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From: Cancer Drug Discovery and Development:
Combination Cancer Therapy: Modulators and Potentiators Edited by: G. K. Schwartz © Humana Press Inc., Totowa, NJ
8
INTRODUCTION
For nearly 50 yr, cisplatin has been a mainstay of treatment of multiple can- cers, including ovarian, lung, and testicular cancer. However, the development of resistance impacts the utility of the drug in many patients, particularly in the setting of recurrent disease. Although the exact mechanism of cisplatin resis- tance needs to be delineated, it is widely accepted that multiple factors may contribute to cisplatin resistance.
We will discuss the history and clinical pharmacology of cisplatin and discuss the mechanisms of resistance, placing particular emphasis on the NF-gB pathway, which in multiple cancers has been shown to be associated with drug resistance.
Finally, we will discuss novel approaches that may enable the circumventing of platinum resistance.
Targeting NF-gB
to Increase the Activity
of Cisplatin in Solid Tumors
Don S. Dizon, MD, Carol Aghajanian, MD, X. Jun Yan, MD, and David R. Spriggs, MD
CONTENTS
INTRODUCTION
HISTORY
MECHANISM OF ACTION
RESISTANCE
NF-gBIN PLATINUM RESISTANCE
DEGRADATION OF IgB:THE UBIQUITIN–PROTEASOME PATHWAY
MODIFYING PLATINUM RESISTANCE—PROTEASOME INHIBITION
HISTORY
The discovery of cis-diaminedichloroplatinum (II) (CDDP) dates back to 1845, when Peyrone first synthesized the compound later known as Peyrone’s chloride. However, it was not until 1983 that the chemical structure was eluci- dated by Alfred Werner. Its role as a chemotherapeutic agent was first discovered in the 1960s in a series of experiments unrelated to cancer therapy by Dr. Saul Rosenberg (1).
As part of the preliminary investigations into the effect of magnetic fields on cellular growth, Rosenberg applied a platinum electrode to a culture dish con- taining Escherichia coli. With activation, he observed that bacterial growth ap- peared to decrease in the presence of platinum. He realized that the effect of cisplatin was inhibitory to cellular division. From this original observation came the discovery that a chemical reaction between the platinum electrode and ammonium chloride produced diamine-dichloroplatinum (DDP) and that activ- ity was confined to the cis-isomer of the molecule. In 1968, Rosenberg and colleagues proved the antiproliferative effect of the drug in the murine sarcoma 180 model (2). This was eventually followed by the publication of the first phase I clinical trial by the National Cancer Institute (NCI) in 1970, followed by the introduction of cisplatin into widespread clinical practice in 1978 (3,4).
Although numerous compounds have been synthesized, only carboplatin (CBDCA) has succeeded in gaining approval in the United States. Carboplatin differed from cisplatin in structure by the incorporation of a cyclobutane ring ligand in place of the two chloride groups of cisplatin. This resulted in a more stable molecule and was felt to be less reactive and likely less toxic. The initial phase I trial, conducted by Calvert and colleagues, revealed that carboplatin produced no neurotoxicity, ototoxicity, or nephrotoxicity in the first 60 patients treated, a vast improvement over the toxicity seen with cisplatin (5,6). Delayed myelosuppression, particularly thrombocytopenia, proved to be the dose-limit- ing toxicity. Clinical activity was seen in several tumor types, including ovarian cancer, prompting subsequent investigations. However, although safer than cisplatin, it did not solve the problem of cisplatin resistance.
MECHANISM OF ACTION
Within the extracellular hyperchloremic compartment, cisplatin exists in a stable, nonreactive form. Once it crosses cellular membranes, a series of aquation reactions occurs, displacing each chloride ion. With the initial loss of the first chloride ion, it binds to DNA preferentially by linking N(7) sites at GC sequences or GG, respectively (7,8). Following the second aquation, the remaining chloride is lost and the Pt-DNA adduct is closed, creating a bifunctional lesion. It is important to note that the diaquated platinum species does not interact with DNA alone, but can also react with RNA and proteins.
