Differential requirement of Hippo cascade during CTNNB1 or AXIN1 mutation-driven hepatocarcinogenesis.

BACKGROUND AND AIMS: Gain-of-function (GOF) mutations of CTNNB1 and loss-of-function (LOF) mutations of AXIN1 are recurrent genetic alterations in hepatocellular carcinoma (HCC). We aim to investigate the functional contribution of Hippo/YAP/TAZ in GOF CTNNB1 or LOF AXIN1 mutant HCCs. APPROACH AND RESULTS: The requirement of YAP/TAZ in c-Met/ß-Catenin and c-Met/sgAxin1-driven HCC was analyzed using conditional Yap , Taz , and Yap;Taz knockout (KO) mice. Mechanisms of AXIN1 in regulating YAP/TAZ were investigated using AXIN1 mutated HCC cells. Hepatocyte-specific inducible TTR-CreER T2KO system was applied to evaluate the role of Yap;Taz during tumor progression. Cabozantinib and G007-LK combinational treatment were tested in vitro and in vivo . Nuclear YAP/TAZ was strongly induced in c-Met/sgAxin1 mouse HCC cells. Activation of Hippo via overexpression of Lats2 or concomitant deletion of Yap and Taz significantly inhibited c-Met/sgAxin1 driven HCC development, whereas the same approaches had mild effects in c-Met/ß-Catenin HCCs. YAP is the major Hippo effector in c-Met/ß-Catenin HCCs, and both YAP and TAZ are required for c-Met/sgAxin1-dependent hepatocarcinogenesis. Mechanistically, AXIN1 binds to YAP/TAZ in human HCC cells and regulates YAP/TAZ stability. Genetic deletion of YAP/TAZ suppresses already formed c-Met/sgAxin1 liver tumors, supporting the requirement of YAP/TAZ during tumor progression. Importantly, tankyrase inhibitor G007-LK, which targets Hippo and Wnt pathways, synergizes with cabozantinib, a c-MET inhibitor, leading to tumor regression in the c-Met/sgAxin1 HCC model. CONCLUSIONS: Our studies demonstrate that YAP/TAZ are major signaling molecules downstream of LOF AXIN1 mutant HCCs, and targeting YAP/TAZ is an effective treatment against AXIN1 mutant human HCCs.

TBX3 functions as a tumor suppressor downstream of activated CTNNB1 mutants during hepatocarcinogenesis.

BACKGROUND & AIMS: Gain of function (GOF) mutations in the CTNNB1 gene are one of the most frequent genetic events in hepatocellular carcinoma (HCC). T-box transcription factor 3 (TBX3) is a liver-specific target of the Wnt/ß-catenin pathway and thought to be an oncogene mediating activated ß-catenin-driven HCC formation. METHODS: We evaluated the expression pattern of TBX3 in human HCC specimens. Tbx3 was conditionally knocked out in murine HCC models by hydrodynamic tail vein injection of Cre together with c-Met and ΔN90-ß-catenin (c-Met/ß-catenin) in Tbx3flox/flox mice. TBX3 was overexpressed in human HCC cell lines to investigate the functions of TBX3 in vitro. RESULTS: A bimodal expression pattern of TBX3 in human HCC samples was detected: high expression of TBX3 in GOF CTNNB1 HCC and downregulation of TBX3 in non-CTNNB1 mutant tumors. High expression of TBX3 was associated with increased differentiation and decreased expression signatures of tumor growth. Using Tbx3flox/flox mice, we found that ablation of Tbx3 significantly accelerates c-Met/ß-catenin-driven HCC formation. Moreover, Tbx3(-) HCC demonstrated increased YAP/TAZ activity. The accelerated tumor growth induced by loss of TBX3 in c-Met/ß-catenin mouse HCC was successfully prevented by overexpression of LATS2, which inhibited YAP/TAZ activity. In human HCC cell lines, overexpression of TBX3 inhibited HCC cell growth as well as YAP/TAZ activation. A negative correlation between TBX3 and YAP/TAZ target genes was observed in human HCC samples. Mechanistically, phospholipase D1 (PLD1), a known positive regulator of YAP/TAZ, was identified as a novel transcriptional target repressed by TBX3. CONCLUSION: Our study suggests that TBX3 is induced by GOF CTNNB1 mutants and suppresses HCC growth by inactivating PLD1, thus leading to the inhibition of YAP/TAZ oncogenes. LAY SUMMARY: TBX3 is a liver-specific target of the Wnt/ß-catenin pathway and thought to be an oncogene in promoting liver cancer development. Herein, we demonstrate that TBX3 is in fact a tumor suppressor gene that restricts liver tumor growth. Strategies which increase TBX3 expression and/or activities may be effective for HCC treatment.

