Sex disparity in clinical characteristics and long-term prognosis after liver resection for patients with intrahepatic cholangiocarcinoma: A propensity score matching analysis.

Aim: To compare the survival outcomes between male and female patients with intrahepatic cholangiocarcinoma who underwent liver resection. Methods: Data from 976 consecutive intrahepatic cholangiocarcinoma patients undergoing liver resection between January 2005 and May 2013 at the Eastern Hepatobiliary Surgery Hospital were prospectively collected and retrospectively reviewed. Patient clinicopathological characteristics, overall survival, and cumulative recurrence rates were compared between male and female patients using propensity score matching. Results: Propensity score matching generated 313 matched pairs of patients. Among the entire cohort, the 1-, 3-, and 5-year overall survival and recurrence rates of the male and female patients were 60.2 %, 37.3 %, and 27.7 % vs. 65.8 %, 40.4 %, and 31.0 % (P = 0.380) and 50.6 %, 67.4 %, and 74.2 % vs. 44.4 %, 63.5 %, and 69.6 % (P = 0.123), respectively. In the matched cohort, the 1-, 3-, and 5-year overall survival and recurrence rates of the male and female patients were 60.6 %, 35.9 % and 22.4 % vs. 66.4 %, 40.6 % and 31.1 % (P = 0.041) and 51.5 %, 69.3 % and 83.9 % vs. 44.3 %, 63.6 %, and 69.9 % (P = 0.041), respectively. After adjustment for other confounding variables by multivariate Cox regression analysis, male sex was independently associated with worse overall survival (hazard ratio = 1.322, 95 % confidence interval: 1.079-1.621, P = 0.007) and tumor recurrence (hazard ratio = 1.337, 95 % confidence interval: 1.088-1.645, P = 0.006). A subgroup analysis of patients younger than 55 years old after propensity score matching showed that male patients had significantly worse overall survival and higher recurrence rates than female patients after surgery, while no significant difference in long-term overall survival and recurrence was observed between male and female patients older than 55 years old after propensity score matching. Conclusion: Male sex was an independent risk factor for overall survival and tumor recurrence in patients after liver resection for intrahepatic cholangiocarcinoma.

m6A RNA methylation-mediated upregulation of HLF promotes intrahepatic cholangiocarcinoma progression by regulating the FZD4/ß-catenin signaling pathway.

Hepatic leukemia factor (HLF) is aberrantly expressed in human malignancies. However, its role in regulating intrahepatic cholangiocarcinoma (ICC) remains unclear. This study aimed to define the role of HLF in ICC progression. Here, we showed that HLF expression is upregulated in ICC and predicts the poor prognosis in patients. Mechanistically, HLF activation in ICC is mediated by METTL3-dependent m6A methylation of the HLF mRNA. As per the results from the loss- or gain-of-function experiments, HLF promoted the self-renewal, tumorigenicity, proliferation and metastasis of ICC cells. RNA-seq and CUT&Tag analyses showed that frizzled-4 (FZD4) and forkhead box Q1 (FOXQ1) are target genes of HLF. Moreover, FOXQ1 transcriptionally activates METTL3 expression, forming a positive feedback loop, which subsequently activates WNT/ß-catenin signaling and downstream tumor stemness. Furthermore, HLF expression was positively correlated with METTL3, IGF2BP3, FZD4 and FOXQ1 expression in ICC tissues in a large ICC cohort. The combined IHC panels exhibited a better prognostic value for patients with ICC than any of these components alone. In conclusions, these findings demonstrated that the METTL3/HLF/FOXQ1 regulatory circuit drove FZD4-mediated WNT/ß-catenin activation in ICC progression, suggesting that targeting this axis could be novel therapeutic strategy for ICC.

High expression of aurora kinase B predicts poor prognosis in hepatocellular carcinoma after curative surgery and its effects on the tumor microenvironment.

