TCF Reporter Activity Research Articles

Targeting transcription factor TCF4 by γ-Mangostin, a natural xanthone.

Given that colon cancer is the third most common cancer in incidence and cause of death in the United States, and current treatment modalities are insufficient, there is a need to develop novel agents. Towards this, here we focus on γ-Mangostin, a bioactive compound present in the Mangosteen (Garcinia mangostana) fruit. γ-Mangostin suppressed proliferation and colony formation, and induced cell cycle arrest and apoptosis of colon cancer cell lines. Further, γ-Mangostin inhibited colonosphere formation. Molecular docking and CETSA (Cellular thermal shift assay) binding assays demonstrated that γ-Mangostin interacts with transcription factor TCF4 (T-Cell Factor 4) at the β-catenin binding domain with the binding energy of -5.5 Kcal/mol. Moreover, γ-Mangostin treatment decreased TCF4 expression and reduced TCF reporter activity. The compound also suppressed the expression of Wnt signaling target proteins cyclin D1 and c-Myc, and stem cell markers such as LGR5, DCLK1 and CD44. To determine the effect of γ-Mangostin on tumor growth in vivo, we administered nude mice harboring HCT116 tumor xenografts with 5 mg/Kg of γ-Mangostin intraperitoneally for 21 days. γ-Mangostin treatment significantly suppressed tumor growth, with notably lowered tumor volume and weight. In addition, western blot analysis revealed a significant decrease in the expression of TCF4 and its downstream targets such as cyclin D1 and c-Myc. Together, these data suggest that γ-Mangostin inhibits colon cancer growth through targeting TCF4. γ-Mangostin may be a potential therapeutic agent for colon cancer.

Discovery of Novel Spiroindoline Derivatives as Selective Tankyrase Inhibitors.

The canonical WNT pathway plays an important role in cancer pathogenesis. Inhibition of poly(ADP-ribose) polymerase catalytic activity of the tankyrases (TNKS/TNKS2) has been reported to reduce the Wnt/β-catenin signal by preventing poly ADP-ribosylation-dependent degradation of AXIN, a negative regulator of Wnt/β-catenin signaling. With the goal of investigating the effects of tankyrase and Wnt pathway inhibition on tumor growth, we set out to find small-molecule inhibitors of TNKS/TNKS2 with suitable drug-like properties. Starting from 1a, a high-throughput screening hit, the spiroindoline derivative 40c (RK-287107) was discovered as a potent TNKS/TNKS2 inhibitor with >7000-fold selectivity against the PARP1 enzyme, which inhibits WNT-responsive TCF reporter activity and proliferation of human colorectal cancer cell line COLO-320DM. RK-287107 also demonstrated dose-dependent tumor growth inhibition in a mouse xenograft model. These observations suggest that RK-287107 is a promising lead compound for the development of novel tankyrase inhibitors as anticancer agents.

Targeting DDX3 in Medulloblastoma Using the Small Molecule Inhibitor RK-33

Medulloblastoma is the most common malignant tumor that arises from the cerebellum of the central nervous system. Clinically, medulloblastomas are treated by surgery, radiation, and chemotherapy, all of which result in toxicity and morbidity. Recent reports have identified that DDX3, a member of the RNA helicase family, is mutated in medulloblastoma. In this study, we demonstrate the role of DDX3 in driving medulloblastoma. With the use of a small molecule inhibitor of DDX3, RK-33, we could inhibit growth and promote cell death in two medulloblastoma cell lines, DAOY and UW228, with IC50 values of 2.5 μM and 3.5 μM, respectively. Treatment of DAOY and UW228 cells with RK-33 caused a G1 arrest, resulted in reduced TCF reporter activity, and reduced mRNA expression levels of downstream target genes of the WNT pathway, such as Axin2, CCND1, MYC, and Survivin. In addition, treatment of DAOY and UW228 cells with a combination of RK-33 and radiation exhibited a synergistic effect. Importantly, the combination of RK-33 and 5 Gy radiation caused tumor regression in a mouse xenograft model of medulloblastoma. Using immunohistochemistry, we observed DDX3 expression in both pediatric (55%) and adult (66%) medulloblastoma patients. Based on these results, we conclude that RK-33 is a promising radiosensitizing agent that inhibits DDX3 activity and down-regulates WNT/β-catenin signaling and could be used as a frontline therapeutic strategy for DDX3-expressing medulloblastomas in combination with radiation.

