Abstract PS16-15: The influence of mechanical and chemical insult on the integrity of vascular endothelial cells and associated impact on epithelial protein lost in neoplasm (EPLIN) and potential partner protein network

Abstract

Abstract Background. Vascular endothelium is the frontline structure in the body facing constant high velocity flow and external harmful agents absorbed within the circulation. It is not fully understood how endothelial cells respond to such external insults. We have previously shown that a cytoskeleton binding protein, Epithelial Protein Lost in Neoplasm (EPLIN) is an essential protein in regulating the migration and adhesiveness of vascular endothelial cells and in doing so, contributing to the normal process of angiogenesis and its responsiveness to complex environments, thereby influencing tumour development (1). The portraited role of EPLIN in the intracellular process has led to the hypothesis that EPLIN has an important role to play during the response of endothelial cells to external hostile factors by providing a stable and coordinated mechanism. The current study explores the influence of EPLIN and its associated protein network in the endothelial cell response to physical injury and extrinsic carcinogenic agents. Method. Human primary cultured vascular endothelial cells and immortalised endothelial cells were used as cell models. The cells were subject to either physical wounding injuries or exposure to a carcinogenic chemical, namely diethystibestrol (DIET). Gene expression patterns from the endothelial cells were subject to analysis by gene microarray. The cellular response of endothelial cells was explored using proliferation and migration assays. The potential paracrine impact on cancer cells in close proximity to the endothelial cells was tested using endothelial cell conditioned media. Results. Both mechanical injury and chemical challenge (at non-toxic concentration range) resulted in mild yet statistically insignificant reductions in EPLIN expression in both endothelial models. A number of the proposed EPLIN network partners in immortalised endothelial cells were affected by mechanic injuries, most notably CTNNA1, CTNNAL1, CTNNA3, CTNNB1 and the caveolins. Chemical injury resulted in more drastic reductions of caveolin and its variant2 (CAV2) (p<0.001 for both). In the cell model, neither mechanical nor chemical insults caused significant changes in the regulatory elements of the network, namely signal-induced proliferation-associated gene 1 (SIPA1) nor its variants. Likewise, other network members closely linked to the cytoskeleton, including gamma-catenin and paxillin, were also unaffected. A similar response was seen with primary culture endothelial cells. Endothelial cells, following mechanical injury had a stimulatory effect on the growth of breast cancer cells (MDA MB-231 and BT20) due largely to a rapid rise in G2 and S phase of cell cycle and migration of breast cancer cells, in a co-culture assay. However, the effects of endothelial cells treated with DIET elicited more profound effects on the migration of cancer cells. Discussion. Our results demonstrate that vascular endothelial cells are sensitive to the external mechanical and harmful chemical damages. Such damage appears to influence breast cancer cells in a paracrine fashion. Similarly, such damage resulted in altered expression of a range of cell adhesion molecules but did not significantly influence EPLIN. EPLIN, a central element of this network may be involved in co-ordinating this response though further study is needed. Reference1 Sanders AJ, Ye L, Mason MD, et al. The impact of EPLINα (Epithelial protein lost in neoplasm) on endothelial cells, angiogenesis and tumorigenesis. Angiogenesis. 2010 Dec;13(4):317-26. Citation Format: Jianyuan Zeng, Andrew James Sanders, Tracey Amanda Martin, Wen Guo Jiang. The influence of mechanical and chemical insult on the integrity of vascular endothelial cells and associated impact on epithelial protein lost in neoplasm (EPLIN) and potential partner protein network [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-15.