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Wnt signalling is a critical cellular communication pathway controlling cell fate in all metazoan organisms. Timely activation of this pathway is crucial to coordinate development, control homeostasis of adult tissues, and to avoid cancer. Wnt signal transduction depends primarily on the activities of three multiprotein complexes; the ‘degradasome’, which targets the central effector β-catenin for degradation in the absence of Wnt; the ‘signalosome’, which is assembled by Dishevelled upon Wnt-receptor binding to inactivate the degradasome, thus allowing β-catenin to accumulate; and the ‘enhanceosome’, which captures β-catenin, granting it access to target genes and relieving their transcriptional repression by Gro/TLE. Many of the components of these complexes have now been identified, but details of their regulation, and in particular the mechanisms by which they are switched on and off, remain poorly understood. The majority of this thesis is concerned with the mechanism by which β-catenin relieves the transcriptional repression imposed upon Wnt target genes, and thereby activates the Wnt ‘transcriptional switch’. In Chapter 2, I present data showing that apposition of Gro/TLE and UBR5, a HECT E3 ubiquitin ligase, by β-catenin promotes Gro/TLE ubiquitylation, earmarking it for extraction by the VCP/p97 ATPase and ultimately leading to inactivation of its repressive function. In Chapter 3, I present the results of a different, ongoing study to identify the mechanism by which a cytoplasmic negative regulator, Naked, acts to interfere with the function of Dishevelled, promoting the switching of signalosomes and the termination of canonical Wnt signalling. These findings advance our understanding of the mechanisms by which the Wnt signalling pathway is switched on and off, and suggest new targets for therapeutic intervention in Wnt- driven cancers.

// Yan Jia 1, 2, * , Baoping Cao 1, 3, * , Yunsheng Yang 1 , Enqiang Linghu 1 , Qimin Zhan 4 , Youyong Lu 5 , Yingyan Yu 6 , James G. Herman 7 , Mingzhou Guo 1 1 Department of Gastroenterology & Hepatology, Chinese PLA General Hospital, Beijing 100853, China 2 Department of Breast Oncology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, and Key Laboratory of Cancer Prevention and Therapy, Tianjin 300060, China 3 Medical College of NanKai University, Tianjin 300071, China 4 State Key Laboratory of Molecular Oncology, Cancer Institute and Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100021, China 5 Laboratory of Molecular Oncology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital/Institute, Beijing 100142, China 6 Shanghai Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200240, China 7 The Hillman Cancer Center, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, USA * These authors have contributed equally to this work Correspondence to: Mingzhou Guo, e-mail: mzguo@hotmail.com James G. Herman, e-mail: Hermanj3@upmc.edu Keywords: NKD1, NKD2, SOX18, DNA methylation, gastric cancer Received: April 23, 2015 Accepted: September 02, 2015 Published: September 14, 2015 ABSTRACT Naked cuticle homolog2 ( NKD2 ) is located in chromosome 5p15.3, which is frequently loss of heterozygosity in human colorectal and gastric cancers. In order to understand the mechanism of NKD2 in gastric cancer development, 6 gastric cancer cell lines and 196 cases of human primary gastric cancer samples were involved. Methylation specific PCR (MSP), gene expression array, flow cytometry, transwell assay and xenograft mice model were employed in this study. The expression of NKD1 and NKD2 was silenced by promoter region hypermethylation. NKD1 and NKD2 were methylated in 11.7% (23/196) and 53.1% (104/196) in human primary gastric cancer samples. NKD2 methylation is associated with cell differentiation, TNM stage and distant metastasis significantly (all P < 0.05), and the overall survival time is longer in NKD2 unmethylated group compared to NKD2 methylated group ( P < 0.05). Restoration of NKD2 expression suppressed cell proliferation, colony formation, cell invasion and migration, induced G2/M phase arrest, and sensitized cancer cells to docetaxel. NKD2 inhibits SOX18 and MMP-2,7,9 expression and suppresses BGC823 cell xenograft growth. In conclusion, NKD2 methylation may serve as a poor prognostic and chemo-sensitive marker in human gastric cancer. NKD2 impedes gastric cancer metastasis by inhibiting SOX18.

