L’isoforme p200CUX1 est un répresseur transcriptionnel qui est clivé dans les NIH3T3 par la cathepsin L en isoforme pllOCUXl laquelle acquiert alors la capacité d’activer la transcription des gènes cibles. Dans le système digestif, CUX1 est exprimé dans l’iléon et le côlon proximal chez la souris adulte. Aucun rôle dans le contexte de l’épithélium colique n’a été attribué à ce jour aux isoformes p200CUX1 et p110CUX1. Le but de cette Thèse a donc été d’investiguer l’implication de p200CUX1 et p110CUX1 dans l’épithélium colique ainsi que dans la carcinogenèse colorectale. Une étude différentielle par criblage de micropuces à ADN a permis d’identifier le gène PLZF comme cible transcriptionnelle de CUX1 dans le côlon. Une analyse informatique a prédit 15 sites de liaison potentiels pour CUX1 dans la portion 5’-UTR et dans une région de 776 p.b. du promoteur du gène PLZF. Des essais de gel de rétention et d’immunoprécipitation de la chromatine ont démontré une interaction de CUX1 avec certains sites prédits du gène PLZF in vitro et in vivo. Des essais transcriptionnels ont rapporté une diminution de deux fois de l’activité transcriptionnelle du promoteur du gène PLZF par CUX1. Les sites # 13, 14 et 15 du promoteur du gène PLZF ont été identifiés comme étant les sites préférentiellement liés par CUX1. Une analyse Western a permis d’établir un patron d’expression réciproque entre les cellules qui expriment CUX1 et celles qui expriment PLZF. Ces travaux ont également permis d’identifier un nouveau mécanisme intracellulaire impliqué dans la production de l’isoforme p11OCUX1. Un traitement des cellules 293T, T84 et Caco2/15 avec deux inhibiteurs du protéasome, soit le MG132 et le bortezomib, résulte en une diminution de l’expression de l’isoforme p11OCUX1. Une rapide accumulation de l’isoforme p200CUX1 est observée après 4 heures de traitement des cellules T84 au MG132. L’utilisation d’extraits de protéines cytoplasmiques et nucléaires suggère que la protéolyse limitée de l’isoforme p200CUX1 en p11OCUX1 prend place dans le noyau. Une expérience de dégradation in vitro confirme que le protéasome est la seule protéase responsable du clivage protéolytique. Une analyse informatique de la séquence en acides aminés de CUX1 a prédit quatre régions susceptibles d’être clivées par le protéasome. Enfin, le présent travail a tenté de démystifier le rôle de p11OCUX1 dans la prolifération des cellules épithéliales intestinales et coliques. Les résultats montrent que l’isoforme p110CUX1 n’est pas un agent transformant. Sa surexpression dans les cellules IEC-6 et DLD-1 ne mène pas à une modulation de la vitesse de prolifération cellulaire. Aucune polype n’est visible au niveau de l’intestin grêle et/ou du côlon suite à la surexpression de p11OCux1 et ce, même après une période latente de 12 mois. L’abolition de l’expression des isoformes de CUX1 par interférence à l’ARN dans les cellules IEC-6 conduit à une diminution significative de la prolifération cellulaire, alors que dans les cellules T84 et DLD-1, il y a un ralentissement modeste de la croissance cellulaire. Les cellules DLD-1 shCUX1 ne forment pas moins de colonies en indépendance d'ancrage. Les résultats de cette étude démontrent que le gène PLZF est une cible de CUX1, que le protéasome est impliqué dans la protéolyse limitée de p200CUX1 en p110CUX1 et que p110CUX1 n’est pas impliquée dans la prolifération des cellules épithéliales intestinales et coliques et de cancer du côlon chez l’humain.

