Ubiquitin-like protein FAT10 suppresses SIRT1-mediated autophagy to protect against ischemic myocardial injury.

Autophagy plays a deleterious role in ischemic myocardial injury. The deacetylase SIRT1 is a well-established regulator of autophagy that can be modified by the ubiquitin-like protein SUMO1. Our previous work demonstrated that another ubiquitin-like protein, FAT10, exerts cardioprotective effects against myocardial ischemia by stabilizing the caveolin-3 protein; however, the effects of FAT10 on autophagy through SIRT1 are unclear. Here, we constructed a Fat10-knockout rat model to evaluate the role of FAT10 in autophagy. In vivo and in vitro assays confirmed that FAT10 suppressed autophagy to protect the heart from ischemic myocardial injury. Mechanistically, FAT10 was mainly involved in the regulation of the autophagosome formation process. FAT10 affected autophagy through modulating SIRT1 degradation, which resulted in reduced SIRT1 nuclear translocation and inhibited SIRT1 activity via its C-terminal glycine residues. Notably, FAT10 competed with SUMO1 at the K734 modification site of SIRT1, which further reduced LC3 deacetylation and suppressed autophagy. Our findings suggest that FAT10 inhibits autophagy by antagonizing SIRT1 SUMOylation to protect the heart from ischemic myocardial injury. This is a novel mechanism through which FAT10 regulates autophagy as a cardiac protector.

Modified median hepatic fissure approach for resection of liver tumours located in the angle between the root of the middle and right hepatic veins.

BACKGROUND: Liver tumours between the root angle of the middle and right hepatic veins are a special type of liver segment VIII tumour. In this study, we designed a modified median hepatic fissure approach to remove these tumours. The safety and effectiveness of the approach were evaluated. MATERIALS AND METHODS: From April 2015 to November 2019, 11 patients with liver tumours between the angle of the middle and right hepatic veins underwent this modified median hepatic fissure approach. We retrospectively analysed data from the perioperative periods of these 11 patients, including general condition, operation time, intraoperative bleeding, and postoperative complications. Disease-free survival and overall survival were assessed. RESULTS: Of the 11 patients, 9 patients had primary hepatocellular carcinoma and 2 had colorectal liver metastases. The average intraoperative blood loss was 285 mL (150-450 mL). Two patients developed postoperative bile leakage, but there were no significant serious complications, such as intraabdominal bleeding and liver failure, in any of the patients. The liver function returned to the normal range on the 5th day after surgery. Of the 11 patients, 5 have survived for more than 3 years (45.5%), and 4 have been disease-free for more than 3 years (36.3%). CONCLUSIONS: For liver tumours between the root angle of the middle and right hepatic veins, the modified median hepatic fissure approach is a safe and feasible method.

Human Leukocyte Antigen F Locus Adjacent Transcript 10 Overexpression Disturbs WISP1 Protein and mRNA Expression to Promote Hepatocellular Carcinoma Progression.

Recently, studies on transcriptome-proteome relationships have revealed mRNA/protein expression discordance for certain genes and speculated that protein posttranslational modification (PTM) may be involved. However, there is currently no evidence to support this hypothesis. Wnt-induced secreted protein-1 (WISP1) is the downstream target gene of ß-catenin and plays an important role in tumorigenesis and progression, but the expression and role of WISP1 in different tumor types are controversial. Here, we first confirmed that WISP1 protein expression was significantly down-regulated in hepatocellular carcinoma (HCC) tissue and could be an independent predictor of poor prognosis for patients with HCC. In vivo and in vitro evidence was provided that WISP1 can suppress HCC cell proliferation. Further studies have found that low WISP1 protein expression was related to expression of human leukocyte antigen F locus adjacent transcript 10 (FAT10), a specific ubiquitin-like protein with both degradation and stabilization functions, which plays an important role in PTM. FAT10 overexpression facilitated WISP1 degradation by FAT10ylation to decrease WISP1 protein expression, thus promoting HCC proliferation. Interestingly, we found and demonstrated that FAT10 overexpression could result in WISP1 protein/mRNA expression discordance, with protein expression decreasing while mRNA expression increased. The underlying mechanism is that FAT10 exerts substrate stabilization and degradation functions simultaneously, while FAT10 overexpression promotes WISP1 mRNA expression by stabilizing ß-catenin and directly degrades WISP1 protein. Conclusion: Our study demonstrated that overexpression of FAT10 results in expression discordance between WISP1 protein and mRNA, thereby promoting HCC progression by down-regulating WISP1 protein expression.

