Gemcitabine Inhibits the Progression of Pancreatic Cancer by Restraining the WTAP/MYC Chain in an m6A-Dependent Manner.

PURPOSE: Pancreatic cancer (PC) is a common malignant tumor of the digestive system, and its 5-year survival rate is only 4%. N6-methyladenosine (m6A) RNA methylation is the most common post-transcriptional modification and dynamically regulates cancer development, while its role in PC treatment remains unclear. MATERIALS AND METHODS: We treated PC cells with gemcitabine and quantified the overall m6A level with m6A methylation quantification. Real-time quantitative reverse transcription polymerase chain reaction and Western blot analyses were used to detect expression changes of m6A regulators. We verified the m6A modification on the target genes through m6A-immunoprecipitation (IP), and further in vivo experiments and immunofluorescence (IF) assays were applied to verify regulation of gemcitabine on Wilms' tumor 1-associated protein (WTAP) and MYC. RESULTS: Gemcitabine inhibited the proliferation and migration of PC cells and reduced the overall level of m6A modification. Additionally, the expression of the "writer" WTAP was significantly downregulated after gemcitabine treatment. We knocked down WTAP in cells and found target gene MYC expression was significantly downregulated, m6A-IP also confirmed the m6A modification on MYC. Our experiments showed that m6A-MYC may be recognized by the "reader" IGF2BP1. In vivo experiments revealed gemcitabine inhibited the tumorigenic ability of PC cells. IF analysis also showed that gemcitabine inhibited the expression of WTAP and MYC, which displayed a significant trend of co-expression. CONCLUSION: Our study confirmed that gemcitabine interferes with WTAP protein expression in PC, reduces m6A modification on MYC and RNA stability, thereby inhibiting the downstream pathway of MYC, and inhibits the progression of PC.

IGF2BP2 promotes pancreatic carcinoma progression by enhancing the stability of B3GNT6 mRNA via m6A methylation.

BACKGROUND: Pancreatic carcinoma (PC) is a highly lethal cancer with an increasing mortality rate, its five-year survival rate is only approximately 4%. N6-methyladenosine (m6A) modification is the most common posttranscriptional modification of RNA, it could affect tumor formation by regulating m6A modifications in the mRNA of key oncogenes or tumor suppressor genes. However, its role in PC remains unclear. METHODS: We combined bioinformatic analysis with in vitro and in vivo experiments to investigate the expression profile of methylation modulators and identify key m6A regulators in the progression of PC. Further study focused on exploring the target genes binding to the regulators through RIP and immunofluorescence staining experiment. RESULTS: TCGA and Gene Expression Omnibus (GEO) analyses revealed an overall increasing trend in the expression of m6A regulators in PC, and consensus clustering analysis of m6A modification showed that the expression of regulators was negatively correlated with the survival rate. LASSO-Cox regression analysis revealed that IGF2BP2, METTL3, ALKBH5 and KIAA1429 were associated with hazard ratios (HR), but only IGF2BP2 was sufficiently appropriate for the m6A survival prognosis model. The IHC and WB results verified high protein expression of IGF2BP2 in PC, and IGF2BP2 knockdown inhibited the proliferation and migration of PC cells. We predicted and verified B3GNT6 was observably regulated by IGF2BP2 via RIP assays. In addition, IF staining confirmed the co-expression of IGF2BP2 and B3GNT6. The tumor-promoting effect of IGF2BP2 and its co-expression with B3GNT6 were verified in an animal model. CONCLUSIONS: Elevated m6A levels promote PC progression. IGF2BP2 is a credible marker and modulates B3GNT6 mRNA stability, indicating that IGF2BP2 is a potential prognostic marker and therapeutic target in PC progression.

Glycolytic and fatty acid oxidation genes affect the treatment and prognosis of liver cancer.

