Activation Of Canonical Wnt Pathway Research Articles

SETD3 regulates endoderm differentiation of mouse embryonic stem cells through canonical Wnt signaling pathway.

With self-renewal and pluripotency features, embryonic stem cells (ESCs) provide an invaluable tool to investigate early cell fate decisions. Pluripotency exit and lineage commitment depend on precise regulation of gene expression that requires coordination between transcription (TF) and chromatin factors in response to various signaling pathways. SET domain-containing 3 (SETD3) is a methyltransferase that can modify histones in the nucleus and actin in the cytoplasm. Through an shRNA screen, we previously identified SETD3 as an important factor in the meso/endodermal lineage commitment of mouse ESCs (mESC). In this study, we identified SETD3-dependent transcriptomic changes during endoderm differentiation of mESCs using time-course RNA-seq analysis. We found that SETD3 is involved in the timely activation of the endoderm-related gene network. The canonical Wnt signaling pathway was one of the markedly altered signaling pathways in the absence of SETD3. The assessment of Wnt transcriptional activity revealed a significant reduction in Setd3-deleted (setd3∆) mESCs coincident with a decrease in the nuclear pool of the key TF β-catenin level, though no change was observed in its mRNA or total protein level. Furthermore, a proximity ligation assay (PLA) found an interaction between SETD3 and β-catenin. We were able to rescue the differentiation defect by stably re-expressing SETD3 or activating the canonical Wnt signaling pathway by changing mESC culture conditions. Our results suggest that alterations in the canonical Wnt pathway activity and subcellular localization of β-catenin might contribute to the endoderm differentiation defect of setd3∆ mESCs.

SMAD4 and KCNQ3 alterations are associated with lymph node metastases in oesophageal adenocarcinoma

Metastasis in oesophageal adenocarcinoma (OAC) is an important predictor of survival. Radiological staging is used to stage metastases in patients, and guide treatment selection, but is limited by the accuracy of the approach. Improvements in staging will lead to improved clinical decision making and patient outcomes. Sequencing studies on primary tumours and pre-cancerous tissue have revealed the mutational landscape of OAC, and increasingly cheap and widespread sequencing approaches offer the potential to improve staging assessment. In this work we present an analysis of lymph node metastases found by radiological and pathological sampling, identifying new roles of the genes SMAD4 and KCNQ3 in metastasis. Through transcriptomic analysis we find that both genes are associated with canonical Wnt pathway activity, but KCNQ3 is uniquely associated with changes in planar cell polaritiy associated with non-canonical Wnt signalling. We go on to validate our observations in KCNQ3 in cell line and xenograph systems, showing that overexpression of KCNQ3 reduces wound closure and the number of metastases observed. Our results suggest both genes as novel biomarkers of metastatic risk and offer new potential routes to drug targeting.

AXIN1 bi-allelic variants disrupting the C-terminal DIX domain cause craniometadiaphyseal osteosclerosis with hip dysplasia

Sclerosing skeletal dysplasias result from an imbalance between bone formation and resorption. We identified three homozygous, C-terminally truncating AXIN1 variants in seven individuals from four families affected by macrocephaly, cranial hyperostosis, and vertebral endplate sclerosis. Other frequent findings included hip dysplasia, heart malformations, variable developmental delay, and hematological anomalies. In line with AXIN1 being a central component of the β-catenin destruction complex, analyses of primary and genome-edited cells harboring the truncating variants revealed enhanced basal canonical Wnt pathway activity. All three AXIN1-truncating variants resulted in reduced protein levels and impaired AXIN1 polymerization mediated by its C-terminal DIX domain but partially retained Wnt-inhibitory function upon overexpression. Addition of a tankyrase inhibitor attenuated Wnt overactivity in the AXIN1-mutant model systems. Our data suggest that AXIN1 coordinates the action of osteoblasts and osteoclasts and that tankyrase inhibitors can attenuate the effects of AXIN1 hypomorphic variants.

MiR-455-3p inhibits gastric cancer progression by repressing Wnt/β-catenin signaling through binding to ARMC8

