SMAD4 regulates the progression of cholangiocarcinoma by modulating the expression of STING1.

SMAD4 is a tumour suppressor and an important regulator of tumour immune scape which is downregulated in cholangiocarcinoma (CCA). STING1 is a vital sensing factor of abnormal DNA; however, the correlation between SMAD4 and STING1 and the role of the SMAD4-STING1 interaction in the progression of CCA have not yet been evaluated. Public database was analysed to reveal the expression of SMAD4 and STING1. A cohort comprising 50 iCCA, 113 pCCA and 119 dCCA patients was assembled for the study. Immunohistochemistry was employed to evaluate the expression levels of STING1 and SMAD4. In vitro transwell and CCK8 assays, along with luciferase reporter assay, were conducted to analyse the potential regulatory mechanisms of SMAD4 on the expression of STING1. Expression of SMAD4 and STING1 were downregulated in CCA tumours and STING1 expression correlated with SMAD4 expression. The overexpression of SMAD4 was found to suppress the migration, invasion and proliferation capabilities of CCA cells; whereas, the knockdown of SMAD4 enhanced these abilities. Furthermore, it was observed that SMAD4 translocated into the nucleus following TGF-ß1 stimulation. Knockdown of SMAD4 resulted in the inhibition of STING1 transcriptional activity, whereas the overexpression of SMAD4 promoted the transcriptional activity of STING1. Clinically, low STING1 and SMAD4 expression indicated poor prognosis in CCA, and simultaneously low expression of STING1 and SMAD4 predicts poorer patient survival. SMAD4 regulates the expression of STING1 through its transcription regulating function. Dual low expression of STING1 and SMAD4 had more power in predicting patient survival. These results indicate that SMAD4-silenced CCA may downregulate its STING1 expression to adapt to the immune system.

PTP-MEG2 regulates quantal size and fusion pore opening through two distinct structural bases and substrates.

Tyrosine phosphorylation of secretion machinery proteins is a crucial regulatory mechanism for exocytosis. However, the participation of protein tyrosine phosphatases (PTPs) in different exocytosis stages has not been defined. Here we demonstrate that PTP-MEG2 controls multiple steps of catecholamine secretion. Biochemical and crystallographic analyses reveal key residues that govern the interaction between PTP-MEG2 and its substrate, a peptide containing the phosphorylated NSF-pY83 site, specify PTP-MEG2 substrate selectivity, and modulate the fusion of catecholamine-containing vesicles. Unexpectedly, delineation of PTP-MEG2 mutants along with the NSF binding interface reveals that PTP-MEG2 controls the fusion pore opening through NSF independent mechanisms. Utilizing bioinformatics search and biochemical and electrochemical screening approaches, we uncover that PTP-MEG2 regulates the opening and extension of the fusion pore by dephosphorylating the DYNAMIN2-pY125 and MUNC18-1-pY145 sites. Further structural and biochemical analyses confirmed the interaction of PTP-MEG2 with MUNC18-1-pY145 or DYNAMIN2-pY125 through a distinct structural basis compared with that of the NSF-pY83 site. Our studies thus provide mechanistic insights in complex exocytosis processes.

Identification of a benzo imidazole thiazole derivative as the specific irreversible inhibitor of protein tyrosine phosphatase.

Protein tyrosine phosphatases (PTPs) play key roles in many physiological processes, including cell proliferation, differentiation, immune responses and neural activities. Inappropriate regulation of the PTP activity could lead to human diseases, such as cancer or diabetes. Functional studies of PTP can be greatly facilitated by chemical probes that covalently label the active site of a PTP through an activity-dependent chemical reaction. Here, we characterize compound E4 as a new class of PTP activity probes. Compound E4 inactivate STEP in a time- and concentration-dependent fashion. Further study showed that compound E4 inhibits a series of PTPs in a time dependent manner, whereas it shows little or no inhibition toward metal dependent protein phosphatases. Collectively, this new identified covalent inhibitor of PTPs has the potential to be developed to an active site Cys directed PTP probes to study the active properties of the PTPs in cell signaling.

