Interactions between genistein and Wnt pathway in colon adenocarcinoma and early embryos.

The Wnt signaling pathway is one of the most ancient and pivotal signaling cascades, governing diverse processes in development and cancer regulation. Within the realm of cancer treatment, genistein emerges as a promising candidate due to its multifaceted modulation of various signaling pathways, including the Wnt pathway. Despite promising preclinical studies, the precise mechanisms underlying genistein's therapeutic effects via Wnt modulation remain elusive. In this study, we unveil novel insights into the therapeutic mechanisms of genistein by elucidating its inhibitory effects on Wnt signaling through macropinocytosis. Additionally, we demonstrate its capability to curtail cell growth, proliferation, and lysosomal activity in the SW480 colon adenocarcinoma cell model. Furthermore, our investigation extends to the embryonic context, where genistein influences gene regulatory networks governed by endogenous Wnt pathways. Our findings shed light on the intricate interplay between genistein, Wnt signaling, membrane trafficking, and gene regulation, paving the way for further exploration of genistein's therapeutic potential in cancer treatment strategies.

Na,K-ATPase activity promotes macropinocytosis in colon cancer via Wnt signaling.

Recent research has shown that membrane trafficking plays an important role in canonical Wnt signaling through sequestration of the ß-catenin destruction complex inside multivesicular bodies (MVBs) and lysosomes. In this study, we introduce Ouabain, an inhibitor of the Na,K-ATPase pump that establishes electric potentials across membranes, as a potent inhibitor of Wnt signaling. We find that Na,K-ATPase levels are elevated in advanced colon carcinoma, that this enzyme is elevated in cancer cells with constitutively activated Wnt pathway and is activated by GSK3 inhibitors that increase macropinocytosis. Ouabain blocks macropinocytosis, which is an essential step in Wnt signaling, probably explaining the strong effects of Ouabain on this pathway. In Xenopus embryos, brief Ouabain treatment at the 32-cell stage, critical for the earliest Wnt signal in development-inhibited brains, could be reversed by treatment with Lithium chloride, a Wnt mimic. Inhibiting membrane trafficking may provide a way of targeting Wnt-driven cancers.

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 ß-catenin. The generally accepted view is that the canonical effects of Wnt growth factors are caused by the transcription of ß-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 ß-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 PMA phorbol ester tumor promoter increases canonical Wnt signaling via macropinocytosis.

Activation of the Wnt pathway lies at the core of many human cancers. Wnt and macropinocytosis are often active in the same processes, and understanding how Wnt signaling and membrane trafficking cooperate should improve our understanding of embryonic development and cancer. Here, we show that a macropinocytosis activator, the tumor promoter phorbol 12-myristate 13-acetate (PMA), enhances Wnt signaling. Experiments using the Xenopus embryo as an in vivo model showed marked cooperation between the PMA phorbol ester and Wnt signaling, which was blocked by inhibitors of macropinocytosis, Rac1 activity, and lysosome acidification. Human colorectal cancer tissue arrays and xenografts in mice showed a correlation of cancer progression with increased macropinocytosis/multivesicular body/lysosome markers and decreased GSK3 levels. The crosstalk between canonical Wnt, focal adhesions, lysosomes, and macropinocytosis suggests possible therapeutic targets for cancer progression in Wnt-driven cancers.

Addition of exogenous diacylglycerol enhances Wnt/ß-catenin signaling through stimulation of macropinocytosis.

Activation of Wnt signaling triggers macropinocytosis and drives many tumors. We now report that the exogenous addition of the second messenger lipid sn-1,2 DAG to the culture medium rapidly induces macropinocytosis. This is accompanied by potentiation of the effects of added Wnt3a recombinant protein or the glycogen synthase kinase 3 (GSK3) inhibitor lithium chloride (LiCl, which mimics Wnt signaling) in luciferase transcriptional reporter assays. In a colorectal carcinoma cell line in which mutation of adenomatous polyposis coli (APC) causes constitutive Wnt signaling, DAG addition increased levels of nuclear ß-catenin, and this increase was partially inhibited by an inhibitor of macropinocytosis. DAG also expanded multivesicular bodies marked by the tetraspan protein CD63. In an in vivo situation, microinjection of DAG induced Wnt-like twinned body axes when co-injected with small amounts of LiCl into Xenopus embryos. These results suggest that the DAG second messenger plays a role in Wnt-driven cancer progression.

