Pan-Cancer Analysis Reveals Disulfidoptosis-Associated Genes as Promising Immunotherapeutic Targets: Insights Gained from Bulk Omics and Single-Cell Sequencing Validation.

Disulfidoptosis, a novel form of cell death, is distinct from other well-known cell death mechanisms. Consequently, a profound investigation into disulfidoptosis elucidates the fundamental mechanisms underlying tumorigenesis, presenting promising avenues for therapeutic intervention. Comprehensive analysis of disulfidoptosis-associated gene (DRG) expression in pan cancer utilized TCGA, GEO, and ICGC datasets, including survival and Cox-regression analyses for prognostic evaluation. We analyzed the association between DRG expression and both immune cell infiltration and immune-related gene expression using the ESTIMATE and TISDIB datasets. We obtained our single-cell RNA sequencing (scRNA-seq) data from the GEO repository. Subsequently, we assessed disulfidoptosis activity in various cell types. Evaluation of immune cell infiltration and biological functions was analyzed via single-sample gene set enrichment (ssGSEA) and gene set variation analysis (GSVA). For in vitro validation experiments, the results from real-time PCR (RT-qPCR) and Western blot were used to explore the expression of SLC7A11 in hepatocellular carcinoma (HCC) tissues and different cancer cell lines, while siRNA-mediated SLC7A11 knockdown effects on HCC cell proliferation and migration were examined. Expression levels of DRGs, especially SLC7A11, were significantly elevated in tumor samples compared to normal samples, which was associated with poorer outcomes. Except for SLC7A11, DRGs consistently exhibited high CNV and SNV rates, particularly in HCC. In various tumors, DRGs were negatively associated with DNA promoter methylation. TME analyses further illustrated a negative correlation of DRG expression with ImmuneScore and StromalScore and a positive correlation with tumor purity. Our analysis unveiled diverse cellular subgroups within HCC, particularly focusing on Treg cell populations, providing insights into the intricate interplay of immune activation and suppression within the tumor microenvironment (TME). These findings were further validated through RT-qPCR, Western blot analyses, and immunohistochemical analyses. Additionally, the knockdown of SLC7A11 induced a suppression of proliferation and migration in HCC cell lines. In conclusion, our comprehensive pan-cancer analysis research has demonstrated the significant prognostic and immunological role of disulfidoptosis across a spectrum of tumors, notably HCC, and identified SLC7A11 as a promising therapeutic target.

IKZF4/NONO-RAB11FIP3 axis promotes immune evasion in gastric cancer via facilitating PD-L1 endosome recycling.

As an immune checkpoint protein expressed by diverse cancer cells, programmed death ligand 1 (PD-L1) facilitates immune evasion by interacting with programmed cell death-1 (PD-1) on T cells. Despite the clinical benefits observed in various cancer types, strategies targeting PD-1/PD-L1 have demonstrated limited efficacy in gastric cancer (GC). Furthermore, the regulation of PD-L1, especially at post-translational modification levels, remains largely unknown. Therefore, it is crucial to elucidate the mechanisms governing PD-L1 expression to enhance anti-tumor immunity. In this study, we have identified that IKAROS family zinc finger 4 (IKZF4) and Non-POU domain-containing octamer-binding (NONO) synergistically regulate and enhance the expression of RAB11 family-interacting protein 3 (RAB11FIP3) in GC. The IKZF4/NONO-RAB11FIP3 axis facilitates the endosomal recycling of PD-L1, particularly on the cell membrane of GC cells. Moreover, overexpression of RAB11FIP3 mitigates the hypo-expression of PD-L1 protein resulting from IKZF4 or NONO deletion. Functionally, the silencing of RAB11FIP3 or IKZF4 promotes T cell proliferation, and enhances T-cell cytotoxicity towards GC cells in vitro, which further inhibits tumor immune evasion in mice via increasing the infiltration of CD8+ T cells into the tumor microenvironment (TME) to suppress GC progression. Our study suggests that the IKZF4/NONO-RAB11FIP3 axis promotes immune evasion by facilitating PD-L1 endosome recycling, thus presenting a potential therapeutic target for GC treatment.

