Vascular endothelial growth factor is an autocrine growth factor, signaling through neuropilin-1 in non-small cell lung cancer

Background The VEGF pathway has become an important therapeutic target in lung cancer, where VEGF has long been established as a potent pro-angiogenic growth factor expressed by many types of tumors. While Bevacizumab (Avastin) has proven successful in increasing the objective tumor response rate and in prolonging progression and overall survival in patients with NSCLC, the survival benefit is however relatively short and the majority of patients eventually relapse. The current use of tyrosine kinase inhibitors alone and in combination with chemotherapy has been underwhelming, highlighting an urgent need for new targeted therapies. In this study, we examined the mechanisms of VEGF-mediated survival in NSCLC cells and the role of the Neuropilin receptors in this process. Methods NSCLC cells were screened for expression of VEGF and its receptors. The effects of recombinant VEGF and its blockade on lung tumor cell proliferation and cell cycle were examined. Phosphorylation of Akt and Erk1/2 proteins was examined by high content analysis and confocal microscopy. The effects of silencing VEGF on cell proliferation and survival signaling were also assessed. A Neuropilin-1 stable-transfected cell line was generated. Cell growth characteristics in addition to pAkt and pErk1/2 signaling were studied in response to VEGF and its blockade. Tumor growth studies were carried out in nude mice following subcutaneous injection of NP1 over-expressing cells. Results Inhibition of the VEGF pathway with anti-VEGF and anti-VEGFR-2 antibodies or siRNA to VEGF, NP1 and NP2 resulted in growth inhibition of NP1 positive tumor cell lines associated with down-regulation of PI3K and MAPK kinase signaling. Stable transfection of NP1 negative cells with NP1 induced proliferation in vitro, which was further enhanced by exogenous VEGF. In vivo, NP1 over-expressing cells significantly increased tumor growth in xenografts compared to controls. Conclusions Our data demonstrate that VEGF is an autocrine growth factor in NSCLC signaling, at least in part, through NP1. Targeting this VEGF receptor may offer potential as a novel therapeutic approach and also support the evaluation of the role of NP1 as a biomarker predicting sensitivity or resistance to VEGF and VEGFR-targeted therapies in the clinical arena.

The tumour-promoting receptor tyrosine kinase, EphB4, regulates expression of Integrin-β8 in prostate cancer cells

Background The EphB4 receptor tyrosine kinase is overexpressed in many cancers including prostate cancer. The molecular mechanisms by which this ephrin receptor influences cancer progression are complex as there are tumor-promoting ligand-independent mechanisms in place as well as ligand-dependent tumor suppressive pathways. Methods We employed transient knockdown of EPHB4 in prostate cancer cells, coupled with gene microarray analysis, to identify genes that were regulated by EPHB4 and may represent linked tumor-promoting factors. We validated target genes using qRT-PCR and employed functional assays to determine their role in prostate cancer migration and invasion. Results We discovered that over 500 genes were deregulated upon EPHB4 siRNA knockdown, with integrin β8 (ITGB8) being the top hit (29-fold down-regulated compared to negative non-silencing siRNA). Gene ontology analysis found that the process of cell adhesion was highly deregulated and two other integrin genes, ITGA3 and ITGA10, were also differentially expressed. In parallel, we also discovered that over-expression of EPHB4 led to a concomitant increase in ITGB8 expression. In silico analysis of a prostate cancer progression microarray publically available in the Oncomine database showed that both EPHB4 and ITGB8 are highly expressed in prostatic intraepithelial neoplasia, the precursor to prostate cancer. Knockdown of ITGB8 in PC-3 and 22Rv1 prostate cancer cells in vitro resulted in significant reduction of cell migration and invasion. Conclusions These results reveal that EphB4 regulates integrin β8 expression and that integrin β8 plays a hitherto unrecognized role in the motility of prostate cancer cells and thus targeting integrin β8 may be a new treatment strategy for prostate cancer.

