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Introduction

In 1982, Nusse and Varmus identified a novel gene named, integration site 1 (Int-1), whilst investigating oncogenes utilizing the mouse mammary tumor virus. Int-1 was later confirmed to be a homolog of the Drosophila Wingless gene. To minimize the nomenclature conclusion, a consensus was reached in 1991 to designate genes of the Int-1/Wingless family uniformly as ‘Wnt’ (1).

The Wnt signaling pathway can be subdivided into two major classes: Canonical and non-canonical signaling pathways. In the canonical Wnt pathway, β-catenin serves a central regulatory role by binding T cell-specific transcription factor (TCF)/lymphoid enhancer-binding factor 1 to regulate the expression of downstream genes, which controls cell fate, proliferation and differentiation (2). The role of the canonical Wnt signaling pathway in tumorigenesis has been extensively reported. Mutations in β-catenin or loss of function of upstream regulatory factors, such as adenomatous polyposis coli, Axin and glycogen synthase kinase 3β, all lead to increased nuclear accumulation of β-catenin, activating downstream oncogenes, such as c-myc, cyclin D1 and vascular endothelial growth factor, and promoting tumor cell proliferation, survival and invasion (3). By contrast, non-canonical Wnt pathways are independent of β-catenin nuclear translocation and primarily include the Wnt/Ca2+ pathway and the Wnt/planar cell polarity (PCP) pathway. Growing research has highlighted the significance of the Wnt/Ca2+ pathway in tumor biology, revealing that its effects can differ substantially among several tumor types. The present review aims to summarize this research regarding Wnt/Ca2+ signaling, to systematically discuss its multiple roles and diverse mechanisms in several tumors, and to provide new insights into the diagnosis and therapeutic strategies of these malignancies.

Overview of the Wnt/Ca2+ signaling pathway

Core components of the Wnt/Ca2+ pathway

A vast body of research has revealed the core components of the Wnt/Ca2+ pathway, including extracellular ligands, transmembrane receptors and intracellular signaling molecules (4,5). Specifically, extracellular signaling is primarily mediated by the following: Wnt proteins; the transmembrane receptors, R-spondin-2-leucine rich repeat containing G protein-coupled receptor 5 complex and frizzled (FZD)-2 receptor; and the intracellular signaling molecules, heterotrimeric guanine nucleotide-binding protein (G protein), disheveled (Dvl), phospholipase C (PLC), phosphatidylinositol 4,5-bisphosphate (PIP2), protein kinase C (PKC), calmodulin (CaM)-dependent protein kinase II (CaMKII) and calcineurin (CaN). Although these molecules are independent of the nuclear translocation of β-catenin, they serve an integral and pivotal role in the Wnt/Ca2+ pathway (6).

Extracellular ligands of the Wnt signaling pathway

Extracellular ligands of the Wnt signaling pathway consist of a highly conserved family of secreted glycoproteins known as Wnt proteins. Currently, 19 Wnt family members have been identified in mammals. Wnt ligands of the Wnt1 class, including Wnt2, Wnt3, Wnt3a and Wnt8a, predominantly activate the canonical Wnt/β-catenin pathway, whereas ligands of the Wnt5a class, comprising Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a and Wnt11, primarily trigger the non-canonical Wnt pathways. Specifically, the Wnt/Ca2+ signaling pathway is mainly activated by Wnt5a and Wnt11 (7).

After being synthesized in the endoplasmic reticulum (ER), Wnt proteins undergo glycosylation and palmitoylation modifications prior to their transport to the extracellular space where they can interact with receptors, subsequently activating downstream signaling pathways (2). Palmitoylation, which is crucial for Wnt protein secretion, is catalyzed by porcupine O-acyltransferase in the ER, attaching the palmitoleic acid acyl group to the Wnt protein (8). Subsequently, the palmitoylated Wnt binds to the transmembrane protein Wntless/Evi in the Golgi apparatus, which encapsulates Wnt within vesicles and facilitates its secretion into the extracellular space for interaction with receptors on target cells (9). Although Wnt signaling primarily occurs between adjacent cells, the precise mechanism by which extracellular Wnt is delivered to target cells remains unclear. Previous studies have demonstrated that the protein core of glypican Dlp can form a groove-like structure that binds to and shields the palmitoylated moiety of Wnt, increasing its water solubility and facilitating its extracellular diffusion and receptor binding, thereby enabling efficient Wnt signal transmission (10). However, the intricate mechanisms underlying this process are still not fully understood and require further exploration.

Transmembrane receptors for Wnt

FZD proteins constitute seven-pass transmembrane receptors resembling classic G-protein-coupled receptors, which function as membrane receptors for Wnt secreted glycoproteins (11). The fundamental structure of FZD proteins includes an extracellular N-terminal region, seven transmembrane helixes and an intracellular C-terminal tail (12). As a unique feature of this protein subfamily, the N-terminal region contains a conserved cysteine-rich structural domain (CRD), which is tethered to the first transmembrane helix via a flexible linker. The CRD possesses a binding site for the Wnt protein, recognizing both the palmitoleate moiety at the N-terminus and the hydrophobic amino acid motifs at the C-terminus of Wnt. The C-terminal intracellular domain, situated within the plasma membrane (PM), exhibits variability in length and lacks high conservation among diverse FZD receptors. It is characterized by a fully conserved KTXXXW motif, which engages with the post-synaptic density protein/discs large/zona occludens-1 domain of Dvl proteins, thereby facilitating intracellular signal transduction (13). There are 19 Wnt genes and 12 FZD receptors in vertebrates. These FZD receptors exhibit differential affinity for Wnt ligands and, in combination with different co-receptors, can selectively activate their respective unique Wnt signaling pathways (14).