90% of adducts formed are 1,2-intrastrand crosslinks between purine resi- dues. The antitumor efficacy of each crosslink has not been established, but the intrastrand adduct, which causes local kinking and unwinding of the DNA, is felt to be an important mediator of the cytotoxicity. The interstrand adduct may also be important, as it has been shown to block both DNA and RNA polymerases (9).
Furthermore, increased DNA repair activity of the interstrand adducts within ovarian cancer cell lines has also been linked to resistance (10).
There is now substantial evidence that cisplatin kills cells by activating apoptosis. Overexpression of anti-apoptotic proteins bcl-xl and bcl-2 can pre- vent cisplatin-induced cell death (11,12). However, cell-cycle arrest at G2 has also been associated with cytotoxicity.
RESISTANCE
Multiple, independent mechanisms have been implicated in both intrinsic and acquired cisplatin resistance. The multiplicity of these mechanisms is a mark of the kind of resistance seen in DNA damaging agent chemotherapeutics. Each mechanism described below has generally been characterized as important but insufficient to explain all of the observed resistant phenotype.
Studies in cell lines with acquired platinum resistance have revealed that it is likely multifactorial. Decreased intracellular drug accumulation has been shown to develop rapidly in vivo (13). Gately has proposed a mechanism of cellular accumulation of cisplatin based on several lines of data suggesting both passive diffusion and the existence of a protein-mediated transport mechanism (14). He proposed that cisplatin entry occurs by both mechanisms and that the transport is mediated by a phosphorylation cascade, based on evidence that cisplatin up- take is modifiable with agents that inhibit the Na-K ATPase and the membrane surface. More recently, a copper transporter has been implicated (15,16). The regulation of expression for these transporters may be an potential target for intervention.
Another mechanism involves increased levels of glutathiones (GSH) or glu- tathione-S-transferase activity. Cisplatin acts as an electrophile that is particu- larly reactive with sulfur-containing nucleophiles. Binding to these molecules may be an important method of detoxification. In several cisplatin-resistant cell lines, including the ovarian cancer cell line SKOV-3R, high levels of intracellu- lar thiol have been documented (17). However, the degree to which intracellular platinum interacts with glutathione in vivo is still unclear. This mechanism has been targeted by clinical agents depleting glutathione (18). Other studies have utilized a prodrug cleaved by glutathione S-transferase pi (19,20).
Another important mechanism is that of mismatch repair (MMR). Alteration of MMR may confer intrinsic resistance to cisplatin and carboplatin (21). Fink and colleagues showed that loss of the MMR protein MSH2 resulted in twofold
resistance to cisplatin that was sufficient to impair response to cisplatin in vivo (22). The rationale behind this is that MMR acts as a detector system for DNA damage. When it senses damage, it may generate a series of events ultimately leading to apoptosis. This association between loss of MMR and decreased cell apoptosis has been shown in ovarian cancer cell lines (23,24). It is also widely recognized that loss of MMR proteins is associated with resistance to a number of chemotherapeutic agents, including cisplatin.
Cells may also be able to replicate DNA past the site of the platinum-DNA adduct in a process termed replicative bypass. This may prove to be another im- portant mechanism of clinical resistance. The data for this come from preclinical studies in both murine and human cancer cell lines. Gibbons et al. studied the effect of two types of platinum adducts—ethylenediamminedichloroplatinum(II) (en-Pt) and diamminecyclohexylamine-platinum (DACH-Pt)—on both a parental and resistant murine leukemia cell line (L1210) with resistance to either cisplatin (L1210/DDP) or DACH-Pt compounds (L1210/DACH) (25). In the L1210/DDP cell line, the en-Pt was fourfold less effective than the DACH-Pt adduct in inhibi- tion of chain elongation. In the L1210/DACH cell line, the reverse was true: the DACH-Pt was 2.7-fold less effective than the en-Pt adduct in inhibition, suggesting that replicative bypass was carrier specific. This work was subsequently confirmed in a parental and cisplatin-resistant ovarian cancer cell line (26).