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Every year, more than a million people are diagnosed with colorectal cancer (CRC) that develops in the large intestine. It is one of the most studied cancers in the world but still more knowledge about how this cancer develops and acts is needed in order to use more effective ways to treat CRC. Autophagy is a vital mechanism in cells that is also suggested to maintain cancer cell survival. In normal cells, it plays an important role by removing damaged cells and organelles as well as eliminating pathogens. Under metabolic stress this mechanism is induced to provide enough nutrients and energy for the cell to survive. Cancer cells are exposed to greater environmental stress than normal cells and therefore, cancer cells exhibit higher levels of autophagy suggesting it to be a crucial mechanism for their survival. Gaining a deeper understanding of this essential mechanism and its activation might provide new insights and improved treatments for the fight against colorectal cancer. In situ Proximity Ligation Assay (PLA) is a protein detection method that enables sensitive and specific detection of proteins and protein-protein interactions (PPIs) in cell lines and tissue samples. The method uses simultaneous recognition of two independent antigens on a protein or protein complex together with a rolling circle amplification (RCA) to form a rolling circle product (RCP) on top of the target. By using fluorescent oligonucleotides, RCP can be visualized and is seen as a bright spot that enables sensitive detection of the target at single-molecule resolution. The aim of this study was to develop assays to detect endogenous molecular events known to be biomarkers of autophagy in situ in order to study autophagy mechanism in CRC patient samples. We focused our research on two PPIs that were known to interact when autophagy is induced. The first investigated interaction was between microtubule-associated protein 1A/1B- light chain 3 (LC3) and sequestome-1 (SQSTM1), an interaction that occurs during autophagy initiation. The second interaction was between B-cell lymphoma 2 (Bcl-2) and Bcl-2/adenovirus E1B 19-kDa interacting protein 3 (BNIP3) that takes place during hypoxia-induced autophagy. To study whether these PPIs can be used as a detection method to monitor autophagy, we used a well- established cell model based on serum starvation and CoCl2 - an hypoxic mimetic- treatment of the intestinal cancer cell line Caco-2 in comparison to normal culture condition. According to isPLA quantification, detection of both PPIs was distinctly higher in treated cells compared to untreated cells giving promising results and suggesting that they can be potentially used as suitable assays to monitor these biomarkers of autophagy. For development of an improved protein detection method that enables the study of several PPIs simultaneously in a tissue sample (In situ Multiplexing), we conjugated directly a short oligonucleotide strand to the primary antibodies. These formed proximity probes could later be used in in situ for multiplexing.

DKK1, SFRP1, WIF-1, and Wnt5a encode Wnt pathway genes that are frequently silenced by promoter hypermethylation in colorectal cancer. Despite attractive biological consequences of these events, it is unclear whether they contribute to patient prognostication or may influence tumour cell biology within distinct patient subsets. I sought to determine the prognostic roles of these methylation events in a large cohort of colorectal carcinomas from Ontario and Newfoundland. Methylation was quantified and associated with patient clinicopathlogical features. Methylation was present in cancer tissue. DKK1, Wnt5a, and SFRP1 were strongly and independently associated with tumour subtype in a manner that suggested subtype-specific activity of Wnt signaling. Methylation of DKK1 was a borderline prognosticator of favourable outcome. These results offer intriguing insight into subtype-specific biology and lead to a proposed model whereby methylation-induced Wnt bias may contribute to patient outcome.

Colorectal cancer (CRC) is common in Ontario. This study described the processes of care following CRC resection, and identified CRC relapse from administrative data. Methods: CRC patients aged 18-80 from 1996-2001 with a colorectal resection were identified from the Ontario Cancer Registry. Linked discharge abstracts and physician billings were examined for physician visits, body imaging and endoscopy over the 5 year follow-up period. Administrative codes suggesting disease relapse were compared with patient charts. Results: Overall, 12,804 patients were identified and 8,804 had no evidence of relapse. Most (96.2%) patients had general practitioner follow-up, while 49.3% had medical oncology and 80.4% had general surgery follow-up. Greater than 90% of patients received endoscopy, while only 68.7% of patients received body imaging. Detecting disease relapse was 87.5% sensitive and 93.0% specific. Conclusions: There is potential for improving post-resectional follow-up in CRC patients. It is possible to detect relapse through administrative databases.