Contrast-enhanced CT-based radiomic analysis for determining the response to anti-programmed death-1 therapy in esophageal squamous cell carcinoma patients: A pilot study.

BACKGROUND: In view of the fact that radiomics features have been reported as predictors of immunotherapy to various cancers, this study aimed to develop a prediction model to determine the response to anti-programmed death-1 (anti-PD-1) therapy in esophageal squamous cell carcinoma (ESCC) patients from contrast-enhanced CT (CECT) radiomics features. METHODS: Radiomic analysis of images was performed retrospectively for image samples before and after anti-PD-1 treatment, and efficacy analysis was performed for the results of two different time node evaluations. A total of 68 image samples were included in this study. Quantitative radiomic features were extracted from the images, and the least absolute shrinkage and selection operator method was applied to select radiomic features. After obtaining selected features, three classification models were used to establish a radiomics model to predict the ESCC status and efficacy of therapy. A cross-validation strategy utilizing three folds was employed to train and test the model. Performance evaluation of the model was done using the area under the curve (AUC) of receiver operating characteristic, sensitivity, specificity, and precision metric. RESULTS: Wavelet and area of gray level change (log-sigma) were the most significant radiomic features for predicting therapy efficacy. Fifteen radiomic features from the whole tumor and peritumoral regions were selected and comprised of the fusion radiomics score. A radiomics classification was developed with AUC of 0.82 and 0.884 in the before and after-therapy cohorts, respectively. CONCLUSIONS: The combined model incorporating radiomic features and clinical CECT predictors helps to predict the response to anti-PD-1therapy in patients with ESCC.

Down-Regulated CLDN10 Predicts Favorable Prognosis and Correlates With Immune Infiltration in Gastric Cancer.

Background: CLDN10, an important component of the tight junctions of epithelial cells, plays a crucial role in a variety of tumors. The effect of CLDN10 expression in gastric cancer, however, has yet to be elucidated. Methods: Differential expression of CLDN10 at the mRNA and protein levels was evaluated using Oncomine, ULCAN, HPA and TIMER2.0 databases. Real-time polymerase chain reaction (RT-PCR) was utilized to further verify the expression of CLDN10 in vitro. Correlations between CLDN10 expression and clinical outcomes of gastric cancer were explored by Kaplan-Meier Plotter. Gene set enrichment analysis (GSEA) and protein-protein interaction (PPI) were performed via LinkedOmics and GeneMANIA. The correlations between CLDN10 expression and immune cell infiltration and somatic copy number alternations (SCNA) in gastric cancer were explored by TIMER2.0 and GEPIA2.0. Results: CLDN10 expression was lower in gastric cancer compared to adjacent normal tissues, and associated with better prognosis. CLDN10 also showed significant differences at different T stages, Lauren classification, treatments and HER2 status. PPI and GSEA analysis showed that CLDN10 might be involved in signal transmission, transmembrane transport and metabolism. In some major immune cells, low expression of CLDN10 was associated with increased levels of immune cell infiltration. In addition, it was found that different SCNA status in CLDN10 might affect the level of immune cell infiltration. Furthermore, the expression of CLDN10 was significantly associated with the expression of several immune cell markers, especially B cell markers, follicular helper T cell (Tfh) markers and T cell exhaustion markers. Conclusion: Down-regulated CLDN10 was associated with better overall survival (OS) in gastric cancer. And CLDN10 may serve as a potential prognostic biomarker and correlate to immune infiltration levels in gastric cancer.

Post treatment NLR is a predictor of response to immune checkpoint inhibitor therapy in patients with esophageal squamous cell carcinoma.

