Nedd9 regulates metastasis of non-small cell lung cancer through activation of epithelial-mesenchymal transition and tumor cells migration

Y.A. Topchu*, A.M. Mazitova**, M.V. Tikhomirova***, Z.I. Abramova****, A.Y. Deneka*****

Kazan Federal University, Kazan, 420008 Russia

E-mail: *topchu_1993@mail.ru, **sashamazitova@mail.ru, ***mary20011994@mail.ru, ****ziabramova@mail.ru, *****AYDeneka@kpfu.ru

Received April 26, 2019

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DOI: 10.26907/2542-064X.2020.1.123-133

For citation: Topchu Y.A., Mazitova A.M., Tikhomirova M.V., Abramova Z.I., Deneka A.Y. Nedd9 regulates metastasis of non-small cell lung cancer through activation of epithelial-mesenchymal transition and tumor cells migration. Uchenye Zapiski Kazanskogo Universiteta. Seriya Estestvennye Nauki, 2020, vol. 162, no. 1, pp. 123–133. doi: 10.26907/2542-064X.2020.1.123-133. (In Russian)

Abstract

Non-small cell lung cancer (NSCLC) has a low survival rate, with metastasis contributing to the vast majority of deaths. Elevated expression of the protein Nedd9 (neural precursor cell expressed, developmentally down-regulated 9) has been reported in a large subset of lung cancers and other malignancies as a promotor of aggressive phenotypes and drug resistance. This study was performed to identify the mechanisms by which Nedd9 regulates invasion and metastasis of non-small cell lung cancer in the transgenic murine model.

Aiming to address the research goals, we performed a set of in vivo and in vitro experiments with the help of such methods as magnetic resonance imaging (MRI), immunohistochemical staining of tissues and microscopy, western blotting.

We found that Nedd9 constitutive null genotype enhanced tumor growth in an inducible Kras/Trp53 model, in which Kras mutation is induced specifically in the lung tissue by inhalation of adenovirus. Pathological examination of the tissues demonstrated that Nedd9 null genotype also was associated with higher invasive capacity in vivo, including direct invasion to the heart. We carried out a set of experiments to unveil the mechanism underlying the phenotype discovered.

Overall, our data support the model in which Nedd9 provides critical support for early stages of the NSCLC growth, and progression beyond this early stage in the absence of Nedd9 requires extensive intracellular protein signaling reprogramming, allowing tumor cells to acquire mesenchymal properties, such as increased mobility and invasion due to a compensatory rearrangement of intracellular protein signaling pathways and activation of the epithelial-mesenchymal transition (EMT). These results are novel and have not been previously described in the literature.

The biological mechanism by which Nedd9 regulates growth, invasion, and metastasis of NSCLC has been poorly investigated, and its study carries not only fundamental implication – discovery of novel mechanisms driving tumor development, but also significant practical importance: assessment of levels of Nedd9 activity in NSCLC patients can potentially serve as a biomarker of response to chemotherapy.

Keywords: lung cancer, metastasis, epithelial to mesenchymal transition, immunohistochemistry, transgenic murine model

Acknowledgments. The research was funded by the Russian Science Foundation (project no. 18-75-00104). The part of this study was supported by Russian Government Program of Competitive Growth of Kazan Federal University.

Author contribution statement. Y.A. Topchu – conducting experiments, interpretation of the results, writing of the manuscript; A.M. Mazitova – conducting experiments, interpretation of the results, writing of the manuscript; M.V. Tikhomirova – conducting experiments, interpretation of the results; Z.I. Abramova – reviewing and editing of the manuscript; A.Y. Deneka – project development, conceptualization, original draft preparation.

Figure Captions

Fig. 1. Comparative analysis of tumor burden in the lungs of mice with the intact Nedd9 and null Nedd9 (Nedd9–/–) genotype. Representative MRI images (a) and quantitation of tumor volume (b) based on MRI scans of the lungs of mice aged 29 weeks. Arrows indicate tumors in the lungs.

Fig. 2. Hematoxylin- and eosin-stained lung tissues from mice with intact Nedd9 and null Nedd9 (Nedd9–/–) genotype. Arrow indicates direct invasion into the heart tissue.

Fig. 3. Absence of Nedd9 increases the metastatic activity of tumor cells through activation of epithelial-mesenchymal transition (EMT). a) Immunofluorescent immunohistochemical (IF-IHC) staining of tumor sections from mice with intact Nedd9 and null Nedd9 (Nedd9–/–) genotype for mesenchymal marker vimentin (orange) and macrophage marker F4-80 (green) and DAPI (blue). b) Representative images from Western blot analysis for EMT marker vimentin in murine tumor tissue lysates (four mice with null Nedd9 and four control mice).

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