Aberrant Β-catenin Signaling Research Articles

Abstract 280: Aberrant β-catenin signaling from the bone marrow niche leads to transformation of IDH1/2-mutant hematopoietic stem cells and compromises response to IDH1/2 inhibitors

Abstract Our purpose is to identify stromal-derived signals required for IDH1/2-mut hematopoietic stem cells (HSCs) to become leukemogenic. Mutations in IDH genes are found in 20% of acute myeloid leukemia (AML) patients and are not transformative. They produce the oncometabolite 2HG which inhibits key epigenetic regulators. We explored whether epigenetic changes in the bone marrow (BM) niche could provide cooperative signals to IDH-mut HSCs leading to their transformation and disease development. We found that 2HG is elevated in the BM plasma of MDS/AML patients with IDH1/2 mutations as compared to IDH-wt patients. Interestingly, 2HG is taken up by BM stromal cells (BMSCs) suggesting it may induce aberrant methylation patterns in them. Indeed, methylome analysis showed hypermethylation in BMSCs of AML patients with IDH1/2 mutations compared to wt. Among the most hypermethylated genes in the IDH-mut stroma are AXIN1, AXIN2 and NDK2, all negative regulators of β-catenin; this pathway activation was previously shown to cause MDS/AML in mice and present in 38% of patients. In agreement with these findings βcat activation in the stroma of IDH-mut vs IDH-wt AML patients showed a strong positive correlation as 50% of IDH-mut patients had activated βcat in their BMSCs. Inquiring whether stroma methylation precedes HSC transformation, we analyzed samples from healthy individuals with IDH-mut clonal hematopoiesis. They exhibited increased global and AXIN1 promoter methylation compared to samples without such clones, but not active βcat in osteoblasts, indicating this might be a later step along with disease development. In fact, transplantation of HSCs from Idh2-mut mice to recipients expressing active βcat in osteoblasts led to increase in blasts and shortened survival compared to wt mice transplanted with Idh2-mut HSCs. As many patients relapse following treatment with IDH inhibitors, we investigated a potential role for βcat signaling in treatment outcome. IDH-mut AML patients who do not respond to treatment with the IDHi present more frequently with activated βcat in osteoblasts compared to responders. Analyzing samples from the same patient pre- and post-treatment with IDHi, we found before treatment most patients were positive for βcat stromal activation, whereas after treatment, all patients with complete response were negative, while patients with progressive disease remained positive. Our model suggests IDH-mut HSCs secrete 2HG inducing epigenetic alterations in the niche, some of which cause activation of the βcat pathway. In turn, this additional signal leads to the transformation of HSCs to dysplastic cells and MDS/AML development. This work uncovers a new mechanism for transformation to MDS/AML and resistance to IDHi treatment. Thus, targeting βcat activation in the stroma, in combination with IDHi, could improve treatment outcome. Citation Format: Paraskevi Vgenopoulou, Luis Flores, Ioanna Mosialou, Junfei Zhao, X. Shawn Liu, Pamela Sung, Virginie Penard-Lacronique, Martin Carroll, Stavroula Kousteni. Aberrant β-catenin signaling from the bone marrow niche leads to transformation of IDH1/2-mutant hematopoietic stem cells and compromises response to IDH1/2 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 280.

Mitoquinone ameliorated airway inflammation by stabilizing β-catenin destruction complex in a steroid-insensitive asthma model

Background and purposeMitochondrial dysfunction is an essential part of the pathophysiology of asthma, and potential treatments that target the malfunctioning mitochondria have attracted widespread attention. We have previously demonstrated that aberrant epithelial β-catenin signaling played a crucial role in a toluene diisocyanate (TDI)-induced steroid-insensitive asthma model. The objective of this study was to determine if the mitochondrially targeted antioxidant mitoquinone(MitoQ) regulated the activation of β-catenin in TDI-induced asthma. MethodMice were sensitized and challenged with TDI to generate a steroid-insensitive asthma model. Human bronchial epithelial cells (16HBE) were exposed to TDI-human serum albumin (HSA) and ethidium bromide(EB) to simulate the TDI-induced asthma model and mitochondrial dysfunction. ResultsMitoQ dramatically attenuated TDI-induced AHR, airway inflammation, airway goblet cell metaplasia, and collagen deposition and markedly protected epithelial mitochondrial functions by preserving mass and diminishing the production of reactive oxygen species (ROS). MitoQ administration stabilized β‐catenin destruction complex from disintegration and inhibited the activation of β‐catenin. Similarly, YAP1, an important constituent of β‐catenin destruction complex, was inhibited by Dasatinib, which alleviated airway inflammation and the activation of β‐catenin, and restored mitochondrial mass. In vitro, treating 16HBE cells with EB led to the activation of YAP1 and β-catenin signaling, decreased the expression of glucocorticoid receptors and up-regulated interleukin (IL)-1β, IL6 and IL-8 expression. ConclusionOur results indicated that mitochondria mediates airway inflammation by regulating the stability of the β-catenin destruction complex and MitoQ might be a promising therapeutic approach to improve airway inflammation and severe asthma. Availability of data and materialsThe data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.

Transcriptome sequencing analysis of the role of β-catenin in F-actin reorganization in embryonic palatal mesenchymal cells.

