Transcription Of Wnt Target Genes Research Articles

An investigation on the alterations in Wnt signaling in ADHD across developmental stages

The canonical Wnt signaling pathway plays a vital role in the developmental processes of the Central Nervous System throughout both prenatal and postnatal stages, as well as in maintaining homeostasis during adulthood. Its complex intracellular cascade involves the participation of key proteins (i.e., GSK3β and β-catenin) to activate the transcription of Wnt target genes. These genes subsequently control processes like cell proliferation, maturation, and the determination of cell fate. Previous studies suggest that this pathway can also be associated with Attention-Deficit Hyperactivity Disorder (ADHD), a neurodevelopmental disorder with multifactorial etiology. This study aimed to clarify if and at what developmental stage the Wnt pathway is altered in ADHD. Accordingly, we carried out proteomic and functional assessments of the Wnt pathway using Western Blot and reporter assays, respectively. These assessments were performed at the induced pluripotent stem cell (iPSC), neural stem cell (NSC), and neuronal phases. IPSCs were generated from somatic cells retrieved from 5 controls and 5 patients diagnosed with ADHD. As opposed to the developmental stage of iPSCs, ADHD NSCs showed alterations in the protein expression of both GSK3β and β-catenin, suggesting increased Wnt activity in the ADHD group. Moreover, Wnt reporter assays confirmed higher Wnt activity in ADHD NSCs. Our molecular findings in NSCs correlated with genetic predisposition to ADHD and clinical traits displayed by their respective donors. In conclusion, these results suggest that a crucial cellular pathway is disrupted in patient-specific NSCs, potentially explaining the developmental deficits clinically exhibited by ADHD patients.

RNF2 regulates Wnt/ß-catenin signaling via TCF7L1 destabilization

The Wnt signaling pathway is a crucial regulator of various biological processes, such as development and cancer. The downstream transcription factors in this pathway play a vital role in determining the threshold for signaling induction and the length of the response, which vary depending on the biological context. Among the four transcription factors involved in canonical Wnt/ß-catenin signaling, TCF7L1 is known to possess an inhibitory function; however, the underlying regulatory mechanism remains unclear. In this study, we identified the E3 ligase, RNF2, as a novel positive regulator of the Wnt pathway. Here, we demonstrate that RNF2 promotes the degradation of TCF7L1 through its ubiquitination upon activation of Wnt signaling. Loss-of-function studies have shown that RNF2 consistently destabilizes nuclear TCF7L1 and is required for proper Wnt target gene transcription in response to Wnt activation. Furthermore, our results revealed that RNF2 controls the threshold, persistence, and termination of Wnt signaling by regulating TCF7L1. Overall, our study sheds light on the previously unknown degradation mechanism of TCF7L1 by a specific E3 ligase, RNF2, and provides new insights into the variability in cellular responses to Wnt activation.

When You Come to a Fork in the Road, Take It: Wnt Signaling Activates Multiple Pathways through the APC/Axin/GSK-3 Complex.

The Wnt signaling pathway is a highly conserved regulator of metazoan development and stem cell maintenance. Activation of Wnt signaling is an early step in diverse malignancies. Work over the past four decades has defined a "canonical" Wnt pathway that is initiated by Wnt proteins, secreted glycoproteins that bind to a surface receptor complex and activate intracellular signal transduction by inhibiting a catalytic complex composed of the classical tumor suppressor Adenomatous Polyposis Coli (APC), Axin, and Glycogen Synthase Kinase-3 (GSK-3). The best characterized effector of this complex is β-catenin, which is stabilized by inhibition of GSK-3, allowing β-catenin entrance to the nucleus and activation of Wnt target gene transcription, leading to multiple cancers when inappropriately activated. However, canonical Wnt signaling through the APC/Axin/GSK-3 complex impinges on other effectors, independently of β-catenin, including the mechanistic Target of Rapamycin (mTOR), regulators of protein stability, mitotic spindle orientation, and Hippo signaling. This review focuses on these alternative effectors of the canonical Wnt pathway and how they may contribute to cancers.

LEF1 enhances β-catenin transactivation through IDR-dependent liquid-liquid phase separation.

