Pathology - Theses
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ItemInvestigating the role of hypoxia in tumour progression in breast cancer( 2015)Metastasis is a major cause of morbidity and mortality in breast cancer patients. The molecular processes and mediators that underpin this process have yet to be completely delineated. Hypoxia, the state of reduced oxygen conditions, occurs frequently in solid tumours and is a factor of poor prognosis for patient outcome. The upregulation of HIF-1α, the main mediator of the hypoxic response pathway, has been implicated in several different facets of tumour progression, including tumour growth, angiogenesis, therapy resistance and metastasis. Hypoxia has been shown to induce Epithelial-to-Mesenchymal Transition (EMT), a highly conserved developmental program that facilitates tumour cell dissemination. It is thought that EMT is co-opted by epithelial tumour cells in order to acquire a degree of plasticity, allowing them to undergo a number of genetic, biochemical and morphological changes to adopt a mesenchymal phenotype. This results in the loss of polarity, and the gain of migratory and invasive capabilities. EMT is regulated by a core cassette of transcription factors, including Snail, Slug, Twist, Zeb1 and Zeb2. Zeb1 is the most proximal transcription factor, however, how hypoxia modulates Zeb1 expression is not known. This study demonstrates that Siah, a family of E3 ubiquitin ligases and a master regulator of HIF-1α protein expression, binds to and targets Zeb1 for proteasomal degradation. Loss of Siah2 is sufficient to cause spontaneous EMT in tumour cells derived from the PyMT murine model of breast cancer. On the other hand, EMT induction led to the decrease in Siah protein expression. This work is the first to describe a post-translational mechanism of regulation of Zeb1 and further defines the relationship between hypoxia and EMT. There are, in fact, two forms of hypoxia in a growing tumour, chronic hypoxia and intermittent hypoxia. Chronic hypoxia describes the long-term limitations on oxygen diffusion caused by abnormal tumour vasculature. While intermittent hypoxia refers to the fluctuations of oxygen tension in a tumour, caused by the aberrant and temporary closing and reopening of tumour-supplying blood vessels. The consequences of these two different types of hypoxia in breast cancer have not yet been well characterised. Using the orthotopic, syngeneic PyMT murine model of breast cancer, it was found that intermittent hypoxia-treated cells gave rise to a greater number of larger lung metastases in vivo. This was facilitated by an enhanced ability for anchorage-independent growth, increased clonogenicity, the induction of a pro-tumourigenic gene expression and secretory profile, and the increase in tumour-initiating capacity through the gain of cancer stem cell properties. RNA sequencing of hypoxia-treated cells found distinct gene expression patterns between treatment groups. While, pathway analysis revealed a marked enrichment of immune-related pathways and a downregulation of DNA replication and cell cycle pathways, by both chronic and intermittent hypoxia. Interestingly, chronic hypoxia also upregulated extracellular matrix degradation pathways, in spite of the lack of an overt EMT in cells. These results unveil novel mechanisms and pathways involved in hypoxia-mediated metastasis while highlighting the extensive effects of hypoxia signalling in cancer. Taken together, this work demonstrates the complexity of hypoxia signalling in tumour progression. Not only does it endow tumour cells with an aggressive, tumour-initiating phenotype, but it also contributes to the priming of the tumour microenvironment to be pro-inflammatory and immunosuppressive and ultimately, tumour-promoting.