Molecular mechanism of ERK dephosphorylation by striatal-enriched protein tyrosine phosphatase.

Striatal-enriched tyrosine phosphatase (STEP) is an important regulator of neuronal synaptic plasticity, and its abnormal level or activity contributes to cognitive disorders. One crucial downstream effector and direct substrate of STEP is extracellular signal-regulated protein kinase (ERK), which has important functions in spine stabilisation and action potential transmission. The inhibition of STEP activity toward phospho-ERK has the potential to treat neuronal diseases, but the detailed mechanism underlying the dephosphorylation of phospho-ERK by STEP is not known. Therefore, we examined STEP activity toward para-nitrophenyl phosphate, phospho-tyrosine-containing peptides, and the full-length phospho-ERK protein using STEP mutants with different structural features. STEP was found to be a highly efficient ERK tyrosine phosphatase that required both its N-terminal regulatory region and key residues in its active site. Specifically, both kinase interaction motif (KIM) and kinase-specific sequence of STEP were required for ERK interaction. In addition to the N-terminal kinase-specific sequence region, S245, hydrophobic residues L249/L251, and basic residues R242/R243 located in the KIM region were important in controlling STEP activity toward phospho-ERK. Further kinetic experiments revealed subtle structural differences between STEP and HePTP that affected the interactions of their KIMs with ERK. Moreover, STEP recognised specific positions of a phospho-ERK peptide sequence through its active site, and the contact of STEP F311 with phospho-ERK V205 and T207 were crucial interactions. Taken together, our results not only provide the information for interactions between ERK and STEP, but will also help in the development of specific strategies to target STEP-ERK recognition, which could serve as a potential therapy for neurological disorders. Regulation of phospho-ERK by STEP underlies important neuronal activities. A detailed enzymologic characterisation and cellular studies of STEP revealed that specific residues in KIM and active site mediated ERK recognition. Structural differences between the KIM-ERK interfaces and the active site among different ERK phosphatases could be targeted to develop specific STEP inhibitor, which has therapeutic potential for neurological disorders. PKA, protein kinase A & NGF, nerve growth factor.

Case report: Obstructive jaundice caused by biliary cystadenoma.

Biliary cystadenoma (also called mucinous cystic neoplasm with low-grade intraepithelial neoplasia) is a rare cystic tumor that arises from the biliary epithelium. The cause of biliary cystadenoma is still unclear. Jaundice is a rare presentation of intrahepatic biliary cystadenoma, which can lead to a diagnostic dilemma. Herein, we present a case of intrahepatic biliary cystadenoma that primarily exhibited as jaundice. A 56-year-old woman has suffered from yellow staining of her skin and sclera for more than 1 month. She had a poor appetite and mild epigastric pain. Laboratory examination showed elevated levels of total bilirubin and elevated carbohydrate antigen 19-9 (CA19-9). A contrast-enhanced computed tomography of the abdomen showed a 7.4 * 5.3-cm, oval, low-density lesion in the left liver parenchyma with a clear boundary and visible septa. The common bile duct was obviously dilated with wall thickening. On magnetic resonance imaging, the lesion in the liver showed a multilocular cystic, unenhanced long T2 signal. There was local thickening of the common bile duct wall with short T2-like filling defects and high signal intensity on diffusion-weighted imaging (DWI). The patient had no history of other malignant tumors and adjuvant therapy such as radiotherapy and chemotherapy. She was clinically suspected of having either biliary cystadenoma or a malignancy; hence, resection was performed. Macroscopically, the excised tissue specimen showed a polypoid mass in the common bile duct, which extended along the bile duct to the intrahepatic bile duct. There was a cystic and solid mass in the left liver with yellow turbid fluid, which was associated with the polypoid mass in the common bile duct. Histopathology suggests mucinous cystadenoma of the liver and hilar bile duct. The differential diagnosis of biliary cystadenoma and treatment selection have been discussed.