Wnt/β-catenin signaling plays a crucial role in cancer development, primarily activated by β-catenin forming a transcription complex with LEF/TCF in the nucleus and initiating the transcription of Wnt target genes. Here, we report that LEF1, a member of the LEF/TCF family, can form intrinsically disordered region (IDR)-dependent condensates with β-catenin both in vivo and in vitro, which is required for β-catenin-dependent transcription. Notably, LEF1 with disrupted IDR lost its promoting activity on tumor proliferation and metastasis, which can be restored by substituting with FUS IDR. Our findings provide new insight into the essential role of liquid-liquid phase separation in Wnt/β-catenin signaling and present a potential new target for cancer therapy.

The Multi-CDK Inhibitor Dinaciclib Shows Synergistic Activity with the BET Inhibitor PLX51107 and Reverses BET Resistance through Inhibition of Canonical Wnt Signaling in Acute Myeloid Leukemia (AML)

PLX51107 is a novel BET inhibitor (BETi) with antineoplastic activity in pre-clinical models of both aggressive B-cell malignancies and AML. Differentiating it from other BET inhibitors currently under investigation, PLX51107 has a unique binding site in the acetylated lysine binding pocket of BRD4. Through interaction with pTEFb, which is comprised of CDK9 and several cyclin T1 heterodimers, BRD4 promotes phosphorylation of RNA polymerase II, ultimately influencing gene transcription. Dinaciclib is a potent inhibitor of multiple cyclin dependent kinases including CDK9 and has been shown to have pre-clinical activity in AML. Therefore, we hypothesized that PLX51107 would have synergistic activity when combined with dinaciclib. We found synergistic effects on proliferation in primary AML cells when PLX51107 was combined with dinaciclib in stromal co-cultures across a range of dose combinations below the IC50 of either compound alone. Synergistic activity was also present in cytokine support culture conditions, colony forming assays, and synergy assays utilizing COMbenefit software. Interestingly, the highly conserved canonical β-catenin dependent Wnt signaling pathway is a known BETi resistance mechanism in AML. Therefore, destruction of β-catenin and inhibition of Wnt signaling could represent a novel therapeutic target to circumvent development of BET-mediated resistance. We found dinaciclib treatment reduced protein levels of total β-catenin in primary AML cells and AML cell lines (Figure 1A). Phosphorylated β-catenin and non-phosphorylated "active" β-catenin protein levels were also reduced following dinaciclib treatment (Figure 1A). In addition, mRNA expression of multiple components of the Wnt pathway, including the Wnt co-receptor LRP6, AXIN2, FZD1, and many Wnt target genes were significantly decreased following dinaciclib and combination treatment. Moreover, in a luciferase based Wnt reporter cell line, we found dinaciclib significantly reduced Wnt3a induced luciferase activity, suggesting a direct effect on β-catenin mediated transcription of Wnt target genes. Collectively, this data suggests dinaciclib has multiple inhibitory effects on canonical β-catenin dependent Wnt signaling. We found combination treatment of PLX51107 and dinaciclib produced synergistic reduction in expression of both Wnt signaling components and Wnt target genes. Conversely, β-catenin and Wnt target gene expression levels were either not significantly changed or even increased when treated with PLX51107 alone. In order to examine the effects of dinaciclib in the setting of BET resistance, we developed PLX51107 resistant MOLM-13 and MV4-11 AML cell lines via serial drugging. These resistant cell lines remained sensitive to dinaciclib treatment and to cytotoxic chemotherapy drugs including cytarabine and etoposide while demonstrating resistance to PLX51107 via Annexin PI and MTS assays. We found sensitivity to dinaciclib treatment in these resistant cell lines remained unaffected by BETi resistance and continued to demonstrate inhibition of Wnt signaling, similar to the effects seen in parental cells. Therefore, dinaciclib represents a novel strategy to overcome potential therapy emergent BETi resistance. We next examined the efficacy of PLX51107 in combination with dinaciclib in vivo, utilizing luciferase labeled MOLM-13 AML cells xenotransplanted into NOD / SCID / IL2rgnull (NSG) immunodeficient mice. Daily oral dosing with 20 mg/kg PLX51107 resulted in prolonged survival (median 42 days) compared to vehicle treated control animals (median 28 days, p< 0.001). Weekly intraperitoneal 10 mg/kg dinaciclib did not significantly improve survival (median 30 days, p=0.09) in MOLM-13 xenograft mice. However, mice receiving combination therapy survived a median of 56.5 days, significantly longer than with either drug alone (p<0.05) (Figure 1B). We also found decreased disease burden by bioluminescence and by histopathology in combination treated animals, compared to both vehicle and single agent controls. In conclusion, we have demonstrated in vitro and in vivo synergistic activity of the BET inhibitor PLX51107 in combination with dinaciclib in pre-clinical models of AML and have identified novel inhibition of Wnt / β-catenin signaling by dinaciclib, thereby providing a strategy to circumvent resistance to BET inhibitors in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting Wnt/β-Catenin Signaling by TET1/FOXO4 Inhibits Metastatic Spreading and Self-Renewal of Cancer Stem Cells in Gastric Cancer.

