New insights into the mechanism of resistance to lenvatinib and strategies for lenvatinib sensitization in hepatocellular carcinoma.

Lenvatinib is a multikinase inhibitor that suppresses vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor α (PDGFRα), as well as the proto-oncogenes RET and KIT. Lenvatinib has been approved by the US Food and Drug Administration (FDA) for the first-line treatment of hepatocellular carcinoma (HCC) due to its superior efficacy when compared to sorafenib. Unfortunately, the development of drug resistance to lenvatinib is becoming increasingly common. Thus, there is an urgent need to identify the factors that lead to drug resistance and ways to mitigate it. We summarize the molecular mechanisms that lead to lenvatinib resistance (LR) in HCC, which involve programmed cell death (PCD), translocation processes, and changes in the tumor microenvironment (TME), and provide strategies to reverse resistance.

A combined pre- and intra-operative nomogram in evaluation of degrees of liver cirrhosis predicts post-hepatectomy liver failure: a multicenter prospective study.

Background: Adequate evaluation of degrees of liver cirrhosis is essential in surgical treatment of hepatocellular carcinoma (HCC) patients. The impact of the degrees of cirrhosis on prediction of post-hepatectomy liver failure (PHLF) remains poorly defined. This study aimed to construct and validate a combined pre- and intra-operative nomogram based on the degrees of cirrhosis in predicting PHLF in HCC patients using prospective multi-center's data. Methods: Consecutive HCC patients who underwent hepatectomy between May 18, 2019 and Dec 19, 2020 were enrolled at five tertiary hospitals. Preoperative cirrhotic severity scoring (CSS) and intra-operative direct liver stiffness measurement (DSM) were performed to correlate with the Laennec histopathological grading system. The performances of the pre-operative nomogram and combined pre- and intra-operative nomogram in predicting PHLF were compared with conventional predictive models of PHLF. Results: For 327 patients in this study, histopathological studies showed the rates of HCC patients with no, mild, moderate, and severe cirrhosis were 41.9%, 29.1%, 22.9%, and 6.1%, respectively. Either CSS or DSM was closely correlated with histopathological stages of cirrhosis. Thirty-three (10.1%) patients developed PHLF. The 30- and 90-day mortality rates were 0.9%. Multivariate regression analysis showed four pre-operative variables [HBV-DNA level, ICG-R15, prothrombin time (PT), and CSS], and one intra-operative variable (DSM) to be independent risk factors of PHLF. The pre-operative nomogram was constructed based on these four pre-operative variables together with total bilirubin. The combined pre- and intra-operative nomogram was constructed by adding the intra-operative DSM. The pre-operative nomogram was better than the conventional models in predicting PHLF. The prediction was further improved with the combined pre- and intra-operative nomogram. Conclusions: The combined pre- and intra-operative nomogram further improved prediction of PHLF when compared with the pre-operative nomogram. Trial Registration: Clinicaltrials.gov Identifier: NCT04076631.

18[Formula: see text]-Glycyrrhetinic Acid Inhibits TGF-[Formula: see text]-Induced Epithelial-to-Mesenchymal Transition and Metastasis of Hepatocellular Carcinoma by Targeting STAT3.

18[Formula: see text]-glycyrrhetinic acid (GA) is the active ingredient of the traditional Chinese medicinal herb Glycyrrhizae radix et rhizoma. We previously demonstrated that GA inhibited tumor growth in hepatocellular carcinoma (HCC). However, the effect of GA on transforming growth factor-[Formula: see text] (TGF-[Formula: see text]-induced epithelial-mesenchymal transition (EMT) and metastasis were still unclear. In this study, in vitro transwell assays and immunofluorescence (IF) demonstrated that GA inhibited TGF-[Formula: see text]-induced migration, invasion and EMT of HCC cells. However, it had little effect on the inhibition of proliferation by TGF-[Formula: see text]. Moreover, we confirmed that GA suppressed the metastasis of HCC cells in vivousing an ectopic lung metastasis model. Furthermore, we found that GA inhibited TGF-[Formula: see text]-induced EMT mainly by reducing the phosphorylation of signal transducer and activator of transcription 3 (STAT3), which played an essential role in TGF-[Formula: see text]-induced EMT and cell mobility. Mechanistically, GA inhibited the phosphorylation of STAT3 by increasing the expression of Src homology 2 domain-containing protein tyrosine phosphatases 1 and 2 (SHP1 and SHP2). Therefore, we concluded that GA inhibited TGF-[Formula: see text]-induced EMT and metastasis via the SHP1&SHP2/STAT3/Snail pathway. Our data provide an attractive therapeutic target for future multimodal management of HCC.

