Remodeling of purinergic receptor-mediated Ca 2+ signaling as a consequence of EGF-induced epithelial-mesenchymal transition in breast cancer cells

Background The microenvironment plays a pivotal role in tumor cell proliferation, survival and migration. Invasive cancer cells face a new set of environmental challenges as they breach the basement membrane and colonize distant organs during the process of metastasis. Phenotypic switching, such as that which occurs during epithelial-mesenchymal transition (EMT), may be associated with a remodeling of cell surface receptors and thus altered responses to signals from the tumor microenvironment. Methodology/Principal Findings We assessed changes in intracellular Ca 2+ in cells loaded with Fluo-4 AM using a fluorometric imaging plate reader (FLIPR TETRA) and observed significant changes in the potency of ATP (EC 50 0.175 μM (-EGF) versus 1.731 μM (+EGF), P<0.05), and the nature of the ATP-induced Ca 2+ transient, corresponding with a 10-fold increase in the mesenchymal marker vimentin (P<0.05). We observed no change in the sensitivity to PAR2-mediated Ca 2+ signaling, indicating that these alterations are not simply a consequence of changes in global Ca 2+ homeostasis. To determine whether changes in ATP-mediated Ca 2+ signaling are preceded by alterations in the transcriptional profile of purinergic receptors, we analyzed the expression of a panel of P2X ionotropic and P2Y metabotropic purinergic receptors using real-time RT-PCR and found significant and specific alterations in the suite of ATP-activated purinergic receptors during EGF-induced EMT in breast cancer cells. Our studies are the first to show that P2X 5 ionotropic receptors are enriched in the mesenchymal phenotype and that silencing of P2X 5 leads to a significant reduction (25%, P<0.05) in EGF-induced vimentin protein expression. Conclusions The acquisition of a new suite of cell surface purinergic receptors is a feature of EGF-mediated EMT in MDA-MB-468 breast cancer cells. Such changes may impart advantageous phenotypic traits and represent a novel mechanism for the targeting of cancer metastasis.

The epithelial to mesenchymal transition and metastatic progression in carcinoma

Many data have emerged in support of the concept that the promotion of a migratory phenotype in epithelial cells is the result of an EMT, which leads to the loss of differentiation and to the acquisition of several mesenchymal features. Such an event is likely to occur during the metastatic conversion enabling the metastatic cell to invade the connective tissue and disseminate. Even though it cannot be excluded that some cancer cells might constitutively express a metastatic phenotype, the EMT implicated in the tumor progression process would rather be transient, induced in certain cells of the primary tumor by different factors, and reversed when the cells settle down in secondary organs (as schematically represented in Figure 1). However, it is clear that the phenotype that would emerge from the EMT occurring during tumor progression would also be modulated by the genetic background of the cells and must be to some extent different than the ones observed in normal physiological processes, and must also vary from one tumor to another.

Mesenchymal-epithelial transition (MET) as a mechanism for metastatic colonisation in breast cancer

As yet, there is no cure for metastatic breast cancer. Historically, considerable research effort has been concentrated on understanding the processes of metastasis, how a primary tumour locally invades and systemically disseminates using the phenotypic switching mechanism of epithelial to mesenchymal transition (EMT); however, much less is understood about how metastases are then formed. Breast cancer metastases often look (and may even function) as 'normal' breast tissue, a bizarre observation against the backdrop of the organ structure of the lung, liver, bone or brain. Mesenchymal to epithelial transition (MET), the opposite of EMT, has been proposed as a mechanism for establishment of the metastatic neoplasm, leading to questions such as: Can MET be clearly demonstrated in vivo? What factors cause this phenotypic switch within the cancer cell? Are these signals/factors derived from the metastatic site (soil) or expressed by the cancer cells themselves (seed)? How do the cancer cells then grow into a detectable secondary tumour and further disseminate? And finallyCan we design and develop therapies that may combat this dissemination switch? This review aims to address these important questions by evaluating long-standing paradigms and novel emerging concepts in the field of epithelial mesencyhmal plasticity.