Simple SummaryMetastasis is the main cause of death for patients suffering gastric cancer. Epithelial-mesenchymal transition (EMT) and cancer stem cells (CSC) are critical attributes of metastasis, both of which are regulated tightly by DNA methylation and Wnt/β-catenin signaling. In this study, we unveiled a novel TET1-FOXO4-β-catenin signaling cascade, in which TET1 inhibits β-catenin activity and its nuclear translocation through transactivating FOXO4 expression. TET1 expression can significantly inhibit EMT and stemness properties of gastric cancer cells, while knocking-down endogenous TET1 induces metastasis and enhances self-renewal of CSCs by activating canonical Wnt signaling, which could be fully rescued by modulating FOXO4 expression. Our data also showed that low expression of TET1 or FOXO4 predicts poor survival of gastric cancer patients, suggesting reactivation of TET1 or FOXO4 might be a novel therapeutic approach to prevent gastric cancer metastasis.Metastasis is the main cause of death for patients suffering gastric cancer. Epithelial-mesenchymal transition (EMT) and cancer stem cells (CSC) are critical attributes of metastasis, both of which are regulated tightly by DNA methylation and Wnt/β-catenin signaling. Here, we studied the functions of DNA dioxygenase TET1 in regulating Wnt signaling and in gastric cancer metastasis. Knocking-down and overexpressing TET1 in gastric cancer cells promoted and inhibited metastatic spreading to the liver in immune-deficient mice, respectively. TET1 showed inhibitory effects on metastasis-related features -EMT and CSC, which were reversed by interfering with Wnt/β-catenin signaling. RNA-sequencing identified FOXO4 as a direct transactivating target of TET1. FOXO4 directly interacted with β-catenin and recruited it in the cytoplasm, so as to inhibit β-catenin-mediated transcription of Wnt target genes, including CSC marker EpCAM. Moreover, modulation of FOXO4 could reverse the effects of TET1 manipulation on EMT and self-renewal of CSCs. The analysis with clinical samples confirmed the value of FOXO4 as an independent prognostic predictor of patients’ overall survival. Taken together, regulation of Wnt signaling by TET1/FOXO4 is essential for metastasis-associated cellular properties, and targeting TET1/FOXO4/β-catenin pathway may serve as promising therapeutics in the prevention and treatment of gastric cancer metastasis.

Abstract 1862: Naphthoquinone analogues inhibit Wnt/β-catenin signaling and colorectal cancer tumorigenesis