TGF-ß1 accelerates the hepatitis B virus X-induced malignant transformation of hepatic progenitor cells by upregulating miR-199a-3p.

Increasing evidence has suggested that liver cancer arises partially from transformed hepatic progenitor cells (HPCs). However, the detailed mechanisms underlying HPC transformation are poorly understood. In this study, we provide evidence linking the coexistence of hepatitis B virus X protein (HBx) and transforming growth factor beta 1 (TGF-ß1) with miR-199a-3p in the malignant transformation of HPCs. The examination of liver cancer specimens demonstrated that HBx and TGF-ß1 expression was positively correlated with epithelial cell adhesion molecule (EpCAM) and cluster of differentiation 90 (CD90). Importantly, EpCAM and CD90 expression was much higher in the specimens expressing both high HBx and high TGF-ß1 than in those with high HBx or high TGF-ß1 and the double-low-expression group. HBx and TGF-ß1 double-high expression was significantly associated with poor prognosis in primary liver cancer. We also found that HBx and TGF-ß1 induced the transformation of HPCs into hepatic cancer stem cells and promoted epithelial-mesenchymal transformation, which was further enhanced by concomitant HBx and TGF-ß1 exposure. Moreover, activation of the c-Jun N-terminal kinase (JNK)/c-Jun pathway was involved in the malignant transformation of HPCs. miR-199a-3p was identified as a significantly upregulated microRNA in HPCs upon HBx and TGF-ß1 exposure, which were shown to promote miR-199a-3p expression via c-Jun-mediated activation. Finally, we found that miR-199a-3p was responsible for the malignant transformation of HPCs. In conclusion, our results provide evidence that TGF-ß1 cooperates with HBx to promote the malignant transformation of HPCs through a JNK/c-Jun/miR-199a-3p-dependent pathway. This may open new avenues for therapeutic interventions targeting the malignant transformation of HPCs in treating liver cancer.

Resection might be a meaningful choice for hepatocellular carcinoma with portal vein thrombosis: A systematic review and meta-analysis.

BACKGROUND: According to the Barcelona Clinic Liver Cancer (BCLC) staging system, the presence of portal vein tumor thrombosis (PVTT) is considered to indicate an advanced stage of hepatocellular carcinoma (HCC) with nearly no cure. Hepatic resection and transarterial chemoembolization (TACE) have recently been recommended for treatment of HCC with PVTT. METHODS: We conducted a systematic review to compare the overall survival between patients with HCC and PVTT undergoing hepatectomy, TACE or conservative treatment including sorafenib chemotherapy. The PubMed, Web of Science, and Cochrane Library databases were searched. All relevant studies were considered. Hazard ratios with 95% confidence intervals were calculated for comparison of the cumulative overall survival. Ten retrospective studies met the inclusion criteria and were included in the review. RESULTS: Overall survival was not higher in the hepatectomy group than TACE group. But survival rate was higher in hepatectomy group than conservative group. The subgroup analysis demonstrated that hepatectomy was superior in patients without PVTT in the main trunk than in patients with main portal vein invasion. In patients without main PVTT, hepatectomy has showed more benefit than TACE. However, there has been no significant difference between the hepatectomy and TACE groups among patients with main PVTT. CONCLUSION: For patients with resectable HCC and PVTT, hepatectomy might be more effective in patients without PVTT in the main trunk than TACE or conservative treatment.

