Gene expression changes in HPV16-mediated carcinogenesis: Comparisons between an in vitro cell model and cervical cancer

To study the molecular mechanisms of human papillomavirus 16 (HPV16)-mediated transformation, we previously established an in vitro model system in which normal human keratinocytes (NHKc or N) transfected with HPV16 DNA became immortalized (IM); IM cells were then subjected to selection for growth factor-independence and subsequently for resistance to differentiation induction, yielding differentiation resistant cells (DR). IM cells and DR cells in monolayer culture share many common properties with precancerous lesions in vivo such as insensitivity to growth inhibition by transforming growth factor beta (TGFB1) and over expression of the epidermal growth factor receptor (EGFR). We performed gene expression profile analysis comparing IMs versus NHKc, DR versus IM directly, and DR versus NHKc using IM as a reference. We identified and confirmed increased expression of SIX1 and GDF15 in both DR cells and cervical cancer tissue specimens, and further compared all gene expression changes in the HKc/HPV16 model to a battery of gene expression changes previously described in cervical cancer. Interestingly, unsupervised cluster analysis revealed that discordant changes in DR versus NHKc clustered together; and affected mainly genes whose products are extracellularly localized. The observed discordant changes in extracellularly localized gene products points to a major drawback of traditional monolayer culture: the lack of a proper microenvironment. As more and more studies support an essential role of the tumor microenvironment in carcinogenesis, organotypic cultures that incorporate both extracellular matrix components and fibroblasts provide better models. In fact, when placed in organotypic culture, IM cells reproduce the morphology of dysplasia, while DR cells produce a highly disorganized epithelial layer reminiscent of carcinoma in situ. Therefore we set out to address how organotypic cultures of HPV16-transformed cells recapitulate the molecular features of HPV16-mediated carcinogenesis by comparing genome-wide gene expression profiles of organotypic-cultured cell lines in our in vitro model system to both the battery of known gene expression changes in cervical cancer and to cell lines in monolayer culture. We demonstrate that organotypic culture alters the expression of a cluster of proliferation related genes in a stage specific fashion. Interestingly, many of these genes have been reported to be over expressed in cervical cancer cases with poor outcomes.