Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2007 Jun 15

Ulm, Univ., Diss., 2007.

Introduction: Van Gogh-Like 2 (VANGL2) is a scaffolding planar cell polarity protein involved in non-canonical Wnt signalling. It has been shown to have crucial roles in regulating epithelial development and homeostasis. Moreover, VANGL2 has been implicated in human cancers, with increased expression and copy number amplification seen in several cancer contexts. Many related components within this pathway have also been linked to cancer development, with VANGL2 expression known to regulate factors involved in cell migration and extracellular matrix (ECM) remodelling in cell lines. These cellular processes tend to be erroneously activated in cancer. VANGL2 is known to inhibit the classical driver pathway of colorectal cancer (CRC), canonical, or β- catenin dependant, Wnt signalling, in CRC cell lines. The aim of this thesis is to determine the expression of VANGL2 in CRC, and to investigate how VANGL2 may act to regulate intestinal homeostasis and disease. Methods: Transcriptional verification of VANGL2 expression in the mouse intestine was carried out by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), and transcripts localised within the murine colon using RNA-In Situ Hybridisation (RNAISH). Expression and localisation of the VANGL2 protein and related non-canonical Wnt signalling components was confirmed using immuno-histochemistry (IHC). Furthermore, using a combination of human Tissue Micro-Array (TMA), transcriptional data and genomic data, we determined an association between VANGL2 on tumour grade and disease-free survival. To functionally validate the effects of VANGL2 on colorectal biology, we used a model in which VANGL2 is selectively deleted from the colonic epithelium using Villin-CreERT Vangl2flox mouse lines. Using a combination of molecular biology methods, we identified the ECM as differentially regulated following VANGL2 modulation. To test the role of VANGL2 in colorectal cancer, we used a murine colorectal cancer model in which adenomatous polyposis coli (APC) is deleted from colonic epithelium resulting in the formation of cancer concurrently with deletion of Vangl2. We evaluated survival of these mice as well as tumour number and size. Tumour tissue was analysed using IHC, qRT-PCR and 3-Dimensional organoid culture. Results: Within this thesis I have illustrated that the murine colonic epithelium expresses Vangl2, and other components known to interact with VANGL2 including Vangl1, Wnt5A, and Protein Tyrosine Kinase 7 (Ptk7). I have also shown that VANGL2 is expressed within the human colonic epithelium. I go on to show that 9.2% of human CRC possesses VANGL2 transcriptional alterations which correlates with a worsened disease-free survival (DFS) rate among patients. Using IHC, I also show that higher grade CRC is associated with increased VANGL2 expression. In our murine cancer model, mice with single or dual-copy loss of VANGL2 were found to have a reduced number of colonic tumours, while maintaining similar tumour size. Investigations to identify how VANGL2 may have control of tumour initiation were carried out focussing on the ECM. I found that, contrary to what I have discovered in the healthy murine colon, tumours from VANGL2-deficient mice had increased transcription of the ECM markers Secreted protein acidic and rich in cysteine (Sparc) and Decorin (Dcn), as well as increased expression of the ECM regulators Matrix Metallopeptidase 9 (Mmp9) and Tissue Inhibitor of Metalloproteinases 1 (Timp1). Changes in the ECM was also seen at the protein level, with increases in staining for the ECM components Col1 (Collagen, type I), and Laminin in VANGL2-deficient tissue. The ECM modulator Connective Tissue Growth Factor (Ctgf), is implicated in multiple cancers including CRC and is increased within VANGL2-deficient tumours at both the transcript and protein level, implicating Ctgf in increasing the ECM of these tumours.

published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Includes bibliographical references (p. 204-216).

