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Cheng Zhou,1,* Zhang-Fu Yang,1,* Bao-Ye Sun,1,* Yong Yi,1 Zheng Wang,1 Jian Zhou,1 Jia Fan,1 Wei Gan,1 Ning Ren,1,2 Shuang-Jian Qiu1 1Department of Liver Surgery and Transplantation & Key Laboratory of Carcinogenesis and Cancer Invasion (Ministry of Education), Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, People’s Republic of China; 2Institute of Fudan Minhang Academic Health System & Key Laboratory of Whole-Period Monitoring and Precise Intervention of Digestive Cancer, Minhang Hospital, Fudan University, Shanghai, People’s Republic of China*These authors contributed equally to this workCorrespondence: Shuang-Jian Qiu, Liver Cancer Institute and Zhongshan Hospital, Fudan University, Shanghai, 200030, People’s Republic of China, Tel +86 13916625289, Email qiu.shuangjian@zs-hospital.sh.cnPurpose: Immunogenic cell death (ICD) is a cell death modality that plays a vital role in anticancer therapy. In this study, we investigated whether lenvatinib induces ICD in hepatocellular carcinoma and how it affects cancer cell behavior.Patients and Methods: Hepatoma cells were treated with 0.5 μM lenvatinib for two weeks, and damage-associated molecular patterns were assessed using the expression of calreticulin, high mobility group box 1, and ATP secretion. Transcriptome sequencing was performed to investigate the effects of lenvatinib on hepatocellular carcinoma. Additionally, CU CPT 4A and TAK-242 were used to inhibit TLR3 and TLR4 expressions, respectively. Flow cytometry was used to assess PD-L1 expression. Kaplan–Meier and Cox regression models were applied for prognosis assessment.Results: After treatment with lenvatinib, there was a significant increase in ICD-associated damage-associated molecular patterns, such as calreticulin on the cell membrane, extracellular ATP, and high mobility group box 1, in hepatoma cells. Following treatment with lenvatinib, there was a significant increase in the downstream immunogenic cell death receptors, including TLR3 and TLR4. Furthermore, lenvatinib increased the expression of PD-L1, which was later inhibited by TLR4. Interestingly, inhibiting TLR3 in MHCC-97H and Huh7 cells strengthened their proliferative capacity. Moreover, TLR3 inhibition was identified as an independent risk factor for overall survival and recurrence-free survival in patients with hepatocellular carcinoma.Conclusion: Our study revealed that lenvatinib induced ICD in hepatocellular carcinoma and upregulated PD-L1 expression through TLR4 while promoting cell apoptosis through TLR3. Antibodies against PD-1/PD-L1 can enhance the efficacy of lenvatinib in the management of hepatocellular carcinoma.Graphical Abstract: Keywords: hepatocellular carcinoma, immunogenic cell death, lenvatinib, toll-like receptor

Background Brahma-related gene 1 (BRG1) is essential for embryogenesis and cellular metabolism. A deficiency of BRG1 in vivo decreases lipid droplets, but the molecular mechanism underlying its role in lipid metabolism associated with hepatocellular carcinoma (HCC) remains unknown. Aims We aimed to determine the role of BRG1 in lipid metabolism in HCC. Methods We assessed the differential expression of BRG1 in HCC and adjacent non-tumorous tissues using tissue microarrays. We stained lipid droplets in HCC cells with Bodipy fluorescence and Oil Red O, and verified BRG1 binding to the promoter region of glycosylated lysosomal membrane protein (GLMP) using chromatin immunoprecipitation. Results The expression of GLMP, a potential lipid metabolism regulator, was suppressed by BRG1 via transcriptional activity. Knockdown of BRG1 decreased lipid droplets, increased GLMP expression and altered the phosphoinositide-3-kinase adaptor protein 1 (PIK3AP1)/phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT) pathway in HCC, which further GLMP knockdown partially restored. Thus, GLMP knockdown increased lipid droplets and differentially altered the PI3K/AKT pathway. Conclusions Downregulating BRG1 decreased lipid droplet deposition in HCC cells by upregulating GLMP and altering the PI3K/AKT pathway. Both BRG1 and GLMP might serve as therapeutic targets for disorders associated with dysregulated lipid metabolism, such as NAFLD and NAFLD-associated HCC.

