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.

Anatomical sectionectomy based on Takasaki's segmentation for solitary intrahepatic cholangiocarcinoma: a propensity-matched analysis.

BACKGROUND: Anatomical sectionectomy based on Takasaki's segmentation has shown advantages in hepatocellular carcinoma. However, whether this approach improves the survival of intrahepatic cholangiocarcinoma (ICC) remains unknown. METHODS: A series of 248 consecutive patients with solitary ICCs who underwent hepatectomy were studied retrospectively. The patients were classified into the groups of anatomical sectionectomy based on Takasaki's segmentation (TS group) and non-Takasaki's hepatectomy (NTH group). The bias between the two groups was minimized using propensity score matching (PSM). Recurrence-free survival (RFS) and overall survival (OS) were evaluated with Kaplan-Meier analysis. The Cox proportional hazards model was performed to determine the adverse risk factors associated with survival. RESULTS: After PSM, 67 pairs of patients were compared. Both the RFS and OS rates in the TS group were significantly better than those in the NTH group (23.2 % vs. 16.5 %, and 40.4 % vs. 27.3 %, P = 0.035 and 0.032, respectively). Multivariate analysis showed that NTH was independently associated with worse RFS and OS than TS. The stratified analysis demonstrated that the RFS and OS rates in the TS group with tumor stage I and tumor size ≥3 cm were significantly better than those in the NTH group, while the survival rates for ICC with stage I and tumor size <3 cm or stage II-III showed no significant difference. CONCLUSION: TS was associated with improved RFS and OS in patients with solitary ICC even after PSM. TS may be preferred particularly in patients with tumor stage I and tumor size ≥3 cm.

ß-Catenin Sustains and Is Required for YES-associated Protein Oncogenic Activity in Cholangiocarcinoma.

BACKGROUND & AIMS: YES-associated protein (YAP) aberrant activation is implicated in intrahepatic cholangiocarcinoma (iCCA). Transcriptional enhanced associate domain (TEAD)-mediated transcriptional regulation is the primary signaling event downstream of YAP. The role of Wnt/ß-Catenin signaling in cholangiocarcinogenesis remains undetermined. Here, we investigated the possible molecular interplay between YAP and ß-Catenin cascades in iCCA. METHODS: Activated AKT (Myr-Akt) was coexpressed with YAP (YapS127A) or Tead2VP16 via hydrodynamic tail vein injection into mouse livers. Tumor growth was monitored, and liver tissues were collected and analyzed using histopathologic and molecular analysis. YAP, ß-Catenin, and TEAD interaction in iCCAs was investigated through coimmunoprecipitation. Conditional Ctnnb1 knockout mice were used to determine ß-Catenin function in murine iCCA models. RNA sequencing was performed to analyze the genes regulated by YAP and/or ß-Catenin. Immunostaining of total and nonphosphorylated/activated ß-Catenin staining was performed in mouse and human iCCAs. RESULTS: We discovered that TEAD factors are required for YAP-dependent iCCA development. However, transcriptional activation of TEADs did not fully recapitulate YAP's activities in promoting cholangiocarcinogenesis. Notably, ß-Catenin physically interacted with YAP in human and mouse iCCA. Ctnnb1 ablation strongly suppressed human iCCA cell growth and Yap-dependent cholangiocarcinogenesis. Furthermore, RNA-sequencing analysis revealed that YAP/ transcriptional coactivator with PDZ-binding motif (TAZ) regulate a set of genes significantly overlapping with those controlled by ß-Catenin. Importantly, activated/nonphosphorylated ß-Catenin was detected in more than 80% of human iCCAs. CONCLUSION: YAP induces cholangiocarcinogenesis via TEAD-dependent transcriptional activation and interaction with ß-Catenin. ß-Catenin binds to YAP in iCCA and is required for YAP full transcriptional activity, revealing the functional crosstalk between YAP and ß-Catenin pathways in cholangiocarcinogenesis.

MicroRNA-188-5p inhibits hepatocellular carcinoma proliferation and migration by targeting forkhead box N2.

