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ItemInteraction between p21-activated kinase 1 and beta-cateninLiu, He ( 2012)Colorectal cancer (CRC) was the second most frequently occurring cancer and the second leading cause of cancer death in Australia in 2007 (AIHW2010). Hyper-activation of the Wnt/β-catenin signaling pathway is a hallmark of colorectal cancer. The Wnt signaling pathway plays a critical role in embryonic development and homeostasitic maintenance in mature tissues, particularly in regeneration of intestinal epithelium (Lynch and Lynch 2005). In studies of human colon cancer over the last two decades, mutations have been identified in genes coding for Wnt/β-catenin pathway components, such as axin, adenomatous polyposis coli (APC) and β-catenin, which are known to contribute to tumor progression. Tumor genetic studies have revealed that mutations in these members of the Wnt/β-catenin pathway occur in approximately 90% of colorectal cancers (Bienz and Clevers, 2000; Cottrell et al., 1992; Morin et al., 1997; Polakis, 2000; Powell et al., 1992; Vogelstein and Kinzler, 2004). Under normoxic conditions (having a normal atmospheric oxygen concentration of 20~21%), the transcription factor 4 (TCF4) stably binds to β-catenin in the nuclei of colon carcinoma cells and is constitutively activated. This activation stimulates cell migration and proliferation, and contributes to the development of colorectal tumors (Munemitsu et al., 1995). Under hypoxic conditions β-catenin interacts with the heterodimeric transcription factor hypoxia inducible factor-1α (HIF-1α), enhances HIF-1-mediated transcription, and further promotes cell survival and adaptation to hypoxia (Kaidi et al., 2007). In a mouse model carrying a mutation in the APC gene, the gastrin gene has been identified as a downstream target of the β-catenin/TCF4 signaling pathway (Koh et al., 2000). Similarly the expression of a constitutively active β-catenin causes a threefold increase in gastrin promoter activity (Koh et al., 2000). In previous studies from this laboratory, p21-activated kinase 1 (PAK1) was found to interact with β-catenin and to be required for the regulation of the β-catenin signaling pathway by gastrins (He et al., 2008). PAK1 kinase activity has been implicated in various cellular processes such as gene regulation, cytoskeletal reorganization, cell growth, motility, and morphogenesis (Kumar et al., 2006). PAK1 also functions as a key node in various signaling pathways leading to cell growth, migration and survival. PAK1 has oncogenic functions in a broad range of cancers including CRC and its hyper-activation has been well documented in breast cancer (Dummler et al., 2009; Kumar et al., 2006). PAK1 expression has also been reported to increase in the progression of colorectal carcinomas to metastasis (Carter et al., 2004). However, the specific role of PAK1 in β-catenin signaling and the mechanism by which PAK1 interacts with β-catenin in CRC have not been investigated in detail. The studies in Chapter 3 demonstrate that PAK1 is required for maximal expression of β-catenin and its downstream targets and is important for Wnt signaling pathways in CRC, that β-catenin/TCF4 transcriptional activity is also significantly reduced in PAK1 knockdown cells, and that knocking down PAK1 decreases cell proliferation, migration, HIF-1α expression and cell survival. The mechanism by which PAK1 interacts with β-catenin was further investigated by studying the signaling networks of both proteins in Chapter 4. Cellular β-catenin expression is regulated at the protein level through phosphorylation by glycogen synthase kinase 3-beta (GSK-3β). In the cytoplasm, β-catenin forms a complex with APC, axin, GSK-3β and casein kinase 1 (CK1) (Giles et al., 2003; Kikuchi et al., 2006), and GSK-3β then induces serine-threonine phosphorylation at the amino-terminal of β-catenin, and the phosphorylated β-catenin binds to βTrCP, an E3 ubiquitin ligase that promotes the degradation of β-catenin (Wu et al., 2003). Without phosphorylation by GSK-3β, the stabilized β-catenin accumulates and is translocated to the nucleus, where it interacts with transcription factors of the TCF/LEF-1 family (mainly TCF4), leading to the increased expression of genes which stimulate cell proliferation and migration, and contribute to the development of tumors (He et al., 1998; Shtutman et al., 1999; Tetsu and McCormick, 1999). According to the results of the studies of Chapter 4, there is no change in either expression of GSK-3β protein or in kinase activity of GSK-3β as measured by phosphorylation at Serine 9 in PAK1 knockdown cells. These results indicate that PAK1 may not regulate β-catenin through GSK-3β signaling in CRC cell lines. Other effectors of β-catenin activity have also been studied in Chapter 4. The integrin-linked kinase (ILK) has been reported to be involved in β-catenin/TCF4 signaling through multiple