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INTRODUCTION: The p53 tumor suppressor family is classically activated after DNA damage and plays a central role in cell fate decisions. Although, the p53 family activates many of the same genes in response to DNA damage, p73 plays distinct biological functions in development and metastasis. It is likely that p73 activates a unique transcriptional network which is critical for its anti-metastatic and anti-invasive action. Long non-coding RNAs (lncRNAs) are a class of mRNA-like transcripts longer than 200 nucleotides. They lack protein-coding ability and are believed to be involved in various kinds of biological processes. Increasing evidence suggests that lncRNA are frequently aberrantly expressed in cancers. Therefore, the roles of dysregulated functional lncRNA in human malignant tumors have attracted considerable scientific interest. The objective of our study is to find out novel long non-coding RNAs that can act as transcriptional targets of p73 and to delineate their role in p73-mediated anti-metastatic response. METHODS: For this purpose, we performed transcriptome sequencing in HCT116p73wt and HCT116p73KD cells and screened the data for modulation of expression of lncRNAs in differential manner. Quantitative Real Time PCR was further carried out to validate the data obtained after screening RNA seq Data. Promoter analysis was carried out for the identification of p73 binding sites in the selected upregulated or downregulated lncRNAs which was further confirmed by Luciferase reporter, ChIP and site directed mutagenesis assays. RESULTS: About six lncRNAs were observed to be significantly upregulated while four were down-regulated upon knockdown of p73. The promoters of selected lncRNAs were analysed in silico using TF Bind and JASPAR software for p73 binding sites and luciferase reporter assays suggested regulation of lncRNAs by p73. Chromatin immunoprecipitation showed promoter enrichment of the selected lncRNAs. Site directed mutagenesis further confirmed the exact binding sites of p73 onto the promoters of these novel long non coding RNAs. CONCLUSION: Together, our study provides insights into the differential regulation of long non-coding RNAs in p73 dependent manner which further will provide the mechanism of their action at the genome level. Citation Format: Chanchal Bareja, Apoorva Uboveja, Daman Saluja. Elucidating the differential regulation of novel long non coding RNAs and their mechanism of action in p73 dependent manner [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 1560.

p73 is a member of the p53 tumor suppressor family and exerts its tumor suppressor functions by suppressing metastasis. It is increasingly evident that long noncoding RNAs (lncRNAs) play a significant role in tumor suppression. The present study is aimed to identify novel lncRNAs that play a role in p73-mediated suppression of metastasis in colorectal cancer cells. Transcriptome analysis was performed to detect the differentially expressed lncRNAs in presence and absence of p73. Out of these, FER1L4 lncRNA was found to be significantly induced in a p73-dependent manner. p73 binding to FER1L4 promoter was confirmed through bioinformatic analysis, luciferase reporter, ChIP and site-directed mutagenesis assays. Knockdown of FER1L4 and p73 significantly increased the invasion and migration rate of colorectal cancer cells as confirmed by wound-healing assay. Knockdown of FER1L4 decreased the expression of E-cadherin and increased the expression of prominent EMT markers such as N-cadherin, Snail, Vimentin and Fibronectin. FER1L4 causes a G2/M cell cycle arrest in a p73-dependent manner in HCT116p53-/-p73+/+ 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+/+ cells with increase in time-dependent treatment of etoposide and FER1L4kd inhibited apoptosis even in the presence of p73. The protein expression level of 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. FER1L4 also sponges the expression of miR-1273g-3p, thus inhibiting its oncogenic role. RNA-In situ hybridization (RNA-ISH) confirmed the decreased expression of p73 and FER1L4 mRNA in 30 human metastatic CRC tissues as compared to 30 human non-metastatic CRC tissues. Taken together, we provide conclusive proof that p73 exerts its anti-metastatic function by inducing the expression of lncRNA FER1L4 in response to genotoxic stress. Citation Format: Apoorva Uboveja, Yatendra Kumar Satija, Fouzia Siraj, Chanchal Bareja, Daman Saluja. p73 - lncRNA Fer1l4 axis plays a critical role in suppression of cancer cell migration, invasion and metastasis in a p73-dependent manner via inhibition of miR-1273g-3p [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2388.

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.