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Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins (C.J.S., J.-I.P., D.M.R., S.R.D., D.W.B., B.D.N.) and Department of Medicine, Johns Hopkins University School of Medicine (D.W.B.), Baltimore, Maryland 21231; and Cephalon, Inc. (B.R.), West Chester, Pennsylvania 19380
Address all correspondence and requests for reprints to: Dr. Barry Nelkin, CRB, Room 552, 1650 Orleans Street, Baltimore, Maryland 21231. E-mail: bnelkin{at}jhmi.edu.
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Abstract |
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Objective: The objective of this study was to investigate a systemic therapeutic approach to treat MTC. We studied the sensitivity of an MTC cell line and xenograft to irinotecan, alone and in combination with the tyrosine kinase inhibitor, CEP-751.
Results: In TT cell culture and xenografts, irinotecan treatment was highly effective. This effect was augmented by treatment with CEP-751. Treatment of TT cell xenografts resulted in durable complete remission in 100% of the mice, with median time to recurrence of 70 d for irinotecan alone and more than 130 d for irinotecan plus CEP-751. Although irinotecan induced an S phase checkpoint arrest in TT cells, CEP-751 in combination with irinotecan resulted in a loss of this arrest. CEP-751 induced a loss in the induction of the DNA repair program marked by phospho-H2AX and the checkpoint pathway marked by the activated Chk1 pathway.
Conclusions: Irinotecan treatment was highly effective in a preclinical model of human MTC, resulting in complete remission in 100% of the xenografts treated. The duration of remission was further enhanced by combination with the kinase inhibitor, CEP-751. These results suggest that irinotecan, alone or in combination, may be useful for the treatment of MTC.
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Introduction |
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MTC can be inherited as an autosomal dominant syndrome in about 20% of the cases (7). These inherited forms of MTC occur within three related multiple endocrine neoplasia type 2 (MEN 2) syndromes: MEN 2A, MEN 2B, and familial MTC (7). The germline genetic defects implicated in the development of the MEN 2 syndromes are mutations resulting in activation of the RET receptor tyrosine kinase (7). RET somatic mutations are detectable in approximately 40% of sporadic MTC, and RET is expressed in virtually all MTC tumors (8). The association of activated RET mutants with MTC and the ability of activated RET to transform cells and transgenic animals make RET an attractive target for possible treatment of MTC (9, 10, 11, 12, 13). Recent reports from this laboratory and others have identified pharmacological inhibitors of RET (14, 15, 16, 17, 18, 19). These inhibitors have been shown to inhibit the growth of MTC cells and RET-transformed fibroblasts in vitro and in vivo, suggesting a potential therapeutic utility in cases of MTC with RET mutations. In addition to therapy targeted to RET, there is a need for systemic therapy for the approximately 50% of sporadic MTC patients whose tumors lack RET mutations.
In this report we show that the topoisomerase I poison, irinotecan, is highly effective against TT cells in culture and TT cell xenografts in a nude mouse model. Irinotecan caused complete remission of the xenograft tumors, with a median duration of response of approximately 70 d. We show that irinotecan causes the cells to arrest in early S phase. We also show that the tyrosine kinase inhibitor, CEP-751, acts additively in culture with irinotecan and results in a loss of S phase checkpoint induction. CEP-751 in combination with irinotecan also caused complete remission of the TT xenografts in a nude mouse model, with five of nine mice apparently cured, with complete remission continuing for more than 130 d. Our results suggest that irinotecan may be an effective treatment for MTC, and this treatment may be improved in combination with CEP-751.
