Chronic hepatitis B (CHB) remains a serious global health problem. In our previous investigation, HBV was found to activate a mild ER stress, which facilitated the establishment of persistent HBV infection. However, the role of ER stress manipulation in HBV replication and its underlying mechanisms remain still unclear. Our data showed that mild ER stress inhibited HBV transcription and replication, while severe ER stress enhanced them. Mechanistically, in contrary to the effect on HBV replication, mild ER stress activated whereas severe ER stress inhibited mTOR signaling in HBV-infected cells. Further, mTOR signaling was revealed to be critical for mild ER stress-mediated HBV inhibition. Furthermore, ATF6 but not PERK or IRE1α was found to be involved in mild ER stress-mediated mTOR and the following HBV inhibition. Moreover, ATF6, per se, could inhibit HBV transcription and replication via activating mTOR signaling. Together, ATF6-mediated mild ER stress inhibited HBV transcription and replication through mTOR activation, which might present as an important therapeutic target for CHB patients.

Purpose:To evaluate the short-term clinical efficacy, side effects and risk factors affecting the clinical effectiveness of the combination of lipidol and epirubicin-loaded drug-eluting bead transarterial chemoembolization (DEB-TACE) in the treatment of hepatocellular carcinoma (HCC). Methods:A total of 120 patients with HCC who underwent DEB-TACE plus lipiodol treatment from December 2017 to August 2020 were enrolled. Short-term local tumor response was evaluated using mRECIST. Postoperative complications and liver function disorders were analyzed on the basis of clinical parameters. Results:The median overall survival (OS) was 31.44 months (95% CI: 27.24-35.46 months). According to mRISIST, the disease control rate is 75.8%. The objective response rate was 22.5%. Multivariate analysis showed that tumor size and conversion therapy were the two independent prognostic factors correlated with OS. Postoperatively, liver function showed transient changes and no grade 4 adverse events were observed. Most of the postoperative complications were characterized by post-embolism syndrome. Conclusion:The combination of lipiodol and DEB-TACE offers effective local control and safety for patients with HCC. Lipiodol used in the DEB-TACE procedure provides several additional benefits for drug-eluting beads embolization. The synergistic effect of these two methods enhances therapeutic efficacy through dual antitumor mechanisms.

Myeloid cells are physiologically related to innate immunity and inflammation. Tumor-associated myeloid cells gained increasing interest because of their critical roles in tumor progression and anticancer immune responses in human malignancies. However, the associations between tumor-associated myeloid cell-related genes and hepatocellular carcinoma have yet to be revealed. Here, through the integrating analysis of bulk and single-cell RNA (scRNA) sequencing of public HCC samples, we developed a gene signature to investigate the role of HCC-specific myeloid signature genes in HCC patients. We firstly defined 317 myeloid cell marker genes through analyzing scRNA data of HCC from the GEO dataset. After selecting the differentially expressed genes, eleven genes were also proved prognostic. Then we built a gene signature from the TCGA cohort and verified further with the ICGC dataset by applying the LASSO Cox method. An eight genes signature (FABP5, C15orf48, PABPC1, TUBA1B, AKR1C3, NQO1, AKR1B10, SPP1) was achieved finally. Patients in the high risk group correlated with higher tumor stages and poor survival than those in the low-risk group. The risk score was proved to be an independent risk factor for prognosis. The high risk group had higher infiltrations of dendritic cells, macrophages and Tregs. And the APC co-inhibition, T cell co-inhibition pathways were also activated. Besides, the risk score positively correlated with multidrug resistance proteins. In conclusion, our myeloid cell marker genes related signature can predict patients’ survival and may also indicate the levels of immune infiltration and drug resistance.