The PMA Phorbol Ester Tumor Promoter Increases Canonical Wnt Signaling Via Macropinocytosis.

Activation of the Wnt pathway lies at the core of many human cancers. Wnt and macropinocytosis are often active in the same processes, and understanding how Wnt signaling and membrane trafficking cooperate should improve our understanding of embryonic development and cancer. Here we show that a macropinocytosis activator, the tumor promoter Phorbol 12-myristate 13-acetate (PMA), enhances Wnt signaling. Experiments using the Xenopus embryo as an in vivo model showed marked cooperation between the PMA phorbol ester and Wnt signaling, which was blocked by inhibitors of macropinocytosis, Rac1 activity, and lysosome acidification. Human colorectal cancer tissue arrays and xenografts in mice showed a correlation of cancer progression with increased macropinocytosis/multivesicular body/lysosome markers and decreased GSK3 levels. The crosstalk between canonical Wnt, focal adhesions, lysosomes, and macropinocytosis suggests possible therapeutic targets for cancer progression in Wnt-driven cancers.

Targeting Membrane Trafficking as a Strategy for Cancer Treatment.

Membrane trafficking is emerging as an attractive therapeutic strategy for cancer. Recent reports have found a connection between Wnt signaling, receptor-mediated endocytosis, V-ATPase, lysosomal activity, and macropinocytosis through the canonical Wnt pathway. In macropinocytic cells, a massive internalization of the plasma membrane can lead to the loss of cell-surface cadherins, integrins, and other antigens that mediate cell-cell adhesion, favoring an invasive phenotype. V-ATPase is a key regulator in maintaining proper membrane trafficking, homeostasis, and the earliest developmental decisions in the Xenopus vertebrate development model system. Here, we review how the interference of membrane trafficking with membrane trafficking inhibitors might be clinically relevant in humans.

Wnt, GSK3, and Macropinocytosis.

Here we review the regulation of macropinocytosis by Wnt growth factor signaling. Canonical Wnt signaling is normally thought of as a regulator of nuclear ß-catenin, but emerging results indicate that there is much more than ß-catenin to the Wnt pathway. Macropinocytosis is transiently regulated by EGF-RTK-Ras-PI3K signaling. Recent studies show that Wnt signaling provides for sustained acquisition of nutrients by macropinocytosis. Endocytosis of Wnt-Lrp6-Fz receptor complexes triggers the sequestration of GSK3 and components of the cytosolic destruction complex such as Axin1 inside multivesicular bodies (MVBs) through the action of the ESCRT machinery. Wnt macropinocytosis can be induced both by the transcriptional loop of stabilized ß-catenin, and by the inhibition of GSK3 even in the absence of new protein synthesis. The cell is poised for macropinocytosis, and all it requires for triggering of Pak1 and the actin machinery is the inhibition of GSK3. Striking lysosomal acidification, which requires macropinocytosis, is induced by GSK3 chemical inhibitors or Wnt protein. Wnt-induced macropinocytosis requires the ESCRT machinery that forms MVBs. In cancer cells, mutations in the tumor suppressors APC and Axin1 result in extensive macropinocytosis, which can be reversed by restoring wild-type protein. In basal cellular conditions, GSK3 functions to constitutively repress macropinocytosis.

Lysosomes are required for early dorsal signaling in the Xenopus embryo.

