Wnt signaling in cell adhesion, development, and colon cancer

Wnt signaling is essential for embryonic development, influencing processes such as axis formation, cell proliferation and differentiation, cell fate decisions, and axon guidance. It also plays a role in maintaining tissue homeostasis in adult organisms. The loss of normal cell polarity and adhesion caused by Wnt signaling activation is a fundamental step for tumor progression and metastasis. Activating the canonical Wnt pathway is a driving force in many human cancers, especially colorectal, hepatocellular, and mammary carcinomas. Wnt causes the stabilization and nuclear transport of newly synthesized transcriptional regulator β‐catenin. The generally accepted view is that the canonical effects of Wnt growth factors are caused by the transcription of β‐catenin target genes. Here, we review recent findings that indicate Wnt is a regulator of many other cellular physiological activities, such as macropinocytosis, endosome trafficking, protein stability, focal adhesions, and lysosomal activity. Some of these regulatory responses occur within minutes and do not require new protein synthesis, indicating that there is much more to Wnt beyond the well‐established transcriptional role of β‐catenin. The main conclusion that emerges from these studies is that in basal cell conditions, the activity of the key protein kinase GSK3, which is inhibited by Wnt pathway activation, normally represses the actin machinery that orchestrates macropinocytosis with implications in cancer. These contributions expand our understanding of the multifaceted roles of Wnt signaling in cellular processes, development, and cancer, providing insights into potential therapeutic targets and strategies.

the fundamental role of membrane trafficking, lysosomes, focal adhesion, and macropinocytosis in the Wnt pathway, suggesting a more general role of these signals in regulating cellular processes.[3] As shown in Figure 1, when the Wnt ligand binds to its receptors "Frizzled" and the co-receptor LDLreceptor-related protein 6 (LRP-6), this triggers the formation of the LRP-6 signalosome. 4These co-receptors are part of a destruction complex of β-catenin (a transcription factor and cell adhesion molecule) along with glycogen synthase kinase 3 (GSK3), which F I G U R E 1 Model of Wnt/β-catenin pathway in the presence and absence of Wnt ligand.This model illustrates the Wnt/β-catenin pathway's activity in response to the presence or absence of the Wnt ligand.Without Wnt, the proteins APC, Axin, CK1, DVL, and GSK3 constitute the destruction complex that controls β-catenin levels, influencing Wnt signaling.Upon Wnt ligand binds with the Frizzled (Fz) receptor and the LRP-6 co-receptor, GSK3 translocates to the membrane and is then internalized into an early endosome and subsequently into multivesicular bodies (MVBs).This process, which sequesters GSK3 and the destruction complex, triggers macropinocytosis in the Wnt pathway.In the cell nucleus, β-catenin forms an active complex with LEF (lymphoid enhancer factor) and TCF (T-cell factor) proteins by displacing TLE/Groucho complexes and recruitment of histone-modifying co-activators such as CBP/p300, BRG1, BCL9, and Pygo.Such a transcriptional switch leads to a change in multiple cellular processes, such as the activation of genes involved in proliferation and cell migration.Additionally, the transmembrane E3 ligases RNF43/ZNRF3 represent important elements down regulators of the Wnt signaling through the ubiquitination and degradation of the Fzd/Lrp5/6 receptor complex via the endolysosomal pathway.While β-catenin is recognized as an important oncogene, it is also pivotal for cell adhesion as a structural component of the cadherin complex structure (detailed in Figure 3) (Figure created with BioRender.com,accessed on 11 July 2023.).APC, adenomatous polyposis coli; GSK3, glycogen synthase kinase 3; LRP-6, LDL-receptor-related protein 6.
