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Review
What Makes Tumors Multidrug Resistant? Piet Borst* Jos Jonkers Sven Rottenberg The Netherlands Cancer Institute; Division of Molecular Biology; Amsterdam, The Netherlands *Correspondence to: Piet Borst; The Netherlands Cancer Institute; Division of Molecular Biology; Plesmanlaan 121; 1066 CX Amsterdam, The Netherlands; Tel.: 0031.20.512.2880; Fax: 0031.20.669.1383; Email:
[email protected] Original manuscript submitted: 8/23/07 Manuscript accepted:08/23/07 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/4936
Key words apoptosis, breast cancer, cisplatin, doxorubicin, drug transporters, multidrug resistance, docetaxel, P-glycoprotein, senescence Acknowledgements We thank Dr. Karin de Visser for helpful comments on the paper. Our experimental work was supported by the Dutch Cancer Society (Grant NKI 2001-2472, to Piet Borst and J. Wijnholds; Grant NKI 2002-2635, to Jos Jonkers and A. Berns; Grant NKI 20053379 to Piet Borst, R. Bernards, and R.L. Beijersbergen; Grant NKI 2006-3566, to Piet Borst, Sven Rottenberg, and Jos Jonkers; and Grant NKI-2007-3772, to Jos Jonkers, Sven Rottenberg and J.H.M. Schellens) and the European Union (FP6 Integrated Project 037665-Chemores to Piet Borst and Sven Rottenberg). Sven Rottenberg was supported by fellowships from the Swiss National Science Foundation (PBBEB-104429) and the Swiss Foundation for Grants in Biology and Medicine.
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Abstract Tumors arising “spontaneously” in genetically modified mice now make it possible to study mechanisms of drug resistance in animal tumors resembling their human counterparts. We have studied mouse mammary tumors induced by conditional deletion of Brca1 and p53. These tumors respond to monotherapy with the maximal tolerable dose of doxorubicin, or docetaxel, but eventually always become resistant to the drugs. Resistance in most tumors is caused by upregulation of drug transporters and not by interference with apoptosis/senescence. The tumors also respond to cisplatin, but do not become resistant, even after repeated treatments at the maximum tolerable dose. We conclude that resistance due to interference with cell death effector pathways (apoptosis/ senescence) is not an option in these tumors, re-emphasizing doubts that such mechanisms play a role in epithelial tumors. Tumors responding to drug may shrink to less than 5% of their volume before relapsing. We argue that this resistant remnant fraction may provide a test for the tumor stem cell hypothesis and, more generally, that “spontaneous” mouse tumors resembling their human counterparts provide a useful new tool for drug development and for improving treatment regimens.
Introduction Drug resistance remains one of the most pressing problems in the management of cancer patients, as in the end most patients die because their disseminated cancer has become resistant to all drugs available. It is therefore remarkable that we still do not know what the mechanism of this pan‑resistance is. A lack of suitable animal models has undoubtedly contributed to this ignorance. This gap is now being filled by the “spontaneous” mouse tumors that can be reproducibly generated in genetically modified mice.1,2 In this perspective we discuss our experiments with one of these models, the Brca1‑/‑, p53‑/‑ breast cancer model. These tumors become invariably resistant against the maximum tolerable dose of doxorubicin or docetaxel,3 and for doxorubicin we know why.
