The Prognostic and Therapeutic Role of Histone Acetylation Modification in LIHC Development and Progression.

Background and Objectives: The modification of histone acetylation plays a vital role in regulating tumor occurrence and development, but the interaction between histone acetylation modulator genes and the liver hepatocellular carcinoma (LIHC) microenvironment, as well as immunotherapy, has not been investigated. Materials and Methods: Analysis of all statistical data was carried out using R software (Version 4.2.0) and the online tool Sangerbox. Comprehensive bioinformatics analysis, including signature construction and validation, functional analyses, immune and genomic features analyses, and immunotherapy prediction analyses, were performed to explore the prognostic and therapeutic role of histone acetylation modulator genes in LIHC development and progression. Results: The LIHC cohort from The Cancer Genome Atlas (TCGA) database was selected as the training cohort; the GSE76427 cohort from the Gene Expression Omnibus (GEO) database and the LIRI-JP cohort from the International Cancer Genome Consortium (ICGC) database were selected as the validation cohorts. The histone acetylation modulator gene-based prognostic signature was constructed and validated successfully. Immune infiltration analysis showed that most immune cells and immune functions were enriched in patients with high histone acetylation risk scores (HARS). Additionally, high levels of checkpoint inhibitors (ICIs) and human leukocyte antigens (HLAs) were also observed in high HARS patients. Meanwhile, TIDE algorithm analysis was conducted to explore the relationship between HARS and immunotherapy response, and submap algorithm analysis was used for the verification of the results, from which we found that high HAPS patients were more likely to respond to immunotherapy. Conclusions: Our findings revealed that the histone acetylation modulator genes, particularly for KAT21, SIRT6, and HAT1, may have the potential to function as a new prognostic marker and therapeutic target for LIHC.

Gut microbiota-derived short-chain fatty acids regulate group 3 innate lymphoid cells in HCC.

BACKGROUND AND AIMS: Type 3 innate lymphoid cells (ILC3s) are essential for host defense against infection and tissue homeostasis. However, their role in the development of HCC has not been adequately confirmed. In this study, we investigated the immunomodulatory role of short-chain fatty acids (SCFAs) derived from intestinal microbiota in ILC3 regulation. APPROACH AND RESULTS: We report that Lactobacillus reuteri was markedly reduced in the gut microbiota of mice with HCC, accompanied by decreased SCFA levels, especially acetate. Additionally, transplantation of fecal bacteria from wild-type mice or L. reuteri could promote an anticancer effect, elevate acetate levels, and reduce IL-17A secretion in mice with HCC. Mechanistically, acetate reduced the production of IL-17A in hepatic ILC3s by inhibiting histone deacetylase activity, increasing the acetylation of SRY (sex-determining region Y)-box transcription factor 13 (Sox13) at site K30, and decreasing expression of Sox13. Moreover, the combination of acetate with programmed death 1/programmed death ligand 1 blockade significantly enhanced antitumor immunity. Consistently, tumor-infiltrating ILC3s correlated with negative prognosis in patients with HCC, which could be functionally mediated by acetate. CONCLUSIONS: These findings suggested that modifying bacteria, changing SCFAs, reducing IL-17A-producing ILC3 infiltration, and combining with immune checkpoint inhibitors will contribute to the clinical treatment of HCC.

Hepatic stellate cell activation and senescence induced by intrahepatic microbiota disturbances drive progression of liver cirrhosis toward hepatocellular carcinoma.