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Materials and Methods |
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Irinotecan was purchased from Pfizer (New York, NY) under the brand name Camptosar. CEP-751 was supplied by Cephalon, Inc. (West Chester, PA), and was dissolved in dimethylsulfoxide for cell culture experiments (19, 20). CEP-751 was formulated in a 40% polyethylene glycol 1000, 10% povidone C30, and 2% benzyl alcohol in sterile water for use in nude mouse experiments. The phospho-Chk1 (checkpoint kinase 1) (Ser345) and phospho-cyclin-dependent kinase 2 (phospho-cdk2; Thr160) antibodies were purchased from Cell Signaling Technology (catalog no. 2341 and 2561; Beverly, MA). The cdc25A (cell division cycle 25A) antibody was purchased from Labvision (catalog no. MS-640; Fremont, CA). The phospho-H2AX (histone 2A family member X) and glyceraldehyde-3-phospate dehydrogenase antibodies were purchased from Trevigen (catalog no. 4411-PC and 2275-PC; Gaithersburg, MD). The secondary antibodies were purchased from Santa Cruz Biotechnology (SC-2004 and SC-2005, Santa Cruz, CA). Phosphatase inhibitor and protease inhibitor cocktails were purchased from Sigma-Aldrich Corp. (P2850, P5726, and P8340; St. Louis, MO).
Cell culture
The human MTC cell line, TT, is available from American Type Culture Collection (Manassas, VA). Cells were maintained in RPMI 1640 medium supplemented with 16% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin in a 37 C incubator with 5% CO2. For cell culture experiments, cells were seeded 48 h before use and treated in normal serum conditions.
Western blotting
Cells were treated for 48 h as described above. They were then washed with PBS and harvested by scraping the cells with 1x sodium dodecyl sulfate lysis buffer [2% sodium dodecyl sulfate and 62.5 mM Tris (pH 6.8)]. Lysates were electrophoresed on 4–20% gradient polyacrylamide gels and transferred onto polyvinylidene difluoride. Blots were probed at 4 C overnight with primary antibodies diluted 1:1000 in 5% milk, except for phospho-H2AX, which was diluted in 5% calf serum diluted in Tween/Tris-buffered saline. Secondary antibodies were diluted 1:5000, and blots were visualized using Supersignal Pico Chemiluminescence (Pierce Chemical Co., Rockford, IL).
Growth curves
Growth curves were performed four times using the 3,4,5-dimethylthiazol-2,5-diphenyltetrazolium (MTT) assay (M2128, Sigma-Aldrich Corp.) as described previously (21). Cells were seeded in 24-well plates using phenol red-free RPMI 1640 with 16% FBS. After 48 h, the medium was replaced with normal serum media (16% FBS) containing irinotecan or CEP-751, alone or in combination, at the appropriate concentrations or with dimethylsulfoxide as a vehicle control. In every experiment, each concentration was used in quadruplicate, and the drug-containing medium was replaced every other day.
Combination index
The interaction between CEP-751 and irinotecan was analyzed in three separate experiments using the combination index method of Chou and Talalay (22) to determine whether the combination was additive or synergistic. Cell viability assays (MTT) were performed in quadruplicate on cells at various concentrations of each drug, alone and in combination, at a fixed ratio.
Cell cycle analysis
TT cells were seeded in six-well plates (106 cells/well) and treated for 48 h with drug in normal serum conditions. Treatment groups were untreated, 5 µM irinotecan, 100 nM CEP-751, and irinotecan plus CEP-751. The cells were trypsinized from the plates, then lysed in Hoechst 33258 staining solution for fluorescence-activated cell sorting analysis (0.56% Nonidet P-40, 3.7% formaldehyde, and 11 µg/ml Hoechst 33258 in PBS). The nuclei were analyzed using an LSR Flow Cytometer (BD Biosciences, Franklin Lakes, NJ) gated for single nuclei. The cell cycle profile was determined using 10,000 gated nuclei with CellQuest software (version 3.3, BD Biosciences).
