Early effects of factors released by tumor cells derived from cervical cancer on the morphology and protein profile of vascular endothelial cells

Abstract

Tumor angiogenesis plays a crucial role in cervical cancer (CC) from early stages. Research for potential biomarkers and therapeutictargets has been hampered by heterogeneity, plasticity, and molecular differences of the endothelial cells that form the tumor vasculature.This is due, in part, to the effect of factors released by tumor cells. The aim of this work was to investigate the early effects of factorsreleased by CC-derived tumor cells on the morphology and protein profile of endothelial cells. Treatment with CC-derived HeLa cellsconditioned medium (TCM) for 3 h increased the number of HMEC-1 endothelial cells with cytoplasmic processes and a more elongatedshape. Furthermore, qRT-PCR analysis revealed that HMEC-1 cells exposed to TCM decreased VE-cadherin endothelial marker mRNAand increased α-smooth muscle actin mRNA, a cancer-associated fibroblast marker. These findings could be associated with early stagesof tumor angiogenesis characterized by increased cell migration and a partial transition from endothelial to mesenchymal phenotype(EndoMT). Next, the proteome response of HMEC-1 cells was studied under these experimental conditions, performing a Label-Freequantitative (LFQ) mass spectrometry (MS) (CEQUIBIEM Proteomics Center). Proteins were identified and quantified with theProteome Discoverer software and the Uniprot database. Using the Perseus software, 9 negatively regulated proteins and 24 positivelyregulated proteins were obtained in HMEC-1 cells treated with TCM (P ≤ 0.05 and fold change > 1.5), where clusterin (CLU) presentedthe highest fold change. CLU is a glycoprotein with a key role in cellular stress response and cancer, regulating processes such as cellmigration. In addition, the classification of positively regulated proteins, according to their class, with the PANTHER bioinformaticstool, showed 2 cytoskeletal proteins, actin alpha cardiac muscle 1 (ACTC1) and tubulin beta-8 chain (TUBB8). ACTC1 can promotecell migration and modulate the length of actin tension fibers. qRT-PCR additional analysis also revealed an increased mRNA expressionof CLU and ACTC1 in HMEC-1 cells treated with TCM. These results suggest that the recently identified proteins are involved in theearly stages of biological processes leading to angiogenesis or EndoMT in CC and could be considered as potential biomarkers.