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Encyclopedia of Cancer, 2015
The name Wnt comes from wingless (Wg) and Int-1. Wingless is a Drosophila gene important during development and Int-1 is a gene into which the mouse mammary tumor virus (MMTV) integrated to cause tumors. Wg and Int-1 were subsequently found to be related in sequence. Wnt signaling is the result of binding of Wnt proteins to cell-surface receptors. Wnt family members exist in all multicellular organisms.
Vertebrate Wnts comprise a family of 19 hydrophobic cysteine-rich secreted glycoproteins of ~350 amino acids in length. Their hydrophobicity stems from lipid modification – serine palmitoleoylation – that is essential for signaling activity and secretion. Wnt proteins are quite insoluble and associate with HSPGs (heparan sulfate proteoglycans) in the extracellular matrix, so they often signal only over short distances. Long-range Wnt signaling does take place, however, for example, when Wnts form gradients to pattern developing tissues. Long-range Wnt signaling is mediated by lipoprotein particles and extracellular vesicles called exosomes and is facilitated by the retromer. Wnt signals can elicit a number of cellular responses including proliferation, differentiation, and migration. The nature of the response is dictated by the responding cell and the specific Wnts and Wnt receptors present.
Mutation of Wnt genes is rare, and its significance in cancer is not known. However, Wnt gene expression patterns are frequently altered in tumor cells. Moreover, inhibition of Wnt secretion, using inhibitors of the acyltransferase PORCN to block Wnt palmitoleoylation, or direct inhibition using antibodies, or downregulation using siRNA, can induce apoptosis in some tumor cell types (see Table 1). Both increases and decreases in expression of Wnts have been observed in cancer, perhaps reflecting different functions that are specific to the tissue type or tumor stage.
Wnt signaling pathways
Wnt proteins normally bind to cell-surface receptors of the frizzled (FZD) family, which activate intracellular disheveled (DVL) family proteins. At this stage, the Wnt signals bifurcate and can activate distinct pathways. The best understood of these is the Wnt/β-catenin pathway, also referred to as the canonical pathway (APC/β-catenin pathway) (Fig. 1). In this pathway, Wnt proteins also bind to LRP5/6 receptors, which interact with the β-catenin-binding protein, Axin. In the absence of Wnts, a so-called β-catenin destruction complex controls the level of β-catenin in the cytoplasm. This complex, which includes Axin, glycogen synthase kinase-3 (GSK-3), casein kinase-I (CKI), and adenomatous coli polyposis protein (APC) (APC Gene in familial adenomatous polyposis), facilitates β-catenin phosphorylation, ubiquitination (Ubiquitination), and ultimately degradation in the proteasome. Canonical Wnt signals disrupt this complex by eliciting changes in Axin localization and stability, resulting in the stabilization of β-catenin in the cytoplasm. Stabilized β-catenin enters the nucleus and binds to transcription factors, primarily of the Tcf/LEF family, thereby regulating gene expression. The Wnt/β-catenin pathway is permanently active in many tumor types, in particular in colon cancer, as a result of mutations in APC, β-catenin, or Axin. In addition, the E3 ubiquitin ligases ZNRF3 (zinc/RING finger protein 3) and RNF3 (RING finger protein 43), which inhibit Wnt signaling by promoting FZD degradation, are found mutated in several types of cancer, and chromosomal translocations result in gene fusions that increase expression of R-spondins, which associate with LGR4-6 (leucine-rich repeat containing G protein-coupled receptor family transmembrane receptors) and inhibit ZNRF3/RNF3. In rare cases, inactivation of Wnt/β-catenin signaling leads to tumor formation, for example, in sebaceous tumors with LEF-1 mutations (Table 2).
