NBS1 lactylation is required for efficient DNA repair and chemotherapy resistance.

The Warburg effect is a hallmark of cancer that refers to the preference of cancer cells to metabolize glucose anaerobically rather than aerobically1,2. This results in substantial accumulation of lacate, the end product of anaerobic glycolysis, in cancer cells3. However, how cancer metabolism affects chemotherapy response and DNA repair in general remains incompletely understood. Here we report that lactate-driven lactylation of NBS1 promotes homologous recombination (HR)-mediated DNA repair. Lactylation of NBS1 at lysine 388 (K388) is essential for MRE11-RAD50-NBS1 (MRN) complex formation and the accumulation of HR repair proteins at the sites of DNA double-strand breaks. Furthermore, we identify TIP60 as the NBS1 lysine lactyltransferase and the 'writer' of NBS1 K388 lactylation, and HDAC3 as the NBS1 de-lactylase. High levels of NBS1 K388 lactylation predict poor patient outcome of neoadjuvant chemotherapy, and lactate reduction using either genetic depletion of lactate dehydrogenase A (LDHA) or stiripentol, a lactate dehydrogenase A inhibitor used clinically for anti-epileptic treatment, inhibited NBS1 K388 lactylation, decreased DNA repair efficacy and overcame resistance to chemotherapy. In summary, our work identifies NBS1 lactylation as a critical mechanism for genome stability that contributes to chemotherapy resistance and identifies inhibition of lactate production as a promising therapeutic cancer strategy.

Pleckstrin-2 promotes tumour immune escape from NK cells by activating the MT1-MMP-MICA signalling axis in gastric cancer.

Immune escape is a major challenge in tumour immunotherapy. Pleckstrin-2(PLEK2) plays a critical role in tumour progression, but its role in immune escape in gastric cancer (GC) remains uncharacterized. RNA sequencing was used to explore the differentially expressed genes in a GC cell line that was resistant to the antitumor effect of Natural killer (NK) cells. Apoptosis and the expression of IFN-γ and TNF-α were detected by flow cytometry (FCM). PLEK2 expression was examined by Western blotting and immunohistochemistry (IHC). PLEK2 was upregulated in MGC803R cells that were resistant to the antitumor effect of NK cells. PLEK2 knockout increased the sensitivity of GC cells to NK cell killing. PLEK2 expression was negatively correlated with MICA and positively correlated with MT1-MMP expression both in vitro and in vivo. PLEK2 promoted Sp1 phosphorylation through the PI3K-AKT pathway, thereby upregulating MT1-MMP expression, which ultimately led to MICA shedding. In mouse xenograft models, PLEK2 knockout inhibited intraperitoneal metastasis of GC cells and promoted NK cell infiltration. In summary, PLEK2 suppressed NK cell immune surveillance by promoting MICA shedding, which serves as a potential therapeutic target for GC.

Confounded association between proton pump inhibitor use and risk of biliary tract cancer: Result from three cohorts.

Recent epidemiological studies suggested that proton pump inhibitor (PPI) use was associated with an increased risk of biliary tract cancer (BTC), however, confounders were not adequately controlled. Our study aimed to evaluate PPI use and subsequent risk of BTC and its subtypes in three well-established cohorts. We conducted a pooled analysis of the subjects free of cancers in UK Biobank (n = 463 643), Nurses' Health Study (NHS, n = 80 235) and NHS II (n = 95 869). Propensity score weighted Cox models were used to estimate marginal HRs of PPIs use on BTC risk, accounting for potential confounders. We documented 284 BTC cases in UK Biobank (median follow-up: 7.6 years), and 91 cases in NHS and NHS II cohorts (median follow-up: 15.8 years). In UK biobank, PPI users had a 96% higher risk of BTC compared to nonusers in crude model (HR 1.96, 95% CI 1.44-2.66), but the effect was attenuated to null after adjusting for potential confounders (HR 0.95, 95% CI 0.60-1.49). PPI use was not associated with risk of BTC in the pooled analysis of three cohorts (HR 0.93, 95% CI 0.60-1.43). We also observed no associations between PPI use with risk of intrahepatic (HR 1.00, 95% CI 0.49-2.04), extrahepatic bile duct (HR 1.09, 95% CI 0.52-2.27) and gallbladder cancers (HR 0.66, 95% CI 0.26-1.66) in UK Biobank. In summary, regular use of PPIs was not associated with the risk of BTC and its subtypes.

