Published OnlineFirst August 20, 2019; DOI: 10.1158/0008-5472.CAN-19-1362 Cancer Review Research Wnt Signaling in Cancer: Not a Binary ON:OFF Switch Dustin J. Flanagan1, Elizabeth Vincan2,3, and Toby J. Phesse4,5 Abstract In the March 1 issue of Cancer Research, we identified the polyposis coli (Apc) mutation. Apc is located in the cytoplasm Wnt receptor Fzd7 as an attractive therapeutic target for the downstream of Fzd7 in the Wnt signaling cascade and APC treatment of gastric cancer. In summary, we showed that mutations activate Wnt/b-catenin signaling, therefore, this pharmacological inhibition of Wnt receptors, or genetic dele- result seems counterintuitive. Here, we analyze this result in tion of Fzd7, blocks the initiation and growth of gastric tumors. greater detail in the context of current knowledge of Wnt Inhibiting Fzd receptors, specifically Fzd7, inhibits the growth signaling and discuss the wider implications of this aspect of of gastric cancer cells even in the presence of adenomatous Wnt signaling in other cancers. Regulation of Wnt Signaling Intracellularly including the ligand, receptors, and intracellular transduction and at the Plasma Membrane components (6, 7). In addition to mutations, epigenetic changes are also observed in gastric tumors to Wnt inhibitors such as sFRP The Wnt signaling pathway regulates many cell functions, (binds directly to Wnt ligands) and Dkk1 (binds to and inhibits including proliferation, migration, apoptosis, and differentia- Lrp5/6 receptors), resulting in activation of Wnt signaling at the tion (1). It is essential during embryonic development and also level of the receptor/ligand (1). Indeed, RNF43, an E3 ubiquitin in homeostasis of several adult tissues, including the GI tract (2, 3), ligase that turns over Fzd on the cell surface (8), is mutated in liver, breast, and skin (1), and is deregulated in many cancers, approximately 18% of human gastric tumors, whereas many Wnt including colon, gastric, breast, and liver (4). Wnt ligands are ligands are overexpressed (7). This is around the same frequency secreted glycoproteins that bind to a "U" shaped pocket in the (18%) observed for APC mutations in gastric cancer, approx- dimer of Fzd receptors via a lipid modification of palmitoleic acid imately 37% of which also contain an RNF43 mutation, indicat- to form a receptor complex with co-receptors, including Lrp5/6 ing compound activation of Wnt signaling in the same tumor (7). and Lgr4/5 (1). This complex interacts with components of a This observation that Wnt signaling is regulated intracellularly cytoplasmic "destruction complex" that contains Gsk3, Axin, Ck1, and at the plasma membrane is consistent with our experimental and Apc, to form a large signalosome (1). In the absence of a Wnt data in which we could inhibit the growth of Apc-mutant gastric ligand, newly translated b-catenin proteins are bound and phos- tumors by deleting Fzd7 and prompts a frequently asked question phorylated by the destruction complex. However, in the presence when presenting this work, "how can a cell with mutant Apc of Wnt/Fzd binding, signalosome assembly disrupts this process respond to inhibition of a Fzd receptor upstream?" and b-catenin is free to accumulate, translocate into the nucleus and associate with transcription factors Tcf/Lef to regulate target Heterogenous Wnt signaling regulates distinct cell functions in gene transcription (Fig. 1A; ref. 1). a tumor Nuclear b-catenin, a hallmark of active Wnt signaling (1), is To answer this question, we first need to highlight that Wnt observed in approximately 30% of human gastric tumors (5). signaling is not a binary system whereby the pathway is on or off, Next-generation sequencing has revealed that Wnt signaling is but rather is highly regulated in a compound fashion to provide deregulated in gastric tumors at several points in the pathway, spatial and temporal variation depending on multiple environ- mental factors (9). This was first demonstrated by Thomas Bra- bletz in 1998, who showed heterogenous expression of nuclear b 1Cancer Research UK Beatson Institute, Glasgow, United Kingdom. 2University of -catenin (a surrogate marker of active Wnt signaling) within Melbourne and Victorian Infectious Diseases Reference Laboratory, Doherty human colorectal cancers, despite all tumor cells harboring APC Institute of Infection and Immunity, Melbourne, Australia. 3School of Pharmacy mutation (Fig. 1B; ref. 10). Furthermore, Wnt signaling is pro- and Biomedical Sciences, Curtin University, Perth, Australia. 