Development and external validation of MRI-based RAS mutation status prediction model for liver metastases of colorectal cancer.

BACKGROUND: The mutation status of rat sarcoma viral oncogene homolog (RAS) has prognostic significance and serves as a key predictive biomarker for the effectiveness of antiepidermal growth factor receptor (EGFR) therapy. However, there remains a lack of effective models for predicting RAS mutation status in colorectal liver metastases (CRLMs). This study aimed to construct and validate a diagnostic model for predicting RAS mutation status among patients undergoing hepatic resection for CRLMs. METHODS: A diagnostic multivariate prediction model was developed and validated in patients with CRLMs who had undergone hepatectomy between 2014 and 2020. Patients from Institution A were assigned to the model development group (i.e., Development Cohort), while patients from Institutions B and C were assigned to the external validation groups (i.e., Validation Cohort_1 and Validation Cohort_2). The presence of CRLMs was determined by examination of surgical specimens. RAS mutation status was determined by genetic testing. The final predictors, identified by a group of oncologists and radiologists, included several key clinical, demographic, and radiographic characteristics derived from magnetic resonance images. Multiple imputation was performed to estimate the values of missing non-outcome data. A penalized logistic regression model using the adaptive least absolute shrinkage and selection operator penalty was implemented to select appropriate variables for the development of the model. A single nomogram was constructed from the model. The performance of the prediction model, discrimination, and calibration were estimated and reported by the area under the receiver operating characteristic curve (AUC) and calibration plots. Internal validation with a bootstrapping procedure and external validation of the nomogram were assessed. Finally, decision curve analyses were used to characterize the clinical outcomes of the Development and Validation Cohorts. RESULTS: A total of 173 patients were enrolled in this study between January 2014 and May 2020. Of the 173 patients, 117 patients from Institution A were assigned to the Model Development group, while 56 patients (33 from Institution B and 23 from Institution C) were assigned to the Model Validation groups. Forty-six (39.3%) patients harbored RAS mutations in the Development Cohort compared to 14 (42.4%) in Validation Cohort_1 and 8 (34.8%) in Validation Cohort_2. The final model contained the following predictor variables: time of occurrence of CRLMs, location of primary lesion, type of intratumoral necrosis, and early enhancement of liver parenchyma. The diagnostic model based on clinical and MRI data demonstrated satisfactory predictive performance in distinguishing between mutated and wild-type RAS, with AUCs of 0.742 (95% confidence interval [CI]: 0.651─0.834), 0.741 (95% CI: 0.649─0.836), 0.703 (95% CI: 0.514─0.892), and 0.708 (95% CI: 0.452─0.964) in the Development Cohort, bootstrapping internal validation, external Validation Cohort_1 and Validation Cohort_2, respectively. The Hosmer-Lemeshow goodness-of-fit values for the Development Cohort, Validation Cohort_1 and Validation Cohort_2 were 2.868 (p = 0.942), 4.616 (p = 0.465), and 6.297 (p = 0.391), respectively. CONCLUSIONS: Integrating clinical, demographic, and radiographic modalities with a magnetic resonance imaging-based approach may accurately predict the RAS mutation status of CRLMs, thereby aiding in triage and possibly reducing the time taken to perform diagnostic and life-saving procedures. Our diagnostic multivariate prediction model may serve as a foundation for prognostic stratification and therapeutic decision-making.

Toxicity of GO to Freshwater Algae in the Presence of Al2O3 Particles with Different Morphologies: Importance of Heteroaggregation.

The roles of Al2O3 particles with different morphologies in altering graphene oxide (GO) toxicity to Chlorella pyrenoidosa were investigated. Algal growth inhibition by GO with coexisting Al2O3 particles was much lower than the sum of inhibitions from the individual materials for all the three Al2O3, showing the toxicity mitigation by Al2O3. The lowest GO toxicity was observed at the concentrations of 300, 150, and 100 mg/L for Al2O3 nanoparticles (NPs, 8-10 nm), bulk particles (BPs, 100-300 nm), and fibers (diameter: 10 nm; length: 400 nm), respectively. GO-Al2O3 heteroaggregation was responsible for the observed toxicity reduction. GO-induced algal membrane damage was suppressed by the three types of Al2O3 due to GO-Al2O3 heteroaggregation, and the reduction in intracellular reactive oxygen species generation and physical contact were confirmed as two main mechanisms. Moreover, the exposure sequence of GO and Al2O3 could highly influence the toxicity, and the simultaneous exposure of individual GO and Al2O3 showed the lowest toxicity due to minimum direct contact with algal cells. Humic acid further decreased GO-Al2O3 toxicity due to enhanced steric hindrance through surface coating of GO-Al2O3 heteroaggregates. This work provides new insights into the role of natural mineral particles in altering the environmental risk of GO.