Background CXCL11 has been reported to be up-regulated in hepatocellular carcinoma (HCC) tissues and cancer-associated fibroblasts (CAFs), and CAF-secreted CXCL11 has been found to promote HCC cell proliferation and migration. Knowledge on how CAFs promote HCC progression is imperative for the future design of anti-tumor drugs addressing the high rates of disease recurrence. Herein, we propose a mechanism by which LINC00152 positively regulates CXCL11 expression and, subsequently, HCC cell phenotypes and growth characteristics via miR-205-5p in CAFs. Methods The expression of LINC00152, miR-205-5p in HCC/non-cancerous tissues, CAFs/NFs and HCC cell lines was determined by RT-qPCR. The CXCL11 expression and secretion were determined by westernblot and ELISA. Different expressions of LINC00152, CXCL11 and miR-205-5p in CAFs were achieved by transfection with corresponding overexpression/knockdown vectors or mimics/inhibitor. The interactions among LINC00152, miR-205-5p and CXCL11 were confirmed by FISH, luciferase, AGO2 and RNA-pulldown assays. Transwell, colony formation and MTT assays were performed to assess the role of CAFs conditioned medium (CM) in HCC cell phenotype. BALB/c nude mice xenografts were used to determine the role of CAFs on HCC growth in vivo. Results We found that in vitro, CM from CAFs transfected with sh-LINC00152 dramatically suppressed HCC cell viability, colony formation and migration, and that CM from CAFs transfected with miR-205-5p inhibitor (CAF-CM (miR-205-5p inhibitor)) exerted opposite effects on HCC cell phenotypes. Exogenous overexpression of CXCL11 in CAFs or CAF-CM (miR-205-5p inhibitor) could partially attenuate the effects of LINC00152 knockdown. In contrast, CM from CAFs transfected with LINC00152 dramatically increased HCC cell viability, colony formation and migration, and CM from CAFs transfected with miR-205-5p mimics (CAF-CM (miR-205-5p mimics)) exerted opposite effects on HCC cell phenotypes. Knockdown of CXCL11 in CAFs or CAF-CM (miR-205-5p mimics) could partially attenuate the effects of LINC00152 overexpression. In vivo, LINC00152 knockdown in CAFs inhibited tumor growth in a mouse model, which could be reversed by CXCL11 overexpression in CAFs. Mechanistically, we found that LINC00152 could act as a ceRNA to counteract miR-205-5p-mediated suppression on CXCL11 by directly binding to miR-205-5p and the 3’UTR of CXCL11. Conclusion Our data indicate that a LINC00152/miR-205-5p/CXCL11 axis in HCC CAFs can affect the proliferative and migrative abilities of HCC cells in vitro and HCC tumor growth in vivo.

Cancer-associated fibroblasts (CAFs) are commonly acquired activated extracellular matrix (ECM)-producing myofibroblasts, a phenotypes with multiple roles in hepatic fibrogenesis and carcinogenesis via crosstalk with cohabitating stromal/cancer cells. Here, we discovered a mechanism whereby CAF-derived cytokines enhance hepatocellular carcinoma (HCC) progression and metastasis by activating the circRNA-miRNA-mRNA axis in tumor cells. CAFs secreted significantly higher levels of CXCL11 than normal fibroblasts (NFs), and CXCL11 also had comparatively higher expressions in HCC tissues, particularly in metastatic tissues, than para-carcinoma tissues. Both CAF-derived and experimentally introduced CXCL11 promoted HCC cell migration. Likewise, CAFs promoted tumor migration in orthotopic models, as shown by an increased number of tumor nodules, whereas CXCL11 silencing triggered a decrease of it. CXCL11 stimulation upregulated circUBAP2 expression, which was significantly higher in HCC tissues than para-carcinoma tissues. Silencing circUBAP2 reversed the effects of CXCL11 on the expression of IL-1β/IL-17 and HCC cell migration. Further downstream, the IFIT1 and IFIT3 levels were significantly upregulated in HCC cells upon CXCL11 stimulation, but downregulated upon circUBAP2 silencing. IFIT1 or IFIT3 silencing reduced the expression of IL-17 and IL-1β, and attenuated the migration capability of HCC cells. Herein, circUBAP2 counteracted miR-4756-mediated inhibition on IFIT1/3 via sponging miR-4756. miR-4756 inhibition reversed the effects induced by circUBAP2 silencing on the IL-17 and IL-1β levels and HCC cell migration. In orthotopic models, miR-4756 inhibition also reversed the effects on metastatic progression induced by silencing circUBAP2.

