Control of Wnt Receptor Turnover by R-spondin-ZNRF3/RNF43 Signaling Module and Its Dysregulation in Cancer

Abstract

Aberrant activation of the Wnt/β-catenin pathway is frequently found in various cancers, often through mutations of downstream components. Inhibiting β-catenin signaling in tumors with downstream pathway mutations remains challenging, due to a lack of favorable targets. On the other hand, targeting upstream components of the Wnt pathway is rather straightforward. However, it is difficult to identify tumors addicted to autocrine or paracrine Wnt signaling. Discovery of the R-spondin-ZNRF3/RNF43 signaling module and its genetic alterations in cancers represents a breakthrough in this area. Membrane E3 ligase ZNRF3 and RNF43 are critical negative feedback regulators of the Wnt pathway, which function through promoting ubiquitination and degradation of Wnt receptors. R-spondin proteins (RSPO1-4) serve as natural antagonists of ZNRF3/RNF43. To maintain strong and sustained Wnt/β-catenin signaling, cancers need to overcome ZNRF3/RNF43-mediated feedback inhibition. Indeed, mutations of RNF43/ZNRF3 and recurrent translocations of RSPO2/RSPO3 have recently been identified in various cancers. Significantly, genetic alterations in RNF43/ZNRF3/RSPO2/RSPO3 have shown promise as predictive biomarkers in pre-clinical models for the efficacy of upstream Wnt inhibitors. In this review, we will discuss the biology of the R-spondin-ZNRF3/RNF43 signaling module, cancer-associated alterations of this signaling module, and their value as biomarkers to identify Wnt-addicted tumors.

Intrinsic disorder in spondins and some of their interacting partners

“…[32][33][34][35][36][37][38][39][40] These proteins have molecular masses of approximately 35 kDa and are characterized by the presence of 2 N-terminal furin-like repeats, which are necessary for Wnt signaling. R-spondins can enhance responses to low-dose Wnt protein and also serves as activators of a canonical Wnt signaling pathway, where they act as ligands for the LGR4-6 receptors.…”

“…55 Besides interaction with the LGR4-6 receptors, Rspo1 regulates the canonical Wnt/b-catenin dependent pathway and non-canonical Wnt signaling by acting as an inhibitor of a transmembrane E3 ubiquitin ligase, zinc and ring finger 3 (ZnRF3), and the E3 ubiquitin-protein ligase RING finger protein 43 (RNF43), which are critical negative feedback regulators of the Wnt pathway, which function through promoting ubiquitination and degradation of Wnt receptors, and thereby playing a crucial role in the control of cancer development. 38 Therefore, Rspo1 serves as an important member of the R-spondinZnRF3/RNF43 signaling module that plays a crucial role in the control of Wnt signaling. 38 In fact, RSpo1 can simultaneously bind to the extracellular domains of ZnRF3/RNF43 and LGR4/5.…”

“…38 Therefore, Rspo1 serves as an important member of the R-spondinZnRF3/RNF43 signaling module that plays a crucial role in the control of Wnt signaling. 38 In fact, RSpo1 can simultaneously bind to the extracellular domains of ZnRF3/RNF43 and LGR4/5. 56 Formation of this complex induces auto-ubiquitination and membrane clearance of ZnRF3/RNF43, leading to the increased levels of frizzled (Fzd).…”

“…The formation of the tripartite ZnRF3/RNF43-Rspo1-LGR4/5 complex is explained by the fact that Rspo1 utilizes its Fu2 domain for interaction with LGR4/5 and binds to ZnRF3/RNF43 via its Fu1 domain. 38 This tripartite complex represents a complex functional state of Rspo1, with LGR4/5 and ZnRF3/ RNF43 acting as the engagement and the efficacy receptors for Rspo1, respectively. 38 Furthermore, Rspo1 can be engaged in the physical binary interactions with the extracellular domains of frizzled-8 (Fzd8) 57 and low-density lipoprotein receptor-related protein 6 (LRP6), 58 which serves as the Wnt coreceptor, without formation of a ternary complex with these 2 proteins.…”

“…They are found in eukaryotic organisms and are grouped into different families, such as the R-spondin, the subcommissural organ (SCO)-spondin, the M-spondin (mindin), and the F-spondin. Spondins are engaged in various vital biological functions, such as regulators of Wnt signaling (R-spondins), [32][33][34][35][36][37][38][39][40] regulation of the developing skeleton, limb formation, and the maintenance of adult bone mass (R-spondins), 39,41 regulation of stem cells (R-spondin), 37,42,43 neuron/ glia interaction and neuronal differentiation and development (SCO-spondin), 44,45 interaction with the b-amyloid precursor protein (APP) and its controlled proteolysis (F-spondin), 46,47 regulation of the accurate path-finding of embryonic axons (F-spondin), 48 and promotion of the neurite outgrowth and inhibition of the angiogenesis (F-spondin and mindin). 48 Being the members of the TSP family, these proteins have complex modular structures.…”