mechanisms (Novak et al., 1998; Tan et al., 2001). The Inhibitor of β-catenin and TCF4 (ICAT) is reported to inhibit β-catenin nuclear signaling by competing with TCF4 for binding with β-catenin (Tago et al., 2000). ICAT is also located downstream of ILK in progastrin-mediated signaling in CRC tumors (Pannequin et al., 2007). Interestingly, PAK1 has been shown to be responsible for phosphorylation-dependent translocation and gene regulation of ILK (Acconcia et al., 2007). The studies of Chapter 4 demonstrate that PAK1 is required for ILK activity, that knocking down PAK1 increases ICAT expression and that PAK1 regulates β-catenin through the ILK/ICAT signaling pathway. After the investigation of the PAK1 and β-catenin interaction in CRC cells in vitro, the role of PAK1 in β-catenin signaling is further explored in vivo using animal models in Chapter 5. In this study, the in vivo role of the PAK1 protein and its interaction with β-catenin was examined using Severe Combined Immunodeficiency (SCID) mice and genetically modified APCΔ14/+ mice, and siRNAs were used as a treatment to inhibit expression of the proteins of interest. The growth of CRC cell lines as xenografts in SCID mice was studied and tumor histology in the genetically modified CRC mouse model APCΔ14/+ mice was analysed. In xenograft studies with human CRC cells in Chapter 5, PAK1 knockdown suppressed tumor growth by inhibition of proliferation and stimulation of apoptosis. In addition, PAK1 siRNA treatment delayed the growth of wildtype human CRC cells at an early stage of tumor development and contributed to tumor necrosis at later stages. In studies with APCΔ14/+ mice, expression of both PAK1 and β-catenin protein was reduced in tumors from APCΔ14/+ mice treated with PAK1 siRNA compared with mice treated with control siRNA. PAK1 siRNA treatment decreased tumor numbers significantly and slowed the bodyweight drop caused by tumor development. This thesis provides detailed information on the Wnt/β-catenin and PAK1 signaling pathways, contributes to understanding of the mechanism of human CRC development, and provides a novel direction for effective CRC treatment. The results presented here indicate that PAK1 could be a suitable target for CRC therapy. Future studies focusing on PAK1 as a drug development target may have promising outcomes in clinical trials as well as in cancer therapy.
ItemThe renin angiotensin system and macrophages in regulation of colorectal liver metastasesWen, Shu Wen ( 2012)Metastasis to the liver is the leading cause of death for colorectal cancer (CRC) patients. The systemic treatment of CRC liver metastases is suboptimal and with limited response rates. Targeting of the renin angiotensin system (RAS) may be a potential adjunct therapeutic strategy in this disease. Blockade of the RAS can inhibit tumour growth in a mouse model of CRC liver metastases. However, the underlying mechanisms remain unclear. Participation of the RAS in inflammatory diseases and in malignancy suggests that macrophages may be a novel mediator of RAS-induced effects. This thesis addressed the role of the RAS in regulating macrophage biology and its consequent impact on the growth of CRC liver metastases. Macrophage depletion studies using an orthotopic murine model of CRC liver metastases demonstrated the bimodal role of macrophages in determining tumour growth. They exhibit an early inhibitory and a later stimulatory effect. Alterations in iNOS- and VEGF- expressing cells, and T-cell responses may be responsible for the observed reduction in tumour burden following depletion of pro-tumour macrophages at the late stage of metastatic growth. Using combined in-vitro with in-vivo experiments the potential of the RAS to alter macrophage function was demonstrated. In-vivo, the anti-tumour affects of ACE inhibition (captopril) on CRC liver metastases was mediated by changes in macrophage biology that inhibited initial tumour seeding and proliferation, as well as promoting macrophage migration. In-vitro, both key RAS peptides, Ang II and Ang-(1–7) were capable of altering tumour-regulatory factors, including iNOS, MMP-9, VEGF and TNF-α in murine macrophages. These factors are equally important in directing macrophage polarisation (M1 or M2 macrophages). Conditioned media from macrophages stimulated with Ang-(1–7) reduced the proliferation and viability of both human and mouse CRC cells, but increased cell migration in-vitro. Supernatant of Ang II-treated macrophages also altered the kinetics of mouse, but not human CRC cells. Interestingly, Ang II and its receptor inhibition did not induce distinct macrophage polarisation. It is clear the RAS has important immunomodulatory roles that can regulate tumour progression and its metastasis. Further understanding of these physiological mechanisms will enable agents targeting the RAS to reach their full therapeutic potential in the treatment of CRC liver metastasis.
