Several animal models have been established in attempts to understand the biology of HPV-mediated tumorigenesis, 
. Two of the clinically relevant mouse models, K14-E7 and K14-HPV16 utilized the K-14 keratin promoter to target transgene expression to the stratified squamous epithelium, including the skin and the cervix. In these systems, both types of transgenic mice develop cervical pathologies following long-term estrogen treatment 
. We took advantage of these mice to test our hypothesis that activation of the Wnt pathway by β-catenin in the presence of HPV-E7 oncogene expression can accelerate the development of cervical cancer. We were able to generate transgenic mice (designated K14-E7/ΔN87βcat), which express the HPV16-E7 oncogene along with constitutively active β-catenin. We found that in this double-transgenic model, mice developed invasive cervical cancers with 94% penetrance as early as seven months of age. In striking contrast, these tumors occurred in only 50% of K14-E7 mice. The latter result conflicts with the studies of Herber et al.
, which reported invasive cervical cancer in 80% of K14-E7 animals by 7 months of age 
. This difference may be related to environmental or dietary factors.
The K14-ΔN87βcat mice used in our study were originally of the CD1 background and were backcrossed into FVB mice for 15 generations before they were used in our study. Therefore, we do not consider differences in genetic background to be a significant difference between our study and the one reported by Herber et al
. Both CD1 and FVB strains are permissive to squamous epithelial malignancies 
. Another factor related to this disparity might be diet, because phytoestrogens in the mouse diet can induce molecular and physiological changes in the uterus 
. Phytoestrogens in laboratory diets can vary between formulations and from batch to batch of the same formulation 
. Phytoestrogens can also mimic the properties of mammalian estrogens. Since estrogen dose is an important parameter in the development of cervical cancer 
, dietary phytoestrogens can influence estrogen-regulated processes and strongly influence studies on comparative carcinogenicity. The animals in our study were fed the PMI Irradiated Rodent 5053 diet from Harlan Laboratories, which contains 380 ppm of phytoestrogens. Although our present results differ from other reports regarding tumor penetrance 
, all of our mice possessed the same genetic background (all F1 littermates), diet, and housing environment. Therefore, our study was internally controlled. We feel confident, therefore, in concluding that our results support our main hypothesis that β-catenin can accelerate cervical cancer progression.
We observed an intriguing difference in total body weights between mice in the groups (). However, we were unable to determine the underlying mechanism. Because K14-E7/ΔN87βcat mice had the lowest body weights and the highest tumor incidence, tumor-related cachexia might have been involved. However, when we compared the weights of K14-E7 animals with tumors (n
8) to ones without (n
8), we did not detect a statistically significant difference (Figure S2
). We could not perform the same comparison for the other groups because the number of animals was insufficient for meaningful statistical analysis. We believe that it is reasonable to conclude that the weight difference resulted from the progressively worsening combined skin phenotype. We initially supposed that the double transgenics would have difficulty in eating or drinking. However, when we assessed food and water intake by members of each group for 6 weeks, we did not observe any statistically significant differences (Figure S3
). Therefore, only the combined skin phenotype remains as a plausible explanation.
Two out of 19 K14-E7/ΔN87βcat, but none of the K14-HPV16/ΔN87β mice (0/10) developed invasive tumors. The K14-ΔN87βcat mice that constitute both double transgenic models are not derived from same genetic background and therefore cannot be compared with each other. Since only F1 littermates were used in the study, K14-ΔN87βcat littermates of K14-HPV16/ΔN87βcat were derived from one of each parental heterozygote, namely K14-ΔN87βcat and K14-HPV16. In contrast, K14-ΔN87βcat littermates of the K14-E7/ΔN87βcat were derived from the parental heterozygotes K14-ΔN87βcat and K14-E7. However, since both K14-HPV16 and K14-E7 possess the FVB/N background, one would have expected no phenotypic difference. To evaluate the genetic backgrounds of K14-HPV16 and K14-E7 mice, we performed SNP analyses on three animals from each group. A 1449 loci medium density SNP linkage analysis showed that K14-E7 and K14-HPV16 mice, respectively, were 100% and 99.83% FVB/NTac. These findings suggested that the alteration in the genetic background of K14-HPV16 animals might have been responsible for the differences between the two K14-ΔN87βcat groups. Furthermore, the same genetic alteration might account for why we observed only dysplasias in K14-HPV16/ΔN87βcat mice compared with invasive cancer and dysplasias no in K14-E7/ΔN87βcat mice.
We performed IHC to evaluate nuclear β-catenin levels (Figure S4
). We did not observe significant nuclear β-catenin staining signal intensity in sections prepared from either group group (Figure S4
). The endogenous level of β-catenin is very high in the mouse cervix (), but IHC was not sensitive enough to detect protein expressed from transgene. However, our published data and other IHC studies demonstrate that β-catenin localizes to the cytoplasm or nucleus in more than two thirds of human invasive cervical carcinoma samples 
, suggesting an active canonical Wnt signaling pathway.
We believe that two mechanisms might account for observed Wnt pathway activation in cervical cancer. Mutations in the β-catenin gene are common in colon cancer, whereas β-catenin mutations in cervical cancer are rare, which suggests activation of the Wnt pathway upstream of β-catenin in cervical carcinoma 
. Epigenetic changes in Wnt pathway regulators in cervical tumor samples and cell lines, as well as in other cancers have been reported, which can explain the activation of the canonical Wnt pathway in the absence of β-catenin mutations 
. Alternatively, HPV oncogenes might directly modulate the Wnt pathway. One recent report provides evidence that HPV16 E6 is able to augment β-catenin/TCF-dependent transcription induced by Wnt3a or β-catenin expression. Because this augmentation is not associated with GSK-3 β activity or major alterations in the levels, stability, or cellular distribution of β-catenin, the authors suggest that this relates to activation of ubiquitin ligase E6AP by E6 
. In another study, Rampias et al.
(2010) reported that in oropharyngeal cancer cells, HPV16 E6 and E7 oncogenes are involved in nuclear accumulation of β-catenin and activation of Wnt signaling mediated by the ubiquitin/proteosome pathway 
Our study provides a potential link between activation of the Wnt signaling pathway and its contribution to HPV-mediated cervical cancer. These results indicate that activation of the canonical Wnt pathway might represent secondary events that are required for malignant transformation of HPV-infected epithelial cells. Targeting the canonical Wnt pathway may therefore provide the basis for developing clinical interventions to prevent disease progression in populations at risk for HPV infection and to treat advanced cervical cancers.