Wnt Inhibitory Factor 1 Overexpression Research Articles

WNT-inhibitory factor 1-mediated glycolysis protects photoreceptor cells in diabetic retinopathy

BackgroundIn diabetic retinopathy (DR), hypoxia-inducible factor (HIF-1α) induces oxidative stress by upregulating glycolysis. This process leads to neurodegeneration, particularly photoreceptor cell damage, which further contributes to retinal microvascular deterioration. Further, the regulation of Wnt-inhibitory factor 1 (WIF1), a secreted Wnt signaling antagonist, has not been fully characterized in neurodegenerative eye diseases. We aimed to explore the impact of WIF1 on photoreceptor function within the context of DR.MethodTwelve-week-old C57BL/KsJ-db/db mice were intravitreally injected with WIF1 overexpression lentivirus. After 4 weeks, optical coherence tomography (OCT), transmission electron microscopy (TEM), H&E staining, and electroretinography (ERG) were used to assess the retinal tissue and function. The potential mechanism of action of WIF1 in photoreceptor cells was explored using single-cell RNA sequencing. Under high-glucose conditions, 661 W cells were used as an in vitro DR model. WIF1-mediated signaling pathway components were assessed using quantitative real-time PCR, immunostaining, and western blotting.ResultTypical diabetic manifestations were observed in db/db mice. Notably, the expression of WIF1 was decreased at the mRNA and protein levels. These pathological manifestations and visual function improved after WIF1 overexpression in db/db mice. TEM demonstrated that WIF1 restored damaged mitochondria, the Golgi apparatus, and photoreceptor outer segments. Moreover, ERG indicated the recovery of a-wave potential amplitude. Single-cell RNA sequencing and in vitro experiments suggested that WIF1 overexpression prevented the expression of glycolytic enzymes and lactate production by inhibiting the canonical Wnt signaling pathway, HIF-1α, and Glut1, thereby reducing retinal and cellular reactive oxygen species levels and maintaining 661 W cell viability.ConclusionsWIF1 exerts an inhibitory effect on the Wnt/β-catenin-HIF-1α-Glut1 glycolytic pathway, thereby alleviating oxidative stress levels and mitigating pathological structural characteristics in retinal photoreceptor cells. This mechanism helps preserve the function of photoreceptor cells in DR and indicates that WIF1 holds promise as a potential therapeutic candidate for DR and other neurodegenerative ocular disorders.Graphical

Epigenetic regulation of human WIF1 and DNA methylation situation of WIF1 and GSTM5 in urothelial carcinoma

WNT inhibitory factor 1 (WIF1) is known to function as a tumor suppressor gene; it inhibits oncogene activation by preventing WNT signaling. This study investigated the epigenetic regulation of WIF1 gene in bladder cancer. We observed a positive relationship between WIF1 mRNA expression and survival probability of bladder cancer patients. The WIF1 gene expression could be enhanced by DNA demethylation drug 5-aza-2′-deoxycytidine (5-aza-dC) and histone deacetylase inhibitor trichostatin A (TSA), suggesting that epigenetic modifications could regulate WIF1 gene expression. Overexpression of WIF1 inhibited cell proliferation and migration in 5637 cells, confirming the tumor suppressor role of WIF1. 5-Aza-dC dose dependently increased WIF1 gene expression while reducing DNA methylation level, suggesting that reversing WIF1 DNA methylation could activate its gene expression. We collected the cancer tissues and urine pellets of bladder cancer patients and only urine pellets from non-bladder cancer volunteers for DNA methylation analysis, but the methylation level of WIF1 gene −184 to +29 did not differ between patients and controls. We also analyzed glutathione S-transferase Mu 5 (GSTM5) gene methylation level because GSTM5 DNA hypermethylation was suggested to be a tumor biomarker in our previous study. It confirmed a higher GSTM5 DNA methylation in bladder cancer patients than in controls. In summary, this study suggests that the 5-aza-dC activated WIF1 gene which showed an anti-cancer effect, while WIF1 promoter −184 to +29 did not provide a suitable methylation assay region in clinical samples. In contrast, GSTM5 promoter −258 to −89 is a useful region for DNA methylation assay because it shows a higher methylation level in bladder cancer patients.

Wnt inhibitory factor 1 ameliorated diabetic retinopathy through the AMPK/mTOR pathway-mediated mitochondrial function.

