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.

Includes summary / Includes bibliography / [586] leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (M.D.)--University of Adelaide, Dept. of Anatomy and Histology, 1988

Thesis (M.Sc.)--Memorial University of Newfoundland, 2005. / Bibliography: leaves 105-133.

Ladministration de spermine aux rats non sevrés, dans des conditions expérimentales précises, constitue un modèle détude de la maturation postnatale de lintestin grêle. Lanalyse des variations survenant au sein du protéome cellulaire, trois jours après lingestion de spermine, met en évidence des protéines impliquées dans le processus de maturation de lintestin grêle. La même étude, réalisée quelques heures après lingestion, précise la phase de desquamation cellulaire initialement observée. Les analyses effectuées révèlent que, six heures après le traitement des ratons à la spermine, la phase de desquamation touche à sa fin et lépithélium se régénère tandis que, dix-huit heures après ce traitement, les résultats indiquent un début de maturation des entérocytes, signalé, notamment, par lapparition, dans liléon, dune isoforme de la phosphatase alcaline, qui est spécifique de lépithélium intestinal mature. Des protéines impliquées dans la conformation de la chromatine et, par là, dans la régulation de lexpression de gènes spécifiques, sont identifiées six heures après lingestion de la polyamine. Devant la diversité des protéines identifiées trois jours après le traitement des animaux, nous nous sommes plus particulièrement intéressée à des modifications survenant au niveau de la cathepsine D et des enzymes du cycle de lurée. Des propriétés des enzymes de ce cycle sont effectivement modifiées lors de la maturation postnatale spontanée de lintestin grêle. Lors de lingestion de spermine, on observe des modifications au niveau de la transcription de gènes codant pour certaines de ces enzymes. Dans le cas de la cathepsine D, lexpression du gène codant pour lenzyme et lactivité de celle-ci sont diminuées dans les entérocytes de liléon suite à lingestion de spermine. Laction de cette polyamine a été reproduite, in vitro, à partir dune culture de cellules de tumeurs mammaires surexprimant le gène de la cathepsine D. Par ailleurs, nos résultats montrent que, dans certains cas, la spermine nagit pas au niveau de lexpression génique mais que des modifications post-traductionnelles des protéines sont à lorigine des variations dactivités enzymatiques observées, comme cest le cas de la phosphatase alcaline intestinale. Les protéines identifiées par lanalyse protéomique, et létude théorique de leur régulation, révèlent que plusieurs voies de signalisation intracellulaire sont affectées par lingestion de spermine : cette polyamine active la voie de signalisation faisant intervenir la PLC ainsi que celle dépendant des glucocorticoïdes. A linverse, la signalisation intracellulaire impliquant la PKA est inhibée. En conclusion, notre approche globale, considérant les protéines dont des propriétés varient de manière majeure dans les entérocytes de rats immatures soumis à laction de la spermine, ouvre plusieurs pistes pour des recherches futures ciblées. La poursuite des investigations, limitées cette fois à considérer une voie métabolique particulière ou des protéines spécifiques, conduira à une compréhension plus complète des modifications survenant dans les entérocytes du rat au moment du sevrage.

published_or_final_version / Surgery / Master / Master of Surgery

Introduction: Colorectal cancer (CRC) is the fourth-most common cancer in Canada with a high mortality rate. Familial adenomatous polyposis (FAP) is a hereditary form of CRC; FAP patients carry germline mutations of the tumour suppressor gene adenomatous polyposis coli (APC). The function of Dlx genes in the gastrointestinal tract (GIT) has not been previously explored. Methods: Immunofluorescence (IF) was performed to identify Dlx2+ intestinal cells. Chromatin immunoprecipitation (ChIP) was performed to identify DLX2-Apc promoter interaction. Quantitative real time polymerase chain reaction (qRT-PCR) was performed on mouse small and large intestines (normal and Dlx1/Dlx2 mutant mice). Electrophoretic mobility shift assays (EMSA) and reporter assays were carried out to investigate direct binding and activity, respectively, of DLX2 on the Apc promoter in-vitro. Dlx2 expression was explored in ApcMIN mice and human CRC tumor specimens. Results: Dlx2 is highly expressed in mouse embryonic and adult intestinal epithelia. Moreover, Dlx2 is expressed in the ApcMIN mice GIT as well as in some human CRC tumor specimens. ChIP, EMSA and reporter gene assays demonstrated that DLX2 protein specifically interacts with the Apc promoter in-situ and activates its expression in vitro. In-vivo and in-vitro, β-catenin protein levels are increased when DLX2 is absent or reduced by shRNA to Dlx2. Conclusions: Regulation of APC expression during development is poorly understood. We have evidence that DLX2 interacts with the Apc promoter in-vivo. We have shown that DLX2 induces Apc transcription by directly binding to the Apc promoter in-vitro. We also showed that β-catenin expression is altered in the Dlx1/Dlx2 mutant GIT. This finding implicates the involvement of DLX2 in the canonical Wnt signalling pathway. Ultimately, restoring APC expression may be a novel strategy towards preventing progression of intestinal polyps to adenocarcinoma. This research will contribute to our knowledge of the genetic and epigenetic regulatory pathways that control intestinal development, mucosal self-renewal and CRC.

No description available.

Introduction: Colorectal cancer (CRC) is the fourth-most common cancer in Canada with a high mortality rate. Familial adenomatous polyposis (FAP) is a hereditary form of CRC; FAP patients carry germline mutations of the tumour suppressor gene adenomatous polyposis coli (APC). The function of Dlx genes in the gastrointestinal tract (GIT) has not been previously explored. Methods: Immunofluorescence (IF) was performed to identify Dlx2+ intestinal cells. Chromatin immunoprecipitation (ChIP) was performed to identify DLX2-Apc promoter interaction. Quantitative real time polymerase chain reaction (qRT-PCR) was performed on mouse small and large intestines (normal and Dlx1/Dlx2 mutant mice). Electrophoretic mobility shift assays (EMSA) and reporter assays were carried out to investigate direct binding and activity, respectively, of DLX2 on the Apc promoter in-vitro. Dlx2 expression was explored in ApcMIN mice and human CRC tumor specimens. Results: Dlx2 is highly expressed in mouse embryonic and adult intestinal epithelia. Moreover, Dlx2 is expressed in the ApcMIN mice GIT as well as in some human CRC tumor specimens. ChIP, EMSA and reporter gene assays demonstrated that DLX2 protein specifically interacts with the Apc promoter in-situ and activates its expression in vitro. In-vivo and in-vitro, β-catenin protein levels are increased when DLX2 is absent or reduced by shRNA to Dlx2. Conclusions: Regulation of APC expression during development is poorly understood. We have evidence that DLX2 interacts with the Apc promoter in-vivo. We have shown that DLX2 induces Apc transcription by directly binding to the Apc promoter in-vitro. We also showed that β-catenin expression is altered in the Dlx1/Dlx2 mutant GIT. This finding implicates the involvement of DLX2 in the canonical Wnt signalling pathway. Ultimately, restoring APC expression may be a novel strategy towards preventing progression of intestinal polyps to adenocarcinoma. This research will contribute to our knowledge of the genetic and epigenetic regulatory pathways that control intestinal development, mucosal self-renewal and CRC.

No description available.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.