phosphorylates β-catenin in the absence of Wnt, leading to ubiquitin-mediated proteolysis of β-catenin.However, in the presence of Wnt, the destruction complex will be recruited to the LRP-6 signalosome, sparing the β-catenin from degradation. 5Instead, the activation of the Wnt pathway helps stabilize β-catenin, allowing it to translocate into the nucleus to interact with other transcriptional regulators, such as LEF1, to trigger the encoding of many different Wnt-related genes important for cell fate determination, including oncogenes. 6The Wnt/β-catenin signaling is negatively regulated by ZNRF3/RNF43, leading to endo-lysosomal degradation of the Wnt receptor/ co-receptor. 7ecent studies found that Wnt treatment, along with mutations in tumor suppressor adenomatous polyposis coli (APC) and Axin1 genes, enhances macropinocytosis and lysosomal activity, impacting cellular nutrition. 1This is accompanied by alternations in the nucleocytoplasmic distribution of focal adhesion proteins, that is, affecting cell migration behaviors. 2This is particularly interesting in the context of oncology because cancer metastasis accounts for more than 90% of all cancer-related deaths but is the least understood biological process in cancer. 7he early phases of metastasis involve increased proteolysis, loss of cell-cell adhesion, migration, and invasion of primary tumor cells into surrounding tissues. 3ne of the critical connections between cells is mediated by a protein called E-cadherin.When E-cadherin stops working, it is like untying a boat from its dock; the cell loses its anchor, and proteins that were held in place, such as β-catenin, are released from the cytoplasmic side of the adhesion complex.Normally, β-catenin is part of a complex that helps cells stick to each other, but when freed, it moves to the nucleus and works with TCF/LEF transcription factors (as shown in Figure 1), to switch on genes that could contribute to cancer progression. 8,9herefore, investigating fundamental biological processes such as membrane trafficking, lysosomes, and focal adhesions under the influence of Wnt signaling could shed light on new mechanisms leading to the dissemination of malignant cells and their seeding of distant organs.
Endocytosis encompasses the cellular process of internalizing substances within vesicles formed from the cell membrane. 10Macropinocytosis, a subtype of endocytosis, involves the uptake of significant extracellular fluid and solutes by forming large vesicles (>0.2 μm) called macropinosomes. 11,12Focal adhesions are specialized structures at the cell-extracellular matrix (ECM) interface, facilitating cell adhesion, migration, and signaling by linking integrin receptors to the actin cytoskeleton and serving as vital hubs for cellular interactions with the environment. 13lterations to the actin-based cytoskeleton are also characteristics of transformed cells.β-Catenin interacts with the cytoplasmic domain of E-cadherin and binds to α-catenin, an actin-binding protein. 14The Wnt signaling pathway directly targets the actin cytoskeleton, disrupting stress fibers. 14Recently, a connection was reported between Wnt signaling, receptor-mediated endocytosis, lysosomal activity, and macropinocytosis in the Wnt pathway.15][16][17] Focal adhesions have also been implicated in this regulation system. 3,15A recent study analyzed Wntinduced Lrp6 interacting proteins using an APEX2-mediated proximity labeling approach and showed that, besides the endocytosis machinery proteins, focal adhesion proteins were also among the list of Wnt-inducible Lrp6 binding partners.This suggests that focal adhesion proteins play an important role in Wnt signaling. 18The same study showed that endocytosis was the top hit after 5 min of Wnt treatment.Tumor cells are softer than normal cells, mainly due to the loss of cytoskeletal support 18 (Figure 2).The loss of stiffness can represent a phenotype of tumor development, which facilitates cancer cell migration and adaptation to other tissues. 19Therefore, finding new strategies to suppress endocytosis/macropinocytosis in Wnt-driven cancers and increasing tumor cell adhesion (thus suppressing metastasis) can be a potential strategy to develop new therapeutic targets in cancer.