Abstract 4760: Wnt/β-catenin signaling mediates resistance of colorectal cancer cell lines to chemoradiotherapy

Abstract Background: Activation of Wnt/β-catenin signaling plays a central role in the development and progression of colorectal cancer (CRC). Previously, we demonstrated that the Wnt transcription factor TCF7L2 was overexpressed in primary rectal cancers that were resistant to chemoradiotherapy (CRT), and that TCF7L2 functionally mediates resistance of CRC to clinically relevant doses of CRT. However, it remained unclear whether the resistance was mediated by a TCF7L2 inherent mechanism or Wnt/β-catenin signaling in general. Methods: We silenced another key-component of canonical Wnt-signaling, CTNNB1 (β-catenin) in rectal cancer cell lines LS1034, SW480, and SW837 by RNAi. Afterwards cells were exposed to varying doses of irradiation (+/- 5 FU) to assess the influence of β-catenin on radiation response. Activation of Wnt-signaling in “normal” epithelial cells (retina pigment epithelial cells RPE) was achieved either by stimulation with Wnt-3A, or stable overexpression of non-degradable β-catenin (S33Y-mutated) and confirmed by a dual luciferase reporter assay. Changes in radio-(chemo-) sensitivity were analyzed again by a colony formation assay. SW1463 were repeatedly irradiated (68Gy) to establish an isogenic radio-resistant cell line. Gene-expression profiles, without or 6h after a single dose of 4Gy, of RPE cells expressing a control vector or overexpressing β-catenin and radioresistant or wildtype SW1463 cells were established using micro Arrays. Results: Silencing of CTNNB1 resulted in (chemo-) radiation-sensitization of all three CRC-cell lines. To further investigate the potential role of Wnt/β-catenin signaling in controlling therapeutic responsiveness, non-tumorigenic RPE cells were stimulated with Wnt-3A, which significantly increased resistance to CRT. This effect could be recapitulated by overexpression of β-catenin (S33Y-mut.), resulting in a significantly increased resistance to CRT. The effect could be rescued by siRNA mediated knockdown of β-catenin. Consistent with these findings, we observed higher expression levels of active (unphosphorylated) β-catenin as well as increased TCF reporter activity in SW1463 cells that were rendered radiation-resistant due to repeated IR treatment. Gene expression profiling of radioresistant and wildtype SW1463 as well as RPE cells overexpressing β-catenin or a control vector revealed significant transcriptomal changes. Most interestingly the resistant cells (radioresistant SW1463 and RPE overexpressing β-catenin) reacted with differential activation of metabolic, inflammatory, cell survival and cell cycle pathways to irradiation. Conclusion: Together, these findings strongly support the interpretation that Wnt/β-catenin signaling plays a central role in mediating resistance of CRC cells to CRT by deregulating essential pathways. Hence, pathway inhibition may represent a promising strategy to increase therapeutic responsiveness to CRT. Citation Format: Georg Emons, Melanie Spitzner, Sebastian Reineke, Noam Auslander, Frank Kramer, Margret Rave-Fraenk, Jochen Gaedcke, Michael Ghadimi, Thomas Ried, Marian Grade. Wnt/β-catenin signaling mediates resistance of colorectal cancer cell lines to chemoradiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4760. doi:10.1158/1538-7445.AM2017-4760

Abstract 1653: A novel role for Wnt/β-catenin signaling in mediating resistance of colorectal cancer to chemoradiotherapy

Abstract Background: Preoperative chemoradiotherapy represents the standard treatment for patients with rectal cancer. However, the clinical response of individual tumors to multimodal treatment is not uniform, and ranges from complete response to complete resistance. Therefore, the identification of novel therapeutic targets whose modification could be harnessed to sensitize a priori resistant tumors is exceedingly important. Previously, we demonstrated that the Wnt transcription factor TCF7L2 (also known as TCF4) was overexpressed in primary rectal cancers that were resistant to chemoradiotherapy (CRT), and that TCF7L2 functionally mediates resistance of CRC cells to clinically relevant doses of ionizing radiation (IR). Methods: Using siRNAs we silenced CTNNB1 (β-catenin), another key-component of canonical Wnt-signaling, in colorectal cancer cell lines LS1034, SW480, and SW837. To asses influence on CRT, cells were exposed to 0, 1, 2, 4, 6 and 8 Gy of X-rays and 5-FU. Wnt- signaling was stimulated in retinal pigment epithelial cells (RPE) either by adding Wnt-3A, or overexpressing non degradable β-catenin (S33Y-mutated) and analyzed changes in CRT. Finally we repetitively irradiated SW1463 (68Gy) to establish an isogenic radio-resistant cell line and examined changes in protein expression. Results: Silencing of CTNNB1 resulted in (chemo-) radiation-sensitization of all three CRC-cell lines. To further investigate the potential role of Wnt/β-catenin signaling in controlling therapeutic responsiveness, non-tumorigenic RPE cells were stimulated with Wnt-3A, which significantly increased resistance to CRT. This effect could be recapitulated by overexpression of β-catenin (S33Y-mutated), resulting in a significantly increased resistance to CRT. The effect could be rescued by siRNA mediated knockdown of β-catenin. Consistent with these findings, we observed higher expression levels of active (unphosphorylated) β-catenin as well as increased TCF reporter activity in SW1463 cells that were rendered radiation-resistant due to repeated IR treatment. Conclusion: Together, these findings strongly support the interpretation that Wnt/β-catenin signaling plays a central role in mediating resistance of CRC cells to CRT. Hence, pathway inhibition may represent a promising strategy to increase therapeutic responsiveness to CRT, which represents the standard treatment for locally advanced rectal cancers. This would have considerable clinical implications. Citation Format: Georg Emons, Melanie Spitzner, Sebastian Reineke, Frank Kramer, Margret Rave-Fraenk, Jochen Gaedcke, Michael Ghadimi, Thomas Ried, Marian Grade. A novel role for Wnt/β-catenin signaling in mediating resistance of colorectal cancer to chemoradiotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1653.

