IGF-1R Targeting Increases the Antitumor Effects of DNA-Damaging Agents in SCLC Model: An Opportunity to Increase the Efficacy of Standard Therapy

IGF-1R and pAkt were frequently expressed in human SCLC tissue specimens

We assessed IGF-1R expression and Akt phosphorylation in one of the largest cohort of SCLC human specimens (83 patients). Positive IGF-1R immunohistochemical staining (Fig. 1A) was observed in 44 patients (53%), whereas pAkt (Fig. 1B) was detected in 31 patients (37%). A total of 17 (21%) and 22 (26%) of all tumors exhibited a minimum of 2+ staining intensity for IGF-1R and pAkt, respectively. Concomitant expression of both proteins was present in 24 samples (29%) suggesting that IGF pathway could be a relevant target in SCLCs.

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Figure 1.

A and B, representative sections of positive IGF1R (A) and pAkt (B) staining in human SCLC specimens at ×200 magnification. Immunohistochemical analyses were conducted using the Discovery XT automated platform (Ventana Medical Systems). C, effect of R1507 on IGF-1R protein expression in H526, H146, and H69 cell lines (Western blot analyses). Cells were exposed to R1507 or to vehicle during 1 hour, following overnight serum starvation. R1507 (robatumumab, a fully human neutralizing IgG1 monoclonal antibody directed against IGF-1R) was provided by Roche Diagnostics and stored at −80°C. D, effect of R1507 on IGF-1R downstream pathways: Akt, pAkt, MAPK, pMAPK, and pMEK in H526, H146 and H69 cell lines (Western blot analyses). Human-established SCLC cell lines NCI-H146, NCI-H69, and NCI-H526 were obtained from the ATCC (ATCC-LGC Standards) and were cultured in the following medium: RPMI-1640 (GIBCO, Invitrogen) supplemented with 1% GlutaMAX, 10% FBS, and 1% penicillin/streptomycin. Cells growing in floating aggregates were maintained in plastic flasks and incubated at 37°C in 5% CO₂ humidified atmosphere. Serum-starved cells were exposed to increasing doses of R1507 and incubated in the presence or absence of IGF-1. All the Western blots displayed are representative of 3 independent experiments and were conducted as previously described (12). The full list of antibodies with their dilutions is provided in Supplementary Information. Signals were detected using the Super Signal West Pico chemoluminescent system (Pierce Biotechnology).

R1507 downregulated IGF-1R protein expression and disrupted its downstream signaling in selected SCLC cell lines

Three SCLC cell lines were selected according to their high basal IGF1R protein expression and their specific mutation profiles: H146 cells are mutated for MAP2K4 (c.814_891del78), whereas H69 cells are mutated for both PIK3CA (c.317_325del9) and c-Met (c.2962C>T) (Cosmic database). Western blot analyses showed a downregulation of IGF-1R protein expression in the presence of R1507 (200 nmol/L) in all the selected cell lines (Fig. 1C).

R1507 decreased IGF-1–induced downstream effector pAKT in H526 and H146 cells but not in H69 cells (Fig. 1D). R1507 also inhibited ligand-induced phosphorylation of MEK in H526 cells. However, the MAPK pathway (pMAPK, pMEK) appeared to be stimulated by R1507 in a dose-dependent manner in H146 cells.

R1507 inhibited colony formation of selected SCLC cell lines in vitro

Exploring the sensitivity of selected SCLC cell lines to R1507, we observed a 35% and 45% relative reduction in colony formation of H526 cells (P < 0.0001) and H146 cells (P < 0.0001), respectively (Fig. 2A). No significant reduction in the colony-forming capacity was observed in H69 cells (data not shown). Although we did not study any SCLC cell lines without IGF-1R basal expression, such feature does not appear to be correlated with sensitivity to R1507. Indeed, R1507 treatment had no effect on H69 cells, whereas these cells present IGF1R basal expression (Fig. 1D). Interestingly, only the cell lines (H526 and H146) with IGF-1R gene amplification exhibited sensitivity to R1507, whereas those devoid of IGF1R gene amplification (H69) did not respond to R1507 (Fig. 2B).

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Figure 2.

