Functional Genomics for Epithelial-Mesenchymal Transition in Breast Cancer.

摘要

Enhanced tumorigenicity of the PMC42-LA cell line after transfection with a library of shRNAmir constructs has been validated in a repeat experiment, and replicate material obtained for next generation sequencing. The methodology for the sequencing has been revised and re-validated, causing some delays, however it is now imminent. It will be possible, using the revised methodology, to sequence the shRNA associated with both altered colony morphology and EpCAM expression in vitro, as well as the formation of tumors in vivo. The in vivo relevance of EpCAM levels has been affirmed. Extension of the boutique library of 4,462 shRNA constructs to a full genome screen in the PMC42-LA cell line will not proceed due to the time required for this model. Instead, more emphasis will be placed on the MDA-MB-468 model where we have extensively characterized the in vivo EMP (two distinct zones at the tumour periphery and stromal interface, respectively), as well as circulating tumor cells and lung micrometastases. Again, metastasis from this model has taken longer than anticipated, resulting in additional delays. Nonetheless, these cells have been tagged with Luciferase2, and will be subjected to in vivo testing after transfection with a full genome library of shRNAmir constructs. The reporter assays required for Aim three have been developed and await the completion of Aims 1 and 2, however, they have already been successfully used in additional screens outside of this grant.