Beta-catenin Destruction Complex Research Articles

SAT509 The Clinical Significance of Cribriform Morular Thyroid Carcinoma

Abstract Disclosure: G. Soh: None. K. Sek: None. S. Yang: None. M. Nga: None. S. Mok: None. We present two young (aged below 30) female patients who were diagnosed with the rare cribriform morular thyroid carcinoma. Both patients presented with neck swellings and underwent total thyroidectomy for the thyroid malignancies. The histological diagnosis prompted subsequent genetic evaluation. Both patients demonstrated genotypic and phenotypic evidence of familial adenomatosis polyposis (FAP) syndrome. One of the patients had a mutation which resulted in a premature translational stop signal in the adenomatous polyposis coli (APC) gene. She was eventually diagnosed with adenocarcinoma in 2 colonic polyps and underwent a curative pan-proctocolectomy. The other patient was diagnosed clinically with FAP as she had multiple (30-40) colonic polyps which were tubular adenomas. She had a variant of unknown significance in the APC gene. She declined surgery and remained on colonoscopic surveillance. Both patients’ thyroid cancers are in clinical remission. While uncommon, diagnosing cribriform-morular thyroid carcinoma is significant as 25 to 30% of patients harbour genetic mutations in the APC gene on chromosome 5q21. This is associated with FAP syndrome, an autosomal dominant genetic syndrome characterized by young-onset colonic polyposis, usually of malignant potential. Additionally, these patients may exhibit features of Gardner’s syndrome, a subset of FAP syndrome in which patients can develop extra-colonic manifestations such as osteomas of the mandible and supernumerary teeth. Histologically, cribriform morular thyroid carcinoma are well circumscribed and show a variable combination of follicular, cribriform, papillary, trabecular, solid and morular patterns. There is nuclear and cytoplasmic staining for beta catenin, as the inactivating APC mutation causes a loss of beta catenin destruction complex, impeding beta catenin degradation and increasing its nuclear translocation. This is a key event in thyroid tumorigenesis. The diagnosis of cribriform morular thyroid carcinoma should alert the clinician about the possibility of FAP with consideration for further clinical and genetic evaluation to confirm the diagnosis. References1)Cameselle-Teijeiro, J. M., Peteiro-González, D., Caneiro-Gómez, J., Sánchez-Ares, M., Abdulkader, I., Eloy, C., Melo, M., Amendoeira, I., Soares, P., & Sobrinho-Simões, M. (2018). Cribriform-morular variant of thyroid carcinoma: A neoplasm with distinctive phenotype associated with the activation of the Wnt/β-catenin pathway. Modern Pathology, 31(8), 1168–1179. https://doi.org/10.1038/s41379-018-0070-2 2)Peiling Yang, S., & Ngeow, J. (2016). Familial non-medullary thyroid cancer: Unraveling the Genetic Maze. Endocrine-Related Cancer, 23(12). https://doi.org/10.1530/erc-16-0067 Presentation Date: Saturday, June 17, 2023

Lipolysis and gestational diabetes mellitus onset: a case-cohort genome-wide association study in Chinese

BackgroundGenetic knowledge of gestational diabetes mellitus (GDM) in Chinese women is quite limited. This study aimed to identify the risk factors and mechanism of GDM at the genetic level in a Chinese population.MethodsWe conducted a genome-wide association study (GWAS) based on single nucleotide polymorphism (SNP) array genotyping (ASA-CHIA Bead chip, Illumina) and a case-cohort study design. Variants including SNPs, copy number variants (CNVs), and insertions-deletions (InDels) were called from genotyping data. A total of 2232 pregnant women were enrolled in their first/second trimester between February 2018 and December 2020 from Anqing Municipal Hospital in Anhui Province, China. The GWAS included 193 GDM patients and 819 subjects without a diabetes diagnosis, and risk ratios (RRs) and their 95% confidence intervals (CIs) were estimated by a regression-based method conditional on the population structure. The calling and quality control of genotyping data were performed following published guidelines. CNVs were merged into CNV regions (CNVR) to simplify analyses. To interpret the GWAS results, gene mapping and overexpression analyses (ORAs) were further performed to prioritize the candidate genes and related biological mechanisms.ResultsWe identified 14 CNVRs (false discovery rate corrected P values < 0.05) and two suggestively significant SNPs (P value < 0.00001) associated with GDM, and a total of 19 candidate genes were mapped. Ten genes were significantly enriched in gene sets related to lipase (triglyceride lipase and lipoprotein lipase) activity (LIPF, LIPK, LIPN, and LIPJ genes), oxidoreductase activity (TPH1 and TPH2 genes), and cellular components beta-catenin destruction complex (APC and GSK3B genes), Wnt signalosome (APC and GSK3B genes), and lateral element in the Gene Ontology resource (BRCA1 and SYCP2 genes) by two ORA methods (adjusted P values < 0.05).ConclusionsGenes related to lipolysis, redox reaction, and proliferation of islet β-cells are associated with GDM in Chinese women. Energy metabolism, particularly lipolysis, may play an important role in GDM aetiology and pathology, which needs further molecular studies to verify.