NF-gB IN PLATINUM RESISTANCE
Based on the diversity of possible resistance mechanisms, it is logical to consider how such diverse processes are induced during the acquisition of resis- tance. Shared transcriptional activation is one possible mechanism. NF-gB is a transcription factor that has been linked to apoptosis rescue, inflammation, cell adhesion, differentiation, and cell growth. Since its discovery in 1986 by Sen and Baltimore, it has been the subject of investigation as a means of controlling various cellular processes, including tumorigenicity (27).
Within the cell, NF-gB is sequestered in the cytoplasm, where it is inactive and bound to IkB protein through binding to the RelA-p50 subunit of NF-gB. This interaction is a key regulatory step in the activation of NF-gB. All stimulatory signals of NF-gB, such as tumor necrosis factor (TNF)-_, growth factors, or cytokines, result in the activation of an IKK dimer that acts in a serine-specific fashion. That is, signal activation of IKK results in the phosphorylation of mem- bers of the IkB family at specific serine residues near the N-terminus, which subsequently target IKB species for degradation via the ubiquitin-proteasome pathway.
When it dissociates from IkB, NF-gB is freed and is transported to the nucleus, where it binds to specific sequences and results in expression of target genes.
Serial experiments have shown that the cellular response to NF-gB is not uniform; instead, there is variability on this response, depending on both the cell
type and the type of stimulus (28). Despite this, constitutive activation of NF-gB has been descried in a number of human cancers, including ovarian (29,30), breast (31,32), colon (33), and cervical cancer (34), to name but a few. NF-gB not only plays a role in these cellular processes, but also is felt to be a potential mediator of both chemosensitivity and drug resistance by blocking apoptosis induced by chemotherapy.
In our studies, we have examined the relationship between NF-gB activation and the acquisition of cisplatin resistance. When we examined a variety of plati- num-sensitive and -resistant cell lines, it was clear that in every case cisplatin resistance was associated with increases in NF-gB binding activity. In Fig. 1A, several different cell lines are examined for NF-gB activity and compared to the platinum-resistant phenotype. In each case, the CDDP-resistant line shows increased NF-gB-binding activity. In Fig. 1B, SK-OV-3 cells are exposed to cisplatin for various times, and an assay of IKK activity is shown for each time period.
It is possible to use the IkB/IKK systems to establish an unequivocal linkage between platinum resistance and activation of the NF-gB system. When a vector expressing IKK_ is transfected into SK-Ov-3 cells, it is possible to select for co- expression of a selectable marker such as Neomycin resistance (Fig. 2A). Those cells with IKK_ expression are substantially more resistant to cisplatin cytotox- icity (Fig. 2B). Similarly, when a mutated IKK_ or IKKB is transfected into the SK-Ov-3 cells, viable clones can be selected (Fig. 3A and 3B, respectively).
These inactive kinases act as dominant-negative genes, silencing the normal IKK dimer and leading to increased platinum sensitivity (Fig. 4).
DEGRADATION OF IKB:
THE UBIQUITIN–PROTEASOME PATHWAY
As previously discussed, the dissociation of IkB and NFgB is the key step in the activation of NFgB. This requires phosphorylation of NFgB, which subse- quently targets it for ubiquination and degradation by the 26S proteasome.
Transfectants with either wild-type or resistant IKK will cause predictable changes in IkB. As shown in Fig. 5, exposure to CDDP or transfection with a wild-type IKK results in a decrease of IkB_ and IkBB by Western blot. In contrast, the mutated IKK_ and IKKB vectors prevent the loss of the IkB family members following platinum treatment.
In normal cells, the ubiquitin-proteasome pathway is responsible for the orderly breakdown of multiple proteins and thereby helps to define protein turn- over rates. Proteasomes, large complexes of proteolytic enzymes, break down these intracellular proteins, recognizing them by their ubiquitin molecular tags.
In addition to degrading unwanted proteins, the proteasome is involved in the regulation of cellular signals that govern the cell-division cycle, as well as cell growth and differentiation.
Selective inhibition of proteasome activity has numerous effects, including attenuating the activity of NF-gB as well as inhibiting the activity of bcl-2, a gene involved in cell survival. Both NF-gB and bcl-2 are used by cancer cells to help them survive treatment with standard chemotherapy agents. In tumor cells, proteasome inhibition produces overwhelming cellular stress by stabilizing cell- cycle regulatory proteins and disrupting cellular proliferation, ultimately leading to apoptosis.