Diabetic retinopathy (DR) is one of the most common complications of diabetes mellitus and will lead to visual impairment. We aim to explore the effects and mechanisms of wnt inhibitory factor 1 (WIF1) in the progression of DR. To establish DR in vitro and in vivo, human retinal pigment epithelium (RPE) cell line ARPE-19 was treated with high-glucose (HG) and diabetic mice models were induced by streptozotocin (STZ), respectively. Different dose of recombinant WIF1 protein was used to treat DR. qRT-PCR and western blotting results demonstrated that WIF1 was downregulated, while VEGFA was upregulated in HG-induced ARPE-19 cells. WIF1 overexpression promoted cell migration. The ARPE-19 cells culture medium treated with WIF1 inhibited retinal endothelial cell tube formation and downregulated VEGFA expression. Moreover, WIF1 decreased the levels of ROS and MDA, while increasing the activity of SOD and GPX. WIF1 increased the ΔΨm in the mitochondria and downregulated the expression of mitochondrial autophagy-related proteins including Parkin, Pink1, LC3-II/LC3-I ratio, cleaved caspase 3, and cyt-c, which ameliorated mitochondrial dysfunction. The in vivo studies further demonstrated the consistent effects of WIF1 in STZ-induced mice. Taken together, WIF1 ameliorated mitochondrial dysfunction in DR by downregulating the AMPK/mTOR pathway.

A study on the mechanism of Wnt inhibitory factor 1 in osteoarthritis.

IntroductionIn our study we aimed to investigate the mechanism of Wnt inhibitory factor 1 (WIF1) on regulating chondrocyte proliferation and apoptosis via reactive oxygen species (ROS) and the Wnt/βcatenin signaling pathway in osteoarthritis (OA).Material and methodsOsteoarthritis chondrocytes were treated with interleukin 1β (IL-1β) to simulate an inflammatory condition. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were applied for detecting WIF1 expression in OA chondrocytes. MTT assay and flow cytometry were carried out to analyze the cell proliferation and apoptosis. Content of ROS was detected using flow cytometry, and activity of the Wnt/βcatenin signaling pathway was detected using immunofluorescence, western blot and luciferase reporter assay. Western blot and enzyme-linked immunosorbent assay (ELISA) were performed to detect the expression of apoptosis-related proteins and secretion of matrix metalloproteinases (MMPs).ResultsWIF1 expression in OA chondrocytes was significantly lower than in normal chondrocytes. After WIF1 cDNA transfection, the aberrantly high ROS level in OA chondrocytes was down-regulated, which led to the increase of proliferation and reduction of apoptosis. The Wnt/βcatenin signaling pathway was suppressed by WIF1 overexpression and the secretion of MMPs was therefore reduced.ConclusionsUp-regulation of WIF1 would promote proliferation and suppress apoptosis of OA chondrocytes through eliminating ROS production and reduce secretion of MMPs via blocking the Wnt/βcatenin signaling pathway.

WIF1 enhanced dentinogenic differentiation in stem cells from apical papilla

BackgroundOdontogenic mesenchymal stem cells (MSCs) isolated from tooth tissues are a reliable resource that can be utilized for dental tissue regeneration. Exploration of the mechanisms underlying the regulation of their differentiation may be helpful for investigating potential clinical applications. The stem cell niche plays an important role in maintaining cell functioning. Previous studies found that Wnt inhibitory factor 1 (WIF1) is more highly expressed in apical papilla tissues than in stem cells from apical papilla (SCAPs) using microarray analysis. However, the function of WIF1 in SCAPs remains unclear. In the present study, we investigated the function of WIF1 during dentinogenic differentiation in SCAPs.MethodsA retrovirus containing HA-WIF1 was used to overexpress WIF1 in SCAPs. Using Western blot analysis, we verified the expression of HA-WIF1. Alkaline phosphatase (ALP) activity assays, Alizarin Red staining and quantitative calcium analysis were performed to investigate the in vitro potential for dentinogenic differentiation in SCAPs. The expression of dentinogenesis-associated genes DSPP, DMP1, Runx2 and OSX were assayed using real-time RT-PCR. Transplantation experiments were used to measure dentinogenesis potential in vivo.ResultsThe real time RT-PCR results showed that WIF1 was more highly expressed in apical papilla tissues than in SCAPs, and its expression was increased during the process of dentinogenic differentiation. Overexpression of WIF1 enhanced ALP activity and mineralization in vitro, as well as the expression of DSPP, DMP1 and OSX in SCAPs. Moreover, in vivo transplantation experiments revealed that dentinogenesis in SCAPs was enhanced by WIF1 overexpression.ConclusionThese results suggest that WIF1 may enhance dentinogenic differentiation potential in dental MSCs via its regulation of OSX and identified potential target genes that could be useful for improving dental tissue regeneration.

