Introduction to Wnt signaling pathway

The Wnt signaling pathway is a highly conserved signal transduction pathway that plays a critical role in cell fate determination, maintaining cell growth and differentiation during tissue and organ development. Due to its strong association with tumorigenesis, it is also a widely studied target for anticancer drug discovery.

Overview and significance

The term Wnt was coined from combining the Wingless (Wg) gene in Drosophila melanogaster and the integration (int-1) proto-oncogene in mammals. Initially identified as an oncogene in mice, Wnt was later found to be conserved across several species.¹'². Thus, the multifaceted role of this gene in embryonic development, cell proliferation, apoptosis and cancer led to the fusion of the terms Wg and Int, forming Wnt, which stands for Wingless-related integration site.

Core components of Wnt signaling

The Wnt signaling pathway comprises the following components:

1. The Wnt proteins, a family of secreted glycoproteins
2. The frizzled protein (FRZ), for which Wnt acts as a ligand
3. Coreceptors required to initiate signaling
4. Surface receptors that bind Wnt, such as the low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/LRP6) or tyrosine kinase orphan receptor 2 (ROR2)
5. The disheveled protein (DVL), a cytoplasmic scaffold protein
6. β-catenin, utilizing either the canonical or non-canonical Wnt signaling pathways

These components collectively activate the transcription factor family TCF/LEF to induce gene transcription in the nucleus.

Classification of the Wnt signaling pathway

Wnt signaling progresses through two pathways: the canonical (β-catenin-dependent) and non-canonical (β-catenin-independent) pathways.

• The Canonical Wnt signaling pathway (β-catenin-dependent) activates Wnt target genes by upregulating β-catenin, which acts as a binding partner for transcription factors, engendering gene transcription necessary for embryogenesis and organogenesis.
• The noncanonical Wnt signaling pathway (β-catenin-independent) refers to the alternative signaling pathways that do not impact β-catenin levels. These include the planar cell polarity (PCP) pathway, which regulates cell migration, and the Wnt/Ca2+ pathway, which modulates intracellular calcium release and inflammation.

Canonical Wnt signaling pathway mechanism

Ligand binding and signal initiation

The canonical Wnt signaling pathway primarily acts on target genes by controlling the β-catenin levels. In the absence of Wnt, β-catenin is degraded by a destruction complex. When present, Wnt serves as a ligand for the Frizzled Class Receptor (FZD) and its coreceptor LRP5/6. This binding triggers the recruitment and activation of DVL, which inhibits the β-catenin destruction complex and prevents its degradation.³

Gene regulation and biological roles

Without the destruction complex, β-catenin can accumulate in the cytoplasm and eventually translocate into the nucleus to bind TCF/LEF (T-cell factor/lymphoid enhancer-binding factor) transcription factors. TCF/LEF activate target genes, such as c-Myc, cyclin D1 and Axin 2, critical regulators of cell proliferation and development in health and oncogenesis.⁴

Noncanonical Wnt signaling and cellular processes

Functional diversity of noncanonical pathways

Noncanonical Wnt signaling pathways do not regulate gene transcription through β-catenin and TCF/LEF factors.

The two major pathways are as follows:

1. The planar cell polarity (PCP) pathway: Similarly to the canonical pathway, Wnt ligands bind FZD. However, the coreceptors required for signal transduction are ROR2 and Tyrosine-protein kinase transmembrane receptor (RYK). The subsequent DVL activation induces directional polarity in cells and regulates cytoskeletal dynamics, which are essential for cell shape and movement.⁵
2. The Wnt/Calcium Pathway has a similar mechanism to PCP; the ligand-receptor interaction activates heterotrimeric G proteins instead of DVL. The resulting signaling cascade triggers the release of Ca2+ from the endoplasmic reticulum into the cytoplasm, which coordinates cell migration, inflammatory response, tissue morphogenesis and apoptosis.⁶

Noncanonical signaling in developmental biology

Collectively, these pathways engender functional diversity by allowing cells to respond to morphological cues for tissue organization. Therefore, noncanonical Wnt signaling is pivotal during embryogenesis, as it ensures ventral/dorsal and anterior/posterior development, bone and muscle development, and organ shaping (e.g., facial structures, heart tube looping and kidney tubule formation).⁷

Molecular players in Wnt signal transduction

The Wnt family and Wnt gene diversity

The proteins within the Wnt family are highly conserved across multicellular eukaryotes, indicating a common ancestral gene, serving developmental functions in humans, mice and flies, among other species. The ubiquity of Wnt across several organisms underscores its biological significance.

Role of receptors and signaling intermediates

Frizzled receptors in Wnt signal transduction are a family of G protein-coupled receptor-like proteins. Their extracellular region has a cysteine-rich domain (CRD) responsible for binding Wnt ligands. Upon ligand binding, FZD undergoes conformational changes that bring DVL close to its cytoplasmic tail.⁸

The coreceptors are key to these conformational changes. DVL is only recruited when FZD is in a tripartite complex with LRP5/6 (for canonical) or ROR2/RYK (for noncanonical). ⁸

The disheveled protein DVL propagates the signal received from FZD into downstream intracellular pathways. It has three significant domains:⁹

1. Postsynaptic density 95/Discs large/ZO-1 (PDZ) facilitates membrane localization and FZD-FVL-downstream protein interactions necessary for signal transduction.
2. Dishevelled-Axin (DIX) facilitates the interaction with the destruction complex to initiate the canonical signaling pathway.
3. Postsynaptic density 95/Discs large/ZO-1 (DEP) is associated with noncanonical signaling pathways.