Budd-Chiari syndrome and its associated hepatocellular carcinoma: Clinical risk factors and potential immunotherapeutic benefit analysis.

Background: Hepatocellular carcinoma (HCC) is a well-described complication of Budd-Chiari syndrome (BCS). However, the risk factors of BCS in developing HCC and clinical characteristics and imaging features of BCS-associated HCC is still to be determined. Methods: Data from 113 consecutive patients with primary BCS in Qilu hospital were retrospectively studied. The clinical features of 12 HCC patients associated with BCS were also analyzed. Chi-square analysis was performed to analyze the differences in clinical characteristics. The treatment regime and CT imaging features of BCS-associated HCC were also illustrated. Results: 113 consecutive patients admitted to our hospital between January 2009 and June 2016 with a primary diagnosis of BCS were enrolled. 10.6% (12/113) was diagnosed with HCC. The BCS patients were mainly male gender with an average age of 49.2 years. Symptom duration longer than one year exhibited decreased serum ALT and AST and increased ascites ratio. BCS-associated HCC patients were presented with IVC block and stricture of the hepatic venous outflow tract. Patients with HCC were older and showed elevated serum AST and total bilirubin. Most nodules of HCC located in the right posterior lobe with heterogeneous enhancement during the arterial phase and washout during the delayed phase. Conclusions: The results indicate that BCS patients with IVC block and stricture of hepatic venous outflow tract seem to be associated with HCC. BCS associated HCC nodules exhibited irregular and heterogeneous enhancement in the arterial phase and washout on the delayed phase.

Physical activity improves outcomes of combined lenvatinib plus anti-PD-1 therapy in unresectable hepatocellular carcinoma: a retrospective study and mouse model.

BACKGROUND: Physical activity is known to have anti-cancer effects, including immunomodulatory actions. This study investigated the hypothesis that physical activity synergizes with combined lenvatinib plus anti-PD-1 therapy to enhance efficacy in patients with unresectable HCC. METHODS: The physical activity levels of patients with unresectable HCC receiving combined lenvatinib plus anti-PD-1 therapy were recorded by questionnaire. Patients were categorized according to physical activity levels (active vs. sedentary). The primary outcome was overall survival (OS). Secondary outcomes included objective response rate (ORR) and progression-free survival (PFS). A subcutaneous syngeneic HCC model was generated in C57BL/6 mice. Mice were randomized to receive placebo, combined lenvatinib plus anti-PD-1 antibodies or combination therapy plus physical activity. Tumors were measured every 3 days and harvested for immunohistochemistry analysis at 20 mm maximum diameter. RESULTS: Fifty-nine patients with unresectable HCC were categorized to active (n = 28) or sedentary (n = 31) groups. The active group had higher albumin and des-γ-carboxy prothrombin levels and lower hepatitis B virus load at baseline; other clinical and oncologic characteristics were comparable between the two groups. Patients in the active group had significantly longer OS (HR = 0.220, 95% CI 0.060-0.799) and PFS (HR = 0.158, 95% CI 0.044-0.562) and higher ORR (OR = 4.571, 95% CI 1.482-14.102) than patients in the sedentary group. Regular physical activity was independently associated with OS, PFS and ORR. The mouse model showed that physical activity significantly suppressed tumor growth and prolonged survival of tumor-bearing mice. Furthermore, physical activity inhibited Treg cell infiltration and immune checkpoint expression (including CTLA4, TIGIT and TIM3) induced by long-term combined lenvatinib plus anti-PD-1 therapy, improving efficacy. CONCLUSIONS: Regular physical activity was associated with improved outcomes in unresectable HCC receiving combined lenvatinib plus anti-PD-1 therapy. Physical activity may improve therapeutic efficacy by reprograming the tumor microenvironment from an immunosuppressive to immunostimulatory phenotype.

Research progress of bile biomarkers and their immunoregulatory role in biliary tract cancers.

