Epithelial—mesenchymal and mesenchymal—epithelial transitions in carcinoma progression

Like a set of bookends, cellular, molecular, and genetic changes of the beginnings of life mirror those of one of the most common cause of death—metastatic cancer. Epithelial to mesenchymal transition (EMT) is an important change in cell phenotype which allows the escape of epithelial cells from the structural constraints imposed by tissue architecture, and was first recognized by Elizabeth Hay in the early to mid 1980's to be a central process in early embryonic morphogenesis. Reversals of these changes, termed mesenchymal to epithelial transitions (METs), also occur and are important in tissue construction in normal development. Over the last decade, evidence has mounted for EMT as the means through which solid tissue epithelial cancers invade and metastasize. However, demonstrating this potentially rapid and transient process in vivo has proven difficult and data connecting the relevance of this process to tumor progression is still somewhat limited and controversial. Evidence for an important role of MET in the development of clinically overt metastases is starting to accumulate, and model systems have been developed. This review details recent advances in the knowledge of EMT as it occurs in breast development and carcinoma and prostate cancer progression, and highlights the role that MET plays in cancer metastasis. Finally, perspectives from a clinical and translational viewpoint are discussed

Analysis of the CDKN2A, CDKN2B and CDK4 genes in 48 Australian melanoma kindreds

Germline mutations within the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene and one of its targets, the cyclin dependent kinase 4 (CDK4) gene, have been identified in a proportion of melanoma kindreds. In the case of CDK4, only one specific mutation, resulting in the substitution of a cysteine for an arginine at codon 24 (R24C), has been found to be associated with melanoma. We have previously reported the identification of germline CDKN2A mutations in 7/18 Australian melanoma kindreds and the absence of the R24C CDK4 mutation in 21 families lacking evidence of a CDKN2A mutation. The current study represents an expansion of these efforts and includes a total of 48 melanoma families from Australia. All of these families have now been screened for mutations within CDKN2A and CDK4, as well as for mutations within the CDKN2A homolog and 9p21 neighbor, the CDKN2B gene, and the alternative exon 1 (E1beta) of CDKN2A. Families lacking CDKN2A mutations, but positive for a polymorphism(s) within this gene, were further evaluated to determine if their disease was associated with transcriptional silencing of one CDKN2A allele. Overall, CDKN2A mutations were detected in 3/30 (10%) of the new kindreds. Two of these mutations have been observed previously: a 24 bp duplication at the 5' end of the gene and a G to C transversion in exon 2 resulting in an M531 substitution. A novel G to A transition in exon 2, resulting in a D108N substitution was also detected. Combined with our previous findings, we have now detected germline CDKN2A mutations in 10/48 (21%) of our melanoma kindreds. In none of the 'CDKN2A-negative' families was melanoma found to segregate with either an untranscribed CDKN2A allele, an R24C CDK4 mutation, a CDKN2B mutation, or an E1beta mutation. The last three observations suggest that these other cell cycle control genes (or alternative gene products) are either not involved at all, or to any great extent, in melanoma predisposition.

Towards local cultures of sustainability : facilitating community created environment education centres through design