Table 1. Examples of Wnt genes with altered expression in cancer
|Wnt||Tumor type||Change in expression||Comments|
|WNT1||Several||Increase||Wnt-1 antibody induces apoptosis|
|WNT11||Colon, breast, prostate||Increase||Wnt-11 antibody and siRNA inhibit migration|
|WNT5A||Stomach, breast, prostate, melanoma, leukemia||Increase (decrease in leukemia)||Increase probably linked to metastasis|
|WNT16||Leukemia, basal cell carcinoma||Increased expression of alternative spliceform||Wnt-16 antibody induces apoptosis|
Fig. 1. A simplified view of Wnt/β-catenin signaling. In the absence of Wnt (left), a protein complex that includes Axin, APC, GSK-3, and CK-Ia promotes the phosphorylation, ubiquitination, and subsequent degradation of β-catenin in the proteasome; Tcf/LEF proteins associate with Groucho/TLE (Transducin-Like Enhancer of Split) family members to repress target gene expression; ZNRF3 and RNF3 promote FZD degradation. On the right, Wnt binds FZD and LRP5/6 resulting in DVL and LRP5/6 phosphorylation and Axin recruitment/degradation. This allows accumulation of β-catenin, which enters the nucleus and associates with Tcf/LEF to activate gene expression; R-Spondin enhances Wnt signaling by binding ZNRF3/ RNF3 and LGR family proteins. For simplicity, several other components of the pathway are not shown
Table 2. Wnt/β-catenin pathway mutations in cancer
|Intracellular component||Common tumor types||Effect of mutations|
|APC||Colon cancer||Stabilization of β-catenin|
|β -catenin||Liver cancer||Stabilization of β-catenin|
|Axin||Medulloblastoma||Stabilization of β-catenin|
|LEF-1||Sebaceous tumors, leukemia||Reduced Wnt/ β-catenin signaling|
Noncanonical Wnts bind to FZD receptors such as FZD7 and receptor tyrosine kinases such as ROR1/2 and Ryk. Noncanonical Wnt signals do not stabilize β-catenin and in some instances inhibit Wnt/β-catenin signals. The planar cell polarity (PCP) pathway is a noncanonical pathway that was first defined in Drosophila, where it controls the uniform orientation of hairs and bristles on the body. PCP signaling has also been studied extensively in frogs and zebrafish, where it regulates convergent extension. A good example of PCP in mammals is the uniform orientation of the hair cell stereociliary bundles within the cochlea. PCP pathway proteins include FZD, DVL, and several proteins not directly involved in other Wnt signaling pathways; those relevant to cancer include VANGL, which is overexpressed in tumors and promotes metastasis, and PTK7, a catalytically inactive transmembrane kinase (receptor tyrosine kinase) that is overexpressed in tumor-derived cell lines. The PCP pathway activates the small GTPases, Rac and Rho (Rho family proteins), and the protein kinases c-Jun NH2-terminal kinase (JNK) and Rho-associated kinase (ROCK) to induce changes in the cytoskeleton. In the Wnt-calcium (Ca2+) pathway (Fig. 2), which has been characterized mainly in frogs and zebrafish, binding of Wnt to FZD leads to activation of G proteins that then activate phospholipase C and phosphodiesterase, leading to increased concentrations of free intracellular Ca2+. These events result in activation of protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) and the Ca2+sensitive protein phosphatase calcineurin. PKC and calcineurin directly and indirectly regulate various transcription factors such as nuclear factor of activated T cells (NF-AT).
There is increasing evidence for the involvement of heterotrimeric G-protein subunits in canonical and noncanonical signaling pathways. For example, certain G-protein subunits participate in Wnt/Ca2+ signaling by activating a phosphodiesterase that lowers cGMP levels, thereby inactivating protein kinase G; Wnt-3a signals through Gaq to activate PKCb; and prostaglandin E2 receptors stimulate proliferation of colon cancer cells through the β-catenin pathway by association of Gas with Axin.
The most studied noncanonical Wnt, Wnt-5a, can have positive and negative effects on tumor progression that depend on the tumor type. For example, WNT5A expression is reduced in human leukemia, and Wnt5a heterozygous mutant mice (Haploinsufficiency) develop B-cell lymphoma. In contrast, the expression of WNT5A is increased in gastric cancer and prostate cancer, where it correlates with tumor aggressiveness. Wnt-5a is likely to have more than one function, controlling cell proliferation and/or differentiation in some tissues and promoting cell migration in others. At the molecular level, these differences might be explained by the ability of Wnt-5a to activate discrete signaling pathways through distinct receptors. For example, in transfected cells, Wnt-5a inhibits Wnt/β-catenin signaling by binding to ROR2 and activates Wnt/β-catenin signaling by binding to FZD4. There are also examples of canonical Wnts that activate β-catenin-independent signals, for example, Wnt-3a activation of PKC and JNK/ATF2. Finally, although Wnt-1 is best known as an activator of Wnt/β-catenin signaling, anti-Wnt-1 antibodies induce apoptosis in cells that do not express β-catenin, suggesting Wnt noncanonical signals are important for cancer cell survival.
