p53 prevents progression of nevi to melanoma predominantly through cell cycle regulation

p53 is the central member of a critical tumor suppressor pathway in virtually all tumor types, where it is silenced mainly by missense mutations. In melanoma, p53 predominantly remains wild type, thus its role has been neglected. To study the effect of p53 on melanocyte function and melanomagenesis, we crossed the 'high-p53'Mdm4+/- mouse to the well-established TP-ras0/+ murine melanoma progression model. After treatment with the carcinogen dimethylbenzanthracene (DMBA), TP-ras0/+ mice on the Mdm4+/- background developed fewer tumors with a delay in the age of onset of melanomas compared to TP-ras0/+ mice. Furthermore, we observed a dramatic decrease in tumor growth, lack of metastasis with increased survival of TP-ras0/+: Mdm4+/- mice. Thus, p53 effectively prevented the conversion of small benign tumors to malignant and metastatic melanoma. p53 activation in cultured primary melanocyte and melanoma cell lines using Nutlin-3, a specific Mdm2 antagonist, supported these findings. Moreover, global gene expression and network analysis of Nutlin-3-treated primary human melanocytes indicated that cell cycle regulation through the p21WAF1/CIP1 signaling network may be the key anti-melanomagenic activity of p53.

Inverse expression states of the BRN2 and MITF transcription factors in melanoma spheres and tumour xenografts regulate the NOTCH pathway

The use of adherent monolayer cultures have produced many insights into melanoma cell growth and differentiation, but often novel therapeutics demonstrated to act on these cells are not active in vivo. It is imperative that new methods of growing melanoma cells that reflect growth in vivo are investigated. To this end, a range of human melanoma cell lines passaged as adherent cultures or induced to form melanoma spheres (melanospheres) in stem cell media have been studied to compare cellular characteristics and protein expression. Melanoma spheres and tumours grown from cell lines as mouse xenografts had increased heterogeneity when compared with adherent cells and 3D-spheroids in agar (aggregates). Furthermore, cells within the melanoma spheres and mouse xenografts each displayed a high level of reciprocal BRN2 or MITF expression, which matched more closely the pattern seen in human melanoma tumours in situ, rather than the propensity for co-expression of these important melanocytic transcription factors seen in adherent cells and 3D-spheroids. Notably, when the levels of the BRN2 and MITF proteins were each independently repressed using siRNA treatment of adherent melanoma cells, members of the NOTCH pathway responded by decreasing or increasing expression, respectively. This links BRN2 as an activator, and conversely, MITF as a repressor of the NOTCH pathway in melanoma cells. Loss of the BRN2-MITF axis in antisense-ablated cell lines decreased the melanoma sphere-forming capability, cell adhesion during 3D-spheroid formation and invasion through a collagen matrix. Combined, this evidence suggests that the melanoma sphere-culture system induces subpopulations of cells that may more accurately portray the in vivo disease, than the growth as adherent melanoma cells.