BACKGROUND: In view of the fact that peripheral blood parameters have been reported as predictors of immunotherapy to various cancers, this study aimed to determine the predictors of response to anti-programmed death-1 (anti-PD-1) therapy in patients with esophageal squamous cell carcinoma (ESCC) from peripheral blood parameters. METHODS: A retrospective analysis was conducted to investigate the predictive value of peripheral blood parameters including neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), monocyte-to-lymphocyte ratio (MLR) and systemic immune-inflammation index (SII) in the response to anti-PD-1 antibody treatment. 119 ESCC patients receiving combined treatment including anti-PD-1 antibody were enrolled in this study. RESULTS: The median progression-free survival (PFS) of all ESCC patients was 3.73 months. PFS rate in ESCC patients with low NLR at 6 weeks post treatment was higher than patients with high NLR (HR = 2.097, 95% CI 0.996-4.417, P = 0.027). However, PFS rate in ESCC patients with low NLR at baseline (HR = 1.060, 95% CI 0.524-2.146, P = 0.869) or 3 weeks post treatment (HR = 1.293, 95% CI 0.628-2.663, P = 0.459) was comparable with high NLR. And no statistically different was found in PFS rate between low PLR and high PLR at baseline (HR = 0.786, 95% CI 0.389-1.589, P = 0.469), 3 weeks post treatment (HR = 0.767, 95% CI 0.379-1.552, P = 0.452) or 6 weeks post treatment (HR = 1.272, 95% CI 0.624-2.594, P = 0.488) in ESCC patients. PFS rate was also comparable between low MLR and high MLR at baseline (HR = 0.826, 95% CI 0.408-1.670, P = 0.587), 3 weeks post treatment (HR = 1.209, 95% CI 0.590-2.475, P = 0.580) or 6 weeks post treatment (HR = 1.199, 95% CI 0.586-2.454, P = 0.596). PFS rate was similar between patients with low SII and high SII at baseline (HR = 1.120, 95% CI 0.554-2.264, P = 0.749), 3 weeks post treatment (HR = 1.022, 95% CI 0.500-2.089, P = 0.951) and 6 weeks post treatment (HR = 1.759, 95% CI 0.851-3.635, P = 0.097). CONCLUSIONS: NLR at 6 weeks post treatment is a predictor of the response to anti-PD-1 treatment in patients with ESCC.

LncRNA CSMD1-1 promotes the progression of Hepatocellular Carcinoma by activating MYC signaling.

Emerging evidence suggests that long non-coding RNAs (lncRNA) play critical roles in the development and progression of diverse cancers including hepatocellular carcinoma (HCC), but the underlying molecular mechanisms of lncRNAs that are involved in hepatocarcinogenesis have not been fully explored. Methods: In this study, we profiled lncRNA expression in 127 pairs of HCC and nontumor liver tissues (a Discovery Cohort) using a custom microarray. The expression and clinical significance of lncCSMD1-1 were then validated with qRT-PCR and COX regression analysis in a Validation Cohort (n=260) and two External Validation Cohorts (n=92 and n=124, respectively). In vitro and in vivo assays were performed to explore the biological effects of lncCSMD1-1 on HCC cells. The interaction of lncCSMD1-1 with MYC was identified by RNA pull-down and RNA immunoprecipitation. The role of LncCSMD1-1 in the degradation of MYC protein was also investigated. Results: With microarray, we identified a highly upregulated lncRNA, lncCSMD1-1, which was associated with tumor progression and poor prognosis in the Discovery Cohort, and validated in another 3 HCC cohorts. Consistently, ectopic expression of lncCSMD1-1 notably promotes cell proliferation, migration, invasion, tumor growth and metastasis of HCC cells in in vitro and in vivo experiments. Gene expression profiling on HCC cells and gene sets enrichment analysis indicated that the MYC target gene set was significantly enriched in HCC cells overexpressing lncCSMD1-1, and lncCSMD1-1 was found to directly bind to MYC protein in the nucleus of HCC cells, which resulted in the elevation of MYC protein. Mechanistically, lncCSMD1-1 interacted with MYC protein to block its ubiquitin-proteasome degradation pathway, leading to activation of its downstream target genes. Conclusion: lncCSMD1-1 is upregulated in HCC and promotes progression of HCC by activating the MYC signaling pathway. These results provide the evidence that lncCSMD1-1 may serve as a novel prognostic marker and potential therapeutic target for HCC.

AMOTL1 enhances YAP1 stability and promotes YAP1-driven gastric oncogenesis.

