PCK1 attenuates tumor stemness via activating the Hippo signaling pathway in hepatocellular carcinoma.

Liver cancer stem cells were found to rely on glycolysis as the preferred metabolic program. Phosphoenolpyruvate carboxylase 1 (PCK1), a gluconeogenic metabolic enzyme, is down-regulated in hepatocellular carcinoma and is closely related to poor prognosis. The oncogenesis and progression of tumors are closely related to cancer stem cells. It is not completely clear whether the PCK1 deficiency increases the stemness of hepatoma cells and promotes the oncogenesis of hepatocellular carcinoma. Herein, the results showed that PCK1 inhibited the self-renewal property of hepatoma cells, reduced the mRNA level of cancer stem cell markers, and inhibited tumorigenesis. Moreover, PCK1 increased the sensitivity of hepatocellular carcinoma cells to sorafenib. Furthermore, we found that PCK1 activated the Hippo pathway by enhancing the phosphorylation of YAP and inhibiting its nuclear translocation. Verteporfin reduced the stemness of hepatoma cells and promoted the pro-apoptotic effect of sorafenib. Thus, combined treatment with verteporfin and sorafenib may be a potential anti-tumor strategy in hepatocellular carcinoma.

Prognostic value of creatine kinase (CK)-MB to total-CK ratio in colorectal cancer patients after curative resection.

OBJECTIVES: This study aimed to evaluate the prognostic significance of postoperative Creatine Kinase type M and B (CK-MB) to total Creatine Kinase (CK) ratio (CK-MB/CK) in colorectal cancer (CRC) patients after radical resection. METHODS: This was a single-center retrospective cohort analysis. Subjects were stage I-III CRC patients hospitalized in Sichuan Cancer Hospital from January 2017 to May 2021. Patients were divided into abnormal group and normal group according to whether the CK-MB/CK ratio was abnormal after surgery. Through a comparative analysis of clinical data, laboratory test results, and prognosis differences between the two groups, we aimed to uncover the potential relationship between abnormal CK-MB > CK results and CRC patients. To gauge the impact of CK-MB/CK on overall survival (OS) and disease-free survival (DFS), we employed the multivariable COX regression and LASSO regression analysis. Additionally, Spearman correlation analysis, logistic regression, and receiver-operating characteristic (ROC) curve analysis were conducted to assess the predictive value of the CK-MB/CK ratio for postoperative liver metastasis. RESULTS: Cox regression analysis revealed that the CK-MB/CK ratio was a stable risk factors for OS (HR = 3.82, p < 0.001) and DFS (HR = 2.31, p < 0.001). To distinguish hepatic metastases after surgery, the ROC area under the curve of CK-MB/CK was 0.697 (p < 0.001), and the optimal cut-off value determined by the Youden index was 0.347. CONCLUSIONS: Postoperative abnormal CK-MB/CK ratio predicts worse prognosis in CRC patients after radical resection and serves as a useful biomarker for detecting postoperative liver metastasis.

Signal amplification strategy of DNA self-assembled biosensor and typical applications in pathogenic microorganism detection.

Biosensors have emerged as ideal analytical devices for various bio-applications owing to their low cost, convenience, and portability, which offer great potential for improving global healthcare. DNA self-assembly techniques have been enriched with the development of innovative amplification strategies, such as dispersion-to-localization of catalytic hairpin assembly, and dumbbell hybridization chain reaction, which hold great significance for building biosensors capable of realizing sensitive, rapid and multiplexed detection of pathogenic microorganisms. Here, focusing primarily on the signal amplification strategies based on DNA self-assembly, we concisely summarized the strengths and weaknesses of diverse isothermal nucleic acid amplification techniques. Subsequently, both single-layer and cascade amplification strategies based on traditional catalytic hairpin assembly and hybridization chain reaction were critically explored. Furthermore, a comprehensive overview of the recent advances in DNA self-assembled biosensors for the detection of pathogenic microorganisms is presented to summarize methods for biorecognition and signal amplification. Finally, a brief discussion is provided about the current challenges and future directions of DNA self-assembled biosensors.

Deficiency of gluconeogenic enzyme PCK1 promotes metabolic-associated fatty liver disease through PI3K/AKT/PDGF axis activation in male mice.

