This application aims to investigate the fundamentals of RNA regulation central to human breast cancer progression. Aberrant gene expression is known to be an important factor in cancer, yet very little is known about the role RNA-binding proteins (RBPs) play in disease onset and progression. Sequence specific RBPs coordinately regulate subsets of functionally related mRNAs as RNA regulons to form ribonucleoprotein complexes (RNPs), which are remodeled during activation of cells with small molecule drugs. The overall objective of this proposal is to determine the dynamics of transient cellular RNP complexes coordinated by the RBP human antigen R (HuR) to promote or suppress tumorigenesis. Regulation by HuR is known to be altered in many cancers. It has been shown to stabilize mRNA targets encoding known cell growth regulators and proto-oncogenes, and studies have demonstrated that targets of HuR are different in normal cells compared to cancer cells. No cancer-linked mutations of HuR have been reported, yet there is a strong correlation between HuR levels and carcinogenesis. In addition, recent studies suggest a mechanism whereby HuR is able to compete with microRNAs to stabilize mRNA targets. It is therefore likely that HuR differentially regulates the stability of mRNAs encoding factors involved in tumorigenesis by repressing typically suppressive microRNAs, resulting in the coordinated synthesis of cancer-associated proteins and enhancement of disease progression. To test this hypothesis, RNP-Immunoprecipitation followed by high-throughput sequencing will be used to identify and map remodeling of mRNA subsets associated with HuR throughout a time course of human breast cancer malignancy. This time course will quantify the stepwise transition from a normal human mammary epithelial cell to the fully malignant phenotype. Similar studies will be conducted in parallel to identify mRNA subsets associated with Argonaute2 protein (a major RBP component of microRNPs), to compare mRNAs targeted by microRNAs during the same time course. Furthermore, a combination of well-established molecular and biochemical techniques, including luciferase constructs, knockout and rescue experiments, immunofluorescence and RNA affinity precipitation assays, will be used to specifically determine the mechanistic details that allow HuR to rescue mRNA targets from microRNA-mediated suppression. This work will provide information about the underlying biology of carcinogenesis at the level of posttranscriptional coordination of gene expression, resulting in a more comprehensive understanding of the many layers of complex gene regulation. The posttranscriptional signatures generated in these studies may be used to identify small molecule effectors of HuR. Therefore, the results from this study may ultimately lead to the ability to counter the regulatory changes that contribute to advanced stages of malignancy.
The ability to specifically target the changes in gene regulation that enhance progression from a normal cell to a cancer cell may prove to be an effective and novel approach to the treatment of cancer. Completion of this project will reveal changes in posttranscriptional regulation that are important for human breast cancer progression. Resulting posttranscriptional expression signatures will allow us to identify small molecule drugs that may be able to slow progression of cancer by countering the regulatory changes that contribute to advanced stages of malignancy.