Abstract Background Polycomb protein suppressor of zeste 12 (SUZ12) has been widely revealed involving in development and diverse physiopathology processes. However, the roles of SUZ12 in hepatocellular carcinoma (HCC) remain largely unknown. Methods Tissue microarray was used to examine the expression level of SUZ12. The gain- and loss- of function analysis were conducted to evaluate the effects of SUZ12 on the proliferation, migration and invasion of HCC cells. Meanwhile, luciferase reporter assay and RT-PCR assay were conducted to examine the effect of SUZ12 on transcriptional activity of chemokine receptors 7 (CXCR7). Results The expression level of SUZ12 was positively associated with HCC development, as revealed by tissue microarray analysis. Further gain- and loss- of function analysis demonstrated that SUZ12 promoted the proliferation, migration and invasion of HCC. Mechanistically, we found that SUZ12 could upregulate the expression of CXCR7 at the transcriptional level in HCC cells, and CXCR7 was revealed to contribute to the tumor-promoting roles of SUZ12. Of interest, luciferase reporter assay revealed SUZ12 positively controlled the CXCR7 via direct promoter combination but not epigenetic suppression. Moreover, high SUZ12 expression was positively correlated with CXCR7 in advanced patients with portal vein tumor thrombus. Conclusion Our findings indicate that SUZ12 plays pro-oncogenic roles in the progression of HCC, partially by activating CXCR7 signaling, especially in HCC patients with portal vein tumor thrombus. The SUZ12/CXCR7 axis may serve as a potential therapeutic target for treatments of advanced HCC patients.

Background:Although transarterial chemoembolization (TACE) has been widely used for treating the spontaneous rupture of hepatocellular carcinoma (HCC), no existing model exists for predicting survival. The aim of this study was thus to develop and validate a nomogram for estimating the prognosis in patients with ruptured HCC upon undergoing TACE treatment.Methods:This study included 55 patients with spontaneously ruptured HCC who underwent TACE treatment between January 2015 and April 2019. The diagnosis of spontaneous HCC rupture was based on the disruption of the peritumoral liver capsule with surrounding fluid in the perihepatic region. The prognostic nomogram was constructed using the independent predictors assessed by the multivariate Cox proportional hazards model.Results:The median overall survival (OS) was 6.4 months, with 6-month and 1-year survival rates of 52.7% and 41.8%, respectively. In the univariate analysis, the size of the largest tumor, total bilirubin (TBIL) levels, and aspartate aminotransferase (AST) levels were associated with the OS of patients. Multivariate analysis suggested that TBIL levels (HR =0.358, P=0.036) and diameter of the largest tumor (HR =1.012, P=0.044) were independent prognostic factors for predicting the OS. Based on these variables, we developed and validated a nomogram for the risk stratification of HCC rupture after TACE treatment for individual patients. According to the nomogram risk assessment, we were able to evaluate the approximate 1- and 2-year survival rates based on patients' tumor diameter and TBIL level after TACE treatment of ruptured HCC. The concordance index for the OS prediction was 0.748 (95% CI: 0.691-0.805). This newly developed nomogram represents an intuitive tool for predicting the OS of patients with ruptured HCC.Conclusions:This study indicated that TBIL levels and diameter of the largest tumor were independent prognostic factors for predicting the OS of ruptured HCC. This study may help maximize favorable TACE treatment outcomes.

Portal vein tumor thrombosis (PVTT), a common complication of advanced hepatocellular carcinoma (HCC), remains the bottleneck of the treatments. Liver cancer cells potentially experienced multi-steps during PVTT process, including cancer cells leave from cancer nest, migrate in extracellular matrix, invade the vascular barrier, and colonize in the portal vein. Accumulated evidences have revealed numerous of molecular mechanisms including genetic and epigenetic regulation, cancer stem cells, immunosuppressive microenvironment, hypoxia, et al. contributed to the PVTT formation. In this review, we discuss state-of-the-art PVTT research on the potential molecular mechanisms and experimental models. In addition, we summarize PVTT-associated clinical trials and current treatments for PVTT and suppose perspectives exploring the molecular mechanisms and improving PVTT-related treatment for the future.