Hippo signaling functions to limit cellular growth, but the aberrant nuclear accumulation of its downstream YAP1 leads to carcinogenesis. YAP1/TEAD complex activates the oncogenic downstream transcription, such as CTGF and c-Myc. How YAP1 is protected in the cytoplasm from ubiquitin-mediated degradation remains elusive. In this study, a member of Angiomotin (Motin) family, AMOTL1 (Angiomotin Like 1), was screened out as the only one to promote YAP1 nuclear accumulation by several clinical cohorts, which was further confirmed by the cellular functional assays. The interaction between YAP1 and AMOTL1 was suggested by co-immunoprecipitation and immunofluorescent staining. The clinical significance of the AMOTL1-YAP1-CTGF axis in gastric cancer (GC) was analyzed by multiple clinical cohorts. Moreover, the therapeutic effect of targeting the oncogenic axis was appraised by drug-sensitivity tests and xenograft-formation assays. The upregulation of AMOTL1 is associated with unfavorable clinical outcomes of GC, and knocking down AMOTL1 impairs its oncogenic properties. The cytoplasmic interaction between AMOTL1 and YAP1 protects each other from ubiquitin-mediated degradation. AMOTL1 promotes YAP1 translocation into the nuclei to activate the downstream expression, such as CTGF. Knocking down AMOTL1, YAP1, and CTGF enhances the therapeutic efficacies of the first-line anticancer drugs. Taken together, AMOTL1 plays an oncogenic role in gastric carcinogenesis through interacting with YAP1 and promoting its nuclear accumulation. A combination of AMOTL1, YAP1, and CTGF expression might serve as a surrogate of Hippo activation status. The co-activation of the AMOTL1/YAP1-CTGF axis is associated with poor clinical outcomes of GC patients, and targeting this oncogenic axis may enhance the chemotherapeutic effects.

The prognostic value of a seven-lncRNA signature in patients with esophageal squamous cell carcinoma: a lncRNA expression analysis.

BACKGROUND: Long non-coding RNAs (lncRNAs) have been reported to be prognostic biomarkers in many types of cancer. We aimed to identify a lncRNA signature that can predict the prognosis in patients with esophageal squamous cell carcinoma (ESCC). METHODS: Using a custom microarray, we retrospectively analyzed lncRNA expression profiles in 141 samples of ESCC and 81 paired non-cancer specimens from Sun Yat-Sen University Cancer Center (Guangzhou, China), which were used as a training cohort to identify a signature associated with clinical outcomes. Then we conducted quantitative RT-PCR in another 103 samples of ESCC from the same cancer center as an independent cohort to verify the signature. RESULTS: Microarray analysis showed that there were 338 lncRNAs significantly differentially expressed between ESCC and non-cancer esophagus tissues in the training cohort. From these differentially expressed lncRNAs, we found 16 lncRNAs associated with overall survival (OS) of ESCC patients using Cox regression analysis. Then a 7-lncRNA signature for predicting survival was identified from the 16 lncRNAs, which classified ESCC patients into high-risk and low-risk groups. Patients with high-risk have shorter OS (HR: 3.555, 95% CI 2.195-5.757, p < 0.001) and disease-free survival (DFS) (HR: 2.537, 95% CI 1.646-3.909, p < 0.001) when compared with patients with low-risk in the training cohort. In the independent cohort, the 7 lncRNAs were detected by qRT-PCR and used to compute risk score for the patients. The result indicates that patients with high risk also have significantly worse OS (HR = 2.662, 95% CI 1.588-4.464, p < 0.001) and DFS (HR 2.389, 95% CI 1.447-3.946, p < 0.001). The univariate and multivariate Cox regression analyses indicate that the signature is an independent factor for predicting survival of patients with ESCC. Combination of the signature and TNM staging was more powerful in predicting OS than TNM staging alone in both the training (AUC: 0.772 vs 0.681, p = 0.002) and independent cohorts (AUC: 0.772 vs 0.660, p = 0.003). CONCLUSIONS: The 7-lncRNA signature is a potential prognostic biomarker in patients with ESCC and may help in treatment decision when combined with the TNM staging system.