The expression, regulation and function of kallikrein 4 in prostate cancer

Prostate cancer is a significant health problem faced by aging men. Currently, diagnostic strategies for the detection of prostate cancer are either unreliable, yielding high numbers of false positive results, or too invasive to be used widely as screening tests. Furthermore, the current therapeutic strategies for the treatment of the disease carry considerable side effects. Although organ confined prostate cancer can be curable, most detectable clinical symptoms occur in advanced disease when primary tumour cells have metastasised to distant sites - usually lymph nodes and bone. Many growth factors and steroids assist the continued growth and maintenance of prostatic tumour cells. Of these mitogens, androgens are important in the development of the normal prostate but are also required to sustain the growth of prostate cancer cells in the early stage of the disease. Not only are androgens required in the early stage of disease, but also many other growth factors and hormones interact to cause uncontrolled proliferation of malignant cells. The early, androgen sensitive phase of disease is followed by an androgen insensitive phase, whereby androgens are no longer required to stimulate the growth of the tumour cells. Growth factors such as transforming growth factor  and  (TGF/), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin-like growth factors (IGFs), Vitamin D and thyroid hormone have been suggested to be important at this stage of disease. Interestingly, some of the kallikrein family of genes, including prostate specific antigen (PSA), the current serum diagnostic marker for prostate cancer, are regulated by androgens and many of the aforementioned growth factors. The kallikrein gene family is a group of serine proteases that are involved in a diverse range of physiological processes: regulation of local blood flow, angiogenesis, tissue invasion and mitogenesis. The earliest members of the kallikrein gene family (KLK1-KLK3) have been strongly associated with general disease states, such as hypertension, inflammation, pancreatitis and renal disease, but are also linked to various cancers. Recently, this family was extended to include 15 genes (KLK1-15). Several newer members of the kallikrein family have been implicated in the carcinogenesis and tumour metastasis of hormone-dependent cancers such as prostate, breast, endometrial and ovarian cancer. The aims of this project were to investigate the expression of the newly identified kallikrein, KLK4, in benign and malignant prostate tissues, and prostate cancer cell lines. This thesis has demonstrated the elevated expression of KLK4 mRNA transcripts in malignant prostate tissue compared to benign prostates. Additionally, expression of the full length KLK4 transcript was detected in the androgen dependent prostate cancer cell line, LNCaP. Based on the above finding, the LNCaP cell line was chosen to assess the potential regulation of full length KLK4 by androgen, thyroid hormone and epidermal growth factor. KLK4 mRNA and protein was found to be up-regulated by androgen and a combination of androgen and thyroid hormone. Thyroid hormone alone produced no significant change in KLK4 mRNA or protein over the control. Epidermal growth factor treatment also resulted in elevated expression levels of KLK4 mRNA and protein. To assess the potential functional role(s) of KLK4/hK4 in processes associated with tumour progression, full length KLK4 was transfected into PC-3 cells - a prostate cancer cell line originally derived from a secondary bone lesion. The KLK4/hK4 over-expressing cells were assessed for their proliferation, migration, invasion and attachment properties. The KLK4 over-expressing clones exhibited a marked change in morphology, indicative of a more aggressive phenotype. The KLK4 clones were irregularly shaped with compromised adhesion to the growth surface. In contrast, the control cell lines (parent PC-3 and empty vector clones) retained a rounded morphology with obvious cell to cell adhesion, as well as significant adhesion to their growth surface. The KLK4 clones exhibited significantly greater attachment to Collagen I and IV than native PC-3s and empty vector controls. Over a 12 hour period, in comparison to the control cells, the KLK4 clones displayed an increase in migration towards PC-3 native conditioned media, a 3 fold increase towards conditioned media from an osteoblastic cell line (Saos-2) and no change in migration towards conditioned media from neonatal foreskin fibroblast cells or 20% foetal bovine serum. Furthermore, the increase in migration exhibited by the KLK4 clones was partially blocked by the serine protease inhibitor, aprotinin. The data presented in this thesis suggests that KLK4/hK4 is important in prostate carcinogenesis due to its over-expression in malignant prostate tissues, its regulation by hormones and growth factors associated with prostate disease and the functional consequences of over-expression of KLK4/hK4 in the PC-3 cell line. These results indicate that KLK4/hK4 may play an important role in tumour invasion and bone metastasis via increased attachment to the bone matrix protein, Collagen I, and enhanced migration due to soluble factors produced by osteoblast cells. This suggestion is further supported by the morphological changes displayed by the KLK4 over-expressing cells. Overall, this data suggests that KLK4/hK4 should be further studied to more fully investigate the potential value of KLK4/hK4 as a diagnostic/prognostic biomarker or in therapeutic applications.

Prognostic significance of IGF and ECM induced signalling proteins in breast cancer patients