Background Accumulating evidence indicates that tumor heterogeneity is characterized by distinct immunosubtypes. However, prior studies have mainly focused on the functions of T cells. The role of tumor-infiltrating B cells in the microenvironment of hepatocellular carcinoma (HCC) requires further investigation. Methods We conducted an integrative analysis of single cell RNA sequencing (scRNA-seq) datasets in HCC tumor samples from Gene Expression Omnibus database. We analyzed the features of B cells in normal liver tissue and HCC. Additionally, we conducted a deconvolution analysis using the matrix of scRNA-seq datasets and the RNA-seq datasets in The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) database. The survival analyses of the TCGA-LIHC cohort with different B cell infiltration rates and was further validated. Finally, we performed immunohistochemistry analysis of primary tumor tissue of HCC patients using antibodies against CD79A and validated the impact of tumor-infiltrating B cells in the prognosis of LIHC. Results We identified several subtypes of B cells in the microenvironment of HCC, including the plasma cells and naïve B cells. The relative ratio of B cells, but not the plasma cells, was significantly decreased in HCC as compared to the normal liver tissue (P<0.05). In addition, genes related to antigen presentation and cell proliferation were decreased in tumor-infiltrating B cells (P<0.05). The observation of B cell infiltration was further validated with the TCGA-LIHC cohort. The overall survival and disease-free survival in HCC patients with higher B-cell infiltration rate were significantly longer than those in the lower infiltration group (P<0.05) in the TCGA-LIHC cohort. Moreover, we demonstrated higher infiltration rates of B cells were significantly associated with a better prognosis of HCC in our cohort. Conclusions Tumor-infiltrating B cells potentially exert a tumor-suppressive function in the microenvironment of HCC and the higher levels of B cell infiltration are associated with a favorable outcome of HCC. Targeted activation of B cells may improve the tumor immune-targeted therapy.

S100 protein family members (S100s) are commonly dysregulated in various tumors including hepatocellular carcinoma (HCC). However, the diverse expression, mutation, prognosis and associations with immune infiltration of S100s in HCC have yet to be analyzed. Herein we investigated the roles of S100s in HCC from the Oncomine, Gene Expression Profiling Interactive Analysis (GEPIA), Human Protein Atlas, Kaplan-Meier Plotter, cBioPortal and TIMER databases. Compared with para-cancer tissues, the expression levels of S100A4/S100A6/S100A10/S100A11/S100A13/S100A14/S100P were higher in HCC tissues, while the expression levels of S100A8/S100A9/S100A12 were decreased in tumor tissues. The mRNA levels of S100A2/S100A7/S100A7A/S100A8/S100A9/S100A11 were correlated with advanced tumor stage. Besides, higher mRNA expressions of S100A6/S100A10/S100A11/S100A13/S100A14/S100P were shown to have shorter overall survival (OS), while higher expression of S100A12 was associated with favorable OS. Further, the mutation rate of S100s was investigated, and the high mutation rate (53%) was associated with shorter OS. Additionally, the expressions of S100s were found to be significantly associated with various immune infiltrating cells. Hence, our results showed that S100A6/S100A10/S100A11/S10012/S100A13/S100A14/S100P may be regarded as new prognostic or therapeutic markers and S100s inhibitors may be helpful in the combination of immunotherapies.

Purpose: Ribosomal protein metallopanstimulin-1 (MPS-1) is implicated in tumorigenesis. However, to date, the underlying role of MPS-1 in the generation, progression and prognosis of hepatocellular carcinoma (HCC) remains unknown. This study aims to investigate the expression of MPS-1 in HCC and its significance for the prognosis of HCC. Methods: The Oncomine and GEPIA databases were used to analyze the expression pattern of MPS-1 in HCC. Immunohistochemical staining was performed on tissue microarrays containing 169 HCC tissue samples to examine the expression of MPS-1. In addition, univariate and multivariate Cox regression analyses and Kaplan-Meier analysis were used to verify the correlation between clinicopathological factors in HCC patients and its clinical prognostic significance. Results: MPS-1 was more highly expressed in HCC than in normal tissues, and MPS-1 expression was correlated with serum AFP levels (P = 0.003), liver cirrhosis (P = 0.024), tumor embolus (P = 0.009) and tumor recurrence (P < 0.003). MPS-1 was an independent prognostic factor for the overall survival of HCC (HR, 1.92; 95% CI, 1.01-3.68), and a higher expression of MPS-1 predicted poorer survival. Furthermore, high expression of MPS-1 indicated a poor prognosis in patients with AFP positivity, cirrhosis or HBsAg positivity. Conclusion: These findings demonstrate that MPS-1 is highly expressed in HCC and serves as an independent prognostic marker, highlighting the potential role of MPS-1 as a novel biomarker and therapeutic target for HCC.