Krüppel-like factor 8 promotes cancer stem cell-like traits in hepatocellular carcinoma through Wnt/ß-catenin signaling.

Krüppel-like factor 8 (KLF8) is highly expressed in hepatocellular carcinoma (HCC) and contributes to tumor initiation and progression by promoting HCC cell proliferation and invasion. However, the role of KLF8 in liver cancer stem cells (LCSCs) is not known. In the current study, we investigated the role of KLF8 in LCSCs to determine if KLF8 is a novel marker of these cells. We found that KLF8 was highly expressed in primary HCC tumors, distant migrated tissues, and LCSCs. Patients with high KLF8 expression had a poor prognosis. KLF8 promoted stem cell-like features through activation of the Wnt/ß-catenin signaling pathway. Cell apoptosis was significantly increased in HCC cells with knockdown of KLF8 compared with the control cells when treated with the same doses of sorafenib or cisplatin. Taken together, our study shows that KLF8 plays a potent oncogenic role in HCC tumorigenesis by maintaining stem cell-like features through activation of the Wnt/ß-catenin signaling pathway and promoting chemoresistance. Thus, targeting KLF8 may provide an effective therapeutic approach to suppress tumorigenicity of HCC. © 2016 Wiley Periodicals, Inc.

TGF-ß regulates hepatocellular carcinoma progression by inducing Treg cell polarization.

BACKGROUND/AIMS: TGF-ß plays a key role in the progression of various tumors. The main objective of our study was to investigate whether TGF-ß is able to regulate N-nitrosodiethylamine (DEN)-induced hepatocellular carcinoma (HCC) progression in a mouse model by inducing Treg cell polarization. METHODS: HCC progression, TGF-ß and Foxp3 expression levels, serum TGF-ß, IL10 and GP73 levels as well as percentage of Treg cells were analyzed in healthy, HCC and HCC+SM-16 mouse groups. The effect of TGF-ß on Treg cell polarization in vitro was measured by flow cytometric analysis. The expression of TGF-ß and IL10 was identified by IHC in HCC patients and the correlation between TGF-ß and IL10 was also assessed. RESULTS: TGF-ß expression is up-regulated in a DEN-induced HCC mouse model. TGF-ß can promote the differentiation of Foxp3(+)CD4(+) T cells (Treg cells) in vitro. However, blocking the TGF-ß pathway with a specific TGF-ß receptor inhibitor, SM-16, reduced HCC progression and the percentage of Treg cells in liver tissue. The correlation between TGF-ß and Treg cells was also confirmed in HCC patients and the expression of both TGF-ß and IL-10 was shown to be associated with HCC progression. CONCLUSION: TGF-ß is necessary for HCC progression, acting by inducing Treg cell polarization.

Development and characterization of a novel neutralizing scFv vectored immunoprophylaxis against botulinum toxin type A.

Botulinum toxin is a protein toxin secreted by Clostridium botulinum that is strongly neurotoxic. Due to its characteristics of being super toxic, quick acting, and difficult to prevent, the currently reported antiviral studies focusing on monoclonal antibodies have limited effectiveness. Therefore, for the sake of effectively prevention and treatment of botulism and to maintain country biosecurity as well as the health of the population, in this study, we intend to establish a single chain antibody (scFv) targeting the carboxyl terminal binding functional domain of the botulinum neurotoxin heavy chain (BONT/AHc) of botulinum neurotoxin type A, and explore the value of a new passive immune method in antiviral research which based on adeno-associated virus (AAV) mediated vector immunoprophylaxis (VIP) strategy. The scFv small-molecular single-chain antibody sequenced, designed, constructed, expressed and purified by hybridoma has high neutralising activity and affinity level, which can lay a good foundation for the modification and development of antibody engineering drugs. In vivo experiments, AAV-mediated scFv engineering drug has good anti-BONT/A toxin neutralisation ability, has advantages of simple operation, stable expression and good efficacy, and may be one of the effective treatment strategies for long-term prevention and protection of BONT/A botulinum neurotoxin.

miR-204 inhibits epithelial to mesenchymal transition by targeting slug in intrahepatic cholangiocarcinoma cells.

