Β-catenin Degradation Complex Research Articles

Screening and identification of miRNAs negatively regulating FAM83A/Wnt/β-catenin signaling pathway in non-small cell lung cancer

The prevalence of non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancers, with the Wnt/β-catenin signaling pathway exhibiting robust activation in this particular subtype. The expression of FAM83A (family with sequence similarity 83, member A) has been found to be significantly upregulated in lung cancer, leading to the stabilization of β-catenin and activation of the Wnt signaling pathway. In this study, we conducted a screening of down-regulated miRNAs in lung cancer with FAM83A as the target. Ultimately, we identified miR-1 as a negative regulator of FAM83A and confirmed that FAM83A is a direct target gene of miR-1 through dual luciferase reporter assays. The overexpression of miR-1 significantly attenuated the expression level of FAM83A and suppressed the Wnt signaling pathway, leading to a reduction in the expression levels of downstream target genes AXIN2, CyclinD1, and C-MYC. Additionally, it decreased the nuclear translocation of β-catenin. In addition, overexpression of miR-1 accelerated the degradation of β-catenin by inhibiting FAM83A, promoted the assembly of β-catenin degradation complex, and inhibited the proliferation, migration and invasion of NSCLC cells. In summary, miR-1 may be a potential candidate miRNA for the treatment of NSCLC.

Dicerandrol C Suppresses Proliferation and Induces Apoptosis of HepG2 and Hela Cancer Cells by Inhibiting Wnt/β-Catenin Signaling Pathway.

Overwhelming evidence points to an aberrant Wnt/β-catenin signaling as a critical factor in hepatocellular carcinoma (HCC) and cervical cancer (CC) pathogenesis. Dicerandrol C (DD-9), a dimeric tetrahydroxanthenone isolated from the endophytic fungus Phomopsis asparagi DHS-48 obtained from mangrove plant Rhizophora mangle via chemical epigenetic manipulation of the culture, has demonstrated effective anti-tumor properties, with an obscure action mechanism. The objective of the current study was to explore the efficacy of DD-9 on HepG2 and HeLa cancer cells and its functional mechanism amid the Wnt/β catenin signaling cascade. Isolation of DD-9 was carried out using various column chromatographic methods, and its structure was elucidated with 1D NMR. The cytotoxicity of DD-9 on HepG2 and HeLa cells was observed with respect to the proliferation, clonality, migration, invasion, apoptosis, cell cycle, and Wnt/β-catenin signaling cascade. We found that DD-9 treatment significantly reduced tumor cell proliferation in dose- and time-dependent manners in HepG2 and HeLa cells. The subsequent experiments in vitro implied that DD-63 could significantly suppress the tumor clonality, metastases, and induced apoptosis, and that it arrested the cell cycle at the G0/G1 phase of HepG2 and HeLa cells. Dual luciferase assay, Western blot, and immunofluorescence assay showed that DD-9 could dose-dependently attenuate the Wnt/β-catenin signaling by inhibiting β-catenin transcriptional activity and abrogating β-catenin translocated to the nucleus; down-regulating the transcription level of β-catenin-stimulated Wnt target gene and the expression of related proteins including p-GSK3-β, β-catenin, LEF1, Axin1, c-Myc, and CyclinD1; and up-regulating GSK3-β expression, which indicates that DD-9 stabilized the β-catenin degradation complex, thereby inducing β-catenin degradation and inactivation of the Wnt/β-catenin pathway. The possible interaction between DD-9 and β-catenin and GSK3-β protein was further confirmed by molecular docking studies. Collectively, DD-9 may suppress proliferation and induce apoptosis of liver and cervical cancer cells, possibly at least in part via GSK3-β-mediated crosstalk with the Wnt/β-catenin signaling axis, providing insights into the mechanism for the potency of DD-9 on hepatocellular and cervical cancer.

RHAMM/hyaluronan inhibit β-catenin degradation, enhance downstream signaling, and facilitate fibrosarcoma cell growth.