What Are the Issues? We discuss only two: (1) What is the mechanism of multidrug resistance of cancer cells? Classically this has been attributed to the upregulation of drug transporters, such as P‑glycoprotein.4‑7 These transporters are able to extrude a range of anti‑cancer drugs out of the cell, lowering their concentration below the level required for cytotoxic effects. More recently, Scott Lowe and Clemens Schmitt and coworkers8‑15 have delineated an alternative cause of multidrug resistance, based on experiments with Myc‑induced lymphomas in mice. In this model interference with putative effector pathways of drug action, apoptosis and senescence, could result in resistance to a variety of drugs and even to irradiation. Table 1 compares this type of resistance with “classical” multidrug resistance. The issue is whether blocks in effector pathways are also important in tumors of epithelial origin, (as implied by Schmitt et al., ref. 11) or not, as argued by other investigators.16‑19 (2) What is the contribution of tumor stem cells to drug resistance? There is evidence that solid tumors do not consist of homogeneous cell populations, but recapitulate the heterogeneity of the tissue from which the tumor arises,20‑23 which contains somatic stem cells, rapidly amplifying transit cells, differentiated tumor cells and stromal cells. If the differentiated epithelial cells that make up the bulk of the tumor are drug‑sensitive, while the tumor stem cell compartment is not, the tumor will initially respond to drug, but Cell Cycle
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Table 1
A comparison of two types of multidrug resistance (MDR)
Type of MDR “Classical” Defective Damage (refs. 5–7) Effector Pathways (refs. 8,12,15) Resistance ‑ to natural product drugs
+
+
‑ to alkylating agents ‑
+
‑ to X‑rays ‑
+
Upregulation drug pumps
+ ‑
Defective apoptosis/senescence ‑ Inducible in all types of tumor cells
+
+ ‑
eventually recur.24 Realistic animal tumor models are in principle able to answer the question whether inability to obtain full eradication of the tumor is due to a resistant tumor fraction with stem cell properties.
A Mouse Breast Cancer Model for Multidrug Resistance In Vivo By selecting cells in culture for resistance to drugs, investigators have obtained information on potential mechanisms of resistance. Until recently, however, no in vivo model was available to critically test these mechanisms for their relevance in real tumors. Attempts to generate multidrug resistance (MDR) in transplantable mouse tumors or in human xenografts in vivo have failed thus far. A reasonable (but untested) explanation for these failures is that the mouse immune system is at fault: Even severely immunocompromised mice still have an innate immune system that can attack immunogenic tumor cells. Once tumor cell multiplication is inhibited by drug, the mouse immune defense will finish the tumor off. This is where “spontaneous” tumors in genetically modified mice enter the scene.1,2 As these tumors arise more or less naturally by deletions of tumor suppressor genes and/or overexpression of oncogenes, they can be expected to be less immunogenic, like most human tumors. We have used a mouse breast cancer model in which the tumor suppressor genes Brca1 and p53 are inactivated in breast epithelium by a tissue‑specific recombination induced in the developing mammary gland by a Cre recombinase gene driven by a Keratin 14 promoter.25 These mouse mammary tumors resemble familial breast cancers in human BRCA1 mutation carriers25 and they are convenient for drug studies: The growth of the, relatively superficial, tumors is easily measured and tumor samples can be taken before and during drug treatment. Tumor fragments can be transplanted into the fat pads of congenic mice and we have shown that the gene expression profiles and the drug responses of the transplanted tumors are remarkably similar to those of the parental tumor, greatly expanding the potential for testing various drug regimens on (pieces of ) the same tumor.3 When these tumor‑bearing mice were treated with the maximum tolerable dose (MTD) of doxorubicin or docetaxel, two interesting results were obtained, as illustrated by the data for doxorubicin treatment shown in Figure 1: (1) The response to drug varies from tumor to tumor. Some tumors, such as tumor T6, nearly completely www.landesbioscience.com
Figure 1. Effect of doxorubicin treatment (5 mg/kg i.v.) on the growth of Brca1‑/‑, p53‑/‑ mammary tumors in mice. The days on which treatment was given are indicated by the arrows. (Taken from ref. 3).
