WNT signals are transduced to the canonical pathway for cell fate determination, and to the noncanonical pathway for control of cell movement and tissue polarity. Canonical WNT signals are transduced through Frizzled family receptors and LRP5/LRP6 coreceptor to the β-catenin signaling cascade. Microtubule affinity–regulating kinase (PAR-1) family kinases, casein kinase Iε (CKIε), and FRAT are positive regulators of the canonical WNT pathway, whereas APC, AXIN1, AXIN2, CKIα, NKD1, NKD2, βTRCP1, βTRCP2, ANKRD6, Nemo-like kinase (NLK), and peroxisome proliferator–activated receptor γ (PPARγ) are negative regulators. Nuclear complex, consisting of T-cell factor/lymphoid enhancer factor, β-catenin, BCL9/BCL9L, and PYGO, activates transcription of canonical WNT target genes such as FGF20, DKK1, WISP1, MYC, CCND1, and Glucagon (GCG). Noncanonical WNT signals are transduced through Frizzled family receptors and ROR2/RYK coreceptors to the Dishevelled-dependent (Rho family GTPases and c-jun NH2-terminal kinase) or the Ca2+-dependent (NLK and nuclear factor of activated T cells) signaling cascades. WNT signals are context-dependently transduced to both pathways based on the expression profile of WNT, SFRP, WIF, DKK, Frizzled receptors, coreceptors, and the activity of intracellular WNT signaling regulators. Epigenetic silencing and loss-of-function mutation of negative regulators of the canonical WNT pathway occur in a variety of human cancer. WNT, fibroblast growth factor (FGF), Notch, Hedgehog, and transforming growth factor β/bone morphogenetic protein signaling network are implicated in the maintenance of tissue homeostasis by regulating self-renewal of normal stem cells as well as proliferation or differentiation of progenitor (transit-amplifying) cells. Breakage of the stem cell signaling network leads to carcinogenesis. Nonsteroidal anti-inflammatory drugs and PPARγ agonists with the potential to inhibit the canonical WNT signaling pathway are candidate agents for chemoprevention. ZTM000990 and PKF118-310 are lead compounds targeted to the canonical WNT signaling cascade. Anti-WNT1 and anti-WNT2 monoclonal antibodies show in vitro effects in cancer treatment. After the optimization, derivatives of small-molecule compound and human monoclonal antibody targeted to the WNT signaling pathway could be used in cancer medicine.
WNT Signaling Pathway
Overview. The human WNT gene family consists of 19 members, encoding evolutionarily conserved glycoproteins with 22 or 24 Cys residues (1). WNT signals are transduced to the canonical pathway for cell fate determination, and to the noncanonical pathway for control of cell movement and tissue polarity (Fig. 1).
Canonical WNT signaling pathway. Canonical WNT signals are transduced through Frizzled (FZD) family receptors and LRP5/LRP6 coreceptor to the β-catenin signaling cascade (2, 3). In the absence of canonical WNT signaling, β-catenin complexed with APC and AXIN is phosphorylated by casein kinase Iα (CKIα) and glycogen synthase kinase 3β (GSK3β) in the NH2-terminal degradation box, which is polyubiquitinated by βTRCP1 or βTRCP2 complex for the following proteasome-mediated degradation (4). In the presence of canonical WNT signaling, Dishevelled (DVL) is phosphorylated by CKIα for high-affinity binding to FRAT. Because canonical WNT signal induces the assembly of FZD-DVL complex and LRP5/6-AXIN-FRAT complex (5, 6), β-catenin is released from phosphorylation by CKIα and GSK3β for stabilization and nuclear accumulation. Nuclear β-catenin is complexed with T-cell factor/lymphoid enhancer factor (TCF/LEF) family transcription factors and also with Legless family docking proteins (BCL9 and BCL9L) associated with PYGO family coactivators (PYGO1 and PYGO2; refs. 7, 8). The TCF/LEF-β-catenin-Legless-PYGO nuclear complex is the effector of the canonical WNT signaling pathway to activate the transcription of target genes such as FGF20, DKK1, WISP1, MYC, and CCND1 (9–12).
