Integrating TCGA and Single-Cell Sequencing Data for Hepatocellular Carcinoma: A Novel Glycosylation (GLY)/Tumor Microenvironment (TME) Classifier to Predict Prognosis and Immunotherapy Response.

The major liver cancer subtype is hepatocellular carcinoma (HCC). Studies have indicated that a better prognosis is related to the presence of tumor-infiltrating lymphocytes (TILs) in HCC. However, the molecular pathways that drive immune cell variation in the tumor microenvironment (TME) remain poorly understood. Glycosylation (GLY)-related genes have a vital function in the pathogenesis of numerous tumors, including HCC. This study aimed to develop a GLY/TME classifier based on glycosylation-related gene scores and tumor microenvironment scores to provide a novel prognostic model to improve the prediction of clinical outcomes. The reliability of the signatures was assessed using receiver operating characteristic (ROC) and survival analyses and was verified with external datasets. Furthermore, the correlation between glycosylation-related genes and other cells in the immune environment, the immune signature of the GLY/TME classifier, and the efficacy of immunotherapy were also investigated. The GLY score low/TME score high subgroup showed a favorable prognosis and therapeutic response based on significant differences in immune-related molecules and cancer cell signaling mechanisms. We evaluated the prognostic role of the GLY/TME classifier that demonstrated overall prognostic significance for prognosis and therapeutic response before treatment, which may provide new options for creating the best possible therapeutic approaches for patients.

[Tea polyphenols inhibits the proliferation of prostate cancer DU145 cells].

OBJECTIVE: To investigate the effect of tea polyphenols on the proliferation of human prostate cancer cells and its possible mechanism. METHODS: We cultured androgen-independent prostate cancer DU145 cells in the medium with different concentrations (50, 100, 250 and 500 microg/ml) of tea polyphenols, and those in the normal medium as the control. After 48 hours of culture, we detected the survival rate of the cells by MTT assay and determined the expression of survivin by Western blot and quantitative RT-PCR. RESULTS: At 48 hours, the survival rates of the prostate cancer DU145 cells were 0.97 +/- 0.12, 0.71 +/- 0.07, 0.20 +/- 0.03 and 0.08 +/- 0.01 in the 50, 100, 250 and 500 microg/ml tea polyphenols treatment groups, all significantly reduced as compared with the control group (P < 0.01) except that of the 50 microg/ml group (P = 0.42). Furthermore, the survival rate continued to decrease with the prolonging of time, dropping below 5% at 96 hours except in the 50 microg/ml group. The grey values of the survivin expression in the 100, 250 and 500 microg/ml tea polyphenols groups were 13 425 +/- 34, 2 017 +/- 24 and 1 274 +/- 22, respectively, at 48 hours, significantly lower than 15 075 +/- 48 in the control group (P < 0.01). Moreover, the content of survivin mRNA at 48 hours was markedly lower in the 50, 100, 250 and 500 microg/ml treatment groups (0.74 +/- 0.03, 0.64 +/- 0.02, 0.52 +/- 0.01 and 0.21 +/- 0.02) than in the control (P < 0.01). CONCLUSION: Tea polyphenols can inhibit the proliferation of human prostate cancer DU145 cells, which may be associated with the decreased expression of the survivin gene.

O1-conotoxin Tx6.7 cloned from the genomic DNA of Conus textile that inhibits calcium currents.