BACKGROUND: The significance of the relationship between the microbiota and diseases is increasingly being recognized. However, the characterization of tumor microbiome and their precise molecular mechanisms through which microbiota promotes hepatocellular carcinoma (HCC) development are still unclear. METHODS: The intrahepatic microbiota was investigated from tumor, normal adjacent tissues in 46 patients with HCC and normal hepatic tissues in 33 patients with hemangioma by 16S rRNA gene sequencing. Taxonomic composition differences in patients were evaluated using Linear discriminant analysis Effect Size (LefSe) and Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) to predict microbial functional pathways. Associations between the most relevant taxa and clinical characteristics of HCC patients were analyzed by Spearman rank correlations. The effects of microbe on hepatic stellate cells (HSCs) activation and HCC progression were examined. RESULTS: We observed intrahepatic microbiota disturbances by reduced microbial diversity in HCC. The tumor microbiota of the HCC patients with cirrhosis showed higher abundance of Stenotrophomonas maltophilia (S. maltophilia). S. maltophilia provoked senescence-associated secretory phenotype (SASP) in HSCs by activating TLR-4-mediated NF-κB signaling pathway, which in turn induced NLRP3 inflammasome complex formation and secreted various inflammatory factors in the liver, thus facilitating HCC progression in mice. Moreover, signs of SASP were also observed in the HSCs in the area of HCC with higher S. maltophilia enrichment arising in patients with cirrhosis. CONCLUSIONS: Our analysis of the hepatic microbiota revealed for the first time that patients with HCC exhibited a dysbiotic microbial community with higher S. maltophilia abundance, which induced the expression SASP factors of HSCs and cirrhosis in the liver, concurring in the process of hepatocarcinogenesis.

Regulatory T Cells Improved the Anti-cirrhosis Activity of Human Amniotic Mesenchymal Stem Cell in the Liver by Regulating the TGF-ß-Indoleamine 2,3-Dioxygenase Signaling.

Liver fibrosis is a progression stage of chronic liver disease, while current therapies cannot cure or attune cirrhosis effectively. Human amniotic mesenchymal stromal cell (hAMSC) presented immunoregulatory and tissue repairability of multiple illnesses. Regulatory T cells (Treg) had been proved to be functional in reducing immune cell activity. We showed that co-infusion of hAMSC and Treg prevented mild liver fibrosis comparing with hAMSC or Treg alone group. In vitro study indicated that the addition of Treg or the supernatant of Treg improved the hepatocyte growth factor (HGF) secreting and cell differentiation ability of hAMSC. Reduction of TGF-ß significantly decreased the HGF secreting and differentiation of hAMSC. Multiple signal neutralizers were added to the culture to understand further the mechanism, which showed that 1-MT, the suppressor of Indoleamine 2,3-dioxygenase (IDO), was involved in the effect of TGF-ß in regulating hAMSC. Depletion of TGF-ß or IDO signaling successfully abolished the effect of Treg in improving hAMSC's function both in vitro and vivo. Finally, our result indicated that Treg improved the function of hAMSC by regulating the TGF-ß-IDO signaling and co-infusion of hAMSC and Treg provided a promising approach for treating liver cirrhosis.

One Single Site Clinical Study: To Evaluate the Safety and Efficacy of Immunotherapy With Autologous Dendritic Cells, Cytokine-Induced Killer Cells in Primary Hepatocellular Carcinoma Patients.

Dendritic cells (DCs) and cytokine-induced killer (CIK) cells play an important role in the anti-tumor immune response. In this study, we evaluated the clinical effectiveness of DC/CIK-CD24 immunotherapies to primary hepatocellular carcinoma patients who received radical resection. 36 resected primary hepatocellular carcinoma (HCC) patients were enrolled from August 2014 to December 2015. All patients received two or four times of DC/CIK immunotherapy after radical resection. 1-4 years patients' survival rates were evaluated during the follow-up. The 4-year survival rate of patients who received two times of immunotherapy was 47.1%, and the rate of those who received four times of immunotherapies was 52.6%. Compared to baseline, after receiving the DC/CIK-CD24 autotransfusion, the serum Treg concentration of the patients decreased, while CD3+, CD4+, CD56+ increased slightly. The adverse effect of immunotherapy was I-II° transient fever and could be tolerable. DC/CIK-CD24 immunotherapy can delay the relapse time.

