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Supplementary Materials from Evaluation of current methods used to analyze the expression profiles of ATP-binding cassette transporters yields an improved drug-discovery database

Background: A major issue in the treatment of acute myeloid leukemia (AML) is resistance to chemotherapeutic drugs. Multidrug resistance can be caused by ATP-binding cassette (ABC) transporters that function as drug efflux pumps. The majority of these proteins have not yet been examined in malignant diseases.Experimental Design: A newly developed microarray for the simultaneous quantification of 38 ABC transporter genes and Taqman real-time PCR was used to analyze the expression of ABC transporters in pediatric AML and healthy bone marrow. Small interfering RNA was used to verify the role of ABCA3 in drug resistance.Results: Using the microarray, we identified four new ABC transporters, which were overexpressed in many AML samples compared with healthy bone marrow: ABCA2, ABCA3, ABCB2, and ABCC10. The overexpression of these four genes was verified by real-time PCR in 42 samples from children with AML and 18 samples of healthy bone marrow. The median expression of ABCA3 was three times higher in 21 patients who had failed to achieve remission after the first course of chemotherapy than in a well-matched group of 21 patients who had achieved remission at this stage (P = 0.023). Incubation of cell lines with a number of different cytostatic drugs induced an up-regulation of ABCA3. Down-regulation of ABCA3 by small interfering RNA sensitized cells to doxorubicin.Conclusion: Our results show that ABCA2, ABCA3, ABCB2, and ABCC10 are overexpressed in childhood AML compared with healthy bone marrow. ABCA3 is the most likely transporter to cause drug resistance.

PDF file, 1202KB, Supplementary Figure 1. Kaplan-Meier Survival Curves. Supplementary Figure 2. Kaplan-Meier Survival Curves. Supplementary Table 1. Univariate analysis of clinical covariates.

Purpose: This study assesses the ability of multidrug resistance (MDR)–associated gene expression patterns to predict survival in patients with newly diagnosed carcinoma of the ovary. The scope of this research differs substantially from that of previous reports, as a very large set of genes was evaluated whose expression has been shown to affect response to chemotherapy.Experimental Design: We applied a customized TaqMan low density array, a highly sensitive and specific assay, to study the expression profiles of 380 MDR-linked genes in 80 tumor specimens collected at initial surgery to debulk primary serous carcinoma. The RNA expression profiles of these drug resistance genes were correlated with clinical outcomes.Results: Leave-one-out cross-validation was used to estimate the ability of MDR gene expression to predict survival. Although gene expression alone does not predict overall survival (OS; P = 0.06), four covariates (age, stage, CA125 level, and surgical debulking) do (P = 0.03). When gene expression was added to the covariates, we found an 11-gene signature that provides a major improvement in OS prediction (log-rank statistic P < 0.003). The predictive power of this 11-gene signature was confirmed by dividing high- and low-risk patient groups, as defined by their clinical covariates, into four specific risk groups on the basis of expression levels.Conclusion: This study reveals an 11-gene signature that allows a more precise prognosis for patients with serous cancer of the ovary treated with carboplatin- and paclitaxel-based therapy. These 11 new targets offer opportunities for new therapies to improve clinical outcome in ovarian cancer. Clin Cancer Res; 18(11); 3197–206. ©2012 AACR.

The development of multidrug resistance (MDR) to chemotherapy remains a major challenge in the treatment of cancer. Resistance exists against every effective anticancer drug and can develop by multiple mechanisms. These mechanisms can act individually or synergistically, leading to MDR, in which the cell becomes resistant to a variety of structurally and mechanistically unrelated drugs in addition to the drug initially administered. Although extensive work has been done to characterize MDR mechanisms in vitro, the translation of this knowledge to the clinic has not been successful. Therefore, identifying genes and mechanisms critical to the development of MDR in vivo and establishing a reliable method for analyzing highly homologous genes from small amounts of tissue is fundamental to achieving any significant enhancement in our understanding of MDR mechanisms and could lead to treatments designed to circumvent it. In this study, we use a previously established database that allows the identification of lead compounds in the early stages of drug discovery that are not ATP-binding cassette (ABC) transporter substrates. We believe this can serve as a model for appraising the accuracy and sensitivity of current methods used to analyze the expression profiles of ABC transporters. We found two platforms to be superior methods for the analysis of expression profiles of highly homologous gene superfamilies. This study also led to an improved database by revealing previously unidentified substrates for ABCB1, ABCC1, and ABCG2, transporters that contribute to MDR. [Mol Cancer Ther 2009;8(7):2057–66]