Neutrophil gelatinase-associated lipocalin (NGAL) an early-screening biomarker for ovarian cancer : NGAL is associated with epidermal growth factor-induced epithelio-mesenchymal transition

The expression of neutrophil gelatinase-associated lipocalin (NGAL) has been shown to be upregulated in ovarian cancer cells. In this study, we report that the expression of immunoreactive NGAL (irNGAL) in ovarian tumors changes with disease grade and that this change is reflected in the concentration of NGAL in peripheral blood. A total of 59 ovarian tissues including normal, benign, borderline malignant and grades 1, 2 and 3 malignant were analyzed using immunohistochemistry. irNGAL was not present in normal ovaries and the NGAL expression was weak to moderate in benign tissues. Both borderline and grade 1 tumors displayed the highest amount of NGAL expression with moderate to strong staining, whereas in grade 2 and 3 tumors, the extent of staining was significantly less (p < 0.01) and staining intensity was weak to moderate. Staining in all cases was confined to the epithelium. NGAL expression was analyzed by ELISA in 62 serum specimens from normal and different grades of cancer patients. Compared to control samples, the NGAL concentration was 2 and 2.6-fold higher in the serum of patients with benign tumors and cancer patients with grade 1 tumors (p < 0.05) and that result was consistent with the expression of NGAL performed by Western blot. NGAL expression was evaluated by Western blot in an immortalized normal ovarian cell line (IOSE29) as well as ovarian cancer cell lines. Moderate to strong expression of NGAL was observed in epithelial ovarian cancer cell lines SKOV3 and OVCA433 while no expression of NGAL was evident in normal IOSE29 and mesenchyme-like OVHS1, PEO.36 and HEY cell lines. NGAL expression was downregulated in ovarian cancer cell lines undergoing epithelio-mesenchymal transition (EMT) induced by epidermal growth factor (EGF). Down-regulation of NGAL expression correlated with the upregulation of vimentin expression, enhanced cell dispersion and downregulation of E-cadherin expression, some of the hallmarks of EMT. EGF-induced EMT phenotypes were inhibited in the presence of AG1478, an inhibitor of EGF receptor tyrosine kinase activity. These data indicate that NGAL may be a good marker to monitor changes of benign to premalignant and malignant ovarian tumors and that the molecule may be involved in the progression of epithelial ovarian malignancies.

The epithelial-mesenchymal transition : new insights in signaling, development, and disease

The conversion of an epithelial cell to a mesenchymal cell is critical to metazoan embryogenesis and a de. ning structural feature of organ development. Current interest in this process, which is described as an epithelial- mesenchymal transition (EMT), stems from its developmental importance and its involvement in several adult pathologies. Interest and research in EMT are currently at a high level, as seen by the attendance at the recent EMT meeting in Vancouver, Canada (October 1-3, 2005). The meeting, which was hosted by The EMT International Association, was the second international EMT meeting, the . rst being held in Port Douglas, Queensland, Australia in October 2003. The EMT International Association was formed in 2002 to provide an international body for those interested in EMT and the reverse process, mesenchymal-epithelial transition, and, most importantly, to bring together those working on EMT in development, cancer, . brosis, and pathology. These themes continued during the recent meeting in Vancouver. Discussion at the Vancouver meeting spanned several areas of research, including signaling pathway activation of EMT and the transcription factors and gene targets involved. Also covered in detail was the basic cell biology of EMT and its role in cancer and . brosis, as well as the identi. cation of new markers to facilitate the observation of EMT in vivo. This is particularly important because the potential contribution of EMT during neoplasia is the subject of vigorous scientific debate (Tarin, D., E.W. Thompson, and D.F. Newgreen. 2005. Cancer Res. 65:5996-6000; Thompson, E.W., D.F. Newgreen, and D. Tarin. 2005. Cancer Res. 65:5991-5995).

Cadherins in the human placenta : epithelial-mesenchymal transition (EMT) and placental development