In vivo tumor growth in nude mice
TT cells suspended in Matrigel (5 x 106 cells/200 µl) were inoculated sc into the right flank of 4- to 6-wk-old male athymic nude (nu/nu) mice (Harlan Laboratories, Indianapolis, IN). Once palpable, tumors were measured at indicated intervals using Vernier calipers. Tumor volumes were calculated using the formula: length x width x height x (0.5236). After a 2- to 3-wk period, tumors reached approximately 0.1 cc in average size, and animals were sorted into groups of 10 to achieve equal distribution of tumor size in all treatment groups. Treatment groups were made up of mice treated with vehicle control (40% polyethylene glycol 1000, 10% povidone C30, and 2% benzyl alcohol in sterile water), CEP-751 (10 mg/kg mouse/dose), irinotecan (10 mg/kg mouse/dose) plus vehicle control, and irinotecan plus CEP-751. Animals received once daily ip injection of irinotecan for two cycles of 2 wk each, separated by a 1-wk rest period, with each cycle consisting of 5 d treatment on, 2 d off, followed by 5 d on treatment. Animals treated with CEP-751 alone or in combination with irinotecan were treated twice daily with sc injections using the same schedule. At the end of the experiments, animals were killed by CO2 asphyxiation. Statistical analysis of differences in tumor volumes was performed using Student’s t test. All animal studies were performed according to protocols approved by the Johns Hopkins Animal Care and Use Committee.
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Results |
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We evaluated the activity of irinotecan against the human MTC cell line, TT. The TT cell line was derived from an apparently sporadic case of MTC harboring a C634W-activating mutation in the RET receptor tyrosine kinase gene (23). In numerous studies, the TT cell line has been shown to reflect the resistance of MTC to chemotherapy and radiation (24, 25, 26, 27). TT cells were treated with irinotecan, and growth was evaluated by MTT assay over a 2-d period. Figure 1
shows that irinotecan inhibited the growth of TT cells even at the lowest concentration of 200 nM and caused significant cytotoxicity at doses greater than 500 nM. The calculated IC50 was determined to be 2 µM. This activity suggests that TT cells are sensitive to inhibition by topoisomerase I.
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In some tumor types, the inhibition of receptor tyrosine kinases or their downstream signal transduction pathways can augment the effects of irinotecan and other chemotherapeutic agents (28). We have previously shown that the tyrosine kinase inhibitors, CEP-701 and CEP-751, target RET and inhibit the growth of TT cells (19). Therefore, we explored the effects of the combination of irinotecan and CEP-751. Figure 2A shows TT cells treated for 48 h with irinotecan at 1 or 5 µM in the presence or absence of 100 nM CEP-751 in normal 16% FBS medium. There was a significant degree of cell death with 5 µM irinotecan treatment of the TT cells, but at 1 µM irinotecan, the cells showed a slightly flatter morphology, with minimal cell death. The growth curve demonstrated that 5 µM irinotecan was cytotoxic, whereas 1 µM irinotecan was cytostatic (Fig. 2B). CEP-751-treated cells showed a cytostatic effect, with some cell death (Fig. 2, A and B), consistent with previous results obtained in this laboratory (19). Addition of 100 nM CEP-751 to 1 µM irinotecan resulted in a significant increase in cell death over that using either agent alone. The combination of 1 µM irinotecan and 100 nM CEP-751 resulted in cell death comparable to that of the 5 µM irinotecan-treated cells (Fig. 2, A and B). When 100 nM CEP-751 was combined with 5 µM irinotecan, there was a minimal increase in cell death over that with irinotecan alone due to the effectiveness of irinotecan alone at this dose (Fig. 2, A and B).
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). Irinotecan treatment is effective against TT xenografts, and this activity is augmented by CEP-751
After determining the effectiveness of these drugs against TT cells in vitro, we examined their activity in a nude mouse model. Mice bearing palpable sc TT xenograft tumors were treated for 5 d/wk for 2 wk, rested for 1 wk, and then treatment was repeated for another 2-wk cycle. Thus, the treatment was completed on d 33. Irinotecan was administered by ip injection of 10 mg/kg·d. CEP-751 was administered at 10 mg/kg by sc injection twice daily. The mice tolerated the treatments well, with only minimal transient weight loss observed. Dramatically, 100% of TT cell xenografts treated with either irinotecan alone or irinotecan plus CEP-751 responded with complete remission (Fig. 3A). The group treated with irinotecan alone had a median duration of remission of 70 d. The effect of irinotecan was significant vs. the control mice for each of these irinotecan-treated groups (P < 0.0001). Treatment with a combination of irinotecan and CEP-751 extended the duration of complete remission to a median of more than 130 d (P < 0.0001 vs. irinotecan alone; Fig. 3B). Indeed, in five of the nine mice in this group, the tumor never recurred and was undetectable upon killing the animal and autopsy on d 160. In contrast, the tumors in untreated mice and mice treated with CEP-751 progressed during the same time period (Fig. 3A). This result shows that irinotecan alone is very effective against TT tumors, and that the inhibition with CEP-751 in combination with this treatment can further increase the efficacy of irinotecan in this xenograft model. These complete responses to irinotecan in this in vivo model of MTC are especially notable, because this model has been resistant to therapy, with few reports of any objective responses (25, 26, 27).