Ecological sustainability has been proposed to address the problem of human impacts increasingly degrading planetary resources and ecosystems, threatening biodiversity, eco-services and human survival. Ecological sustainability is an imperative, with Australia having one of the highest eco-footprints per person worldwide. While significant progress has been made via implementation of ecologically sustainable design in urban communities, relatively little has been undertaken in small, disparate regional communities in Australia. Regional communities are disadvantaged by rural economic decline associated with structural change and inequities of resource transfer. The ecologically sustainable solution is holistic, so all settlements need to be globally wise, richly biodiverse yet locally specific. As a regional solution to this global problem, this research offers the practical means by which a small regional community can contribute. It focuses on the design and implementation of a community centre and the fostering of transformative community learning through an integrated ‘learning community’ awareness of ecologically sustainable best practice. Lessons learned are documented by the participant researcher who as a designer, facilitator, local resident and social narrator has been deeply connected with the Tweed-Caldera region over a period since 1980. The collective action of the local community of Chillingham has been diligently recorded over a decade of design and development. Over this period, several positive elements emerged in terms of improvements to the natural and built environment, greater social cohesion and co-operative learning along with a shift towards a greener local economy. Behavioural changes in the community were noted as residents strived to embrace ecological ideals and reduce fossil fuel dependency. They found attractive local solutions to sourcing of food and using local employment opportunities to up skill their residents via transformative learning as a community in transition. Finally, the catalytic impact of external partnering has also been documented. How well the region as a whole has achieved its ecologically sustainable objectives is measured in terms of the delivered success of private and public partnering with the community, the creation of a community centre cum environment education centre, the restoration of local heritage buildings, the repair of riparian forests and improved water conditions in local river systems, better roads and road safety, local skills and knowledge transfer, support of local food and local/regional growers markets to attract tourists via the integrated trails network. In aggregate, each and every element contributes to a measure of eco-positive development for the built environment, its social organisation and its economy that has guided the local community to find its own pathway to sustainability. Within the Tweed-Caldera bioregion in northern New South Wales, there has been a lack of strategic planning, ecologically sustainable knowledge and facilities in isolated communities that could support the development of a local sustained green economy, provide a hub for socio-cultural activities and ecology based education. The first challenge in this research was to model a whole systems approach to eco-positive development in Chillingham, NSW, a small community where Nature and humanity know no specific boundary. The net result was the creation of a community environment education centre featuring best-affordable ecological practice and regionally distinctive, educational building form from a disused heritage building (cow bale). This development, implemented over a decade, resonated with the later regional wide programs that were linked in the Caldera region by the common purpose of extending the reach of local and state government assistance to regional NSW in economic transition coupled with sustainability. The lessons learned from these linked projects reveal that subsequent programs have been significantly easier to initiate, manage, develop and deliver results. In particular, pursuing collaborative networks with all levels of government and external private partners has been economically effective. Each community’s uniqueness has been celebrated and through drawing out these distinctions, has highlighted local vision, strategic planning, sense of belonging and connection of people with place. This step has significantly reduced the level of friction between communities that comes from natural competition for the finite pool of funds. Following the pilot Tweed-Caldera study, several other NSW regional communities are now undertaking a Community Economic Transition Program based on the processes, trials and positive experiences witnessed in the Tweed-Caldera region where it has been demonstrated that regional community transition programs can provide an opportunity to plan and implement effective long term strategies for sustainability, empowering communities to participate in eco-governance. This thesis includes the design and development of a framework for community created environment education centres to provide an equal access place for community to participate to meet their essential needs locally. An environment centre that facilitates community transition based on easily accessible environmental education, skills and infrastructure is necessary to develop local cultures of sustainability. This research draws upon the literatures of ecologically sustainable development, environmental education and community development in the context of regional community transition towards ‘strong sustainability’. The research approach adapted is best described as a four stage collaborative action research cycle where the participant researcher (me) has a significant involvement in the process to foster local cultures of sustainability by empowering its citizens to act locally and in doing so, become more self reliant and socially resilient. This research also draws upon the many fine working exemplars, such as the resilience of the Cuban people, the transition town initiative in Totnes, U.K. and the models of Australian Community Gardens, such as CERES (Melbourne) and Northey Street (Brisbane). The objectives of this study are to research and evaluate exemplars of ecologically sustainable environment education centres, to facilitate the design and development of an environment education centre created by a small regional community as an ecologically sustainable learning environment; to facilitate a framework for community transition based on environmental education, skills and infrastructure necessary to develop local cultures of sustainability. The research was undertaken as action research in the Tweed Caldera in Northern NSW. This involved the author as participant researcher, designer and volunteer in two interconnected initiatives: the Chillingham Community Centre development and the Caldera Economic Transition Program (CETP). Both initiatives involved a series of design-led participatory community workshops that were externally facilitated with the support of government agency partnerships, steering committees and local volunteers. Together the Caldera research programs involved communities participating in developing their own strategic planning process and outcomes. The Chillingham Community Centre was developed as a sustainable community centre/hub using a participatory design process. The Caldera Economic Transition Program (CETP) prioritised Caldera region projects: the Caldera farmer’s market; community gardens and community kitchens; community renewable energy systems and an integrated trails network. The significant findings were: the CETP projects were capable of moving towards an eco-positive design benchmark through transformative learning. Community transition to sustainability programs need to be underpinned by sustainability and environmental education based frameworks and practical on ground experience in local needs based projects through transformative learning. The actioned projects were successfully undertaken through community participation and teamwork. Ecological footprint surveys were undertaken to guide and assess the ongoing community transition process, however the paucity of responses needs to be revisited. The concept of ecologically sustainable development has been adopted internationally, however existing design and planning strategies do not assure future generations continued access to healthy natural life support systems. Sustainable design research has usually been urban focussed, with little attention paid to regional communities. This study seeks to redress this paucity through the design of ecologically sustainable (deep green) learning environments for small regional communities. Through a design-led process of environmental education, this study investigates how regional communities can be facilitated to model the principles of eco-positive development to support transition to local cultures of sustainability. This research shows how community transition processes and projects can incorporate sustainable community development as transformative learning through design. Regional community transition programs can provide an opportunity to plan long term strategies for sustainability, empowering people to participate in eco-governance. A framework is developed for a community created environment education centre to provide an equal access place for the local community to participate in implementing ways to meet their essential needs locally. A community environment education centre that facilitates community transition based on holistic environmental education, skills and infrastructure is necessary to develop local cultures of sustainability.