Diagnostic and prognostic value of circular RNAs in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the sixth most common malignant tumour, which has posed a heavy health and financial burden worldwide. Due to limited symptoms at the early stage and the limitation in current biomarkers, HCC patients are usually diagnosed at the advanced stage with a pessimistic overall survival rate. Circular RNAs (circRNAs) are a subclass of single-stranded RNAs characterized by a covalently closed loop structure without 3'- or 5'-end. With advances in high-throughput sequencing technology and bioinformatics, accumulating studies have demonstrated the promotor or suppressor roles of circRNAs in the carcinogenesis, progression, and metastasis of HCC. Moreover, circRNAs are characteristic of higher abundance, stability and conservation compared with linear RNAs. Therefore, circRNAs have emerged as one of the most promising diagnostic and prognostic biomarkers for HCC with reliable accuracy, sensitivity and specificity. In this review, we briefly introduce the characteristics of circRNAs and summarize the roles of circRNAs in the biological procedures of HCC. Furthermore, we provide an overview on the potential diagnostic and prognostic value of circRNAs as biomarkers for patients with HCC. Finally, we discuss future perspectives of circRNAs in cancer research.

Long Non-coding RNA SNHG17 Upregulates RFX1 by Sponging miR-3180-3p and Promotes Cellular Function in Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common types of cancer that is associated with poor quality of life in patients and a global health burden. The mechanisms involved in the development and progression of HCC remain poorly understood. METHODS: Hepatocellular carcinoma human samples and cell lines were subjected to qRT-PCR for expression assessment. CCK-8 assay, Transwell migration and invasion assay, were applied for cell function detection. Animal experiment was used to measure the function of SNHG17 on cell growth in vivo. Western blot was conducted to evaluate the level of EMT in cells. RIP, RNA pull-down and luciferase reporter assays were performed to assess the correlation between SNHG17, miR-3180-3p and RFX1. RESULTS: Our study demonstrated that SNHG17 was upregulated in HCC human samples and involved cell proliferation, migration, invasion progress. SNHG17 promoted HCC cell growth and metastasis in vivo. Furthermore, we investigated the downstream factor of SNHG17, SNHG17 acted as a molecular sponge for miR-3180-3p, and SNHG17 regulated RFX1 expression via miR-3180-3p. SNHG17 promotes tumor-like behavior in HCC cells via miR-3180-3p/RFX1. CONCLUSION: We determined RFX1 as the target of miR-3810-3p; SNHG17 enhanced the progression of HCC via the miR-3180-3p/RFX1 axis. Taken together, our findings may provide insight into the molecular mechanism involved in the progression of HCC and develop SNHG17 as a novel therapeutic target against HCC.

The proportion of CD19+CD24hiCD27+ regulatory B cells predicts the occurrence of acute allograft rejection in liver transplantation.

BACKGROUND: Regulatory B cells (Bregs) play an essential role in inflammation and transplant tolerance. Several studies have reported a decreased number of Bregs in renal transplant patients with graft rejection. However, little is known about their role in the liver alloresponse. METHODS: To investigate whether the circulating Bregs have been associated with acute allograft rejection (AR) in liver transplantation patients, 19 patients receiving liver allografts from donation after cardiac death (DCD) donors were retrospectively studied. RESULTS: The postoperative proportions of circulating CD19+CD24hiCD38hi transitional Bregs (tBregs) and CD19+CD24hiCD27+ memory Bregs (mBregs) in patients diagnosed with AR (AR group) and other patients with stable allograft liver function (SF group) were evaluated using flow cytometry (FCM) analysis. Results showed that while no significant changes were found regarding both the tBreg and mBreg, proportions across all time points in the SF group, the AR group showed significantly decreased proportions of mBregs. All of the five AR patients responded fine to the treatments, and the proportions of mBregs increased significantly after anti-rejection therapies. In addition, AR was suspected in four recipients, but gradually they were diagnosed with hemolytic or obstructive jaundice and showed no decrease in the proportion of mBregs. CONCLUSIONS: For the first time, our results suggested the potential role of a decreased proportion of circulating mBregs in predicting AR in patients with post liver transplantation.

Liver Transplantation Using Right Lobe Graft With Focal Nodular Hyperplasia: Report of 2 Cases.