BACKGROUND: This study aimed to identify the biological functions, expression modes, and possible mechanisms underlying the relationship between metastatic human hepatocellular carcinoma (HCC) and MicroRNA-188-5p (miR-188) dysregulation using cell lines. METHODS: A decrease in miR-188 was detected in low and high metastatic HCC cells compared to that in normal hepatic cells and non-invasive cell lines. Gain- and loss-of-function experiments were performed in vitro to investigate the role of miR-188 in cancer cell (Hep3B, HepG2, HLF, and LM3) proliferation and migration. RESULTS: miR-188 mimic transfection inhibited the proliferation of metastatic HLF and LM3 cells but not non-invasive HepG2 and Hep3B cells; nonetheless, miR-188 suppression promoted the growth of HLF and LM3 cells. miR-188 upregulation inhibited the migratory rate and invasive capacity of HLF and LM3, rather than HepG2 and Hep3B cells, whereas transfection of a miR-188 inhibitor in HLF and LM3 cells had the opposite effects. Dual-luciferase reporter assays and bioinformatics prediction confirmed that miR-188 could directly target forkhead box N2 (FOXN2) in HLF and LM3 cells. Transfection of miR-188 mimics reduced FOXN2 levels, whereas miR-188 inhibition resulted in the opposite result, in HLF and LM3 cells. Overexpression of FOXN2 in HLF and LM3 cells abrogated miR-188 mimic-induced downregulation of proliferation, migration, and invasion. In addition, we found that miR-188 upregulation impaired tumor growth in vivo. CONCLUSIONS: In summary, this study showed thatmiR-188 inhibits the proliferation and migration of metastatic HCC cells by targeting FOXN2.

Complexin-1 and synaptotagmin-1 compete for binding sites on membranes containing PtdInsP2.

Complexin-1 is an essential protein for neuronal exocytosis that acts to depress spontaneous fusion events while enhancing evoked neurotransmitter release. In addition to binding soluble N-ethylmaleimide-sensitive factor attachment protein receptors, it is well established that complexin associates with membranes in a manner that depends upon membrane curvature. In the present work, we examine the membrane binding of complexin using electron paramagnetic resonance spectroscopy, fluorescence anisotropy, and total internal reflection fluorescence microscopy. The apparent membrane affinity of complexin is found to strongly depend upon the concentration of protein used in the binding assay, and this is a result of a limited number of binding sites for complexin on the membrane interface. Although both the N- and C-terminal regions of complexin associate with the membrane interface, membrane affinity is driven by its C-terminus. Complexin prefers to bind liquid-disordered membrane phases and shows an enhanced affinity toward membranes containing phosphatidylinositol 4-5-bisphosphate (PI(4,5)P2). In the presence of PI(4,5)P2, complexin is displaced from the membrane surface by proteins that bind to or sequester PI(4,5)P2. In particular, the neuronal calcium sensor synaptotagmin-1 displaces complexin from the membrane but only when PI(4,5)P2 is present. Complexin and synaptotagmin compete on the membrane interface in the presence of PI(4,5)P2, and this interaction may play a role in calcium-triggered exocytosis by displacing complexin from its fusion-inhibiting state.

Loss of Apc Cooperates with Activated Oncogenes to Induce Liver Tumor Formation in Mice.

Hepatocellular carcinoma (HCC) and hepatoblastoma are the major types of primary liver cancer in adulthood and childhood, respectively. Wnt/ß-catenin signaling deregulation is one of the most frequent genetic events in hepatocarcinogenesis. APC regulator of WNT signaling pathway (APC) encodes an inhibitor of the Wnt cascade and acts as a tumor suppressor. Germline defects of the APC gene lead to familial adenomatous polyposis, and its somatic mutations occur in multiple tumor types. However, the contribution of APC in hepatocarcinogenesis remains unclear. Therefore, APC mutations and expression patterns were examined in human HCC and hepatoblastoma samples. Whether loss of Apc alone or in cooperation with other oncogenes triggers liver tumor development in vivo was also investigated. sgApc alone could not drive liver tumor formation, but synergized with activated oncogenes (YapS127A, TazS89A, and c-Met) to induce hepatocarcinogenesis. Mechanistically, Apc deletion induced the activation of ß-catenin and its downstream targets in mouse liver tumors. Furthermore, Ctnnb1 ablation or TCF4-mediated transcription blockade completely prevented liver tumor formation, indicating the requirement of a functional ß-catenin pathway for loss of Apc-driven hepatocarcinogenesis. This study shows that a subset of HCC patients with loss-of-function APC mutations might benefit from therapeutic strategies targeting the Wnt/ß-catenin pathway.