Human mesenchymal stem cell-engineered hepatic cell sheets accelerate liver regeneration in mice.

Mesenchymal stem cells (MSCs) are an attractive cell source for cell therapy. Based on our hypothesis that suppression of Wnt/β-catenin signal enhances hepatic differentiation of human MSCs, we developed human mesenchymal stem cell-engineered hepatic cell sheets by a small molecule compound. Screening of 10 small molecule compounds was performed by WST assay, TCF reporter assay, and albumin mRNA expression. Consequently, hexachlorophene suppressed TCF reporter activity in time- and concentration-dependent manner. Hexachlorophene rapidly induced hepatic differentiation of human MSCs judging from expression of liver-specific genes and proteins, PAS staining, and urea production. The effect of orthotopic transplantation of human mesenchymal stem cell-engineered hepatic cell sheets against acute liver injury was examined in one-layered to three-layered cell sheets system. Transplantation of human mesenchymal stem cell-engineered hepatic cell sheets enhanced liver regeneration and suppressed liver injury. The survival rates of the mice were significantly improved. High expression of complement C3 and its downstream signals including C5a, NF-κB, and IL-6/STAT-3 pathway was observed in hepatic cell sheets-grafted tissues. Expression of phosphorylated EGFR and thioredoxin is enhanced, resulting in reduction of oxidative stress. These findings suggest that orthotopic transplantation of hepatic cell sheets manufactured from MSCs accelerates liver regeneration through complement C3, EGFR and thioredoxin.

Abstract 3570: Identification of the DEAD box RNA helicase DDX3 as a therapeutic target in colorectal cancer

Abstract Over 85% of colorectal cancers is driven by aberrations in the Wnt-signaling pathway. Thus, identifying druggable targets in this pathway can be beneficial for optimizing colorectal cancer treatment. Within this context, a member of the RNA helicase gene family, DDX3, has been identified to exhibit oncogenic properties in breast and lung carcinomas as well as medulloblastomas. Notably, recent studies have identified DDX3 as a multilevel activator of Wnt-signaling in both normal and transformed cells without activating mutations in the Wnt signaling pathway. In this study, we evaluated whether DDX3 also plays a role in the constitutionally activated Wnt-signaling that drives colorectal cancer and therefore could be a potential therapeutic target in this cancer type. To determine if DDX3 is expressed in colorectal cancers, we immunohistochemically stained a cohort of 303 Dutch and German colorectal cancer patients. We found 40.4% of these tumors to overexpress DDX3 in comparison to the surrounding normal tissue. DDX3 expression was found predominantly in the cytoplasm and occasionally in the nucleus. High cytoplasmic DDX3 expression correlated with nuclear Beta-catenin expression, a marker of activated Wnt-signaling. The presence of nuclear DDX3 expression correlated with shorter overall survival (HR = 2.38, 95% CI 1.45-3.93, p < 0.001). Functionally, we validated these findings in vitro and found that inhibition of DDX3 with siRNA resulted in reduced proliferation and a G1-arrest in the HCT116 and HT29 colorectal cancer cell lines. This finding further supports the potential oncogenic role of DDX3 in colorectal cancer. With respect to targeting DDX3, we developed a small molecule inhibitor of DDX3, referred to as RK-33. RK-33 is designed to bind to the ATP-binding site of DDX3 and abrogate its functional activity. As proof of principle, we demonstrated that RK-33 binds preferentially to DDX3 and not to DDX5 and DDX17, other members of the RNA helicase family. Moreover, RK-33 inhibited the helicase activity in an in vitro assay. Furthermore, treatment of colorectal cancer cell lines and patient derived 3D- tumor cell cultures indicated that RK-33 inhibits growth and promotes cell death with IC-50 values ranging from 2.5 to 8 uM. To further elucidate the mechanism of RK-33, we studied if inhibition of DDX3 with RK-33 could cause inhibition of Wnt-signaling in colorectal cancer cell lines. Treatment with RK-33 indeed resulted in reduced TCF-reporter activity and lowered the mRNA expression levels of the Wnt-signaling downstream target genes AXIN-2, C-MYC, CCND1 and BIRC5A. Overall, we conclude that DDX3 has an oncogenic role in colorectal cancer. Inhibition of DDX3 with the small molecule inhibitor RK-33 causes potent inhibition of Wnt-signaling and is a promising future treatment strategy in colorectal cancer. Citation Format: Marise R. Heerma van Voss, Farhad Vesuna, Kari Trumpi, Justin Brilliant, Liudmila L. Kodach, Folkert H.M. Morsink, G. Johan A. Offerhaus, Horst Buerger, Elsken van der Wall, Paul J. van Diest, Venu Raman. Identification of the DEAD box RNA helicase DDX3 as a therapeutic target in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3570. doi:10.1158/1538-7445.AM2015-3570