A, colony-forming assays were conducted on H526, H146, and H69 cells treated or not with R1507. Clonogenic survival assays were conducted in methylcellulose as previously described (12). Colonies consisting of at least 50 viable cells were estimated 21 days posttreatment. The surviving fraction was normalized to the corresponding untreated control. Data presented are means ± SEM from at least 3 independent experiments. Comparisons were computed using the Student t test. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01, respectively. B, FISH analysis shows amplification of IGF-1R (red probes) in H525 and H146 cells but not in H69 cells. C, clonogenic formation assays were conducted on H146 and H526 cells combining R1507 (200 nmol/L) and cisplatin (CDDP; 3 μmol/L). Cisplatin was purchased from Mylan Pharmaceuticals and stored at room temperature, protected from light. D, R1507 (200 nmol/L) and IR (2 Gy). A total irradiation dose of 2 Gy was delivered using a linear accelerator (200-kV X-ray irradiator, dose rate = 0.96 Gy/min) within the hour after seeding. Clonogenic survival assays were conducted in methylcellulose as previously described (12). Colonies consisting of at least 50 cells were counted at day 21. The surviving fraction was compared with the corresponding untreated control. Data presented are means ± SEM from 3 independent experiments. Comparisons were computed using the Student t test. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01, respectively.

R1507 sensitized H146 and H526 cells to cisplatin and IR effects

Because cisplatin (CDDP)-based chemotherapy and IR are the standard treatments for SCLC, we investigated whether R1507 could enhance their efficacy in SCLC cell lines. To formally assess the synergy relationship, 2-way ANOVA were computed. We observed that the combination of R1507 and CDDP decreased their colony-forming capacity by 90% and 87% in H526 and H146 cells, respectively (P < 0.001; Fig. 2C). We observed a synergistic inhibitory effect of the CDDP-R1507 combination on the colony-forming capacity of H146 cells (2-way ANOVA interaction test; P = 0.008) and an additive inhibitory effect in H526 cells (2-way ANOVA interaction test; P = 0.691). The R1507-IR combination decreased the colony-forming capacity by 87% and 88% in H526 and H146 cells, respectively (P < 0.001; Fig. 2D). We also noted a synergistic effect of R1507-IR on the colony-forming capacity of H146 cells (2-way ANOVA interaction test; P = 0.0001) and an additive inhibitory effect in H526 cells (2-way ANOVA interaction test; P = 0.101).

The triple combination of R1507-cisplatin-IR potentiated the antitumor effect of cisplatin-IR (current standard treatment) in H146 and H526 cells in vitro

Cisplatin (CDDP) combined with IR remains the standard of care for the treatment of limited-stage SCLCs. We herein observe that the addition of R1507 to CDDP-IR resulted in a 32% and 45% relative reduction in the colony-forming capacity compared with CDDP-IR in H146 and H526 cells, respectively (P < 0.001 and <.00001; Fig. 3).

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Figure 3.

Clonogenic formation assays were conducted on H146 and H526 cells combining R1507 (200 nmol/L), cisplatin (CDDP; 3 μmol/L), and IR (2 Gy). Colony-forming assays were conducted in methylcellulose as previously described (12). Colonies consisting of at least 50 cells were counted at day 21. The surviving fraction was compared with the corresponding untreated control. Data presented are means ± SEM from 3 independent experiments. Comparisons were computed using the Student t test. ***, P < 0.001; **, P < 0.01, respectively.

The addition of R1507 to CDDP or IR induced nonsignificant delays in tumor growth in vivo

To evaluate the delay in tumor growth according to the various treatment conditions in vivo, we assessed the RTV5 in H526 and H146 xenografts (Fig. 4A and B and Supplementary Figs. S1 and S2). Single-agent R1507 induced a nonsignificant delay in tumor growth in H526 and in H146 cells (Supplementary Table S1). Similarly, the R1507-CDDP and R1507-IR combination produced a nonsignificant delay in tumor growth in both H146 cells and H526 cells (Fig. 4B).

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Figure 4.