| WNT SIGNALING AND CELL ADHESION
The Wnt pathway is essential for multicellular functions [20][21][22][23][24] and has also been previously linked to cancer since an overexpression or insertion of int1, the mouse ortholog of the Drosophila wingless gene, in the Wnt1 region of the genome, led to the formation of tumors. 25,26he activation of Wnt signaling by using a Wnt3a commercial peptide or using pharmacological inhibitors of GSK3 or the loss of tumor suppressor proteins, such as APC and Axin1, resulted in an unexpected increase in the endocytosis and lysosomal degradation of BSA-Dequenched (BSA-DQ), a reagent that fluoresces after degradation by lysosomal proteases increases.Notably, a significant enhancement in lysosomal degradation was observed even when the concentration of BSA-DQ was substantially lower (1000-fold lower) than the quantity of unlabeled BSA present in the 10% serum used in the cultures.These findings imply that Wnt signaling stimulates macropinocytosis, followed by lysosomal degradation, which is normally repressed by the GSK3-Axin1-APC destruction complex. 1,17he emerging connection between Wnt signaling, lysosomal trafficking, and macropinocytosis opens new connections that Wnt signaling regulates focal adhesion proteins by endocytosis/macropinocytosis. 3 Wnt was previously shown to stimulate the Src (Rous sarcoma oncogene)-Dvl (Dishevelled, an intracellular component of the Wnt signaling pathway) interaction 27 ; but Du et al. also reported that ECM stiffness dictates Wnt signaling through the integrin pathway. 28Importantly, Capelluto et al. reported that the DIX domain is involved in the formation of Dvl dynamic polymers, Axin, actin, and associates with the vesicular structures. 29his is also the location of focal adhesion proteins, which mediate the interaction between the cytoskeleton and the ECM. 29More recently, the ESCRT (endosomal sorting complexes required for transport) machinery has been shown to regulate the transit of c-Src from endosomes to the plasma membrane. 30It was reported that Wnt signaling requires endosomes/multivesicular bodies (MVBs) to sequester GSK3 from the cytosol (Figure 1). 31It was also reported that Wnt stimulates macropinocytosis, 1,17,32,33 and blocking the ESCRT machinery not only inhibits macropinocytosis but also the β-catenin stabilization. 1 The association between endocytosis, MVBs, cell polarity, and Wnt, followed up the finding that Wnt regulates focal adhesions.
Wnt and cell-matrix adhesion are often active in the same developmental processes, and crosstalk between them should result in reciprocal regulation.Wnt signaling pathways, canonical or Wnt/β-catenin dependent pathway, and the noncanonical or β-catenin-independent pathway, which includes the Planar Cell Polarity pathway and the Wnt/Ca 2+ pathway, 7 regulate cell adhesion by controlling the stability of proteins like β-catenin, a component of adherents junctions.On the other hand, cellular mechanisms responsible for regulating adhesion and migration can also modulate the activity of several Wnt pathway components. 34For example, the overexpression of cadherins in Xenopus embryos was shown to inhibit β-catenin transcriptional activity. 35In a different study, it was reported that reductions in cadherin levels can release β-catenin bound at the cell surface and enhance nuclear β-catenin signaling events in the presence of Wnt. 36Therefore, it seems likely that cell adhesion is linked in some important way to Wnt signaling and developmental patterning events.Knowing the molecular mechanism in which Wnt signaling and focal adhesions cooperate will improve our understanding of embryonic development and tumorigenesis.It has been proposed that focal adhesion restoration strategies could be useful in the treatment of metastatic cancer. 37,38nderstanding how macropinocytosis, Wnt, and focal adhesions intersect could lead to new targets in cancer treatment.
The Wnt and Integrin signaling pathways are very ancient and were already present in sponges.Integrins are central components of the focal adhesion complex that anchor the actin cytoskeleton to the ECM.Consisting of α and β subunits, there are 24 possible heterodimers, yet the majority share a common β-1 subunit, leading us to focus on Integrin beta 1 (ITGβ1) which has a role as a major adhesion receptor and is associated with cancer.Integrins are constantly endocytosed and recycled back to the plasma membrane through multiple pathways 39,40 (Figure 3).Tight regulation of Integrin turnover from the cell surface is pivotal to a number of biological processes: cell migration, cytokinesis, and metastasis.Integrins are predominantly internalized via clathrinmediated endocytosis.Using a surface biotinylation assay, it was reported that Wnt causes the internalization of focal adhesions and integrins from the cell surface into MVBs 3 (Figure 4).In Xenopus embryos, it was found that using ITGβ1 MO inhibited the effects of Wnt signaling in the embryo.However, this was rescued by the addition of ITGβ1 WT human mRNA, which proved that, in part, Integrin β-1 is required for axis formation mediated by Wnt signaling.These results even suggest that Wnt signaling could be regulated by ITGβ1 and vice versa through a negative feedback loop. 3Overall, the results prove there is a crosstalk between focal adhesions and Wnt signaling.All of these findings support the connection between membrane trafficking, cell adhesion, and Wnt signaling.

