The majority of colorectal cancers have lost/inactivated the p53 tumor suppressor gene. Using isogenic human colon cancer cells that differ only in their p53 status, we demonstrate that loss of p53 renders tumor cells relatively resistant to the topoisomerase I inhibitor, irinotecan. Whereas irinotecan-induced up-regulation of the proapoptotic proteins PUMA and Noxa requires p53, we find that irinotecan inhibits Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 and 5 (STAT3/5) signaling in both p53-proficient and p53-deficient tumor cells. We show that irinotecan inhibits JAK2-STAT3/5-dependent expression of survival proteins (Bcl-xL and XIAP) and cooperates with Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) to facilitate p53-independent apoptosis of colon cancer cells. Whereas xenografts of p53-deficient colon cancer cells are relatively resistant to irinotecan compared with their p53-proficient counterparts, combined treatment with irinotecan and Apo2L/TRAIL eliminates hepatic metastases of both p53-proficient and p53-deficient cancer cells in vivo and significantly improves the survival of animals relative to treatment with either agent alone. Although the synergy between chemotherapy and Apo2L/TRAIL has been ascribed to p53, our data demonstrate that irinotecan enhances Apo2L/TRAIL-induced apoptosis of tumor cells via a distinct p53-independent mechanism involving inhibition of JAK2-STAT3/5 signaling. These findings identify a novel p53-independent channel of cross-talk between topoisomerase I inhibitors and Apo2L/TRAIL and suggest that the addition of Apo2L/TRAIL can improve the therapeutic index of irinotecan against both p53-proficient and p53-deficient colorectal cancers, including those that have metastasized to the liver.
Colorectal cancer is the second most common malignancy and cause of cancer-related mortality. The most common site of colorectal cancer metastases is the liver, and it is often the only site involved. Median overall survival of patients with metastatic colorectal cancer remains ∼2 years after chemotherapy with regimens incorporating the antimetabolite 5-fluorouracil, leucovorin, and the topoisomerase I inhibitor irinotecan (1, 2).
The development of colorectal cancer involves acquisition of multiple genetic aberrations that reduce cellular susceptibility to apoptosis and confer resistance to therapy (3, 4, 5, 6, 7). The vast majority of colorectal cancers have lost or inactivated p53, a tumor suppressor gene that plays a key role in induction of apoptosis in response to several chemotherapeutic agents (8, 9, 10, 11, 12, 13). The mechanism by which p53 induces apoptosis involves transcriptional activation of proapoptotic genes such as the Bcl-2 homology 3 (BH3)-domain containing proteins, PUMA (p53 up-regulated modulator of apoptosis) and Noxa (14, 15, 16, 17, 18, 19). These genes encode proteins that trigger mitochondrial outer membrane permeabilization via the multidomain Bcl-2 family members BAX and BAK (20, 21). In addition, cytosolic p53 promotes mitochondrial permeabilization by transcription-independent activation of BAX, as well as release of proapoptotic multidomain and BH3-only proteins from their sequestration by Bcl-xL, an antiapoptotic member of the Bcl-2 family (22, 23). The consequent mitochondrial release of cytochrome c and Smac/DIABLO (second mitochondria-derived activator of caspase) promotes the transactivation of caspase-9 and downstream effector caspases (-7 and -3) that execute cell death (24). Human colon cancer cells in which p53 is disrupted by gene targeting are relatively resistant to diverse chemotherapeutic agents compared with their isogenic p53-proficient counterparts (13). Inactivating mutations of p53in human colorectal cancers may limit the clinical efficacy of chemotherapeutic regimens and have been correlated with decreased survival in patients with colorectal adenocarcinimas (25, 26, 27, 28).
A distinct death signaling process is initiated by ligation of cell surface death receptors, TRAIL-R1/DR4 and TRAIL-R2/DR5, by their cognate ligand [Apo2L/TRAIL (Apo2 Ligand/tumor necrosis factor-related apoptosis inducing ligand)] or agonistic antibodies (29, 30). Death receptor engagement results in Fas-associated death domain protein-dependent recruitment and autocatalytic activation of caspase-8 (31, 32). Whereas caspase-8 can directly activate procaspase-3 in some cell types, death receptor-induced apoptosis of colon cancer cells requires caspase-8–mediated cleavage of BID (p22), a “BH-3 domain only” prodeath Bcl-2 family member (33, 34, 35). The active truncated form of BID (p15) triggers mitochondrial membrane permeabilization via the activation of BAX and BAK (36, 37, 38, 39). Although Apo2L/TRAIL activates a p53-independent death signaling pathway, its ability to induce apoptosis is counteracted by Bcl-xL, a member of the Bcl-2 family, and by X-linked inhibitor of apoptosis protein (XIAP), a member of the IAP family of caspase inhibitors (34, 40, 41). The expression of Bcl-xL and XIAP is up-regulated by activation of signal transducer and activator of transcription 3/5 (STAT3/5; refs. 42, 43, 44, 45, 46, 47). Phosphorylation of STATs at tyrosine residues by receptor-associated Janus kinases (JAKs) induces their dimerization and translocation to the nucleus, where they bind to specific DNA sequences and modulate expression of target genes involved in cell survival (48). Whereas JAK2-STAT3/5 activity is transient and tightly regulated in normal cells, it is constitutively activated in a wide variety of human cancer cells (49).