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ItemBiomarkers in ductal carcinoma in situ( 2016)Ductal carcinoma in situ (DCIS), a non-invasive form of breast cancer and a non-obligate precursor of invasive carcinoma of the breast, displays heterogeneous behaviour. Most DCIS are adequately managed by local surgical excision alone, but in 20-30% of cases, disease recurrence occurs after local surgical excision either as DCIS or invasive carcinoma. Accurate identification of these two clinical outcome groups at the time of diagnosis is desirable to allow appropriate treatment allocation. In this thesis, genomic and epigenetic alterations in DCIS epithelium, including copy number aberrations, somatic mutations, and DNA methylation were investigated as markers of DCIS biology and outcome. In addition, the expression and significance of LRH-1, a nuclear receptor which acts as a transcription factor, was investigated in both invasive carcinoma and DCIS. Copy number analysis of DCIS of known clinical outcome identified amplification of 20q13 to be associated with disease recurrence, but this was unable to be validated on an independent cohort. Targeted next generation sequencing of a panel of breast cancer-relevant genes revealed that the mutational profile of DCIS was similar to that reported for invasive carcinomas, with the most frequently mutated genes being GATA3, PIK3CA, and TP53. A high prevalence of GATA3 mutations in DCIS was observed and TP53-mutant DCIS was associated with high stromal tumour infiltrating lymphocytes. Mutations of RUNX1 were a novel finding, not previously reported in DCIS. Promoter methylation of a candidate gene panel, consisting predominantly of known tumour suppressor genes, was associated with adverse tumour features in DCIS. Methylation load varied among DCIS cases, suggesting that methylation differs in importance in the tumorigenesis of DCIS, and that assessment of methylation may be useful as a biological classifier of DCIS. Finally, LRH-1 mRNA expression patterns in breast cancers was similar to that reported for breast cancer cell lines and distinct LRH-1 immunohistochemical staining patterns were associated with tumour phenotype in both invasive breast carcinoma and DCIS. The results of this thesis demonstrate that copy number alterations, somatic mutations, DNA methylation, and LRH-1 expression are indicative of DCIS phenotype and hence biology. These markers showed promise as prognostic biomarkers, although validation of their utility was hampered by the small number of pure DCIS cases with both adequate genomic material and long-term clinical outcome data. Nonetheless, the findings of this thesis indicate that assessment of these biomarkers can be performed in routine diagnostic tissue material and provide several avenues for future research.
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ItemA translational analysis of the genomic variation associated with breast cancer in hereditary breast and ovarian cancer families( 2014)Breast cancer remains a significant health issue with the highest incidence and the second highest cause of cancer-related mortality amongst women worldwide. A family history is an important risk factor, with women found to be twice as likely to develop the disease if they have one first-degree relative affected with breast cancer. It is the role of familial cancer clinics (FCCs) and genetic testing services to identify ‘high-risk’ individuals and offer genetic testing to eligible individuals looking for mutations within high-penetrance genes, such as BRCA1 and BRCA2. For mutation carriers, cancer risk assessment and management is based on a well-established foundation of evidence. However, up to 80% of individuals have no identifiable BRCA mutation, resulting in risk assessment and management advice that is based only on family history and is no longer personalised but instead directed by summary data from epidemiological studies of the general population. Researchers have continued the search for the other genetic causes of the heritable fraction of breast cancer through large, collaborative genome-wide association studies (GWAS). To date, these studies have identified over 70 common variants associated with breast cancer risk. Individually, the associations for these variants are too weak to be useful (relative risk <1.5), however, when the risks for these variants are combined multiplicatively (termed polygenic risk) the risk becomes more appreciable. It is estimated that up to 28% of familial breast cancer risk can be explained by common variants. Despite this, genetic testing for common genomic variants has yet to be implemented into routine clinical practice. This study explores the clinical significance and implications of common genomic variants to the assessment and risk management of high-risk breast and ovarian cancer families, to generate the data required to facilitate the translation of common variant genetic testing into clinical practice. To conduct this research, the study utilised the Victorian Familial Breast and Ovarian Cancer Cohort (VFBOCC) which consists of individuals (female index cases) who have been assessed as high-risk for breast and/or ovarian