BACKGROUND: Metabolic reprogramming is a feature of tumour cells and is essential to support their rapid proliferation. The glycolytic activity of liver cancer cells is significantly higher than that of normal liver cells, and the rapidly proliferating tumour cells are powered by aerobic glycolysis. Lipid metabolism reprogramming enables tumour cells to meet their needs for highly proliferative growth and is an important driving force for the development of hepatocellular carcinoma (HCC). AIM: To explore the influence of different metabolic subtypes of HCC and analyse their significance in guiding prognosis and treatment based on the molecular mechanism of glycolysis and fatty acid oxidation (FAO). METHODS: By downloading related data from public databases including the Cancer Genome Atlas (TCGA), the Molecular Signatures Database, and International Cancer Genome Consortium, we utilised unsupervised consensus clustering to divide TCGA Liver Hepatocellular Carcinoma samples into four metabolic subgroups and compared single nucleotide polymorphism, copy number variation, tumour microenvironment, and Genomics of Drug Sensitivity in Cancer and Tumour Immune Dysfunction and Exclusion between different metabolites. The differences and causes of survival and the clinical characteristics between them were analysed, and a prognostic model was established based on glycolysis and FAO genes. Combined with the clinical features, a Norman diagram was created to compare the pros and cons of each model. RESULTS: In the four metabolic subgroups, with the increase in glycolytic expression, the median survival of patients showed the worst results, while FAO showed the best. When comparing the follow-up analysis of each group, we considered that the differences between them might be related to reactive oxygen species, somatic copy number variation of key genes, and immune microenvironment. It was also found that the FAO group and the low-risk group had better efficacy and response to immune checkpoint blockade treatment and anti-tumour drugs. CONCLUSION: There are obvious differences in genes, chromosomes, and clinical characteristics between metabolic subgroups. The establishment of a prognostic model could predict patient prognosis and guide clinical treatment.

A novel prognostic immunoscore based on The Cancer Genome Atlas to predict overall survival in colorectal cancer patients.

Colorectal cancer (CRC) is highly prevalent worldwide. The relationship between the infiltration of immunocytes in CRC and clinical outcome has been investigated in recent years. The present study aims to construct a new prognostic signature using an immunocyte panel. Our novel prognostic immunoscore included 13 types of immunocytes, which were identified by least absolute shrinkage and selection operator (LASSO)-Cox regression. The time-dependent receiver operating characteristic (ROC) curve and Kaplan-Meier survival estimates were applied to evaluate the prognostic ability. Compared with the signature based on a single immune marker (i.e., CD8 mRNA expression and CD8+ expressing T cells), the novel prognostic immunoscore possessed better specificity and sensitivity of prognosis (area under the curves (AUCs) are 0.852, 0.856, and 0.774 for 1-, 2-, and 3-year survival times, respectively). Significant differences were identified between the high and low immunoscore groups in overall survival and disease-free survival in training and validation cohorts. Combining the immunoscore with clinical information may provide a more accurate prognosis for CRC. The immunoscore can identify patients with poor outcomes in the high Tumor Mutational Burden (TMB) group, who may benefit the most from immunotherapy. The immunoscore was also closely related to two immune checkpoints (i.e., PD-L1 and PD-1, r = 0.3087 and r = 0.3341, respectively). Collectively, our study demonstrates that the novel prognostic immunoscore reported here may be useful in distinguishing different prognoses and may improve the clinical management of patients with CRC.

RNA methyltransferase NSUN2 promotes growth of hepatocellular carcinoma cells by regulating fizzy-related-1 in vitro and in vivo.

The aim of the study was to investigate the role of NSUN2 (NOP2/Sun RNA Methyltransferase Family Member 2) in hepatocellular carcinoma (HCC). The expressions of NSUN2 and FZR1 were measured. Cell viability, proliferation, and apoptosis were assessed. HCC xenograft in nude mouse model was established. Tumor weight and volume were examined. Tumor tissues were collected for immunohistochemistry (IHC). TCGA database analysis and clinical sample testing suggested that the transcript levels of NSUN2 and FZR1 were increased in HCC tissues. NSUN2 knockdown inhibited HCC cell viability and proliferation, and promoted cell apoptosis. Moreover, the effects of NSUN2 could be countered by overexpressing FZR1. In animal experiment, NSUN2 silencing suppressed tumor growth in nude mice by downregulating FZR1. In conclusion, NSUN2 has a regulatory effect on HCC cell proliferation and apoptosis. NSUN2 knockout could inhibit cellular processes in HCC and tumor growth, likely via FZR1 inhibition. This finding has not only revealed the role of NSUN2 in HCC growth, but also suggests a promising target for HCC treatment.

Cisplatin-resistant HepG2 cell-derived exosomes transfer cisplatin resistance to cisplatin-sensitive cells in HCC.