Intracellular components and downstream molecules of Wnt

Upon binding of the Wnt protein to the FZD receptor, the intracellular Dvl protein and G protein become activated, subsequently inducing the activation of PLC. However, the mechanisms of Dvl and G proteins in Wnt signaling are complex. In the canonical Wnt/β-catenin signaling pathway, Dvl functions as a linchpin. It stabilizes β-catenin to prevent degradation and facilitates its nuclear translocation, enabling the regulation of downstream target genes transcription (15). By contrast, G proteins do not appear to participate in the canonical Wnt/β-catenin signaling (16). In non-canonical Wnt pathways, such as the Wnt/PCP pathway, Dvl employs a distinct mechanism. It interacts with the disheveled-associated activator of morphogenesis 1 and T-cell lymphoma invasion and metastasis 1 proteins to activate Ras homolog family member A (RhoA) and Rac family small GTPase 1 (Rac1), respectively. Both RhoA and Rac1, belonging to the Rho family of small GTPases, are crucial for regulating actin polymerization and cytoskeletal reorganization (17). Moreover, another mechanism of G protein activation in non-canonical Wnt signaling involves Daple (CCDC88C), a key protein that functions both as a Dvl-binding protein and as a guanine nucleotide exchange factor (GEF) for G proteins. Upon ligand stimulation, Daple dissociates from Dvl and binds to FZDs, forming a Frizzled-Daple-Gαi ternary complex. This complex activates the Gαi subunit, which inhibits cyclic AMP levels and releases the Gβγ heterodimer, enhancing Rac1 and PI3K-Akt signaling (18,19). However, this mechanism has yet to be validated in the Wnt/Ca2+ signaling pathway. Gong et al (20) reported that Sec14-like lipid-binding protein 3 (Sec14L3), a phosphatidylinositol transfer protein, serves as a critical mediator that bridges FZD receptors and Dvl within the Wnt/Ca2+ pathway. As a key component acting as a GTPase in this signaling cascade, Sec14L3 normally exists in an inactive GDP-bound state (Sec14L3-GDP), which allows it to form complexes with FZD receptors and Dvl. Upon stimulation by non-canonical Wnt signaling, Sec14L3 undergoes a conformational change, transitioning to its active GTP-bound form (Sec14L3-GTP). Subsequently, the activated Sec14L3-GTP binds to PLC on the PM, facilitating the activation of PLC and subsequent Wnt/Ca2+ signaling. In this context, it is hypothesized that Dvl acts as a scaffolding protein, recruiting an unknown Sec14L3-GEF to promote the formation of Sec14L3-GTP (20). Despite this, the exact molecular mechanisms involving Dvl and G proteins in activating the Wnt/Ca2+ pathway still require further investigation (20,21). Once PLC is activated, it rapidly elicits a transient elevation of intracellular concentrations of the second messengers diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), which subsequently activate downstream effector molecules, such as PKC, CaMKII and CaN (22).

PLC

PLC is a pivotal phospholipase family localized on the cytoplasmic membrane. It encompasses 13 distinct members, each possessing conserved EF-hand domains, PH domains and C2 domains, which together form the structural basis for the function of PLC (23). Upon activation, PLC mediates the hydrolysis of PIP2 into IP3 and DAG (24). DAG, as a hydrophobic molecule, associates with the conserved region 1 (C1) domain of PKC, thereby facilitating its recruitment and activation at the PM (25). IP3 binds to inositol 1,4,5-trisphosphate receptors (InsP3Rs) on the ER membrane, inducing a conformational change in the InsP3R complex that opens Ca2+ channels and releases Ca2+ into the cytoplasm (26). Ca2+ depletion in the ER triggers the dissociation of Ca2+ from the EF-hand motif of the ER calcium sensor stromal interaction molecule 1 (STIM1). This process leads to a conformational change in STIM1, switching it from a tight, inactive form to an extended, active form. Activated STIM1 then interacts with and activates Orai1 at the ER-PM junction, converting it from a dimer to a tetramer, to form a cytoplasmic calcium release-activated calcium (CRAC) channel (27). The mechanism by which CRAC channels facilitate the influx of extracellular Ca2+ is termed store-operated calcium entry (28).

PKC

PKC, a family of phospholipid-dependent serine/threonine kinases, is subdivided into three subfamilies based on their structural and activation characteristics: Conventional or classic PKC isozymes (cPKCs; α, βI, βII and γ), novel or non-classic PKC isozymes (nPKCs; δ, ε, η and θ), and atypical PKC isozymes (aPKCs; ζ, ι and λ) (29). The members of the PKC family share a conserved structure, consisting of an N-terminal regulatory region and a C-terminal catalytic region, which are connected by the C1-C4 segment and the V0-V5 variable region. Among the three subfamilies, only cPKC has a unique Ca2+-binding C2 structural domain, whereas nPKC and aPKC are Ca2+-insensitive (30). Activation of cPKC necessitates both Ca2+ and DAG. Initially, the binding of Ca2+ to the C2 domain induces a conformational change in cPKC, thereby exposing the PIP2 binding site, which is typically concealed in its autoinhibited state. This newly exposed site then interacts with PIP2 on the PM, facilitating the recruitment of cPKC to the PM. Subsequently, DAG, which is embedded in the membrane, binds to the C1 domain of cPKC, further inducing a conformational change that displaces the pseudosubstrate and ultimately leads to the activation of cPKC (31). The PKC signaling pathway serves a pivotal role in regulating gene expression, cell proliferation, differentiation, migration, survival and apoptosis. Additionally, there is evidence suggesting its crucial involvement in carcinogenesis (32).

