Direct repression of MYB by ZEB1 suppresses proliferation and epithelial gene expression during epithelial-to-mesenchymal transition of breast cancer cells

Introduction Epithelial-to-mesenchymal transition (EMT) promotes cell migration and is important in metastasis. Cellular proliferation is often downregulated during EMT, and the reverse transition (MET) in metastases appears to be required for restoration of proliferation in secondary tumors. We studied the interplay between EMT and proliferation control by MYB in breast cancer cells. Methods MYB, ZEB1, and CDH1 expression levels were manipulated by lentiviral small-hairpin RNA (shRNA)-mediated knockdown/overexpression, and verified with Western blotting, immunocytochemistry, and qRT-PCR. Proliferation was assessed with bromodeoxyuridine pulse labeling and flow cytometry, and sulforhodamine B assays. EMT was induced with epidermal growth factor for 9 days or by exposure to hypoxia (1% oxygen) for up to 5 days, and assessed with qRT-PCR, cell morphology, and colony morphology. Protein expression in human breast cancers was assessed with immunohistochemistry. ZEB1-MYB promoter binding and repression were determined with Chromatin Immunoprecipitation Assay and a luciferase reporter assay, respectively. Student paired t tests, Mann–Whitney, and repeated measures two-way ANOVA tests determined statistical significance (P < 0.05). Results Parental PMC42-ET cells displayed higher expression of ZEB1 and lower expression of MYB than did the PMC42-LA epithelial variant. Knockdown of ZEB1 in PMC42-ET and MDA-MB-231 cells caused increased expression of MYB and a transition to a more epithelial phenotype, which in PMC42-ET cells was coupled with increased proliferation. Indeed, we observed an inverse relation between MYB and ZEB1 expression in two in vitro EMT cell models, in matched human breast tumors and lymph node metastases, and in human breast cancer cell lines. Knockdown of MYB in PMC42-LA cells (MYBsh-LA) led to morphologic changes and protein expression consistent with an EMT. ZEB1 expression was raised in MYBsh-LA cells and significantly repressed in MYB-overexpressing MDA-MB-231 cells, which also showed reduced random migration and a shift from mesenchymal to epithelial colony morphology in two dimensional monolayer cultures. Finally, we detected binding of ZEB1 to MYB promoter in PMC42-ET cells, and ZEB1 overexpression repressed MYB promoter activity. Conclusions This work identifies ZEB1 as a transcriptional repressor of MYB and suggests a reciprocal MYB-ZEB1 repressive relation, providing a mechanism through which proliferation and the epithelial phenotype may be coordinately modulated in breast cancer cells.

Negative affectivity and responses to work stressors : an experimental study

An experiment tested the hypothesis that individuals high in negative affectivity (NA) show increased stress reactivity to stressors. There were three predictor variables: NA (measured 1 week prior to experimental participation), and two manipulated variables—demand (high/low) and behavioral control (high/low). First-year psychology students (n=256) were randomly allocated to one of the four experimental conditions. Measures obtained were initial and post-task negative mood, coping strategies, task satisfaction, and performance (subjective and objective). Participants with high levels of NA reported more post-task negative mood in response to high demand conditions, compared to participants with low NA. A similar pattern of results emerged for task satisfaction, particularly in response to high demand-low behavioral control situations. Mediation analyses suggested this was because participants with high NA used more emotion-focused coping strategies. The study provides support for the stress reactivity role of NA in the stressor-strain process.

Induction of epithelial to mesenchymal transition in PMC42-LA human breast carcinoma cells by carcinoma-associated fibroblast secreted factors

