Nitric oxide deficiency and endothelial–mesenchymal transition of pulmonary endothelium in the progression of 4T1 metastatic breast cancer in mice

Abstract

BackgroundMesenchymal transformation of pulmonary endothelial cells contributes to the formation of a metastatic microenvironment, but it is not known whether this precedes or follows early metastasis formation. In the present work, we characterize the development of nitric oxide (NO) deficiency and markers of endothelial–mesenchymal transition (EndMT) in the lung in relation to the progression of 4T1 metastatic breast cancer injected orthotopically in mice.MethodsNO production, endothelial nitric oxide synthase (eNOS) phosphorylation status, markers of EndMT in the lung, pulmonary endothelium permeability, and platelet activation/reactivity were analyzed in relation to the progression of 4T1 breast cancer metastasis to the lung, as well as to lung tissue remodeling, 1–5 weeks after 4T1 cancer cell inoculation in Balb/c mice.ResultsPhosphorylation of eNOS and NO production in the lungs of 4T1 breast cancer-bearing mice was compromised prior to the development of pulmonary metastasis, and was associated with overexpression of Snail transcription factor in the pulmonary endothelium. These changes developed prior to the mesenchymal phenotypic switch in the lungs evidenced by a decrease in vascular endothelial-cadherin (VE-CAD) and CD31 expression, and the increase in pulmonary endothelial permeability, phenomena which coincided with early pulmonary metastasis. Increased activation of platelets was also detected prior to the early phase of metastasis and persisted to the late phase of metastasis, as evidenced by the higher percentage of unstimulated platelets binding fibrinogen without changes in von Willebrand factor and fibrinogen binding in response to ADP stimulation.ConclusionsDecreased eNOS activity and phosphorylation resulting in a low NO production state featuring pulmonary endothelial dysfunction was an early event in breast cancer pulmonary metastasis, preceding the onset of its phenotypic switch toward a mesenchymal phenotype (EndMT) evidenced by a decrease in VE-CAD and CD31 expression. The latter coincided with development of the first metastatic nodules in the lungs. These findings suggest that early endothelial dysfunction featured by NO deficiency rather than EndMT, might represent a primary regulatory target to prevent early pulmonary metastasis.Electronic supplementary materialThe online version of this article (10.1186/s13058-018-1013-z) contains supplementary material, which is available to authorized users.

Enhanced Prevention of Breast Tumor Metastasis by Nanoparticle‐Delivered Vitamin E in Combination with Interferon‐Gamma

“…Therefore, the H&E staining images of representative sections of lungs are illustrated (Figure 5C). Typical tumor nodules were observed in the saline group, visualizing the metastasized cancer cells and their localization, as reported previously 35,36. Smaller and fewer nodules were found in lungs with IFN‐γ and NP‐TOS15 treatment, indicating the lung metastasis was inhibited to some degree.…”

Retinol-Binding Protein 4 Accelerates Metastatic Spread and Increases Impairment of Blood Flow in Mouse Mammary Gland Tumors

“…Recent studies utilizing the model of mouse mammary gland cancer 4T1, reflecting a basal-like phenotype (in human: negative for nuclear estrogen receptor (ER) α, progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), i.e., triple-negative, and positive for epidermal growth factor receptor (EGFR)) [1][2][3][4], have shown a predominant role of endothelium damage during the metastatic process of these cells [5][6][7]. For instance, it is evidenced that endothelial dysfunction in the lungs, which was assessed as decreased activity and phosphorylation of endothelial nitric oxide synthase (eNOS) resulting in a low nitric oxide (NO) production state, was an early event in breast Literature data [4] and our histopathological analyses confirmed that no cancer cells were detected in the lungs of 67NR tumor-bearing mice ( Figure S1A).…”

“…RBP4 enhances the metastatic potential of breast cancer tumors through a direct effect on cancer cells and through increased endothelial dysfunction and impairment of blood vessels within the tumor.Cancers 2020, 12, 623 2 of 21 cancer pulmonary metastasis. These processes precede the onset of a phenotypic switch in the lung endothelium toward a mesenchymal phenotype (EndMT), which is parallel to the appearance of the first pulmonary metastatic colonies [7]. Therefore, therapeutic strategies that aim to normalize endothelial dysfunction can decrease the metastatic potential of this type of breast cancer [8][9][10].Apart from its involvement in cancer development, endothelial dysfunction plays an important role in the development of cardiovascular diseases and atherosclerosis.…”

“…Cancers 2020, 12, 623 2 of 21 cancer pulmonary metastasis. These processes precede the onset of a phenotypic switch in the lung endothelium toward a mesenchymal phenotype (EndMT), which is parallel to the appearance of the first pulmonary metastatic colonies [7]. Therefore, therapeutic strategies that aim to normalize endothelial dysfunction can decrease the metastatic potential of this type of breast cancer [8][9][10].…”

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“…However, several studies have reported that NOS3 might play an anticancer role in malignant tumours. Research by Smeda et al reported that NOS3 activity and phosphorylation reduction was an early event in the lung metastasis of breast cancer, preceding the onset of the mesenchymal phenotype (EndMT) (25). NOS3 participated in the enhancement of Taxol chemosensitivity with astragaloside IV treatment in breast cancer as a downstream target of caveolin-1 (26).…”