disappear, while others, such as T5, only show a partial response. This diversity in drug response was also seen with docetaxel3 and it reflects the diversity of mutations that the tumor‑initiating cells have accumulated on the long road between the initiating Brca1/p53 deletions and the final tumor. (2) Irrespective of the initial response, all tumors eventually become fully resistant to the MTD of doxorubicin and similar results were obtained with docetaxel.3
Mechanisms of Drug Resistance in the Doxorubicin‑resistant Tumors We used micro‑array gene expression analysis to determine how tumors become drug resistant in the Brca1‑/‑;p53‑/‑ breast cancer model. By comparing RNA from doxorubicin‑resistant and untreated tumors, we found expression of only 45 genes upregulated in the resistant tumors and none downregulated. Most of the upregulated genes were attributable to the stromal compartment (invading macrophages) and only one of the upregulated genes could be linked to drug resistance: Mdr1a, one of the genes encoding the drug-transporting mouse P-glycoproteins. The upregulation of Mdr1a and Mdf1b was verified by a quantitative RNA analysis. The results summarized in Figure 2 show that in 11 of the 13 doxorubicin‑resistant tumors resistance was associated with increased levels of Mdr1a or Mdr1b RNA, or both. The increase varied between 2.5‑ and 90‑fold. The ability to transplant fragments of the resistant tumors made it possible to test for some tumors whether the elevated Mdr1 expression was really the cause of resistance.3 First, we showed that these tumors were cross‑resistant to docetaxel. Simultaneous resistance to doxorubicin (targeting topoisomerase II) and docetaxel (targeting tubulin) is highly characteristic of MDR due to elevated P‑glycoprotein.5‑7 Secondly, we showed that resistant tumors extruded 99m Tc‑sestamibi (a P‑glycoprotein substrate) in vivo at higher rate than sensitive
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Figure 2. Levels of Mdr1a and Mdr1b RNA in doxorubicin‑resistant tumors relative to the corresponding drug‑sensitive tumors before treatment. The RNA levels were determined by a quantitative PCR‑based method. For comparison relative RNA levels are presented for another drug transporter, Mrp1. The mouse Mrp1 does not transport doxorubicin47 and is not affected by the doxorubicin treatment. (Taken in modified form from ref. 3).
tumors. This increased extrusion could be prevented by the coadministration of the P‑glycoprotein inhibitor cyclosporin A. Thirdly, we have found (unpublished) that the doxorubicin resistance was completely reversed by coadministration of doxorubicin with tariquidar, a specific P‑glycoprotein inhibitor.26 It is interesting that in some tumors a modest increase in Mdr1 expression (three‑fold) is sufficient to render a mammary tumor completely resistant against doxorubicin (Fig. 2). These low levels of P‑glycoprotein are barely visible on Western blots and completely undetectable with standard anti‑P‑glycoprotein antibodies, such as C219, in tumor sections.
Do Blocks in Apoptosis/senescence Contribute to Drug Resistance in any Tumors of Epithelial Origin?
Evidence that genetic interference with apoptosis can affect the outcome of conventional cancer therapy was first presented by Lowe et al. (ref. 27), who demonstrated the requirement of functional p53 for efficient killing of mouse embryo fibroblasts (MEFs) by ionizing radiation, 5‑fluoroucil (5‑FU), etoposide, or doxorubicin. This came as a surprise, as screens for a multidrug‑resistant phenotype among established epithelial tumor cell lines had never yielded mutants disturbed in apoptosis. In fact, the type of MDR observed by Lowe and collaborators is very different from the MDR usually found in cell lines, selected for resistance in vitro. This only affects large Do Blocks in Apoptosis/senescence Contribute to Drug Resistance in the Mouse Mammary Tumor Model? amphipathic drugs, transported by P‑glycoprotein, whereas Lowe’s resistance included 5‑FU and radiation, as summarized in Table 1. Although upregulation of Mdr1 appears to explain resistance in In subsequent work Lowe and coworkers turned to an in vivo 11 out of 13 doxorubicin‑resistant tumors, inhibition of apoptosis/ mouse lymphoma model28 to test whether a block in apoptosis senescence might explain the resistance of the remaining two. It does results in drug resistance.12 The mice carry a c‑myc transgene driven not. We did not find any significant alteration in the expression of by the lg‑m promoter‑enhancer, resulting in overexpression of the myc genes known to be involved in cellular apoptosis or senescence in oncogene in the B‑cell lineage. In this system inactivation of p53 or these two tumors (or in any of the tumors resistant to doxorubicin INK4a/ARF or overexpression of Bcl‑2 resulted in a high degree of or docetaxel).3 Why the two doxorubicin‑resistant tumors without resistance of the tumor.12,29 Schmitt et al.12 conclude that their ‘data Mdr1 upregulation are resistant, is not yet known. Notably, we do provide compelling evidence that, when assessed in an appropriate not find a decrease in topoisomerase II expression, a known cause of