Fig. 2. A simplified view of Wnt/β-catenin-independent signaling. In the Wnt-Ca2+ pathway, Wnt binding to FZD activates G proteins, thereby activating a number of downstream signals. The PCP pathway involves activation of FZD and DVL, which together with several additional proteins, including VANGL and PTK7, affect Rac/Rho signaling pathways. For simplicity, several other components of the pathway are not shown
Table 3. Examples of ligands and receptors involved in Wnt signaling
|Ligand||Receptor||Key events||Examples of changes in cancer|
|Wnt (several)||FZD||FZD recruits DVL||FZD7 overexpressed in hepatocellular carcinoma|
|Wnt (several)||LRP5/6||LRP5/6 recruits Axin||–|
|Wnt-5a||ROR2||Inhibits Wnt/β-catenin signal||–|
|Wnt (several)||RYK||RYK also binds FZD8 and DVL; activates Wnt/β-catenin signal||RYK overexpressed in ovarian cancer|
|sFRP family (sFRP1-4)||Also bind to FZD||Inhibit Wnt signals by binding to Wnt||Reduced expression (CpG methylation) (CpG islands)|
|Dickkopf family (DKK-1, -2, -4)||LRP5/6||Inhibit Wnt/β-catenin signal||Reduced expression (CpG methylation)|
|Wnt inhibitory factor 1 (WIF1)||–||Inhibits Wnt signals by binding to Wnt||Reduced expression (CpG methylation)|
|Norrie disease protein (NDP)||FZD4||Activates Wnt/β-catenin signal||–|
|Sclerostin (SOST)||LRP5/6||Inhibits Wnt/β-catenin signal||–|
|R-spondin family (RSPO1-4)||LRP6, FZD8||Activate Wnt/β-catenin signal||Reduced expression|
Other ligands and receptors that directly regulate Wnt signaling
Several other proteins modulate Wnt signaling by binding or modifying Wnts, binding to Wnt receptors or by acting as Wnt receptors (Table 3). The secreted frizzled related protein (sFRP) family and Wnt inhibitory factor 1 (WIF1) interact with Wnt proteins themselves, so have the potential to inhibit all Wnt signals. sFRP proteins also bind to FZD receptors and in some instances augment rather than inhibit Wnt/ β-catenin signaling. The expression of sFRP proteins and WIF1 is downregulated in many tumors as a result of promoter methylation. In contrast to sFRP proteins, Dickkopf (DKK) family members and Sclerostin interact with LRP5/6 and are therefore thought to inhibit only Wnt/β-catenin signaling. The expression of DKK family members is often reduced in tumors as a result of promoter methylation. However, DKK1 is highly expressed in myeloma (Multiple myeloma), where it contributes to osteolytic bone disease by inhibiting the osteoblast differentiation. While DKK1 and DKK4 inhibit Wnt/β-catenin signaling, DKK2 can activate it, while DKK3 does not directly affect Wnt signaling since it does not bind to LRP5/6. DKK1 is itself a bcatenin/Tcf target gene that provides a negativefeedback loop for Wnt/β-catenin signaling. Finally, NDP/Norrin is a secreted protein that activates Wnt/β-catenin signaling by binding to FZD4. Examples of other proteins that function as Wnt receptors are RYK, a catalytically inactive receptor tyrosine kinase that binds Wnt proteins using a domain related to WIF1, and ROR2, which binds to Wnt-5a and some other Wnts. Finally, it is worth noting that Wnt proteins can also be inactivated by the extracellular deacylase NOTUM, which enzymatically removes the Wnt lipid moiety and by the membrane-tethered metalloprotease TIKI, which cleaves the Wnt amino terminal domain.
R-Spondin — The R-spondin family (RSPO1-4) are secreted proteins that activate Wnt signaling by neutralizing the transmembrane E3 ubiquitin ligases RNF43 and ZNRF3 that remove FZD receptors from the cell surface. They are characterized by two furin-like repeats and a thrombospondin domain. Gene fusions involving RSPO2 and RSPO3 occur in 10% of colon tumors and are mutually exclusive with APC mutations.
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