PAWS1/FAM83G, a member of the poorly characterised FAM83 family of proteins that share the conserved domain of unknown function DUF1669, was identified as an interactor of the SMAD1 transcription factor. Because BMP signalling plays a fundamental role during embryogenesis, collaboration with Jim Smith (The Francis Crick Institute, London) led to the discovery that ectopic expression of PAWS1 mRNA in Xenopus embryos leads to a complete duplication of body axis. In similar assays, such a phenotype is associated with either the inhibition of canonical (SMAD4-dependent) BMP signalling or the activation of canonical Wnt/β-catenin signalling. PAWS1 has been reported to modulate only non-canonical BMP signalling and not influence canonical BMP signalling, findings which were also validated in Xenopus embryos and PAWS1-knockout U2OS cells in this thesis. Therefore, focus turned to investigating potential roles of PAWS1 in the regulation of the canonical Wnt/β-catenin signalling pathway. The canonical Wnt/β-catenin signalling pathway plays critical roles during embryogenesis, stem cell self-renewal and in adult tissue homeostasis and is often misregulated in developmental defects, including skin and hair abnormalities, and cancer. In the absence of Wnt signals, sequential phosphorylation of the transcriptional co-factor β-catenin by CK1 and GSK3 results in the ubiquitylation of β-catenin, priming it for degradation via the proteasome. Upon Wnt-activation, β-catenin is stabilised and translocates to the nucleus, where it associates with TCF and LEF and regulates the expression of Wnt-target genes. Although the fundamental steps in Wnt signalling are established, many gaps remain in our understanding of the precise regulation of the pathway. It is demonstrated in this thesis that PAWS1 activates Wnt signalling in both Xenopus embryos and human cells. Furthermore, in PAWS1-knockout U2OS cells Wnt signalling is attenuated. Collectively, these data uncover a role for PAWS1 as a novel regulator of canonical Wnt/β-catenin signalling. In search of molecular mechanisms through which PAWS1 regulates Wnt/β-catenin signalling, a proteomic approach on endogenous PAWS1 revealed the Ser/Thr protein kinase CK1α as a robust PAWS1 interactor. PAWS1 interacts and colocalises with endogenous CK1α. CK1 isoforms are key regulators of Wnt signalling and they phosphorylate many components of the pathway, however their precise regulation in cells, despite being critically important, is poorly understood. After mapping CK1-interaction sites to key residues within the conserved DUF1669 domain of PAWS1, it was possible to demonstrate that the interaction between PAWS1 and CK1α is critical for PAWS1 to activate Wnt signalling in both Xenopus embryos and U2OS cells. Although the phosphorylation of β-catenin on Ser45, which is reported to be phosphorylated by CK1 isoforms, appears to be unaltered by PAWS1-deficiency, the Wnt3A-induced nuclear translocation of β-catenin is slightly inhibited in PAWS1 knockout U2OS cells. It is likely that PAWS1 controls Wnt signalling by directing CK1α to key subcellular locations and substrates upon Wnt stimulation to regulate the nuclear translocation of β-catenin. Consistent with this hypothesis, a global phosphoproteomics analysis of wild type and PAWS1-/- U2OS cells has revealed differential phosphorylation of proteins that may be regulated by the PAWS1:CK1α interaction. Interestingly, PAWS1 appears to control levels of endogenous CK1α protein and vice versa, although the mechanisms by which each achieves this are still unclear. The findings that the DUF1669 domain of PAWS1 interacts with CK1α led to the discovery that all FAM83 members bind to different CK1 isoforms through an identical mechanism. This has led to the hypothesis that FAM83 members serve as anchoring proteins for CK1 isoforms, and in doing so, they regulate CK1 subcellular localisation and substrate accessibility in cells. Regulation of PAWS1 by post-translational modifications remains poorly defined. A proteomic approach identified calcium and calmodulin-dependent kinase isoforms D and G (CaMK2D and CaMK2G) as two novel interactors of PAWS1. CaMK2 enzymes are activated in response to calcium signals to control cytoskeletal rearrangements and cell movement. PAWS1 has been implicated in actin cytoskeletal dynamics and cell migration, through its dynamic interaction with the adapter protein CD2AP at the cell periphery. Here, PAWS1 has been shown to be phosphorylated at Ser356 by CaMK2D in cells and this phosphorylation event is demonstrated to be important for PAWS1-dependent cell migration. Lastly, several PAWS1 mutations have been recently linked with the pathogenesis of skin diseases in dogs and humans. However, how these mutations relate to PAWS1 function in cells and potentially cause the disease phenotypes remain elusive. In this thesis, initial steps have been taken to address the potential impact of these pathogenic mutants on PAWS1 function.