Increased hyaluronan deposition (HA) in various cancer tissues, including sarcomas, correlates with disease progression. The receptor for hyaluronic acid-mediated motility (RHAMM) expression is elevated in most human cancers. β-catenin is a critical downstream mediator of the Wnt signaling pathways, facilitating carcinogenic events characterized by deregulated cell proliferation. We previously showed that low molecular weight (LMW) HA/RHAMM/β-catenin signaling axis increases HT1080 fibrosarcoma cell growth. Here, focusing on mechanistic aspects and utilizing immunofluorescence and immunoprecipitation, we demonstrate that LMW HA treatment enhanced RHAMM intracellular localization (p ≤ 0.001) and RHAMM/β-catenin colocalization in HT1080 fibrosarcoma cells (p ≤ 0.05). Downregulating endogenous HA attenuated the association of RHAMM/β-catenin in HT1080 fibrosarcoma cells (p ≤ 0.0.01). Notably, Axin-2, the key β-catenin degradation complex component, and RHAMM were demonstrated to form a complex primarily to cell membranes, enhanced by LMW HA (p ≤ 0.01). In contrast, LMW HA attenuated the association of β-catenin and Axin-2 (p ≤ 0.05). The utilization of FH535, a Wnt signaling inhibitor, showed that LMW HA partially rescued the Wnt-dependent growth of HT1080 cells and restored the expression of Wnt/β-catenin mediators, cyclin-D1 and c-myc (p ≤ 0.05). B6FS fibrosarcoma cells with different HA metabolism do not respond to the LMW HA growth stimulus (p = NS). The present study identifies a novel LMW HA/RHAMM mechanism in a fibrosarcoma model. LMW HA regulates intracellular RHAMM expression, which acts as a scaffold protein binding β-catenin and Axin-2 at different cellular compartments to increase β-catenin expression, transcriptional activity, and fibrosarcoma growth.

Novel read through agent: ZKN-0013 demonstrates efficacy in APCmin model of familial adenomatous polyposis.

Familial adenomatous polyposis (FAP) is a precancerous, colorectal disease characterized by hundreds to thousands of adenomatous polyps caused by mutations in the tumor suppressor gene adenomatous polyposis coli (APC). Approximately 30% of these mutations are premature termination codons (PTC), resulting in the production of a truncated, dysfunctional APC protein. Consequently, the β-catenin degradation complex fails to form in the cytoplasm, leading to elevated nuclear levels of β-catenin and unregulated β-catenin/wnt-pathway signaling. We present in vitro and in vivo data demonstrating that the novel macrolide, ZKN-0013, promotes read through of premature stop codons, leading to functional restoration of full-length APC protein. Human colorectal carcinoma SW403 and SW1417 cells harboring PTC mutations in the APC gene showed reduced levels of nuclear β-catenin and c-myc upon treatment with ZKN-0013, indicating that the macrolide-mediated read through of premature stop codons produced bioactive APC protein and inhibited the β-catenin/wnt-pathway. In a mouse model of adenomatous polyposis coli, treatment of APCmin mice with ZKN-0013 caused a significant decrease in intestinal polyps, adenomas, and associated anemia, resulting in increased survival. Immunohistochemistry revealed decreased nuclear β-catenin staining in the epithelial cells of the polyps in ZKN-0013-treated APCmin mice, confirming the impact on the β-catenin/wnt-pathway. These results indicate that ZKN-0013 may have therapeutic potential for the treatment of FAP caused by nonsense mutations in the APC gene. KEY MESSAGES: • ZKN-0013 inhibited the growth of human colon carcinoma cells with APC nonsense mutations. • ZKN-0013 promoted read through of premature stop codons in the APC gene. • In APCmin mice, ZKN-0013 treatment reduced intestinal polyps and their progression to adenomas. • ZKN-0013 treatment in APCmin mice resulted in reduced anemia and increased survival.

Rapamycin-induced autophagy in osteosarcoma cells is mediated via the biglycan/Wnt/β-catenin signaling axis.

Biglycan is a class I secreted small leucine-rich proteoglycan (SLRP), which regulates signaling pathways connected to bone pathologies. Autophagy is a vital catabolic process with a dual role in cancer progression. Here, we show that biglycan inhibits autophagy in two osteosarcoma cell lines (P ≤ 0.001), while rapamycin-induced autophagy decreases biglycan expression in MG63 osteosarcoma cells and abrogates the biglycan-induced cell growth increase (P ≤ 0.001). Rapamycin also inhibits β-catenin translocation to the nucleus, inhibiting the Wnt pathway (P ≤ 0.001) and reducing biglycan's colocalization with the Wnt coreceptor LRP6 (P ≤ 0.05). Furthermore, biglycan exhibits protective effects against the chemotherapeutic drug doxorubicin in MG63 OS cells through an autophagy-dependent manner (P ≤ 0.05). Cotreatment of these cells with rapamycin and doxorubicin enhances cells response to doxorubicin by decreasing biglycan (P ≤ 0.001) and β-catenin (P ≤ 0.05) expression. Biglycan deficiency leads to increased caspase-3 activation (P ≤ 0.05), suggesting increased apoptosis of biglycan-deficient cells treated with doxorubicin. Computational models of LRP6 and biglycan complexes suggest that biglycan changes the receptor's ability to interact with other signaling molecules by affecting the interdomain bending angles in the receptor structure. Biglycan binding to LRP6 activates the Wnt pathway and β-catenin nuclear translocation by disrupting β-catenin degradation complex (P ≤ 0.01 and P ≤ 0.05). Interestingly, this mechanism is not followed in moderately differentiated, biglycan-nonexpressing U-2OS OS cells. To sum up, biglycan exhibits protective effects against the doxorubicin in MG63 OS cells by activating the Wnt signaling pathway and inhibiting autophagy.