Palatogenesis is a highly regulated and coordinated developmental process that is coordinated by multiple transcription factors and signaling pathways. Our previous studies identified that defective palatal shelf reorientation due to aberrant mesenchymal β-catenin signaling is associated with Filamentous actin (F-actin) dysregulation. Herein, the underlying mechanism of mesenchymal β-catenin in regulating F-actin cytoskeleton reorganization is further investigated. Firstly, β-catenin silenced and overexpressed mouse embryonic palatal mesenchymal (MEPM) cells were established by adenovirus-mediated transduction. Subsequently, we compared transcriptomes of negative control (NC) group, β-catenin knockdown (KD) group, or β-catenin overexpression group respectively using RNA-sequencing (RNA-seq), and differentially expressed genes (DEGs) screened were further identified by quantitative real-time polymerase chain reaction (qRT-PCR). Finally, in vivo experiments further verified the expression change of critical molecules. We discovered 184 and 522 DEGs in the knockdown and overexpression groups compared to the NC group, respectively (adjusted P<0.05; |fold change| >2.0). Among these, 106 DEGs were altered in both groups. Gene Ontology (GO) enrichment analysis relating to biological functions identified cytokine-cytokine receptor interaction, and positive modulation of cellular migration. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment assessment indicated that these DEGs were chiefly linked by the regulation of signaling receptor activity and chemokine signaling pathways. Quantitative real-time polymerase chain reaction (qRT-PCR) results showed that the similar expression trend of serum amyloid A3 (Saa3) and CXC-chemokine ligand 5 (Cxcl5) possibly involved in cytoskeletal rearrangement with RNA-seq data. Experiments in vivo displayed that no significant expression change of Saa3 and Cxcl5 was observed in β-catenin knockout and overexpressed mouse models. Our study provides an expression landscape of DEGs in β-catenin silenced and overexpressed MEPM cells, which emphasizes the important role of processes such as chemotactic factor and cell migration. Our data gain deeper insight into genes associated with F-actin reorganization that is regulated by β-catenin either directly or by another route, which will contribute to further investigation of the exact mechanism of mesenchymal β-catenin/F-actin in palatal shelf reorientation.

Tcf-1 promotes genomic instability and T cell transformation in response to aberrant β-catenin activation

Understanding the mechanisms promoting chromosomal translocations of the rearranging receptor loci in leukemia and lymphoma remains incomplete. Here we show that leukemias induced by aberrant activation of β-catenin in thymocytes, which bear recurrent Tcra/Myc-Pvt1 translocations, depend on Tcf-1. The DNA double strand breaks (DSBs) in the Tcra site of the translocation are Rag-generated, whereas the Myc-Pvt1 DSBs are not. Aberrantly activated β-catenin redirects Tcf-1 binding to novel DNA sites to alter chromatin accessibility and down-regulate genome-stability pathways. Impaired homologous recombination (HR) DNA repair and replication checkpoints lead to retention of DSBs that promote translocations and transformation of double-positive (DP) thymocytes. The resulting lymphomas, which resemble human T cell acute lymphoblastic leukemia (T-ALL), are sensitive to PARP inhibitors (PARPis). Our findings indicate that aberrant β-catenin signaling contributes to translocations in thymocytes by guiding Tcf-1 to promote the generation and retention of replication-induced DSBs allowing their coexistence with Rag-generated DSBs. Thus, PARPis could offer therapeutic options in hematologic malignancies with active Wnt/β-catenin signaling.

Abstract 952: Attenuation of β-Catenin signaling by ormeloxifene in hepatocellular carcinoma

Abstract Background: Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer with approximately 41,000 new diagnosis and 29,000 fatalities every year only in the U.S. Severe toxicity and poor response to available chemotherapeutic agents make HCC clinical management extremely difficult. Aberrant β-catenin signaling is found to play a critical role in differentiation grades of HCC patients. Thus, attempts to find more potent and non-toxic β-Catenin inhibitors is urgently needed for cancer therapy. Notably, ormeloxifene (ORM), clinically approved selective estrogen receptor modulator with therapeutic index, has shown excellent anticancer activity, rendering it as an ideal candidate for repurposing. In this study, we investigated the anti-cancer potential of ORM against hepatocellular carcinoma. Methodology: Cell proliferation and colony formation assays were performed to assess the therapeutic activity of ORM in human hepatocellular carcinoma (HepG2, SK-HEP-1, Hep3B, and C3A) cells. Boyden chamber and Matrigel assays were carried out for investigating the effect of ORM on migration and invasion abilities of HCC cells, respectively. The effects of ORM on β-catenin and EMT associated proteins were analyzed through Western blotting and qPCR. Confocal microscopy was used to determine the β-catenin nuclear localization following ORM treatment in HCC cells. Results: As compared to vehicle-treated group, ORM treatment (2.5-20 µM) suppressed proliferation and colony formation in human hepatocellular carcinoma cells in a dose and time-dependent manner. Moreover, ORM treatment suppresses the migration and invasion of human hepatocellular carcinoma cells as shown by wound healing and Matrigel invasion assay, respectively. ORM effectively inhibited the protein levels and mRNA expression of total β-catenin. Additionally, our confocal microscopy results further showed reduced nuclear translocation of β-catenin following the ORM treatment. ORM treatment inhibited epithelial-to-mesenchymal transition (EMT) process as evident by repression of N-cadherin, Slug, Snail, vimentin, MMPs (MMP2 and MMP3), and induced the expression of pGSK3β. Experiments are also being conducted to see how ORM affects epigenetic markers linked to EMT and β-catenin signaling. Conclusion: Taken together, ORM inhibited β-catenin signaling and exhibited potent anticancer effects against HCC and could be investigated further as a novel therapeutic modality for HCC treatment. Citation Format: Mohammed Sikander, Shabnam Malik, Anyssa Rodriguez, Molly Vela, Daniel Zubieta, Vivek K. Kashyap, Anupam Dhasmana, Bilal B. Hafeez, Sheema Khan, Fathi T. Halaweish, Subhash C. Chauhan, Meena Jaggi. Attenuation of β-Catenin signaling by ormeloxifene in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 952.

