👤 D M Virshup

Wnt signaling regulates the balance between stemness and differentiation in multiple tissues and in cancer. RNF43-mutant pancreatic cancers are dependent on Wnt production, and pharmacologic blockade of the pathway, e.g., by PORCN inhibitors, leads to tumor differentiation. However, primary resistance to these inhibitors has been observed. To elucidate potential mechanisms, we performed in vivo CRISPR screens in PORCN inhibitor-sensitive RNF43-mutant pancreatic cancer xenografts. As expected, genes in the Wnt pathway whose loss conferred drug resistance were identified, including APC, AXIN1, and CTNNBIP1. Unexpectedly, the screen also identified the histone acetyltransferase EP300 (p300), but not its paralog, CREBBP (CBP). We found that EP300 is silenced due to genetic alterations in all the existing RNF43-mutant pancreatic cancer cell lines that are resistant to PORCN inhibitors. Mechanistically, loss of EP300 directly downregulated GATA6 expression, thereby silencing the GATA6-regulated differentiation program and leading to a phenotypic transition from the classical subtype to the dedifferentiated basal-like/squamous subtype of pancreatic cancer. EP300 mutation and loss of GATA6 function bypassed the antidifferentiation activity of Wnt signaling, rendering these cancer cells resistant to Wnt inhibition. Show less

The Wnt/beta-catenin signaling pathway is critical in both cellular proliferation and organismal development. However, how the beta-catenin degradation complex is inhibited upon Wnt activation remains unclear. Using a directed RNAi screen we find that protein phosphatase 1 (PP1), a ubiquitous serine/threonine phosphatase, is a novel potent positive physiologic regulator of the Wnt/beta-catenin signaling pathway. PP1 expression synergistically activates, and inhibition of PP1 inhibits, Wnt/beta-catenin signaling in Drosophila and mammalian cells as well as in Xenopus embryos. The data suggest that PP1 controls Wnt signaling through interaction with, and regulated dephosphorylation of, axin. Inhibition of PP1 leads to enhanced phosphorylation of specific sites on axin by casein kinase I. Axin phosphorylation markedly enhances the binding of glycogen synthase kinase 3, leading to a more active beta-catenin destruction complex. Wnt-regulated changes in axin phosphorylation, mediated by PP1, may therefore determine beta-catenin transcriptional activity. Specific inhibition of PP1 in this pathway may offer therapeutic approaches to disorders with increased beta-catenin signaling. Show less

Wnt signaling plays a key role in cell proliferation and development. Recently, casein kinase I (CKI) and protein phosphatase 2A (PP2A) have emerged as positive and negative regulators of the Wnt pathway, respectively. However, it is not clear how these two enzymes with opposing functions regulate Wnt signaling. Here we show that both CKI delta and CKI epsilon interacted directly with Dvl-1, and that CKI phosphorylated multiple components of the Wnt-regulated beta-catenin degradation complex in vitro, including Dvl-1, adenomatous polyposis coli (APC), axin, and beta-catenin. Comparison of peptide maps from in vivo and in vitro phosphorylated beta-catenin and axin suggests that CKI phosphorylates these proteins in vivo as well. CKI abrogated beta-catenin degradation in Xenopus egg extracts. Notably, CKI decreased, whereas inhibition of CKI increased, the association of PP2A with the beta-catenin degradation complex in vitro. Additionally, inhibition of CKI in vivo stabilized the beta-catenin degradation complex, suggesting that CKI actively destabilizes the complex in vivo. The ability of CKI to induce secondary body axes in Xenopus embryos was reduced by the B56 regulatory subunit of PP2A, and kinase-dead CKI epsilon acted synergistically with B56 in inhibiting Wnt signaling. The data suggest that CKI phosphorylates and destabilizes the beta-catenin degradation complex, likely through the dissociation of PP2A, providing a mechanism by which CKI stabilizes beta-catenin and propagates the Wnt signal. Show less

Wnt signaling increases beta-catenin abundance and transcription of Wnt-responsive genes. Our previous work suggested that the B56 regulatory subunit of protein phosphatase 2A (PP2A) inhibits Wnt signaling. Okadaic acid (a phosphatase inhibitor) increases, while B56 expression reduces, beta-catenin abundance; B56 also reduces transcription of Wnt-responsive genes. Okadaic acid is a tumor promoter, and the structural A subunit of PP2A is mutated in multiple cancers. Taken together, the evidence suggests that PP2A is a tumor suppressor. However, other studies suggest that PP2A activates Wnt signaling. We now show that the B56, A and catalytic C subunits of PP2A each have ventralizing activity in Xenopus embryos. B56 was epistatically positioned downstream of GSK3beta and axin but upstream of beta-catenin, and axin co-immunoprecipitated B56, A and C subunits, suggesting that PP2A:B56 is in the beta-catenin degradation complex. PP2A appears to be essential for beta-catenin degradation, since beta-catenin degradation was reconstituted in phosphatase-depleted Xenopus egg extracts by PP2A, but not PP1. These results support the hypothesis that PP2A:B56 directly inhibits Wnt signaling and plays a role in development and carcinogenesis. Show less