Biliary tract cancers (BTCs), including cholangiocarcinoma and gallbladder carcinoma, originate from the biliary epithelium and have a poor prognosis. Surgery is the only choice for cure in the early stage of disease. However, most patients are diagnosed in the advanced stage and lose the chance for surgery. Early diagnosis could significantly improve the prognosis of patients. Bile has complex components and is in direct contact with biliary tract tumors. Bile components are closely related to the occurrence and development of biliary tract tumors and may be applied as biomarkers for BTCs. Meanwhile, arising evidence has confirmed the immunoregulatory role of bile components. In this review, we aim to summarize and discuss the relationship between bile components and biliary tract cancers and their ability as biomarkers for BTCs, highlighting the role of bile components in regulating immune response, and their promising application prospects.

Irbesartan inhibits metastasis by interrupting the adherence of tumor cell to endothelial cell induced by angiotensin II in hepatocellular carcinoma.

BACKGROUND: The use of angiotensin II inhibitors is associated with a low risk of recurrence and metastasis in hepatocellular carcinoma (HCC) patients. Vascular cell adhesion molecule-1 (VCAM-1) is a key factor in tumor metastasis. METHODS: The effects of angiotensin II and irbesartan (an angiotensin II inhibitor) on HCC were explored with a xenograft model, microarray analysis and cell adhesion experiments. The relationship between the expression of VCAM-1 in HCC tissues and prognosis was analyzed with public and our institutional clinical databases. The effects of angiotensin II, irbesartan and VCAM-1 on adhesion and metastasis in HCC were explored with a xenograft model and cell adhesion experiments. The regulatory mechanisms were analyzed by Western blot analysis. RESULTS: Angiotensin II type 1 receptor and VCAM-1 were expressed in HCC tissues. Irbesartan inhibited HCC growth and metastasis in vivo and weakened the adhesion of HCC cells to endothelial cells, an effect that was enhanced by angiotensin II. VCAM-1 was found to be an independent risk factor for recurrence and survival in HCC patients with microvascular invasion. Angiotensin II upregulated VCAM-1 expression, and this upregulation was inhibited by irbesartan. Angiotensin II enhanced adhesion mainly by promoting the expression of VCAM-1 in HCC cells. Irbesartan inhibited the expression of VCAM-1 by reducing p38/MAPK phosphorylation activated by angiotensin II in HCC cells. CONCLUSIONS: Irbesartan attenuates metastasis by inhibiting angiotensin II-activated VCAM-1 via the p38/MAPK pathway in HCC.

Adjuvant therapies after curative treatments for hepatocellular carcinoma: Current status and prospects.

Tumor recurrence rate after surgery or ablation of hepatocellular carcinoma (HCC) is as high as 70%. However, there are no widely accepted adjuvant therapies; therefore, no treatment has been recommended by guidelines from the American Association for the Study of Liver Disease or the European Association for the Study of the Liver. All the registered trials failed to find any treatment to prolong recurrence-free survival, which is the primary outcome in most studies, including sorafenib. Some investigator-initiated studies revealed that anti-hepatitis B virus agents, interferon-α, transcatheter chemoembolization, chemokine-induced killer cells, and other treatments prolonged patient recurrence-free survival or overall survival after curative therapies. In this review, we summarize the current status of adjuvant treatments for HCC and explain the challenges associated with designing a clinical trial for adjuvant therapy. Promising new treatments being used as adjuvant therapy, especially anti-PD-1 antibodies, are also discussed.

NT5DC2 promotes tumor cell proliferation by stabilizing EGFR in hepatocellular carcinoma.