Abstract In this study, we performed a structure-activity-relationship study of seven previously identified chloro-naphthoquinone analogs and evaluated their abilities to inhibit the Wnt/β-catenin signaling pathway. The Wnt/β-catenin signaling pathway plays essential roles in colorectal cancer (CRC) initiation, proliferation, and development. While targeting the Wnt/β-catenin pathway has been validated as a very promising approach for CRC treatment, developing a therapeutic for inhibition of this elusive pathway has been very challenging for researchers. Of the seven chloro-naphthoquinone analogues, we found that two compounds, Compound 3 and Compound 6, significantly inhibited Wnt target gene transcription and Wnt-induced colorectal tumorigenesis in vitro. Chromatin immunoprecipitation binding assays and computational modeling analysis revealed that Compound 3 and Compound 6 inhibit the Wnt/β-catenin pathway through disruption of the TCF4-DNA binding, a crucial step for the activation of the Wnt/β-catenin signaling pathway. Lastly, using a patient-derived organoid model and xenograft mouse model, we showed that these compounds inhibit CRC tumorigenesis. Taken together, this study demonstrates a novel mechanism of action for these chloro-naphthoquinone analogs, which can be further explored in future drug design and discovery efforts for small molecules targeting the TCF family proteins for inhibition of the Wnt/β-catenin signaling pathway. Citation Format: Rosalie G. Hoyle, Andrew Morris, Piyusha Pagare, Shadid Uz Zaman, Zhikun Ma, Yan Zhang, Jiong Li. Naphthoquinone analogues inhibit Wnt/β-catenin signaling and colorectal cancer tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1862.

The CK1δ/ϵ-Tip60 Axis Enhances Wnt/β-Catenin Signaling via Regulating β-Catenin Acetylation in Colon Cancer.

Casein kinase 1δ/ϵ (CK1δ/ϵ) are well-established positive modulators of the Wnt/β-catenin signaling pathway. However, the molecular mechanisms involved in the regulation of β-catenin transcriptional activity by CK1δ/ϵ remain unclear. In this study, we found that CK1δ/ϵ could enhance β-catenin-mediated transcription through regulating β-catenin acetylation. CK1δ/ϵ interacted with Tip60 and facilitated the recruitment of Tip60 to β-catenin complex, resulting in increasing β-catenin acetylation at K49. Importantly, Tip60 significantly enhanced the SuperTopFlash reporter activity induced by CK1δ/ϵ or/and β-catenin. Furthermore, a CK1δ/CK1ϵ/β-catenin/Tip60 complex was detected in colon cancer cells. Simultaneous knockdown of CK1δ and CK1ϵ significantly attenuated the interaction between β-catenin and Tip60. Notably, inhibition of CK1δ/ϵ or Tip60, with shRNA or small molecular inhibitors downregulated the level of β-catenin acetylation at K49 in colon cancer cells. Finally, combined treatment with CK1 inhibitor SR3029 and Tip60 inhibitor MG149 had more potent inhibitory effect on β-catenin acetylation, the transcription of Wnt target genes and the viability and proliferation in colon cancer cells. Taken together, our results revealed that the transcriptional activity of β-catenin could be modulated by the CK1δ/ϵ-β-catenin-Tip60 axis, which may be a potential therapeutic target for colon cancer.

Discovery of 1-Benzoyl 4-Phenoxypiperidines as Small-Molecule Inhibitors of the β-Catenin/B-Cell Lymphoma 9 Protein-Protein Interaction.

Structure-based design and optimization were performed to develop small-molecule β-catenin/B-cell lymphoma 9 (BCL9) inhibitors and improve their inhibitory activities. Compound ZL3138 with a novel 1-benzoyl 4-phenoxypiperidine scaffold was discovered to disrupt the β-catenin/BCL9 protein-protein interaction (PPI) with a Ki of 0.96 μM in AlphaScreen competitive inhibition assays and displayed good selectivity for β-catenin/BCL9 over β-catenin/E-cadherin PPIs. The binding mode of new inhibitors was characterized by structure-activity relationship and site-directed mutagenesis studies. Protein pull-down assays indicate that this series of compounds directly binds with β-catenin. Cellular target engagement and co-immunoprecipitation experiments demonstrate that ZL3138 binds with β-catenin and disrupts the β-catenin/BCL9 interaction without affecting the β-catenin/E-cadherin interaction in living cells. Further cell-based studies show that ZL3138 selectively suppresses transactivation of Wnt/β-catenin signaling, regulates transcription and expression of Wnt target genes, and inhibits the growth of Wnt/β-catenin-dependent cancer cells.

Adenomatous polyposis coli in cancer and therapeutic implications.

