Danfang Zhang

Background A hypoxic microenvironment is the most frequent characteristic in tumor microenvironment. Programmed death-ligand 1 (PD-L1) is an important molecule and therapeutic target that mediates the immune response of tumor cells. Previous studies have shown that hypoxia can lead to increased expression of Nucleophosmin 1 (NPM1) and PD-L1. However, the exact regulatory mechanisms of NPM1 and PD-L1 expression under hypoxic conditions are still poorly understood. Methods The relationships among hypoxia, NPM1 and PD-L1 were explored by western blotting, immunofluorescence staining, flow cytometry and chromatin immunoprecipitation-quantitativePCR(ChIP-qPCR). Animal tumor models were established to explore the effect of NPM1 expression on tumor growth. The relationships between NPM1 and breast cancer (BC) clinical features and immune infiltration were revealed by bioinformatics analysis. Results NPM1 mediates increased PD-L1 expression in the hypoxic microenvironment of BC. HIF-1α can increase the expression of NPM1 by activating the p-AKT pathway and binding to the NPM1 promoter. Increased expression of NPM1 can promote tumor growth and inhibit T cell infiltration. Bioinformatics analysis showed that the high expression of NPM1 was associated with poorer survival and immunosuppression in patients with BC. Conclusions The hypoxic microenvironment promotes PD-L1 expression via NPM1 in BC, which may be further associated with the inhibition of tumor immunity. NPM1 may serve as a potential target for modulating PD-L1 immunotherapy.

Necroptosis is a caspase-independent form of programmed cell death. Receptor interacting protein kinase 1 (RIPK1) is a key molecule in the initiation of necroptosis and the formation of the necrotic complex. Vasculogenic mimicry (VM) provides a blood supply to tumor cells that is not dependent on endothelial cells. However, the relationship between necroptosis and VM in triple-negative breast cancer (TNBC) is not fully understood. In this study, we found that RIPK1-dependent necroptosis promoted VM formation in TNBC. Knockdown of RIPK1 significantly suppressed the number of necroptotic cells and VM formation. Moreover, RIPK1 activated the p-AKT/eIF4E signaling pathway during necroptosis in TNBC. eIF4E was blocked by knockdown of RIPK1 or AKT inhibitors. Furthermore, we found that eIF4E promoted VM formation by promoting epithelial-mesenchymal transition (EMT) and the expression and activity of MMP2. In addition to its critical role in necroptosis-mediated VM, eIF4E was essential for VM formation. Knockdown of eIF4E significantly suppressed VM formation during necroptosis. Finally, through clinical significance, the results found that eIF4E expression in TNBC was positively correlated with the mesenchymal marker vimentin, the VM marker MMP2, and the necroptosis markers MLKL and AKT. In conclusion, RIPK1-dependent necroptosis promotes VM formation in TNBC. Necroptosis promotes VM formation by activating RIPK1/p-AKT/eIF4E signaling in TNBC. eIF4E promotes EMT and MMP2 expression and activity, leading to VM formation. Our study provides a rationale for necroptosis-mediated VM and also providing a potential therapeutic target for TNBC.

Ring finger protein 31 (RNF31) has been found to play an important role in tumor immunity. However, the role of RNF31 in liver hepatocellular carcinoma (LIHC) has not been reported. Therefore, we investigated the expression and prognostic value of RNF31 in patients with LIHC and explored its relationship with immune cell infiltration. The Cancer Genome Atlas liver hepatocellular carcinoma (TCGA-LIHC) dataset was downloaded to analyse the impact of RNF31 on the prognosis and immune cell infiltration of LIHC. The Tumor Immune Estimation Resource (TIMER) database was used to analyse the correlation between RNF31 and tumor immune cell infiltration in LIHC. Additionally, we analysed the relationship between RNF31 and tumor necrosis factor (TNF) as well as the interferon-gamma (IFN-γ) signaling pathway. The expression of RNF31 in LIHC was significantly higher than that in normal tissues. Increased RNF31 expression was associated with decreased overall survival (OS) and relapse-free survival (RFS). An increase in RNF31 expression was closely related to the infiltration levels of immune cells (e.g., natural killer (NK) cells, CD8 + T cells, and B cells). RNF31 was also positively correlated with the expression of immune checkpoint genes in LIHC. Moreover, RNF31 may participate in TNF and IFN-γ signaling pathways. In conclusion, RNF31 is a potentially valuable prognostic biomarker in LIHC. RNF31 is also associated with immune cell infiltration in LIHC. RNF31 may be a potential target for immunotherapy of LIHC.