Background & aims Hepatocellular carcinoma (HCC) is a fatal disease characterized by vast molecular heterogeneity. Although major advances in tumour genetics has led to the identification of new biomarkers, the prognosis of patients with HCC remains dismal. Methods Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and western blot (WB) were used to evaluate meiosis-specific nuclear structural 1 (MNS1) expression in HCC cells. Immunohistochemistry staining was used to evaluate MNS1 expression in HCC tissues. Clinical significance of MNS1 was evaluated by Cox regression analysis. Transwell assays were conducted to assess cells migration ability. Cell counting kit-8 and colony formation assays were performed to detect cells proliferation ability. NOD/SCID/γc(null) (NOG) mice model was adopted to investigate functions of MNS1 in vivo. Results The expression of MNS1, which is elevated in most HCC tissues, correlated with poor survival in HCC patients. Functional experiments revealed the oncogenic role of MNS1, which promotes HCC growth and metastasis through AKT-dependent modulation of β-catenin. β-Catenin expression was crucial for MNS1's oncogenic effects. MNS1 indirectly translocated β-catenin from the cytoplasm to the nucleus via the MNS1-GSK3β axis. Conclusions MNS1 promotes HCC growth and metastasis via activating PI3K/AKT signalling and may serve as an important prognostic biomarker as well as potential novel therapeutic target for HCC.

Hepatic stellate cells (HSCs) play vital roles in tumorigenesis and progression of hepatocellular carcinoma (HCC). However, there remains a lack of high-throughput studies on gene expression alterations in HCC cells in response to direct interactions with HSCs. In this study, we established a direct co-culture model of HSCs and HCC cells. We found that the expression of a set of miRNAs, most notably miR-1246, was triggered by HSCs. RORα was confirmed as the target gene of miR-1246. Either overexpression of miR-1246 or knockdown of RORα enhanced the proliferation, invasiveness, and metastatic capability of HCC both in vitro and in vivo, through Wnt/β-catenin pathway activation and promotion of epithelial-mesenchymal transition (EMT). Moreover, upregulation of miR-1246 and repression of RORα were prominent features of aggressive clinical HCC. The miR-1246-RORα-Wnt/β-catenin axis is a novel pathway through which HSCs accelerate HCC progression.

Background Mixed evidence challenges preoperative alpha-fetoprotein (AFP) as an independent prognostic factor for patients with hepatocellular carcinoma (HCC) after hepatectomy. Results Daily post-operative decrease of AFP by 9% as compared to the preoperative level (A09) were selected as the Cut-off. The Kaplan-Meier curve showed that A09 was significantly different for OS (P=0.043) and RFS (P=0.03). A decrease in risk by 54% was observed for OS and 32% for RFS in the at-risk population (A09>9%). A better concordance was observed after adding A09 into TNM and BCLC staging systems. Moreover, a consistent concordance was observed in the internal (FDZS5:0.63; FDZS3:0.608) and external (FDZS5:0.85; FDZS3:0.762) validation cohorts, suggesting its prognostic value in HCC population with elevated AFP. Conclusions Decrease in perioperative serum AFP rather than preoperative AFP is an independent prognostic factor for HCC patients after hepatectomy. Cut-off A09 significantly discriminates overall and recurrence-free survival and could be interpret into TNM and BCLC staging systems to improve the stratification power for HCC patients with elevated AFP. Methods Kaplan-Meier curve depicted the differences of overall survival (OS) and recurrence-free survival (RFS). Nomogram and concordance were employed to evaluate the superiority of the current staging system.