Background Breast carcinoma is accompanied by changes in the acellular and cellular components of the microenvironment, the latter typified by a switch from fibroblasts to myofibroblasts. Methods: We utilised conditioned media cultures, Western blot analysis and immunocytochemistry to investigate the differential effects of normal mammary fibroblasts (NMFs) and mammary cancer-associated fibroblasts (CAFs) on the phenotype and behaviour of PMC42-LA breast cancer cells. NMFs were obtained from a mammary gland at reduction mammoplasty, and CAFs from a mammary carcinoma after resection. Results We found greater expression of myofibroblastic markers in CAFs than in NMFs. Medium from both CAFs and NMFs induced novel expression of α-smooth muscle actin and cytokeratin-14 in PMC42-LA organoids. However, although conditioned media from NMFs resulted in distribution of vimentin-positive cells to the periphery of PMC42-LA organoids, this was not seen with CAF-conditioned medium. Upregulation of vimentin was accompanied by a mis-localization of E-cadherin, suggesting a loss of adhesive function. This was confirmed by visualizing the change in active β-catenin, localized to the cell junctions in control cells/ cells in NMF-conditioned medium, to inactive β-catenin, localized to nuclei and cytoplasm in cells in CAF-conditioned medium. Conclusion We found no significant difference between the influences of NMFs and CAFs on PMC42-LA cell proliferation, viability, or apoptosis; significantly, we demonstrated a role for CAFs, but not for NMFs, in increasing the migratory ability of PMC42-LA cells. By concentrating NMF-conditioned media, we demonstrated the presence of factor(s) that induce epithelial-mesenchymal transition in NMF-conditioned media that are present at higher levels in CAF-conditioned media. Our in vitro results are consistent with observations in vivo showing that alterations in stroma influence the phenotype and behaviour of surrounding cells and provide evidence for a role for CAFs in stimulating cancer progression via an epithelial-mesenchymal transition. These findings have implications for our understanding of the roles of signalling between epithelial and stromal cells in the development and progression of mammary carcinoma.

Whole-genome multiparametric screening to identify modulators of epithelial-to-mesenchymal transition

Metastasis accounts for the poor prognosis of the majority of solid tumors. The phenotypic transition of nonmotile epithelial tumor cells to migratory and invasive “mesenchymal” cells (epithelial-to-mesenchymal transition [EMT]) enables the transit of cancer cells from the primary tumor to distant sites. There is no single marker of EMT; rather, multiple measures are required to define cell state. Thus, the multiparametric capability of high-content screening is ideally suited for the comprehensive analysis of EMT regulators. The aim of this study was to generate a platform to systematically identify functional modulators of tumor cell plasticity using the bladder cancer cell line TSU-Pr1-B1 as a model system. A platform enabling the quantification of key EMT characteristics, cell morphology and mesenchymal intermediate filament vimentin, was developed using the fluorescent whole-cell-tracking reagent CMFDA and a fluorescent promoter reporter construct, respectively. The functional effect of genome-wide modulation of protein-coding genes and miRNAs coupled with those of a collection of small-molecule kinase inhibitors on EMT was assessed using the Target Activation Bioapplication integrated in the Cellomics ArrayScan platform. Data from each of the three screens were integrated to identify a cohort of targets that were subsequently examined in a validation assay using siRNA duplexes. Identification of established regulators of EMT supports the utility of this screening approach and indicated capacity to identify novel regulators of this plasticity program. Pathway analysis coupled with interrogation of cancer-related expression profile databases and other EMT-related screens provided key evidence to prioritize further experimental investigation into the molecular regulators of EMT in cancer cells.

Industry-school partnerships: An ecological case study to understand operational dynamics

This thesis is an ecological systems case study of an industry-school partnership. It examines a minerals and energy sector partnership with Queensland schools and explains the operational dynamics. In doing so, an original contribution to theory and practice was presented, together with implications for the impact of industry on education.

Using a free online questionnaire to determine the skills, competencies and knowledge required to work in a digital library environment in Australia

This paper discusses the data collection technique used to determine the skills and knowledge required of academic librarians working in a digital library environment in Australia. The research was undertaken as part of the researcher’s master’s thesis conducted at Tallinn University. The data collection instrument used was a freely available online survey tool, and its advantages and disadvantages are discussed in terms of the desired outcomes and circumstances surrounding the thesis project. Decisions regarding the design of the questionnaire are also discussed.

