Sculpting conducting nanopore size and shape through de novo protein design.

Transmembrane ß-barrels have considerable potential for a broad range of sensing applications. Current engineering approaches for nanopore sensors are limited to naturally occurring channels, which provide suboptimal starting points. By contrast, de novo protein design can in principle create an unlimited number of new nanopores with any desired properties. Here we describe a general approach to designing transmembrane ß-barrel pores with different diameters and pore geometries. Nuclear magnetic resonance and crystallographic characterization show that the designs are stably folded with structures resembling those of the design models. The designs have distinct conductances that correlate with their pore diameter, ranging from 110 picosiemens (~0.5 nanometer pore diameter) to 430 picosiemens (~1.1 nanometer pore diameter). Our approach opens the door to the custom design of transmembrane nanopores for sensing and sequencing applications.

Overexpression of TBX3 suppresses tumorigenesis in experimental and human cholangiocarcinoma.

TBX3 behaves as a tumor suppressor or oncoprotein across cancer. However, TBX3 function remains undetermined in intrahepatic cholangiocarcinoma (iCCA), a deadly primary liver malignancy with few systemic treatment options. This study sought to investigate the impact of TBX3 on iCCA. We found that overexpression of TBX3 strongly inhibited human iCCA cell growth. In the Akt/FBXW7ΔF mouse iCCA model, overexpression of Tbx3 reduced cholangiocarcinogenesis in vivo, while inducible genetic knockout of Tbx3 accelerated iCCA growth. RNA-seq identified MAD2L1 as a downregulated gene in TBX3-overexpressing cells, and ChIP confirmed that TBX3 binds to the MAD2L1 promoter. CRISPR-mediated knockdown of Mad2l1 significantly reduced the growth of two iCCA models in vivo. Finally, we found that TBX3 expression is upregulated in ~20% of human iCCA samples, and its high expression is associated with less proliferation and better survival. MAD2L1 expression is upregulated in most human iCCA samples and negatively correlated with TBX3 expression. Altogether, our findings suggest that overexpression of TBX3 suppresses CCA progression via repressing MAD2L1 expression.

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.

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.

Targeting Iron - Respiratory Reciprocity Promotes Bacterial Death.

Discovering new bacterial signaling pathways offers unique antibiotic strategies. Here, through an unbiased resistance screen of 3,884 gene knockout strains, we uncovered a previously unknown non-lytic bactericidal mechanism that sequentially couples three transporters and downstream transcription to lethally suppress respiration of the highly virulent P. aeruginosa strain PA14 - one of three species on the WHO's 'Priority 1: Critical' list. By targeting outer membrane YaiW, cationic lacritin peptide 'N-104' translocates into the periplasm where it ligates outer loops 4 and 2 of the inner membrane transporters FeoB and PotH, respectively, to suppress both ferrous iron and polyamine uptake. This broadly shuts down transcription of many biofilm-associated genes, including ferrous iron-dependent TauD and ExbB1. The mechanism is innate to the surface of the eye and is enhanced by synergistic coupling with thrombin peptide GKY20. This is the first example of an inhibitor of multiple bacterial transporters.

Scientific Hepatectomy for Hepatocellular Carcinoma.

With advances in imaging technology and surgical instruments, hepatectomy can be perfectly performed with technical precision for hepatocellular carcinoma (HCC). However, the 5-year tumor recurrence rates remain greater than 70%. Thus, the strategy for hepatectomy needs to be reappraised based on insights of scientific advances. Scientific evidence has suggested that the main causes of recurrence after hepatectomy for HCC are mainly related to underlying cirrhosis and the vascular spread of tumor cells that basically cannot be eradicated by hepatectomy. Liver transplantation and systemic therapy could be the solution to prevent postoperative recurrence in this regard. Therefore, determining the severity of liver cirrhosis for choosing the appropriate surgical modality, such as liver transplantation or hepatectomy, for HCC and integrating newly emerging immune-related adjuvant and/or neoadjuvant therapy into the strategy of hepatectomy for HCC have become new aspects of exploration to optimize the strategy of hepatectomy. In this new area, hepatectomy for HCC has evolved from a pure technical concept emphasizing anatomic resection into a scientific concept embracing technical considerations and scientific advances in underlying liver cirrhosis, vascular invasion, and systemic therapy. By introducing the concept of scientific hepatectomy, the indications, timing, and surgical techniques of hepatectomy will be further scientifically optimized for individual patients, and recurrence rates will be decreased and long-term survival will be further prolonged.