Because chemotherapeutic agents and Apo2L/TRAIL use different BH3-domain–containing proteins to activate BAX and BAK, the simultaneous delivery of both death signals may converge to promote apoptosis of tumor cells (29, 30, 35). In addition to activating transcription of PUMA and Noxa, the elevation of p53 in response to chemotherapeutic agents could facilitate Apo2L/TRAIL-induced apoptosis by releasing both BID and BAX from their sequestration by Bcl-xL (22). Chemotherapeutic agents might also augment Apo2L/TRAIL-induced apoptosis by inducing p53-dependent expression of death receptors, TRAIL-R1 and TRAIL-R2 (35, 50, 51). Although p53 has a multifunctional role in chemosensitization of tumor cells to death ligands, it is not known whether or how such synergistic cytotoxicity between chemotherapeutic agents and Apo2L/TRAIL can be exerted against the vast majority of colorectal cancers that have lost or inactivated p53. It is also not known if the addition of Apo2L/TRAIL can improve the efficacy of chemotherapeutic agents against hepatic metastases of p53-deficient colorectal cancers without prohibitive hepatotoxicity in vivo. These central questions are addressed in this study.
We find that irinotecan inhibits JAK2-STAT3/5–dependent expression of survival proteins, such as Bcl-xL and XIAP, and cooperates with Apo2L/TRAIL to facilitate p53-independent apoptosis of colon cancer cells. Whereas xenografts of p53-deficient colon cancer cells are relatively resistant to irinotecan compared with their p53-proficient counterparts, combined treatment with irinotecan and Apo2L/TRAIL eliminates hepatic metastases of both p53-proficient and p53-deficient cancer cells in vivo and significantly improves the survival of animals relative to treatment with either agent alone. These findings identify a novel p53-independent channel of cross-talk between topoisomerase I inhibitors and Apo2L/TRAIL and suggest that the addition of Apo2L/TRAIL can improve the therapeutic index of irinotecan against not only p53-proficient tumors but also the majority of colorectal cancers that have lost or inactivated p53.
MATERIALS AND METHODS
Cells and Transfection.
The HCT116 human colon adenocarcinoma cell line containing wild-type p53 (p53+/+) and isogenic p53-deficient (p53−/−) derivatives of HCT116 cells were generated by disruption of p53 alleles by gene targeting (13). HCT116 cells (p53+/+) were infected with a retroviral vector encoding green fluorescent protein (GFP) and Bcl-xL (pMX/IRES-GFP:pMIG), and cells overexpressing Bcl-xL (p53+/+Bcl-xL) were isolated by selection of GFP-positive cells using fluorescence-activated cell sorter. Cells were cultured at 37°C and 5% CO2 in McCoy’s 5A medium supplemented with 10% fetal calf serum, penicillin (100 units/mL), and streptomycin (100 μg/mL). Fresh normal human hepatocytes (In Vitro Technologies, Baltimore, MD) were cultured at 37°C and 5% CO2 in Hepatocyte Culture Medium.
Treatment with Recombinant Human Apo2L/TRAIL.
Exponentially growing cells (2 × 105 per well) were incubated with nontagged soluble recombinant human Apo2L/TRAIL (0.1–1 μg/mL; Genentech, Inc., San Francisco, CA) at 37°C for the indicated time intervals.
Treatment with Topoisomerase I Inhibitors.
Cells were incubated with camptothecin (50, 100, or 200 ng/mL, Sigma-Aldrich, St. Louis, MO) or irinotecan hydrochloride (CPT-11, Camptosar, 25, 50, or 100 μg/mL, Pharmacia-Upjohn, Kalamazoo, MI) at 37°C.
Treatment with the JAK2 Kinase Inhibitor AG490.
Cells were incubated with the JAK2 inhibitor tyrphostin AG490 (50–100 μmol/L, Calbiochem, La Jolla, CA).
Expression of JAK2, STAT3, or STAT5 was inhibited using vector-expressed short-interfering RNA (TranSilent JAK2 siRNA, TranSilent STAT3 siRNA, or TranSilent STAT5a siRNA, Panomics, Inc., Redwood City, CA). HCT116 cells (12-well plates, 50% confluency) were cotransfected with a GFP-vector and TranSilent JAK2 siRNA, TranSilent STAT3 siRNA, TranSilent STAT5a siRNA, or TranSilent Control siRNA, using Lipofectamine 2000. Cells were harvested 48 hours after transfection for immunoblot analysis of JAK2, STAT3, STAT5, and Bcl-xL.
