A majority of the eukaryotic genome is noncoding and only 2% represents protein-coding sequences. Among the several types of non-coding RNAs, microRNAs (miRNAs) and long non-coding (lncRNAs) have gained significant attention due to their roles in vital cellular functions, including proliferation and survival. miRNAs directly bind to mRNAs to inhibit mRNA stability and/or translation. lncRNAs can activate or repress gene expression via diverse mechanisms including transcription and splicing. Deregulation of miRNAs and lncRNAs has been correlated with human diseases, including cancer. Recently, we and others have identified specific miRNAs and lncRNAs transactivated by p53, the most frequently mutated gene in human cancer. A deeper understanding of the biology of these miRNAs and lncRNAs is critical in determining their potential in cancer therapy.

The goal of my laboratory is to investigate the functions of Regulatory RNAs in the p53 pathway and in colorectal cancer differentiation, an intriguing question in cancer biology and therapeutics. Mammalian miRNAs bind to target mRNAs via partial complementarity. A single miRNA can regulate the expression of hundreds of mRNAs. However, due to partial complementarity between a miRNA and its target miRNA, identifying miRNA targets is challenging (Thomas et al., Nat Struct Mol Biol, 2010). We developed a biochemical approach to identify endogenous mRNAs bound to a transfected biotinylated-miR-34a (Lal et al., PLOS Genet, 2011). Combining this strategy with bioinformatics identified a network of genes and canonical pathways regulated by the p53-regulated miRNA miR-34a and the growth suppressor miR-24 (Lal et al., Mol Cell, 2009; Lal et al., Nat Struct Mol Biol, 2009). In addition, we have identified tumor suppressive functions of specific miRNAs in the p53 pathway and in colorectal cancer cell differentiation (Li et al., Mol Cell Biol, 2013, Jones et al, Cell Death Diff, 2015, Subramanian et al, Oncogene, 2015, Jones et al., PNAS, 2015).  

More recently, we have identified several lncRNAs regulated by p53 during DNA damage in colorectal cancer cells. One such lncRNA that we named PINCR (p53-induced noncoding RNA) is in an intergenic region on the X-Chromosome. PINCR is a direct target of p53. Its expression is almost undetectable in untreated cells but it is induced about 100-fold after DNA damage (Chaudhary et al., eLife, 2017). We found that this lncRNA exerts a critical prosurvival function in colorectal cancer cells in vitro and tumor growth in vivo. Targeted deletion of PINCR significantly impaired G1 arrest and induced hypersensitivity to chemotherapeutic drugs. PINCR regulates the induction of a subset of p53 targets involved in G1 arrest and apoptosis, including BTG2, RRM2B and GPX1. Using a novel RNA pulldown approach that utilized endogenous S1-tagged PINCR, we found that PINCR associates with the enhancer region of these genes by binding to RNA- and DNA-binding protein Matrin3 that, in turn, associates with p53. Our findings on the initial characterization if PINCR demonstrate a prosurvival function of a p53/PINCR/Matrin3 axis in response to DNA damage. Our studies on another p53-induced lncRNA that we termed PURPL (p53 upregulated regulator of p53 levels) revealed a p53-PURPL auto-regulatory feedback loop and demonstrated a role for PURPL in maintaining basal p53 levels (Li et al., Cell Reports, 2017). 

lncRNAs should be truly noncoding but there is increasing evidence that some lncRNA genes encode small proteins or micropeptides. Our initial characterization of LINC00675 that we termed FORCP (FOXA1-Regulated Conserved Small Protein), demonstrated that FORCP encodes a novel, highly conserved, naturally occurring small protein of 79 amino acids (Li et al, eLife, 2020). We found that the FORCP protein is localized to the endoplasmic reticulum and  regulates apoptosis and tumorigenicity in well-differentiated colorectal cancer cells. 

Our recent work on RNA-binding proteins (RBPs) in the p53 pathway and in colorectal cancer has revealed key functions of RBPs. For instance, we discovered that the p53-induced RBP ZMAT3 functions as a key splicing factor (Muys et al., Genes Dev, 2021) and that the RBP Matrin3 controls mitotic spindle dynamics in colorectal cancer by functioning as a splicing repressor for CDC14B (Muys et al., Cell Reports, 2023). Ongoing studies on RBPs have uncovered novel functions of RBPs in the p53 pathway and in colorectal cancer.    

The utilization of cell and molecular biological approaches to investigate the functions and mechanisms of lncRNAs and RBPs, combined with analysis of their expression in patient samples and in vivo studies in mice will help better understand their role in cancer pathogenesis.