Introduction: Hypoxia is an important characteristic of solid tumors. However, spatial transcriptomics (ST) of hypoxia-associated heterogeneity is not clear.Methods: This study integrated Spatial Transcriptomics (ST) with immunofluorescence to demonstrate their spatial distribution in human claudin-low breast cancer MDA-MB-231 engraft. ST spots were clustered with differentially expression genes. The data were combined with hypoxia-specific marker and angiogenesis marker-labeled serial sections to indicate the spatial distribution of hypoxia and hypoxia-inducted transcriptional profile. Moreover, marker genes, cluster-specific hypoxia genes, and their co-essential relationship were identified and mapped in every clusters. The clinicopathological association of marker genes of hypoxia-dependent spatial clusters was explored in 1904 breast cancers from METABRIC database.Results: The tumor from center to periphery were enriched into five hypoxia-dependent subgroups with differentially expressed genes, which were matched to necrosis, necrosis periphery, hypoxic tumor, adaptive survival tumor, and invasive tumor, respectively. Different subgroups demonstrated distinct hypoxia condition and spatial heterogeneity in biological behavior and signaling pathways. Cox regression analysis showed that the invasive tumor (cluster 0) and hypoxic tumor (cluster 6) score could be served as independent prognostic factors in claudin-low patients. KM analysis indicated that high invasive tumor (cluster 0) and hypoxic tumor (cluster 6) score was associated with poor prognoses of claudin-low patients. Further analysis showed that hypoxia-induced immune checkpoints, such as CD276 and NRP1, upregulation in invasive tumor to block infiltration and activation of B cells and CD8+ T cells to change tumor immune microenvironment. Discussion: This study reveals hypoxia-dependent spatial heterogeneity in claudin-low breast cancer and highlights its potential value as a predictive biomarker of clinical outcomes and immunotherapy response. The molecules found in this study also provided potential molecular mechanisms and therapeutic targets for subsequent studies.

Objective: Hypoxia is an important feature of pancreatic ductal adenocarcinoma (PDAC). Previously, we found that hypoxia promotes ENO1 expression and PDAC invasion. However, the underlying molecular mechanism was remains unclear. Methods: The relationship between ENO1 expression and clinicopathological characteristics was analyzed in 84 patients with PADC. The effects of CoCl₂-induced hypoxia and ENO1 downregulation on the apoptosis, invasion, and proliferation of PDAC cells were evaluated in vitro and in vivo. Hypoxia- and ENO1-induced gene expression was analyzed by transcriptomic sequencing. Results: The prognosis of PDAC with high ENO1 expression was poor (P < 0.05). High ENO1 expression was closely associated with histological differentiation and tumor invasion in 84 PDAC cases (P < 0.05). Hypoxia increased ENO1 expression in PDAC and promoted its migration and invasion. Apoptotic cells and the apoptosis marker caspase-3 in the CoCl₂-treated ENO1-sh group were significantly elevated (P < 0.05). Transcriptomic sequencing indicated that CoCl₂-induced PDAC cells initiated MAPK signaling. Under hypoxic conditions, PDAC cells upregulated ENO1 expression, thereby accelerating ERK phosphorylation and inhibiting apoptosis (P < 0.05). Consistent results were also observed in a PDAC-bearing mouse hindlimb ischemia model. Conclusions: Hypoxia-induced ENO1 expression promotes ERK phosphorylation and inhibits apoptosis, thus leading to PDAC survival and invasion. These results suggest that ENO1 is a potential therapeutic target for PDAC.

Background: The expression and activation of eukaryotic translation initiation factor 4E (eIF4E) is associated with cell transformation and tumor initiation, but the functional role and the mechanism whereby it drives immune cell infiltration in breast cancer (BRCA) remain uncertain. Methods: Oncomine, Timer and UALCAN were used to analyze the expression of eIF4E in various cancers. PrognoScan, Kaplan–Meier plotter, and GEPIA were utilized to analyze the prognostic value of eIF4E in select cancers. In vitro cell experiments were used to verify the role of eIF4E in promoting the progression of BRCA. ImmuCellAI and TIMER database were used to explore the relationship between eIF4E and tumor infiltrating immune cells. The expression of a macrophage marker (CD68⁺) and an M2-type marker (CD163⁺) was evaluated using immunohistochemistry in 50 invasive BRCA samples on tissue microarrays. The Human Protein Atlas (HPA) database was used to show the expression of eIF4E and related immune markers. LinkedOmics and NetworkAnalyst were used to build the signaling network. Results: Through multiple dataset mining, we found that the expression of eIF4E in BRCA was higher than that in normal tissues, and patients with increased eIF4E expression had poorer survival and a higher cumulative recurrence rate in BRCA. At the cellular level, BRCA cell migration and invasion were significantly inhibited after eIF4E expression was inhibited by siRNA. Immune infiltration analysis showed that the eIF4E expression level was significantly associated with the tumor purity and immune infiltration levels of different immune cells in BRCA. The results from immunohistochemical (IHC) staining further proved that the expression of CD68⁺ and CD163⁺ were significantly increased and correlated with poor prognosis in BRCA patients (P < 0.05). Finally, interaction network and functional enrichment analysis revealed that eIF4E was mainly involved in tumor-related pathways, including the cell adhesion molecule pathway and the JAK-STAT signaling pathway. Conclusions: Our study has demonstrated that eIF4E expression has prognostic value for BRCA patients. eIF4E may act as an essential regulator of tumor macrophage infiltration and may participate in macrophage M2 polarization.