Antigen presentation capability and AP-1 activation accompany methotrexate-induced colon cancer cell senescence in the context of aberrant β-catenin signaling

Reversible cellular senescence was demonstrated previously to constitute colon cancer cell response to methotrexate. The current study presents a comparison of two senescent states of colon cancer cells, arrested and reversing, resulting from respectively, 120 h exposure to the drug, and 48 h exposure followed by 96 h regrowth in drug-free media. The upregulation of immunoproteasome subunit-coding genes and the increase in human leukocyte antigen HLA-A/B/C membrane level indicated MHC-I-restricted antigen presentation as common to both senescent states. Nuclear factor NF-κB p65 level decreased and activating protein AP-1: c-Jun, Fra2 and JunB nuclear levels increased in both senescent cell populations. Notably, the increase in AP-1- dependent transcription occurred after 48 h exposure to methotrexate. β-catenin nuclear level increased after 48 h exposure to the drug and remained as such only in senescence-arrested cells. β-catenin level was found uncoupled from the protein phosphorylation status indicating the deregulation of β-catenin signaling in colon cancer cells employed in the study. These findings carry implications for both, a general mechanism of senescence establishment and putative advantages for colon cancer treatment.

Abstract 2599: Pleiotropic effects of PPARD accelerate colorectal tumorigenesis progression and invasion

Abstract APC mutations activate aberrant β-catenin signaling to drive colorectal cancer (CRC) initiation. CRC progression however requires additional molecular mechanisms. PPAR-delta (PPARD) is a downstream target of β-catenin and upregulated in CRC. PPARD germline genetic deletion has however been reported to promote intestinal tumorigenesis in Apcmin mice, which has questioned PPARD effects on β-catenin aberrant activation and CRC. Addressing this knowledge gap is important because PPARD is a druggable protein. Mice with targeted PPARD overexpression/deletion into intestinal epithelial cells (IECs) were bred with mice with intestinally targeted ApcΔ580 mutation via CDX2-cre (ApcΔ580) or CDX2-cre-ERT2 (ApcΔ580-TMX). APCΔ580 mice treated with PPARD agonist (GW501516) or antagonist (GSK3787), and human CRC organoid cells were also tested. PPARD expression was examined in human CRC invasive fronts and their paired colorectal adenomas and cancer centers. PPARD’s mechanisms to promote CRC were screened by Functional Proteomics Reverse Phase Protein Assay (RPPA) and subsequently validated in in-vitro and in-vivo studies. PPARD overexpression/deletion in IECs in mice augmented/suppressed β-catenin activation via upregulation/downregulation of BMP7/TAK1 signaling and strongly promoted/suppressed CRC .PPARD downregulation by siRNA in human CRC organoid cells inhibited BMP7/β-catenin signaling and suppressed organoid self-renewals. GW501516 enhanced while GSK3787 suppressed CRC tumorigenesis in ApcΔ580 mice. PPARD expression was significantly higher in human CRC invasive fronts versus their paired invasive tumor centers and adenomas. RPPA and validation studies identified PPARD upregulation of multiple other pro-invasive pathways: PDGFRβ, connexin 43, AKT1, EIF4G1 and CDK1, Our data demonstrate that PPARD strongly potentiates multiple pro-tumorigenic pathways to promote CRC progression and invasiveness. Citation Format: Yi Liu, Yasunori Deguchi, Rui Tian, Daoyan Wei, Ling Wu, Weidong Chen, Weiguo Xu, Min Xu, Fuyao Liu, Shen Gao, Jonathan C. Jaoude, Sarah P. Chrieki, Micheline J. Moussalli, Mihai Gagea, Jeffrey S. Morris, Russell Broaddus, Xiangsheng Zuo, Imad Shureiqi. Pleiotropic effects of PPARD accelerate colorectal tumorigenesis progression and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2599.

Pleiotropic Effects of PPARD Accelerate Colorectal Tumorigenesis, Progression, and Invasion.