Abstract 5843: Truncated APC &amp; AXIN2 expression contribute to maintaining β-catenin/WNT signaling at the “Just Right” level for CRC cell proliferation

Abstract Adenomatous polyposis coli (APC) mutation leads to increased WNT signaling and stem cell overpopulation, which drives development of colorectal cancer (CRC). APC mutations often encode a truncated protein that has loss of APC’s domains that mediate APC interaction with AXIN2. A substantial body of scientific evidence indicates that truncated APC is essential for CRC cell proliferation by maintaining beta-catenin levels and WNT signaling at the “just right” level (known as the “Just Right” Hypothesis). Thus, AXIN2 is of particular interest because both wild-type APC and AXIN2 are part of the beta-catenin destruction complex, which is essential for tumor-suppressing ability. Hypothesis: the degree of AXIN2 expression, in the context of truncated APC, is critical to maintain WNT-signaling activity at the “just right” level which is optimal for CRC cell proliferation. Accordingly, we evaluated the effect of AXIN2 levels in CRC cells that contain APC protein-truncating mutations on WNT signaling (Top Flash assay), expression of WNT-targeted genes, and CRC cell proliferation and differentiation. We found that modulating AXIN2 expression altered WNT signaling, expression of WNT-targeted genes, and CRC cell proliferation in CRC cell lines with different APC-truncation causing mutations. Indeed, western blot results showed that knock down of AXIN2 gene expression increased truncated-APC and AXIN1 protein expression, and decreased expression of several WNT-target genes (e.g. LGR5, CD44, c-Jun, c-Myc, Cyclin D1), particularly genes that have reported roles in cell cycle regulation. These findings were consistent with our independent bioinformatics analyses of NCI TCGA RNA-seq data on CRC patients. Overall, our study suggests that i) The beta-catenin destruction complex is still functional at a certain level despite APC-truncating mutations; ii) Perturbing AXIN2 expression leads to changes in WNT-signaling activity that deviate from a level that is “just right” for malignant cell proliferation, likely by affecting the function of the destruction complex; iii) Deviation from “just right” WNT signaling levels alters WNT-target gene expression and diminishes the ability of APC-mutant CRC cells to maintain uncontrolled proliferation. Citation Format: Chi Zhang, Caroline O. Facey, Katherine Funk, Lynn M. Opdenaker, Bruce M. Boman. Truncated APC &amp; AXIN2 expression contribute to maintaining β-catenin/WNT signaling at the “Just Right” level for CRC cell proliferation [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 5843.

APC Mutation Marks an Aggressive Subtype of BRAF Mutant Colorectal Cancers.

Background: WNT activation is a hallmark of colorectal cancer. BRAF mutation is present in 15% of colorectal cancers, and the role of mutations in WNT signaling regulators in this context is unclear. Here, we evaluate the mutational landscape of WNT signaling regulators in BRAF mutant cancers. Methods: we performed exome-sequencing on 24 BRAF mutant colorectal cancers and analyzed these data in combination with 175 publicly available BRAF mutant colorectal cancer exomes. We assessed the somatic mutational landscape of WNT signaling regulators, and performed hotspot and driver mutation analyses to identify potential drivers of WNT signaling. The effects of Apc and Braf mutation were modelled, in vivo, using the Apcmin/+ and BrafV637/Villin-CreERT2/+ mouse, respectively. Results: RNF43 was the most frequently mutated WNT signaling regulator (41%). Mutations in the beta-catenin destruction complex occurred in 48% of cancers. Hotspot analyses identified potential cancer driver genes in the WNT signaling cascade, including MEN1, GNG12 and WNT16. Truncating APC mutation was identified in 20.8% of cancers. Truncating APC mutation was associated with early age at diagnosis (p < 2 × 10−5), advanced stage (p < 0.01), and poor survival (p = 0.026). Apcmin/+/BrafV637 animals had more numerous and larger SI and colonic lesions (p < 0.0001 and p < 0.05, respectively), and a markedly reduced survival (median survival: 3.2 months, p = 8.8 × 10−21), compared to animals with Apc or Braf mutation alone. Conclusions: the WNT signaling axis is frequently mutated in BRAF mutant colorectal cancers. WNT16 and MEN1 may be novel drivers of aberrant WNT signaling in colorectal cancer. Co-mutation of BRAF and APC generates an extremely aggressive neoplastic phenotype that is associated with poor patient outcome.

TM4SF1, a binding protein of DVL2 in hepatocellular carcinoma, positively regulates beta-catenin/TCF signalling.