Breast cancer is a leading contributor to the burden of disease in Australia. Fortunately, the recent introduction of diverse therapeutic strategies have improved the survival outcome for many women. Despite this, the clinical management of breast cancer remains problematic as not all approaches are sufficiently sophisticated to take into account the heterogeneity of this disease and are unable to predict disease progression, in particular, metastasis. As such, women with good prognostic outcomes are exposed to the side effects of therapies without added benefit. Furthermore, women with aggressive disease for whom these advanced treatments would deliver benefit cannot be distinguished and opportunities for more intensive or novel treatment are lost. This study is designed to identify novel factors associated with disease progression, and the potential to inform disease prognosis. Frequently overlooked, yet common mediators of disease are the interactions that take place between the insulin-like growth factor (IGF) system and the extracellular matrix (ECM). Our laboratory has previously demonstrated that multiprotein insulin-like growth factor-I (IGF-I): insulin-like growth factor binding protein (IGFBP): vitronectin (VN) complexes stimulate migration of breast cancer cells in vitro, via the cooperative involvement of the insulin-like growth factor type I receptor (IGF-IR) and VN-binding integrins. However, the effects of IGF and ECM protein interactions on the dissemination and progression of breast cancer in vivo are unknown. It was hypothesised that interactions between proteins required for IGF induced signalling events and those within the ECM contribute to breast cancer metastasis and are prognostic and predictive indicators of patient outcome. To address this hypothesis, semiquantitative immunohistochemistry (IHC) analyses were performed to compare the extracellular and subcellular distribution of IGF and ECM induced signalling proteins between matched normal, primary cancer, and metastatic cancer among archival formalin-fixed paraffin-embedded (FFPE) breast tissue samples collected from women attending the Princess Alexandra Hospital, Brisbane. Multivariate Cox proportional hazards (PH) regression survival models in conjunction with a modified „purposeful selection of covariates. method were applied to determine the prognostic potential of these proteins. This study provides the first in-depth, compartmentalised analysis of the distribution of IGF and ECM induced signalling proteins. As protein function and protein localisation are closely correlated, these findings provide novel insights into IGF signalling and ECM protein function during breast cancer development and progression. Distinct IGF signalling and ECM protein immunoreactivity was observed in the stroma and/or in subcellular locations in normal breast, primary cancer and metastatic cancer tissues. Analysis of the presence and location of stratifin (SFN) suggested a causal relationship in ECM remodelling events during breast cancer development and progression. The results of this study have also suggested that fibronectin (FN) and ¥â1 integrin are important for the formation of invadopodia and epithelial-to-mesenchymal transition (EMT) events. Our data also highlighted the importance of the temporal and spatial distribution of IGF induced signalling proteins in breast cancer metastasis; in particular, SFN, enhancer-of-split and hairy-related protein 2 (SHARP-2), total-akt/protein kinase B 1 (Total-AKT1), phosphorylated-akt/protein kinase B (P-AKT), extracellular signal-related kinase-1 and extracellular signal-related kinase-2 (ERK1/2) and phosphorylated-extracellular signal-related kinase-1 and extracellular signal-related kinase-2 (P-ERK1/2). Multivariate survival models were created from the immunohistochemical data. These models were found to fit well with these data with very high statistical confidence. Numerous prognostic confounding effects and effect modifications were identified among elements of the ECM and IGF signalling cascade and corroborate the survival models. This finding provides further evidence for the prognostic potential of IGF and ECM induced signalling proteins. In addition, the adjusted measures of associations obtained in this study have strengthened the validity and utility of the resulting models. The findings from this study provide insights into the biological interactions that occur during the development of breast tissue and contribute to disease progression. Importantly, these multivariate survival models could provide important prognostic and predictive indicators that assist the clinical management of breast disease, namely in the early identification of cancers with a propensity to metastasise, and/or recur following adjuvant therapy. The outcomes of this study further inform the development of new therapeutics to aid patient recovery. The findings from this study have widespread clinical application in the diagnosis of disease and prognosis of disease progression, and inform the most appropriate clinical management of individuals with breast cancer.

