Exosomes from cervical cancer cells facilitate pro-angiogenic endothelial reconditioning through transfer of Hedgehog–GLI signaling components

Abstract

Background Angiogenic switch is a hallmark feature of transition from low-grade to high-grade cervical intraepithelial neoplasia (CIN) in cervical cancer progression. Therefore, early events leading to locally-advanced cervical metastatic lesions demand a greater understanding of the underlying mechanisms. Recent leads indicate the role of tumor-derived exosomes in altering the functions of endothelial cells in cervical cancer, which needs further investigation. Methods Exosomes isolated from cervical cancer cell lines were assessed for their angiogenic effect on the human umbilical vein endothelial cells (HUVEC) using tube formation and wound healing assay. The exosomal uptake by HUVEC cells was monitored using PKH-67 labelling followed by fluorescence microscopy. Alterations in Hh-GLI signaling components, PTCH1 and GLI1, in HUVEC were measured by immunoblotting. Changes in angiogenesis-related transcripts of vascular endothelial growth factor VEGF-A, VEGF-B, VEGFR2 and angiopoietin-1, angiopoietin-2, osteopontin were measured in exosome-treated HUVEC and in the exosomal RNA by RT-PCR. Results Enhanced tube formation, with an increased number of nodes and branching was observed in HUVEC’s treated with exosomes derived from different cervical cancer cell lines. HPV-positive (SiHa and HeLa) cells’ exosomes were more angiogenic. Exosome-treated HUVEC showed increased migration rate. PKH-67 labelled exosomes were found internalized in HUVEC. A high level of PTCH1 protein was detected in the exosome—treated endothelial cells. Subsequent RT-PCR analysis showed increased transcripts of Hh-GLI downstream target genes VEGF-A, VEGFR2, angiopoietin-2, and decreased expression of VEGF-B, and angiopoietin-1, suggestive of active Hh-GLI signaling. These effects were more pronounced in HUVEC’s treated with exosomes of HPV-positive cells. However, these effects were independent of tumor-derived VEGF-A as exosomal cargo lacked VEGF-A transcripts or proteins. Conclusion Overall, the data showed cervical cancer exosomes promote pro-angiogenic response in endothelial cells via upregulation of Hh-GLI signaling and modulate downstream angiogenesis-related target genes. The study provides a novel exosome-mediated mechanism potentially favoring cervical angiogenesis and thus identifies the exosomes as potential pharmacological targets against locally-advanced metastatic cervical lesions. Graphic abstract

CD63+ and MHC Class I+ Subsets of Extracellular Vesicles Produced by Wild-Type and CD47-Deficient Jurkat T Cells Have Divergent Functional Effects on Endothelial Cell Gene Expression

“…Extracellular vesicles play important roles in cell-cell communication, mediated in part by the transfer of mRNAs and miRNAs via several uptake mechanisms [ 1 , 2 , 3 ] that alter the fate of acceptor cells [ 4 , 5 , 6 ]. EVs are heterogeneous and bear various surface markers, including CD63, major histocompatibility complex class-I (MHC-I), and CD47 [ 7 , 8 , 9 , 10 ].…”

Emerging role of exosomes in cancer progression and tumor microenvironment remodeling

“…The same phenomenon occurs in cervical cancer. It has been reported that cervical cancer cell-derived exosomes can promote angiogenesis via Hedgehog-GLI signaling and enhancement of VEGF-A, VEGFR2, and angiopoietin-2 expression [ 501 ]. Loading dendritic cells with exosomes derived from HeLa cells stimulates anti-tumor immunity by increasing T-cell cytotoxicity [ 502 ].…”

Untold story of human cervical cancers: HPV-negative cervical cancer

“…Tumor cells can secrete tumor-specific exosomes such as dysfunctional miRNAs involved in cancer pathogenesis ( 66 ). As exosomes can exist in body fluids, exosomal miRNAs can be used as non-invasive diagnostic biomarkers ( 67 ) and novel therapeutic targets to treat cervical cancer.…”

“…Recently, Bhat et al . have suggested that there are quantitative and qualitative differences in exosomal cargos between HPV-positive and HPV-negative cervical cancer cells ( 67 ). Using HPV-positive and HPV-negative cell lines, they showed that HPV-positive exosomes induced relatively more migration and angiogenesis, highlighting the role of exosomes as potential pharmacological targets for metastasis of cervical cancer.…”