The interaction between Axin and DVL2 is critical for the breaking down of the beta‐catenin destruction complex and the activation of the Wnt/beta‐catenin cascade. However, this biological process remains poorly understood. In the present study, TM4SF1 was identified as the interacting partner of DVL2 and positively regulated as Wnt/beta‐catenin signalling by strengthening the DVL2‐Axin interaction. The expression levels of TM4SF1 were elevated in hepatocellular carcinoma (HCC) and were induced by Kras signalling. The overexpression of TM4SF1 promoted the growth and motility of HCC cells, and up‐regulated the target genes (Axin2 and cyclin D1). The down‐regulation of TM4SF1 impaired the capability of HCC cells for growth, migration and metastasis. In addition, the down‐regulation of TM4SF1 promoted the ubiquitination of beta‐catenin. In summary, these results reveal the oncogenic functions of TM4SF1 in HCC progression and suggest that TM4SF1 might be a target for treatment.

Inhibition of Acute Leukemia By a Small Molecule KYA1797K Which Destabilize RAS and β-Catenin

Introduction: Signaling pathways in acute leukemia are aberrantly activated to cause leukemogenesis and relapse after treatment. Like in many other malignancies, upregulation of WNT/beta-catenin pathway and hyperactive RAS is known to be associated with treatment resistance in leukemia. However, there are little studies about RAS singaling pathyway and leukemia, and it is a field of study that needs to be revealed. KYA1797K is a recently developed small molecule, binds directly to RGS domain of axin and enhances the beta-catenin destruction complex which activates GSK3beta and results in degradation of beta-catenin and RAS. In the current study, we tried to find the role of RAS inhibition by KYA1797K in leukemic cell lines and in patient's BM samples. Moreover, other small molecule PCK412 (Midosaturin) was also used for comparison. Materials &amp; Methods: Leukemic cells (MOLT-4, THP-1, MOLT-4, Jurka, KG-1, MV4-11, RS4-11) were cultured in RPMI1640 media under various concentration (0.1-10µM for KYA1797K, 0.5-500nM for PKC412) for 48h and with Erlotinib (1µM) for comparison. Cell proliferation assay on each leukemic cell was done and immunoblotting for β-catenin, GSK3β, Pan-RAS, N-RAS was checked. Downstream targets of Wnt pathway (c-Myc, CD44, LEF1, Met, TCF1/TCF7) were studied by immunoblotting. MOLT-4 was stimulated with Wnt3a (200ng/mL, 4h) and changes in Wnt pathway were observed. Bone marrow samples of AML and ALL patients were evaluated for β-catenin and RAS. KYA1797K (Nat Chem Biol 2016, 12:593) was kindly provided by Prof. Kang-Yell Choi. Results: Suppression of leukemic cells by KYA1797K was evident starting from the concentration of 5 microM. (fig.1) Beta-catenin was down regulated in all cell lines by KYA1797K. Pan-RAS decreased in MOLT-4 and THP-1. All the downstream targets evaluated were down regulated by KYA1797K in MOLT-4 culture, and was evident at the concentration of 5microM. (fig.2) Stimulation of Wnt pathway by Wnt3a was inhibited by KYA1797K. (fig.3) Bone marrow samples from 7 ALL patients showed various status of β-catenin and RAS expression. Two high-risk patients showed suppression of β-cateinin and N-RAS by KYA1797K. (fig.4) In MV4-11 (FLT3 mutant) and RS4-11 (FLT3 wild type), IC50 for PKC412 was higher in RS4-11 compared to MV4-11 while KYA1797K showed same IC50 in both cell lines. β-catenin and RAS downregulation was observed by KYA1797K. Effects of KYA1797K analyzed by qRT-PCR and immunoblot for FLT3, N-RAS, MEK, ERK, ETS2 showed that KYA1797K downregulates FLT3 in MV4-11 even though FLT3 is not the main target of action. It was less effective on RS4-11. (fig.5) In bone marrow samples of ALL patient with FLT3 mutation, KYA1797K 1µM showed effect in reducing leukemia cells. (fig.6) Conclusion: This preclinical study suggests that KYA1797K may be an option for patients with acute leukemia. KYA1797K effectively destabilized β-catenin and RAS in acute leukemia even under Wnt pathway activation. FLT3, N-RAS, MEK, ERK, ETS2 were down regulated by KYA1797K, hence KYA1797K has a potential application for acute leukemia with FLT3 mutation. Extended studies including further in vivo study are needed to build up a strategy in small molecule therapy to target RAS in acute leukemia. Although with limitation, we suggest RAS inhibitor as a potential drug for leukemia. Disclosures No relevant conflicts of interest to declare.

Divergent Axin and GSK-3 paralogs in the beta-catenin destruction complexes of tapeworms.

