p53 mutants with a single amino acid substitution are common in a majority of human cancers. Although the frequent p53 mutations abrogate the tumor suppressor function of wild-type p53 (WTp53), there is mounting evidence that mutant p53 proteins produced in cancer cells also contribute actively to tumorigenesis through gain-of-function. Indeed, it has been shown by several groups that H1299 cells (p53 null) expressing mutant p53 divide faster and are more invasive than the control cell line. MicroRNAs are abundant trans-acting factors that regulate gene expression by binding to target mRNAs via partial complementarity. They are capable of regulating at least 30% of the protein-coding genes in mammals. Although some microRNAs act as fine tuning rheostats to adjust gene expression subtly, there is increasing evidence that others, such as let-7, act as master regulators of key biological processes. Recently, we and others have shown that several microRNAs influence cellular proliferation and invasion. It is plausible that MTp53-regulated microRNAs could play a major role in the gain-of-function phenotype of MTp53 cells. Using deep sequencing we have recently found that a subset of cellular microRNAs are differentially expressed between H1299 cells stably transfected with vector control and H1299 cells stably expressing MTp53. Among these, 2 members of the let-7 family of microRNAs were the most down-regulated in MTp53-expressing H1299 cells. We hypothesize that down-regulation of let-7 (a tumor suppressor microRNA) by the oncogenic MTp53 protein could be an important event in MT-p53-induced increased proliferation and invasive phenotypes. Indeed, our preliminary results in FY11 and FY12 are consistent with this hypothesis when we found that introduction of let-7 in MTp53-expressing H1299 cells decreases cellular proliferation and migration. Importantly, in FY12, we found that decrease in cell migration in let-7 over-expressing cells occurs before the onset of cell cycle block, suggesting that the observed effect on cell migration is not a consequence of inhibition of cell cycle proliferation. Furthermore, we have found that the two MTp53-regulated let-7 family members are also less abundant in MTp53 expressing lung and breast cancer cell lines as compared to lung cancer cell lines that express WTp53. Taken together, our results suggest that decreased abundance of let-7 could play a crucial role in mutant p53-mediated tumorigenesis. The goal of this project is to determine the mechanism by which down-regulation of the MTp53-regulated microRNA, let-7, increases cell cycle progression and metastasis. To investigate this, we need to identify the target genes and pathways regulated by the let-7. Mammalian microRNAs may regulate hundreds of genes, but identifying the critical genes that are regulated by a microRNA is not straightforward. Current approaches to identify microRNA targets fall short of the task. The common tools that have been employed in most studies are (1) bioinformatic algorithms that predict potential target genes that contain conserved complementary sequences in their 3-UTR to a seed region at the 5-end of the microRNA, and (2) analysis of mRNAs that are down-regulated when a microRNA is over-expressed. The bioinformatic approach is hampered by the fact that the existing algorithms have a high margin of error (the majority of predicted genes are not real targets and some of the key targets, such as RAS for let-7, are not predicted). For many microRNAs, current algorithms predict hundreds or even thousands of potential targets, making it difficult to identify the most important targets. Gene expression array analysis does not readily distinguish direct mRNA targets from mRNAs down-regulated through secondary effects and misses most target genes that are regulated by blocking translation rather than by mRNA degradation. To circumvent these problems, we have developed a new biochemical approach to identify microRNA targets by isolating endogenous mRNAs that directly bind to a biotinylated microRNA and applied it to understanding the function of miR-34a. We have shown that combining this method with bioinformatics analysis of mRNAs enriched in the microRNA pull down is a powerful new tool for identifying genes and cellular pathways regulated by miR-34a (Lal et al., Plos Genetics, 2011). In addition to miR-34a, in FY12, we have successfully employed this method to identify the genome-wide targets of the oncogenic miR-21 (Kang et al., J Biol Chem 2012) and the tumor suppressor miR-519 (Abdelmohsen et al., Mol Cell Biol 2012), suggesting that this is a robust method that can be used to identify targets of any microRNA in multiple cell types. We are currently employing our pulldown approach to investigate the mechanism by which introduction of let-7 in MTp53-H1299 cells inhibits proliferation and migration. Furthermore, by comparing the transcripts pulled down by let-7 in vector-transfected H1299 cells and in MTp53-H1299 cells, we may be able to identify genes specifically involved in MTp53-mediated tumorigenesis. To complement the pull-down data, in FY12 we have used microarray analysis to analyze changes in mRNA expression in MTp53-H1299 cells upon let-7 over-expression. The gene expression analysis has identified known and novel targets down-regulated by let-7 in mutant p53 expressing H1299 cells. Our results have enabled us uncover novel let-7 regulated pathways that we are currently investigating. We anticipate that combining the pulldowns with these results from microarrays will help elucidate the genome-wide targets of let-7 and also enable us to determine the relative role of mRNA degradation versus translational inhibition in let-7 function. Finally, we will investigate whether the anti-proliferative and anti-metastatic effect of let-7 can be employed to inhibit tumor formation and tumorigenesis in vivo by employing xenograft approaches in mice. We are also interested in further analyzing the utility of let-7-based therapeutics in mouse models of lung cancer. We anticipate that over-expression of let-7 in MTp53 cells will reduce tumor formation and metastasis in vivo.
|Subramanian, M; Francis, P; Bilke, S et al. (2015) A mutant p53/let-7i-axis-regulated gene network drives cell migration, invasion and metastasis. Oncogene 34:1094-104|
|Li, Xiao Ling; Jones, Matthew F; Subramanian, Murugan et al. (2014) Mutant p53 exerts oncogenic effects through microRNAs and their target gene networks. FEBS Lett 588:2610-5|