Intrahepatic bile duct exploration lithotomy is a useful adjunctive hepatectomy method for bilateral primary hepatolithiasis: an eight-year experience at a single centre.

BACKGROUND: To evaluate the perioperative and long-term results of intrahepatic bile duct exploration lithotomy (IHBDIL) combined with hepatectomy for patients with complicated bilateral primary hepatolithiasis. METHODS: A study was conducted involving 56 patients with complicated bilateral primary hepatolithiasis who underwent IHBDIL combined with hepatectomy at our hospital from January 2006 to December 2014. The perioperative and long-term outcomes that were retrospectively analysed included the stone clearance rate, operative morbidity and mortality, and stone recurrence rate. Patients with a preoperative diagnosis of cholangiocarcinoma were excluded. RESULTS: In all 56 patients, hepatic duct stones were located in the bilateral IHBD. The surgical method was IHBDIL combined with hepatectomy. Postoperative complications occurred in 15 patients (26.8%), 14 patients responded to conservative management, and there was 1 case of postoperative mortality because of hepatic failure. The overall initial success rate of stone clearance was 85.7%, and the final clearance rate was 92.9% following postoperative choledochoscopic lithotripsy. The stone recurrence rate was 13.5%, and the occurrence of postoperative cholangitis was 10.9% during the follow-up period. CONCLUSION: IHBDIL combined with hepatectomy is a safe, effective, and promising treatment for patients with complicated bilateral primary hepatolithiasis. The perioperative and long-term outcomes are satisfactory for complicated bilateral primary hepatolithiasis.

Learning curve for performing choledochotomy bile duct exploration with primary closure after laparoscopic cholecystectomy.

BACKGROUND: Primary closure after laparoscopic cholecystectomy (LC) and laparoscopic common bile duct exploration (LCBDE) is a safe and effective approach for treating cholecystolithiasis with choledocholithiasis. The aim of this study was to evaluate the learning curve of performing primary closure after LC+LCBDE. METHODS: We retrospectively identified all patients who underwent primary closure after LC+LCBDE performed by a single surgeon from January 2009 to April 2015 in our institution, and analyzed preoperative, intraoperative, and postoperative data using the cumulative sum (CUSUM) analysis to evaluate the learning curve for this procedure. RESULTS: Overall, there were 390 patients. The total postoperative complications rate was 7.2%, including bile leakage in 9 (2.3%) patients and retained common bile duct stone in 3 (0.8%) patients. The CUSUM operating time (OT) learning curve was best modeled by the equation: CUSUMOT = 312.209 × procedure0.599 × e(-0.011×procedure) + 122.608 (R2 = 0.96). The learning curve was composed of two phases, phase 1 (the initial 54 patients) and phase 2 (the remaining 336 patients). A significant decrease in the OT (116.8 ± 22.4 vs. 93.8 ± 17.8 min; p < 0.001) and complication rate (16.7 vs. 5.7%; p < 0.01) including the rate of bile leakage (7.4 vs. 1.5%; p < 0.01) and retained stone (3.7 vs. 0.3%; p < 0.01) was observed between the two phases. In addition, 20 patients had conversion to open surgery. Impacted stones were independently associated with conversion, as indicated by a multivariable analysis. CONCLUSION: The data suggest that the learning curve of this procedure was achieved in approximately 54 cases. An impacted stone was the only risk factor that affected the conversion rate.