The Wnt/beta-catenin pathway has many key roles in the development of animals, including a conserved and central role in the specification of the primary (antero-posterior) body axis. The posterior expression of Wnt ligands and the anterior expression of secreted Wnt inhibitors are known to be conserved during the larval metamorphosis of tapeworms. However, their downstream signaling components for Wnt/beta-catenin signaling have not been characterized. In this work, we have studied the core components of the beta-catenin destruction complex of the human pathogen Echinococcus multilocularis, the causative agent of alveolar echinococcosis. We focused on two Axin paralogs that are conserved in tapeworms and other flatworm parasites. Despite their divergent sequences, both Axins could robustly interact with one E. multilocularis beta-catenin paralog and limited its accumulation in a heterologous mammalian expression system. Similarly to what has been described in planarians (free-living flatworms), other beta-catenin paralogs showed limited or no interaction with either Axin and are unlikely to function as effectors in Wnt signaling. Additionally, both Axins interacted with three divergent GSK-3 paralogs that are conserved in free-living and parasitic flatworms. Axin paralogs have highly segregated expression patterns along the antero-posterior axis in the tapeworms E. multilocularis and Hymenolepis microstoma, indicating that different beta-catenin destruction complexes may operate in different regions during their larval metamorphosis.

Supramolecular assembly of the beta-catenin destruction complex and the effect of Wnt signaling on its localization, molecular size, and activity in vivo.

Wnt signaling provides a paradigm for cell-cell signals that regulate embryonic development and stem cell homeostasis and are inappropriately activated in cancers. The tumor suppressors APC and Axin form the core of the multiprotein destruction complex, which targets the Wnt-effector beta-catenin for phosphorylation, ubiquitination and destruction. Based on earlier work, we hypothesize that the destruction complex is a supramolecular entity that self-assembles by Axin and APC polymerization, and that regulating assembly and stability of the destruction complex underlie its function. We tested this hypothesis in Drosophila embryos, a premier model of Wnt signaling. Combining biochemistry, genetic tools to manipulate Axin and APC2 levels, advanced imaging and molecule counting, we defined destruction complex assembly, stoichiometry, and localization in vivo, and its downregulation in response to Wnt signaling. Our findings challenge and revise current models of destruction complex function. Endogenous Axin and APC2 proteins and their antagonist Dishevelled accumulate at roughly similar levels, suggesting competition for binding may be critical. By expressing Axin:GFP at near endogenous levels we found that in the absence of Wnt signals, Axin and APC2 co-assemble into large cytoplasmic complexes containing tens to hundreds of Axin proteins. Wnt signals trigger recruitment of these to the membrane, while cytoplasmic Axin levels increase, suggesting altered assembly/disassembly. Glycogen synthase kinase3 regulates destruction complex recruitment to the membrane and release of Armadillo/beta-catenin from the destruction complex. Manipulating Axin or APC2 levels had no effect on destruction complex activity when Wnt signals were absent, but, surprisingly, had opposite effects on the destruction complex when Wnt signals were present. Elevating Axin made the complex more resistant to inactivation, while elevating APC2 levels enhanced inactivation. Our data suggest both absolute levels and the ratio of these two core components affect destruction complex function, supporting models in which competition among Axin partners determines destruction complex activity.

Abstract P1-04-08: Non-nuclear SUMO dynamics regulate mammary epithelial cell transformation

Abstract Objective: The reversible SUMO-posttranslational modification of protein substrates regulates various cellular processes and consistently is important for normal cell physiology. Disruption of SUMO enzymatic components supports onset of various pathophysiological disorders, including cancer. Our recent study identified a splicing event that differentially modulates expression of 2 SENP7 isoforms. The novel SENP7 variant SENP7S is the predominant SUMO protease in normal mammary epithelia; however onset of precancerous ductal carcinoma in situ (DCIS) reduces SENP7S significantly and stays low in all breast cancer (BCa) subtypes. Inversely, the full-length SENP7L isoform is upregulated in BCa and directly leads to BCa metastasis. Unlike SENP7L, SENP7S isoform contribution to carcinogenesis is unclear. Our objective is to define the biological function of this novel deSUMOylase SENP7S in normal versus cancerous epithelial cells. Results: Consistently with mRNA levels, protein levels of the 2 SENP7 isoforms are also inversely expressed in human BCa versus normal mammary epithelia. SENP7S is localized in the cytosol of MCF10-2A unlike other SUMO proteases including SENP7L that are predominantly nuclear enzymes. Beta-catenin and a component of the Beta-catenin destruction complex, Axin1 are substrates for SENP7S catalytic activity as in the absence of SENP7S, Beta-catenin and Axin 1 are both SUMOylated. Consistently, SENP7S regulates Beta-catenin signaling pathway. SUMOylated Axin1 loses its interaction with Beta-catenin, allowing the Beta-catenin to escape ubiquitylation and further proteasomal degradation. SUMOylated Beta-catenin translocates to the nucleus and activates multiple target genes that potentiate cell proliferation. Increase in cell proliferation and anchorage dependent growth of non-cancerous MCF10-2A cells was observed with inhibition of SENP7S. Additionally, SENP7S depletion potentiates anchorage independent growth of MCF10-2A with significantly greater number and size of spheroids. In comparison to the control. Loss of SENP7S also potentiates the self-renewal properties of the cells, indicative of mammary epithelial cell transformation. Conclusion: SENP7S modulates Beta-catenin stability and signaling and consequently is critical for normal mammary epithelial cell physiology. Loss of SENP7S, as observed in DCIS, initiates mammary epithelial cell transformation. Citation Format: Karami S, Lin F-M, Kumar S, Ren J, Bahnassy S, Bawa-Khalfe T. Non-nuclear SUMO dynamics regulate mammary epithelial cell transformation [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-04-08.