Electromobility Shift Assays.
Nuclear extracts were prepared using NE-PER buffer (Pierce, Rockford, IL). Nuclear protein (5–10 μg) were incubated in a final volume of 20 μL of electrophoretic mobility shift analysis buffer [10 mmol/L HEPES (pH 7.9), 80 mmol/L NaCl, 10% glycerol, 0.5 mmol/L DTT, 1 mmol/L EDTA, and 100 μg/mL poly(deoxyinosinic-deoxycytidylic acid)] with 32P-labeled double-stranded oligonucleotide probe that binds either the STAT5 or STAT3 consensus-binding motif (Santa Cruz Biotechnology, Santa Cruz, CA). Gel supershift assays were performed by preincubation (30–45 min) with 2 μL of a specific antibody against either STAT5 or STAT3 (TransCruz gel supershift antibodies, Santa Cruz Biotechnology). The DNA-protein complexes were resolved on a 5% nondenaturing polyacrylamide gel and visualized by autoradiography.
Protein Tyrosine Phosphatase Assay.
Cell lysates were prepared in phosphate-free lysis buffer [50 mmol/L HEPES (pH 7.4), 60 mmol/L NaCl, 60 mmol/L KCl, 0.1 mmol/L phenylmethylsulfonyl fluoride, 10 μg/mL aprotinin, 10 μg/mL leupeptin, and 10 μg/mL pepstatin]. Protein tyrosine phosphatase activity in cell lysates (5 μg) was measured by dephosphorylation of a tyrosine phosphopeptide or hydrolysis of p-Nitrophenyl phosphate using a protein tyrosine phosphatase assay kit (Upstate Biotechnology, Lake Placid, NY). The protein tyrosine phosphatase inhibitor sodium orthovanadate (vanadate) was used to confirm the specificity of protein tyrosine phosphatase activity in cell lysates.
Cell lysates were prepared, and 50 to 100 μg of protein were resolved by SDS-PAGE, transferred onto Immobilon-P polyvinylidene difluoride membrane (Millipore, Bedford, MA), and probed with antibodies against p53 (Ab-6), p21WAF1/CIP1, PUMA (Ab-1), Noxa (Ab-1, Oncogene Research Products, San Diego, CA), BAX (N-20), Bcl-xL (S-18), caspase-8 (C-20), caspase-9 (H-170), caspase-7 (N-17), pJAK2 (Tyr 1007/Tyr 1008), JAK2, STAT5, STAT3, actin (C-11, Santa Cruz Biotechnology), pSTAT5 (Tyr 694), pSTAT3 (Tyr 705, Cell Signaling Technology, Beverly, MA), BID (IMGENEX, San Diego, CA), XIAP (Sigma-Aldrich, St. Louis, MO), and poly(ADP-ribose) polymerase (PARP, Ab-2, Calbiochem). Immunoreactive protein complexes were visualized by enhanced chemiluminescence (Amersham, Arlington Heights, IL).
RNA Extraction and Northern Blot Hybridization.
Total RNA was extracted using Trizol (Life Technologies, Inc.). RNA samples (20 μg) were analyzed in 1.2% agarose-formaldehyde gels, transferred onto Zeta probe membranes, and UV cross-linked with a Stratalinker. The membranes were hybridized to 32P-labeled probes for human TRAIL-R1/DR4 or human TRAIL-R2/DR5 (Alexis Corporation, San Diego, CA). Membranes were visualized by autoradiography.
Analysis of Cell Death.
Cells were assessed for morphologic features of apoptosis (condensed chromatin and micronucleation) by microscopic visualization. Cell viability was assessed at the indicated intervals by analysis of Annexin V staining (Biovision Inc., Mountain View, CA) of harvested cells (adherent + floating in the medium) using a flow cytometer (Becton Dickinson, Palo Alto, CA). The average percentage of viability (mean ± SE) was calculated from three different experiments.
Female athymic nu/nu mice were inoculated s.c. with 5 × 106 HCT116 cells of either genotype (p53+/+ or p53−/−). Three days after tumor cell inoculation, tumor-bearing mice were randomly distributed (7 per group) for i.p. treatment with irinotecan (50 mg/kg, CPT-11/Camptosar) and/or Apo2L/TRAIL (50 mg/kg) as indicated. Tumor size was measured twice per week.
Hepatic Metastases Model of Colon Cancer.