Acute portal vein thrombosis after hepatectomy in a patient with hepatolithiasis: A case report and review of the literature.

RATIONALE: Portal vein thrombosis is defined as any thrombosis that develops in the portal vein system. It is considered a very rare and extremely lethal complication of hepatopancreatobiliary surgery. PATIENT CONCERNS: Acute portal vein thrombosis after hepatectomy in patients with hepatolithiasisis very rare. Acute portal vein thrombosis is considered as a dangerous complication after hepatectomy. It is easy to ignore the symptom of acute portal vein thrombosis. Once the appropriate time of treatment is past, it would lead to patients' death. DIAGNOSE: Acute portal vein thrombosis after hepatectomy in a patient with hepatolithiasis INTERVENTIONS:: We consider anticoagulation therapy and percutaneous transhepatic portal vein puncture and thrombectomy once the diagnosis of acute portal vein thrombosis is confirmed. OUTCOMES: The patient's liver function continued to deteriorate, eventually resulting in death. LESSONS: Acute portal vein thrombosis after hepatectomy is difficult to diagnose. The management of acute portal vein thrombosis remains controversial according to its severity, location or time of discovering.

Association of PD-L1 and HIF-1α Coexpression with Poor Prognosis in Hepatocellular Carcinoma.

OBJECTIVE: To investigate the correlation between the expression of PD-L1 and HIF-1α in hepatocellular carcinoma (HCC) tissue and further analyze the association with clinical parameters and the prognostic value of coexpression in HCC patients. METHODS: We assessed the expression of PD-L1 and HIF-1α by immunohistochemistry in tumor tissue from 90 HCC patients who underwent curative hepatectomy. The results were validated in an independent cohort of additional 90 HCC patients. RESULTS: PD-L1 and HIF-1α exhibited in tumor tissue high expression rates of 41.11% (37/90) and 43.33% (43/90), respectively, and their expressions were positively correlated (r = 0.563, P < .01). High expression of PD-L1 was significantly associated with low albumin levels (P < .05); high expression of HIF-1α was significantly correlated with high alpha-fetoprotein (AFP) levels and low albumin levels (P < .05); high expression of both PD-L1 and HIF-1α was also significantly associated with high AFP levels and low albumin levels (P < .05). High expression of PD-L1, HIF-1α, as well as both PD-L1 and HIF-1 α was respectively significantly associated with worse overall survival (OS) and disease-free survival (DFS) (P < .05). Patients with co-overexpression of PD-L1 and HIF-1α had the worst prognosis compared with other groups. Additionally, multivariate Cox regression models suggested that high expression of PD-L1, HIF-1α, as well as both PD-L1 and HIF-1α was an independent prognostic factor for OS and DFS (P < .05). Furthermore, the positive correlation and prognostic values of PD-L1 and HIF-1α were validated in an independent data set. CONCLUSION: We demonstrated that HCC patients with co-overexpression of PD-L1 and HIF-1α in tumor tissue had a significantly higher risk of recurrence or metastasis and death compared with others. Therefore, more frequent follow-up is needed for patients with co-overexpression of PD-L1 and HIF-1α. At the same time, a combinational therapy with HIF-1α inhibitors in conjunction with PD-L1 blockade may be beneficial for HCC patients with co-overexpression in the future.

Laparoscopic versus traditional open splenectomy for hepatocellular carcinoma with hypersplenism.

This study aimed to examine the efficacy of the laparoscopic vs. traditional open splenectomy for hepatocellular carcinoma (HCC) with hypersplenism. Between 2002 and 2013, 51 Chinese HCC patients with hypersplenism underwent either simultaneous laparoscopic splenectomy plus anticancer therapies (Lap-S&A) (n=25) or traditional open splenectomy plus anti-cancer therapies (TOS&A) (n=26). The outcomes were reviewed during and after the operation. Anti-cancer therapies for HCC included laparoscopic hepatectomy (LH) and laparoscopic microwave ablation (LMA). The results showed that there was no significant difference in the operating time between the two groups, but the blood loss and blood transfusion were less, pain intensity after surgery was weaker, the time to first bowel movement, time to the first flatus and postoperative hospital stay were shorter, and the postoperative complication rate and the readmission rate were lower in the Lap-S&A group than in the TO-S&A group. Two patients in the Lap-S&A group and one patient in the TO-S&A group died 30 days after surgery. However, no significant difference in the mortality rate was noted between the two groups. It was concluded that simultaneous Lap-S&A holds the advantages of more extensive indications, lower complication incidence and less operative expenditure than conventional open approach and it is a feasible and safe approach for HCC with hypersplenism.