CaMKII

CaMKII is a family of serine/threonine kinases activated by Ca2+ and CaM. Under commonly physiological conditions, each CaMKII holoenzyme typically consists of 12 subunits, with each subunit containing an N-terminal catalytic domain, a central regulatory domain and a C-terminal association domain (33). In its resting state, the catalytic domain of CaMKII is inhibited by an autoinhibitory sequence within the regulatory domain. Upon an increase in intracellular Ca2+ levels, Ca2+ binds to CaM, forming a Ca2+/CaM complex. Subsequently, this complex binds to the CaMKII regulatory domain, inducing a conformational change that disrupts the interaction between the autoinhibitory region and the catalytic domain, thereby relieving the autoinhibition of the kinase. However, this activation is reversible: When Ca2+ levels fall, CaM dissociates from CaMKII and kinase activity is re-inhibited. Additionally, the interaction between the Ca2+/CaM complex and the regulatory domain exposes the phosphorylation site of Thr287 (Thr286 in CaMKIIα), facilitating autophosphorylation. If intracellular Ca2+ concentrations remain elevated, phosphorylation at Thr287 enhances the binding affinity of CaM for CaMKII, preventing the re-association of the catalytic domain with the autoinhibitory region and sustaining CaMKII activation (34,35). CaMKII serves a pivotal role not only in learning and memory, but also in regulating cancer progression (36). Within the non-canonical Wnt/Ca2+/CaMKII pathway, CaMKII activates the transforming growth factor-β-activated kinase 1 (TAK1)-Nemo-like kinase (NLK)-MAPK cascade, resulting in the phosphorylation of TCF, which prevents the binding of the β-catenin-TCF complex to DNA. This, in turn, antagonizes the canonical Wnt/β-catenin signaling pathway and inhibits cancer progression (37).

CaN

CaN, a unique calcium and CaM-dependent serine/threonine phosphatase, serves a crucial role in cellular signal transduction by converting calcium signals into specific cellular responses (38). CaN is a heterodimeric enzyme composed of CaNA and CaNB. CaNA contains a catalytic domain and three regulatory domains: A CaNB-binding domain, a CaM-binding domain and an autoinhibitory domain. CaNB contains four EF-hand motifs for Ca2+ binding (39). In its resting state, the catalytic center of CaN is inhibited by the autoinhibitory domain, rendering the enzyme inactive. Upon signal stimulation, increased cellular cytosolic Ca2+ binds to the EF-hand motifs of CaNB, causing the dissociation of the CaM-binding domain from the B subunit regulatory domain. Subsequently, CaM binds to the CaM-binding domain of CaNA, causing displacement of the autoinhibitory domain from the catalytic center and effectively activating CaN. Notably, even in the absence of CaM, the binding of Ca2+ alone to the EF-hand motifs of CaNB can partially activate CaN (38). The activation of CaN results in the dephosphorylation of several substrates, among which the most extensively studied is nuclear factor of activated T cells (NFAT). Following dephosphorylation, NFAT translocates to the nucleus and activates transcription programs, exerting notable influences on T cell activation and cancer progression. These effects include promoting cancer cell proliferation, metastasis, angiogenesis, inflammatory responses and cancer stem cell (CSC) activity (40,41).

Activation of the Wnt/Ca2+ pathway

The Wnt/Ca2+ pathway, as a crucial branch of the Wnt signaling pathway, belongs to the category of non-canonical Wnt signaling pathways. Its activation primarily depends on the ligands Wnt5a and Wnt11 (7). When these ligands bind to the FZD receptor on the cell membrane, a series of intracellular molecular cascade reactions are triggered (19). Initially, the G protein and the FZD adapter protein Dvl are activated, leading to the activation of PLC, which further cleaves PIP2 to generate IP3 and DAG (24). Subsequently, IP3 binds to InsP3Rs on the ER membrane, triggering the release of Ca2+ from the ER (26). In response to the decrease in ER Ca2+ levels, STIM1 on the ER membrane activates the orai protein in the PM, which tetramerizes to form the CRAC channel, facilitating the entry of extracellular Ca2+ into the cytoplasm (27). The elevated cytoplasmic Ca2+ levels primarily regulate three pathways: i) Together with DAG, Ca2+ activates PKC, which subsequently stimulates cell division cycle 42, inducing actin polymerization and promoting cell polarization and migration (32). This pathway also interacts with the Wnt/PCP pathway (42); ii) Ca2+ activates CaMKII, stimulating TAK1, which then activates NLK to phosphorylate TCF, inhibiting the β-catenin/TCF complex and blocking gene transcription (37); and iii) Ca2+ activates CaN, resulting in the phosphorylation of NFAT and the promotion of downstream gene transcription (40,41) (Fig. 1).

Figure 1. Schematic diagram of the Wnt/Ca2+ signaling pathway, created in BioRender. Jing, L. (2025) https://BioRender.com/t20q988. The Wnt/Ca2+ pathway is primarily activated by Wnt5a and Wnt11 ligands, which bind to the FZD receptor on the cell membrane. This interaction triggers the activation of G proteins and the FZD adapter protein Dvl, which mediates the activation of PLC. PLC then cleaves PIP2 into IP3 and DAG. Subsequently, IP3 binds to InsP3R on the ER membrane, causing the release of Ca2+ from the ER. In response to the decreased Ca2+ levels in the ER, STIM1 proteins on the ER membrane activate the Orai protein in the PM. The Orai protein tetramerizes to form the CRAC channel, which mediates the influx of extracellular Ca2+ into the cytoplasm. The increased cytoplasmic Ca2+ primarily regulates three distinct pathways: i) With DAG, it activates PKC, further stimulating CDC42, inducing actin polymerization and promoting cell polarization and migration. This pathway also cascades through the Wnt/PCP pathway; ii) it activates CaMKII, stimulating TAK1, which then activates NLK to phosphorylate TCF, inhibiting the β-catenin/TCF complex and blocking gene transcription; and iii) it activates CaN, phosphorylating NFAT and promoting downstream gene transcription. FZD, frizzled; G protein, heterotrimeric guanine nucleotide-binding protein; Dvl, disheveled; PLC, phospholipase C; PIP2, phosphatidylinositol 4,5-bisphosphate; IP3, inositol 1,4,5-trisphosphate; DAG, diacylglycerol; InsP3R, inositol 1,4,5-trisphosphate receptors; ER, endoplasmic reticulum; STIM1, stromal interaction molecule 1; PM, plasma membrane; CRAC, calcium release-activated calcium; CDC42, cell division cycle 42; Wnt/PCP, Wnt/planar cell polarity; CaMKII, calmodulin-dependent protein kinase II; TAK1, transforming growth factor-β-activated kinase 1; NLK, Nemo-like kinase;NFAT, nuclear factor of activated T cells; TCF, T Cell Factor; CaN, calcineurin; NFAT, nuclear factor of activated T cells.