Background: Currently, the only broadly used biomarker for hepatocellular carcinoma (HCC), alpha fetoprotein (AFP), has multiple limitations and the need for novel biomarkers is urgent. Aurora kinase B (AURKB) is a key mitotic protein kinase which performs a critical function in cell cycle progression. Nonetheless, neither the function nor the mechanism of AURKB in HCC following curative surgery is fully grasped at this time. This study sought to evaluate the impact of AURKB on prognosis and the tumor immune microenvironment (TIME) in HCC. Methods: We evaluated both the expression profile of AURKB in HCC and its clinical value using online databases and clinical specimens. The prognostic value of AURKB was studied by Kaplan-Meier survival analysis, and the link between AURKB and tumor-infiltrating immune cells (TIICs) were analyzed. Results: We found the mRNA expression patterns of AURKB were remarkably upregulated in HCC in contrast with adjoining normal tissues (P<0.001). Upregulation of the AURKB protein in HCC was additionally verified by clinical samples. The expression of AURKB was substantially associated with Child-Pugh, microvascular invasion (MVI), Edmondson-Steiner grade, and tumor recurrence. Furthermore, patients diagnosed with HCC who had a low AURKB expression had a better. Our data suggested age [hazard ratio (HR): 1.34], alanine aminotransferase (ALT) (HR: 1.65), tumor size (HR: 1.99), mor number (HR: 1.60), MVI (HR: 1.93), grade (HR: 5.58), and AURKB expression (HR: 3.63) independently functioned as prognostic risk indicators for HCC (P<0.05). Importantly, we also found AURKB expression was inversely linked to resting natural killer (NK) cells, M2 macrophages, activated mast cells, and naive B cells, and positively linked to M0 macrophages, T follicular helper cells (Tfh), regulatory T cells (Treg), and resting myeloid dendritic cells. In addition, AURKB expression was also positively linked to the immune checkpoints of PDCD1, CD274, CTLA4, and LAG3. Finally, 1,696 DEGs were discovered, and were predominantly implicated in chromosome segregation, cell cycle, xenobiotic metabolic process, calcium signaling pathway, bile secretion, tyrosine metabolism, and DNA replication. Conclusions: AURKB may be a potential prognostic biomarker for HCC after curative surgery, which correlates with MVI and the TIME in HCC.

Antibody array-based proteomic screening of novel biomarkers in malignant biliary stricture.

BACKGROUND: Distinguishing between benign and malignant bile duct strictures has long been a diagnostic challenge in clinical practice. OBJECTIVE: This study aimed to discover novel biomarkers in bile to improve the diagnostic accuracy of malignant biliary strictures. METHODS: Bile samples were collected from 6 patients with malignant or benign biliary stricture, respectively. Protein profiles of the bile were analyzed with a semi-quantitative human antibody array of 440 proteins. Then the differential expressed proteins were screened by Venn diagram analysis. Following this, the accuracy of these potential biomarkers for discriminating between malignant and non-malignant biliary strictures was validated in a larger (n= 40) group of patients using ROC analysis and the best biomarker combination was further selected by lasso analysis. RESULTS: Twenty proteins were found differentially expressed in malignant versus benign biliary strictures, 6 of which were identified by Venn diagram analysis to be up-regulated regardless of the location of biliary strictures. Among the 6 biomarkers, bile lipocalin-2, P-cadherin, and adipsin showed better diagnostic utility than that of bile CA19-9. Lasso analysis identified that lipocalin-2, P-cadherin and CA19-9 as a group of makers best distinguished malignant from benign strictures. CONCLUSIONS: Lipocalin-2 and P-cadherin measurements in bile could be clinically useful for the detection of malignant biliary strictures.

ACADL plays a tumor-suppressor role by targeting Hippo/YAP signaling in hepatocellular carcinoma.