| WNT-STOP AND FOCAL ADHESIONS
Wnt signaling not only stabilizes β-catenin but also many other substrates phosphorylated by GSK3.Up to 20% of the human proteome contains three or more GSK3 sites in a row (consensus S/TXXXS/T), and the addition of Wnt prolongs the total half-life of HeLa cell proteins by 25%. 30This phenomenon, in which GSK3 substrate proteins are stabilized by Wnt, is designated Wnt-STabilization Of Proteins, or Wnt-STOP. 31,41Since Wnt signaling is maximal at cell cycle phases G2 and M, Wnt-STOP plays an important role in achieving an increase in cell size in preparation for cell division. 412][43][44][45] Wnt-STOP has been also implicated in the regulation of focal adhesions.It is known that some of the focal adhesions contain GSK3 phosphorylation sites such as Vinculin, Zyxin, FAK, and many others.Therefore, there is a possibility that GSK3 could be a major regulator of focal adhesions via the Wnt-STOP. 3,18,31

| WNT SIGNALING AND DEVELOPMENT
The Xenopus embryo has served as a powerful model system to understand the fundamental molecular mechanisms of Wnt signaling that functions universally in different tissues and animal systems. 15,33During the development of the embryo, maternal dorsal determinants are relocalized to the future dorsal side at the one-cell stage.This cortical rotation that is triggered by microtubules results in the stabilization and nuclear localization of β-catenin, which allows the formation of the Spemann Organizer (a cell group in the dorsal region [mesoderm] of the gastrula embryo), giving rise to the embryonic axis.7][48][49][50] The organizer also secretes Wnt antagonists such as Dkk1, Frzb1, and Crescent, and multivalent inhibitors such as Cerberus. 51These proteins determine the dorsal-ventral (D-V) and anteriorposterior (A-P) patterning fields 49,52 (Figure 5).Therefore, understanding the signaling pathways that induce the formation of organizer tissue is of vital importance for understanding vertebrate morphogenesis.Interfering with this cortical rotation at the early cleavage stage with nocodazole, or when the β-catenin expression is knocked down by morpholino (MO, to block mRNA translation) injection (Movie S1), or ultraviolet light results in the inhibition of Wnt and embryos develop without dorsal-anterior structures such as the head or neural tube. 53It was recently reported that lysosomal degradation and membrane trafficking are required for early dorsal axis development in Xenopus.It was shown that expansion of the MVB compartment with low-dose hydroxychloroquine (HCQ) greatly potentiated the dorsalizing effects of the Wnt agonist lithium chloride (LiCl) in embryos; this effect required macropinocytosis.Formation of the dorsal axis required lysosomes, as indicated by brief treatments with the vacuolar ATPase (V-ATPase) inhibitors Bafilomycin A1 (Baf) or Concanamycin A (Con A) at the 32-cell stage.Inhibiting the MVB-forming machinery with a dominant-negative point mutation in Vacuolar Protein Sorting 4 (Vps4-EQ) interfered with the endogenous dorsal axis.Moreover, the overexpression of Wnt activators such as LiCl which is a GSK3 inhibitor (or 6-bromoindirubin-3-oxime [BIO] or CHIR99021 [CHIR]) and mimics Wnt signaling, dorsalized the embryos.This effect can be also blocked by interfering with the lysosomal formation with Baf or Con A. 16 This is also supported by the finding of the V-ATPase activity being involved in the Wnt pathway; the activated Wnt Lrp6 receptor binds to the peripheral V-ATPase protein ATP6AP2 (also called Prorenin receptor).ATP6AP2 binds to transmembrane protein 9 (TMEM9), which binds to and activates the V-ATPase and regulates vesicle acidification.TMEM9 is an MVB protein that induces axial duplication in Xenopus assays.Genetic ablation of TMEM9 inhibits colorectal cancer. 54 way to further investigate the molecular mechanism of lysosomes during development would be to isolate lysosome or MVBs fractions from embryos and then inject these fractions ventrally to see whether they generate a secondary axis, but this is difficult in yolky eggs such as Xenopus.The evidence presented here suggests that membrane trafficking, V-ATPase, and lysosomes are essential for the earliest developmental decisions in the Xenopus vertebrate development model system.Moreover, it was also found that macropinocytosis activators enhance the Wnt pathway 2 (such as Phorbol 12-myristate 13-acetate [PMA] 6 and DAG [diacylglycerol]).We conclude that lysosome function is required to activate the Wnt pathway in Xenopus.The results highlight the intertwining between membrane trafficking, lysosomes, and vertebrate axis formation.The dorsal development of the Xenopus embryo not only depends on β-catenin but depends on macropinocytosis as a necessity.In mammalian cells, low-dose HCQ increased Wnt signaling but only within narrow concentration ranges.It is conceivable that some of the clinical prophylactic antimalarial and anti-autoimmune effects of HCQ might be mediated by increased macropinocytosis and lysosomal activity in small populations of Wntactivated immune cells rather than lysosomal inhibition as is currently thought.The role of focal adhesions in Xenopus development has also been reported. 