Overexpression of Mothers Against Decapentaplegic Homolog 7 Activates the Yes-Associated Protein/NOTCH Cascade and Promotes Liver Carcinogenesis in Mice and Humans.

BACKGROUND AND AIMS: Mothers against decapentaplegic homolog (SMAD) 7 is an antagonist of TGF-ß signaling. In the present investigation, we sought to determine the relevance of SMAD7 in liver carcinogenesis using in vitro and in vivo approaches. APPROACH AND RESULTS: We found that SMAD7 is up-regulated in a subset of human HCC samples with poor prognosis. Gene set enrichment analysis revealed that SMAD7 expression correlates with activated yes-associated protein (YAP)/NOTCH pathway and cholangiocellular signature genes in HCCs. These findings were substantiated in human HCC cell lines. In vivo, overexpression of Smad7 alone was unable to initiate HCC development, but it significantly accelerated c-Myc/myeloid cell leukemia 1 (MCL1)-induced mouse HCC formation. Consistent with human HCC data, c-Myc/MCL1/Smad7 liver tumors exhibited an increased cholangiocellular gene expression along with Yap/Notch activation and epithelial-mesenchymal transition (EMT). Intriguingly, blocking of the Notch signaling did not affect c-Myc/MCL1/Smad7-induced hepatocarcinogenesis while preventing cholangiocellular signature expression and EMT, whereas ablation of Yap abolished c-Myc/MCL1/Smad7-driven HCC formation. In mice overexpressing a myristoylated/activated form of AKT, coexpression of SMAD7 accelerated carcinogenesis and switched the phenotype from HCC to intrahepatic cholangiocarcinoma (iCCA) lesions. In human iCCA, SMAD7 expression was robustly up-regulated, especially in the most aggressive tumors, and directly correlated with the levels of YAP/NOTCH targets as well as cholangiocellular and EMT markers. CONCLUSIONS: The present data indicate that SMAD7 contributes to liver carcinogenesis by activating the YAP/NOTCH signaling cascade and inducing a cholangiocellular and EMT signature.

Cabozantinib-based combination therapy for the treatment of hepatocellular carcinoma.

OBJECTIVE: Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer with limited treatment options. Cabozantinib, an orally bioavailable multikinase inhibitor is now approved by Food and Drug Administration (FDA) for HCC patients. We evaluated the therapeutic efficacy of cabozantinib, either alone or in combination, in vitro and in vivo. DESIGN: Human HCC cell lines and HCC mouse models were used to assess the therapeutic efficacy and targeted molecular pathways of cabozantinib, either alone or in combination with the pan-mTOR inhibitor MLN0128 or the checkpoint inhibitor anti-PD-L1 antibody. RESULTS: Cabozantinib treatment led to stable disease in c-Met/ß-catenin and Akt/c-Met mouse HCC while possessing limited efficacy on Akt/Ras and c-Myc liver tumours. Importantly, cabozantinib effectively inhibited c-MET and ERK activity, leading to decreased PKM2 and increased p21 expression in HCC cells and in c-Met/ß-catenin and Akt/c-Met HCC. However, cabozantinib was ineffective in inhibiting the Akt/mTOR cascade. Intriguingly, a strong inhibition of angiogenesis by cabozantinib occurred regardless of the oncogenic drivers. However, cabozantinib had limited impact on other tumour microenvironment parameters, including tumour infiltrating T cells, and did not induce programmed death-ligand 1 (PD-L1) expression. Combining cabozantinib with MLN0128 led to tumour regression in c-Met/ß-catenin mice. In contrast, combined treatment with cabozantinib and the checkpoint inhibitor anti-PD-L1 antibody did not provide any additional therapeutic benefit in the four mouse HCC models tested. CONCLUSION: c-MET/ERK/p21/PKM2 cascade and VEGFR2-induced angiogenesis are the primary targets of cabozantinib in HCC treatment. Combination therapies with cabozantinib and mTOR inhibitors may be effective against human HCC.