Lysosomes are the digestive center of the cell and play important roles in human diseases, including cancer. Previous work has suggested that late endosomes, also known as multivesicular bodies (MVBs), and lysosomes are essential for canonical Wnt pathway signaling. Sequestration of Glycogen Synthase 3 (GSK3) and of ß­catenin destruction complex components in MVBs is required for sustained canonical Wnt signaling. Little is known about the role of lysosomes during early development. In the Xenopus egg, a Wnt-like cytoplasmic determinant signal initiates formation of the body axis following a cortical rotation triggered by sperm entry. Here we report that cathepsin D was activated in lysosomes specifically on the dorsal marginal zone of the embryo at the 64-cell stage, long before zygotic transcription starts. Expansion of the MVB compartment with low-dose hydroxychloroquine (HCQ) greatly potentiated the dorsalizing effects of the Wnt agonist lithium chloride (LiCl) in embryos, and 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 or Concanamycin 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. The Wnt-like activity of the dorsal cytoplasmic determinant Huluwa (Hwa), and that of microinjected xWnt8 messenger RNA, also required lysosome acidification and the MVB-forming machinery. We conclude that lysosome function is required for early dorsal axis development in Xenopus. The results highlight the intertwining between membrane trafficking, lysosomes, and vertebrate axis formation.

Canonical Wnt signaling induces focal adhesion and Integrin beta-1 endocytosis.

During canonical Wnt signaling, the Wnt receptor complex is sequestered together with glycogen synthase kinase 3 (GSK3) and Axin inside late endosomes, known as multivesicular bodies (MVBs). Here, we present experiments showing that Wnt causes the endocytosis of focal adhesion (FA) proteins and depletion of Integrin ß 1 (ITGß1) from the cell surface. FAs and integrins link the cytoskeleton to the extracellular matrix. Wnt-induced endocytosis caused ITGß1 depletion from the plasma membrane and was accompanied by striking changes in the actin cytoskeleton. In situ protease protection assays in cultured cells showed that ITGß1 was sequestered within membrane-bounded organelles that corresponded to Wnt-induced MVBs containing GSK3 and FA-associated proteins. An in vivo model using Xenopus embryos dorsalized by Wnt8 mRNA showed that ITGß1 depletion decreased Wnt signaling. The finding of a crosstalk between two major signaling pathways, canonical Wnt and focal adhesions, should be relevant to human cancer and cell biology.

Cell Biology of Canonical Wnt Signaling.

Wnt signaling has multiple functions beyond the transcriptional effects of ß-catenin stabilization. We review recent investigations that uncover new cell physiological effects through the regulation of Wnt receptor endocytosis, Wnt-induced stabilization of proteins (Wnt-STOP), macropinocytosis, increase in lysosomal activity, and metabolic changes. Many of these growth-promoting effects of canonical Wnt occur within minutes and are independent of new protein synthesis. A key element is the sequestration of glycogen synthase kinase 3 (GSK3) inside multivesicular bodies and lysosomes. Twenty percent of human proteins contain consecutive GSK3 phosphorylation motifs, which in the absence of Wnt can form phosphodegrons for polyubiquitination and proteasomal degradation. Wnt signaling by either the pharmacological inhibition of GSK3 or the loss of tumor-suppressor proteins, such as adenomatous polyposis coli (APC) and Axin1, increases lysosomal acidification, anabolic metabolites, and macropinocytosis, which is normally repressed by the GSK3-Axin1-APC destruction complex. The combination of these cell physiological effects drives cell growth.

Wnt-inducible Lrp6-APEX2 interacting proteins identify ESCRT machinery and Trk-fused gene as components of the Wnt signaling pathway.

The canonical Wnt pathway serves as a hub connecting diverse cellular processes, including ß-catenin signaling, differentiation, growth, protein stability, macropinocytosis, and nutrient acquisition in lysosomes. We have proposed that sequestration of ß-catenin destruction complex components in multivesicular bodies (MVBs) is required for sustained canonical Wnt signaling. In this study, we investigated the events that follow activation of the canonical Wnt receptor Lrp6 using an APEX2-mediated proximity labeling approach. The Wnt co-receptor Lrp6 was fused to APEX2 and used to biotinylate targets that are recruited near the receptor during Wnt signaling at different time periods. Lrp6 proximity targets were identified by mass spectrometry, and revealed that many endosomal proteins interacted with Lrp6 within 5 min of Wnt3a treatment. Interestingly, we found that Trk-fused gene (TFG), previously known to regulate the cell secretory pathway and to be rearranged in thyroid and lung cancers, was strongly enriched in the proximity of Lrp6. TFG depletion with siRNA, or knock-out with CRISPR/Cas9, significantly reduced Wnt/ß-catenin signaling in cell culture. In vivo, studies in the Xenopus system showed that TFG is required for endogenous Wnt-dependent embryonic patterning. The results suggest that the multivesicular endosomal machinery and the novel player TFG have important roles in Wnt signaling.

