[Cancer Biology & Therapy 3:4, 382-384, April 2004]; ©2004 Landes Bioscience
The Molecular Mysteries Underlying P-Glycoprotein-Mediated Multidrug Resistance Commentary
Chemotherapy is still one of the most effective ways to treat disseminated cancer. Unfortunately, even when multiple agents are used simultaneously, the effectiveness of chemotherapy is limited by the multidrug resistance of cancer cells. Classical multidrug resistance (MDR) is frequently associated with the decreased cellular accumulation of anti-cancer drugs and the elevated expression of an ATP-dependent drug-efflux pump, termed P-glycoprotein (P-gp).1 As shown in Figure 1, cells may be intrinsically resistant to chemotherapy in cancers originating from tissues already expressing P-gp or as a result of events related to malignant transformation. Alternatively, cancer cells may acquire resistance through selection or the induction of P-gp expression. P-gp, also known as ABCB1, belongs to the superfamily of ATP-binding cassette (ABC) transporters. ABC transporters are integral membrane proteins that transport a wide variety of substrates across cell membranes. Substrates include metabolic products, lipids, sterols, and xenobiotics such as chemotherapeutic drugs. There are 48 ABC proteins encoded in the human genome, grouped into 7 subfamilies ranging from A to G.2 At present, apart from ABCB1 (MDR1/Pgp), two other members, ABCC1 (MRP1) and ABCG2 (MXR) have been demonstrated to contribute to the multidrug resistance of tumors. In addition, further members of the A, B, C and G subfamilies that have been shown to actively transport various compounds may also contribute to selected cases of multidrug resistance. MDR affects many anticancer drugs including hydrophobic natural product drugs (e.g., doxorubicin and paclitaxel), new hydrophobic synthetic agents (e.g., imatinib mesylate),3 and more water-soluble drugs, such as cisplatin and methotrexate. Recently, to determine whether ABC transporters other than P-gp are involved in drug resistance in cancer cells, we have characterized the expression profile of the 48 ABC transporters in the National Cancer Institute-60 cancer cell panel to predict drug response among 100,000 chemical compounds. One of the major conclusions of the study was that ABCB1/MDR1 stands out among ABC transporters by conferring the strongest resistance against the greatest variety of compounds in vitro (Szakacs G, Annereau J-P, Lababidi S, Shankavaram U, Bussey K, Reinhold W, Guo Y, Kruh GD, Weinstein J, Gottesman MM, unpublished data). Undoubtedly, MDR1-mediated resistance in vitro is due to the wide substrate recognition and the high efficiency of the pump. In addition, the clinical (in vivo) predominance of MDR1 is ensured by mechanisms regulating its expression. Effective regulation not only provides the background for MDR1 expression, but also coordinates the detoxification machinery that involves phase I (oxidation, reduction or hydrolysis), phase II (conjugation), and phase III (efflux) systems. However, while a large body of information has been collected about the mechanism of action, less is known about the regulatory mechanisms that govern the tissue- and time-specific expression of MDR1/P-gp. Regulation of MDR1/P-gp is amazingly complex, and may include different regulatory mechanisms in normal tissues and in cancer cells. The global genomic instability of cancer cells may either give rise to MDR1 gene amplification or shape the chromatin structure of the MDR1 locus, all of which can lead to either the overexpression of MDR1 or the activation of the silent MDR1 gene. Furthermore, mutations may change the expression and activity of transcription factors, and gene rearrangements may result in the juxtaposition of the MDR1 gene to a constitutively active promoter.4 For a comprehensive summary of the regulation of ABC transporters, we refer the interested reader to the excellent recent review by Scotto.5 In this commentary, we merely list some of the most relevant cis-acting elements and their corresponding trans-acting proteins found within the MDR1 promoter. In most cases, MDR1 expression in cancer cells occurs by transcriptional activation rather than by the amplification of the gene, a phenomenon often observed in cell lines selected for resistance.6,7 Normal and malignant cells often rely on the same transcriptional machinery, defined by the DNA binding motifs within the MDR1 promoter. The MDR1 gene, as is true for all of the human multidrug transporters examined to date, has a “TATA-less”
KEY WORDS multidrug resistance, P-glycoprotein, MDR1, ZNRD1, transcriptional regulation
Commentary to:
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Overexpression of ZNRD1 Promotes Multidrug-Resistant Phenotype of Gastric Cancer Cells Through Upregulation of P-Glycoprotein
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Previously published online as a Cancer Biology & Therapy E-publication: http://www.landesbioscience.com/journals/cbt/abstract.php?id=743
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Received 01/29/04; Accepted 01/29/04
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*Correspondence to: Michael M. Gottesman; Laboratory of Cell Biology; National Cancer Institute; NIH; 37 Convent Drive; Room 1A09; Bethesda, Maryland 208924254 USA; Tel.: 301.496.1530; Fax: 301.402.0450; Email:
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Laboratory of Cell Biology; Center for Cancer Research; National Cancer Institute; NIH; Bethesda, Maryland USA
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Gergely Szakács G. Kevin Chen Michael M. Gottesman*
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Yongquan Shi, Yumei Zhang, Yanqiu Zhao, Liu Hong, Na Liu, Hizohang Jin, Yanglin Pan and Daiming Fan
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promoter, where, instead of a TATA box, the transcription complex is controlled by an initiator (Inr) element. The transcriptional factors acting on the MDR1 promoter can be classified into several major groups, which include the inverted CCAAT-box (Y-box) binding proteins (NF-Y, YB-1), the CAAT-box interacting proteins (c-fos, NF-kappaB), and the GC-box interacting proteins (Sp1-3, EGR1, WT1).5 Careful studies established the role of both the inverted CCAAT-box and GC-rich elements in either the constitutive or inducible expression of MDR1. Mutations within the inverted CCAAT-box eliminate basal transcriptional activity of the MDR1 promoter in SW620 cells and abolish MDR1 activation by C/EBPβ in MCF-7 cells, suggesting that CCAAT-binding proteins are critical in mediating MDR-related signals to the MDR1 promoter.8,9 The GCrich binding site serves as a docking area for a large number of transcription factors. It seems that Sp1 regulates many other ABC transporters and the specificity of the regulation may rely on the interaction between Sp family members and other transcriptional factors.5 EGR1 was shown to modulate MDR1 promoter activity in hematopoietic cells, and the Wilms’ tumor (WT) suppressor, WT1, has the ability to suppress TPA-mediated MDR1 activation in K562 cells.10,11 These results suggest a coordinated regulation of MDR1 promoter activity, with several overlapping binding sites for many different transcription factors. It is likely that through the competition for binding sites, these transcription factors regulate transcription in a highly complex and interactive manner, where the combination, rather than the individual elements of the myriads of factors shape the ultimate response and provide specificity. In this issue of Cancer Biology & Therapy, Shi et al. add yet another layer to the complexity by establishing the role of ZNDR1 in promoting the up-regulation of MDR1 in gastric cancer cells.12 ZNDR1 is a recently cloned zinc ribbon gene, which is believed to contain two zinc-ribbon domains.13 The authors speculate that the effect of ZNDR1 is mediated through the binding of its zinc fingers to the MDR1 promoter, analogous to the mechanism demonstrated for Sp1 or WT1. While this hypothesis awaits confirmation, the notion remains exciting given the possibility of targeting the MDR1 promoter in cancer therapy. Despite promising in vitro results, successful modulation of clinical MDR through the chemical blockade of drug efflux from cancer cells remains elusive.14 Over the years, several generations of MDR1 modulators have raised hopes only to fail in clinical trials. The negative results may be explained by several factors, such as the intrinsic toxicity of the modulators and the unwelcome inhibition of
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Figure 1. Development of multidrug resistant cancer. Although chemotherapy remains one of the most effective treatments of cancer, cells often become resistant to the cytotoxic agents applied. Intrinsic or primary resistance is characteristic of cancers originating from tissues constitutively expressing high levels of P-gp, such as the epithelia of the kidney, liver and intestines, the endothelial cells of the brain, ovary, testis and the adrenal cortex and placenta. Since P-gp is also expressed in hematopoietic stem cells and other circulating blood cells, intrinsic resistance is also found in several hematological malignancies.1 These localizations are consistent with the concept that P-gp plays a major role in the normal uptake and excretion of many different drugs and also serves as blood-brain, blood-testis, blood-ovary, and blood-fetus barriers for cytotoxic drugs. There are several paths a “naïve” cell might take to increase expression of P-gp. Cells enduring chronic environmental (“xenobiotic”) stress increase their P-gp expression, as has been shown in experiments applying heat shock, or partial hepatectomy in various models.17-19 Molecular events occurring during malignant transformation (such as mutations in p53) may be sufficient to produce increased MDR1 expression.20-22 More importantly, due to their inherent genetic instability and heightened frequency of mutation, cancer cells are frequently heterogeneous with respect to MDR1 expression and such cells have a selective advantage during adaptation to stress, such as hypoxia or inflammation. As a result of these mechanisms, a significant portion of malignant cells may be already prepared for defense against chemotherapy at the time of diagnosis. In addition, as a last resort, cells may “mobilize” MDR1 in direct response to drug exposure (induction).