UPA affects the CRSsNP nasal mucosa epithelium apoptosis by regulating WIF1

Chronic rhinosinusitis without nasal polyps (CRSsNP) is the main type of Chronic rhinosinusitis (CRS) and is a common otorhinolaryngologic disease worldwide. However, the mechanisms of CRSsNP remain poorly understood. In this study, C57BL/6J wild-type and urokinase-type plasminogen activator (uPA) gene knockout (uPA-/-) mice were used to construct the CRSsNP model. Primary human nasal epithelial cells (HNEC) were isolated from CRSsNP patient and treated with uPA knockdown/overexpression lentivirus. CCK-8 and Annexin-V/PI staining were used to detected cell proliferation and apoptosis. In vivo, we found that uPA depletion alleviated mucosal inflammation in the CRSsNP mice model. Wnt inhibitory factor 1 (WIF1) was upregulated in the uPA-/- CRSsNP mice model. In vitro, inhibition of uPA increased cell proliferation and decreased cell apoptosis. Mechanistically, uPA depletion upregulated WIF1 and BCL2 expression, and reduced the expression level of BAX in CRSsNP HNEC. In contrast, decreased cell proliferation and increased cell apoptosis were observed after uPA overexpression. Consistently, a reduction in WIF1 and BCL2 expression levels and an increase in the BAX expression level were observed upon uPA ectopic expression. Furthermore, WIF1 overexpression rescued the effects caused by uPA overexpression in vitro. In conclusion, uPA affects the CRSsNP nasal mucosal epithelium cell apoptosis by upregulating WIF1. To our knowledge, this is the first study to explore the role of uPA in CRSsNP to date.

The cardiac microenvironment uses non-canonical WNT signaling to activate monocytes after myocardial infarction.

A disturbed inflammatory response following myocardial infarction (MI) is associated with poor prognosis and increased tissue damage. Monocytes are key players in healing after MI, but little is known about the role of the cardiac niche in monocyte activation. This study investigated microenvironment‐dependent changes in inflammatory monocytes after MI. RNA sequencing analysis of murine Ly6Chigh monocytes on day 3 after MI revealed differential regulation depending on location. Notably, the local environment strongly impacted components of the WNT signaling cascade. Analysis of WNT modulators revealed a strong upregulation of WNT Inhibitory Factor 1 (WIF1) in cardiomyocytes—but not fibroblasts or endothelial cells—upon hypoxia. Compared to wild‐type (WT) littermates, WIF1 knockout mice showed severe adverse remodeling marked by increased scar size and reduced ejection fraction 4 weeks after MI. While FACS analysis on day 1 after MI revealed no differences in neutrophil numbers, the hearts of WIF1 knockouts contained significantly more inflammatory monocytes than hearts from WT animals. Next, we induced AAV‐mediated cardiomyocyte‐specific WIF1 overexpression, which attenuated the monocyte response and improved cardiac function after MI, as compared to control‐AAV‐treated animals. Finally, WIF1 overexpression in isolated cardiomyocytes limited the activation of non‐canonical WNT signaling and led to reduced IL‐1β and IL‐6 expression in monocytes/macrophages. Taken together, we investigated the cardiac microenvironment's interaction with recruited monocytes after MI and identified a novel mechanism of monocyte activation. The local initiation of non‐canonical WNT signaling shifts the accumulating myeloid cells toward a pro‐inflammatory state and impacts healing after myocardial infarction.

Hypermethylation of WIF1 and its inhibitory role in the tumor growth of endometrial adenocarcinoma.

Endometrial carcinoma is the most common malignancy of the female genital tract and is the fourth most common malignancy among women worldwide. Endometrial adenocarcinoma (EAC) accounts for ~80% of endometrial carcinoma cases. Numerous critical genetic events have been determined to serve an essential role in EAC progression; however, the precise molecular mechanisms underlying EAC progression remain unclear. Pyrosequencing and methylation-specific PCR were used to detect the methylation status of Wnt inhibitory factor 1 (WIF1). Immunohistochemistry and western blot were used to detect the expression of WIF1, Wnt family member 1 and other related pathways. The anticancer role of WIF1 in EAC was investigated in vitro and in vivo. Two of the three EAC cases exhibited significantly high methylation in five CpG sites, and the WIF1 methylation rate in EAC and endometrial tissues was 43.4 and 8%, respectively (P<0.05). The kappa consistency coefficient was −0.369 between methylation and mRNA expression (P<0.05) and WIF1 expression levels were significantly downregulated in EAC tissues compared with non-tumorous tissues (P<0.01). The 5-year overall survival rates were significantly lower for patients with tumors that negatively expressed WIF1 when compared with the 77.9% exhibited by those with positive WIF1 expression. Furthermore, the proliferation rate of KLE cells was significantly reduced following 5-aza-20-deoxycytidine treatment or WIF1 overexpression in vitro and in vivo, which may be associated with downregulated c-Myc and phosphorylated-extracellular signal-regulated kinase expression. These results demonstrated the important role of WIF1 in EAC tumorigenesis, and suggested that WIF1 may be a potential drug target in EAC treatment.