// Barbara Fazi 1 , Sabrina Garbo 1 , Nicola Toschi 1, 2, 3 , Annunziato Mangiola 4 , Malinska Lombari 1 , Daria Sicari 1, 7 , Cecilia Battistelli 5 , Silvia Galardi 1 , Alessandro Michienzi 1 , Gianluca Trevisi 4 , Rona Harari-Steinfeld 6 , Carla Cicchini 5 and Silvia Anna Ciafre 1 1 Department of Biomedicine and Prevention, University of Rome “Tor Vergata”, Rome, Italy 2 Department of Radiology, Athinoula A. Martinos Center for Biomedical Imaging, Boston, MA, USA 3 Harvard Medical School, Boston, MA, USA 4 Department Head and Neck, Institute of Neurosurgery, Catholic University of Sacred Heart, Rome, Italy 5 Department of Cellular Biotechnologies and Haematology, Sezione di Genetica Molecolare, Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome, Italy 6 Goldyne Savad Institute of Gene Therapy, Hadassah University Hospital, Hebrew University, Jerusalem 7 Present address: Laboratorio Nazionale CIB (LNCIB), AREA Science Park, Trieste, Italy Correspondence to: Silvia Anna Ciafre, email: ciafre@uniroma2.it Keywords: glioblastoma; lncRNA; H19; NKD1; EZH2 Received: July 21, 2017 Accepted: February 10, 2018 Epub: February 14, 2018 Published: March 20, 2018 ABSTRACT The still largely obscure molecular events in the glioblastoma oncogenesis, a primary brain tumor characterized by an inevitably dismal prognosis, impel for investigation. The importance of Long noncoding RNAs as regulators of gene expression has recently become evident. Among them, H19 has a recognized oncogenic role in several types of human tumors and was shown to correlate to some oncogenic aspects of glioblastoma cells. Here we, hypothesyze that in glioblastoma H19 exerts its function through the interaction with the catalytic subunit of the PRC2 complex, EZH2. By employing a factor analysis on a SAGE dataset of 12 glioblastoma samples, we show that H19 expression in glioblastoma tissues correlates with that of several genes involved in glioblastoma growth and progression. H19 knock-down reduces viability, migration and invasiveness of two distinct human glioblastoma cell lines. Most importantly, we provide a mechanistic perspective about the role of H19 in glioblastoma cells, by showing that its expression is inversely linked to that of NKD1, a negative regulator of Wnt pathway, suggesting that H19 might regulate NKD1 transcription via EZH2-induced H3K27 trimethylation of its promoter. Indeed, we showed that H19 binds EZH2 in glioblastoma cells, and that EZH2 binding to NKD1 and other promoters is impaired by H19 silencing. In this work we describe H19 as part of an epigenetic modulation program executed by EZH2, that results in the repression of Nkd1. We believe that our results can provide a new piece to the complex puzzle of H19 function in glioblastoma.

The activity of the Wnt pathway undergoes complex regulation to ensure proper functioning of this principal signaling mechanism during development of adult tissues. The regulation may occur at several levels and includes both positive and negative feedback loops. In the present study we employed one of such negative feedback regulators, naked cuticle homolog 1 (Nkd1), to follow the Wnt pathway activity in the intestine and liver and in neoplasia originated in these organs. Using lineage tracing in transgenic mice we localized Nkd1 mRNA to the bottom parts of the small intestinal crypts and hepatocytes surrounding the central vein of the hepatic lobule. Furthermore, in two mouse models of intestinal tumorigenesis, Nkd1 expression levels were elevated in tumors when compared to healthy tissue. We utilized a collection of human intestinal polyps and carcinomas to confirm that NKD1 represents a robust marker of neoplastic growth. In addition, expression analysis of NKD1 in liver cancer showed that high expression levels of the gene distinguish a subclass of hepatocellular carcinomas related to aberrant Wnt signaling. Finally, our results were confirmed by bioinformatic analysis of large publicly available datasets that included gene expression profiling and high-throughput sequencing data of human colon and liver cancer specimens.

Background: Naked cuticle Drosophila 1 (NKD1) has been related to non-small cell lung cancer in that decreased NKD1 levels have been associated with both poor prognosis and increased invasive quality. Methods: Forty cases of lung adenocarcinoma staged as Tis or T1a were selected. Cases were subclassified into adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA), and small adenocarcinoma (SAD). Immunohistochemical studies for NKD1 were performed. Results: Forty samples comprised five cases of AIS (12.5%), eight of MIA (20.0%), and 27 of SAD (67.5%). AIS and MIA showed no lymph node metastasis and 100% disease-free survival, whereas among 27 patients with SAD, 2 (7.4%) had lymph node metastasis, and 3 (11.1%) died from the disease. Among the 40 cases, NKD1-reduced expression was detected in 8 (20%) samples, whereas normal expression was found in 15 (37.5%) and overexpression in 17 (42.5%). Loss of NKD1 expression was significantly associated with lymph node metastasis (p = 0.001). All cases with predominant papillary pattern showed overexpression of NKD1 (p= 0.026). Conclusions: Among MIA and SAD, MIA had better outcomes than SAD. Down-regulated NKD1 expression was closely associated with nodal metastasis, and overexpression was associated with papillary predominant adenocarcinoma.