Inactivating mutations of the adenomatous polyposis coli (APC) gene and consequential upregulation of the Wnt signaling pathway are critical initiators in the development of colorectal cancer (CRC), the third most common cancer in the United States for both men and women. Emerging evidence suggests APCmutations are also found in gastric, breast and other cancers. The APC gene, located on chromosome 5q, is responsible for negatively regulating the b-catenin/Wnt pathway by creating a destruction complex with Axin/Axin2, GSK-3b, and CK1. In the event of an APC mutation, b-catenin accumulates, translocates to the cell nucleus and increases the transcription of Wnt target genes that have carcinogenic consequences in gastrointestinal epithelial stem cells. A literature review was conducted to highlight carcinogenesis related to APC mutations, as well as preclinical and clinical studies for potential therapies that target steps in inflammatory pathways, including IL-6 transduction, and Wnt pathway signaling regulation. Although a range of molecular targets have been explored in murine models, relatively few pharmacological agents have led to substantial increases in survival for patients with colorectal cancer clinically. This article reviews a range of molecular targets that may be efficacious targets for tumors with APC mutations.

Pannexin 1 binds β-catenin to modulate melanoma cell growth and metabolism

Melanoma is the most aggressive skin malignancy with increasing incidence worldwide. Pannexin1 (PANX1), a member of the pannexin family of channel-forming glycoproteins, regulates cellular processes in melanoma cells including proliferation, migration, and invasion/metastasis. However, the mechanisms responsible for coordinating and regulating PANX1 function remain unclear. Here, we demonstrated a direct interaction between the C-terminal region of PANX1 and the N-terminal portion of β-catenin, a key transcription factor in the Wnt pathway. At the protein level, β-catenin was significantly decreased when PANX1 was either knocked down or inhibited by two PANX1 blockers, Probenecid and Spironolactone. Immunofluorescence imaging showed a disrupted pattern of β-catenin localization at the cell membrane in PANX1-deficient cells, and transcription of several Wnt target genes, including MITF, was suppressed. In addition, a mitochondrial stress test revealed that the metabolism of PANX1-deficient cells was impaired, indicating a role for PANX1 in the regulation of the melanoma cell metabolic profile. Taken together, our data show that PANX1 directly interacts with β-catenin to modulate growth and metabolism in melanoma cells. These findings provide mechanistic insight into PANX1-mediated melanoma progression and may be applicable to other contexts where PANX1 and β-catenin interact as a potential new component of the Wnt signaling pathway.

IOX1 Suppresses Wnt Target Gene Transcription and Colorectal Cancer Tumorigenesis through Inhibition of KDM3 Histone Demethylases.

Epigenetic activation of Wnt/β-catenin signaling plays a critical role in Wnt-induced tumorigenesis, notably in colorectal cancers. KDM3 and KDM4 histone demethylases have been reported to promote oncogenic Wnt signaling through demethylation of H3K9 on Wnt target gene promoters and are suggested to be potential therapeutic targets. However, potent inhibitors for these regulators are still not available. In addition, which family is most responsible for activation of Wnt target genes and Wnt-induced oncogenesis is not well documented, specifically in colorectal cancer. In this study, we characterized the functional redundancy and differences between KDM3 and KDM4 in regard to regulating Wnt signaling. Our data suggest that KDM3 may play a more essential role than KDM4 in regulating oncogenic Wnt signaling in human colorectal cancer. We also identified that IOX1, a known histone demethylase inhibitor, significantly suppresses Wnt target gene transcription and colorectal cancer tumorigenesis. Mechanistically, IOX1 inhibits the enzymatic activity of KDM3 by binding to the Jumonji C domain and thereby preventing the demethylation of H3K9 on Wnt target gene promoters. Taken together, our data not only identified the critical mechanisms by which IOX1 suppressed Wnt/β-catenin signaling and colorectal cancer tumorigenesis through inhibition of KDM3, but also suggested that IOX1 may represent an attractive small molecule lead for future drug design and discovery.