Laparotomy was performed on anesthetized female athymic nu/nu mice (∼6 to 8 weeks of age), and the spleen was divided into two hemi-spleens using titanium clips, leaving the vascular pedicles intact. After injection of 5 × 106 HCT116 colon cancer cells of either genotype (p53+/+ or p53−/−) into one of the hemi-spleens, the tumor cell-contaminated hemi-spleen was surgically removed, leaving a functional hemi-spleen free of tumor cells. The injected tumor cells flow into the splenic and portal veins and form tumor deposits in the liver. Five days after tumor challenge, mice were randomly distributed (10 per group) for treatment with vehicle (controls), irinotecan (50 mg/kg, CPT-11), Apo2L/TRAIL (50 mg/kg), or the combination of irinotecan and Apo2L/TRAIL. Three mice from each group were euthanized using CO2 inhalation at 35 days and 70 days after tumor challenge. The livers were sectioned and H&E stained to determine the presence and size of gross and microscopic tumor burden and evaluation of hepatic toxicity. Tumor-free survival was recorded for the remaining animals in each group.
Tumor growth was compared using random effects linear models where the log of tumor size is modeled over time in weeks. The log transform was used for variance stabilization. The random effects account for the correlation of the serial observations made on the same mice. Quadratic models and models including indicator variables for week of observation were fit, and it was found that the quadratic models were sufficient for describing the data yet require fewer parameters to be estimated. Residual plots were used to assess assumptions. Models including and excluding terms for treatment were compared to assess statistical significance of treatment. AIC was used to determine the best fitting model, and P values for model comparisons were calculated using χ2 tests comparing model deviances. Survival of mice carrying hepatic xenografts of p53+/+ or p53−/− HCT116 cells was analyzed using Kaplan-Meier curves of time to death and Cox proportional hazards model. P values reported are from Cox proportional hazards model and also from log rank tests. Proportionality of hazards was assessed using Schoenfeld residuals.
All of the animal protocols were approved by the Institutional Animal Care and Use Committee of the Johns Hopkins Medical Institutions.
Role of p53 in the Response of Colon Cancer Cells to the Topoisomerase I Inhibitor Irinotecan.
We evaluated the role of p53 in the response of colon cancer cells to the topoisomerase I inhibitor camptothecin or its derivative, irinotecan (CPT-11), by comparing p53-proficient (p53+/+) HCT116 colon cancer cells with isogenic p53-deficient (p53−/−) derivatives of HCT116 cells that were generated by targeted inactivation of both p53 alleles via homologous recombination (13). Whereas treatment of p53+/+ HCT116 cells with either irinotecan or camptothecin resulted in dose-dependent induction of apoptosis, p53−/− HCT116 cells were relatively resistant to either agent (Fig. 1,A and B). Exposure of p53+/+ HCT116 cells to irinotecan resulted in increased levels of p53 and its transcriptional targets, p21WAF1/CIP1, and the proapoptotic BH3-domain–containing proteins, PUMA and Noxa (Fig. 1,C). However, the induction of p21WAF1/CIP1, PUMA, or Noxa in response to irinotecan was impaired in p53−/− HCT116 cells (Fig. 1,C). In contrast to these p53-dependent responses, treatment with irinotecan reduced the expression of Bcl-xL and XIAP in both p53+/+ and p53−/− HCT116 cells (Fig. 1 C).
Irinotecan Inhibits JAK2-STAT3/5–Dependent Expression of Bcl-xL and XIAP in Colon Cancer Cells Independently of p53.
The JAK and STAT families are key components of shared signaling pathways activated by diverse cytokines and growth factor receptors. After tyrosine phosphorylation by JAKs, STAT monomers dimerize via reciprocal phosphotyrosine-SH2 interactions and translocate to the nucleus, where they bind DNA and regulate transcription of target genes, such as Bcl-xL and XIAP. The phosphotyrosine residue Tyr1007 in the activation loop is critical for activation of JAK2, which, in turn, phosphorylates the tyrosine residues required for activation of STAT3 (Tyr705) and STAT5 (Tyr694). Immunoblot analysis showed constitutive tyrosine phosphorylation of JAK2 (Tyr1007/Tyr1008), STAT3 (Tyr705), and STAT5 (Tyr694) in both p53+/+ and p53−/− HCT116 cells (Fig. 2,A and B). Treatment with irinotecan increased protein tyrosine phosphatase activity (Fig. 2,C) and resulted in the rapid dephosphorylation of tyrosine residues of JAK2, STAT3, and STAT5 in both p53+/+ and p53−/− HCT116 cells (Fig. 2,A and B). Irinotecan-mediated dephosphorylation of JAK2 was counteracted by pretreatment of cells with the protein tyrosine phosphatase inhibitor sodium vanadate (Fig. 2,A). Consistent with its ability to dephosphorylate JAK2, electrophoretic mobility shift assays showed that irinotecan reduced STAT3- and STAT5-DNA binding activity in both p53+/+ and p53−/− HCT116 cells (Fig. 2 D and E).