We described 2 cases of adult-to-adult liver transplantation using right lobe grafts donated by 2 patients suffering from focal nodular hyperplasia, the volumes of which were 360 cm3 and 220 cm3. This study was performed in compliance with the Declaration of the Helsinki. For preparation of the graft, back-table hepatic venous outflow reconstruction, including replacement of the retrohepatic inferior vena cava and bridging of hepatic segment V5/V8, was performed by using prosthetic vessel grafts. The liver grafts after subtracting the weight of focal nodular hyperplasia functioned well without serious small-for-size syndrome or graft dysfunction in spite of graft-to-recipient weight ratio less than 0.8%. This suggested the functioning of hepatocyte within focal nodular hyperplasia, which avoided graft deficiency. These 2 recipients were given not only conventional immunosuppressants (tacrolimus and mycophenolate sodium) but also anticoagulants (low molecular weight heparin and warfarin). Thrombosis of the prosthetic inferior vena cava 1 month after transplant in recipient 1 was treated by placement of intravascular stent. Biliary duct anastomotic stricture 2 weeks after transplant in recipient 2 was treated by placement of biliary stent. These 2 recipients remain well at more than 2 years post-transplant. These successful explorative cases highlight the safe use of a graft containing focal nodular hyperplasia. The partial liver resection grafts with focal nodular hyperplasia could be applied to the patients on the waiting list for liver transplantation.

Identification of TAF1, HNF4A, and CALM2 as potential therapeutic target genes for liver fibrosis.

The molecular mechanism of liver fibrosis caused by hepatitis C virus (HCV) is not clear. The aim of this study is to understand the molecular mechanism of liver fibrosis induced by HCV and to identify potential therapeutic targets for hepatic fibrosis. We analyzed gene expression patterns between high liver fibrosis and low liver fibrosis samples, and identified genes related to liver fibrosis. We identified TAF1, HNF4A, and CALM2 were related to the development of liver fibrosis. HNF4A is important for hepatic fibrogenesis, and upregulation of HNF4A is an ideal choice for treating liver fibrosis. The gene expression of CALM2 is significantly lower in liver fibrosis samples than nonfibrotic samples. TAF1 may serve as a biomarker for liver fibrosis. The results were further validated by an independent data set GSE84044. In summary, our study described changes in the gene expression during the occurrence and development of liver fibrosis. The TAF1, HNF4A, and CALM2 may serve as novel targets for the treatment of liver fibrosis.

Liver transplantation using the otherwise-discarded partial liver resection graft with hepatic benign tumor: Analysis of a preliminary experience on 15 consecutive cases.

RATIONALE: The shortage of available donor organs limits the development of liver transplantation. This case-serial study presents a novel way to expand the donor pool by using the other-wise discarded partial liver resection graft with hepatic benign tumor. PATIENT CONCERNS: From 2012 to 2016, 15 patients with hepatic lesions were admitted to our hospital. 12 patients suffered from right epigastric discomfort and 3 patients worried about uncertain diagnosis. INTERVENTIONS: Regular hepatic lobectomy was performed for all patients and after back-table management the resected partial liver grafts were used for patients with end-stage liver disease for liver transplantation. OUTCOMES: All patients had improved liver function within 1 week of transplantation. Patients had no serious small-for-size syndrome despite graft-to-recipient weight ratio less than 0.8%. Back-table hepatic venous reconstruction with prosthetic vascular grafts was performed without serious early complications, and late thrombosis in vessel graft did not affect liver function. Postoperative computed tomography scans demonstrated a remarkable growth in graft volume and a continuous decrease in hemangioma in recipients using the grafts with hemangioma. One patient died from pulmonary embolism on day 7 after transplant, and the rest of 14 recipients had been surviving well, especially recipient 1 for more than 4 years, although 3 recipients had tumor recurrence and had been treated with sorafenib. DIAGNOSES: The postoperative pathological diagnosis reported cavernous hemangioma (n = 11), perivascular epithelioid cell tumor (n = 2), inflammatory pseudotumor for (n = 1), and focal nodular hyperplasia (n = 1). LESSONS: The partial liver grafts with hepatic benign tumors are safe for liver transplantation. In addition, prosthetic vascular grafts can be used for hepatic venous outflow reconstruction, especially in right lobe liver transplantation.

