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 as peroxidases. Studies on the normal function of P-gp suggest that it is involved in normal uptake and distribution of many drugs. C11-desmethoxy-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 barrier based on the fact that luciferin is an ABCG2 substrate at the blood-brain barrier and its passage into the brain can be detected in transgenic mice in which luciferase is expressed in mouse glioma cells. We have also shown that the specificity of mouse and human ABCG2 are very similar. 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. In collaboration with T.M. Przytycka (NLM), we have modeled how changes in single nucleotides can affect the structure of RNA. 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. Preliminary 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;in addition, P-gp-expressing cells appear in some instances to be more resistant to killing caused by exogenous TRAIL.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC005598-24
Application #
8762997
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
24
Fiscal Year
2013
Total Cost
$905,230
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
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
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
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
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:
Gottesman, Michael M; Pastan, Ira H (2015) The Role of Multidrug Resistance Efflux Pumps in Cancer: Revisiting a JNCI Publication Exploring Expression of the MDR1 (P-glycoprotein) Gene. J Natl Cancer Inst 107:
Pluchino, Kristen M; Esposito, Dominic; Moen, Janna K et al. (2015) Identification of a Cryptic Bacterial Promoter in Mouse (mdr1a) P-Glycoprotein cDNA. PLoS One 10:e0136396
Souza, Paloma S; Madigan, James P; Gillet, Jean-Pierre et al. (2015) Expression of the multidrug transporter P-glycoprotein is inversely related to that of apoptosis-associated endogenous TRAIL. Exp Cell Res 336:318-28
Weidner, Lora D; Zoghbi, Sami S; Lu, Shuiyu et al. (2015) The Inhibitor Ko143 Is Not Specific for ABCG2. J Pharmacol Exp Ther 354:384-93
Zheng, Ming; Zhang, Haili; Dill, David L et al. (2015) The role of Abcb5 alleles in susceptibility to haloperidol-induced toxicity in mice and humans. PLoS Med 12:e1001782
Keniya, Mikhail V; Holmes, Ann R; Niimi, Masakazu et al. (2014) Drug resistance is conferred on the model yeast Saccharomyces cerevisiae by expression of full-length melanoma-associated human ATP-binding cassette transporter ABCB5. Mol Pharm 11:3452-62

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