BackgroundGlobally, gastric cancer (GC) is one of the world’s most widespread malignancies, with persistent high mortality and morbidity rates. Increasing evidence now suggests that microRNAs (miRNAs) participate in many biological processes, with miR-455-3p having key roles in the progression of diverse cancers. Nevertheless, miR-455-3p function and expression in GC remain unclear.MethodsWe explored miR-455-3p expression in GC using quantitative polymerase chain reaction (qPCR). To further examine the effect of miR-455-3p in GC, after transfecting miR-455-3p mimics or inhibitors into GC cells, 5-ethynyl-2’-deoxyuridine (EdU) incorporation and colony formation assays were performed to examine cell proliferation. Flow cytometry was used to detect apoptosis, and expression levels of Bax, Bcl-2, Snail, N-cadherin, E-cadherin, and Caspase-3 were assessed by western blotting (WB). Using online databases and luciferase assays, we identified armadillo repeat-containing protein 8 (ARMC8) as a promising target of miR-455-3p. A mouse tumor model was established to investigate actions of miR-455-3p in vivo. Expression levels of C-myc, cyclinD1, and β-catenin were examined using WB and immunofluorescence.ResultsMiR-455-3p expression was attenuated in GC tissue and cell lines. MiR-455-3p overexpression inhibited GC cell proliferation, epithelial-mesenchymal transition (EMT), as well as facilitated apoptosis, while suppression of miR-455-3p had the opposite effects. From luciferase assays, we confirmed that ARMC8 was a novel and direct downstream target gene of miR-455-3p, and that the tumor suppressive role of miR-455-3p was in part reversed due to ARMC8 overexpression. Moreover, miR-455-3p inhibited GC growth in vivo via ARMC8. We also observed that miR-455-3p repressed canonical Wnt pathway activation by binding to ARMC8.ConclusionsMiR-455-3p exerted tumor inhibitory effects in GC by targeting ARMC8. Therefore, intervening in the miR-455-3p/ARMC8/Wnt/βcatenin axis could be a promising novel treatment strategy for GC.

RNA-binding Protein QKI Inhibits Osteogenic Differentiation Via Suppressing Wnt Pathway

Dysregulation of MSCs differentiation is associated with many pathophysiological processes. Genetically modified MSCs transplantation helps restore bone loss efficiently. BMSCs-specific QKI overexpressing and knockdown mice were built to explore QKI's role in bone formation and fat accumulation. Primary BMSCs with QKI overexpression and knockout were subjected to osteogenic and adipogenic differentiation. ALP staining and oil red O staining were performed to evaluate the differences between the groups. RNA immunoprecipitation was performed to identify the QKI-related pathway. QKI deficient BMSCs were transplanted into mice with glucocorticoid-induced osteoporosis to evaluate its therapeutic potential. Mice harboring BMSC-specific transgenic QKI exhibited reduced bone mass, while BMSC-specific QKI-deficient mice showed an increase in bone mass. Osteogenic differentiation of QKI deficient BMSCs was promoted and adipogenic differentiation was inhibited, while QKI overexpression in BMSCs displayed the opposite effects. To define the underlying mechanisms, RIP sequencing was performed. Wnt pathway-related genes were the putative direct target mRNAs of QKI, Canonical Wnt pathway activation was involved in QKI's effects on osteogenic differentiation. RNA immunoprecipitation quantitative real-time Polymerase Chain Reaction (PCR) and RNA fluorescence in situ hybridization experiments further validated that QKI repressed the expressions of Wnt5b, Fzd7, Dvl3 and β-catenin via direct binding to their putative mRNA specific sites. Glucocorticoid-induced osteoporotic mice transplanted with QKI deficient BMSCs exhibited less bone loss compared with mice transplanted with control BMSCs. QKI suppressed BMSCs osteogenic differentiation by downregulating the expressions of Wnt5b, Fzd7, Dvl3 and β-catenin. Loss of QKI in BMSCs transplantation may provide a new strategy for the treatment of orthopedic diseases such as osteoporosis.

Arjunolic acid modulate pancreatic dysfunction by ameliorating pattern recognition receptor and canonical Wnt pathway activation in type 2 diabetic rats

BackgroundArjunolic acid (AA) is a potent phytochemical with multiple therapeutics effects. In this study, AA is evaluated on type 2 diabetic (T2DM) rats to understand the mechanism of β-cell linkage with Toll-like receptor 4 (TLR-4) and canonical Wnt signaling. However, its role in modulating TLR-4 and canonical Wnt/β-catenin crosstalk on insulin signaling remains unclear during T2DM.AimThe current study is aimed to examine the potential role of AA on insulin signaling and TLR-4-Wnt crosstalk in the pancreas of type 2 diabetic rats. MethodMultiple methods were used to determine molecular cognizance of AA in T2DM rats, when treated with different dosage levels. Histopathological and histomorphometry analysis was conducted using masson trichrome and H&E stains. While, protein and mRNA expressions of TLR-4/Wnt and insulin signaling were assessed using automated Western blotting (jess), immunohistochemistry, and RT-PCR. ResultsHistopathological findings revealed that AA had reversed back the T2DM-induced apoptosis and necrosis caused to rats pancreas. Molecular findings exhibited prominent effects of AA in downregulating the elevated level of TLR-4, MyD88, NF-κB, p-JNK, and Wnt/β-catenin by blocking TLR-4/MyD88 and canonical Wnt signaling in diabetic pancreas, while IRS-1, PI3K, and pAkt were all upregulated by altering the NF-κB and β-catenin crosstalk during T2DM. ConclusionOverall results, indicate that AA has potential to develop as an effective therapeutic in the treatment of T2DM associated meta-inflammation. However, future preclinical research at multiple dose level in a long-term chronic T2DM disease model is warranted to understand its clinical relevance in cardiometabolic disease.

