Vascular endothelial growth factor (VEGF) up-regulates epidermal growth factor receptor (EGF-R) in cervical cancer in vitro: this action is mediated through HPV-E6 in HPV-positive cancers

Abstract

Objectives Epidermal Growth Factor Receptor (EGF-R) up-regulation in cervical cancer cells leads to an increase in cell proliferative Insulin-like Growth Factor II (IGF-II) and Vascular Endothelial Growth Factor (VEGF) and a decrease of the anti-proliferative IGF-binding protein-3 (IGF-BP3). The objectives for this study are: (a) to find if VEGF, in turn, up-regulates EGF-R and down-regulates IGF-BP3; (b) to determine if human papilloma virus (HPV-E6) mediates this action of VEGF in HPV-positive cells; and (c) to verify if these effects are reflected in changes in cell proliferation Methods We used HPV-positive HeLa (Black), ME-180 and CaSki (Caucasian) and HPV-negative HT-3 (Caucasian) cell lines. (a) Levels of HPV-E6 in the HPV-positive cells were enumerated after treating the cells for 24 h with 20 ng/ml of VEGF using our semi-quantitative immunofluorescent antibody assay. (b) Cellular levels of EGF-R, HPV-E6, IGF-II and IGF-BP3 were enumerated in ME-180 and CaSki cells incubated for 24 h with 5, 10 and 20 ng/ml of VEGF. (c) HPV-negative HT-3 and HPV-positive ME-180 and CaSki cells were incubated with 20 ng/ml VEGF alone or in combination with antibodies to HPV-E6 and EGF-R. HPV-E6 (measured only in HPV-positive cells), EGF-R, IGF-II and IGF-BP3 levels were measured. (d) Cell proliferation was determined using cell proliferation Bradykinine-U colorimetric assay, in HT-3, HeLa and ME-180 cell lines in the presence of VEGF alone and with HPV-E6 antibodies. Results (a) In all the HPV-positive cell lines, 20 ng/ml VEGF significantly increased (30-50%; P < 0.0001) the HPV-E6. (b) In the ME-180 and CaSki cells, VEGF treatment up-regulated EGF-R, IGF-II and HPV-E6 and down-regulated IGF-BP3 in a dose-dependent manner ( P < 0.001). (c) These effects of VEGF were eliminated when the HPV-positive cells were co-incubated with antibodies to HPV-E6 or EGF-R. In the HPV-negative HT-3 cells, VEGF decreased IGF-BP3 while increasing EGF-R and IGF-II levels. Antibodies to EGF-R eliminated these effects ( P < 0.0001). (d) Treatment with VEGF resulted in increased cell proliferation in HT-3, HeLa and ME-180 cells; co-incubation with HPV-E6 antibodies abrogated this effect only in the HPV-positive cells. Conclusions In cervical cancer, VEGF up-regulates EGF-R and down-regulates IGF-BP3, thus amplifying the cell proliferative activity of EGF-R. This action of VEGF seems to be mediated, directly through EGF-R or indirectly through HPV-E6 in the HPV-positive cancers, while EGF-R up-regulation appears to play a major role in the HPV-negative cervical cancers.