HPV16 E6-mediated stabilization of ErbB2 in neoplastic transformation of human cervical keratinocytes

Abstract

Whether ErbB2 receptor tyrosine kinase contributes to cervical cancer is controversial. We have examined the effects of E6 and E7 genes of human papillomaviruses type 16 (HPV-16) on ErbB2 expression in primary human cervical keratinocytes (HCK) immortalized with hTERT (HCK1T). In E6-positive cells (HCK1T-E6 and HCK1T-E6E7), ErbB2 expression levels increased with the cell density. HCK1T-E6E7 showed impaired contact inhibition and anchorage-independent growth in soft agar which were abrogated with introduction of ErbB2-specific short hairpin RNA (shRNA) or an ErbB2 specific inhibitor AG825. Furthermore, increased ErbB2 expression was also observed in HPV16 positive cervical cancer cell lines and this was diminished by introduction of HPV16E6-or E6AP-shRNA. At post-confluence cell densities, ErbB2 protein was stabilized in the presence of E6 whereas increased ErbB2 expression was not obvious with E6 mutants incapable of degrading p53. Furthermore, introduction of p53-shRNA to HCK1T resulted in increased ErbB2 protein stability, indicating possible ErbB2 regulation through p53. Finally, we showed that tumor formation of ErbB2-shRNA introduced SiHa cells were almost abolished. Taken together, these data indicate an important role of ErbB2 regulation by HPV16 E6 in oncogenic transformation of human cervical keratinocytes.

An In vitro Multistep Carcinogenesis Model for Human Cervical Cancer

“…HCK1, HCK4, HCK7, HCK8, HCK9, and HCK12 cells derived from different donors were maintained in low-calcium serum-free keratinocyte-growth medium (KGM; Epilife-KG2 KURABO Industries) unless otherwise described. HCK1T cells were established by transduction of hTERT into HCK1 cells (12). These HCK cells were transduced with HPV16 E6E7 followed by the oncogene(s) of interest.…”

“…Cells were seeded at 5 Â 10 4 cells per 35-mm dish (BD-Falcon 3046) in an appropriate medium. Colonies over 50 Am in diameter were counted after 3 wk as previously described (12).…”

“…Western blotting was conducted as described previously (12). Antibodies used were listed in the Supplementary Materials and Methods.…”

“…Thus, we chose normal human cervical keratinocytes (HCK; ref. 12) and established an in vitro model for cervical cancer by sequential transduction of defined genetic elements. The results with this model strongly indicate that only one or two genetic alterations might be sufficient to promote full transformation of HCK on a background of HPV16 E6 and E7 expression.…”

Proteomic Analysis of Normal and Cancer Cervical Cell Lines Reveals Deregulation of Cytoskeleton-associated Proteins

“…HeLa (HPV 18+), SiHa (HPV 16+) and C33A (HPV-negative) cervical cancer cell lines, were purchased from ATCC (Manassas, VA, USA) and cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) (Gibco-Invitrogen, Waltham, MA, USA) at 37˚C, 5% CO 2 as previously described (9). ΗCK1T cells were a kind offer of Tohru Kiyono (10) and were cultured as proposed (11) in Defined Keratinocyte Serum-Free Medium (SFM) (Gibco BRL, San Francisco, CA, USA) supplemented with 5 ng/ml Epidermal Growth Factor (EGF) (Gibco BRL) and 50 μg/ml of Bovine Pituitary Extract (BPE) (Gibco BRL). When the cells reached a concentration of 106 cells per ml, they were trypsinized and harvested, and the pellets were washed in Phosphate Buffered Saline (PBS) 3 times.…”