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To explore the mechanism underlying the additive effects of CEP-751 and irinotecan, we performed cell cycle analysis using fluorescence-activated cell sorting. Irinotecan, as a topoisomerase I poison, induces double-stranded DNA breaks in a largely DNA replication-dependent manner. Figure 4A shows the cell cycle analysis of TT cells treated with 5 µM irinotecan for 48 h. A dramatic increase in the proportion of cells accumulating in early S phase was observed, similar to the effect of irinotecan in other cell types (29). Treatment with CEP-751 alone resulted in the accumulation of cells in G2/M, as shown previously (19). When CEP-751 was added in combination with 5 µM irinotecan, the cell cycle profile was significantly changed from that when either of the compounds used alone. The cells no longer accumulated in early S phase or G2/M, as seen with either compound alone. Instead, the cells now exhibited a cell cycle profile much more similar to the normal untreated TT cells. This result suggests that CEP-751 acts to block the early S phase arrest that occurs in TT cells treated with irinotecan alone, thus allowing the cells to continue through the cell cycle while accumulating DNA damage. We suspected, therefore, that CEP-751 acts to block the normal checkpoints induced upon DNA damage with irinotecan. Interestingly, there did not seem to be any significant induction of classic apoptotic cells in any of the treated cells, as determined by a sub-G0 peak, suggesting the possibility of an alternative mechanism for cell death. This lack of apoptosis is also supported by a lack of cleaved poly(ADP-ribose) polymerase (data not shown).
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Previous reports have shown that double-strand breaks of DNA induced by irinotecan treatment result in a DNA repair pathway marked by phosphorylation of histone H2AX (30). In this study we show that TT cells treated with irinotecan exhibit increased phosphorylation of histone H2AX, and this phosphorylation was significantly inhibited when the cells were treated with irinotecan in the presence of CEP-751 (Fig. 4B). Previous reports have also shown activation of Chk1 by phosphorylation in response to irinotecan-mediated DNA damage (31). We show that irinotecan treatment resulted in increased Chk1 phosphorylation in TT cells, and that this activation is also blocked when the cells are treated with irinotecan in the presence of CEP-751 (Fig. 4B). One of the effects of Chk1 is an S phase block is phosphorylation of cdc25A, resulting in its degradation (29). Cdc25A is a phosphatase that promotes the activation of cdk2. In this study we show that cdc25A is degraded in response to irinotecan treatment, as would be expected in a cdk2-dependent S phase block (Fig. 4B). CEP-751 in combination with irinotecan also acts to block this degradation (Fig. 4B), possibly resulting in a release of the S phase arrest shown in Fig. 4A. Cdk2 is phosphorylated on Thr160, and this effect is also blocked by CEP-751. These results are consistent with involvement of chk1, cdc25A, and H2AX in the S phase checkpoint induction and suggest that CEP-751 may act to block this checkpoint. CEP-751 is an effective RET inhibitor (IC50, 
200 nM) (19), but like most tyrosine kinase inhibitors, CEP-751 also inhibits other kinases, including FLT3, TrkA, and KDR (20, 32, 33). Therefore, it is possible that CEP-751 may affect the S phase checkpoint through kinase activities other than RET. To determine whether the effect on these DNA damage checkpoint proteins is dependent on RET inhibition, cell lines that do not express RET were treated with irinotecan and CEP-751. Indeed, in the colon cancer cell line, HCT-116, and the nonsmall cell lung cancer cell line, H1299, CEP-751 blocked irinotecan-induced chk1 phosphorylation (data not shown). Together, these results support the possibility that inhibition of another, as yet unidentified, kinase may help to sensitize these cells to irinotecan treatment by allowing progression through cell cycle checkpoints without repair of DNA damage.
