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Mtorc1 Signaling Suppresses Wnt/Β-Catenin Signaling Through DVL-Dependent Regulation of Wnt Receptor FZD Level

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Mtorc1 Signaling Suppresses Wnt/Β-Catenin Signaling Through DVL-Dependent Regulation of Wnt Receptor FZD Level mTORC1 signaling suppresses Wnt/β-catenin signaling through DVL-dependent regulation of Wnt receptor FZD level Hao Zenga,BoLua, Raffaella Zamponia, Zinger Yanga, Kristie Wetzela, Joseph Loureiroa, Sina Mohammadia, Martin Beibelb, Sebastian Berglingb, John Reece-Hoyesa, Carsten Russa, Guglielmo Romab, Jan S. Tchorzb, Paola Capodiecia, and Feng Conga,1 aChemical Biology and Therapeutics, Novartis Institutes for Biomedical Research, Novartis Pharma AG, Cambridge, MA 02139; and bChemical Biology and Therapeutics, Novartis Institutes for Biomedical Research, Novartis Pharma AG, 4056 Basel, Switzerland Edited by Roeland Nusse, Stanford University School of Medicine, Stanford, CA, and approved September 14, 2018 (received for review May 18, 2018) Wnt/β-catenin signaling plays pivotal roles in cell proliferation and expected that other signaling pathways might impact Wnt tissue homeostasis by maintaining somatic stem cell functions. The signaling through regulation of FZD turnover. Nevertheless, mammalian target of rapamycin (mTOR) signaling functions as an whether and how other pathways control FZD turnover is integrative rheostat that orchestrates various cellular and meta- unknown. bolic activities that shape tissue homeostasis. Whether these two The mammalian target of rapamycin complex 1 (mTORC1) fundamental signaling pathways couple to exert physiological signaling coordinates anabolic and catabolic metabolism to functions still remains mysterious. Using a genome-wide CRISPR- support cell growth and tissue homeostasis (11–13). The Cas9 screening, we discover that mTOR complex 1 (mTORC1) sig- mTORC1 activity is controlled by various upstream signals, in- naling suppresses canonical Wnt/β-catenin signaling. Deficiency in cluding growth factors, nutrients, and stress. Numerous growth tuberous sclerosis complex 1/2 (TSC1/2), core negative regula- factor and mitogen-dependent pathways converge on Tuberous tors of mTORC1 activity, represses Wnt/β-catenin target gene Sclerosis Complex (TSC) to impact mTORC1 signaling trans- expression, which can be rescued by RAD001. Mechanistically, duction (12). TSC is composed of TSC1, TSC2, and TBC1D7, mTORC1 signaling regulates the cell surface level of Wnt receptor among which TSC2 is the catalytic subunit with GTPase- Frizzled (FZD) in a Dishevelled (DVL)-dependent manner by influ- activating protein (GAP) activity toward the small GTPase encing the association of DVL and clathrin AP-2 adaptor. Sustained Rheb which binds and directly activates mTORC1 activity β mTORC1 activation impairs Wnt/ -catenin signaling and causes loss through unclear mechanisms (14). The physical interaction of of stemness in intestinal organoids ex vivo and primitive intestinal β – TSC1 and TSC2 is also essential for maintaining the protein progenitors in vivo. Wnt/ -catenin dependent liver metabolic stability of TSC2 (15, 16). Activated mTORC1 phosphorylates a zonation gene expression program is also down-regulated by mTORC1 variety of downstream effectors, such as S6K, 4EBP1, ULK1, activation. Our study provides a paradigm that mTORC1 signaling cell TFEB, and Grb10, and consequently controls a number of cel- autonomously regulates Wnt/β-catenin pathway to influence stem lular processes, including protein synthesis, autophagy, and cell maintenance. CRISPR screen | mTORC1 signaling | intestinal stem cells | Frizzled | Significance organoids The Wnt/β-catenin signaling pathway plays prominent roles nt signaling is critical for development and tissue ho- during embryonic development and adult tissue homeostasis Wmeostasis, and its deregulation has been implicated in a by maintaining somatic stem cell functions. The mammalian variety of diseases (1–4). The canonical Wnt/β-catenin signaling target of rapamycin complex 1 (mTORC1) signaling pathway pathway is initiated upon binding of Wnt ligand to the seven-pass has also been implicated in regulating stem cell functions in transmembrane receptor FZD and the single-pass low-density multiple tissue types. However, the crosstalk between these lipoprotein receptor-related protein 5 or 6 (LRP5/6) on the cell two pathways remains largely unclear. Herein, using in vitro surface. This event further triggers phosphorylation of LRP5/6, in- cell lines, ex vivo organoids, and an in vivo mouse model, we activation of β-catenin destruction complex, and accumulation of made striking findings in support of a paradigm that mTORC1 β active β-catenin and partnering with TCF/LEF transcription factors, signaling cell autonomously suppresses Wnt/ -catenin signal- leading to the activation of Wnt/β-catenin target gene expression ing through down-regulating the Wnt receptor FZD level to (2, 5). Although the core Wnt/β-catenin pathway components have influence stem cell functions, with implications in the aging been extensively investigated over the past decades, additional process. contributors and the crosstalk with other signaling pathways await Author contributions: H.Z. and F.C. designed research; H.Z., B.L., R.Z., K.W., S.M., J.R.