Long-chain acyl-CoA dehydrogenase (ACADL) is a mitochondrial enzyme that catalyzes the initial step of fatty acid oxidation, but the role of ACADL in tumor biology remains largely unknown. Here, we found that ACADL was frequently downregulated in hepatocellular carcinoma (HCC), and its low expression was significantly correlated with poor clinical prognosis of HCC patients. Restoring the expression of ACADL in HCC cells resulted cell cycle arrest and growth suppression through suppressing Hippo/YAP signaling evidenced by decreased YAP nuclear accumulation and downstream target genes expression. Reactivation of YAP by XMU-MP-1 diminished the inhibitory effect of ACADL on HCC growth. More importantly, the nuclear accumulation of YAP was negatively correlated with ACADL expression levels in HCC specimens, and YAP inhibitor verteporfin effectively suppressed growth of HCC organoids with low ACADL expression. Together, our findings highlight a novel function of ACADL in regulating HCC growth and targeting ACADL/Yap may be a potential strategy for HCC precise treatment.

High Serum Levels of Cholesterol Increase Antitumor Functions of Nature Killer Cells and Reduce Growth of Liver Tumors in Mice.

BACKGROUND AND AIMS: The relationship between serum cholesterol level and development of hepatocellular carcinoma (HCC) remains unclear. We investigated the effects of serum cholesterol level on development of liver tumors in mice. METHODS: We performed studies with C57BL/6J mice, mice with disruption of the low-density lipoprotein receptor gene (Ldlr-/-mice), and mice with conditional deletion of nature killer (NK) cells (NKdele mice). Some C57BL/6J and NKdele mice were given injections of diethylinitrosamine to induce liver tumor formation. Mice were placed on a normal diet (ND) or high-cholesterol diet (HCD) to induce high serum levels of cholesterol. We also studied mice with homozygous disruption of ApoE (ApoE-/- mice), which spontaneously develop high serum cholesterol. C57BL/6J and NKdele mice on the ND or HCD were implanted with Hep1-6 (mouse hepatoma) cells and growth of xenograft tumors and lung metastases were monitored. Blood samples were collected from mice and analyzed by biochemistry and flow cytometry; liver and tumor tissues were collected and analyzed by histology, immunohistochemistry, and RNA-sequencing analysis. NK cells were isolated from mice and analyzed for cholesterol content, lipid raft formation, immune signaling, and changes in functions. We obtained matched tumor tissues and blood samples from 30 patients with HCC and blood samples from 40 healthy volunteers; levels of cholesterol and cytotoxicity of NK cells were measured. RESULTS: C57BL/6J mice on HCD and ApoE-/- mice with high serum levels of cholesterol developed fewer and smaller liver tumors and lung metastases after diethylinitrosamine injection or implantation of Hep1-6 cells than mice on ND. Liver tumors from HCD-fed mice and ApoE-/- mice had increased numbers of NK cells compared to tumors from ND-fed mice. NKdele mice or mice with antibody-based depletion for NK cells showed similar tumor number and size in ND and HCD groups after diethylinitrosamine injection or implantation of Hep1-6 cells. NK cells isolated from C57BL/6J mice fed with HCD had increased expression of NK cell-activating receptors (natural cytotoxicity triggering receptor 1 and natural killer group 2, member D), markers of effector function (granzyme B and perforin), and cytokines and chemokines compared with NK cells from mice on ND; these NK cells also had enhanced cytotoxic activity against mouse hepatoma cells, accumulated cholesterol, increased lipid raft formation, and immune signaling activation. NK cells isolated from HCD-fed Ldlr-/- mice did not have increased cholesterol content or cytotoxic activity against mouse hepatoma cells compared with ND-fed Ldlr-/- mice. Serum levels of cholesterol correlated with number and activity of NK cells isolated from human HCCs. CONCLUSIONS: Mice with increased serum levels of cholesterol due to an HCD or genetic disruption of ApoE develop fewer and smaller tumors after injection of hepatoma cells or a chemical carcinogen. We found cholesterol to accumulate in NK cells and activate their effector functions against hepatoma cells. Strategies to increase cholesterol uptake by NK cells can be developed for treatment of HCC.

Impact of Anatomical Versus Non-anatomical Liver Resection on Short- and Long-Term Outcomes for Patients with Intrahepatic Cholangiocarcinoma.