TM9SF4 is a novel regulator in lineage commitment of bone marrow mesenchymal stem cells to either osteoblasts or adipocytes

BackgroundOsteoporosis is a common bone disease in elderly population caused by imbalanced bone formation and bone resorption. Mesenchymal stem cells (MSCs) are responsible for maintaining this bone homeostasis. The phenotype of transmembrane 9 superfamily 4 (TM9SF4) knockout mice suggests a relationship between TM9SF4 proteins and bone homeostasis. But the effect of TM9SF4 in osteology has never been reported. In the present study, we investigated the function of TM9SF4 in MSC differentiation commitment, as well as its role in osteoporosis.MethodsPrimary bone marrow MSCs, isolated from TM9SF4 wildtype (TM9SF4+/+) and knockout (TM9SF4−/−) mice, were induced to differentiate into osteoblasts or adipocytes, respectively. The osteogenesis was examined by qRT-PCR detection of osteogenic markers, ALP staining and Alizarin Red S staining. The adipogenesis was tested by qRT-PCR quantification of adipogenic markers and Oil Red O staining. The cytoskeletal organization of MSCs was observed under confocal microscope. The osteoporotic model was induced by ovariectomy in TM9SF4+/+ and TM9SF4−/− mice, followed by Toluidine blue and H&E staining to assess lipid accumulation in trabecular bones, as well as micro-computed tomography scanning and immunohistochemistry staining for bone mass density assessment. The experiments on signaling pathways were conducted using qRT-PCR, Western blot and Alizarin Red S staining.ResultsWe determined the role of TM9SF4 in MSC differentiation and found that TM9SF4−/− MSCs had higher potential to differentiate into osteoblasts and lower capability into adipocytes, without affecting osteoclastogenesis in vitro. In ovariectomy-induced osteoporotic model, TM9SF4−/− mice retained higher bone mass and less lipid accumulation in trabecular bones, indicating an important role of TM9SF4 in the regulation of osteoporosis. Mechanistically, TM9SF4-depleted cells showed elongated actin fibers, which may act through mTORC2/Akt/β-catenin pathway to promote their commitment into osteoblasts. Furthermore, TM9SF4-depleted cells showed higher activity of canonical Wnt pathway, suggesting the participation of Wnt/β-catenin during TM9SF4-regulated osteogenesis.ConclusionsOur study demonstrates TM9SF4 as a novel regulator for MSC lineage commitment. Depletion of TM9SF4 preferentially drives MSCs into osteoblasts instead of adipocytes. Furthermore, TM9SF4−/− mice show delayed bone loss and reduced lipid accumulation during ovariectomy-induced osteoporosis. Our results indicate TM9SF4 as a promising target for the future clinical osteoporotic treatment.

Investigation of the Epithelial to Mesenchymal Transition (EMT) Process in Equine Papillomavirus-2 (EcPV-2)-Positive Penile Squamous Cell Carcinomas.

Equine penile squamous cell carcinoma (epSCC) is the most frequent tumor of the external male genitalia, representing 67.5% of equine genital cancers. epSCC is associated with papilloma virus (PV) infection and has been recently proposed as a model for human PV-induced squamous cell carcinomas. It has already been suggested that epSCC might undergo epithelial-to-mesenchymal transition (EMT). This work aims to investigate in detail this process and the possible role of PV oncoproteins in epSCC. For this purpose, 18 penile SCCs were retrospectively selected and tested for both EcPV2 presence and oncoproteins (EcPV2 E6 and EcPV2 E7) expression. Moreover, immunohistochemical EMT characterization was carried out by analyzing the main epithelial markers (E-cadherin, β-catenin, and pan-cytokeratin AE3/AE1), the main mesenchymal markers (N-cadherin and vimentin), and the main EMT-related transcription factors (TWIST-1, ZEB-1). PCR analysis was positive for EcPV2 in 16 out of 18 samples. EMT was investigated in epSCC positive for EcPV2. The immunohistochemistry results suggested the presence of EMT processes in the neoplastic cells at the tumor invasive front. Moreover, the significant upregulation of RANKL, together with BCATN1, LEF1, and FOSL1 genes, might suggest a canonical Wnt pathway activation, similarly to what is reported in human penile squamous cell carcinomas

Wnt3a and ASCs are capable of restoring mineralization in staph aureus-infected primary murine osteoblasts.

Bone infections are one of the main reasons for impaired bone regeneration and non-union formation. In previous experimental animal studies we could already demonstrate that bone defects due to prior infections showed a markedly reduced healing capacity, which could effectively be enhanced via application of Wnt3a and Adipose-derived stromal cells (ASCs). For a more in-depth analysis, we investigated proliferation and mineralization of cultured osteoblasts infected with staph aureus and sought to investigate effects of Wnt3a and ASCs on infected osteoblasts. Primary murine osteoblasts were isolated from calvariae and infected with staph aureus. Infected osteoblasts received treatment via application of recombinant Wnt3a, ASC conditioned medium and were furthermore cocultured with ASCs. Osteoblasts were evaluated by Alamar blue assay for metabolic activity, TUNEL-assay for apoptosis, ALP and Alizarin Red staining for mineralization. In addition, immunoflourescent staining (IF) and qRT-PCR analyses were performed. Infected osteoblasts showed a markedly reduced ability for mineralization and increased apoptosis, which could be restored to physiological levels by Wnt3a and ASC treatment. Interestingly, metabolic activity of osteoblasts seemed to be unaffected by staph aureus infection. Additional analyses of Wnt-pathway activity revealed effective enhancement of canonical Wnt-pathway activity in Wnt3a-treated osteoblasts. In summary, we gained further osteoblast-related insights into pathomechanisms of reduced bone healing capacity upon infections.

