The University of Wisconsin Bioengineering Research Partnership (UW-BRP) is developing new molecular analysis tools to identify and validate biological endpoints whereby the activity of novel anti-cancer agents can be more accurately and rapidly evaluated as to their molecular mechanism(s) and clinical relevance. The work is focused on epidermal growth factor receptor (EGFR), given that its overexpression and mutation has been closely-associated with some of the most incurable cancers. However, the tools are sufficiently versatile to be applicable to other key signaling molecules. Whereas the basis of many existing screens restricts their application to in vitro molecular analyses of enriched preparations of receptors or other signaling molecules, the UW-BRP seeks to establish principles for tools that can also be applied to the analysis of samples from cultured cells, and from biopsies of xenographs and spontaneous tumor tissues. This capability will ultimately enable a fundamental approach that will span the molecular, cellular and tissue levels and will be used in both basic research and in animal and human clinical trials. Over the past 4 years, the UW-BRP has defined key analytic characteristics of liquid crystal-based tools for molecular analysis, and demonstrated reporting of EGFR expression and activation (phosphorylation) as well as inhibition of EGFR tyrosine kinase activity by small molecules (in complex samples such as membrane extracts and cell lysates). The present proposal seeks continued support for the multi-disciplinary team of researchers with diverse expertise in chemical and biological engineering, chemistry and biochemistry, and the biomolecular and biomedical sciences to develop further this broadly-applicable bioanalytical approach via integration of advances in the following areas: a) identification and optimization of uniformly immobilized, single chain antibodies that recognize epitopes of key EGFR mutants in cell lysates, b) integration of liquid crystal-based analytic methodologies and sample preparation for quantification of expression, activation and kinase inhibition of wild-type and mutant EGFRs using small samples (a few cells), and c) the investigation of key cell signaling proteins that participate in processes associated with carcinogenesis. Specifically, the UW-BRP molecular analysis tools will be compared to conventional methods in a study that will a) rapidly and sensitively assess the levels and activity of wild-type and mutant human EGFRs in biological samples, b) test the hypothesis that wild-type and oncogenic forms of the EGFR will exhibit differential inhibitor specificity, and c) assess if agents that potently inhibit EGF-mediated events in vitro will also exhibit a capacity to antagonize EGFR expression and/or activity in cell culture. In the long term, these new tools should be useful for the assessment of the molecular mechanisms and consequences of anti-cancer agents, thereby facilitating their research from basic biology through to clinical assessment of efficacy. PUBLIC
The University of Wisconsin Bioengineering Research Partnership is focused on the development of an integrated bioengineering systems approach that will provide the foundations for new molecular tools to analyze the protein regulatory pathways that underlie cellular transformation into cancerous states. These tools have the potential to accelerate the identification and validation of biological endpoints whereby the activity of novel anti-cancer agents can be more accurately and rapidly evaluated for their molecular mechanisms and clinical relevance.
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