Canonical Wingless-type MMTV Integration Site Research Articles

Signal transduction pathway activity in high-grade serous carcinoma, its precursors and Fallopian tube epithelium

ObjectiveTo determine the activity of key signal transduction pathways in serous tubal intraepithelial carcinoma (STIC) and concurrent high-grade serous carcinoma (HGSC) and compare this to pathway activity in normal Fallopian tube epithelium (FTE). MethodsWe assessed mRNA expression levels of pathway-specific target genes with RT-qPCR in STIC and concurrent HGSC (n = 8) and normal FTE (n = 8). Subsequently, signal transduction pathway assays were used to assess functional activity of the androgen (AR) and estrogen receptor (ER), phosphoinositide-3-kinase (PI3K), Hedgehog (HH), transforming growth factor beta (TGF-β) and canonical wingless-type MMTV integration site (Wnt) pathways. ResultsThere were no statistically significant differences in pathway activity between STIC and HGSC, but STIC and HGSC demonstrated significantly lower ER and higher PI3K and HH pathway activity in comparison to normal FTE, suggesting these pathways as putative early drivers. In addition, we determined FOXO3a protein expression by immunohistochemistry and found loss of FOXO3a protein expression in STIC and HGSC compared to normal FTE. This observation confirmed that activation of PI3K signaling by loss of FOXO is an early hallmark of serous carcinogenesis. Furthermore, HGSC demonstrated significant loss of AR and Wnt pathway activity in relation to FTE, suggesting these pathways contribute to disease progression. ConclusionOur observations, together with the previously described associations between p53 signaling and both PI3K and HH pathway activity, provide evidence that increased PI3K and HH pathway activity and loss of ER pathway activity may be underlying events contributing to neoplastic transformation of FTE into STIC.

Survival Is Related to Estrogen Signal Transduction Pathway Activity in Postmenopausal Women Diagnosed with High-Grade Serous Ovarian Carcinoma.

Simple SummaryAll cells have a complex internal network of ‘communication chains’ called signal transduction pathways (STPs). Through interaction of different proteins in STPs, they are partly responsible for the behavior of a cell. In our study, we investigated the activity of six STPs in 85 women with advanced stage high-grade serous ovarian cancer (HGSC). To investigate the relation between and differences in survival and STP activity, women with a short disease-free survival (below 12 months) and a long disease-free survival (above 24 months) were included. We found no differences in mean STP activity between short-term survivors (52 women) and long-term survivors (33 women). However, when we analyzed postmenopausal women, we found that both disease-free and overall survival were related to estrogen receptor (ER) pathway signaling. This indicates that a better survival outcome was related to a more active ER pathway in this subgroup.High-grade serous ovarian carcinoma (HGSC), the most common subtype of ovarian cancer, has a high mortality rate. Although there are some factors associated with survival, such as stage of disease, there are remarkable differences in survival among women diagnosed with advanced stage disease. In this study, we investigate possible relations between survival and signal transduction pathway (STP) activity. We assessed the functional activity of the androgen receptor (AR), estrogen receptor (ER), phosphoinositide-3-kinase (PI3K), Hedgehog (HH), transforming growth factor beta (TGF-β) and canonical wingless-type MMTV integration site (Wnt) pathway in 85 primary tumor samples of patients with FIGO stage IIIC to IVB HGSC and disease-free survival (DFS) below 12 (n = 52) or over 24 months (n = 33). There were no significant differences in median pathway activity between patients with a short and long DFS. In univariate Cox proportional hazards analysis, ER pathway activity was related to a favorable DFS and overall survival (OS) in postmenopausal women (p = 0.033 and p = 0.041, respectively), but not in premenopausal women. We divided the postmenopausal group into subgroups based on ER pathway activity quartiles. Survival analysis revealed that postmenopausal women in the lowest ER quartile had a shorter DFS and OS (log-rank p = 0.006 and p < 0.001, respectively). Furthermore, we were able to form subgroups of patients based on an inverse relation between ER and PI3K pathway activity. In conclusion, in postmenopausal patients with advanced stage HGSC, a poorer survival outcome was associated with low functional ER pathway activity.

High-mobility group AT-Hook 1 mediates the role of nuclear factor I/X in osteogenic differentiation through activating canonical Wnt signaling.