Expression of LGR5 in mammary myoepithelial cells and in triple-negative breast cancers

Lineage tracing in mice indicates that LGR5 is an adult stem cell marker in multiple organs, such as the intestine, stomach, hair follicles, ovary, and mammary glands. Despite many studies exploring the presence of LGR5 cells in human tissues, little is known about its expression profile in either human mammary tissue or pathological lesions. In this study we aim to investigate LGR5 expression in normal, benign, and malignant lesions of the human breast using RNA in situ hybridization. LGR5 expression has not been observed in normal lactiferous ducts and terminal duct lobular units, whereas LGR5-positive cells have been specifically observed in the basal myoepithelium of ducts in the regenerative tissues, ductal carcinoma in situ, and in ducts surrounded by invasive cancer cells. These findings suggest LGR5 marks facultative stem cells that are involved in post injury regeneration instead of homeostatic stem cells. LGR5 positivity was found in 3% (9 of 278 cases) of invasive breast cancers (BC), and it showed positive associations with higher histologic grades (P = 0.001) and T stages (P < 0.001), while having negative correlations with estrogen receptor (P < 0.001) and progesterone receptor (P < 0.001) expression. Remarkably, all LGR5-positive BC, except one, belong to triple-negative BC (TNBC), representing 24% (9 of 38 cases) of all of them. LGR5 histoscores have no correlations with EGFR, CK5/6, Ki-67, or P53 expression. Additionally, no β-catenin nuclear localization was observed in LGR5-positive BC, indicating that canonical Wnt pathway activation is less likely involved in LGR5 expression in BC. Our results demonstrate that LGR5 expression is induced in regenerative conditions in the myoepithelium of human mammary ducts and that its expression is only observed in TNBC subtype among all invasive BC. Further studies regarding the functional and prognostic impact of LGR5 in TNBC are warranted.

FSH stimulated bovine granulosa cell steroidogenesis involves both canonical and noncanonical WNT signaling

Gonadotrophins play key roles in follicular development; however, the underlying molecular mechanisms are not fully understood. Follicle stimulating hormone (FSH) regulation of aromatase and subsequent estradiol (E2) production relies on β-catenin, a key effector of WNT signaling. We previously demonstrated that treatment with the canonical WNT inhibitor, IWR-1, reduced FSH induced bovine granulosa cell E2 production in vitro. Here we demonstrated that intrafollicular injection in vivo with IWR-1 alters steroidogenesis and triggers a significant decrease in estrogen to progesterone ratio in the IWR-1 treated follicles compared to diluent injected control follicles. We next examined markers of canonical and noncanonical WNT signaling in dominant and subordinate follicles collected at different stages of follicular development and showed that protein for both CTNNB1 (canonical pathway) and phosphorylated (p)-LEF1 (noncanonical pathway) was significantly elevated in dominant compared to subordinate follicles at the early dominance stage of development. Therefore, we hypothesized that canonical and/or noncanonical WNT ligands modulate FSH stimulated E2 production. Hence, we examined the effects of specific WNT ligands on FSH stimulated E2 production in the absence of endogenous WNT production in vitro. Universal WNT signaling inhibitor, LGK-974 was able to inhibit FSH stimulation of E2 and reduce the abundance of proteins linked to canonical and noncanonical WNT pathway activation. Supplementation with the canonical ligand WNT2b did not affect the inhibitory effects of LGK-974 on FSH stimulated E2 production but rescued the LGK-974 mediated inhibition of CTNNB1 (canonical pathway) but not p-LEF1, p-JNK or p-P38 abundance (noncanonical pathway) abundance. In contrast, WNT5a treatment rescued FSH stimulated estradiol production and indices of activation of both the canonical (CTNNB1) and noncanonical (p-LEF1, p-JNK and p-P38) WNT signaling pathways in LGK-974 treated granulosa cells. Taken together, these results suggest that both canonical and noncanonical WNT pathways activation is linked to FSH stimulation of E2 production by bovine granulosa cells.

Odontoblast differentiation is regulated by an interplay between primary cilia and the canonical Wnt pathway.

Primary cilium is a protruding cellular organelle that has various physiological functions, especially in sensory reception. While an avalanche of reports on primary cilia have been published, the function of primary cilia in dental cells remains to be investigated. In this study, we focused on the function of primary cilia in dentin-producing odontoblasts. Odontoblasts, like most other cell types, possess primary cilia, which disappear upon the knockdown of intraflagellar transport protein 88. In cilia-depleted cells, the expression of dentin sialoprotein, an odontoblastic marker, was elevated, while the deposition of minerals was slowed. This was recapitulated by the activation of canonical Wnt pathway, also decreased the ratio of ciliated cells. In dental pulp cells, as they differentiated into odontoblasts, the ratio of ciliated cells was increased, whereas the canonical Wnt signaling activity was repressed. Our results collectively underscore the roles of primary cilia in regulating odontoblastic differentiation through canonical Wnt signaling. This study implies the existence of a feedback loop between primary cilia and the canonical Wnt pathway.