Supplementary Figures 1-2 from ABCA3 as a Possible Cause of Drug Resistance in Childhood Acute Myeloid Leukemia

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The lack of markers to predict chemotherapy responses in patients poses a major handicap in cancer treatment. We searched for gene expression patterns that correlate with docetaxel or cisplatin response in a mouse model for breast cancer associated with BRCA1 deficiency. Array-based expression profiling did not identify a single marker gene predicting docetaxel response, despite an increase in Abcb1 (P-glycoprotein) expression that was sufficient to explain resistance in several poor responders. Intertumoral heterogeneity explained the inability to identify a predictive gene expression signature for docetaxel. To address this problem, we used a novel algorithm designed to detect differential gene expression in a subgroup of the poor responders that could identify tumors with increased Abcb1 transcript levels. In contrast, standard analytical tools, such as significance analysis of microarrays, detected a marker only if it correlated with response in a substantial fraction of tumors. For example, low expression of the Xist gene correlated with cisplatin hypersensitivity in most tumors, and it also predicted long recurrence-free survival of HER2-negative, stage III breast cancer patients treated with intensive platinum-based chemotherapy. Our findings may prove useful for selecting patients with high-risk breast cancer who could benefit from platinum-based therapy. Cancer Res; 72(9); 2350–61. ©2012 AACR.

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Reactive oxygen species (ROS) are constantly generated in mitochondria as a consequence of normal cellular metabolic reactions and play vital roles in cell signaling and homeostasis. Prolong production of elevated levels of ROS may lead to oxidative damage, genetic instability, and genetic mutation causing cancer initiation and progression. One proposed therapeutic strategy is to alter oxidative stress from a ROS-induced tumor promoting event to a ROS-induced apoptotic signaling to kill cancer cells. Most ROS inducing therapeutic agents initially induce ROS to kill cancer cells by apoptosis; however, prolonged exposure of such drugs may increase ROS scavenging enzymes resulting in a reduced ROS levels and an adaptive microenvironment that makes cancer cells resistant to the same ROS-inducing therapeutic agents. Chronic exposure of ROS may also upregulate the multidrug resistance associated protein expression in cancer cells. We hypothesize that induction of oxidative stress may sensitize drug resistant cancer cells leading to cell death. We used two pairs of cell lines, a mitoxantrone sensitive and mitoxantrone resistant MCF7 human breast adenocarcinoma (MCF7/MCF7MX) and a H460 non-small lung cancer cell line (H460/H460MX) for our experiments to study the impact of oxidative stress on drug resistance. We employed western blot, ELISA assays, and FACS analysis to determine the total and specific protein oxidative carbonylation, protein aggregation, and cell death by apoptosis or necrosis. At basal level, both mitoxantrone resistant MCF7MX and H460MX cells showed slightly lower level of total protein carbonylation compared to their mitoxantrone sensitive counterparts. Chemical induction of oxidative stress by external sources increased the total protein carbonylation and cell death in both mitoxantrone sensitive and resistance cancer cells. We also observed an increase in protein aggregation with increasing oxidative stress. Our results indicate that oxidative stress-induced protein oxidation by carbonylation and aggregation of oxidized protein which may sensitize drug resistant cancer cells leading to cell death. Citation Format: Baikuntha P. Aryal, Jean-Pierre Gillet, V Ashutosh Rao. Oxidative stress and cell death in multidrug-resistant cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 70.