Fig. 1. (A) Gel-binding studies of the cell lines indicated. In each case, the wild-type cell is paired with the cisplatin-resistant clone. (B) In vitro kinase assay using cell extracts from SK-Ov-3 cells, treated with CDDP for times indicated. Phosphorylation on IkB was measured by densitometery to provide the bar graph below. Note that the resistant line is not treated with CDDP.
Fig. 2. (A) Transfected SK-Ov-3 cells using a selectable IKK_ expression vector with a neomycin resistance marker. (B) The effect of the IKK_ expression vector on CDDP killing, as measured by vital dye conversion.
Fig. 3. (A) Transfection with a kinase inactive mutated IKK_ expression vector selected by neomycin resistance. The Western blot illustrates the presence of the mutated kinase.
(B) Transfection with a kinase-inactive mutated IKK` expression vector selected by neomycin resistance. The Western blot illustrates the presence of the mutated kinase.
MODIFYING PLATINUM RESISTANCE—
PROTEASOME INHIBITION
Given the importance of the RelA–IkB complex in inhibiting NF-gB from translocating into the nucleus, there has been a significant effort into blocking this dissociation. One of these has been to block the 26S proteasome, which degrades IkB.
Of the agents in clinical trial, PS-341 (bortezomib, Velcade®, Millenium Pharmaceuticals) is the furthest in development. PS-341 is a dipeptidyl boronic acid inhibitor with high specificity for the proteasome. PS-341 acts through the ubiquitin-proteasome pathway to alter the expression of important proteins nec- essary for accelerated and uncontrolled cellular division. Because it targets the proteasome, it is not specifically targeting the NF-gB signaling pathway. How- ever, this effect is felt to be central to the activity of this drug, particularly in melanoma.
The phase I trials of this agent have been completed in both solid and hema- tologic malignancies. In the phase I trial in solid tumors, patients received esca- lating doses of PS-341 twice weekly for 2 wk followed by a 1-wk break.
Forty-three patients were treated, and PK data demonstrated a dose-related inhi- bition of the 20S proteasome with increasing doses. Evidence of tumor activity was seen in this cohort, with one patient with non-small-cell lung carcinoma exhibiting a partial response (35).
In the phase I trial for refractory hematological malignancies, patients were treated twice weekly for 4 wk followed by a 2-wk rest period. Twenty-seven patients were treated; PD data showed that 20S proteasome inhibition occurred in a time-dependent fashion and that this was dose-related. Complete responses Fig. 4. Effect of mutated IKK_ and mutated IKK` on platinum sensitivity in a 96-well vital dye cytoxicity assay.
were seen in patients with heavily pre-treated plasma cell dyscrasias. Other responses were seen in patients with mantle-cell and follicular lymphoma (36).
Based on this preliminary evidence, the drug has been launched into phase II and III trials in multiple disease sites. Preliminary phase II results of PS-341 in patients with melanoma who had failed two prior regimens were presented at the 2002 annual meeting of the American Society of Clinical Oncology (37). Sev- enty-eight patients received PS-341 as a twice-weekly infusion for 2 wk fol- lowed by a week off. The authors reported that 77% had a reduction or stabilization in their M protein, which was used as a surrogate for their tumor burden. This included 20% who had 90% reduction in their M protein.
Bortezomib is now approved for use in myeloma and under study in a variety of other settings.
CONCLUSION
NF-gB is a key regulator in multiple processes from inflammation to tumori- genicity and chemotherapy resistance. We have only begun to explore methods of manipulating this pathway. PS-341 can serve as proof of principle that block- ing the activation of NF-gB can lead to responses. Clinical trials of PS-341 and cisplatin are ongoing in ovarian cancer to evaluate the theory that blocking the proteasome will decrease cisplatin resistance.
Fig. 5. Effect of cisplatin and the various expression vectors on IkB family member protein levels before and after CDDP.
REFERENCES
1. Rosenberg B, Van Camp L, Grimley EB, Thomson AJ. The inhibition of growth or cell division in Escherichia coli by different ionic species and platinum (IV) complexes. J Biol Chem 1967;242(6):1347–1352.