Effect of Tumor Size on Cancer-Specific Survival in Small Hepatocellular Carcinoma.

OBJECTIVE: To further clarify the prognosis of small hepatocellular carcinoma (HCC) in different subgroups and to assess the connection between the tumor cutoff point and its impact on prognostic significance. PATIENTS AND METHODS: In this study, Surveillance, Epidemiology, and End Results Program-registered patients with small HCC and patients from the Liver Transplantation Center of Nanjing Medical University (LTCNJMU) were combined and analyzed. The optimal cutoff point of the tumor size was determined by using the "X-tile" program. The 60-month, cancer-specific survival data were obtained. Kaplan-Meier methods and multivariable Cox regression models were used to analyze long-term hepatocellular carcinoma-specific survival (HCSS) outcomes and risk factors. RESULTS: There were significant differences among these different tumor size subgroups with regard to 60-month HCSS rates (P<.001). The X-tile program indicated that the difference in survival was most significant (maximum of χ(2) log-rank values) (P<.001) for the tumor size 35 mm. A stratified analysis of the effect of tumor size on 60-month HCSS rate found that only localized and regional stages could be validated as independent predictors, but not advanced stages. CONCLUSION: These results confirmed that patients with small HCC are essentially a heterogeneous group. Tumor size is still an independent prognostic factor for resected small HCC, and patients with tumors of 0- to 35-mm diameter have a better 60-month HCSS rate than do those with larger tumors (36-50 mm).

MiR-135a functions as a tumor suppressor in epithelial ovarian cancer and regulates HOXA10 expression.

The activation of homeobox A10 (HOXA10) has been proved to be an important event in epithelial ovarian carcinogenesis, yet its regulation in epithelial ovarian cancer (EOC) is still not fully understood. Here, we aimed to reveal the mechanism that a predicted target miRNA regulates HOXA10 expression and the association of its expression with progression of EOC. Here, by using computer-assisted algorithms from PicTar, TargetScan, and miRBase, we identified that the predicted target miRNA of HOXA10 was miR-135a. MiR-135a expression in EOC tissues and controls was measured with quantitative RT-PCR. The role of miR-135a and HOXA10 in the growth and survival of several EOC cell lines was determined with several in vitro approaches. We found that miR-135a expression was downregulated in an EOC patient cohort. Also, patients with low miR-135a expression had shorter overall survival and progression-free survival durations than those with high expression. Functional analysis of three EOC-derived cell lines (SKOV-3, HEY, and OVCAR-3) demonstrated that miR-135a directly regulated HOXA10 expression by targeting its 3'-UTR. Inhibition of HOXA10 expression with miR-135a mimics and HOXA10 siRNA consistently resulted in cell apoptosis with concomitant enhancement of caspase-3, increase of p53 expression and reduction of Bcl-2 expression, and also suppressed cell growth and adhesion. These findings suggest that ubiquitous loss of miR-135a expression is a critical mechanism for the overexpression of HOXA10 in EOC cells, which is implicated in epithelial ovarian carcinogenesis. Furthermore, miR-135a may be predictive of EOC prognosis.

TGF-ß signaling is often attenuated during hepatotumorigenesis, but is retained for the malignancy of hepatocellular carcinoma cells.