Background: Conotoxins exhibit great potential as neuropharmacology tools and therapeutic candidates due to their high affinity and specificity for ion channels, neurotransmitter receptors or transporters. The traditional methods to discover new conotoxins are peptide purification from the crude venom or gene amplification from the venom duct. Methods: In this study, a novel O1 superfamily conotoxin Tx6.7 was directly cloned from the genomic DNA of Conus textile using primers corresponding to the conserved intronic sequence and 3' UTR elements. The mature peptide of Tx6.7 (DCHERWDWCPASLLGVIYCCEGLICFIAFCI) was synthesized by solid-phase chemical synthesis and confirmed by mass spectrometry. Results: Patch clamp experiments on rat DRG neurons showed that Tx6.7 inhibited peak calcium currents by 59.29 ± 2.34% and peak potassium currents by 22.33 ± 7.81%. In addition, patch clamp on the ion channel subtypes showed that 10 µM Tx6.7 inhibited 56.61 ± 3.20% of the hCaV1.2 currents, 24.67 ± 0.91% of the hCaV2.2 currents and 7.30 ± 3.38% of the hNaV1.8 currents. Tx6.7 had no significant toxicity to ND7/23 cells and increased the pain threshold from 0.5 to 4 hours in the mouse hot plate assay. Conclusion: Our results suggested that direct cloning of conotoxin sequences from the genomic DNA of cone snails would be an alternative approach to obtaining novel conotoxins. Tx6.7 could be used as a probe tool for ion channel research or a therapeutic candidate for novel drug development.

Single-cell transcriptome analysis reveals the metabolic changes and the prognostic value of malignant hepatocyte subpopulations and predict new therapeutic agents for hepatocellular carcinoma.

Background: The development of HCC is often associated with extensive metabolic disturbances. Single cell RNA sequencing (scRNA-seq) provides a better understanding of cellular behavior in the context of complex tumor microenvironments by analyzing individual cell populations. Methods: The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) data was employed to investigate the metabolic pathways in HCC. Principal component analysis (PCA) and uniform manifold approximation and projection (UMAP) analysis were applied to identify six cell subpopulations, namely, T/NK cells, hepatocytes, macrophages, endothelial cells, fibroblasts, and B cells. The gene set enrichment analysis (GSEA) was performed to explore the existence of pathway heterogeneity across different cell subpopulations. Univariate Cox analysis was used to screen genes differentially related to The Overall Survival in TCGA-LIHC patients based on scRNA-seq and bulk RNA-seq datasets, and LASSO analysis was used to select significant predictors for incorporation into multivariate Cox regression. Connectivity Map (CMap) was applied to analysis drug sensitivity of risk models and targeting of potential compounds in high risk groups. Results: Analysis of TCGA-LIHC survival data revealed the molecular markers associated with HCC prognosis, including MARCKSL1, SPP1, BSG, CCT3, LAGE3, KPNA2, SF3B4, GTPBP4, PON1, CFHR3, and CYP2C9. The RNA expression of 11 prognosis-related differentially expressed genes (DEGs) in normal human hepatocyte cell line MIHA and HCC cell lines HCC-LM3 and HepG2 were compared by qPCR. Higher KPNA2, LAGE3, SF3B4, CCT3 and GTPBP4 protein expression and lower CYP2C9 and PON1 protein expression in HCC tissues from Gene Expression Profiling Interactive Analysis (GEPIA) and Human Protein Atlas (HPA) databases. The results of target compound screening of risk model showed that mercaptopurine is a potential anti-HCC drug. Conclusion: The prognostic genes associated with glucose and lipid metabolic changes in a hepatocyte subpopulation and comparison of liver malignancy cells to normal liver cells may provide insight into the metabolic characteristics of HCC and the potential prognostic biomarkers of tumor-related genes and contribute to developing new treatment strategies for individuals.

Corrigendum: Single-cell transcriptome analysis reveals the metabolic changes and the prognostic value of malignant hepatocyte subpopulations and predict new therapeutic agents for hepatocellular carcinoma.

[This corrects the article DOI: 10.3389/fonc.2023.1104262.].

O-GlcNAcylation is a novel regulator of lung and colon cancer malignancy.