The niche of hepatic cancer stem cell and cancer recurrence.

Currently, surgical resection is one of only a few options for treating hepatocellular carcinoma (HCC). Unfortunately, postoperative tumor recurrence remains almost inevitable despite additional radiation or chemotherapy treatment following radical resection. Clinical observations and a growing body of experimental evidence have led to speculation that there is a population of persistent hepatic cancer stem cells (HCSCs), which are difficult to completely remove surgically. HCSCs are most often in a quiescent state and thought to reside in a specific microenvironment known as a niche that provides the cues necessary for HCSCs to maintain a balance of self-renewal and differentiation. Residual HCSCs following surgery may alter their fate by invading into the blood circulation. Furthermore, it remains to be determined if hepatectomy render the postoperative niche more favorable for the survival and growth of HCSCs, and therefore the recurrence of HCC. A better understanding of the mechanisms for HCSCs self-renewal, invasion and recurrence may provide new insights into curative strategies for treating HCC.

Oval cell response is attenuated by depletion of liver resident macrophages in the 2-AAF/partial hepatectomy rat.

BACKGROUND/AIMS: Macrophages are known to play an important role in hepatocyte mediated liver regeneration by secreting inflammatory mediators. However, there is little information available on the role of resident macrophages in oval cell mediated liver regeneration. In the present study we aimed to investigate the role of macrophages in oval cell expansion induced by 2-acetylaminofluorene/partial hepatectomy (2-AAF/PH) in rats. METHODOLOGY/PRINCIPAL FINDINGS: We depleted macrophages in the liver of 2-AAF/PH treated rats by injecting liposome encapsulated clodronate 48 hours before PH. Regeneration of remnant liver mass, as well as proliferation and differentiation of oval cells were measured. We found that macrophage-depleted rats suffered higher mortality and liver transaminase levels. We also showed that depletion of macrophages yielded a significant decrease of EPCAM and PCK positive oval cells in immunohistochemical stained liver sections 9 days after PH. Meanwhile, oval cell differentiation was also attenuated as a result of macrophage depletion, as large foci of small basophilic hepatocytes were observed by day 9 following hepatectomy in control rats whereas they were almost absent in macrophage depleted rats. Accordingly, real-time polymerase chain reaction analysis showed lower expression of albumin mRNA in macrophage depleted livers. Then we assessed whether macrophage depletion may affect hepatic production of stimulating cytokines for liver regeneration. We showed that macrophage-depletion significantly inhibited hepatic expression of tumor necrosis factor-α and interleukin-6, along with a lack of signal transducer and activator of transcription 3 phosphorylation during the early period following hepatectomy. CONCLUSIONS: These data indicate that macrophages play an important role in oval cell mediated liver regeneration in the 2-AAF/PH model.

Hepatic oval cell lines generate hepatocellular carcinoma following transfection with HBx gene and treatment with aflatoxin B1 in vivo.