Background & Aims: Preoperative prediction of microvascular invasion (MVI) is critical for treatment strategy making in patients with hepatocellular carcinoma (HCC). We aimed to develop a deep learning (DL) model based on preoperative dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) to predict the MVI status and clinical outcomes in patients with HCC. Methods We retrospectively included a total of 321 HCC patients with pathologically confirmed MVI status. Preoperative DCE-MRI of these patients were collected, annotated and further analyzed by DL in this study. A predictive model for MVI integrating DL-predicted MVI status (DL-MVI) and clinical parameters was constructed with multivariate logistic regression. Results Of 321 HCC patients, 136 patients were pathologically MVI absent and 185 patients were MVI present. Recurrence-free survival (RFS) and overall survival (OS) were significantly different between the DL-predicted MVI-absent and MVI-present. Among all clinical variables, only DL-predicted MVI status and AFP were independently associated with MVI: DL-MVI (odds ratio [OR]=35.738; 95% confidence interval [CI]: 14.027-91.056; p

We aimed to investigate whether improvements in the prognosis of patients with hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) who have undergone hepatectomy are associated with reductions in the liver inflammation and fibrosis by antiviral therapy (AVT).Patients who underwent hepatectomy and re-hepatectomy for HBV-related HCC between 2010 and 2019 were divided into two groups. Histological changes in liver were compared between initial and recurrence stages within each group. Propensity score matching (PSM) analysis was performed to compare prognostic outcomes.After PSM, AVT group showed a significantly better prognosis than did non-AVT group (RFS: 19.1% vs. 5.8%, P = 0.001; OS: 64.0% vs. 43.2%, P 0.001). The improvements in G and S were independent protective factors for RFS (G: P 0.001; S: P 0.001) and OS (G: P = 0.013; S: P 0.001).The application of AVT after initial surgery improved liver inflammation and fibrosis, further benefiting long-term outcomes of patients with HBV-related HCC.

Background: Recently, more and more treatment strategies for Hepatocellular carcinoma (HCC) have emerged, but the therapeutic effect is still not satisfactory. This study is aimed to explore the mechanism of Interleukin 35 (IL-35) in promoting the progression of liver cancer and to explore the application value of IL-35 in the treatment of HCC.Methods: We used clinical tissue microarray (TMA) immunohistochemistry (IHC) to explore the prognostic value of IL-35 expression in patients with HCC. The effect of IL-35 on the function of HCC was explored by functional experiments including wound-healing assay, transwell, cell counting kit-8, cell adhesion assay and endothelial tube formation assay in vitro and mouse xenografts in vivo. And flow cytometry was used to study the effect of IL-35 on infiltrating immune cells in tumor. The molecular mechanism of the function of IL-35 on the progression of HCC was explored by sequencing, ELISA, WB, PCR and other technical means. Finally, through in vivo tumor animal experiments to explore the value of anti-IL-35 antibody and combined with anti-PD-1 antibody in the treatment of liver cancer.Results: High expression of IL-35 in patients with HCC were identified to be associated with poor prognosis. And we have found that IL-35 facilitated tumor progression by affecting neutrophil infiltration, angiogenesis, and CD8+ T-cell infiltration in a mouse model. Additionally, on the one hand C-C motif chemokine ligand 3 (CCL3) has been found to be a key factor mediating the recruitment of neutrophils by IL-35, on the other hand fibroblast growth factor 2 (FGF2) secreting by neutrophil when stimulated by IL-35 was also found to be the core cytokine to promote intratumoral angiogenesis. And IL-35 was also discovered to facilitated the adhesion of tumor to endothelial cells, with neutrophils further enhancing this effect in vitro and vivo. More important, anti-IL-35 antibody was found to be a valid treatment for HCC in xenograft tumor model, and it could give full play to the curative effect of 1:1＞2 when combination therapy with PD-1 antibody.Conclusion: Our data show that the expression of IL-35 in patients with HCC is an important tumor promoting factor. The application of anti-IL-35 antibody and treatment combined anti-IL-35 antibody with anti-PD-1 antibody have potential therapeutic value in the treatment of liver cancer.