Casein kinase 1α regulates murine spermatogenesis via p53-Sox3 signaling.

Casein kinase 1α (CK1α), acting as one member of the β-catenin degradation complex, negatively regulates the Wnt/β-catenin signaling pathway. CK1α knockout usually causes both Wnt/β-catenin and p53 activation. Our results demonstrated that conditional disruption of CK1α in spermatogonia impaired spermatogenesis and resulted in male mouse infertility. The progenitor cell population was dramatically decreased in CK1α conditional knockout (cKO) mice, while the proliferation of spermatogonial stem cells (SSCs) was not affected. Furthermore, our molecular analyses identified that CK1α loss was accompanied by nuclear stability of p53 protein in mouse spermatogonia, and dual-luciferase reporter and chromatin immunoprecipitation assays revealed that p53 directly targeted the Sox3 gene. In addition, the p53 inhibitor pifithrin α (PFTα) partially rescued the phenotype observed in cKO mice. Collectively, our data suggest that CK1α regulates spermatogenesis and male fertility through p53-Sox3 signaling, and they deepen our understanding of the regulatory mechanism underlying the male reproductive system.

MEDB-01. Management of the risk of medulloblastoma associated to familial adenomatous polyposis and dysregulated Wnt signalosome

APC is the key gene of the familial adenomatous polyposis (FAP). This tumor suppressor gene functions by negatively regulating the β-catenin protein and the majority of APC mutations disrupt the β-catenin degradation complex signalosome leading to the activation of the canonical Wnt pathway. Pathogenic APC mutations were reported in association to medulloblastoma. In this study, we report rare mutations of the APC gene detected in Tunisian families from the governorate of Sfax presenting clinically with various digestive and extra-digestive manifestations. Our goal was to assess the oncogenic risks encountered by our pediatric carriers to offer an accurate genetic counselling, particularly at the neurologic level. Molecular investigation of all members of two families was conducted, using bidirectional sequencing of all 15 exons of the APC gene. A phenotype-genotype correlation was conducted to elucidate the mutational pathophysiological mechanism. Two rare mutations were revealed in our familial study. The first mutation was located at exon 13 and was a missense mutation at codon 1690. The second mutation was a deletion identified at codon 4652 in exon 15. The mutations resulted both in truncated gene products. Clinical manifestations closely depending on the position of the mutation were respectively colic polyposis for the first mutation and soft tissue fibromatous tumors for the second. The localization of the APC mutations allows better targeting of surveillance for clinical manifestations that may be included in FAP. Mutations that remove the Axin-binding sites, as is the case for the first mutation, lead to severe clinical pictures whereas mutations that retain one or two of the Axin binding sites are associated with other features such as desmoid tumors. The risk of the Wnt- medulloblastoma subtype, higher among patients with FAP should be considered with a more awareness of signs and symptoms related to CNS tumors in the FAP context.

Tankyrase Regulates Neurite Outgrowth through Poly(ADP-ribosyl)ation-Dependent Activation of β-Catenin Signaling.

Poly(ADP-ribosyl)ation is a post-translational modification of proteins by transferring poly(ADP-ribose) (PAR) to acceptor proteins by the action of poly(ADP-ribose) polymerase (PARP). Two tankyrase (TNKS) isoforms, TNK1 and TNK2 (TNKS1/2), are ubiquitously expressed in mammalian cells and participate in diverse cellular functions, including wnt/β-catenin signaling, telomere maintenance, glucose metabolism and mitosis regulation. For wnt/β-catenin signaling, TNKS1/2 catalyze poly(ADP-ribosyl)ation of Axin, a key component of the β-catenin degradation complex, which allows Axin’s ubiquitination and subsequent degradation, thereby activating β-catenin signaling. In the present study, we focused on the functions of TNKS1/2 in neuronal development. In primary hippocampal neurons, TNKS1/2 were detected in the soma and neurites, where they co-localized with PAR signals. Treatment with XAV939, a selective TNKS1/2 inhibitor, suppressed neurite outgrowth and synapse formation. In addition, XAV939 also suppressed norepinephrine uptake in PC12 cells, a rat pheochromocytoma cell line. These effects likely resulted from the inhibition of β-catenin signaling through the stabilization of Axin, which suggests TNKS1/2 enhance Axin degradation by modifying its poly(ADP-ribosyl)ation, thereby stabilizing wnt/β-catenin signaling and, in turn, promoting neurite outgrowth and synapse formation.

