Wnt Isoform-Specific Interactions with Coreceptor Specify Inhibition or Potentiation of Signaling by LRP6 Antibodies

Abstract

β-catenin-dependent Wnt signaling is initiated as Wnt binds to both the receptor FZD and coreceptor LRP5/6, which then assembles a multimeric complex at the cytoplasmic membrane face to recruit and inactivate the kinase GSK3. The large number and sequence diversity of Wnt isoforms suggest the possibility of domain-specific ligand-coreceptor interactions, and distinct binding sites on LRP6 for Wnt3a and Wnt9b have recently been identified in vitro. Whether mechanistically different interactions between Wnts and coreceptors might mediate signaling remains to be determined. It is also not clear whether coreceptor homodimerization induced extracellularly can activate Wnt signaling, as is the case for receptor tyrosine kinases. We generated monoclonal antibodies against LRP6 with the unexpected ability to inhibit signaling by some Wnt isoforms and potentiate signaling by other isoforms. In cell culture, two antibodies characterized further show reciprocal activities on most Wnts, with one antibody antagonizing and the other potentiating. We demonstrate that these antibodies bind to different regions of LRP6 protein, and inhibition of signaling results from blocking Wnt binding. Antibody-mediated dimerization of LRP6 can potentiate signaling only when a Wnt isoform is also able to bind the complex, presumably recruiting FZD. Endogenous autocrine Wnt signaling in different tumor cell lines can be either antagonized or enhanced by the LRP6 antibodies, indicating expression of different Wnt isoforms. As anticipated from the roles of Wnt signaling in cancer and bone development, antibody activities can also be observed in mice for inhibition of tumor growth and in organ culture for enhancement of bone mineral density. Collectively, our results indicate that separate binding sites for different subsets of Wnt isoforms determine the inhibition or potentiation of signaling conferred by LRP6 antibodies. This complexity of coreceptor-ligand interactions may allow for differential regulation of signaling by Wnt isoforms during development, and can be exploited with antibodies to differentially manipulate Wnt signaling in specific tissues or disease states.

LRP5/6 directly bind to Frizzled and prevent Frizzled-regulated tumour metastasis

“…Increased autocrine canonical Wnt ligands and LRP5/6 receptors at the cell surface have been associated with tumour development and progression 3,4,7,39 . These previous observations have provided rationale for the development of anti-LRP6 antibodies aimed at inhibiting LRP6 function 40,41 . According to our present LRP5/6-Frz interaction model, the application of anti-LRP6 antibodies that block Wnt ligand-mediated b-catenin signalling requires possible reconsideration because competitive interaction of these antibodies might separate Frz from LRP6 to activate the Frz-regulated non-canonical pathway and result in tumour metastasis.…”

Frizzled and LRP5/6 Receptors for Wnt/ -Catenin Signaling

“…Unexpectedly, however, Wnt3a and Wnt9b were shown to preferentially interact with P3E3 -P4E4 and P1E1-P2E2, respectively, and a Wnt3a -Wnt9b -LRP6 (extracellular domain) complex could be detected in vitro , suggesting a possibility that a single LRP6 may engage two different Wnt proteins simultaneously. Furthermore, functional-blocking monoclonal antibodies (mAbs) against epitopes in P1 and P3, respectively, show a distinct inhibition profile toward different Wnt proteins, presumably via blocking Wnt binding to either P1 or P3 (Ettenberg et al 2010;Gong et al 2010). These data infer that many Wnts (Wnt1, Wnt2, Wnt2b, Wnt6, Wnt8a, Wnt9a, Wnt9b, and Wnt10a) interact with P1, whereas Wnt3 and Wnt3a prefer P3 ( Fig.…”

“…2A). Other Wnts (Wnt7a, Wnt7b, and Wnt10a) could not be assigned into either group because they were not inhibited by the mAbs alone or in combination (Gong et al 2010), raising the possibility that these Wnts may bind to regions outside P1 and P3. The remaining Wnts have not been tested in these assays.…”

Targeting Wnt-driven cancer through the inhibition of Porcupine by LGK974

“…Indeed, multiple attempts to develop antibodies against key Wnt coreceptors, including LRP6 and FZD, have been reported (11,35,36) with varying degrees of success. A PORCN inhibitor, IWP2, has been reported to show good potency and specificity in inhibiting Wnt signaling in vitro (37,38).…”