[Construction and Evaluation of a Prognostic Risk Prediction Model of Pancreatic Ductal Adenocarcinoma Based on Immune-Related Genes].

Objective To construct a risk prediction model by integrating the molecular subtypes of pancreatic ductal adenocarcinoma (PDAC) and immune-related genes.Methods With GSE71729 data set (n=145) as the training set,the differentially expressed genes and differential immune-related genes between the squamous and non-squamous subtypes of PDAC were integrated to construct a regulatory network,on the basis of which five immune marker genes regulating the squamous subtype were screened out.An integrated immune score (IIS) model was constructed based on patient survival information and immune marker genes to predict the clinical prognosis of PDAC patients,and its predictive performance was tested with 5 validation sets (n=758).Results PDAC patients were assigned into high risk and low risk groups according to the IIS.In both training and validation sets,the overall survival of patients in the high risk group was shorter than that in the low risk group (both P<0.001).The multivariable Cox regression showed that IIS was an independent prognostic factor for PDAC (HR=2.16,95%CI=1.50-3.10,P<0.001).Conclusion IIS can be used for risk stratification of PDAC patients and may become a potential prognostic marker for PDAC.

A novel sphingolipid metabolism-related long noncoding RNA signature predicts the prognosis, immune landscape and therapeutic response in pancreatic adenocarcinoma.

Sphingolipid metabolism affects prognosis and resistance to immunotherapy in patients with cancer and is an emerging target in cancer therapy with promising diagnostic and prognostic value. Long noncoding ribonucleic acids (lncRNAs) broadly regulate tumour-associated metabolic reprogramming. However, the potential of sphingolipid metabolism-related lncRNAs in pancreatic adenocarcinoma (PAAD) is poorly understood. In this study, coexpression algorithms were employed to identify sphingolipid metabolism-related lncRNAs. The least absolute shrinkage and selection operator (LASSO) algorithm was used to develop a sphingolipid metabolism-related lncRNA signature (SMLs). The prognostic predictive stability of the SMLs was validated using Kaplan-Meier. Univariate and multivariate Cox, receiver operating characteristic (ROC) and clinical stratification analyses were used to comprehensively assess the SMLs. Gene set variation analysis (GSVE), gene ontology (GO) and tumor mutation burden (TMB) analysis explored the potential mechanisms. Additionally, single sample gene set enrichment analysis (ssGSEA), ESTIMATE, immune checkpoints and drug sensitivity analysis were used to investigate the potential predictive function of the SMLs. Finally, an SMLs-based consensus clustering algorithm was utilized to differentiate patients and determine the suitable population for immunotherapy. The results showed that the SMLs consists of seven sphingolipid metabolism-related lncRNAs, which can well determine the clinical outcome of individuals with PAAD, with high stability and general applicability. In addition, the SMLs-based consensus clustering algorithm divided the TCGA-PAAD cohort into two clusters, with Cluster 1 showing better survival than Cluster 2. Additionally, Cluster 1 had a higher level of immune cell infiltration than Cluster 2, which combined with the higher levels of immune checkpoints in Cluster 1 suggests that Cluster 1 is more consistent with an immune 'hot tumor' profile and may respond better to immune checkpoint inhibitors (ICIs). This study offers new insights regarding the potential role of sphingolipid metabolism-related lncRNAs as biomarkers in PAAD. The constructed SMLs and the SMLs-based clustering are valuable tools for predicting clinical outcomes in PAAD and provide a basis for clinical selection of individualized treatments.

G protein subunit gamma 5 promotes the proliferation, metastasis and glycolysis of breast cancer cells through the Wnt/ß-catenin pathway.

GNG5 is suggested to exert a critical effect on tumor development in human beings; however, its function and related mechanism within breast cancer (BC) are still unclear. In this regard, the present work focused on identifying and evaluating GNG5's function and revealing its possible molecular mechanism. Subcutaneous tumorigenesis model of nude mice and in-vitro cell model was established. The relationship between GNG5 expression and BC was studied through knockdown and overexpression experiments. The proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of liver cancer cell lines overexpressing or silencing GNG5 were detected. Furthermore, the pathway mechanism of GNG5 was evaluated at the molecular level and was performed to further verify the possible targets and mechanisms of action. In comparison with that in normal tissue, GNG5 level within BC tissue was higher. In addition, GNG5 overexpression stimulated BC cell proliferation, invasion, migration and EMT. BC cells with reduced GNG5 expression exhibited significant decreases in glucose uptake, lactate levels, and ATP concentrations. In addition, GNG5 knockdown inhibited Wnt/ß-catenin signaling. This study indicates that GNG5 may generate a vital function in BC. The results of the current work demonstrated GNG5's effect on BC pathological process, also providing a reference for developing new targeted therapies for BC.

Comprehensive Analysis of Somatic Reversion Mutations in Homologous Recombination Repair (HRR) Genes in A Large Cohort of Chinese Pan-cancer Patients.