Hepatic oval cells (HOC) are considered to be the stem cells of the liver and have been linked to the development of hepatic malignancies. Studies have demonstrated that chronic hepatitis B virus (HBV) infection and dietary aflatoxin B1 (AFB1) exposure are among the most important risk factors for the development of hepatocellular carcinoma (HCC). However, little research has been done to evaluate the role of oval cells in these two environmental factors on hepatocarcinogenesis. In this study, partial transformation of rat HOC (LE/6) were accomplished by transfected HBV x gene (HBx), and then transfected cells were implanted both intra-hepatically and subcutaneously into nude mice treated with AFB1 in vivo. We found the oval cells produced tumors (4/24 of the animals) in liver following transfection with HBx gene and treatment with AFB1. These intrahepatic tumors included HCC cells (immunopositive for HepParl, ALB, CK8 and AFP) and mesenchymal cells (immunopositive for Vimentin and SMA). Whereas mesenchymal tumors were observed at the subcutaneous tissue with a similar rate in all controls treated with cell lines (10/24 in HBx-oval cells/AFB1 group, 8/20 in HBx-oval cells/non-AFB1 group, 10/20 in non-HBx/AFB1 group; 9/20 in non-HBx/non-AFB1 group). Conversely, none of the controls developed intrahepatic tumors. These results provide an evidence that oval cells have the capacity to generate HCC through the combined effects of the HBx and AFB1 in the liver microenvironment.

[Differentiation of hepatic oval cell into mature hepatocyte induced by hepatic stellate cells].

OBJECTIVE: To investigate the role of hepatic stellate cells in the differentiation of hepatic oval cells into adult hepatocyte. METHODS: The oval cell were cocultured with primary hepatic stellate cells (HSC) in the same well (M-coculture) or separately cultured with HSC by millIcell (S-coculture). Oval cells were cultured alone as control; the expression of adult hepatocyte marker HNF-4alpha, albumin, and oval cell marker AFP, CK-19 in each group were detected by real-time PCR and western-blot. Phenotype changes were observed by transmission electron microscope (TEM); PAS staining was used to detect the quantity of glycogen granule in oval cell. Albumin level in supernatant was detected using ELISA kit. RESULT: (1) The relative level of HNF-4alpha and albumin mRNA expression compared with pre-coculture: M-coculture: HNF-4a: 1.9+/-0.2, 10.7+/-1.2, 12.0+/-1.3; albumin: 5.7+/-1.6, 110.7+/-13.7, 173.6+/-22.3. S-coculture: 1.4+/-0.1, 3.2+/-0.6, 8.9+/-1.4 times; albumin: 2.9+/-1.4, 22.3+/-8.5, 96.3+/-16.3. The relative level of HNF-4a and albumin mRNA expression in coculture group (M- and S-coculture) were higher than control group (LSD-t: 32.98, 10.08, 13.38, 7.96; P less than 0.01); and a higher level of HNF-4a and albumin was found in M-coculture group compared to S-coculture group (LSD-t: 32.98, 25.65; P less than 0.01). The relative level of AFP and CK-19 mRNA expression compared with pre-coculture: M-coculture: 1.1+/-0.2, 0.2+/-0.0, 0.0+/-0.0; S-coculture group: AFP: 1.0+/-0.2, 0.2+/-0.1, 0.1+/-0.0; CK-19: 0.6+/-0.1, 0.1+/-0.0, 0.0+/-0.0; control group: AFP: 1.0+/-0.1, 1.0+/-0.1, 1.1+/-0.1, CK-19: 1.0+/-0.1, 1.1+/-0.1, 1.0+/-0.1. The relative level of AFP and CK-19 mRNA expression in coculture group (M- and S-coculture) were lower than that in control group (LSD-t: 37.99, 34.50, 13.59, 22.46; P less than 0.01). (2) The albumin secretion was detected in M-coculture: 14 day: (15.30+/-0.09) ng/ml, 21: (20.98+/-0.12) ng/ml; S-coculture: 14 day: (11.41+/-0.13) ng/ml, 21 day:(15.12+/-0.17) ng/ml. (3) It showed more organelles such as endoplasmic reticulum, mitochondrion and Golgi apparatus in oval cells cocultured with HSC. And cholangiole-like structure appeared between oval cells cocultured with HSC. (4) PAS staining showed glycogen granules could be observed in coculture groups. CONCLUSION: HSC can induce differentiation of oval cell into mature hepatocyte.

The epithelial-mesenchymal transition promotes transdifferentiation of subcutaneously implanted hepatic oval cells into mesenchymal tumor tissue.

