Uboveja Apoorva, Satija Yatendra Kumar, Siraj Fouzia, Sharma Ira, Saluja Daman
Dr. B.R. Ambedkar Centre for Biomedical Research, University of Delhi, New Delhi, 110007, India.
CSIR-Indian Institute of Toxicology Research, Lucknow, 226001, India.
Oncogenesis. 2020 Feb 6;9(2):12. doi: 10.1038/s41389-020-0193-4.
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是p53肿瘤抑制家族的成员，可反式激活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，但其确切机制仍不清楚。在这里，我们鉴定出微管结合蛋白Navigator-3（NAV3）是p73的一个新的转录靶点，它在DNA损伤时以p73依赖的方式上调，并在p73介导的抑制癌细胞侵袭、迁移和转移中起重要作用。DNA损伤诱导p73会导致内源性NAV3 mRNA和蛋白水平迅速增加。通过生物信息学分析，我们在NAV3启动子中鉴定出两个p73结合位点。与此一致，通过荧光素酶、染色质免疫沉淀和定点诱变分析证实了p73与NAV3启动子的结合。伤口愈合、细胞侵袭和细胞迁移分析证实，NAV3和p73表达的缺失显著提高了结肠癌细胞的侵袭和迁移率。此外，敲低NAV3会降低E-钙黏蛋白的表达，并增加其他突出的间充质标志物如N-钙黏蛋白、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表达均下调。此外，在非转移性和转移性人类结肠癌组织样本中，NAV3和p73的表达模式显示出广泛的显著相关性。综上所述，我们的研究提供了确凿的证据，证明Navigator-3是p73的直接转录靶点，并且在p73介导的抑制癌细胞侵袭、迁移和转移的基因毒性应激反应中起关键作用。