Colonisation of the maternal uterine wall by the trophoblast involves a series of alterations in the behaviour and morphology of trophoblast cells. Villous cytotrophoblast cells change from a well-organised coherently layered phenotype to one that is extravillous, acquiring a proliferative, migratory and invasive capacity, to facilitate fetal-maternal interaction. These changes are similar to those of other developmental processes falling under the umbrella of an epithelial-mesenchymal transition (EMT). Modulation of cell adhesion and cell polarity occurs through changes in cell-cell junctional molecules, such as the cadherins. The cadherins, particularly the classical cadherins (e.g. Epithelial-(E)-cadherin), and their link to adaptors called catenins at cell-cell contacts, are important for maintaining cell attachment and the layered phenotype of the villous cytotrophoblast. In contrast, reduced expression and re-organization of cadherins from these cell junctional regions promote a loosened connection between cells, coupled with reduced apico-basal polarity. Certain non-classical cadherins play an active role in cell migration processes. In addition to the classical cadherins, two other cadherins which have been reported in placental tissues are vascular endothelial (VE) cadherin and cadherin-11. Cadherin molecules are well placed to be key regulators of trophoblast cell behaviour, analogous to their role in other developmental EMTs. This review addresses cadherin expression and function in normal and diseased human placental tissues, especially in fetal growth restriction and pre-eclampsia where trophoblast invasion is reduced.

Epithelial mesenchymal transition traits in human breast cancer cell lines parallel the CD44HI/CD24lO/-stem cell phenotype in human breast cancer

We review here the recently emerging relationship between epithelial-mesenchymal transition (EMT) and breast cancer stem cells (BCSC), and provide analyses of published data on human breast cancer cell lines, supporting their utility as a model for the EMT/BCSC state. Genome-wide transcriptional profiling of these cell lines has confirmed the existence of a subgroup with mesenchymal tendencies and enhanced invasive properties ('Basal B'/Mesenchymal), distinct from subgroups with either predominantly luminal ('Luminal') or mixed basal/luminal ('Basal A') features (Neve et al. Cancer Cell, 2006). A literature-derived EMT gene signature has shown specific enrichment within the Basal B subgroup of cell lines, consistent with their over-expression of various EMT transcriptional drivers. Basal B cell lines are found to resemble BCSC, being CD44highCD24low. Moreover, gene products that distinguish Basal B from Basal A and Luminal cell lines (Basal B Discriminators) showed close concordance with those that define BCSC isolated from clinical material, as reported by Shipitsin et al. (Cancer Cell, 2007). CD24 mRNA levels varied across Basal B cell lines, correlating with other Basal B Discriminators. Many gene products correlating with CD24 status in Basal B cell lines were also differentially expressed in isolated BCSC. These findings confirm and extend the importance of the cellular product of the EMT with Basal B cell lines, and illustrate the value of analysing these cell lines for new leads that may improve breast cancer outcomes. Gene products specific to Basal B cell lines may serve as tools for the detection, quantification, and analysis of BCSC/EMT attributes.

Epidermal growth factor-induced epithelio-mesenchymal transition in human breast carcinoma cells

PMC42-LA cells display an epithelial phenotype: the cells congregate into pavement epithelial sheets in which E-cadherin and β-catenin are localized at cell-cell borders. They abundantly express cytokeratins, although 5% to 10% of the cells also express the mesenchymal marker vimentin. Stimulation of PMC42-LA cells with epidermal growth factor (EGF) leads to epithelio-mesenchymal transition-like changes including up-regulation of vimentin and down-regulation of E-cadherin. Vimentin expression is seen in virtually all cells, and this increase is abrogated by treatment of cells with an EGF receptor antagonist. The expression of the mesenchyme-associated extracellular matrix molecules fibronectin and chondroitin sulfate proteoglycan also increase in the presence of EGF. PMC42-LA cells adhere rapidly to collagen I, collagen IV, and laminin-1 substrates and markedly more slowly to fibronectin and vitronectin. EGF increases the speed of cell adhesion to most of these extracellular matrix molecules without altering the order of adhesive preference. EGF also caused a time-dependent increase in the motility of PMC42-LA cells, commensurate with the degree of vimentin staining. The increase in motility was at least partly chemokinetic, because it was evident both with and without chemoattractive stimuli. Although E-cadherin staining at cell-cell junctions disappeared in response to EGF, β-catenin persisted at the cell periphery. Further analysis revealed that N-cadherin was present at the cell-cell junctions of untreated cells and that expression was increased after EGF treatment. N- and E-cadherin are not usually coexpressed in human carcinoma cell lines but can be coexpressed in embryonic tissues, and this may signify an epithelial cell population prone to epithelio-mesenchymal-like responses.

