Interleukin-11 Receptor Is a Candidate Target for Ligand-Directed Therapy in Lung Cancer
2016; Elsevier BV; Volume: 186; Issue: 8 Linguagem: Inglês
10.1016/j.ajpath.2016.04.013
ISSN1525-2191
AutoresMarina Cardó‐Vila, Serena Marchiò, Masanori Sato, Fernanda I. Staquicini, Tracey L. Smith, Julianna Bronk, Guosheng Yin, Amado J. Zurita, Menghong Sun, Carmen Behrens, Richard L. Sidman, J. Jack Lee, Waun Ki Hong, Ignacio I. Wistuba, Wadih Arap, Renata Pasqualini,
Tópico(s)HER2/EGFR in Cancer Research
ResumoWe previously isolated an IL-11–mimic motif (CGRRAGGSC) that binds to IL-11 receptor (IL-11R) in vitro and accumulates in IL-11R–expressing tumors in vivo. This synthetic peptide ligand was used as a tumor-targeting moiety in the rational design of BMTP-11, which is a drug candidate in clinical trials. Here, we investigated the specificity and accessibility of IL-11R as a target and the efficacy of BMTP-11 as a ligand-targeted drug in lung cancer. We observed high IL-11R expression levels in a large cohort of patients (n = 368). In matching surgical specimens (i.e., paired tumors and nonmalignant tissues), the cytoplasmic levels of IL-11R in tumor areas were significantly higher than in nonmalignant tissues (n = 36; P = 0.003). Notably, marked overexpression of IL-11R was observed in both tumor epithelial and vascular endothelial cell membranes (n = 301; P < 0.0001). BMTP-11 induced in vitro cell death in a representative panel of human lung cancer cell lines. BMTP-11 treatment attenuated the growth of subcutaneous xenografts and reduced the number of pulmonary tumors after tail vein injection of human lung cancer cells in mice. Our findings validate BMTP-11 as a pharmacologic candidate drug in preclinical models of lung cancer and patient-derived tumors. Moreover, the high expression level in patients with non-small cell lung cancer is a promising feature for potential translational applications. We previously isolated an IL-11–mimic motif (CGRRAGGSC) that binds to IL-11 receptor (IL-11R) in vitro and accumulates in IL-11R–expressing tumors in vivo. This synthetic peptide ligand was used as a tumor-targeting moiety in the rational design of BMTP-11, which is a drug candidate in clinical trials. Here, we investigated the specificity and accessibility of IL-11R as a target and the efficacy of BMTP-11 as a ligand-targeted drug in lung cancer. We observed high IL-11R expression levels in a large cohort of patients (n = 368). In matching surgical specimens (i.e., paired tumors and nonmalignant tissues), the cytoplasmic levels of IL-11R in tumor areas were significantly higher than in nonmalignant tissues (n = 36; P = 0.003). Notably, marked overexpression of IL-11R was observed in both tumor epithelial and vascular endothelial cell membranes (n = 301; P < 0.0001). BMTP-11 induced in vitro cell death in a representative panel of human lung cancer cell lines. BMTP-11 treatment attenuated the growth of subcutaneous xenografts and reduced the number of pulmonary tumors after tail vein injection of human lung cancer cells in mice. Our findings validate BMTP-11 as a pharmacologic candidate drug in preclinical models of lung cancer and patient-derived tumors. Moreover, the high expression level in patients with non-small cell lung cancer is a promising feature for potential translational applications. 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Combinatorial screenings in patients: the interleukin-11 receptor alpha as a candidate target in the progression of human prostate cancer.Cancer Res. 2004; 64: 435-439Crossref PubMed Scopus (122) Google Scholar This formulation, termed BMTP-11, underwent evaluation in a first-in-man phase zero clinical trial, aimed at defining drug specificity, maximum tolerated dose, and limiting toxicities with promising pilot results.22Pasqualini R. Millikan R.E. Christianson D.R. Cardó-Vila M. Driessen W.H. Giordano R.J. Hajitou A. Hoang A.G. Wen S. Barnhart K.F. Baze W.B. Marcott V.D. Hawke D.H. Do K.A. Navone N.M. Efstathiou E. Troncoso P. Lobb R.R. Logothetis C.J. Arap W. Targeting the interleukin-11 receptor alpha in metastatic prostate cancer: a first-in-man study.Cancer. 