APC mutations activate aberrant β-catenin signaling to drive initiation of colorectal cancer; however, colorectal cancer progression requires additional molecular mechanisms. PPAR-delta (PPARD), a downstream target of β-catenin, is upregulated in colorectal cancer. However, promotion of intestinal tumorigenesis following deletion of PPARD in Apcmin mice has raised questions about the effects of PPARD on aberrant β-catenin activation and colorectal cancer. In this study, we used mouse models of PPARD overexpression or deletion combined with APC mutation (ApcΔ580 ) in intestinal epithelial cells (IEC) to elucidate the contributions of PPARD in colorectal cancer. Overexpression or deletion of PPARD in IEC augmented or suppressed β-catenin activation via up- or downregulation of BMP7/TAK1 signaling and strongly promoted or suppressed colorectal cancer, respectively. Depletion of PPARD in human colorectal cancer organoid cells inhibited BMP7/β-catenin signaling and suppressed organoid self-renewal. Treatment with PPARD agonist GW501516 enhanced colorectal cancer tumorigenesis in ApcΔ580 mice, whereas treatment with PPARD antagonist GSK3787 suppressed tumorigenesis. PPARD expression was significantly higher in human colorectal cancer-invasive fronts versus their paired tumor centers and adenomas. Reverse-phase protein microarray and validation studies identified PPARD-mediated upregulation of other proinvasive pathways: connexin 43, PDGFRβ, AKT1, EIF4G1, and CDK1. Our data demonstrate that PPARD strongly potentiates multiple tumorigenic pathways to promote colorectal cancer progression and invasiveness. SIGNIFICANCE: These findings address long-standing, important, and unresolved questions related to the potential role of PPARD in APC mutation-dependent colorectal tumorigenesis by showing PPARD activation enhances APC mutation-dependent tumorigenesis.

Non-canonical activation of β-catenin by PRL-3 phosphatase in acute myeloid leukemia.

Aberrant activation of Wnt/β-catenin signaling pathway is essential for the development of AML; however, the mechanistic basis for this dysregulation is unclear. PRL-3 is an oncogenic phosphatase implicated in the development of LSCs. Here, we identified Leo1 as a direct and specific substrate of PRL-3. Serine-dephosphorylated form of Leo1 binds directly to β-catenin, promoting the nuclear accumulation of β-catenin and transactivation of TCF/LEF downstream target genes such as cyclin D1 and c-myc. Importantly, overexpression of PRL-3 in AML cells displayed enhanced sensitivity towards β-catenin inhibition in vitro and in vivo, suggesting that these cells are addicted to β-catenin signaling. Altogether, our study revealed a novel regulatory role of PRL-3 in the sustenance of aberrant β-catenin signaling in AML. PRL-3 may serve as a biomarker to select for the subset of AML patients who are likely to benefit from treatment with β-catenin inhibitors. Our study presents a new avenue of cancer inhibition driven by PRL-3 overexpression or β-catenin hyperactivation.

XY oocytes of sex-reversed females with a Sry mutation deviate from the normal developmental process beyond the mitotic stage†.

The fertility of sex-reversed XY female mice is severely impaired by a massive loss of oocytes and failure of meiotic progression. This phenomenon remains an outstanding mystery. We sought to determine the molecular etiology of XY oocyte dysfunction by generating sex-reversed females that bear genetic ablation of Sry, a vital sex determination gene, on an inbred C57BL/6 background. These mutant mice, termed XYsry− mutants, showed severe attrition of germ cells during fetal development, resulting in the depletion of ovarian germ cells prior to sexual maturation. Comprehensive transcriptome analyses of primordial germ cells (PGCs) and postnatal oocytes demonstrated that XYsry− females had deviated significantly from normal developmental processes during the stages of mitotic proliferation. The impaired proliferation of XYsry− PGCs was associated with aberrant β-catenin signaling and the excessive expression of transposable elements. Upon entry to the meiotic stage, XYsry− oocytes demonstrated extensive defects, including the impairment of crossover formation, the failure of primordial follicle maintenance, and no capacity for embryo development. Together, these results suggest potential molecular causes for germ cell disruption in sex-reversed female mice, thereby providing insights into disorders of sex differentiation in humans, such as “Swyer syndrome,” in which patients with an XY karyotype present as typical females and are infertile.

Abstract 4374: Non-canonical activation of β-catenin by PRL-3 phosphatase in acute myeloid leukemia

Abstract Aberrant activation of Wnt/β-catenin signaling pathway is essential for the development of AML; however, the mechanistic basis for the dysregulation is unclear. PRL-3 is an oncogenic phosphatase implicated in the development of LSCs. Here, we identified Leo1 as a direct and specific substrate of PRL-3. Serine-dephosphorylated form of Leo1 binds directly to β-catenin, promoting the nuclear accumulation of β-catenin and transactivation of TCF/LEF downstream target genes such as cyclin D1 and c-myc. Importantly, overexpression of PRL-3 in AML cells displayed enhanced sensitivity towards β-catenin inhibition in vitro and in vivo, suggesting that these cells are addicted to β-catenin signaling. Altogether, our study revealed a novel regulatory role of PRL-3 in the sustenance of aberrant β-catenin signaling in AML. PRL-3 may serve as a biomarker to select for the subset of AML patients subset who are likely to benefit from treatment with β-catenin inhibitors. Our study presents a new avenue of cancer inhibition driven by PRL-3 overexpression or β-catenin hyperactivation. Citation Format: Phyllis SY Chong, Jianbiao Zhou, Jing Yuan Chooi, Wee Joo Chng, Jayantha Gunaratne, Qi Zeng. Non-canonical activation of β-catenin by PRL-3 phosphatase in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4374.