Abstract 4394: Genistein downregulates onco-miR-1260b and inhibits Wnt-signaling in renal cancer cells

Abstract Introduction and Objective: Wnt signaling plays an important role in renal cancer and that is modulated by genistein in other cancers. Recently microRNAs have emerged as new regulators of gene expression. Thus we focused on microRNAs to examine the regulatory mechanism of genistein on the Wnt signaling pathway in RCC. Methods: Initially we investigated the effect of genistein on Wnt signaling (TCF reporter assays) in renal cancer cells and using microarray identified candidate miRNAs whose expression was decreased by genistein. We performed functional analyses and investigated the relationship between miRNA expression and renal cancer patient outcomes. We also did 3′UTR luciferase assays to look at direct miRNA regulation of Wnt signaling related genes. Results: Genistein promoted apoptosis while inhibiting RCC cell proliferation and invasion. Genistein also decreased TCF reporter activity in RCC cells. We found that miR-1260b was highly expressed and significantly down-regulated by genistein in RCC cells. The expression of miR-1260b was significantly higher in renal cancer tissues compared to normal and significantly related to over-all shorter survival. Additionally miR-1260b promoted renal cancer cell proliferation and invasion in RCC cells. The 3′UTR luciferase activity of target genes (sFRP1, Dkk2, Smad4) was significantly decreased and their protein expression significantly up-regulated in miR-1260b inhibitor transfected renal cancer cells. Conclusions: Our data suggests that genistein inhibited Wnt signaling by regulating miR-1260b expression in renal cancer cells. Source of Funding: Grants RO1CA138642, RO1CA130860, RO1CA160079, I01BX001123, VA Merit Review and VA Program Project (PI: R. Dahiya). Citation Format: Hiroshi Hirata, Koji Ueno, Yuichiro Tanaka, Z.Laura Tabatabai, Koichi Nakajima, Yuji Hinoda, Nobuhisa Ishii, Rajvir Dahiya. Genistein downregulates onco-miR-1260b and inhibits Wnt-signaling in renal cancer cells. [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 4394. doi:10.1158/1538-7445.AM2014-4394

Ligand-independent canonical Wnt activity in canine mammary tumor cell lines associated with aberrant LEF1 expression.

Pet dogs very frequently develop spontaneous mammary tumors and have been suggested as a good model organism for breast cancer research. In order to obtain an insight into underlying signaling mechanisms during canine mammary tumorigenesis, in this study we assessed the incidence and the mechanism of canonical Wnt activation in a panel of 12 canine mammary tumor cell lines. We show that a subset of canine mammary cell lines exhibit a moderate canonical Wnt activity that is dependent on Wnt ligands, similar to what has been described in human breast cancer cell lines. In addition, three of the tested canine mammary cell lines have a high canonical Wnt activity that is not responsive to inhibitors of Wnt ligand secretion. Tumor cell lines with highly active canonical Wnt signaling often carry mutations in key members of the Wnt signaling cascade. These cell lines, however, carry no mutations in the coding regions of intracellular Wnt pathway components (APC, β-catenin, GSK3β, CK1α and Axin1) and have a functional β-catenin destruction complex. Interestingly, however, the cell lines with high canonical Wnt activity specifically overexpress LEF1 mRNA and the knock-down of LEF1 significantly inhibits TCF-reporter activity. In addition, LEF1 is overexpressed in a subset of canine mammary carcinomas, implicating LEF1 in ligand-independent activation of canonical Wnt signaling in canine mammary tumors. We conclude that canonical Wnt activation may be a frequent event in canine mammary tumors both through Wnt ligand-dependent and novel ligand–independent mechanisms.

Genistein downregulates onco-miR-1260b and upregulates sFRP1 and Smad4 via demethylation and histone modification in prostate cancer cells.

