When we mention Epithelial-to-mesenchymal transition (EMT), most people will immediately think of tumors. We often say that tumors have EMT characteristics. However, the EMT is an indispensable mechanism during morphogenesis, as without mesenchymal cells, tissues and organs will never be formed^[1].

The EMT process describes the differentiation of stationary epithelial cells towards a mesenchymal, motile phenotype, which was initially observed in early development^[1]. Later, it was found that EMT is not only critical in development and wound healing, but also represents a salient property of primary tumor formation and metastasis^[2].

The EMT process can promote cancer stem-like properties, immune evasion, multi-drug resistance, invasive phenotype, mediate the plasticity of cancer cells, allowing them to persistently and irreversibly adapt to changing conditions. EMT is inseparably linked with tumor invasion, metastasis, drug resistance, and immune escape^[3][4][5].

EMT is regulated by various signaling pathways such as the TGF-β, Wnt/β-catenin, Hedgehog, and Notch signaling pathway. These pathways trigger EMT by stimulating transcription factors including Snail, Twist, and ZEB1/2. Among all the signaling pathways, the Wnt/β-catenin pathway shows its pivotal role in the regulation of EMT^[6].

• Activation of Wnt/β-catenin induces EMT

Under normal circumstances, negative regulators of the Wnt/β-catenin signaling pathway, such as adenomatous polyposis coli protein (APC), glycogen synthase kinase 3 protein (GSK-3), and Axin, can bind with β-catenin to form a complex resulting in phosphorylation, and further degradation.

However, mutations and deletions of these negative regulator genes of the Wnt/β-catenin pathway have been found in various cancer cells. β-catenin accumulates in large quantities in the cytoplasm and then moves into the nucleus to form complexes with transcription factors such as T cell factor/Lymphoid enhancing factor (TCF/LEF), activating downstream target genes to promote cell cycle development or produce abnormal proteins, inducing EMT, and leading to cell carcinogenesis.

• Pathways of Wnt activation

The activation of Wnt is achieved by different Wnt-protein ligands binding to the Frizzled family cell surface receptor, thereby transmitting biological signals into the cell, including three pathways.

(1) Canonical Wnt pathway (Wnt/β-catenin): The Wnt gene is abnormally activated in tumor cells, activating the phosphorylation of Dsh protein in the cytoplasm, inhibiting the key component GSK3β activity in the GSK3β/APC/Axin complex, preventing GSK3β's phosphorylation and ubiquitination of β-catenin, reducing the phosphorylation degradation of β-catenin^[9]. β-catenin accumulates into the cytosol and then translocates to the nucleus to bind the LEF-TCF co-transcription factors, thereby affecting cell adhesion, tissue morphogenesis, and tumor development^[10].

(2) Non-canonical Wnt/PCP pathway: On one hand, the Fz receptor binds to the Wnt ligand and causes Dvl to be phosphorylated. On the other hand, Smurf ubiquitinates Prickle (a protein that typically inhibits Wnt/PCP signaling). The degradation of Prickle allows Dvl to bind with DAAM, activating Rac1, Profilin, and RhoA. Rac1 activates JNK, which in turn phosphorylates c-Jun and CapZIP. Then c-Jun enters the cell nucleus to stimulate gene transcription. Meanwhile, RhoA activates DIA1 and ROCK, which in turn activates MRLC. CapZIP, MRLC, DIA1 and Profilin all stimulate actin polymerization, thereby affecting cell polarity and migration^[7].

(3) Non-canonical Wnt/Ca²⁺ pathway: The binding of Wnt to the Fz receptor leads to G protein-mediated activation of PLC, stimulating Ca²⁺ release. DAG and Ca²⁺ together activate protein kinase C (PKC) to stimulate Cdc42, leading to actin polymerization, promoting cell polarization and migration. At the same time, the binding of IP3 with InsP3R leads to an increase in cytoplasmic Ca²⁺, calcineurin activates the nuclear factor of activated T cells (NFAT), which causes gene transcription^[7].

A large amount of research shows that dysregulation of the Wnt/β-catenin signaling pathway can lead to EMT, but who is involved in the regulation of Wnt/β-catenin?

• miRNA targets Wnt/β-catenin to regulate EMT

The core of Wnt/β-catenin signal transduction activation is the accumulation of β-catenin in the cytoplasm. Therefore, miRNA targeting β-catenin may inhibit EMT by targeting the Wnt signaling pathway or its downstream transcription factors.

In August of this year, Dongsheng Zhu and others revealed that miR-199b-3p plays a key role in the formation and progression of osteosarcoma (OS). miR-199b-3p can bind to the 3' untranslated region (UTR) of CCDC88A, downregulating the expression level of CCDC88A, inhibiting EMT and the Wnt/β-catenin signaling pathway, thereby mediating its tumour-suppressive effect on OS cell proliferation and invasion^[11].

Additionally, research by Ling has proven that miR-145 inhibits EMT in lung cancer cells via targeting the Oct4 mediated Wnt/β‑catenin signaling pathway^[12]. MiR-33b also binds to ZEB1 3'-UTR region and suppresses the activity of WNT/β-catenin signaling in lung adenocarcinoma cells and in turn suppressed tumor cell growth and EMT in vitro and in vivo^[13].

• WNT3A-RIP1-β-catenin pathway induces EMT

In July 2023, A-Ram Kang revealed new potential roles of RIP1 in controlling WNT/β-catenin canonical signaling to enhance metastasis of colorectal cancer (CRC)^[14].

In the absence of WNT ligands, phosphorylated β-catenin is recognized and bound by β-TrCP. Ubiquitination of β-catenin and regulation of RIP1 ubiquitination by cIAP1/2 subsequently lead to the degradation of β-catenin protein.

However, WNT3A treatment induced cIAP1/2 degradation, abrogated the recruitment of β-TrCP to β-catenin and sequentially blocked β-catenin ubiquitination. RIP1 and β-catenin binded and stabilized one another. This binding of RIP1 and β-catenin also stimulated the dissociation of the β-catenin–β-TrCP complex (but does not change the protein levels of β-catenin and β-TrCP) and the inhibition of β-catenin ubiquitination, thereby stimulating EMT induction, enhancing the in vitro migration and invasion ability of CRC cells^[14].

In addition, many oncogenic signals, such as receptor tyrosine kinase (RTKs) family, PI(3)K/Akt, MAPK etc., inhibit GSK-3β activity, and thus leading to a large accumulation of β-catenin and triggering cell migration and EMT^[15]. The Wnt family will produce crosstalk with Ras/Raf/MEK/ERK, TGFβ/Smad and other pathways, mutually influencing and cooperatively regulating genes related to cell invasion and metastasis, jointly participating in mediating the EMT process of tumor cells.

There are also many protein targets that affect the EMT process of tumor cells by participating in the regulation of Wnt/β-catenin. For example, IL-1β secreted by TAMs increases the availability of β-catenin in colorectal cancer cells by phosphorylating GSK3β, hindering the function of the β-catenin destruction complex^[16]. MRGBP promotes CRC progression via DKK1/Wnt/β-catenin and NF-kB/p65 pathways mediated EMT^[17].