Noncanonical WNT signaling pathway. Noncanonical WNT signals are transduced through FZD family receptors and coreceptors, such as ROR2 and RYK (13–15). Small G proteins (RHOA, RHOU, RAC, and CDC42) and c-jun NH2-terminal kinase are the DVL-dependent effector molecules of the noncanonical pathway (16, 17), whereas Nemo-like kinase (NLK) and nuclear factor of activated T cells (NFAT) are the Ca2+-dependent effector molecules of noncanonical pathway (18, 19). Small G proteins are implicated in the cytoskeletal reorganization during invasion and metastasis. NLK phosphorylates TCF/LEF family transcription factors to inhibit the canonical WNT signaling pathway. NFAT transcription factor is implicated in the convergent extension during early embryogenesis as well as in the metastasis during carcinogenesis. Noncanonical WNT signaling pathway, transduced to a variety of DVL- or Ca2+-dependent cascades, is overlapping with the planar cell polarity signaling pathway (20).
WNT Signaling Pathway and Carcinogenesis
Secreted-type WNT signaling inhibitors. SFRP1, SFRP2, SFRP3, SFRP4, SFRP5, WIF1, DKK1, DKK2, DKK3, and DKK4 are secreted-type WNT signaling inhibitors (21). SFRP family members and WIF1 are WNT antagonists that inhibit WNT binding to FZD family receptors. DKK family members interact with LRP5/LRP6 coreceptor and trigger its endocytosis to prevent formation of the WNT-FZD-LRP5/LRP6 complex for the canonical WNT signaling.
Intracellular-type canonical WNT signaling inhibitors. APC, AXIN1, AXIN2, CKIα, GSK3β, NKD1, NKD2, ANKRD6, and NLK are negative regulators of the canonical WNT signaling pathway. APC, AXIN1, and AXIN2 are scaffold proteins of the β-catenin destruction complex, whereas CKIα and GSK3β are serine/threonine kinases that phosphorylate β-catenin to trigger degradation.
Canonical WNT signaling activation during carcinogenesis. Genetic predisposition, environmental factor, and aging are risk factors of human cancer (22). Transcriptional activation of canonical WNTs occurs during tissue regeneration associated with chronic persistent inflammation, and up-regulation of Wnt1, Wnt3, or Wnt10b due to mouse mammary tumor virus (MMTV) integration leads to mammary carcinogenesis (23, 24).
Epigenetic silencing of SFRP1, SFRP2, DKK1, WIF1, and AXIN2 genes occurs in premalignant tissues associated with chronic inflammation or in human cancer, whereas mutation of APC, AXIN1, and AXIN2 genes occurs in human cancer (24–27). Genes encoding canonical WNT signaling inhibitors are down-regulated during carcinogenesis due to epigenetic silencing and genetic alteration.
Network between WNT and Other Oncodevelopmental Pathways
Fibroblast growth factor signaling pathway. Fibroblast growth factor (FGF) signals are transduced through FGF receptors to the phosphatidylinositol 3-kinase-AKT, Ca2+, and mitogen-activated protein kinase signaling cascades. Mammary carcinogenesis in MMTV-Wnt1 transgenic mice is accelerated by MMTV integration around Fgf3-Fgf4 or Fgf8 loci, and that in MMTV-Fgf3 transgenic mice by MMTV integration around Wnt1-Wnt10b locus (23, 24). FGF20 is up-regulated in human colorectal cancer as the target gene of the canonical WNT signaling pathway (9). Although the mechanisms are not the same, FGF and canonical WNT signaling pathways inhibit GSK3β activity and activate Ca2+ signaling. FGF and canonical WNT signaling pathways converge to the β-catenin signaling cascade and the epithelial-mesenchymal transition signaling cascade through GSK3β down-regulation (24), and also to the Ca2+-dependent NFAT signaling cascade. Cross-talk of FGF and WNT signaling pathways in tumors leads to more malignant phenotype through the potentiation of β-catenin, epithelial-mesenchymal transition, and NFAT signaling cascades.
Notch signaling pathway. Notch ligand binding to Notch family receptor induces transcriptional activation of Notch target genes through the nuclear translocation of Notch intracellular domain. Among Notch ligand genes, JAG1 gene is predicted as an evolutionarily conserved target of the canonical WNT signaling pathway based on the conservation of double TCF/LEF–binding sites within the 5′ promoter region of mammalian JAG1 orthologues (28). Notch4 is activated due to MMTV integration during mammary carcinogenesis (29), wherein WNT signaling pathway is also activated. Notch and WNT signaling pathways are both necessary for the self-renewal of hematopoietic stem cells (30). Notch and WNT signaling pathways synergize to inhibit terminal differentiation of intestinal epithelial cells partially through down-regulation of ATOH1/HATH1 bHLH transcription factor. Together, these facts indicate that Notch and WNT signaling pathways keep the homeostasis of stem and progenitor (transit-amplifying) cells through the inhibition of terminal differentiation.