2015; 121: 2411-2421Crossref PubMed Scopus (38) Google Scholar Here, we evaluate IL-11R as a molecular target in human lung cancer and propose a therapeutic approach based on the selective targeting of IL-11R. In human samples, we found increased expression of IL-11R in tumors compared with adjacent nonmalignant tissues. Overexpressed IL-11R was exposed on the tumor cell membrane and was present also in tumor vascular endothelial cells, supporting the ligand-directed accessibility of this receptor from the circulation. We demonstrated that in vitro BMTP-11 efficiently targets and kills a panel of lung cancer cell lines derived from adenocarcinoma (ADC), squamous cell carcinoma (SCC), or large cell lung carcinoma. When BMTP-11 was systemically administered in vivo, we observed a significant delay in the growth of subcutaneous tumor xenografts plus a reduction in the establishment of pulmonary tumors after tail vein injection of human lung cancer cells. Taken together, these results suggest that BMTP-11 could potentially serve as an active targeted drug candidate to be evaluated in patients with lung cancer. The human lung cancer cell lines A549, H226, H460, and H522 were from the ATCC (Manassas, VA); HOP62 and HOP92 were from the National Cancer Institute, Center for Cancer Research cell repository (Bethesda, MD). Cells were cultured at 37°C in 5% CO2 and maintained in RPMI 1640 supplemented with 5% fetal bovine serum (ThermoFisher Scientific, Waltham, MA), 5 mmol/L l-glutamine, and antibiotics (100 U/mL penicillin G and 100 mg/mL streptomycin; ThermoFisher Scientific). Exponentially growing cells were harvested, washed with phosphate-buffered saline (PBS), and resuspended in ice-cold PBS that contained 1% bovine serum albumin. Human Fc Receptor Blocking Reagent (Miltenyi Biotec, San Diego, CA) was added to each cell suspension (20 μL per 1 × 107 cells) and incubated for 10 minutes on ice. Next, 20 μL of either anti-human IL-11Rα-phycoerythrin–labeled antibody (Clone N-20; Santa Cruz Biotechnology, Dallas, TX) or phycoerythrin-conjugated rabbit IgG isotype control (GeneTex, Irvine, CA) was added per 1 × 106 cells in 100 μL of PBS that contained 1% bovine serum albumin and incubated at 4°C for 1 hour. Subsequently, cells were washed with PBS that contained 1% bovine serum albumin and analyzed with a BD FACSCanto flow cytometer (BD Biosciences, Franklin Lakes, NJ). Cells were grown to 95% confluence in 96-well plates (Fisher Scientific, Pittsburgh, PA). Serial molar concentrations of either BMTP-11 or nontargeted D[KLAKLAK]2 control were added to each well to a final molar concentration of 1 to 1000 μmol/L. After incubation at 37°C for 18 hours, cell viability was assessed with the WST-1 assay (Roche Life Sciences, Indianapolis, IN). Values obtained in test conditions were normalized to negative controls and were expressed as percentage values. Apoptotic cell death was evaluated with the Annexin V/PI kit (BD Biosciences, San Jose, CA) according to the manufacturer's instructions. In essence, 15,000 cells were seeded into 96-well plates, treated with serial molar concentrations of either peptide (1 to 1000 μmol/L) for 6 hours, followed by the addition of 4 μL/well of the Annexin V antibody. Fluorescence was quantified in a ClarioStar plate reader (BMG Labtech, Ortenberg, Germany). For both assays, reverse sigmoidal dose–response curves with variable slopes were fitted, and concentration that inhibits 50% (IC50) values were calculated with the Prism software version 5.0 (GraphPad Software, Inc., La Jolla, CA). Female nude BALB/c mice were from Harlan Laboratories (Indianapolis, IN). The H460 human lung cancer cell line is a