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.

Postoperative adjuvant tyrosine kinase inhibitors combined with anti-PD-1 antibodies improves surgical outcomes for hepatocellular carcinoma with high-risk recurrent factors.

Background: The clinical value of postoperative adjuvant therapy (PAT) for hepatocellular carcinoma (HCC) remains unclear. This study aimed to explore the effect of PAT with tyrosine kinase inhibitors (TKIs) and anti-PD-1 antibodies on the surgical outcomes of HCC patients with high-risk recurrent factors (HRRFs). Methods: HCC patients who underwent radical hepatectomy at Tongji Hospital between January 2019 and December 2021 were retrospectively enrolled, and those with HRRFs were divided into PAT group and non-PAT group. Recurrence-free survival (RFS) and overall survival (OS) were compared between the two groups after propensity score matching (PSM). Prognostic factors associated with RFS and OS were determined by Cox regression analysis, and subgroup analysis was also conducted. Results: A total of 250 HCC patients were enrolled, and 47 pairs of patients with HRRFs in the PAT and non-PAT groups were matched through PSM. After PSM, the 1- and 2-year RFS rates in the two groups were 82.1% vs. 40.0% (P < 0.001) and 54.2% vs. 25.1% (P = 0.012), respectively. The corresponding 1- and 2-year OS rates were 95.4% vs. 69.8% (P = 0.001) and 84.3% vs. 55.5% (P = 0.014), respectively. Multivariable analyses indicated that PAT was an independent factor related to improving RFS and OS. Subgroup analysis demonstrated that HCC patients with tumor diameter > 5 cm, satellite nodules, or vascular invasion could significantly benefit from PAT in RFS and OS. Common grade 1-3 toxicities, such as pruritus (44.7%), hypertension (42.6%), dermatitis (34.0%), and proteinuria (31.9%) were observed, and no grade 4/5 toxicities or serious adverse events occurred in patients receiving PAT. Conclusions: PAT with TKIs and anti-PD-1 antibodies could improve surgical outcomes for HCC patients with HRRFs.

Radiation therapy in the era of immune treatment for hepatocellular carcinoma.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment in recent years and provide new opportunities to treat hepatocellular carcinoma (HCC). To date, several ICIs have been approved by the FDA for advanced HCC in first-line or second-line therapy. Downstaging conversion therapy for potentially resectable HCC to provide opportunities for surgical intervention is challenging. ICIs have become a hot spot in this field due to their high response rate. However, HCC has various etiologies and can evade the immune system through multiple mechanisms, which limit the efficacy of ICI monotherapy and demand novel combination strategies. Radiation therapy (RT) is also a candidate for conversion therapy in HCC and is currently gaining increasing attention as a good combination partner with ICIs due to its ability to modulate the tumor microenvironment. In this review, we illustrate the current indications for ICIs and RT in HCC, the rationale for their synergistic combination, and the current clinical trials in combination therapy. We also speculate on predictive biomarkers and novel future strategies to further enhance the efficacy of this combination. This review aims to provide references for future research on radiation and immunotherapy to arrive at a promising new era of HCC treatment.

HBcAb positivity increases the risk of postoperative complications after extended hemihepatectomy for hilar cholangiocarcinoma.

