Abstract 1552: Deciphering the mechanism of action of Long non-coding RNA fer1l4 in the suppression of invasion, migration and metastasis of colon cell carcinoma

Abstract Introduction: p73 transcription factor belongs to the p53 tumor suppressor family. Recent studies revealed that p73 wields its tumor suppressor properties by inhibiting metastasis. Although the literature on the p73 transcriptional circuit has chiefly concentrated on protein-coding genes, it has been progressively pointed out that p73 is also able to transcriptionally modulate non-coding RNA (ncRNA) members. These involve microRNAs (miRNAs) and many p53-regulated long non-coding RNAs (lncRNAs). Methods: Identification of p73 binding sites in the FER1L4 promoter region was made by JASPER and TF BIND which was further confirmed by chromatin immunoprecipitation (ChIP) and site-directed mutagenesis experiments. Effect of FER1L4 in p73 mediated cell cycle arrest and apoptosis was checked by cell cycle analysis, Annexin-V/PI, and TUNEL apoptosis assays. Depletion of FER1L4 enhanced cell proliferation, migration, and invasion in a p73-dependent manner. Furthermore, RNA-In situ hybridization (RNA-ISH) analysis of non-metastatic and metastatic human colon cancer tissue samples was carried out to compare the levels of FER1L4 and p73 in metastatic and non-metastatic tumor tissue samples. We also checked the expression of different miRNA including miR1273g-3p and its effect on PTEN expression. Results: We have identified lncRNA FER1L4 as a novel p73 transcriptional target that gets induced as a result of genotoxic stress. The binding of p73 to FER1L4 promoter was established by bioinformatics analysis, luciferase reporter, and ChIP assays. Both FER1L4 and p73 knockdown enhanced the migration and invasion rate of colorectal cancer cells. FER1L4 knockdown reduced E-cadherin expression and enhanced the expression of N-cadherin, Vimentin, Snail, and Fibronectin. Cell cycle assays revealed that FER1L4 induces a G2/M cell cycle arrest in a p73-dependent manner. Annexin V/PI and TUNEL assays revealed FER1L4 induced apoptosis in HCT116p53-/-p73+/+ colon cancer cells under genotoxic stress and FER1L4 knockdown inhibited apoptosis even in the presence of p73. The expression of pro-apoptotic markers such as Bad, Bax, Bik, Bim, Bid, Bak and PUMA decreased upon FER1L4 and p73 knockdown. FER1L4 sponges the expression of miR-1273g-3p, which in turn increases PTEN expression leading to cell cycle arrest. RNA In-situ hybridization revealed the down-regulation of both p73 and FER1L4 expression in metastatic colon cancer tissue as compared to non-metastatic tissue. Conclusion: We provide conclusive proof that p73 exerts its anti-metastatic properties by inducing lncRNA FER1L4 in response to genotoxic stress which in turn sponges the expression of miR1273g-3p, a regulator of PTEN. Citation Format: Daman Saluja, Apoorva Uboveja, Yatendra kumar Satija, Fouzia Siraj. Deciphering the mechanism of action of Long non-coding RNA fer1l4 in the suppression of invasion, migration and metastasis of colon cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1552.