2,55The Focal Adhesion Kinase (FAK) is a key regulator of FA maintenance and signaling and is known to interact with the Wnt pathway in several ways. 56In cultured cells, the FAK inhibitor strongly decreased β-catenin signaling and, in the Xenopus embryo, resulted in ventralized embryos with reduced dorsal-anterior structures.These findings support the critical role of membrane trafficking and focal adhesions in the Wnt pathway (Figure 6), highlighting that membrane trafficking is emerging as a critical target to block the activation of the Wnt pathway.

| WNT SIGNALING, COLON CANCER, AND MACROPINOCYTOSIS
The cellular homeostasis of the intestinal epithelium is maintained with the balance between continuous cell division, cell differentiation, and apoptosis, with Wnt signaling as the dominant force maintaining homeostasis along the crypt.Axin and APC are two major tumor suppressors, and their loss of function, which leads to increased nuclear β-catenin, lies at the heart of cancers such as hepatocellular and colorectal carcinomas 2 (Figure 7).
Knowing how the Wnt pathway works is important because 80% of all colon cancer cases have mutations in the tumor suppressor APC. 57Wnt signaling is important in the initiation of cancer, and over time, these initiated cells can develop additional changes.These changes lead to the beginning of uncontrolled growth called an adenomatous polyp, a small growth of cell material associated with the colon's wall.But this tumor is not yet cancer, it is an adenoma, which is easy to remove surgically.Over time, however, the tumor can progress and acquire properties where it can invade and become a carcinoma and, with additional mutations, even begin to spread or metastasize. 2ndocytosis plays a role in cancer by causing loss of cell adhesion or morphological polarity, which can lead to malignant transformation of cells. 580][61][62] For example, the overexpression of Rab proteins such as Rab3d is seen F I G U R E 5 Genetic expression in dorsal and ventral regions of the early Xenopus laevis gastrula.Genes specifically expressed in the dorsal and ventral fragments of the early Xenopus laevis gastrula have produced a plethora of new genes involved in embryonic patterning.Many components of the dorsal and ventral region of the Spemann organizer in the Xenopus gastrula have been a rich source of new molecules and developmental mechanisms.Many of the novel genes identified encoded secreted antagonists of the BMP and the Wnt pathways.Diagram based and modified from De Robertis and Tejeda-Muñoz. 47n breast and lung cancer. 63In addition, Wnt signaling is also known to increase endocytosis, which can also implicate a possible connection between Wnt signaling and cancer.This increase in endocytosis in Wnt signaling led to further investigation, revealing that Wnt signaling utilizes macropinocytosis. 1 Macropinocytosis is the large nonselective uptake of molecules such as nutrients and other macromolecules in the cell's environment.The macropinosomes that allow the uptake of water and other molecules have membranes that are made up of the cell's plasma membrane as well as the actin-rich membrane, called ruffles.The ruffles undergo protrusive movements allowing the vesicle to close and take up the contents, which are either transported to the lysosome for degradation or to the cell surface for exocytosis. 64,65Wnt signaling utilizes macropinocytosis by transporting the contents from the cell surface in MVBs to the lysosomes in the cell for degradation.Additionally, colorectal cancer cells are known for their robust macropinocytosis and increased nuclear β-catenin when APC, a destruction complex protein, is missing.Studies showed that macropinocytosis is required for Wnt signaling.This was demonstrated by the decrease in nuclear β-catenin when colorectal cancer cells were treated with macropinocytosis inhibitors such as ethylisopropyl amiloride (EIPA). 