GSK3 Inhibits Macropinocytosis and Lysosomal Activity through the Wnt Destruction Complex Machinery.

Canonical Wnt signaling is emerging as a major regulator of endocytosis. Here, we report that Wnt-induced macropinocytosis is regulated through glycogen synthase kinase 3 (GSK3) and the ß-catenin destruction complex. We find that mutation of Axin1, a tumor suppressor and component of the destruction complex, results in the activation of macropinocytosis. Surprisingly, inhibition of GSK3 by lithium chloride (LiCl), CHIR99021, or dominant-negative GSK3 triggers macropinocytosis. GSK3 inhibition causes a rapid increase in acidic endolysosomes that is independent of new protein synthesis. GSK3 inhibition or Axin1 mutation increases lysosomal activity, which can be followed with tracers of active cathepsin D, ß-glucosidase, and ovalbumin degradation. Microinjection of LiCl into the blastula cavity of Xenopus embryos causes a striking increase in dextran macropinocytosis. The effects of GSK3 inhibition on protein degradation in endolysosomes are blocked by the macropinocytosis inhibitors EIPA or IPA-3, suggesting that increases in membrane trafficking drive lysosomal activity.

Functional Interaction of Hypoxia-Inducible Factor 2-Alpha and Autophagy Mediates Drug Resistance in Colon Cancer Cells.

Hypoxia and the accumulation of hypoxia-inducible factors (HIFs) in tumors have been associated with therapeutic resistance and with autophagy establishment. We examined the effects of stable knockdown of HIF-1α or HIF-2α expression on autophagy and drug resistance in colon cancer cells. We found that under normoxic conditions, malignant cells exhibit increased basal levels of autophagy, compared with non-malignant cells, in addition to the previously reported coexpression of HIF-1α and HIF-2α. Knockdown of HIF-1α or HIF-2α expression resulted in increased autophagic and apoptotic cell death, indicating that the survival of cells is HIF-dependent. Cytotoxic-induced cell death was significantly increased by knockdown of HIFs but not by autophagy inhibition. Strikingly, although malignancy-resistant cells were sensitized to death by nutrient stress, the combination with HIF-2α depletion, but not with HIF-1α depletion, induced severe cell death. Oxidative stress levels were significantly increased as a result of HIF-2α specific inhibition or silencing suggesting that this may contribute to sensitize cells to death. The in vitro results were confirmed in vivo using a xenograft mouse model. We found that coordinated autophagy and mTOR inhibition enhanced cell death and induced tumor remission only in HIF-2α-silenced cells. Finally, using a specific HIF-2α inhibitor alone or in combination with drugs in patient-derived primary colon cancer cells, overcame their resistance to 5-FU or CCI-779, thus emphasizing the crucial role played by HIF-2α in promoting resistance and cell survival.

Wnt canonical pathway activates macropinocytosis and lysosomal degradation of extracellular proteins.