Identification and validation of a PD-L1-related signature from mass spectrometry in gastric cancer.

BACKGROUND: According to the guidelines, PD-L1 expression is a critical indicator for guiding immunotherapy application. According to certain studies, regardless of PD-L1 expression, immunotherapy could be advantageous for individuals with gastric cancer. Therefore, new scoring systems or biomarkers are required to enhance treatment strategies. METHODS: Mass spectrometry and machine learning were used to search for strongly related PD-L1 genes, and the NMF approach was then used to separate gastric cancer patients into two categories. Differentially expressed genes (DEGs) between the two subtypes identified in this investigation were utilized to develop the UBscore predictive model, which was verified by the Gene Expression Omnibus (GEO) database. Coimmunoprecipitation, protein expression, and natural killing (NK) cell coculture experiments were conducted to validate the findings. RESULTS: A total of 123 proteins were identified as PD-L1 interactors that are substantially enriched in the proteasome complex at the mRNA level. Using random forest, 30 UPS genes were discovered in the GSE66229 cohort, and ANAPC7 was experimentally verified as one of 123 PD-L1 interactors. Depending on the expression of PD-L1 and ANAPC7, patients were separated into two subgroups with vastly distinct immune infiltration. Low UBscore was related to increased tumor mutation burden (TMB) and microsatellite instability-high (MSI-H). In addition, chemotherapy medications were more effective in individuals with a low UBscore. Finally, we discovered that ANAPC7 might lead to the incidence of immunological escape when cocultured with NK-92 cells. CONCLUSION: According to our analysis of the PD-L1-related signature in GC, the UBscore played a crucial role in prognosis and had a strong relationship with TMB, MSI, and chemotherapeutic drug sensitivity. This research lays the groundwork for improving GC patient prognosis and treatment response.

Complex in vitro 3D models of digestive system tumors to advance precision medicine and drug testing: Progress, challenges, and trends.

Digestive system cancers account for nearly half of all cancers around the world and have a high mortality rate. Cell culture and animal models represent cornerstones of digestive cancer research. However, their ability to enable cancer precision medicine is limited. Cell culture models cannot retain the genetic and phenotypic heterogeneity of tumors and lack tumor microenvironment (TME). Patient-derived xenograft mouse models are not suitable for immune-oncology research. While humanized mouse models are time- and cost-consuming. Suitable preclinical models, which can facilitate the understanding of mechanisms of tumor progression and develop new therapeutic strategies, are in high demand. This review article summarizes the recent progress on the establishment of TME by using tumor organoid models and microfluidic systems. The main challenges regarding the translation of organoid models from bench to bedside are discussed. The integration of organoids and a microfluidic platform is the emerging trend in drug screening and precision medicine. A future prospective on this field is also provided.

Cross-Talk of Focal Adhesion-Related Gene Defines Prognosis and the Immune Microenvironment in Gastric Cancer.

Background: Focal adhesion, as the intermediary between tumor cells and extracellular matrix communication, plays a variety of roles in tumor invasion, migration, and drug resistance. However, the potential role of focal adhesion-related genes in the microenvironment, immune cell infiltration, and drug sensitivity of gastric cancer (GC) has not yet been revealed. Methods: The genetic and transcriptional perspectives of focal adhesion-related genes were systematically analyzed. From a genetic perspective, the focal adhesion index (FAI) was constructed based on 18 prognosis-related focus adhesion-related genes to evaluate the immune microenvironment and drug sensitivity. Then three prognosis-related genes were used for consistent clustering to identify GC subtypes. Finally, use FLT1, EGF, COL5A2, and M2 macrophages to develop risk signatures, and establish a nomogram together with clinicopathological characteristics. Results: Mutations in the focal adhesion-related gene affect the survival time and clinical characteristics of GC patients. FAI has been associated with a shorter survival time, immune signaling pathways, M2 macrophage infiltration, epithelial-mesenchymal transition (EMT) signaling, and diffuse type of GC. FAI recognizes ALK, cell cycle, and BMX signaling pathways inhibitors as sensitive agents for the treatment of GC. FLT1, EGF, and COL5A2 may distinguish GC subtypes. The established risk signature is of great significance to the prognostic evaluation of GC based on FLT1, EGF, and COL5A2 and M2 macrophage expression. Conclusion: The focal adhesion-related gene is a potential biomarker for the evaluation of the immune microenvironment and prognosis. This work emphasizes the potential impact of the focal adhesion pathway in GC therapy and highlights its guiding role in prognostic evaluation.

