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  • br Acknowledgements We thank Dr Stefan Schulte Merker and hi

    2023-01-24


    Acknowledgements We thank Dr. Stefan Schulte-Merker and his group members at the Hubrecht Institute (Utrecht, the Netherlands) for their invaluable support of the zebrafish studies. Our work is supported by grants from the Dutch Cancer Society (KWF) and the Netherlands Organisation for Scientific Research (NWO).
    Introduction The development of drug resistance remains a barrier to the successful long term treatment of many cancer patients. In the case of ovarian cancer, 20% of patients exhibit intrinsic drug resistance and do not respond to initial therapy, while the majority of the patients that initially respond eventually acquire drug resistance and only 30% of patients with advanced disease survive for more than 5years after diagnosis [1]. Drug resistance is well documented as a limiting factor which affects response to cytotoxic therapies such as carboplatin and paclitaxel, but also to newly emerging molecularly targeted therapies [2]. The identification of genes which confer drug resistance may provide novel therapeutic targets that can be exploited to develop drugs which re-sensitize tumor azd9291 to chemotherapeutic agents [3]. Increasingly, new drug targets are being identified by functional genomic approaches which utilize high-throughput screens in which genes are identified by virtue of their biological function. Genome-wide functional screening to identify new drug targets has become feasible (for example, [4], [5]). Additionally, numerous microarray studies have already identified large sets of genes whose expression is altered in drug-resistant cells and there is a pressing need to evaluate these genes for their contribution to drug resistance. To address this need, we have established an RNAi screen to azd9291 identify genes affecting drug resistance. Although many groups are using RNAi to identify genes affecting cell growth and survival [6], functional genomic screens to identify genes regulating drug resistance are beginning to be reported [7], [8], [9], [10]. To establish a screen to identify genes which contribute to drug resistance we set several goals. The screen should be: amenable to high-throughput screening (HTS); use a limited number of drug concentrations to achieve reasonable throughput; applicable to several cell lines to maximize the chance of providing an appropriate genetic background — different cell lines may use different resistance mechanisms; provide a means to rank the genes by providing an estimate of the changes in IC50 following down-regulation of the gene; and be capable of detecting small (2-fold) changes in drug sensitivity comparable to those commonly observed during drug combination experiments. The cell lines used comprise those ovarian cancer lines in the “NCI-60” and include lines which were isolated from patients either prior to (OVCAR-5, [11]) or after (OVCAR-3, OVCAR-4, OVCAR-8, IGROV-1, Sk-Ov-3) treatment with chemotherapy. We validated our screen by showing that the inhibition of the expression of FLIP increases sensitivity to paclitaxel and the inhibition of expression of Bcl-XL increases sensitivity to carboplatin. To conduct the screen, 90 genes were selected which have previously been shown to be over-expressed in tumors isolated after relapse following carboplatin/paclitaxel combination chemotherapy [12]. These genes are, therefore, candidate genes which may contribute to acquired drug resistance. One of the hits identified in the screen was ENPP2. This gene encodes autotaxin, an enzyme which functions as a lysophospholipase D and catalyzes the production of the survival factors lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) [13]. Ectopic expression of autotaxin has recently been shown to be sufficient to induce mammary cancer [14] and autotaxin inhibits paclitaxel-induced apoptosis in breast cancer cells [15]. Furthermore, a combined autotaxin and LPA-receptor antagonist is able to inhibit the growth of MDA-MB-231 xenografts [16]. We observed that the inhibition of autotaxin using both siRNA and a chemical inhibitor of autotaxin accelerates apoptosis induced by carboplatin in ovarian cancer cells while increasing the expression of autotaxin delays apoptosis.