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MDR1 residing in pharmacological barriers, resulting in the altered distribution of the simultaneously administered chemotherapy. Selective manipulation of the regulatory network underlying MDR offers a new strategy to developing new types of anticancer drugs. Indeed, using peptide combinatorial libraries expressed in yeast, Bartsevich et al. have identified novel zinc finger proteins that selectively bind to the overlapped regulatory site (EGR1/SP1/ WT1), and the same proteins were shown to selectively inhibit MDR1 expression in highly resistant cells.15,16 Reversal of MDR is the ultimate frontier in the fight against cancer. Even the best targeted chemotherapy is doomed to fail if the cytotoxic molecules don’t accumulate in the cancer cells. As more details of the signaling pathways are revealed and additional targets are identified, the promise of tumor-specific inhibition of MDR1 may become a clinical reality.
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12. Shi Y, Zhang Y, Zhao Y, Hong L, Liu N, Jin H, Yan J, Pan Y, Fan D. Overexpression of ZNRD1 promotes multidrug-resistant phenotype of gastric cancer cells through upregulation of p-glycoprotein; Cancer Biol Ther 2004; 3:377-81. 13. Fan W, Wang Z, Kyzysztof F, Prange C, Lennon G. A new zinc ribbon gene (ZNRD1) is cloned from the human MHC class I region. Genomics 2000; 63:139-41. 14. Sikic BI. Modulation of multidrug resistance: a paradigm for translational clinical research. Oncology 1999; 13:183-7. 15. Bartsevich VV, Juliano RL. Regulation of the MDR1 gene by transcriptional repressors selected using peptide combinatorial libraries. Mol Pharmacol 2000; 58:1-10. 16. Xu D, Ye D, Fisher M, Juliano RL. Selective inhibition of P-glycoprotein expression in multidrug-resistant tumor cells by a designed transcriptional regulator. J Pharmacol Exp Ther 2002; 302:963-71. 17. Chin KV, Tanaka S, Darlington G, Pastan I, Gottesman MM. Heat shock and arsenite increase expression of the multidrug resistance (MDR1) gene in human renal carcinoma cells. J Biol Chem 1990; 265:221-6. 18. Thorgeirsson SS, Huber BE, Sorrell S, Fojo A, Pastan I, Gottesman MM. Expression of the multidrug-resistance gene in hepatocarcinogenesis and regenerating rat liver. Science 1987; 236:1120-2. 19. Marino PA, Gottesman MM, Pstan I. Regulation of the multidrug resistance gene in regenerating rat liver. Cell Growth Differ 1990; 1:57-62. 20. Chin KV, Ueda K, Pastan I, Gottesman MM. Modulation of activity of the promoter of the human MDR1 gene by Ras and p53. Science 1992; 255:459-62. 21. Zastawny RL, Salvino R, Chen J, Benchimol S, Ling V. The core promoter region of the P-glycoprotein gene is sufficient to confer differential responsiveness to wild-type and mutant p53. Oncogene 1993; 8:1529-35. 22. Thottassery JV, Zambetti GP, Arimori K, Schuetz EG, Schuetz JD. p53-dependent regulation of MDR1 gene expression causes selective resistance to chemotherapeutic agents. Proc Natl Acad Sci USA 1997; 94:11037-42.
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