Dickkopf-related protein-1 inhibition attenuates amyloid-beta pathology associated to Alzheimer's disease

Alzheimer's disease (AD) constitutes the leading cause of dementia worldwide. It is associated to amyloid-β (Aβ) aggregation and tau hyper-phosphorylation, accompanied by a progressive cognitive decline. Evidence suggests that the canonical Wnt pathway is deregulated in AD. Pathway activity is mediated by β-catenin stabilization in the cytosol, and subsequent translocation to the nucleus to regulate the expression of several genes implicated in brain homeostasis and functioning. It was recently proposed that Dickkopf-related protein-1 (DKK1), an endogenous antagonist of the pathway, might be implicated in AD pathogenesis. Here, we hypothesized that canonical Wnt pathway deactivation associated to DKK1 induction contributes to late-onset AD pathogenesis, and thus DKK1 neutralization could attenuate AD pathology. For this purpose, human post-mortem AD brain samples were used to assess pathway activity, and aged APPswe/PS1 mice were used to investigate DKK1 in late-onset AD-like pathology and therapy. Our findings indicate that β-catenin levels progressively decrease in the brain of AD patients, correlating with the duration of symptoms. Next, we found that Aβ pathology in APPswe/PS1 mediates DKK1 induction in the brain. Pharmacological neutralization of DKK1's biological activity in APPswe/PS1 mice restores pathway activity by stabilizing β-catenin, attenuates Aβ pathology, and ameliorates the memory of mice. Attenuation of AD-like pathology upon DKK1 inhibition is accompanied by a reduced protein expression of beta-site amyloid precursor protein (APP) cleaving enzyme-1 (BACE1). Moreover, DKK1 inhibition enhances vascular density, promotes blood-brain barrier (BBB) integrity by increasing claudin 5, glucose transporter-1 (GLUT1), and ATP-binding cassette sub-family B member-1 (ABCB1) protein expression, as well as ameliorates synaptic plasticity by increasing brain-derived neurotrophic factor (BDNF), and postsynaptic density protein-95 (PSD-95) protein expression. DKK1 conditional induction reduces claudin 5, abcb1, and psd-95 mRNA expression, validating its inhibition effects. Our results indicate that neutralization of DKK1's biological activity attenuates AD-like pathology by restoring canonical Wnt pathway activity.

Abstract 4917: Estrogen receptor D538G mutation promotes cell migration via hyperactivation of Wnt signaling pathway

Abstract Background: Hotspot estrogen receptor-α (ERα/ESR1) mutations occur in 30-40% endocrine resistant ER+ breast cancer, with Y537S and D538G as the two most frequent hotspot mutations. A mostly unanswered question is and how these mutations facilitate metastatic processes. Methods: Frequencies of different ESR1 hotspot mutations were compared within three publicly available cohorts. Y537S and D538G genome-edited MCF7 and T47D cell models were used for in vitro characterization. Wound scratching, spheroid collective migration, chemotaxis assays were applied to examine different metastatic properties. Tail vein injection in nude mice followed by human CK19 lung section immune staining was used to characterize in vivo metastasis. Canonical Wnt pathway activity was studied using top-flash reporter assays, immunoblotting, and overexpression of dominant negative TCF4. ChIP-seq and ATAC-seq were utilized to profile ER global binding patterns and chromatin accessibility in cell models, respectively. Porcupine inhibitor-LGK974 was used to evaluate the efficiency of Wnt-targeted therapy. Results: D538G mutations were more frequent in ER+ metastatic lesions compared to Y537S among three metastatic breast cancer cohorts with unbiased SNV detection. In line with this, T47D-D538G ESR1 mutant cells showed strongly enhanced cell migration in vitro which was not observed in Y537S cells. We also observed increased lung micro-metastasis in vivo after tail vein injection of the D538G cells. Cell line transcriptomic analysis revealed uniquely hyperactivated Wnt pathway in D538G mutant cells, which was further confirmed in vitro by top-flash reporter and immunoblot. Suppression of canonical Wnt pathways blocked T47D-D538G specific cell migration. Combination treatment of fulvestrant and LGK974 synergistically inhibited D538G specific migration. Mechanistically, multiple Wnt regulator genes were found uniquely upregulated in D538G cells. Interestingly, none of these targets gained ER binding peaks at proximal regulatory regions, suggesting potential epigenetic regulation, which was confirmed in ATAC-seq results. Knockdown of FOXA1, a well-characterized pioneer factor, decreased canonical Wnt activity and abrogated the D538G-unique cell migration in short-term. Conclusion: T47D-D538G cells showed uniquely enhanced cell migration via Wnt hyperactivation, potentially mediated via epigenetic remodeling. These findings suggest the further study of potential combinatorial targeting of Wnt and ER signaling in D538G ESR1 mutant tumors. Citation Format: Zheqi Li, Kevin M. Levine, Spencer Arneson, Kristofer C. Berrett, Nolan M. Priedigkeit, Amir Bahreini, Jian Chen, Li Zhu, Jason S. Carroll, George C. Tseng, Peter C. Lucas, Jennifer M. Atkinson, Jason Gertz, Adrian V. Lee, Steffi Oesterreich. Estrogen receptor D538G mutation promotes cell migration via hyperactivation of Wnt signaling pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4917.