2. Rosenberg B, VanCamp L. The successful regression of large solid sarcoma 180 tumors by platinum compounds. Cancer Res 1970;30:1799–1802.
3. Talley RW, O’Bryan RM, Gutterman JU, Brownlee RW, McCredie KB. Clinical evaluation of toxic effects of cis-diamminedichloroplatinum (NSC119875)—Phase I clinical study.
Cancer Chemother Rep 1973;57(4):465–471.
4. Lebwohl D, Canetta R. Clinical development of platinum complexes in cancer therapy: an historical perspective and an update. Eur J Cancer 1998;34(10):1522–1534.
5. Calvert A, Harland SJ, Newell DR, et al. Early clinical Studies of cis-diammine-1,1- cyclobutane dicarboxylate platinum II. Cancer Chemother Pharmacol 1982;9(3):140–147.
6. Calvert AH, Harland SJ, Newell DR, Siddik ZH, Harrap KR. Phase I studies with carboplatin at the Royal Marsden Hospital. Cancer Treat Rev 1985;12 Suppl A:51–57.
7. Eastman A. Reevaluation of interaction of cis-dichloro(ethylenediamine)platinum(II) with DNA. Biochemistry 1986;25(13):3912–3915.
8. Lemaire MA, Schwartz A, Rahmouni AR, Leng M. Interstrand cross-links are preferentially formed at the d(GC) sites in the reaction between cis-diamminedichloroplatinum (II) and DNA. Proc Natl Acad Sci USA 1991;88(5):1982–1985.
9. Corda Y, Job C, Anin MF, Leng M, Job D. Spectrum of DNA—platinum adduct recognition by prokaryotic and eukaryotic DNA-dependent RNA polymerases. Biochemistry 1993;32(33):8582–8588.
10. Zhen W, Link CJ Jr, O’Connor PM, et al. Increased gene-specific repair of cisplatin interstrand cross-links in cisplatin-resistant human ovarian cancer cell lines. Mol Cell Biol 1992;12(9):3689–3698.
11. Minn AJ, Rudin CM, Boise LH, Thompson CB. Expression of bcl-xL can confer a multidrug resistance phenotype. Blood 1995;86(5):1903–1910.
12. Miyashita T, and Reed JC. Bcl-2 oncoprotein blocks chemotherapy-induced apoptosis in a human leukemia cell line. Blood 1993;81(1):151–157.
13. Andrews PA, Jones JA, Varki NM, Howell SB. Rapid emergence of acquired cis- diamminedichloroplatinum(II) resistance in an in vivo model of human ovarian carcinoma.
Cancer Commun 1990;2(2):93–100.
14. Gately DP, and Howell SB. Cellular accumulation of the anticancer agent cisplatin: a review.
Br J Cancer 1993;67(6):1171–1176.
15. Komatsu M, Sumizawa T, Mutoh M, et al. Copper-transporting P-type adenosine triphos- phatase (ATP7B) is associated with cisplatin resistance. Cancer Res 2000;60(5):1312–1316.
16. Katano K, Kondo A, Safaei R, et al. Acquisition of resistance to cisplatin is accompanied by changes in the cellular pharmacology of copper. Cancer Res 2002;62(22):6559–6565.
17. Mistry P, Kelland LR, Abel G, Sidhar S, Harrap KR. The relationships between glutathione, glutathione-S-transferase and cytotoxicity of platinum drugs and melphalan in eight human ovarian carcinoma cell lines. Br J Cancer 1991;64(2):215–220.
18. Bailey HH, Mulcahy RT, Tutsch KD, et al. Phase I clinical trial of intravenous L-buthionine sulfoximine and melphalan: an attempt at modulation of glutathione. J Clin Oncol 1994;12(1):194–205.
19. Rosen LS, Laxa B, Boulos L, et al. Phase I study of TLK286 (glutathione S-transferase P1- 1 activated glutathione analogue) in advanced refractory solid malignancies. Clin Cancer Res 2003;9(5):1628–1638.