Impact of the Wnt/Ca2+ signaling pathway on the development and prognosis of several types of cancers

Dichotomous roles of Wnt/Ca2+ signaling in tumor progression

The Wnt/Ca2+ signaling pathway exerts a marked influence on the proliferation, invasion and migration of tumor cells by precisely regulating critical cellular processes, such as cell cycle progression, apoptosis, tumor angiogenesis and epithelial-mesenchymal transition (EMT) (43). This pathway serves as a notable driver of tumor malignant transformation. Specifically, in prostate cancer (PCa) (44), melanoma (45), colorectal cancer (CRC) (46), ovarian cancer (OC) (47) and lung cancer (48), the Wnt/Ca2+ signaling pathway acts as a tumor-promoting factor, accelerating tumor progression. Conversely, in other types of cancer, such as acute leukemia (AL) (49–51), thyroid cancer (TC) (52) and hepatocellular carcinoma (HCC) (53), the pathway exerts tumor suppressor gene functions by inhibiting malignant behaviors, including tumor cell proliferation, invasion and migration, thereby contributing to improved cancer prognosis to a certain extent.

Wnt/Ca2+/CaMKII axis promotes migration and drug resistance in PCa

PCa is a malignancy that specifically impacts humans, which serves a substantial role in elevating male mortality rates worldwide (54). A previous study reported that the mRNA expression levels of Wnt5a were markedly elevated in PCa specimens compared with those in healthy controls; therefore, this may have potential as a genetic marker for PCa diagnosis (55). Furthermore, elevated expression of Wnt5a and its receptor FZD2 were reported in an enzalutamide-resistant PCa cell line, specifically in C4-2B multi-drug-resistant variant (MDVR) cells. Blocking the expression of Wnt5a and FZD2 not only restored the sensitivity of C4-2B MDVR cells to enzalutamide, but also downregulated the expression of genes involved in regulating tumor cell survival and proliferation (56). Further research reported that Wnt5a promoted the migration of PCa cells via the Wnt/Ca2+/CaMKII pathway. In comparison with benign prostate tissue, malignant prostate tissue exhibited abnormally high levels of Wnt5a protein expression, which activated the Wnt/Ca2+/CaMKII pathway. The activated CaMKII then facilitated the reorganization of the cytoskeleton in PCa cells through a series of complex signal transduction mechanisms, ultimately enhancing their migratory capacity (44).

Wnt/Ca2+ pathway activates CaMKII and PKC, enhancing the invasion, migration and anti-apoptosis characteristics of melanoma

Melanoma is a malignant tumor originating from melanocytes, which ranks third in incidence among skin malignancies (57). Immunohistochemical analysis has demonstrated that the majority of metastatic melanomas exhibit intense Wnt5a staining compared with benign tumors (58). Further investigation has revealed that, by activating the non-canonical Wnt/Ca2+ signaling pathway, Wnt5a facilitates the phosphorylation of the downstream molecule CaMKII. It not only augments the angiogenic capacity of melanoma, but also promotes the formation of vasculogenic mimicry (VM), which in turn accelerates the processes of tumor cell proliferation, invasion and migration (59). Additionally, activation of the Wnt5a/FZD-5 pathway promotes Ca2+-dependent phosphorylation of PKC in melanoma cells, mediating the invasion and migration of melanoma cells by downregulating metastasis suppressor genes (KiSS-1 metastasis suppressor and cadherin 1) and upregulating metastasis-associated molecules (CD44 and SNAIL) (60). A previous study reported that Wnt5a-induced PKC signaling promoted the phosphorylation of the endogenous substrate myristoylated alanine-rich C-kinase substrate, which is essential for melanoma cell invasion (45). Furthermore, non-canonical Wnt/Ca2+ signaling serves a crucial role in inhibiting apoptosis in melanoma cells. TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, can induce apoptosis in melanoma cells. However, Wnt5a-mediated Wnt/Ca2+ signaling protects melanoma cells from apoptosis by activating CaMKII to upregulate the expression of cellular Fas-associated death domain-like interleukin-1β converting enzyme inhibitory protein (c-FLIP), thereby inhibiting TRAIL-mediated cleavage of caspase-8 and caspase-10. By contrast, the CaMKII inhibitor KN-93 can reduce the expression of c-FLIP and restore the sensitivity of resistant cells to TRAIL-induced apoptosis (61).

Wnt/Ca2+ pathway activates NFAT and CaMKII, accelerating the proliferation and migration of CRC cells

CRC ranks as the third most prevalent cancer worldwide and is the second leading cause of global cancer-related mortality (62). In a tumor sphere formation assay, Wnt3a and Wnt5a ligands were reported to promote the formation of colon CSC spheroids and cell proliferation by activating PLC and NFAT. Specific inhibition of PLC or NFAT can notably impair sphere formation capacity, suggesting that Wnt/Ca2+ signaling is important for maintaining the self-renewal of colon CSCs (63). The Wnt/Ca2+ signaling pathway also promotes CRC cell migration. Research has demonstrated that both Wnt3a and Wnt5a ligands are capable of activating the Wnt/Ca2+ signaling pathway, which subsequently induces PLC-dependent colon cancer cell migration (64). Furthermore, tumor-associated macrophages (TAMs), particularly M2-polarized macrophages, highly express Wnt5a. In co-culture systems of macrophages and CRC cells, Wnt5a has been reported to promote the expression of C-C chemokine ligand 2 in TAMs through the Wnt5a/CaMKII/ERK pathway, thereby enhancing the proliferation and migration of CRC cells (46). In addition, the expression levels of Wnt11 are markedly elevated in CRC, and are closely associated with tumor aggressiveness and patient prognosis (65). Notably, Wnt11 may enhance the invasiveness of CRC by promoting the morphological transformation of intestinal epithelial cells and the EMT process by enhancing the activation of PKC and CaMKII (66).