Divergent Epigenomes in Pediatric and Adult Acute Myeloid Leukemia Implicate Cell of Origin and Transcriptional Silencing of Immune Responses As Sources of Clinically Relevant Heterogeneity: A Report from the Children's Oncology Group and NCI/COG Therapeutically Applicable Research to Generate Effective Treatments (TARGET) Initiative

Acute myeloid leukemia (AML) carries a poor prognosis across age groups. In children, AML has become the leading cause of leukemia mortality, with only 60% of cases securing long-term remission. In adults, outcomes are far worse, with 5 year survival approaching 24%. The mutational and transcriptional characterization of AML1has not yet translated into improved outcomes for most patients.The TARGET AML project is an effort of Children's Oncology Group (COG) and the National Cancer Institute to characterize molecular abnormalities in pediatric AML. 197 cases were selected for whole genome sequencing (WGS) of diagnostic specimens, 284 cases for mRNA sequencing, 289 cases for DNA methylation arrays, and 721 cases for targeted sequencing (182 assayed by WGS). Most patients (93%) were uniformly treated on COG study AAML0531 or AAML03P1. The Cancer Genome Atlas (TCGA) AML project1characterized 177 comparable adult AMLs with identical assays.DNA methylation changes radically during differentiation of blood cells2, and recurrent pre-leukemic mutations in adult AML3affect DNA methylation and chromatin modifiers. We thus investigated whether differences in cell-of-origin, immune signalling, and regulatory aberrations were captured by focal or regional differences in DNA methylation, within or between adult and pediatric AML patients.In cytogenetically similar TARGET and TCGA AML cases, striking differences in DNA methylation emerge (fig. 1). Pediatric FLT3-mutant AMLs dominate a cluster with normal-progenitor-like DNA methylation. Mutant DNMT3A, RUNX1, and TP53, which selectively favor preleukemic hematopoietic stem cells3,4,5 (HSCs), are common in adult AML, rare in pediatric AML, and tend towards HSC-like hypermethylation. Transcriptional & epigenetic signatures of the cell of origin persist even after leukemic transformation6. Thus we sought to identify the most likely cell of origin for each case. Previous studies of mRNA7 and DNA methylation8 differences in HSCs and progenitor cells (HSPCs), leukemic stem cells (LSCs), and AML blasts allowed us to model these differences in TCGA and TARGET AMLs. RNAseq results revealed many LSC-like cases with aberrant β-catenin signaling and TP53 regulation, distinct from blasts and normal HSPCs (fig. 2a). DNA methylation segregated cases resembling granulocyte/monocyte progenitors (GMPs) from those resembling other HSPC subsets (fig. 2b). DNMT3A mutants strongly associated with HSC/LSC-like mRNA expression, as did most MLL-rearranged AMLs. Nearly all TP53 and RUNX1 mutants presented LSC-like mRNA expression and retained HSC-like methylomes. These results suggest that decades of selective HSC attrition enable cooperating adult-specific mutations to initiate leukemia, while the timescales in pediatric AML favor fusion genes capable of transforming progenitors as well as HSCs.With matched mRNA expression & DNA methylation data from 256 TARGET cases and 156 TCGA cases, we found over 100 genes where DNA methylation accompanied loss of transcription (silencing) in AML but not in normal HSPCs (fig. 3a). Many such genes lie in regions affected by recurrent copy number aberrations, most notably chromosome arms 5q and 19q. Recurrently mutated or deleted genes such as DNMT3A, TET2, SPRY4, and CDKN2A/B are silenced, some mutually exclusively with mutations or CNV. Functional enrichment analyses of silenced genes with DAVID9revealed 4 clusters: NK-cell signaling, innate immune response regulation, transcriptional regulation, and (on chromosome 19q) zinc finger genes involved in Toll-like receptor signaling. Some silencing co-occurs with specific molecular features, but no event was perfectly predicted by any molecular or cytogenetic feature (fig. 3b).Drug-gene interaction mining with DGIDb10 suggests silencing may inform treatment. Silencing of the mitotic checkpoint gene CHFR may confer sensitivity to microtubule inhibitors11, silencing of MGMT suggests greater benefit from alkylating agents12, and demethylating agents may benefit cases with silenced immune response13. Biomarker driven clinical trials will be needed to evaluate these and other markers in pediatric and adult AML, but evidence of independent genetic and epigenetic evolution in AML14supports their continued investigation.This work is dedicated to the late Robert J. Arceci, without whom none of this would have been possible. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Chronic exposure to endosulfan induces inflammation in murine colon via β-catenin expression and IL-6 production