Focal adhesion kinase (FAK) promotes cholangiocarcinoma development and progression via YAP activation.

BACKGROUND & AIMS: Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is upregulated in many tumor types and is a promising target for cancer therapy. Herein, we elucidated the functional role of FAK in intrahepatic cholangiocarcinoma (iCCA) development and progression. METHODS: Expression levels and activation status of FAK were determined in human iCCA samples. The functional contribution of FAK to Akt/YAP murine iCCA initiation and progression was investigated using conditional Fak knockout mice and constitutive Cre or inducible Cre mice, respectively. The oncogenic potential of FAK was further examined via overexpression of FAK in mice. In vitro cell line studies and in vivo drug treatment were applied to address the therapeutic potential of targeting FAK for iCCA treatment. RESULTS: FAK was ubiquitously upregulated and activated in iCCA lesions. Ablation of FAK strongly delayed Akt/YAP-driven mouse iCCA initiation. FAK overexpression synergized with activated AKT to promote iCCA development and accelerated Akt/Jag1-driven cholangiocarcinogenesis. Mechanistically, FAK was required for YAP(Y357) phosphorylation, supporting the role of FAK as a central YAP regulator in iCCA. Significantly, ablation of FAK after Akt/YAP-dependent iCCA formation strongly suppressed tumor progression in mice. Furthermore, a remarkable iCCA growth reduction was achieved when a FAK inhibitor and palbociclib, a CDK4/6 inhibitor, were administered simultaneously in human iCCA cell lines and Akt/YAP mice. CONCLUSIONS: FAK activation contributes to the initiation and progression of iCCA by inducing the YAP proto-oncogene. Targeting FAK, either alone or in combination with anti-CDK4/6 inhibitors, may be an effective strategy for iCCA treatment. LAY SUMMARY: We found that the protein FAK (focal adhesion kinase) is upregulated and activated in human and mouse intrahepatic cholangiocarcinoma samples. FAK promotes intrahepatic cholangiocarcinoma development, whereas deletion of FAK strongly suppresses its initiation and progression. Combined FAK and CDK4/6 inhibitor treatment had a strong anti-cancer effect in in vitro and in vivo models. This combination therapy might represent a valuable and novel treatment against human intrahepatic cholangiocarcinoma.

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.

Liver Collagen Contents Are Closely Associated with the Severity of Cirrhosis and Posthepatectomy Liver Failure in Patients with Hepatocellular Carcinoma and Child-Pugh Grade A Liver Function.

BACKGROUND: Hepatocellular carcinoma (HCC) is usually accompanied by different severities of cirrhosis, which is a risk factor for posthepatectomy liver failure (PHLF). Collagen proportional area (CPA) measurements can quantitatively determine the collagen contents of liver tissue. This study explored the impact of CPA on PHLF, and further investigated the correlation between CPA and a non-invasive method, namely cirrhotic severity scoring (CSS), previously proposed by our team. METHODS: A total of 224 HCC patients with Child-Pugh grade A liver function undergoing hepatectomy between 2017 and 2019 were retrospectively studied. Quantitative digital image analysis of resected liver tissues was used for the CPA measurement. Risk factors for PHLF were subjected to univariate and multivariate analyses, and the correlation between CPA and CSS was analyzed. RESULTS: Overall, 28 (12.5%) patients experienced PHLF. Patients with PHLF had higher CPA values than those without PHLF (p < 0.001). Multivariate analysis showed CPA and extent of hepatectomy to be independent risk factors for PHLF. CPA values were divided into four stages based on their quartiles (C1: < 6.6%; C2: 6.6-10.7%; C3: 10.7-18.0%; C4: ≥ 18.0%). The incidence of PHLF increased with increasing CPA stages (p < 0.001). Furthermore, CSS was significantly correlated with CPA (r = 0.720; p < 0.001). The incidence of PHLF also increased with increasing severity of cirrhosis evaluated by CSS (p < 0.001). CONCLUSIONS: In HCC patients with Child-Pugh grade A liver function, cirrhosis could be staged by liver collagen contents, which significantly influenced PHLF. Furthermore, CSS was useful in the preoperative evaluation of cirrhotic severity.

