Resistance to chemotherapy occurs in cancer cells because of intrinsic or acquired changes in expression of specific proteins. We have studied resistance to natural product chemotherapeutic agents such as doxorubicin, Vinca alkaloids, and taxol. In most cases, cells become simultaneously resistant to multiple drugs because of reductions in intracellular drug concentrations. For the natural product drugs, this cross-resistance is frequently due to expression of an energy-dependent drug efflux system (ABC transporter) known as P-glycoprotein (P-gp), the product of the MDR1 or ABCB1 gene, or to other members of the ABC transporter family, including ABCB5. To explore the possibility that other members of the ABC family of transporters may be involved in drug resistance in cancer, we have developed real-time PCR for detection of most of the 48 known ABC transporters; these techniques have been used to correlate expression of novel ABC transporters in cancer cell lines of known drug resistance. Expression of approximately 30 ABC transporters has been shown to correlate in the NCI-60 cell lines with resistance to specific cytotoxic drugs. Furthermore, this analysis has revealed that some drugs are more toxic to P-gp-expressing cells than to non-expressors, suggesting a novel approach to treatment of MDR cancers. Several different chemical classes with this property, including thiosemicarbazides (e.g., NSC73306), have been identified. A quantitative structure activity analysis of NSC73306 analogs, a further correlation analysis in the NCI-60 cell lines, and a high-throughput screen for compounds in the U.S. Pharmacopeia that kill P-gp-expressing cells have yielded many additional compounds with improved ability to kill selectively P-gp-expressing cells, but also with improved solubility properties. In addition, the compound tiopronin, which is a sulfhydryl donor used clinically to treat cystinosis, and some of its derivatives have been shown to be powerful selective agents for killing MDR cells. In this case, expression of P-gp is not required for multidrug-resistant cells to be sensitive to tiopronin, which appears to kill by targeting glutathione peroxidase. Studies on the normal function of P-gp suggest that it is involved in normal uptake and distribution of many drugs. C11-desmethyl-loperamide has been developed in collaboration with Robert Innis in NIMH to PET image distribution of this specific P-gp substrate in cancers and in the brain, with and without treatment with potent inhibitors of P-gp such as tariquidar. PET ligands that are weak bases are trapped in lysosomes, amplifying the uptake signal in the brain, in some normal tissues, and in cancers. We have shown that among three most prominent transporters at the blood-brain barrier (ABCB1, ABCC1, ABCG2), this compound is specific for ABCB1 (P-gp). We have developed a system for analysis of ABCG2 expression at the blood-brain and the blood-placental barriers based on the fact that luciferin is an ABCG2 substrate at these barriers and its passage into the brain or into developing fetuses can be detected in transgenic mice in which luciferase is expressed at the blood-brain barrier or blood-placental barrier. We are now extending this model to examine the role of transporters in brain tumors and in the placenta. We have also shown that the specificity of mouse and human ABCG2 are very similar, meaning that mouse models adequately reflect the function of human ABCG2. A synonymous polymorphism of P-gp (C3435T, no amino acid change) in the setting of a specific P-gp haplotype can affect efficiency of P-gp pumping by altering the rhythm of protein folding and changing substrate and inhibitor interactions with P-gp. The C3435T haplotype appears to change mRNA folding, and cause a major translational delay, which results in altered conformation of P-gp. Stable transfectants of porcine LLC-PK1 cells with the haplotype form of P-gp show altered drug resistance and inhibitor sensitivity compared to wild-type P-gp transfectants. We have created a highly sensitive, quantitative assay for ABC transporter mRNAs and other mRNAs associated with drug resistance in cultured cancer cells. We have studied samples from human ovarian cancer, hepatocellular