Abstract Four breast cancer cell lines (MCF-7, BT-549, MDA-MB-231 and T-47D) and four ovarian cancer cell lines (1A9/A2780, ES-2, MES-OV and OVCAR-3) were selected for taxane resistance by exposing cells to either docetaxel or paclitaxel in the presence of the P-glycoprotein inhibitor, PSC-833 (valspodar, 2 μM). All of these co-selected variants are MDR1/ABCB1(-), and the resistance to taxanes is not transporter-mediated. We have previously reported elevated class III β-tubulin (TUBB3), reduced BRCA1, elevated CDKN1A (p21), and altered epithelial-mesenchymal transition (EMT) genes in the majority of the non-MDR1 taxane variants. Inhibitors of apoptosis (IAP) proteins directly bind and inhibit several caspases, and may play a critical role in determining cell fate after exposure to chemotherapeutic agents. In this study, we profiled the expression of IAP proteins and found elevated content in this panel of taxane-resistant human breast and ovarian cancer cell lines. In both the paclitaxel- (TP) and docetaxel-selected (TxTP) ovarian cancer cell lines we observed significantly overexpressed cIAP1 (BIRC2), cIAP2 (BIRC3), XIAP (BIRC4), and Livin (BIRC7) relative to parental cell lines. Expression of cIAP2 was undetectable in OVCAR-3 and its taxane resistant cell lines, OVCAR-3/TP20 and OVCAR-3/TxTP5. Elevated cIAP1 and XIAP levels were detected in the human breast cancer variant BT-549/TxTP50, and Livin was overexpressed in all taxane-resistant breast cancer cell models. Survivin (BIRC5) is highly expressed in cancers and has been associated with taxane resistance via its effects on apoptosis and the cell cycle. We observed reduced Survivin content in the majority of the variants established in our laboratory except for the ovarian ES-2/TP80 cell line where we found slightly elevated expression resulting from paclitaxel selection relative to its parental control. In addition to these alterations in IAP content, we observed elevated Bcl-2 in ovarian (MES-OV/TP40 and MES-OV/TxTP50) and breast (MCF-7/TxTP50, BT-549/TxTP50, and MDA231/TxTP50) cancer cell lines at the transcript level by rt-PCR and confirmed by immunoblotting. Specific gene silencing by RNAi and treatment with small molecule inhibitors will test the functional significance of IAP alterations in these models of taxane resistance. Citation Format: George E. Duran, Anamaria Brozovic, Francisco J. Martinez, E Brian Francisco, Yan C. Wang, Branimir I. Sikic. Overexpression of inhibitors of apoptosis (IAP) family members in human breast and ovarian cancer models of non-MDR1 taxane resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 901. doi:10.1158/1538-7445.AM2013-901
Abstract In order to identify determinants of cellular response to this new taxane, we derived a resistant variant from the MCF-7 human breast cancer cell line by stepwise selection in cabazitaxel. The 33-fold resistance to cabazitaxel in this variant, MCF-7/CTAX, was associated with ABCB1/P-glycoprotein (P-gp) activation, but also 3-fold residual resistance to taxanes after modulation with the P-gp inhibitor PSC-833, 2 μM. Another variant was established by co-selecting in the presence of PSC-833 (MCF-7/CTAX-P). This variant was negative for ABCB1 transcripts and accumulated parental levels of rhodamine-123, BODIPY-labeled paclitaxel and [3H]-docetaxel, indicating that the taxane resistance (9-fold) observed in this cell line is not mediated by transporters. However, we observed a 34% reduction in bound fluorescent-labeled paclitaxel in MCF-7/CTAX-P relative to parental controls by flow cytometry. These cells are hypersensitive to depolymerizing agents (vincas and colchicine) indicating a change in tubulin dynamic instability, and we observed reduced baseline tubulin polymer in untreated MCF-7/CTAX-P cells, and impaired tubulin polymerization in response to taxane exposure (cabazitaxel or docetaxel) relative to MCF-7 parental cells. Quantitative PCR and immunoblotting confirmed elevated levels of the class III (TUBB3) β-tubulin isotype in both MCF-7/CTAX and MCF-7/CTAX-P. Reduced BRCA1 content was observed in both MCF-7 variants, which could affect taxane response via the regulation of the mitotic spindle checkpoint. Gene silencing with specific siRNAs confirmed that reduced TUBB3 sensitizes cells to cabazitaxel, and reduced BRCA1 results in taxane resistance. In addition, altered epithelial-mesenchymal transition markers (elevated Vimentin, reduced E-cadherin) are associated with cabazitaxel resistance in these MCF-7 variants. 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. Citation Format: George E. Duran, Yan C. Wang, E Brian Francisco, Francisco J. Martinez, Branimir I. Sikic. Resistance to cabazitaxel is associated with ABCB1/P-glycoprotein activation, alterations in β-tubulin content and dynamics, reduced BRCA1, and a mesenchymal phenotype in MCF-7 human breast cancer variants. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 763. doi:10.1158/1538-7445.AM2014-763
<p>The supplementary data include one table and 5 figures. Table S1 presents cabazitaxel cytotoxic activity in the human breast cancer cell line MCF-7, uterine sarcoma cell line MES-SA, and ovarian cancer cell lines ES-2, MES-OV, and OVCAR-3. Figure S1 presents the molecular structure of cabazitaxel and cytotoxicity curves for three taxanes in MCF-7 cells. Figure S2 shows an immunoblot of drug transporter proteins in parental and drug resistant variants of MCF-7. Figure S3 shown rhodamine123 and BODIPY-paclitaxel accumulation by flow cytometry in parental and resistant variants. Figure S4 shows polymerized versus soluble tubulin in parental vs resistant variants. Figure S5 shows expression of apoptotic regulators.</p>
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.
ABCB1 expression is uncommon in ovarian cancers in the clinical setting so we investigated non-MDR mechanisms of resistance to taxanes. 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. 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. These alterations may serve as biomarkers for predicting taxane effectiveness in ovarian cancer and should be considered as therapeutic targets.
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