In vitro and in vivo studies on protease-activated receptor-2

Protease-activated receptor-2 (PAR2) is a G protein coupled receptor (GPCR) that is activated by proteolytic cleavage of its amino terminal domain by trypsin-like serine proteases. Cleavage of this receptor exposes a neoepitope, termed the tethered ligand (TL), which binds intramolecularly within the receptor to stimulate signal transduction via coupled G proteins. PAR2-mediated signal transduction is also experimentally stimulated by hexapeptides (agonist peptides; APs) that are homologous to the TL sequence. Due to the irreversible nature of PAR2 proteolysis, downstream signal transduction is tightly regulated. Following activation, PAR2 is rapidly uncoupled from downstream signalling by the post-translational modifications phosphorylation and ubiquination which facilitate interactions with â- arrestin. This scaffolding protein couples PAR2 to the internalisation machinery initiating its desensitisation and trafficking through the early and late endosomes followed by receptor degradation. PAR2 is widely expressed in mammalian tissues with key roles for this receptor in cardiovascular, respiratory, nervous and musculoskeletal systems. This receptor has also been linked to pathological states with aberrant expression and signalling noted in several cancers. In prostate cancer, PAR2 signalling induces migration and proliferation of tumour derived cell lines, while elevated receptor expression has been noted in malignant tissues. Importantly, a role for this receptor has also been suggested in prostate cancer bone metastasis as coexpression of PAR2 and a proteolytic activator has been demonstrated by immunohistochemical analysis. Based on these data, the primary focus of this project has been on two aspects of PAR2 biology. The first is characterisation of cellular mechanisms that regulate PAR2 signalling and trafficking. The second aspect is the role of this receptor in prostate cancer bone metastasis. In addition, to permit these studies, it was first necessary to evaluate the specificity of the commercially available anti-PAR2 antibodies SAM11, C17, N19 and H99. The evaluation of the four commercially available antibodies was assessed using four techniques: immunoprecipitation; Western blot analysis; immunofluorescence; and flow cytometry. These approaches demonstrated that three of the antibodies efficiently detect ectopically expressed PAR2 by each of these techniques. A significant finding from this study was that N19 was the only antibody able to specifically detect N-glycosylated endogenous PAR2 by Western blot analysis. This analysis was performed on lysates from prostate cancer derived cell lines and tissue derived from wildtype and PAR2 knockout mice. Importantly, further evaluation demonstrated that this antibody also efficiently detects endogenous PAR2 at the cell surface by flow cytometry. The anti-PAR2 antibody N19 was used to explore the in vitro role of palmitoylation, the post-translational addition of palmitate, in PAR2 signalling, trafficking, cell surface expression and desensitization. Significantly, use of the palmitoylation inhibitor 2-bromopalmitate indicated that palmitate addition is important in trafficking of PAR2 endogenously expressed by prostate cancer cell lines. This was supported by palmitate labelling experiments using two approaches which showed that PAR2 stably expressed by CHO cells is palmitoylated and that palmitoylation occurs on cysteine 361. Another key finding from this study is that palmitoylation is required for optimal PAR2 signalling as Ca2+ flux assays indicated that in response to trypsin agonism, palmitoylation deficient PAR2 is ~9 fold less potent than wildtype receptor with a reduction of about 33% in the maximum signal induced via the mutant receptor. Confocal microscopy, flow cytometry and cell surface biotinylation analyses demonstrated that palmitoylation is required for efficient cell surface expression of PAR2. Importantly, this study also identified that palmitoylation of this receptor within the Golgi apparatus is required for efficient agonist-induced rab11amediated trafficking of PAR2 to the cell surface. Interestingly, palmitoylation is also required for receptor desensitization, as agonist-induced â-arrestin recruitment and receptor degradation were markedly reduced in CHO-PAR2-C361A cells compared with CHO-PAR2 cells. Collectively, these data provide new insights on the life cycle of PAR2 and demonstrate that palmitoylation is critical for efficient signalling, trafficking, cell surface localization and degradation of this receptor. This project also evaluated PAR2 residues involved in ligand docking. Although the extracellular loop (ECL)2 of PAR2 is known to be required for agonist-induced signal transduction, the binding pocket for receptor agonists remains to be determined. In silico homology modelling, based on a crystal structure for the prototypical GPCR rhodopsin, and ligand docking were performed to identify PAR2 transmembrane (TM) amino acids potentially involved in agonist binding. These methods identified 12 candidate residues that were mutated to examine the binding site of the PAR2 TL, revealed by trypsin cleavage, as well as of the soluble ligands 2f-LIGRLO-NH2 and GB110, which are both structurally based on the AP SLIGRLNH2. Ligand binding was evaluated from the impact of the mutated residues on PAR2-mediated calcium mobilisation. An important finding from these experiments was that mutation of residues Y156 and Y326 significantly reduced 2f-LIGRLO-NH2 and GB110 agonist activity. L307 was also important for GB110 activity. Intriguingly, mutation of PAR2 residues did not alter trypsin-induced signalling to the same extent as for the soluble agonists. The reason for this difference remains to be further examined by in silico and in vitro experimentation and, potentially, crystal structure studies. However, these findings identified the importance of TM domains in PAR2 ligand docking and will enhance the design of both PAR2 agonists and potentially agents to inhibit signalling (antagonists). The potential importance of PAR2 in prostate cancer bone metastasis was examined using a mouse model. In patients, prostate cancer bone metastases cause bone growth by disrupting bone homeostasis. In an attempt to mimic prostate cancer growth in bone, PAR2 responsive 22Rv1 prostate cancer cells, which form mixed osteoblastic and osteolytic lesions, were injected into the proximal aspect of mouse tibiae. A role for PAR2 was assessed by treating these mice with the recently developed PAR2 antagonist GB88. As controls, animals bearing intra-tibial tumours were also treated with vehicle (olive oil) or the prostate cancer chemotherapeutic docetaxel. The effect of these treatments on bone was examined radiographically and by micro-CT. Consistent with previous studies, 22Rv1 tumours caused osteoblastic periosteal spicule formation and concurrent osteolytic bone loss. Significantly, blockade of PAR2 signalling reduced the osteoblastic and osteolytic phenotype of 22Rv1 tumours in bone. No bone defects were detected in mice treated with docetaxel. These qualitative data will be followed in the future by quantitative micro-CT analysis as well as histology and histomorphometry analysis of already collected tissues. Nonetheless, these preliminary experiments highlight a potential role for PAR2 in prostate cancer growth in bone. In summary, in vitro studies have defined mechanisms regulating PAR2 activation, downstream signalling and trafficking and in vivo studies point to a potential role for this receptor in prostate cancer bone metastasis. The outcomes of this project are that a greater understanding of the biology of PAR2 may lead to the development of strategies to modulate the function of this receptor in disease.