A, effect of R1507 (18 mg/kg i.p. qw), cisplatin (3mg/kg i.p. days 1, 8, 15), IR (4 fractions of 2 Gy; days 1–4), and their combinations on H526 xenografts in nude mice. Mice were injected with 5 × 10⁶ H526 cells into the flank. Animals were randomized to different treatment groups of 5 to 7 mice each when xenograft diameters attained 5 mm. R1507 was administered via an intraperitoneal injection once weekly at a dose of 18 mg/kg and continued until the end of the experiment. Cisplatin was administered at a dose of 3 mg/kg i.p. at days 1, 8, and 15. IR was delivered in 4 fractions of 2 Gy on days 1 to 4 (A 200-kV X-ray irradiator, dose rate = 0.45 Gy/min), using mouse jigs to exclusively expose the tumor beds. Bidimensional tumor measurement on each mouse was conducted twice weekly to measure the tumor volume using the following formula: (length × width²)/2. Animals were sacrificed when the main tumor volume per group had attained 2,200 to 2,800 mm³. Data presented are means ± SEM of xenograft tumor volumes. B, Kaplan–Meier plots showing the tumor progression of nude mice with H526 xenografts. median RTV5: R1507-cisplatin-IR = 50 days vs. cisplatin-IR = 34 days, P = 0.006. The endpoint, RTV5, is defined as the moment when tumor volume reached 5 times the baseline volume. Differences in median RTV5 were computed with log-rank test.

The triple combination CDDP-IR-R1507 caused a dramatic delay in tumor growth in H526 xenografts as compared with the CDDP-IR combination

The triple combination R1507-CDDP-IR produced a significant delay in tumor growth compared with the standard CDDP-IR regimen in H526 cells (median RTV5: R1507-cisplatin-IR = 50 days vs. cisplatin-IR = 34 days, P = 0.006; Fig. 4A and B). Notably, the experimental regimen yielded an 84% (P = 0.01) reduction of the mean tumor volume compared with the CDDP-IR regimen (tumor assessment at day 42; Supplementary Table S1).

Integrated genomic and functional analysis revealed adaptive mechanisms under R1507

Because R1507 as a single-agent showed clear antitumor activity in vitro but not in vivo, we hypothesized that (i) R1507 as a single agent would not be sufficient to inhibit IGF-1R in vivo and/or (ii) molecular adaptive mechanisms would occur under anti-IGF-1R treatment in vivo.

To evaluate whether R1507 could efficiently inhibit IGF-1R in tumor tissue, we examined IGF-1R expression in H526-xenografted mice at different treatment times. We observed an expected decrease in IGF-1R expression on day 2, but a secondary increase on day 7 (Fig. 5A). This did not appear to be attributable to a lack of R1507 availability on day 7, as the product half-life in mice is estimated at 10 days (11).

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Figure 5.

A, representative sections of IGF-1R staining in H526 xenografts according to various treatment times: baseline (no treatment), days 2 and 7 after R1507. Nude mice bearing H526 xenografts were treated with R1507 (18 mg/kg, i.p.) at day 1 only. Immunohistochemical analyses were conducted using the Discovery XT automated platform (Ventana Medical Systems). B, gene expression profiles (Human Genome U133 Plus 2.0 Array, Affymetrix) derived from H526 xenografts treated according to different time-related conditions: vehicle, 24 hours after R1507 treatment (R1507 day 1), and 7 days after R1507 treatment (R1507 day 7). Microarray probe sets down- or upregulated (2-fold change, P < 0.05, Benjamini–Hochberg adjusted) in R1507 day 1 and R1507 day 7 groups were compared using Venn diagrams. Numbers of probe sets are indicated. C, low-scale protein antibody array. H526 cells were exposed to R1507 treatment (200 nmol/L) for 3 hours (ST-R1507) or for 4 weeks (LT-R1507). The phosphorylation of 28 receptor tyrosine kinases and 11 signaling nodes are detected using the PathScan RTK Signaling Antibody Array Kit. Data represented are the means ± SEM of the fold changes in relative pixel intensities (quantification of the protein pixel intensities relative to the vehicle treatment group) of 3 independent experiments.

We thus hypothesized that some adaptive mechanisms occurred upon R1507 exposure and resulted in the secondary increase in IGF1R expression on day 7. The overall regulation of gene expression induced by R1507 in H526 xenografts at the following time points: baseline (vehicle), R1507 day 1, and R1507 day 7. A total of 2,396 probe sets were differentially expressed at R1507 day 1, whereas only 254 probe sets were differentially expressed at R1507 day 7. Interestingly, only 6.7% (n = 156) of the initially deregulated probe sets (R1507 day 1) remained differentially expressed at R1507 day 7 (Fig. 5B, Supplementary Fig. S3). To investigate the biologic basis of the gene expression alterations, we computed the overlap between the differentially expressed probes and gene set databases. The following pathways were enriched in the R1507-day 1 condition: cell-to-cell adhesion signaling, MAPK pathway, β-arrestin, and vitamin D receptor (Supplementary Table S2). Interestingly, the downregulation of the MAPK pathway for R1507 day 1 was consistent with our previous results in vitro (Fig. 1D). However, some pathways like MAPK signaling appeared to be inversely deregulated between R1507-day 1 and R1507-day 7 (Supplementary Table S2), suggesting a role in the escape to R1507 alone in vivo.