Wnt/Ca2+ pathway activates NFAT and PKCα, promoting EMT, angiogenesis and invasion in OC

OC has the third highest incidence among malignant tumors of the female reproductive system and epithelial OC (EOC) constitutes the most prevalent subtype (67). The Wnt5a/Ca2+ signaling pathway serves a pivotal role in OC invasion. A large-scale study involving 623 patients revealed that the protein expression levels of Wnt5a in patients with EOC were markedly higher than in patients with benign and borderline ovarian tumors, as well as in healthy control subjects. The upregulation of Wnt5a suppresses the canonical Wnt pathway in EOCs, induces non-canonical Wnt signaling, augments the expression of NFAT, JNK and PKCα, diminishes cell adhesion, and accelerates the EMT (68). Research by Abedini et al (69) further supported the aforementioned findings. In mouse ovarian surface epithelium (OSE) cells, Wnt5a activated CaMKII via Wnt/Ca2+ signaling, upregulating vimentin and CD44 to facilitate EMT, and transforming OSE cells into a mesenchymal cell morphology. The expression of Wnt5a varies across different OC cell lines, with lower expression observed in the well-differentiated OC cell line SKOV3 relative to the OVCAR3 cell line. It has been reported that overexpression of Wnt5a in SKOV3 cells activates PKCα, upregulates vimentin and suppresses the expression of E-cadherin, thereby enhancing the EMT and vasculogenic capacity of OC cells in vitro. Notably, the application of a PKCα inhibitor can reverse these effects (47). In addition, the Wnt5a/PKC/CAMP responsive element binding protein 1 axis of the non-canonical Wnt signaling pathway serves an important role in the self-renewal and dedifferentiation of ovarian CSCs (70).

Wnt/Ca2+ pathway activates PKC, promoting the proliferation and invasion of lung cancer cells

Lung cancer has the second highest incidence and first highest mortality rate among all types of cancer (71). Research has reported that the expression levels of Wnt5a in non-small cell lung cancer (NSCLC) tissues are markedly higher than those in corresponding adjacent normal tissues, and that Wnt5a is associated with a poor prognosis in NSCLC (72). Wnt5a can activate the Wnt/Ca2+/PKC signaling pathway, substantially enhancing the proliferation, migration, invasion and colony formation of lung cancer cells, whilst suppressing apoptosis. Notably, the PKC inhibitor GF109203X has been reported to notably decrease the proportion of aldehyde dehydrogenase-positive lung CSCs in the cisplatin-resistant A549/DPP cell line, and to reverse the suppressive effect of Wnt5a on cell apoptosis (48). Previous investigations have reported that the expression levels of activating transcription factor-2 (ATF-2) in NSCLC were also markedly higher than in normal bronchial epithelial cells. High expression of ATF-2 can augment the activity of the Wnt/Ca2+ signaling pathway, boosting the proliferation and invasion of NSCLC cells. However, knockdown of ATF-2 in A549 cells can inhibit the activity of the Wnt/Ca2+ signaling pathway, and substantially reduce the proliferation and invasion of NSCLC cells (73).

Wnt/Ca2+ pathway activates CaMKII and PKC, regulating the differentiation and inhibiting abnormal proliferation of AL cells

AL is the most common hematopoietic stem cell malignancy in children and has the highest incidence rate among pediatric oncological disorders (74). A study encompassing 86 pediatric specimens of acute lymphoblastic leukemia (ALL) reported that the Wnt5a promoter was highly methylated in both B cell ALL and T cell ALL (T-ALL) patient samples, with a decrease in mRNA expression levels observed in both. Notably, this decrease was more pronounced in patients with T-ALL (75). In B-cell tumors, Wnt5a inhibits the expression of cyclin D1 by activating the Wnt/Ca2+ pathway, thereby negatively regulating B-cell proliferation (49). Regarding acute myeloid leukemia (AML), a previous study reported that among 11 AML cell lines and 252 samples from patients with AML, seven cell lines and 43% (107/252) of patient samples exhibited high methylation of the Wnt5a promoter. Compared with unmethylated patients, Wnt5a-methylated patients exhibited downregulated Wnt5a expression and upregulated cyclin D1 transcription levels (50). The Wnt/Ca2+ signaling pathway is pivotal for the differentiation of AML cells and subpopulations induced by 6-benzylthioinosine (6-BT). Following treatment with 6-BT, HL-60 cells have been reported to exhibit decreased β-catenin levels and elevated Ca2+ concentrations, as well as increased phosphorylation levels of CaMKII and PKC. By contrast, pretreatment with the PKC inhibitor bisindolylmaleimide suppresses the Wnt/Ca2+ signaling and impedes cell differentiation (51). Foxy-5, a novel and innovative Wnt5a-mimicking compound, effectively inhibits the abnormally activated β-catenin, PI3K/AKT and MAPK/ERK signaling pathways in AML, thereby inducing leukemia cell differentiation and markedly inhibiting their aberrant proliferation, cell survival and self-renewal potential (76).

Wnt/Ca2+ pathway inhibits TC cell proliferation via CaMKII activation and β-catenin phosphorylation

TC is a prevalent malignancy of the endocrine system and there has been a substantial rise in its incidence globally over the past few decades (77). Studies have reported that Wnt5a exhibits either low or no expression in normal thyroid tissue and undifferentiated TC, but demonstrates high expression in differentiated TC (78,79). Further research has reported that when human follicular TC cells (FTC-133) are stimulated with recombinant Wnt5a, or when Wnt5a is overexpressed in FTC-133 cells, the intracellular Ca2+ levels rapidly elevate, subsequently activating CaMKII downstream of the Wnt/Ca2+ signaling pathway. Activated CaMKII facilitates the phosphorylated degradation of β-catenin, leading to the downregulation of nuclear c-myc expression, thereby inhibiting the proliferative effect of canonical Wnt signaling on FTC-133 cells. This effect can be blocked by the CaM inhibitor W-7 or the CaMKII inhibitor KN-93 (52). However, previous studies have also reported that the expression levels of receptor tyrosine kinase-like orphan receptor 2 (ROR2) and Wnt5a in papillary thyroid carcinoma (PTC) tissues are markedly higher than those in adjacent normal tissues, and their expression levels are closely associated with tumor staging and lymph node metastasis (79). Long-stranded non-coding RNA FAM230B is highly expressed in PTC tissues and functions as a microRNA (miR) sponge, protecting miR-378a-3p from degrading Wnt5a mRNA through a competitive mechanism, thereby upregulating Wnt5a expression and enhancing the migration and invasion of PTC cells (80). However, there remains a lack of definitive evidence regarding whether the oncogenic role of Wnt5a in TC is mediated through the Wnt5a/Ca2+ signaling pathway.

