Function Of Wnt Signaling Research Articles

A Foxf1-Wnt-Nr2f1 cascade promotes atrial cardiomyocyte differentiation in zebrafish.

Nr2f transcription factors (TFs) are conserved regulators of vertebrate atrial cardiomyocyte (AC) differentiation. However, little is known about the mechanisms directing Nr2f expression in ACs. Here, we identified a conserved enhancer 3' to the nr2f1a locus, which we call 3'reg1-nr2f1a (3'reg1), that can promote Nr2f1a expression in ACs. Sequence analysis of the enhancer identified putative Lef/Tcf and Foxf TF binding sites. Mutation of the Lef/Tcf sites within the 3'reg1 reporter, knockdown of Tcf7l1a, and manipulation of canonical Wnt signaling support that Tcf7l1a is derepressed via Wnt signaling to activate the transgenic enhancer and promote AC differentiation. Similarly, mutation of the Foxf binding sites in the 3'reg1 reporter, coupled with gain- and loss-of-function analysis supported that Foxf1 promotes expression of the enhancer and AC differentiation. Functionally, we find that Wnt signaling acts downstream of Foxf1 to promote expression of the 3'reg1 reporter within ACs and, importantly, both Foxf1 and Wnt signaling require Nr2f1a to promote a surplus of differentiated ACs. CRISPR-mediated deletion of the endogenous 3'reg1 abrogates the ability of Foxf1 and Wnt signaling to produce surplus ACs in zebrafish embryos. Together, our data support that downstream members of a conserved regulatory network involving Wnt signaling and Foxf1 function on a nr2f1a enhancer to promote AC differentiation in the zebrafish heart.

Targeting Wnt Pathways with Small Molecules as New Approach in Cardiovascular Disease.

The increasing incidences of morbidity and mortality associated with cardiovascular diseases represent significant difficulties for clinical treatment and have a major impact on patient health. Wnt signaling pathways are highly conserved and are well known for their regulatory roles in embryonic development, tissue regeneration, and adult tissue homeostasis. Wnt signaling is classified into two distinct pathways: canonical Wnt/β-catenin signaling and noncanonical pathways, including planar cell polarity and Wnt/Ca2+ pathways. A growing body of experimental evidence suggests the involvement of both canonical and non-canonical Wnt signaling pathways in the development of cardiovascular diseases, including myocardial hypertrophy, arrhythmias, diabetic cardiomyopathy, arrhythmogenic cardiomyopathy, and myocardial infarction. Thus, to enhance patient quality of life, diagnosing and treating cardiac illnesses may require a thorough understanding of the molecular functions played by the Wnt pathway in these disorders. Many small-molecule inhibitors specifically target various components within the Wnt signaling pathways, such as Frizzled, Disheveled, Porcupine, and Tankyrase. This study aims to present an overview of the latest findings regarding the functions of Wnt signaling in human cardiac disorders and possible inhibitors of Wnt, which could lead to novel approaches for treating cardiac ailments.

Exploring DIX-DIX Homo- and Hetero-Oligomers in Wnt Signaling with AlphaFold2.

Wnt signaling is involved in embryo development and cancer. The binding between the DIX domains of Axin1/2, Dishevelled1/2/3, and Coiled-coil-DIX1 is essential for Wnt/β-catenin signaling. Structural and biological studies have revealed that DIX domains are polymerized through head-to-tail interface interactions, which are indispensable for activating β-catenin Wnt signaling. Although different isoforms of Dvl and Axin proteins display both redundant and specific functions in Wnt signaling, the specificity of DIX-mediated interactions remains unclear due to technical challenges. Using AlphaFold2(AF2), we predict the structures of 6 homodimers and 22 heterodimers of DIX domains without templates and compare them with the reported X-ray complex structures. PRODIGY is used to calculate the binding affinities of these DIX complexes. Our results show that the Axin2 DIX homodimer has a stronger binding affinity than the Axin1 DIX homodimer. Among Dishevelled (Dvl) proteins, the binding affinity of the Dvl1 DIX homodimer is stronger than that of Dvl2 and Dvl3. The Coiled-coil-DIX1(Ccd1) DIX homodimer shows weaker binding than the Axin1 DIX homodimer. Generally, heterodimer interactions tend to be stronger than those of homodimers. Our findings provide insights into the mechanism of the Wnt signaling pathway and highlight the potential of AF2 and PRODIGY for studying protein-protein interactions in signaling pathways.