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Discussion |
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The mechanism by which CEP-751 sensitizes MTC cells to irinotecan is not certain. However, because inhibition of receptor tyrosine kinase targets in other cancers can also sensitize cells to irinotecan, this suggests that a common mechanism may be widely used in cancer (28, 35). In other cell lines, irinotecan treatment of cells has been shown to induce an S phase or G2/M checkpoint (31, 36). Activation of the S phase checkpoint is mediated by the ATM (ataxia telangiectasia mutated)- and Rad3-related (ATR) checkpoint pathway and its downstream effectors, such as H2AX, Chk1, cdc25A, and cdk2 (30, 36), allowing damage to be repaired before completion of cell division. We showed that CEP-751 interferes with the induction of the S phase checkpoint, potentially preventing DNA repair before completion of cell division. Our data suggest that this blocking of checkpoint induction may be mediated through the ATM/ATR pathway, because downstream effectors of the ATM/ATR pathway, Chk1 and cdc25A, are affected by CEP-751. The mechanism by which the ATM/ATR signaling pathway is blocked is as yet unknown, but the determination of such connections could be extremely beneficial in our understanding of this disease as well as the mechanisms of resistance to radiation and chemotherapy in this cancer model. In addition to the interaction with the checkpoint pathways, CEP-751 inhibition of RET or other tyrosine kinases could also modulate cellular sensitivity through other pathways. For example, RET has been shown to induce nuclear factor-
B and Akt, which can activate cell survival signaling pathways (37, 38). It is also possible that inhibition of RET and other kinases may activate an ABC cellular transport mechanism; such a mechanism has been reported recently for EGFR inhibition by Iressa in pediatric tumor cell lines (39).
Irinotecan is already approved for clinical use in metastatic colorectal cancer, either as a combination with 5-fluorouracil/leucovorin for the first-line therapy or as a monotherapy for relapse after 5-fluorouracil treatment (40). In colorectal tumor cell lines, the IC50 of irinotecan has been reported to be between 2.5 and greater than 20 µM (41). In our MTC cell line, the IC50 is even lower at 2 µM, suggesting that MTC may be at least as sensitive as colorectal cancer to irinotecan. In light of the preclinical results reported in this paper, a phase II clinical trial of irinotecan for advanced MTC has been initiated. The implications of the results of the combination treatment also suggest the potential for possible future clinical utility of a combination of a tyrosine kinase inhibitor and irinotecan for treatment of MTC.
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Acknowledgments |
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Footnotes |
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First Published Online November 1, 2005
Abbreviations: ATM, Ataxia-telangiectasia mutated; ATR, ATM- and Rad3-related; cdk2, cyclin-dependent kinase 2; Chk1, checkpoint kinase 1; FBS, fetal bovine serum; MEN 2, multiple endocrine neoplasia type 2; MTC, medullary thyroid cancer; MTT, 3,4,5-dimethylthiazol-2,5-diphenyltetrazolium.
Received August 22, 2005.
Accepted October 20, 2005.
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References |
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B is constitutively active in C-cell carcinoma and required for RET-induced transformation. Cancer Res 61:4526–4535This article has been cited by other articles:
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, Treatment cycles. Tumors were measured at the time points shown and are the averages of all of tumors in the group. B, Kaplan-Meier curve showing time to progression of TT xenografts to a 4-fold size cutoff from their initial size on d 0.
H2AX induction in response to DNA damage, whereas CEP-751 blocks some of this induction. The Chk1 pathway is also induced by irinotecan treatment, and this is also blocked by CEP-751 treatment.