Jean-Pierre Gillet

Mechanisms of multidrug resistance in cancer.

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 numerous mechanisms including decreased drug uptake, increased drug efflux, activation of detoxifying systems, activation of DNA repair mechanisms, evasion of drug-induced apoptosis, etc. In the first part of this chapter, we briefly summarize the current knowledge on individual cellular mechanisms responsible for MDR, with a special emphasis on ATP-binding cassette transporters, perhaps the main theme of this textbook. Although extensive work has been done to characterize MDR mechanisms in vitro, the translation of this knowledge to the clinic has not been crowned with success. Therefore, identifying genes and mechanisms critical to the development of MDR in vivo and establishing a reliable method for analyzing clinical samples could help to predict the development of resistance and lead to treatments designed to circumvent it. Our thoughts about translational research needed to achieve significant progress in the understanding of this complex phenomenon are therefore discussed in a third section. The pleotropic response of cancer cells to chemotherapy is summarized in a concluding diagram.

Redefining the relevance of established cancer cell lines to the study of mechanisms of clinical anti-cancer drug resistance

Although in vitro models have been a cornerstone of anti-cancer drug development, their direct applicability to clinical cancer research has been uncertain. Using a state-of-the-art Taqman-based quantitative RT-PCR assay, we investigated the multidrug resistance (MDR) transcriptome of six cancer types, in established cancer cell lines (grown in monolayer, 3D scaffold, or in xenograft) and clinical samples, either containing >75% tumor cells or microdissected. The MDR transcriptome was determined a priori based on an extensive curation of the literature published during the last three decades, which led to the enumeration of 380 genes. No correlation was found between clinical samples and established cancer cell lines. As expected, we found up-regulation of genes that would facilitate survival across all cultured cancer cell lines evaluated. More troubling, however, were data showing that all of the cell lines, grown either in vitro or in vivo, bear more resemblance to each other, regardless of the tissue of origin, than to the clinical samples they are supposed to model. Although cultured cells can be used to study many aspects of cancer biology and response of cells to drugs, this study emphasizes the necessity for new in vitro cancer models and the use of primary tumor models in which gene expression can be manipulated and small molecules tested in a setting that more closely mimics the in vivo cancer microenvironment so as to avoid radical changes in gene expression profiles brought on by extended periods of cell culture.

Microarray-based Detection of Multidrug Resistance in Human Tumor Cells by Expression Profiling of ATP-binding Cassette Transporter Genes

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.

Prolonged Drug Selection of Breast Cancer Cells and Enrichment of Cancer Stem Cell Characteristics

BACKGROUND Cancer stem cells are presumed to have virtually unlimited proliferative and self-renewal abilities and to be highly resistant to chemotherapy, a feature that is associated with overexpression of ATP-binding cassette transporters. We investigated whether prolonged continuous selection of cells for drug resistance enriches cultures for cancer stem-like cells. METHODS Cancer stem cells were defined as CD44+/CD24⁻ cells that could self-renew (ie, generate cells with the tumorigenic CD44+/CD24⁻ phenotype), differentiate, invade, and form tumors in vivo. We used doxorubicin-selected MCF-7/ADR cells, weakly tumorigenic parental MCF-7 cells, and MCF-7/MDR, an MCF-7 subline with forced expression of ABCB1 protein. Cells were examined for cell surface markers and side-population fractions by microarray and flow cytometry, with in vitro invasion assays, and for ability to form mammospheres. Xenograft tumors were generated in mice to examine tumorigenicity (n = 52). The mRNA expression of multidrug resistance genes was examined in putative cancer stem cells and pathway analysis of statistically significantly differentially expressed genes was performed. All statistical tests were two-sided. RESULTS Pathway analysis showed that MCF-7/ADR cells express mRNAs from ABCB1 and other genes also found in breast cancer stem cells (eg, CD44, TGFB1, and SNAI1). MCF-7/ADR cells were highly invasive, formed mammospheres, and were tumorigenic in mice. In contrast to parental MCF-7 cells, more than 30% of MCF-7/ADR cells had a CD44+/CD24⁻ phenotype, could self-renew, and differentiate (ie, produce CD44+/CD24⁻ and CD44+/CD24+ cells) and overexpressed various multidrug resistance-linked genes (including ABCB1, CCNE1, and MMP9). MCF-7/ADR cells were statistically significantly more invasive in Matrigel than parental MCF-7 cells (MCF-7 cells = 0.82 cell per field and MCF-7/ADR = 7.51 cells per field, difference = 6.69 cells per field, 95% confidence interval = 4.82 to 8.55 cells per field, P < .001). No enrichment in the CD44+/CD24⁻ or CD133+ population was detected in MCF-7/MDR. CONCLUSION The cell population with cancer stem cell characteristics increased after prolonged continuous selection for doxorubicin resistance.

