Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2007. / Includes bibliographical references (leaves 75-94). Also available in electronic version.

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Thesis (Ph. D.)--University of Sydney, 2007. / Title from title screen (viewed Aug. 30, 2007). Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Public Health, Faculty of Medicine. Includes bibliography.

Thesis (Ph.D.)--University of Western Australia, 2007.

Thesis (Ph.D.)--Indiana University, 2009. / Title from screen (viewed on August 28, 2009). Department of Biochemistry and Molecular Biology, Indiana University-Purdue University Indianapolis (IUPUI). Advisor(s): William Bosron. Includes vita. Includes bibliographical references (leaves 102-111).

Le cancer colorectal métastatique est l’une des premières causes de décès par cancer dans les pays occidentaux, malgré le développement récent de nouveaux traitements ciblés. L’amélioration de la survie des patients passe par une meilleure compréhension des mécanismes moléculaires impliqués dans la progression tumorale et la formation des métastases. Compte tenu de l’importance du rôle des axes mTOR/HIF1α et CXCL12/CXCR4/CXCR7 dans le processus métastatique, nos objectifs ont été : i) d’analyser de façon extensive le statut de CXCL12 dans une collection de polypes et de tumeurs coliques de tous stades et phénotypes en comparaison avec la muqueuse saine, puis de comprendre les mécanismes régulant l’expression de la chimiokine dans les cellules tumorales ; ii) d’étudier in vitro le rôle de l’hypoxie dans la régulation de la signalisation induite par CXCL12 via CXCR4 et CXCR7 et l’intérêt d’une inhibition des axes mTOR/HIF-1α et CXCL12/CXCR4/CXCR7 en particulier sur les capacités migratoires des cellules tumorales.Nous avons montré que l’extinction du gène CXCL12 est un évènement précoce et systématique au cours de la cancérogenèse colique et que cette perte d’expression pourrait être régulée par un mécanisme d’acétylation au niveau des histones. Une expression différentielle de CXCR4 et CXCR7 au sein des tumeurs du colon a été mise en évidence. L’augmentation d’expression de CXCR7 dans les métastases par rapport aux stades précoces montre l’importance de cet axe dans le processus métastatique. Le maintien de l’expression à la surface cellulaire de CXCR4 en normoxie pendant au moins 24h après un bref passage en hypoxie n’avait pas encore été décrit. Ceci pourrait expliquer le « homing » des cellules tumorales circulantes dans les niches métastatiques selon un gradient de CXCL12. L’utilisation combinée d’irinotécan et de chalcone permettant d’inhiber la migration des cellules tumorales est une approche originale in vitro. Enfin, nous avons initié le développement de modèles métastatiques de cancer du colon par greffe orthotopique sur le caecum de souris NUDE dans l’objectif de tester de nouvelles approches thérapeutiques ciblées in vivo. / Despite the recent development of new targeted chemotherapies, metastatic colorectal cancer is still one of the leading causes of cancer related deaths in western countries. A better understanding of metastatic process would improve survival. Since the role of mTOR/HIF1α and CXCL12/CXCR4/CXCR7 axes in metastasis formation, our objectives were: i) to analyze extensively CXCL12 status in a collection of polyps and colon tumors whatever stages and phenotypes; ii) to study the role of hypoxia in CXCL12/CXCR4/CXCR7 signaling pathway in vitro and on tumor cells migration. We have shown that the CXCL12 extinction is a systematic early event during colorectal carcinogenesis. CXCL12 loss expression may be regulated by histone acetylation mechanism. There is a differential CXCR4 and CXCR7 expression in colon tumors. Increased CXCR7 expression in metastasis compared to early stages underlines the importance of this axis in metastatic process. We have shown for the first time that CXCR4 expression remained stabilized at the cell membrane 24 hours after a transient passage in hypoxia. It could explain circulating cells are attracted in metastatic niches under CXCL12 gradient. Drug combinations with chalcone and irinotecan are an original approach for inhibiting cell migration in vitro. Finally, we have initiated the development of a metastatic model of colon cancer with orthotopic colon human tumors xenograft in NUDE mice to test new therapeutic approaches in vivo.