DNA methylation is increasingly recognised as a significant epigenetic event that may initiate and drive the process of neoplasia in humans. In the colon, DNA methylation of key genes is common in a subset of colorectal cancers. The extent to which DNA methylation at various genes contributes to initiation of colorectal neoplasms is less clear. This study sought to clarify the biological and clinicopathological significance of methylation of various genes in the development of sporadic and familial colorectal neoplasia. Quantitative methylation-specific PCR (qMSP) assays (capable of detecting down to a measureable proportion of 0.1% of the total input DNA) were developed to determine the presence of CpG methylation at a given gene. Methylation of MLH1-C was found in the apparently normal mucosa samples from seven of 104 (7%) of individuals with sporadic colorectal cancer (CRC) showing microsatellite instability (MSI). No methylation of MLH1-C was found in the biological samples of individuals with microsatellite stable (MSS) counterparts (n=131). MLH1-C methylation may be a field defect that predisposes to the development of sporadic colorectal neoplasia, particularly those demonstrating MSI. Methylation of three of five genes within the 3p22 region including AB002340, MLH1, ITGA9, PLCD1 and DLEC1 (regional 3p22 methylation) was found in 83% of sporadic MSI (n=86) and 12% of MSS cancers demonstrating BRAF V600E mutation (n=42). Regional 3p22 correlated strongly with CpG island methylator phenotype (CIMP), and other clinicopathological characteristics typical of CIMP. Thus, regional 3p22 methylation and CIMP may be overlapping phenomena. Regional 3p22 methylation and the BRAF V600E mutation were found in normal colonic mucosa of four individuals with sporadic MSI CRC, and these cases also had multiple synchronous serrated polyps. These molecular aberrancies may predispose some individuals to the development of metachronous serrated neoplasia. Germline epimutations of APC do not contribute towards the development of FAP, AFAP, or hyperplastic polyposis syndromes. However, APC methylation in normal colonic mucosa of these individuals may represent a field defect in the development of futher neoplasms. In conclusion, different patterns of DNA methylation in normal colonic mucosa may represent a field defect important in the development of different subtypes of colorectal neoplasia.

Background: It is well recognized that not all patients with stage C colorectal cancer (CRC) derive a survival benefit from adjuvant chemotherapy. It would therefore be advantageous to identify factors that define a target group for treatment. It has been suggested that those most likely to benefit are women with proximal tumours. Recent work has suggested microsatellite instability (MSI) may be a useful marker however the limited studies performed are conflicting. Aim: To determine if gender, site, tumour histology or microsatellite (MSI) status predict survival benefit from 5FU-based adjuvant chemotherapy in stage C CRC. Method: Data was collated on stage C colorectal cancer cases that underwent curative resection over a 20-year period (inclusive of years prior to standard chemotherapy). Pathology was re-evaluated, DNA extracted from the formalin fixed paraffin specimen and MSI status established. Primary endpoint was cancer-related death. Kaplan-Meier curves were constructed for univariate analysis and differences analysed by log rank test. Multivariate analysis was performed using Cox proportional hazard model adjusting for age, gender, site, distinct pathological variables and MSI. A compounding effect between these factors and chemotherapy benefit was measured by interaction testing Results: 811 unselected cases were included in the study. Thirty-seven percent received chemotherapy. Chemotherapy significant improved cancer-specific survival (HR of dying 0.66 (95% CI 0.52-0.83 p=0.0003). Female gender offered a survival advantage overall (HR 0.81 95% CI 0.68-0.97; p=0.02) however site did not influence outcome (HR 1.03). On interaction testing, gender, site and tumour histology did not significantly influence the survival effect of chemotherapy. 