Wnt/Ca2+ pathway activates PKC and induces β-catenin phosphorylation, inhibiting the proliferation of HCC cells

Liver cancer is a prevalent global health problem, with an escalating incidence and substantial mortality rates. It has risen to become the sixth most common cancer and the third leading cause of cancer-related deaths worldwide. Moreover, HCC comprises ~90% of all liver cancer cases (71). Studies have reported that, in comparison with normal tissues adjacent to human HCC, the mRNA and protein expression levels of Wnt11 are markedly downregulated in HCC tissues (53,81). Further in vitro experiments have reported that Wnt11 effectively inhibits canonical Wnt signaling by facilitating β-catenin phosphorylation through activation of the Wnt/Ca2+/PKC signaling pathway, thereby substantially suppressing HCC cell proliferation. Notably, this inhibitory effect can be reversed by the PKC inhibitor bisindolylmaleimide I (53). Additionally, it has been reported that Wnt5a expression levels are notably lower in HCC tissues compared with those in adjacent non-cancerous tissues, and are closely associated with disease progression and a poor prognosis (82,83). In HCC cell lines, Wnt5a overexpression not only enhances β-catenin phosphorylation, but also elevates E-cadherin levels, effectively inhibiting the proliferation and migration of HCC cells (84,85). Although the inhibitory effect of Wnt5a on canonical Wnt signaling in HCC cells has been previously reported (86), the specific mechanisms remain unclear.

Challenges and future trends of Wnt/Ca2+ signaling in cancer therapy

The Wnt/Ca2+ signaling pathway serves a pivotal role in tumorigenesis, metastasis and therapeutic resistance by regulating cellular migration, invasion and remodeling of the tumor microenvironment (TME) (43). Epigenetic modifications, particularly the dynamic regulation of DNA methylation (87–90) and histone modifications (91–93), markedly impact the activity of Wnt/Ca2+ signaling, offering novel therapeutic strategies centered on epigenetic regulation for cancer treatment.

At the DNA methylation level, aberrant methylation is a key driver of cancer progression. Notably, hypermethylation of the SFRP promoter, which encodes a Wnt antagonist, leads to gene silencing and subsequently disinhibition of Wnt ligand activity, promoting aberrant pathway activation in CRC, breast cancer (BC) and other malignancies (87). Conversely, hypermethylation of Wnt ligand genes (such as WNT5A) suppresses transcription, resulting in reduced Wnt/Ca2+ signaling pathway activity (88,89). Additionally, methylation-induced silencing of calcium channel subunit genes (such as the calcium channel α2-Δ3 subunit) disrupts intracellular Ca2+ signaling, impairing downstream Wnt/Ca2+-dependent effects, including CaN-mediated apoptosis, fostering oncogenesis or neurodevelopmental defects (90). Notable progress has been made in developing targeted drugs against DNA methyltransferases. Hypomethylating agents such as azacitidine and decitabine have demonstrated substantial clinical importance in treating high-risk myelodysplastic syndromes, chronic myelomonocytic leukemia and AML (94).

Complementing the DNA methylation-based strategy, histone modification regulation emerges as another critical mechanism in reshaping Wnt/Ca2+ signaling and tumor phenotypes. Histone deacetylase (HDAC) inhibitors enhance histone acetylation, upregulating Wnt ligand expression (such as Wnt4, Wnt5a and Wnt11) and strengthening Wnt/Ca2+ signaling, promoting cellular migration and differentiation (91). For example, the HDAC6 inhibitor BML-281 increases Wnt5a expression and activates downstream Wnt/Ca2+ signaling (92). Furthermore, HDAC inhibitors (HDACis) restore the expression of the secretory pathway Ca2+-ATPase secretory pathway calcium ATPase 2 (SPCA2) in triple-negative BC (TNBC), elevating intracellular Ca2+ levels and inducing a less aggressive tumor phenotype via non-canonical Wnt/Ca2+ signaling (93). Currently, five HDACis (vorinostat, romidepsin, belinostat, panobinostat and tucidinostat) are approved for treating T-cell lymphomas and multiple myeloma by the US Food and Drug Administration. Ongoing clinical trials continue to explore their potential in AML, B-cell lymphomas and several solid tumors (95,96).

Post-translational modification emerges as another critical dimension in modulating this pathway. Among these, palmitoylation is a key post-translational modification for Wnt protein secretion, ensuring correct Wnt ligand localization. Porcupine inhibitors (such as WNT974, ETC-159, RXC004 and CGX1321) block WNT ligand O-palmitoylation, suppressing both canonical and non-canonical Wnt signaling. These agents have shown preclinical promise in CRC, pancreatic cancer and HCC, with several entering Phase I trials (97,98).

Beyond epigenetic therapies, researchers have developed ligands and receptor-targeted agents against the Wnt/Ca2+ signaling pathway. For example, two Wnt5a mimetic peptides, Foxy-5, a WNT5A agonist and Box-5, a WNT5A antagonist, modulate Wnt5a-dependent signaling to reduce tumor metastasis. Foxy-5 specifically binds to FZD-5 receptors in BC, rapidly activating membrane calcium signaling without affecting canonical β-catenin or JNK pathways (99). Preclinical and clinical studies have demonstrated its potent anti-metastatic activity, reducing tumor metastasis by 70–80% in WNT5A-deficient BC models (100). Moreover, Box-5 inhibits Wnt5a-induced Ca2+/PKC signaling, exhibiting unique anti-invasive properties in melanoma (101).

In addition to peptide-based agents, monoclonal antibodies targeting Wnt receptors have shown promise. Vantictumab (OMP-18R5), a fully humanized monoclonal antibody, targets multiple FZD receptors, blocking Wnt ligand binding and inhibiting canonical and non-canonical pathways. Preclinical investigations have revealed its antiproliferative efficacy across diverse human tumor models, with improved therapeutic outcomes observed when combined with chemotherapeutic regimens (102–104).