Purpose: Mutations leading to homologous recombination deficiency (HRD) increase the tumor sensitivity to platinum-based chemotherapy and PARP inhibitors. However, reversion mutations often develop conferring acquired drug resistance. There is still a lack of comprehensive investigation on HRR reversion mutations in large pan-cancer cohorts, especially in the Eastern Asian population. This study aims to characterize reversion mutations in homologous recombination repair (HRR)-related genes in a large cohort of Chinese pan-cancer patients. Methods: Sequencing data from 23,375 patients across over 17 cancer types were retrospectively analyzed for pathogenic/likely pathogenic (P/LP) germline mutations in 15 HRR genes. Somatic mutations detected in tumor or circulating cell-free DNA predicted to restore the open reading frame of the deleterious allele were subsequently identified as reversion mutations. Results: 654 cases out of 23,375 (2.8%) unselected pan-cancer patients were identified with HRR germline mutations. Secondary somatic mutations were further analyzed in their matched tumor/plasma samples. The overall frequency of reversion mutation was 1.7% (11/654). The reversion mutations occurred only in 3 out of the 15 HRR genes: BRCA1 (3.8%), BRCA2 (3.5%) and PALB2 (2.0%) from 11 patients (6 breast cancers, 1 ovarian cancer, 1 pancreatic cancer, 1 lung cancer and 2 breast and ovarian dual cancers). We identified total 25 reversion events (BRCA1, n=9; BRCA2, n=8; PALB2, n=8), including 12 pure deletions, 10 missense single nucleotide variants, 2 insertions and 1 splice site mutation. Besides, we detected microhomology length >1bp in seven out of the reversion deletions (58.3%), suggestive of microhomology-mediated end-joining (MMEJ) repair signature. Intriguingly, a positive correlation (r=0.85, p=0.001) between the length of deletion and the microhomology length was also observed. We obtained disease courses from 6/11 patients with reversion events. Four acquired reversions after the failure of the PARP inhibitor treatment. Two patients had somatic reversion mutations identified after progressing on platinum-based treatment. Conclusion: This study comprehensively depicts the prevalence and characteristics of HRR reversion mutation of germline mutations in an unselected Chinese pan-cancer cohort. The reversion mutations predominantly occurred in BRCA1, BRCA2 and PALB2. The results revealed that reversion mutations frequently occurred after resistance to platinum-based chemotherapy and/or PARP inhibitor. Our study provides insight into the underlying mechanism of drug resistance in HRD tumors and suggests that monitoring HRR mutation status along the disease course could be beneficial especially for informing resistance mechanisms and guiding subsequent therapies.

miRNA-144 targeting DNAJC3-AS1 reverses the resistance of the breast cancer cell line Michigan Cancer Foundation-7 to doxorubicin.

This study investigated the role of miRNA-144 (miR-144) targeting of the long noncoding DNAJC3-AS1 in regulating breast cancer chemosensitivity. Real-time quantitative polymerase chain reaction was employed to detect the levels of miR-144 in different drug-resistant cells. MTT assays were used to measure the proliferation of cells in different treatment groups. The apoptosis rate of transfected cells was detected by flow cytometry. Western blotting was used to detect levels of DNAJC3-AS1 protein and of autophagy-related proteins. A double luciferase report experiment was performed to evaluate the targeting effect of miR-144 on DNAJC3-AS1. The level of miR-144 was significantly downregulated in MCF-7 doxorubicin-resistant cells. Upregulated expression of miR-144 increased the doxorubicin sensitivity of drug-resistant cells and the rate of apoptosis. DNAJC3-AS1 was the direct target of miR-144; overexpression of DNAJC3-AS1 significantly rescued the apoptosis induced by miR-144 and reversed the inhibition of autophagy by miR-144. Overexpression of miR-144 can reduce drug resistance in breast cancer cells by inhibiting autophagy or targeting DNAJC3-AS1 for downregulation. miR-144/DNAJC3-AS1 provide a new target for reducing drug resistance in breast cancer.

Role of the MAPK pathway in the observed bystander effect in lymphocytes co-cultured with macrophages irradiated with γ-rays or carbon ions.

AIMS: The radiation-induced bystander effect (RIBE) has potential implications in cancer risks from space particle radiation; however, the mechanisms underlying RIBE are unclear. The role of the MAPK pathway in the RIBEs of different linear energy transfer (LET) was investigated. MAIN METHODS: Human macrophage U937 cells were irradiated with γ-rays or carbon ions and then co-cultured with nonirradiated HMy2.CIR (HMy) lymphocytes for different periods. The activation of MAPK proteins and the generation of intracellular nitric oxide (NO) and reactive oxygen species (ROS) in the irradiated U937 cells were measured. Micronuclei (MN) formation in the HMy cells was applied to evaluate the bystander damage. Some U937 cells were pretreated with different MAPK inhibitors before irradiation. KEY FINDINGS: Additional MN formation was induced in the HMy cells after co-culturing with irradiated U937 cells, and the yield of this bystander MN formation was dependent on the co-culture period with γ-ray irradiation but remained high after 1h of co-culture with carbon irradiation. Further investigations disclosed that the time response of the RIBEs had a relationship with LET, where ERK played a different role from JNK and p38 in regulating RIBEs by regulating the generation of the bystander signaling factors NO and ROS. SIGNIFICANCE: The finding that the RIBE of high-LET radiation could persist for a much longer period than that of γ-rays implies that particle radiation during space flight could have a high risk of long-term harmful effects. An appropriate intervention targeting the MAPK pathway may have significant implications in reducing this risk.