Downregulation of CXXC Finger Protein 4 Leads to a Tamoxifen-resistant Phenotype in Breast Cancer Cells Through Activation of the Wnt/β-catenin Pathway

Tamoxifen is a successful endocrine therapy drug for estrogen receptor–positive (ER+) breast cancer. However, resistance to tamoxifen compromises the efficacy of endocrine treatment. In the present study, we identified potential tamoxifen resistance–related gene markers and investigated their mechanistic details. First, we established two ER + breast cancer cell lines resistant to tamoxifen, named MCF-7/TMR and BT474/TMR. Gene expression profiling showed that CXXC finger protein 4 (CXXC4) expression is lower in MCF-7/TMR cells than in MCF-7 cells. Furthermore, CXXC4 mRNA and protein expression are lower in the resistant cell lines than in the corresponding parental cell lines. We also investigated the correlation between CXXC4 and endocrine resistance in ER + breast cancer cells. CXXC4 knockdown accelerates cell proliferation in vitro and in vivo and renders breast cancer cells insensitive to tamoxifen, whereas CXXC4 overexpression inhibits cancer cell growth and increases tamoxifen sensitivity of resistant cells. In addition, we demonstrated that CXXC4 inhibits Wnt/β-catenin signaling in cancer cells by modulating the phosphorylation of GSK-3β, influencing the integrity of the β-catenin degradation complex. Silencing the CXXC4 gene upregulates expression of cyclinD1 and c-myc (the downstream targets of Wnt signaling) and promotes cell cycle progression. Conversely, ectopic expression of CXXC4 downregulates the expression of these proteins and arrests the cell cycle in the G0/G1 phase. Finally, the small-molecule inhibitor XAV939 suppresses Wnt signaling and sensitizes resistant cells to tamoxifen. These results indicate that components of Wnt pathway that are early in response to tamoxifen could be involved as an intrinsic factor of the transition to endocrine resistance, and inhibition of Wnt signaling may be an effective therapeutic strategy to overcome tamoxifen resistance.

Hsa-miR-942 fingerprint in colorectal cancer through Wnt signaling pathway

The Wnt signaling pathway has been identified for its function in carcinogenesis and embryonic development. It is known to play a vital role in the initiation and development of colorectal cancer (CRC). Therefore, it is of great importance for CRC research to illuminate the mechanisms which regulate Wnt pathway activity. Here, we intended to examine the effect of hsa-miR-942 (miR-942) on the Wnt signaling activity, cell cycle progression, and its expression in CRC tissues. RT-qPCR results indicated that miR-942 is significantly upregulated in colorectal cancer. Then, overexpression of miR-942 promoted, whereas its inhibition decreased the Wnt signaling activity, detected by RT-qPCR and Top/Fop flash assay. Inhibition of Wnt signaling by using PNU-74654 or IWP-2 small molecules indicated that miR-942 applies its effect to the β-catenin degradation complex level. Then, RT-qPCR and dual luciferase assay showed that miR-942 upregulated Wnt signaling through direct targeting of APC, which is a tumor suppressor in Wnt signaling pathway. Furthermore, the western blotting analysis indicated that β.catenin, as a main member of Wnt signaling pathway is upregulated following the overexpression of miR-942. Finally, miR-942 overexpression resulted in cell cycle progression in SW480 cells. Taken together, our findings established an oncogenic role for miR-942 in CRC and indicated that this miRNA might be a crucial target for CRC therapy.