A comparative analysis of the conservative bridge transition design approaches

Differential settlement at the bridge approach between the deck and rail track on ground is often considered as a source of challenging technical and economical problem. This caused by the sudden stiffness changes between the bridge deck and the track on ground, and changes in soil stiffness of backfill and sub-grade with soil moisture content and loading history. To minimise the negative social and economic impacts due to poor performances of railway tracks at bridge transition zones, it is important, a special attention to be given at design, construction and maintenance stages. It is critically challenging to obtain an appropriate design solution for any given site condition and most of the existing conventional design approaches are unable to address the actual on-site behaviour due to their inherent assumptions of continuity and lack of clarifying of the local effects. An evaluation of existing design techniques is considered to estimate their contributions to a potential solution for bridge transition zones. This paper analyses five different approaches: the Chinese Standard, the European Standard with three different approaches, and the Australian approach. Each design approach is used to calculate the layer thicknesses, accounting critical design features such as the train speed, the axle load, the backfill subgrade condition, and the dynamic loading response. Considering correlation between track degradation and design parameters, this paper concludes that there is still a need of an optimised design approach for bridge transition zones.

Conceptualising, evaluating and reporting social resilience in vulnerable regional and remote communities facing climate change in tropical Queensland : marine and tropical sciences research facility transition project final report