High threshold of β1 integrin inhibition required to block collagen I-induced membrane type-1 matrix metalloproteinase (MT1-MMP) activation of matrix metalloproteinase 2 (MMP-2)

Background Matrix metalloproteinase-2 (MMP-2) is an endopeptidase that facilitates extracellular matrix remodeling and molecular regulation, and is implicated in tumor metastasis. Type I collagen (Col I) regulates the activation of MMP-2 through both transcriptional and post-transcriptional means; however gaps remain in our understanding of the involvement of collagen-binding ?1 integrins in collagen-stimulated MMP-2 activation. Methods Three ?1 integrin siRNAs were used to elucidate the involvement of ?1 integrins in the Col I-induced MMP-2 activation mechanism. ?1 integrin knockdown was analyzed by quantitative RT-PCR, Western Blot and FACS analysis. Adhesion assay and collagen gel contraction were used to test the biological effects of ?1 integrin abrogation. MMP-2 activation levels were monitored by gelatin zymography. Results All three ?1 integrin siRNAs were efficient at ?1 integrin knockdown and FACS analysis revealed commensurate reductions of integrins ?2 and ?3, which are heterodimeric partners of ?1, but not ?V, which is not. All three ?1 integrin siRNAs inhibited adhesion and collagen gel contraction, however only the siRNA showing the greatest magnitude of ?1 knockdown inhibited Col I-induced MMP-2 activation and reduced the accompanying upregulation of MT1-MMP, suggesting a dose response threshold effect. Re-transfection with codon-swapped ?1 integrin overcame the reduction in MMP-2 activation induced by Col-1, confirming the ?1 integrin target specificity. MMP-2 activation induced by TPA or Concanavalin A (Con A) was not inhibited by ?1 integrin siRNA knockdown. Conclusion Together, the data reveals that strong abrogation of ?1 integrin is required to block MMP-2 activation induced by Col I, which may have implications for the therapeutic targeting of ?1 integrin.

Stable expression of silencing-suppressor protein enhances the performance and longevity of an engineered metabolic pathway

Transgenic engineering of plants is important in both basic and applied research. However, the expression of a transgene can dwindle over time as the plant's small (s)RNA-guided silencing pathways shut it down. The silencing pathways have evolved as antiviral defence mechanisms, and viruses have co-evolved viral silencing-suppressor proteins (VSPs) to block them. Therefore, VSPs have been routinely used alongside desired transgene constructs to enhance their expression in transient assays. However, constitutive, stable expression of a VSP in a plant usually causes pronounced developmental abnormalities, as their actions interfere with endogenous microRNA-regulated processes, and has largely precluded the use of VSPs as an aid to stable transgene expression. In an attempt to avoid the deleterious effects but obtain the enhancing effect, a number of different VSPs were expressed exclusively in the seeds of Arabidopsis thaliana alongside a three-step transgenic pathway for the synthesis of arachidonic acid (AA), an ω-6 long chain polyunsaturated fatty acid. Results from independent transgenic events, maintained for four generations, showed that the VSP-AA-transformed plants were developmentally normal, apart from minor phenotypes at the cotyledon stage, and could produce 40% more AA than plants transformed with the AA transgene cassette alone. Intriguingly, a geminivirus VSP, V2, was constitutively expressed without causing developmental defects, as it acts on the siRNA amplification step that is not part of the miRNA pathway, and gave strong transgene enhancement. These results demonstrate that VSP expression can be used to protect and enhance stable transgene performance and has significant biotechnological application.

The social tasks of learning to become a physical education teacher: Considering the HPE subject department as a community of practice

Initial teacher education (ITE) students participate in various workplaces within schools and in doing so, form understandings about the numerous, and at times competing, expectations of teachers’ work. Through these experiences they form understandings about themselves as health and physical education (HPE) teachers. This paper examines the ways communities of practice within HPE subject department offices function as sites of workplace learning for student teachers. In particular this research focused on how ITE students negotiate tacit and contradictory expectations as well as social tasks during the practicum and the ways in which their understandings are mediated through participation in the workspace. Qualitative methods of survey and semi-structured interview were used to collect data on a cohort of student teachers during and following their major (10 week) practicum experience. Analysis was informed by theories of communities of practice (Wenger, 1998), workplace learning (Billett, 2001), and social task systems (Doyle, 1977). It was evident that considerable effort, attention, and energy was expended on various interrelated social tasks aimed at building positive relationships with their supervisor and other HPE teachers at the school. The social dynamics were highly nuanced and required a game-like approach. In our view the complexity that student teachers must negotiate in striving for an excellent evaluation warrants specific attention in physical education teacher education (PETE) programs. This study raises questions regarding our responsibilities in sending student teachers into contexts that might even be described as toxic. We offer some suggestions for how PETE might better support students going into practicum contexts that might be regarded as problematic workplaces.