BACKGROUND: Hepatitis B core antibody (HBcAb) positivity is considered a prior hepatitis B virus (HBV) infection. However, little is known about the effect of HBcAb positivity on surgical safety for hilar cholangiocarcinoma (hCCA). The present study aims to investigate the role of HBcAb positivity on postoperative complications of hCCA. METHODS: A retrospective analysis was performed on the status of HBcAb positivity, liver fibrosis, perioperative surgical complications, and long-term outcomes of hCCA patients with Hepatitis B surface antigen (HBsAg) negativity who underwent surgical treatment in Tongji Hospital from April 2012 to September 2019. RESULTS: HBcAb positivity with negative HBsAg occurs in 137 hCCA patients (63.1%). A total of 99 hCCA patients with negative HBsAg underwent extended hemihepatectomy, of whom 69 (69.7%) and 30 (30.3%) were HBcAb-positive and HBcAb-negative, respectively. Significant fibrosis was detected in 63.8% of the patients with HBcAb-positive, which was markedly higher than those with HBcAb-negative (36.7%) (p = 0.016). The postoperative complications and 90-day mortality rates were 37.4% (37/99) and 8.1% (8/99), respectively. The incidence of postoperative complications in HBcAb-positive patients (44.9%) was significantly higher than that in HBcAb-negative patients (20.0%) (p = 0.018). All the patients who died within 30-day after surgery were HBcAb-positive. Multivariate analysis showed that the independent risk factors for complications were HBcAb positivity, preoperative cholangitis, portal occlusion >15 min, and significant fibrosis. There were no significant differences in recurrence-free survival (RFS) and overall survival (OS) between HBcAb-positive and HBcAb-negative patients (p = 0.642 and p = 0.400, respectively). CONCLUSIONS: HBcAb positivity is a common phenomenon in hCCA patients from China, a country with highly prevalent HBcAb positivity. The status of HBcAb-positive markedly increases the incidence of postoperative complications after extended hemihepatectomy for hCCA patients.

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.

Sculpting conducting nanopore size and shape through de novo protein design.

Transmembrane ß-barrels (TMBs) are widely used for single molecule DNA and RNA sequencing and have considerable potential for a broad range of sensing and sequencing applications. Current engineering approaches for nanopore sensors are limited to naturally occurring channels such as CsgG, which have evolved to carry out functions very different from sensing, and hence provide sub-optimal starting points. In contrast, de novo protein design can in principle create an unlimited number of new nanopores with any desired properties. Here we describe a general approach to the design of transmembrane ß-barrel pores with different diameter and pore geometry. NMR and crystallographic characterization shows that the designs are stably folded with structures close to the design models. We report the first examples of de novo designed TMBs with 10, 12 and 14 stranded ß-barrels. The designs have distinct conductances that correlate with their pore diameter, ranging from 110 pS (~0.5 nm pore diameter) to 430 pS (~1.1 nm pore diameter), and can be converted into sensitive small-molecule sensors with high signal to noise ratio. The capability to generate on demand ß-barrel pores of defined geometry opens up fundamentally new opportunities for custom engineering of sequencing and sensing technologies.

Machine learning predicts portal vein thrombosis after splenectomy in patients with portal hypertension: Comparative analysis of three practical models.

BACKGROUND: For patients with portal hypertension (PH), portal vein thrombosis (PVT) is a fatal complication after splenectomy. Postoperative platelet elevation is considered the foremost reason for PVT. However, the value of postoperative platelet elevation rate (PPER) in predicting PVT has never been studied. AIM: To investigate the predictive value of PPER for PVT and establish PPER-based prediction models to early identify individuals at high risk of PVT after splenectomy. METHODS: We retrospectively reviewed 483 patients with PH related to hepatitis B virus who underwent splenectomy between July 2011 and September 2018, and they were randomized into either a training (n = 338) or a validation (n = 145) cohort. The generalized linear (GL) method, least absolute shrinkage and selection operator (LASSO), and random forest (RF) were used to construct models. The receiver operating characteristic curves (ROC), calibration curve, decision curve analysis (DCA), and clinical impact curve (CIC) were used to evaluate the robustness and clinical practicability of the GL model (GLM), LASSO model (LSM), and RF model (RFM). RESULTS: Multivariate analysis exhibited that the first and third days for PPER (PPER1, PPER3) were strongly associated with PVT [odds ratio (OR): 1.78, 95% confidence interval (CI): 1.24-2.62, P = 0.002; OR: 1.43, 95%CI: 1.16-1.77, P < 0.001, respectively]. The areas under the ROC curves of the GLM, LSM, and RFM in the training cohort were 0.83 (95%CI: 0.79-0.88), 0.84 (95%CI: 0.79-0.88), and 0.84 (95%CI: 0.79-0.88), respectively; and were 0.77 (95%CI: 0.69-0.85), 0.83 (95%CI: 0.76-0.90), and 0.78 (95%CI: 0.70-0.85) in the validation cohort, respectively. The calibration curves showed satisfactory agreement between prediction by models and actual observation. DCA and CIC indicated that all models conferred high clinical net benefits. CONCLUSION: PPER1 and PPER3 are effective indicators for postoperative prediction of PVT. We have successfully developed PPER-based practical models to accurately predict PVT, which would conveniently help clinicians rapidly differentiate individuals at high risk of PVT, and thus guide the adoption of timely interventions.