Expression of PTRF in PC3 cells modulates cholesterol dynamics and the actin cytoskeleton impacting secretion pathways

Expression of caveolin-1 is up-regulated in prostate cancer metastasis and is associated with aggressive recurrence of the disease. Intriguingly, caveolin-1 is also secreted from prostate cancer cell lines and has been identified in secreted prostasomes. Caveolin-1 is the major structural component of the plasma membrane invaginations called caveolae. Co-expression of the coat protein Polymerase I and transcript release factor (PTRF) is required for caveolae formation. We recently found that expression of caveolin-1 in the aggressive prostate cancer cell line PC-3 is not accompanied by PTRF, leading to noncaveolar caveolin-1 lipid rafts. Moreover, ectopic expression of PTRF in PC-3 cells sequesters caveolin-1 into caveolae. Here we quantitatively analyzed the effect of PTRF expression on the PC-3 proteome using stable isotope labeling by amino acids in culture and subcellular proteomics. We show that PTRF reduced the secretion of a subset of proteins including secreted proteases, cytokines, and growth regulatory proteins, partly via a reduction in prostasome secretion. To determine the cellular mechanism accounting for the observed reduction in secreted proteins we analyzed total membrane and the detergent-resistant membrane fractions. Our data show that PTRF expression selectively impaired the recruitment of actin cytoskeletal proteins to the detergent-resistant membrane, which correlated with altered cholesterol distribution in PC-3 cells expressing PTRF. Consistent with this, modulating cellular cholesterol altered the actin cytoskeleton and protein secretion in PC-3 cells. Intriguingly, several proteins that function in ER to Golgi trafficking were reduced by PTRF expression. Taken together, these results suggest that the noncaveolar caveolin-1 found in prostate cancer cells generates a lipid raft microenvironment that accentuates secretion pathways, possibly at the step of ER sorting/exit. Importantly, these effects could be modulated by PTRF expression.

Macrophage inhibitory cytokine-1 (MIC-1/GDF15) slows cancer development but increases metastases in TRAMP prostate cancer prone mice

Macrophage inhibitory cytokine-1 (MIC-1/GDF15), a divergent member of the TGF-β superfamily, is over-expressed by many common cancers including those of the prostate (PCa) and its expression is linked to cancer outcome. We have evaluated the effect of MIC-1/GDF15 overexpression on PCa development and spread in the TRAMP transgenic model of spontaneous prostate cancer. TRAMP mice were crossed with MIC-1/GDF15 overexpressing mice (MIC-1fms) to produce syngeneic TRAMPfmsmic-1 mice. Survival rate, prostate tumor size, histopathological grades and extent of distant organ metastases were compared. Metastasis of TC1-T5, an androgen independent TRAMP cell line that lacks MIC-1/GDF15 expression, was compared by injecting intravenously into MIC-1fms and syngeneic C57BL/6 mice. Whilst TRAMPfmsmic-1 survived on average 7.4 weeks longer, had significantly smaller genitourinary (GU) tumors and lower PCa histopathological grades than TRAMP mice, more of these mice developed distant organ metastases. Additionally, a higher number of TC1-T5 lung tumor colonies were observed in MIC-1fms mice than syngeneic WT C57BL/6 mice. Our studies strongly suggest that MIC-1/GDF15 has complex actions on tumor behavior: it limits local tumor growth but may with advancing disease, promote metastases. As MIC-1/GDF15 is induced by all cancer treatments and metastasis is the major cause of cancer treatment failure and cancer deaths, these results, if applicable to humans, may have a direct impact on patient care.