Pyroptosis is a novel kind of cellular necrosis and shown to be involved in cancer progression. However, the diverse expression, prognosis and associations with immune status of pyroptosis-related genes in Hepatocellular carcinoma (HCC) have yet to be analyzed. Herein, the expression profiles and corresponding clinical characteristics of HCC samples were collected from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Then a pyroptosis-related gene signature was built by applying the least absolute shrinkage and selection operator (LASSO) Cox regression model from the TCGA cohort, while the GEO datasets were applied for verification. Twenty-four pyroptosis-related genes were found to be differentially expressed between HCC and normal samples. A five pyroptosis-related gene signature (GSDME, CASP8, SCAF11, NOD2, CASP6) was constructed according to LASSO Cox regression model. Patients in the low-risk group had better survival rates than those in the high-risk group. The risk score was proved to be an independent prognostic factor for overall survival (OS). The risk score correlated with immune infiltrations and immunotherapy responses. GSEA indicated that endocytosis, ubiquitin mediated proteolysis and regulation of autophagy were enriched in the high-risk group, while drug metabolism cytochrome P450 and tryptophan metabolism were enriched in the low-risk group. In conclusion, our pyroptosis-related gene signature can be used for survival prediction and may also predict the response of immunotherapy.

The relationship between immune and inflammatory responses in hepatocellular carcinoma (HCC) has garnered significant interest. In the peripheral blood and tumour microenvironment (TME), neutrophils, which are innate immune cells, crucially respond to various inflammatory factors, leading to tumour progression. To some extent, they affect the clinical treatment strategy and survival among HCC patients. A high circulating neutrophil-to-lymphocyte ratio is a reliable factor that can be used to predict poor outcomes in HCC patients. However, the mechanisms underlying the protumoural effects of circulating neutrophils remain poorly understood. Besides, the distinct role and function of neutrophils at the site of HCC remain relatively unclear, which is partially attributed to their substantial heterogeneity compared with other immune cells. In this review, we firstly discuss the current information available, detailing distinct subsets, functional phenotypes, and the impact of circulating and tumour-infiltrating neutrophils on tumourigenesis in HCC. Furthermore, we describe recent pre-clinical and clinical studies concerning neutrophils for evaluating the feasibility of targeting diverse protumoural aspects to improve therapeutic efficacy, thus paving the way for neutrophil-based treatment, especially in combination with immunotherapy.

目的:分析肝细胞癌(HCC)患者经导管动脉化疗栓塞术(TACE)术前后甲胎蛋白(AFP)的变化,来预测肿瘤的变化和患者的生存期.方法:评估自2005年1月至2007年12月在复旦大学附属中山医院肝癌研究所接受T ACE治疗的HCC患者的资料.根据患者TACE术前及术后1个月AFP浓度的变化,把患者分成3组:明显下降组、小幅下降组和无下降组.比较3组在生存期和疾病控制率(DCR)方面的差异,同时完成单因素和多因素变量分析.结果:在338例患者中,明显下降组患者113例、小幅下降组41例、无下降组184例.明显下降组、小幅下降组、无下降组中位生存期(月)和DCR(％)分别为:16.0和56.2;8.0和37.5;6.0和29.9,明显下降组患者的DCR和预后更优(P<0.001).单因素和多因素分析提示,AFP下降幅度可作为预测总体生存期的独立因素(P<0.001).结论:AFP下降幅度可作为HCC患者接受TACE治疗后,反映患者预后的一个临床指标.