Reactive oxygen species (ROS) are constantly produced under normal as well as stress conditions and play important roles in cell signaling and homeostasis. An elevated level of ROS has been reported in most cancers which may play a critical role in both cancer progression or cancer suppression. One proposed therapeutic strategy is to alter the oxidative stress from ROS-induced tumor promoting event to a ROS-induced apoptotic signaling. Most ROS inducing therapeutic agents initially induce ROS to kill cancer cells by apoptosis; however, prolonged exposure of drugs may result in reduced ROS level and an adaptive microenvironment that makes cancer cells resistant to the same ROS-inducing therapeutic drugs. Chronic exposure of ROS may also upregulate the multidrug resistance protein expression in cancer cells. We hypothesize that differences in oxidative stress and the antioxidant response between drug sensitive and drug resistance cells may play a key role in drug resistance to redox-active drugs. We used two pairs of cell lines, a mitoxantrone sensitive and resistance MCF7 human breast adenocarcinoma (MCF7/MCF7MX) and H460 non-small lung cancer cell lines (H460/H460MX) for our experiments. We employed western blot and ELISA assays to determine the total and specific protein oxidation by carbonylation. Both MCF7MX and H460MX showed slightly lower level of total protein carbonylation compared to their mitoxantrone sensitive counterparts. In western blot analysis, we observed two specific proteins that were differentially carbonylated in MCF7MX compared to and MCF7. Both cytoskeletal and multifunctional proteins were identified by mass spectrometry and confirmed by two-color western blot analysis. Investigation of the potential roles of these specific protein carbonylation in drug resistance will be presented. Citation Format: Baikuntha P. Aryal, Jean-Pierre Gillet, V Ashutosh Rao. Oxidative stress and protein carbonylation towards multi-drug resistance in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5842.

A major issue in the treatment of T-cell acute lymphoblastic leukemia (T-ALL) is resistance to chemotherapeutic drugs. Multidrug resistance can be caused by ATP-binding cassette (ABC) transporters. The majority of these proteins have not yet been examined in T-ALL. Using a newly developed microarray for the simultaneous quantification of 38 ABC transporter genes, we observed a consistent overexpression of ABCA2/ABCA3 in clinical samples of ALL. Therefore, we analyzed the association of these two genes with drug resistance. Treatment of CCRF-CEM and Jurkat cells with methotrexate, vinblastine, or doxorubicin led to an induction of ABCA3 expression, whereas a significant increase of ABCA2 expression was only observed in Jurkat cells. To study the causal relationship of ABCA2/A3 overexpression with drug resistance, we applied RNA interference (RNAi) technology. RNAi specific for ABCA2 or ABCA3 led to a partial decrease of expression in these two ABC transporters. Upon cotreatment of RNAi for ABCA2 with methotrexate and vinblastine, a partial decrease of ABCA2 expression as well as a simultaneous increase of ABCA3 expression was observed. Vice versa, ABCA3 RNAi plus drugs decreased ABCA3 and increased ABCA2 expression. This indicates that down-regulation of one ABC transporter was compensated by the up-regulation of the other. Application of RNAi for both ABCA2 and ABCA3 resulted in a more efficient reduction of the expression of both transporters. As a consequence, a significant sensitization of cells to cytostatic drugs was achieved. In conclusion, ABCA2 and ABCA3 are expressed in many T-ALL and contribute to drug resistance. [Mol Cancer Ther 2006;5(8):1986–94]

Background: A major issue in the treatment of acute myeloid leukemia (AML) is resistance to chemotherapeutic drugs. Multidrug resistance can be caused by ATP-binding cassette (ABC) transporters that function as drug efflux pumps. The majority of these proteins have not yet been examined in malignant diseases. Experimental Design: A newly developed microarray for the simultaneous quantification of 38 ABC transporter genes and Taqman real-time PCR was used to analyze the expression of ABC transporters in pediatric AML and healthy bone marrow. Small interfering RNA was used to verify the role of ABCA3 in drug resistance. Results: Using the microarray, we identified four new ABC transporters, which were overexpressed in many AML samples compared with healthy bone marrow: ABCA2, ABCA3, ABCB2, and ABCC10. The overexpression of these four genes was verified by real-time PCR in 42 samples from children with AML and 18 samples of healthy bone marrow. The median expression of ABCA3 was three times higher in 21 patients who had failed to achieve remission after the first course of chemotherapy than in a well-matched group of 21 patients who had achieved remission at this stage (P = 0.023). Incubation of cell lines with a number of different cytostatic drugs induced an up-regulation of ABCA3. Down-regulation of ABCA3 by small interfering RNA sensitized cells to doxorubicin. Conclusion: Our results show that ABCA2, ABCA3, ABCB2, and ABCC10 are overexpressed in childhood AML compared with healthy bone marrow. ABCA3 is the most likely transporter to cause drug resistance.