BACKGROUNDS: Cancer cell resistance to chemotherapy drugs such as Gemcitabine, Oxaliplatin, Cisplatin, Doxorubicin, and 5-fluorouracil account for the main reason of chemotherapy failure for HCC patients, especially for those with advanced HCC or metastasis patients. This emerging resistance limits the effectiveness and clinical application of these chemotherapy drugs. Previous studies reported that drug-resistant tumor cell-derived exosomes could transfer their resistance property to tumor sensitive cells in some cancer, including lung and gastric cancer. This study sought to explore whether HepG2/DDP cell-derived exosomes transmit cisplatin (DDP) resistance to HepG2 and other HCC sensitive cells, and provide considerable guidance for HCC nursing with Cisplatin DDP clinically. METHODS: The HepG2 DDP-resistant cell line (HepG2/DDP) was established, and the exosomes from both HepG2/DDP and HepG2 cells were isolated and named ES-2, ES-1, respectively. HepG2 or SMMC-7721 or Huh7 cells were treated with DDP or DDP + ES-2, and HepG2/DDP cells were treated with ES-1. Then, the activation of drug resistance via HepG2/DDP exosomes transfer to HepG2, SMMC-7721 and Huh7 cells were assessed by cell viability assay and ROS formation. Moreover, the relative expression of P-glycoprotein (P-gp) was measured by western blot analysis. RESULTS: HepG2/DDP cell-derived exosomes were successfully isolated from cisplatin-resistant HepG2 cells, and named ES-2. Cell viability of HepG2 or SMMC-7721 or Huh7 cells treated with DDP + ES-2 was enhanced compared with that of DDP treatment group. Also, the concentration of ROS generated in cells under DDP or DDP + ES-2 treatment was strongly increased compared with that of control, although the concentration of ROS was clearly smaller in DDP + ES-2 treatment group compared with DDP treatment. At the same time, the expression of P-gp was upregulated on the ES-2 surface. CONCLUSION: The results mentioned above clarified that HepG2/DDP cell-derived exosomes conferred cisplatin resistance to HepG2 and other HCC cell lines, and provided a new significance for improving the effectiveness of DDP in treating HCC.

ß-arrestin2 Inhibits Apoptosis and Liver Inflamation Induced by Ischemia-reperfusion in Mice via AKT and TLR4 Pathway.

BACKGROUND: Liver ischemia and reperfusion (I/R) is a common but severe clinical problem. Previous studies have revealed that the expression level of ß-arrestin2 affects serum deprivation (SD)-induced cell apoptosis and was involved in lipopolysaccharide (LPS) stimulated TLR4 signaling pathway. However, little is known about ß-arrestin2 in liver apoptosis and immune response induced by I/R. METHODS: A non-lethal model of segmental (70%) hepatic ischemia was utilized. Histology examination, cell apoptosis and cytokine levels were measured using H&E staining, TUNEL assay, and ELISA, respectively. Apoptosis-related protein and gene level of cytokines were respectively detected using Western blot and Real-time PCR. RESULTS: Our data showed that knockout (KO) of ß-arrestin2 gene significantly deteriorated the injury of liver caused by I/R according to liver histology, higher serum liver enzyme, and increased level of cell apoptosis. ß-arrestin2 KO could result in increased level of apoptosis related protein and decreased level of Akt phosphorylation. Furthermore, decreased levels of Bcl-2 and Bad phosphorylation, but increased level of Bax were found in ß-arrestin2 KO group. In addition, the levels of p-ERK1/2, p-p38MAPKs, and p-NF-κB in ß-arrestin2 KO group were significantly higher than that in WT group. CONCLUSIONS: ß-arrestin2 protected liver from I/R injury and this effect may be due to the regulating of Akt pathway, Bcl-2/Bax ratio, MAPKs and NF-κB pathway.

EXO1 overexpression is associated with poor prognosis of hepatocellular carcinoma patients.

