Long Noncoding Rna Fer1l4 Is A Novel P73 Transcriptional Target And Inhibits Colon Cancer Cell Migration And Invasion In A P73-Dependent Manner

p73 is a member of the p53 tumor suppressor family, which transactivates p53-responsive genes and mediates DNA damage response. Recent evidences suggest that p73 exerts its tumor suppressor functions by suppressing metastasis, but the exact mechanism remains unknown. It is increasingly evident that long non-coding RNAs (lncRNAs) play a significant role in tumor suppression. However, we still have a limited knowledge of the clinical significance of lncRNAs in colorectal cancer. The present study is aimed to identify novel lncRNAs that play a role in p73-mediated suppression of metastasis in colorectal cancer cells. p73 was knocked down in HCT116p53−/−p73+/+ cells and transcriptome analysis was performed to detect the differentially expressed lncRNAs in presence and absence of p73. The top two up-regulated and down-regulated lncRNAs were selected for further analysis based on the false discovery rate (FDR), fold-change (FC) and P-values. Out of these, FER1L4 lncRNA was found to be significantly induced by DNA damage in a p73-dependent manner. Through bioinformatics analysis, we identified two p73-binding sites in FER1L4 promoter. Consistent with this, p73 binding to FER1L4 promoter was confirmed through luciferase reporter assays and Chromatin Immunoprecipitation (ChIP) assays. Site-directed mutagenesis of both the binding sites totally abrogated p73 responsiveness, indicating that both the sites are equally responsible and essential for p73 binding. In addition, Real-time quantitative PCR demonstrated a rapid increase in endogenous FER1L4 mRNA upon induction of p73. Furthermore, knockdown of FER1L4 and p73 significantly increased the invasion and migration rate of colorectal cancer cells as confirmed by wound-healing assay. Also, knockdown of FER1L4 decreased the expression of E-cadherin, a cancer metastasis suppressor, and increased the expression of other prominent EMT markers such as N-cadherin, Snail, Vimentin and Fibronectin. Cell functional assays further revealed that FER1L4 causes a G2/M cell cycle arrest in a p73-dependent manner in HCT116p53−/−p73+/+ colon cancer cells and upon FER1L4kd, normal cell cycle progression was observed. Annexin V/PI and TUNEL apoptosis assays revealed that FER1L4 induced apoptosis in HCT116p53−/−p73+/+ colon cancer cells with increase in time-dependent treatment of etoposide and FER1L4kd inhibited apoptosis even in the presence of p73. The protein expression level of several pro-apoptotic genes such as Bad, Bax, Bik, Bim, BID, Bak and PUMA decreased upon FER1L4kd and p73kd, confirming that FER1L4 plays a role in p73-mediated apoptosis and cell cycle arrest. Taken together, we provide evidence that FER1L4 is a direct transcriptional target of p73 and p73 exerts its anti-metastatic function by inducing the expression of FER1L4 in response to genotoxic stress. Citation Format: Apoorva Uboveja, Yatendra Kumar Satija, Chanchal Bareja, Daman Saluja. Long noncoding RNA FER1L4 is a novel p73 transcriptional target and inhibits colon cancer cell migration and invasion in a p73-dependent manner [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4707.