Cell metabolism disorder is considered as both direct and indirect consequence of oncogenic mutations for tumoigenesis. Autophagy is a metabolic stress response and a mechanism of natural cellular degradation. It is believed that autophagy, as well as metabolism, is a crucial process for the adaptation of cancer cells to changes in nutrient availability. Understanding the relationship between metabolism and autophagy and targeting on the key steps are regarded as a promising strategy to treat cancer. Halofuginone (HF), a semisynthetic quinazolinone alkaloid originally derived from the plant Dichroa febrifuga Lour., has gained attention for its potential therapeutic effects in a variety of cancers. We hypothesize that HF may inhibit cancer cell proliferation by inducing changes in glucose metabolism and modulating related autophagy. A series of studies, from in vitro to in vivo, were designed to approve this concept in colorectal cancer (CRC);Firstly, we found that HF inhibited human CRC cell proliferation and induced the generation of reactive oxygen species (ROS) and apoptosis. As expected, a reduced level of NADPH was also observed, at least in part due to inactivation of glucose-6-phosphate dehydrogenase (G6PD) in pentose phosphate pathway (PPP) upon HF treatment. Given these findings, we further investigated metabolic regulation of HF through Akt/mTORC1-mediated aerobic glycolysis and found that HF downregulated the Akt/mTORC1 signaling pathway. Moreover, metabolomics found slower rates in both glycolytic flux and glucose-derived tricarboxylic acid cycle flux. Meanwhile, both glucose transporter GLUT1 and hexokinase-2 in glycolysis were suppressed in CRC cells by HF. These findings support our notion that HF regulates the Akt/mTORC1 signaling pathway to dampen glucose uptake and glycolysis in CRC cells. Furthermore, HF retarded tumor growth in nude mice inoculated with HCT116 cells and reduced the viability of primary cells from the tissues of CRC patients. This finding further confirmed our hypothesis that HF inhibits CRC cell growth through metabolic regulation of Akt/mTORC1. Because mTORC1 can inhibit autophagy through phosphorylation and inactivation of the initiating kinase ULK1 in cancer cells, we further studied the HF effects on CRC in different nutritional conditions. The results showed that HF in nutrient-rich conditions could reduce SQSTM1/p62 through mTORC1-mediated phosphorylation at Ser757 of ULK1. More interestingly, HF elevated SQSTM1 protein level in low nutrient condition through AMPK-mediated phosphorylation at Ser317/777 of ULK1. It showed that HF could regulate nutrient-sensing mTORC1-ULK1 or AMPK-ULK1 to dually modulate autophagy in CRC cells. Further study by using a variety of methods, including mRFP-GFP-LC3 puncta formation, transmission electron microscope (TEM) analysis and monodansylcadaverine (MDC) staining, found that HF could induce autophagosome formation and inhibit autophagosome membrane elongation, depending on nutrient-sensing pathways. Furthermore, we found HF pronouncedly enhanced expression level of Atg7 under nutrient-rich conditions while it decreased Atg7 in CRC cells under nutrient-poor conditions. These two findings imply that Atg7 is required in dual regulation of autophagic flux depending on nutrient conditions. This conclusion was then validated by comparing with autophagy-related proteins in Atg7 knockout (KO) MEFs and Wild-type (WT) MEFs upon HF treatment. Importantly, through analysis of metabolome and metabolic enzymes, we found that HF inhibited glycolysis under nutrient-rich conditions while it inhibited gluconeogenesis under nutrient-poor conditions in an Atg7-dependent manner. In subsequent studies, we found that caloric restriction (CR) in a xenograft mouse model, which mimics low nutrition in vitro, enhanced the anticancer activity of HF. Further analysis of the expression of TQTSM1 and LC3 in tumor tissues demonstrated that HF is an autophagic inducer in xenograft-bearing nude mice with ad libitum feeding, whereas it is an autophagic inhibitor when using CR.;In summary, this study explains how HF controls CRC cell growth through its influences on glucose metabolism and autophagy regulation. HF not only dually regulates autophagy in vitro and in vivo to inhibit cancer cell growth and proliferation through nutrient-sensing pathways under different conditions, but also modulates glycolysis/gluconeogenesis through an autophagic pathway. These results suggest that HF could turn out to be a potent therapeutic drug for treating CRC.