Most hepatocellular carcinoma (HCC) patients are diagnosed at an advanced stage; however, the effect of systemic therapy on advanced HCC remains undetermined. Therefore, new treatment targets must be identified. We analyzed Gene Expression Omnibus datasets from two HCC patient cohorts and found that NT5DC2 was associated with vascular invasion and poor survival. In two hepatoma cell lines, NT5DC2 overexpression promoted HCC cell proliferation and clone formation in vitro and promoted tumor growth in vivo. Coimmunoprecipitation assays and liquid chromatography with tandem mass spectrometry analysis revealed that NT5DC2 bound directly to epidermal growth factor receptor (EGFR). NT5DC2 upregulated EGFR expression by downregulating EGFR ubiquitination and preventing its degradation via the ubiquitin-proteasome pathway but did not upregulate its transcription. EGFR upregulation activated downstream signal transduction, which played a critical role in the protumor effects of NT5DC2. Erlotinib, a small-molecule inhibitor of EGFR, blocked the effect of NT5DC2 in promoting HCC cell proliferation. In a cohort of 79 patients who underwent curative resection for HCC, NT5DC2 expression in the tumors was associated with larger tumors and microvascular invasion. NT5DC2 expression was also independently associated with recurrence-free survival. The present study demonstrated for the first time that NT5DC2 promotes tumor cell proliferation in HCC and may serve as a potential molecular target for treating HCC. EGFR blockage could be used to treat selected patients with NT5DC2 upregulation.

Renin-angiotensin inhibitors were associated with improving outcomes of hepatocellular carcinoma with primary hypertension after hepatectomy.

BACKGROUND: The activation of the renin-angiotensin system (RAS) promotes tumor progression. In this study, we aimed to assess whether RAS inhibitors (RASIs) could improve the outcome of hepatocellular carcinoma (HCC) patients with primary hypertension after curative liver resection. METHODS: Data on 387 consecutive patients with primary hypertension who underwent curative liver resection for HCC were reviewed. The study population was divided into two groups based on the type of anti-hypertensive medications: the RASI group (patients using RASIs) and the non-RASI group (patients using other anti-hypertensive drugs but not RASIs). Kaplan-Meier curves, log-rank tests and cox proportional hazards regression models were used to analyze time to recurrence (TTR) and overall survival (OS). RESULTS: There were 144 (37.2%) patients in RASI group and 243 (62.8%) in non-RASI group. The preoperative clinicopathological features were comparable between the two groups. Kaplan-Meier curves demonstrated HCC patients with RASIs had a longer TTR and OS than the patients with non-RASIs (both P<0.001). On multivariate analysis, RASIs administration was identified as an independent prognostic factor for TTR [hazard ratio (HR) =0.52, 95% confidence interval (CI), 0.38-0.70, P<0.001] and OS (HR =0.50, 95% CI, 0.34-0.74, P<0.001). Patients in the RASI group had lower rates of extrahepatic metastases than patients in the non-RASI group (2.8% vs. 7.8%, P<0.042). CONCLUSIONS: Targeting the RAS was associated with a reduced risk of recurrence, decreased rate of extrahepatic metastases and prolonged survival of HCC patients with primary hypertension after curative liver resection.

PFKFB3 blockade inhibits hepatocellular carcinoma growth by impairing DNA repair through AKT.

Overexpression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a key molecule of glucose metabolism in cytoplasm, has been found in various tumors. Emerging evidence has suggested that PFKFB3 is also located in the nucleus and apparent in regulatory functions other than glycolysis. In this study, we found that PFKFB3 expression is associated with hepatocellular carcinoma (HCC) growth and located mainly in the nucleus of tumor cells. PFKFB3 overexpression was associated with large tumor size (p = 0.04) and poor survival of patients with HCC (p = 0.027). Knockdown of PFKFB3 inhibited HCC growth, not only by reducing glucose consumption but also by damaging the DNA repair function, leading to G2/M phase arrest and apoptosis. In animal studies, overexpression of PFKFB3 is associated with increased tumor growth. Mechanistically, PFKFB3 silencing decreased AKT phosphorylation and reduced the expression of ERCC1, which is an important DNA repair protein. Moreover, PFK15, a selective PFKFB3 inhibitor, significantly inhibited tumor growth in a xenograft model of human HCC. PFKFB3 is a potential novel target in the treatment of HCC.

