Brief Article pubs.acs.org/molecularpharmaceutics
Enhanced Antitumor Activity of Cetuximab in Combination with the Jak Inhibitor CYT387 against Non-Small-Cell Lung Cancer with Various Genotypes Yuan Hu,*,†,‡ Xian-Zhe Dong,†,‡ Xu Liu,† Ping Liu,† and Yi-Bang Chen§ †
Department of Clinical Pharmacology, Pharmacy Care Center, Chinese PLA General Hospital, Beijing 100853, China Department of Pharmacology and System Therapeutics, Mount Sinai School of Medicine; New York, New York 10029, United States
§
ABSTRACT: Cetuximab, an epidermal growth factor receptor (EGFR) inhibitor, is effective in the treatment of non-small-cell lung cancers (NSCLCs). However, resistance to EGFR inhibitors limits its effectiveness. In this study, we investigated the effectiveness of Jak-2 inhibitor, CYT387, in combination with cetuximab. Xenograft animal models were administered with cetuximab or CYT387 or their combination. It was observed that NSCLC cells exhibited enormous differences in responses to cetuximab; cell lines were more intrinsically resistant to cetuximab. In resistant cell lines (H1975 and H1650), the efficacy of cetuximab was increased when combined with CYT387, whereas CYT387 alone in low doses exhibited little effect on NSCLC cell proliferation. In addition, the antitumor activity of cetuximab was increased in H1975 resistant model in spite of low efficacy of cetuximab treatment alone in. Jak/STAT signaling was suppressed effectively by the combination of cetuximab and CYT387. In summary, our findings indicated that CYT387 has a potent indirect antitumor activity, and it is also synergistic in its activity in combination with cetuximab against NSCLC tumors, especially with cetuximab intrinsic-resistance tumors. These indications were mediated via Janus kinase (Jak)-signal transducer and transcription (STAT) pathway activator. Our results strongly and consistently supported the potential synergism of CYT387 as Jak inhibitor for anti-NSCLC therapy with EGFR-targeting agents. KEYWORDS: resistant, Jak/STAT, cetuximab
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STAT, could be attributed to this variability.10,11 EGFR mediates intracellular signaling through RAS-RAF-MEKMAPK pathway, PI3K PTEN-AKT pathway upon activation in NSCLC. EGFR can also activate STAT3 in both a Jakdependent and a Jak-independent manner.7,10,12 Phosphorylation of pSTAT3 is correlated with phosphorylaion of pEGFR in NSCLC patients.13 Reports have also demonstrated enhanced antitumor activity and reduced drug resistance, which is attributed to the effects of the combination of Jak/ STAT pathway inhibitor, EGFR target (Erlotinib) drug, or chemotherapy drug (doxorubicin), which could enhance antitumor activity13 or reduce the drug resistance14−17 or both. Although Jak inhibitors are still in early clinical trials, very limited data is available to validate their efficacy. CYT387, an ATP-competitive inhibitor of Jak1 and Jak2, is more effective against Jak2, which inhibits the cell proliferation in the cell lines.18 Most of the cell lines were established in the context of viral carcinogenesis due to limited number of NSCLC cell lines. These cell lines enabled a biased estimation of different clinical
INTRODUCTION Lung cancer is the second most commonly occurring solid tumor in China, while it is the sixth most commonly occurring tumor worldwide.1,2 Non-small-cell lung cancer (NSCLC) accounts for approximately 85%3,4 of all the cancers. Though immunotherapy is an effective strategy in the treatment of lung cancer, chemotherapy remains the standard treatment for NSCLC patients at the advanced stages. Clinical data has demonstrated that the effectiveness of chemotherapies is very limited due to multiple side effects.5Cetuximab is a chimeric, monoclonal EGFR-specific IgG1 antibody, which has 5−10fold higher affinity toward EGFR as compared to endogenous ligands. It also inhibits EGFR phosphorylation.6 However, owing to the low efficacy, cetuximab is not suited for monotherapy in patients with advanced NSCLC.7 Therefore, the development of an effective cetuximab-based multidrug combination therapy is required. Jak/STAT3 signal transduction pathway is downstream of cytokine receptors. This pathway is also involved and activated in hematologic malignancies and varied solid tumors, such as head and neck squamous cell carcinoma (HNSCC), NSCLC, and SCLC.8,9 Clinical data has demonstrated that the inhibition of Jak/STAT influences NSCLC cells. Cell line specific effects and/or the signaling pathways, both upstream and downstream from Jak/ © 2015 American Chemical Society