Abstract DDT01-01: BI 905677: A first-in-class LRP5/6 antagonist targeting Wnt-driven proliferation and immune escape

Abstract Aberrant Wnt/β-catenin signalling has been shown to play a key role in tumorigenesis and resistance to immunotherapy in several tumor indications. Wnt ligand-mediated signals are transduced by two distinct receptor types, the serpentine receptor Frizzled (FZD) and the closely related single-span transmembrane proteins LRP5 and LRP6. Formation of the FZD-Wnt-LRP5/6 trimeric complex induces phosphorylation of LRP5 or LRP6 intracellular domains leading to inactivation of the β-catenin degradation complex, allowing stabilized β-catenin to enter the nucleus, bind to the TCF transcription factors, and act as a transcriptional activator of Wnt target genes. We have developed a first-in-class LRP5/6 antagonist, a bi-paratopic antibody comprising two modules binding to distinct epitopes of LRP5 (or LRP6). BI 905677 is a highly potent blocker of signalling induced by the Wnt family of ligands and shows anti-tumor activity in cancer models harbouring genomic alterations in upstream regulators of the Wnt pathway, such as RNF43 mutations or RSPO fusions. Furthermore, BI 905677 in combination with an anti-PD-1 immune checkpoint inhibitor induces dendritic cell activation and T cell infiltration in tumor tissues leading to complete responses in syngeneic tumor models. A Phase I clinical trial is underway in patients with advanced cancer to evaluate safety, tolerability, pharmacokinetic and pharmacodynamic properties, and efficacy of BI 905677 (NCT03604445). Citation Format: Vittoria Zinzalla, Barbara Drobits-Handl, Alexander Savchenko, Jörg Rinnenthal, Markus Johann Bauer, Michael Sanderson, Sophia Maria Blake, Norbert Schweifer, Robert Gerhardus Jacob Vries, Hans Clevers, Norbert Kraut. BI 905677: A first-in-class LRP5/6 antagonist targeting Wnt-driven proliferation and immune escape [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 DDT01-01.

MiR-15/16 mediate crosstalk between the MAPK and Wnt/β-catenin pathways during hepatocyte differentiation from amniotic epithelial cells

MiR-15/16 play an important role in liver development and hepatocyte differentiation, but the mechanisms by which these miRNAs regulate their targets and downstream genes to influence cell fate are poorly understood. In this study, we showed up-regulation of miR-15/16 during HGF- and FGF4-induced hepatocyte differentiation from amniotic epithelial cells (AECs). To elucidate the role of miR-15/16 and their targets in hepatocyte differentiation, we investigated the roles of miR-15/16 in both the MAPK and Wnt/β-catenin pathways, which were predicted to be involved in miR-15/16 signaling. Our results demonstrated that the transcription of miR-15/16 was enhanced by c-Fos, c-Jun, and CREB, important elements of the MAPK pathway, and miR-15/16 in turn directly targeted adenomatous polyposis coli (APC) protein, a major member of the β-catenin degradation complex. MiR-15/16 destroyed these degradation complexes to activate β-catenin, and the activated β-catenin combined with LEF/TCF7L1 to form a transcriptional complex that enhanced transcription of hepatocyte nuclear factor 4 alpha (HNF4α). HNF4α also bound the promoter region of miR-15/16 and promoted its transcription, thereby forming a regulatory circuit to promote the differentiation of AECs into hepatocytes. Endogenous miRNAs are, therefore, involved in hepatocyte differentiation from AECs and should be considered during the development of an effective hepatocyte transplant therapy for liver damage.

Axin-2 knockdown promote mitochondrial biogenesis and dopaminergic neurogenesis by regulating Wnt/β-catenin signaling in rat model of Parkinson's disease

Wnts and the components of Wnt/β-catenin signaling are widely expressed in midbrain and required to control the fate specification of dopaminergic (DAergic) neurons, a neuronal population that specifically degenerate in Parkinson's disease (PD). Accumulating evidence suggest that mitochondrial dysfunction plays a key role in pathogenesis of PD. Axin-2, a negative regulator of Wnt/β-catenin signaling affects mitochondrial biogenesis and death/birth of new DAergic neurons is not fully explored. We investigated the functional role of Axin-2/Wnt/β-catenin signaling in mitochondrial biogenesis and DAergic neurogenesis in 6-hydroxydopamine (6-OHDA) induced rat model of PD-like phenotypes. We demonstrate that single unilateral injection of 6-OHDA into the medial forebrain bundle (MFB) potentially dysregulates Wnt/β-catenin signaling in substantia nigra pars compacta (SNpc). We used shRNA lentiviruses to genetically knockdown Axin-2 to up-regulate Wnt/β-catenin signaling in SNpc in parkinsonian rats. Genetic knockdown of Axin-2 up-regulates Wnt/β-catenin signaling by destabilizing the β-catenin degradation complex in SNpc in parkinsonian rats. Axin-2 shRNA mediated activation of Wnt/β-catenin signaling improved behavioural functions and protected the nigral DAergic neurons by increasing mitochondrial functionality in parkinsonian rats. Axin-2 shRNA treatment reduced apoptotic signaling, autophagy and ROS generation and improved mitochondrial membrane potential which promotes mitochondrial biogenesis in SNpc in parkinsonian rats. Interestingly, Axin-2 shRNA-mediated up-regulation of Wnt/β-catenin signaling enhanced net DAergic neurogenesis by regulating proneural genes (Nurr-1, Pitx-3, Ngn-2, and NeuroD1) and mitochondrial biogenesis in SNpc in parkinsonian rats. Therefore, our data suggest that pharmacological/genetic manipulation of Wnt signaling that enhances the endogenous regenerative capacity of DAergic neurons may have implication for regenerative approaches in PD.