Metabolic associated fatty liver disease (MAFLD) encompasses a broad spectrum of hepatic disorders, including steatosis, nonalcoholic steatohepatitis (NASH) and fibrosis. We demonstrated that phosphoenolpyruvate carboxykinase 1 (PCK1) plays a central role in MAFLD progression. Male mice with liver Pck1 deficiency fed a normal diet displayed hepatic lipid disorder and liver injury, whereas fibrosis and inflammation were aggravated in mice fed a high-fat diet with drinking water containing fructose and glucose (HFCD-HF/G). Forced expression of hepatic PCK1 by adeno-associated virus ameliorated MAFLD in male mice. PCK1 deficiency stimulated lipogenic gene expression and lipid synthesis. Moreover, loss of hepatic PCK1 activated the RhoA/PI3K/AKT pathway by increasing intracellular GTP levels, increasing secretion of platelet-derived growth factor-AA (PDGF-AA), and promoting hepatic stellate cell activation. Treatment with RhoA and AKT inhibitors or gene silencing of RhoA or AKT1 alleviated MAFLD progression in vivo. Hepatic PCK1 deficiency may be important in hepatic steatosis and fibrosis development through paracrine secretion of PDGF-AA in male mice, highlighting a potential therapeutic strategy for MAFLD.

Obatoclax inhibits SARS-CoV-2 entry by altered endosomal acidification and impaired cathepsin and furin activity in vitro.

Coronavirus disease 2019 (COVID-19) caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has set off a global pandemic. There is an urgent unmet need for safe, affordable, and effective therapeutics against COVID-19. In this regard, drug repurposing is considered as a promising approach. We assessed the compounds that affect the endosomal acidic environment by applying human angiotensin-converting enzyme 2 (hACE2)- expressing cells infected with a SARS-CoV-2 spike (S) protein-pseudotyped HIV reporter virus and identified that obatoclax resulted in the strongest inhibition of S protein-mediated virus entry. The potent antiviral activity of obatoclax at nanomolar concentrations was confirmed in different human lung and intestinal cells infected with the SARS-CoV-2 pseudotype system as well as clinical virus isolates. Furthermore, we uncovered that obatoclax executes a double-strike against SARS-CoV-2. It prevented SARS-CoV-2 entry by blocking endocytosis of virions through diminished endosomal acidification and the corresponding inhibition of the enzymatic activity of the endosomal cysteine protease cathepsin L. Additionally, obatoclax impaired the SARS-CoV-2 S-mediated membrane fusion by targeting the MCL-1 protein and reducing furin protease activity. In accordance with these overarching mechanisms, obatoclax blocked the virus entry mediated by different S proteins derived from several SARS-CoV-2 variants of concern such as, Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2). Taken together, our results identified obatoclax as a novel effective antiviral compound that keeps SARS-CoV-2 at bay by blocking both endocytosis and membrane fusion. Our data suggested that obatoclax should be further explored as a clinical drug for the treatment of COVID-19.

Potent SARS-CoV-2 neutralizing antibodies with protective efficacy against newly emerged mutational variants.

Accumulating mutations in the SARS-CoV-2 Spike (S) protein can increase the possibility of immune escape, challenging the present COVID-19 prophylaxis and clinical interventions. Here, 3 receptor binding domain (RBD) specific monoclonal antibodies (mAbs), 58G6, 510A5 and 13G9, with high neutralizing potency blocking authentic SARS-CoV-2 virus display remarkable efficacy against authentic B.1.351 virus. Surprisingly, structural analysis has revealed that 58G6 and 13G9 both recognize the steric region S470-495 on the RBD, overlapping the E484K mutation presented in B.1.351. Also, 58G6 directly binds to another region S450-458 in the RBD. Significantly, 58G6 and 510A5 both demonstrate prophylactic efficacy against authentic SARS-CoV-2 and B.1.351 viruses in the transgenic mice expressing human ACE2 (hACE2), protecting weight loss and reducing virus loads. Together, we have evidenced 2 potent neutralizing Abs with unique mechanism targeting authentic SARS-CoV-2 mutants, which can be promising candidates to fulfill the urgent needs for the prolonged COVID-19 pandemic.