Different mechanisms of drug resistance, including ATP-binding cassette (ABC) transporters, are responsible for treatment failure of tumors. We developed a low-density DNA microarray which contains 38 genes of the ABC transporter gene family. This tool has been validated with three different multidrug-resistant sublines (CEM/ADR5000, HL60/AR, and MCF7/CH1000) known to overexpress either the ABCB1 (MDR1), ABCC1 (MRP1), or ABCG2 (MXR and BCRP) genes. When compared with their drug-sensitive parental lines, we observed not only the overexpression of these genes in the multidrug-resistant cell lines but also of other ABC transporter genes pointing to their possible role in multidrug resistance. These results were corroborated by quantitative real-time reverse transcription-PCR. As the microarray allows the determination of the expression profile of many ABC transporters in a single hybridization experiment, it may be useful as a diagnostic tool to detect drug resistance in clinical samples.

No effective systemic therapy exists for patients with advanced hepatocellular carcinoma (HCC). Surgery is the mainstay treatment for patients with early stage HCC tumors. Either resection or transplantation is advocated if the HCC is within the Milan criteria. Nonsurgical treatments including percutaneous ethanol injection, radiofrequency ablation, and transcatheter arterial chemoembolization (TACE) are used as adjuvant therapy to surgery but also to treat unresectable HCC. Although a meta-analysis showed a beneficial survival effect for patients with intermediate HCC treated by TACE using doxorubicin and cisplatin, the survival benefit of systemic chemotherapy for the treatment of liver cancer is marginal at best. Although Sorafenib, a multi-tyrosine kinase inhibitor, is becoming established as the first line of therapy for advanced HCC, it was recently shown to be a substrate of ABCB1 and ABCG2, two major transporters involved in multidrug resistance (MDR) and expressed in hepatocytes. In this study, the MDR transcriptome was analyzed in a cohort of 38 patients using TaqMan-based qRT-PCR, with the aim of finding ways to sensitize this intrinsically MDR cancer. The study reveals a 45-gene signature, validated using an independent cohort of 53 patients, that predicts overall survival (OS). We used the Connectivity Map tool, designed to reveal connections among drugs, genes, and pathological states, to identify three compounds that converted the gene expression profile of HCC cell lines from one matching patients with poor OS to one with good OS. These compounds increased histone acetylation, resulting in the sensitization of tumor cells to conventional chemotherapy including cisplatin, sorafenib and 5-FU. This work provides new treatment strategies based on gene expression profiling and connectivity data for a disease for which no effective systemic treatments exist. More importantly, the current study reveals a new strategy to sensitize any type of refractory cancer to chemotherapy. Citation Format: Jean-Pierre Gillet, Jesper B. Andersen, James P. Madigan, Sudhir Varma, Chung-Pu Wu, Anna M. Calcagno, Suresh V. Ambudkar, Snorri S. Thorgeirsson, Michael M. Gottesman. Multidrug resistance transcriptome analysis highlights compounds that sensitize resistant hepatocellular carcinoma through increased histone acetylation. [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 954. doi:10.1158/1538-7445.AM2013-954

Inhibition of histone deacetylase (HDAC) enzymes represents a promising therapeutic approach in clinical oncology, as aberrant gene expression and alterations in histone acetylation due to HDACs have been implicated in tumor development and progression. Even though several histone deacetylase inhibitors (HDIs) are currently in clinical trials, so far only the HDIs romidepsin and vorinostat have been approved by the U.S. Food and Drug Administration for the treatment of cutaneous T-cell lymphoma (CTCL). During clinical trials with romidepsin in CTCL, disease progression was noted in some patients who initially responded to therapy, while the disease in other patients did not respond to therapy suggesting that both de novo and acquired resistance to romidepsin were observed. To identify molecular determinants of resistance, we selected HuT78 CTCL cells with romidepsin in the presence of inhibitors of P-glycoprotein (Pgp) to prevent upregulation of Pgp as a mechanism of resistance. Resistant sublines were approximately 250- to 385-fold resistant to romidepsin; the Pgp inhibitor tariquidar did not significantly reverse resistance. The sublines also exhibited resistance to apoptosis following treatment with the HDIs apicidin, belinostat, entinostat, panobinostat, and vorinostat. A custom gene-expression array detected elevated expression of insulin receptor (INSR) in romidepsin resistant cells compared to parental cells. Immunoblot analysis of downstream effectors of the IR pathway demonstrated a 4- to 8-fold increase in mitogen-activated protein kinase (MAPK) kinase (MEK) phosphorylation. Even though resistant cells did not respond to 48 h treatment with inhibitors of the insulin receptor, they exhibited exquisite sensitivity to treatment with as little as 1 nM of the MEK inhibitor PD0325901. Sensitivity to MEK inhibition in resistant cells was associated with restoration of the pro-apoptotic protein Bim. Combined treatment of romidepsin with MEK inhibitors also significantly yielded greater apoptosis in resistant cells compared to romidepsin and MEK inhibitor treatment alone. Gene expression analysis of circulating tumor samples obtained from patients with CTCL enrolled on the NCI 1312 Phase II romidepsin study suggested interaction of romidepsin with the MAPK pathway, indicated by altered expression of genes demonstrated to be under its control. These findings implicate activation of MEK as a resistance mechanism to romidepsin, and suggest combination of romidepsin with MEK inhibitors in clinical trials. Citation Format: Arup R. Chakraborty, Rob Robey, Zhirong Zhan, Victoria Luchenko, Michael Gottesman, Nathan Collie, Jean-Pierre Gillet, Richard Piekarz, Andrew Kossenkov, Louise Showe, Susan Bates. Resistance to the histone deacetylase inhibitor romidepsin is associated with degradation of Bim following MAPK pathway activation. [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 3399. doi:10.1158/1538-7445.AM2013-3399