Endosulfan (ENDO) is a widely used organochlorine (OC) pesticide and persistent organo-pollutant. Epidemiological studies have shown that high levels of OC exposure were related to colorectal cancer (CRC) incidence. The objectives of the present study were to evaluate histological changes in the colon, as well as in in situ expression of β-catenin and P-selectin, and serum levels of select pro-inflammatory cytokines in mice administered ENDO; there is a relationship between increased serum IL-6 and P-selectin levels in CRC patients and aberrant β-catenin signaling is important in initiation/maintenance of most CRCs. Mice were exposed to ENDO (at dose < LD50) orally once a week for up to 24 weeks, and monitored (inclusive) for a total of 42 weeks. The experiment was comprised of three groups, one that did not receive ENDO (olive oil vehicle), one administered 2 mg ENDO/kg/week and a positive control (for induction of CRC) given a weekly 20 mg 1,2-dimethylhydrazine (DMH)/kg injection. The results indicated that oral administration of ENDO provoked moderate inflammation starting at six weeks, and severe colonic inflammation with an appearance of dysplastic formations (aberrant crypts) in mice treated with ENDO (or DMH) for 12 weeks or longer. Serum IL-6 levels significantly increased starting at six weeks and rose to a peak of 15-fold higher than in controls at 42 weeks; TNFα levels likewise significantly increased, with a later peak (≈four-fold higher than controls) at 30–42 weeks. Immunohistochemical analysis of the colon also showed that expression of β-catenin and P-selectin increased with length of exposure to ENDO. Taken together, the results indicate that continued repeated oral exposure to ENDO induces increased expression of β-catenin and P-selectin, inflammation in the colon, and, ultimately, local tissue dysplasia.

Gtpbp2 is a positive regulator of Wnt signaling and maintains low levels of the Wnt negative regulator Axin.

BackgroundCanonical Wnt signals, transduced by stabilized β-catenin, play similar roles across animals in maintaining stem cell pluripotency, regulating cell differentiation, and instructing normal embryonic development. Dysregulated Wnt/β-catenin signaling causes diseases and birth defects, and a variety of regulatory processes control this pathway to ensure its proper function and integration with other signaling systems. We previously identified GTP-binding protein 2 (Gtpbp2) as a novel regulator of BMP signaling, however further exploration revealed that Gtpbp2 can also affect Wnt signaling, which is a novel finding reported here.ResultsKnockdown of Gtpbp2 in Xenopus embryos causes severe axial defects and reduces expression of Spemann-Mangold organizer genes. Gtpbp2 knockdown blocks responses to ectopic Wnt8 ligand, such as organizer gene induction in ectodermal tissue explants and induction of secondary axes in whole embryos. However, organizer gene induction by ectopic Nodal2 is unaffected by Gtpbp2 knockdown. Epistasis tests, conducted by activating Wnt signal transduction at sequential points in the canonical pathway, demonstrate that Gtpbp2 is required downstream of Dishevelled and Gsk3β but upstream of β-catenin, which is similar to the previously reported effects of Axin1 overexpression in Xenopus embryos. Focusing on Axin in Xenopus embryos, we find that knockdown of Gtpbp2 elevates endogenous or exogenous Axin protein levels. Furthermore, Gtpbp2 fusion proteins co-localize with Dishevelled and co-immunoprecipitate with Axin and Gsk3b.ConclusionsWe conclude that Gtpbp2 is required for canonical Wnt/β-catenin signaling in Xenopus embryos. Our data suggest a model in which Gtpbp2 suppresses the accumulation of Axin protein, a rate-limiting component of the β-catenin destruction complex, such that Axin protein levels negatively correlate with Gtpbp2 levels. This model is supported by the similarity of our Gtpbp2-Wnt epistasis results and previously reported effects of Axin overexpression, the physical interactions of Gtpbp2 with Axin, and the correlation between elevated Axin protein levels and lost Wnt responsiveness upon Gtpbp2 knockdown. A wide variety of cancer-causing Wnt pathway mutations require low Axin levels, so development of Gtpbp2 inhibitors may provide a new therapeutic strategy to elevate Axin and suppress aberrant β-catenin signaling in cancer and other Wnt-related diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-016-0138-x) contains supplementary material, which is available to authorized users.

A kinome siRNA screen identifies HGS as a potential target for liver cancers with oncogenic mutations in CTNNB1.

BackgroundAberrant activation of the Wnt/β-catenin pathway is a major and frequent event in liver cancer, but inhibition of oncogenic β-catenin signaling has proven challenging. The identification of genes that are synthetically lethal in β-catenin-activated cancer cells would provide new targets for therapeutic drug design.MethodsWe transfected the parental HuH6 hepatoblastoma cell line with a doxycycline-inducible shRNA against CTNNB1 (gene coding for β-catenin) to obtain an isogenic cell line pair with or without aberrant β-catenin signaling. Using this hepatoblastoma isogenic cell line pair, we performed a human kinome-wide siRNA screen to identify synthetic lethal interactions with oncogenic CTNNB1. The phenotypic readouts of the screen were cell proliferation, cell cycle arrest and apoptosis, which were assessed by image-based analysis. In addition, apoptosis was assessed by flow cytometric experiments and immunoblotting. The potential synthetic lethal relationship between candidates genes identified in the screen and oncogenic CTNNB1 was also investigated in a different cellular context, a colorectal HCT116 isogenic cell line pair.ResultsWe first determined the experimental conditions that led to the efficient expression of shRNA against CTNNB1 and maximal reduction of β-catenin signaling activity in response to doxycycline treatment. Following high throughput screening in which 687 genes coding for kinases and proteins related to kinases (such as pseudokinases and phosphatases) were targeted, we identified 52 genes required for HuH6 survival. The silencing of five of these genes selectively impaired the viability of HuH6 cells with high β-catenin signaling: HGS, STRADA, FES, BRAF and PKMYT1. Among these candidates, HGS depletion had the strongest inhibitory effect on cell growth and led to apoptosis specifically in HuH6 with high β-catenin activity, while HuH6 with low β-catenin activity were spared. In addition, HGS was identified as a potential synthetic lethal partner of oncogenic CTNNB1 in the HCT116 colorectal isogenic cell line pair.ConclusionsThese results demonstrate the existence of crosstalk between β-catenin signaling and HGS. Importantly, HGS depletion specifically affected cells with uncontrolled β-catenin signaling activity in two different types of cancer (Hepatoblastoma HuH6 and colorectal HCT116), and thus may represent a new potential target for novel therapeutic strategies in liver and colorectal cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-2037-8) contains supplementary material, which is available to authorized users.