cancer based on their personal and family history of cancer. In Chapter 3, the development of a database that systematically incorporated and allowed for the well-defined characterisation of the VFBOCC is discussed. This database framework facilitated a detailed analysis of the cohort; it identified that females in this cohort have an average breast cancer age of onset of 44.9 years, which is much lower than the average age of onset observed in the general population, and that only 22% of the VFBOCC had a BRCA1 or BRCA2 mutation identified by diagnostic genetic testing. The establishment of this database enabled the VFBOCC data to be utilised in common genomic variant research as part of this thesis as well as within several collaborations which are also investigating the potential role of recently discovered rare genetic variations to familial breast cancer risk. In Chapter 4, the association of 62 common genomic variants with a range of clinical features from 1,136 women affected with breast cancer from the VFBOCC is examined. This study found the associations of common variants were stronger in the familial setting when compared to unselected breast cancer cases from the general population, highlighting the significance of hereditary factors in the aetiology of breast cancer. A polygenic risk score (PRS) was then calculated for each woman and the mean PRS was found to be significantly higher in breast cancer cases with no identifiable BRCA mutation when compared to population controls (P= 4.44x10-40) and women who had a BRCA1 or BRCA2 mutation (P=0.0002). These findings illustrate that common variants can help to predict a group of individuals who are at high risk of developing breast cancer but unlikely to harbour a mutation within the BRCA1 or BRCA2 genes. The next step was to investigate the additional effect of the PRS on the outputs from clinical tools used by FCCs to predict the likelihood of a high-risk individual harbouring a BRCA1 or BRCA2 mutation (Chapter 5). This research found that the addition of common variants to the tools did not significantly improve their performance. In Chapter 6, the focus of this study shifts to the clinical implications of introducing common variant genetic testing into clinical practice. It outlines the development of a personalised breast cancer risk report incorporating common variant data, which was evaluated by a sample of health professionals (oncologists, clinical geneticists and genetic counsellors) in a series of hypothetical patient scenarios to determine the potential impact of this additional genetic information on risk assessment and management recommendations of high-risk women with no identifiable BRCA mutation. This study found common variant information assisted in determining an individual woman’s risk of developing a breast cancer, with health professionals recommending increased screening, risk-reducing medications (Tamoxifen) and prophylactic surgery to women with a high PRS. In addition, health professionals identified the need for further research, the development of education programs, and clinical guidelines for risk management recommendations if this new type of genetic information was to be implemented as part of routine clinical practice.
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ItemInvestigating MYB in the context of mammary gland biology, transformation and as a therapeutic target in breast and colon cancer( 2014)Breast cancer is the second most common form of malignancy diagnosed in Australian women, imposing an enormous social and economic burden on society. If the cancer spreads to secondary locations, patient survival decreases dramatically. Therapeutic strategies for treatment of metastatic disease are desperately needed in breast cancer. Recently we have shown that when Myb is conditionally deleted from the mammary gland of MMTV-Neu and MMTV-PyMT mice, tumour formation is ablated. To provide further insight into the function of Myb in mammary gland development, cre-mediated conditional knock out of c-myb in the mouse mammary gland was examined. The conditional deletion of c-myb led to a reduction in branching and terminal end bud formation. These data indicate that if MYB could be inhibited for breast cancer therapy, there are potentially few side effects on the normal mammary gland. The ability to inhibit DNA binding transcription factors is a long sort after goal in oncology, as their importance in disease initiation and progression is well documented. To target MYB, we have developed a DNA vaccine. Preclinical studies have largely examined the MYB DNA vaccine in the context of colon cancer models using prophylactic vaccination. However, preliminary data indicate that it may also be effective in reducing the metastatic burden in preclinical breast cancer models. This thesis aims to further investigate the role of MYB during mammary gland development and provide insights into its involvement in tumourigenesis. Furthermore, the MYB DNA vaccine will be assessed for its effectiveness as a therapeutic treatment in clinically relevant surgical models of metastatic breast cancer, as well as further development as a therapeutic in the setting of colon cancer.