It was previously reported that the loss of the transcription factor nuclear factor I/X (NFIX) gene in mice impaired endochondral ossification and mineralization in bone. However, the cellular and molecular basis for the defect remains unexplored. In this study, we investigated if and how NFIX regulates osteoblast differentiation. Nfix mRNA was induced during osteogenic and adipogenic differentiation of progenitor cells. Loss-of-function and gain-of-function studies revealed that NFIX induced osteoblast differentiation and impaired adipocyte formation from progenitor cells. RNA-seq and promoter analysis revealed that NFIX transcriptionally stimulated the expression of high-mobility group AT-Hook 1 (HMGA1). We then demonstrated that HMGA1 stimulated osteogenic differentiation of progenitor cells at the expense of adipogenic differentiation. The effect of Nfix siRNA on the differentiation of progenitor cells could be attenuated when HMGA1 was simultaneously overexpressed. Further investigations revealed the stimulatory effect of NFIX and HMGA1 on canonical wingless-type MMTV integration site family (Wnt) signaling. HMGA1 transcriptionally activates the expression of low-density lipoprotein receptor-related protein 5. Finally, in vivo transfection of Nfix siRNA to the marrow of mice reduced osteoblasts and increased fat accumulation in the marrow, and inactivated HMGA1/β-catenin signaling in bone marrow mesenchymal stem cells. This study suggests that HMGA1 plays a role in osteoblast commitment and mediates the function of NFIX through transcriptionally activating canonical Wnt signaling.

WNT signaling in pre-granulosa cells is required for ovarian folliculogenesis and female fertility.

ABSTRACTIn mammalian ovaries, immature oocytes are reserved in primordial follicles until their activation for potential ovulation. Precise control of primordial follicle activation (PFA) is essential for reproduction, but how this is achieved is unclear. Here, we show that canonical wingless-type MMTV integration site family (WNT) signaling is pivotal for pre-granulosa cell (pre-GC) activation during PFA. We identified several WNT ligands expressed in pre-GCs that act in an autocrine manner. Inhibition of WNT secretion from pre-GCs/GCs by conditional knockout (cKO) of the wntless (Wls) gene led to female infertility. In Wls cKO mice, GC layer thickness was greatly reduced in growing follicles, which resulted in impaired oocyte growth with both an abnormal, sustained nuclear localization of forkhead box O3 (FOXO3) and reduced phosphorylation of ribosomal protein S6 (RPS6). Constitutive stabilization of β-catenin (CTNNB1) in pre-GCs/GCs induced morphological changes of pre-GCs from a squamous into a cuboidal form, though it did not influence oocyte activation. Our results reveal that canonical WNT signaling plays a permissive role in the transition of pre-GCs to GCs, which is an essential step to support oocyte growth.

A novel Sprouty4-ERK1/2-Wnt/β-catenin regulatory loop in marrow stromal progenitor cells controls osteogenic and adipogenic differentiation

BackgroundSprouty (SPRY) proteins play critical roles in controlling cell proliferation, differentiation, and survival by inhibiting receptor tyrosine kinase (RTK)-mediated extracellular signal-regulated kinase (ERK) signaling. Recent studies have demonstrated that SPRY4 negatively regulates angiogenesis and tumor growth. However, whether SPRY4 regulates osteogenic and/or adipogenic differentiation of mesenchymal stem cells remains to be explored. ResultsIn this study, we investigated the expression pattern of Spry4 and found that its expression was regulated during the differentiation of mouse marrow stromal progenitor cells and increased in the metaphysis of ovariectomized mice. In vitro loss-of-function and gain-of-function studies demonstrated that SPRY4 inhibited osteogenic differentiation and stimulated adipogenic differentiation of progenitor cells. In vivo experiments showed that silencing of Spry4 in the marrow of C57BL/6 mice blocked fat accumulation and promoted osteoblast differentiation in ovariectomized mice. Mechanistic investigations revealed the inhibitory effect of SPRY4 on canonical wingless-type MMTV integration site (Wnt) signaling and ERK pathway. ERK1/2 was shown to interact with low-density lipoprotein receptor-related protein 6 (LRP6) and activate the canonical Wnt signaling pathway. Inactivation of Wnt signaling attenuated the inhibition of adipogenic differentiation and stimulation of osteogenic differentiation by Spry4 small interfering RNA (siRNA). Finally, promoter study revealed that β-catenin transcriptionally inhibited the expression of Spry4. ConclusionsOur study for the first time suggests that a novel SPRY4-ERK1/2-Wnt/β-catenin regulatory loop exists in marrow stromal progenitor cells and plays a key role in cell fate determination. It also highlights the potential of SPRY4 as a novel therapeutic target for the treatment of metabolic bone disorders such as osteoporosis.