Regional and remote communities in tropical Queensland are among Australia’s most vulnerable in the face of climate change. At the same time, these socially and economically vulnerable regions house some of Australia’s most significant biodiversity values. Past approaches to terrestrial biodiversity management have focused on tackling biophysical interventions through the use of biophysical knowledge. An equally important focus should be placed on building regional-scale community resilience if some of the worst biodiversity impacts of climate change are to be avoided or mitigated. Despite its critical need, more systemic or holistic approaches to natural resource management have been rarely trialed and tested in a structured way. Currently, most strategic interventions in improving regional community resilience are ad hoc, not theory-based and short term. Past planning approaches have not been durable, nor have they been well informed by clear indicators. Research into indicators for community resilience has been poorly integrated within adaptive planning and management cycles. This project has aimed to resolve this problem by: * Reviewing the community and social resilience and adaptive planning literature to reconceptualise an improved framework for applying community resilience concepts; * Harvesting and extending work undertaken in MTSRF Phase 1 to identifying the learnings emerging from past MTSRF research; * Distilling these findings to identify new theoretical and practical approaches to the application of community resilience in natural resource use and management; * Reconsidering the potential interplay between a region’s biophysical and social planning processes, with a focus on exploring spatial tools to communicate climate change risk and its consequent environmental, economic and social impacts, and; * Trialling new approaches to indicator development and adaptive planning to improve community resilience, using a sub-regional pilot in the Wet Tropics. In doing so, we also looked at ways to improve the use and application of relevant spatial information. Our theoretical review drew upon the community development, psychology and emergency management literature to better frame the concept of community resilience relative to aligned concepts of social resilience, vulnerability and adaptive capacity. Firstly, we consider community resilience as a concept that can be considered at a range of scales (e.g. regional, locality, communities of interest, etc.). We also consider that overall resilience at higher scales will be influenced by resilience levels at lesser scales (inclusive of the resilience of constituent institutions, families and individuals). We illustrate that, at any scale, resilience and vulnerability are not necessarily polar opposites, and that some understanding of vulnerability is important in determining resilience. We position social resilience (a concept focused on the social characteristics of communities and individuals) as an important attribute of community resilience, but one that needs to be considered alongside economic, natural resource, capacity-based and governance attributes. The findings from the review of theory and MTSRF Phase 1 projects were synthesized and refined by the wider project team. Five predominant themes were distilled from this literature, research review and an expert analysis. They include the findings that: 1. Indicators have most value within an integrated and adaptive planning context, requiring an active co-research relationship between community resilience planners, managers and researchers if real change is to be secured; 2. Indicators of community resilience form the basis for planning for social assets and the resilience of social assets is directly related the longer term resilience of natural assets. This encourages and indeed requires the explicit development and integration of social planning within a broader natural resource planning and management framework; 3. Past indicator research and application has not provided a broad picture of the key attributes of community resilience and there have been many attempts to elicit lists of “perfect” indicators that may never be useful within the time and resource limitations of real world regional planning and management. We consider that modeling resilience for proactive planning and prediction purposes requires the consideration of simple but integrated clusters of attributes; 4. Depending on time and resources available for planning and management, the combined use of well suited indicators and/or other lesser “lines of evidence” is more flexible than the pursuit of perfect indicators, and that; 5. Index-based, collaborative and participatory approaches need to be applied to the development, refinement and reporting of indicators over longer time frames. We trialed the practical application of these concepts via the establishment of a collaborative regional alliance of planners and managers involved in the development of climate change adaptation strategies across tropical Queensland (the Gulf, Wet Tropics, Cape York and Torres Strait sub-regions). A focus on the Wet Tropics as a pilot sub-region enabled other Far North Queensland sub-region’s to participate and explore the potential extension of this approach. The pilot activities included: * Further exploring ways to innovatively communicate the region’s likely climate change scenarios and possible environmental, economic and social impacts. We particularly looked at using spatial tools to overlay climate change risks to geographic communities and social vulnerabilities within those communities; * Developing a cohesive first pass of a State of the Region-style approach to reporting community resilience, inclusive of regional economic viability, community vitality, capacitybased and governance attributes. This framework integrated a literature review, expert (academic and community) and alliance-based contributions; and * Early consideration of critical strategies that need to be included in unfolding regional planning activities with Far North Queensland. The pilot assessment finds that rural, indigenous and some urban populations in the Wet Tropics are highly vulnerable and sensitive to climate change and may require substantial support to adapt and become more resilient. This assessment finds that under current conditions (i.e. if significant adaptation actions are not taken) the Wet Tropics as a whole may be seriously impacted by the most significant features of climate change and extreme climatic events. Without early and substantive action, this could result in declining social and economic wellbeing and natural resource health. Of the four attributes we consider important to understanding community resilience, the Wet Tropics region is particularly vulnerable in two areas; specifically its economic vitality and knowledge, aspirations and capacity. The third and fourth attributes, community vitality and institutional governance are relatively resilient but are vulnerable in some key respects. In regard to all four of these attributes, however, there is some emerging capacity to manage the possible shocks that may be associated with the impacts of climate change and extreme climatic events. This capacity needs to be carefully fostered and further developed to achieve broader community resilience outcomes. There is an immediate need to build individual, household, community and sectoral resilience across all four attribute groups to enable populations and communities in the Wet Tropics region to adapt in the face of climate change. Preliminary strategies of importance to improve regional community resilience have been identified. These emerging strategies also have been integrated into the emerging Regional Development Australia Roadmap, and this will ensure that effective implementation will be progressed and coordinated. They will also inform emerging strategy development to secure implementation of the FNQ 2031 Regional Plan. Of most significance in our view, this project has taken a co-research approach from the outset with explicit and direct importance and influence within the region’s formal planning and management arrangements. As such, the research: * Now forms the foundations of the first attempt at “Social Asset” planning within the Wet Tropics Regional NRM Plan review; * Is assisting Local government at regional scale to consider aspects of climate change adaptation in emerging planning scheme/community planning processes; * Has partnered the State government (via the Department of Infrastructure and Planning and Regional Managers Coordination Network Chair) in progressing the Climate Change adaptation agenda set down within the FNQ 2031 Regional Plan; * Is informing new approaches to report on community resilience within the GBRMPA Outlook reporting framework; and * Now forms the foundation for the region’s wider climate change adaptation priorities in the Regional Roadmap developed by Regional Development Australia. Through the auspices of Regional Development Australia, the outcomes of the research will now inform emerging negotiations concerning a wider package of climate change adaptation priorities with State and Federal governments. Next stage research priorities are also being developed to enable an ongoing alliance between researchers and the region’s climate change response.

Treatment with the vascular disruptive agent OXi4503 induces an immediate and widespread epithelial to mesenchymal transition in the surviving tumor