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ItemThe role of hypoxic signalling in the primary tumour microenvironment and metastasis in breast cancer( 2013)Intratumoural hypoxia is a poor prognostic factor associated with reduced disease-free survival in many cancer types, including breast cancer. Under hypoxic conditions, tumour cells signal via the hypoxic response pathway and stabilisation of the HIF-1 transcription factor to increase the expression of genes that promote survival, proliferation, angiogenesis, invasion and metastatic spread. The factors secreted from hypoxic tumour cells help to recruit various stromal and bone marrow-derived cell (BMDC) lineages that together create a growth-supporting primary tumour microenvironment. The aims of this thesis are twofold; to investigate novel treatments targeting HIF-1 in the primary tumour microenvironment, and to investigate the effects of hypoxic signalling on pre-metastatic niche formation in breast cancer. Antioxidants have been identified as potential anti-cancer drugs that reduce tumour growth and metastasis in a HIF-1-dependent manner. N-acetylcysteine (NAC) is one such antioxidant that has a promising but somewhat controversial role in cancer therapy. This study utilised three orthotopic, syngeneic murine models of breast cancer, the PyMT, EO771 and 4T1.2 models, to investigate the ability of NAC to regulate the hypoxic response in breast tumour cells, and prevent tumour growth and metastasis. NAC demonstrated an ability to prevent HIF-1 stabilisation and signalling via the hypoxic response pathway in breast tumour cells in vitro. When administered in vivo however, NAC treatment did not inhibit primary tumour growth or metastasis. Furthermore, NAC treatment promoted metastases formation in an experimental metastasis model. This work brings into question the validity of NAC as an anti-tumourigenic agent in breast cancer, and highlights the need to further investigate its properties in vivo in different cancer models. Recently, it has been demonstrated that a primary tumour promotes metastasis through the formation of supportive microenvironments, termed pre-metastatic niches, in secondary organs predisposed to metastases. Cytokines, chemokines and growth factors secreted from tumour cells promote the mobilisation and recruitment of BMDCs to pre-metastatic organs, where they interact with the extracellular matrix and other stromal components to create tumour-promoting niches. While hypoxic tumour cells themselves have an increased ability to metastasize, they also secrete a variety of factors independently identified in pre-metastatic niches. This study investigated the combinatorial effect of factors secreted from hypoxic breast tumour cells on pre-metastatic niche formation in distant organs. Using immune competent eGFP bone marrow chimeric mice and different syngeneic mammary cancer models, hypoxic factors were shown to create an immunosuppressed environment in pre-metastatic organs. A subset of granulocytic CD11b+/Ly6Cmed/Ly6G+ myeloid-derived suppressor cells with increased CCR2 expression and STAT3 signalling were identified in the pre-metastatic niche. As were CD3-/NK1.1+ Natural Killer cells with reduced maturity, cytotoxicity, and expression of the activating receptor NKG2D. Together, factors secreted from hypoxic breast tumour cells promote metastasis by creating immunosuppressed pre-metastatic niches in distant organs. This work identifies novel components of the pre-metastatic niche that provide potential avenues for therapeutic intervention in metastatic disease.
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ItemGenetic variants, phenotypic spectrum and breast cancer risk associated with germline mutations in PALB2: identifying female PALB2 mutation carriers at time of diagnosis( 2013)PALB2 is a breast cancer susceptibility gene which encodes a protein that is fundamental to the maintenance of genome stability via the homologous recombination DNA repair pathway. Mutations in PALB2 are rare, varying from 0.1% to 1.5% depending upon the population, but there are substantial breast cancer risks associated with at least some of the mutations in PALB2. Risk-reducing clinical screening strategies and prophylactic treatments are currently available for women who are at high risk of breast cancer. There are also potential therapies that target DNA repair dysfunction which have been shown to be effective in PALB2-deficient cells. Therefore, it is of high importance that female carriers of high-risk PALB2 mutations are identified so that these clinical benefits can be made available to them. Due to the rarity of PALB2 mutations, using current clinical screening platforms, it would be optimal to enrich for women most likely to be mutation carriers prior to genetic testing. The main objectives of the research described in this thesis were to determine the prevalence and penetrance associated with PALB2 mutations in the Australian population and to identify tumour morphological features associated with PALB2 mutation status that could facilitate the identification of female carriers of pathogenic germline mutations of PALB2 at the time of breast cancer diagnosis. A nonsense mutation, PALB2 c.3113G>A, was identified through mutation screening of the coding regions of PALB2 in 0.3% of women that were recruited via population-based sampling of young women with breast cancer without selection for their family history of the disease. Modified segregation analysis was used to estimate that this germline mutation was associated with 91% cumulative risk of breast cancer to the age of 70 years (95% confidence interval = 44-100). Further mutation screening was performed to assess the prevalence of PALB2 mutations in high-risk breast and/or ovarian cancer families attending Familial Cancer Clinics in Australasia. Four PALB2 mutations, including PALB2 c.3113G>A, were found to contribute to breast cancer risk in approximately 1.5% of these families. Tumour morphologies of 28 invasive breast cancers arising in women who carry a germline loss-of-function PALB2 mutation were compared to breast tumours of a population-based sample of 828 non-carriers. Minimal sclerosis was identified as a key morphological feature that not only distinguishes carriers of PALB2 mutations from non-carriers of high-risk mutations in BRCA1, BRCA2, ATM, TP53 and PALB2 (p < 0.001) but also distinguishes PALB2 mutation carriers from BRCA1 (p = 0.05) and BRCA2 (p = 0.04) mutation carriers. In addition to the main objectives of this research, we have used massively parallel sequencing to interrogate the exonic sequences of four individuals of a multiple-case family that segregates PALB2 c.3113G>A to identify other genetic risk factors that may be segregating through this family. We have identified a genetic variant, XAF1 c.343G>T, that has the potential of being involved in breast cancer predisposition or in breast cancer progression. This study brings together several lines of evidence to indicate that genetic information about PALB2 is relevant to breast cancer clinical genetics services. It has identified a PALB2 germline mutation that is associated with high breast cancer risk. This supports the reports of recent population-based studies of breast cancer that at least some PALB2 mutations are associated with breast cancer risks comparable to that of the average pathogenic mutation in BRCA2: 45% (95% confidence interval = 31%-56%). It has also identified a tumour morphological feature, predictive of PALB2 mutation status, which could be used to facilitate the identification of carriers of PALB2 mutations at the time of diagnosis, irrespective of family history. The important information generated by this study could expedite PALB2 mutation testing and translation of genetic information about PALB2 mutation status into clinical practice in Australia.