Several type 2 diabetes-associated variants in genes annotated to WNT signaling interact with dietary fiber in relation to incidence of type 2 diabetes.

BackgroundTCF7L2 is a central transcription factor in the canonical wingless-type MMTV integration site (WNT) signaling pathway, and genetic variants in TCF7L2 have been found to interact with dietary fiber intake on type 2 diabetes risk. Here, we investigate whether other type 2 diabetes genes could be involved in the WNT signaling pathway and whether variants in such genes might interact with dietary fiber on type 2 diabetes incidence.ResultsWe included 26,905 individuals without diabetes from the Malmö Diet and Cancer Study cohort. Diet data was collected at baseline using a food frequency questionnaire, a 7-day food record, and an interview. Altogether, 51 gene loci were analyzed for putative links to WNT signaling. Over a mean follow-up period of 14.7 years, 3132 incident cases of type 2 diabetes were recorded. Seven genes (nine single nucleotide polymorphisms (SNPs)) were annotated as involved in WNT signaling including TCF7L2 (rs7903146 and rs12255372), HHEX (rs1111875), HNF1A (rs7957197), NOTCH2 (rs10923931), TLE4 (rs13292136), ZBED3 (rs4457053), and PPARG (rs1801282 and rs13081389). SNPs in TCF7L2, NOTCH2, and ZBED3 showed significant interactions with fiber intake on type 2 diabetes incidence (Pinteraction = 0.034, 0.005, 0.017, and 0.002, respectively). The magnitude of the association between the TCF7L2 risk allele and incident type 2 diabetes increased from the lowest to the highest quintiles of fiber intake. Higher fiber associated with lower type 2 diabetes risk only among risk allele carriers of the NOTCH2 variant and homozygotes of the risk allele of the ZBED3 variant.ConclusionsOur results suggest that several type 2 diabetes susceptibility SNPs in genes involved in WNT signaling may interact with dietary fiber intake on type 2 diabetes incidence.Electronic supplementary materialThe online version of this article (doi:10.1186/s12263-016-0524-4) contains supplementary material, which is available to authorized users.

Expression of osterix Is Regulated by FGF and Wnt/β-Catenin Signalling during Osteoblast Differentiation.

Osteoblast differentiation from mesenchymal cells is regulated by multiple signalling pathways. Here we have analysed the roles of Fibroblast Growth Factor (FGF) and canonical Wingless-type MMTV integration site (Wnt/β-Catenin) signalling pathways on zebrafish osteogenesis. We have used transgenic and chemical interference approaches to manipulate these pathways and have found that both pathways are required for osteoblast differentiation in vivo. Our analysis of bone markers suggests that these pathways act at the same stage of differentiation to initiate expression of the osteoblast master regulatory gene osterix (osx). We use two independent approaches that suggest that osx is a direct target of these pathways. Firstly, we manipulate signalling and show that osx gene expression responds with similar kinetics to that of known transcriptional targets of the FGF and Wnt pathways. Secondly, we have performed ChIP with transcription factors for both pathways and our data suggest that a genomic region in the first intron of osx mediates transcriptional activation. Based upon these data, we propose that FGF and Wnt/β-Catenin pathways act in part by directing transcription of osx to promote osteoblast differentiation at sites of bone formation.

Canonical Wnt suppressor, Axin2, promotes colon carcinoma oncogenic activity

Aberrant activation of canonical Wingless-type MMTV integration site family (Wnt) signaling is pathognomonic of colorectal cancers (CRC) harboring functional mutations in either adenomatous polyposis coli or β-catenin. Coincident with Wnt cascade activation, CRCs also up-regulate the expression of Wnt pathway feedback inhibitors, particularly the putative tumor suppressor, Axin2. Because Axin2 serves as a negative regulator of canonical Wnt signaling in normal cells, recent attention has focused on the utility of increasing Axin2 levels in CRCs as a means to slow tumor progression. However, rather than functioning as a tumor suppressor, we demonstrate that Axin2 acts as a potent promoter of carcinoma behavior by up-regulating the activity of the transcriptional repressor, Snail1, inducing a functional epithelial-mesenchymal transition (EMT) program and driving metastatic activity. Silencing Axin2 expression decreases Snail1 activity, reverses EMT, and inhibits CRC invasive and metastatic activities in concert with global effects on the Wnt-regulated cancer cell transcriptome. The further identification of Axin2 and nuclear Snail1 proteins at the invasive front of human CRCs supports a revised model wherein Axin2 acts as a potent tumor promoter in vivo.