Depletion of VPS35 attenuates metastasis of hepatocellular carcinoma by restraining the Wnt/PCP signaling pathway.

Vesicle Protein Sorting 35 (VPS35) is a novel oncogene that promotes tumor growth through the PI3K/AKT signaling in hepatocellular carcinoma (HCC). However, the role of VPS35 in HCC metastasis and the underlying mechanisms remain largely unclear. In this study, we observed that overexpression of VPS35 enhanced hepatoma cell invasion and metastasis by inducing epithelial-mesenchymal transition (EMT)-related gene expression. Conversely, knockout of VPS35 significantly inhibited hepatoma cell migration and invasion. Furthermore, depletion of VPS35 decreased the lung metastasis of HCC in nude mice. By transcriptome analysis, we determined that VPS35 promoted HCC metastasis by activating the Wnt/non-canonical planar cell polarity (PCP) pathway. Mechanistically, VPS35 activated the PCP pathway by regulating membrane sorting and trafficking of Frizzled-2 (FZD2) and ROR1 in hepatoma cells. Collectively, our results indicate that VPS35 promotes HCC metastasis via enhancing the Wnt/PCP signaling, thus providing a potential prognostic marker and therapeutic target for HCC.

A Rapid and Efficient Screening System for Neutralizing Antibodies and Its Application for SARS-CoV-2.

After the pandemic of COVID-19, neutralizing antibodies (NAbs) against SARS-CoV-2 have been developed for the prophylactic and therapeutic purposes. However, few methodologies are described in detail on how to rapidly and efficiently generate effective NAbs to SARS-CoV-2. Here, we integrated and optimized a strategically screening method for NAbs, which has enabled us to obtain SARS-CoV-2 receptor-binding domain (RBD) specific NAbs within 6 days, followed by additional 9 days for antibody production and function analysis. Using this method, we obtained 198 specific Abs against SARS-CoV-2 RBD from the blood samples of COVID-19 convalescent patients, and 96 of them showed neutralizing activity. At least 20% of these NAbs exhibited advanced neutralizing potency and high affinity, with the top two NAbs showing half-maximal inhibitory concentration (IC50) to block authentic SARS-CoV-2 at 9.88 and 11.13 ng/ml, respectively. Altogether, our study provides an effective methodology with high applicable value for discovering potential preventative and therapeutic NAbs for the emerging infectious diseases.

Longitudinal Dynamics of the Neutralizing Antibody Response to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is a global pandemic with no licensed vaccine or specific antiviral agents for therapy. Little is known about the longitudinal dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific neutralizing antibodies (NAbs) in patients with COVID-19. METHODS: Blood samples (n = 173) were collected from 30 patients with COVID-19 over a 3-month period after symptom onset and analyzed for SARS-CoV-2-specific NAbs using the lentiviral pseudotype assay, coincident with the levels of IgG and proinflammatory cytokines. RESULTS: SARS-CoV-2-specific NAb titers were low for the first 7-10 days after symptom onset and increased after 2-3 weeks. The median peak time for NAbs was 33 days (interquartile range [IQR], 24-59 days) after symptom onset. NAb titers in 93.3% (28/30) of the patients declined gradually over the 3-month study period, with a median decrease of 34.8% (IQR, 19.6-42.4%). NAb titers increased over time in parallel with the rise in immunoglobulin G (IgG) antibody levels, correlating well at week 3 (r = 0.41, P < .05). The NAb titers also demonstrated a significant positive correlation with levels of plasma proinflammatory cytokines, including stem cell factor (SCF), TNF-related apoptosis-inducing ligand (TRAIL), and macrophage colony-stimulating factor (M-CSF). CONCLUSIONS: These data provide useful information regarding dynamic changes in NAbs in patients with COVID-19 during the acute and convalescent phases.

DNA and RNA sequencing identified a novel oncogene VPS35 in liver hepatocellular carcinoma.