Anatomical liver resection improves surgical outcomes for combined hepatocellular-cholangiocarcinoma: A propensity score matched study.

Background: The efficacies of anatomical resection (AR) and non-anatomical resection (NAR) in the treatment of combined hepatocellular-cholangiocarcinoma (cHCC-CCA) remain unclear. This study aimed to compare the prognostic outcomes of AR with those of NAR for cHCC-CCA. Method: Patients diagnosed with pathology-confirmed cHCC-CCA, and who underwent curative resection at Tongji hospital between January 2010 and December 2019 were included in this retrospective study. A one-to-one propensity score matching (PSM) analysis was used to compare the long-term outcomes of AR to those of NAR. Results: A total of 105 patients were analyzed, of whom 48 (45.7%) and 57 (54.3%) underwent AR and NAR, respectively. There were no significant differences in short-term outcomes between the two groups, including duration of postoperative hospital stay, the incidence of perioperative complications, and incidence of 30-day mortality. However, both, the 5-year overall survival (OS) and recurrence-free survival (RFS) rates of AR were significantly better than those of NAR (40.5% vs. 22.4%, P=0.002; and 37.3% vs. 14.4%, P=0.002, respectively). Multivariate analysis showed that NAR, multiple tumors, larger-sized tumors (>5 cm), cirrhosis, lymph node metastasis, and vascular invasion were independent risk factors for poor prognoses. Stratified analysis demonstrated similar outcomes following AR versus NAR for patients with tumors > 5cm in diameter, while AR had better survival than NAR in patients with tumors ≤5 cm in diameter. After PSM, when 34 patients from each group were matched, the 5-year OS and RFS rates of AR were still better than those of NAR. Conclusion: Patients with cHCC-CCA who underwent AR had better long-term surgical outcomes than those who underwent NAR, especially for those with tumors ≤5 cm in diameter. However, no differences in the risk of surgical complications were detected between the two groups.

Histological Severity of Cirrhosis Influences Surgical Outcomes of Hepatocellular Carcinoma After Curative Hepatectomy.

Background: Hepatocellular carcinoma (HCC) is frequently associated with cirrhosis. The present study investigated the impact of histological severity of cirrhosis on surgical outcomes for HCC and further developed novel nomograms to predict postoperative recurrence and survival. Methods: A total of 1524 consecutive patients undergoing curative hepatectomy for HCC between 1999 and 2015 were retrospectively studied. Cirrhotic severity was histologically staged according to the Laennec staging system. Short- and long-term outcomes were investigated. Recurrence-free survival (RFS) and overall survival (OS) predictive nomograms were constructed based on the results of multivariate analysis. The predictive accuracy of the nomograms was measured by the concordance index (C-index) and calibration. Results: Patients in the severe cirrhosis group had significantly higher morbidity and mortality rates than patients in the no, mild, and moderate cirrhosis groups. The 5-year RFS and OS rates were 36.8% and 64.5%, respectively, in the no cirrhosis group, compared to 34.8% and 60.4% in the mild cirrhosis group, 17.3% and 43.4% in the moderate cirrhosis group, and 6.1% and 20.1% in the severe cirrhosis group. Long-term survival outcomes were significantly worse as cirrhotic severity was increased. The C-index was 0.727 for the RFS nomogram and 0.746 for the OS nomogram. Calibration curves showed good agreement between actual observations and nomogram predictions. The 2 nomograms had a superior discriminatory ability to predict RFS and OS compared to other staging systems. Conclusion: Histological severity of cirrhosis significantly affected surgical outcomes in HCC patients undergoing curative hepatectomy. The novel nomograms, including histological severity of cirrhosis, showed an accurate prediction of postoperative recurrence and survival.