BACKGROUND/AIMS: MicroRNAs (miRNAs) play critical roles during carcinogenesis and cancer progression. Down-regulation of miR-204 has been frequently observed in various cancers. In this study, we investigated the roles and mechanisms of miR-204 in human intrahepatic cholangiocarcinoma (ICC). METHODS: The relative expression of miR-204 in ICC tissues and cell lines was monitored by qRT-PCR. Effects of miR-204 were studied in human ICC cell lines HuH28 and HuCCT1, and cells were analyzed for proliferation, migration and invasion. Expression levels of miR-204 target gene Slug and EMT markers (E-cadherin and vimentin) in ICC cell lines and tissues were measured by qRT-PCR, western blotting and immunofluorescence. RESULTS: miR-204 was frequently downregulated in human ICC, and the low-level expression of miR-204 was significantly associated with lymph node metastasis. Overexpression of miR-204 dramatically suppressed ICC cell migration and invasion, as well as the epithelial-mesenchymal transition process (EMT). Slug was identified as a direct target of miR-204, and its downregulation by miR-204 in HuH28 cells reversed EMT, as shown by the increased expression of the epithelial marker E-cadherin and decreased expression of the mesenchymal marker vimentin. CONCLUSION: These findings suggest that miR-204 plays negative roles in the invasive and/or metastatic potential of ICC, and that its suppressive effects are mediated by repressing Slug expression.

Sorafenib sensitizes hepatocellular carcinoma cell to cisplatin via suppression of Wnt/ß-catenin signaling.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Systemic chemotherapy plays an important role in the treatment of patients with advanced liver cancer. However, chemoresistance to cisplatin is a major limitation of cisplatin-based chemotherapy in the clinic, and the underlying mechanism of such resistance is not fully understood. In this study, we found that nuclear accumulation of ß-catenin was higher in cisplatin-resistant Huh7 cells than in Huh7 cells, indicating that Wnt signaling was activated in cisplatin-resistant cells. Wnt signaling inhibition increased cisplatin-induced growth inhibition in hepatoma cell. We further demonstrated that sorafenib could inhibit Wnt signaling in Huh7 cells and cisplatin-resistant Huh7 cells. Co-treatment with cisplatin and sorafenib was more effective in inhibiting cancer cell proliferation than cisplatin alone in vitro and in vivo, whereas Wnt3a (Wnt activator) treatment abrogated sorafenib-induced growth inhibition. These data demonstrated that sorafenib sensitizes human HCC cell to cisplatin via suppression of Wnt/ß-catenin signaling, thus offering a new target for chemotherapy of HCC.

Plantamajoside promotes metformin-induced apoptosis, autophagy and proliferation arrest of liver cancer cells via suppressing Akt/GSK3ß signaling.

Metformin, a well-known antidiabetic drug, exhibits anticancer effect in a variety of cancers, including liver cancer. Plantamajoside (PMS), a phenylethanoid glycoside compound isolated from Plantago asiatica, is proved to possess anticancer effects, too. In our study, we hypothesized that PMS might promote metformin mediated anticancer effects on liver cancer. The half maximal inhibitory concentration (IC50) of metformin was evaluated by cell viability assay. The influence of PMS on proliferation, migration, invasion and apoptosis of metformin-treated cells was evaluated by BrdU incorporation assay, flow cytometry, western blot, wound scratch healing assay, transwell cell migration assay and immunofluorescence. A fasting/feeding mouse model was built to evaluate the influence of PMS on metformin sensitivity in vivo. PMS (2.5, 10 or 40 µg/mL) treatment reduced the IC50 of metformin under different glucose concentrations. PMS (10 µg/mL) promoted metformin (5 mm) induced apoptosis and autophagy, and inhibition on proliferation, migration and invasion of HepG2 and HuH-7 cells. In the fasting/feeding mouse model, PMS (50 mg/kg) promoted metformin (200 mg/kg) induced proliferation arrest and apoptosis in vivo. Meanwhile, PMS reduced the level of pAkt(ser473) and GSK3ß(ser9) in HepG2 and HuH-7 cells. Restoration of Akt/GSK3ß signaling by a constitutively activated myr-Akt1 abrogated the effects of PMS on metformin-treated liver cancer cells. Our results demonstrated that PMS promoted the anticancer effects of metformin on liver cancer in vitro and in vivo.

