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.

中文翻译：

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p73-NAV3轴在抑制结肠癌转移中起关键作用。

p73是p53抑癌家族的成员，该家族可激活p53反应基因并介导DNA损伤反应。最近的证据表明p73通过抑制转移发挥其肿瘤抑制功能，但确切的机制尚不清楚。在这里，我们确定Navigator-3（NAV3），一种微管结合蛋白，是p73的新型转录靶标，它以依赖p73的方式被DNA损伤上调，并且在p73介导的癌细胞抑制中起着至关重要的作用。入侵，迁移和转移。响应DNA损伤而诱导p73导致内源性NAV3 mRNA和蛋白质水平快速增加。通过生物信息学分析，我们确定了NAV3启动子中的两个p73结合位点。与此相一致，通过萤光素酶，染色质免疫沉淀，和定点诱变分析。通过伤口愈合，细胞侵袭和细胞迁移测定证实，NAV3和p73表达的废除显着增加了结肠直肠癌细胞的侵袭和迁移速率。同样，敲低NAV3降低了E-钙黏着蛋白的表达，并增加了其他重要的间充质标记，例如N-钙黏着蛋白，Snail，波形蛋白和纤连蛋白的表达。免疫组织化学分析显示，与非转移性癌组织相比，转移性结肠癌组织中NAV3和p73的表达均下调。另外，NAV3和p73的表达模式在非转移性和转移性人结肠癌组织样品中均显示出广泛的显着相关性。在一起