| Summary: | The aim of this work was to identify novel predictors of paclitaxel response in ovarian cancer. I have used expression profiling as a tool to study gene expression profiles of paclitaxel resistance in vivo and in vitro. To do this I studied the sources of bias in microarray experiments. Using analysis of variance methods I found that image segmentation was a significant source of bias. This finding had direct practical implications on gene discovery in microarray experiments. I have designed in vitro experiments to study a cell line model of paclitaxel resistance comprising a parent paclitaxel-sensitive cell line (SKOV3) and its daughter paclitaxel-resistant line (TRSKOV3) that was derived by prolonged and repeated exposure to the drug. Microarray expression analysis identified transforming growth factor beta induced gene (<i>TGFBI) </i>as the most differentially underexpressed in the TRSKOV3 cell line. Importantly, selective down-regulation of the gene using RNA interference resulted in the acquisition of resistance in the parental SKOV3 line. Studying a panel of 20 additional breast and ovarian established cancer cell lines confirmed the association between <i>TGFBI</i> underexpression and paclitaxel-resistance. Moreover, silencing of <i>TGFBI</i> using RNA interference in SKOV3, HCT116 (a colon cancer cell line) and CAL51 (a breast cancer cell line) resulted in significant mitotic abnormalities and centrosome amplification. This indicated loss of cell cycle checkpoints as a plausible cause of paclitaxel resistance following <i>TGFBI </i>silencing. In parallel with this in vitro work I have completed a prospective randomized study (CTCR-OV01) of sequential chemotherapy to study response in patients with advanced epithelial ovarian cancer to neoadjuvant single agent carboplatin or paclitaxel. Paired samples (before and after neoadjuvant treatment) were collected to correlate molecular markers with chemo-therapy response. These samples were used to validate the results from my in vitro work. <i>TGFBI</i> was found to be significantly underexpressed in paclitaxel-resistant patients. Importantly, paclitaxel-resistant patients showed significant centrosome amplification that negatively correlated with <i>TGFBI</i> expression levels which confirmed the results obtained following <i>TGFBI</i> silencing in tissue culture. Samples obtained from CTCR-OV01 study were also used for gene expression profiling using oligonucleotide arrays which also confirmed downregulation of <i>TGFBI</i> in paclitaxel-resistant patients. Furthermore, 28 genes were differentially expressed in the paclitaxel-resistant patients, of which 16 were extracellular matrix (ECM)-related genes. Importantly, <i>TGFBI</i> expression showed a significant overall correlation (<i>r</i> = 0.89) with ECM-related genes indicating a possible class effect of ECM-related genes on response to paclitaxel treatment.
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