model, Bcl‑2 can act as a potent multi‑drug resistance gene,’ and doxorubicin resistance in cultured cells. that disruption of apoptosis can have profound effects on treatment In an attempt to force these mammary tumors to become outcome’. In subsequent work Schmitt et al.9 showed that a block resistant by inactivating drug‑damage effector pathways (apop- in apoptosis induced by Bcl-2 overexpression was less efficient than tosis/senescence), we treated the mice with cisplatin, which is not a loss of p53 and INK4a in protecting the Em‑myc lymphoma against P‑glycoprotein substrate. All tumors nicely responded to this drug, cyclophosphamide. They also found that p53 and INK4a promote the tumor often became unpalpable, but it always relapsed, even cellular senescence, if the cells are not killed by apoptosis. Hence, after the most dose‑intensive treatment of tumor transplanted into both resistance to cellular senescence and to apoptosis can contribute wild‑type mice.3 Whereas we were unable to eradicate these tumors to therapy resistance. An interesting addition to these results was the with cisplatin, we were also unable to get fully relapsed tumors that demonstration that apoptosis in the Em‑myc model can be prevented had become resistant to cisplatin, even after up to six treatments.3 by signaling through Akt (PKB) (which is known to prevent apopApparently these tumors cannot generate cisplatin resistance in all tosis) and that inhibition of Akt signaling by rapamycin can sensitize tumor cells. the cells to drug treatment.30 This shows that understanding the We conclude that resistance due to blocks in apoptosis/senescence mechanism of drug resistance could help in devising rational syneris not an available option for these mammary tumors. getic drug combinations that overcome resistance. The question is whether the Em‑myc lymphoma model, which is highly sensitive to apoptosis,31 is representative for human tumors, at large, which are for >90% of epithelial origin and devoid of a functional p53 pathway.16‑18 In our mammary tumor model, which 2784
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also lacks p53, we did not find any changes in the expression of genes involved in apoptosis, such as Bcl-2, Apaf1 or caspase genes.3 We were even unable to induce full‑blown resistance against cisplatin,3 which should have occurred if interference with cell death pathways would allow these tumor cells to escape drug‑induced death. As pointed out by Brown and coworkers16,18,19 the evidence that tumors of epithelial origin are killed by drug‑induced apoptosis/ senescence is not persuasive. Moreover, cells doomed by excessive damage will die anyhow. If cells are so severely damaged by irradiation or drugs that they cannot repair the damage, they will even die if apoptotic and senescence pathways are fully blocked.18 When cell‑cycle checkpoints are inactivated in tumor cells, the damaged cells will enter mitosis and die through mitotic catastrophe.32,33 Even if the cells do not enter mitosis, they will eventually lyse through necrosis.18
Drug‑resistant “Remnants” and the Cancer Stem Cell Hypothesis Figure 1 shows that some BRCA1‑ and p53‑deficient tumors nearly completely disappear after doxorubicin treatment, for instance tumor T6. Results with cisplatin are even more striking. After treatment with this drug tumors may become unpalpable for weeks before relapsing.3 Why are the cells in these “remnants” not killed, like the rest of the tumor cells? They could be poorly accessible to drug, or represent a noncycling sub‑population. Anatomically the remnants do not look like necrotic, fibrotic, drug‑impermeable sanctuaries, however, and they have resisted all dose‑intensive drug regimens.3 This raises the possibility that the remnants are not made up of a random sample of the cells in the original untreated tumor, but of cells that differ from the bulk of the tumor cells in their response to drug. We are tempted to think that the remnants consist of tumor‑initiating (stem?) cells resistant to cisplatin, whereas the differentiated cells in the growing tumor are sensitive. This interpretation can also be applied to the doxorubicin‑treated tumors. In some of these tumors the differentiated tumor cells would also initially be sensitive to drug, but the tumor stem (?) cell would then undergo an (epi)genetic alteration resulting in a fully resistant tumor. As the resistant tumors retain resistance on transplantation, the alteration must occur in the tumor‑initiating cell compartment rather than in a rapidly amplifying transit population. Whatever the nature of remnants, they provide a unique opportunity for learning more about intractable forms of resistance. In unpublished experiments with M. Pajic in our lab, we have shown that our mammary tumors can be dissociated into single cell suspensions by standard protocols developed for normal mouse mammary glands.34,35 These cell suspensions can be FACS‑sorted using surface markers and give rise to tumors when transplanted into mouse mammary pads. In theory it should therefore be possible to find out whether “remnants” are enriched in tumor‑initating “stem” cells and why these cells resist drug.