Background:Recently several microRNAs (miRNAs) have been found to be regulated by genistein in cancer cells. In this study, we focused on the gene regulatory effect of genistein on microRNA and its target genes in prostate cancer (PC).Methods:Initially, we investigated the effect of genistein on prostate cancer cells and identified that the expression of miRNA-1260b was decreased by genistein. We performed functional analyses and investigated the relationship between miRNA-1260b expression and prostate cancer patient outcomes. Two target genes (sFRP1 and Smad4) of miR-1260b were identified based on computer algorithm and 3′UTR luciferase assay was carried out to determine direct miRNA regulation of the genes.Results:Genistein promoted apoptosis while inhibiting prostate cancer cell proliferation, invasion and TCF reporter activity in PC cells. MiR-1260b was highly expressed in prostate cancer tissues and significantly downregulated by genistein in PC cells. After knocking down miR-1260b, cell proliferation, invasion, migration and TCF reporter activity were decreased in PC cells. Western analysis and 3′UTR luciferase assay showed that the two target genes (sFRP1 and Smad4) were directly regulated by miR-1260b. The expression of sFRP1 and Smad4 was significantly decreased in prostate cancer tissues. Genistein also increased expression of these two genes via DNA demethylation and histone modifications.Conclusions:Our data suggest that genistein exerts its anti-tumour effect via downregulation of miR-1260b that targeted sRRP1 and Smad4 genes in prostate cancer cells. The expression of sFRP1 and Smad4 was also modulated by genistein via DNA methylation or histone modifications in PC cell lines.

Genistein downregulates onco-miR-1260b and inhibits Wnt-signalling in renal cancer cells

Background:Wnt-signalling has an important role in renal cancer and it is modulated by genistein in other cancers. Recently, microRNAs (miRNAs) have emerged as new regulators of gene expression. Thus, we focused on miRNAs to examine the regulatory mechanism of genistein on the Wnt-signalling pathway in renal cell carcinoma (RCC).Methods:Initially, we investigated the effect of genistein on Wnt-signalling (TOPflash reporter assay (TCF reporter assays)) in renal cancer cells, and using microarray identified candidate miRNAs whose expression was decreased by genistein. We performed functional analyses and investigated the relationship between miRNA expression and renal cancer patient outcomes. We also did 3′UTR luciferase assays to look at direct miRNA regulation of Wnt-signalling-related genes.Results:Genistein promoted apoptosis while inhibiting RCC cell proliferation and invasion. Genistein also decreased TCF reporter activity in RCC cells. We found that miR-1260b was highly expressed and significantly downregulated by genistein in RCC cells. The expression of miR-1260b was significantly higher in renal cancer tissues compared with normal, and significantly related to overall shorter survival. In addition, miR-1260b promoted renal cancer cell proliferation and invasion in RCC cells. The 3′UTR luciferase activity of target genes (sFRP1, Dkk2, Smad4) was significantly decreased and their protein expression significantly upregulated in miR-1260b inhibitor-transfected renal cancer cells.Conclusion:Our data suggest that genistein inhibited Wnt-signalling by regulating miR-1260b expression in renal cancer cells.

Plakoglobin at adherens junctions after β‐catenin loss: Mechanism & biological implications in hepatocellular carcinoma

β‐Catenin (β‐cat) is important in liver homeostasis as a part of Wnt signaling & cell‐cell adhesions, while aberrant activation is observed in hepatocellular carcinoma (HCC). We previously reported that hepatocyte‐specific β‐cat knockout mice (KO) lack adhesive defects, since its loss is compensated by increased plakoglobin (PG; γ‐catenin) at adherens junctions (AJ) over wildtype (WT) livers. Since PG is a desmosomal protein we asked if its increase in KOs might affect the junction. We saw no change in desmosomal ultrastructure (via TEM), proteins, or their association with PG, besides an increase in Plakophilin‐3. We then utilized an in vitro model to further elucidate the role & regulation of PG after β‐cat knockdown (KD). PG is unable to rescue β‐cat‐ TCF reporter activity; however, it suitably compensates for β‐cat loss at AJs as assessed by scratch‐wound assay, centrifugal assay for cell adhesion & hanging‐drop assay. PG increase is observed in HCC cells only upon decrease in β‐cat protein levels, not after blockade of β‐cat's transactivational activity. There was enhanced serine & threonine phosphorylation of PG after β‐cat KD which was abrogated by inhibiting protein kinases A (PKA) & PKG. In conclusion, PG compensates for β‐cat loss at the AJ without affecting desmosomes, but is unable to fulfill functions in Wnt signaling. Catenin‐sensing mechanism depends on β‐cat protein levels & not activity. Anti‐β‐cat therapies for HCC that affect β‐cat levels may target aberrant Wnt signaling without negatively impacting intercellular adhesion, if mechanisms responsible for PG stabilization are spared.