Overexpression of eEF1A1 regulates G1-phase progression to promote HCC proliferation through the STAT1-cyclin D1 pathway.

Hepatocellular carcinoma (HCC) is a common cancer worldwide with an aggressive and highly proliferative activity. Studies had confirmed that HCC cell proliferation is associated with the cell cycle's G1 phase, but the detailed molecular mechanism has not been thoroughly elucidated to date. Eukaryotic translation elongation factor 1A1 (eEF1A1) is an evolutionarily conserved elongation factor protein and is involved in tumor cell proliferation. However, which phase of the cell cycle is regulated by eEF1A1 to influence cell proliferation in HCC and its detailed molecular mechanism remain unclear. In this study, we observed that eEF1A1 influences HCC cell proliferation by regulating the cell cycle's G1 phase. In addition, eEF1A1 influences G1 phase by regulating cyclin D1 expression, promoting HCC cell proliferation both in vitro and in vivo. Moreover, our results indicated that eEF1A1 regulates cyclin D1 expression through STAT1 signaling. STAT1 increases the transcriptional activity of cyclin D1 by binding to the cyclin D1 promoter. Taken together, these findings enabled us to identify a novel mechanism by which eEF1A1 regulates the cell cycle's G1 phase to promote tumor proliferation by regulating cyclin D1 expression through STAT1 signaling in HCC.

Blockage of glycolysis by targeting PFKFB3 alleviates sepsis-related acute lung injury via suppressing inflammation and apoptosis of alveolar epithelial cells.

Sepsis-related acute lung injury (ALI) is characterized by excessive lung inflammation and apoptosis of alveolar epithelial cells resulting in acute hypoxemic respiratory failure. Recent studies indicated that anaerobic glycolysis play an important role in sepsis. However, whether inhibition of aerobic glycolysis exhibits beneficial effect on sepsis-induced ALI is not known. In vivo, a cecal ligation and puncture (CLP)-induced ALI mouse model was set up and mice treated with glycolytic inhibitor 3PO after CLP. The mice treated with the 3PO ameliorated the survival rate, histopathological changes, lung inflammation, lactate increased and lung apoptosis of mice with CLP-induced sepsis. In vitro, the exposure of human alveolar epithelial A549 cells to lipopolysaccharide (LPS) resulted in cell apoptosis, inflammatory cytokine production, enhanced glycolytic flux and reactive oxygen species (ROS) increased. While these changes were attenuated by 3PO treatment. Sequentially, treatment of A549 cells with lactate caused cell apoptosis and enhancement of ROS. Pretreatment with N-acetylcysteine (NAC) significantly lowered LPS and lactate-induced the generation of ROS and cell apoptosis in A549 cells. Therefore, these results indicate that anaerobic glycolysis may be an important contributor in cell apoptosis of sepsis-related ALI. Moreover, LPS specifically induces apoptotic insults to A549 cell through lactate-mediated enhancement of ROS.

Rock2 promotes RCC proliferation by decreasing SCARA5 expression through ß-catenin/TCF4 signaling.

Rho-associated coiled-coil forming protein kinase 2 (Rock2), as a key effector of the small GTPase RhoA, is involved in tumor development. Scavenger receptor class A member 5 (SCARA5) is an important regulator of biological processes in cancer cells. However, the roles and relationship of Rock2 and SCARA5 in renal cell carcinoma (RCC) remain unclear. In this study, we found that Rock2 expression was markedly increased in clinical RCC tissues compared with that in adjacent non-cancerous tissues. High expression of Rock2 was inversely correlated with patient survival in RCC, which indicated that Rock2 may be a prognostic marker in human RCC. In addition, Rock2 knockdown increased SCARA5 expression and suppressed RCC cell proliferation both in vitro and in vivo. Furthermore, we found that the ß-catenin/TCF4 pathway contributed to the effect of Rock2 on SCARA5-mediated RCC proliferation. Taken together, these results suggest that this newly identified Rock2-ß-catenin/TCF4-SCARA5 axis will provide novel insight into the understanding of the regulatory mechanisms of proliferation in human RCC.

