Combined beta1 integrin/EGFR targeting of head and neck cancers <em>in-vitro</em> and <em>in-vivo</em>.

Tumor cell resistance to radiotherapy and chemotherapy including novel targeted drugs is a great challenge for cancer treatment. One resistance mechanism found in cancer cells is the activation of bypass signaling which reduces the efficacy of the applied therapy. Therefore combined targeting of multiple pathways might increase tumor radiosensitivity and cell death. In this study, we examined the effect of dual beta1 integrin/EGFR inhibition in combination with irradiation in 10 head and neck squamous cell carcinoma (HNSCC) cell lines using 3D cell cultures and xenograft models. &nbsp; 3D colony formation assays, Western blotting, sequential immunoprecipitation-mass spectrometry, immunofluorescence staining, phosphoproteome arrays (including Reactome-based bioinformatics), and proximity ligation assays were performed. Downregulation of specific proteins was achieved by siRNA technology. Additionally, wildtype and constitutively active forms of Focal Adhesion Kinase (FAK) tagged to GFP were stably expressed in HNSCC cells. With regard to beta1 integrin targeting, one responder HNSCC model (UTSCC15) and one non-responder model (SAS) were transplanted subcutaneously on the legs of nude mice and treated with monotherapy or combination therapy of AIIB2 (monoclonal anti-beta1 integrin antibody) and Cetuximab plus/minus radiotherapy (20 Gy single dose). Tumor control and growth delay as well as signaling were assessed. The level of significance was determined by unpaired, 2-sided Student&acute;s t-test and Mann-Whitney-U-test using Microsoft Excel 2003 or log-rank test (actuarial estimates for time to local tumor recurrence were obtained using the Kaplan-Meier method). &nbsp; beta1 integrin targeting resulted in activation of Erk1/2 signaling pathway via FAK. We were able to prevent this activation by additional Cetuximab treatment, which enhanced both cytotoxicity and radiosensitization of AIIB2 monotherapy in-vitro and in-vivo. In-vitro, the ratio of responder to non-responder models for radiosensitization by beta1 integrin targeting was 8:2. Intriguingly and in contrast to SAS non-responder tumor xenografts, UTSCC15 responder tumor xenografts were completely controlled upon AIIB2/Cetuximab/radiotherapy. Mechanistically, we found a protein complex consisting of FAK and Erk1 that connects beta1 integrin and EGFR downstream signaling pathways. Targeting of beta1 integrin induced dissociation of this complex and hyperphosphorylation of the Ras-Raf-MEK-Erk signaling axis downstream of EGFR. To effectively prevent this bypass signaling, we simultaneously blocked beta1 integrin and EGFR, which could be causatively linked to a significantly higher radiosensitization as compared to monotherapies. Reactome bioinformatics clearly revealed a higher number of deactivated signaling hubs upon combined treatment relative to single treatments. As FAK seemed central to beta1 integrin and EGFR signal transduction, we used our FAK transfectants and found that the constitutively active form of FAK decreased the cellular radiosensitivity, abrogated HNSCC cell susceptibility to both AIIB2 and Cetuximab, and was downstream of Erk1. &nbsp; In summary, our data provide evidence for the superiority of simultaneous beta1 integrin/EGFR targeting over single targeting with regard to cytotoxicity and radiosensitization of HNSCC. In general, inhibition of overexpressed transmembrane receptors enables the deactivation of large parts of the prosurvival signaling network and, thus, represents a promising approach for patients with HNSCC tumors

Simultaneous &beta;1 integrin-EGFR targeting and radiosensitization of human head and neck cancer.

BACKGROUND: Signaling from integrins and receptor tyrosine kinases (RTKs) contributes substantially to therapy resistance of malignant tumors. We investigated simultaneous &beta;1 integrin-epidermal growth factor receptor (EGFR) targeting plus radiotherapy in human head and neck squamous cell carcinomas (HNSCCs). METHODS: Ten HNSCC cell lines were grown in three-dimensional laminin-rich extracellular matrix cell cultures and two of them as tumor xenografts in nude mice (n = 12-16 per group). Targeting of &beta;1 integrin and EGFR with monoclonal inhibitory antibodies (AIIB2 and cetuximab, respectively) was combined with x-ray irradiation. Clonogenic survival, tumor growth, and tumor control (evaluated by Kaplan-Meier analysis), apoptosis, phosphoproteome (interactome, network betweeness centrality analysis), receptor expression (immunohistochemistry), and downstream signaling (western blotting) were assessed. Various mutants of the integrin signaling mediator focal adhesion kinase (FAK) were employed for mechanistic studies. All statistical tests were two-sided. RESULTS: Compared with &beta;1 integrin or EGFR single inhibition, combined &beta;1 integrin-EGFR targeting resulted in enhanced cytotoxicity and radiosensitization in eight out of 10 tested HNSCC cell lines, which responded with an FAK dephosphorylation after &beta;1 integrin inhibition. In vivo, simultaneous anti-&beta;1 integrin/anti-EGFR treatment and radiotherapy of UTSCC15 responder xenografts enabled better tumor control compared with anti-EGFR monotherapy and irradiation (hazard ratio [HR] = 6.9, 95% confidence interval [CI] = 1.6 to 30.9, P = .01), in contrast to the SAS nonresponder tumor model (HR = 0.9, 95% CI = 0.4 to 2.3, P = .83). Mechanistically, a protein complex consisting of FAK- and Erk1-mediated prosurvival signals for radiation resistance, which was effectively compromised by &beta;1 integrin and EGFR blocking. CONCLUSIONS: Concomitant targeting of &beta;1 integrin and EGFR seems a powerful and promising approach to overcome radioresistance of HNSCCs

Methodology for measuring environmental health within Europe. Health Risk from Environmental Pollution Levels in Urban Systems (HEREPLUS)

Background: The European Commission funds a European research project titled "Health Risk from Environmental Pollution Levels in Urban Systems" (HEREPLUS) that focuses on environmental health within Europe. The HEREPLUS project was presented at the 16th EUPHA conference in Lisbon in November 2008 within a workshop named "The assessment of the effect of air pollution on population and environmental health: the integration of epidemiology and geographical information system (GIS)". Methods: The HEREPLUS project aims to measure the correlation between air pollution (especially ozone and particulate matter), meteorology, vegetation and human health in four European cities (Rome, Madrid, Athens and Dresden) by using a Geoinformation System to develop risk maps and subsequently guidelines to reduce air pollution and number of diseases. Results: The project started in September 2008 and a large, structured, relational database has been developed and completed. A literature review including national as well as international scientific literature goes on and will be completed in April 2009. Final results will be presented and published in 2011. Conclusions: Detailed scientific knowledge is important and needed to implement environmental programmes with the overall aim to protect human population against environmental related diseases

Tobacco use among medical students in Europe: results of a multicentre study using the Global Health Professions Student Survey

To examine smoking prevalence, knowledge and attitudes, and tobacco cessation training among university students attending European medical schools using the Global Health Professional Students Survey approach