Axis inhibition protein 1 (Axin1) Deletion-Induced Hepatocarcinogenesis Requires Intact ß-Catenin but Not Notch Cascade in Mice.

Inactivating mutations of axis inhibition protein 1 (AXIN1), a negative regulator of the Wnt/ß-Catenin cascade, are among the common genetic events in human hepatocellular carcinoma (HCC), affecting approximately 10% of cases. In the present manuscript, we sought to define the genetic crosstalk between Axin1 mutants and Wnt/ß-catenin as well as Notch signaling cascades along hepatocarcinogenesis. We discovered that c-MET activation and AXIN1 mutations occur concomitantly in ~3%-5% of human HCC samples. Subsequently, we generated a murine HCC model by means of CRISPR/Cas9-based gene deletion of Axin1 (sgAxin1) in combination with transposon-based expression of c-Met in the mouse liver (c-Met/sgAxin1). Global gene expression analysis of mouse normal liver, HCCs induced by c-Met/sgAxin1, and HCCs induced by c-Met/∆N90-ß-Catenin revealed activation of the Wnt/ß-Catenin and Notch signaling in c-Met/sgAxin1 HCCs. However, only a few of the canonical Wnt/ß-Catenin target genes were induced in c-Met/sgAxin1 HCC when compared with corresponding lesions from c-Met/∆N90-ß-Catenin mice. To study whether endogenous ß-Catenin is required for c-Met/sgAxin1-driven HCC development, we expressed c-Met/sgAxin1 in liver-specific Ctnnb1 null mice, which completely prevented HCC development. Consistently, in AXIN1 mutant or null human HCC cell lines, silencing of ß-Catenin strongly inhibited cell proliferation. In striking contrast, blocking the Notch cascade through expression of either the dominant negative form of the recombinant signal-binding protein for immunoglobulin kappa J region (RBP-J) or the ablation of Notch2 did not significantly affect c-Met/sgAxin1-driven hepatocarcinogenesis. Conclusion: We demonstrated here that loss of Axin1 cooperates with c-Met to induce HCC in mice, in a ß-Catenin signaling-dependent but Notch cascade-independent way.

Use of Direct Liver Stiffness Measurement in Evaluating the Severity of Liver Cirrhosis in Patients with Hepatocellular Carcinoma.

BACKGROUND: Severity of liver cirrhosis plays an important role in determining the safe extents of hepatectomy in patients with hepatocellular carcinoma (HCC). The aim of this study was to investigate whether direct liver stiffness measurement can help surgeons to evaluate the severity of liver cirrhosis in HCC patients. METHODS: Overall, 119 HCC patients who underwent open hepatectomy were retrospectively studied. The severity of liver cirrhosis was histologically staged using the Laennec staging system. Direct liver stiffness measurement was performed during operation using a sclerometer device named LX-C Shaw hardmeter, and its efficacy in assessing the severity of liver cirrhosis was compared with that of transient elastography (TE) and cirrhotic severity scoring (CSS) previously proposed by our team. RESULTS: Liver stiffness measured by LX-C Shaw hardmeter was significantly correlated with the severity of liver cirrhosis. Spearman correlation coefficients for the correlation between the severity of liver cirrhosis and direct liver stiffness measurement, TE, and CSS were 0.751, 0.454, and 0.705, respectively (all P < 0.001). The areas under the receiver operating characteristic curves (AUCs) of direct liver stiffness measurement were 0.891 for moderate cirrhosis and 0.944 for severe cirrhosis and superior to those of TE (0.735 and 0.776, respectively) and CSS (0.888 and 0.905, respectively). CONCLUSIONS: Direct liver stiffness measurement is a useful method in evaluating the severity of liver cirrhosis in HCC patients.

Effectiveness comparison of indocyanine green retention test with the cirrhotic severity scoring in evaluating the pathological severity of liver cirrhosis in patients with hepatocellular carcinoma and Child-Pugh grade A liver function.