Despite the aforementioned advances, challenges remain in optimizing Wnt/Ca2+-targeted therapies, including toxicities and tumor heterogeneity. Whilst epigenetic drugs are generally less toxic than traditional chemotherapeutics, they may still cause myelosuppression, gastrointestinal disturbances and hepatorenal toxicity. Additionally, their efficacy in solid tumors often lags behind that in hematological malignancies, with variable patient responses. Combining epigenetic drugs with other antitumor agents offers a promising strategy to enhance efficacy and reduce toxicity, yet optimal dosing and regimen design require further refinement (94,96). Moreover, Porcupine inhibitors (such as WNT974 and ETC-159) and pan-FZD antibodies (such as Vantictumab) have shown dose-limiting toxicities related to loss of bone mass, likely due to the essential role of Wnt signaling in osteoblast differentiation and regulation of bone homeostasis (105). Therefore, by integrating the expression profiles of Wnt ligands and receptors in tumor cells and the TME, it may be possible to develop inhibitors targeting specific Wnt ligands and receptors and minimize off-target effects on bone and other normal tissues, thus preserving skeletal integrity and avoiding unnecessary side effects.

Discussion

In recent years, integrating interdisciplinary research methods and the widespread application of innovative technologies have markedly advanced tumor diagnosis and treatment (106,107). Tumorigenesis and tumor progression are governed by intricate biological processes involving numerous signaling pathways, among which the Wnt signaling pathway serves as a core regulatory factor and has therefore attracted notable scientific interest. Until now, several studies have performed in-depth explorations of the role of the canonical Wnt/β-catenin signaling pathway in tumorigenesis. This pathway exerts crucial regulatory effects on multiple biological processes of tumor cells, including proliferation, differentiation and apoptosis, as well as invasion and metastasis (108–111). However, the specific role of the Wnt/Ca2+ signaling pathway, as an important non-canonical branch of the Wnt signaling pathway, in tumorigenesis and tumor development has not been fully explored. Therefore, the present review systematically introduces the research progress of the Wnt/Ca2+ signaling pathway and explores its biological effects and potential mechanisms in several tumors (Table I and Fig. 1). Furthermore, it provides a comprehensive overview of existing therapeutic strategies targeting this pathway in cancer and discusses associated challenges.

Table I. Wnt/Ca2+ signaling pathway and cancer.

Table I.

Wnt/Ca2+ signaling pathway and cancer.

Cancer type	Function/mechanisms	(Refs.)
PCa	Wnt5a is highly expressed in metastatic PCa; Wnt5a/Ca2+ activates CaMKII, regulating the cytoskeleton and promoting migration of PCa cells.	(44,55,56)
Melanoma	Wnt5a is highly expressed in metastatic melanoma; Wnt5a/Ca2+ signaling activates CaMKII and PKC to inhibit the canonical Wnt signaling, enhancing melanoma angiogenesis and vasculogenic mimicry formation, and promoting melanoma cell migration and invasion; Wnt5a/Ca2+ signaling activates CaMKII and inhibits TNF-related apoptosis-inducing ligand-induced apoptosis.	(45,58–61)
CRC	Wnt/Ca2+ signaling activates PLC and nuclear factor of activated T cells, promoting the proliferation of colon cancer stem cells, and accelerating the proliferation and migration of CRC cells; the Wnt5a/CaMKII/ERK pathway enhances C-C chemokine ligand 2 expression in tumor-associated macrophages, promoting CRC cell proliferation and migration; Wnt11/Ca2+ signaling activates PKC and CaMKII, stimulating proliferation of intestinal epithelial cells, reducing E-cadherin contacts and promoting CRC metastasis.	(46,63–66)
OC	Wnt5a is highly expressed in epithelial OC; Wnt5a/Ca2+ signaling activates PKCα and promotes the progression of epithelial-mesenchymal transition and VM in OC cells; Wnt5a/PKC/CREB1 signaling promotes the proliferation of ovarian cancer stem cells.	(47,68–70)
Lung cancer	Wnt5a is highly expressed in non-small cell lung cancer; Wnt5a/Ca2+ signaling activates PKCα and CaMKII, and promotes the proliferation and invasion of non-small cell lung cancer cells.	(48,72,73)
AL	Wnt5a has a low expression in AL; Wnt5a/Ca2+ signaling downregulates cyclin D1, inhibiting B-cell proliferation; 6-benzylthioinosine upregulates Wnt/Ca2+ signaling to activate CaMKII and PKC, promoting differentiation of AML cells; Foxy-5 blocks abnormally activated β-catenin, PI3K/AKT, MAPK/ERK and other signaling pathways in AML to inhibit its progression.	(49–51,75,76)
TC	Wnt5a has a low expression in undifferentiated TC; Wnt5a/Ca2+ signaling activates CaMKII, promoting β-catenin degradation, downregulating c-myc and inhibiting the proliferation of FTC-133 cells.	(52,78–80)
HCC	Wnt11 has a low expression in HCC tissues; Wnt11/Ca2+ activates PKC, downregulates canonical Wnt signaling and inhibits HCC cell proliferation; Wnt5a has a low expression in HCC tissues; Wnt5a inhibits canonical Wnt signaling and reduces the proliferation and migration of HCC cell lines.	(53,81–86)

[i] PCa, prostate cancer; CaMKII, calmodulin-dependent protein kinase II; PKC, protein kinase C; CRC, colorectal cancer; PLC, phospholipase C; OC, ovarian cancer; VM, vasculogenic mimicry; CREB1, CAMP responsive element binding protein 1; AL, acute leukemia; AML, acute myeloid leukemia; TC, thyroid cancer; HCC, hepatocellular carcinoma.