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.

Verifying a Student Engagement, Success and Retention Maturity Model (SESR-MM): A reality check of the model using data derived from three universities

Australian higher education institutions (HEIs) have entered a new phase of regulation and accreditation which includes performance-based funding relating to the participation and retention of students from social and cultural groups previously underrepresented in higher education. However, in addressing these priorities, it is critical that HEIs do not further disadvantage students from certain groups by identifying them for attention because of their social or cultural backgrounds, circumstances which are largely beyond the control of students. In response, many HEIs are focusing effort on university-wide approaches to enhancing the student experience because such approaches will enhance the engagement, success and retention of all students, and in doing so, particularly benefit those students who come from underrepresented groups. Measuring and benchmarking student experiences and engagement that arise from these efforts is well supported by extensive collections of student experience survey data. However no comparable instrument exists that measures the capability of institutions to influence and/or enhance student experiences where capability is an indication of how well an organisational process does what it is designed to do (Rosemann & de Bruin, 2005). We have proposed that the concept of a maturity model (Marshall, 2010; Paulk, 1999) may be useful as a way of assessing the capability of HEIs to provide and implement student engagement, success and retention activities and we are currently articulating a Student Engagement, Success and Retention Maturity Model (SESR-MM), (Clarke, Nelson & Stoodley, 2012; Nelson, Clarke & Stoodley, 2012). Our research aims to address the current gap by facilitating the development of an SESR-MM instrument that aims (i) to enable institutions to assess the capability of their current student engagement and retention programs and strategies to influence and respond to student experiences within the institution; and (ii) to provide institutions with the opportunity to understand various practices across the sector with a view to further improving programs and practices relevant to their context. Our research extends the generational approach which has been useful in considering the evolutionary nature of the first year experience (FYE) (Wilson, 2009). Three generations have been identified and explored: First generation approaches that focus on co-curricular strategies (e.g. orientation and peer programs); Second generation approaches that focus on curriculum (e.g. pedagogy, curriculum design, and learning and teaching practice); and third generation approaches—also referred to as transition pedagogy—that focus on the production of an institution-wide integrated holistic intentional blend of curricular and co-curricular activities (Kift, Nelson & Clarke, 2010). Our research also moves beyond assessments of students’ experiences to focus on assessing institutional processes and their capability to influence student engagement. In essence, we propose to develop and use the maturity model concept to produce an instrument that will indicate the capability of HEIs to manage and improve student engagement, success and retention programs and strategies. The issues explored in this workshop are (i) whether the maturity model concept can be usefully applied to provide a measure of institutional capability for SESR; (ii) whether the SESR-MM can be used to assess the maturity of a particular set of institutional practices; and (iii) whether a collective assessment of an institution’s SESR capabilities can provide an indication of the maturity of the institution’s SESR activities. The workshop will be approached in three stages. Firstly, participants will be introduced to the key characteristics of maturity models, followed by a discussion of the SESR-MM and the processes involved in its development. Secondly, participants will be provided with resources to facilitate the development of a maturity model and an assessment instrument for a range of institutional processes and related practices. In the final stage of the workshop, participants will “assess” the capability of these practices to provide a collective assessment of the maturity of these processes. References Australian Council for Educational Research. (n.d.). Australasian Survey of Student Engagement. Retrieved from http://www.acer.edu.au/research/ausse/background Clarke, J., Nelson, K., & Stoodley, I. (2012, July). The Maturity Model concept as framework for assessing the capability of higher education institutions to address student engagement, success and retention: New horizon or false dawn? A Nuts & Bolts presentation at the 15th International Conference on the First Year in Higher Education, “New Horizons,” Brisbane, Australia. Department of Education, Employment and Workplace Relations. (n.d.). The University Experience Survey. Advancing quality in higher education information sheet. Retrieved from http://www.deewr.gov.au/HigherEducation/Policy/Documents/University_Experience_Survey.pdf Kift, S., Nelson, K., & Clarke, J. (2010) Transition pedagogy - a third generation approach to FYE: A case study of policy and practice for the higher education sector. The International Journal of the First Year in Higher Education, 1(1), pp. 1-20. Marshall, S. (2010). A quality framework for continuous improvement of e-Learning: The e-Learning Maturity Model. Journal of Distance Education, 24(1), 143-166. Nelson, K., Clarke, J., & Stoodley, I. (2012). An exploration of the Maturity Model concept as a vehicle for higher education institutions to assess their capability to address student engagement. A work in progress. Submitted for publication. Paulk, M. (1999). Using the Software CMM with good judgment, ASQ Software Quality Professional, 1(3), 19-29. Wilson, K. (2009, June–July). The impact of institutional, programmatic and personal interventions on an effective and sustainable first-year student experience. Keynote address presented at the 12th Pacific Rim First Year in Higher Education Conference, “Preparing for Tomorrow Today: The First Year as Foundation,” Townsville, Australia. Retrieved from http://www.fyhe.com.au/past_papers/papers09/ppts/Keithia_Wilson_paper.pdf