Disclaimer In this brief review we have discussed only two aspects of drug resistance: the causes of multidrug resistance and the potential role of stem cells. We only reviewed in vivo experiments with two mouse tumor models. Much more is known. For some drugs the cause of www.landesbioscience.com
resistance is fully known, even in human patients. An example is imatinib (Gleevec), a drug targeting the ABL protein kinase essential for survival of most Chronic Myeloid Leukaemia cells. Amino acid substitutions in the kinase explain acquired resistance in most patients.36 However, our knowledge of imatinib resistance is more the exception than the rule. Even for one of the oldest drugs in oncology, methotrexate (MTX), we do not know exactly what causes resistance in all patients. Several interesting resistance mechanisms have been found in cultured cells selected for MTX resistance, but the relative contributions of these mechanisms to resistance in patients is still in dispute.37 Mechanisms of MTX resistance have been analyzed in resistant L1210 mouse leukemic lines derived in vivo,38 but not in spontaneous tumors. We expect that mouse tumor models that resemble human tumors could help to determine which resistance mechanisms actually apply in real tumors, not only for MDR drugs, but also for other drugs used in patients.
What Next in Drug Resistance? In the final analysis, mechanisms of drug resistance in human tumors will have to be studied in patients, not mice. We think, however, that realistic and versatile mouse models can help to hone ideas and tools. To give a few examples: (1) We find that upregulation of Mdr1 genes is the main mechanism causing resistance to doxorubicin and docetaxel in our mammary tumor model. Whether this mechanism is important in drug resistance of human breast cancer remains controversial.39 This is not the end of the road, however. We are crossing in null alleles for the mouse Mdr1 genes40 into our tumor model. These P‑glycoprotein‑deficient mice will allow us to study other mechanisms of drug resistance. (2) Optimizing the toolkit. Methods for studying drug resistance have been developed using cell lines selected for resistance in vitro. These methods may not always be suitable for studying resistance in real tumors. A case in point is our experience with the C219 monoclonal antibody commonly used for immunohistochemical detection of P‑glycoprotein in tissues.41 We were unable to detect the increased levels of P‑glycoprotein in doxorubicin‑resistant tumors, although we had no difficulty in seeing endogenous P‑glycoprotein in sections of mouse liver or gut with the same batch of antibody (ref. 3 and Pajic M, Rottenberg S, unpublished). Obviously, P‑glycoprotein levels that make the tumor drug‑resistant are too low to be detected with available antibodies. (3) Only a fraction of human tumors responds to standard chemotherapy. The holy grail of chemotherapy is to identify the sensitive tumors before treatment is started. Gene expression profiles should in principle be able to allow clinicians to make the distinction between sensitive and resistant, but the results of this approach have been modest thus far. A possible explanation could be that the genetic variation in human tumors is so enormous that the relevant differences drown in the noise. In contrast, mouse tumors are genetically rather homogeneous and our tumors all originate by deletion of Brca1 and p53.25 Nevertheless the initial response of these tumors to doxorubicin varies and the variations in response to docetaxel are even more pronounced.3 We think that it should be possible to find gene expression profiles predicting drug sensitivity in such sets of genetically homogeneous tumors. A complication of BRCA1‑deficient