Wnt signaling modulates the response to DNA damage in the Drosophila wing imaginal disc by regulating the EGFR pathway.

Despite the deep conservation of the DNA damage response (DDR) pathway, cells in different contexts vary widely in their susceptibility to DNA damage and their propensity to undergo apoptosis as a result of genomic lesions. One of the cell signaling pathways implicated in modulating the DDR is the highly conserved Wnt pathway, which is known to promote resistance to DNA damage caused by ionizing radiation in a variety of human cancers. However, the mechanisms linking Wnt signal transduction to the DDR remain unclear. Here, we use a genetically encoded system in Drosophila to reliably induce consistent levels of DNA damage in vivo, and demonstrate that canonical Wnt signaling in the wing imaginal disc buffers cells against apoptosis in the face of DNA double-strand breaks. We show that Wg, the primary Wnt ligand in Drosophila, activates epidermal growth factor receptor (EGFR) signaling via the ligand-processing protease Rhomboid, which, in turn, modulates the DDR in a Chk2-, p53-, and E2F1-dependent manner. These studies provide mechanistic insight into the modulation of the DDR by the Wnt and EGFR pathways in vivo in a highly proliferative tissue. Furthermore, they reveal how the growth and patterning functions of Wnt signaling are coupled with prosurvival, antiapoptotic activities, thereby facilitating developmental robustness in the face of genomic damage.

Engineering a targeted and safe bone anabolic gene therapy to treat osteoporosis in alveolar bone loss

Alveolar bone loss in elderly populations is highly prevalent and increases the risk of tooth loss, gum disease susceptibility, and facial deformity. Unfortunately, there are very limited treatment options available. Here, we developed a bone-targeted gene therapy that reverses alveolar bone loss in patients with osteoporosis by targeting the adaptor protein Schnurri-3 (SHN3). SHN3 is a promising therapeutic target for alveolar bone regeneration, because SHN3 expression is elevated in human and mouse mandible tissues with osteoporosis while deletion of SHN3 in mice greatly increases alveolar bone and tooth dentin mass. We used a bone-targeted recombinant adeno-associated virus (rAAV) carrying an artificial microRNA (miRNA) that silences SHN3 expression to restore alveolar bone loss in mouse models of both postmenopausal and senile osteoporosis by enhancing WNT signaling and osteoblast function. Additionally, rAAV-mediated silencing of SHN3 enhanced bone formation and collagen production of human skeletal organoids in xenograft mice. Finally, rAAV expression in the mandible was tightly controlled via liver- and heart-specific miRNA-mediated repression or via a vibration-inducible mechanism. Collectively, our results demonstrate that AAV-based bone anabolic gene therapy is a promising strategy to treat alveolar bone loss in osteoporosis.

Systems genetics analysis of human body fat distribution genes identifies adipocyte processes.

Excess abdominal fat is a sexually dimorphic risk factor for cardio-metabolic disease and is approximated by the waist-to-hip ratio adjusted for body mass index (WHRadjBMI). Whereas this trait is highly heritable, few causal genes are known. We aimed to identify novel drivers of WHRadjBMI using systems genetics. We used two independent cohorts of adipose tissue gene expression and constructed sex- and depot-specific Bayesian networks to model gene-gene interactions from 8,492 genes. Using key driver analysis, we identified genes that, in silico and putatively in vitro, regulate many others. 51-119 key drivers in each network were replicated in both cohorts. In other cell types, 23 of these genes are found in crucial adipocyte pathways: Wnt signaling or mitochondrial function. We overexpressed or down-regulated seven key driver genes in human subcutaneous pre-adipocytes. Key driver genes ANAPC2 and RSPO1 inhibited adipogenesis, whereas PSME3 increased adipogenesis. RSPO1 increased Wnt signaling activity. In differentiated adipocytes, MIGA1 and UBR1 down-regulation led to mitochondrial dysfunction. These five genes regulate adipocyte function, and we hypothesize that they regulate fat distribution.