Circulating tumour cells, their role in metastasis and their clinical utility in lung cancer

Circulating tumour cells (CTCs) have attracted much recent interest in cancer research as a potential biomarker and as a means of studying the process of metastasis. It has long been understood that metastasis is a hallmark of malignancy, and conceptual theories on the basis of metastasis from the nineteenth century foretold the existence of a tumour "seed" which is capable of establishing discrete tumours in the "soil" of distant organs. This prescient "seed and soil" hypothesis accurately predicted the existence of CTCs; microscopic tumour fragments in the blood, at least some of which are capable of forming metastases. However, it is only in recent years that reliable, reproducible methods of CTC detection and analysis have been developed. To date, the majority of studies have employed the CellSearch™ system (Veridex LLC), which is an immunomagnetic purification method. Other promising techniques include microfluidic filters, isolation of tumour cells by size using microporous polycarbonate filters and flow cytometry-based approaches. While many challenges still exist, the detection of CTCs in blood is becoming increasingly feasible, giving rise to some tantalizing questions about the use of CTCs as a potential biomarker. CTC enumeration has been used to guide prognosis in patients with metastatic disease, and to act as a surrogate marker for disease response during therapy. Other possible uses for CTC detection include prognostication in early stage patients, identifying patients requiring adjuvant therapy, or in surveillance, for the detection of relapsing disease. Another exciting possible use for CTC detection assays is the molecular and genetic characterization of CTCs to act as a "liquid biopsy" representative of the primary tumour. Indeed it has already been demonstrated that it is possible to detect HER2, KRAS and EGFR mutation status in breast, colon and lung cancer CTCs respectively. In the course of this review, we shall discuss the biology of CTCs and their role in metastagenesis, the most commonly used techniques for their detection and the evidence to date of their clinical utility, with particular reference to lung cancer.

Studios for the masses : can student colaboration replace the master-apprentice relationship in design instruction?

This action research project investigated the use of a collaborative learning approach for addressing issues associated with teaching urban design to large, diverse cohorts. As a case study, I observed two semesters of an urban design unit that I revised between 2011 and 2012 to incorporate collaborative learning activities. Data include instructional materials, participant observations, peer-reviews of collaborative learning activities, feedback from students and instructors and student projects. Themes that emerged through qualitative analysis include the challenge of removing inequalities inherent in the diverse cohort, the challenge of unifying project guidance and marking criteria, and the challenge of providing project guidance for a very large cohort. Most notably, the study revealed a need to clarify learning objectives relating to design principles in order to fully transition to and benefit from a collaborative learning model.