cancer (HCC), and acute myelogenous leukemia (AML) in some detail. In ovarian cancer, there is an 11-gene MDR signature associated with poor response to chemotherapy. In HCC, the signature is more complex, but accurately distinguishes poor prognosis vs. better prognosis cancer. Agents that change the pattern of gene expression from poor prognosis to better prognosis patterns also sensitize cultured HCC cells to anti-cancer drugs. For AML, samples from the same patients before and after chemotherapy were studied. In this case, resistance in each case shows a different pattern of expression of ABC genes and other MDR genes, suggesting that individualized approaches to resistance to therapy will be needed. Studies in collaboration with Kevin Chen (NINDS) on regulation of expression of ABCG2 during development indicate that it is regulated during development post-transcriptionally by a microRNA (miRNA) that targets the 3 prime non-coding region of ABCG2 mRNA. ABCG2 mRNA levels are high in pluripotent stem cells, but are not expressed as protein until the cells begin to differentiate. One of the interesting phenotypes frequently associated in tissue culture with expression of ABCB1 is altered response to agents that induce apoptosis. Studies using tet-inducible P-gp KB cell lines indicate that the apoptosis-inducing ligand TRAIL is decreased in expression when P-gp is expressed in KB cells. This provides a potential mechanism for altered sensitivity of KB cells to exogenous TRAIL. ABCB5 is a close molecular relative of ABCB1. It is expressed in pigmented cells in the brain and eye, and in melanoma. In collaboration with Richard Cannon (University of Otega, New Zealand) we have shown that when expressed in yeast, ABCB5 is a multidrug transporter. ABCB5 knock-out mice are sensitive to the major tranquilizer haloperidol, consistent with a role of this transporter in the brain (with Gary Peltz, Stanford School of Medicine).

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC005598-26
Application #
9153480
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
26
Fiscal Year
2015
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Kannan, Pavitra; Schain, Martin; Kretzschmar, Warren W et al. (2017) An automated method measures variability in P-glycoprotein and ABCG2 densities across brain regions and brain matter. J Cereb Blood Flow Metab 37:2062-2075
Esser, Lothar; Zhou, Fei; Pluchino, Kristen M et al. (2017) Structures of the Multidrug Transporter P-glycoprotein Reveal Asymmetric ATP Binding and the Mechanism of Polyspecificity. J Biol Chem 292:446-461
Kumar, Jeyan S; Wei, Bih-Rong; Madigan, James P et al. (2016) Bioluminescent imaging of ABCG2 efflux activity at the blood-placenta barrier. Sci Rep 6:20418
Bakhsheshian, Joshua; Wei, Bih-Rong; Hall, Matthew D et al. (2016) In Vivo Bioluminescent Imaging of ATP-Binding Cassette Transporter-Mediated Efflux at the Blood-Brain Barrier. Methods Mol Biol 1461:227-39
Weidner, Lora D; Fung, King Leung; Kannan, Pavitra et al. (2016) Tariquidar Is an Inhibitor and Not a Substrate of Human and Mouse P-glycoprotein. Drug Metab Dispos 44:275-82
Fung, King Leung; Kapoor, Khyati; Pixley, Jessica N et al. (2016) Using the BacMam Baculovirus System to Study Expression and Function of Recombinant Efflux Drug Transporters in Polarized Epithelial Cell Monolayers. Drug Metab Dispos 44:180-8
Pluchino, Kristen M; Hall, Matthew D; Moen, Janna K et al. (2016) Human-Mouse Chimeras with Normal Expression and Function Reveal That Major Domain Swapping Is Tolerated by P-Glycoprotein (ABCB1). Biochemistry 55:1010-23
Liow, Jeih-San; Zoghbi, Sami S; Hu, Shuo et al. (2016) (18)F-FCWAY, a serotonin 1A receptor radioligand, is a substrate for efflux transport at the human blood-brain barrier. Neuroimage 138:134-140
Kumar, Jeyan S; Miller Jenkins, Lisa M; Gottesman, Michael M et al. (2016) The Drug Excipient Cyclodextrin Interacts With d-Luciferin and Interferes With Bioluminescence Imaging. Mol Imaging 15:
Greene, James M; Levy, Doron; Herrada, Sylvia P et al. (2016) Mathematical Modeling Reveals That Changes to Local Cell Density Dynamically Modulate Baseline Variations in Cell Growth and Drug Response. Cancer Res 76:2882-90

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