OBJECTIVE: The aim of this study was to examine the impact of anatomical resection (AR) versus non-anatomical resection (NAR) on the survival outcomes in patients with intrahepatic cholangiocarcinoma (ICC). PATIENTS AND METHODS: Data on 702 consecutive patients who underwent either AR (n = 319) or NAR (n = 383) for ICC were reviewed. Disease-free survival (DFS) and overall survival (OS) following AR versus NAR was compared using propensity score matching (PSM). Subgroups of patients who benefited from AR versus NAR were examined after being stratified by the 8th TNM staging of ICC. RESULTS: AR and NAR had similar complication rates (26.6% vs. 25.1%, p = 0.634). AR was associated with better 1-, 3-, and 5-year DFS and OS rates compared with NAR after PSM (58.1%, 35.7% and 28.1% vs. 44.1%, 23.9% and 18.0%; 72.9%, 45.7% and 36.0% vs. 62.0%, 30.8% and 25.3%; both p = 0.002). On multivariate analysis, NAR was associated with worse DFS and OS than AR [hazard ratio (HR) 1.461 and 1.488; 95% confidence interval (CI) 1.184-1.804 and 1.189-1.863, respectively]. Stratified analysis demonstrated similar outcomes following AR versus NAR for ICC at stages IA, II with vascular invasion, and III with visceral peritoneum perforation, local extrahepatic invasion and nodal metastasis, while NAR was associated with worse DFS and OS versus AR for stages IB (HR 1.897 and 2.321; 95% CI 1.179-3.052 and 1.376-3.914, respectively) or II ICC without vascular invasion (2.071 and 2.077; 95% CI 1.239-3.462 and 1.205-3.579, respectively). CONCLUSIONS: AR was associated with better survival outcomes compared with NAR in ICC patients with stage IB or II tumors without vascular invasion.

Diet-induced hepatic steatosis activates Ras to promote hepatocarcinogenesis via CPT1α.

Aberrant activation of the RAS cascade ubiquitously occurs in human hepatocellular carcinomas (HCC), regardless of rare mutations of RAS. However, the association between the Ras cascade and hepatic steatosis during hepatocarcinogenesis remains under-investigated. Here, the variation in the constitutive activity of Ras signaling and HCC incidence was found in a nonalcoholic fatty liver disease (NAFLD)-HCC mouse model, and Ras activity was induced by hepatic steatosis. Even in hepatocyte-specific expression of KrasG12D (Alb-Cre/KrasG12D, Krashep) mice, mutagenic activation of Ras signaling was still significantly enhanced by NAFLD, with downregulation of negative regulators. Interestingly, hepatic steatosis could be alleviated by persistent activation of Ras, whereas Ras accelerated DNA damage and HCC progression through Carnitine palmitoyltransferase 1A (CPT1α). A close correlation between active Ras and CPT1α was also shown in clinical steatosis peri-tumor tissues of HCC samples and experimental models. CPT1α inhibitor etomoxir (ETO) largely ameliorated active Ras-drived HCC. These findings can provide a novel link between steatosis and Ras activity in liver cancer.

Long-term survival after partial hepatectomy for sub-stage patients with intermediate stage hepatocellular carcinoma.

