The discovery of new classes of regulatory RNA species and the factors that control their biogenesis and function has revealed unexpected new mechanisms of gene regulation that affect normal development and tumorigenesis. Project 2 has three Aims that focus on different aspects of regulatory RNAs and cancer. Research in Aim 1 concerns a miRNA cluster containing two miRNAs, mir-143 and -145, which are frequently down-regulated in human cancer. We have constructed a mouse strain carrying a conditional mutant allele of the mir-143~145 cluster and will use the strain and cells derived from it to study the functions of these miRNAs in normal cells and in tumor development Of particular interest is the role of mir-143-145 in 5q- syndrome, colon cancer, and lung cancer, all of which will be examined in in vivo models. The tools developed in this project will also be extremely useful in defining targets of these miRNAs.
In Aim 2, we will explore the potential use of tumor suppressor miRNAs in cancer treatment. We have generated considerable data that ectopic expression of both let-7g and mlr-S4a can inhibit lung tumor development in mice. In addition to refining this analysis with additional methods of conditional miRNA expression in an autochthonous model of advanced adenocarcinoma of the lung, we will use novel, nanoparticle-based miRNA delivery methods to treat such established tumors. The effects of delivery of let-7g, mir-S4a and combinations will be evaluated for effects on proliferation, cellular senescence and cell death of tumor cells;tumor progression;and survival of treated animals.
Aim 3 is directed at a distinct class of regulatory RNA, the large intervening non-coding RNAs or lincRNAs. We have shown that several members of this newly characterized class of RNAs are regulated by the tumor suppressor p53, including lincRNA-p21 and linc4S. Using a combination of whole-animal experimentation, including the development of mouse strains carrying conditional mutant alleles of these genes, as well as cell-based studies, we will investigate the function of lincRNA-p21 and Iinc43 on p53 responses to DNA damage and oncogene-induced stress. Deep RNA sequencing will be used to determine the changes in gene expression associated with loss-of-function of these lincRNAs, and biochemical methods will be employed to explore their potential roles in regulating gene expression through inducing alterations in chromatin structure of target genes. Finally, mutant mouse strains will be used to determine whether mutation of these lincRNAs predisposes to cancer.

Public Health Relevance

The elucidation of the molecular genetic events that underlie tumor development is critical for the effective treatment and prevention of cancer. This research is directed at understanding the function of new classes of regulatory RNA molecules, termed miRNAs and lincRNA, in the development of cancer and in normal physiology. The research will also lead to the development of powerful new tools to study cancer-relevant genes and pathways.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Program Projects (P01)
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Massachusetts Institute of Technology
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Zamudio, Jesse R; Kelly, Timothy J; Sharp, Phillip A (2014) Argonaute-bound small RNAs from promoter-proximal RNA polymerase II. Cell 156:920-34
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