VBP1 modulates Wnt/β-catenin signaling by mediating the stability of the transcription factors TCF/LEFs

The Wnt/β-catenin pathway is one of the major pathways that regulates embryonic development, adult homeostasis, and stem cell self-renewal. In this pathway, transcription factors T-cell factor and lymphoid enhancer factor (TCF/LEF) serve as a key switch to repress or activate Wnt target gene transcription by recruiting repressor molecules or interacting with the β-catenin effector, respectively. It has become evident that the protein stability of the TCF/LEF family members may play a critical role in controlling the activity of the Wnt/β-catenin signaling pathway. However, factors that regulate the stability of TCF/LEFs remain largely unknown. Here, we report that pVHL binding protein 1 (VBP1) regulates the Wnt/β-catenin signaling pathway by controlling the stability of TCF/LEFs. Surprisingly, we found that either overexpression or knockdown of VBP1 decreased Wnt/β-catenin signaling activity in both cultured cells and zebrafish embryos. Mechanistically, VBP1 directly binds to all four TCF/LEF family members and von Hippel-Lindau tumor-suppressor protein (pVHL). Either overexpression or knockdown of VBP1 increases the association between TCF/LEFs and pVHL and then decreases the protein levels of TCF/LEFs via proteasomal degradation. Together, our results provide mechanistic insights into the roles of VBP1 in controlling TCF/LEFs protein stability and regulating Wnt/β-catenin signaling pathway activity.

Bile salt-dependent lipase promotes the barrier integrity of Caco-2 cells by activating Wnt/β-catenin signaling via LRP6 receptor.

Bile salt-dependent lipase (BSDL) within intestinal lumen can be endocytosed by enterocytes and support the intestinal barrier function. However, the epithelial-supporting effect of this protein has not been verified in a human cell line and neither the direct signaling pathway nor the function of endocytosis in this process has been clearly identified. We sought to investigate the signaling pathway and the membrane receptor through which BSDL might exert these effects using intestinal epithelial cells. Caco-2 cells were treated with recombinant BSDL, and the barrier function, cell proliferation, and activation of the Wnt signaling pathway were assessed. The effect of Wnt signaling activation induced by BSDL and BSDL endocytosis was investigated in LRP6-silenced and non-silenced cells. Moreover, caveolae- and clathrin-dependent endocytosis inhibitors were also applied respectively to analyze their effects on Wnt signaling activation induced by BSDL. BSDL treatment increased the barrier function but not proliferation of Caco-2 cells. It also induced β-catenin nuclear translocation and activated Wnt target gene transcription. Moreover, in the Wnt pathway, BSDL increased the levels of non-phosphorylated-β-catenin (Ser33/37/Thr41) and phosphorylated-β-catenin (Ser552). Notably, the silencing of LRP6 expression impaired BSDL endocytosis and decreased BSDL-induced β-catenin nuclear translocation. The inhibition of BSDL endocytosis induced by caveolae-mediated endocytosis inhibitor was stronger than that by clathrin-mediated endocytosis inhibitor, and the Wnt signaling activation associated with its endocytosis was also most likely caveolae-dependent. Our findings suggested that LRP6, a canonical Wnt pathway co-receptor, can mediate BSDL endocytosis and then activate Wnt signaling in Caco-2 cells.

TBX3 acts as tissue-specific component of the Wnt/β-catenin transcriptional complex.

BCL9 and PYGO are β-catenin cofactors that enhance the transcription of Wnt target genes. They have been proposed as therapeutic targets to diminish Wnt signaling output in intestinal malignancies. Here we find that, in colorectal cancer cells and in developing mouse forelimbs, BCL9 proteins sustain the action of β-catenin in a largely PYGO-independent manner. Our genetic analyses implied that BCL9 necessitates other interaction partners in mediating its transcriptional output. We identified the transcription factor TBX3 as a candidate tissue-specific member of the β-catenin transcriptional complex. In developing forelimbs, both TBX3 and BCL9 occupy a large number of Wnt-responsive regulatory elements, genome-wide. Moreover, mutations in Bcl9 affect the expression of TBX3 targets in vivo, and modulation of TBX3 abundance impacts on Wnt target genes transcription in a β-catenin- and TCF/LEF-dependent manner. Finally, TBX3 overexpression exacerbates the metastatic potential of Wnt-dependent human colorectal cancer cells. Our work implicates TBX3 as context-dependent component of the Wnt/β-catenin-dependent transcriptional complex.