The roles of exonuclease 1 (EXO1) in hepatocellular carcinoma (HCC) tumorigenesis and progression remain unclear. This study aimed to assess the prognostic value and therapeutic potential of EXO1 in HCC. Exo1 gene copy numbers were obtained from three Oncomine microarray datasets (n = 447). EXO1 mRNA expression was validated by semi-quantitative PCR and QuantiGene® 2.0 assays. Cell growth curve and colony formation were performed to asses the cell proliferation. Clonogenic assay, flow cytometry, and immunofluorescence were adopted to acess the effects of EXO1 knockdown and radiation on cell survival, cell cycle distribution and DNA repair. Western blots were performed to reveal the related mechanism. A significant copy number variation (CNV) of the Exo1 gene was found in HCC specimens in three separate sets of published microarray data. In the 143 cases treated by our team, EXO1 expression levels were elevated (86.71%, 124/143). In addition, EXO1 overexpression was correlated with larger tumor size (P = 0.002), increased lymph node metastasis (P=0.033) and lower Edmondson grade (P = 0.018). High EXO1 expression unfavorably affected overall survival (OS) (P = 0.009). Both univariate and multivariate Cox regression analyses identified EXO1 as an independent predictor of OS (univariate, P = 0.012; multivariate, P = 0.039). Silencing of EXO1 in vitro reduced cell proliferation. EXO1 knockdown further suppressed clonogenic cell survival, abrogated radiation-induced G2/M phase arrest, and enhanced γ-H2AX foci after exposure to irradiation. The accumulation of ataxiatelangiectasia mutated (ATM) might partially regulate the EXO1 related radiosensitivity. In summary, EXO1 could be a promising prognostic marker, with a potential therapeutic value in HCC.

High expression of piwi-like RNA-mediated gene silencing 1 is associated with poor prognosis via regulating transforming growth factor-ß receptors and cyclin-dependent kinases in breast cancer.

Previous studies have demonstrated that abnormal expression levels of PIWI may serve a crucial role in tumorigenesis. However, the pathological role and its association with prognosis remains to be fully elucidated. In the present study, the expression levels of piwi­like RNA­mediated gene silencing 1 (HIWI) and piwi­like RNA­mediated gene silencing 2 (HILI) in breast cancer tissues were reported to be high. The high expression levels of HIWI are correlated with poor prognosis in detected patients. In addition, by overexpression and interference, it was demonstrated that HIWI promotes the activity of breast cancer cells while depression of HIWI may induce apoptosis of breast cancer cells. It was additionally identified that suppression of HIWI may arrest the cells at the G2/M stage. The expression levels of transforming growth factor­ß receptor (TßR)I, TßRII, cyclin­dependent kinase (CDK)4, CDK6 and CDK8 were observed to be regulated by HIWI, which indicated a novel mechanism of HIWI in the regulation of breast cancer progression. The present study provides novel insight into the HIWI expression in breast cancer, providing a potential biomarker for assessment of prognosis and therapy of breast cancer.

Epithelial but not stromal expression of collagen alpha-1(III) is a diagnostic and prognostic indicator of colorectal carcinoma.

Colorectal cancer (CRC) is the third most common cancer in males and the second in females worldwide with very poor prognosis. Collagen alpha-1(III) (COL3A1) gene, encoding an extracellular matrix protein, is upregulated in human cancers. Here, we revealed that COL3A1 was increased in CRC by analysis of five Oncomine gene expression datasets (n = 496). Immunohistochemistry analysis of a tissue microarray (n = 90) demonstrated that cancer epithelial but not stromal COL3A1 was significantly upregulated comparing with the normal counterparts. High COL3A1 mRNA and/or protein expression was accompanied with high stage, T stage, Dukes stage, grade and older age, as well as smoking and recurrence status. Upregulated COL3A1 predicted poor overall (p = 0.003) and disease-free (p = 0.025) survival. Increased epithelial but not stromal COL3A1 protein predicted worse outcome (p = 0.03). Older patients (age>65) with high COL3A1 had worse survival than younger (age≤65) with high COL3A1. Plasma COL3A1 was increased in CRC patients (n = 86) by 5.4 fold comparing with healthy individuals, enteritis and polyps patients. Plasma COL3A1 had an area under curve (AUC) of 0.92 and the best sensitivity/specificity of 98.8%/69.1%. While plasma CEA had a poorer prediction power (AUC = 0.791, sensitivity/selectivity = 70.2%/73.0%). Older patients (age≥60) had higher plasma COL3A1 than younger patients. The epithelial COL3A1 protein had an AUC of 0.975 and the best sensitivity/specificity of 95.2%/91.1%. Silencing of COL3A1 suppressed CRC cell proliferation in in vitro MTT assay and in in vivo Zebra fish xenograft model by downregulation of PI3K/AKT and WNT signaling. COL3A1 was a novel diagnosis and prognosis marker of CRC.