Herpes virus entry mediator (HVEM) is overexpressed in several malignancies, including hepatocellular carcinoma (HCC). However, to the best of our knowledge, the clinical significance of HVEM in hepatitis B virus (HBV)-related HCC remains unclear. Thus, the present study aimed to explore the clinical significance of HVEM in HBV-related HCC. In the present study, HVEM expression was evaluated in HCC cell lines and HCC frozen samples. The prognostic value of HVEM was assessed in a cohort of 221 patients with HBV-related HCC, following radical resection. B- and T-lymphocyte attenuator (BTLA) expression in subsets of CD8(+) T cells was determined via flow cytometry analysis. The results demonstrated high HVEM expression in HCC cell lines, and in HCC tissues compared with paired non-cancerous liver tissues. HVEM expression was demonstrated to be significantly associated with tumor encapsulation and vascular invasion. Furthermore, tumor HVEM status was significantly associated with infiltration of regulatory T cells, but not with CD8(+) T cells. Notably, high HVEM expression in HCC was determined to be an independent predictor of an unfavorable outcome of patients with HCC following radical resection. Higher BTLA expression (the receptor of HVEM) was observed in both HCC-infiltrating CD8(+) effector memory (CCR7(−) CD45RA(−)) and CD45RA(+) effector memory (CCR7(−) CD45RA(+)) T cells in HCC tissues and blood compared with those in paired peritumor tissues or peripheral blood. Taken together, the results of the present study suggest that HVEM may serve a critical role in HBV-related HCC, most likely by promoting tumor progression and tumor immune evasion, thus the HVEM/BLTA signaling pathway may be a potential target in tumor immunotherapy.

Hepatocarcinogenesis is a complicated process that is affected by a variety of microenvironmental factors, such as secretory chemokines and cell-extracellular matrix (ECM). Retinoic acid receptor-related orphan receptor (ROR)-α has been shown to attenuate tumor invasiveness by inducing suppressive cell microenvironment, and its low expression was associated with a worse prognosis in HCC patients. In the present study, we attempted to investigate the role and mechanism of the dominant transcript of ROR-α, ROR-α-1, in HCC development and progression. Among the four transcripts (ROR-α-1/-2/-3/-4), overexpression of ROR-α-1 dramatically suppressed the capacity of MHCC97H cells to proliferate, migrate and invade. We analyzed the differentially expressed genes in ROR-α-1-overexpressed and non-overexpressed MHCC97H cells, performed Gene Ontology (GO) enrichment analysis on these differentially-expressed genes, and found out that factors involved in the tumor microenvironment and ECM are related to the anti-tumor effects of ROR-α-1. Among these factors, chemokine CXCL5 was significantly downregulated by ROR-α-1 overexpression. Overexpression of ROR-α-1 remarkably inhibited the capacity of HCC cells to proliferate, migrate, invade, and downregulated the protein levels of β-catenin, c-Myc, Cyclin D1, and N-cadherin, suggesting the tumor-suppressive role of ROR-α-1 in MHCC97H cells. Moreover, overexpression of CXCL5 dramatically attenuated the suppressive effects of cell proliferation, migration and invasion induced by ROR-α-1 overexpression in MHCC97H, suggesting that ROR-α-1 exerts its anti-tumor effects via downregulating CXCL5. In conclusion, we demonstrate the tumor-suppressive role of ROR-α-1 in MHCC97H cells and that ROR-α-1 might play a tumor-suppressive role via regulation of chemokine CXCL5.