O-GlcNAc is a monosaccharide attached to serine or threonine hydroxyl moieties on numerous nuclear and cytoplasmic proteins; O-GlcNAcylation is dynamically regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Although recent studies have shown that O-GlcNAcylation plays essential roles in breast cancer progression, it is also necessary to know whether O-GlcNAcylation is involved in other types of human cancer. In this study, O-GlcNAcylation levels and the expressions of OGT and OGA in human lung and colon cancer tissues were examined by immunohistochemistry analysis. We found that O-GlcNAcylation as well as OGT expression was significantly elevated in the cancer tissues compared with that in the corresponding adjacent tissues. Additionally, the roles of O-GlcNAcylation in the malignancy of lung and colon cancer were investigated in vitro. The results showed that O-GlcNAcylation markedly enhanced the anchorage-independent growth of lung and colon cancer cells; O-GlcNAcylation could also enhance lung and colon cancer invasion in a context-dependent manner. All together, this study suggests that O-GlcNAcylation might play important roles in lung and colon cancer formation and progression, and may be a valuable target for diagnosis and therapy of cancer.

O-GlcNAcylation of SIRT1 enhances its deacetylase activity and promotes cytoprotection under stress.

SIRT1 is the most evolutionarily conserved mammalian sirtuin, and it plays a vital role in the regulation of metabolism, stress responses, genome stability, and ageing. As a stress sensor, SIRT1 deacetylase activity is significantly increased during stresses, but the molecular mechanisms are not yet fully clear. Here, we show that SIRT1 is dynamically modified with O-GlcNAc at Ser 549 in its carboxy-terminal region, which directly increases its deacetylase activity both in vitro and in vivo. The O-GlcNAcylation of SIRT1 is elevated during genotoxic, oxidative, and metabolic stress stimuli in cellular and mouse models, thereby increasing SIRT1 deacetylase activity and protecting cells from stress-induced apoptosis. Our findings demonstrate a new mechanism for the activation of SIRT1 under stress conditions and suggest a novel potential therapeutic target for preventing age-related diseases and extending healthspan.

Inhibition of E-cadherin/catenin complex formation by O-linked N-acetylglucosamine transferase is partially independent of its catalytic activity.

p120-catenin (p120) contains a large central armadillo repeat domain, via which it binds to E­cadherin to stabilize the latter, thereby regulating cell­to­cell adhesion. A previous study by our group demonstrated that O­linked N­acetylglucosamine (O­GlcNAc) is involved in the regulation of the interaction between p120 and E­cadherin. As O­GlcNAc transferase (OGT) is able to directly bind to the majority of its target proteins, the present study hypothesized that OGT may additionally regulate the formation of the E­cadherin/catenin complex independent of its catalytic activity. To verify this hypothesis, a catalytically inactive OGT mutant was expressed in H1299 cells, and its effects on the formation of the E­cadherin/catenin complex were assessed. A cytoskeleton­binding protein extraction assay confirmed that OGT inhibited the formation of the E­cadherin/catenin complex independent of its catalytic activity. In addition, co­immunoprecipitation and pull­down assays were used to evaluate the interaction between OGT and p120. Immunoblotting indicated that OGT was able to directly bind to p120. To determine the domain of p120 involved in binding to OGT, a series of deletion mutants of p120 were constructed and subjected to protein binding assays by pull­down assays. Immunoblotting showed that OGT bound to the regulatory and armadillo domains of p120, which might interfere with the interaction between p120 and E­cadherin. Finally, OGT, p120 and E­cadherin cytoplasmic domains (ECD) were recombinantly expressed in BL21 (DE3) recombinant E. coli cells, and a glutathione S­transferase (GST) pull­down assay was performed to assess the interactions among the purified recombinant proteins. Immunoblotting indicated that maltose­binding protein (MBP)­OGT inhibited the binding of His­p120 to GST­ECD in a dose­dependent manner. All of these results suggested that OGT inhibited the formation of the E­cadherin/catenin complex through reducing the interaction between p120 and E­cadherin. The present study provided a novel underlying mechanism of the regulation of the interaction between p120 and E­cadherin, and thus E­cadherin­mediated cell­cell adhesion, which has essential roles in cancer development and progression.