MicroRNA-18a promotes cancer progression through SMG1 suppression and mTOR pathway activation in nasopharyngeal carcinoma.

miR-18a has been reported to be upregulated in nasopharyngeal carcinoma (NPC) tissues by microarray assays. However, the roles and the underlying mechanisms of miR-18a in NPC remain poorly understood. Here we demonstrated by real-time RT-PCR that miR-18a expression is upregulated in NPC tissues, and positively correlated with tumor size and TNM stage. Moreover, miR-18a expression could be upregulated by NF-κB activation or Epstein-Barr virus encoded latent membrane protein 1 expression. The ectopic expression of miR-18a promoted NPC cell proliferation, migration and invasion, while the repression of miR-18a had opposite effects. Candidate genes under regulation by miR-18a were screened out through a whole-genome microarray assay, further identified by a reporter assay and verified in clinical samples. SMG1, a member of the phosphoinositide 3-kinase-related kinases family and an mTOR antagonist, was identified as functional target of miR-18a. Our results confirmed that miR-18a exerts its oncogenic role through suppression of SMG1 and activation of mTOR pathway in NPC cells. Importantly, in vivo xenograft tumor growth in nude mice was effectively inhibited by intratumor injection of miR-18a antagomir. Our data support an oncogenic role of miR-18a through a novel miR-18a/SMG1/mTOR axis and suggest that the antitumor effects of antagomir-18a may make it suitable for NPC therapy.

miR-665 expression predicts poor survival and promotes tumor metastasis by targeting NR4A3 in breast cancer.

Cancer metastasis is the main cause of death in breast cancer (BC) patients. Therefore, prediction and treatment of metastasis is critical for enhancing the survival of BC patients. In this study, we aimed to identify biomarkers that can predict metastasis of BC and elucidate the underlying mechanism of the functional involvement of such markers in metastasis. miRNA expression profile was analyzed using a custom microarray system in 422 BC tissues. The relationship between the upregulated miR-665, metastasis and survival of BC was analyzed and verified in another set of 161 BC samples. The biological function of miR-665 in BC carcinogenesis was explored with in vitro and in vivo methods. The target gene of miR-665 and its signaling cascade were also analyzed. There are 399 differentially expressed miRNAs between BC and noncancerous tissues, of which miR-665 is the most upregulated miRNA in the BC tissues compared with non-tumor breast tissues (P < 0.001). The expression of miR-665 predicts metastasis and poor survival in 422 BC patients, which is verified in another 161 BC patients and 2323 BC cases from online databases. Ectopic miR-665 expression promotes epithelial-mesenchymal transition (EMT), proliferation, migration and invasion of BC cells, and increases tumor growth and metastasis of BC in mice. Bioinformatics, luciferase assay and other methods showed that nuclear receptor subfamily 4 group A member 3 (NR4A3) is a target of miR-665 in BC. Mechanistically, we demonstrated that miR-665 promotes EMT, invasion and metastasis of BC via inhibiting NR4A3 to activate MAPK/ERK kinase (MEK) signaling pathway. Our study demonstrates that miR-665 upregulation is associated with metastasis and poor survival in BC patients, and mechanistically, miR-665 enhances progression of BC via NR4A3/MEK signaling pathway. This study provides a new potential prognostic biomarker and therapeutic target for BC patients.

Long Non-Coding RNA XLOC_006753 Promotes the Development of Multidrug Resistance in Gastric Cancer Cells Through the PI3K/AKT/mTOR Signaling Pathway.