Radiation enhances the invasiveness of irradiated and nonirradiated bystander hepatoma cells through a VEGF-MMP2 pathway initiated by p53.

Recent evidence has shown that irradiation can promote the invasiveness of hepatocellular carcinoma cells and have an impact on the invasive behavior of nonirradiated surrounding cancer cells, which may enhance overall tumor aggressiveness. However, the role of the TP53 tumor suppressor gene in the invasion of irradiated hepatoma cells and their nonirradiated bystanders remain largely unknown. In the present study, we found that irradiation increased the invasiveness of human hepatoma HepG2 cells, and pretreatment of the cells with SU1498 (an inhibitor of vascular endothelial growth factor receptor 2, VEGFR2) and GM6001 (an inhibitor of matrix metalloproteinases 2, MMP2) demonstrated that radiation-enhanced invasiveness is associated with the interplay between MMP2 and VEGF signaling. In addition, while radiation-induced expression and phosphorylation of p53, inhibition of p53 function with pifithrin-α or transfection of cells with p53 siRNA significantly reduced the activation of both MMP2 and VEGF and resulted in a reduction of radiation-induced invasiveness. Interestingly, we also found that the invasiveness of the nonirradiated bystander cells was also elevated after co-culturing with irradiated cells and that bystander invasive potential was regulated paracrine in a manner by MMP2 and VEGF from the irradiated cells through a p53-dependent mechanism. Taken together, our data demonstrate that radiation-induced up-regulation of p53 is responsible for the promotion of VEGF-MMP2 pathway involved in the enhancement of invasiveness of both irradiated and bystander hepatoma cells.

Dose response of micronuclei induced by combination radiation of α-particles and γ-rays in human lymphoblast cells.

Combination radiation is a real situation of both nuclear accident exposure and space radiation environment, but its biological dosimetry is still not established. This study investigated the dose-response of micronuclei (MN) induction in lymphocyte by irradiating HMy2.CIR lymphoblast cells with α-particles, γ-rays, and their combinations. Results showed that the dose-response of MN induced by γ-rays was well-fitted with the linear-quadratic model. But for α-particle irradiation, the MN induction had a biphasic phenomenon containing a low dose hypersensitivity characteristic and its dose response could be well-stimulated with a state vector model where radiation-induced bystander effect (RIBE) was involved. For the combination exposure, the dose response of MN was similar to that of α-irradiation. However, the yield of MN was closely related to the sequence of irradiations. When the cells were irradiated with α-particles at first and then γ-rays, a synergistic effect of MN induction was observed. But when the cells were irradiated with γ-rays followed by α-particles, an antagonistic effect of MN was observed in the low dose range although this combination radiation also yielded a synergistic effect at high doses. When the interval between two irradiations was extended to 4h, a cross-adaptive response against the other irradiation was induced by a low dose of γ-rays but not α-particles.

SirT1 regulates radiosensitivity of hepatoma cells differently under normoxic and hypoxic conditions.

Intratumoral hypoxic cells are more resistant to radiotherapy due to a reduction in lifespan of DNA-damaging free radicals and augmentation of post-irradiation molecular restoration. SirT1, a member of the mammalian sirtuin family, deacetylates various transcription factors to trigger cell defense and survival in response to stresses and DNA damage. In this study, we provide new evidence indicating that overexpression of SirT1 in hepatoma HepG2 cells allowed the cells to become much more resistant to irradiation under hypoxia than under normoxia. When SirT1 was knocked down in both HepG2 and SK-Hep-1 cells, the radiosensitivity was increased, especially under hypoxia. But this enhanced radiosensitivity in SirT1-deficient cells was extensively decreased by infecting cells with c-Myc siRNA. Furthermore, the expression of c-Myc protein and its acetylation were increased in the SirT1 knockdown cells and these increments under hypoxic conditions were much more notable than under normoxia. In addition, c-Myc interference significantly suppressed phosphorylated p53 protein expression after irradiation, especially under hypoxic conditions. The current findings indicate that SirT1 confers a higher radioresistance in hypoxic cells than in normoxic cells due to the decreased levels of c-Myc protein and its acetylation, and that a c-Myc-dependent radiation-induced phosphorylated p53 may be involved. SirT1 could serve as a novel target of radiation damage and thus as a potential strategy to advance the efficiency of radiotherapy in hepatoma entities.