Canonical Wnt signaling is emerging as a major regulator of endocytosis. Wnt treatment markedly increased the endocytosis and degradation in lysosomes of BSA. In this study, we report that in addition to receptor-mediated endocytosis, Wnt also triggers the intake of large amounts of extracellular fluid by macropinocytosis, a nonreceptor-mediated actin-driven process. Macropinocytosis induction is rapid and independent of protein synthesis. In the presence of Wnt, large amounts of nutrient-rich packages such as proteins and glycoproteins were channeled into lysosomes after fusing with smaller receptor-mediated vesicles containing glycogen synthase kinase 3 (GSK3) and protein arginine ethyltransferase 1 (PRMT1), an enzyme required for canonical Wnt signaling. Addition of Wnt3a, as well as overexpression of Disheveled (Dvl), Frizzled (Fz8), or dominant-negative Axin induced endocytosis. Depletion of the tumor suppressors adenomatous polyposis coli (APC) or Axin dramatically increased macropinocytosis, defined by incorporation of the high molecular weight marker tetramethylrhodamine (TMR)-dextran and its blockage by the Na+/H+ exchanger ethylisopropyl amiloride (EIPA). Macropinocytosis was blocked by dominant-negative vacuolar protein sorting 4 (Vps4), indicating that the Wnt pathway is dependent on multivesicular body formation, a process called microautophagy. SW480 colorectal cancer cells displayed constitutive macropinocytosis and increased extracellular protein degradation in lysosomes, which were suppressed by restoring full-length APC. Accumulation of the transcriptional activator ß-catenin in the nucleus of SW480 cells was inhibited by methyltransferase inhibition, EIPA, or the diuretic amiloride. The results indicate that Wnt signaling switches metabolism toward nutrient acquisition by engulfment of extracellular fluids and suggest possible treatments for Wnt-driven cancer progression.

Glycogen Synthase Kinase 3ß Is Positively Regulated by Protein Kinase Cζ-Mediated Phosphorylation Induced by Wnt Agonists.

The molecular events that drive Wnt-induced regulation of glycogen synthase kinase 3ß (GSK-3ß) activity are poorly defined. In this study, we found that protein kinase Cζ (PKCζ) and GSK-3ß interact mainly in colon cancer cells. Wnt stimulation induced a rapid GSK-3ß redistribution from the cytoplasm to the nuclei in malignant cells and a transient PKC-mediated phosphorylation of GSK-3ß at a different site from serine 9. In addition, while Wnt treatment induced a decrease in PKC-mediated phosphorylation of GSK-3ß in nonmalignant cells, in malignant cells, this phosphorylation was increased. Pharmacological inhibition and small interfering RNA (siRNA)-mediated silencing of PKCζ abolished all of these effects, but unexpectedly, it also abolished the constitutive basal activity of GSK-3ß. In vitro activity assays demonstrated that GSK-3ß phosphorylation mediated by PKCζ enhanced GSK-3ß activity. We mapped Ser147 of GSK-3ß as the site phosphorylated by PKCζ, i.e., its mutation into alanine abolished GSK-3ß activity, resulting in ß-catenin stabilization and increased transcriptional activity, whereas phosphomimetic replacement of Ser147 by glutamic acid maintained GSK-3ß basal activity. Thus, we found that PKCζ phosphorylates GSK-3ß at Ser147 to maintain its constitutive activity in resting cells and that Wnt stimulation modifies the phosphorylation of Ser147 to regulate GSK-3ß activity in opposite manners in normal and malignant colon cells.

Glycogen synthase kinase 3 in Wnt signaling pathway and cancer.

Glycogen synthase kinase 3 (GSK-3) was first discovered in 1980 as one of the key enzymes of glycogen metabolism. Since then, GSK-3 has been revealed as one of the master regulators of a diverse range of signaling pathways, including those activated by Wnts, participating in the regulation of numerous cellular functions, suggesting that its activity is tightly regulated. Numerous studies have pointed to an association of GSK-3 dysregulation with the onset and progression of human diseases, including diabetes mellitus, obesity, inflammation, neurological illnesses, and cancer. Therefore, GSK-3 is recognized as an attractive therapeutic target in multiple disorders. However, the great number of substrates that are phosphorylated by GSK-3 has raised the question of whether this limits its feasibility as a therapeutic target because of the potential disruption of many cellular processes and also by the fear that inhibition of GSK-3 may stimulate or aid in malignant transformation, as GSK-3 can phosphorylate pro-oncogenic factors. This mini review focuses on the role played by GSK-3 in Wnt signaling pathway and cancer using as model colon cancer.