Identification of an Immune-Related Long Noncoding RNA Pairs Model to Predict Survival and Immune Features in Gastric Cancer.

Background: Gastric cancer (GC) remains one of the most malignant tumors around the world, and an accurate model that reliably predicts survival and therapeutic efficacy is urgently needed. As a novel predictor for prognosis in a variety of cancers, immune-related long noncoding RNA pairs (IRlncRNAPs) have been reported to predict tumor prognosis. Herein, we integrated an IRlncRNAPs model to predict the clinical outcome, immune features, and chemotherapeutic efficacy of GC. Methods: Based on the GC data obtained from The Cancer Genome Atlas (TCGA) database and the Immunology Database and Analysis Portal (ImmPort), differentially expressed immune-related long noncoding RNAs (DEIRlncRNAs) were identified. Least absolute shrinkage and selection operator (LASSO) regression and Cox regression analysis were used to select the most appropriate overall survival (OS)-related IRlncRNAPs to develop a prognostic signature. The riskScore of each sample was calculated by comparing the long noncoding RNA expression level in each IRlncRNAP. Based on the riskScore for each patient, GC patients were divided into high- and low-risk groups. Then, the correlation of the signature and riskScore with OS, clinical features, immune cell infiltration, immune-related gene (IRG) expression and chemotherapeutic efficacy in GC was analyzed. Results: A total of 107 DEIRlncRNAs were identified which formed 4297 IRlncRNAPs. Fifteen OS-related IRlncRNAPs were selected to develop a prognostic model. GC patients could be accurately classified into high- and low-risk groups according to the riskScore of the prognostic model. The 1-, 2-, 3-, and 5-year receiver operating characteristic (ROC) curves for the riskScore were drawn and the area under the curve (AUC) values were found to be 0.788, 0.810, 0.825, and 0.868, respectively, demonstrating a high sensitivity and accuracy of this prognostic signature. Moreover, the immune-related riskScore was an independent risk factor. Patients showed a poorer outcome within the high-risk group. In addition, the riskScore was found to be significantly correlated with the clinical features, immune infiltration status, IRG expression, and chemotherapeutic efficacy in GC. Conclusion: The prognostic model of IRlncRNAPs offers great promise in predicting the prognosis, immune infiltration status, and chemotherapeutic efficacy in GC, which might be helpful for the selection of chemo- and immuno-therapy of GC.

Identification of Stemness Characteristics Associated With the Immune Microenvironment and Prognosis in Gastric Cancer.

BACKGROUND: Gastric cancer (GC) is a highly heterogeneous disease. In recent years, the prognostic value of the mRNA expression-based stemness index (mRNAsi) across cancers has been reported. We intended to identify stemness index-associated genes (SI-genes) for clinical characteristic, gene mutation status, immune response, and tumor microenvironment evaluation as well as risk stratification and survival prediction. METHODS: The correlations between the mRNAsi and GC prognosis, clinical characteristics, gene mutation status, immune cell infiltration and tumor microenvironment were evaluated. Weighted gene correlation network analysis (WGCNA) was performed to identify SI-genes from differentially expressed genes (DEGs) in The Cancer Genome Atlas (TCGA). Single-sample gene set enrichment analysis (ssGSEA) was employed to calculate the sample SI-gene-based ssGSEA score according to the SI-genes. Then, the correlations between the ssGSEA score and GC prognosis, clinical characteristics, gene mutation status, immune cell infiltration and tumor microenvironment were analyzed. Finally, the least absolute shrinkage and selection operator (LASSO) Cox regression algorithm was used to construct a prognostic signature with prognostic SI-genes. The ssGSEA score and prognostic signature were validated using the Gene Expression Omnibus (GEO) database. RESULTS: The mRNAsi could predict overall survival (OS), clinical characteristics, the gene mutation status, immune cell infiltration, and the tumor microenvironment composition. Fourteen positive SI-genes and 178 negative SI-genes were screened out using WGCNA. The ssGSEA score, similar to the mRNAsi, was found to be closely related to OS, clinical characteristics, the gene mutation status, immune cell infiltration, and the tumor microenvironment composition. Finally, a prognostic signature based on 18 prognostic SI-genes was verified to more accurately predict GC 1-year, 3-year, and 5-year OS than traditional clinical prediction models. CONCLUSION: The ssGSEA score and prognostic signature based on 18 prognostic SI-genes are of great value for immune response evaluation, risk stratification and survival prediction in GC and suggest that stemness features are crucial drivers of GC progression.