well-established model, known to produce consistent subcutaneous tumors and lung tumors after intravenous administration. A subcutaneous model was obtained by implantation of 106 H460 cells into the flank; expression of IL-11R in these tumors was confirmed by immunohistochemistry (IHC). Treatment consisted of injections of 10 mg/kg of either BMTP-11 or nontargeted D[KLAKLAK]2 peptide (n = 10 per group) at days 3 and 10 after tumor implantation. Tumor sizes were serially measured with a digital caliper, and tumor volumes were calculated.23Marchiò S. Lahdenranta J. Schlingemann R.O. Valdembri D. Wesseling P. Arap M.A. Hajitou A. Ozawa M.G. Trepel M. Giordano R.J. Nanus D.M. Dijkman H.B. Oosterwijk E. Sidman R.L. Cooper M.D. Bussolino F. Pasqualini R. Arap W. Aminopeptidase A is a functional target in angiogenic blood vessels.Cancer Cell. 2004; 5: 151-162Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 24Arap M.A. Lahdenranta J. Mintz P.J. Hajitou A. Sarkis A.S. Arap W. Pasqualini R. 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Orthotopic reconstitution of human small-cell lung carcinoma after intravenous transplantation in SCID mice.Anticancer Res. 1992; 12: 1407-1410PubMed Google Scholar Again, expression of IL-11R was confirmed in lung tumors by IHC staining with an anti–IL-11R antibody. Mice were treated with BMTP-11 (n = 10) or nontargeted D[KLAKLAK]2 peptide (n = 9) as described above in this section and sacrificed 15 days after tumor cell injection. Lungs were recovered, weighed on a digital scale, formalin-fixed, and paraffin-embedded. Ten sections corresponding to different levels of the lungs were obtained, which were stained with hematoxylin and eosin. The number of tumors in each slide was determined by a pathologist. All animal protocols were approved by the Institutional Animal Care and Use Committee at The University of Texas MD Anderson Cancer Center, and all experiments were completed in accordance with institutional guidelines. The retrospective study was approved by the Institutional Review Board at The University of Texas MD Anderson Cancer Center. Surgical lung cancer specimens were obtained from the Lung Cancer Specialized Program of Research Excellence Tissue Bank. A panel of patient-derived samples (n = 368) was collected from 1997 to 2001. A tissue microarray was constructed from triplicate 1-mm–diameter cores, punctured from central, intermediate, and peripheral regions of primary lung cancers (n = 327), as described.28Raso M.G. Behrens C. Herynk M.H. Liu S. Prudkin L. Ozburn N.C. Woods D.M. Tang X. Mehran R.J. Moran C. Lee J.J. Wistuba I.I. Immunohistochemical expression of estrogen and progesterone receptors identifies a subset of NSCLCs and correlates with EGFR mutation.Clin Cancer Res. 2009; 15: 5359-5368Crossref PubMed Scopus (145) Google Scholar, 29Behrens C. Feng L. Kadara H. Kim H.J. Lee J.J. Mehran R. Hong W.K. Lotan R. Wistuba I.I. Expression of interleukin-1 receptor-associated kinase-1 in non-small cell lung carcinoma and preneoplastic lesions.Clin Cancer Res. 2009; 16: 34-44Crossref PubMed Scopus (27) Google Scholar IHC was performed on 4-μm formalin-fixed paraffin-embedded tissue sections. Antigen retrieval was achieved by heating in 1 mmol/L EDTA pH 8.0 (ThermoFisher Scientific), followed by blocking in 3% H2O2 for 15 minutes and Protein Block Solution (Dako Corp., Carpinteria, CA; Code X0909) for 30 minutes. The primary goat anti-human (clone K20; Santa Cruz Biotechnology; used for human tissues) or rabbit (clone C20; Santa Cruz Biotechnology; used for mouse tissues) anti–IL-11Rα antibody was incubated for 2 hours. Staining was revealed with a secondary antibody and Envision Plus Dual Link-labeled polymer (Dako Corp.) for 30 minutes, after which time diaminobenzidine was applied for 5 minutes. Slides were counterstained with hematoxylin. IL-11R expression was evaluated by two independent expert pathologists as