Adsorption and catalytic degradation of preservative parabens by graphene-family nanomaterials.

Parabens pose increasing threats to human health due to endocrine disruption activity. Adsorption and degradation of parabens by three types of graphene-family nanomaterials (GFNs) were therefore investigated. For a given paraben, the maximum adsorption capacities (Q0) followed the order of reduced graphene oxide (RGO) > multilayered graphene (MG) > graphene oxide (GO); for a given GFN, Q0 followed the order of butylparaben (BuP) > propylparaben (PrP) > ethylparaben (EtP) > methylparaben (MeP), dominated by hydrophobic interaction. MeP removal by all the three GFNs was highly enhanced (0.55-4.37 times) with the assistance of H2O2 due to additional catalytic degradation process, and MG showed the highest removal enhancement. ∙OH was confirmed as the dominant radicals responsible for parabens degradation. For MG and RGO, the metal impurities (Fe, Cu, Mn, and Co) initiated Fenton-like reaction with H2O2 to generate ∙OH. GO contained oxygen-centered free radicals, which were responsible for ∙OH formation via transferring electron to H2O2. Four degradation byproducts of MeP were identified, including oxalic, propanedioic, fumaric, and 2,5-dihydroxybenzoic acids. Combined with density function theory calculations, the degradation sites and pathways were identified and confirmed. These findings provide useful information on mechanistic understanding towards the adsorption and degradation of parabens by GFNs.

TIGIT-Fc Promotes Antitumor Immunity.

T-cell immunoreceptor with Ig and ITIM domains (TIGIT) is a checkpoint receptor that mediates both T-cell and natural killer (NK)-cell exhaustion in tumors. An Fc-TIGIT fusion protein was shown to induce an immune-tolerance effect in a previous report, but the relevance of the TIGIT-Fc protein to tumor immunity is unknown. Here, we found that TIGIT-Fc promotes, rather than suppresses, tumor immunity. TIGIT-Fc treatment promoted the effector function of CD8+ T and NK cells in several tumor-bearing mouse models. TIGIT-Fc treatment resulted in potent T cell- and NK cell-mediated tumor reactivity, sustained memory-induced immunity in tumor rechallenge models, enhanced therapeutic effects via an antibody against PD-L1, and induction of Th1 development in CD4+ T cells. TIGIT-Fc showed a potent antibody-dependent cell-mediated cytotoxicity effect but had no intrinsic effect on tumor cell development. Our findings elucidate the role of TIGIT-Fc in tumor immune reprogramming, suggesting that TIGIT-Fc treatment alone or in combination with other checkpoint receptor blockers is a promising anticancer therapeutic strategy.

LncRNA MFI2-AS1 promotes HCC progression and metastasis by acting as a competing endogenous RNA of miR-134 to upregulate FOXM1 expression.

Hepatocellular carcinoma (HCC) is worldwide accepted most common malignancies, as well as the second major cause of death among Chinese with cancer. There is an increasing evidence that could prove the potential effect of long non-coding RNAs (lncRNAs) to the biological performance of HCC. In present study, with high expression level in The Cancer Genome Atlas (TCGA) HCC samples, lncRNA MFI2 Antisense RNA 1 (MFI2-AS1) was closely related to poor prognosis and advanced stage among patients with HCC. In addition, up-regulation of MFI2-AS1 was further comfirmed in HCC tissues and HCC cell line. Ectopic expression of MFI2-AS1 stimulated the proliferation and metastasis of HCC cells, but knockdown MFI2-AS1 suppressed HCC cell proliferation and metastasis, indicating that MFI2-AS1 exerted oncogenic functions in the tumorigenesis of HCC. Simultaneously, compared with the negative control group, xenograft tumors in MFI2-AS1 group were characterized with poor growth, smaller volumes and less liver metastases. The post-transcriptional regulation of FOXM1 by MFI2-AS1 occured mechanistically, playing a role of competing with endogenous RNA (ceRNA) in HCC to sponge miR-134. Over-expression of MFI2-AS1 increased FOXM1 expression both at mRNA and protein level, whereas it was reducd by miR-134. Meanwhile, knockdown of miR-134 abolished the repression of shMFI2-AS1 on FOXM1 expression. Furthermore, we demonstrated that miR-134 reverses the impact of MFI2-AS1 on HCC proliferation and metastasis through regulation on FOXM1. Collectively, we determined that MFI2-AS1 crucially acted in HCC progression via functioning as miR-134 sponge to upregulating FOXM1 expression, and was conducive to the promotion of better understanding the direct diagnostics and iatreusiology of lncRNA in HCC.

