Abstract 3161: The RNA binding protein HuR stabilizes the ornithine decarboxylase (ODC) transcript in non-melanoma skin cancer

Abstract

Abstract Non-melanoma skin cancer (NMSC), comprised of both basal cell carcinoma and squamous cell carcinoma, is the most common malignancy in the United States. The work described here focuses on understanding the underlying mechanisms of this disease using a mouse keratinocyte model comprised of two cells lines that represent distinct stages in skin carcinogenesis, initiation and progression, denoted as C5N and A5 respectively. Previous results have shown that induction of ornithine decarboxylase (ODC) enzyme activity is both necessary and sufficient for the promotion of skin tumors. As expected, we saw a dramatic increase in ODC enzyme activity in A5 spindle carcinoma cells when compared to C5N keratinocytes. Interestingly, this increase in ODC enzyme activity correlates with an increase in ODC stability in A5 cells compared to C5N cells. Thus, we sought to investigate whether ODC mRNA interacted with the RNA binding protein HuR, which is known to bind to and stabilize its target mRNA transcripts. HuR has been shown by others to localize to the cytoplasm where it stabilizes numerous transcripts corresponding to proteins that are important in the development of a variety of malignancies. Moreover, studies involving human tumor samples from breast cancer, ovarian cancer, and Merkel cell carcinoma patients have shown a correlation between high cytoplasmic HuR presence and increased tumor grade. By conducting biotin-pulldown assays, which use a synthetic ODC transcript to test for RNA-protein interactions, as well as mRNP assays, which identify endogenous RNA-protein interactions, we confirmed that HuR is able to bind to the ODC 3’UTR in A5 cells but not in C5N cells. Immunofluorescence results showed that HuR localization between the two cell lines was different, with A5 cells displaying a diffuse HuR presence in both the nucleus and cytoplasm while C5N cells exhibited nuclear localization of HuR. In order to definitively link HuR to ODC mRNA stability, knockdown experiments were conducted using a short interfering RNA (siRNA) that was specific for the coding region of HuR. These knockdown experiments confirmed our hypothesis that HuR is able to bind to and stabilize ODC mRNA in A5 cells, as a 60% knockdown of HuR protein correlated with a profound decrease in ODC mRNA half-life and reduced intracellular ODC protein. These results suggest that the stabilization of ODC mRNA in A5 spindle carcinoma cells is regulated directly by changes in localization of the HuR protein. These data are novel, as they show for the first time that an RNA binding protein is able to bind to and regulate ODC mRNA stability. Knowledge from these and subsequent studies could be used to design RNA binding protein-targeted therapies to combat not only NMSC but other epithelial cancers and/or non-malignant anomalies that display an increased presence of cytoplasmic HuR. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3161.