Abstract 3071: Differential susceptibility of breast cancer cells to mitochondrial dysfunction induced by Wnt inhibitors

Abstract Advances in research have shown that cancer cells have distinctive bioenergetic alterations to meet their energy requirements. It has been established that certain cancers are significantly reliant on mitochondrial respiration, suggesting that modulation of mitochondrial function may have therapeutic value. The Wnt signaling pathway is a potential therapeutic target of cancer, including breast and colorectal cancers. Recent findings suggest that bi-directional crosstalk between Wnt signaling and mitochondrial function plays a crucial role in tumorigenesis, as well as differentiation and self-renewal of stem cells. In this study, 71 agents known to alter Wnt signaling were screened for effects on mitochondrial function in BT474 and Hs578T breast cancer cell lines, which have highly oxidative and highly glycolytic bioenergetic phenotypes, respectively. This screen was performed using the Agilent Seahorse XF Mito Tox assay, which enables sensitive detection and characterization of mitochondrial modulators via a standardized, quantitative parameter, the Mito Tox Index (MTI). The MTI facilitates the comparison of drug efficacy by detecting modes of mitochondrial toxicity, inhibition, and uncoupling. The MTI-based screening of the Wnt modulators identified one inhibitor and two uncouplers as potent agents inducing mitochondrial dysfunction for both BT474 and Hs578T. Despite their distinctive bioenergetic phenotypes, MTI-based dose-response assays revealed no significant differences in the efficacy of these modulators between BT474 and Hs578T cell types. In contrast, significant differential effects on ATP production rates were detected, showing cellular energy depletion induced by these agents. Using the Agilent Seahorse XF Real-Time ATP Rate assay, BT474 cells showed significant metabolic energy crisis in the presence of all 3 compounds, whereas Hs578T cells maintained stable total ATP production. This differential impact on ATP production rate corresponds directly to differences in cell viability, with BT474 showing higher susceptibility to these compounds. These results suggest that the anti-cancer efficacy of mitochondrial modulators can be different depending on the metabolic phenotype of the target cell and should be considered in therapeutic design. Citation Format: Yoonseok Kam, Lisa Winer, George W. Rogers, Natalia Romero. Differential susceptibility of breast cancer cells to mitochondrial dysfunction induced by Wnt inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3071.

Loss of dlx5a/dlx6a Locus Alters Non-Canonical Wnt Signaling and Meckel's Cartilage Morphology.

The dlx genes encode transcription factors that establish a proximal-distal polarity within neural crest cells to bestow a regional identity during craniofacial development. The expression regions of dlx paralogs are overlapping yet distinct within the zebrafish pharyngeal arches and may also be involved in progressive morphologic changes and organization of chondrocytes of the face. However, how each dlx paralog of dlx1a, dlx2a, dlx5a and dlx6a affects craniofacial development is still largely unknown. We report here that the average lengths of the Meckel's, palatoquadrate and ceratohyal cartilages in different dlx mutants were altered. Mutants for dlx5a-/- and dlx5i6-/-, where the entire dlx5a/dlx6a locus was deleted, have the shortest lengths for all three structures at 5 days post fertilization (dpf). This phenotype was also observed in 14 dpf larvae. Loss of dlx5i6 also resulted in increased proliferation of neural crest cells and expression of chondrogenic markers. Additionally, altered expression and function of non-canonical Wnt signaling were observed in these mutants suggesting a novel interaction between dlx5i6 locus and non-canonical Wnt pathway regulating ventral cartilage morphogenesis.

Predicting Modifiers of Genotype-Phenotype Correlations in Craniofacial Development.