Porcupine and CBP/β-catenin are the most suitable targets for the inhibition of canonical Wnt signaling in colorectal carcinoma cell lines*

Aim: The activation of canonical Wnt pathway is etiologically associated with the development of colorectal cancers. There are many possible molecular targets for the therapeutic abrogation of Wnt/β-catenin signaling. The aim of this study was to select the best molecular targets for the attenuation of β-catenin-dependent gene expression in colorectal cancer cell lines. Material/Methods: An siRNA screen was used for the selection of the best molecular targets for the down-regulation of TCF/LEF-dependent GFP expression in HCT116 cells. The level of the expression of β-catenin target genes was analyzed by qPCR. The effect of the tested chemicals on cell migration, cell cycle and apoptosis was assessed by the wound healing assay, flow cytometric analysis of propidium iodide stained cells and flow cytometric analysis of the activity of caspases-3/7, respectively. Results: Of the forty three genes which were tested in the screening stage, eight (KDM6A, KDM1A, PORCN, KDM4C, CARM1, DVL1, CBP, KMT2A) were selected as most promising. Small molecule inhibitors of these proteins (GSK-J4, GSK-LSD1, IWP-2, ML324, MS049, Dvl-PDZ Domain Inhibitor II, PRI-724, MM-102) were further used. The inhibitors of Porcupine (IWP-2) and CBP (PRI-724) were most effective in the down-regulation of the expression of β-catenin target genes and the induction of apoptosis in HCT116 cells, but showed weaker effects in DLD-1 cells. Conclusions: The inhibition of CBP and Porcupine is the most effective in attenuating canonical Wnt signaling in colon cancer cells. Future studies should determine which factors affect the sensitivity towards these promising anti-cancer agents.

A transcriptional and post-transcriptional dysregulation of Dishevelled 1 and 2 underlies the Wnt signaling impairment in type I Gaucher disease experimental models.

Bone differentiation defects have been recently tied to Wnt signaling alterations occurring in vitro and in vivo Gaucher disease (GD) models. In this work, we provide evidence that the Wnt signaling multi-domain intracellular transducers Dishevelled 1 and 2 (DVL1 and DVL2) may be potential upstream targets of impaired beta glucosidase (GBA1) activity by showing their misexpression in different type 1 GD in vitro models. We also show that in Gba mutant fish a miR-221 upregulation is associated with reduced dvl2 expression levels and that in type I Gaucher patients single-nucleotide variants in the DVL2 3' untranslated region are related to variable canonical Wnt pathway activity. Thus, we strengthen the recently outlined relation between bone differentiation defects and Wnt/β-catenin dysregulation in type I GD and further propose novel mechanistic insights of the Wnt pathway impairment caused by glucocerebrosidase loss of function.

Abstract LB-094: Systemic RSPO1 delivery leads to regression of intestinal adenomas via activation of the TGFβ/SMAD pathway

Abstract Colorectal cancers (CRCs) most commonly develop from adenomas harboring a mutant APC gene that leads to aberrant activation of the canonical Wnt/ß-catenin pathway. One of the mechanisms of adenoma progression involves the PROX1 transcription factor that promotes tumor dysplasia and progression in CRC. In the normal intestinal epithelial cells, ligands of the R-Spondin (RSPO) family are able to enhance the canonical Wnt pathway activity by binding to receptor proteins of the LGR family, such as LGR5. Tumorigenic RSPO gene fusions have recently been discovered in CRCs, but the role of RSPOs in cancer is incompletely known. We previously reported that TGFß induced apoptosis of APC-mutant organoids, including the LGR5+ stem cells, was mediated by the proapoptotic protein BIM, whereas wild-type intestinal crypts were markedly less sensitive to TGFß. The KRAS oncogene increased apoptosis resistance in the adenomas via activation of the Erk1/2 kinase pathway, which led to Bim down-regulation and increased resistance to TGFß. A subsequent study showed that RSPO/LGR5 can inhibit the in vitro growth of cultured human CRC cell line by activating the TGFß/SMAD signaling pathway. Because we were interested in analyzing the effect of exogenous Wnt signals on PROX1 expression in intestinal tumorigenesis, we constructed AAV-vectors for systemic expression of a soluble RSPO1 protein in ApcMin/+ mice. We found that the RSPO1-Fc fusion protein suppresses the Wnt/ß-catenin signaling activity in intestinal adenomas and in adenoma-derived intestinal organoids ex vivo, but not in normal intestinal epithelial cells. In the Apc mutant cells, the RSPO1-Fc fusion protein activated the TGFß/SMAD signaling pathway to suppress several Wnt target genes and adenoma growth, which effect was rescued suppressed by the TGFß receptor kinase inhibitor SB-431542. Simultaneously, RSPO1-Fc induced proliferation of the normal intestinal stem cells, giving them a growth advantage over the mutant cells, which enabled the intestinal epithelium to eventually outgrow the adenoma cells. Prolonged systemic expression of AAV-RSPO1-Fc decreased significantly the number of the intestinal adenomas and improved the overall survival of ApcMin/+ mice. Thus RSPO1-Fc provides the normal intestinal epithelial cells a growth advantage when compared to the adenoma cells, which eventually leads to the extrusion of the adenomatous tissue. An attractive idea now is to exploit such differential response of normal vs. cancer cells in cancer therapy. Citation Format: Marianne Lähde, Sarika Heino, Jenny Högström, Veli-Matti Leppänen, Seppo Kaijalainen, Olli Ritvos, Owen Sansom, Kari Alitalo. Systemic RSPO1 delivery leads to regression of intestinal adenomas via activation of the TGFβ/SMAD pathway [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 LB-094.