Oncogenic Mutations in Armadillo Repeats 5 and 6 of ß-Catenin Reduce Binding to APC, Increasing Signaling and Transcription of Target Genes.

BACKGROUND & AIMS: The ß-catenin signaling pathway is one of the most commonly deregulated pathways in cancer cells. Amino acid substitutions within armadillo repeats 5 and 6 (K335, W383, and N387) of ß-catenin are found in several tumor types, including liver tumors. We investigated the mechanisms by which these substitutions increase signaling and the effects on liver carcinogenesis in mice. METHODS: Plasmids encoding tagged full-length ß-catenin (CTNNB1) or ß-catenin with the K335I or N387K substitutions, along with MET, were injected into tails of FVB/N mice. Tumor growth was monitored, and livers were collected and analyzed by histology, immunohistochemistry, and quantitative reverse-transcription polymerase chain reaction. Tagged full-length and mutant forms of ß-catenin were expressed in HEK293, HCT116, and SNU449 cells, which were analyzed by immunoblots and immunoprecipitation. A panel of ß-catenin variants and cell lines with knock-in mutations were analyzed for differences in N-terminal phosphorylation, half-life, and association with other proteins in the signaling pathway. RESULTS: Mice injected with plasmids encoding K335I or N387K ß-catenin and MET developed larger, more advanced tumors than mice injected with plasmids encoding WT ß-catenin and MET. K335I and N387K ß-catenin bound APC with lower affinity than WT ß-catenin but still interacted with scaffold protein AXIN1 and in the nucleus with TCF7L2. This interaction resulted in increased transcription of genes regulated by ß-catenin. Studies of protein structures supported the observed changes in relative binding affinities. CONCLUSION: Expression of ß-catenin with mutations in armadillo repeats 5 and 6, along with MET, promotes formation of liver tumors in mice. In contrast to N-terminal mutations in ß-catenin that directly impair its phosphorylation by GSK3 or binding to BTRC, the K335I or N387K substitutions increase signaling via reduced binding to APC. However, these mutant forms of ß-catenin still interact with the TCF family of transcription factors in the nucleus. These findings show how these amino acid substitutions increase ß-catenin signaling in cancer cells.

Mammalian Target of Rapamycin Complex 2 Signaling Is Required for Liver Regeneration in a Cholestatic Liver Injury Murine Model.

Cholestatic liver injury may lead to a series of hepatobiliary syndromes, which can progress to cirrhosis and impaired liver regeneration, eventually resulting in liver-related death. Mammalian target of rapamycin complex 2 (mTORC2) is a major regulator of liver metabolism and tumor development. However, the role of mTORC2 signaling in cholestatic liver injury has not been characterized to date. In this study, we generated liver-specific Rictor knockout mice to block the mTORC2 signaling pathway. Mice were treated with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) to induce cholestatic liver injury. DDC feeding induced cholestatic liver injury and ductular reaction as well as activation of the mTORC2/Akt signaling pathway in wild-type mice. Loss of mTORC2 led to significantly decreased oval cell expansion after DDC feeding. Mechanistically, this phenotype was independent of mTORC1/fatty acid synthase cascade (Fasn) or yes-associated protein (Yap) signaling. Notch pathway was instead strongly inhibited during DDC-induced cholestatic liver injury in liver-specific Rictor knockout mice. Furthermore, mTORC2 deficiency in adult hepatocytes did not inhibit ductular reaction in this cholestatic live injury mouse model. Our results indicated that mTORC2 signaling effectively regulates liver regeneration by inducing oval cell proliferation. Liver progenitor cells or bile duct cells, rather than mature hepatocytes, would be the major source of ductular reaction in DDC-induced cholestatic liver injury.