Autophagy-related genes prognosis signature as potential predictive markers for immunotherapy in hepatocellular carcinoma.

Autophagy-related genes (ATGs) depress tumorigenesis. However, in tumor tissue, it promotes tumor progression. Here, we demonstrated that 63 ATGs were differentially expressed in normal tissues and tumor tissues of hepatocellular carcinoma (HCC), and seven prognostic-related genes were chosen to establish prognostic risk signatures. It is not just an independent prognostic factor for HCC, but also closely related to the degree of malignancy of HCC. Further, the hallmarks of PI3K-AKT-mTOR signaling was significantly enriched in the high-risk group. Moreover, AKT-pS473 and mTOR-pS2448 expression was down-regulated and correlated with patient prognosis in high-risk group. Finally, we demonstrate that the prognosis signature of ATGs is closely related to immune cell infiltration and PD-L1 expression. In conclusion, ATGs are a crucial factor in the malignant progression of HCC and will be a new prognostic marker for diagnosis and treatment. ATGs prognostic signatures are potentially useful for predicting PD-L1 therapeutic effects.

Long Noncoding RNA GM16343 Promotes IL-36ß to Regulate Tumor Microenvironment by CD8+T cells.

OBJECTIVE: To investigate the effect of long noncoding RNA GM16343 on interleukin 36ß promotion of CD8+T cells in tumor microenvironment regulation. METHODS: The differentially expressed long noncoding RNA in interleukin 36ß-stimulated mouse CD8+T cells was screened by gene chip technology, and the significant differentially expressed long noncoding RNAs were verified by real-time polymerase chain reaction. The lentiviral vector that overexpresses or knockdown GM16343 was constructed, transfected into CD8+T cells, and stimulated with interleukin 36ß, and the amount of interferon γ secreted was detected by enzyme-linked immunosorbent assay. A mouse subcutaneous xenograft model that stably express interleukin 36ß was established, and the tumor size and mouse survival time were observed by stimulation with CD8+T cells overexpression or knockdown of GM16343. RESULTS: A total of 12 long noncoding RNAs with significant differences were screened by gene chip analysis. Real-time polymerase chain reaction showed that the difference in GM16343 was larger, and the difference between the groups was observed to be the most significant. Compared to control group, CD8+T cells overexpressing GM16343 increased the secretion of interferon γ, and the tumor diameter of the mice after stimulation showed significant reduction, and the survival time showed significant prolongation. Compared to control group, the CD8+T cells after GM16343 were knocked down. The interferon γ secretion was decreased, and no significant change in tumor diameter and survival time was observed. CONCLUSION: Interleukin 36ß may enhance antitumor immune response of CD8+T cells by regulating GM16343.

Lentivirus-mediated RNA interference targeting programmed death receptor ligand 1 increases the immunologic anti-tumor effect of dendritic cell vaccination against pancreatic cancer in SCID-hu mice.