802 cases were included in the MSI analysis of which 77 exhibited MSI. MSI status did not influence prognosis (HR of cancer death 1.45, 95% CI 0.90-2.21; p= 0.13). However, in the non-chemotherapy cohort, MSI conferred a significantly less favourable outcome (HR 1.89, 95%CI 1.13-3.16; p= 0.02). Chemotherapy produced a survival benefit in both the MSI (HR 0.08 95% CI 0.02-0.27; p=<0.0001) and the microsatellite stable (MSS) cohort (HR 0.62, 95% CI 0.47-0.81; p=0.001). On interaction testing, neither compounded the benefit of chemotherapy, however of all the tested parameters, MSI came closest to significance (p=0.08). Conclusion: These results suggest that 5FU-based adjuvant chemotherapy for stage C colorectal cannot be targeted using gender, tumour site, histological characteristics or MSI. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1522132 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2010

Research Doctorate - Doctor of Philosophy (PhD) / A number of oncolytic picornaviruses are currently under evaluation as potential therapeutic agents for a range of human malignancies. In particular, a subset of naturally occurring human C-cluster enteroviruses; Coxsackievirus A13 (CVA13), Coxsackievirus A15 (CVA15), Coxsackievirus A18 (CVA18) and Coxsackievirus A21 (CVA21) and the human B-cluster enterovirus, Echovirus 1 (EV1), display promising pre-clinical oncolytic activity against a wide variety of neoplastic cells. CVA21 is currently under clinical evaluation for the control of melanoma, breast, prostate and head/neck cancer. The preferential targeting of cancer cells by this subset of viruses is based on extracellular capsid interactions with specific viral receptors (intercellular adhesion molecule-1 [ICAM-1], decay-accelerating factor [DAF] or integrin α2β1), on the surface of malignant cells. In the present study, the therapeutic potential of this subset of enteroviruses was evaluated as a novel treatment strategy for the control of human malignancies of the gastrointestinal system. In Chapter 3, the capacity of the aforementioned enteroviruses for oncolytic activity was assessed in a panel of in vitro human gastric cancer cell cultures. Flow cytometric analysis revealed low-to-medium levels of ICAM-1, in addition to abundant α2β1 and DAF expression on the surface of gastric cancer cell lines. Cell monolayer lytic infectivity assays demonstrated that, of the viruses under evaluation, EV1 displayed the most potent and widespread in vitro lytic activity against the gastric cancer cell lines. Monoclonal antibody blockade confirmed the specific integrin α2β1-mediated route of EV1 cell infection in the gastric cancer MKN-45 cell line. Subsequently, an in vivo dose ranging study assessing the efficacy of oncolytic EV1 was undertaken in an immune-compromised MKN-45-Luc mouse model of human gastric cancer peritoneal carcinomatosis (PC). In this model, an intra-peritoneal dose of as little as 1x103 TCID50 EV1 resulted in a significant reduction in peritoneal tumour burden. In Chapter 4, the oncolytic capacity of this enterovirus subset was further evaluated, as a potential therapeutic option for the control of colorectal cancer (CRC). Flow cytometric analysis of a panel of CRC cell lines demonstrated abundant levels of DAF and integrin α2β1, and low-to-moderate levels of ICAM-1 expression on the surface of CRC cells. Of the subset of viruses examined, a DAF-using variant of CVA21 (CVA21-DAFv) displayed the most potent and widespread oncolytic activity against in vitro CRC cell cultures. Consequently, the potential in vivo oncolytic capacity of CVA21-DAFv and the wild-type CVA21 was evaluated in three individual immune-compromised mouse sub-cutaneous xenograft models of human CRC. However, despite the immunohistochemical detection of ICAM-1/DAF on cells of the CRC xenografts, and the detection of infectious virus in the blood of treated tumour-bearing mice, a detectable reduction in tumour burden was not observed. On account of the varying degrees of oncolytic efficacy observed in colorectal and gastric cancers, global gene expression profiling was employed in Chapters 5 and 6, to further elucidate the molecular mechanisms of enterovirus-mediated tumour cell tropism and cell death. As the most extensively characterised