Double primary hepatic cancer (hepatocellular carcinoma and intrahepatic cholangiocarcinoma) originating from hepatic progenitor cell: a case report and review of the literature.

BACKGROUND: Synchronous development of primary hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) in different sites of the liver have rarely been reported before. The purpose of this study is to investigate the clinicopathological characteristics of synchronous double cancer of HCC and ICC. CASE PRESENTATION: A 56-year-old Chinese man without obvious liver cirrhosis was preoperation diagnosed with multiple HCC in segments VI (SVI) and VII (SVII) by the abdominal computed tomography (CT) and contrast-enhanced ultrasonography (CEUS). We performed hepatic resection of both segments. The tumors in SVI and SVII were pathologically diagnosed as ICC and HCC, respectively. Immunohistochemically, the HCC in SVII was positive for HepPar-1 and negative for CK19, while the ICC in SVI tumor was positive for CK19 and negative for HepPar-1. Interestingly, the immunohistochemical results also showed that the classic hepatic progenitor cell (HPCs) markers CD34 and CD117 were both positive of the two tumors. The patient still survived and at a 1-year follow-up did not show evidence of metastasis or new recurrent lesions. We speculate that the two masses may have originated from HPCs based on the findings of this patient. CONCLUSIONS: Synchronous development of HCC and ICC is very rare with unique clinical and pathological features. The correct preoperative diagnosis of double hepatic cancer of HCC and ICC is difficult. Hepatitis B virus (HBV) and hepatitis C virus (HCV) infection were both the independent risk factor to the development of double liver cancer. Hepatic resection is the preferred and most effective treatment choice. The prognosis of synchronous occurrence of double hepatic cancer was poorer than for either HCC or ICC, and the origin of it needs further study.

Rock2 stabilizes ß-catenin to promote tumor invasion and metastasis in colorectal cancer.

Rho-associated coiled-coil-containing protein kinase 2 (Rock2) is an effector for the small GTPase Rho and plays an important role in tumor progression and metastasis. However, the effect of Rock2 in colorectal cancer (CRC) still remains unclear. In this study, we found that Rock2 expression was markedly increased in clinical CRC tissues compared with adjacent non-cancerous tissues. High expression of Rock2 was correlated with tumor metastasis and poor prognosis in CRC. In addition, the knockdown of Rock2 suppressed the invasion and metastasis of CRC cells both in vitro and in vivo. Furthermore, we found that the ß-catenin/TCF4 pathway contributed to the effects of Rock2 in CRC cells, and Rock2 stabilized ß-catenin by preventing its ubiquitination and degradation. Taken together, this novel pathway for ß-catenin control plays a biologically relevant role in CRC metastasis.

Cathepsin S-mediated autophagic flux in tumor-associated macrophages accelerate tumor development by promoting M2 polarization.