-H., further exploration. C.R., G.R., J.S.T., and P.C. performed research; H.Z., Z.Y., J.L., M.B., and S.B. analyzed data; As the core receptor of Wnt proteins, the strength of the Wnt/ and H.Z. and F.C. wrote the paper. β -catenin signaling pathway is exquisitely sensitive to the level of Conflict of interest statement: All authors were employees of Novartis Pharma AG when FZD on the plasma membrane. Regulation of FZD turnover has the research was conducted. emerged as a key regulatory node of the Wnt signaling pathway. This article is a PNAS Direct Submission. For instance, FZD is degraded by membrane E3 ligase Published under the PNAS license. ZNRF3 and RNF43, and this activity of ZNRF3 and RNF43 is Data deposition: The raw RNA-seq reads are available in the NCBI Short Read Archive blocked by secreted R-spondin proteins (6, 7). Mutations of under the accession no. SRP140499 (for liver RNA-seq samples) and no. SRP155719 (for RNF43 and ZNRF3 and amplification of R-spondin genes are intestinal organoid samples). found in various tumors (8, 9). Later, DVL was identified to act 1To whom correspondence should be addressed. Email: [email protected]. as a negative regulator of FZD turnover, largely through re- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cruitment of ZNRF3/RNF43 to FZD receptors (10). Consider- 1073/pnas.1808575115/-/DCSupplemental. ing the critical role of FZD in Wnt signaling, it would be Published online October 8, 2018. E10362–E10369 | PNAS | vol. 115 | no. 44 www.pnas.org/cgi/doi/10.1073/pnas.1808575115 Downloaded by guest on September 24, 2021 growth factor signaling, among others (12). Recent studies have wide CRISPR-Cas9 screening (Fig. 1A). First, we engineered − − highlighted the roles of mTORC1 signaling in stem cell (SC) Tp53 / mouse embryonic fibroblast (MEF) cells to express functions and aging (11–13). For example, Tsc1 deletion in he- Cas9 and Super-Topflash green fluorescent protein (STF-GFP) matopoietic stem cells (HSCs) led to elevated mTORC1 activity reporter (MEF–Cas9–STF-GFP). The MEF–Cas9–STF-GFP and concomitant loss of stemness in HSCs (17). Despite some cells were then infected with a pooled lentiviral sgRNA library similarities in regulating diverse fundamental cellular processes, covering 18,360 genes (5 sgRNAs per gene) at a sufficiently low whether Wnt and mTORC1 signaling pathways couple to exert multiplicity of infection to bias for integration of a single len- physiological functions is still unclear. tiviral sgRNA cassette per cell. After puromycin selection and In this study, we discovered a surprising inhibitory effect of multiple passages to allow gene editing to take place, STF-GFP mTORC1 signaling on Wnt/β-catenin signaling through an un- reporter activity was induced by Wnt3a. Cells were then sorted biased CRISPR screen. Mechanistic studies revealed that for populations with the lowest 25% (low GFP) and the mTORC1 signaling decreases the expression of membrane FZD highest 25% (high GFP) of the GFP signal and subjected to through enhancing DVL-mediated FZD turnover. Activation of next-generation sequencing (NGS) and informatics analysis to mTORC1 signaling in vivo inhibits Wnt signaling in the intestine identify positive and negative regulators of Wnt/β-catenin and liver. Our findings provide a mechanistic connection of these signaling, respectively (Fig. 1A). two fundamental signaling pathways and enhance our un- Inspection of the list of genes targeted by sgRNAs that are derstanding of these pathways in stem cell maintenance and recovered from low GFP and high GFP populations revealed tissue homeostasis. several genes encoding known core positive (Lrp6 and Ctnnb1) and negative regulators (Znrf3 and Apc) of Wnt/β-catenin Results pathway (Fig. 1B). Interestingly, sgRNAs targeting Tsc1/2 which A Genome-Wide CRISPR-Cas9 Screening Reveals that mTORC1 Signaling encode core negative regulators of the mTORC1 signaling and Negatively Modulates Wnt/β-Catenin Signaling. We set out to identify Mtor itself were identified in the low GFP and high GFP pop- regulators of Wnt/β-catenin signaling by carrying out a genome- ulations (Fig. 1B), respectively, suggesting a suppressive role of AB E Tp53 P Tsc1 Tsc2 Mtor Gusb Lrp6 Tsc2 Tsc2 Ctnnb1 Axin2 Znrf3 Apc C Tsc2 Tsc2 F Tsc2 Tsc2 Tsc2 Tsc2 M D Tsc2 Tsc2 Fig. 1. A genome-wide CRISPR-Cas9 screening reveals that mTORC1 signaling negatively modulates Wnt/β-catenin signaling. (A) Workflow of the CRISPR- Cas9 screening process. STF-GFP
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