BACKGROUND: Evaluating cirrhotic severity is essential for individualizing surgical modalities for patients with hepatocellular carcinoma (HCC). Our previous study proposed a non-invasive method named cirrhotic severity scoring (CSS) to stage liver cirrhosis. Indocyanine green retention rate at 15 min (ICG-R15) has been widely used for the preoperative evaluation of hepatic functional reserve; however, whether ICG-R15 is well correlated with cirrhotic severity, and especially whether comparable with CSS in predicting cirrhotic severity in HCC patients with Child-Pugh grade A liver function remains unknown. METHODS: Overall, 510 HCC patients with Child-Pugh grade A liver function undergoing hepatectomy between January 2011 and December 2014 were retrospectively studied. Cirrhotic severity was pathologically assessed using the Laennec staging system. The correlations between ICG-R15, CSS, and cirrhotic severity were analyzed. Furthermore, the performance of ICG-R15 and CSS in predicting posthepatectomy liver failure (PHLF) and 90-day mortality was compared. RESULTS: Patients with no, mild, moderate, and severe cirrhosis accounted for 15.9%, 29.2%, 35.9%, and 19.0%, respectively, in the entire cohort. ICG-R15 was found to be less than 10% in 100%, 93.3%, 86.3%, and 70.1% of the patients with no, mild, moderate, and severe cirrhosis, respectively. There was only a weak correlation between ICG-R15 and the pathological severity of liver cirrhosis (r = 0.325; P < 0.001). However, CSS showed a strong correlation with the pathological severity of liver cirrhosis (r = 0.788; P < 0.001). For those with ICG-R15 in the normal range, the accuracy of CSS in diagnosing no/mild, moderate, and severe cirrhosis was 89.1%, 72.8%, and 72.1%, respectively. In addition, CSS was superior to ICG-R15 in predicting PHLF and 90-day mortality. CONCLUSIONS: CSS was more useful than ICG-R15 in the preoperative assessment of cirrhotic severity in HCC patients with Child-Pugh grade A liver function. More studies are needed to further validate CSS in patients with different Child-Pugh grades.

Complexin Binding to Membranes and Acceptor t-SNAREs Explains Its Clamping Effect on Fusion.

Complexin-1 is a SNARE effector protein that decreases spontaneous neurotransmitter release and enhances evoked release. Complexin binds to the fully assembled four-helical neuronal SNARE core complex as revealed in competing molecular models derived from x-ray crystallography. Presently, it is unclear how complexin binding to the postfusion complex accounts for its effects upon spontaneous and evoked release in vivo. Using a combination of spectroscopic and imaging methods, we characterize in molecular detail how complexin binds to the 1:1 plasma membrane t-SNARE complex of syntaxin-1a and SNAP-25 while simultaneously binding the lipid bilayer at both its N- and C-terminal ends. These interactions are cooperative, and binding to the prefusion acceptor t-SNARE complex is stronger than to the postfusion core complex. This complexin interaction reduces the affinity of synaptobrevin-2 for the 1:1 complex, thereby retarding SNARE assembly and vesicle docking in vitro. The results provide the basis for molecular models that account for the observed clamping effect of complexin beginning with the acceptor t-SNARE complex and the subsequent activation of the clamped complex by Ca2+ and synaptotagmin.

Asymmetric Phosphatidylethanolamine Distribution Controls Fusion Pore Lifetime and Probability.

Little attention has been given to how the asymmetric lipid distribution of the plasma membrane might facilitate fusion pore formation during exocytosis. Phosphatidylethanolamine (PE), a cone-shaped phospholipid, is predominantly located in the inner leaflet of the plasma membrane and has been proposed to promote membrane deformation and stabilize fusion pores during exocytotic events. To explore this possibility, we modeled exocytosis using plasma membrane SNARE-containing planar-supported bilayers and purified neuroendocrine dense core vesicles (DCVs) as fusion partners, and we examined how different PE distributions between the two leaflets of the supported bilayers affected SNARE-mediated fusion. Using total internal reflection fluorescence microscopy, the fusion of single DCVs with the planar-supported bilayer was monitored by observing DCV-associated neuropeptide Y tagged with a fluorescent protein. The time-dependent line shape of the fluorescent signal enables detection of DCV docking, fusion-pore opening, and vesicle collapse into the planar membrane. Four different distributions of PE in the planar bilayer mimicking the plasma membrane were examined: exclusively in the leaflet facing the DCVs; exclusively in the opposite leaflet; equally distributed in both leaflets; and absent from both leaflets. With PE in the leaflet facing the DCVs, overall fusion was most efficient and the extended fusion pore lifetime (0.7 s) enabled notable detection of content release preceding vesicle collapse. All other PE distributions decreased fusion efficiency, altered pore lifetime, and reduced content release. With PE exclusively in the opposite leaflet, resolution of pore opening and content release was lost.