LGR5 is Expressed by Ewing Sarcoma and Potentiates Wnt/β-Catenin Signaling

Ewing sarcoma (ES) is an aggressive bone and soft tissue tumor of putative stem cell origin that predominantly occurs in children and young adults. Although most patients with localized ES can be cured with intensive therapy, the clinical course is variable and up to one third of patients relapse following initial remission. Unfortunately, little is yet known about the biologic features that distinguish low-risk from high-risk disease or the mechanisms of ES disease progression. Recent reports have suggested that putative cancer stem cells exist in ES and may contribute to an aggressive phenotype. The cell surface receptor leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) is a somatic stem cell marker that functions as an oncogene in several human cancers, most notably colorectal carcinoma. LGR5 is a receptor for the R-spondin (RSPO) family of ligands and RSPO-mediated activation of LGR5 potentiates Wnt/β-catenin signaling, contributing to stem cell proliferation and self-renewal. Given its presumed stem cell origin, we investigated whether LGR5 contributes to ES pathogenesis. We found that LGR5 is expressed by ES and that its expression is relatively increased in cells and tumors that display a more aggressive phenotype. In particular, LGR5 expression was increased in putative cancer stem cells. We also found that neural crest-derived stem cells express LGR5, raising the possibility that expression of LGR5 may be a feature of ES cells of origin. LGR5-high ES cells showed nuclear localization of β-catenin and robust activation of TCF reporter activity when exposed to Wnt ligand and this was potentiated by RSPO. However, modulation of LGR5 or exposure to RSPO had no impact on proliferation confirming that Wnt/β-catenin signaling in ES cells does not recapitulate signaling in epithelial cells. Together these studies show that the RSPO-LGR5-Wnt-β-catenin axis is present and active in ES and may contribute to tumor pathogenesis.

Identification of the Wnt Signalling Protein, TCF7L2 As a Significantly Overexpressed Transcription Factor in AML

AbstractAbstract 1281Acute Myeloid Leukaemia (AML) is one of the most genetically heterogeneous malignancies with the defining feature of inhibition of terminal myeloid development arising from a disordered development programme. To discover the most commonly dysregulated genes in this disease we compared Affymetrix GC-RMA normalized mRNA expression data of normal human hematopoietic progenitor (CD34+) cells with diagnostic samples from 223 AML patients previously enrolled in AML NCRI-MRC UK Trials. We used Mixed Model ANOVA and MetaCore™ to identify dysregulated gene networks. Processes associated with Wnt signalling were found to be the most significantly dysregulated: Wnt and cytoskeletal remodelling (p=1.16−26) and; developmental Wnt signalling (p=2.91−16). Interactome analysis identified TCF7L2 (aka TCF4) as the most significantly dysregulated Wnt factor (p=7.56−25; z-score = 19.87) which showed elevated expression in 78 percent of AML patients. The TCF7L2/β-catenin complex is a nuclear regulator of canonical Wnt signalling that is involved in the development of several cancers. To validate this result we examined the expression of TCF7L2 by RQ-PCR using matched AML samples (n=13). These data significantly correlated with the normalized array expression data (R= 0.711; p=0.006) which in turn correlated with total protein expression determined by Western blotting (n=16);R=0.598; p= 0.014 (see also below). To gain further insights into the prognostic impact of TCF7L2 expression at diagnosis in AML, we next examined a number a variables including patients’ age, leukocytes count, prognostic group, gender, WHO performance in multivariate analysis. We have shown that TCF7L2 over expression was found to be independently prognostic for reduced complete remission rate after the adjustment for these co-variables (p=0.0144), OR=5.19 [95% C.I.=1.39–19.39].The TCF7L2 gene undergoes exon splicing which yields multiple isoforms which have been reported to yield functionally distinct proteins. We therefore compared TCF7L2 mRNA isoform expression in AML patients with normal human bone marrow and progenitor cells. As expected we found extensive variability of TCF7L2 splicing particularly at the 3’ end of the gene (exon 13–18). Bone marrow and progenitor cells isoforms had distinct exon composition (with respect to exon 4) suggesting developmental regulation of isoform expression. AML patients were heterogeneous in the isoform expression pattern, but aberrant exon composition (compared to normal cells) was not observed. At the protein level we detected expression of 72kDa (corresponding to predicted exon composition 1–15 and 18) and 58kDa (exons 1–13 and 18) protein isoforms. In both normal and AML cells, expression of the 58kDa form was dominant.To determine the functional significance of TCF7L2 overexpression we employed lentiviral shRNA vectors targeting TCF7L2 isoforms in leukemic cell lines (K562 and U937) engineered to co-express a TCF-GFP reporter which enabled simultaneous analysis of the effect on TCF-dependent transcription. TCF reporter activity was inhibited by shRNA expression and the cells became non-responsive to Wnt agonists (Wnt3A and BIO) demonstrating that canonical Wnt signalling is dependent on TCF7L2 expression in these cells. Phenotypically, shRNA expression was found to strongly inhibit proliferation and to promote apoptosis indicating that TCF7L2 expression is required for the proliferation and survival of myeloid leukemia cells. In summary these data identify Wnt signalling as the most commonly dysregulated process in AML and show that one of key mediator of canonical Wnt signalling, TCF7L2, is overexpressed in AML, is associated with poor clinical outcome, and is functionally required to maintain the proliferation and viability of myeloid leukemia cells. Disclosures:No relevant conflicts of interest to declare.