Hepatic oval cells are thought to represent facultative hepatic epithelial stem cells in liver in which damage inhibits hepatocyte proliferation and liver regeneration. The LE/6 hepatic stem cell line was derived from the liver of male Sprague-Dawley rats fed a choline-deficient diet containing 0.1% ethionine. They are histochemically characterized by their expression of hepatocytic (hepPar1), cholangiocytic cytokeratin (CK19), hepatic progenitor cell (OV-6), and hematopoietic stem cell (c-kit) markers. In this study, we transplanted LE/6 cells by subcutaneous injection into adult female nude mice, and examined their engraftment and differentiation potential in the subcutaneous microenvironment in vivo. Our results demonstrated that following subcutaneous transplantation, differentiation of LE/6 cells into mesenchymal tumor tissue (MTT) was associated with reduced E-cadherin expression, upregulation of E-cadherin repressor molecules (Snail proteins), and increased expression of vimentin and N-cadherin, all of these events are characteristic of the epithelial-mesenchymal transition (EMT).

Hepatic non-parenchymal cells and extracellular matrix participate in oval cell-mediated liver regeneration.

AIM: To elucidate the interaction between non-parenchymal cells, extracellular matrix and oval cells during the restituting process of liver injury induced by partial hepatectomy (PH). METHODS: We examined the localization of oval cells, non-parenchymal cells, and the extracellular matrix components using immunohistochemical and double immunofluorescent analysis during the proliferation and differentiation of oval cells in N-2-acetylaminofluorene (2-AAF)/PH rat model. RESULTS: By day 2 after PH, small oval cells began to proliferate around the portal area. Most of stellate cells and laminin were present along the hepatic sinusoids in the periportal area. Kupffer cells and fibronectin markedly increased in the whole hepatic lobule. From day 4 to 9, oval cells spread further into hepatic parenchyma, closely associated with stellate cells, fibronectin and laminin. Kupffer cells admixed with oval cells by day 6 and then decreased in the periportal zone. From day 12 to 15, most of hepatic stellate cells (HSCs), laminin and fibronectin located around the small hepatocyte nodus, and minority of them appeared in the nodus. Kupffer cells were mainly limited in the pericentral sinusoids. After day 18, the normal liver lobule structures began to recover. CONCLUSION: Local hepatic microenvironment may participate in the oval cell-mediated liver regeneration through the cell-cell and cell-matrix interactions.

[Expressions of ATP binding cassette transporter genes in rat hepatic oval cells].

OBJECTIVE: Liver regeneration occurs through hepatocytes after acute liver injury. However, severe liver injury activates bipotential oval cells from canals of Hering which can differentiate into hepatocytes and biliary epithelial cells. Most models of oval cell activation have employed potential carcinogens to inhibit hepatocyte replication in the face of a regenerative stimulus. Oval cells must be able to withstand the toxic milieu of the damaged liver. ATP binding cassette transporters are cytoprotective efflux pumps that may contribute to the protection of these cells. The aim of this study was to determine the ABC transporter expressions in hepatic oval cells. METHODS: A rat model was established by feeding 2-acetylaminofluorene combined with partial hepatectomy to activate hepatic oval cells. Oval cells were isolated and purified using selective enzymatic digestion and density gradient centrifugation from the heterogeneous hepatic cell population. The expressions of ABC transporter gene, including MDR1, MRP1 and Bcrp1, in isolated hepatic oval cells and hepatocytes were measured by quantitative real-time reverse transcription-polymerase chain reaction and those in rat liver tissues were measured by immunohistochemistry. RESULTS: Compared to those in the rat hepatocytes, mRNA expressions of the genes encoding MDR1, MRP1 and Bcrp1 were increased up to 9-, 1.5- and 13.8-folds in hepatic oval cells. Immunohistochemical staining of rat liver slides demonstrated that the expression of MDR1 proteins was found around periportal areas, and Bcrp1 protein was found located on cell membranes. CONCLUSION: Hepatic oval cells express high levels of the ABC transporter gene that may have cytoprotective functions during severe hepatotoxicity.