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tumors is that they are defective in DNA double‑strand break repair and that the genetic noise level can therefore be higher than in repair‑proficient tumors.25 We have not encountered this complication yet in our drug resistance studies, but it could presumably be circumvented by studying repair‑proficient tumor models. (4) Ways could be explored to use the mouse mammary fat pad system for analysis of human tumors. It has been shown, for instance, that seeding of mouse mammary fat pads with immortalized human stromal cells, allows outgrowth of human breast cancer pieces transplanted into this humanized fat pad.42 This system might be suitable for testing the initial response of human tumors to drug, although it would not be suitable for studying drug resistance arising later, because of immunological complications. (5) “Spontaneous” mouse tumors are potentially interesting for optimizing drug treatment before going into patients. New drugs, new combinations, new drug schedules, attempts to circumvent resistance mechanisms, can all be tested in mouse models. We are studying, for instance, an inhibitor of Poly‑ADP‑Ribose Polymerase1 (PARP) developed by Kudos/AstraZeneca.43 This inhibitor of DNA single‑strand break repair has the potential to synergize with BRCA1‑deficiency in tumors.43‑45 (6) The Brca1‑/‑, p53‑/‑ mouse mammary tumors have obvious advantages as an experimental system, but also drawbacks. The tumors are more sensitive to drug monotherapy than the corresponding human tumors and they do not metastasize. However, the number of mouse tumor models designed to resemble human counterparts is rapidly increasing1,2 and some of these models will be suitable for studying aspects of drug chemotherapy that cannot be optimally addressed in the Brca1‑/‑ model. We have found, for instance, that mouse mammary tumors induced by deletion of p53 and E-cadherin46 are more resistant than the Brca1‑/‑, p53‑/‑ model discussed here (unpublished results).
Concluding Remarks Tumor cells are opportunistic. They will try to do what it takes to become resistant to chemotherapy. As we have shown, a modest three‑fold increase in Mdr1 gene expression suffices to make mouse mammary tumors completely resistant to a drug transported by P‑glycoprotein. It is therefore conceivable that defects in cell death effector pathways could contribute to chemotherapy resistance in some human tumors, as postulated by Lowe and coworkers. These defects could specifically contribute to primary resistance of tumors, an area that has not been accessible to biochemical experiments. However, the evidence that such defects can lead to complete multidrug resistance in real tumors is not conclusive in our opinion. The range of mouse models now available may eventually settle the issue. References 1. Jonkers J, Berns A. Animal models of cancer. In: Knowles M, Selby P, eds. Introduction to the Cellular and Molecular Biology of Cancer. Oxford University, 2005:317‑36. 2. Frese KK, Tuveson DA. Maximizing mouse cancer models. Nat Rev Cancer 2007, (in press). 3. Rottenberg S, Nygren AOH, Pajic M, Van Leeuwen FWB, Van der Heijden I, Van de Wetering K, Liu X, de Visser KE, Gilhuijs KG, Van Tellingen O, Schouten JP, Jonkers J. Selective induction of chemotherapy resistance of mammary tumors in a conditional mouse model for hereditary breast cancer. Proc Natl Acad Sci USA 2007; 104:12117‑22. 4. Juliano RL, Ling V. A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochim Biophys Acta 1976; 455:152‑62. 5. Borst P, Oude Elferink R. Mammalian ABC transporters in health and disease. In: Richardson CC, Kornberg R, Raetz CHR, Thorstensen K, eds. Ann Rev Biochem. California: Science, 2002:537‑92.
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