BACKGROUND: Tumor-associated macrophages (TAMs) are the major component of tumor-infiltrating leukocytes. TAMs are heterogeneous, with distinct phenotypes influenced by the microenvironment surrounding tumor tissues, but relatively little is known about the key molecular in these cells that contribute to malignant phenotypes. Autophagic activity is a critical factor in tumor development that contributes to enhancing cellular fitness and survival in the hostile tumor microenvironment. However, the molecular basis and relations between autophagy and TAMs polarization remain unclear. METHODS: Cathepsin S (Cat S) expression was analyzed in human colon carcinoma and normal colon tissues. In vivo effects were evaluated using PancO2 subcutaneous tumor model and SL4 hepatic metastasis model. Immunofluorescence staining, flow cytometry and real-time PCR were done to examine TAMs polarization. Western blotting assay, transmission electron microscopy, mCherry-GFP-LC3 transfection and DQ-BSA degradation assays were carried out to determine its role in regulating autophagy. RESULTS: In the present study, we showed that the enhanced expression of Cat S correlated with the severity of histologic grade as well as clinical stage, metastasis, and recurrence, which are known indicators of a relatively poor prognosis of human colon carcinoma. Cat S knockout led to decreased tumor growth and metastasis. Moreover, Cat S knockout inhibited M2 macrophage polarization during tumor development. We further demonstrated that Cat S was required for not only autophagic flux but also the fusion processes of autophagosomes and lysosomes in TAMs. Importantly, we found that Cat S contributed to tumor development by regulating the M2 phenotype of TAMs through the activation of autophagy. CONCLUSIONS: These results indicated that Cat S-mediated autophagic flux is an important mechanism for inducing M2-type polarization of TAMs, which leads to tumor development. These data provide strong evidence for a tumor-promoting role of autophagy in TAMs and suggest Cat S could be a potential target for cancer therapy.

Rock2 promotes the invasion and metastasis of hepatocellular carcinoma by modifying MMP2 ubiquitination and degradation.

Rho-associated coiled-coil-containing protein kinase 2 (Rock2) is a downstream effector of Rho that plays an important role in the tumorigenesis and progression of hepatocellular carcinoma (HCC). Matrix metalloproteinase 2 (MMP2) is a master regulator of tumor metastasis. In this study, we investigated the collections of Rock2 and MMP2 in HCCs and determined the potential role and molecular mechanism of Rock2 in MMP2-mediated invasiveness and metastasis. We found that Rock2 and MMP2 were markedly overexpressed in HCCs compared with the corresponding adjacent tissues, where a positive correlation in their expression was found. The knockdown of Rock2 significantly decreased MMP2 expression and inhibited the invasion and metastasis of HCC in vitro and in vivo. Additionally, the upregulation of MMP2 rescued the decreased migration and invasion induced by the knockdown of Rock2, whereas the knockdown of MMP2 decreased Rock2-enhanced HCC migration and invasion. Mechanistically, Rock2 stabilized MMP2 by preventing its ubiquitination and degradation. Together, our results link two drivers of invasion and metastasis in HCC and identify a novel pathway for MMP2 control.

Overexpression of HOXB9 promotes metastasis and indicates poor prognosis in colon cancer.

OBJECTIVE: Homeobox B9 (HOXB9) is proposed to be involved in tumor angiogenesis and metastasis. We investigated the role of HOXB9 in the progression of colon cancer. METHODS: HOXB9 expression was investigated by immunohistochemically and Western blotting in 128 colon cancer patients and the results were analyzed statistically associated with clinicopathological data and survival of the patients. The effect of HOXB9 on cell invasion and metastases abilities were analyzed in vitro and in vivo. RESULTS: HOXB9 is overexpressed in colon cancer tissues and significantly correlated with metastasis and poor survival of patients (P<0.05, respectively). Additionally, high levels of expression of HOXB9 were observed in metastatic lymph nodes. The down-regulation of HOXB9 expression can inhibit the migration and invasive ability of colon cancer cells, while exogenous expression of HOXB9 in colon cancer cells enhanced cell migration and invasiveness. Moreover, stable knockdown of HOXB9 reduced the liver and lung metastasis of colon cancer in vivo. CONCLUSIONS: HOXB9 may play an important role in the invasion and metastasis of colon cancer cells and may be a useful biomarker for metastasis and prognostic of colon cancer.

Cuproptosis-related molecular classification and gene signature of hepatocellular carcinoma and experimental verification.

