Exploring the Role of POF1B in the Carcinogenesis of Squamous Cell Carcinoma Models
Open AccessThe objective of this study is to characterize a novel c-Rel target gene, premature ovarian failure 1B (POF1B) and investigate its function in epithelial cancers. As the second most common cause of death internationally, cancer is a major public health concern. Approximately 90% of cancers originate from epithelial tissues. Squamous cell carcinomas (SCC) are a type of epithelial cancer, and our lab uses primary murine keratinocytes, which we genetically modify, as a model to study squamous epithelial cancers. Using this model to overexpress ΔNp63α, a p53 homolog overexpressed in human SCCs, the laboratory previously showed that: in vivo overexpression of ΔNp63α with the v-RasHA oncogene yields 100% conversion of benign papillomas to carcinomas; and that in vitro, ΔNp63α overexpressing keratinocytes accumulate nuclear c-Rel, which is critical for ΔNp63α mediated override of the normal growth arrest response. Furthermore, co-localization of ΔNp63α and c-Rel was seen in human HNSCC tumors and co-regulation of genes in HNSCC cell lines. POF1B was identified as a c-Rel target gene in a microarray study of ΔNp63α/v-RasHA expressing keratinocytes in which c-Rel expression was modulated. This finding was validated by RT-PCR. Here we show that POF1B is also positively regulated by c-Rel at the protein level. POF1B has been linked to cytoskeleton regulation and planar cellular polarity. Given the direct correlation with c-Rel expression we explored if either POF1B, or its upstream regulator, c-Rel, are involved in migration in ΔNp63α/v-RasHA transduced keratinocytes. Using a wounding “scratch” assay in ΔNp63α/v-RasHA transduced keratinocytes with modulated c-Rel levels, we observed a positive correlation with c-Rel expression and wound healing. In contrast, wound healing negatively correlated with POF1B expression in the same assay. To address if different results would be obtained in an established tumor cell line, we performed a transwell migration assay using the UM-SCC-38 cell line. Consistent with the results in ΔNp63α/v-RasHA transduced keratinocytes, POF1B negatively correlated with migration, suggesting that despite their co-modulation in our system, c-Rel and POF1B differentially impact migration. The impact of c-Rel will need to be assessed in the UM-SCC-38 cell line. If consistent with the mouse model, future studies will dissect the different roles of these proteins in migration and given literature reports will address a potential role for POF1B in collective migration. Immunostaining was used to characterize POF1B expression and the impact of POF1B depletion on morphology. In preliminary findings, depletion of POF1B in ΔNp63α/v-RasHA transduced keratinocytes resulted in altered cellular morphology with reduced cell-cell contact. This robust effect of POF1B depletion was not observed in the UM-SCC-38 cell line, however the cellular organization appeared somewhat less organized than in the control. Furthermore, in this cell line POF1B exhibited localization at the cell borders, consistent with cell-cell adhesion. Future studies will focus on further elucidating the potential role of the POF1B protein in squamous cell carcinomas.
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