GPR84 sustains aberrant β-catenin signaling in leukemic stem cells for maintenance of MLL leukemogenesis

Abstractβ-catenin is required for establishment of leukemic stem cells (LSCs) in acute myeloid leukemia (AML). Targeted inhibition of β-catenin signaling has been hampered by the lack of pathway components amenable to pharmacologic manipulation. Here we identified a novel β-catenin regulator, GPR84, a member of the G protein-coupled receptor family that represents a highly tractable class of drug targets. High GPR84 expression levels were confirmed in human and mouse AML LSCs compared with hematopoietic stem cells (HSCs). Suppression of GPR84 significantly inhibited cell growth by inducing G1-phase cell-cycle arrest in pre-LSCs, reduced LSC frequency, and impaired reconstitution of stem cell–derived mixed-lineage leukemia (MLL) AML, which represents an aggressive and drug-resistant subtype of AML. The GPR84-deficient phenotype in established AML could be rescued by expression of constitutively active β-catenin. Furthermore, GPR84 conferred a growth advantage to Hoxa9/Meis1a-transduced stem cells. Microarray analysis demonstrated that GPR84 significantly upregulated a small set of MLL-fusion targets and β-catenin coeffectors, and downregulated a hematopoietic cell-cycle inhibitor. Altogether, our data reveal a previously unrecognized role of GPR84 in maintaining fully developed AML by sustaining aberrant β-catenin signaling in LSCs, and suggest that targeting the oncogenic GPR84/β-catenin signaling axis may represent a novel therapeutic strategy for AML.

Abstract 1938: Inhibition of both physiological and aberrant Wnt/β-catenin signaling activities associated with stemness in nasopharyngeal carcinoma

Abstract We previously demonstrated that basic or physiological levels of β-catenin signaling and tissue context play decisional roles in the regulation of self-renewal networks in nasopharyngeal carcinoma (NPC) HONE1 cells. Introduction of physiological levels of β-catenin signaling in HONE1 hybrid cells plays a central role in the control of other pathway activities, such as TGF-β, Activin, and pluripotency maintenance (LIFR and IL6ST), during the stemness transition process. These results revealed novel regulatory relationships among Wnt signaling, stemness, epithelial-mesenchymal transition (EMT), tumor suppressive pathways and expression of common cancer stem cell surface markers, such as CD44 and CD133, in human tumor cells. In current studies, we found that several NPC cell lines, including CNE1, HK1, HNE1, SUNE1, and C666-1, have aberrant and significant up-regulation of β-catenin expression, when compared to HONE1 cells that have low levels endogenous β-catenin expression. In these NPC cells with aberrant β-catenin signaling activities, the Wnt pathway (Axin2, APC, TCF1 and TCF3), stemness factors (Nanog, and Oct4) and pluripotency markers (IFITM1 and GRB7) are strongly overexpressed, compared to HONE1 cells. Our findings suggest that aberrant Wnt/β-catenin signaling activities may play a prominent role in promoting tumor progression in NPC cells. To further verify the role of aberrant β-catenin signaling in NPC, we utilize shRNA to knock down β-catenin expression in these cells, including CNE1 and HK1. As found in HONE1 hybrids, the diminished expression of core stem cell factors (Nanog, Oct4, Klf4, and Sox2) is also detected in both CNE1 and HK1 cell lines. Concomitantly, the gene expression of EMT markers (E-cadherin and Zeb1) is also inhibited when β-catenin is knocked down by shRNA β-catenin infection. These preliminary results suggest that β-catenin signaling plays a dominant role in regulating both physiological and aberrant Wnt/β-catenin signaling activities as well as EMT events in NPC development. Citation Format: Yue Cheng, Yee Peng Phoon, Maria L. Lung. Inhibition of both physiological and aberrant Wnt/β-catenin signaling activities associated with stemness in nasopharyngeal carcinoma. [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 1938. doi:10.1158/1538-7445.AM2014-1938

Dietary compound isoliquiritigenin targets GRP78 to chemosensitize breast cancer stem cells via β-catenin/ABCG2 signaling.

