RACK1 suppresses gastric tumorigenesis by stabilizing the β-catenin destruction complex.
ABSTRACT Dysregulation of Wnt signaling has been involved in gastric tumorigenesis by mechanisms that are not fully understood. The receptor for activated protein kinase C (RACK1, GNB2L1) is involved in development of different tumor types, but its expression and function have not been investigated in gastric tumors.
We analyzed expression of RACK1 in gastric tumor samples and their matched normal tissues from 116 patients using immunohistochemistry. Effects of knockdown with small interfering RNAs or overexpression of RACK1 in gastric cancer cell lines were evaluated in cell growth and tumor xenograft. RACK1 signaling pathways were investigated in cells and zebrafish embryos using immunoblot, immunoprecipitation, microinjection, and in situ hybridization assays.
Expression of RACK1 was reduced in gastric tumor samples and correlated with depth of tumor infiltration and poor differentiation. Knockdown of RACK1 in gastric cancer cells accelerated their anchorage-independent proliferation in soft agar, whereas overexpression of RACK1 reduced their tumorigenicity in nude mice. RACK1 formed a complex with glycogen synthase kinase Gsk3β and Axin to promote the interaction between Gsk3β and β-catenin and thereby stabilized the β-catenin destruction complex. On stimulation of Wnt3a, RACK1 repressed Wnt signaling by inhibiting recruitment of Axin by Dishevelled 2 (Dvl2). Moreover, there was an inverse correlation between expression of RACK1 and localization of β-catenin to the cytoplasm/nucleus in human gastric tumor samples.
RACK1 negatively regulates Wnt signaling pathway by stabilizing the β-catenin destruction complex and act as a tumor suppressor in gastric cancer cells.
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ABSTRACT: Studies have shown that fish oils, containing n-3 fatty acids, have protective effects against ischemia-induced, fatal cardiac arrhythmias in animals and perhaps in humans. In this study we used the whole-cell voltage-clamp technique to assess the effects of dietary, free long-chain fatty acids on the Na+ current (INa,alpha) in human embryonic kidney (HEK293t) cells transfected with the alpha-subunit of the human cardiac Na+ channel (hH1alpha). Extracellular application of 0.01 to 30 microM eicosapentaenoic acid (EPA, C20:5n-3) significantly reduced INa,alpha with an IC50 of 0.51 +/- 0.06 microM. The EPA-induced suppression of INa,alpha was concentration- and voltage-dependent. EPA at 5 microM significantly shifted the steady-state inactivation relationship by -27.8 +/- 1.2 mV (n = 6, P < 0.0001) at the V1/2 point. In addition, EPA blocked INa,alpha with a higher "binding affinity" to hH1alpha channels in the inactivated state than in the resting state. The transition from the resting state to the inactivated state was markedly accelerated in the presence of 5 microM EPA. The time for 50% recovery from the inactivation state was significantly slower in the presence of 5 microM EPA, from 2.1 +/- 0.8 ms for control to 34.8 +/- 2.1 ms (n = 5, P < 0.001). The effects of EPA on INa,alpha were reversible. Furthermore, docosahexaenoic acid (C22:6n-3), alpha-linolenic acid (C18:3n-3), conjugated linoleic acid (C18:2n-7), and oleic acid (C18:1n-9) at 5 microM and all-trans-retinoic acid at 10 microM had similar effects on INa,alpha as EPA. Even 5 microM of stearic acid (C18:0) or palmitic acid (C16:0) also significantly inhibited INa, alpha. In contrast, 5 microM EPA ethyl ester did not alter INa,alpha (8 +/- 4%, n = 8, P > 0.05). The present data demonstrate that free fatty acids suppress INa,alpha with high "binding affinity" to hH1alpha channels in the inactivated state and prolong the duration of recovery from inactivation.Proceedings of the National Academy of Sciences 03/1998; 95(5):2680-5. · 9.74 Impact Factor
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ABSTRACT: Axin promotes the phosphorylation of beta-catenin by GSK-3beta, leading to beta-catenin degradation. Wnt signals interfere with beta-catenin turnover, resulting in enhanced transcription of target genes through the increased formation of beta-catenin complexes containing TCF transcription factors. Little is known about how GSK-3beta-mediated beta-catenin turnover is regulated in response to Wnt signals. We have explored the relationship between Axin and Dvl-2, a member of the Dishevelled family of proteins that function upstream of GSK-3beta. Expression of Dvl-2 activated TCF-dependent transcription. This was blocked by co-expression of GSK-3beta or Axin. Expression of a 59 amino acid GSK-3beta-binding region from Axin strongly activated transcription in the absence of an upstream signal. Introduction of a point mutation into full-length Axin that prevented GSK-3beta binding also generated a transcriptional activator. When co-expressed, Axin and Dvl-2 co-localized within expressing cells. When Dvl-2 localization was altered using a C-terminal CAAX motif, Axin was also redistributed, suggesting a close association between the two proteins, a conclusion supported by co-immunoprecipitation data. Deletion analysis suggested that Dvl-association determinants within Axin were contained between residues 603 and 810. The association of Axin with Dvl-2 may be important in the transmission of Wnt signals from Dvl-2 to GSK-3beta.The EMBO Journal 06/1999; 18(10):2823-35. · 9.82 Impact Factor
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ABSTRACT: Wnt signaling has an important role in both embryonic development and tumorigenesis. beta-Catenin, a key component of the Wnt signaling pathway, interacts with the TCF/LEF family of transcription factors and activates transcription of Wnt target genes. Here, we identify a novel beta-catenin-interacting protein, ICAT, that was found to inhibit the interaction of beta-catenin with TCF-4 and represses beta-catenin-TCF-4-mediated transactivation. Furthermore, ICAT inhibited Xenopus axis formation by interfering with Wnt signaling. These results suggest that ICAT negatively regulates Wnt signaling via inhibition of the interaction between beta-catenin and TCF and is integral in development and cell proliferation.Genes & Development 08/2000; 14(14):1741-9. · 12.44 Impact Factor