Expression of human papillomavirus type 16 E6 and E7 oncoproteins in primary foreskin keratinocytes is sufficient to alter the expression of angiogenic factors

Abstract

Human papillomavirus (HPV) 16 is involved in causing cervical cancer. The E6 and E7 proteins of HPV 16 immortalize human keratinocytes and this is due, at least in part, to inactivation of the tumor suppressor proteins p53 and pRB. These tumor suppressor proteins also regulate the expression of pro-and antiangiogenic factors by cells. For this reason, experiments were conducted to determine whether the expression of E6 and E7 in primary keratinocytes alters the phenotype of these cells such that they express diminished levels of antiangiogenic factors and/or increased levels of proangiogenic factors. To avoid variances in experimental observations, pools of human foreskin keratinocytes from multiple sources were infected with recombinant retrovirus expressing HPV 16 E6 and E7 or control retrovirus. Gene array analysis, RT-PCR, ELISAs and Western blotting showed that in cells expressing HPV 16 E6 and E7, expression levels of two potent angiogenesis inhibitors, thrombospondin-1 and maspin, were lower compared to controls. Additionally, major angiogenesis inducers, interleukin-8 and vascular endothelial growth factor (VEGF), were increased relative to controls. VEGF can be produced as multiple splice variants, all of which are required for the formation of patent blood vessels. The expression of HPV 16 E6 and E7 in keratinocytes augmented expression of VEGF 121, 145, 165 and 189. These observations show that HPV 16 E6 and E7 alter the phenotype of primary keratinocytes, diminishing expression of inhibitors and increasing expression of inducers of angiogenesis. This altered phenotype may permit keratinocytes infected by HPV 16 to play a role in the progression of cancer by permitting tumors to acquire a blood supply permissive of growth and spread.

Inhibition of cervical cancer cell growth in vitro and in vivo with dual shRNAs

“…A few studies have shown that HPV E6/E7 could regulate VEGF expression. [36][37][38] However, the current result indicates that VEGF may also modulate HPV E7 expression.…”

“…46 It is also known that expression of HPV 16 E6/E7 in human primary keratinocytes increased the VEGF level in these cells. 38 These reports suggest that there is a relationship between oncogene E6/E7 and angiogenesis factor VEGF with the former modulating the expression of the latter. However, in this study, we showed that silencing VEGF could also affect E7 expression.…”

Chemotherapeutic alteration of VEGF-/PDGF- and PDGF-Rα/β expression by imatinib in HPV-transformed squamous cell carcinoma compared to HPV-negative HNSCC in vitro

“…This is in contrast to oropharyngeal cancer, which is induced by repeated alcohol and tobacco exposure and is characterized by mutational loss of p16 and p53 genes, TP53 mutation and further down-regulation of CDKN2A (30)(31)(32)(33), leading to uncontrolled cellular growth in up to 80% of HNSCC. The tumor suppressor gene p53 positively regulates the expression of angiogenic inhibitors and inhibits pro-angiogenic factors (34)(35)(36).…”

Molecular targets for therapeutic interventions in human papillomavirus-related cancers (Review)

“…Among the most studied pro-angiogenic factors, VEGF is consistently overexpressed in cervical neoplastic lesions (77). Overexpression of E6 is associated to higher production of VEGF in cervical cancer (22,77). Studies using transgenic mice as well as samples of human cervical tissue suggest that both E6 and E7, besides hypoxia, can stimulate VEGF production (78,79).…”

“…In contrast, expression of the anti-angiogenic factors trombospondin (TSP)-1 and 2 were decreased in cells infected by HPV. It demonstrates that cervical cancer expression of HPV-16 integrated genes is able to contribute to a pro-angiogenic phenotype (referred as angiogenic switch) that might support tumor growth and invasion (22,74,80).…”