20. Townsend DM, Shen H, Staros AL, Gate L, Tew KD. Efficacy of a glutathione S-transferase pi-activated prodrug in platinum-resistant ovarian cancer cells. Mol Cancer Ther 2002;1(12):1089–1095.
21. Fink D, Nebel S, Aebi S, et al. The role of DNA mismatch repair in platinum drug resistance.
Cancer Res 1996;56(21):4881–4886.
22. Fink D, Zheng H, Nebel S, et al. In vitro and in vivo resistance to cisplatin in cells that have lost DNA mismatch repair. Cancer Res 1997;57(10):1841–1845.
23. Toney JH, Donahue BA, Kellett PJ, Bruhn SL, Essigmann JM, Lippard SJ. Isolation of cDNAs encoding a human protein that binds selectively to DNA modified by the anticancer drug cis-diamminedichloroplatinum(II). Proc Natl Acad Sci USA 1989;86(21):8328–8332.
24. Pil PM, and Lippard SJ. Specific binding of chromosomal protein HMG1 to DNA damaged by the anticancer drug cisplatin. Science 1992;256(5054):234–237.
25. Gibbons GR, Kaufmann WK, Chaney SG. Role of DNA replication in carrier-ligand-specific resistance to platinum compounds in L1210 cells. Carcinogenesis 1991;12(12):2253–2257.
26. Mamenta EL, Poma EE, Kaufmann WK, Delmastro DA, Grady HL, Chaney SG. Enhanced replicative bypass of platinum-DNA adducts in cisplatin-resistant human ovarian carcinoma cell lines. Cancer Res 1994;54(13):3500–3505.
27. Sen R, and Baltimore D. Inducibility of kappa immunoglobulin enhancer-binding protein Nf- kappa B by a posttranslational mechanism. Cell 1986;47(6):921–928.
28. Bours V, Bonizzi G, Bentires-Alj M, et al. NF-kappaB activation in response to toxical and therapeutical agents: role in inflammation and cancer treatment. Toxicology 2000;153(1- 3):27–38.
29. Huang S, Robinson JB, Deguzman A, Bucana CD, Fidler IJ. Blockade of nuclear factor- kappaB signaling inhibits angiogenesis and tumorigenicity of human ovarian cancer cells by suppressing expression of vascular endothelial growth factor and interleukin 8. Cancer Res 2000;60(19):5334–5339.
30. Dejardin E, Deregowski V, Chapelier M, et al. Regulation of NF-kappaB activity by I kappaB- related proteins in adenocarcinoma cells. Oncogene 1999;18(16):2567–2577.
31. Sovak MA, Bellas RE, Kim DW, et al. Aberrant nuclear factor-kappaB/Rel expression and the pathogenesis of breast cancer. J Clin Invest 1997;100(12):2952–2960.
32. Nakshatri H, Bhat-Nakshatri P, Martin DA, Goulet RJ Jr, Sledge GW Jr. Constitutive activa- tion of NF-kappaB during progression of breast cancer to hormone-independent growth. Mol Cell Biol 1997;17(7):3629–3639.
33. Lind DS, Hochwald SN, Malaty J, et al. Nuclear factor-kappa B is upregulated in colorectal cancer. Surgery 2001;130(2):363–369.
34. Nair A, Venkatraman M, Maliekal TT, Nair B, Karunagaran D. NF-kappaB is constitutively activated in high-grade squamous intraepithelial lesions and squamous cell carcinomas of the human uterine cervix. Oncogene 2003;22(1):50–58.
35. Aghajanian C, Soignet S, Dizon DS, et al. A phase I trial of the novel proteasome inhibitor PS341 in advanced solid tumor malignancies. Clin Cancer Res 2002;8(8):2505–2511.
36. Orlowski RZ, Stinchcombe TE, Mitchell BS, et al. Phase I trial of the proteasome inhibitor PS-341 in patients with refractory hematologic malignancies. J Clin Oncol 2002;20(22):4420–4427.
37. Richardson PG, Barlogie B, Berenson J, et al. A phase 2 study of bortezomib in relapsed, refractory myeloma. N Engl J Med 2003;348(26):2609–2617.