4European Cancer moted by macrophage-derived TNF-a (11) and H. pylori infection Stem Cell Research Institute, Cardiff University, Cardiff, United Kingdom. can activate the Wnt pathway via Fzd7 (12), highlighting a role for 5Doherty Institute of Infection and Immunity, Melbourne, Australia. the microenvironment for variable Wnt activity in tumors. This Corresponding Authors: Toby J. Phesse, Cell Signalling and Cancer Laboratory, indicates that additional factors can regulate Wnt signal strength European Cancer Stem Cell Research Institute, Cardiff University, Hadyn Ellis even in APC-mutant cells. This can occur because the APC gene is Building, Maindy Road, Cardiff CF24 4HQ, United Kingdom. Phone: 44-0-29- not deleted but truncated and mutant APC is still transcribed and 2068-8495; E-mail: [email protected]; and Elizabeth Vincan, University of Melbourne, Melbourne, Australia. E-mail: [email protected] translated into a protein that is able to partially function in the destruction complex and respond to WNT ligands, albeit at a Cancer Res 2019;XX:XX–XX reduced level (13, 14). Indeed, Wnt3 secretion maintains high doi: 10.1158/0008-5472.CAN-19-1362 Wnt signaling in APC-mutant colon cancer cells (14), whereas Ó2019 American Association for Cancer Research. APC is methylated, and subsequently inhibited further during www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst August 20, 2019; DOI: 10.1158/0008-5472.CAN-19-1362 Flanagan et al. A Active Wnt signaling Misconceived Wnt signaling Actual Wnt signaling in in Apc-mutant cells Apc-mutant cells Ligand/receptor Fzd inhibitor inhibitors (including sFRP, Wnt RSpo Wnt RSpo Wnt RSpo Fzd Lgr4/5 Fzd LLgr4/5 Fzd Lgr4/5 Dkk) LRP LRP LRP Dvl Dvl Dvl β-Catenin degradation complex Axin Inactive β-catenin Compromised, yet still degradation complex, no role Axin β Axin functional, β-catenin GSK3 for upstream pathway Apc Apcmut degradation complex can be GSK3β GSK3β inhibited by upstream pathway β-cat β-cat β-cat β-cat β-cat β-cat β-cat β-cat β-cat β-cat Nucleus Nucleus Nucleus β-cat β-cat β-cat TCF/LEF TCF/LEF TCF/LEF B β-Catenin immunohistochemistry Colorectal carcinoma Invasive front Tumor center Figure 1. Wnt signaling with mutant Apc. A, The levels of cytoplasmic b-catenin are regulated by the degradation complex that is inhibited when Wnt signaling is active (left). In Apc-mutant cells, it is often misconceived that Apc is completely deleted and therefore the degradation complex is nonfunctional and Wnt signaling cannot be regulated upstream of the degradation complex at the level of the receptor/ligand (illustration faded out; middle). However, mutant Apc is transcribed and translated, resulting in a compromised, yet functional, b-catenin degradation complex that explains how upstream factors, including sFRP, Dkk, and Fzd inhibitors, can still modulate Wnt signal activity (right). B, Immunohistochemistry for b-catenin in a human colorectal carcinoma showing increased nuclear localization, as a surrogate marker of active Wnt, in the invasive front compared with the tumor center. B is reprinted with permission from Brabletz et al. Pathol Res Pract. 1998;194:701–4 (10). tumor progression in colon tumors, highlighting variable cancer and colorectal cancer preferentially select different Wnt activity as a feature of colon tumors (15). This explains why mechanisms of optimal, "just right," levels of Wnt signaling APC-mutant cells are sensitive to Fzd inhibition in our work, but required for tumor growth and progression. The "just right" what is the function of this variable Wnt signal? model of Wnt signaling proposes APC mutations are selected for Approximately 37% of APC-mutant gastric tumors also contain sub-maximal levels of Wnt signaling to provide a Wnt signal that RNF43 mutations, indicating compound activation of Wnt sig- is sufficient to transform cells, but not excessive and cytotox- naling in the same tumor (3). However, only 5.5% of colon ic (16). Experimental evidence from in vivo data helped confirm 1322T/þ tumors have APC and RNF43 mutations, suggesting that gastric these models. For instance, the Apc mouse model retains a OF2 Cancer Res; 2019 Cancer Research Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst August 20, 2019; DOI: 10.1158/0008-5472.CAN-19-1362 Wnt Signaling in Apc-Mutant Cells Liver Stomach Lung CRC Intracellular = 46.8% Intracellular = 39.1% Intracellular = 21.8% Intracellular = 82.5% Receptor = 48.7% Receptor = 57.0% Receptor = 59.1% Receptor = 38.0% GBM Breast Melanoma Prostate Intracellular = 8.4% Intracellular = 13.8% Intracellular = 26.5% Intracellular = 22.7% Receptor = 19.0% Receptor = 43.6% Receptor = 58.9% Receptor = 40.9% Intracellular Amplification Deletion Multiple alteration Head & Neck Ovarian Bladder Mutation Intracellular = 31.38% Intracellular = 30.1% Intracellular