described.21Zurita A.J. Troncoso P. Cardó-Vila M. Logothetis C.J. Pasqualini R. Arap W. Combinatorial screenings in patients: the interleukin-11 receptor alpha as a candidate target in the progression of human prostate cancer.Cancer Res. 2004; 64: 435-439Crossref PubMed Scopus (122) Google Scholar, 30Sun M. Behrens C. Feng L. Ozburn N. Tang X. Yin G. Komaki R. Varella-Garcia M. Hong W.K. Aldape K.D. Wistuba I.I. HER family receptor abnormalities in lung cancer brain metastases and corresponding primary tumors.Clin Cancer Res. 2009; 15: 4829-4837Crossref PubMed Scopus (131) Google Scholar Cytoplasmic scores were obtained by multiplying a 4-value intensity score (0, none; 1+, weak; 2+, moderate; 3+, strong) by the percentage of positive area. The membrane expression of IL-11R was quantified as percentage of membrane-positive cells over a total of at least 200 cells evaluated (Supplemental Table S1). To analyze IHC data, Wilcoxon signed rank tests were conducted to compare expressions of IL-11R in tumor specimens with nonmalignant bronchial epithelium. Linear regression models for continuous variables assuming a normal distribution and logistic regression models for binary variables were used to study the association of IL-11R expression with histology for all of the cohort patients. For patients with NSCLC (n = 301), either linear regression models or logistic regression models were built to characterize the association between IL-11R expression and clinical factors, including tumor pathology and smoking history. Regression analyses of survival data based on the Cox proportional hazards model were conducted on overall survival, defined as time from surgery to death or to the end of the study. The Student's t-test was applied to analyze all of the other data. All of the statistical tests were two-sided. P values < 0.05 were considered statistically significant. Statistical analyses were performed with the Prism software version 5.0 (GraphPad Software, Inc.). To begin to define the feasibility of a BMTP-11–based therapy in human lung cancer, we first examined the expression of IL-11R in a panel (n = 41) of primary formalin-fixed paraffin-embedded tumors by IHC with a specific anti–IL-11Rα antibody. This analysis revealed high cytoplasmic positivity in both SCC (Figure 1, A–C) and ADC (Figure 1D). In samples with grade 3+ cytoplasmic positivity, we also observed membrane IL-11R staining and grade 3+ positivity in blood vessels into the tumor stroma (Figure 1, E and F). For a subset of patient-derived samples (n = 18), matching nonmalignant lung tissues were also available; the expression of IL-11R in these tumor-adjacent lung tissues was comparable with that in lung tissue from a healthy donor (Figure 1G), except for a specific region corresponding to the bronchiolar wall below the basement membrane (Figure 1H). Cytoplasmic IL-11R levels were quantified and compared between tumor and nonmalignant tissues (Figure 2), confirming the observed higher expression in tumor cells (P = 0.003).Figure 2IL-11R expression scores in tumor and adjacent nonmalignant tissue. Comparison of the cytoplasm expression scores between lung cancer and matched adjacent nonmalignant bronchial epithelium. n = 18 lung cancer samples; n = 18 nonmalignant bronchial epithelium samples. *P < 0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT) These promising results prompted us to extend the analysis to a larger panel of lung cancer tissue microarray specimens (pilot, n = 327) (Table 1, Supplemental Table S1). In this cohort of patients, the cytoplasmic levels of IL-11R were significantly higher in NSCLC than in SCLC (ADC or SCC versus SCLC, P < 0.0001) (Table 2). The successive analyses were therefore focused exclusively on NSCLC samples (n = 301). The presence of IL-11R on the cell membrane was evaluated in 200 cells/tissue sections, revealing well-detectable positivity in approximately 25% of the