Implication of dysregulation of the canonical wingless-type MMTV integration site (WNT) pathway in diabetic nephropathy

The wingless-type MMTV integration site (WNT) pathway mediates multiple physiological and pathological processes, such as inflammation, angiogenesis and fibrosis. The aim of this study was to investigate whether canonical WNT signalling plays a role in the pathogenesis of diabetic nephropathy. Expression of WNT ligands and frizzled receptors in the canonical WNT pathway in the kidney was compared at the mRNA level using real-time RT-PCR between Akita mice, streptozotocin-induced diabetic rats and db/db mice and their respective non-diabetic controls. Renal function was evaluated by measuring the urine albumin excretion. Human renal proximal tubular epithelial cells were treated with high-glucose medium and 4-hydroxynonenal (HNE). Levels of β-catenin, connective tissue growth factor and fibronectin were determined by western blot analysis. Some of the WNT ligands and frizzled receptors showed increased mRNA levels in the kidneys of Akita mice, streptozotocin-induced diabetic rats and db/db mice compared with their non-diabetic controls. Renal levels of β-catenin and WNT proteins were upregulated in these diabetic models. Lowering the blood glucose levels by insulin attenuated the activation of WNT signalling in the kidneys of Akita mice. In cultured human renal proximal tubular epithelial cells, both high glucose and HNE activated WNT signalling. Inhibition of WNT signalling with a monoclonal antibody blocking LDL-receptor-related protein 6 ameliorated renal inflammation and fibrosis and reduced proteinuria in Akita mice. The WNT pathway is activated in the kidneys of models of both type 1 and 2 diabetes. Dysregulation of the WNT pathway in diabetes represents a new pathogenic mechanism of diabetic nephropathy and renders a new therapeutic target.

CCN proteins in normal and injured liver

CCN proteins are small secreted cysteine-rich proteins containing up to four individual structural modules including an insulin-like growth factor binding domain, a von Willebrand Factor type C motif, a thrombospondin type I module and a carboxyl-terminal cystine knot. Actually, there is a large body of evidence suggesting that members of the CCN protein family encompass an expansive repertoire of functions in crucial areas including control of development, cell fate, angiogenesis, tumorigenesis, osteogenesis, cell adhesion, mitogenesis, migration, chemotaxis, and cell survival. Moreover, this family is supposed to modulate signalling of integrins, transforming growth factor-betas, bone morphogenetic proteins, vascular endothelial growth factor, Notch and factors that mediate signals via the canonical Wingless-type MMTV integration site family. However, several of these properties are not substantiated by experimental data but were deduced from proteins sharing one or more of the structural modules with these proteins. In this review, the actual knowledge of biological activities and molecular involvement of CCN proteins in maintenance of liver health and in initiation and progression of hepatic diseases is summarized and discussed.

The role of lipid peroxidation products and oxidative stress in activation of the canonical wingless-type MMTV integration site (WNT) pathway in a rat model of diabetic retinopathy.

Aims/hypothesisOur recent studies suggest that activation of the wingless-type MMTV integration site (WNT) pathway plays pathogenic roles in diabetic retinopathy and age-related macular degeneration. Here we investigated the causative role of oxidative stress in retinal WNT pathway activation in an experimental model of diabetes.MethodsCultured retinal pigment epithelial cells and retinal capillary endothelial cells were treated with a lipid peroxidation product, 4-hydroxynonenal (HNE), and an antioxidant, N-acetyl-cysteine (NAC). In vivo, rats with streptozotocin-induced diabetes were treated by NAC for 8 weeks. Activation of the canonical WNT pathway was measured by TOPFLASH assay and by western blot analysis of WNT pathway components and a WNT target gene, Ctgf. Oxidative stress in the retina was evaluated by immunostaining of HNE and 3-nitrotyrosine.ResultsLevels of phosphorylated and total LDL receptor-related protein (LRP)6, and cytosolic β-catenin, as well as transcriptional activity of T cell factor (TCF)/β-catenin were significantly increased by HNE. The production of connective tissue growth factor (CTGF) was also upregulated by HNE. NAC blocked the WNT pathway activation induced by HNE. Furthermore, LRP6 stability was increased by HNE and decreased by NAC. Retinal levels of HNE and 3-nitrotyrosine were significantly increased in diabetic rats, compared with those in non-diabetic rats. In the same diabetic rat retinas, levels of LRP6, cytosolic β-catenin and CTGF were significantly increased. NAC treatment reduced HNE and 3-nitrotyrosine levels and attenuated the upregulation of LRP6, β-catenin and CTGF in diabetic rat retina.Conclusions/interpretationLipid peroxidation products activate the canonical WNT pathway through oxidative stress, which plays an important role in the development of retinal diseases.

