title: Regulation of CD44E by DARPP-32-dependent activation of SRp20 splicing factor in gastric tumorigenesis.
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.
authors.affiliation: Department of Surgery, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: S
authors.initials: S
authors.lastname: Zhu
authors.affiliation: Department of Surgery, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Department of Surgery, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: A
authors.initials: A
authors.lastname: Katsha
authors.affiliation: Department of Surgery, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: J
authors.initials: J
authors.lastname: Hong
authors.affiliation: Department of Surgery, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: A
authors.initials: A
authors.lastname: Belkhiri
authors.affiliation: Department of Surgery, Vanderbilt University Medical Center, Nashville, TN, USA.|Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN, USA.|Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN, USA.
authors.forename: W
authors.initials: W
authors.lastname: El-Rifai
mesh_terms: D017398:Alternative Splicing / Q000235:genetics; D000818:Animals; D063646:Carcinogenesis / Q000235:genetics*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32 / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D018960:Hyaluronan Receptors / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D016601:RNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D000068103:Serine-Arginine Splicing Factors; D015398:Signal Transduction / Q000235:genetics; D013274:Stomach Neoplasms / Q000235:genetics* / Q000473:pathology; D023041:Xenograft Model Antitumor Assays
chemical_list: C497483:CD44 protein, human; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32; D018960:Hyaluronan Receptors; C497928:PPP1R1B protein, human; D016601:RNA-Binding Proteins; C491388:SRSF3 protein, human; D000068103:Serine-Arginine Splicing Factors