Porcupine (PORCN) is a membrane bound O-acyltransferase that is required for Wnt palmitoylation, secretion, and biologic activity. All evaluable human Wnts require PORCN for their activity, suggesting that inhibition of PORCN could be an effective treatment for cancers dependent on excess Wnt activity. In this study, we evaluated the PORCN inhibitor Wnt-C59 (C59), to determine its activity and toxicity in cultured cells and mice. C59 inhibits PORCN activity in vitro at nanomolar concentrations, as assessed by inhibition of Wnt palmitoylation, Wnt interaction with the carrier protein Wntless/WLS, Wnt secretion, and Wnt activation of b-catenin reporter activity. In mice, C59 displayed good bioavailability, as once daily oral administration was sufficient to maintain blood concentrations well above the IC 50 . C59 blocked progression of mammary tumors in MMTV-WNT1 transgenic mice while downregulating Wnt/b-catenin target genes. Surprisingly, mice exhibit no apparent toxicity, such that at a therapeutically effective dose there were no pathologic changes in the gut or other tissues. These results offer preclinical proof-of-concept that inhibiting mammalian Wnts can be achieved by targeting PORCN with small-molecule inhibitors such as C59, and that this is a safe and feasible strategy in vivo. Cancer Res; 73(2); 502-7. Ó2012 AACR.

Wnt/␤-catenin signaling is initiated at the cell surface by association of secreted Wnt with its receptors Frizzled (Fz) and low density lipoprotein receptor-related protein 5/6 (LRP5/6). The study of these molecular interactions has been a significant technical challenge because the proteins have been inaccessible in sufficient purity and quantity. In this report we describe insect cell expression and purification of soluble mouse Fz8 cysteine-rich domain and human LRP6 extracellular domain and show that they inhibit Wnt/␤-catenin signaling in cellular assays. We determine the binding affinities of Wnts and Dickkopf 1 (Dkk1) to the relevant co-receptors and reconstitute in vitro the Fz8 CRD⅐Wnt3a⅐LRP6 signaling complex. Using purified fragments of LRP6, we further show that Wnt3a binds to a region including only the third and fourth ␤-propeller domains of LRP6 (E3E4). Surprisingly, we find that Wnt9b binds to a different part of the LRP6 extracellular domain, E1E2, and we demonstrate that Wnt3a and Wnt9b can bind to LRP6 simultaneously. Dkk1 binds to both E1E2 and E3E4 fragments and competes with both Wnt3a and Wnt9b for binding to LRP6. The existence of multiple, independent Wnt binding sites on the LRP6 co-receptor suggests new possibilities for the architecture of Wnt signaling complexes and a model for broad-spectrum inhibition of Wnt/␤-catenin signaling by Dkk1.Since its discovery more than 30 years ago, the Wnt signaling pathway has captured the interest of researchers due to its role in development, progression of cancer, and self-renewal and differentiation of stem cells (1, 2). Wnt signaling is initiated at the cell surface where secreted Wnt glycoprotein forms a complex with the receptors Frizzled (Fz) 2 (3) and low density lipoprotein receptor-related proteins 5 or 6 (LRP5/6) (4, 5). This results in stabilization of intracellular ␤-catenin ("Wnt/␤-catenin pathway") and its translocation to the nucleus. Nuclear ␤-catenin then initiates T-cell factor-dependent transcription of downstream Wnt target genes (6, 7). The interactions of LRP5/6 and Fz with Wnt are critical for mediating Wnt/␤-catenin signaling (5,8,9). Underscoring the central role that these receptors play in Wnt signaling, suppression of either Fz or LRPs has been shown to generate phenotypes associated with Wnt mutations (10). Furthermore, mechanisms have evolved to regulate the Wnt⅐Fz⅐LRP complex through secreted proteins that interfere with these extracellular interactions. One such example is Dickkopf-1 (Dkk1), which inhibits Wnt signaling by binding to LRP6 (9,11,12). Dkk1 has been shown to play a crucial role in bone formation, vertebrate embryogenesis, and suppression of cancer cell proliferation (13-15). Therefore, Wnt ligands, receptors Fz and LRP, and their natural inhibitors are all of great therapeutic interest. Indeed, recent findings show that the soluble extracellular domain of Fz8 interferes with Wnt-driven tumor growth in vivo (16).The detailed molecular arrangement of the Wnt⅐Fz⅐LRP ternary complex at the cell surface is ...

Axon degeneration initiated by trophic factor withdrawal shares many features with programmed cell death, but many prior studies discounted a role for caspases in this process, particularly Caspase-3. Recently, Caspase-6 was implicated based on pharmacological and knockdown evidence, and we report here that genetic deletion of Caspase-6 indeed provides partial protection from degeneration. However, we find at a biochemical level that Caspase-6 is activated effectively only by Caspase-3 but not other “upstream” caspases, prompting us to revisit the role of Caspase-3. In vitro, we show that genetic deletion of Caspase-3 is fully protective against sensory axon degeneration initiated by trophic factor withdrawal, but not injury-induced Wallerian degeneration, and we define a biochemical cascade from pro-survival Bcl2 family regulators to Caspase-9, then Caspase-3, and then Caspase-6. Only low levels of active Caspase-3 appear to be required, helping explain why its critical role has been obscured in prior studies. In vivo, Caspase-3 and Caspase-6 knockout mice show a delay in developmental pruning of retinocollicular axons, thereby implicating both Caspase-3 and Caspase-6 in axon degeneration that occurs as a part of normal development.