BACKGROUND: The current study aimed to examine the long-term survival after partial hepatectomy for patients with BCLC intermediate stage hepatocellular carcinoma (HCC) stratified by the Bolondi's sub-staging model. MATERIALS AND METHODS: This cohort consisted of 360 patients with BCLC intermediate stage HCC who underwent partial hepatectomy between January 2008 and February 2010. Patients were stratified into 3 subgroups (B1-B3) based on the Bolondi's sub-staging model. The last follow-up was conducted at February 2014. RESULTS: Of these patients, 166, 171 and 23 patients had B1, B2, and B3 sub-stage HCC, respectively. The postoperative 5-year Overall survival (OS) rate for patients with these three sub-stages was 49.5%, 33.7% and 12.9%, respectively (P < 0.001). Compared with the reported survival outcomes from previous studies which used transarterial chemoembolization (TACE) as first-line treatment, hepatectomy had a better median survival than TACE in B1 and B2 patients. On multivariable analysis, presence of esophageal and gastric varices, higher NDR score, presence of microvascular invasion, differentiation grade III-IV, and patterns of AFP decreases after surgery were the independent risk factors of OS in the sub-stages B1 and B2 patients. A nomogram which integrated all these independent risk factors was developed, with a C-index of 0.71 for OS prediction. The calibration curve showed an optimal agreement between prediction by the nomogram and actual observation. CONCLUSIONS: The patients with intermediate stage HCC clarified as sub-stages B1 and B2 according to Bolondi's model had an optimal long-term survival following partial hepatectomy than TACE. Their postoperative prognosis could be accurately predicted by our proposed nomogram.

Cholesterol inhibits hepatocellular carcinoma invasion and metastasis by promoting CD44 localization in lipid rafts.

Cholesterol plays a vital role in modulating the action of membrane proteins critical to cellular function. The effect of serum cholesterol on the prognosis of hepatocellular carcinoma (HCC) patients remains uncertain. Here, we report that high levels of cholesterol predict good survival and low disease recurrence after surgery. Cholesterol could significantly suppress migration and invasion of HCC cells and restrain metastasis of HCC in mice. High levels of cholesterol promoted CD44 translocation into lipid rafts and attenuated CD44-Ezrin binding, which are crucial for cell migration and cancer metastasis. The suppressive effect of cholesterol on HCC metastasis was abolished by the downregulation of CD44 or its palmitoylation inhibitor, which blocked CD44 localization in lipid rafts. Furthermore, pharmacologically promoting CD44 retention inside lipid rafts obviously attenuated HCC migration and invasion, providing a potential therapeutic strategy to prolong the survival of HCC patients.

Contradictory effects of mitochondria- and non-mitochondria-targeted antioxidants on hepatocarcinogenesis by altering DNA repair in mice.

Conflicting effects of antioxidant supplementation on cancer prevention or promotion is of great concern to healthy people and cancer patients. Despite recent studies about antioxidants accelerating the progression of lung cancer and melanoma, antioxidants may still play a role in cancer prevention. Both tumor and antioxidants types influence the actual efficacy. However, little is known about the impact of different types of antioxidants on primary hepatocellular carcinoma (HCC), including non-mitochondrial- and mitochondrial-targeted antioxidants. Utilizing mouse models of chemical hepatocarcinogenesis, we showed that administration of non-mitochondria-targeted antioxidants N-acetylcysteine (NAC) and the soluble vitamin E analog, Trolox, prevented tumorigenesis, whereas administration of mitochondria-targeted antioxidants SS-31 (the mitochondria-targeted peptide) and Mito-Q (a derivative of ubiquinone) facilitated tumorigenesis. RNA sequencing revealed that NAC and SS-31 caused very different changes in the oxidation-reduction state and DNA damage response. In diethylnitrosamine (DEN)-treated primary hepatocytes, NAC and Trolox alleviated DNA damage by activating ataxia-telangiectasia mutated (ATM)/ATM and Rad3-related (ATR) for DNA repair whereas SS-31 and Mito-Q aggravated damage by inactivating them. Interestingly, partial recovery of SS-31-scavengened mitochondrial reactive oxygen species (mtROS) could alleviate SS-31-aggravated DNA damage. Localization of ATM between mitochondria and nuclei was altered after NAC and SS-31 treatment. Furthermore, blockage of phospho-ATR (p-ATR) led to the recurrence of NAC-ameliorated DEN HCC. In contrast, reactivation of p-ATR blocked SS-31-promoted DEN HCC. Conclusion: These results demonstrate that the type of antioxidants plays a previously unappreciated role in hepatocarcinogenesis, and provide a mechanistic rationale for exploring the therapeutic use of antioxidants for liver cancer. (Hepatology 2018;67:623-635).