Structural and functional implications of plakoglobin compensation due to β‐catenin loss in the liver

β‐Catenin is important in liver homeostasis as a part of Wnt signaling and cell‐cell adhesions. We previously reported that hepatocyte‐specific β‐catenin knockout (KO) mice show higher plakoglobin (PG; γ‐catenin) levels over wild‐type (WT) livers and also maintain intact adherens junctions (AJ) as a result of PG compensation. Since PG is a desmosomal protein we wanted to see if its increase in the KOs comes at the expense of desmosomes. Here we show no change in the desmosomes in the KO livers by protein analysis and TEM. Next we wanted to address functionality of PG's compensation for β‐catenin loss especially in Wnt signaling. We show that knocking down PG in Hep3B human hepatoma cells only slightly decreases TCF reporter (TOPflash) activity, indicating that PG is able bind TCF and participate in Wnt signaling. However, knock down of β‐catenin shows a dramatically greater drop in TOPflash activity. Next, we wanted to determine if changes in PG affect proliferation and migration. Changes in PG levels do not affect thymidine incorporation rates. However, scratch‐wound assays reveal that β‐catenin knock down (and subsequent PG increase) increases migration of Hep3Bs. Also, PG knock down decreases migration, indicating that PG may play a role in cell motility. Thus, β‐catenin loss is compensated by PG at the AJ without affecting desmosomes; however, the functions of β‐catenin as a part of the Wnt pathway remain unfulfilled by PG. Moreover, changes in PG levels also affect the migratory potential of human hepatoma cells. Research support: NIH grant 1R01DK62277.

High-Frequency Canonical Wnt Activation in Multiple Sarcoma Subtypes Drives Proliferation through a TCF/β-Catenin Target Gene, CDC25A

Wnt canonical signaling is critical for normal development as well as homeostasis of several epithelial tissues, and constitutive activation of this pathway is commonly observed in carcinomas. We show here that 50% of human sarcomas (n = 45) and 65% of sarcoma cell lines (n = 23) of diverse histological subtypes exhibit upregulated autocrine canonical Wnt signaling. Furthermore, in Wnt autocrine cell lines, we identify alterations including overexpression or gene amplification of Wnt ligands and/or LRP5/6 coreceptors and epigenetic silencing of different cell surface Wnt antagonists. Mutations in adenomatous polyposis coli (APC) gene were observed in two nonautocrine Wnt-positive sarcoma cell lines. Finally, downregulation of the activated Wnt pathway inhibited sarcoma cell proliferation both in vitro and in vivo by a mechanism involving the downregulation of CDC25A.

Abstract 5002: Functional significance and mechanisms of inactivation of Secreted Wnt antagonist DICKKOPF-3 (DKK-3) gene in human renal cell carcinoma

Abstract Introduction: The Wnt signaling pathway is activated in most cancers while Wnt antagonist genes are inactivated. It has been reported that mRNA expression of Wnt antagonists is regulated by promoter methylation in many cancers. However the functional significance and mechanisms of inactivation of Secreted Wnt antagonist DKK-3 gene in kidney cancer has not been reported. Methods : DKK-1, DKK-2 and DKK-3 mRNA expression levels were detected by using real-time PCR. RCC cells were incubated with 5-aza-2′-deoxycytidine or with TSA. The mRNA expression of apoptosis and cell cycle genes analyzed by real-time PCR 72 hr after transfection. Chromatin DNA was immunoprecipitated with antibodies for histone H3 (H3), and tri-methyl-histone H3 (Lys4) (H3K4). RCC cell lines were transiently cotransfected with either mock or DKK-3, TOPflash reporter plasmid, and pRL-TK plasmid encoding Renilla luciferase as an internal control for transfection efficiency. Cell lysates were measured for relative luciferase activities. Cell viability was analyzed by the MTS assay after transfection. Stable transfectants were used for cell cycle and apoptosis analysis using a Cell Lab Quanta SC Flow Cytometer. Mock and DKK-3 stable cells were injected subcutaneously into the left and right back side flanks of nu/nu mice, respectively. Results : DKK-3 mRNA expression in Caki-2, A498 and 769-P RCC cells was lower than that of normal kidney HK-2 cells. The DKK-3 promoter was methylated in RCC cells, but unmethyalted in normal kidney HK-2 cells. Expression of DKK-1 and DKK-2 mRNA was upregulated after treating RCC cells with 5-Aza-2′-deoxycytidine, but DKK-3 mRNA levels were not affected. In contrast, the level of DKK-3 mRNA was upregulated after TSA treatment, whereas these of DKK-1 and DKK-2 were not. The level of H3K4 in A-498 and 769-P cells was higher compared with HK-2 cells after TSA treatment, while that of H3 in A-498 and 769-P cells were not changed. Expression of cell cycle and apoptosis related genes showed that p21, MDM-2, Puma and Bik genes were increased after transfecting RCC cell lines with a DKK-3 expression plasmid. DKK-3 did not inhibit the Tcf-reporter transcriptional activity in RCC cells, but decreased the cell viability of A-498 and 769-P cells. DKK-3 stable transfectants increased the rate of apoptosis and induced G2/M arrest together with an increase in p21 expression. Growth of stable DKK-3 transfected cells in nude mice was decreased compared to controls. Conclusions : Our data suggests for the first time that mRNA expression of DKK-3 is regulated by histone modification and that DKK-3 inhibits cell growth and induces apoptosis in RCC cell. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5002.