Abstract 2428: Redox regulation of β-catenin in colorectal cancer

Abstract Colorectal cancer (CRC) is the second most newly diagnosed cancer in the United States. β-catenin is the downstream effector of the Wnt/β-catenin signaling pathway. Loss of function (LOF) mutations on adenomatous polyposis coli (APC), a critical member of the β-catenin degradation complex leads to constitutively elevated β-catenin as an early oncogenic event in 85% of CRCs and is associated with decreased CRC patient survival. Increased level of β-catenin augments the nuclear accumulation of β-catenin, where it acts a co-activator of a number of transcription factors leading to the expression of downstream target genes that promote cancer cell proliferation, survival, and migration. There is no effective therapy for β-catenin as a cause of cancer. In addition to CRC, elevated levels of Wnt signaling have been reported in leukemia, melanoma, and breast cancers highlighting the need for novel therapies that attenuate aberrant β-catenin mediated transcription. Here we report on the redox dependency of the Wnt/β-catenin signaling pathway, identified through a forward genetic screen in Drosophila, and identify thioredoxin reductase-1 (TXNR1) as a potential target for the inhibition of aberrant β-catenin transcriptional activity through in vivo studies with the APCmin/+ mouse model. Drosophila harboring a mutation in their thioredoxin reductase (trxr-1) gene and a partial lethality phenotype were used in an enhancer/suppressor forward genetic screen. Increased lethality was observed when the trxr-1 mutation (trxr-1481) was combined with heterozygous mutations and deletions in wnt2, while wnt2 deletion and mutations alone had no effect on lethality. To test the translational value of these findings, APCmin/+ mice were treated with PX-12, a human TXNR1 inhibitor, daily by gavage from day 40 to time of death. We observed a dose dependent reduction in the number and size of intestinal polyps by PX-12 treatment, and an increased survival in treated mice. Furthermore, mechanistic studies utilizing human CRC cell lines harboring LOF mutations to the β-catenin degradation complex revealed β-Catenin mediated transcription, as measured by TOP Flash luciferase assay, was diminished when TXNR1 was silenced by siRNA. Additionally, we observed decreased β-catenin protein level in CRC cell lines treated with PX-12 or siRNA targeting TXNRD1 and its functional partner thioredoxin (TXN). The addition of the pan caspase inhibitor ZVAD but not the proteasome inhibitor MG132 was able to stabilize β-catenin protein upon treatment with PX-12 suggesting β-catenin destabilization is occurring via a caspase dependent mechanism. These results suggest a conserved mechanism for the redox regulation of β-catenin involving TXNR1; furthermore, inhibition of TXNR1 activity destabilizes β-catenin protein and decreases β-catenin mediated transcription in human CRC cell lines and inhibits intestinal neoplasia development in an experimental mouse model for CRC. Citation Format: Alejandro D. Campos, Brian James, Petrus De Jong, Robert Lemos, Nikolas Marino, Garth Powis. Redox regulation of β-catenin in colorectal cancer [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 2428.

Introduction of hsa-miR-103a and hsa-miR-1827 and hsa-miR-137 as new regulators of Wnt signaling pathway and their relation to colorectal carcinoma.