ItemThe role of the renin-angiotensin system in liver regeneration and colorectal cancer liver metastasesKOH, SHIR LIN ( 2012)Background: Colorectal cancer (CRC) is the second most common cause of cancer related death in Australia with over 4700 deaths reported annually. CRC liver metastasis (CRCLM) contributes to over 70% of the disease mortality. While unresected patients rarely survive beyond 2 years, partial hepatectomy (PH) improves their survival to 25%-60% at 5 years. Blockade of the renin-angiotensin system(RAS) has been shown to enhance liver regeneration and, separately, to inhibit CRCLM. Targeting the RAS may offer a unique synergistic anti-cancer therapy by inhibiting CRCLM tumour growth while simultaneously enhancing liver regeneration following PH. Aim: This study investigated the expression of the RAS during liver regeneration and in CRCLM. The effects of RAS blockade on liver regeneration and CRCLM in the regenerating liver were determined to investigate its potential benefits as a therapeutic avenue for CRCLM patients. Methods: Male CBA mice (10-12 weeks) were used in this study. After 70% partial hepatectomy (PH) alone, captopril (750mg/kg) or saline (control), were administered intraperitoneally on a daily basis until the endpoints (days 1, 2, 4, 6 and 8 post-surgery). A mouse model of CRCLM in the regenerating liver was developed. Mice induced with CRCLM and subjected to 70% PH were treated with captopril (250mg/kg) daily until the endpoints (days 2, 6, 16 and 21). At study endpoints, liver regeneration was assessed by measuring the liver-to-body weight ratio. CRCLM tumour burden (percentage of liver metastases) was calculated using total liver and tumour volumes using quantitative stereology. Liver function tests were performed on mouse serum collected from days 2 and 6. The expression of the RAS components, cell proliferation, apoptosis, hepatic stellate cells (HSC) and liver endothelial cell densities, matrix metalloproteinase (MMP)-9, transforming growth factor (TGF)-β were quantified. Statistical analyses were performed using 2-sample independent T-test, one-way ANOVA with post-hoc analysis, or Kruskal Wallis followed by Mann-Whitney U tests as appropriate (SPSS v.18). P-value of <0.05 was considered statistically significant. Results: Captopril significantly inhibited CRCLM tumour growth and increased tumour cell apoptosis in the regenerating liver at day 21. Captopril also enhanced early liver regeneration and this was associated with an increase in hepatocyte proliferation at 6 hours after PH as well as an increase in HSC density and MMP-9 levels 2 days after PH. The decrease in hepatocyte proliferation at day 2 was transient. By day 4 onwards there was no significant difference between control and treated livers. Captopril also decreased the hepatocyte injury marker, alanine transaminase. The ability of captopril to increase human hepatocyte proliferation was confirmed in vitro. The RAS was expressed in the liver and tumours during liver regeneration and tumour growth phases. Liver and tumour differed in their RAS expression; tumour AT1R expression levels were lower than normal liver, while tumour MasR and AT2R levels were upregulated during cancer progression. Conclusion: This thesis showed a tumour-specific RAS expression which could be targeted to inhibit tumour growth while allowing the liver to regenerate following PH. This is supporting by my findings that RAS blockade with captopril following PH was associated with a reduction in CRCLM tumour growth without impairing liver regeneration. Thus, captopril may offer a new avenue to improve CRCLM patient outcomes by inhibiting tumour growth whilst enhancing the early stage of liver regeneration.