1,2,13,31The study shows macropinocytosis is important for Wnt signaling since nuclear β-catenin accumulation is a marker for Wnt signaling.However, that was reduced without cell drinking.There are different activators of macropinocytosis, such as Ras, which is part of the Ras to ERK pathway, which consists of Ras GTPases and three kinases RAF, MEK, and ERK. 667][68] Ras is one of the most frequently mutated protooncogenes in cancer. 64ike the Wnt/β-catenin pathway, the Ras to ERK pathway is also implicated in colorectal cancer.It has been shown that Wnt/β-catenin pathway mutations, like in the APC and Ras/ERK pathway mutation in KRas, occurring together induce an increase in initiation and growth of colorectal adenocarcinoma which was not possible when the mutations acted alone without each other. 69,70he direct connection between Ras and Wnt signaling is through the pathway Wnt-STOP. 31,71This is when proteins like Ras are stabilized by the sequestration and decrease in GSK3 because it cannot phosphorylate proteins like Ras as it would normally. 31Ras itself contains two threonine residues phosphorylated by GSK3 that generate a phosphodegron leading to degradation via the b-TrCP-E3 polyubiquitin ligase in a process enhanced by Axin1.The fact that Ras is a direct substrate of GSK3 is interesting and could be essential for targeting Wnt signaling.Even though this connection is known between Ras and Wnt signaling, the relationship between Ras and Wnt signaling, specifically through membrane trafficking, remains elusive.Rac1 is another macropinocytosis activator in a family of small Rho GTPases. 72Rac is generally known to be associated with the regulation of actin filaments at the cell periphery to produce lamellipodia and membrane ruffles. 73Specifically, Rac1 is important for membrane ruffling (formation of protrusions [or projections]) and macropinosome in some cells such F I G U R E 7 Progression of colon cancer from normal crypt to dysregulated signaling.This figure illustrates the progression from a normal intestinal crypt to colorectal cancer (CRC), highlighting the crucial role of APC (adenomatous polyposis coli) mutations in approximately 85% of colon cancer cases.In a normal crypt, a balance of signaling gradients, including BMP, TGF-β, and Wnt, maintain intestinal stem cell functionality and healthy tissue architecture.APC mutations disrupt this equilibrium.Initially, the loss of APC function leads to benign polyps characterized by epithelial dysplasia.0][81][82] These mutations, alongside disruptions in normal signaling gradients, transform normal cells into cancer-associated fibroblasts (CAFs) and colon cancer stem cells, leading to a dysregulated signaling environment conducive to tumor growth.The figure also delineates the rise in Wnt signaling and subsequent decrease in BMP signaling, contributing to increased cancer stemness in the tumor microenvironment.2][83] Even in these CRC cases, the Wnt pathway is frequently activated, whether through mutations in Axin1, Axin2, RNF43, or ZNRF3, overexpression of R-Spondin 2 or 3, or mutations that stabilize β-catenin (Figure created  as dendritic cells, macrophages, fibroblasts, etc. 72 Rac1 has also increased Wnt signaling by stimulating β-catenin-dependent transcription of Wnt target genes. 74Even though there is a known connection between Wnt signaling and Rac1 in the context of β-catenin, it is still unknown if Rac1 or other macropinocytosis activators can induce a Wnt phenotype in model organisms.Further, Taelman et al. reported a list of possible Wnt-STOP targets, such as PAK1-3, which has three putative GSK3 phosphorylation sites in a row that have been highly conserved in the evolution. 31Stabilization of PAK1 by Wnt-STOP could trigger macropinocytosis.Other intriguing Wnt-STOP targets identified are activators of CDC42 (the guanine nucleotide exchange factors Dock9/10/11) that could activate macropinocytosis if stabilized.The extent to which this link between Wnt signaling/GSK3/Wnt-STOP and macropinocytosis contributes to disease is unknown.These topics could link macropinocytosis events downstream of Wnt signaling with malignant relevant cytoskeletal rearrangements.Elucidating the novel concept that focal adhesions are regulated by Wnt signaling could provide critical insight into the complexities through which focal adhesions are misregulated during the pathogenesis of disease and could contribute key details to target this process via drug treatments interfering with macropinocytosis, focal adhesions, lysosomal activity, and membrane trafficking.