The long non-coding RNA HOTAIR affects the radiosensitivity of pancreatic ductal adenocarcinoma by regulating the expression of Wnt inhibitory factor 1

Pancreatic ductal adenocarcinoma (PDAC) is seriously resistant to radiotherapy and the mechanism is largely unknown. HOX transcript antisense intergenic RNA (HOTAIR) is overexpressed in PDAC. However, the function of HOTAIR has never been related to the radiosensitivity of PDAC. In this present study, the expression of HOTAIR in the PDAC cell lines and tissues was measured by quantitative real-time PCR (qRT-PCR), and the association between HOTAIR expression levels and X-ray treatment in PDAC cell lines was investigated. Additionally, the influence of HOTAIR knockdown on radiosensitivity, proliferation, and apoptosis of PDAC cells after radiation was evaluated by colony formation assays, Cell Counting Kit-8 (CCK-8) assays, and flow cytometry, respectively. Furthermore, the correlation between HOTAIR and Wnt inhibitory factor 1 (WIF-1) expression in PDAC cell lines and tissues was studied to assess the role of HOTAIR and WIF-1 in the radiosensitivity of PDAC. The results confirmed that HOTAIR expression was significantly increased in the PDAC cell lines and tissues (n = 90) compared with human normal pancreatic ductal epithelial cell line (HPDE) and matched adjacent normal tissues (n = 90). Functionally, HOTAIR knockdown enhanced the radiosensitivity of PDAC cells, reduced the proliferation, and increased the apoptosis of cells after radiation. And HOTAIR silencing increased the expression of WIF-1. Furthermore, the overexpression of WIF-1 revealed that HOTAIR modulated the radiosensitivity of PDAC cells by regulating the expression of WIF-1. These data reveals that HOTAIR can affect the radiosensitivity of PDAC cells partly via regulating the expression of WIF-1, and HOTAIR-WIF-1 axis is a potential target for PDAC radiotherapy.

Abstract 3314: Co-regulation of srGAP1 by Wnt and androgen receptor signaling in castration resistant prostate cancer

Abstract Androgen receptor (AR) and Wnt signaling both play a critical role during prostate cancer progression to castration-resistance prostate cancer (CRPC). Over expression of AR can activate transcriptional activities of Wnt signaling pathway and promote CRPC. Recent data in our laboratory showed a potential co-regulation of srGAP1 (Slit-Robo-GTPase activating protein1) and active Wnt and AR signaling in CRPC. srGAP1 is a downstream component of Slit-Robo signaling. Our data showed that srGAP1 is overexpressed in CRPC cell lines (androgen-insensitive prostate cancer) while absent in both androgen-sensitive cells and in normal prostate epithelial cells. We also detected increased srGAP1 expression in human CRPC tissues compared with normal epithelial prostate and androgen sensitive prostate cancer tissues by immunohistochemistry analysis. When androgen-sensitive LNCaP cells were grown under androgen deprived condition, we observed induced expression of srGAP1. Interestingly, srGAP1 expression was also increased when LNCaP cells were grown under Wnt stimulated conditions. And, srGAP1 expression was decreased when Wnt signaling was inhibited by a Wnt antagonist Wnt-inhibitory factor-1 (WIF-1) in CRPC cell lines. Functional studies using chromatin immmunoprecipitation and luciferase assays were performed to examine whether Wnt transcription factor LEF-1 or TCF-4 binds and activates the srGAP1 promoter. Overexpression of WIF-1 inhibited the promoter activity of srGAP1. Taken together, our results indicated that srGAP1 is co-regulated by both Wnt and AR signaling and srGAP1 may be a new potential Wnt target gene in castration-resistance prostate cancer. Citation Format: Noriko N. Yokoyama, Toshinori Sakai, Zheng Sun, Shujuan Shao, Jiaoti Huang, Bang H. Hoang, Xiaolin Zi. Co-regulation of srGAP1 by Wnt and androgen receptor signaling in castration resistant prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3314. doi:10.1158/1538-7445.AM2014-3314

Abstract B08: Triptolide has antitumor effects in lung cancer via Wnt pathway inhibition.