The Zinc Finger Transcription Factor Growth Factor Independence 1b (Gfi1b) Regulates The Wnt/Beta-Catenin Signaling Pathway In Hematopoietic Stem Cells Through Interaction With Inhibitory Proteins

Hematopoietic stem cells (HSCs) reside in the bone marrow in specific niches at the border between bone cells and the bone marrow (endosteal niche) or around blood vessels (perivascular niche). In the endosteal niche, HSCs are maintained at low oxygen levels in a quiescent (dormant) state by adhesion to niche cells. We have previously shown that Gfi1b restricts the expansion and proliferation of HSCs as well as their mobilization or re-localization into peripheral blood. We have proposed that Gfi1b exerts this function by regulating the expression of surface molecules such as integrins on HSCs that are required to maintain them in their bone marrow niche at a quiescent state. The objective of this study was to gain more insight into the precise molecular mechanisms by which Gfi1b regulates HSCs dormancy and mobilization and to obtain insights that may be exploited in the future to improve stem cell therapies or the expansion of human hematopoietic stem cells for clinical use.Immune precipitation and mass spectrometry identified a series of Gfi1b interacting proteins, most notably a group of regulators of the canonical Wnt/beta-catenin pathway. Independent protein IP validation of these findings suggested that Gfi1b can interact with several inhibitors of the canonical Wnt/beta catenin pathway namely with APC (Adenomatous polyposis coli) a tumor suppressor protein and important factor in the beta-catenin destruction complex, with the DNA helicase and chromatin remodeling factor CHD8, which silences beta catenin mediated transcription, with CtBP which antagonizes beta-catenin activity and is part of the LSD1/CoRest histone demethylase complex and with the direct beta-catenin inhibitors TLE1 and TLE3 (also called Groucho). Of particular interest was that the interactions between the Groucho proteins and Gfi1b were dependent on a previously unidentified Groucho binding domain (GBD) in Gfi1b. This is a well-conserved six-amino acid stretch that is found in the middle part of the Gfi1b protein. In addition, the binding of CtBP was dependent on the presence of the 20 amino acid N-terminal SNAG domain in Gfi1b that also mediates LSD1 binding. Using luciferase reporter gene assays (TOP/FOP reporter assay), we found that Gfi1b was able to significantly up-regulates TCF/beta-catenin-dependent transcription upon activation by LiCl or Wnt3A in HEK293 cells. This activity of Gfi1b was dependent on both the presence of the SNAG domain and the newly identified Groucho binding domain. Also, Gfi1b was able to reverse partially the inhibitory effect of CtBP and TLE3 on beta-catenin activity in the TOP/FOP reporter assays. To obtain further evidence that Gfi1b is indeed implicated in regulating the Wnt/beta catenin signaling pathway in hematopoietic stem cells, we FACS sorted Lin-Kit1+Sca+ hematopoietic progenitors (LSK cells) from wt and Gfi1b deficient mice and tested them for expression of Wnt effector genes using a Wnt signaling specific PCR array. We observed that the majority of Wnt target genes were significantly down regulated in Gfi1 deficient LSKs compared to wt LSKs. Among the genes affected the most were typical Wnt targets such as Axin2, Frz7, Tcf4, Klf5, Vegfa and Ccnd1. To show that Gfi1b is able to regulate Wnt pathway effectors in vivo in HSCs, we crossed Gfi1b flox/flox, Mx-Cre mice with animals that carry a NLS-lacZ reporter gene under the control of the endogenous Axin2 promoter/enhancer region. Treatment with pIpC, which deletes Gfi1b correlated with a significant decrease of Axin2 expression in HSCs and MPP1, which are high Gfi1b expressing cells. The Axin2 reporter was not affected by Gfi1b deletion in MPP2 or GMPs, which express low levels or no Gfi1b.The canonical Wnt/b-catenin signaling pathway is recognized as one of the elements that are critically important in the regulation of HSC function. Here we have identified Gfi1b as a potential new player in the Wnt-beta catenin signaling pathway. Our data suggest that Gfi1b acts on at least two inhibitory complexes of this pathway, on the TLE family of Groucho proteins and the CtBP/LSD1 complex and regulates effectors of the Wnt/beta-catenin signaling cascade. We propose therefore that Gfi1b may titer the level of activation of the Wnt/beta-catenin signaling pathway in HSCs, which offers an explanation of the hematopoietic stem cell phenotype seen in mice lacking Gfi1b. Disclosures:No relevant conflicts of interest to declare.