Liver hepatocellular carcinoma (LIHC) is the second leading cause of cancer mortality worldwide. Although cancer driver genes identified so far have been considered to be saturated or nearly saturated, challenges remain in discovering novel genes underlying carcinogenesis due to significant tumor heterogeneity. Here, in a small cohort of hepatitis B virus (HBV)-associated LIHC, we investigated the transcriptional patterns of tumor-mutated alleles using both whole-exome and RNA sequencing data. A graph clustering of the transcribed tumor-mutated alleles characterized overlapped functional clusters, and thus prioritized potentially novel oncogenes. We validated the function of the potentially novel oncogenes in vitro and in vivo. We showed that a component of the retromer complex-the vacuolar protein sorting-associated protein 35 (VPS35)-promoted the proliferation of hepatoma cell through the PI3K/AKT signaling pathway. In VPS35-knockout hepatoma cells, a significantly reduced distribution of membrane fibroblast growth factor receptor 3 (FGFR3) demonstrated the effects of VPS35 on sorting and trafficking of transmembrane receptor. This study provides insight into the roles of the retromer complex on carcinogenesis and has important implications for the development of personalized therapeutic strategies for LIHC.

SLC27A5 deficiency activates NRF2/TXNRD1 pathway by increased lipid peroxidation in HCC.

Solute carrier family 27 member 5 (SLC27A5/FATP5) is involved in fatty acid transport and bile acid metabolism; however, little is known about its role in human diseases. Here, we first show that SLC27A5 expression is downregulated in hepatocellular carcinoma (HCC) by DNA hypermethylation, and reduced SCL27A5 expression contributes to tumor progression and poor prognosis. Both gain- and loss-of-function studies demonstrated that SLC27A5 has an antiproliferative effect on HCC cells in vitro and in vivo. Knockout of SLC27A5 increases polyunsaturated lipids, leading to increased NADP+/NADPH ratio, ROS production as well as lipid peroxidation and the subsequent accumulation of 4-hydroxy-2-nonenal (4-HNE) in hepatoma cells. Mass spectrometry analysis found that 4-HNE directly modifies cysteine residues (Cys513, 518) on KEAP1, thus leading KEAP1/NRF2 pathway activation and increases the expression levels of NRF2 target genes, such as TXNRD1. Further, SLC27A5 expression negatively correlates with TXNRD1 expression in hepatoma cells and clinical HCC samples, and blockade of NRF2/TXNRD1 using genetic approaches or inhibitors sensitizes SLC27A5-deficient hepatoma cells to sorafenib treatment. Collectively, we demonstrated that SLC27A5 acts as a novel tumor suppressor by suppressing TXNRD1 expression via the KEAP1/NRF2 pathway in HCC. Combination therapy of sorafenib and NRF2/TXNRD1 inhibitors may be a promising strategy in personalized HCC treatment.

PCK1 Downregulation Promotes TXNRD1 Expression and Hepatoma Cell Growth via the Nrf2/Keap1 Pathway.

Gluconeogenesis, generates glucose from small carbohydrate substrates, and drives the metabolic flux in parallel but opposite to glycolysis. The cytoplasmic isoform of phosphoenolpyruvate carboxykinase (PCK1 or PEPCK-C), a rate-limiting enzyme in gluconeogenesis, initiates the gluconeogenesis process and is reportedly dysregulated in multiple types of cancer. Gluconeogenesis mainly occurs in the liver during fasting, and previous studies have demonstrated that PCK1 acts as a tumor suppressor in hepatocellular carcinoma (HCC); however, the role of PCK1 in cancer progression remains incompletely understood. In the current study, we found that PCK1 expression was decreased in HCC as compared to adjacent normal liver tissues, and low PCK1 expression correlated with poor patient prognosis. Furthermore, overexpression of PCK1 suppressed reactive oxygen species (ROS) production and nuclear translocation of Nrf2 in hepatoma cells. In addition, thioredoxin reductase 1 (TXNRD1), an antioxidant enzyme regulated by the Nrf2/Keap1 pathway, was downregulated upon overexpression of PCK1 in HCC cell lines. Furthermore, we verified this axis using nude mouse xenograft model. Finally, we found that auranofin, a TXNRD1 inhibitor, enhanced the sensitivity of PCK1-knockout hepatoma cells to sorafenib-induced apoptosis. Taken together, our findings suggest that PCK1 deficiency promotes hepatoma cell proliferation via the induction of oxidative stress and the activation of transcription factor Nrf2, and that targeting the TXNRD1 antioxidant pathway sensitizes PCK1-knockout hepatoma cells to sorafenib treatment in vitro.