Background: Hepatocellular carcinoma (HCC) is a highly heterogeneous malignancy with poor overall prognosis. Cuproptosis, a recently proposed mode of copper-dependent cell death, plays a critical role in the malignant progression of various tumors; however, the expression and prognostic value of cuproptosis-related regulatory genes in HCC remain unclear. Methods: Genomic, genetic, and expression profiles of ten key cuproptosis-related regulatory genes were analyzed using The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) dataset and protein expression data from the Human Protein Atlas (HPA) database. Unsupervised clustering of HCC patients based on these ten key cuproptosis-related regulatory genes was used to identify different HCC subtypes and analyze the differences in clinical and immune characteristics among subtypes. Subsequently, univariate Cox and least absolute shrinkage and selection operator (LASSO) Cox analyses were used to establish a cuproptosis-related prognostic signature, and the accuracy of prognostic signature prediction was internally validated by Kaplan-Meier survival analysis and time-dependent receiver operating characteristic curve in TCGA training and testing cohorts. The prognostic signature was externally validated using TCGA-LIHC entire cohort and International Cancer Genome Consortium Liver Cancer (ICGC-LIRI) cohorts. Finally, the expression landscape of cuproptosis-related regulatory genes in prognostic signature was explored by quantitative real-time polymerase chain reaction (qRT-PCR), western blotting and immunohistochemistry (IHC) experiments. Results: Ten cuproptosis-related genes were differentially expressed in normal and HCC tissues. Unsupervised clustering identified two subtypes and HCC patients with these two subtypes had different clinical prognoses and immune characteristics, as well as different degrees of response to immunotherapy. Lipoyltransferase 1 (LIPT1), dihydrolipoamide s-acetyltransferase (DLAT), and cyclin dependent kinase inhibitor 2A (CDKN2A) were selected to construct a prognostic signature, which significantly distinguished HCC patients with different survival periods in the TCGA training and testing cohorts and was well validated in both the TCGA-LIHC entire cohort and ICGC-LIRI cohort. The risk score of the prognostic signature was confirmed to be an independent prognostic factor, and nomograms were generated to effectively predict the probability of HCC patient survival. The qRT-PCR, western blotting and IHC results also revealed a significant imbalance in the expression of these cuproptosis-related genes in HCC. Conclusions: The classification and prognostic signature based on cuproptosis-related regulatory genes helps to explain the heterogeneity of HCC, which may contribute to the individualized treatment of patients with the disease.

FAT10 mediates the sorafenib-resistance of hepatocellular carcinoma cells by stabilizing E3 ligase NEDD4 to enhance PTEN/AKT pathway-induced autophagy.

Although sorafenib is the first-line therapeutic agent for advanced hepatocellular carcinoma (HCC), the development of drug resistance in HCC cells limits its clinical efficacy. However, the key factors involved in mediating the sorafenib resistance of HCC cells and the underlying mechanisms have not been elucidated. In this study, we generated sorafenib-resistant HCC cell lines, and our data demonstrate that HLA-F locus-adjacent transcript 10 (FAT10), a ubiquitin-like protein, is markedly upregulated in sorafenib-resistant HCC cells and that reducing the expression of FAT10 in sorafenib-resistant HCC cells increases sensitivity to sorafenib. Mechanistically, FAT10 stabilizes the expression of the PTEN-specific E3 ubiquitin ligase NEDD4 that causes downregulation of PTEN, thereby inducing AKT-mediated autophagy and promoting the resistance of HCC cells to sorafenib. Moreover, we screened the small molecule Compound 7695-0983, which increases the sensitivity of sorafenib-resistant HCC cells to sorafenib by inhibiting the expression of FAT10 to inhibit NEDD4-PTEN/AKT axis-mediated autophagy. Collectively, our preclinical findings identify FAT10 as a key factor in the sorafenib resistance of HCC cells and elucidate its underlying mechanism. This study provides new mechanistic insight for the exploitation of novel sorafenib-based tyrosine kinase inhibitor (TKI)-targeted drugs for treating advanced HCC.

FAT10 protects cardiac myocytes against apoptosis.