Assembly and Comparison of Plasma Membrane SNARE Acceptor Complexes.

Neuronal exocytotic membrane fusion occurs on a fast timescale and is dependent on interactions between the vesicle SNARE synaptobrevin-2 and the plasma membrane SNAREs syntaxin-1a and SNAP-25 with a 1:1:1 stoichiometry. Reproducing fast fusion rates as observed in cells by reconstitution in vitro has been hindered by the spontaneous assembly of a 2:1 syntaxin-1a:SNAP-25 complex on target membranes that kinetically alters the binding of synaptobrevin-2. Previously, an artificial SNARE acceptor complex consisting of 1:1:1 syntaxin-1a(residues 183-288):SNAP-25:syb(residues 49-96) was found to greatly accelerate the rates of lipid mixing of reconstituted target and vesicle SNARE proteoliposomes. Here we present two (to our knowledge) new procedures to assemble membrane-bound 1:1 SNARE acceptor complexes that produce fast and efficient fusion without the need of the syb(49-96) peptide. In the first procedure, syntaxin-1a is purified in a strictly monomeric form and subsequently assembled with SNAP-25 in detergent with the correct 1:1 stoichiometry. In the second procedure, monomeric syntaxin-1a and dodecylated (d-)SNAP-25 are separately reconstituted into proteoliposomes and subsequently assembled in the plane of merged target lipid bilayers. Examining single particle fusion between synaptobrevin-2 proteoliposomes and planar-supported bilayers containing the two different SNARE acceptor complexes revealed similar fast rates of fusion. Changing the stoichiometry of syntaxin-1a and d-SNAP-25 in the target bilayer had significant effects on docking, but little effect on the rates of synaptobrevin-2 proteoliposome fusion.

Molecular Interactions of Lipopolysaccharide with an Outer Membrane Protein from Pseudomonas aeruginosa Probed by Solution NMR.

Pseudomonas aeruginosa is an opportunistic human pathogen causing pneumonias that are particularly severe in cystic fibrosis and immunocompromised patients. The outer membrane (OM) of P. aeruginosa is much less permeable to nutrients and other chemical compounds than that of Escherichia coli. The low permeability of the OM, which also contributes to Pseudomonas' significant antibiotic resistance, is augmented by the presence of the outer membrane protein H (OprH). OprH directly interacts with lipopolysaccharides (LPS) that constitute the outer leaflet of the OM and thus contributes to the structural stability of the OM. In this study, we used solution NMR spectroscopy to characterize the interactions between LPS and OprH in molecular detail. NMR chemical shift perturbations observed upon the addition of LPS to OprH in DHPC micelles indicate that this interaction is predominantly electrostatic and localized to the extracellular loops 2 and 3 and a number of highly conserved basic residues near the extracellular barrel rim of OprH. Single-site mutations of these residues were not enough to completely abolish binding, but OprH with cumulative mutations of Lys70, Arg72, and Lys103 no longer binds LPS. The dissociation constant (∼200 µM) measured by NMR is sufficient to efficiently bind LPS to OprH in the OM. This work highlights that solution NMR is suitable to study specific interactions of lipids with integral membrane proteins and provides a detailed molecular model for the interaction of LPS with OprH; i.e., an interaction that contributes to the integrity of the OM of P. aeruginosa under low divalent cation and antibiotic stress conditions. These methods should thus be useful for screening antibiotics that might disrupt OprH-LPS interactions and thereby increase the permeability of the OM of P. aeruginosa.