Endoglin Modulates TGFβR2 Induced VEGF and Proinflammatory Cytokine Axis Mediated Angiogenesis in Prolonged DEHP-Exposed Breast Cancer Cells.

Mahendra Jadhao,Li-Fang Wang,Dhanashri Deshmukh,Eing-Mei Tsai,Chun-Lin Chen,Chien-Chih Chiu,Jeff Yi-Fu Chen,Wei-Ting Liao,Wangta Liu,Ritesh Urade,Sheng-Kai Hsu

doi:10.3390/biomedicines10020417

Abstract

Angiogenesis is the process of vascular network development and plays a crucial role in cancer growth, progression, and metastasis. Phthalates are a class of environmental pollutants that have detrimental effects on human health and are reported to increase cancer risk. However, the interplay between phthalate exposure and angiogenesis has not been investigated thoroughly. In this study, we investigated the effect of prolonged di (2-ethylhexyl) phthalate (DEHP) treatment on the angiogenic potential of triple-negative breast cancer. MDA-MB-231 cells were exposed to physiological concentrations of DEHP for more than three months. Prolonged DEHP exposure induced angiogenesis in breast cancer cells. Endoglin (ENG)/CD105 is a membrane glycoprotein and an auxiliary receptor of the TGFβ receptor complex. In endothelial cells, ENG is highly expressed and it is a prerequisite for developmental angiogenesis. A literature review highlights endoglin as a well-known mesenchymal stem cell marker responsible for vascular development and angiogenesis. NGS analysis showed that endoglin overexpression in DEHP-exposed MDA-MB-231 cells correlated with tumor development and growth. An in vivo zebrafish xenograft assay showed that VEGFA induced sprouting of the subintestinal vein (SIV) in embryos injected with DEHP-exposed cells. Endoglin knockdown reduced SIV sprouting and VEGFA expression in zebrafish embryos. An in vitro HUVEC tube formation assay showed that endoglin depletion reversed DEHP-induced VEGF-mediated HUVEC tube formation in coculture. DEHP-induced endoglin activated TGFβ/SMAD3/VEGF and MAPK/p38 signaling in MDA-MB-231 cells. A cytokine angiogenesis antibody array showed induced expression of the inflammatory cytokines IL1α, IL1β, IL6, and IL8, along with GMCSF and VEGF. Endoglin knockdown reversed DEHP-induced activation of the TGFβ/SMAD3/VEGF signaling axis, MAPK/p38 signaling, and cytokine regulation, limiting angiogenesis potential both in vivo and in vitro. Targeting endoglin might serve as a potential alternative treatment to control angiogenesis, leading to metastasis and limiting cancer progression.

Highlights

In the growth and progression of breast cancer, new blood vessel generation from preexisting vessels is pivotal and is known as neovascularization
We demonstrated the detailed mechanism of endoglin-mediated regulation of tumor growth factor-β (TGFβ), Mitogen-activated protein kinase (MAPK)/p38 signaling, and cytokines controlling angiogenesis in prolonged di (2-ethylhexyl) phthalate (DEHP)-exposed MDA-MB-231 cells in vitro and in vivo
TGFβ showingshowing a positive correlawith DEHP-treated MDA-MB-231 cells. (B) Blue–Pink O’ gram showing core tion with DEHP-treated MDA-MB-231 cells. (B) Blue–Pink O’ gram showing core enrichment of individual genes in the TGFβ signaling gene set. (C–F) Quantitative Polymerase Chain Reaction (qPCR) analysis showing the mRNA expression of endoglin, TGFβRII, SMAD3, and vascular endothelial growth factor (VEGF)

Summary

Introduction

In the growth and progression of breast cancer, new blood vessel generation from preexisting vessels is pivotal and is known as neovascularization. Angiogenic cues or ischemia increase endothelial permeability, giving rise to matrix metalloproteins for extracellular matrix degradation and relieving pericyte-EC contact [2] This provides space and a chance for the adjacent cells to influx fluids and/or macromolecules due to the absence of cell-to-cell contact. Endothelial cells tend to proliferate and migrate toward promigratory phenomena (such as VEGF) to reach their final destination, where they undergo morphogenesis to form a lumen and branches [3]. Several factors, such as VEGF, thrombin, and sphingosine 1 phosphate, control EC permeability and lead to reversible loss of junctional integrity [4]. Activation of the VEGF receptor by its ligand leads to activation of a number of downstream signaling pathways, such as PI3K, MAPK, and PLCγ, which mostly occur during angiogenesis [5]

Methods

Results

Discussion

Conclusion