A highly effective and adjustable dual plasmid system for O-GlcNAcylated recombinant protein production in E. coli.

O-GlcNAcylation is a ubiquitous, dynamic and reversible post-translational protein modification in metazoans, and it is catalysed and removed by O-GlcNAc transferase (OGT) and O-GlcNAcase, respectively. Prokaryotes lack endogenous OGT activity. It has been reported that coexpression of mammalian OGT with its target substrates in Escherichia coli produce O-GlcNAcylated recombinant proteins, but the plasmids used were not compatible, and the expression of both OGT and its target protein were induced by the same inducer. Here, we describe a compatible dual plasmid system for coexpression of OGT and its target substrate for O-GlcNAcylated protein production in E. coli. The approach was validated using the CKII and p53 protein as control. This compatible dual plasmid system contains an arabinose-inducible OGT expression vector with a pUC origin and an isopropyl ß-d-thiogalactopyranoside-inducible OGT target substrate expression vector bearing a p15A origin. The dual plasmid system produces recombinant proteins with varying O-GlcNAcylation levels by altering the inducer concentration. More importantly, the O-GlcNAcylation efficiency was much higher than the previously reported system. Altogether, we established an adjustable compatible dual plasmid system that can effectively yield O-GlcNAcylated proteins in E. coli.

O-GlcNAcylation is increased in prostate cancer tissues and enhances malignancy of prostate cancer cells.

O-GlcNAc is an O-linked ?-N-acetylglucosamine moiety attached to the side-chain hydroxyl of a serine or threonine residue in numerous cytoplasmic and nuclear proteins. In this study, we detected the level of O-GlcNAc in prostate, liver and pancreatic cancer tissues, and found that the global O-GlcNAc modification also known as O-GlcNAcylation, is specifically increased in prostate cancer tissues compared to corresponding adjacent tissues. In addition, we found that global O-GlcNAcylation is increased in prostate cancer cells and not in benign prostatic hyperplasia (BPH) epithelial cells. O-GlcNAc enhanced the anchorage-independent growth and the migratory/invasive ability of prostate cancer cells. More importantly, we provide here, for the first time to the best of our knowledge, direct evidence that increased O-GlcNAcylation induces malignant transformation of nontumorigenic (BPH) cells. Furthermore, our study suggested that inhibiting the formation of the E-cadherin/catenin/cytoskeleton complex may underly the O-GlcNAc-induced prostate cancer progression. Overall, these findings indicated that O-GlcNAcylation is increased in prostate, but not in liver and pancreatic cancer tissues, and that O-GlcNAc can enhance the malignancy of prostate cancer cells.

GlcNAcylation plays an essential role in breast cancer metastasis.

GlcNAcylation, a dynamic posttranslational modification, is involved in a wide range of biological processes and some human diseases. Although there is emerging evidence that some tumor-associated proteins are modified by GlcNAcylation, the role of GlcNAcylation in tumor progression remains unclear. Here, we show that GlcNAcylation enhances the migration/invasion of breast cancer cells in vitro and lung metastasis in vivo. The decrease of cell surface E-cadherin is the molecular mechanism underlying GlcNAcylation-induced breast cancer metastasis. p120 and beta-catenin, but not E-cadherin, are GlcNAcylated; the GlcNAcylation of p120 and beta-catenin might play roles in the decrease of cell surface E-cadherin. Moreover, immunohistochemistry analysis indicated that the global GlcNAcylation level in breast tumor tissues is elevated significantly as compared with that in the corresponding adjacent tissues; further, GlcNAcylation was significantly enhanced in metastatic lymph nodes compared with their corresponding primary tumor tissues. This is the first report to clearly elucidate the roles and mechanisms whereby GlcNAcylation influences the malignant properties of breast cancer cells. These results also suggest that GlcNAcylation might be a potential target for the diagnosis and therapy of breast cancer.