Programmed death receptor ligand 1 (PD-L1), which belongs to the B7 family, is overexpressed in a variety of human cancer types and serves a crucial role in immune escape by malignant cells. Programmed death receptor 1 (PD-1) is a specific PD-L1 receptor. PD-1/PD-L1 signaling inhibits the antitumor effects of dendritic cell (DC) immunization for tumor treatment. The aim of the present study was to determine whether inhibiting PD-L1 may increase the immunologic anti-tumor effect of dendritic cells against pancreatic cancer. In the present study, PD-L1 levels in non-cancerous and malignant tissue samples were compared, and the impact of PD-L1 downregulation on human pancreatic cancer PaTu8988 cells was determined by lentivirus-based RNA interference and DC immunotherapy. PD-L1 expression in pancreatic specimens was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry. PaTu8988 cells expressing reduced levels of PD-L1 were generated by lentivirus-based knockdown to assess the mechanism by which the inhibition of PD-L1 signaling in DC immunization affects therapeutic outcomes in pancreatic cancer-bearing SCID-hu mice. PD-L1 levels were markedly elevated in pancreatic adenocarcinoma samples compared with in non-cancerous tissue. PD-L1 silencing in pancreatic adenocarcinoma cells resulted in improved treatment outcomes of DC immunization in vitro and in vivo compared with traditional DC immunization. PD-L1 silencing enhances the antitumor response of cytotoxic T cells by increasing interferon γ production in vitro. In vivo, this method prevented tumor growth and lung metastasis, and prolonged survival in the SCID-hu model. In conclusion, the results of the present study suggested that suppressing PD-L1 in malignant cells during DC immunization may be a useful tool for immunotherapy in pancreatic adenocarcinoma.

IL-9 Exerts Antitumor Effects in Colon Cancer and Transforms the Tumor Microenvironment In Vivo.

As a newly discovered cytokine, interleukin 9 was initially considered a T-lymphocyte growth factor. Interleukin 9 affects target cells by binding to a member of the γc-family of receptors and is involved in inflammation, autoimmune diseases, and other ailments. In recent years, mounting evidence reveals that interleukin 9 exerts antitumor effects, which has attracted considerable attention. Many previous studies were performed in vivo by establishing a mouse model of melanoma. Here, interleukin 9 protein and messenger RNA expression levels were both low in colon carcinoma tissue specimens, as assessed by immunohistochemistry and quantitative real-time polymerase chain reaction. In addition, interleukin 9 expression in these samples was correlated with TNM staging, Dukes staging, lymph node metastasis, and good prognosis, but not with gender, age, tumor size, tumor differentiation, and hepatic metastasis. In vivo, by establishing a mouse subcutaneous allograft model, we found that interleukin 9 overexpression inhibited tumor growth and resulted in longer survival time. Then, antitumor immune responses were increased by interleukin 9 as demonstrated by flow cytometry. Furthermore, interleukin 9 was shown to exert antitumor effects by regulating T-cell function and killing tumor cells in the tumor microenvironment. Overall, this study revealed that interleukin 9 exerts robust antitumor effects in colon cancer and transforms the tumor microenvironment in vivo.

[Expression of interleukin-9 in colon cancer tissues and its clinical significance].

OBJECTIVE: To investigate the expression of interleukin-9 (IL-9) in colon cancer tissues and its clinical significance. METHODS: Immunohistochenmistry and qRT-PCR were used to detect the expressions of IL-9 protein and mRNA in 92 colon cancer tissues and paired adjacent normal tissues. The correlation of IL-9 expressions with the clinicopathological features and prognosis of the patients was analyzed. RESULTS: IL-9 protein and mRNA expressions were significantly higher in adjacent normal tissues than in the colon cancer tissues (P < 0.001). In colon cancer patients, IL-9 expression was significantly correlated with TNM stage (P=0.013), Ducks stage (P=0.025) and lymph node metastasis (P=0.004) but not with gender, age, tumor size, differentiation or hepatic metastasis (P > 0.05). The survival time of colon cancer patients with positive IL-9 expression was significantly longer than that of patients negative for IL-9 expression (P=0.015). CONCLUSIONS: IL-9 expression is lowered in colon cancer tissues compoved with in the adjacent normal tissues. IL-9 expression is negatively correlated with TNM staging, Ducks staging and lymph node metastasis but positively with good prognosis, suggesting its important role in the tumor microenvironment of colon cancer.