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.

Prognostic Nomograms Based on the Cirrhotic Severity Scoring for Preoperative Prediction of Long-Term Outcomes in Patients with HBV-Related Hepatocellular Carcinoma and Child-Pugh Grade A Liver Function.

Background: Cirrhotic severity scoring (CSS) is a noninvasive method that can predict histological severity of cirrhosis. This study is aimed at assessing the predictive value of CSS on long-term outcomes after curative hepatectomy for patients with hepatitis B virus- (HBV-) related hepatocellular carcinoma (HCC) and Child-Pugh grade A liver function and further developing novel nomograms to preoperatively predict posthepatectomy recurrence and survival. Methods: Consecutive patients who underwent curative hepatectomy for HCC between 2008 and 2014 were retrospectively studied. According to the CSS, patients were subclassified into 3 groups: no/mild, moderate, and severe cirrhosis. The impact of CSS on recurrence-free survival (RFS) and overall survival (OS) was assessed. Furthermore, RFS and OS nomograms were developed. Results: The 5-year RFS and OS rates were 36.1% and 62.8% in the no/mild cirrhosis group, compared with 28.4% and 56.2% in the moderate cirrhosis group, and 16.2% and 33.0% in the severe cirrhosis group. Long-term survival outcomes were significantly worse with the increment of cirrhotic severity. CSS, alpha-fetoprotein level, tumor size, tumor number, and macrovascular invasion were identified as independent predictors of both RFS and OS. Besides, albumin-bilirubin grade was an independent risk factor of OS not RFS. RFS- and OS-predictive nomograms based on these preoperative variables were built. For these 2 nomograms, the C-indexes were 0.696 and 0.732, respectively. Calibration curves exhibited good agreement between actual observation and nomogram prediction. Conclusions: CSS was a predictor for long-term outcomes in HCC patients after curative hepatectomy. The novel nomograms exhibited accurate preoperative prediction of posthepatectomy recurrence and OS.

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.

Tumor size may influence the prognosis of solitary hepatocellular carcinoma patients with cirrhosis and without macrovascular invasion after hepatectomy.

Hepatocellular carcinoma (HCC) is usually associated with varying degrees of cirrhosis. Among cirrhotic patients with solitary HCC in the absence of macro-vascular invasion, whether tumor size drives prognosis or not after hepatectomy remains unknown. This study aimed to investigate the prognostic impact of tumor size on long-term outcomes after hepatectomy for solitary HCC patients with cirrhosis and without macrovascular invasion. A total of 813 cirrhotic patients who underwent curative hepatectomy for solitary HCC and without macrovascular invasion between 2001 and 2014 were retrospectively studied. We set 5 cm as the tumor cut-off value. Propensity score matching (PSM) was performed to minimize the influence of potential confounders including cirrhotic severity that was histologically assessed according to the Laennec staging system. Recurrence-free survival (RFS) and overall survival (OS) were compared between the two groups before and after PSM. Overall, 464 patients had tumor size ≤ 5 cm, and 349 had tumor size > 5 cm. The 5-year RFS and OS rates were 38.3% and 61.5% in the ≤ 5 cm group, compared with 25.1% and 59.9% in the > 5 cm group. Long-term survival outcomes were significantly worse as tumor size increased. Multivariate analysis indicated that tumor size > 5 cm was an independent risk factor for tumor recurrence and long-term survival. These results were further confirmed in the PSM cohort of 235 pairs of patients. In cirrhotic patients with solitary HCC and without macrovascular invasion, tumor size may significantly affect the prognosis after curative hepatectomy.

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.