Treatment with the JAK-specific inhibitor tyrphostin AG490 resulted in the rapid dephosphorylation of STAT3 and STAT5 and a corresponding reduction in their DNA binding activity in both p53+/+ and p53−/− HCT116 cells (Fig. 2,B, D, and E). Treatment with AG490 or inhibition of JAK2, STAT3, or STAT5 by their respective siRNA vectors resulted in decreased expression of Bcl-xL in cells of either genotype (Fig. 2,F–I). Inhibition of STAT5 expression by STAT5 siRNA also resulted in reduced expression of XIAP (Fig. 2,I). Consistent with its ability to inhibit JAK2-STAT3/5 signaling, irinotecan decreased the expression of Bcl-xL and XIAP in both p53+/+ and p53−/− HCT116 cells (Fig. 1,C) but was less effective in doing so after pretreatment with vanadate (Fig. 2,A). Treatment with camptothecin resulted in a similar p53-independent inhibition of JAK2-STAT3/5 signaling (data not shown) and corresponding reduction in Bcl-xL and XIAP expression (Fig. 3 A).
Apo2L/TRAIL and Topoisomerase I Inhibitors Cooperate to Induce p53-Independent Apoptosis of Colon Cancer Cells.
Unlike the p53-dependent induction of PUMA and Noxa in response to topoisomerase I inhibitors, BID undergoes p53-independent activation in response to engagement of death receptors (TRAIL-R1 and TRAIL-R2) with Apo2L/TRAIL. Apo2L/TRAIL induces caspase-8–mediated cleavage of BID (p22) to an active truncated form (p15), which triggers mitochondrial activation of caspases (-9, -7, and -3) via the activation of BAX and BAK. However, Apo2L/TRAIL-induced death signaling in colon cancer cells is counteracted by sequestration of truncated form of Bid by Bcl-xL or inhibition of caspases by XIAP (34, 40, 41). Because topoisomerase I inhibitors decreased JAK2-STAT3/5–dependent expression of Bcl-xL and XIAP in a p53-independent manner (Fig. 2), we investigated whether they cooperate with Apo2L/TRAIL to induce apoptosis of p53+/+ and p53−/− colon cancer cells.
Treatment of p53+/+ HCT116 cells with topoisomerase I inhibitors (camptothecin or irinotecan) resulted in activation of caspase-9 and caspase-7 and cleavage of the caspase-3 substrate PARP (Fig. 3,A). Unlike p53+/+ HCT116 cells, p53−/− HCT116 cells failed to activate caspase-9 or caspase-7 and were relatively resistant to either camptothecin- or irinotecan-induced apoptosis (Fig. 3,A-D and Fig. 4,A-C). Whereas exposure of p53+/+ HCT116 cells to topoisomerase I inhibitors resulted in increased levels of death receptors for Apo2L/TRAIL (TRAIL-R1 and TRAIL-R2), the induction of TRAIL-R1 and TRAIL-R2 in response to the same treatment was impaired in p53−/− HCT116 cells (Fig. 3,B). However, Apo2/TRAIL synergized with either camptothecin or irinotecan to promote activation of caspases (-9 and -7) and cleavage of PARP independently of p53 (Fig. 3,A and Fig. 4,A). Accordingly, Apo2L/TRAIL cooperated with either camptothecin or irinotecan to augment apoptosis of p53+/+ as well as p53−/− HCT116 cells (Fig. 3,C and D and Fig. 4,B and C). These results indicate that the relative resistance of p53-deficient colon cancer cells to topoisomerase I inhibitors is circumvented by sequential treatment with Apo2L/TRAIL. They additionally demonstrate that irinotecan-mediated augmentation of Apo2L/TRAIL-induced apoptosis does not require p53-dependent up-regulation of death receptor expression. Unlike their p53-deficient counterparts, HCT116 cells forced to overexpress Bcl-xL by stable introduction of an expression vector encoding Bcl-xL [Bcl-xL(p53+/+)] did not exhibit cleavage of caspase-9, caspase-7, or PARP and remained relatively resistant to combinatorial treatment with irinotecan and Apo2L/TRAIL (Fig. 4 A–C).
Because irinotecan reduced JAK2-STAT3/5 signaling, we investigated whether such inhibition is sufficient to augment Apo2L/TRAIL-induced apoptosis of colon cancer cells independently of p53. To determine the effect of JAK2 inhibition on the sensitivity of tumor cells to Apo2L/TRAIL-induced apoptosis, p53+/+ and p53−/− HCT116 cells were cotransfected with a GFP vector and either JAK2 siRNA or control siRNA vector. Analysis of GFP-positive cells showed that expression of JAK2 siRNA but not control siRNA augmented the sensitivity of p53+/+ as well as p53−/− HCT116 cells to Apo2L/TRAIL-induced apoptosis (Fig. 5,A and B). Likewise, treatment with AG490 (100 μmol/L) for 24 hours significantly increased apoptosis of either cell population in response to Apo2L/TRAIL (Fig. 5 C). Together, these data indicate that inhibition of JAK2-STAT3/5 signaling provides a p53-independent mechanism by which irinotecan augments Apo2L/TRAIL-induced apoptosis of colon cancer cells.
Addition of Apo2L/TRAIL Overcomes the Relative Resistance of p53-Deficient Colon Cancer Cells to Irinotecan In vivo.