virus in pre-clinical studies, and the only virus of this subset under current clinical evaluation, CVA21 was selected as the challenge virus for analysis of the transcriptional response to enterovirus infection. Malignant cells that displayed reproducible susceptibility to in vitro and in vivo lytic CVA21 challenge were necessary for extensive characterisation, therefore, melanoma SK-Mel-28 and breast cancer MDA-MB-231-Luc cell lines, rather than CRC cell lines, were utilised. In Chapter 5, the response of SK-Mel-28 and MDA-MB-231-Luc cell monolayers, and a supporting panel of malignant and normal cell lines, to in vitro CVA21 challenge was assessed. In Chapter 6, the transcriptional response of immune-compromised mouse SK-Mel-28 and MDA-MB-231-Luc xenograft cells to systemic CVA21 administration was characterised. The transcriptional response of cells propagated as in vitro monolayers differed markedly when compared to that of in vivo xenografts generated from the same cell lines. In Chapter 5, a delayed rate of CVA21 replication and cell lysis was observed in normal cell cultures, as compared to malignant cell lines. Gene expression profiling suggested that the normal human lung fibroblast cell line, MRC-5, mounted an interferon (IFN)-mediated innate immune response against CVA21 challenge, a phenomenon not observed following challenge of the malignant cell line panel. Such findings suggest a potential role for the functional status of the IFN-mediated innate immune system in the tumour cell tropism of oncolytic CVA21. Somewhat surprisingly, in Chapter 6, an IFN-mediated transcriptional response was observed in the SK-Mel-28/MDA-MB-231-Luc xenograft cells, potentially attributed to the ‘priming’ effects of in vivo endogenous murine IFN activity. Furthermore, in Chapters 5 and 6, the potential contributions of transcriptionally regulated genes, in respect to their biological roles in cell cycle regulation, apoptosis, oxidative stress, stimulation of anti-tumoural immunity, and inhibition of angiogenesis in CVA21-mediated oncolysis were considered. Moreover, in Chapter 6, a distinct genetic signature of infection was identified, comprising a total of 9 individual genes, significantly upregulated in response to infection in each xenograft model at 24 and 72 h following the systemic administration of CVA21. The identified genes involved in this core transcriptional response to infection may serve as effective molecular biomarkers for the evaluation of oncolytic CVA21 efficacy.

Research Doctorate - Doctor of Philosophy (PhD) / A number of oncolytic picornaviruses are currently under evaluation as potential therapeutic agents for a range of human malignancies. In particular, a subset of naturally occurring human C-cluster enteroviruses; Coxsackievirus A13 (CVA13), Coxsackievirus A15 (CVA15), Coxsackievirus A18 (CVA18) and Coxsackievirus A21 (CVA21) and the human B-cluster enterovirus, Echovirus 1 (EV1), display promising pre-clinical oncolytic activity against a wide variety of neoplastic cells. CVA21 is currently under clinical evaluation for the control of melanoma, breast, prostate and head/neck cancer. The preferential targeting of cancer cells by this subset of viruses is based on extracellular capsid interactions with specific viral receptors (intercellular adhesion molecule-1 [ICAM-1], decay-accelerating factor [DAF] or integrin α2β1), on the surface of malignant cells. In the present study, the therapeutic potential of this subset of enteroviruses was evaluated as a novel treatment strategy for the control of human malignancies of the gastrointestinal system. In Chapter 3, the capacity of the aforementioned enteroviruses for oncolytic activity was assessed in a panel of in vitro human gastric cancer cell cultures. Flow cytometric analysis revealed low-to-medium levels of ICAM-1, in addition to abundant α2β1 and DAF expression on the surface of gastric cancer cell lines. Cell monolayer lytic infectivity assays demonstrated that, of the viruses under evaluation, EV1 displayed the most potent and widespread in vitro lytic activity against the gastric cancer cell lines. Monoclonal antibody blockade confirmed the specific integrin α2β1-mediated route of EV1 cell infection