Abstract A134: Functional perturbation of PARP6 affects centromeric proteins and is associated with increased multi-polar spindles in breast cancer cell lines.

Abstract Poly(ADP-ribose) polymerases (PARP) are a family of 17 enzymes that catalyze the transfer of the ADP-ribose from NADH to post-translationally modify (PTM) acceptor proteins. Currently, the PTM regulation of proteins by PARPs have been demonstrated to regulate numerous signaling cascades including but not limited to DNA damage response and tumor development for PARP1-3 as well as telomere maintenance, spindle assembly, vesicular movement, and regulation of beta-catenin destruction complex for PARP5a-5b. Although progress on the biological role and therapeutic potential for some PARPs has been made, the function of and possible therapeutic application for other PARPs are not fully understood. Using cell free enzyme and cell-base multi-polar spindle assays, semi-selective PARP6 inhibitors AZ482 and AZ108 were identified. From in vitro PARP6 knock-down and AZ108 pharmacological studies in breast cancer cell lines, PARP6 perturbation was demonstrated to disrupt spindle pole clustering. To identify PARP6 protein substrate that may contribute to the spindle pole clustering defect, an in vitro acceptor protein substrate screen was performed using high-density protein microarrays containing more than 8,000 unique proteins. From this screen, an enrichment for centromeric and microtubule organizing proteins was identified. Of these centromeric PARP6 protein substrate, CHEK1 and CENP were shown to be modulated by AZ108 treatment in breast cancer cell lines. These results implicate PARP6 in a role for stablization of the spindle assembly during mitosis. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A134. Citation Format: SHAUN GROSSKURTH, Philip Petteruti, Xin Wang, Keith Mikule. Functional perturbation of PARP6 affects centromeric proteins and is associated with increased multi-polar spindles in breast cancer cell lines. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A134.

Role of the Beta Catenin Destruction Complex in Mediating Chemotherapy-Induced Senescence-Associated Secretory Phenotype

Cellular senescence is considered as a tumor suppressive mechanism. Recent evidence indicates however that senescent cells secrete various growth factors and cytokines, some of which may paradoxically promote cancer progression. This phenomenon termed senescence-associated secretory phenotype (SASP) must be inhibited in order for anti-proliferative agents to be effective. The present study was designed to determine whether the β-catenin destruction complex (BCDC), known to integrate the action of various growth factors and cytokines, would represent a suitable target to inhibit the activity of SASP components. For this, we carried out experiments to determine the effect of drug-induced senescence on secretion of SASP, β-catenin transactivation, and the relationship between these processes. Moreover, genetic and pharmacological approaches were used to define the implication of BCDC in mediating the effects of SASP components on cell migration and resistance to drugs. The findings indicate that drug-induced senescence was associated with expression of various Wnt ligands in addition to previously known SASP components. Beta catenin transactivation and expression of genes implicated in epithelial-mesenchymal transition (EMT) also increased in response to drug-induced SASP. These effects were prevented by Pyrvinium, a recently described activator of BCDC. Pyrvinium also suppressed the effects of SASP on cell migration and resistance to doxorubicin. Together, these findings provide insights on the potential role of BCDC in mediating the effects of drug-induced SASP on cancer cell invasion and resistance to therapy, and suggest that targeting this pathway may represent an effective approach to enhance the activity of current and prospective anti-cancer therapeutics.

Assembly of Dishevelled 3-based supermolecular complexes via phosphorylation and Axin

BackgroundDishevelled-3 (Dvl3) is a multivalent scaffold essential to cell signaling in development. Dsh/Dvls enable a myriad of protein-protein interactions in Wnt signaling. In the canonical Wnt/β-catenin pathway specifically, Dvl3 polymerizes to form dynamic protein aggregates, so-called “signalsomes”, which propagate signals from the Wnt receptor Frizzled to downstream elements.ResultsVery large Dvl3-based supermolecular complexes form in response to Wnt3a. These complexes are identified by steric-exclusion chromatography, affinity pull-downs, proteomics, and fluorescence correlation microscopy (fcs). In the current work, the roles of Dvl3 phosphorylation and of Axin in the assembly of Dvl3-based supermolecular complexes in response to Wnt3a are probed in totipotent mouse F9 teratocarcinoma cells. Point mutations of phosphorylation sites of Dvl3 which interfere with Lef/Tcf-sensitive transcriptional activation by Wnt3a are shown to interfere more proximally with the assembly of Dvl3-based supermolecular complexes. Axin, a Dvl-interacting protein, plays a central role in organizing the beta-catenin destruction complex. The assembly of Dvl3-based supermolecular complexes is blocked either by depletion of Axin or by mutation of Axin sites necessary for polymerization in response to Wnt3a.ConclusionThese data demonstrate that Wnt3a activation of the canonical pathway requires specific phosphorylation events as well as Axin to assemble very large, Dvl3-based supermolecular complexes; these complexes are a prerequisite to activation of Lef/Tcf-sensitive transcription.

