The human papillomavirus E6 oncogene dysregulates the cell cycle and contributes to cervical carcinogenesis through two independent activities

Abstract

Cervical cancer is a leading cause of death due to cancer among women worldwide. Using transgenic mice to dissect the contributions of the human papillomavirus (HPV) 16 E6 and E7 oncogenes in cervical cancer, E7 was identified previously to be the dominant oncogene. Specifically, when treated with exogenous estrogen for 6 months, E7 transgenic mice developed cancer throughout the reproductive tract, but E6 transgenic mice did not. E6 contributed to carcinogenesis of the reproductive tract, as E6/E7 double transgenic mice treated for 6 months with estrogen developed larger cancers than E7 transgenic mice. In the current study, we investigated whether the E6 oncogene alone could cooperate with estrogen to induce cervical cancer after an extended estrogen treatment period of 9 months. We found that the E6 oncogene synergizes with estrogen to induce cervical cancer after 9 months, indicating that E6 has a weaker but detectable oncogenic potential in the reproductive tract compared with the E7 oncogene. Using transgenic mice that express mutant forms of HPV16 E6, we determined that the interactions of E6 with cellular alpha-helix and PDZ partners correlate with its ability to induce cervical carcinogenesis. In analyzing the tumors arising in E6 transgenic mice, we learned that E6 induces expression of the E2F-responsive genes, Mcm7 and cyclin E, in the absence of the E7 oncogene. E6 also prevented the expression of p16 in tumors of the reproductive tract through a mechanism mediated by the interaction of E6 with alpha-helix partners.

Figures

Figure 1

Comparison of E6 expression in K14E6 transgenic mice and human cervical cancer cell lines. A, levels of E6 in HPV-positive and HPV-negative cervical cell lines. Immunoblots probed with antibodies specific for either E6 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The GAPDH-specific immunoblot was done to confirm equivalence in loading and was done for all experiments displayed in this figure, although shown only for the top-most one. For each sample, 200 µg of total soluble protein were analyzed. The top blot was loaded with samples from HPV-negative C33A, HPV18-positive HeLa, and HPV16-positive SiHa and Caski human cervical cancer–derived cell lines. The second blot was loaded with samples from various clonal populations of W12E (20850 and 20963) and W12I (20861, 20822, 20862, and 201402) cell lines derived from a HPV16-positive CINI lesion. W12E clones harbor the viral genome in the extrachromosomal state. W12I clones harbor the genome in a chromosomally integrated state. B, levels of E6 in the dorsal skin of K14E6WT and K14E6mutant transgenic mice at age postnatal day 9 or 10. In this immunoblot, 200 µg (top) or 250 µg (bottom) of total cellular protein from each mouse tissue sample and 100 or 125 µg of SiHa and Caski extracts were analyzed. Extracts from different animals (A and B) of the same genotype were loaded to assess reproducibility of findings. Bottom, levels of E6 protein in E6 homozygous transgenic mice in both skin and ear. C, levels of E6 in the reproductive tract of K14E6WT and K14E6mutant transgenic mice. Top, 200 µg of total cellular protein from each mouse tissue sample were analyzed. As above, extracts from different animals (A, B, and C) of the same genotype were loaded to assess reproducibility of findings. In the bottom blot assessing relative amounts of E6 protein in K14E6WT and K14E6Δ146–151 mice, the amount of protein loaded is indicated in each lane. Mice were treated with estrogen to synchronize them in estrus, thereby eliminating variability in cervical epithelial thickness.

Figure 2

Characterization of reproductive tumors and the proliferative index of the cervix. A, comparison of tumor sizes between K14E6WT, K14E6I128T, and K14E6Δ146–151 transgenic mice. B, comparison of tumor sizes between K14E7WT, K14E6WT/K14E7WT, and K14E6mutant/K14E7WT transgenic mice. C, classification of reproductive tumors by location. Middle, a cartoon representation of the murine reproductive tract and identifies the approximate borders for determining tumor location used in histopathologic diagnosis. Left (for K14E6WT) and right (for K14E6WT/K14E7WT), the breakdown of total percentage of tumor development by location (top) and the percentage of total area these tumors encompassed (bottom). In K14E6I128T and K14E6Δ146–151, the percentage of tumors arising in the vagina was 0% and 4%, respectively (data not shown). In K14E6I128T/K14E7WT and K14E6Δ146–151/K14E7WT transgenic mice, the tumors that developed in the vagina were 45% and 38%, respectively (data not shown). D, quantification of epithelial hyperplasia in the cervix. The average percentage of basal and suprabasal BrdUrd-positive cells was obtained from eight (×40) microscopic fields per mouse. An average of at least three mice per genotype were used to calculate the percentage.

Figure 3

Evaluation of E2F-responsive gene expression in the estrogen-treated epithelium and tumors from the reproductive tract. Columns 1 and 2, MCM7 staining (brown staining nuclei), which is up-regulated in singly or double transgenic reproductive epithelia and tumors, whereas MCM7 expression is restricted to the basal and parabasal layers of nontransgenic epithelium. Columns 3 and 4, cyclin E staining (brown nuclei). Cyclin E expression, similar to MCM7 expression, is also up-regulated in both single and double transgenic mice. Magnification, ×40.

Figure 4

p53, p16, and pRb status in the reproductive tract. Images from sections stained with antibodies to p53, p16, or pRb (brown) and counterstained with hematoxylin (blue). Column 1, p53 expression in the cervical epithelium from various genotypes. The nontransgenic (NTG; top) sample is from a mouse exposed to 5 Gy of ionizing radiation and is used as a positive control to show p53-positive staining, which is primarily observed in the basal and parabasal strata. The cervical epithelium from unirradiated nontransgenic mice (data not shown) is p53 negative. All other panels are from unirradiated mice. Columns 2 to 4, the status of p53, p16, and retinoblastoma (pRB) expression in reproductive tumors from various genotypes. Tumors expressing the E7 oncogene generally displayed variable positivity for p53. Shown in the panel from the E6/E7 tumor is an area of high sporadic p53 positivity. p16 was up-regulated in tumors expressing E7 or K14E6I128T. Retinoblastoma expression was inversely correlated to p16 in tumors expressing either HPV E6 or E7. Magnification, ×40.