Yan C. Wang

Regional activation of chromosomal arm 7q with and without gene amplification in taxane-selected human ovarian cancer cell lines.

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. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045‐2257/suppmat.

Mechanisms of resistance to cabazitaxel.

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. Mol Cancer Ther; 14(1); 193–201. ©2014 AACR.

The miR‐200 family differentially regulates sensitivity to paclitaxel and carboplatin in human ovarian carcinoma OVCAR‐3 and MES‐OV cells

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.

Enhancement of paclitaxel and carboplatin therapies by CCL2 blockade in ovarian cancers

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.

Decreased levels of baseline and drug-induced tubulin polymerisation are hallmarks of resistance to taxanes in ovarian cancer cells and are associated with epithelial-to-mesenchymal transition

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.

Abstract 817: Enhancement of paclitaxel and carboplatin therapy by CCL2 blockade in ovarian cancers

Ovarian cancer is strongly associated with a pro-inflammatory leukocyte infiltrate, and very high levels of chemokines are found in ascites, including CCL2. CNTO 888, a neutralizing anti-CCL2 antibody, could inhibit the pro-tumor inflammatory infiltrate and tumor growth, thus providing clinical benefit to ovarian cancer patients. It was reported that CCL2 stimulated tumor cells to produce pro-angiogenic factors instead of directly stimulating bone marrow endothelial cells (Zhang J et al. 2009). The anti-CCL2 antibody CNTO 888 also had no effect as a monotherapy on capillary tube formation; however, CCL2 increased VEGF-A mRNA expression levels in PC-3 cells after 4-6 h of treatment. The induction of VEGF-A mRNA expression in PC-3 cells was blocked by pretreatment with a neutralizing antibody, indicating this induction was mediated by CCL2. CCL2 has been shown to be overexpressed in tumor cell lines resistant to taxanes. CCL2 may be induced by chemotherapy and mediate chemoresistance to taxanes. Moreover, this upregulation of CCL2 by taxane-based chemotherapy has been shown to occur via the JNK pathway. Study design: Our overall hypothesis is that CCL2 neutralization can inhibit tumor growth of taxane resistant metastatic ovarian cancer, and that stroma plays an important role in promoting tumor growth. This project is focused on three pairs of taxane resistant variants developed in the lab: ES-2/TP, MES-OV/TP and OVCAR-3/TP. These three cell models exhibit alterations in tubulin expression and dynamics along with other non-MDR1/P-glycoprotein mechanisms of resistance to taxanes. Using quantitative PCR, we observed elevated CCL2 expression (7x, 45x and 9x, respectively) in these cell models relative to parental controls, and confirmed these findings at the protein level by ELISA. CCR2 expression has been determined as well, by qPCR and by FACS analysis. In order to assess tumor growth by bioluminescent imaging, our taxane variants were transduced with the pHR2-gfp/luc lentiviral vector and were sorted in order to obtain homogenous gfp/luc positive population. These cells have been implanted intraperitoneally (i.p.) and subcutaneously (s.c.) in athymic nude, adult female mice. A pilot study was done to determine the growth of our three cell lines pairs injected subcutaneously or intraperitoneally into mice. Mice were then treated with antibodies to neutralize both the human tumor-derived CCL2 (CNTO 888) and the mouse orthologue of human CCL2, MCP-1 (C1142), with and without chemotherapy (paclitaxel or carboplatin). Bioluminescence images have been acquired in order to evaluate tumor growth during treatment. We observed a significant additive effect on efficacy of paclitaxel and carboplatin when the CCL2 blockade is added, compared to the chemotherapy alone (percent tumor burden at week 7 compared with week 3: paclitaxel 82%, paclitaxel + CNTO888/C1142 19%, carboplatin 74%, carboplatin + CNTO888/C1142 13%). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 817. doi:1538-7445.AM2012-817

Reannotation and Analysis of Clinical and Chemotherapy Outcomes in the Ovarian Data Set From The Cancer Genome Atlas

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.

Abstract 4001: The anti-CD47 antibody Hu5F9-G4 activates macrophages and inhibits ovarian cancer xenografts, alone and in combination with chemotherapy or immunotherapy

Macrophage activation by inhibition of CD47 signaling is a new therapeutic approach to cancers. Binding of CD47 to its receptor SIRPα on macrophages initiates a signal cascade that inhibits phagocytosis. Blocking CD47 from interaction with SIRPα by the CD47 antibody Hu5F9-G4 results in phagocytosis of cancer cells, and anticancer activity in xenografts of human cancers. We developed taxane resistant variants from human ovarian carcinoma (OC) cell lines (ES-2, MES-OV and OVCAR-3) by step-wise exposure to paclitaxel (Ptx) and the transport inhibitor valspodar (6x resistance in ES-2/TP, 9x in MES-OV/TP, and 30x in OVCAR-3/TP). These 6 lines were transduced to establish stable luciferase expression for OC xenograft (OCX) imaging. All express CD47 by immunoblotting and flow cytometry, and all express calreticulin (CRT). CRT is a prophagocytic signal that promotes phagocytosis in the presence of CD47 blockade. CRT levels are lower in the Ptx-resistant variants (p Citation Format: Rishil J. Kathawala, David C. Mundy, George E. Duran, Jens-Peter Volkmer, Kevin S. Kao, Yan C. Wang, Aditi Bellary, Irving L. Weissman, Branimir I. Sikic. The anti-CD47 antibody Hu5F9-G4 activates macrophages and inhibits ovarian cancer xenografts, alone and in combination with chemotherapy or immunotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4001.

Abstract 763: 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

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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