Epithelial to mesenchymal transition (EMT) is considered an important mechanism in tumor resistance to drug treatments; however, in vivo observation of this process has been limited. In this study we demonstrated an immediate and widespread EMT involving all surviving tumor cells following treatment of a mouse model of colorectal liver metastases with the vascular disruptive agent OXi4503. EMT was characterized by significant downregulation of E-cadherin, relocation and nuclear accumulation of b-catenin as well as significant upregulation of ZEB1 and vimentin. Concomitantly, significant temporal upregulation in hypoxia and the pro-angiogenic growth factors hypoxia-inducible factor 1-alpha, hepatocyte growth factor, vascular endothelial growth factor and transforming growth factor-beta were seen within the surviving tumor. The process of EMT was transient and by 5 days after treatment tumor cell reversion to epithelial morphology was evident. This reversal, termed mesenchymal to epithelial transition (MET) is a process implicated in the development of new metastases but has not been observed in vivo histologically. Similar EMT changes were observed in response to other antitumor treatments including chemotherapy, thermal ablation, and antiangiogenic treatments in our mouse colorectal metastasis model and in a murine orthotopic breast cancer model after OXi4503 treatment. These results suggest that EMT may be an early mechanism adopted by tumors in response to injury and hypoxic stress, such that inhibition of EMT in combination with other therapies could play a significant role in future cancer therapy.

Electron-conduction mechanism and specific heat above transition temperature in LaFeAsO and BaFe2As2 superconductors

The ionization energy theory is used to calculate the evolution of the resistivity and specific heat curves with respect to different doping elements in the recently discovered superconducting pnictide materials. Electron-conduction mechanism in the pnictides above the structural transition temperature is explained unambiguously, which is also consistent with other strongly correlated materials, such as cuprates, manganites, titanates and magnetic semiconductors.

Effects of wall transition regions on surface-wave propagation in a plasma layer bounded by a metal

Effect of near-wall transition regions on the surface wave propagation in a magnetoactive plasma layer bounded by a metal. It is shown that the account for inhomogeneities of plasma density or magnetic field causes an appearance of coupling between surface waves, propagating across magnetic field and localized near difference boundaries of the structure. The resonance damping of surface waves is analyzed too.

Epithelial-mesenchymal transition and mesenchymal-epithelial transition are essential for the acquisition of stem cell properties in hTERT-immortalised oral epithelial cells

BACKGROUND INFORMATION: Evidence has shown that mesenchymal-epithelial transition (MET) and epithelial-mesenchymal transition (EMT) are linked to stem cell properties. We currently lack a model showing how the occurrence of MET and EMT in immortalised cells influences the maintenance of stem cell properties. Thus, we established a project aiming to investigate the roles of EMT and MET in the acquisition of stem cell properties in immortalised oral epithelial cells. RESULTS: In this study, a retroviral transfection vector (pLXSN-hTERT) was used to immortalise oral epithelial cells by insertion of the hTERT gene (hTERT(+)-oral mucosal epithelial cell line [OME]). The protein and RNA expression of EMT transcriptional factors (Snail, Slug and Twist), their downstream markers (E-cadherin and N-cadherin) and embryonic stem cell markers (OCT4, Nanog and Sox2) were studied by reverse transcription PCR and Western blots in these cells. Some EMT markers were detected at both mRNA and protein levels. Adipocytes and bone cells were noted in the multi-differentiation assay, showing that the immortal cells underwent EMT. The differentiation assay for hTERT(+)-OME cells revealed the recovery of epithelial phenotypes, implicating the presence of MET. The stem cell properties were confirmed by the detection of appropriate markers. Altered expression of alpha-tubulin and gamma-tubulin in both two-dimensional-cultured (without serum) and three-dimensional-cultured hTERT(+)-OME spheroids indicated the re-programming of cytoskeleton proteins which is attributed to MET processes in hTERT(+)-OME cells. CONCLUSIONS: EMT and MET are essential for hTERT-immortalised cells to maintain their epithelial stem cell properties.

Iron(II) spin transition complexes with dendritic ligands, Part I

The ligands G1- and G2-oligo (benzyl ether) (PBE) dendrons and their iron(II) complexes [Fe(Gn-PBE)3]A2·xH2O (with n = 1, 2 and A = triflate, tosylate) were prepared. The magnetic properties of the complexes were investigated by a SQUID magnetometer. All complexes exhibit gradual spin transition below room temperature. At very low temperatures the magnetic behaviour reflects zero-field splitting (ZFS) effects. 57Fe-Mössbauer spectroscopy was performed to distinguish between ZFS of high spin species and spin state conversion into the low spin state. Further characterisation was carried out by thermogravimetric analysis (TGA) and FT-IR spectroscopy. Structural features have been determined by powder XRD measurements.