The Wnt/β-catenin signaling pathway is often deregulated in cancer. Here we investigate Wnt/β-catenin signaling, aberrant accumulation of β-catenin, and epigenetic deregulation in breast cancer and parathyroid tumours. An aberrantly spliced Wnt coreceptor LRP5 (LRP5Δ) is important for accumulation of nonphosphorylated active β-catenin and tumour growth in parathyroid tumours. Paper I demonstrated frequent expression of LRP5Δ in breast tumours and substantiated that breast tumour cell growth was dependent on continuous activation of the Wnt/β-catenin pathway by LRP5Δ. A LRP5 antibody reduced the levels of active β-catenin, inhibited tumour cell growth and caused apoptosis in breast cancer cells. Antibody therapy may have a significant role in the treatment of breast cancer. Paper II revealed lost expression of the tumour suppressor gene APC in parathyroid carcinomas, likely due to CpG methylation. Also accumulation of nonphosporylated active β-catenin was observed, indicating activation of Wnt/β-catenin signaling. Treatment of primary parathyroid carcinoma cells with the demethylating agent 5-aza-2’-deoxycytidine reduced the levels of active β-catenin, inhibited cell growth and caused apoptosis, suggesting that adjuvant epigenetic therapy could be considered in patients with metastatic or recurrent parathyroid carcinoma. In paper III we showed that the expression of the tumour suppressor gene HIC1 was generally reduced in parathyroid tumours of primary and secondary origin, and parathyroid carcinomas. Overexpressing HIC1 reduced cell viability and suppressed colony formation, supporting a tumour suppressor role in the parathyroid gland. Results suggested that the observed underexpression of HIC1 could be explained by epigenetic deregulation involving histone methylation rather than CpG methylation. Paper IV demonstrated increased expression of the histone methyltransferase EZH2 in parathyroid tumours of primary and secondary origin, and most apparent in parathyroid carcinomas. Decreasing EZH2 resulted in reduced cell viability and colony formation capacity suggesting that EZH2 may function as an oncogene in parathyroid tumours. Furthermore, depletion of EZH2 also reduced the amount of active β-catenin. EZH2 may represent a novel therapeutic target in parathyroid tumours. The fact that HIC1 was underexpressed and EZH2 overexpressed in parathyroid tumours regardless of the hyperparathyroid disease state may represent a possibility for a common pathway in parathyroid tumour development.

Wnt signalling is involved throughout development and is inappropriately activated in a variety of human cancers. In the canonical pathway, secreted Wnt proteins induce the stabilization of b-catenin. Drosophila Naked (Nkd), or Nkd1 and Nkd2 in vertebrates, is believed to antagonize canonical Wnt signalling through an interaction with Dishevelled (Dvl). Analysis of a high-throughput protein-protein interaction screen conducted in our lab led to the identification of novel Nkd1 interacting proteins, including Nkd1/2 and Axin1. Mapping of Nkd1 regions required for these novel interactions and functional studies, including transcriptional and siRNA mediated knockdown assays, were performed to examine the role of Nkd1 in canonical Wnt signalling. Previous work suggests that Nkd1 functions only through Dvl, but this work suggests that Nkd1 acts via a more complex mechanism. In addition to serving as an antagonist to regulate the canonical Wnt pathway, we propose that Nkd1 may also act positively to promote signalling.

Wnt signalling is involved throughout development and is inappropriately activated in a variety of human cancers. In the canonical pathway, secreted Wnt proteins induce the stabilization of b-catenin. Drosophila Naked (Nkd), or Nkd1 and Nkd2 in vertebrates, is believed to antagonize canonical Wnt signalling through an interaction with Dishevelled (Dvl). Analysis of a high-throughput protein-protein interaction screen conducted in our lab led to the identification of novel Nkd1 interacting proteins, including Nkd1/2 and Axin1. Mapping of Nkd1 regions required for these novel interactions and functional studies, including transcriptional and siRNA mediated knockdown assays, were performed to examine the role of Nkd1 in canonical Wnt signalling. Previous work suggests that Nkd1 functions only through Dvl, but this work suggests that Nkd1 acts via a more complex mechanism. In addition to serving as an antagonist to regulate the canonical Wnt pathway, we propose that Nkd1 may also act positively to promote signalling.