| CONCLUSION
The complex interplay between Wnt signaling and membrane trafficking presents exciting new research areas.Understanding the underlying molecular mechanisms and functional implications of this interaction holds the potential to understand developmental processes further and implement novel therapeutic strategies in cancer.The findings presented here underscore the importance of Wnt signaling beyond β-catenin-focused cancer treatments that traditionally focus on cancer cell proliferation over metastasis.The delineated connections between focal adhesions, macropinocytosis, lysosomes, and Wnt signaling are important, given their discrete influences on cancer progression.For instance, the loss of focal adhesions decreases the cell-matrix adhesion and promotes cell migration, a critical aspect of cancer metastasis. 75Meanwhile, Wnt signaling, when unregulated, can lead to increased transcription of oncogenes such as those involved in colon cancer. 76Additionally, the role of macropinocytosis is strongly implicated in cancer growth through increased intake of extracellular nutrients is noteworthy. 77Thus, the association between focal adhesions and Wnt signaling may guide the development of new targeted therapies that suppress cancer metastasis.Similarly, insights into Wnt signaling and macropinocytosis could refine new therapy strategies for restricting cancer cell nutrient uptake.Proximity Ligation Assays reveal the further potential for early cancer detection via Wnt-induced changes in focal adhesions sequestered inside MVB vesicles as biomarkers.A protein interaction approach, such as APEX2, can be used to study these dynamic integrin and Wnt signaling pathways.The effect of integrin or focal adhesion depletion on Wnt signaling in culture cells on soft and hard substrates with high-resolution live-imaging techniques could allow us to visualize key changes in localization or movements that can be combined with genomic approaches to impact cancer detection, diagnosis, treatment, and in the end prevention.Exploring the effect of macropinocytosis inhibitors such as EIPA on focal adhesion proteins in cancer cells could help us understand how cell adhesion is affected during cancer and how focal adhesions act as a tumor suppressor in the Wnt pathway.Malignant cells are softer than normal cells, mainly due to the loss of cytoskeletal support. 19,78The loss of stiffness can represent a phenotype of tumor development, which facilitates malignant cell migration and adaptation to other tissues.In summary, this timely review sets the stage for high-impact research at the crossroads of cancer cell adhesion, endocytosis, lysosomal activity, and growth factor signaling.The collecting findings herald a potential shift in the perceived role of Wnt signaling in cancer cell growth and nutrition, paving the way for novel cancer treatments.