Nuclear Regulation of Wnt/β-Catenin Signaling: It's a Complex Situation.

Wnt signaling is an evolutionarily conserved metazoan cell communication pathway required for proper animal development. Of the myriad of signaling events that have been ascribed to cellular activation by Wnt ligands, the canonical Wnt/β-catenin pathway has been the most studied and best understood. Misregulation of Wnt/β-catenin signaling has been implicated in developmental defects in the embryo and major diseases in the adult. Despite the latter, no drugs that inhibit the Wnt/β-catenin pathway have been approved by the FDA. In this review, we explore the least understood step in the Wnt/β-catenin pathway—nuclear regulation of Wnt target gene transcription. We initially describe our current understanding of the importation of β-catenin into the nucleus. We then focus on the mechanism of action of the major nuclear proteins implicated in driving gene transcription. Finally, we explore the concept of a nuclear Wnt enhanceosome and propose a modified model that describes the necessary components for the transcription of Wnt target genes.

Wnt-Induced Stabilization of KDM4C Is Required for Wnt/β-Catenin Target Gene Expression and Glioblastoma Tumorigenesis.

Wnt/β-catenin signaling activates the transcription of target genes to regulate stem cells and cancer development. However, the contribution of epigenetic regulation to this process is unknown. Here, we report that Wnt activation stabilizes the epigenetic regulator KDM4C that promotes tumorigenesis and survival of human glioblastoma cells by epigenetically activating the transcription of Wnt target genes. KDM4C protein expression was upregulated in human glioblastomas, and its expression directly correlated with Wnt activity and Wnt target gene expression. KDM4C was essential for Wnt-induced gene expression and tumorigenesis of glioblastoma cells. In the absence of Wnt3a, protein kinase R phosphorylated KDM4C at Ser918, inducing KDM4C ubiquitination and degradation. Wnt3a stabilized KDM4C through inhibition of GSK3-dependent protein kinase R activity. Stabilized KDM4C accumulated in the nucleus and bound to and demethylated TCF4-associated histone H3K9 by interacting with β-catenin, promoting HP1γ removal and transcriptional activation. These findings reveal that Wnt-KDM4C-β-catenin signaling represents a novel mechanism for the transcription of Wnt target genes and regulation of tumorigenesis, with important clinical implications. SIGNIFICANCE: These findings identify the Wnt-KDM4C-β-catenin signaling axis as a critical mechanism for glioma tumorigenesis that may serve as a new therapeutic target in glioblastoma.

Parkinson's disease, aging and adult neurogenesis: Wnt/β-catenin signalling as the key to unlock the mystery of endogenous brain repair.

A common hallmark of age‐dependent neurodegenerative diseases is an impairment of adult neurogenesis. Wingless‐type mouse mammary tumor virus integration site (Wnt)/β‐catenin (WβC) signalling is a vital pathway for dopaminergic (DAergic) neurogenesis and an essential signalling system during embryonic development and aging, the most critical risk factor for Parkinson's disease (PD). To date, there is no known cause or cure for PD. Here we focus on the potential to reawaken the impaired neurogenic niches to rejuvenate and repair the aged PD brain. Specifically, we highlight WβC‐signalling in the plasticity of the subventricular zone (SVZ), the largest germinal region in the mature brain innervated by nigrostriatal DAergic terminals, and the mesencephalic aqueduct‐periventricular region (Aq‐PVR) Wnt‐sensitive niche, which is in proximity to the SNpc and harbors neural stem progenitor cells (NSCs) with DAergic potential. The hallmark of the WβC pathway is the cytosolic accumulation of β‐catenin, which enters the nucleus and associates with T cell factor/lymphoid enhancer binding factor (TCF/LEF) transcription factors, leading to the transcription of Wnt target genes. Here, we underscore the dynamic interplay between DAergic innervation and astroglial‐derived factors regulating WβC‐dependent transcription of key genes orchestrating NSC proliferation, survival, migration and differentiation. Aging, inflammation and oxidative stress synergize with neurotoxin exposure in “turning off” the WβC neurogenic switch via down‐regulation of the nuclear factor erythroid‐2‐related factor 2/Wnt‐regulated signalosome, a key player in the maintenance of antioxidant self‐defense mechanisms and NSC homeostasis. Harnessing WβC‐signalling in the aged PD brain can thus restore neurogenesis, rejuvenate the microenvironment, and promote neurorescue and regeneration.