Most human birth defects are phenotypically variable even when they share a common genetic basis. Our understanding of the mechanisms of this variation is limited, but they are thought to be due to complex gene-environment interactions. Loss of the transcription factor Gata3 associates with the highly variable human birth defects HDR syndrome and microsomia, and can lead to disruption of the neural crest-derived facial skeleton. We have demonstrated that zebrafish gata3 mutants model the variability seen in humans, with genetic background and candidate pathways modifying the resulting phenotype. In this study, we sought to use an unbiased bioinformatic approach to identify environmental modifiers of gata3 mutant craniofacial phenotypes. The LINCs L1000 dataset identifies chemicals that generate differential gene expression that either positively or negatively correlates with an input gene list. These chemicals are predicted to worsen or lessen the mutant phenotype, respectively. We performed RNA-seq on neural crest cells isolated from zebrafish across control, Gata3 loss-of-function, and Gata3 rescue groups. Differential expression analyses revealed 551 potential targets of gata3. We queried the LINCs database with the 100 most upregulated and 100 most downregulated genes. We tested the top eight available chemicals predicted to worsen the mutant phenotype and the top eight predicted to lessen the phenotype. Of these, we found that vinblastine, a microtubule inhibitor, and clofibric acid, a PPAR-alpha agonist, did indeed worsen the gata3 phenotype. The Topoisomerase II and RNA-pol II inhibitors daunorubicin and triptolide, respectively, lessened the phenotype. GO analysis identified Wnt signaling and RNA polymerase function as being enriched in our RNA-seq data, consistent with the mechanism of action of some of the chemicals. Our study illustrates multiple potential pathways for Gata3 function, and demonstrates a systematic, unbiased process to identify modifiers of genotype-phenotype correlations.

Dendrobium officinale Polysaccharide (DOP) Promotes Hair Regrowth in Testosterone-Induced Bald Mice.

Androgenetic alopecia can affect up to 70% of males and 40% of females; however, certain therapeutic medications offer partial and transitory improvement but with major side effects. Dendrobium officinale polysaccharide (DOP) has been reported to improve androgen-related hair loss in mice, but the molecular mechanism remains unclear. To explore the effects of DOP on androgenetic alopecia. In this study, testosterone was subcutaneously administered to shave dorsa skin of mice to establish androgenetic alopecia; the effects of DOP in androgenetic alopecia were explored by DOP administration. Testosterone treatment extended the time of skin growing dark and hair growing, decreased the mean numbers of follicles in skin tissues, decreased β-catenin and cyclin D1 levels, and elevated testosterone, DHT (dihydrotestosterone), and 5α-reductase levels. In contrast, DOP administration shortened skin growing dark and hair growing times, promoted follicle cell proliferation, increased follicle numbers, increased β-catenin and cyclin D1 levels, and decreased testosterone, DHT, and 5α-reductase levels. DOP application significantly improved testosterone-induced hair follicle miniaturization and hair loss, possibly through affecting the Wnt signaling and hair follicle stem cell functions. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Mechanisms of mutant β-catenin in endometrial cancer progression.

Endometrial carcinoma (EC) is the most diagnosed gynecological malignancy in Western countries. Both incidence and mortality rates of EC have steadily risen in recent years. Despite generally favorable prognoses for patients with the endometrioid type of EC, a subset of patients has been identified with decreased progression-free survival. Patients in this group are distinguished from other endometrioid EC patients by the presence of exon 3 hotspot mutations in CTNNB1, the gene encoding for the β-catenin protein. β-catenin is an evolutionarily conserved protein with critical functions in both adherens junctions and Wnt-signaling. The exact mechanism by which exon 3 CTNNB1 mutations drive EC progression is not well understood. Further, the potential contribution of mutant β-catenin to adherens junctions’ integrity is not known. Additionally, the magnitude of worsened progression-free survival in patients with CTNNB1 mutations is context dependent, and therefore the importance of this subset of patients can be obscured by improper categorization. This review will examine the history and functions of β-catenin, how these functions may change and drive EC progression in CTNNB1 mutant patients, and the importance of this patient group in the broader context of the disease.

Wnt5a-Vangl1/2 signaling regulates the position and direction of lung branching through the cytoskeleton and focal adhesions.

Lung branching morphogenesis requires reciprocal interactions between the epithelium and mesenchyme. How the lung branches are generated at a defined location and projected toward a specific direction remains a major unresolved issue. In this study, we investigated the function of Wnt signaling in lung branching in mice. We discovered that Wnt5a in both the epithelium and the mesenchyme plays an essential role in controlling the position and direction of lung branching. The Wnt5a signal is mediated by Vangl1/2 to trigger a cascade of noncanonical or planar cell polarity (PCP) signaling. In response to noncanonical Wnt signaling, lung cells undergo cytoskeletal reorganization and change focal adhesions. Perturbed focal adhesions in lung explants are associated with defective branching. Moreover, we observed changes in the shape and orientation of the epithelial sheet and the underlying mesenchymal layer in regions of defective branching in the mutant lungs. Thus, PCP signaling helps define the position and orientation of the lung branches. We propose that mechanical force induced by noncanonical Wnt signaling mediates a coordinated alteration in the shape and orientation of a group of epithelial and mesenchymal cells. These results provide a new framework for understanding the molecular mechanisms by which a stereotypic branching pattern is generated.