Background: ITGA3 is a member of the integrin family, a cell surface adhesion molecule that can interact with extracellular matrix (ECM) proteins. The purpose of this study was to explore the significance of ITGA3 expression in the prognosis and clinical diagnosis of breast cancer patients.Methods: Oncomine, the Human Protein Atlas (HPA) and UALCAN were used to analyze the expression of ITGA3 in various cancers. PrognoScan, GEPIA, Kaplan–Meier plotter and Easysurv were utilized to analyze the prognosis of ITGA3 in certain cancers. Based on TCGA data, a receiver operating characteristic (ROC) curve was used to evaluate the diagnostic performance of ITGA3 expression. cBio-Portal and MethSurv were used to evaluate the genomic mechanism. LinkedOmics, NetworkAnalyst and Metascape were used to build the signaling network. TIMER is a web server for comprehensive analysis of tumor infiltrating immune cells and tumor infiltrating lymphocytes (TILs).Results: The expression of ITGA3 in normal breast tissues was greater than that in breast cancer tissues at both the mRNA and protein levels. High expression of ITGA3 was associated with better prognosis of breast cancer patients. ROC analysis indicated that ITGA3 had significant diagnostic value. Genomic analysis revealed that promoter methylation of ITGA3 leads to transcriptional silencing, which may be one of the mechanisms underlying ITGA3 downregulation in BRCA. Immune infiltration analysis showed that ITGA3 may be involved in the recruitment of immune cells.Conclusions: This study identified ITGA3 as a novel biomarker to estimate the diagnosis and prognosis of breast cancer. In addition, ITGA3 is involved in ECM regulation and immune cell infiltration.

Background The expression and activation of Eukaryotic translation initiation factor 4E (eIF4E) is related to tumor transformation and genesis, but the functional role and the mechanism whereby it drives immune infiltration in breast cancer remain uncertain. Methods Multiple databases were used for assessing the expression and clinic-pathological features of eIF4E, after which ImmuCellAI and TIMER database were used to explore the relationship between eIF4E and immune infiltration and clinicopathological information were collected from clinical cases of breast cancer. Finally, the genes co-expressed with eIF4E were identified by LinkedOmics. These genes were analyzed protein interaction network and TF-miRNA interaction network by Networkanalyst, and enriched by GO and KEGG. The key genes of immune markers were functionally annotated. Results High expression of eIF4E was associated with poor overall survival (OS) and relapse-free survival (RFS) in breast cancer samples from multiple databases. It is notable that the expression of eIF4E is positively correlated with the infiltration of CD8⁺T cells, macrophages, neutrophils and dendritic cells. The expression of eIF4E is closely related to many immune markers in breast cancer. Immunohistochemical analysis showed that the patients with high expression of eIF4E related macrophage marker (CD68⁺) and M2 type marker (CD163⁺) in tumor stroma(TS) were significantly correlated with poor prognosis (P < 0.05). High infiltration density of CD163⁺ had significant difference in TNM staging compared with those with low(P < 0.05).Functional analysis of co-expressed genes showed that they were involved in the biological process of human immune response. GO analysis showed that co-expressed immune marker genes were involved in human immune response, adaptive immune response, macrophage activation, extracellular structure organization and regulation of DNA metabolism process. KEGG enrichment showed that it was involved in inflammatory bowel disease, cell adhesion molecule pathway, JAK-STAT signal pathway, T cell receptor signal pathway and so on. Conclusions These results suggest that high expression of eIF4E regulates immune cell infiltration, especially promotes macrophage M2 polarization by JAK/STAT6 and PI3K/AKT pathway, which is associated with poor prognosis of breast cancer patients. It is a valuable prognostic biomarker for breast cancer patients, and may be a potential therapeutic target in the future.