FOXC2 expression links epithelial-mesenchymal transition and stem cell properties in breast cancer

Resistance to chemotherapy and metastases are the major causes of breast cancer-related mortality. Moreover, cancer stem cells (CSC) play critical roles in cancer progression and treatment resistance. Previously, it was found that CSC-like cells can be generated by aberrant activation of epithelial–mesenchymal transition (EMT), thereby making anti-EMT strategies a novel therapeutic option for treatment of aggressive breast cancers. Here, we report that the transcription factor FOXC2 induced in response to multiple EMT signaling pathways as well as elevated in stem cell-enriched factions is a critical determinant of mesenchymal and stem cell properties, in cells induced to undergo EMT- and CSC-enriched breast cancer cell lines. More specifically, attenuation of FOXC2 expression using lentiviral short hairpin RNA led to inhibition of the mesenchymal phenotype and associated invasive and stem cell properties, which included reduced mammosphere-forming ability and tumor initiation. Whereas, overexpression of FOXC2 was sufficient to induce CSC properties and spontaneous metastasis in transformed human mammary epithelial cells. Furthermore, a FOXC2-induced gene expression signature was enriched in the claudin-low/basal B breast tumor subtype that contains EMT and CSC features. Having identified PDGFR-β to be regulated by FOXC2, we show that the U.S. Food and Drug Administration-approved PDGFR inhibitor, sunitinib, targets FOXC2-expressing tumor cells leading to reduced CSC and metastatic properties. Thus, FOXC2 or its associated gene expression program may provide an effective target for anti-EMT-based therapies for the treatment of claudin-low/basal B breast tumors or other EMT-/CSC-enriched tumors.

Quantifying the roles of cell motility and cell proliferation in a circular barrier assay

Moving fronts of cells are essential features of embryonic development, wound repair and cancer metastasis. This paper describes a set of experiments to investigate the roles of random motility and proliferation in driving the spread of an initially confined cell population. The experiments include an analysis of cell spreading when proliferation was inhibited. Our data have been analysed using two mathematical models: a lattice-based discrete model and a related continuum partial differential equation model. We obtain independent estimates of the random motility parameter, D, and the intrinsic proliferation rate, λ, and we confirm that these estimates lead to accurate modelling predictions of the position of the leading edge of the moving front as well as the evolution of the cell density profiles. Previous work suggests that systems with a high λ/D ratio will be characterized by steep fronts, whereas systems with a low λ/D ratio will lead to shallow diffuse fronts and this is confirmed in the present study. Our results provide evidence that continuum models, based on the Fisher–Kolmogorov equation, are a reliable platform upon which we can interpret and predict such experimental observations.

Nanotechnology in the targeted drug delivery for bone diseases and bone regeneration

Nanotechnology is a vigorous research area and one of its important applications is in biomedical sciences. Among biomedical applications, targeted drug delivery is one of the most extensively studied subjects. Nanostructured particles and scaffolds have been widely studied for increasing treatment efficacy and specificity of present treatment approaches. Similarly, this technique has been used for treating bone diseases including bone regeneration. In this review, we have summarized and highlighted the recent advancement of nanostructured particles and scaffolds for the treatment of cancer bone metastasis, osteosarcoma, bone infections and inflammatory diseases, osteoarthritis, as well as for bone regeneration. Nanoparticles used to deliver deoxyribonucleic acid and ribonucleic acid molecules to specific bone sites for gene therapies are also included. The investigation of the implications of nanoparticles in bone diseases have just begun, and has already shown some promising potential. Further studies have to be conducted, aimed specifically at assessing targeted delivery and bioactive scaffolds to further improve their efficacy before they can be used clinically

In vitro functional characterisation of IGF-I : VN-induced breast cancer progression

