We used DNA microarrays to characterize the global gene expression patterns in surface epithelial cancers of the ovary. We identified groups of genes that distinguished the clear cell subtype from other ovarian carcinomas, grade I and II from grade III serous papillary carcinomas, and ovarian from breast carcinomas. Six clear cell carcinomas were distinguished from 36 other ovarian carcinomas (predominantly serous papillary) based on their gene expression patterns. The differences may yield insights into the worse prognosis and therapeutic resistance associated with clear cell carcinomas. A comparison of the gene expression patterns in the ovarian cancers to published data of gene expression in breast cancers revealed a large number of differentially expressed genes. We identified a group of 62 genes that correctly classified all 125 breast and ovarian cancer specimens. Among the best discriminators more highly expressed in the ovarian carcinomas were PAX8 (paired box gene 8), mesothelin, and ephrin-B1 (EFNB1). Although estrogen receptor was expressed in both the ovarian and breast cancers, genes that are coregulated with the estrogen receptor in breast cancers, including GATA-3, LIV-1, and X-box binding protein 1, did not show a similar pattern of coexpression in the ovarian cancers.

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.

The origin of drug-resistant cells in human cancers has been a fundamental problem of cancer pharmacology. Two major contrasting hypotheses (genetics versus epigenetics) have been proposed to elucidate the mechanisms of acquired drug resistance. In this study, we answer these fundamental questions through investigation of the genetic and epigenetic pathways that control the origin of ABCB1 (MDR1) gene activation with acquired multidrug resistance in drugsensitive human sarcoma (MES-SA cells). The genetic and epigenetic bases of this selected activation involve the initiation of transcription at a site 112 kb upstream of the ABCB1 proximal promoter (P1) in the drug-resistant cells. This activation was associated with a chromatin-remodeling process characterized by an increase in acetylated histone H3 within a 968-bp region 5V of the ABCB1 upstream promoter. These alterations provide both genetic and epigenetic susceptibility for ABCB1 expression in drug-resistant cells. Complete activation of the ABCB1 gene through the coding region was proposed by interactions of selected trans-alterations or epigenetic changes on the ABCB1 proximal promoter, which occurred during initial drug exposure. Thus, our data provide evidence for a major genomic alteration that changes the chromatin structure of the ABCB1 upstream promoter via acetylation of histone H3 initiating ABCB1 activation, further elucidating the genetic and epigenetic bases that determine chemotherapeutic response in drug-resistant derivatives of MES-SA cells. (Cancer Res 2005; 65(20): 9388-97)

Taxanes are important drugs in the treatment of ovarian and other cancers, but their efficacy is limited by intrinsic and acquired drug resistance. Expression of the multidrug transporter P-glycoprotein, encoded by the MDR1 (ABCB1) gene, is one of the causes of clinical drug resistance to taxanes. To study the mechanisms of MDR1 activation related to taxanes, we established 11 multidrug-resistant variants from six ovarian cancer cell lines by continuous exposure to either paclitaxel or docetaxel. We profiled gene expression and gene copy number alterations in these cell lines using cDNA microarrays and identified a cluster of genes coactivated with MDR1 in 7q21.11-13. Regional activation was evident in nine resistant variants displaying a coexpression pattern of up to 22 genes over an 8-Mb area, including SRI, MGC4175, CLDN12, CROT, and CDK6. In six of these variants, regional activation was driven by gene copy number alterations, with low-level gains or high-level amplifications spanning the involved region. However, three variants displayed regional increases in gene expression even without concomitant gene copy number changes. These results suggest that regional gene activation may be a fundamental mechanism for acquired drug resistance, with or without changes in gene dosage. In addition to numerical and structural chromosomal changes driven by genome instability in cancer cells, other mechanisms might be involved in MDR1 regional activation, such as chromatin remodeling and DNA or histone modifications of the 7q21 region.

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.

Purpose The ovarian cancer data set from The Cancer Genome Atlas integrates genomic and proteomic data with clinical annotations based on chart abstractions. We aimed to develop an algorithm to create a matching, more accessible clinical data set cataloging time to treatment failure (TTF) of sequential lines of treatment in patients with serous ovarian cancers. Materials and Methods The master data set of 587 patients with serous ovarian cancer was condensed into a more homogeneous and clinically relevant population comprised of high-risk patients with both grade 3 cancers and stage III or IV disease, resulting in a subgroup of 450 patients. We quantified the TTF of different lines of therapy as well as different therapeutic combinations by extrapolating from the time of starting one therapy to the time of starting a subsequent therapy. Results The overall survival (OS) of patients was highly related to platinum sensitivity status, with median OS times of 56.6, 27.0, and 11.6 months in patients who had platinum-sensitive, -resistant, or -refractory disease, respectively. In high-risk patients, the median TTFs were 14.8, 10.2, 5.7, and 4.1 months with the first, second, third, and fourth lines of chemotherapy, respectively. Patients with stable disease after first-line therapy had similar OS outcomes as patients with partial remissions (34.4 v 33.7 months, respectively). Conclusion This new data set enhances the clinical annotation by providing exploitable chemotherapy benefit data that can now be paired with genomic and proteomic data within The Cancer Genome Atlas data. The major determinant of OS in this study was platinum sensitivity status. TTF decreased with each successive line of therapy. However, patients who achieved only stable disease with first-line therapy had OS similar to those with partial remission.