Colorectal cancer (CRC) is the third most prevalent and second leading causes of cancer-associated deaths globally. Over the last few decades, ent-kaurane diterpenes have been widely investigated for their anticancer potentials. Flexicaulin A (9) is a naturally occurring ent-kaurane. Previously, our lab modified the structure of 9 by replacing the C-11 hydroxyl group with carbonyl group and obtained a novel compound oxoflexicaulin A (11). However, anticancer activities and mechanistic pathway of these two compounds are yet to be explored. In the current thesis, we evaluated the cytotoxicity of compounds 9 and 11 in A549 lung, A375 melanoma, PANC1 pancreatic, HCT-116 and HT-29 colon cancer and 293T human embryonic kidney cells as well as compared the activity with a number of known natural ent-kauranes to describe their structure-activity relationship. Our study found that the presence of α, β-unsaturated ketone groups in ent-kaurane structure acted as the pharmacophore. The replacement of C-11 hydroxyl group by carbonyl in 9 (IC50: 3.68 µM) gives a novel potent anticancer compound 11 (IC50: 0.77 µM). Considering the novelty and superior activity of 11, its mechanistic pathway was studied in HCT-116 cells and compared with the natural scaffold 9. Flow cytometry analysis by Annexin V/PI staining along with fluorescent staining by DAPI showed that both compounds induced apoptosis in HCT-116 cells. Induction of apoptosis is mediated through up-regulation of tumor suppressor protein p53 and pro-apoptotic protein Bax, Bak and puma as well as promoting the cleavage of PARP while down-regulation of anti-apoptotic protein Bcl-2 and PARP. Apart from their effect on apoptosis, compounds 9 and 11 stimulated the event of senescence, a process of cellular aging, as confirmed by β-galactosidase assay. Induction of senescence is related with up-modulation of p21 and p27 while down-modulation of p16, Rb and its transcription factor E2F1. Moreover, immunofluorescence staining showed translocation of p21 and p27 from the cytoplasm to nucleus after treatment with 9 and 11. Further study found that the two ent-kauranes inhibited the protein level of two NF-κB sub-units p65 and p50 in the nucleus as well suppressed the cytoplasmic level of NF-κB inhibitor IkB-α. Both compounds also inhibited the expression and phosphorylation of STAT3 in the cytoplasm and nucleus, so as for the expression and phosphorylation of Src, an up-stream kinase of STAT3. In xenograft nude mice model, compound 11 remarkably inhibited tumor growths (volume and size) but the body weights of the mice were also reduced (p < 0. 05). Therefore, we designed to synthesis a series of prodrug analogs from 11 by adding acetal protecting group to reduce the toxicity. One of the prodrug (37) significantly attenuated the tumor volume and size (p < 0.05) at 50 mg/kg without any toxicity (p > 0.05). The prodrug 37 is actually released as compound 11 in the mice due to its cleavage in the acidic microenvironment of tumor. Taken together, antitumor effect of compound 11 in CRC model is supposed to be mediated through induction of apoptosis and senescence via modulation of NF-κB and STAT3 signaling pathway. Keywords: Colorectal cancer, ent-kaurane, flexicaulin A, oxoflexicaulin A, cytotoxicity, anticancer, apoptosis, pathway, NF-κB, STAT3.

