The role of long noncoding RNA CRNDE in normal physiology and cancer

PROJECT SUMMARY A growing body of evidence indicates that long noncoding RNAs (lncRNAs), a diverse class of non-protein- coding transcripts >200 nucleotides in length, play important roles in the initiation and progression of cancer. LncRNAs have been proposed to regulate all cancer hallmarks, but, in the vast majority of cases, their molecular mechanisms of action remain poorly understood. This knowledge gap is a major impediment towards realizing the potential of lncRNAs as therapeutic targets in cancer and other diseases. As in many human malignancies, lncRNAs are frequently dysregulated in renal cell carcinoma (RCC). RCC is the most common type of kidney cancer and the most lethal malignant urological tumor, with approximately 70,000 new cases diagnosed annually in the United States. To date, most genomic studies of RCC (and other cancers) have focused on identifying disease-associated alterations of protein-coding genes. Our understanding of the molecular pathways regulated by lncRNAs in RCC, and the functional roles of these transcripts in this malignancy, remains limited. We mined RNA-seq data from RCC patients to identify a set of 805 lncRNAs that are commonly overexpressed in this tumor type. We generated a custom CRISPR interference (CRISPRi) library targeting these lncRNAs and performed screens in multiple RCC cell lines to identify lncRNAs that are essential for RCC cell proliferation. These experiments revealed that the lncRNA Colorectal neoplasia differentially expressed (CRNDE) is required for growth of all tested RCC cell lines. Although this lncRNA has been shown to be overexpressed and is associated with poor patient survival in RCC and other types of cancer, its molecular function remains unclear. We identified a critical region of CRNDE that is necessary for RCC cell proliferation and we identified proteins that interact with this sequence. We also generated novel genetically-engineered alleles in mice that enable constitutional or conditional deletion of critical Crnde sequences. In this proposal, we will leverage our new understanding of this lncRNA, and the novel resources we have generated, in order to dissect the molecular function of CRNDE and define its role in normal physiology and in RCC pathogenesis in vivo. These experiments will take advantage of our extensive experience, and that of our collaborators, in evaluating noncoding RNA functions and RCC biology using cellular and animal models. Successful completion of the proposed research will address two major knowledge gaps in the fields of RNA biology and cancer biology: 1) our limited understanding of the molecular mechanisms-of-action of lncRNAs; and 2) how these mechanisms are co-opted by cancer cells to promote tumor growth, particularly in RCC. We anticipate that the principles revealed by these studies will be broadly applicable to our understanding of the roles of other lncRNAs in cancer cells and may set the stage for developing therapeutics that target CRNDE or the pathways it controls.