Abstract Introduction: Triptolide is an extract of the herb Tripterygium wilfordii (Tw), which has been used to treat autoimmune disorders in traditional Chinese medicine. It has also been reported to have anti-cancer effects in vitro, although little is known about its mechanism of action. The Wnt/B-catenin pathway has been implicated in regulating cancer stem cells in lung cancer, making this pathway a potential target for novel lung cancer therapies. Methods: Here we tested the efficacy of triptolide in several lung cancer cell lines. We elucidated a Wnt pathway mechanism for triptolide effect in lung cancer, first by using a Wnt focused cDNA array in vitro. We examined the effect of triptolide on tumor cell death using flow cytomotery and a beta-galactosidase assay. We tested the ability of triptolide to prevent development of lung tumors in the E160D transgenic mouse model of lung cancer, which carries a somatic mutation in the DNA repair enzyme flap endonuclease-1 (FEN-1). Twelve month old mice were fed triptolide 1mg/kg daily for 2 months and were sacrificed at 18 months along with a control group. Results: Triptolide had more potent therapeutic efficacy in multiple lung cancer cell lines compared with cisplatin and paclitaxel, in vitro. In H460 and A549 cell lines, we found that triptolide resulted in Wnt pathway silencing via overexpression of Wnt inhibitory factor-1 (WIF-1), which occurs due to promoter demethylation. Addition of recombinant Wnt3a, which causes Wnt pathway activation, partially rescues the effect of triptolide on lung cancer cell lines. Triptolide resulted in cellular senescence as demonstrated by beta-galactosidase assay. Finally, in the E160D transgenic mouse model of lung cancer, a two month course of oral triptolide treatment decreased the development of lung tumors from 70% to 30% at 18 months (p=0.01). There were no observed clinical toxicities at the dosage of triptolide used. Conclusions: Triptolide has potent anti-cancer effects in vitro in lung cancer cell lines, and significantly decreases lung tumor development in a transgenic mouse model. Triptolide suppresses the Wnt pathway by causing upregulation of WIF-1a by promoter demethylation. Future studies are needed to test this promising and novel Wnt-targeting therapy in lung cancer animal models, and to identify biomarkers which may predict sensitivity in particular subgroups of lung cancer patients. Citation Format: Youqiang Li, Li Zheng, Binghui Shen, Jae Kim, Dan Raz. Triptolide has antitumor effects in lung cancer via Wnt pathway inhibition. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B08.

Wnt Inhibitory Factor 1 Deficiency Uncouples Cartilage and Bone Destruction in Tumor Necrosis Factor α–Mediated Experimental Arthritis

Wnt signaling plays a pivotal role in skeletal development and in the control of cartilage and bone turnover. We have recently shown that the secreted Wnt antagonist Wnt inhibitory factor 1 (WIF-1) is mainly expressed in the upper layers of epiphyseal and articular cartilage and, to a lesser extent, in bone. Nevertheless, WIF-1(-/-) mice develop normally. In light of these findings, we undertook this study to analyze the role of WIF-1 in arthritis. Expression analyses for WIF-1 were performed by real-time reverse transcription-polymerase chain reaction (RT-PCR). WIF-1(-/-) and tumor necrosis factor (TNF)-transgenic mice were crossbred, and the progression of arthritis in TNF-transgenic WIF-1(-/-) mice and littermate controls was evaluated. Structural joint damage was analyzed by histologic staining, histomorphometry, and micro-computed tomography. Wnt/β-catenin signaling was investigated by real-time RT-PCR and immunofluorescence on primary chondrocytes. WIF-1 expression was repressed by TNFα in chondrocytes and osteoblasts and down-regulated in experimental arthritis and in articular cartilage from patients with rheumatoid arthritis. WIF-1 deficiency partially protected TNF-transgenic mice against bone erosion and loss of trabecular bone, probably as a result of less osteoclast activity. In contrast, arthritis-related cartilage damage was aggravated by WIF-1 deficiency, while overexpression of WIF-1 attenuated cartilage degradation in TNF-transgenic mice. In chondrocytes, TNFα stimulated canonical Wnt signaling, which could be blocked by WIF-1, indicating a direct effect of TNFα and WIF-1 on Wnt signaling in this system. These data suggest that WIF-1 may take part in the fine-tuning of cartilage and bone turnover, promoting the balance of cartilage versus bone anabolism.

Abstract 3883: Robo1, a new target, by Wnt and androgen receptor signaling in castration resistant prostate cancer.

