Assay of Rab25 function in ovarian and breast cancers

Abstract

There is a multitude of critical steps during the pathogenesis of cancer that allow cells to acquire the ability to escape from normal controls on cell growth, to avoid programmed cell death, and to become malignant. Here, we describe a molecular approach that can be broadly applied to identify drivers of genomic aberrations in cancer development. In the process, areas of genomic aberrations and genes that are dysregulated by genomic amplification are identified by array comparative genomic hybridization (CGH) and transcription profiling, respectively, with major emphasis on coordinating amplification at the CGH and RNA level and on correlation with patient's outcomes. Once candidate genes are identified, we perform functional genomics by manipulating levels in normal and tumor cells using RNAi or transfection, and assessing a battery of cellular functions including proliferation, anti-apoptosis, loss of contact inhibition, changes in cell signaling or transcriptional profiles, anchorage-independent growth, and in vivo tumor growth. We have successfully used this approach to identify the RAB25 gene that has been implicated in the progression and aggressiveness of ovarian and breast cancers.