Association of sporadic chondrocalcinosis with a -4-basepair G-to-A transition in the 5′-untranslated region of ANKH that promotes enhanced expression of ANKH protein and excess generation of extracellular inorganic pyrophosphate

Objective Certain mutations in ANKH, which encodes a multiple-pass transmembrane protein that regulates inorganic pyrophosphate (PPi) transport, are linked to autosomal-dominant familial chondrocalcinosis. This study investigated the potential for ANKH sequence variants to promote sporadic chondrocalcinosis. Methods ANKH variants identified by genomic sequencing were screened for association with chondrocalcinosis in 128 patients with severe sporadic chondrocalcinosis or pseudogout and in ethnically matched healthy controls. The effects of specific variants on expression of common markers were evaluated by in vitro transcription/translation. The function of these variants was studied in transfected human immortalized CH-8 articular chondrocytes. Results Sporadic chondrocalcinosis was associated with a G-to-A transition in the ANKH 5′-untranslated region (5′-UTR) at 4 bp upstream of the start codon (in homozygotes of the minor allele, genotype relative risk 6.0, P = 0.0006; overall genotype association P = 0.02). This -4-bp transition, as well as 2 mutations previously linked with familial and sporadic chondrocalcinosis (+14 bp C-to-T and C-terminal GAG deletion, respectively), but not the French familial chondrocalcinosis kindred 143-bp T-to-C mutation, increased reticulocyte ANKH transcription/ANKH translation in vitro. Transfection of complementary DNA for both the wild-type ANKH and the -4-bp ANKH protein variant promoted increased extracellular PPi in CH-8 cells, but unexpectedly, these ANKH mutants had divergent effects on the expression of extracellular PPi and the chondrocyte hypertrophy marker, type X collagen. Conclusion A subset of sporadic chondrocalcinosis appears to be heritable via a -4-bp G-to-A ANKH 5′-UTR transition that up-regulates expression of ANKH and extracellular PPi in chondrocyte cells. Distinct ANKH mutations associated with heritable chondrocalcinosis may promote disease by divergent effects on extracellular PPi and chondrocyte hypertrophy, which is likely to mediate differences in the clinical phenotypes and severity of the disease.

MicroRNA regulation of epithelial-to-mesenchymal transition during re-epithelialisation: Assessing an open wound

It is becoming increasing clear that microRNAs contribute to the regulation of many biological processes, including wound healing. After injury, keratinocytes need to undergo what is known as an epithelial-to-mesenchymal transition (EMT) to initiate re-epithelialisation. During this process, keratinocytes reduce their attachment to the underlying matrix, extend membrane protrusions, become motile and migrate over the wound bed, affecting wound closure. MicroRNAs that regulate EMT are aberrantly upregulated in keratinocytes at the edge of non-healing wounds and potentially play a role in the chronicity of these wounds. In vitro and in vivo, downregulation of these microRNAs promotes EMT and migration, facilitating re-epithelialisation in wound models. This review will focus on the role of microRNAs that regulate or have potential to regulate EMT and re-epithelialisation during wound healing

“We have to work within the system!”: Staff perceptions of organizational barriers to decision making for older adults with dementia in Australian aged care facilities

The current study explored the perceptions of direct care staff working in Australian residential aged care facilities (RACFs) regarding the organizational barriers that they believe prevent them from facilitating decision making for individuals with dementia. Normalization process theory (NPT) was used to interpret the findings to understand these barriers in a broader context. The qualitative study involved semi-structured interviews (N = 41) and focus groups (N = 8) with 80 direct care staff members of all levels working in Australian RACFs. Data collection and analysis were conducted in parallel and followed a systematic, inductive approach in line with grounded theory. The perceptions of participants regarding the organizational barriers to facilitating decision making for individuals with dementia can be described by the core category, Working Within the System, and three sub-themes: (a) finding time, (b) competing rights, and (c)not knowing. Examining the views of direct care staff through the lens of NPT allows possible areas for improvement to be identified at an organizational level and the perceived barriers to be understood in the context of promoting normalization of decision making for individuals with dementia.

