GSK3β is a multifunctional serine/threonine kinase that regulates various cellular pathways, depending on its substrates for phosphorylation. It is evident that regulation of Wnt/β-catenin signaling is only one of its diverse functions. Since oncogenic transcription factors (e.g., c-Jun, c-Myc) and proto-oncoproteins (i.e., β-catenin, Gli proteins) are putative GSK3β substrates for phosphorylation-dependent inactivation, it is hypothesized that GSK3β interferes with cellular neoplastic transformation and tumor development, as exemplified by its activity in Wnt/β-catenin signaling. However, only a few studies have addressed its role(s) in human cancer, and these studies have reported differing effects of GSK3β on cancer cells. Using GSK3β deficient mouse embryonic fibroblasts, it was shown that GSK3β plays a crucial role in cell survival mediated by the nuclear factor-kappaB (NF-κB) pathway (Nature 2000; 406:86-90). Interestingly, we have recently shown that the Wnt/β-catenin and NF-κB pathways were co-activated in colorectal cancer by dysregulation in the ubiquitin system (J Natl Cancer Inst 2004; 96:1161-70). Thus, these observations bring forward apparently opposing notions regarding the functions of GSK3β in neoplastic cells on the one hand, removing a neoplastic trigger by phosphorylation-dependent degradation of β-catenin oncoprotein in the ubiquitin system, and on the other, contributing to a cell proliferation and survival pathways by regulating NF-κB. The present study was therefore undertaken to clarify the role of GSK3β in cancer by analyzing expression and activity of this kinase in colon cancer cells and clinical colorectal cancers and by investigating its effects on cancer cells. In colon cancer cell lines and colorectal cancer patients, levels of GSK3β expression and its active form were higher in tumor cells than in their normal counterparts; these findings were independent of nuclear accumulation of β-catenin oncoprotein in the tumor cells. Inhibition of GSK3β activity by its Ser9 phosphorylation was defective in colorectal cancers but preserved in non-neoplastic cells and tissues. Strikingly, inhibition of GSK3β activity by chemical inhibitors and its expression by RNA interference targeting GSK3β induced apoptosis and attenuated proliferation of colon cancer cells in vitro. Our findings demonstrate an unrecognized role of GSK3β in tumor cell survival and proliferation and warrant proposing this kinase as a novel and potential therapeutic target in colorectal cancer.

Although constitutive activation of beta-catenin/Tcf signalling is implicated in the development of human cancers, the mechanisms by which the beta-catenin/Tcf pathway promotes tumorigenesis are incompletely understood. Messenger RNA turnover has a major function in regulating gene expression and is responsive to developmental and environmental signals. mRNA decay rates are dictated by cis-acting elements within the mRNA and by trans-acting factors, such as RNA-binding proteins (reviewed in refs 2, 3). Here we show that beta-catenin stabilizes the mRNA encoding the F-box protein betaTrCP1, and identify the RNA-binding protein CRD-BP (coding region determinant-binding protein) as a previously unknown target of beta-catenin/Tcf transcription factor. CRD-BP binds to the coding region of betaTrCP1 mRNA. Overexpression of CRD-BP stabilizes betaTrCP1 mRNA and elevates betaTrCP1 levels (both in cells and in vivo), resulting in the activation of the Skp1-Cullin1-F-box protein (SCF)(betaTrCP) E3 ubiquitin ligase and in accelerated turnover of its substrates including IkappaB and beta-catenin. CRD-BP is essential for the induction of both betaTrCP1 and c-Myc by beta-catenin signalling in colorectal cancer cells. High levels of CRD-BP that are found in primary human colorectal tumours exhibiting active beta-catenin/Tcf signalling implicates CRD-BP induction in the upregulation of betaTrCP1, in the activation of dimeric transcription factor NF-kappaB and in the suppression of apoptosis in these cancers.

Purpose: Glycogen synthase kinase-3β (GSK3β) regulates multiple cell signaling pathways and has been implicated in glucose intolerance, neurodegenerative disorders, and inflammation. We investigated the expression, activity, and putative pathologic role of GSK3β in gastrointestinal, pancreatic, and liver cancers. Experimental Design: Colon, stomach, pancreatic, and liver cancer cell lines; nonneoplastic HEK293 cells; and matched pairs of normal and tumor tissues of stomach and colon cancer patients were examined for GSK3β expression and its phosphorylation at serine 9 (inactive form) and tyrosine 216 (active form) by Western immunoblotting and for GSK3β activity by in vitro kinase assay. The effects of small-molecule GSK3β inhibitors and of RNA interference on cell survival, proliferation, and apoptosis were examined in vitro and on human colon cancer cell xenografts in athymic mice. The effects of GSK3β inhibition on human telomerase reverse transcriptase (hTERT) expression and telomerase activity were compared between colon cancer and HEK293 cells. Results: Cancer cell lines and most cancer tissues showed increased GSK3β expression and increased tyrosine 216 phosphorylation and activity but decreased serine 9 phosphorylation compared with HEK293 cells and nonneoplastic tissues. Inhibition of GSK3β resulted in attenuated cell survival and proliferation and increased apoptosis in most cancer cell lines and in HT-29 xenografts in rodents but not in HEK293 cells. GSK3β inhibition in colon cancer cells was associated with decreased hTERT expression and telomerase activity. Conclusion:The results indicate that deregulated GSK3β sustains gastrointestinal cancer cells survival through modulation of hTERT and telomerase. (Clin Cancer Res 2009;15(22):6810-9)