Abstract Androgen receptor (AR) and β-catenin/TCF-4 signaling both play a critical role during prostate cancer progression to castration-resistance and cross-talk with each other. Therefore, identifying novel down-stream targets that are co-regulated by AR and β-catenin/TCF-4 signaling is important for understanding the development of castration-resistant prostate cancer (CRPC). Robo1, a transmembrane receptor of Slit, is known for its roles in neurogenesis and angiogenesis. Previous studies have shown that Robo/Slits are aberrantly expressing in different cancers. We have shown that Robo1 is over-expressed in androgen-insensitive prostate cancer cell lines (C4-2B, PC3, 22RV1) compared to androgen-sensitive lines (LNCaP and LAPC-4), and absent in normal prostate epithelial cells. Androgen deprivation resulted in enhanced expression of Robo1 and addition of synthetic androgen R1881decreased Robo1 expression in LNCaP cells. In addition, Wnt 3A and 5A conditioned medium stimulation increased Robo1 mRNA expression in LNCaP cells. Inhibition of Wnt signaling by overexpression of secreted Wnt antagonists Wnt-inhibitory factor-1 (WIF-1) and Frzb/sFRP3 in two CRPC cell lines, decreased the expression of Robo1. Using GenBank and MatInspector software, we have identified potential binding sites for TCF-4 in the 5’ flanking promoter region of the ROBO1 gene. Chromatin immunoprecipitation followed by real-time PCR analysis revealed that TCF-4 binds to the ROBO1 promoter at two predicted sites, and that WIF-1 and Frzb/sFRP3 expression inhibited its promoter binding. Overexpression of WIF-1 and Frzb/sFRP3 also inhibited the promoter activity of Robo1. Knock-down of Robo1 by short-hairpin RNA in PC3 cells resulted in dramatic morphological changes with increased cell aggregation and adhesion. Taken together, our results indicate that Robo1 is a co-target of Wnt and AR signaling. Further studies are in progress to define the functional role of Robo1 in CRPC. Citation Format: Noriko N. Yokoyama, Zheng Sun, Toshinori Sakai, Bang H. Hoang, Xiaolin Zi. Robo1, a new target, by Wnt and androgen receptor signaling in castration resistant prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3883. doi:10.1158/1538-7445.AM2013-3883

Abstract 4298: microRNA-200c mediates the tumor suppressive effects of Wnt inhibitory factor 1 in human malignant salivary gland cells.

Abstract Background: The important role of Wingless-type (Wnt)/β-catenin pathway is well established in various human tumors. Previously, we have demonstrated that Wnt inhibitory factor 1 (WIF1) is significantly downregulated in human salivary gland tumors. The tumor suppressor microRNA, miR-200c, is downregulated in many cancers and is known to repress migration and invasion of cancer cells. Aims: To determine the tumor suppressive effects of WIF1 and delineate the mechanisms by which WIF1 regulates microRNAs in human malignant salivary gland cells. Methods: Human malignant salivary gland cells (Carcinoma ExPleomorphic adenoma, CaExPA79) were transiently transfected with either empty vector or pCI blast-WIF1 and cell proliferation was determined by MTT assay at various time points. CaExPA79 cells transfected with WIF1 for 48 h were used for total RNA isolation and gene expression studies. To determine the effect of WIF1 restoration on salivary gland cancer cells, CaExPA79 cells were treated with 50 μM of 5-aza-2′-deoxycytidine (DAC), a demethylating agent, for 4 days. After DAC treatment, the cells were either used for cell cycle analysis or total RNA isolation and real-time RT-PCR analysis. To determine the effect of WIF1 on miR-200c, CaExPA79 cells were transfected with pCI blast-WIF1 along with miR200c-Luc reporter vector and Renilla luciferase expressing plasmid pRL-TK for 24 h. Luciferase activity was determined using Dual-Luciferase Reporter Assay System. Results: WIF1 significantly inhibited the proliferation of CaExPA79 at 24, 48 and 72 h. Treatment with DAC increased the WIF1 mRNA expression and resulted in a significant accumulation of cells in S phase. DAC treatment also resulted in a significant increase in miR-200c expression. WIF1 over-expression resulted in a significant increase in miR-200c expression and led to a significant reduction in the expression of miR-200c targeted genes as documented by luciferase reporter gene assays. Conclusions: WIF1 inhibits salivary gland cancer cell proliferation and induces cell cycle arrest. Most importantly, our findings suggest that WIF1 is a positive transcriptional regulator of the tumor suppressor microRNA miR-200c and leads to a corresponding downregulation of its downstream targets. Taken together, this study reveals a novel mechanism by which WIF1 elicits it's tumor suppressive effects on salivary gland cancer cells. Grant support: This work was supported by the Oklahoma Center for the Advancement of Science &amp; Technology (LQ) (HR08-018). LQ holds a Presbyterian Health Foundation Endowed Chair in Otorhinolaryngology. Citation Format: Ilangovan Ramachandran, David Obeso, Sripathi M. Sureban, Elangovan Thavathiru, Antonio Reis, Lurdes Queimado. microRNA-200c mediates the tumor suppressive effects of Wnt inhibitory factor 1 in human malignant salivary gland cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4298. doi:10.1158/1538-7445.AM2013-4298

