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Regulation of CD44E by DARPP-32-dependent activation of SRp20 splicing factor in gastric tumorigenesis

Oncogene volume 35, pages 1847–1856 (2016)Cite this article

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Abstract

CD44E is a frequently overexpressed variant of CD44 in gastric cancer. Mechanisms that regulate CD44 splicing and expression in gastric cancer remain unknown. Herein, we investigated the role of DARPP-32 (dopamine and cyclic adenosine monophosphate-regulated phosphoprotein, Mr 32000) in promoting tumor growth through regulation of CD44 splicing. Using western blot and quantitative real-time PCR analysis, our results indicated that knockdown of endogenous DARPP-32 markedly reduces the expression of CD44 V8-V10 (CD44E). Using a quantitative splicing luciferase reporter system, we detected a significant increase in the reporter activity following DARPP-32 overexpression (P<0.001). Conversely, knocking down endogenous DARPP-32 significantly attenuated the splicing activity (P<0.001). Further experiments showed that DARPP-32 regulates the expression of SRp20 splicing factor and co-exists with it in the same protein complex. Inhibition of alternative splicing with digitoxin followed by immunoprecipitation and immunoblotting indicated that DARPP-32 has an important role in regulating SRp20 protein stability. The knockdown of endogenous DARPP-32 confirmed that DARPP-32 regulates the SRp20-dependent CD44E splicing. Using tumor xenograft mouse model, knocking down endogenous DARPP-32 markedly reduced SRp20 and CD44E protein levels with a decreased tumor growth. The reconstitution of SRp20 expression in these cells rescued tumor growth. In addition, we also demonstrated frequent co-overexpression and positive correlation of DARPP-32, SRp20 and CD44E expression levels in human gastric primary tumors. Our novel findings establish for the first time the role of DARPP-32 in regulating splicing factors in gastric cancer cells. The DARPP-32–SRp20 axis has a key role in regulating the CD44E splice variant that promotes gastric tumorigenesis.

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Acknowledgements

This study was supported by grants from the National Institutes of Health (R01CA93999); Vanderbilt SPORE in Gastrointestinal Cancer (P50 CA95103); Vanderbilt Ingram Cancer Center (P30 CA68485); the Vanderbilt Digestive Disease Research Center (DK058404), and the Department of Veterans Affairs. The contents of this work are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health, Department of Veterans Affairs or Vanderbilt University.

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Authors and Affiliations

  1. Department of Surgery, Vanderbilt University Medical Center, Nashville, TN, USA

    S Zhu, Z Chen, A Katsha, J Hong, A Belkhiri & W El-Rifai

  2. Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN, USA

    W El-Rifai

  3. Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN, USA

    W El-Rifai

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  1. S Zhu
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Correspondence to W El-Rifai.

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Zhu, S., Chen, Z., Katsha, A. et al. Regulation of CD44E by DARPP-32-dependent activation of SRp20 splicing factor in gastric tumorigenesis. Oncogene 35, 1847–1856 (2016). https://doi.org/10.1038/onc.2015.250

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