Members of the insulin-like growth factor (IGF) family have been shown to play critical roles in normal growth and development, as well as in tumour biology. The IGF system is complex and the biological effects of the IGFs are determined by their diverse interactions between many molecules, including their interactions with extracellular matrix (ECM) proteins. Recent studies have demonstrated that IGFs associate with the ECM protein vitronectin (VN) through IGF-binding proteins (IGFBP) and that this interaction modulates IGF-stimulated biological functions, namely cell migration and cell survival through the cooperative involvement of the type-I IGF receptor (IGF-1R) and VN-binding integrins. Since IGFs play important roles in the transformation and progression of breast cancer and VN has been found to be over-expressed at the leading edge of breast tumours, this project aimed to describe the effects of IGF-I:VN interactions on breast cell function. This was undertaken to dissect the molecular mechanisms underlying IGF-I:VN-induced responses and to design inhibitors to block the effects of such interactions. The studies described herein demonstrate that the increase in migration of MCF-7 breast cancer cells in response to the IGF-I:IGFBP-5:VN complex is accompanied by differential expression of genes known to be involved in migration, invasion and/or survival, including Tissue-factor (TF), Stratifin (SFN), Ephrin-B2, Sharp-2 and PAI-1. This „migration gene signature‟ was confirmed using real-time PCR analysis. Substitution of the native IGF-I within the IGF-I:IGFBP:VN complex with the IGF-I analogue, \[L24]\[A31]-IGF-I, which has a reduced affinity for the IGF-1R, failed to stimulate cell migration and interestingly, also failed to induce the differential gene expression. This supports the involvement of the IGF-1R in mediating these changes in gene expression. Furthermore, lentiviral shRNA-mediated stable knockdown of TF and SFN completely abrogated the increased cell migration induced by IGF-I:IGFBP:VN complexes in MCF-7 cells. Indeed, when these cells were grown in 3D Matrigel™ cultures a decrease in the overall size of the 3D spheroids in response to the IGF-I:IGFBP:VN complexes was observed compared to the parental MCF-7 cells. This suggests that TF and SFN have a role in complex-stimulated cell survival. Moreover, signalling studies performed on cells with the reduced expression of either TF or SFN had a decreased IGF-1R activation, suggesting the involvement of signalling pathways downstream of IGF-1R in TF- and/or SFN-mediated cell migration and cell survival. Taken together, these studies provide evidence for a common mechanism activated downstream of the IGF-1R that induces the expression of the „migration gene signature‟ in response to the IGF-I:IGFBP:VN complex that confers breast cancer cells the propensity to migrate and survive. Given the functional significance of the interdependence of ECM and growth factor (GF) interactions in stimulating processes key to breast cancer progression, this project aimed at developing strategies to prevent such growth factor:ECM interactions in an effort to inhibit the downstream functional effects. This may result in the reduction in the levels of ECM-bound IGF-I present in close proximity to the cells, thereby leading to a reduction in the stimulation of IGF-1R present on the cell surface. Indeed, the inhibition of IGF-I-mediated effects through the disruption of its association with ECM would not alter the physiological levels of IGF-I and potentially only exert effects in situations where abnormal over expression of ECM proteins are found; namely carcinomas and hyperproliferative diseases. In summary, this PhD project has identified novel, innovative and realistic strategies that can be used in vitro to inhibit the functions exerted by the IGF-I:IGFBP:VN multiprotein complexes critical for cancer progression, with a potential to be translated into in vivo investigations. Furthermore, TF and SFN were found to mediate IGF-I:IGFBP:VN-induced effects, thereby revealing their potential to be used as therapeutic targets or as predictive biomarkers for the efficacy of IGF-1R targeting therapies in breast cancer patients. In addition to its therapeutic and clinical scope, this PhD project has significantly contributed to the understanding of the role of the IGF system in breast tumour biology by providing valuable new information on the mechanistic events underpinning IGF-I:VN-mediated effects on breast cell functions. Furthermore, this is the first instance where favourable binding sites for IGF-II, IGFBP-3 and IGFBP-5 on VN have been identified. Taken together, this study has functionally characterised the interactions between IGF-I and VN and through innovative strategies has provided a platform for the development of novel therapies targeting these interactions and their downstream effects.

Heparan sulfate proteoglycans, tumour progression and the cancer stem cell niche

The cancer stem cell hypothesis states that tumours arise from cells with the ability to self-renew and differentiate into multiple cell types, and that these cells persist in tumors as a distinct population that can cause disease relapse and hence metastasis. The crux of this hypothesis is that these cells are the only cells capable of, by themselves, giving rise to new tumours. What proportion of a tumour consists of these stem cells, where are they localised, how are they regulated, and how can we identify them? The stromal cells embedded within the extracellular matrix (ECM) not only provide a scaffold but also produce ECM constituents for use by stem cells. Heparan sulfate proteoglycans (HSPGs) are ubiquitous to this cell niche and interact with a large number of ligands including growth factors, their receptors, and ECM structural components. It is still unclear whether ECM degradation and subsequent metastasis is a result of proteases produced by the tumour cells themselves or by cells within the stromal compartment. The identification of the cellular origin of cancer stem cells along with microenvironmental changes involved in the initiation, progression and the malignant conversion of all cancers is critical to the development of targeted therapeutics. As ubiquitous members of the ECM microenvironment and hence the cancer cell niche, HSPGs are candidates for a central role in these processes.

