinsic TLR9 activation induces immune escape through PD-L1 upregulation in hepatocellular carcinoma.

A TLR9 agonist in combination with a PD-1 inhibitor produced powerful antitumor responses in a clinical trial despite TLR9 agonists as monotherapies failing to generate systemic antitumor immune responses due to immunosuppressive effects. However, the mechanism involved in the improved response induced by their combination remains unknown. Methods: Subcutaneous and orthotopic Hepa1-6 tumor model was used for single-drug and combined-drug treatment. We used TLR9 agonist stimulation or lentiviral vectors to overexpress TLR9 and activate TLR9 signaling. We next investigated the crosstalk between PARP1 autoPARylation and ubiquitination and between STAT3 PARylation and phosphorylation mediated by TLR9. Tissue chips were used to analyze the relationships among TLR9, PARP1, p-STAT3 and PD-L1 expression. Results: In this study, we found that the TLR9 agonist in combination with anti-PD-1 therapy or anti-PD-L1 therapy yielded an additive effect that inhibited HCC growth in mice. Mechanistically, we found that TLR9 promoted PD-L1 transcription by enhancing STAT3 Tyr705 phosphorylation. Then, we observed that TLR9 negatively regulated PARP1 expression, which mediated a decrease in STAT3 PARylation and an increase in STAT3 Tyr705 phosphorylation. Moreover, we found that TLR9 enhanced PARP1 autoPARylation by inhibiting PARG expression, which then promoted the RNF146-mediated ubiquitination and subsequent degradation of PARP1. Finally, we observed positive associations between TLR9 and p-STAT3 (Tyr705) or PD-L1 expression and negative associations between TLR9 and PARP1 in HCC patient samples. Conclusions: We showed that hepatoma cell-intrinsic TLR9 activation regulated the crosstalk between PARP1 autoPARylation and ubiquitination and between STAT3 PARylation and phosphorylation, which together upregulated PD-L1 expression and finally induces immune escape. Therefore, combination therapy with a TLR9 agonist and an anti-PD-1 antibody or anti-PD-L1 had much better antitumor efficacy than either monotherapy in HCC.

Disulfiram combined with copper induces immunosuppression via PD-L1 stabilization in hepatocellular carcinoma.

As a potential antitumor drug and chemotherapeutic sensitizer, disulfiram combined with Copper (DSF/Cu2+) does not exert considerable antitumor effects on an immunocompetent hepatocellular carcinoma (HCC) model. In this article, we will explore the mechanism underlying the resistance to DSF in HCC. We analyzed the antitumor effect of DSF/Cu2+ in vivo studies. Tumor and immune cells collected from mice were analyzed by flow cytometry. Then, we analyzed the transcriptional changes in liver cancer cells after DSF/Cu2+ treatment by transcriptional expression profiling. The expression of PD-L1 was detected by real-time PCR, Western blotting and flow cytometry. The expression of PARP1 and GSK3ß was knocked down by small interfering RNAs (siRNAs). A subcutaneous Hepa1-6 tumor model was used for single-drug or combined-drug studies. Tissue chips (268 samples of liver cancer tissue) were used to analyze the relationship among PARP1, p-GSK3ß and PD-L1. We found that DSF/Cu2+ failed to inhibit HCC tumor growth in C57BL/6 mice. DSF/Cu2+ upregulated PD-L1 expression by inhibiting PARP1 activity and enhancing GSK3ß phosphorylation at Ser9 and ultimately inhibited T cell infiltration. The combination of DSF/Cu2+ and an anti-PD-1 antibody produced an additive effect that slowed HCC growth in mice. In addition, we observed negative associations between PARP1 and p-GSK3ß (Ser9) or PD-L1 expression in tumor tissue samples from HCC patients. Through in vitro and in vivo studies, we found that DSF/Cu2+ could restrain GSK3ß activity by inhibiting PARP1, leading to the upregulation of PD-L1 expression. Combination therapy with DSF/Cu2+ and an anti-PD-1 antibody showed much better antitumor efficacy than monotherapy.

Effect of emodin on T cell subsets in NOD mice with NaI­induced experimental autoimmune thyroiditis.

Chronic lymphocytic thyroiditis (CLT), also known as Hashimoto's thyroiditis, is an autoimmune disease in which the thyroid gland is gradually destroyed. To date, only a limited number of agents can effectively suppress thyroiditis development in CLT patients. The aim of the current study was to investigate the protective effect of emodin on experimental autoimmune thyroiditis (EAT) in mice, which is considered an excellent model for CLT. NaI was used to induce the EAT model in non­obese diabetic (NOD) mice. An ELISA method was employed to detect the TgAb level (thyroid inflammation) in the serum of the EAT mice. The T cell subsets in peripheral blood and spleen were detected by flow cytometry. The histopathological study revealed that the thyroid inflammatory cell infiltration was significantly reduced by emodin compared with the model group. In addition, ELISA assays indicated that the NaI­induced serum TgAb upregulation was dramatically revered by emodin. Moreover, the level of serum IFN­Î³ and the cell populations of CD3+CD4+IL­4+, CD3+CD4+ IFN­Î³+, CD3+CD8+IL­4+, CD3+CD8+ IFN­Î³+ T cells in peripheral blood monocytes and splenic lymphocytes were significantly increased by NaI in the model group compared with in the normal group. Nevertheless, this type of increase was markedly attenuated by emodin. To conclude, the EAT model was successfully established by treating NOD mice with NaI. Emodin indicated an inhibitory effect on the autoimmune response that was significantly different in EAT compared with control mice. Furthermore, the anti­inflammatory action of emodin on EAT mice may be mediated via the inhibition of the secretion of IFN­Î³ and the cell numbers of CD3+CD4+IL­4+, CD3+CD4+ IFN­Î³+, CD3+CD8+IL­4+ and CD3+CD8+ IFN­Î³+ T cells in the peripheral blood monocytes and splenic lymphocytes. Therefore, the data may offer valuable insight on the efficacy of treatment of CLT with emodin.