Because irinotecan and Apo2L/TRAIL exhibited synergistic killing of tumor cells in a p53-independent fashion in vitro, we tested the individual and combined effects of irinotecan and Apo2L/TRAIL against p53+/+ and p53−/− HCT116 cells injected s.c. in athymic nude mice. Treatment of mice with three doses of irinotecan after injection with p53+/+ HCT116 cells resulted in a marked inhibition of tumor growth compared with untreated controls (Fig. 6,A–C). Tumor formation was prevented during irinotecan treatment, and tumor outgrowth was delayed until 10 days after the last dose of irinotecan. However, treatment with irinotecan was less effective against p53−/− HCT116 tumors (Fig. 6,A–C). p53−/− tumors exhibited continuous growth in irinotecan-treated animals, albeit at an attenuated rate compared with untreated controls. A comparison of the growth of s.c. tumor xenografts of p53+/+ and p53−/− HCT116 cells in mice treated with irinotecan (CPT-11) revealed a marked difference in tumor growth (P < 0.0001; Fig. 6,C). In contrast to the differential response of p53+/+ and p53−/− tumors to irinotecan, treatment of mice with Apo2L/TRAIL resulted in an equivalent inhibition of tumor cells of either genotype (Fig. 6,A-C). Although Apo2L/TRAIL retarded the rate of tumor growth compared with untreated controls, it failed to arrest the growth of either p53+/+ or p53−/− tumors. We next examined the effect of combinatorial treatment with irinotecan and Apo2L/TRAIL. Sequential treatment with Apo2L/TRAIL improved the already robust antitumor effect of irinotecan against p53+/+ cells, with complete inhibition of tumor formation (Fig. 6,A-C). Comparing tumor growth of p53+/+ HCT116 cells in mice treated with irinotecan versus the combination of irinotecan and Apo2L/TRAIL, we found a significant difference in the growth trajectories (P < 0.0001). A similar pattern was seen in the growth of p53−/− HCT116 tumors (Fig. 6,A–C). Unlike treatment with irinotecan or Apo2L/TRAIL alone, combinatorial treatment with irinotecan and Apo2L/TRAIL arrested the growth of p53−/− tumors, with a marked delay in tumor outgrowth after cessation of treatment (Fig. 6 A-C). Although the overall growth of p53−/− tumors tends to be faster, the difference in tumor growth trajectories in mice treated with irinotecan versus the combination of irinotecan and Apo2L/TRAIL was also highly statistically significant (P < 0.0001). Therefore, Apo2L/TRAIL augmented the sensitivity of p53-proficient as well as p53-deficient tumor cells to irinotecan in vivo.
Addition of Apo2L/TRAIL to Irinotecan Prolongs Survival of Animals with Experimental Hepatic Metastases of p53-Proficient and p53-Deficient Colon Cancers.
The most common site of colorectal cancer metastases is the liver, and often it is the only organ involved. In addition to overt distant metastases (Tumor-Node- Metastasis stage IV), patients are also at risk of hepatic metastases after resection of apparently localized colorectal cancer. The successful systemic therapy of colorectal cancers is contingent upon the ability of anticancer agents to eliminate hepatic metastases without prohibitive hepatotoxicity. We examined whether the addition of Apo2L/TRAIL to irinotecan improves the therapeutic index and increases overall animal survival in an experimental hepatic metastasis model of colon cancer (Fig. 7 A).
BALB/c-nude mice carrying hepatic xenografts of p53+/+ and p53−/− HCT116 human colorectal cancer cells were either left untreated or treated with irinotecan or Apo2L/TRAIL alone or the combination of both agents. Untreated control mice developed multiple large tumor masses in the livers at 5 weeks after splenic implantation of HCT116 cells of either genotype (p53+/+ and p53−/−; Fig. 7,B). Seven of 7 untreated mice developed progressive ascites and weight loss and became premorbid within 5 to 6 weeks after implantation of HCT116 cells of either genotype (p53+/+ and p53−/−; Fig. 7,C). Treatment of mice with three doses of irinotecan after implantation of p53+/+ HCT116 cells resulted in a marked inhibition of tumor growth compared with untreated controls. Gross tumor nodules were not evident at 5 weeks after implantation of tumor cells but became apparent at 10 weeks (Fig. 7,B). In contrast to the delayed appearance of p53+/+ tumors in response to irinotecan, p53−/− HCT116 cells formed gross tumor nodules within 5 weeks and large necrotic tumor masses at 10 weeks despite treatment with irinotecan (Fig. 7,B). Although treatment with irinotecan alone prolonged overall survival in mice challenged with either p53+/+ or p53−/− HCT116 cells, only 3 of 7 mice implanted