Polymorphisms of the SIPA1 gene and sporadic breast cancer susceptibility

Background The novel breast cancer metastasis modulator gene signal-induced proliferation-associated 1 (Sipa1) underlies the breast cancer metastasis efficiency modifier locus Mtes 1 and has been shown to influence mammary tumour metastatic efficiency in the mouse, with an ectopically expressing Sipa1 cell line developing 1.5 to 2 fold more surface pulmonary metastases. Sipa1 encodes a mitogen-inducible GTPase activating (GAP) protein for members of the Ras-related proteins; participates in cell adhesion and modulates mitogen-induced cell cycle progression. Germline SIPA1 SNPs showed association with positive lymph node metastasis and hormonal receptor status in a Caucasian cohort. We hypothesized that SIPA1 may also be correlated to breast carcinoma incidence as well as prognosis. Therefore, this study investigated the potential relationship of SIPA1 and human breast cancer incidence by a germline SNP genotype frequency association study in a case-control Caucasian cohort in Queensland, Australia. Methods The SNPs genotyped in this study were identified in a previous study and the genotyping assays were carried out using TaqMan SNP Genotyping Assays. The data were analysed with chi-square method and the Monte Carlo style CLUMP analysis program. Results Results indicated significance with SIPA1 SNP rs3741378; the CC genotype was more frequently observed in the breast cancer group compared to the disease-free control group, indicating the variant C allele was associated with increased breast cancer incidence. Conclusion This observation indicates SNP rs3741378 as a novel potential sporadic breast cancer predisposition SNP. While it showed association with hormonal receptor status in breast cancer group in a previous pilot study, this exonic missense SNP (Ser (S) to Phe (F)) changes a hydrophilic residue (S) to a hydrophobic residue (F) and may significantly alter the protein functions of SIPA1 in breast tumourgenesis. SIPA1 SNPs rs931127 (5' near gene), and rs746429 (synonymous (Ala (A) to Ala (A)), did not show significant associations with breast cancer incidence, yet were associated with lymph node metastasis in the previous study. This suggests that SIPA1 may be involved in different stages of breast carcinogenesis and since this study replicates a previous study of the associated SNP, it implicates variants of the SIPA1 gene as playing a potential role in breast cancer.

Matrix metalloproteinase localisation by in situ-RT-PCR in archival human breast biopsy material

Utilising archival human breast cancer biopsy material we examined the stromal/epithelial interactions of several matrix metalloproteinases (MMPs) using in situ-RT-PCR (IS-RT-PCR). In breast cancer, the stromal/epithelial interactions that occur, and the site of production of these proteases, are central to understanding their role in invasive and metastatic processes. We examined MT1-MMP (MMP-14, membrane type-1-MMP), MMP-1 (interstitial collagenase) and MMP-3 (stromelysin-1) for their localisation profile in progressive breast cancer biopsy material (poorly differentiated invasive breast carcinoma (PDIBC), invasive breast carcinomas (IBC) and lymph node metastases (LNM)). Expression of MT1-MMP, MMP-1 and MMP-3 was observed in both the tumour epithelial and surrounding stromal cells in most tissue sections examined. MT1-MMP expression was predominantly localised to the tumour component in the pre-invasive lesions. MMP-1 gene expression was relatively well distributed between both tissue compartments, while MMP-3 demonstrated highest expression levels in the stromal tissue surrounding the epithelial tumour cells. The results demonstrate the ability to distinguish compartmental gene expression profiles using IS-RT-PCR. Further, we suggest a role for MT1-MMP in early tumour progression, expression of MMP-1 during metastasis and focal expression pattern of MMP-3 in areas of expansion. These expression profiles may provide markers for early breast cancer diagnoses and present potential therapeutic targets.

Polymorphic variants of NFKB1 and its inhibitory protein NFKBIA, and their involvement in sporadic breast cancer

Nuclear factor kappa-beta (NF-kappaB) is a transcription factor responsible for modulating the expression of many genes involved in cell proliferation, differentiation, apoptosis and metastasis. NF-kappaB interacts with IkappaB inhibitory proteins to regulate gene expression. This study investigated common variants within the genes coding for NF-kappaB and IkappaB, NFKB1 and NFKBIA, for involvement in sporadic breast cancer. Genotypes were determined in a population of breast cancer affected individuals and age-matched controls. Results do not support an involvement of the tested NFKB1 and NFKBIA polymorphisms in susceptibility to sporadic breast cancer, in the tested Caucasian population.