Received: December 11, 2015 Accepted: December 19, 2015 Published: December 19, 2015 689
DOI: 10.1021/acs.molpharmaceut.5b00927 Mol. Pharmaceutics 2016, 13, 689−697
Brief Article
Molecular Pharmaceutics
stained with crystal violet (0.1% in 20% methanol). Digital images of the plates were obtained as a permanent record before colony counting. Xenograft Models and Efficacy Studies. All the animal studies were given official approval by the Chinese PLA General Hospital Institutional Animal Care and Use Committee, China. Cancer cells (0.75 ± 0.25 × 106) were injected subcutaneously into the right flank of athymic nude (BALB/c nu/nu) mice (ShangHai Slac Laboratory Animal CO. LTD, China). Tumor volume (V) was obtained by multiplying the three measured dimensions by 0.5 (V = 0.5 × l × w × h). The mice were injected intraperitoneally (IP), once every other day, as soon as the tumors reached a minimum size of 60 mm3; the dosing regimen: vehicle or cetuximab (5 mg/kg) or combination cetuximab (5 mg/kg) plus CYT387 (orally 10 mg/kg). All the studies were done in blinding. Tumor Histology. At the end of the experiment, the tumor was extracted. Following the resection and freezing of tumors in isopentane, the cells were sectioned into positive slides. The frozen sections unstained were fixed in ice-cold acetone for 15 min, which was then rehydrated in PBS and blocked in TBS, containing 10% goat serum, 1% BSA, and goat anti-mouse Fab (Jackson Immunoresearch, West Grove, PA). The whole process was followed by overnight incubation at 4 °C with primary antibodies for cleaved caspase-3. Alexafluor 568-goat anti-rabbit secondary antibodies (Invitrogen, USA) were incubated for 1 h at room temperature following the washing. This was followed by Hoechst 33342 (Molecular Probes, USA) staining. The slides were mounted using VectaShield (Vector Laboratories, Burlingame, CA, USA) and were observed with a nonconfocal fluorescence microscope at 100× magnification (Olympus, Japan). The pictures were developed and analyzed using ImagePro-Plus software (Media Cybernetics, Silver Spring, MD, USA). For caspase-3 activity, a 2−3 mm crosssectional tumor slice was lysed in RIPA buffer by sonication. Colorimetric assays were used for the analysis of the resulting lysates according to the manufacturer’s protocol. Pharmacokinetic Analysis. To characterize the PK of cetuximab, 1650 tumor mice (n = 3 per time points) were bled by cardiac puncture, following a single cetuximan or cetuximab plus CYT387 administration, at the time points of 0, 0.5, 1, 3, 7 24, 48, and 72 h. The blood was collected and centrifuged, and the plasma was pooled and stored at −80 °C until analysis by enzyme-linked immunosorbent assay (ELISA).21 Recombinant human EGFR (extracellular domain) was used in ELISA, which was absorbed onto a microtiter plate. The EGFR captured cetuximab in 10% mouse plasma, which was detected using a peroxidase-conjugated affinipure rabbit anti-human IgG Fc fragment. The assay had a calibration range of 0.1−6 ng/mL (1−60 ng/mL in 100% mouse plasma). Statistical Analysis. Plots and statistics were generated using Prism 5.0 (GraphPad Software, Inc.). Student’s t-test or Tukey’s test were used to analyze significant differences between the groups. P < 0.05 was considered as statistically significant.
situations and variability. Therefore, individually exploring the activity of cetuximab in a more clinically relevant setting is required. Here, we investigated the efficacy of cetuximab treatment both in vitro and in vivo coadministered with CYT387, and its antitumor activity in NSCLC cell lines with various genetic backgrounds and cetuximab-insensitive NSCLC xenograft models.
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METHODS Cell Culture and Reagents. Cetuximab and CYT387 were obtained from Sigma (St. Louis, MO) and Selleck (Tianjin, PR China). The compounds and the stock solutions were stored at −20 °C in dimethyl sulfoxide (DMSO). The compounds were diluted to achieve a final concentration with fresh medium before each experiment.19 Since some cell lines were sensitive to DMSO, the final concentration of DMSO was maintained