A systematic review of studies identifying predictors of poor return to work outcomes following workplace injury

BACKGROUND: Injuries occurring in the workplace can have serious implications for the health of the individual, the productivity of the employer and the overall economic community. OBJECTIVE: The objective of this paper is to increase the current state of understanding of individual demographic and psychosocial characteristics associated with extended absenteeism from the workforce due to a workplace injury. METHODS: Studies included in this systematic literature review tracked participants' return to work status over a minimum of three months, identified either demographic, psychosocial or general injury predictors of poor return to work outcomes and included a heterogeneous sample of workplace injuries. RESULTS: Identified predictors of poor return to work outcomes included older age, female gender, divorced marital status, two or more dependent family members, lower education levels, employment variables associated with reduced labour market desirability, severity or sensitive injury locations, negative attitudes and outcome perceptions of the participant. CONCLUSIONS: There is a need for clear and consistent definition and measurement of return to work outcomes and a holistic theoretical model integrating injury, psychosocial and demographic predictors of return to work. Through greater understanding of the nature of factors affecting return to work, improved outcomes could be achieved.

Seamless journeys : enhancing accessibility to work through digital technologies for people with mobility-related impairments

Numerous disruptions and barriers are encountered by persons with mobility-related disabilities in their daily’s experience of going to work and the pressure these exert on gaining and maintaining their employment. The nature and extent of their difficulties to workforce participation entails a requirement for extensive planning and also strategies to address problems of being stranded (for example, when the bus they are waiting for is not accessible). This paper presents the conceptualisation and methods of understanding workforce participation as a journey, and a discussion on the role digital technologies play in helping people with mobility-related disabilities in their journeys to work and mitigating disruptions when these occur. This is presented through an initial case study that helped identify the sequence of supports needed to be in place to make the work journey possible. Importantly, the paper also highlights points of intervention for the use of digital technologies and where design can potentially help to enhance accessibility to work for people with mobility-related impairments by making journeys to work seamless.

60Sox Report Volume 1: from education to work in Australia's digital content industries. The opinions and practices of aspiring creatives in the creative industries

This report presents findings from the largest survey of aspiring creatives who work or intend to work in the digital content industries ever undertaken in Australia. Survey respondents included those with aspirations to work in the publicly-supported, less commercial end of the Creative Industries spectrum as well as those with aspirations to work in the digital content industries. The survey gathered rich data on their characteristics, skills and attributes, barriers to employment, workforce mobility, career intentions, professional development, mentors and industry supports, and participation in communities of practice. The survey sought to determine if aspiring creatives have the necessary skills and attributes to work effectively in the digital content industries. This task also involved finding out how they develop their skills and attributes, and what they need to develop them further.

From education to work in Australia’s creative digital industries : comparing the opinions and practices of employers and aspiring creatives

This report represents the second of two reports that aim to explore views about the employability of aspiring creatives. The first report, released in June 2009, presented findings from a survey of 507 aspiring creatives, defined as recent graduates and/or people with less than two years industry experience. It presents findings from a project that administered an survey to employers in Australia’s Creative Digital Industries. The survey included questions on employer characteristics, recruitment and training practices, employers’ views of the capabilities of aspiring creatives, and participation in communities of interest/networks, mentoring and internships. The main purpose of the project was to identify capability gaps of aspiring creatives as well as those factors that enhance or inhibit employers’ views of the capabilities of aspiring creatives – both of which impact on the ability of aspiring creatives to find work in their preferred occupations in Australia’s Creative Digital Industries.

Providing youth with skills, training and employment opportunities through ICT Initiatives