in the gastric cancer MKN-45 cell line. Subsequently, an in vivo dose ranging study assessing the efficacy of oncolytic EV1 was undertaken in an immune-compromised MKN-45-Luc mouse model of human gastric cancer peritoneal carcinomatosis (PC). In this model, an intra-peritoneal dose of as little as 1x103 TCID50 EV1 resulted in a significant reduction in peritoneal tumour burden. In Chapter 4, the oncolytic capacity of this enterovirus subset was further evaluated, as a potential therapeutic option for the control of colorectal cancer (CRC). Flow cytometric analysis of a panel of CRC cell lines demonstrated abundant levels of DAF and integrin α2β1, and low-to-moderate levels of ICAM-1 expression on the surface of CRC cells. Of the subset of viruses examined, a DAF-using variant of CVA21 (CVA21-DAFv) displayed the most potent and widespread oncolytic activity against in vitro CRC cell cultures. Consequently, the potential in vivo oncolytic capacity of CVA21-DAFv and the wild-type CVA21 was evaluated in three individual immune-compromised mouse sub-cutaneous xenograft models of human CRC. However, despite the immunohistochemical detection of ICAM-1/DAF on cells of the CRC xenografts, and the detection of infectious virus in the blood of treated tumour-bearing mice, a detectable reduction in tumour burden was not observed. On account of the varying degrees of oncolytic efficacy observed in colorectal and gastric cancers, global gene expression profiling was employed in Chapters 5 and 6, to further elucidate the molecular mechanisms of enterovirus-mediated tumour cell tropism and cell death. As the most extensively characterised virus in pre-clinical studies, and the only virus of this subset under current clinical evaluation, CVA21 was selected as the challenge virus for analysis of the transcriptional response to enterovirus infection. Malignant cells that displayed reproducible susceptibility to in vitro and in vivo lytic CVA21 challenge were necessary for extensive characterisation, therefore, melanoma SK-Mel-28 and breast cancer MDA-MB-231-Luc cell lines, rather than CRC cell lines, were utilised. In Chapter 5, the response of SK-Mel-28 and MDA-MB-231-Luc cell monolayers, and a supporting panel of malignant and normal cell lines, to in vitro CVA21 challenge was assessed. In Chapter 6, the transcriptional response of immune-compromised mouse SK-Mel-28 and MDA-MB-231-Luc xenograft cells to systemic CVA21 administration was characterised. The transcriptional response of cells propagated as in vitro monolayers differed markedly when compared to that of in vivo xenografts generated from the same cell lines. In Chapter 5, a delayed rate of CVA21 replication and cell lysis was observed in normal cell cultures, as compared to malignant cell lines. Gene expression profiling suggested that the normal human lung fibroblast cell line, MRC-5, mounted an interferon (IFN)-mediated innate immune response against CVA21 challenge, a phenomenon not observed following challenge of the malignant cell line panel. Such findings suggest a potential role for the functional status of the IFN-mediated innate immune system in the tumour cell tropism of oncolytic CVA21. Somewhat surprisingly, in Chapter 6, an IFN-mediated transcriptional response was observed in the SK-Mel-28/MDA-MB-231-Luc xenograft cells, potentially attributed to the ‘priming’ effects of in vivo endogenous murine IFN activity. Furthermore, in Chapters 5 and 6, the potential contributions of transcriptionally regulated genes, in respect to their biological roles in cell cycle regulation, apoptosis, oxidative stress, stimulation of anti-tumoural immunity, and inhibition of angiogenesis in CVA21-mediated oncolysis were considered. Moreover, in Chapter 6, a distinct genetic signature of infection was identified, comprising a total of 9 individual genes, significantly upregulated in response to infection in each xenograft model at 24 and 72 h following the systemic administration of CVA21. The identified genes involved in this core transcriptional response to infection may serve as effective molecular biomarkers for the evaluation of oncolytic CVA21 efficacy.

Diss. (sammanfattning) Stockholm : Karol. inst., 2001. / Härtill 7 uppsatser.