Reduced WIF-1 Expression Stimulates Skin Hyperpigmentation in Patients with Melasma

The expression of Wnt inhibitory factor-1 (WIF-1) gene, which was detected by a microarray analysis of hyperpigmented and normally pigmented skin sets of melasma patients, was significantly reduced in the hyperpigmented skin from melasma patients, but not in healthy controls, regardless of UV irradiation. Wnt signals regulate skin pigmentation; however, WIF-1 is expressed in cultured skin keratinocytes and fibroblasts, but not in melanocytes. Therefore, we examined whether WIF-1 knockdown in neighboring keratinocytes and fibroblasts plays a role in melasma. Additionally, the effect of WIF-1 overexpression on the amelioration of hyperpigmentation was examined. WIF-1 knockdown, either in fibroblasts or in keratinocytes, significantly stimulated tyrosinase expression and melanosome transfer, whereas melanocytes with WIF-1 overexpression significantly reduced those parameters. The WIF-1 knockdown decreased glycogen synthase kinase-3β (GSK-3β), β-catenin, and NFATc2 (nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 2) phosphorylation and increased microphthalmia-associated transcription factor (MITF) expression as in melanocytes with Wnt-1 overexpression, whereas the WIF-1 overexpression reversed the results. Expression of Wnts, both canonical and noncanonical, was increased in the hyperpigmented skin of melasma patients. Collectively, WIF-1 downregulation, which may occur in epidermal keratinocytes and in dermal fibroblasts, is involved in melasma development because of the stimulation of melanogenesis and melanosome transfer through upregulation of the canonical and the noncanonical Wnt signaling pathway.

The Wnt inhibitory factor 1 (WIF1) is targeted in glioblastoma and has a tumor suppressing function potentially by induction of senescence

Gene expression-based prediction of genomic copy number aberrations in the chromosomal region 12q13 to 12q15 that is flanked by MDM2 and CDK4 identified Wnt inhibitory factor 1 (WIF1) as a candidate tumor suppressor gene in glioblastoma. WIF1 encodes a secreted Wnt antagonist and was strongly downregulated in most glioblastomas as compared with normal brain, implying deregulation of Wnt signaling, which is associated with cancer. WIF1 silencing was mediated by deletion (7/69, 10%) or epigenetic silencing by promoter hypermethylation (29/110, 26%). Co-amplification of MDM2 and CDK4 that is present in 10% of glioblastomas was associated in most cases with deletion of the whole genomic region enclosed, including the WIF1 locus. This interesting pathogenetic constellation targets the RB and p53 tumor suppressor pathways in tandem, while simultaneously activating oncogenic Wnt signaling. Ectopic expression of WIF1 in glioblastoma cell lines revealed a dose-dependent decrease of Wnt pathway activity. Furthermore, WIF1 expression inhibited cell proliferation in vitro, reduced anchorage-independent growth in soft agar, and completely abolished tumorigenicity in vivo. Interestingly, WIF1 overexpression in glioblastoma cells induced a senescence-like phenotype that was dose dependent. These results provide evidence that WIF1 has tumor suppressing properties. Downregulation of WIF1 in 75% of glioblastomas indicates frequent involvement of aberrant Wnt signaling and, hence, may render glioblastomas sensitive to inhibitors of Wnt signaling, potentially by diverting the tumor cells into a senescence-like state.

Wnt Inhibitory Factor 1 Decreases Tumorigenesis and Metastasis in Osteosarcoma

It has been reported that the progression of osteosarcoma was closely associated with the aberrant activation of canonical Wnt signaling. Wnt inhibitory factor-1 (WIF-1) is a secreted Wnt inhibitor whose role in human osteosarcoma remains unknown. In this study, WIF-1 expression in NHOst and osteosarcoma cell lines was determined by real-time reverse transcription-PCR, methylation-specific PCR, and Western blotting analysis. In addition, tissue array from patient samples was examined for WIF-1 expression by immunohistochemistry. Compared with normal human osteoblasts, WIF-1 mRNA and protein levels were significantly downregulated in several osteosarcoma cell lines. The downregulation of WIF-1 mRNA expression is associated with its promoter hypermethylation in these tested cell lines. Importantly, WIF-1 expression was also downregulated in 76% of examined osteosarcoma cases. These results suggest that the downregulation of WIF-1 expression plays a role in osteosarcoma progression. To further study the potential tumor suppressor function of WIF-1 in osteosarcoma, we established stable 143B cell lines overexpressing WIF-1. WIF-1 overexpression significantly decreased tumor growth rate in nude mice as examined by the s.c. injection of 143B cells stably transfected with WIF-1 and vector control. WIF-1 overexpression also markedly reduced the number of lung metastasis in vivo in an orthotopic mouse model of osteosarcoma. Together, these data suggest that WIF-1 exerts potent antiosteosarcoma effect in vivo in mouse models. Therefore, the reexpression of WIF-1 in WIF-1-deficient osteosarcoma represents a potential novel treatment and preventive strategy.