The Wnt/Ca2+ signaling pathway demonstrates remarkable functional heterogeneity in tumors, acting either as a tumor promoter or suppressor. Such context-dependent effects may be attributed to differences in the ligand selection of the signaling pathway, receptor combination and the TME. The tumor suppressive mechanism of the Wnt/Ca2+ signaling pathway may involve several reasons. First, the Wnt/Ca2+ signaling pathway antagonizes the canonical Wnt/β-catenin pathway. Specifically, the Wnt/Ca2+ pathway inhibits the transcriptional activity of β-catenin by activating the CaMKII and TAK-NLK pathway, thereby limiting tumor proliferation and invasion driven by the canonical Wnt/β-catenin pathway (112). For example, in TNBC, HDACi triggers an increase in resting Ca2+ levels by upregulating the SPCA2 gene. High levels of Ca2+ activate CAMKII, a downstream component of non-canonical Wnt/Ca2+ signaling, leading to phosphorylation of β-catenin and inhibition of its nuclear translocation. This ultimately leads to the conversion of cancer cells to a less aggressive ‘epithelial’ state (93). Secondly, the Wnt/Ca2+ signaling pathway can induce cell apoptosis. High concentrations of intracellular Ca2+, induced by Wnt5a and Wnt11 binding with FZD-2, activate calcium-dependent phosphatases, such as CaN, dephosphorylates the pro-apoptotic protein Bad, promotes the release of cytochrome C, and further induces apoptosis (113). Simultaneously, Ca2+ activates calpain, a CaM-degrading enzyme near the ER, which acts on caspase-12, activating it and releasing it into the cytoplasm, thus inducing apoptosis (114). By contrast, the Wnt/Ca2+ signaling pathway may also promote tumor development, mainly by enhancing the migration and invasion of tumor cells. In ameloblastoma, it has been reported that Wnt5a drives mitochondrial energy production and increases the number of mitochondria, but reduces their size. Moreover, it also increases Ca2+ levels, directly leading to altered mitochondrial dynamics, interactions between the cytoskeleton and mitochondria, and promotion of cell migration via the nuclear factor of activated T cells 2 (NFAT2)-coronin 1A-F-actin axis (115).

In several solid tumors such as PCa, CRC, OC and lung cancer, the Wnt/Ca2+ signaling pathway markedly promotes tumor metastasis through processes such as EMT and VM. This is consistent with the tumor-promoting role of the canonical Wnt/β-catenin pathway in these tumors, and the two pathways work synergistically to promote tumor progression (111,116). This synergistic effect is also reflected in their joint regulation of the TME, particularly by influencing the polarization state of TAMs, thereby promoting the occurrence and development of malignant tumors (117). However, in tumors such as AL, TC and HCC, the Wnt/Ca2+ signaling pathway exhibits distinct biological effects. In these tumors, this pathway is often silenced due to epigenetic modifications of its ligands. Upon activation, it can exert tumor-suppressive effects by inhibiting the Wnt/β-catenin signaling pathway (78,82,118). Nevertheless, this inhibitory effect does not always lead to tumor suppression. For example, in melanoma, Wnt/Ca2+ signaling promotes the transition of tumor cells from a proliferative state to a highly invasive phenotype by inhibiting Wnt/β-catenin signaling, ultimately enhancing their metastatic ability (119). In general, the tendency of the Wnt/Ca2+ pathway to act as a tumor suppressor or a tumor promoter in different contexts is influenced by multiple factors, including tumor type-specific characteristics, TME factors, the dynamic balance between signaling pathways and epigenetic regulatory mechanisms. The complex interactions among these factors determine the functional output of this pathway in different tumors, providing important clues for explaining its spatiotemporal heterogeneity.

Despite considerable advances in elucidating the mechanisms of the Wnt/Ca2+ signaling pathway, several challenges remain to comprehensively understand its molecular intricacies. Notably, the precise timing of the interaction between Dvl and G proteins within the signaling cascade, along with its kinetic characteristics, requires further investigation to identify additional potential therapeutic targets for tumors. The therapeutic landscape of Wnt/Ca2+ pathway-targeted treatments is further complicated by the challenges of achieving sustained therapeutic doses of single-agent epigenetic therapies. Emerging evidence suggests that delivering epigenetic therapies at low doses or short intervals may act as an adjuvant strategy to augment the effects of other oncology treatments. This method shows promise in enhancing the efficacy of existing oncological treatments, though optimal dosing and treatment regimens require continued refinement and systematic optimization (94,96). Profound tumor heterogeneity adds another layer of complexity, manifested through the variable expression levels of Wnt ligands, FZD receptors and their co-receptors (120,121). The signaling outcomes critically depend on the predominant co-receptor context (122). In scenarios where ROR2 serves as the primary co-receptor, Wnt5a facilitates the degradation of β-catenin via FZD2 or FZD5 signaling, thereby inhibiting cancer progression. Conversely, when the low-density lipoprotein receptor related protein 6 (LRP6) co-receptor prevails, Wnt5a forms a complex with FZD4/LRP6, leading to the stabilization of β-catenin and fostering cancer metastasis (123,124). Consequently, by integrating the expression profiles of Wnt ligands and receptors in tumor cells and the TME, it is feasible to identify the Wnt signaling subtypes that dominate during tumorigenesis. This approach enables precise selection of drugs targeting specific Wnt ligands, receptors or co-receptors, thus improving the therapeutic efficacy. Furthermore, synergistic or antagonistic effects between canonical and non-canonical Wnt pathways can be harnessed to develop targeted therapies focusing on shared upstream regulators or downstream effectors, thereby minimizing unnecessary side effects.

Acknowledgements

Not applicable.

Funding

The present work was supported by grants from the National Natural Science Foundation of China (grant nos. 82071738, 81671541 and 81701545), Jiangsu Natural Science Foundation (grant no. BK20231236) and the Medical Leadership Program of Jiangsu College of Nursing (grant no. 2021L001).

Availability of data and materials

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Authors' contributions

LJ and SX designed the review framework, drafted the manuscript, and created and refined the figures and tables. HW and QS reviewed and revised the manuscript, and provided financial support for its publication. All authors read and approved the final manuscript. Data authentication is not applicable.

Ethics approval and consent to participate

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Patient consent for publication

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Competing interests

The authors declare that they have no competing interests.

References