Acquired and intrinsic resistance to histone deacetylase inhibitors (HDIs), a new targeted group of anti-tumor agents, limits their clinical efficacy. A detailed understanding of the mechanisms of resistance to HDIs may lead to strategies designed to increase clinical efficacy. To investigate molecular mechanisms of resistance to the HDI romidepsin (Dp), we studied a cutaneous T-cell lymphoma (CTCL) cell line, HuT 78, independently selected in verapamil (Vp) or valspodar (PSC833) to prevent the emergence of P-glycoprotein (Pgp), a known resistance mechanism. The HuT 78 sublines DpVp 50 and DpP 75 display 100-200-fold resistance to romidepsin, not due to Pgp expression. A custom-made Taqman low density gene expression array detected increased expression of insulin receptor (IR) in the resistant cells. Real-time PCR analysis confirmed the results of gene array and detected more than 50-fold upregulation of IR in the romidepsin-selected cells compared to the parental cells. Increased phosphorylation (5- to 8- fold) of mitogen activated protein kinase kinase (MEK), a downstream effector of the IR pathway, was also observed in the resistant cells compared to the parental cells. While HuT 78 cells were insensitive to MEK inhibition, resistant cells were found to be exquisitely sensitive to MEK inhibition (IC50 < 10 nM) but not to phosphatidylinositol 3-kinase (PI3K) inhibition. Combined treatment of romidepsin with low concentrations (1- 3 nM) of MEK inhibitor also resulted in increased cell death in romidepsin-resistant HuT 78 cells. The exquisite sensitivity to MEK inhibition in the resistant sublines was found to correlate with restoration of the expression of Bim (BCL2L11), a Bcl-2-homology domain-3 only (BH-3) proapoptotic protein. In the resistant cells, the MAPK pathway appeared to regulate Bim expression posttranslationally as we did not detect any gene induction of BCL2L11 or FOXO3, a transcription factor known to activate BCL2L11 expression, following MEK inhibition. These findings implicate increased activation of the MAPK pathway as a mechanism of resistance to romidepsin, and suggest that combining romidepsin with MEK inhibitors may be an effective strategy to overcome resistance.