BACKGROUND/AIMS: The development of multidrug resistance (MDR), which results in disease recurrence and metastasis, is a crucial obstacle to successful chemotherapy for patients with gastric cancer (GC). Long non-coding RNAs (lncRNAs) have been found to play various roles in cancer. This study aimed to investigate the effect of XLOC_006753 on the development of MDR in GC cells. METHODS: The expression levels of XLOC_006753 in GC patients and MDR GC cell lines (SGC-7901/5-FU and SGC-7901/DDP cell line) were assessed by qRT-PCR. Statistical analyses were conducted to determine the relationship between XLOC_006753 expression and clinical features and to assess the prognostic value of XLOC_006753 for overall survival and progression-free survival. Then, a CCK-8 assay was used to detect cell proliferation ability and chemosensitivity. Flow cytometry was used to detect cell cycle and cell apoptosis. A wound-healing assay and transwell assay were used to detect cell migration. The expression of markers for MDR, G1/S transition, epithelial-mesenchymal transition (EMT) and PI3K/ AKT/mTOR signaling pathway were examined by western blot. RESULTS: XLOC_006753 was highly expressed in GC patients and MDR GC cell lines (SGC-7901/5-FU and SGC-7901/DDP cell lines), and its high expression was positively associated with metastasis, TNM stage, tumor size, and poor survival in GC patients. Moreover, XLOC_006753 was an independent prognostic biomarker of overall survival and progression-free survival for gastric cancer patients. Knocking down XLOC_006753 in the two MDR GC cell lines significantly inhibited cell proliferation, cell viability, cell cycle G1/S transition, and migration. XLOC_006753 knockdown also promoted apoptosis. Furthermore, western blots showed that XLOC_006753 knockdown decreased some markers of MDR, G1/S transition, and EMT expression, while increasing caspase9 expression and inhibiting the PI3K/AKT/mTOR signaling pathway in SGC-7901/5-FU and SGC-7901/DDP cells. CONCLUSION: High expression of XLOC_006753 promoted the development of MDR, which was activated by the PI3K/AKT/mTOR pathway in GC cells.

MicroRNA­342­3p suppresses proliferation and invasion of nasopharyngeal carcinoma cells by directly targeting Cdc42.

Deregulated microRNAs play an important role in the development and progression of various types of cancer. In our previous study, we observed that microRNA­342­3p (miR­342­3p) was one of the most markedly downregulated microRNAs in two nasopharyngeal carcinoma (NPC) cell lines compared to non­neoplastic cells by using whole genome small RNA sequencing. In the present study, we confirmed that the expression of miR­342­3p was significantly reduced in NPC tissues compared with normal nasopharyngeal epithelial tissues. Overexpression of miR­342­3p inhibited proliferation, epithelial­mesenchymal transition (EMT), migration and invasiveness of NPC cells. In addition, we observed that Cdc42, a Rho GTPase family member involved in cell proliferation and metastasis, is a direct target of miR­342­3p. Additionally, ML141, a small­molecule inhibitor of Cdc42, efficiently suppressed the invasion of NPC cells compared with the control cells. Finally, we analyzed NPC tissues derived from 10 NPC patients and subjected them to quantitative RT­PCR and immunohistochemistry assays for concomitant determination of the expression levels of miR­342­3p and Cdc42. Our results revealed that miR­342­3p levels were significantly inversely correlated with the protein levels of its target Cdc42. The results of the present study indicated that miR­342­3p inhibited NPC tumor growth and invasion by directly targeting the Cdc42 pathway.

Low serum level of miR-485-3p predicts poor survival in patients with glioblastoma.

MicroRNAs (miRNAs) are short noncoding RNAs that play critical roles in human malignancies and can be used as biomarkers for cancer. Until now, a number of biomarkers for prognosis of glioblastoma (GBM) have been reported in tumor tissues but only a few biomarkers in circulating fluid. Using a custom microarray, we previously identified 19 differentially expressed miRNAs in serum of patients with GBM. In this study, we investigated whether 3 of the 19 miRNAs in serum could be used as prognostic biomarkers for patients with GBM. We first validated the serum levels of 3 candidate miRNAs in an independent cohort of 24 GBM patients and 12 healthy volunteers by real-time quantitative reverse transcription PCR (qRT-PCR), and then evaluated the prognostic value of these miRNAs in a total of 36 GBM patients. The results show that the serum levels of the 3 miRNAs (miR-451a, miR-485-3p and miR-4298) determined by qRT-PCR are significantly different between 24 GBM patients and 12 healthy volunteers (all P <0.05) and are in concordance with the results of microarray analysis. High serum level of miR-451a is correlated with positive tumor O(6)-methylguanine-DNA methyltransferase (MGMT) expression (P = 0.040). Survival analysis showed that low serum miR-485-3p level is associated with poor progression-free survival (PFS) (P < 0.004) and overall survival (OS) (P < 0.023). Furthermore, univariate and multivariate Cox analyses demonstrated that that serum miR-485-3p expression is a significant independent prognostic factor for PFS and OS in GBM patients. In conclusion, serum miR-485-3p level is reduced and might be a potential prognostic biomarker in GBM patients.