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ItemThe contribution of stromal caveolin-1 to breast cancer metastasis( 2012)In a previous study, our group has shown that the expression of caveolin-1 (CAV1) in normal tissue surrounding a primary breast cancer is a powerful prognostic indicator of subsequent metastatic disease. While reports linking expression of CAV1 within breast tumour cells to clinical outcome have not led to any clear conclusions, the finding of a strong positive correlation between loss of CAV1 expression in breast tumour stroma and poor prognosis by our group and others, is novel and exciting as it offers the potential of a reliable prognostic indicator of metastatic disease. These clinical observations have led us to propose the hypothesis that breast tumour cells decrease stromal CAV1 expression, and a reduction in stromal CAV1 expression leads to promotion of tumour growth and metastasis. The aims of this project were to model the progression in breast cancer following stromal CAV1 loss, and determine the underlying breast tumour-stroma paracrine metastasis. Tumours arising from weakly metastatic mammary cell lines 66cl4 and 4T1ch5, grow at a faster or similar rate respectively, when co-injected with CAV1 null mammary fibroblasts. In contrast, co-injection with CAV1 expressing mammary fibroblasts has a suppressive effect on tumour growth. Metastasis to lung was significantly higher in mice with resected primary tumours that arose from co-inoculation of 4T1ch5 cells and CAV1 null mammary fibroblasts, in addition to a significant increase in individual lung tumour nodule size. However no significant difference in immune infiltrate in these resected tumours was observed in preliminary flow cytometry analysis. No significant differences in primary tumour growth or metastasis were observed in human xenograft models. To mimic the phenotype observed in vivo, 3D co-culture assays were developed. Although these assays demonstrated a positive effect of fibroblasts on tumour cell invasion and proliferation, no stromal CAV1 specific effect was observed in response to fibroblast co-culture or conditioned medium. To further understand the consequences of the loss of stromal CAV1, profiling of CAV1 expressing and null mouse mammary fibroblasts was conducted using cytokine arrays and cDNA microarrays. A significant increase in Gas6 and a decrease in RANTES cytokine secretion were observed as a result of CAV1 loss, with no significant changes in their transcript levels. In summary, results from this project demonstrate that stromal CAV1 is an important prognostic factor in breast cancer progression. Based on these findings, a stromal targeted therapy to that restores or substitutes for CAV1 activity in stromal cells, or that targets CAV1 regulated cytokines such as Gas6, may be a viable therapy in the treatment of breast cancer metastasis.