Wnt Signaling Upregulates Teneurin-3 Expression via Canonical and Non-canonical Wnt Pathway Crosstalk.

Teneurins (Tens) are a highly conserved family of proteins necessary for cell-cell adhesion. Tens can be cleaved, and some of their proteolytic products, such as the teneurin c-terminal associated-peptide (TCAP) and the intracellular domain (ICD), have been demonstrated to be biologically active. Although Tens are considered critical for central nervous system development, they have also been demonstrated to play important roles in adult tissues, suggesting a potential link between their deregulation and various pathological processes, including neurodegeneration and cancer. However, knowledge regarding how Ten expression is modulated is almost absent. Relevantly, the functions of Tens resemble several of the effects of canonical and non-canonical Wnt pathway activation, including the effects of the Wnt pathways on neuronal development and function as well as their pivotal roles during carcinogenesis. Accordingly, in this initial study, we decided to evaluate whether Wnt signaling can modulate the expression of Tens. Remarkably, in the present work, we used a specific inhibitor of porcupine, the key enzyme for Wnt ligand secretion, to not only demonstrate the involvement of Wnt signaling in regulating Ten-3 expression for the first time but also reveal that Wnt3a, a canonical Wnt ligand, increases the expression of Ten-3 through a mechanism dependent on the secretion and activity of the non-canonical ligand Wnt5a. Although our work raises several new questions, our findings seem to demonstrate the upregulation of Ten-3 by Wnt signaling and also suggest that Ten-3 modulation is possible because of crosstalk between the canonical and non-canonical Wnt pathways.

GPNMB augments Wnt-1 mediated breast tumor initiation and growth by enhancing PI3K/AKT/mTOR pathway signaling and β-catenin activity.

Glycoprotein Nmb (GPNMB) is overexpressed in triple-negative and basal-like breast cancers and its expression is predictive of poor prognosis within this aggressive breast cancer subtype. GPNMB promotes breast cancer growth, invasion, and metastasis; however, its role in mammary tumor initiation remains unknown. To address this question, we overexpressed GPNMB in the mammary epithelium to generate MMTV/GPNMB transgenic mice and crossed these animals to the MMTV/Wnt-1 mouse model, which is known to recapitulate features of human basal breast cancers. We show that GPNMB alone does not display oncogenic properties; however, its expression dramatically accelerates tumor onset in MMTV/Wnt-1 mice. MMTV/Wnt-1 × MMTV/GPNMB bigenic mice also exhibit a significant increase in the growth rate of established primary tumors, which is attributable to increased proliferation and decreased apoptosis. To elucidate molecular mechanisms underpinning the tumor-promoting effects of GPNMB in this context, we interrogated activated pathways in tumors derived from the MMTV/Wnt-1 and MMTV/Wnt-1 × MMTV/GPNMB mice using RPPA analysis. These data revealed that MMTV/Wnt-1 × MMTV/GPNMB bigenic tumors exhibit a pro-growth signature characterized by elevated PI3K/AKT/mTOR signaling and increased β-catenin activity. Furthermore, we extended these observations to an independent Wnt-1 expressing model of aggressive breast cancer, and confirmed that GPNMB enhances canonical Wnt pathway activation, as evidenced by increased β-catenin transcriptional activity, in breast cancer cells and tumors co-expressing Wnt-1 and GPNMB. GPNMB-dependent engagement of β-catenin occurred, in part, through AKT activation. Taken together, these data ascribe a novel, pro-growth role for GPNMB in Wnt-1 expressing basal breast cancers.