F I G U R E 2
Actin cytoskeleton and focal adhesion response to Wnt3a in human corneal stromal fibroblasts.This figure captures the significant changes in the actin cytoskeleton and focal adhesions of human corneal stromal fibroblasts (HCSFs) after a 20-min exposure to Wnt3a.(A) Untreated HCSF shows that the cells adhere normally to the ECM and exhibit less cell movement and abundant focal adhesion sites.(B) Wnt3a treatment leads to a reduction in cell adherence to the ECM, with actin filaments being disassembled and no longer associated with actin, consequently increasing cell motility.The treated cells demonstrated fewer and shorter adhesion sites, a potential factor in the enhanced mobility observed in cancer cells due to the loss of focal adhesions influenced by Wnt signaling.Notably, enlarged lysosomes are observed after Wnt treatment (indicated by the arrow).(C) The diagram illustrates a model for the endocytosis of the focal adhesion upon heightened Wnt signaling.Activation of Wnt signaling (step 1) prompts the macropinocytosis-mediated internalization of Lrp6/Fz/Wnt/GSK3 signalosomes, along with the master regulator of cell adhesion ITGβ1 and other focal adhesion plaques (step 2).This endocytosis process also sequestration of GSK3, and the destruction complex activates the Wnt/β-catenin pathway (Step 3), as per findings reported in Tejeda-Muñoz et al. 3 ECM, extracellular matrix; GSK3, glycogen synthase kinase 3; ITGβ1, Integrin beta 1.

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I G U R E 3 Dynamics of integrin-mediated cell-extracellular matrix adhesions and cell migration.This figure delineates the process of integrin-mediated cell adhesion to the extracellular matrix (ECM) and subsequent cell migration, which is crucial for various physiological processes and plays a significant role in cancer metastasis.Focal adhesions (FA), which link the cytoskeleton with the extracellular matrix, are orchestrated through the integrin receptor, mediating bidirectional signaling (detailed in the Zoomed-in panel).The assembly and disassembly of focal adhesions (a key aspect in integrin trafficking) are critical in effective cell migration.As shown in steps 1 to 4 (left panel), cells develop a polarized phenotype and then disassemble FA at the rear position, propelling the cell body forward and forming new adhesion points at the leading edge, coordinating the turnover and creation of adhesions during migration (Figure created with BioRender.com, accessed on 11 July 2023).

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I G U R E 4 Wnt treatment triggers macropinocytosis.(A) The gold standard method to study macropinocytosis is dextran of above 200 nm in diameter.Therefore, the high molecular weight marker tetramethylrhodamine (TMR)-dextran TMR 70 K was tested after Wnt3a protein (100 ng/mL from Peprotech) treatment after 20 min (B), resulting in an increase in the uptake of macropinocytosis.(C) Diagram of macropinocytosis is an actin cytoskeleton-based phenomenon driven by the activation of p21-activated kinase-1 (Pak1) that involves large endocytic vesicles (diameter over 0.2 μm).Its inhibition by amiloride derivatives such as EIPA constitutes the modern method for macropinocytosis.EIPA, ethylisopropyl amiloride.

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I G U R E 6 There are different ways to activate Wnt signaling, one of them is through GSK3 inhibition by treatment with LiCl or BIO, or CHIR which mimics Wnt signaling.Incubating the Xenopus embryos at the 32-cell stage with the GSK3 inhibitor LiCl (300 mM, panel D) for 7 min or 6-bromoindirubin-3 0 -oxime (BIO, 30 mM, panel E) and then removing at stage 9.5 and further cultured to the tadpole stage in 0.1 MMR solution, or overnight treatment with CHIR99021 (CHIR, panel F), has very potent phenotypic effects by increasing the early Wnt signal, resulting in an expansion of dorsal-anterior tissues, with a loss of tail structures.Inhibiting V-ATPase (Con A or Baf, panel B and C) results in microcephalic embryos with increased ventral structures, compared with the control embryo in panel A. These indicate that the cell biology of lysosomes, macropinocytosis, membrane trafficking, and ESCRT machinery plays an essential role in vertebrate development and Wnt signaling.(G) The qRT-PCR of blastula stage 9.5 embryos shows that interfering with membrane trafficking with Baf or Con A decreases Wnt target genes Siamois and Xnr3 normalized for ornithine decarboxylase (ODC).An opposite effect is observed with Wnt activators such as LiCl, BIO, and CHIR.(H and I) Example of an in situ hybridization in Xenopus embryos incubated with Baf showing a reduction of the forebrain and midbrain marker otx2 (arrowhead).BIO, 6-bromoindirubin-3-oxime; ESCRT, endosomal sorting complexes required for transport; GSK3, glycogen synthase kinase 3.