Mutant huntingtin-impaired degradation of β-catenin causes neurotoxicity in Huntington's disease

Huntington's disease (HD) is a fatal neurodegenerative disorder causing selective neuronal death in the brain. Dysfunction of the ubiquitin-proteasome system may contribute to the disease; however, the exact mechanisms are still unknown. We report here a new pathological mechanism by which mutant huntingtin specifically interferes with the degradation of beta-catenin. Huntingtin associates with the beta-catenin destruction complex that ensures its equilibrated degradation. The binding of beta-catenin to the destruction complex is altered in HD, leading to the toxic stabilization of beta-catenin. As a consequence, the beta-transducin repeat-containing protein (beta-TrCP) rescues polyglutamine (polyQ)-huntingtin-induced toxicity in striatal neurons and in a Drosophila model of HD, through the specific degradation of beta-catenin. Finally, the non-steroidal anti-inflammatory drug indomethacin that decreases beta-catenin levels has a neuroprotective effect in a neuronal model of HD and in Drosophila and increases the lifespan of HD flies. We thus suggest that restoring beta-catenin homeostasis in HD is of therapeutic interest.

Xenopus axin‐related protein: A link between its centrosomal localization and function in the Wnt/β‐catenin pathway

The Wnt/beta-catenin signaling pathway regulates cell proliferation and cell fate determination in multiple systems. However, the subcellular localization of Wnt pathway components and the significance of this localization for the pathway regulation have not been extensively analyzed. Here we report that Xenopus Axin-related protein (XARP), a component of the beta-catenin destruction complex, is localized to the centrosome. This localization of XARP requires the presence of the DIX domain and an adjacent region. Since other components of the Wnt pathway have also been shown to associate with the centrosome, we tested a hypothesis that the beta-catenin destruction complex operates at the centrosome. However, XARP mutants with poor centrosomal localization revealed an enhanced rather than decreased ability to antagonize the Wnt/beta-catenin pathway. Our data are consistent with the idea that the inactivation of XARP at the centrosome is an important regulatory point in Wnt signaling.

The Terminal Region of β-Catenin Promotes Stability by Shielding the Armadillo Repeats from the Axin-scaffold Destruction Complex

Post-translational stabilization of beta-catenin is a key step in Wnt signaling, but the features of beta-catenin required for stabilization are incompletely understood. We show that forms of beta-catenin lacking the unstructured C-terminal domain (CTD) show faster turnover than full-length or minimally truncated beta-catenins. Mutants that exhibit faster turnover show enhanced association with axin in co-transfected cells, and excess CTD polypeptide can compete binding of the beta-catenin armadillo (arm) repeat domain to axin in vitro, indicating that the CTD may restrict beta-catenin binding to the axin-scaffold complex. Fluorescent resonance energy transmission (FRET) analysis of cyan fluorescent protein (CFP)-arm-CTD-yellow fluorescent protein beta-catenin reveals that the CTD of beta-catenin can become spatially close to the N-terminal arm repeat region of beta-catenin. FRET activity is strongly diminished by the coexpression of beta-catenin binding partners, indicating that an unliganded groove is absolutely required for an orientation that allows FRET. Amino acids 733-759 are critical for beta-catenin FRET activity and stability. These data indicate that an N-terminal orientation of the CTD is required for beta-catenin stabilization and suggest a model where the CTD extends toward the N-terminal arm repeats, shielding these repeats from the beta-catenin destruction complex.

A Lentivirus-Mediated Genetic Screen Identifies Dihydrofolate Reductase (DHFR) as a Modulator of β-Catenin/GSK3 Signaling