To confirm the microarray results at the protein level, we conducted low-scale protein arrays (Fig. 5C, Supplementary Fig. S4). H526 cells were previously cultured with continuous 4-week R1507 exposure (200 nmol/L) as follows: (i) vehicle, (ii) short-term R1507 exposure (ST-R1507: R1507 200 nmol/L—3-hour exposure), (iii) long-term R1507 exposure (LT-R1507: R1507 200 nmol/L—4-week exposure). We observed several striking results: (i) a transient inhibitory effect on the IGF-1R axis after ST-R1507 exposure, with reduced expression of the phosphorylated forms of IGF-1R (insulin receptor; INSR) and Akt (Ser 473), respectively. After LT-R1507 exposure, we observed a return to the baseline expression of the activated forms of IGF-1R and INSR. This is consistent with our previous results in vivo (Fig. 5A). (ii) After LT-R1507 exposure, we observed a major increase in the expression of receptors and signaling proteins involved in angiogenesis (PDGFR, VEGFR2, EphA2, EphA3, EphB3, EphB4), in proliferation (Akt, MAPK, S6 ribosomal protein, STAT3, ALK), and in survival (Akt, ALK). In this respect, the low-scale tyrosine kinase array results are also consistent with our previous genomic analyses (Supplementary Table S2).

Integrated genomic and functional analysis revealed the involvement of nucleotide excision repair and mitochondrial apoptotic pathways in the response to CDDP- and IR-R1507 combinations

To identify the molecular mechanisms involved in the response to CDDP- and IR-R1507 combinations, we conducted global gene expression profiling on mice bearing H526 xenografts treated with the following treatment conditions: vehicle, R1507 CDDP, IR, CDDP-R1507, and IR-R1507 (Fig. 6A and B, Supplementary Figs. S5 and S6, Supplementary Table S2). To investigate the biologic basis of the gene expression variation, we computed the overlap between the differentially expressed probes and gene set databases.

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Figure 6.

Venn diagrams derived from gene expression profiles (Human Genome U133 Plus 2.0 Array, Affymetrix) of H526 xenografts treated according to the following conditions: vehicle, R1507 alone (18 mg/kg, day 1, i.p.), cisplatin alone (2 mg/kg, day 1, i.p.), IR alone (2 Gy, day 1), and the combination of both. All tumors were removed 7 days after treatment. A, microarray probe sets down- or upregulated (2-fold change, P < 0.05, Benjamini–Hochberg adjusted) in the R1507, cisplatin, and R1507 + cisplatin conditions were compared using Venn diagrams. The numbers of probe sets are indicated. B, microarray probe sets down- or upregulated (2-fold change, P < 0.05, Benjamini–Hochberg adjusted) in the R1507, IR, and R1507 + IR conditions were compared using Venn diagrams. The numbers of probe sets are indicated.

The probes differentially expressed exclusively in the CDDP-R1507 combination were associated with downregulation of the nucleotide excision repair (NER) pathway (P = 0.02; through decreases in ERCC1, XAB2, and RAD23A gene expression) and upregulation of the apoptotic mitochondrial response to DNA damage pathway (through gene expression modifications in BCL2L1 and IFI6; P = 0.04; Fig. 6A, Supplementary Table S2, Supplementary Fig. S5).

The probes that were exclusively differentially expressed in the R1507-IR combination were associated with a downregulation of the TGF-β pathway (Fig. 6B, Supplementary Table S2, Supplementary Fig. S6), suggesting that the IR-R1507 combination induced the downregulation of this pathway.

We then showed that the R1507-CDDP combination decreased ERCC1 but not XPA protein expression in H526 cells (Supplementary Fig. S7A) and also decreased cleaved caspase-9 expression (Supplementary Fig. S7C). When we examined the effect of R1507-IR on the TGF-β pathway, we did not observe any impact on SMAD4 protein expression (Supplementary Fig. S7D). We next investigated whether the R1507-IR combination could modify ERCC1 (and XPA at the protein expression level. Interestingly, the R1507-IR combination also decreased ERCC1 but not XPA protein expression in H526 cells (Supplementary Fig. S7C).