Abstract
BackgroundAngiogenic 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.MethodsExosomes 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.ResultsEnhanced 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.ConclusionOverall, 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
Highlights
Angiogenic switch is a hallmark feature of transition from low-grade to high-grade cervical intraepithelial neoplasia (CIN) in cervical cancer progression
We showed that cervical cancer cells possess an active Hh-Glioma-associated oncogene (GLI) signaling, and human papillomavirus (HPV) E6 played an instrumental role in its constitutive activation [36]
Materials Human cervical cancer cell lines with known HPV positivity for HPV type 16—SiHa and HPV type 18—HeLa; and HPV-negative C33a were originally procured from ATCC, human umbilical vein endothelial cells (HUVEC; #CL002) were procured from HiMedia Laboratories Pvt Ltd. (Mumbai, India)
Summary
Angiogenic switch is a hallmark feature of transition from low-grade to high-grade cervical intraepithelial neoplasia (CIN) in cervical cancer progression. Early events leading to locally-advanced cervical metastatic lesions demand a greater understanding of the underlying mechanisms. A detailed understanding of HPV biology over the last 50 years has improved our knowledge of cervical carcinogenesis, the disease is far from eliminated and ranked as the fourth topmost reported malignancy globally [3]. Invasive squamous cell carcinoma develops over a long period via initiation of precursor metastatic lesions [4], wherein productive interactions of cancer cells with their immediate environment determine the outcome. Knowledge of these interactions is very limited and requires deeper understanding for developing therapeutics for management and control of tumor progression
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
