We studied the role of miRNA‐200 family members in cellular sensitivity to paclitaxel and carboplatin, using two ovarian cancer cell lines, OVCAR‐3 and MES‐OV, and their paclitaxel resistant variants OVCAR‐3/TP and MES‐OV/TP. Both resistant variants display a strong epithelial‐mesenchymal transition (EMT) phenotype, with marked decreases in expression of miR‐200c and miR‐141 in OVCAR‐3/TP, and down‐regulation of all five members of the miR‐200 family in MES‐OV/TP. Lentiviral transfection of inhibitors of miR‐200c or miR‐141 in parental OVCAR‐3 triggered EMT and rendered the cells resistant to paclitaxel and carboplatin. Conversely, the infection of OVCAR‐3/TP cells with retroviral particles carrying the miR‐200ab429 and 200c141 clusters triggered a partial mesenchymal to epithelial transition (MET). This partial MET was not sufficient to re‐sensitize OVCAR‐3/TP cells to paclitaxel. However, the miR‐200c/miR‐141 cluster transfectants became 6–8x resistant to carboplatin, an unexpected result, whereas miR‐200a/miR‐200b/miR‐429 had no effect. Transfecting the OVCAR‐3/TP GFP cells with specific miRNA mimics confirmed these data. MiR‐200c and miR‐141 mimics conferred resistance to carboplatin in MES‐OV/TP cells, similar to OVCAR‐3/TP, but sensitized MES‐OV to paclitaxel. Several genes involved in balancing oxidative stress were altered in OVCAR‐3/TP 200c141 cells compared to controls. The miR‐200 family plays major, cell‐context dependent roles in regulating EMT and sensitivity to carboplatin and paclitaxel in OVCAR‐3 and MES‐OV cells.

Ovarian cancer is associated with a leukocyte infiltrate and high levels of chemokines such as CCL2. We tested the hypothesis that CCL2 inhibition can enhance chemotherapy with carboplatin and paclitaxel. Elevated CCL2 expression was found in three non-MDR paclitaxel resistant ovarian cancer lines ES-2/TP, MES-OV/TP and OVCAR-3/TP, compared to parental cells. Mice xenografted with these cells were treated with the anti-human CCL2 antibody CNTO 888 and the anti-mouse MCP-1 antibody C1142, with and without paclitaxel or carboplatin. Our results show an additive effect of CCL2 blockade on the efficacy of paclitaxel and carboplatin. This therapeutic effect was largely due to inhibition of mouse stromal CCL2. We show that inhibition of CCL2 can enhance paclitaxel and carboplatin therapy of ovarian cancer.

We studied mechanisms of resistance to the novel taxane cabazitaxel in established cellular models of taxane resistance. We also developed cabazitaxel-resistant variants from MCF-7 breast cancer cells by stepwise selection in drug alone (MCF-7/CTAX) or drug plus the transport inhibitor PSC-833 (MCF-7/CTAX-P). Among multidrug resistant (MDR) variants, cabazitaxel was relatively less cross-resistant than paclitaxel and docetaxel (15 vs. 200-fold in MES-SA/Dx5 and 9 vs. 60-fold in MCF-7/TxT50, respectively). MCF-7/TxTP50 cells that were negative for MDR but had 9-fold resistance to paclitaxel were also 9-fold resistant to cabazitaxel. Selection with cabazitaxel alone (MCF-7/CTAX) yielded 33-fold resistance to cabazitaxel, 52-fold resistance to paclitaxel, activation of ABCB1, and 3-fold residual resistance to cabazitaxel with MDR inhibition. The MCF-7/CTAX-P variant did not express ABCB1, nor did it efflux rhodamine-123, BODIPY-labeled paclitaxel, and [3H]-docetaxel. These cells are hypersensitive to depolymerizing agents (vinca alkaloids and colchicine), have reduced baseline levels of stabilized microtubules, and impaired tubulin polymerization in response to taxanes (cabazitaxel or docetaxel) relative to MCF-7 parental cells. Class III β-tubulin (TUBB3) RNA and protein were elevated in both MCF-7/CTAX and MCF-7/CTAX-P. Decreased BRCA1 and altered epithelial-mesenchymal transition (EMT) markers are also associated with cabazitaxel resistance in these MCF-7 variants, and may serve as predictive biomarkers for its activity in the clinical setting. In summary, cabazitaxel resistance mechanisms include MDR (although at a lower level than paclitaxel and docetaxel), and alterations in microtubule dynamicity, as manifested by higher expression of TUBB3, decreased BRCA1, and by the induction of EMT.

Background:ABCB1 expression is uncommon in ovarian cancers in the clinical setting so we investigated non-MDR mechanisms of resistance to taxanes.Methods:We established eight taxane-resistant variants from the human ovarian carcinoma cell lines A2780/1A9, ES-2, MES-OV and OVCAR-3 by selection with paclitaxel or docetaxel, with counter-selection by the transport inhibitor valspodar.Results:Non-MDR taxane resistance was associated with reduced intracellular taxane content compared to parental controls, and cross-resistance to other microtubule stabilising drugs. Collateral sensitivity to depolymerising agents (vinca alkaloids and colchicine) was observed with increased intracellular vinblastine. These variants exhibited marked decreases in basal tubulin polymer and in tubulin polymerisation in response to taxane exposure. TUBB3 content was increased in 6 of the 8 variants. We profiled gene expression of the parental lines and resistant variants, and identified a transcriptomic signature with two highly significant networks built around FN1 and CDKN1A that are associated with cell adhesion, cell-to-cell signalling, and cell cycle regulation. miR-200 family members miR-200b and miR-200c were downregulated in resistant cells, associated with epithelial to mesenchymal transition (EMT), with increased VIM, FN1, MMP2 and/or MMP9.Conclusions:These alterations may serve as biomarkers for predicting taxane effectiveness in ovarian cancer and should be considered as therapeutic targets.

Identification of novel drug targets in ovarian cancers has focused on expression of genes in the tyrosine kinase family. We have previously shown that the spleen tyrosine kinase (SYK) gene was expressed in 73% of 55 ovarian cancer specimens, compared to 26% of 60 breast cancer specimens. Previous studies established the role of SYK in tumor progression. Among its many targets, SYK can bind microtubules and other molecules associated with the cytoskeleton. We found SYK to be expressed in 6 of 10 ovarian carcinoma cell lines. Because of the ability of SYK to bind to microtubules, we studied the relationship of SYK expression to cellular migration. We examined several ovarian cancer cell lines that variably express SYK to elucidate the potential effects of SYK on cell migration. Our results demonstrate that knockdown of SYK expression by siRNA resulted in a decreased capability of cells to migrate using a traditional wound scratch assay. The duration of this effect was dependant upon cell line used suggesting that additional protein targets may be involved in the migratory process. The potential effects of SYK expression on invasion are currently being investigated. We will evaluate potential upstream and downstream targets to elucidate the pathway(s) responsible for SYK effects and compare within the cell lines examined. Understanding the growth signals and proteins potentially responsible for invasion and metastasis in ovarian cancer cell lines and tumor samples may eventually lead to new therapeutic approaches for ovarian cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1401. doi:10.1158/1538-7445.AM2011-1401