Background: Although transarterial chemoembolization（TACE）has been widely used for spontaneously ruptured hepatocellular carcinoma (HCC), no existing models can be used for survival prediction. Thus, the aim of the present study was to develop and validate a nomogram for risk prediction of HCC rupture after TACE treatment. Methods: The medical records of patients who underwent transarterial chemoembolization therapy from January 2015 to April 2019 at our Hospital were retrospectively screened. HCC was diagnosed by either imaging or histological evaluation according to the AASLD guideline. Factors that were found to be significant in univariate analysis were subjected to multivariate analysis. Factors in the nomogram were identified by multivariate Cox hazard analysis. The concordance index was calculated. Results: This study consisted of 55 patients with spontaneously ruptured HCC receiving TACE at our center from January 2015 to April 2019. The median overall survival was 6.4 months, with 0.5-year and 1-year survival being 52.7% and 41.8%, respectively. In univariate analysis, largest tumor size, total bilirubin level, and aspartate aminotransferase level were associated with overall survival. Multivariate analysis suggested that total bilirubin level and the max diameter of the tumors were independent prognostic factors to predict overall survival. Based on these variables, we developed and validated a nomogram for individual patient risk stratification of HCC rupture after TACE treatment. The concordance index for overall survival prediction was 0.748 (95% CI: 0.691-0.805). Conclusions: Based on the nomogram, we have provided an intuitive tool for estimating the prediction of ruptured HCC patients. This study may help to improve the perioperative strategy and treatment outcome.

目的 探讨SUZ12蛋白在肝癌组织中的表达及其临床意义.方法 采用组织芯片技术联合免疫组织化学法检测SUZ12蛋白在88例肝癌及27例正常肝组织中的表达水平,通过统计学方法分析其潜在的临床意义.结果 SUZ12在肝癌组织中的表达水平高于正常肝组织,其表达阳性率分别为89.24％和56.65％.Kaplan-Meier分析表明,SUZ12低表达患者的术后生存时间明显延长;Cox回归分析提示,SUZ12蛋白高表达组在肝癌AJCC分期(P<0.001)、肿瘤>5 cm(P=0.006)、低分化(P=0.048)、血管侵犯(P<0.001)与SUZ12低表达组有显著统计学差异,并且SUZ12蛋白表达水平可能是肝癌术后预后的独立危险因素.结论 SUZ12蛋白在肝癌及癌旁中的表达明显高于正常肝组织,其高表达与促进肝癌发生、发展相关,并提示SUZ12是一个潜在促癌基因.

Spontaneous rupture is one of the complications of hepatocellular carcinoma (HCC) associated with a high mortality rate. Transcatheter arterial chemoembolization (TACE) has been widely used in patients with ruptured liver tumors. The aim of the present study was to evaluate the benefits and safety of conventional TACE and the disease prognosis following TACE and surgery with regard to the progression of spontaneously ruptured HCC. The clinical data of 70 patients diagnosed with spontaneous rupture of HCC were retrospectively reviewed. The majority of adverse reactions that occurred following treatment were Grade 2 or below. Grade 3/4 events occurred in 20 patients (14.3%), which included gastrointestinal hemorrhage, cardiac failure, pulmonary embolism, shock and recurrent tumor rupture. All of these patients recovered and were discharged following symptomatic and supportive treatment, with the exception of two cases of severe hemorrhagic shock and hepatic failure prior to TACE treatment. These patients did not survive during the period of hospitalization. Multivariate analysis identified that a maximum tumor size >10 cm and a high serum total bilirubin level >30 µmol/l were independent factors for determining overall patient survival rate. Additionally, the overall survival rates at 1, 6 and 12 months were 92.3, 53.8 and 46.2% in the TACE group and 100, 87.1 and 54.8% in the surgery group, respectively. The overall survival rates at 1 and 6 months following TACE were lower than those of the surgery group (P<0.05). However, the overall survival rates at 12 months were similar (P>0.05). Patients in the TACE group had a shorter hospital admission compared with those in the resection group (median 7 vs. 13 days; P<0.01). Therefore, the data demonstrated that conventional TACE therapy was safe and effective for the treatment of spontaneously ruptured HCC. In addition, this type of therapy conferred a similar long-term survival rate with that of open surgery.