Wnt signaling in development and disease

The Wnt signaling pathways are prominent in developmental biology, while their dysregulation has severe implications for genetic disorders and cancer.

Developmental biology

In embryonic development, Wnt signaling is involved in establishing the body axes, gastrulation, neural tube formation and the development of several organs, including the brain, heart, lungs and kidneys.¹⁰

Their functions are not limited to embryonic development. Wnt signaling pathways also maintain the stem cell niches in various tissues to guide repair after injury. For instance, the canonical pathway can drive the regeneration of hair follicles, muscles and bones.¹¹'¹²

Wnt achieves these functions by promoting stem and progenitor cell differentiation and proliferation while refining their orientation and movement. The β-catenin-dependent canonical pathway directs gene expression to determine cell fate and identity, while the noncanonical pathways govern cell polarity and movement.¹³'¹⁴

Cancer and genetic diseases

Aberrant Wnt signaling is strongly associated with cancer and genetic diseases. Cancer cells can exhibit uncontrolled division and resistance to cell death by hijacking the Wnt mechanisms involved in determining cell fate.

Mutations in the destruction complex and β-catenin lead to β-catenin accumulation, nuclear translocation and persistent activation of oncogenic Wnt target genes.¹⁵ Nearly 90% of colorectal cancer cases involve a mutation in the Adenomatous Polyposis Coli (APC) gene, which maintains cytoplasmic β-catenin levels by degrading excessive β-catenin.¹⁶ Furthermore, dysregulations in the noncanonical pathways, such as Wnt5a-ROR2-mediated signaling, can enhance tumors' invasive and metastatic potential.¹⁷

While excessive Wnt activation can potentially lead to some forms of cancer, loss-of-function in Wnt and its receptors underlie developmental genetic disorders, such as WNT3 mutations in tetra-amelia syndrome,¹⁸ LRP5 mutations in bone-density disorders¹⁹ and PCP mutations in skeletal dysplasias.²⁰

Modulation and inhibition of Wnt signaling

Pathway activation and dysregulation

Overactivation or loss of function can impair tissue development and lead to various degenerative diseases and cancer. Therapeutic strategies aim to inhibit aspects of the Wnt pathways to restore homeostasis.

Inhibitory strategies

Mechanisms to inhibit Wnt signaling include:

1. Wnt inhibitors block Wnt expression and secretion, preventing the initiation of the signaling cascade. Porcupine inhibitors block the porcupine (PORCN) enzyme responsible for Wnt cellular trafficking and secretion.²¹
2. Wnt antagonists mimic Wnt ligands or receptors to prevent Wnt-Frizzled-coreceptor complex formation. These antagonists include Dickkopf-1 (DKK1), which binds LRP5/6 to inhibit its interaction with the Wnt-Frizzled complex,²² and Secreted Frizzled-Related Proteins (SFRP), which mimic the Frizzled extracellular domain and bind Wnt to sequester it from Frizzled.²³
3. Wnt inhibitory small molecules target the intracellular components to disrupt signal transduction in the canonical pathway. Tankyrase inhibitors stabilize the destruction complex to enhance β-catenin destruction, while inhibitors like ICG-001 and PRI-724 interfere with β-catenin-mediated gene transcription in the nucleus.²⁴'²⁶

Emerging Research and Advances

Despite the potential of inhibition strategies, Wnt-targeted therapy faces significant challenges, such as off-target effects, toxicity and drug resistance. Wnt-targeting is considered for degenerative diseases, which may involve different approaches from anticancer strategies.

Advances in pathway targeting

While blocking β-catenin overactivation is essential, therapeutic strategies must maintain β-catenin levels in the cytoplasm to preserve its regulatory functions for cell maintenance. Novel inhibitors target specific β-catenin transcriptional activity without interfering with its role in membrane-level signaling.²⁷ Thus, the side effects can be mitigated.

MicroRNAs regulate various aspects of the Wnt pathway, presenting therapeutic opportunities through gene therapies. miR-34a is a critical tumor suppressor that inhibits β-catenin-transcription factor interactions, while it is significantly downregulated in cancers.²⁸ Synthetic miRNA mimics for miR-34a are currently under investigation for their potential to restore tumor-suppressor function.²⁹ Furthermore, chemically modified oligonucleotides are studied for their ability to inhibit oncogenic miRNAs, such as miR-135b.³⁰

Regenerative medicine strategies seek to boost Wnt signaling to promote cell proliferation and differentiation. For instance, the Wnt agonist R-spondin1 was shown to induce stem cell renewal and regenerate the intestinal lining in mouse models with intestinal injuries due to radiation or chemotherapy.³¹ Another example is the osteoporosis medication romosozumab, which blocks the endogenous Wnt inhibitor sclerostin to restore bone mineral density.³²

Research areas under investigation

Further research is essential to better understand the role of Wnt signaling in health and disease and to develop effective treatment methods. Like many other pathways and biological networks in our cells, the Wnt signaling pathway does not function in isolation. Extensive research revealed its crosstalk with the Notch, Hedgehog, PI3K/Akt, and MAPK pathways.³³

Biomaterial scaffold development and Wnt ligand delivery methods require more elaborate research for regenerative medicine. With correct design and delivery methods, Wnt activity can be used in bone and tissue regeneration in clinical applications and organoid development in research applications.

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