Becoming a health and physical education teacher: Student teacher ‘performances’ in the HPE subject department office

This study considered how physical education teacher education students ‘perform’ their ‘selves’ within subject department offices during the practicum or ‘teaching practice’. The research was framed by a conceptual framework informed by the work of Goffman on ‘performance’ and ‘front’. The findings revealed three common performances across the whole group across all sites. These were: performance of sports talk, bodily performances, and performance of masculine repertoires. Such performances were considered to be inconsistent with the coursework ideals and principles within the teacher education programme but in step with the general ethos of most PE department offices.

Creating physical education in remote Australian schools: Overcoming the tyranny of distance through communities of practice

This paper reports on a qualitative case study undertaken in a remote part of Queensland, Australia. While there is some modest agreement about the capacity of contemporary information technologies to overcome the problems of schooling in areas of extreme remoteness, generally, children educated in such contexts are considered to be disadvantaged. The experiential areas of the curriculum, which often require specific teaching expertise, present the greatest challenge to teachers, and of these, physical education is perhaps the most problematic. This research reports on a case study of three remote Queensland multi-age primary (elementary) schools that come together to form a community of practice to overcome the problems of teaching physical education in such difficult circumstances. Physical education is constructed in these contexts by blurring the school and community boundaries, by contextualizing the subject content to make it relevant, and by adjusting the school day to accommodate potential physical education experiences. Each community gathers its collective experience to ensure the widest possible experiences are made available for the children. In doing so, the children develop a range of competencies that enable seamless transition to boarding high schools.

With the best of intentions: A critical discourse analysis of HPE curriculum materials

Much of physical education curriculum in the developed world and specifically in Australia tends to be guided in principle by syllabus documents that represent, in varying degrees, some form of government education priorities. Through the use of critical discourse analysis we analyze one such syllabus example (an official syllabus document of one of the Australian States) to explore the relationships between the emancipatory/social justice expectations presented in the rubric of and introduction to the official syllabus document, and the language details of learning outcomes that indicate how the expectations might be satisfied. Given the complexity and multilevel pathways of message systems/ideologies we question the efficacy of such documents oriented around social justice principles to genuinely deliver more radical agendas which promote social change and encourage a preparedness to engage in social action leading to a betterment of society.

Social processes of health and physical education teachers’ identity formation: Reproducing and changing culture

This paper examines Initial Teacher Education students’ experiences of participation in health and physical education (HPE) subject department offices and the impact on their understandings and identity formation. Pierre Bourdieu’s concepts of habitus, field, and practice along with Wenger’s communities of practice form the theoretical frame used in the paper. Data were collected using surveys and interviews with student‐teachers following their teaching practicum and analysed using coding and constant comparison. Emergent themes revealed students’ participation in masculine‐dominated sports, gendered body constructions, and repertoires of masculine domination. Findings are discussed in relation to their impact on student‐teachers’ learning, identity formation, and marginalizing practices in the department offices. Implications for teacher education and HPE are explored.

Abnormal levels of heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) in tumour tissue and blood samples from patients diagnosed with lung cancer

Lung cancer is the second most common type of cancer in the world and is the most common cause of cancer-related death in both men and women. Research into causes, prevention and treatment of lung cancer is ongoing and much progress has been made recently in these areas, however survival rates have not significantly improved. Therefore, it is essential to develop biomarkers for early diagnosis of lung cancer, prediction of metastasis and evaluation of treatment efficiency, as well as using these molecules to provide some understanding about tumour biology and translate highly promising findings in basic science research to clinical application. In this investigation, two-dimensional difference gel electrophoresis and mass spectrometry were initially used to analyse conditioned media from a panel of lung cancer and normal bronchial epithelial cell lines. Significant proteins were identified with heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1), pyruvate kinase M2 isoform (PKM2), Hsc-70 interacting protein and lactate dehydrogenase A (LDHA) selected for analysis in serum from healthy individuals and lung cancer patients. hnRNPA2B1, PKM2 and LDHA were found to be statistically significant in all comparisons. Tissue analysis and knockdown of hnRNPA2B1 using siRNA subsequently demonstrated both the overexpression and potential role for this molecule in lung tumorigenesis. The data presented highlights a number of in vitro derived candidate biomarkers subsequently verified in patient samples and also provides some insight into their roles in the complex intracellular mechanisms associated with tumour progression.