Role of Wnt inhibitory factor‐1 and Wnt/wingless signaling in choroid plexus tumors

Little is known on pathways involved in the pathogenesis of choroid plexus tumors (CPTs). The finding of overexpression of Wnt inhibitory factor-1 (Wif-1) prompted us to investigate the functional role of Wif-1 as well as nuclear accumulation of beta-catenin in CPT. In Z310 neoplastic choroid plexus epithelial cells, silencing of Wif1 expression increased proliferative activity not associated with increased canonical Wnt signaling. Nuclear beta-catenin accumulation was also lacking in a series of 16 CPT. In conclusion, our data show that Wif-1 inhibits proliferation of neoplastic choroid plexus epithelial cells, but argue against a role of canonical Wnt/wingless signaling in CPT.

Adiponectin stimulates Wnt inhibitory factor-1 expression through epigenetic regulations involving the transcription factor specificity protein 1

Adiponectin (ADN) is an adipokine possessing growth inhibitory activities against various types of cancer cells. Our previous results demonstrated that ADN could impede Wnt/beta-catenin-signaling pathways in MDA-MB-231 human breast carcinoma cells [Wang,Y. et al. (2006) Adiponectin modulates the glycogen synthase kinase-3 beta/beta-catenin signaling pathway and attenuates mammary tumorigenesis of MDA-MB-231 cells in nude mice. Cancer Res., 66, 11462-11470]. Here, we extended our studies to elucidate the effects of ADN on regulating the expressions of Wnt inhibitory factor-1 (WIF1), a Wnt antagonist frequently silenced in human breast tumors. Our results showed that ADN time dependently stimulated WIF1 gene and protein expressions in MDA-MB-231 cells. Overexpression of WIF1 exerted similar inhibitory effects to those of ADN on cell proliferations, nuclear beta-catenin activities, cyclin D1 expressions and serum-induced phosphorylations of Akt and glycogen synthase kinase-3 beta. Blockage of WIF1 activities significantly attenuated the suppressive effects of ADN on MDA-MB-231 cell growth. Furthermore, our in vivo studies showed that both supplementation of recombinant ADN and adenovirus-mediated overexpression of this adipokine substantially enhanced WIF1 expressions in MDA-MB-231 tumors implanted in nude mice. More interestingly, we found that ADN could alleviate methylation of CpG islands located within the proximal promoter region of WIF1, possibly involving the specificity protein 1 (Sp1) transcription factor and its downstream target DNA methyltransferase 1 (DNMT1). Upon ADN treatment, the protein levels of both Sp1 and DNMT1 were significantly decreased. Using silencing RNA approaches, we confirmed that downregulation of Sp1 resulted in an increased expression of WIF1 and decreased methylation of WIF1 promoter. Taken together, these data suggest that ADN might elicit its antitumor activities at least partially through promoting WIF1 expressions.

Inhibition of Wnt signaling downregulates Akt activity and induces chemosensitivity in PTEN-mutated prostate cancer cells.

The cross-talk between Wnt signaling and the Akt pathway in prostate cancer (Pca) is still unclear. In the present study, we found that WIF-1 downregulates the Akt pathway and also enhances chemosensitivity in PTEN-null Pca cells. Wnt inhibitory factor-1 (WIF-1), an inhibitor of Wnt proteins, was transfected into PC-3 and DU145 Pca cells. Akt was phosphorylated in PTEN-null PC-3 cells but underphosphorylated in PTEN-expressed DU145 cells. The levels of phosphorylated Akt in WIF-1 overexpressing PC-3 cells were lower than those in native or control vector-transfected PC-3 cells. However, WIF-1 showed no additional inhibition of already reduced Akt activity in DU145 cells. Overexpression of WIF-1 resulted in sensitizing PC-3 cells for paclitaxel to induce apoptosis. DU145 cells were more sensitive to paclitaxel but were not affected by WIF-1 transfection. The PI3K inhibitor LY294002 seemed to restore the chemosensitivity of native PC-3 cells like WIF-1 did. Our results show that Wnt signaling is involved in Akt activation in Pca cells. Our data also indicate the possibility that Wnt and its signaling pathway can be therapeutic targets for PTEN-mutated advanced Pca.