The role of transforming growth factor-beta (TGF-ß) signaling in hepatocarcinogenesis remains controversial. We aimed to reveal TGF-ß signaling status in human and murine tissues of hepatocellular carcinoma (HCC) and the mechanisms that mediate TGF-ß's role in regulating HCC malignancy. Here, TGF-ß pathway component expression and activation in human and murine HCC tissues were measured with quantitative RT-PCR and Western blotting assays. The role of TGF-ß receptor and Smad signaling in the growth and survival of several HCC cell lines was determined with several in vitro and in vivo approaches. We found that TGF-ß receptor II (TßRII) expression was downregulated in two different HCC patient cohorts. Consistently, Smad3 phosphorylation was also downregulated in HCC tissues in comparison to that in adjacent normal tissues. Interestingly, many HCC cell lines were sensitive to TGF-ß and growth-inhibited by exogenous TGF-ß. However, stable knockdown of TßRII inhibited cell growth on plastic and in soft agar, and induced apoptosis resulting in suppressed subcutaneous tumor growth and metastatic potential in vivo. Furthermore, knockdown of Smad4 also led to a significant inhibition of growth on plastic and in soft agar with concomitant increase of apoptosis, PTEN expression, and reduced nuclear accumulation of linker region-phosphorylated Smad3. Taken together, TGF-ß signaling pathway plays a dichotomous role in hepatocellular carcinogenesis. It appears to suppress HCC development, but is retained for HCC cell survival and malignancy. Furthermore, Smad4 can mediate both growth inhibitory activity induced by exogenous TGF-ß and the survival activity induced by autocrine TGF-ß revealing a delicate selection of the two opposing activities of TGF-ß during HCC evolution.

Mutant p53 disrupts role of ShcA protein in balancing Smad protein-dependent and -independent signaling activity of transforming growth factor-ß (TGF-ß).

Biomarkers are lacking for identifying the switch of transforming growth factor-ß (TGF-ß) from tumor-suppressing to tumor-promoting. Mutated p53 (mp53) has been suggested to switch TGF-ß to a tumor promoter. However, we found that mp53 does not always promote the oncogenic role of TGF-ß. Here, we show that endogenous mp53 knockdown enhanced cell migration and phosphorylation of ERK in DU145 prostate cancer cells. Furthermore, ectopic expression of mp53 in p53-null PC-3 prostate cancer cells enhanced Smad-dependent signaling but inhibited TGF-ß-induced cell migration by down-regulating activated ERK. Reactivation of ERK by the expression of its activator, MEK-1, restored TGF-ß-induced cell migration. Because TGF-ß is known to activate the MAPK/ERK pathway through direct phosphorylation of the adaptor protein ShcA and MAPK/ERK signaling is pivotal to tumor progression, we investigated whether ShcA contributed to mp53-induced ERK inhibition and the conversion of the role of TGF-ß during carcinogenesis. We found that mp53 expression led to a decrease of phosphorylated p52ShcA/ERK levels and an increase of phosphorylated Smad levels in a panel of mp53-expressing cancer cell lines and in mammary glands and tumors from mp53 knock-in mice. By manipulating ShcA levels to regulate ERK and Smad signaling in human untransformed and cancer cell lines, we showed that the role of TGF-ß in regulating anchorage-dependent and -independent growth and migration can be shifted between growth suppression and migration promotion. Thus, our results for the first time suggest that mp53 disrupts the role of ShcA in balancing the Smad-dependent and -independent signaling activity of TGF-ß and that ShcA/ERK signaling is a major pathway regulating the tumor-promoting activity of TGF-ß.

Evaluation of Epstein-Barr virus latent membrane protein 2 specific T-cell receptors driven by T-cell specific promoters using lentiviral vector.

Transduction of latent membrane protein 2 (LMP2)-specific T-cell receptors into activated T lymphocytes may provide a universal, MHC-restricted mean to treat EBV-associated tumors in adoptive immunotherapy. We compared TCR-specific promoters of distinct origin in lentiviral vectors, that is, Vß6.7, delta, luria, and Vß5.1 to evaluate TCR gene expression in human primary peripheral blood monocytes and T cell line HSB2. Vectors containing Vß 6.7 promoter were found to be optimal for expression in PBMCs, and they maintained expression of the transduced TCRs for up to 7 weeks. These cells had the potential to recognize subdominant EBV latency antigens as measured by cytotoxicity and IFN-γ secretion. The nude mice also exhibited significant resistance to the HLA-A2 and LMP2-positive CNE tumor cell challenge after being infused with lentiviral transduced CTLs. In conclusion, LMP2-specific CTLs by lentiviral transduction have the potential use for treatment of EBV-related tumors.