// Han Guan 1, 2, * , Chunhui Liu 1, 2, * , Fang Fang 3, * , Yeqing Huang 1, 2, * , Tao Tao 1, 2 , Zhixin Ling 1, 2 , Zonghao You 1, 2 , Xu Han 1, 2 , Shuqiu Chen 1, 2 , Bin Xu 1, 2 , Ming Chen 1, 2 1 Department of Urology, Affiliated Zhongda Hospital of Southeast University, Nanjing, China 2 Surgical Research Center, Institute of Urology, Medical School, Nanjing, China 3 Department of Immunology, Bengbu Medical College, Bengbu, China * These authors contributed equally to this work Correspondence to: Bin Xu, email: njxb1982@126.com Ming Chen, email: mingchenseu@126.com Keywords: miR-744, prostate cancer, Wnt/β-catenin signaling, NKD1, tumorigenesis Received: October 21, 2016 Accepted: January 09, 2017 Published: January 18, 2017 ABSTRACT Accumulated evidence indicate that miR-744 functions as either tumor suppressor or oncogene in the progression of a variety of tumors, with a tumor type-specific way. However, little is known about how miR-744 impacts on the tumorigenesis of human prostate cancer. In this study, employing the analyses of microarray, qRT-PCR and re-analysis of MSKCC data, we found that CRPC tissues expressed much more miR-744 than ADPC tissues did, and the expression level of miR-744 was inversely associated with survival of CRPC patients. In vitro studies revealed that miR-744 promotes PCa cells proliferation, enhances migration, invasion; in vivo results demonstrated that silencing of miR-744 mediated by shRNA dramatically reduces PCa xenograft tumor growth. Importantly, through human gene expression array, pathway enrichment analysis and Western blot, we identified that miR-744 dramatically activated Wnt/β-catenin pathway by targeting multiple negative regulators of Wnt/β-catenin signaling, including SFRP1, GSK3β, TLE3 and NKD1. At molecular level, we further defined that NKD1 is a major functional target of miR-744. Our findings indicate that miR-744 acts as one of oncogenic factor in the progression of CRPC by recruiting a mechanism of aberrant activation of Wnt/β-catenin signaling.

Wnt signaling is an evolutionarily conserved pathway involved in many aspects of development and stem cell homeostasis. Activating mutations within the Wnt pathway are key in the initiation of colorectal cancers. Nkd1 is a negative feedback regulator of Wnt signaling with a unique method of antagonism that could potentially block the effect of the activating mutations. However, our understanding of the molecular function of Nkd1 has been derived mainly through zebrafish studies. To ultimately better understand the role of Nkd1 in human disease, I first employed the Intestinal Epithelial Cells line 18 (IEC-18) and Human Embryonic Kidney cells (HEK293) as mammalian models for the investigation of Nkd1 function. Using these two cell lines, I characterized the biochemical and genetic status of Wnt signaling in IEC-18 and HEK293 cells, followed by an analysis of Nkd1 function as it relates to previous work performed in zebrafish. My results indicate that while both cells respond biochemically and genetically to Wnt signaling, there are significant differences in the level of response at both levels that are not congruent with the current dogma. In addition, I found that while Nkd1 activity in these mammalian cell types shares some similarities with our previous findings in zebrafish, there exists cell-dependent differences in Nkd1 function. NSERC CGS-M, Ontario Graduate Scholarship

The Wnt signaling pathway is a highly conserved pathway critical for development and homeostasis of stem cells. Aberrant Wnt signaling causes developmental defects and disease. Over 90% of colorectal cancers (CRC) contain mutations in Wnt signaling which constitutively activate the Wnt pathway independent of a Wnt ligand. Nkd1 is a negative feedback regulator of Wnt signaling and functions by binding and preventing nuclear accumulation of the central signaling component, β-catenin. Nkd1 is upregulated in a large number of CRC but its function in CRC's is unknown. As NKD1 function is dependent on membrane localization it is hypothesized that Nkd1 is activated by the binding of a Wnt ligand to its receptors. To explore NKD1's function and activation, cancerous and mammalian cell lines were used to examine NKD1's response to Wnt. Results show that, under Wnt stimulation the distribution of NKD1 changes and interaction between NKD1 and β-catenin increases. Natural Sciences and Engineering Research Council of Canada

Nkd1 is a negative feedback inhibitor of Wnt signaling known to antagonize Wnt signaling in the cytoplasm by inhibiting the nuclear accumulation of the central signaling component β-catenin. In this thesis, I test the hypothesis that Nkd1 needs to be activated by a Wnt ligand in order for proper function. By stimulating Wnt signaling by a Wnt ligand or a constitutively active receptor, I found that only in the presence of a Wnt ligand does the cellular distribution of Nkd1 change from large punctae to smaller punctae distributed throughout the cytoplasm. Moreover, only in the presence of a Wnt ligand does Nkd1 have the ability to antagonize Wnt signaling and block the nuclear accumulation of β-catenin. Taken together, my data suggests that Nkd1 function and localization are critically dependent on the presence of a Wnt ligand.