Dkk1 Controls Cell-Cell Interaction through Regulation of Non-nuclear β-Catenin Pools.

SummaryDickkopf-1 (Dkk1) is a secreted Wnt antagonist with a well-established role in head induction during development. Numerous studies have emerged implicating Dkk1 in various malignancies and neurodegenerative diseases through an unknown mechanism. Using zebrafish gastrulation as a model for collective cell migration, we unveil such a mechanism, identifying a role for Dkk1 in control of cell connectivity and polarity in vivo, independent of its known function. We find that Dkk1 localizes to adhesion complexes at the plasma membrane and regions of concentrated actomyosin, suggesting a direct involvement in regulation of local cell adhesion. Our results show that Dkk1 represses cell polarization and integrity of cell-cell adhesion, independently of its impact on β-catenin protein degradation. Concurrently, Dkk1 prevents nuclear localization of β-catenin by restricting its distribution to a discrete submembrane pool. We propose that redistribution of cytosolic β-catenin by Dkk1 concomitantly drives repression of cell adhesion and inhibits β-catenin-dependent transcriptional output.

Abstract 3446: STRAP is involved in mutated Apc-induced stemness and tumorigenesis in colon cancer

Abstract Inactivation of the Apc gene is a critical early event in the development of sporadic colorectal cancer (CRC). Loss of its normal function leads to constitutive activation of β-catenin/T-cell factor 4 signaling and hence transcription of Wnt target genes. The signaling scaffold protein STRAP is upregulated in several cancers, where it promotes tumorigenicity and stemness. Here, we tested the effect of conditional deletion of Strap on intestinal tumor formation and growth by crossing ApcMin/+ mice with mice that are null for Strap in the intestinal epithelium, Strapfl/fl;Vil-Cre mice (StrapIEKO). We observed a dramatic decrease in the number of intestinal polyps in the ApcMin/+;Strapfl/fl;Vil-Cre mice (called StrapIEKO;ApcMin/+) compared to mice with intact Strap (Strapfl/fl;ApcMin/+). In an attempt to understand the mechanism of function of STRAP, we have observed that colon tumors from StrapIEKO;ApcMin/+ mice displayed a decrease in β-catenin nuclear localization and downregulation of Wnt targets such as c-Myc, AXIN2, and CCND1. Enteroids prepared from StrapIEKO;ApcMin/+ mice exhibited more budding compared to the ApcMin/+ enteroids, indicating the role of STRAP in stemness. STRAP knockdown in human CRC cells significantly diminished β-catenin/TCF4 transcriptional activity. Specifically, STRAP knockdown impaired the assembly of β-catenin/TCF4 complex and recruitment of the complex to the promoters of β-catenin target genes. Functional proteomic analyses of crypt cells from different genetic mice suggested that the MEK/ERK signaling pathway is suppressed in StrapIEKO;ApcMin/+ mice that is required for the effect of STRAP on Wnt/β-catenin signaling. We have shown that STRAP associates with MEK1/2 and promotes binding between MEK1/2 and ERK1/2 and subsequently induces the phosphorylation of ERK1/2. Given that MEK/ERK signaling is found to promote Wnt/β-catenin signaling, our results suggest that STRAP indirectly induce Wnt/β-catenin signaling through activating MEK/ERK pathway. Importantly, STRAP is found to be a novel target of Wnt/β-catenin signaling as ChIP assays determined putative binding sites of β-catenin/TCF4 complex on Strap promoter. Finally, we showed that forced expression of STRAP in colon cancer organoids derived from patients with colon cancer can induce the growth of colon tumor organoids through promoting Wnt/β-catenin signaling. Altogether, these results suggest that STRAP is involved in mutated APC-induced tumor initiation through a novel feed-forward STRAP/MEK-ERK/β-catenin/STRAP regulatory axis. Citation Format: Trung Vu. STRAP is involved in mutated Apc-induced stemness and tumorigenesis in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3446.