In my laboratory, we study cellular mechanisms that regulate the production of many important gene products, including oncoproteins, inflammatory mediators, and lipoprotein receptors. In particular, we focus on protein factors that regulate cytoplasmic mRNA turnover rates, and the signaling systems that may transiently modulate the activity of these factors. Our experimental approaches vary from cell and molecular biology (cultured cell systems, transfection, RNA interference) to biochemical (gel mobility shift, protein-protein and protein-RNA cross-linking), and biophysical systems (fluorescence anisotropy, resonance energy transfer). Some current foci of interest include:
Trans-acting factors that regulate decay of oncoprotein and inflammatory mediator mRNAs
Many mRNAs encoding oncoproteins and inflammatory mediators contain AU-rich mRNA-destabilizing elements (AREs) in their 3’-untranslated regions. AREs regulate mRNA decay by interacting with cellular ARE-binding factors. For example, tristetraprolin (TTP) or selected AUF1 isoforms induce decay of some ARE-containing mRNAs, while other proteins, like HuR, protect these transcripts from degradation. Recent data also show that microRNAs (miRNAs) and associated proteins can influence ARE-directed mRNA decay. In humans, over 4000 different transcripts contain AREs, yet these sequences share few common features. However, AREs from homologous mRNAs are highly conserved, indicating that the unique sequence composition of each ARE serves one or more essential functions. Our long-term objective is to determine how the size and sequence diversity of AREs directs post-transcriptional regulation at the gene-specific level.
The role of post-transcriptional gene regulatory circuits in tumor development
In 2009, we reported that expression of the mRNA-destabilizing protein tristetraprolin (TTP) was significantly repressed in most human cancers, including aggressive tumors of the breast and prostate. Furthermore, analyses of gene array datasets revealed that suppression of TTP is a negative prognostic indicator in breast cancer, since patients with low TTP expression in breast tumors prior to surgical excision were more likely to present: (i) increased pathological tumor grade, (ii) increased expression of the pro-angiogenic factor VEGF, and (iii) increased risk of death from recurrent breast cancer. Subsequently, we showed that restoring TTP expression in a cultured cancer cell line suppressed three key tumorgenic phenotypes: (i) cell proliferation, (ii) resistance to pro-apoptotic stimuli, and (iii) expression of VEGF mRNA. These findings are consistent with a model whereby cancer cells that lower TTP production develop more aggressive characteristics, likely as a result of increased expression of mRNAs that would otherwise be suppressed by TTP. Ongoing studies in the lab on this project are asking: (i) what TTP-targeted mRNAs are responsible for enhancing tumorigenic phenotypes? and (ii) what cellular mechanisms are suppressing TTP expression in aggressive tumors?
mRNA stability as a novel mechanism to enhance hepatic LDL receptor expression
Hepatic low density lipoprotein (LDL) receptors are essential for efflux of cholesterol-rich LDL particles from the circulation and subsequent cholesterol excretion via bile acid formation. High circulating LDL levels are a prime risk factor for the development of atherosclerotic plaques, contributing to myocardial infarction and related mortality. As such, hepatic regulatory systems that control LDL receptor production are promising targets for the development of novel, aggressive therapies to lower plasma LDL concentrations. We have found that activation of the protein kinase C system stabilizes LDL receptor mRNA in cultured liver cell models, leading to enhanced production of LDL receptor protein and cellular LDL binding. This mRNA stabilization event requires the JNK kinase cascade, but does not appear to involve any known mRNA stability determinants. Ongoing work in the lab is focused on identifying specific LDL receptor mRNA sequences required for JNK-dependent stabilization, and trans-acting factors that functionally interact with this element.