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ItemUnderstanding mammographic density and breast cancer risk: from histology to genomics( 2012)This thesis describes a novel multi-disciplinary strategy that advances our understanding of mammographic density (MD) biology and genetics and its relationship to breast cancer risk. Studies have indicated that women with high MD are at 4 to 6 fold increased risk of getting breast cancer. MD is also highly heritable. Hence, a greater understanding of MD biology and genetics would improve our knowledge about breast biology and potentially identify new cancer predisposition genes. Nonetheless, MD biology remains controversial and much is unknown about the genetic basis of this trait. This thesis revisits MD histopathology using a novel methodology of sampling high and low MD tissue within the breast of an individual. This method samples tissue guided by its true MD, as determined by real-time mammography imaging - in contrast to previous studies that examined MD histopathology relying on “random” sampling of breast tissue. Studies have suggested that dense regions of the breast are potentially associated with breast cancer risk on the basis that percent MD (PMD) adjusted for body mass index (BMI) and age is a better measure of breast cancer risk. Hence, this method also allows assessment of tissue most pertinent to MD associated breast cancer risk. Furthermore, the use of sampling within an individual allows control of all potential confounders that helps improve the power of analysis. The histopathological analysis revealed that high MD regions were significantly associated with greater composition of dense connective tissue stroma and lesser composition of adipose tissue while no difference in glandular areas between high and low MD tissue was observed. However, it was noted that high MD tissue tended to have smaller and lower complexity glands compared to low MD tissue. This raises the possibility of high MD regions being associated with stem-like cells and their niche compared to low MD regions. Whole-genome expression profiling of accrued high and low MD tissue were interrogated to further our understanding of the genetic and biological bases of MD. Currently, only two other studies have investigated gene expression in MD tissue and these studies have generally failed to account for all potential unwanted variation in the data that could impact on the validity of the analysis outcomes. The present work differs from previous studies in that careful quality assessments and analyses were performed to improve sensitivity and power of the study. The use of precisely sampled tissue also allowed a better representation of the MD expression profile. Both single-gene and gene-set based analyses of adjusted MD expression dataset concurred with the histopathological correlates of high versus low MD tissue. Interestingly, these analyses also showed that high MD regions correlated with a cancer-signature and a CD24 (i.e. luminal epithelial)-signature; while the observed anti-correlation with the CD44-signature was postulated to be of stromal origin. The association of high versus low MD tissue with a cancer-signature agrees with the general direction of MD associated breast cancer risk. Furthermore, immunohistochemical examination of the tissue uncovered a trend of potential “stemness” in high versus low MD tissue that suggests the CD24-signature being of progenitor origin. This supports the hypothesis of high MD tissue being associated with “stemness” (and their niche), as proposed from the initial histopathological examination of MD. To further understand MD biology and its genetic basis, both univariate and pathway-based analyses of genome-wide association (GWA) study data were performed. One major issue with such pathway-based analysis is the assignment of SNPs to their respective gene(s). Increasingly, studies suggest that conventional assignment of SNPs to their nearest gene may be inaccurate. This was the impetus to develop a novel framework using expression profiling analysis of high and low MD tissue to guide mapping of SNPs to their genes. The methodology helped improve the biological relevance and reproducibility of the significant pathways, and also identified the mitogen-activated protein kinase (MAPK) signalling pathway as the most significantly associated with the MD trait. This outcome corresponds to the results obtained from a recent study of breast cancer GWA studies. Taken together, this multi-disciplinary approach has given potential insight into the biological and genetic basis of MD, allowing inference to be made about the increased risk associated with high MD.