PKG inhibits TCF signaling in colon cancer cells by blocking β-catenin expression and activating FOXO4

Activation of cGMP-dependent protein kinase (PKG) has anti-tumor effects in colon cancer cells but the mechanisms are not fully understood. This study has examined the regulation of beta-catenin/TCF signaling, as this pathway has been highlighted as central to the anti-tumor effects of PKG. We show that PKG activation in SW620 cells results in reduced beta-catenin expression and a dramatic inhibition of TCF-dependent transcription. PKG did not affect protein stability, nor did it increase phosphorylation of the amino-terminal Ser33/37/Thr41 residues that are known to target beta-catenin for degradation. However, we found that PKG potently inhibited transcription from a luciferase reporter driven by the human CTNNB1 promoter, and this corresponded to reduced beta-catenin mRNA levels. Although PKG was able to inhibit transcription from both the CTNNB1 and TCF reporters, the effect on protein levels was less consistent. Ectopic PKG had a marginal effect on beta-catenin protein levels in SW480 and HCT116 but was able to inhibit TCF-reporter activity by over 80%. Investigation of alternative mechanisms revealed that cJun-N-terminal kinase (JNK) activation was required for the PKG-dependent regulation of TCF activity. PKG activation caused beta-catenin to bind to FOXO4 in colon cancer cells, and this required JNK. Activation of PKG was also found to increase the nuclear content of FOXO4 and increase the expression of the FOXO target genes MnSOD and catalase. FOXO4 activation was required for the inhibition of TCF activity as FOXO4-specific short-interfering RNA completely blocked the inhibitory effect of PKG. These data illustrate a dual-inhibitory effect of PKG on TCF activity in colon cancer cells that involves reduced expression of beta-catenin at the transcriptional level, and also beta-catenin sequestration by FOXO4 activation.

Procaine and procainamide inhibit the Wnt canonical pathway by promoter demethylation of WIF-1 in lung cancer cells

Secreted Wingless type (Wnt) ligands have previously been shown to be involved in tumor developmental processes and oncogenesis. Aberrant promoter methylation of Wnt inhibitory factor-1 (WIF-1) is a fundamental mechanism of epigenetic silencing in human cancers. Procaine, a local anesthetic drug, and procainamide, a drug for the treatment of cardiac arrhythmias, have been reported as inhibitors of DNA methylation, causing demethylation and reactivation of methylation-silenced genes such as RARbeta and GSTP1. The promoter demethylation of WIF-1 has not previously been reported on. We demonstrated previously that WIF-1 is silenced due to promoter hypermethylation in lung cancer cell lines. In this study, we demonstrate promoter demethylation of WIF-1; restoration of WIF-1 expression, and underexpression of cytosolic beta-catenin protein and TCF reporter activity, after procaine and procainamide treatment in H460 and A549 cell lines. Our results provide the first evidence that procaine and procainamide reactivate WIF-1 in these cancer cells and downregulate the Wnt canonical pathway. These results further suggest that procaine and procainamide may have a potential use for preventing the development of lung cancer.

The canonical Wnt signaling pathway plays an important role in lymphopoiesis and hematopoiesis

The evolutionarily conserved canonical Wnt-beta-catenin-T cell factor (TCF)/lymphocyte enhancer binding factor (LEF) signaling pathway regulates key checkpoints in the development of various tissues. Therefore, it is not surprising that a large body of gain-of-function and loss-of-function studies implicate Wnt-beta-catenin signaling in lymphopoiesis and hematopoiesis. In contrast, recent papers have reported that Mx-Cre-mediated conditional deletion of beta-catenin and/or its homolog gamma-catenin (plakoglobin) did not impair hematopoiesis or lymphopoiesis. However, these studies also report that TCF reporter activity remains active in beta-catenin- and gamma-catenin-deficient hematopoietic stem cells and all cells derived from these precursors, indicating that the canonical Wnt signaling pathway was not abrogated. Therefore, these studies in fact show that the canonical Wnt signaling pathway is important in hematopoiesis and lymphopoiesis, even though the molecular basis for the induction of the reporter activity is currently unknown. In this perspective, we provide a broad background to the field with a discussion of the available data and create a framework within which the available and future studies may be evaluated.