FAT10 is a new member of the ubiquitin-like protein family with yet-to-be defined biological functions in the heart. Our objective was to determine the role of FAT10 in the heart. FAT10 is expressed in the normal human and murine hearts, as detected by qPCR and Western blotting. Expression of FAT10 is increased in the heart at the border zone of myocardial infarction and in cultured neonatal rat cardiac myocytes (NRCM) subjected to hypoxia/reoxygenation (H/R) stress. Lentiviral-mediated overexpression of FAT10 in NRCM reduced p53 (TP53) and its target miR-34a levels, while BCL2 level, a target of miR-34a, was increased and BAX level, a pro-apoptotic protein, was reduced. These changes were associated with reduced apoptosis, detected by FACS analysis of annexin-V expression and TUNEL assay, in response to H/R injury. Knock down of FAT10 by shRNA targeting had the opposite effects. Likewise, lentiviral mediated expression of miR-34a was associated with reduced BCL2 and increased BAX levels in NRCM and also reversed changes in BCL-2 and BAX levels observed upon over-expression of FAT10. Treatment of NRCM with proteasome inhibitor MG132 increased p53 and miR-34a levels and reduced BLC2/BAX ratio. These changes were not reversed upon over-expression of FAT10. Thus, FAT10 is upregulated in the heart and NRCM in response to H/R stress, which protects cardiac myocytes against apoptosis. The anti-apoptotic effects of FAT10 are associated with suppression of p53, probably through fatylation and proteasomal degradation, reduced miR-34a expression, and a shift in the BCL2/BAX proteins against apoptosis. Thus, FAT10 is a cardioprotective protein.

Chemokine receptor CX3CR1 contributes to macrophage survival in tumor metastasis.

BACKGROUND: Macrophages, the key component of the tumor microenvironment, are differentiated mononuclear phagocyte lineage cells that are characterized by specific phenotypic characteristics that have been implicated in tumor growth, angiogenesis, and invasion. CX3CR1, the chemoattractant cytokine CX3CL1 receptor, plays an important role in modulating inflammatory responses, including monocyte homeostasis and macrophage phenotype and function. However, the role of CX3CR1 in the regulation of the tumor inflammatory microenvironment is not fully understood. METHODS: Using in vivo hepatic metastasis model, human colon carcinoma specimens, immunohistochemical staining, TUNEL staining, flow cytometry analysis, Western blotting assay and co-culture in three-dimensional peptide gel, we determined the effects of CX3CR1 on angiogenic macrophage survival and tumor metastasis. RESULTS: In this study, we found that CX3CR1 was expressed in human colon carcinomas in a histologic grade- and stage-dependent manner, and CX3CR1 upregulation in TAMs was correlated with poor prognosis. Furthermore, we showed that in a microenvironment lacking CX3CR1, the liver metastasis of colon cancer cells was significantly inhibited. The underlying mechanism is associated with decrease accumulation of angiogenic macrophages that can be partly attributed to increased apoptosis in the tumor microenvironment, thus leading to impaired tumor angiogenesis in the liver and suppressed tumor metastasis. CONCLUSIONS: Our results suggest a role of CX3CR1 in angiogenic macrophage survival in the tumor microenvironment contributing to tumor metastasis.

Rock2 regulates Cdc25A through ubiquitin proteasome system in hepatocellular carcinoma cells.

Rho-associated coiled-coil containing protein kinase 2 (Rock2) belongs to a family of serine/threonine kinases which are actived via interaction with Rho GTPases. Recently, overexpression of Rock2 has been demonstrated in human hepatocellular carcinoma (HCC), but the potential role of Rock2 in tumorigenesis remains unclear. Cdc25A acts as a key checkpoint during the G1/S phase and has also been found to be overexpressed in HCC. Here, we report that Rock2 regulates cell cycle progression via ubiquitination of Cdc25A in HCC. In HCC tissues, Rock2 and Cdc25A were aberrantly upregulated and revealed a significantly positive correlation. Knockdown of Rock2 inhibited HCC cell growth and promoted cell-cycle arrest at the G1/S phase via regulation of Cdc25A. When cells were exposed to DNA damage, Rock2 increased cell survival by regulating Cdc25A. Co-immunoprecipitation and immunofluorescence analyses indicated that Rock2 regulated Cdc25A via direct binding. Furthermore, knockdown of Rock2 activated Cdc25A ubiquitination and promoted its degradation. Our results defined a role for Rock2 in modulation of Cdc25A ubiquitination, indicating a novel mechanism of Cdc25A regulation and a potential function for Rock2 in the development of HCC.