Youth population is increasing explosively particularly in developing countries as a result of rapid urbanization. This increase is bringing large number of social and economic problems. For instance the impacts of job and training availability, and the physical, social and cultural quality of urban environment on young people are enormous, and affect their health, lifestyles, and well-being (Gleeson and Sipe 2006). Besides this, globalization and technological developments are affecting youth in urban areas in all parts of the world, both positively and negatively (Robertson 1995). The rapidly advancing information and communications technologies (ICTs) helps in addressing social and economic problems caused by the rapid growth of urban youth populations in developing countries. ICTs offer opportunities to young people for learning, skill development and employment. But there are downsides: young people in many developing countries lack of having broad access to these new technologies, they are vulnerable to global market changes, and ICTs link them into global cultures which promote consumer goods, potentially eroding local cultures and community values (Manacorda and Petrongolo 1999). However we believe that the positives outweigh such negatives. At the beginning of the twenty-first century, the world’s young population number more than they ever have. There are over a billion young people between the ages of 15 and 24, which 85 per cent of them live in developing countries and mainly in urban environments. Many of these young people are in the process of making, or have already made, the transition from school to work. During the last two decades all around the world, these young people, as new workers, have faced a number of challenges associated with globalization and technological advances on labour markets (United Nations 2004). The continuous decrease in the manufacturing employment is made many of the young people facing three options: getting jobs in the informal economy with insecurity and poor wages and working conditions, or getting jobs in the low-tier service industries, or developing their vocational skills to benefit from new opportunities in the professional and advanced technical/knowledge sectors. Moreover in developing countries a large portion of young people are not even lucky enough to choose among any of these options, and consequently facing long-term unemployment, which makes them highly vulnerable. The United Nations’ World Youth Employment report (2004) indicates that in almost all countries, females tend to be far more vulnerable than males in terms of long-term unemployment, and young people who have advanced qualifications are far less likely to experience long-term unemployment than others. In the limited opportunities of the formal labour market, those with limited vocational skills resort to forced entrepreneurship and selfemployment in the informal economy, often working for low pay under hazardous conditions, with only few prospects for the future (United Nations 2005a). The International Labour Organization’s research (2004) revealed that the labour force participation rates for young people decreased by almost four per cent (which is equivalent of 88 million young people) between 1993 and 2003. This is largely as a result of the increased number of young people attending school, high overall unemployment rates, and the fact that some young people gave up any hope of finding work and dropped out of the labour market. At the regional level, youth unemployment was highest in Middle East and North Africa (MENA) (25.6%) and sub-Saharan Africa (21%) and lowest in East Asia (7%) and the industrialized economies(13.4%) (International Labour Organization 2004). The youth in economically disadvantaged regions (e.g. the MENA region) face many challenges in education and training that delivers them the right set of skills and knowledge demanded by the labour market. As a consequence, the transition from school to work is mostly unsuccessful and young population end up either unemployed or underemployed in the informal sectors (United Nations 2005b). Unemployment and lack of economic prospects of the urban youth are pushing many of them into criminal acts, excessive alcohol use, substance addiction, and also in many cases resulting in processes of social or political violence (Fernandez-Maldonado 2004; United Nations 2005a). Long-term unemployment leads young people in a process of marginalisation and social exclusion (United Nations 2004). The sustained high rates of long-term youth unemployment have a number of negative effects on societies. First, it results in countries failing to take advantage of the human resources to increase their productive potential, at a time of transition to a globalized world that inexorably demands such leaps in productive capacity. Second, it reinforces the intergenerational transmission of poverty. Third, owing to the discrepancy between more education and exposure to the mass media and fewer employment opportunities, it may encourage the spread of disruptive behaviours, recourse to illegal alternatives for generating income and the loss of basic societal values, all of which erode public safety and social capital. Fourth, it may trigger violent and intractable political conflicts. And lastly, it may exacerbate intergenerational conflicts when young people perceive a lack of opportunity and meritocracy in a system that favours adults who have less formal education and training but more wealth, power and job stability (Hopenhayn 2002). To assist in addressing youth’s skill training and employment problems this paper scrutinises useful international practices, policies, initiatives and programs targeting youth skill training, particularly in ICTs. The MENA national governments and local authorities could consider implementing similar initiative and strategies to address some of the youth employment issues. The broader aim of this paper is to investigate the successful practice and strategies for the information and communication related income generation opportunities for young people to: promote youth entrepreneurship; promote public-private partnerships; target vulnerable groups of young people; narrow digital divide; and put young people in charge. The rest of this paper is organised in five parts. First, the paper provides an overview of the literature on the knowledge economy, skill, education and training issues. Secondly, it reviews the role of ICTs for vocational skill development and employability. Thirdly, it discusses the issues surrounding the development of the digital divide. Fourthly, the paper underlines types and the importance of developing ICT initiatives targeting young people, and reviews some of the successful policy implementations on ICT-based initiatives from both developed and developing countries that offer opportunities to young people for learning, skill development and employment. Then the paper concludes by providing useful generalised recommendations for the MENA region countries and cities in: advocating possible opportunities for ICT generated employment for young people; and discussing how ICT policies could be modified and adopted to meet young people’s needs.