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  • br Materials and methods br Results br Discussion We have

    2024-04-18


    Materials and methods
    Results
    Discussion We have developed a high-throughput functional genomic screen that allows the identification of genes that confer drug resistance. Several factors have been identified which must be taken into account to develop a reliable screen. In particular, consideration must be given to the timing of drug addition, the drug concentrations used and the choice of cell line. We validated our methodology by testing the effect of reducing the expression of FLIP upon the sensitivity of AZD6738 to paclitaxel. Paclitaxel has been found to decrease the expression of endogenous FLIP while ectopic expression of FLIP has been shown to decrease sensitivity to paclitaxel in CCRF-HSB2 leukemia cells [22]. Previously, FLIP siRNA has been shown to cause sensitization of colon cancer cells to several cytotoxic drugs [20]. In ovarian cancer cells, the expression of FLIP has been linked to resistance to cisplatin [30]. We tested 6 ovarian cancer cell lines and found that the inhibition of FLIP expression increased sensitivity to paclitaxel in 4 cell lines. To our knowledge, this is the first demonstration that FLIP regulates the sensitivity of ovarian cancer cells to paclitaxel. In OVCAR-3 cells there was no increase in sensitivity to paclitaxel, despite this being the cell line in which we achieved the most effective knockdown of the FLIP mRNA. Thus, the differences in the contribution of FLIP to paclitaxel sensitivity observed in the different cell lines are not solely a result of the different extent of knockdown. The lack of the apparent contribution of FLIP to paclitaxel sensitivity in 2 cell lines highlights the need to use several cell lines when conducting a thorough screen for genes contributing to drug resistance. A particular resistance mechanism may not be operating in each cell line and when a limited number of cell lines are screened, some mechanisms may go undetected. Functional genomic screens are unlikely to provide a comprehensive list of potential drug-resistance genes unless several cell lines are used. We conducted a screen in which we inhibited the expression of 90 genes in 6 cell lines and measured the change in sensitivity to two chemotherapeutic agents. These genes are more likely to contribute to acquired rather than intrinsic drug resistance because the genes used in this study were identified on the basis of their increased expression in samples collected from patients with recurrent disease after chemotherapy. Some of the genes identified as hits in this screen have previously been implicated in drug resistance. These include EDNRA which encodes an endothelin receptor and antagonists of this receptor increase the sensitivity of ovarian cancer cells to paclitaxel [31]. A TGFβ regulated gene, TGBI, has recently been linked to resistance to paclitaxel [32] and several other genes in the TGF signaling axis were hits in our screen including TGFBR2, TGFBR3, TGFB3 and TGF1I4. CYR61, an extracellular matrix protein which has previously been implicated in resistance to chemotherapeutic agents [23], [24], was also identified as a hit. The fact that several genes previously linked to drug resistance were identified provides confidence that our approach has the potential to identify novel genes which contribute to drug resistance. In support of this, relatively few hits were obtained from OVCAR-5 cells, a cell line that was established from a tumor collected prior to chemotherapy. It is also important to note that some genes were identified as hits in some cell lines and not in others. This further underlines the importance of using several cell lines in a screen to identify drug-resistance genes. One of the genes identified in the screen encoded autotaxin. The expression of autotaxin is increased in several tumor types [13] and autotaxin has been linked to mammary tumorigenesis [14]. Autotaxin is a secreted lysophospholipase involved in the hydrolysis of lysophosphatidylcholine to LPA and the hydrolysis of sphingosine phosphorylcholine to S1P. Autotaxin also functions as oligonucleotide phosphodiesterase in vitro. LPA has been well characterized as a survival factor, and S1P is also able to inhibit apoptosis [33]. Autotaxin inhibits apoptosis in fibroblasts by catalyzing the production of LPA [34]. Deregulation of the LPA signaling pathway has been well documented in ovarian cancer. For example, high concentrations of LPA are observed in the peritoneal ascites fluid of patients with ovarian cancer [35], [36]. Ectopic expression of LPP1, an enzyme responsible for metabolizing LPA, decreases the growth and survival of ovarian cancer cells [37]. LPA can directly inhibit anoikis (cell death induced by detachment from extracellular matrix) in ovarian cancer cells [35] as well as stimulating migration [38] and invasion [39] of ovarian cancer cells. Increased expression in ovarian cancer of the receptors for LPA has also been reported [35]. Thus, there is significant evidence linking the deregulation of the autotaxin/LPA signaling pathway to ovarian cancer.