The TP53 gene encodes a transcription factor that plays a pivotal role in many cellular responses through direct regulation of hundreds of target genes. Most human tumors carry missense mutations in the TP53 gene and a majority of the tumors retain the full-length p53 protein. Interestingly, multiple recent studies have shown that these p53 mutations not only result in loss of tumor suppressive functions, but also gain of oncogenic functions that lead to enhanced proliferation and metastasis. However, the molecular basis by which MTp53 acquires novel oncogenic functions has not been completely elucidated. MicroRNAs are an abundant class of small non-coding RNAs that play key roles in gene regulation. Each microRNA can regulate the expression of hundreds of genes by blocking mRNA translation and/or inducing mRNA decay. Recent studies have shown that microRNAs regulate several cancer-related biological processes, including enhanced proliferation, tumor angiogenesis and metastasis. MicroRNA genes reside in regions of the genome as distinct transcriptional units as well as in clusters of polycistronic units. RNA polymerase II transcribes microRNA genes, generating long primary transcripts. Subsequently, the pri-microRNAs are processed to mature microRNAs in a two step process involving RNase-III enzymes (Drosha and Dicer). Among the most pressing questions regarding this unusual class of regulatory microRNA-encoding genes is how their expression is regulated. In normal cells, wild-type p53 has been shown to enhance the biogenesis of some microRNAs during DNA damage. In cancer cells, the MTp53 protein influences the transcriptome by interacting with several transcription factors including E2F1, ETS1 and the p53 family members, p63 and p73. Since the mechanism of transcription of microRNA genes is similar to mRNAs, we hypothesize that in cancer cells, MTp53 alters the abundance of a subset of cellular microRNAs by regulating the activity of the above-mentioned transcription factors. The overall goal of this project is to understand how gain-of-function mutations in TP53 alters microRNA expression in cancer cells. To test the hypothesis that mutations in p53 can regulate transcription of microRNAs in cancer cells, we decided to use stable cell lines expressing MTp53 in p53-null H1299 lung cancer cells. Consistent with the known oncogenic function of mutant p53, the presence of mutant p53 in H1299 cells induced an increased growth rate and random migration. To begin our investigation, in FY11, we employed deep sequencing to compare the abundance of microRNAs between vector-transfected H1299 cells and MTp53 expressing cells. This approach allowed us to identify microRNAs that are specifically regulated by MTp53 in H1299 cells. Using this strategy, we found that MTp53 expressing H1299 cells had significantly increased expression of several oncogenic microRNAs and decreased abundance of select tumor suppressor microRNAs. Our findings suggest that the increased invasiveness of MTp53 cells could be partly mediated by increased levels of oncogenic microRNAs and down-regulation of tumor suppressor microRNAs. To further establish the role of MTp53 in regulating microRNAs expression, we will knockdown endogenous MTp53 in lung, breast and colorectal cancer cell lines and perform deep sequencing. We expect that most microRNAs regulated by MTp53 in H1299-MTp53 cells will also be altered after knockdown of MTp53 in the above-mentioned cell lines. In FY12 we have been able to silence mutant p53 in multiple cell lines using 2 different shRNAs directed against p53. Among the miRNAs up-regulated in H1299-MTp53 cells and these cell lines expressing p53shRNAs, was the oncogenic miRNA miR-155, a recently discovered target of MTp53. Because miR-155 is expressed at reasonably high levels only in immune cells, we hypothesize that regulation of miR-155 by mutant p53 may have a functional effect only in immune cells. We also found that the tumor suppressor miRNA, let-7, was significantly down-regulated by mutant p53 in all the cell lines that tested. Our results also suggest that regulation of let-7 by mutant p53 plays an important role in driving cellular proliferation and increasing cell migration and invasion. The identification of microRNAs regulated by multiple p53 mutants in a panel of cell lines will be indicative of key pathways relevant to mutant p53-mediated tumorigenesis. Having identified the common set of microRNAs regulated by MTp53, we will next investigate (1) whether candidate microRNAs are also regulated by other aggressive mutants of p53 (such as R175H) (2) changes in transcription and regulation of candidate microRNA genes by MTp53 and MTp53-interacting transcription factors such as E2F1, ETS1 and p63/p73. This study will identify a novel role of MTp53 in regulating microRNA expression and will also provide the framework for the design of novel cancer therapies.
|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|