This thesis describes a three year study which explored perceptions and experiences of being involved in self care and perceptions of control and self-efficacy over time amongst patients receiving a six month course of chemotherapy treatment for colorectal cancer. The study was underpinned by Leventhal’s Self Regulation Model and aimed to explore how patients undergoing chemotherapy for colorectal cancer perceived the meaning of self care, what they did as part of their self care in managing the effects of their treatment and whether this changed between the beginning and end of their six month course of chemotherapy. The study also set out to explore the relationship between patients’ perceptions of control and self efficacy and their involvement in self care. The study adopted a patient focussed, mixed method, longitudinal approach for complementarity and expansion purposes in which the qualitative findings formed the focus of the investigation, supplemented by the quantitative findings. This was important to provide a greater breadth and range to the study and to obtain a realistic understanding of patients’ perceptions and experiences of being involved in self care during their six month course of chemotherapy treatment and the influence of their perceptions of control and self efficacy on their involvement in self care. Thirty one patients participated in the study and data were collected using qualitative semi structured interviews (with a subsample of patients who participated in the study) and quantitative questionnaires (Illness Perception Questionnaire-revised and the Strategies Used by People to Promote Health) and prospective self care diaries with the full study sample. Data were collected at several time points over the course of patients’ chemotherapy treatment (beginning, middle and end of treatment) and were analysed and integrated in accordance with Tashakkori and Teddlie (1998)’s guidance for integrating qualitative and quantitative findings in a mixed methods study. The study findings revealed that the use of a mixed method, longitudinal study design was a valuable approach for understanding patients’ involvement in self care during chemotherapy for colorectal cancer and the influence of factors, such as their perceptions of control, on their subsequent involvement in their self care. In particular, the principal findings suggested that self care held a range of meanings to the patients in this study. Principally, patients’ self care consisted of two components; physical self care, carried out to manage the physical impact of undergoing treatment, and emotional self care, carried out to manage their emotional response to being diagnosed with, and undergoing treatment for, cancer. The findings suggested that there was no association between patients’ perceptions of control and the degree of self care that they carried out identified in the quantitative analysis. However, in the qualitative analysis, it was revealed that patients’ perceptions of control were likely to influence their attitudes towards their active involvement in self care and the importance with which they viewed this role. In particular, patients who considered themselves to have a high degree of control during their treatment were more likely to believe that they could limit the impact of the treatment through their own actions, that being actively involved in their self care was important and were interested in taking on this role, and that they would use a greater range of self care strategies in helping to manage the impact of their treatment. Conversely patients who considered themselves to have a lower degree of control during their treatment were less likely to believe that they could limit the impact of the treatment through their own actions, that their active involvement in self care was important and were less likely to expect to take on an active role, preferring to leave the management of treatment-related effects to health professionals, whom they regarded as being the “experts”. The findings from this study have implications for nursing practice because they reinforce the importance of the listening to the patient’s experience and how this approach can contribute to a fuller and more accurate understanding of how patients become involved in their self care and the factors that influence this. This is important so that nurses can provide holistic care, tailored to meet their patients’ self care needs and preferences, and to encourage partnership working between patients, nurses, allied health professionals and other agencies in promoting involvement in self care. The findings also have implications for theories relating to self care in emphasising the importance of patient centred models of care and for Leventhal’s Self Regulation Model in adding further support for the components of the model yet also offering a greater understanding of how the model fits with patients’ emotional responses to the effects of illness and its’ treatments. Finally, the study findings have implications for future research, calling for further research to focus on the meaning of constructs such as self care and control from the patients’ perspective and to further explore the use of the mixed methodology in researching and understanding patients’ involvement in self care and the factors that influence this.

The cancer stem cell (CSC) model of carcinogenesis and progression posits that within a tumor lies a subpopulation of cells that solely possess the ability to initiate a tumor and to differentiate into tumor cell lineages. Although the behavior of such cells is known, the challenge is to identify factors that characterize the CSC subpopulation. In this thesis, cell lines were identified that, when grown in three-dimensions, gave rise to organized colonies containing lumens originating from differentiating cells (“lumen lines”) and to densely-packed, spherical colonies originating from non-differentiating cells (“dense lines”). A microarray comparison of the pair identified genes upregulated in dense lines, including CD55 and BMI1, and in lumen lines, including CDX1 (Chapter 3). CD55 was used to isolate CD55high CSCs via flow cytometry that are able to self-renew, differentiate, initiate more colonies, proliferate more rapidly and exhibit an increased G2/M cell cycle population as opposed to unfractionated cells. Furthermore, the CD55high cells were able to give rise to more differentiated, lumen colonies in vitro, indicating that CD55 enriches for cells possessing a capacity to differentiate, and were able to enrich the CD24highCD44high putative CSC population further (Chapter 4). CDNA induction of BMI1 and CDX1 expression led to increased clonogenicity/proliferation and decreased clonogenicity/proliferation, respectively, and incorporation of a CDX1 reporter construct into the SW1222 cell line identified CDX1+ cells as a low-expressing population of CD55 (Chapter 5). Finally, co-culture of cell lines in an in vivo-like environment with intestinal myofibroblasts promoted the CSC population by enhancing clonogenicity, proliferation and expression of CD55 (Chapter 6). The results of this thesis implicate CD55 as a potent marker of colorectal cancer stemness, link the expression of BMI1 and CDX1 to cancer stemness and differentiation, respectively, and identify a role for the in vivo stem cell niche in maintaining the CSC population.