Cyclin-D1 Modulation of Vitamin-D-Induced Gene Expression in Oral Keratinocytes

Background: Oral cancer is the sixth most frequent cancer worldwide. Ninety percent of the newly diagnosed oral cancers (+400,000/ year) are characterized as squamous cell carcinomas (SCC). SCC, which develops in the cells of the oral epithelium, is a highly aggressive and destructive cancer with a propensity for invasion of surrounding tissues and recurrence. Consequently, little is known about the preventive factors that modulate the risk of oral carcinogenesis. Recently however, two epidemiological studies investigating the predictive factors of cancer, found their strongest association between low levels of vitamin D and oro-pharyngeal cancer. It is our labs goal to further investigate this matter in a cellular model that parallels human oro-pharyngeal carcinogenesis. Recently, our lab identified a novel interaction between Cyclin D1 and a nuclear hormone receptor that modulates gene expression, known as the vitamin D receptor (VDR). Objectives: The objective of our research is to investigate the effect of vitamin D treatment on human keratinocytes engineered to overexpress an oncogene (Cyclin D1) commonly amplified in SCC. We intend to explore vitamin D's effect by monitoring changes in the localization of key components in both the VDR and Cyclin D1 pathways. Our findings should begin address the question of whether the interaction of Cyclin D1 and VDR has any impact on key components in either pathway. Methods: Transgenic keratinocytes over expressing cyclin D1 (OKF6-D1) will be used to examine the relationship between the VDR and Cyclin D1 pathways. Immuno-staining of key pathway components will be used to localize these components to nuclear or cytoplasmic compartments that can be visualized by immunofluorescence (IF) . Lastly, western blot analysis of fractionated lysates (cytoplasmic and nuclear) from transgenic and control keratinocytes will be used to confirm any IF findings . Results: Immunohistochemistry points toward a difference in the localization of VDR when transgenic cells are ligand treated. Lastly, reduced levels of VDRs heterodimeric partner (RXR) were identified when transgenic keratinocytes were treated with vitamin D