The Clinical Relevance of Cancer Cell Lines

Although advances in genomics during the last decade have opened new avenues for translational research and allowed the direct evaluation of clinical samples, there is still a need for reliable preclinical models to test therapeutic strategies. Human cancer-derived cell lines are the most widely used models to study the biology of cancer and to test hypotheses to improve the efficacy of cancer treatment. Since the development of the first cancer cell line, the clinical relevance of these models has been continuously questioned. Based upon recent studies that have fueled the debate, we review the major events in the development of the in vitro models and the emergence of new technologies that have revealed important issues and limitations concerning human cancer cell lines as models. All cancer cell lines do not have equal value as tumor models. Some have been successful, whereas others have failed. However, the success stories should not obscure the growing body of data that motivates us to develop new in vitro preclinical models that would substantially increase the success rate of new in vitro-assessed cancer treatments.

Drug resistance: Still a daunting challenge to the successful treatment of AML

Resistance to chemotherapy remains a challenging issue for patients and their physicians. P-glycoprotein (Pgp, MDR1, ABCB1), as well as a family of structurally and functionally related proteins, are plasma membrane transporters able to efflux a variety of substrates from the cell cytoplasm, including chemotherapeutic agents. The discovery of ABCB1 made available a potential target for pharmacologic down-regulation of efflux-mediated chemotherapy resistance. In patients with acute myeloid leukemia (AML), a neoplasm characterized by proliferation of poorly differentiated myeloid progenitor cells, leukemic cells often express ABCB1 at high levels, which may lead to the development of resistance to chemotherapy. Thus, AML seemed to be a likely cancer for which the addition of drug efflux inhibitors to the chemotherapeutic regimen would improve outcomes in patients. Despite this rational hypothesis, the majority of clinical trials evaluating this strategy have failed to reach a positive endpoint, most recently the Eastern Cooperative Oncology Group E3999 trial. Here we review data suggesting the importance of ABCB1 in AML, address the failure of clinical trials to support a therapeutic strategy aimed at modulating ABCB1-mediated resistance, and consider the type of research that should be conducted in this field going forward.

Involvement of ABC transporters in melanogenesis and the development of multidrug resistance of melanoma

Because melanomas are intrinsically resistant to conventional radiotherapy and chemotherapy, many alternative treatment approaches have been developed such as biochemotherapy and immunotherapy. The most common cause of multidrug resistance (MDR) in human cancers is the expression and function of one or more ATP‐binding cassette (ABC) transporters that efflux anticancer drugs from cells. Melanoma cells express a group of ABC transporters (such as ABCA9, ABCB1, ABCB5, ABCB8, ABCC1, ABCC2, and ABCD1) that may be associated with the resistance of melanoma cells to a broad range of anticancer drugs and/or of melanocytes to toxic melanin intermediates and metabolites. In this review, we propose a model (termed the ABC‐M model) in which the intrinsic MDR of melanoma cells is at least in part because of the transporter systems that may also play a critical role in reducing the cytotoxicity of the melanogenic pathway in melanocytes. The ABC‐M model suggests molecular strategies to reverse MDR function in the context of the melanogenic pathway, which could open therapeutic avenues towards the ultimate goal of circumventing clinical MDR in patients with melanoma.

ABCA3 as a Possible Cause of Drug Resistance in Childhood Acute Myeloid Leukemia

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.

Advances in the molecular detection of ABC transporters involved in multidrug resistance in cancer.

ATP-Binding Cassette (ABC) transporters are important mediators of multidrug resistance (MDR) in patients with cancer. Although their role in MDR has been extensively studied in vitro, their value in predicting response to chemotherapy has yet to be fully determined. Establishing a molecular diagnostic assay dedicated to the quantitation of ABC transporter genes is therefore critical to investigate their involvement in clinical MDR. In this article, we provide an overview of the methodologies that have been applied to analyze the mRNA expression levels of ABC transporters, by describing the technology, its pros and cons, and the experimental protocols that have been followed. We also discuss recent studies performed in our laboratory that assess the ability of the currently available high-throughput gene expression profiling platforms to discriminate between highly homologous genes. This work led to the conclusion that high-throughput TaqMan-based qRT-PCR platforms provide standardized clinical assays for the molecular detection of ABC transporters and other families of highly homologous MDR-linked genes encoding, for example, the uptake transporters (solute carriers-SLCs) and the phase I and II metabolism enzymes.

Toward a Better Understanding of the Complexity of Cancer Drug Resistance

Resistance to anticancer drugs is a complex process that results from alterations in drug targets; development of alternative pathways for growth activation; changes in cellular pharmacology, including increased drug efflux; regulatory changes that alter differentiation pathways or pathways for response to environmental adversity; and/or changes in the local physiology of the cancer, such as blood supply, tissue hydrodynamics, behavior of neighboring cells, and immune system response. All of these specific mechanisms are facilitated by the intrinsic hallmarks of cancer, such as tumor cell heterogeneity, redundancy of growth-promoting pathways, increased mutation rate and/or epigenetic alterations, and the dynamic variation of tumor behavior in time and space. Understanding the relative contribution of each of these factors is further complicated by the lack of adequate in vitro models that mimic clinical cancers. Several strategies to use current knowledge of drug resistance to improve treatment of cancer are suggested.