ItemEffects of chemotherapy on colorectal liver metastasesNguyen, Linh My ( 2012)Background: Colorectal cancer (CRC) is the fourth most frequently occurring cancer in the world. Despite optimum surgical endeavours, many patients will develop disease recurrence. Treatments available for patients who do not qualify for surgical resection are limited and mainly consist of chemotherapy or radiotherapy. Recent innovative options focus on selective targeting of the tumour blood supply, as a means of achieving greater tumour destruction or slowing overall tumour progression. Two main classes of drugs have been used both clinically and experimentally: the angioinhibitory agents (AIA) that inhibit the formation of new vessels, and the vascular disruptive agents (VDA) that target endothelial cells in immature tumour vessels, causing vessel collapse, tumour hypoxia and death. Treatment by VDAs are characterised by rapid and extensive destruction of tumour limited only by the persistence of a viable rim of tumour cells in the periphery, which subsequently leads to recurrence. The VDA OXi4503 is one of the most potent VDAs being tested, and our own research has demonstrated a rapid onset of microvascular thrombosis leading to tumour necrosis in excess of 90% of the tumour. Despite this efficacy, complete tumour eradication was not achieved as a thin rim of viable tumour invariably survived in the periphery giving rise to recurrence. Understanding the mechanisms that enable tumour to survive in the periphery could lead to formulation of drug combinations for total tumour eradication. Based on the finding that only tumour cells in the periphery survive the VDA treatment this study tests the following hypotheses: • Morphological and molecular differences in the tumour contribute to drug resistance in the tumour periphery (Chapter 4). • Treatment with OXi4503 promotes molecular and morphological changes in the residual tumour rendering the tumour resistant to cytotoxic treatments (Chapter 5 and Chapter 6). • Combination treatment using AIA (Sunitinib) and VDA (OXi4503) may produce complete destruction of colorectal liver tumours and hence improve treatment outcomes (Chapter 7). Experimental Design: Using a murine colorectal liver metastases model, inherent morphological and molecular differences within the periphery and the centre of the tumour that may account for differences in resistance to OXi4503 treatment were investigated. H&E staining and immunostaining were used to examine spatial differences in vessel maturity and stability, accumulation of immune cells, the expression of proangiogenic factors/receptors (HIF-1α, VEGF and ATR1) and the expression of epithelial to mesenchymal transition (EMT) markers (ZEB1, vimentin, E-cadherin and β-catenin) between the periphery and the centre of established tumours. The effects of single dose OXi4503 treatment on tumour vessels, cell kinetics, changes in growth factors (HIF-1α, VEGF and ATR1) and EMT markers (ZEB1, vimentin, E-cadherin and β-catenin) were also investigated by H&E and immunohistochemical staining, western blotting and RT-PCR techniques. A combination study testing the combined efficacy of OXi4503 and Sunitinib (AIA) was conducted and the effects were investigated by macroscopic stereology and immunohistochemistry. Results: In this study, significant differences were found between the tumour periphery and the central regions of mouse colorectal liver metastases, including association of the periphery with mature vessels, higher accumulation of immune cells, increased growth factor expression, minimal levels of hypoxia and increased EMT evidence. OXi4503 treatment resulted in collapse of tumour vessels in the tumour centre; however in the periphery the vasculature remained patent. Similarly, tumour apoptosis and proliferation were differentially modulated between centre and periphery after treatment. Significant increases in hypoxia and up-regulation of the pro-angiogenic growth factors HIF-1α, VEGF, and AT1R were detected within the viable periphery of the tumour. Simultaneously there was a significant down-regulation of E-cadherin, relocation and nuclear accumulation of β -catenin and up-regulation of ZEB1 and vimentin. These changes are strongly suggestive of EMT occurring in the surviving tumour in the periphery. This is the first direct evidence of in vivo EMT occurring almost immediately following treatment and involving all the surviving tumour cells. The data presented demonstrated a possible mechanism employed by tumour cells to evade drug treatment and metastasize, and may be targeted for more effective clinical outcomes. Sunitinib/OXi4503 combination treatment produced significantly lower tumour burden compared to either treatment alone. However, tumour kinetic studies revealed higher tumour proliferation compared to tumour apoptosis and the evidence of widespread EMT, suggesting the combination treatment in this model may successfully delay tumour regrowth but disease recurrence seems likely. Conclusion: The molecular and morphological differences seen between the periphery and the bulk of the tumour may account for the observed differential resistance to OXi4503 treatment. Growth factor and EMT changes following OXi4503 treatment further contribute to tumour resistance by providing escape mechanisms, re-vascularisation and tumour regrowth. Combination treatment targeting two different aspects of the tumour vasculature while more effective than either treatment alone, did not achieve complete tumour eradication. One exciting finding in this study is the observation that all surviving tumour cells undergo EMT in all three treatments holding the promise that EMT inhibitors in combination with VDAs or chemotherapies may result in total tumour eradication.