cases. These same samples also exhibited higher cytoplasmic levels of IL-11R (P < 0.0001) (Table 3), suggesting that increased overall expression of IL-11R favors its allocation to the plasma membrane, where it can be targeted by ligand-directed therapeutic agents (such as BMTP-11). Then, to determine whether a subset of patients would specifically benefit from BMTP-11 therapy, we first assessed IL-11R expression in association to specific clinicopathologic features. This analysis revealed that patients with ADC had higher cytoplasmic levels of IL-11R than patients with SCC (P < 0.0001), and patients who had ever smoked (either former or current) had higher cytoplasmic levels of IL-11R than patients who never smoked (P = 0.005) (Table 3). These findings encourage the use of increased expression of IL-11R as a biomarker to identify patients who might benefit from IL-11R–targeted therapy.Table 1Multivariate Analysis for Patients Included in the TMACharacteristicFrequency countTotal frequency, %Histology ADC19258.72 SCLC267.95 SCC10933.33Sex NA267.95 Female16249.54 Male13942.51Race NA267.95 Caucasian26581.04 Other3611.01Type of smoker NA267.95 Current8225.08 Former12036.70 Never9729.66 Unknown20.61Pathologic M NA267.95 M029590.21 M161.83Pathologic N NA267.95 N021264.83 N15516.82 N23410.40Pathologic T NA267.95 T111434.86 T216048.93 T3164.89 T4113.36ADC, adenocarcinoma; NA, not analyzed (low target expression); SCC, squamous cell carcinoma; SCLC, small cell lung cancer; TMA, tissue microarray. Open table in a new tab Table 2Correlation of IL-11R Cytoplasmic Expression with Tumor HistologyVariableDFEstimateSEMWald 95% confidence limitsχ2P > χ2LowerUpperIntercept1109.722213.640182.9880136.456464.71 χ2IL-11R localization123.56<0.001Histology115.62<0.001Type of smoker210.490.0053DF, degrees of freedom. Open table in a new tab ADC, adenocarcinoma; NA, not analyzed (low target expression); SCC, squamous cell carcinoma; SCLC, small cell lung cancer; TMA, tissue microarray. ADC, adenocarcinoma; DF, degrees of freedom; SCC, squamous cell carcinoma; SCLC, small cell lung cancer. DF, degrees of freedom. Having determined the pathologic presence of IL-11R as a molecular target in lung cancer, we next evaluated the activity of BMTP-11 as a ligand-directed therapy in tissue culture and preclinical mouse models. We used the human lung cancer-derived cell lines A549, H226, H460, HOP62, and HOP92. All these NSCLC lines express IL-11R on their cell surface membranes as evaluated by flow cytometry (Table 4). H522 cells were used as a negative control, showing undetectable surface expression of IL-11R. Cultured cells were treated with serial concentrations of BMTP-11 or untargeted D[KLAKLAK]2 (control), followed by evaluation of both viability (by WST-1 assay at 18 hours after treatment) and apoptosis (by Annexin V/PI assay at 6 hours after treatment). Plots were fitted to reverse sigmoidal dose-response curves, showing that BMTP-11 reduced cell viability by inducing apoptosis in all tumor cell lines examined (Figure 3, A–E). H460 (Figure 3A), HOP62 (Figure 3B), and HOP92 (Figure 3C) cells exhibited almost no residual viability when treated with 100 μmol/L BMTP-11, while retaining approximately 100% viability in the presence of equimolar amounts of the control peptide (P < 10−8). Similar results were obtained with H226 (Figure 3D) and A549 (Figure 3E) cells at higher BMTP-11 concentrations (100 to 300 μmol/L). No effect was observed in the IL-11R− cell line H522 (Figure 3F). In all other tumor cell lines examined, the IC50 for BMTP-11 was 6.3- to 11.5-fold lower than the control (Table 5). These results show that BMTP-11 is active against IL-11R–expressing human lung cancer cells in vitro, with the advantage of suitable dose range, wide therapeutic window, and broad applicability in cells derived from different lung cancer types.Table
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