Activation of Canonical Wingless-type MMTV Integration Site Family (Wnt) Signaling in Mature Adipocytes Increases β-Catenin Levels and Leads to Cell Dedifferentiation and Insulin Resistance

Canonical Wnt ligands are secreted by several cell types in the adipose tissue. We examined if mature adipocytes can also be target cells and found that canonical Wnt activation by Wnt3a induced a marked dedifferentiation of both 3T3-L1 and human adipocytes. Typical adipogenic markers were reduced while undifferentiated cell markers like Pref-1/Dlk1, Wnt10b, and Gata2 were increased. The cells also became insulin-resistant with impaired upstream insulin signaling and reduced glucose uptake. Wnt3a stabilized beta-catenin in the absence of the LRP6 receptor and with maintained axin and Dickkopf-1 protein expression. PPARgamma was repressed and PPARgamma ligands could not restore the adipogenic markers or reduce the beta-catenin levels. The dedifferentiated adipocytes expressed the myofibroblast marker alpha-smooth muscle actin and were also susceptible to osteogenic transdifferentiation. These results identify a novel pathway in mature adipose cells that is critical for maintaining the normal adipocyte phenotype and insulin sensitivity.

Thiazolidinediones increase the wingless-type MMTV integration site family (WNT) inhibitor Dickkopf-1 in adipocytes: a link with osteogenesis

Dickkopf-1 (DKK1) is a secreted inhibitor of canonical wingless-type MMTV integration site family (WNT) signalling; the key pathway for cell fate and development. Inhibition of WNT signalling by DKK1 in precursor cells promotes adipogenesis and inhibits osteogenesis. Previous studies have shown that treatment of type 2 diabetic patients with thiazolidinediones (TZDs) reduces bone density and increases risk of bone fractures, while body fat is increased. We examined the effect of TZDs on secretion and DKK1 levels in pre-adipocytes and mature adipose cells and also measured circulating DKK1 levels in 11 patients with type 2 diabetes before and after treatment with the TZD rosiglitazone for 90 days. TZDs added in vitro rapidly increased DKK1 protein levels and secretion in both fully differentiated adipose cells and pre-adipocytes undergoing differentiation. In parallel, beta-catenin levels, a marker of canonical WNT signalling, were reduced. Serum levels of DKK1 were also increased in several of the patients with type 2 diabetes after treatment with rosiglitazone for 90 days. These results provide a novel mechanism whereby peroxisome proliferator-activated receptor-gamma activation can terminate WNT signalling and promote adipogenesis. Furthermore, they provide an explanation for why TZD treatment can lead to reduced bone formation and increased risk of fractures, since inhibited WNT signalling in progenitor cells promotes adipogenesis while osteogenesis is reduced.

Regulation of wingless-type MMTV integration site family (WNT) signalling in pancreatic islets from wild-type and obese mice

TCF7L2 is a type 2 diabetes susceptibility gene and downstream effector of canonical wingless-type MMTV integration site family (WNT) signalling. However, it is unknown whether this pathway is active in adult pancreatic islets in vivo, and whether it is regulated in obesity. We analysed activation of endogenous WNT signalling in the endocrine pancreas from wild-type and obese mice (ob/ob) using a reporter transgene (Topgal). Regulation of WNT signalling was compared using gene chip experiments from isolated pancreatic islets. Activation of canonical WNT signalling in pancreatic islets and the mouse beta cell line MIN6 was measured using immunoblotting for cytosolic beta-catenin. Endogenous canonical WNT signalling was absent in the adult endocrine pancreas in both wild-type and obese mice. We identified WNT4 as an abundant WNT signalling molecule in adult pancreatic islets that is induced in two different insulin-resistant mouse models. Increased expression of WNT4 inhibited canonical WNT signalling in pancreatic islets and MIN6 cells. Canonical WNT signalling is not active in adult beta cells in vivo. WNT4 provides a potential mechanism for suppression of canonical WNT signalling in obese mice.