Impact of Surveillance in Chronic Hepatitis B Patients on Long-Term Outcomes After Curative Liver Resection for Hepatocellular Carcinoma.

BACKGROUND: Clinical guidelines recommend surveillance in high-risk population to early detect hepatocellular carcinoma (HCC), when curative treatment such as liver resection can be applied. However, it is largely unknown whether surveillance would provide long-term survival benefits to these high-risk patients who have received curative liver resection for HCC. METHODS: A prospectively maintained database on patients with chronic hepatitis B infection who underwent curative liver resection for HCC from 2003 to 2014 was reviewed. Patients' overall survival and recurrence were compared between the groups of patients whose HCCs were diagnosed by surveillance or non-surveillance, as well as between the groups of patients operated in the first (2003-2008) and second (2009-2014) 6-year periods. RESULTS: Of 1075 chronic hepatitis B patients with HCC, 452 (42.0%) patients were diagnosed by preoperative surveillance. Compared with the non-surveillance group, the OS and RFS rates were significantly better in the surveillance group (both P < 0.001). Surveillance was associated with a 55% decrease in the overall survival risk and a 48% decrease in the recurrence risk (HR 0.45, 95% CI 0.38-0.53, and HR 0.52, 95% CI 0.44-0.61). Compared with the first period, a significant reduction of 12% and 19% in the overall death and recurrence risks, respectively, was observed in the second period (HR 0.88, 95% CI 0.78-0.97, and HR 0.81, 95% CI 0.70-0.95). CONCLUSION: Surveillance for HCC was associated with favorable long-term overall and recurrence-free survival rates after curative liver resection of HCC in patients with chronic hepatitis B.

MicroRNA-506 suppresses invasiveness and metastasis of human hepatocellular carcinoma cells by targeting IL8.

MicroRNAs (miRNAs) have been reported to be involved in tumor metastasis. In this study, we investigated the function of miR-506 in the metastasis of human hepatocellular carcinoma (HCC). We found that miR-506 is significantly downregulated in the primary tissue of metastatic HCC and in highly metastatic HCC cell lines. Overexpression of miR-506 suppressed HCC cell migration, invasion, and metastasis both in vitro and in vivo. Furthermore, miR-506 was found to specifically target the 3' untranslated region (3'-UTR) of interleukin 8 (IL8) mRNA. Spearman's correlation analysis revealed that miR-506 expression inversely correlated with IL8 mRNA and protein expression in HCC tissue samples. IL8 treatment reversed miR-506-induced suppression of HCC cell migration and invasiveness. Thus, miR-506 acts as a tumor suppressor that may inhibit the migration, invasiveness, and metastasis of HCC cells by targeting IL8.

TGF-ß upregulates miR-182 expression to promote gallbladder cancer metastasis by targeting CADM1.

UNLABELLED: Transforming growth factor ß (TGF-ß) plays important roles in tumor metastasis by regulating miRNAs expression. miR-182 is an important molecule in the regulation of cancer progression. The aim of the study is to assess the role of miR-182 in TGF-ß-induced cancer metastasis. In the present study, we found that miR-182 levels are significantly upregulated in GBC tissues compared with normal controls, and miR-182 expression is remarkably increased in primary tumors that subsequently metastasized, when compared to those primary tumors that did not metastasize. TGF-ß induces miR-182 expression in GBC cells, and overexpression of miR-182 promotes GBC cell migration and invasion, whereas miR-182 inhibition suppresses TGF-ß-induced cancer cell migration and invasion. The blockage of miR-182 by a specific inhibitor effectively inhibits pulmonary metastases in vivo. We further identified that the cell adhesion molecule1 (CADM1) is a new target gene of miR-182. miR-182 negatively regulates CADM1 expression in vitro and in vivo. Importantly, re-expression of CADM1 in GBC cells partially abrogates miR-182-induced cell invasion. CONCLUSIONS: miR-182 is an important mediator of GBC metastasis, thus offering a new target for the development of therapeutic agents against GBC.