Other signaling pathways. WNT signaling pathway also networks with Eph, vascular endothelial growth factor, Hedgehog, and transforming growth factor β/bone morphogenetic protein (TGFβ/BMP) signaling pathways, which are reviewed elsewhere.
Recent Advances in WNT Signaling Pathway
Context-dependent WNT signaling. During the early period of WNT research when β-catenin and TCF/LEF were not the “star players” of the canonical WNT signaling pathway, Xenopus axis duplication assay was mainly used to measure the canonical WNT signaling activity. Because injection of synthetic Wnt1 or Wnt3a mRNA into the ventral marginal zone of Xenopus embryos at the four-cell stage induces axis duplication, Wnt1 and Wnt3a were previously characterized as the canonical WNTs (1). Recently, Wnt1 and Wnt3a were characterized to activate not only the canonical WNT signaling pathway but also the noncanonical WNT signaling pathway based on the cell or tissue type (15, 17). On the other hand, Wnt4, Wnt5a, and Wnt11 were previously characterized as noncanonical WNTs. Although injection of Wnt4, Wnt5a, or Wnt11 mRNA did not induce Xenopus axis duplication, co-injection of one of these noncanonical Wnt mRNA with FZD5 mRNA did induce Xenopus axis duplication (31, 32). Therefore, WNT signals are context-dependently transduced to the canonical and noncanonical pathways based on the expression profile of WNT, SFRP, WIF, DKK, FZD family receptors, coreceptors, and the activity of cytoplasmic WNT signaling regulators.
Peroxisome proliferator–activated receptor γ. WNT10B activates the canonical WNT signaling pathway to inhibit adipogenesis (33), whereas Wnt5b partially inhibits the canonical WNT signaling pathway to promote adipogenesis (34). Peroxisome proliferator–activated receptor γ (PPARγ) is a nuclear receptor predominantly expressed in adipose tissues to induce adipocyte differentiation and β-catenin degradation (35), and is also expressed in intestinal epithelium. PPARγ counteracts the canonical WNT signaling during adipocyte differentiation and colorectal carcinogenesis. PPARγ agonist is one of the promising drugs to inhibit the canonical WNT signaling pathway; however, the mode of small molecules on PPARγ activity in adipose tissues is different from that in colorectal epithelium or colorectal cancer. Therefore, PPARγ agonist inhibiting the canonical WNT signaling pathway in human colorectal epithelium should be developed for clinical application to cancer patients.
Cancer stem cells. Hematologic stem cells and mammary gland stem cells have been isolated for their characterization (30, 36), whereas intestinal stem cells remain to be isolated (37). However, it is generally believed that the WNT, FGF, Notch, Hedgehog, and TGFβ/BMP signaling network is implicated in the maintenance of tissue homeostasis by regulating self-renewal of normal stem cells as well as proliferation or differentiation of progenitor cells (9, 28, 30, 36–39). Breakage of the regulation for “stem cell signaling” network in normal stem cells leads to the transformation to cancer stem cells. Alternatively, acquisition of self-renewal potential in progenitor cells due to epigenetic change or genetic alteration of stem cell signaling–related genes gives rise to cancer stem cells. Detailed analyses on the dysregulation of WNT, FGF, Notch, Hedgehog, and TGFβ/BMP signaling pathways in cancer stem cells derived from a various type of human tissues or organ should be systematically investigated for better understanding of cancer stem cells themselves as well as the scenario of carcinogenesis.
Prevention.Helicobacter pylori, hepatitis viruses, and papilloma viruses are causative agents for gastric cancer, liver cancer, and cervical cancer, respectively. Because chronic persistent inflammation is a causative factor for a variety of human cancer, eradication and vaccination are realistic methods for cancer prevention. However, eradication and/or vaccination could fail to prevent cancer in cases with persistent inflammation with tissue reorganization and epigenetic changes. Because up-regulation of WNT family ligands and down-regulation of WNT inhibitors occur during the early stage of carcinogenesis, nonsteroidal anti-inflammatory drugs and PPARγ agonists with the potential to inhibit the canonical WNT signaling pathway are candidate agents for chemoprevention.
Therapeutics. Small-molecule compounds and human (or humanized) monoclonal antibodies are promising drugs in the post-genome era (22). ZTM000990 and PKF118-310 are lead compounds targeted to the canonical WNT signaling cascade, whereas anti-WNT1 and anti-WNT2 monoclonal antibodies show in vitro effects in cancer treatment (40). After the optimization, derivatives of small-molecule compound and human monoclonal antibody targeted to the WNT signaling pathway could be used in cancer medicine.