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. Here, we identify Navigator-3 (NAV3), a microtubule-binding protein, as a novel transcriptional target of p73, which gets upregulated by DNA damage in a p73-dependent manner and plays a vital role in p73-mediated inhibition of cancer cell invasion, migration, and metastasis. Induction of p73 in response to DNA damage leads to rapid increase in endogenous NAV3 mRNA and protein levels. Through bioinformatic analysis, we identified two p73-binding sites in NAV3 promoter. Consistent with this, p73 binding to NAV3 promoter was confirmed through luciferase, Chromatin Immunoprecipitation, and site-directed mutagenesis assays. Abrogation of NAV3 and p73 expression significantly increased the invasion and migration rate of colorectal cancer cells as confirmed by wound-healing, cell invasion, and cell migration assays. Also, knockdown of NAV3 decreased the expression of E-cadherin and increased the expression of other prominent mesenchymal markers such as N-cadherin, Snail, Vimentin, and Fibronectin. Immunohistochemistry analysis revealed the downregulation of both NAV3 and p73 expression in metastatic colon cancer tissues as compared to non-metastatic cancer tissues. Additionally, the expression pattern of NAV3 and p73 showed extensively significant correlation in both non-metastatic and metastatic human colon cancer tissue samples. Taken together, our study provide conclusive evidence that Navigator-3 is a direct transcriptional target of p73 and plays crucial role in response to genotoxic stress in p73-mediated inhibition of cancer cell invasion, migration, and metastasis.
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.
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