The multi-protein β-catenin destruction complex tightly regulates β-catenin protein levels by shuttling β-catenin to the proteasome. Glycogen synthase kinase 3β (GSK3β), a key serine/threonine kinase in the destruction complex, is responsible for several phosphorylation events that mark β-catenin for ubiquitination and subsequent degradation. Because modulation of both β-catenin and GSK3β activity may have important implications for treating disease, a complete understanding of the mechanisms that regulate the β-catenin/GSK3β interaction is warranted. We screened an arrayed lentivirus library expressing small hairpin RNAs (shRNAs) targeting 5,201 human druggable genes for silencing events that activate a β-catenin pathway reporter (BAR) in synergy with 6-bromoindirubin-3′oxime (BIO), a specific inhibitor of GSK3β. Top screen hits included shRNAs targeting dihydrofolate reductase (DHFR), the target of the anti-inflammatory compound methotrexate. Exposure of cells to BIO plus methotrexate resulted in potent synergistic activation of BAR activity, reduction of β-catenin phosphorylation at GSK3-specific sites, and accumulation of nuclear β-catenin. Furthermore, the observed synergy correlated with inhibitory phosphorylation of GSK3β and was neutralized upon inhibition of phosphatidyl inositol 3-kinase (PI3K). Linking these observations to inflammation, we also observed synergistic inhibition of lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (TNFα, IL-6, and IL-12), and increased production of the anti-inflammatory cytokine IL-10 in peripheral blood mononuclear cells exposed to GSK3 inhibitors and methotrexate. Our data establish DHFR as a novel modulator of β-catenin and GSK3 signaling and raise several implications for clinical use of combined methotrexate and GSK3 inhibitors as treatment for inflammatory disease.

SUMOylation target sites at the C terminus protect Axin from ubiquitination and confer protein stability

Axin is a scaffold protein for the beta-catenin destruction complex, and a negative regulator of canonical Wnt signaling. Previous studies implicated the six C-terminal amino acids (C6 motif) in the ability of Axin to activate c-Jun N-terminal kinase, and identified them as a SUMOylation target. Deletion of the C6 motif of mouse Axin in vivo reduced the steady-state protein level, which caused embryonic lethality. Here, we report that this deletion (Axin-DeltaC6) causes a reduced half-life in mouse embryonic fibroblasts and an increased susceptibility to ubiquitination in HEK 293T cells. We confirmed the C6 motif as a SUMOylation target in vitro, and found that mutating the C-terminal SUMOylation target residues increased the susceptibility of Axin to polyubiquitination and reduced its steady-state level. Heterologous SUMOylation target sites could replace C6 in providing this protective effect. These findings suggest that SUMOylation of the C6 motif may prevent polyubiquitination, thus increasing the stability of Axin. Although C6 deletion also caused increased association of Axin with Dvl-1, this interaction was not altered by mutating the lysine residues in C6, nor could heterologous SUMOylation motifs replace the C6 motif in this assay. Therefore, some other specific property of the C6 motif seems to reduce the interaction of Axin with Dvl-1.

Recruitment of adenomatous polyposis coli and β-catenin to axin-puncta

The adenomatous polyposis coli (APC) tumour suppressor is a multifunctional protein involved in the regulation of Wnt signalling and cytoskeletal dynamics. Little is known about how APC controls these disparate functions. In this study, we have used APC- and axin-fluorescent fusion proteins to examine the interactions between these proteins and show that the functionally distinct populations of APC are also spatially separate. Axin-RFP forms cytoplasmic punctate structures, similar to endogenous axin puncta. Axin-RFP recruits beta-catenin destruction complex proteins, including APC, beta-catenin, glycogen synthase kinase-3-beta (GSK3-beta) and casein kinase-1-alpha (CK1-alpha). Recruitment into axin-RFP puncta sequesters APC from clusters at cell extensions and this prevents its microtubule-associated functions. The interaction between APC-GFP and axin-RFP within the cytoplasmic puncta is direct and dramatically alters the dynamic properties of APC-GFP. However, recruitment of APC to axin puncta is not absolutely required for beta-catenin degradation. Instead, formation of axin puncta, mediated by the DIX domain, is required for beta-catenin degradation. An axinDeltaDIX mutant did not form puncta, but still mediated recruitment of destruction complex proteins and phosphorylation of beta-catenin. We conclude that there are distinct pools of APC and that the formation of axin puncta, rather than the axin/APC complex, is essential for beta-catenin destruction.

Protein phosphatase 1 regulates assembly and function of the β-catenin degradation complex

The Wnt/beta-catenin signaling pathway is critical in both cellular proliferation and organismal development. However, how the beta-catenin degradation complex is inhibited upon Wnt activation remains unclear. Using a directed RNAi screen we find that protein phosphatase 1 (PP1), a ubiquitous serine/threonine phosphatase, is a novel potent positive physiologic regulator of the Wnt/beta-catenin signaling pathway. PP1 expression synergistically activates, and inhibition of PP1 inhibits, Wnt/beta-catenin signaling in Drosophila and mammalian cells as well as in Xenopus embryos. The data suggest that PP1 controls Wnt signaling through interaction with, and regulated dephosphorylation of, axin. Inhibition of PP1 leads to enhanced phosphorylation of specific sites on axin by casein kinase I. Axin phosphorylation markedly enhances the binding of glycogen synthase kinase 3, leading to a more active beta-catenin destruction complex. Wnt-regulated changes in axin phosphorylation, mediated by PP1, may therefore determine beta-catenin transcriptional activity. Specific inhibition of PP1 in this pathway may offer therapeutic approaches to disorders with increased beta-catenin signaling.