Epigallocatechin gallate (EGCG), a major polyphenol in green tea, exhibits diverse biological activities. Previous studies show that EGCG could effectively suppress HBV gene expression and replication, but the role of EGCG in HBV replication and its underlying mechanisms, especially the signaling pathways involved, remain unclear. In this study we investigated the mechanisms underlying EGCG inhibition on HBV replication with a focus on the signaling pathways. We showed that EGCG (12.5−50 μM) dose-dependently inhibited HBV gene expression and replication in HepG2.2.15 cells. Similar results were observed in HBV mice receiving EGCG (25 mg· kg −1 · d −1 , ip) for 5 days. In HepG2.2.15 cells, we showed that EGCG (12.5−50 μM) significantly activate ERK1/2 MAPK signaling, slightly activate p38 MAPK and JAK2/STAT3 signaling, while had no significant effect on the activation of JNK MAPK, PI3K/AKT/mTOR and NF-κB signaling. By using specific inhibitors of these signaling pathways, we demonstrated that ERK1/2 signaling pathway, but not other signaling pathways, was involved in EGCG-mediated inhibition of HBV transcription and replication. Furthermore, we showed that EGCG treatment dose-dependently decreased the expression of hepatocyte nuclear factor 4α (HNF4α) both at the mRNA and protein levels, which could be reversed by pretreatment with the ERK1/2 inhibitor PD98059 (20 μM). Moreover, we revealed that EGCG treatment dose-dependently inhibited the activity of HBV core promoter and the following HBV replication. In summary, our results demonstrate that EGCG inhibits HBV gene expression and replication, which involves ERK1/2-mediated downregulation of HNF4α.These data reveal a novel mechanism for EGCG to inhibit HBV gene expression and replication.

e16115 Background: Patients with advanced stage hepatocellular carcinoma (HCC) have a dismal prognosis. The Barcelona Clinic Liver Cancer (BCLC) staging system does not recommend transarterial chemoembolization (TACE) for treating BCLC C HCC. However, numerous Asian-Pacific institutes still perform TACE for this patient group. There remains a paucity of large real-world data characterizing treatment outcomes of these patients.We aimed to share experiences of 885 cases of BCLC C stage HCC patients treated by TACE in real-world practice. Methods: 885 patients were included in this real-world study who received TACE based monotherapy or combination therapy in liver cancer institute, Fudan University between January 2008 and December 2015. We investigated the clinical outcomes of TACE-based treatment in BCLC C stage HCC. Results: In this cohort of 885 BCLC C HCC patients, vascular invasion was present in 582 patients (65.8%), lymph node involvement was present in 108 patients(12.2%), extrahepatic metastases was present in 232 patients (26.2%).The mean number of TACE per patient was 3.0±1.9 sessions. Median overall survival was 8.0 months (range 3-37) for the entire study population. Of all the patients, 600 patients received TACE-alone treatment, 137 patients received TACE combined Sorafenib treatment, 41 patients received TACE combined local regional treatment, 59 patients received TACE with chemotherapy, 123 patients received TACE with radiotherapy. The median survival time was 6.0 months for TACE-alone group, 11.5 months for TACE+Sorafenib group, 19.8 months for TACE+local regional treatment group, 12.0 month for TACE+chemotherapy group and 12.0 months for TACE+radiotherapy group respectively.These data showed that TACE-based combination therapy can improve overall survival in BCLC C stage HCC, in comparison with TACE alone treatment.After propensity score matching and adjusting for potential confounders, there were still significant survival differences between TACE-alone group and TACE-based combination treatments mentioned above. Conclusions: TACE-based combination therapy can improve overall survival in BCLC C stage HCC patients.