Accumulating evidence suggests that β-catenin signaling in breast cancer stem cells (CSCs) is closely correlated to chemoresistance and adenosine triphosphate (ATP)-binding cassette subfamily G2 (ABCG2) expression. Targeting the aberrant β-catenin signaling in CSCs has become a promising strategy to improve chemosensitivity in cancer treatment. In a pilot screening study, we found that the natural compound isoliquiritigenin (ISL) blocked β-catenin transcription activity with the highest inhibition ratio. Here, we investigated the chemosensitizing effects of ISL on breast CSCs and the underlying mechanisms regulating the β-catenin pathway. ISL could have synergistic effects with chemotherapeutic drugs to inhibit breast cancer cell proliferation and colony formation. In addition, ISL could significantly limit the side population and CSC ratios in breast cancer cells, accompanied by inhibited self-renewal and multidifferentiation abilities. A mechanistic study revealed that ISL could inhibit β-catenin/ABCG2 signaling by activating the proteasome degradation pathway. The drug affinity responsive target stability strategy further identified GRP78 as the direct target of ISL. Subsequent molecular docking analysis and functional studies demonstrated that ISL could dock into the ATP domain of GRP78 and thereby inhibit its ATPase activity, resulting in its dissociation from β-catenin. An in vivo study also suggested that ISL could chemosensitize breast CSCs via the GRP78/β-catenin/ABCG2 pathway, with little toxicity in normal tissues and mammary stem cells. Taken together, the data from this study not only suggest ISL as a natural candidate to enhance breast CSC chemosensitivity but also highlight the significance of GRP78 in mediating cancer drug resistance and β-catenin signaling in CSCs.

Inhibition of β-Catenin Signaling Improves Alveolarization and Reduces Pulmonary Hypertension in Experimental Bronchopulmonary Dysplasia

Bronchopulmonary dysplasia (BPD) is the most common and serious chronic lung disease of preterm infants. The development of pulmonary hypertension (PH) significantly increases the mortality and morbidity of this disease. β-Catenin signaling plays an important role in tissue development and remodeling. Aberrant β-catenin signaling is associated with clinical and experiment models of BPD. To test the hypothesis that inhibition of β-catenin signaling is beneficial in promoting alveolar and vascular development and preventing PH in experimental BPD, we examined the effects of ICG001, a newly developed pharmacological inhibitor of β-catenin, in preventing hyperoxia-induced BPD in neonatal rats. Newborn rat pups were randomized at postnatal day (P)2 to room air (RA) + DMSO (placebo), RA + ICG001, 90% FiO2 (O2) + DMSO, or O2 + ICG001. ICG001 (10 mg/kg) or DMSO was given by daily intraperitoneal injection for 14 days during continuous exposure to RA or hyperoxia. Primary human pulmonary arterial smooth muscle cells (PASMCs) were cultured in RA or hyperoxia (95% O2) in the presence of DMSO or ICG001 for 24 to 72 hours. Treatment with ICG001 significantly increased alveolarization and reduced pulmonary vascular remodeling and PH during hyperoxia. Furthermore, administering ICG001 decreased PASMC proliferation and expression of extracellular matrix remodeling molecules in vitro under hyperoxia. Finally, these structural, cellular, and molecular effects of ICG001 were associated with down-regulation of multiple β-catenin target genes. These data indicate that β-catenin signaling mediates hyperoxia-induced alveolar impairment and PH in neonatal animals. Targeting β-catenin may provide a novel strategy to alleviate BPD in preterm infants.

The Cdc42/Rac Nucleotide Exchange Factor Protein β1Pix (Pak-interacting Exchange Factor) Modulates β-Catenin Transcriptional Activity in Colon Cancer Cells: EVIDENCE FOR DIRECT INTERACTION OF β1PIX WITH β-CATENIN

Wnt/β-catenin signaling is highly regulated and critical for intestinal epithelial development and repair; aberrant β-catenin signaling is strongly associated with colon cancer. The small GTPase Rac1 regulates β-catenin nuclear translocation and signaling. Here we tested the hypothesis that β1Pix, a Cdc42/Rac guanine nucleotide exchange factor (GEF), regulates β-catenin-dependent transcriptional activity and cell function. We report the novel observations that β1Pix binds directly to β-catenin, an action requiring both the β1Pix DH and dimerization domains but not β1Pix GEF activity. In human colon cancer cells, activation of β-catenin signaling with LiCl decreased β1Pix/β-catenin association in the cytosol and increased nuclear binding of β-catenin to β1Pix. Nuclear association of β1Pix and β-catenin was independent of Rac1 expression and activation; down- and up-regulating Rac1 expression levels did not alter nuclear β1Pix/β-catenin association. Ectopic β1Pix expression enhanced LiCl-induced β-catenin transcriptional activity. Conversely, siRNA knockdown of β1Pix attenuated both LiCl-induced β-catenin transcriptional activity and colon cancer cell proliferation. Ectopic expression of β1Pix stimulated β-catenin transcriptional activity, whereas β1PixΔ(602-611), which is unable to bind β-catenin, had no effect. Altogether, these findings suggest that β1Pix functions as a transcriptional regulator of β-catenin signaling through direct interaction with β-catenin, an action that may be functionally relevant to colon cancer biology.