ROR2-Vangl2 complexes direct ovarian cancer cell motility in response to Wnt5a (265)

<b>Objectives:</b> In both developmental and cancer contexts, collective migration has shown to be at least partially dependent upon asymmetric localization of core protein complexes within leader and follower cells. A critical function of non-canonical Wnt signaling in vertebrates is to regulate planar cell polarity (PCP), the polarity axis that organizes the plane of a tissue. Cells at the invasive front of cellular aggregates exhibit elevated expression of genes associated with motility pathways, including Wnt/PCP signaling. Using microfluidic devices, we have previously shown the soluble non-canonical Wnt ligand, <i>WNT5A</i>, influencing the directional motion of ovarian cancer cells via the ROR2 receptor. We sought to determine whether asymmetrical localization of ROR2 could guide <i>WNT5A-</i>directed motility. <b>Methods:</b> A DNA sequence encoding a fusion protein of ROR2 with a C-terminal Emerald (Em) fluorescent protein tag was introduced into ES2 ovarian cancer cells. Aggregated spheroids of ES2 ROR2-Em cells were plated onto a fibronectin-coated surface, and time-lapse fluorescent images were obtained of the radial migration in the presence or absence of <i>WNT5A</i>. Based on the direction of motion, cells were divided into leading or trailing half for analysis of the localization of ROR2 protein. Data were presented as mean +/- SEM. For co-immunoprecipitation experiments, ES2 cells expressing a Myctagged ROR2 DNA construct were utilized, with Myc epitope used for pulldown. <b>Results:</b> In the absence of <i>WNT5A</i>, ROR2 was uniformly distributed among the leading and lagging edge of the migrating cells (46.9% +/-2.452 ROR2 localized on the leading edge). In the presence of <i>WNT5A</i>, ROR2 was preferentially located to the leading edge of migrating cells (64.6% +/- 3.908 ROR2 localized on the leading edge). <i>VANGL2</i>, a core component in planar cell polarity, formed Wnt-induced receptor complexes with ROR2 in developing limb buds and other tumor types. <i>VANGL2</i> had been shown to reside at the protrusive edge of leader cells. To address whether <i>VANGL2</i>-ROR2 complexes contributed to the leading-edge localization of ROR2 in ovarian cancer cells, co-IP experiments were performed. Complex formation of ROR2-<i>VANGL2</i> was present in ovarian cancer cells at baseline (<i>VANGL2</i>:ROR2 IP ratio 1), yet was increased in the presence of <i>WNT5A</i> (<i>VANGL2</i>:ROR2 IP ratio 1.5). <b>Conclusions:</b> We have previously shown that <i>WNT5A</i> promotes the motility of ovarian cancer cells by influencing both directionality of motion as well as the degree of displacement in a ROR2 dependent manner. Asymmetric localization of Wnt/PCP core component complexes within leader and follower cells is crucial for cell motility, an intrinsic metastatic property. Our studies suggest that under the influence of <i>WNT5A</i>, ROR2 localizes to the leading edge of migrating cells. Additionally, ROR2 complexes with <i>VANGL2</i> in ovarian cancer cells, and this complex is enhanced in the presence of <i>WNT5A</i>. As <i>WNT5A</i> is produced at high levels by peritoneal mesothelial cells and omental adipose tissue in the microenvironment of ovarian cancer metastasis, disruption of the ROR2-<i>VANGL2</i> complex represents an attractive target for disrupting metastatic colonization of ovarian tumors.

LSM12 facilitates the progression of colorectal cancer by activating the WNT/CTNNB1 signaling pathway.