with p53+/+ tumor cells and 1 of 7 mice implanted with p53−/− tumor cells remained alive at 17 weeks (Fig. 7,C). Unlike the differential response of hepatic metastases of p53+/+ and p53−/− tumor cells to irinotecan, treatment of mice with Apo2L/TRAIL resulted in equivalent attenuation of the size of metastatic tumors formed by HCT116 cells of either genotype (p53+/+ and p53−/−). In mice treated with Apo2L/TRAIL alone, small tumor nodules were evident at 5 weeks, and much larger tumors were apparent at 10 weeks after implantation of tumor cells (Fig. 7,B). Although treatment with Apo2L/TRAIL prolonged survival, all of the mice challenged with HCT116 cells of either genotype (p53+/+ and p53−/−) succumbed to progressive hepatic metastases within 10 to 11 weeks (Fig. 7,C). Significantly, hepatic metastases of either p53+/+ or p53−/− HCT116 cells were eliminated by combinatorial treatment with irinotecan and Apo2L/TRAIL (Fig. 7,B). Histologic analyses of livers harvested at 5 weeks after injection of tumor cells showed microscopic tumor foci in untreated controls, as well as in mice treated with either irinotecan or Apo2L/TRAIL alone (Fig. 7,D). However, there was no evidence of metastatic tumor foci in the livers of mice that received combinatorial treatment with Apo2L/TRAIL and irinotecan after implantation of either p53+/+ or p53−/− HCT116 cells (Fig. 7,D). Seven of 7 mice injected with p53+/+ HCT116 cells and 6 of 7 mice injected with p53−/− tumor cells remained alive at 17 weeks in response to the combination of irinotecan and Apo2L/TRAIL (Fig. 7 B).
We compared the survival rates of mice carrying hepatic xenografts of p53+/+ or p53−/− HCT116 cells treated with irinotecan versus the combination of irinotecan and Apo2L/TRAIL (Fig. 6). The hazard ratio for death in mice carrying p53−/− HCT116 cells was 3.13 (95% confidence interval, 1.06–9.09, P = 0.04), indicating that mice treated with irinotecan alone were at three times the risk of death at any point in time compared with mice treated with the combination of irinotecan and Apo2L/TRAIL. For mice carrying hepatic xenografts of p53+/+ HCT116 cells, the hazard ratio was estimated to be infinite, because no deaths occurred in the combination arm by the end of the study (17 weeks). On the basis of the log rank test, the difference in survival following irinotecan versus the combination (Irinotecan + Apo2L/TRAIL) in mice carrying p53+/+ HCT116 cells was statistically significant (P = 0.0006). Combining the survival of mice carrying either p53+/+ or p53−/− HCT116 cells together, the estimated hazard ratio after treatment with irinotecan versus the combination of irinotecan and Apo2L/TRAIL was 3.70 (95% confidence interval, 1.33–10.0, P = 0.01).
Addition of Apo2L/TRAIL Improves the Antitumor Efficacy of Irinotecan without Increasing Hepatotoxicity.
In addition to allowing an assessment of the antitumor effects of the Apo2L/TRAIL-irinotecan regimen against colorectal cancer cells residing in a situation that mimics hepatic metastases in patients, our experimental hepatic metastasis model of colon cancer enabled simultaneous examination of the toxicity of each regimen on normal hepatocytes in the identical microenvironment in vivo. Histologic analyses of livers harvested from mice after treatment with Apo2L/TRAIL, irinotecan, or the combination of irinotecan and Apo2L/TRAIL revealed normal lobular architecture and hepatocyte morphology (Fig. 8 A). The elimination of hepatic metastases of p53+/+ or p53−/− colon cancer cells without prohibitive hepatocyte degeneration or lobular disarray suggests that the addition of Apo2L/TRAIL can improve the therapeutic index of irinotecan in the systemic treatment of colon cancer.
We also examined the susceptibility of normal human hepatocytes to the individual and combined effects of irinotecan and Apo2L/TRAIL. Normal hepatocytes were highly susceptible to an agonistic antibody against Fas/CD95 (CH-11), with almost complete elimination of the population by 20 hours (Fig. 8,B). However, incubation of normal human hepatocytes with irinotecan (50 μg/mL), Apo2L/TRAIL (1 μg/mL), or both agents did not significantly augment the levels of spontaneous apoptosis after culture for 20 hours (Fig. 8,B). Similarly, the addition of Apo2L/TRAIL to irinotecan did not promote apoptosis of cynomolgus hepatocytes in vitro or induce significant changes in serum liver enzyme activities, bilirubin or albumin in vivo (data not shown). In contrast, the combination of irinotecan and Apo2L/TRAIL eliminated >80% of p53+/+ or p53−/− HCT116 cells (Fig. 4 B) or CT26 mouse colon cancer cells (data not shown).