Elevated expression of phosphorylated mitogen activated protein kinase kinase (MEK) as a mechanism of resistance to the histone deacetylase inhibitor romidepsin in HUT 78 cutaneous T-cell lymphoma cells. Histone deacetylase inhibitors (HDIs) have shown promise in the treatment of T-cell lymphomas including cutaneous and peripheral T-cell lymphomas. However, resistance to romidepsin limits its activity in some patients. A detailed understanding of the mechanisms of resistance to HDIs may lead to strategies designed to increase clinical efficacy. To study mechanisms of resistance to the HDI romidepsin, the HUT78 cutaneous T-cell lymphoma cell line was exposed to increasing concentrations of romidepsin in the presence of the P-glycoprotein (P-gp) inhibitors verapamil or valspodar (PSC-833) to prevent the emergence of P-gp, a known resistance mechanism. The DpVp35 and DpVp50 sublines are maintained in 35 ng/ml and 50 ng/ml romidepsin, respectively, in the presence of 5 µg/ml verapamil while DpP75 is maintained in 75 ng/ml romidepsin and 3 µg/ml valspodar. In 4-day cytotoxicity assays, the sublines are approximately 55-fold resistant to romidepsin and are not cross resistant to the HDIs belinostat, panobinostat or vorinostat. Low but detectable levels of P-gp do not explain the resistance. We used a custom drug resistance gene expression array and found increased expression of insulin receptor (IR) in the resistant cells that was confirmed by immunoblot analysis. Elevated expression of phosphorylated mitogen activated protein kinase kinase (MEK), a downstream effector of the IR pathway, was also observed in the resistant cells compared to the parental cells. Interestingly, resistant cells were found to be exquisitely sensitive to MEK inhibition, as significant apoptosis was observed after 48 h in the presence of 5 nM of the MEK inhibitor PD0325901 and 10 nM of the MEK inhibitor AZD 6244 as measured by flow cytometry with annexin V and by immunoblot examining poly (ADP-ribose) polymerase (PARP) cleavage. No significant apoptosis was observed in parental cells at concentrations up to 500 nM. Resistant cells were not, however, sensitive to extracellular related kinase (ERK) inhibition or phosphatidylinositol 3-kinase (PI3K) inhibition in as determined by annexin V assay. In summary, we hypothesize that activated MEK can mediate resistance to romidepsin, but may also lead to collateral sensitivity to MEK inhibitors. The emerging role of activated MEK as a mechanism of resistance to romidepsin suggests combination of romidepsin with MEK inhibitors in future clinical trials. 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 2624. doi:10.1158/1538-7445.AM2011-2624

Background: Cancer stem cells (CSCs) are presumed to possess the virtually unlimited ability to proliferate while preserving self-renewal. These cells are also highly resistant to chemotherapeutics, a feature which correlates with overexpression of ATP-binding cassette (ABC) transporters. No reports to our knowledge have shown that prolonged continuous selection of cells for drug resistance enriches cancer stem-like cells. Methods: We examined parental and drug-selected breast cancer cell lines to determine whether there is a relationship between drug resistance and the presence of cells with a CSC phenotype. MCF-7/ADR cells, generated from weakly tumorigenic parental MCF7 cells by continuous exposure to increasing concentrations of doxorubicin, were examined by microarray analysis, flow cytometry, invasion, in vivo tumorigenicity and other CSC characteristics. The putative CSC population was also examined for expression of multidrug resistance (MDR) genes using TaqMan Low Density Arrays. Results: Pathway analysis showed that MCF-7/ADR cells express ABCB1 as well as genes with patterns similar to breast CSCs. MCF-7/ADR cells grow in 3D cultures as mammospheres, are highly invasive and tumorigenic. In contrast to parental cells, over 30% of MCF-7/ADR cells are CD44+/CD24-. The CD44+/CD24- cells have the ability to self-renew and overexpress many MDR genes when compared to the CD44+/CD24+ population. No enrichment in CD44+/CD24- or CD133+ populations was detected in MCF-7/MDR or other cells with forced expression of the ABCB1 transporter or MDR genes. Conclusions: Cells with characteristics of CSCs increased in number following selection for doxorubicin resistance. Based on our findings, we speculate that prolonged drug treatment in vivo may result in expansion of a CSC-like population. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-261.

We have been developing tools to reproducibly correlate the expression of multidrug-resistance (MDR)-associated genes with response to chemotherapy in primary cancers. This study reports the results of a novel MDR gene expression analysis of primary serous carcinoma of the ovary utilizing a TaqMan Low Density Array (TLDA) chip which includes 380 previously characterized multidrug resistance (MDR)-associated genes that were initially identified in cultured cancer cells. Primary tumor samples from 133 patients from 4 sites were studied. All patients were subsequently treated with standard chemotherapy and had known clinical outcome. A 13 gene signature was identified whose expression added statistical power to the risk status of the patients based on standard clinical parameters (age, CA125, and success of surgical debulking) (log-rank statistic p=0.02). When subsets of patients with defined clinical risk were studied, we found that a subset of clinically high risk patients that had low expression of the 13 gene signature had a better outcome than would be predicted by purely clinical criteria. Similarly, clinically low risk patients with high expression of the 13 gene signature had a poorer prognosis. Since the mechanism of action of the 13 MDR genes involved in the signature are well-understood, it might be possible to devise therapeutic strategies to some of these targets with the goal of improving clinical outcome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-51.