Aberrant activation of the WNT signaling pathway is a joint event in colorectal cancer (CRC), but the molecular mechanism is still unclear. Recently, RNA-splicing factor LSM12 (like-Sm protein 12) is highly expressed in CRC tissues. This study aimed to verify whether LSM12 is involved in regulating CRC progression via regulating the WNT signaling pathway. Here, we found that LSM12 is highly expressed in CRC patient-derived tissues and cells. LSM12 is involved in the proliferation, invasion, and apoptosis of CRC cells, similar to the function of WNT signaling in CRC. Furthermore, protein interaction simulation and biochemical experiments proved that LSM12 directly binds to CTNNB1 (also known as β-Catenin) and regulates its protein stability to affect the CTTNB1-LEF1-TCF1 transcriptional complex formation and the associated WNT downstream signaling pathway. LSM12 depletion in CRC cells decreased the in vivo tumor growth through repression of cancer cell growth and acceleration of cancer cell apoptosis. Taken together, we suggest that the high expression of LSM12 is a novel factor leading to aberrant WNT signaling activation, and that strategies targeting this molecular mechanism may contribute to developing a new therapeutic method for CRC.

Oxidative Distress Induces Wnt/β-Catenin Pathway Modulation in Colorectal Cancer Cells: Perspectives on APC Retained Functions.

Colorectal cancer (CRC) is a multistep process that arises in the colic tissue microenvironment. Oxidative stress plays a role in mediating CRC cell survival and progression, as well as promoting resistance to therapies. CRC progression is associated with Wnt/β-Catenin signaling dysregulation and loss of proper APC functions. Cancer recurrence/relapse has been attributed to altered ROS levels, produced in a cancerous microenvironment. The effect of oxidative distress on Wnt/β-Catenin signaling in the light of APC functions is unclear. This study evaluated the effect of H2O2-induced short-term oxidative stress in HCT116, SW480 and SW620 cells with different phenotypes of APC and β-Catenin. The modulation and relationship of APC with characteristic molecules of Wnt/β-Catenin were assessed in gene and protein expression. Results indicated that CRC cells, even when deprived of growth factors, under acute oxidative distress conditions by H2O2 promote β-Catenin expression and modulate cytoplasmic APC protein. Furthermore, H2O2 induces differential gene expression depending on the cellular phenotype and leading to favor both Wnt/Catenin-dependent and -independent signaling. The exact mechanism by which oxidative distress can affect Wnt signaling functions will require further investigation to reveal new scenarios for the development of therapeutic approaches for CRC, in the light of the conserved functions of APC.

Co-activation of Sonic hedgehog and Wnt signaling in murine retinal precursor cells drives ocular lesions with features of intraocular medulloepithelioma

Intraocular medulloepithelioma (IO-MEPL) is a rare embryonal ocular neoplasm, prevalently occurring in children. IO-MEPLs share histomorphological features with CNS embryonal tumors with multilayered rosettes (ETMRs), referred to as intracranial medulloepitheliomas. While Sonic hedgehog (SHH) and WNT signaling pathways are crucial for ETMR pathogenesis, the impact of these pathways on human IO-MEPL development is unclear. Gene expression analyses of human embryonal tumor samples revealed similar gene expression patterns and significant overrepresentation of SHH and WNT target genes in both IO-MEPL and ETMR. In order to unravel the function of Shh and Wnt signaling for IO-MEPL pathogenesis in vivo, both pathways were activated in retinal precursor cells in a time point specific manner. Shh and Wnt co-activation in early Sox2- or Rax-expressing precursor cells resulted in infiltrative ocular lesions that displayed extraretinal expansion. Histomorphological, immunohistochemical, and molecular features showed a strong concordance with human IO-MEPL. We demonstrate a relevant role of WNT and SHH signaling in IO-MEPL and report the first mouse model to generate tumor-like lesions with features of IO-MEPL. The presented data may be fundamental for comprehending IO-MEPL initiation and developing targeted therapeutic approaches.