Median overall survival of patients with metastatic colorectal cancer remains ∼2 years after systemic chemotherapeutic regimens incorporating the antimetabolite 5-fluorouracil, leucovorin, and the topoisomerase I inhibitor, irinotecan (1, 2). Using isogenic colon cancer cells that differ only in their p53 status, we demonstrate that loss of p53 renders colon cancer cells relatively resistant to irinotecan- induced apoptosis. Because p53 deficiency attenuates the induction of apoptosis in response to irinotecan as well as 5-fluorouracil, the frequent loss or inactivation of p53 in colorectal cancers may limit the efficacy of chemotherapeutic regimens in the vast majority of patients (8, 13). Unraveling mechanisms to unleash the death program in p53-deficient tumor cells could aid the design of effective therapeutic interventions against colon cancers that resist chemotherapy.
We show that p53 is required for irinotecan-induced expression of the BH3-domain–containing proteins, PUMA and Noxa, which promote apoptosis through the multidomain Bcl-2 family members BAX and BAK (Fig. 9). Unlike the p53-dependent induction of PUMA and Noxa in response to topoisomerase I inhibitors, the BH-3 domain only protein BID undergoes p53-independent caspase-8–mediated activation in response to Apo2L/TRAIL. Although Apo2L/TRAIL triggers BID-mediated mitochondrial activation of caspases (-9, -7, and -3), this death signaling pathway is interrupted by expression of Bcl-xL or XIAP (34, 52). Our data indicate that endogenous expression of Bcl-xL and XIAP via constitutive activation of the JAK2-STAT3/5 pathway limits the susceptibility of colon cancer cells to Apo2L/TRAIL-induced apoptosis (Fig. 9). Conversely, inhibition of JAK2-STAT3/5 signaling promotes Apo2L/TRAIL-induced apoptosis of colon cancer cells independently of p53.
JAK/STAT signaling is activated via phosphorylation of tyrosine residues by receptor/nonreceptor protein tyrosine kinases and is inhibited by several protein tyrosine phosphatases (48, 53). Whereas JAK2-STAT3/5 activity is normally controlled by the balance between protein tyrosine kinases and protein tyrosine phosphatase assays, it is constitutively activated in cancer cells via oncogenic receptor/nonreceptor tyrosine kinases or inactivating mutations in tyrosine phosphatases. In addition to the expression/activation of epidermal growth factor receptor tyrosine kinase in up to 75% of colorectal cancers, somatic mutations in protein tyrosine phosphatase assays have been identified in 26% of colorectal cancers (54). Our data demonstrate that treatment with irinotecan increases protein tyrosine phosphatase activity and inhibits JAK2-STAT3/5 signaling in both p53-proficient and p53-deficient tumor cells. We show that irinotecan-mediated inhibition of JAK2-STAT3/5 reduces the expression of survival proteins, such as Bcl-xL and XIAP, and cooperates with Apo2L/TRAIL to facilitate p53-independent apoptosis of colon cancer cells (Fig. 9). In addition to the reduction of Bcl-xL and XIAP, repression of other JAK2-STAT3/5-dependent survival proteins may also be involved in irinotecan-mediated sensitization of tumor cells to Apo2L/TRAIL. The sensitization of tumor cells to Apo2L/TRAIL by irinotecan has been attributed previously to p53-dependent up-regulation of death receptors or proapoptotic Bcl-2 family proteins (35). Our data demonstrate that irinotecan enhances Apo2L/TRAIL-induced apoptosis of colon cancer cells via a distinct p53-independent mechanism involving inhibition of JAK2-STAT3/5 activity.
One direct implication of our data is that combined treatment with irinotecan and Apo2L/TRAIL could overcome the resistance of cancer cells to either treatment alone. Our results suggest that the relative resistance of p53-deficient colon cancer cells to topoisomerase I inhibitors can be circumvented by sequential treatment with Apo2L/TRAIL. Conversely, the intrinsic resistance of colon cancer cells to Apo2L/TRAIL may be counteracted by irinotecan-mediated inhibition of JAK2-STAT3/5 activity. Our results demonstrate that the combination of irinotecan and Apo2L/TRAIL can eliminate hepatic metastases of either p53-proficient or p53-deficient human colon cancer xenografts and markedly improve overall animal survival. These findings suggest that the addition of Apo2L/TRAIL can improve the therapeutic index of irinotecan-based regimens against not only p53-proficient tumors but also the vast majority of colon cancers that have lost or inactivated p53.
Grant support: NIH (National Cancer Institute) and a translational research award from the Virginia and D.K. Ludwig Fund for Cancer Research (A. Bedi and R. Ravi).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Atul Bedi, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, 487, The Bunting Family ♦ The Family of Jacob and Hilda Blaustein Building For Cancer Research, 1650 Orleans Street, Baltimore, Maryland 21231-1000. Phone: 410-955-8784; Fax: 410-502-7163; E-mail: firstname.lastname@example.org
The authors thank Dr. Bert Vogelstein for providing isogenic p53-proficient and p53-deficient HCT116 cell lines, Dr. Jeffrey Summers for the retrovirus carrying Bcl-xL, Dr. Sanju Jalla for technical assistance, and Dr. Norman Barker for photomicrography.