Clinical Characteristics and Prognosis of Biliary Atresia with Low Serum Matrix Metalloproteinase-7 Levels.

PURPOSE: Serum matrix metalloproteinase-7 (MMP-7) levels can precisely differentiate biliary atresia (BA) from non-BA cholestasis. However, serum MMP-7 levels of some BA patients were within normal range or slightly elevated. This study aimed to investigate the clinical characteristics and prognosis of biliary atresia with low serum MMP-7 levels. METHOD: This is a retrospective cohort study. Cases of BA from July 2020 to December 2022 were consecutively enrolled. They were divided into low-MMP-7 group (MMP-7 ≤ 25 ng/ml) and high-MMP-7 group (MMP-7 > 25 ng/ml) according to serum MMP-7 levels preoperatively. The perioperative clinical characteristics, the 3-month and 6-month jaundice clearance rate post-Kasai procedure, and the native liver survival were compared between the two groups. RESULTS: A total of 329 cases were included in this study, 40 of which were divided into the low-MMP-7 group. Preoperative GGT and direct bilirubin levels in the low-MMP-7 group were significantly lower than those in the high-MMP-7 group (258.6 U/L, interquartile range [IQR]: 160.4411.6 vs. 406.8 IU/L, IQR: 215,655.0, P = 0.0076; 103.8 µmol/L, IQR: 79.0,121.4 vs. 115.3 µmol/L, IQR: 94,138.8, P = 0.0071), while the gender, the day at surgery and preoperative ALT, AST, TBA, total bilirubin levels showed no significant differences (P > 0.05). The 3-month and 6-month jaundice clearance rate post-Kasai procedure in the low-MMP-7 group were lower than those in the high-MMP-7 group (29.73% vs. 53.09%, P = 0.049; 32.14% vs. 54.73%, P = 0.023). The 1-year native liver survival rate was 29.63% for the low-MMP-7 group and 53.02% for the high-MMP-7 group (P = 0.022). CONCLUSION: Preoperative clinical characteristics were similar between low-MMP-7 group and high-MMP-7 group, while patients with low serum MMP-7 levels showed worse prognosis, indicating that this might be listed as a new clinical subtype of BA which could contribute to designing new treatment strategies for BA in the future. STUDY TYPE: Cohort Study. LEVEL OF EVIDENCE: Level II.

Efficacy and Safety of Endoscopic Retrograde Cholangiopancreatography in Children of Pancreaticobiliary Maljunction Without Obvious Biliary Dilatation.