Identification of TIM-3/Gal-9 Autocrine Loop As a Novel Wnt-Ligands Independent Machinery for the Constitutive Activation of Canonical Wnt Pathway in AML-LSCs

We originally identified T-cell immunoglobulin mucin-3 (TIM-3) as a leukemic stem cells (LSCs)-specific surface molecule and a useful marker for discriminating LSCs from hematopoietic stem cells (HSCs). Furthermore, we recently identified an unique autocrine loop composed of TIM-3 and its ligand galectin-9 (Gal-9). This TIM-3/Gal-9 autocrine loop enhances self-renewal capacity of AML-LSCs and contributes to leukemia progression(Kikushige et al., Cell Stem Cell 2015).To clarify the molecular mechanism how TIM-3 signaling enhances stem cell properties of AML-LSCs, we performed shRNA-mediated knock-down(KD) of TIM-3 in KASUMI-3 (TIM-3+ AML cell line). As a result, TIM-3-KD significantly reduce the proliferation of KASUMI-3. To investigate how TIM-3-KD attenuated leukemia propagation, we compared gene expression profile of scramble-infected control and TIM-3-KD KASUMI-3 cells. Gene Set Enrichment Analysis (GSEA) revealed TIM-3-KD resulted in the significaltly attenuated expression of HSCs and LSCs-related genes (Eppert et al., Nature Med 2011) and canonical Wnt pathway-related genes (BioCarta). Since canonical Wnt pathway is known to regulate stem cell properties via inducing the nucleus accumulation ofβ-catenin in many types of stem cells including HSCs, we next examined the β-catenin level in TIM-3-KD-KASUMI-3 cells. Array scan analysis revealed that KD of TIM-3 significantly impaired the the nucleus accumulation of β-catenin in KASUMI-3 cells. In various types of cancers, the constitutive activation of β-catenin is one of the most common oncogenic signatures, and it is mainly driven by somatic mutations in the genes related to the canonical Wnt pathway. However, such mutations are rarely detected in AML.We, therefore, hypothesized that TIM-3/Gal-9 autocrine loop signaling might be a novel mutation-independent machinery for the constitutive activation of canonical Wnt pathway in primary AML.To test this hypothethis, we first evaluated the phosphorylation status of LDL receptor-related protein 6 (LRP6), because LRP6 phosphorylation is a crucial step for canonical Wnt pathway activation. Surprisingly, Gal-9 stimulation induced phosphorylation of LRP6 and subsequent β-catenin accumulation in primary TIM-3+ AML cells even in the absence of Wnt ligand. On the other hand, LRP6 phosphorylation induced by Gal-9 stimulation was canceled by the blocking antibody to TIM-3 (F38-2E2). These results indicate that Gal-9 stimulation should induce canonical Wnt pathway activation via TIM-3 dependent manner in primary AML cells. Since Gal-9 ligation to TIM-3 is known to induce phosphorylation of tyrosine residues of TIM-3 cytoplasmic tail, leading to the recruitment and activation of several Src family kinases (SFKs) for downstream signal transduction. We found that TIM-3/Gal-9 interaction recruited hematopoietic cell kinase (HCK), a member of SFKs, to TIM-3 and activated HCK by phosphorylation of the SH1 domain (Y410 in HCK) in TIM-3+ AML cells. Furthermore, we confirmed a specific HCK inhibitor A-419259 efficiently blocked the LRP6 phosphorylation and subsequent β-catenin accumulation induced by TIM-3/Gal-9 interaction in primary AML cells. These results suggest that the canonical Wnt pathway activation induced by Gal-9 ligation to TIM-3 is mainly mediated by HCK in primary AML cells.We next focused on p120-catenin as a bridging molecule between HCK activation and LRP6 phosphorylation because, as previously reported, this molecule can be directly activated by SFKs and the activation of this molecule is an initial step for canonical Wnt pathway activation preceding LRP6 phosphorylation. We found that Gal-9 ligation to TIM-3 immediately induced the direct association of HCK and p120-catenin, leading to the activation of p120-catenin in TIM-3+ AML cells. We also confirmed that specific inhibition of HCK completely abrogated the activation of p120-catenin and subsequent canonical Wnt pathway activation in primary TIM-3+ AML cells, indicating that the activation of p120-catenin mediated by HCK is a bridging moleculer machinery for connecting TIM-3 signaling and previously identified canonical Wnt pathway. Since TIM-3 as well as HCK is specifically expressed in LSCs but not in HSCs, we conclude that TIM-3/Gal-9 autocrine loop is a novel Wnt-ligands independent machinery for the constitutive activation of canonical Wnt pathway specific for AML-LSCs. (summarized in figure) [Display omitted] DisclosuresAkashi:Celgene: Research Funding, Speakers Bureau; Pfizer: Research Funding; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Kyowa Hakko Kirin: Research Funding, Speakers Bureau; Taiho Pharmaceutical: Research Funding; MSD: Research Funding; Asahi-kasei: Research Funding; Chugai Pharma: Research Funding; Ono Pharmaceutical: Research Funding; Otsuka Pharmaceutical: Research Funding; Eli Lilly Japan: Research Funding; Novartis pharma: Research Funding; Eisai: Research Funding; sanofi: Research Funding; Astellas Pharma: Research Funding.