Parsing β-catenin’s cell adhesion and Wnt signaling functions in malignant mammary tumor progression

During malignant progression, epithelial cancer cells dissolve their cell-cell adhesion and gain invasive features. By virtue of its dual function, β-catenin contributes to cadherin-mediated cell-cell adhesion, and it determines the transcriptional output of Wnt signaling: via its N terminus, it recruits the signaling coactivators Bcl9 and Pygopus, and via the C terminus, it interacts with the general transcriptional machinery. This duality confounds the simple loss-of-function analysis of Wnt signaling in cancer progression. In many cancer types including breast cancer, the functional contribution of β-catenin's transcriptional activities, as compared to its adhesion functions, to tumor progression has remained elusive. Employing the mouse mammary tumor virus (MMTV)-PyMT mouse model of metastatic breast cancer, we compared the complete elimination of β-catenin with the specific ablation of its signaling outputs in mammary tumor cells. Notably, the complete lack of β-catenin resulted in massive apoptosis of mammary tumor cells. In contrast, the loss of β-catenin's transcriptional activity resulted in a reduction of primary tumor growth, tumor invasion, and metastasis formation in vivo. These phenotypic changes were reflected by stalled cell cycle progression and diminished epithelial-mesenchymal transition (EMT) and cell migration of breast cancer cells in vitro. Transcriptome analysis revealed subsets of genes which were specifically regulated by β-catenin's transcriptional activities upon stimulation with Wnt3a or during TGF-β-induced EMT. Our results uncouple the signaling from the adhesion function of β-catenin and underline the importance of Wnt/β-catenin-dependent transcription in malignant tumor progression of breast cancer.

SOX17 Antagonizes the WNT Signaling Pathway and is Epigenetically Inactivated in Clear-Cell Renal Cell Carcinoma.

BackgroundSRY-box containing gene 17 (SOX17) was reported to be a candidate tumor suppressor gene in multiple tumors. Little is known about its role in clear-cell renal cell carcinoma (ccRCC). This study aims to identify the epigenetic regulation and tumor-suppressive function of SOX17 in ccRCC.Patients and MethodsFifty-five human ccRCC tissue samples, ten adjacent non-malignant kidney tissue samples, 20 paired paraffin section tissues and seven RCC cell lines were obtained. Immunohistochemistry (IHC) and real-time PCR were used to examine the expression of the target genes at the mRNA and protein levels. The methylation of SOX17 was analyzed using methylation-specific PCR (MSP) and bisulfite genomic sequencing (BGS) assay. The functions of SOX17 were examined by using CCK8, colony formation, wound healing assay and Matrigel invasion assays. Luciferase assay was used to analyze the function of SOX17 in the WNT signaling pathway.ResultsWe investigated the SOX17 expression in ccRCC tissues and adjacent non-malignant kidney tissues using PCR and IHC. The expression of SOX17 was lower in ccRCC tissues. Next, we analyzed the DNA promoter methylation of SOX17 in 55 human ccRCC tissues, 10 adjacent non-malignant kidney tissues and RCC cell lines using MSP. DNA methylation of the SOX17 promoter region occurred in 60% of ccRCC tissues and 10% of adjacent non-malignant kidney tissues. In vitro experiments showed that SOX17 suppressed the proliferation of RCC cells. Furthermore, SOX17 inhibited the migration of RCC cells as shown in the wound healing and migration assays. In addition, we found that SOX17 overexpression affected the WNT signaling pathway by downregulating c-myc and cyclinD1.ConclusionIn summary, our study showed that SOX17 is downregulated in ccRCC and the loss of SOX17 expression is regulated via epigenetic mechanisms in ccRCC. In addition, SOX17 negatively regulates the WNT signaling pathway and function as a tumor suppressor in ccRCC.

Abstract PD15-03: LGR4 engages a wnt-independent mechanism to enhance EGFR signaling and promote metastasis of triple-negative breast cancer