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ItemIntegrin β3 as a therapeutic target for breast cancer metastasis to bone( 2011)Breast cancer is the most common cancer in women, with patients currently having high survival rates if the disease is confined to the breast. However, these survival rates drop drastically if the cancer has metastasised to distant sites. Previous studies have shown both tumour and stromal integrin β3 are involved in breast cancer metastasis, yet questions remain regarding its specific role in the process. In particular, whether integrin β3 is essential for metastasis and if so, to which organ and at which stage of the metastatic cascade it is required. Furthermore, the relative contribution of stromal and tumour integrin β3 in tumour growth and metastasis needs to be clarified. To address these questions, the transplantable 4T1 model of breast cancer with spontaneous metastasis was utilised. RNA interference was used to suppress tumour integrin β3 expression in the highly metastatic 4T1.2 and 4T1BM2 cells, and investigate whether tumour integrin β3 is essential for metastasis. Additionally, the contribution of stromal cell populations expressing integrin β3 was investigated in integrin β3 knockout mice. Lastly, the therapeutic potential of targeting integrin β3 was investigated using the disintegrin DisBa-01. Downregulation of tumour integrin β3 expression induced a coordinated decrease in the surface level of the integrin αv subunit. Functional assays revealed integrin αvβ3 dependent decreases in adhesion, migration and MMP9 secretion. In vivo, downregulation of integrin αvβ3 had no affect on primary tumour growth, but significantly reduced spontaneous metastasis to bone, lung and other soft tissues, indicating integrin β3 is required for metastasis to multiple sites. Surprisingly, unlike studies in other tumour types, integrin β3 suppression in 4T1.2 cells did not impact on experimental breast cancer metastasis to lung or bone, suggesting it is acting at an early stage of metastasis. Unlike reports in other models, primary tumour growth was not affected by the loss of stromal integrin β3, indicating orthotopic growth of breast tumours is not dependent on the expression of integrin β3 in stromal cell populations. However, metastasis to bone, but not lung, was reduced in integrin β3 null mice. These observations allow the reconciliation of previous studies that reported conflicting conclusions with regard to the role of stromal integrin β3 in tumour growth and metastasis. In vitro treatment of 4T1BM2 cells with DisBa-01 achieved similar results to down-regulation of the protein by shRNA, suppressing integrin αvβ3 dependent adhesion, proliferation, migration and MMP9 secretion. Unfortunately, these promising results did not translate to effects on tumour growth and metastasis in vivo, and neither experimental nor spontaneous metastasis were suppressed by DisBa-01 treatment under the conditions tested. However, the results obtained provide valuable information with regards to future protocol design. This study demonstrates that tumour expressed integrin β3 is essential for spontaneous breast cancer metastasis to multiple organs, and provides evidence the protein acts at an early stage in the process. It also supports the role of stromal integrin β3 in metastasis to bone, but not to lung. Taken together, these data suggest both tumour and stromal integrin β3 are potential therapeutic targets, with tumour integrin β3 contributing to the process to a greater degree.
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ItemContribution of tumour derived cysteine cathepsin B to breast cancer metastasis( 2012)The major cause of mortality associated with breast cancer is the development of distant meatastases to sites such as lung and bone. Approximately 70% of patients who die from breast cancer have evidence of metastases in the skeleton. The consequences of bone metastasis are always devastating. The clinical outcomes of severe pain, pathologic fractures, spinal cord and nerve compression, leading to hypercalcemia and acid/base imbalance severely diminish the quality of life. Once tumour cells home to bone, curative therapy is no longer possible in most patients and only palliative therapy is available. This emphasises the importance of understanding the mechanisms of primary tumour cell invasion and spread to bone, to be able to identify molecular drivers of bone metastasis as new therapeutic targets. Proteases are known to contribute to tumour cell invasion and angiogenesis and are commonly associated with metastasis. Recent studies in our laboratory support a role for cysteine cathepsin proteases in bone metastasis. Using our unique 4T1.2 syngeneic model of spontaneous bone metastasis, we identified the endogenous cysteine cathepsin inhibitor stefin A as a metastasis suppressor. Tumour cell expression of stefin A significantly reduced bone metastasis in the murine model. The fact that stefin A is a potent metastasis suppressor indicates that its targets, the cysteine cathepsins, have essential roles in distant metastasis. Of the cysteine cathepsins, cathepsin B was co-expressed with stefin A in primary tumours and metastases, suggesting that the mechanism of metastasis suppression by stefin A was through inhibition of cathepsin B. Cathepsin B is highly upregulated in a wide variety of cancers, including breast and prostate cancer and is linked to enhanced tumourigenesis. In this study, using the 4T1.2 spontaneous bone metastasis model, we evaluated the function of cathepsin B in bone metastasis and the potential of selectively targeting this protease as a novel therapeutic. Cathepsin B was abundant in 4T1.2 mammary tumours and matched spine metastases, mimicking that of the human disease. We have demonstrated a critical function for tumour-derived cathepsin B in bone disease. Stable knockdown of cathepsin B in tumour cells significantly reduced collagen I degradation in vitro and bone metastasis in vivo. Additionally, use of a highly selective cathepsin B inhibitor CA-074 significantly reduced metastasis to lung and bone, a reduction that was not observed when using a broad spectrum cysteine cathepsin inhibitor. This study reveals the pro-metastatic role of cathepsin B in distant metastasis and the therapeutic benefit of its selective inhibition in a murine model of breast cancer.