Laparoscopic hepatectomy is associated with a higher incident frequency in hepatolithiasis patients.

PURPOSES: The primary concern regarding laparoscopic hepatectomy in hepatolithiasis patients is surgical safety, which may be high in current practice. METHODS: Hepatolithiasis patients who underwent laparoscopic and laparotomic hepatectomies were retrospectively studies after being matched for age, location of gallstones, liver resection and underlying liver conditions at a ratio of 1:1 (n = 44 in each group). The rates of intraoperative incidents and postoperative complications were examined using validated classification and grading systems. The primary outcome measure was the procedure-related complication/mortality rate. RESULTS: Laparoscopy was converted to open surgery in three patients (6.8 %). The length of the operation for laparoscopic hepatectomy was significantly longer than that for laparotomic hepatectomy (277.5 min [range, 190-410 min] vs. 212.5 min [140-315 min], P < 0.001). The two groups had similar intraoperative blood loss (367.5 mL [150-1200 mL] vs. 392.5 mL [200-1400 mL], P > 0.05) and transfusion frequencies (13.6 vs. 18.2 %, P > 0.05). The laparoscopy group had a higher percentage of patients with at least one intraoperative incident compared with the laparotomy group (22.7 vs. 6.8 %; P < 0.05). Vascular events occurred in nine patients (20.5 %) undergoing laparoscopy and two patients (4.5 %) undergoing laparotomy (OR 5.4 [95 %CI, 1.1-26.7], P < 0.05). CONCLUSIONS: Laparoscopic hepatectomy is associated with a higher risk of intraoperative vascular incidents in hepatolithiasis patients compared wit laparotomy.

The ubiquitin-like protein FAT10 in hepatocellular carcinoma cells limits the efficacy of anti-VEGF therapy by accelerating VEGF-independent angiogenesis.

INTRODUCTION: The efficacy of anti-vascular endothelial growth factor (VEGF) therapy is limited. However, the key factors involved in limiting the efficacy of anti-VEGF therapy and the underlying mechanisms remain unclear. OBJECTIVES: To investigate the effects and mechanisms of human leukocyte antigen F locus-adjacent transcript 10 (FAT10), a ubiquitin-like protein, in limiting the efficacy of anti-VEGF therapy in hepatocellular carcinoma (HCC) cells. METHODS: FAT10 was knocked out in HCC cells using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 technology. Bevacizumab (BV), an anti-VEGF monoclonal antibody, was used to evaluate the efficacy of anti-VEGF therapy in vivo. Mechanisms of FAT10 action were assessed by RNA sequencing, glutathione S-transferase pulldown assays and in vivo ubiquitination assays. RESULTS: FAT10 accelerated VEGF-independent angiogenesis in HCC cells which limited BV efficacy and BV-aggravated hypoxia and inflammation promoted FAT10 expression. FAT10 overexpression increased levels of proteins involved in several signaling pathways in HCC cells, resulting in upregulation of VEGF and multiple non-VEGF proangiogenic factors. Upregulation of multiple FAT10-mediated non-VEGF signals compensated for the inhibition of VEGF signaling by BV, enhancing VEGF-independent angiogenesis and promoting HCC growth. CONCLUSIONS: Our preclinical findings identify FAT10 in HCC cells as a key factor limiting the efficacy of anti-VEGF therapy and elucidate its underlying mechanisms. This study provides new mechanistic insights into the development of antiangiogenic therapies.