Wnt signaling is hyper-activated in most of human cancers including colorectal carcinoma (CRC). Therefore, the introduction of new regulators for Wnt pathway possesses promising diagnostic and therapeutic applications in cancer medicine. Bioinformatics analysis introduced hsa-miR-103a, hsa-miR-1827, and hsa-miR-137 as potential regulators of Wnt signaling pathway. Here, we intended to examine the effect of these human miRNAs on Wnt signaling pathway components, on the cell cycle progression in CRC originated cell lines and their expression in CRC tissues. RT-qPCR results indicated upregulation of hsa-miR-103a, hsa-miR-1827, and downregulation of hsa-miR-137 in CRC tissues. Overexpression of hsa-miR-103a and hsa-miR-1827 in SW480 cells resulted in elevated Wnt activity, detected by both Top/Flash assay and RT-qPCR analysis. Inhibition of Wnt signaling by using PNU-74654 or IWP-2 small molecules suggested that these miRNAs exerts their effect at the β-catenin degradation complex level. Then, RT-qPCR, dual luciferase assay, and western blotting analysis indicated that APC and APC2 transcripts were targeted by hsa-miR-103a, hsa-miR-1827 while, Wnt3a and β-catenin genes were upregulated. However, hsa-miR-137 downregulated Wnt3a and β-catenin genes. Further, hsa-miR-103a and hsa-miR-1827 overexpression resulted in cell cycle progression and reduced apoptotic rate in SW480 cells, unlike hsa-miR-137 overexpression which resulted in cell cycle suppression, detected by flowcytometry and Anexin analysis. Overall, our data introduced hsa-miR-103a, hsa-miR-1827 as onco-miRNAs and hsa-miR-137 as tumor suppressor which exert their effect through regulation of Wnt signaling pathway in CRC and introduced them as potential target for therapy.

Blocking Wnt Secretion Reduces Growth of Hepatocellular Carcinoma Cell Lines Mostly Independent of β-Catenin Signaling

Aberrant activation of Wnt/β-catenin signaling plays a key role in the onset and development of hepatocellular carcinomas (HCC), with about half of them acquiring mutations in either CTNNB1 or AXIN1. However, it remains unclear whether these mutations impose sufficient β-catenin signaling or require upstream Wnt ligand activation for sustaining optimal growth, as previously suggested for colorectal cancers. Using a panel of nine HCC cell lines, we show that siRNA-mediated knockdown of β-catenin impairs growth of all these lines. Blocking Wnt secretion, by either treatment with the IWP12 porcupine inhibitor or knockdown of WLS, reduces growth of most of the lines. Unexpectedly, interfering with Wnt secretion does not clearly affect the level of β-catenin signaling in the majority of lines, suggesting that other mechanisms underlie the growth-suppressive effect. However, IWP12 treatment did not induce autophagy or endoplasmic reticulum (ER) stress, which may have resulted from the accumulation of Wnt ligands within the ER. Similar results were observed for colorectal cancer cell lines used for comparison in various assays. These results suggest that most colorectal and liver cancers with mutations in components of the β-catenin degradation complex do not strongly rely on extracellular Wnt ligand exposure to support optimal growth. In addition, our results also suggest that blocking Wnt secretion may aid in tumor suppression through alternative routes currently unappreciated.

1142 Protein kinase A directly phosphorylates GSK3β, and regulates β-catenin via phosphorylation in granulosa cells

Estradiol serves an important role in female fertility and FSH drives estradiol production. Beta-catenin is a transcriptional co-factor that is required for FSH-induced estradiol production. Beta-catenin is activated via phosphorylation at Ser552 and Ser675 by protein kinase A (PKA) dependent event. Wingless-type mouse mammary tumor virus (WNT) also regulates the β-catenin pathway. Glycogen Synthase Kinase-3 β (GSK3β) is a component of the β-catenin degradation complex; canonical WNT signaling pathway phosphorylates protein kinase B (AKT) and inhibits GSK3β resulting in β-catenin accumulation in the cytoplasm. Work in our laboratory demonstrated that WNT downregulates steroidogenesis. Furthermore, AKT is required for β-catenin accumulation and FSH-induced estradiol production, suggesting convergence of the WNT and FSH pathways. We hypothesize that WNT inhibition of FSH signaling occurs through modulation of phosphorylation patterns on β-catenin. The objective of these experiments was to evaluate the phosphorylation pattern of β-catenin in response to PKA, AKT, and WNT signaling pathways. Granulosa cells (KGN cell line) were cultured and treated with vehicle control, phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (LY) (30 μ M; 30 min), Forskolin (FSK) (10 μ M; 1.5 h), WNT (50 ng/mL, 30 min) and the combination of these treatments. Western blot was used to detect total and phosphorylated β-catenin and phosphorylated GSK3β. Protein abundances were analyzed using densitometry software and densitometry values for treatments were analyzed using the GLM procedure of SAS. When significant model interactions were detected, means were separated using PDIFF. Treatment of FSK combined with WNT and LY enhanced GSK3β phosphorylation compared to control (P < 0.05). Similarly, FSK alone or in combination with WNT and LY enhanced (P < 0.05) phosphorylation of β-catenin at Ser675 and Ser552, but WNT did not alter β-catenin phosphorylation after FSK stimulation (P > 0.10). However, when the AKT pathway was blocked with LY, FSK, and WNT treatment increased phosphorylation of GSK3β and subsequently β-catenin at Ser552 suggesting that PKA can directly phosphorylate GSK3β. Results from these experiments demonstrate that WNT does not alter FSH-stimulated phosphorylation patterns of β-catenin indicating that WNT inhibits FSH signaling via other unknown mechanisms.