CD31 regulates metastasis by inducing epithelial-mesenchymal transition in hepatocellular carcinoma via the ITGB1-FAK-Akt signaling pathway.

Platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31) is a well-known marker of endothelial cells and a key factor for adhesion and accumulation of platelets. CD31 plays roles in cell proliferation, apoptosis, migration, and cellular immunity. CD31 is also expressed on tumor cells, such as breast cancer cells and non-Hodgkin's lymphomas, and contributes to tumor cell invasion. Here, our experiments show that CD31 promotes metastasis by inducing the epithelial-mesenchymal transition in hepatocellular carcinoma by up-regulating integrin ß1 via the FAK/Akt signaling pathway.

miR-182-5p promotes hepatocellular carcinoma progression by repressing FOXO3a.

BACKGROUND: High frequency of recurrence is the major cause of the poor outcomes for patients with hepatocellular carcinoma (HCC). microRNA (miR)-182-5p emerged as a high-priority miRNA in HCC and was found to be related to HCC metastasis. Whether the expression of miR-182-5p in tumor tissue correlated with early recurrence in HCC patients underwent curative surgery was unknown. METHODS: Real-time PCR (RT-PCR) and in situ hybridization (ISH) were conducted to assess the expression of miR-182-5p in HCC cells and tissues. Cell Counting Kit-8 (CCK-8), transwell assays were performed to detected cells proliferation and migration ability. Flow cytometry assays were used to detect cell apoptosis rate, and xenograft model was employed to study miR-182-5p in HCC growth and lung metastasis. The target of miR-182-5p was validated with a dual-luciferase reporter assay and western blotting. Immunohistochemistry, immumoblotting, and immunoprecipitation were performed to test relative protein expression. RESULTS: We showed that high expression of miR-182-5p in tumor tissues correlated with poor prognosis as well as early recurrence in HCC patients underwent curative surgery. miR-182-5p enhanced motility and invasive ability of HCC cells both in vitro and in vivo. miR-182-5p directly targets 3'-UTR of FOXO3a and repressed FOXO3a expression, activating AKT/FOXO3a pathway to promote HCC proliferation. Notably, miR-182-5p activated Wnt/ß-catenin signaling by inhibiting the degradation of ß-catenin and enhancing the interaction between ß-catenin and TCF4 which was mediated by repressed FOXO3a. CONCLUSIONS: Consistently, miR-182-5p can be a potential predictor of early recurrence for HCC patients underwent curative surgery, and FOXO3a plays a key mediator in miR-182-5p induced HCC progression.

Correction to: miR-182-5p promotes hepatocellular carcinoma progression by repressing FOXO3a.

The original article [1] contains an error in Fig. 5a whereby the Western blot bands representing CyclinD1 have mistakenly been duplicated over the Western blot bands intended to represent SGK.

Identification of para-Substituted Benzoic Acid Derivatives as Potent Inhibitors of the Protein Phosphatase Slingshot.

Slingshot proteins form a small group of dual-specific phosphatases that modulate cytoskeleton dynamics through dephosphorylation of cofilin and Lim kinases (LIMK). Small chemical compounds with Slingshot-inhibiting activities have therapeutic potential against cancers or infectious diseases. However, only a few Slingshot inhibitors have been investigated and reported, and their cellular activities have not been examined. In this study, we identified two rhodanine-scaffold-based para-substituted benzoic acid derivatives as competitive Slingshot inhibitors. The top compound, (Z)-4-((4-((4-oxo-2-thioxo-3-(o-tolyl)thiazolidin-5-ylidene)methyl)phenoxy)methyl)benzoic acid (D3) had an inhibition constant (Ki) of around 4 µm and displayed selectivity over a panel of other phosphatases. Moreover, compound D3 inhibited cell migration and cofilin dephosphorylation after nerve growth factor (NGF) or angiotensin II stimulation. Therefore, our newly identified Slingshot inhibitors provide a starting point for developing Slingshot-targeted therapies.