Agents that target angiogenesis have shown limited efficacy for human triple-negative breast cancer (TNBC) in clinical trials. Along with endothelium-dependent vessels, there is also vasculogenic mimicry (VM) in the microcirculation of malignant tumors. The role of VM is not completely understood regarding anti-angiogenic treatment. In this study, human TNBC MDA-MB-231 and Hs578T and non-TNBC MCF-7 and BT474 tumor-bearing mice were treated with sunitinib, an anti-angiogenic drug, using a clinically relevant schedule. The drug was administered for one week and then discontinued. Tumor growth and invasion were observed, and the microcirculation patterns were detected with PAS/endomucin staining. Moreover, hypoxia and VM-associated proteins were evaluated with Hypoxyprobe kits and immunohistochemistry, respectively. Sunitinib significantly inhibited tumor growth in the TNBC and non-TNBC tumors. However, MDA-MB-231 and Hs578T tumors regrew and were more aggressive when the treatment was stopped. The discontinuation had no significant effect on the behavior of the non-TNBC MCF-7 and BT474 tumors. The growth of endothelium-dependent vessels in the TNBC MDA-MB-231 and Hs578T tumors were blocked by sunitinib, during which the number of VM channels significantly increased and resulted in a rebound of endothelium-dependent vessels after sunitinib discontinuation. Moreover, the VM-associated proteins VE-cadherin and Twist1 upregulated in the sunitinib-treated MDA-MB-231 and Hs578T tumors. Furthermore, the clinical significance of this upregulation was validated in 174 human breast cancers. The results from human breast cancer specimens indicated that there were more VM-positive TNBC cases than those in non-TNBC cases. HIF-1α, MMP2, VE-cadherin, and Twist1 were also expressed in a higher level in human TNBC compared with non-TNBC. In aconclusion, sunitinib promoted TNBC invasion by VM. The VM status could be helpful to predict the efficacy of anti-angiogenic therapy in patients with TNBC.

Cancer stem-like cells directly participate in vasculogenic mimicry channels in triple-negative breast cancer

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.

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.

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.

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.