Differential gene expression in breast cancer cell lines and stroma-tumor differences in microdissected breast cancer biopsies revealed by display array analysis

To examine gene-expression patterning in late-stage breast cancer biopsies, we used a microdissection technique to separate tumor from the surrounding breast tissue or stroma. A DD-PCR protocol was then used to amplify expressed products, which were resolved using PAGE and used as probe to hybridize with representative human arrays and cDNA libraries. The probe derived from the tumor–stroma comparison was hybridized with a gene array and an arrayed cDNA library derived from a GCT of bone; 21 known genes or expressed sequence tags were detected, of which 17 showed differential expression. These included factors associated with epithelial to mesenchymal transition (vimentin), the cargo selection protein (TIP47) and the signal transducer and activator of transcription (STAT3). Northern blot analysis was used to confirm those genes also expressed by representative breast cancer cell lines. Notably, 6 genes of unknown function were restricted to tumor while the majority of stroma-associated genes were known. When applied to transformed breast cancer cell lines (MDA-MB-435 and T47D) that are known to have different metastatic potential, DD array analysis revealed a further 20 genes; 17 of these genes showed differential expression. Use of microdissection and the DD-PCR array protocol allowed us to identify factors whose localized expression within the breast may play a role in abnormal breast development or breast carcinogenesis.

School industry partnerships : An innovative strategy for vocational education

One of the challenges confronting contemporary education internationally is to ensure that students are provided with opportunities to make informed choices about future careers and to acquire the capacity to transition into these careers. Schools need to manage their curricula, teacher capacity, timetables, and diversity of student populations by offering pathways that are seen as engaging and meaningful to life beyond schooling. Traditionally, education in the senior years has privileged those students who intend to progress to advanced studies at university or in other professional careers. In more recent times, in response the need for more sophisticated technical knowledge in the trades and a growing skills shortages in these fields, schools have paid more attention to vocational education. It has been argued that the vocational aspect of the school curriculum is less well understood and poorly implemented in comparison with the traditional academic curricula. One attempt to address this issue is through the establishment of school-industry partnerships. This paper explores the process of knowledge transfer between industry and schools in these partnerships. The paper theorises how knowledge that is valued and foundational in workplace employment can inform school curricula and pedagogical practices. The paper draws on theories of organisational knowledge, workplace learning and experiential learning to explore strategies that enhance school-to-employment transition outcomes.

Considering the economic value of natural design elements at city scale

With increasing signs of climate change and the influence of national and international carbon-related laws and agreements, governments all over the world are grappling with how to rapidly transition to low-carbon living. This includes adapting to the impacts of climate change that are very likely to be experienced due to current emission levels (including extreme weather and sea level changes), and mitigating against further growth in greenhouse gas emissions that are likely to result in further impacts. Internationally, the concept of ‘Biophilic Urbanism’, a term coined by Professors Tim Beatley and Peter Newman to refer to the use of natural elements as design features in urban landscapes, is emerging as a key component in addressing such climate change challenges in rapidly growing urban contexts. However, the economics of incorporating such options is not well understood and requires further attention to underpin a mainstreaming of biophilic urbanism. Indeed, there appears to be an ad hoc, reactionary approach to creating economic arguments for or against the design, installation or maintenance of natural elements such as green walls, green roofs, streetscapes, and parklands. With this issue in mind, this paper will overview research as part of an industry collaborative research project that considers the potential for using a number of environmental economic valuation techniques that have evolved over the last several decades in agricultural and resource economics, to systematically value the economic value of biophilic elements in the urban context. Considering existing literature on environmental economic valuation techniques, the paper highlights opportunities for creating a standardised language for valuing biophilic elements. The conclusions have implications for expanding the field of environmental economic value to support the economic evaluations and planning of the greater use of natural elements in cities. Insights are also noted for the more mature fields of agricultural and resource economics.

Identification of Mendel's white flower character

Background The genetic regulation of flower color has been widely studied, notably as a character used by Mendel and his predecessors in the study of inheritance in pea. Methodology/Principal Findings We used the genome sequence of model legumes, together with their known synteny to the pea genome to identify candidate genes for the A and A2 loci in pea. We then used a combination of genetic mapping, fast neutron mutant analysis, allelic diversity, transcript quantification and transient expression complementation studies to confirm the identity of the candidates. Conclusions/Significance We have identified the pea genes A and A2. A is the factor determining anthocyanin pigmentation in pea that was used by Gregor Mendel 150 years ago in his study of inheritance. The A gene encodes a bHLH transcription factor. The white flowered mutant allele most likely used by Mendel is a simple G to A transition in a splice donor site that leads to a mis-spliced mRNA with a premature stop codon, and we have identified a second rare mutant allele. The A2 gene encodes a WD40 protein that is part of an evolutionarily conserved regulatory complex.