LIMA, M. R. Efeito da calendula officinalis em ratos submetidos a periodontite experimental: participação das vias RANK-RANKL-OPG e WNT B-CATENINA. 2016. 66 f. Dissertação (Mestrado em Ciências Morfofuncionais) - Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2016. / Submitted by Erika Fernandes (erikaleitefernandes@gmail.com) on 2017-01-20T16:06:05Z No. of bitstreams: 1 2016_dis_mrlima.pdf: 2579356 bytes, checksum: 5976091267e9fb5683cbc5c4fc27cee2 (MD5) / Approved for entry into archive by Erika Fernandes (erikaleitefernandes@gmail.com) on 2017-01-20T16:06:13Z (GMT) No. of bitstreams: 1 2016_dis_mrlima.pdf: 2579356 bytes, checksum: 5976091267e9fb5683cbc5c4fc27cee2 (MD5) / Made available in DSpace on 2017-01-20T16:06:13Z (GMT). No. of bitstreams: 1 2016_dis_mrlima.pdf: 2579356 bytes, checksum: 5976091267e9fb5683cbc5c4fc27cee2 (MD5) Previous issue date: 2016-12-07 / Periodontitis is an infecto-inflammatory disease that leads to connective tissue and alveolar bone loss. Calendula officinalis (CLO) has been used due to its anti-inflammatory effects. Therefore the aim of this study was to evaluate the effect of CLO on alveolar bone loss (ABL) in rats focusing on RANK-RANKL-OPG and WNT signaling pathways. Experimental periodontitis (EP) was induced through placement of a nylon ligature around the upper left 2nd molar, and the hemimaxilla used as control. The animals were divided in groups: Normal, subjected to no treatment; Saline (SAL), that received 2 ml/kg of 0,9% saline solution orally; or CLO at 90 mg/kg orally, 30 minutes before EP and daily for 11 days until euthanasia. In order to evaluate the periodontal tissue, it macroscopic, micro-tomographic, electron scanning microscopy (SEM), confocal microscopy and polarized light microscopy analyses were performed, as well as immunohistochemistry for WNT 10b, β-catenin, DKK-1, RANK, RANKL, and OPG. During euthanasia the gingival tissue was removed for malonaldehyde (MDA) assay. Treatment with CLO significantly prevented ABL, preserved bone internal microstructure (p<0.05) and topography, and also preserved collagen fibers from the periodontal ligament, when compared to SAL. CLO significantly increased the number of immunopositive cells for WNT 10b, β-catenin and OPG and reduced DKK-1, RANK (p>0.05) and RANKL. CLO reduced the gingival levels of MDA compared to SAL (p<0.05). In this way, we can conclude that CLO prevented ABL via RANK-RANKL-OPG and WNT signaling pathway. / A periodontite é uma doença infecto-inflamatória que causa perda de tecido conjuntivo e osso alveolar. A Calendula officinalis (CLO) tem sido utilizada pelos seus efeitos anti-inflamatórios. Nesse contexto, o objetivo deste trabalho foi avaliar efeito da CLO na perda óssea alveolar (POA) em ratos com foco na participação do eixo RANK-RANKL-OPG e da via WNT/β-catenina. A periodontite experimental (PE) foi induzida através da inserção do fio (nailon 3.0) em torno do 2º molar superior esquerdo, e hemiarcada contralateral usada como controle. Os animais foram divididos em grupos: Normal, não submetido a nenhum procedimento; Salina (SAL), que receberam 2 ml/kg de solução salina 0,9% - v.o.; ou CLO na dose de 90 mg/kg - v.o. 30 min antes da PE e diariamente durante por 11 dias até eutanásia. Para avaliação do tecido periodontal realizaram-se análises macroscópica, por microtomografia computadorizada, por microscopia eletrônica de varredura (MEV), microscopia confocal e microscopia por luz polarizada, imunohistoquímica para WNT 10b, β-catenina, DKK-1, RANK, RANKL e OPG. Por ocasião da eutanásia foi removido tecido gengival para avaliação dos níveis de malondialdeído (MDA). O tratamento com CLO preveniu de forma significante a POA, preservou a microestrutura interna (p<0,05) e topografia do tecido ósseo, e preservou também as fibras colágenas do ligamento periodontal, quando comparado a SAL. A CLO provocou aumento significante de células imunopositivas para WNT 10b, β-catenina e OPG e redução na imunomarcação de DKK-1, RANK (p>0,05) e RANKL. CLO reduziu os níveis de MDA gengivais comparados a SAL (p<0,05). Desta forma, podemos concluir que a CLO previne a POA com participação do eixo RANK-RANKL-OPG e da via WNT/β-catenina.