New regulatory mechanisms of WNT signaling in development, stem cells and cancer

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

New regulatory mechanisms in WNT signaling: Through forward genetics screens in haploid human cells harboring a fluorescent reporter of WNT signaling, we discovered new regulators of ligand-induced signaling and regulators selectively required to sustain hyperactive, oncogenic signaling. This study (Lebensohn et al., eLife 2016) comprised seven genome-wide screens systematically interrogating the pathway, including screens for positive, negative and attenuating regulators of ligand-induced signaling, as well as suppressor screens following disruption of key regulators commonly mutated in WNT-driven tumors, such as the tumor suppressor APC. A comparative analysis of the screens revealed new regulatory mechanisms for ligand reception, signal transduction and transcriptional activation, as well as new gene regulatory elements (Patel, Lebensohn et al., PLoS One 2019). Our current goal is to elucidate the molecular underpinnings of these new regulatory mechanisms, understand their physiological functions and evaluate their potential as therapeutic targets. The following research projects are aimed at elucidating some of these new regulatory mechanisms and evaluating their therapeutic potential. 1) The transcription factor TFAP4 is a new limiting component of the WNT signaling pathway. Our genetic screens uncovered a basic helix-loop-helix leucine zipper transcription factor called TFAP4 as a potential regulator of WNT signaling (Lebensohn et al., eLife 2016). We have shown that TFAP4 acts downstream of b-catenin, the principal transcriptional co-activator in the WNT pathway, and is a limiting component for signaling activity. Furthermore, excess TFAP4 can promote ectopic activation of WNT signaling during Xenopus laevis development, causing the formation of a secondary body axis. Interestingly, TFAP4 expression is tightly linked to malignancy in gastrointestinal cancers: TFAP4 is upregulated in colorectal cancer, where it mediates epithelial-mesenchymal transition (EMT) and metastasis, and its down-regulation in gastric cancer cells inhibits proliferation, induces cell cycle arrest and promotes apoptosis. We are dissecting the molecular mechanism by which TFAP4 regulates WNT signaling, and evaluating its potential as a therapeutic target. 2) A new function of the b-catenin destruction complex regulating WNT signaling through the ubiquitin ligase HUWE1. HUWE1 is a HECT-domain ubiquitin ligase involved in dozens of cellular processes through the ubiquitination of diverse substrates. Both oncogenic and tumor suppressive functions have been ascribed to HUWE1. HUWE1 has been postulated as a negative regulator of WNT signaling through at least two distinct mechanisms. However, in our unbiased forward genetic screen for mediators of hyperactive WNT signaling induced by loss of the b-catenin destruction complex kinase casein kinase 1a, we identified HUWE1 as a positive regulator of the WNT pathway (Lebensohn et al., eLife 2016). We also demonstrated that HUWE1 potentiates WNT signaling in cells and in Xenopus laevis embryos. We have now found that HUWE1 promotes WNT/b-catenin signaling by regulating b-catenin sub-cellular localization. This mechanism is independent of the control of b-catenin protein stability. Potentiation of WNT signaling by HUWE1 requires its ubiquitin ligase activity and a subset of b-catenin destruction complex components. These results reveal a new role for some destruction complex components in regulating b-catenin sub-cellular localization and WNT signaling downstream of HUWE1, distinct from their established activity in controlling b-catenin stability. New regulatory mechanisms in R-spondin signaling: Some WNT responses during embryonic development and in stem cell compartments depend on a second signal provided by the R-spondin family of secreted growth factors, only present in vertebrates. R-spondins are key regulators of WNT signaling strength, but the mechanisms by which they transduce signals are not fully understood, and how the four different members of the family control distinct physiological functions is unknown. We discovered that R-spondins 2 and 3 can uniquely potentiate WNT signaling in cells lacking their only known cell-surface receptors, LGRs 4-6, through an alternative interaction with heparan sulfate proteoglycans (HSPGs) (Lebensohn & Rohatgi, eLife 2018; Dubey et al., eLife 2020, reviewed in Lebensohn et al. Curr Top Dev Biol 2022). This finding is transformative because LGRs were thought to be required to transduce all R-spondin signals and hence determine their site of action, but now we know that R-spondins can also signal in tissues where LGRs are not expressed. The following research projects are aimed at elucidating the molecular mechanisms and physiological functions of HSPG-dependent signaling by R-spondins. 3) Molecular mechanisms of LGR-independent signaling by R-spondins. A central question regarding any signal transduction mechanism is how binding of the ligand to the receptor communicates the signal across the plasma membrane and into the cell. In the prevailing model, simultaneous binding of R-spondins to their LGR receptors and to the transmembrane ubiquitin ligases ZNRF3 or RNF43 triggers their internalization. This prevents ZNRF3 and RNF43 from targeting WNT receptors for ubiquitin-mediated degradation, thus increasing their abundance on the cell surface and enhancing sensitivity to WNT ligands. We are investigating the molecular mechanisms whereby R-spondins transduce signals in the absence of LGRs through their alternative HSPG receptors to potentiate WNT signaling. 4) Physiological roles for LGR-dependent and LGR-independent signaling by R-spondins during embryonic development and in stem cells. Gene amplifications followed by neo- or sub-functionalization of different gene family members underlie much of the functional diversification generated during evolution. Yet, paralogues are often deemed to be redundant, and drastically different phenotypes resulting from their loss are simply attributed to tissue-specific patterns of expression. This is the prevailing view regarding the four members of the R-spondin family, which potentiate WNT signaling during embryonic development and in adult tissue stem cells. However, the markedly distinct phenotypes caused by the loss of different R-spondins or their LGR receptors could also be due to intrinsic differences in the capacity of these R-spondins to signal through LGR-dependent and HSPG-dependent mechanisms. We are using transgenic mouse models to investigating in what contexts these two modes of signaling are used during development, tissue homeostasis and regeneration. We are also trying to understand what unique properties make LGR-dependent and HSPG-dependent R-spondin signaling specifically suited to their physiological functions.