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CD44s is a crucial ATG7 downstream regulator for stem-like property, invasion, and lung metastasis of human bladder cancer (BC) cells

Abstract

Over half a million US residents are suffering with bladder cancer (BC), which costs a total $4 billion in treatment annually. Although recent studies report that autophagy-related gene 7 (ATG7) is overexpressed in BCs, the regulatory effects of ATG7 on cancer stem-like phenotypes and invasion have not been explored yet. Current studies demonstrated that the deficiency of ATG7 by its shRNA dramatically reduced sphere formation and invasion in vitro, as well as lung metastasis in vivo in human invasive BC cells. Further studies indicated that the knockdown of ATG7 attenuated the expression of CD44 standard (CD44s), while ectopic introduction of CD44s, was capable of completely restoring sphere formation, invasion, and lung metastasis in T24T(shATG7) cells. Mechanistic studies revealed that ATG7 overexpression stabilized CD44s proteins accompanied with upregulating USP28 proteins. Upregulated USP28 was able to bind to CD44s and remove the ubiquitin group from CD44s’ protein, resulting in the stabilization of CD44s protein. Moreover, ATG7 inhibition stabilized AUF1 protein and thereby reduced tet1 mRNA stability and expression, which was able to demethylate usp28 promoter, reduced USP28 expression, finally promoting CD44s degradation. In addition, CD44s was defined to inhibit degradation of RhoGDIβ, which in turn promotes BC invasion. Our results demonstrate that CD44s is a key ATG7 downstream regulator of the sphere formation, invasion, and lung metastasis of BCs, providing significant insight into understanding the BC invasions, metastasis, and stem-like properties.

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Acknowledgements

We thank Dr. Martin Eilers from the Institute of Molecular Biology and Tumor Research, Marburg, Germany, and Dr. James M. Angelastro from the Department of Molecular Biosciences, University of California at Davis for their generous gifts of the Flag-tagged USP28 plasmid, the pcDNA3.1/NT-GFP-CD133, and pcDNA3.1/NT-GFP plasmids. We also thank Ms. Nedda Tichi for her critical reading of the manuscript. This work was partially supported by grants from NIH/NCI CA177665, CA165980, CA217923, and NIH/NIEHS ES000260.

Author contributions

CH, HH, and JZ conceived and designed the studies. HY, YL, JL, XC, HL, CH, and JZ detected the cells’ biological function, conducted the RT-PCR assays, carried out the Western Blot and luciferase reporter assays, and performed the statistical analysis. HY, ZT, JX, and XH carried out the animal studies. CH, JZ, and WD drafted the manuscript. All authors read and approved the final manuscript.

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Zhu, J., Huang, G., Hua, X. et al. CD44s is a crucial ATG7 downstream regulator for stem-like property, invasion, and lung metastasis of human bladder cancer (BC) cells. Oncogene 38, 3301–3315 (2019). https://doi.org/10.1038/s41388-018-0664-7

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