PURPOSE: There is no standard surgical approach for pancreaticobiliary maljunction (PBM) without congenital biliary dilatation (CBD). This study aimed to compare outcomes between therapeutic endoscopic retrograde cholangiopancreatography (ERCP) and laparoscopic hepaticojejunostomy (LH) for pediatric patients of PBM without obvious biliary dilatation (PBM-nonOBD). METHODS: We retrospectively reviewed demographic and clinical data of pediatric patients with PBM-nonOBD from 2015 to 2021. There were 33 patients in ERCP group and 35 patients in LH group. Primary outcomes included treatment efficiency, postoperative recovery, and postoperative complications. Univariate analysis was further used to explore prognostic factors for ERCP. RESULTS: The mean diameter of the common bile duct in LH group was larger than that in ERCP group (8.6 ± 1.3 mm vs. 6.9 ± 2.1 mm, p = 0.003), while there were no significant differences between the two groups in age, gender, clinical manifestations, complications, and other imaging findings. Compared with LH group, ERCP group had a shorter operation time and postoperative recovery time. The treatment effective rate of ERCP was inferior to that of LH (45.4 % vs. 85.7 %, p＜0.001). For postoperative adverse events, post-ERCP pancreatitis (15.1 %) was most common in the ERCP group. 30.3 % of patients eventually required LH. Intestinal obstruction (5.7 %), recurrent cholangitis (5.7 %), gastrointestinal bleeding (2.8 %), and anastomotic stenosis (2.8 %) were observed in LH group and 8.6 % of patients required a reoperation. A long common channel may be associated with poor prognosis after ERCP. CONCLUSIONS: ERCP is associated with less surgical trauma, shorter recovery time, and fewer serious complications than LH, while the treatment effective rate of ERCP is inferior to LH. The indications for endoscopic sphincterotomy and the timing of radical surgery need to be further explored. LEVEL OF EVIDENCE: Ⅲ STUDY TYPE: Retrospective Comparative Study.

The microbial metabolite trimethylamine N-oxide promotes antitumor immunity in triple-negative breast cancer.

Immunotherapy has achieved limited success in patients with triple-negative breast cancer (TNBC), an aggressive disease with a poor prognosis. Commensal microbiota have been proven to colonize the mammary gland, but whether and how they modulate the tumor microenvironment remains elusive. We performed a multiomics analysis of a cohort of patients with TNBC (n = 360) and found genera under Clostridiales, and the related metabolite trimethylamine N-oxide (TMAO) was more abundant in tumors with an activated immune microenvironment. Patients with higher plasma TMAO achieved better responses to immunotherapy. Mechanistically, TMAO induced pyroptosis in tumor cells by activating the endoplasmic reticulum stress kinase PERK and thus enhanced CD8+ T cell-mediated antitumor immunity in TNBC in vivo. Collectively, our findings offer new insights into microbiota-metabolite-immune crosstalk and indicate that microbial metabolites, such as TMAO or its precursor choline, may represent a novel therapeutic strategy to promote the efficacy of immunotherapy in TNBC.

A retrospective clinical analysis of pediatric paragonimiasis in a Chinese children's hospital from 2011 to 2019.

Diagnosis of pediatric paragonimiasis is difficult because of its non-specific clinical manifestations. We retrospectively reviewed the records of pediatric paragonimiasis in Children's Hospital of Fudan University from January 2011 to May 2019. The confirmed diagnosis of paragonimiasis was based on positive anti-parasite serological tests from the local Center for Disease Control (CDC). A total of 11 patients (mean age: 7.7 ± 3.1, male-female ratio: 7:4) diagnosed as paragonimiasis were included. 81.8% were from endemic areas such as Sichuan and Yunnan, and 36% had a clear history of raw crab or crayfish consumption. The characteristic clinical features of pediatric paragonimiasis were eosinophilia (100%), pleural effusion (81.8%), hepatomegaly (54.5%), ascites (54.5%), and subcutaneous nodules (45.5%). Misdiagnosed with other diseases including tuberculosis (18.2%), pneumonia (9.1%), intracranial space-occupying lesions (9.1%) and brain abcess (9.1%) led to rehospitalization and prolonged hospitalization. For treatment, a 3-day course of 150 mg/kg praziquantel (PZQ) didn't show ideal treatment effectivity and 63.6% needed more than one course of PZQ, while triclabendazole in a total dose of 10 mg/kg had a better efficacy to stubborn manifestations. This study indicated that pediatric paragonimiasis was often misdiagnosed, and the treatment with a 3-day course of 150 mg/kg PZQ had a high rate of failure.