Molecular hydrogen suppresses activated Wnt/β-catenin signaling.

Molecular hydrogen (H2) is effective for many diseases. However, molecular bases of H2 have not been fully elucidated. Cumulative evidence indicates that H2 acts as a gaseous signal modulator. We found that H2 suppresses activated Wnt/β-catenin signaling by promoting phosphorylation and degradation οf β-catenin. Either complete inhibition of GSK3 or mutations at CK1- and GSK3-phosphorylation sites of β-catenin abolished the suppressive effect of H2. H2 did not increase GSK3-mediated phosphorylation of glycogen synthase, indicating that H2 has no direct effect on GSK3 itself. Knock-down of adenomatous polyposis coli (APC) or Axin1, which form the β-catenin degradation complex, minimized the suppressive effect of H2 on β-catenin accumulation. Accordingly, the effect of H2 requires CK1/GSK3-phosphorylation sites of β-catenin, as well as the β-catenin degradation complex comprised of CK1, GSK3, APC, and Axin1. We additionally found that H2 reduces the activation of Wnt/β-catenin signaling in human osteoarthritis chondrocytes. Oral intake of H2 water tended to ameliorate cartilage degradation in a surgery-induced rat osteoarthritis model through attenuating β-catenin accumulation. We first demonstrate that H2 suppresses abnormally activated Wnt/β-catenin signaling, which accounts for the protective roles of H2 in a fraction of diseases.

A High-Content Imaging Screen for Cellular Regulators of β-Catenin Protein Abundance

Abnormal accumulation of β-catenin protein, a key transcriptional activator required for Wnt signaling, is the hallmark of many tumor types, including colon cancer. In normal cells, β-catenin protein level is tightly controlled by a multiprotein complex through the proteosome pathway. Mutations in the components of the β-catenin degradation complex, such as adenomatous polyposis coli (APC) and Axin, lead to β-catenin stabilization and the constitutive activation of target genes. Since the signal transduction of Wnt/β-catenin is mainly mediated by protein–protein interactions, this pathway has been particularly refractory to conventional target-based small-molecule screening. Here we designed a cellular high-content imaging assay to detect β-catenin protein through immunofluorescent staining in the SW480 colon cancer cell line, which has elevated β-catenin endogenously. We demonstrate that the assay is robust and specific to screen a focused biologically diverse chemical library set against known targets that play diverse cellular functions. We identified a number of hits that reduce β-catenin levels without causing cell death. These hits may serve as tools to understand the dynamics of β-catenin degradation. This study demonstrates that detecting cell-based β-catenin protein stability is a viable approach to identifying novel mechanisms of β-catenin regulation as well as small molecules of therapeutic potential.

HSP105 recruits protein phosphatase 2A to dephosphorylate β-catenin.

The Wnt/β-catenin pathway causes accumulation of β-catenin in the cytoplasm and its subsequent translocation into the nucleus to initiate the transcription of the target genes. Without Wnt stimulation, β-catenin forms a complex with axin (axis inhibitor), adenomatous polyposis coli (APC), casein kinase 1α (CK1α), and glycogen synthase kinase 3β (GSK3β) and undergoes phosphorylation-dependent ubiquitination. Phosphatases, such as protein phosphatase 2A (PP2A), interestingly, also are components of this degradation complex; therefore, a balance must be reached between phosphorylation and dephosphorylation. How this balance is regulated is largely unknown. Here we show that a heat shock protein, HSP105, is a previously unidentified component of the β-catenin degradation complex. HSP105 is required for Wnt signaling, since depletion of HSP105 compromises β-catenin accumulation and target gene transcription upon Wnt stimulation. Mechanistically, HSP105 depletion disrupts the integration of PP2A into the β-catenin degradation complex, favoring the hyperphosphorylation and degradation of β-catenin. HSP105 is overexpressed in many types of tumors, correlating with increased nuclear β-catenin protein levels and Wnt target gene upregulation. Furthermore, overexpression of HSP105 is a prognostic biomarker that correlates with poor overall survival in breast cancer patients as well as melanoma patients participating in the BRIM2 clinical study.