肝细胞癌（hepatocellular carcinoma,HCC）的发病率和死亡率在世界范围内仍居高不下,且呈全球化增长的趋势。欧洲肝脏研究学会（EASL）最近发布了2018年版肝细胞癌临床实践指南,依据近年来大量最新循证医学证据,对2012年EASL与欧洲癌症治疗研究组织（EORTC）联合发布的肝细胞癌诊疗指南进行全面更新和充实,旨在优化HCC患者的诊断和治疗。新版指南对我国临床医师具有较大的参考价值和指导

BACKGROUND:Higher matrix stiffness affects biological behavior of tumor cells, regulates tumor-associated gene/miRNA expression and stemness characteristic, and contributes to tumor invasion and metastasis. However, the linkage between higher matrix stiffness and pre-metastatic niche in hepatocellular carcinoma (HCC) is still largely unknown.METHODS:We comparatively analyzed the expressions of LOX family members in HCC cells grown on different stiffness substrates, and speculated that the secreted LOXL2 may mediate the linkage between higher matrix stiffness and pre-metastatic niche. Subsequently, we investigated the underlying molecular mechanism by which matrix stiffness induced LOXL2 expression in HCC cells, and explored the effects of LOXL2 on pre-metastatic niche formation, such as BMCs recruitment, fibronectin production, MMPs and CXCL12 expression, cell adhesion, etc. RESULTS: Higher matrix stiffness significantly upregulated LOXL2 expression in HCC cells, and activated JNK/c-JUN signaling pathway. Knockdown of integrin β1 and α5 suppressed LOXL2 expression and reversed the activation of above signaling pathway. Additionally, JNK inhibitor attenuated the expressions of p-JNK, p-c-JUN, c-JUN and LOXL2, and shRNA-c-JUN also decreased LOXL2 expression. CM-LV-LOXL2-OE and rhLOXL2 upregulated MMP9 expression and fibronectin production obviously in lung fibroblasts. Moreover, activation of Akt pathway contributed to LOXL2-induced fibronectin upregulation. LOXL2 in CM as chemoattractant increased motility and invasion of BMCs, implicating a significant role of LOXL2 in BMCs recruitment. Except that, CM-LV-LOXL2-OE as chemoattractant also increased the number of migrated HCC cells, and improved chemokine CXCL12 expression in lung fibroblasts. The number of HCC cells adhered to surface of lung fibroblasts treated with CM-LV-LOXL2-OE was remarkably higher than that of the control cells. These results indicated that the secreted LOXL2 facilitated the motility of HCC cells and strengthened CTCs settlement on the remodeled matrix "soil".CONCLUSION:Integrin β1/α5/JNK/c-JUN signaling pathway participates in higher matrix stiffness-induced LOXL2 upregulation in HCC cells. The secreted LOXL2 promotes fibronectin production, MMP9 and CXCL12 expression and BMDCs recruitment to assist pre-metastatic niche formation.

The general prognosis of patients with hepatocellular carcinoma (HCC) remains extremely dismal, due to the high frequency of metastasis. Since 2003, our research group has explored the gene expression profiles of metastasized HCC tissue samples and identified a significant upregulation of CCN3. However, the role and precise pathological function of CCN3 remains elusive. We showed that CCN3 is associated with the poor prognosis of patients with HCC, the malignant phenotype of HCC, and vascular thrombosis. We further evaluated the negative roles of CCN3 in vitro and in vivo, and identified osteopontin (OPN), and coagulation factors tissue factor (TF) and thrombin as the leading genes downstream of CCN3, that are positively associated with HCC cell stemness. We demonstrated that overexpressed CCN3 in HCC cells leads to enhanced survival and increased number of pulmonary metastases in vivo. The elevated levels of OPN and TF were associated with signal activation of nuclear factor κB (NFκB) and extracellular signal-regulated kinases (ERK). Our findings suggest CCN3 is a potential therapeutic target that would affect the upregulation of OPN and coagulation factors, which would lead to an enhanced stemness and blood coagulation microenvironment in HCC tissue.