Staurosporine augments EGF-mediated EMT in PMC42-LA cells through actin depolymerisation, focal contact size reduction and Snail1 induction : a model for cross-modulation

Background A feature of epithelial to mesenchymal transition (EMT) relevant to tumour dissemination is the reorganization of actin cytoskeleton/focal contacts, influencing cellular ECM adherence and motility. This is coupled with the transcriptional repression of E-cadherin, often mediated by Snail1, Snail2 and Zeb1/δEF1. These genes, overexpressed in breast carcinomas, are known targets of growth factor-initiated pathways, however it is less clear how alterations in ECM attachment cross-modulate to regulate these pathways. EGF induces EMT in the breast cancer cell line PMC42-LA and the kinase inhibitor staurosporine (ST) induces EMT in embryonic neural epithelial cells, with F-actin de-bundling and disruption of cell-cell adhesion, via inhibition of aPKC. Methods PMC42-LA cells were treated for 72 h with 10 ng/ml EGF, 40 nM ST, or both, and assessed for expression of E-cadherin repressor genes (Snail1, Snail2, Zeb1/δEF1) and EMT-related genes by QRT-PCR, multiplex tandem PCR (MT-PCR) and immunofluorescence +/- cycloheximide. Actin and focal contacts (paxillin) were visualized by confocal microscopy. A public database of human breast cancers was assessed for expression of Snail1 and Snail2 in relation to outcome. Results When PMC42-LA were treated with EGF, Snail2 was the principal E-cadherin repressor induced. With ST or ST+EGF this shifted to Snail1, with more extreme EMT and Zeb1/δEF1 induction seen with ST+EGF. ST reduced stress fibres and focal contact size rapidly and independently of gene transcription. Gene expression analysis by MT-PCR indicated that ST repressed many genes which were induced by EGF (EGFR, CAV1, CTGF, CYR61, CD44, S100A4) and induced genes which alter the actin cytoskeleton (NLF1, NLF2, EPHB4). Examination of the public database of breast cancers revealed tumours exhibiting higher Snail1 expression have an increased risk of disease-recurrence. This was not seen for Snail2, and Zeb1/δEF1 showed a reverse correlation with lower expression values being predictive of increased risk. Conclusion ST in combination with EGF directed a greater EMT via actin depolymerisation and focal contact size reduction, resulting in a loosening of cell-ECM attachment along with Snail1-Zeb1/δEF1 induction. This appeared fundamentally different to the EGF-induced EMT, highlighting the multiple pathways which can regulate EMT. Our findings add support for a functional role for Snail1 in invasive breast cancer.

Molecular and cellular analysis of basement membrane invasion by human breast cancer cells in Matrigel-based in vitro assays

In vitro analyses of basement membrane invasiveness employing Matrigel (a murine tumor extract rich in basement membrane components) have been performed on human breast cancer model systems. Constitutive invasiveness of different human breast cancer (HBC) cell lines has been examined as well as regulation by steroid hormones, growth factors, and oncogenes. Carcinoma cells exhibiting a mesenchymal-like phenotype (vimentin expression, lack of cell border associated uvomorulin) show dramatically increased motility, invasiveness, and metastatic potential in nude mice. These findings support the hypothesis that epithelial to mesenchymal transition (EMT)-like events may be instrumental in the metastatic progression of human breast cancer. The MCF-7 subline MCF-7ADR appears to have undergone such a transition. The importance of such a transition may be reflected in the emergence of vimentin expression as an indicator of poor prognosis in HBC. Matrix degradation and laminin recognition are highlighted as potential targets for antimetastatic therapy, and analyses of laminin attachment and the matrix metalloproteinase (MMP) family in HBC cell lines are summarized. Matrigel-based assays have proved useful in the study of the molecular mechanisms of basement membrane invasiveness, their regulation in HBC cells, and their potential as targets for antimetastatic therapy.