Cancer cell resistance to paclitaxel continues to be a major clinical problem. most discriminating miRNA was evaluated in a mouse model of paclitaxel resistance. Results miRNA-135a is upregulated in various paclitaxel-resistant cell lines A screen to identify miRNAs involved in paclitaxel resistance was performed in four cell lines that were made paclitaxel-resistant by continuous exposure to paclitaxel expression in paclitaxel-sensitive and -resistant cell lines. Analysis of mRNA expression levels revealed a 2.6-fold downregulation in A549TR cells (P<0.001) and a 7-fold downregulation in MES-SADX5 cells (Figure 4C, P<0.001). A concordant downregulation of APC was also observed at the protein level (Figure 4C). Transfection with the anti-miR-135a inhibitor restored expression both at the mRNA (Figure 4D) and protein level (Figure 4E). Figure 4 miR-135a modulates APC expression in paclitaxel-resistant cancer cells miR-135a-induced paclitaxel resistance is partly mediated by APC downregulation To test the role of APC in the cellular response to paclitaxel, expression was suppressed in parental A549 and MES-SA cells. Transfection with siRNA against APC completely suppressed expression in both cell lines (Figure 5A) and led to a decrease in paclitaxel-induced cytotoxicity in both cell lines (Figures 5B and 5C). Similar results were obtained in A549 cells stably expressing shRNA against APC (Figures 5D and 5E). These results suggest that downregulation of APC may be partly responsible for the effects of miR-135a on paclitaxel sensitivity in these cell lines. Figure 5 miR-135a-mediated APC suppression contributes to paclitaxel resistance The generation of paclitaxel-resistant cell lines than either parental A549 cells (white bars) or vehicle-treated tumors (striped bars, Figure 6C). Treatment of xenotransplanted mice with 10 and 12.5 mg/kg paclitaxel did not generate any paclitaxel-refractory tumors (Supplementary Figure S4). Figure 6 Establishment of A549 paclitaxel-resistant cells (Figure 7C, P=0.01). The maintenance of paclitaxel resistance upon retransplantation suggests that paclitaxel resistance is associated with stable changes in the tumor rather than in transient changes induced by exposure to the host environment. Figure 7 Establishment of an A549 paclitaxel resistance model: retransplantation miR-135a is upregulated in paclitaxel resistance In our experiments we established a role for miR-135a in the cellular response to paclitaxel. To explore the role of GSK1292263 miR-135a expression passage (P=0.030) and to 126.6 nM after re-injection (P=0.008, Figure 8A). As shown in Figure 8B, decreased paclitaxel response is significantly associated with increased miR-135a expression in tumors selected for paclitaxel resistance passage (round 1) and a 4.1-fold higher expression of miR-135a compared to the parental A549 cells (P=0.004). The correlation between miR-135a expression and paclitaxel response was observed in all cell lines derived from these tumors (Figure 8C, P<0.001). Transfection with a miR-135a inhibitor modestly increased paclitaxel-induced cytotoxicity in paclitaxel-resistant cells (Figure 8D). Furthermore, transfection of a miR-135a mimic suppressed paclitaxel-induced cytotoxicity in paclitaxel-sensitive cells (Figure 8E). Together, these results show that miR-135a is involved in the paclitaxel sensitivity of the cell lines established after long-term exposure to paclitaxel paclitaxel resistance miR-135a modulates tumor response to paclitaxel To this aim, A549 cells stably expressing miR-135a were generated, which showed expression of miR-135a approximating that of A549TR cells (P<0.01) (Figure 9A). As expected, miR-135a knockdown attenuated paclitaxel-induced cell death (Figure 9B) and APC protein expression (Figure 9C) relative to control transfected A549 cells relative to control untreated cells. growth of miR-135a GSK1292263 overexpression was not reduced by paclitaxel exposure (Figure 9D). This result demonstrates that miR-135a overexpression alone induces taxane resistance that is maintained during tumor growth models where resistance is induced by repeated or prolonged exposure of cultured cells to gradually increasing drug concentrations. Although this methodology has imparted important knowledge, it has potential disadvantages. First, cells in solid tumors tend to be more drug-resistant than the same cells grown in a monolayer (Hoffman, 1991; Kobayashi pharmacokinetics, and does not allow the formation of active metabolites that may be produced by metabolic activity. Third, the situation does not address the role of the tumor microenvironment GSK1292263 in the generation of drug resistance. To confirm the role of miRNA-135a in paclitaxel resistance, we established a GSK1292263 new Rabbit Polyclonal to MRPL46 mouse model of paclitaxel resistance. One way to circumvent this problem is to use mice that develop spontaneous tumors as a consequence of conditional tissue-specific mutations in proto-oncogenes and tumor suppressor genes. Using this approach, Rottenberg generated docetaxel-resistant Brca1?/?;p53?/? mammary tumors (Rottenberg selection of paclitaxel-resistant tumors. In our model, mice were inoculated with paclitaxel-sensitive tumor cells and then treated with paclitaxel 3x weekly, consistent with current clinical practice. Similar approaches have been employed by others to establish drug-resistant.