Abstract Leucine-rich repeat-containing G-protein-coupled receptors 4/5/6 (LGR4/5/6) have crucial functions in embryonic development, adult tissue homeostasis, and diseases. LGR4/5/6 also play important roles in cancer initiation and progression. LGR4 is highly expressed in multiple types of cancer and associated with poor patient outcome. LGR4 promotes tumorigenesis and metastasis and modulates cancer stem cells, while LGR5 and LGR6 mark cancer stem cells and progenitor cells that contribute to tumor initiation. The biological functions of LGR4/5/6 are primarily attributed to their roles in potentiating Wnt signaling, which requires the binding of their four R-spondin ligands (RSPO1-RSPO4). However, it is unclear whether these proteins have Wnt signaling-independent functions. We have previously reported that LGR4 is important in mammary gland development and breast cancer progression and metastasis. Here, we demonstrate that LGR4 enhances triple-negative breast cancer (TNBC) metastasis independently of Wnt signaling. We first found that LGR4 was highly expressed in the TNBC subtype and associated with poor metastasis-free survival in TNBC patients, while a well-validated Wnt signature failed to correlate with metastasis-free survival of this same subset of TNBC patients. These bioinformatic data suggested divergent functions of LGR4 versus Wnt signaling in TNBC progression. Then we showed that depletion of LGR4 inhibited, while inducible expression of LGR4 promoted TNBC cell migration and invasion, without added exogenous ligands and any detectable impact on baseline Wnt signaling. Importantly, blockade of Wnt signaling by Wnt inhibitors or by genetic depletion of Wntless cannot abolish the effect of LGR4 on TNBC cell metastatic ability. Furthermore, we have generated non-RSPO-binding LGR4 mutants that failed to activate Wnt signaling. These LGR4 mutants that are uncoupled from Wnt signaling can still promote TNBC cell migration and invasion in vitro and TNBC lung metastasis and bone metastasis in vivo, as potently as wild-type LGR4. To identify the molecular mechanisms by which LGR4 promotes TNBC metastasis, we did computational analyses of TCGA data to search for candidate proteins associated with LGR4 and proteomics screenings to uncover protein pathways regulated by LGR4 knockdown in TNBC cells. Both in silico and proteomic analyses identified EGFR as a downstream component of LGR4 signaling in TNBC. We confirmed LGR4 enhanced EGFR signaling by immunoblot analyses and found that EGFR was the crucial mediator of LGR4’s role in TNBC metastasis. Ectopic EGFR rescued migration and invasion of LGR4 knockout cells, while EGFR knockdown and the EGFR inhibitor erlotinib attenuated LGR4-induced cell migration and invasion. In an intra-iliac injection model of bone metastasis, erlotinib also suppressed LGR4-induced TNBC bone metastasis in vivo. Mechanistically, LGR4 did not affect EGFR mRNA levels but EGFR protein stability. LGR4 interacted with EGFR and blocked EGFR ubiquitination and degradation, resulting in persistent EGFR activation. Together, these data uncover a new signaling pathway controlled by LGRs with broad implications for cancer progression and targeted therapy. Citation Format: Fei Yue, Weiyu Jiang, Amy T Ku, Adelaide IJ Young, Weijie Zhang, Eric P Souto, Yankun Gao, Zhihan Yu, Yi Wang, Chad J Creighton, Chandandeep Nagi, Tao Wang, Xin-Hua Feng, Shixia Huang, Cristian Coarfa, Xiang H.-F. Zhang, Qingyun Liu, Xia Lin, Yi Li. LGR4 engages a wnt-independent mechanism to enhance EGFR signaling and promote metastasis of triple-negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD15-03.

Human myelomeningocele risk and ultra-rare deleterious variants in genes associated with cilium, WNT-signaling, ECM, cytoskeleton and cell migration

Myelomeningocele (MMC) affects one in 1000 newborns annually worldwide and each surviving child faces tremendous lifetime medical and caregiving burdens. Both genetic and environmental factors contribute to disease risk but the mechanism is unclear. This study examined 506 MMC subjects for ultra-rare deleterious variants (URDVs, absent in gnomAD v2.1.1 controls that have Combined Annotation Dependent Depletion score ≥ 20) in candidate genes either known to cause abnormal neural tube closure in animals or previously associated with human MMC in the current study cohort. Approximately 70% of the study subjects carried one to nine URDVs among 302 candidate genes. Half of the study subjects carried heterozygous URDVs in multiple genes involved in the structure and/or function of cilium, cytoskeleton, extracellular matrix, WNT signaling, and/or cell migration. Another 20% of the study subjects carried heterozygous URDVs in candidate genes associated with gene transcription regulation, folate metabolism, or glucose metabolism. Presence of URDVs in the candidate genes involving these biological function groups may elevate the risk of developing myelomeningocele in the study cohort.