Katsutoshi Goishi

Cisplatin influences acquisition of resistance to molecular-targeted agents through epithelial-mesenchymal transition-like changes.

Chemotherapy with platinum agents is the standard of care for non‐small‐cell lung cancer (NSCLC); however, novel molecular‐targeted agents like gefitinib have been approved for advanced NSCLCs, including recurrent cases previously treated with platinum‐based chemotherapy. Although these agents show antitumor activity through distinct mechanisms and elicit positive initial responses, tumors invariably develop resistance. Recent studies have revealed mechanisms by which both types of agents induce acquired resistance. However, little is known about whether first‐line treatment with either type of agent affects cancer cell susceptibility and development of resistance against subsequent treatment with the other. Using in vitro drug‐resistant NSCLC cell models, we provide evidence that acquired cisplatin resistance may reduce the sensitivity of cancer cells to subsequent treatment with a molecular‐targeted agent. In addition, first‐line cisplatin treatment influenced the mechanism by which cancer cells developed resistance to subsequent treatment with a molecular‐targeted agent. The influence of cisplatin on acquisition of resistance to a molecular‐targeted agent was associated with epithelial–mesenchymal transition (EMT)‐like alterations such as increased expression of mesenchymal markers, morphological change, and AXL tyrosine kinase‐mediated increased cell motility. Our findings indicate that the influence of platinum‐based chemotherapy on molecular‐targeted therapies and the involvement of EMT and EMT‐related effectors should be considered when developing therapeutic strategies using antitumor agents, especially in the context of sequential therapy.

Noncompetitive Immunoassay Detection System for Haptens on the Basis of Antimetatype Antibodies

BACKGROUND Small molecules classified as haptens are generally measured by competitive immunoassay, which is theoretically inferior to noncompetitive sandwich immunoassay in terms of sensitivity and specificity. We created a method for developing sandwich immunoassays to measure haptens on the basis of antimetatype antibodies. METHODS We generated antimetatype monoclonal antibodies against a hapten-antibody immunocomplex using an ex vivo antibody development system, the Autonomously Diversifying Library (ADLib) system. We selected 2 haptens, estradiol (E2) and 25-hydroxyvitamin D [25(OH)D], as analytes. Sandwich immunoassays for these 2 haptens were developed by use of a 96-well microtiter plate and a fully automated chemiluminescence analyzer, and the performances of these immunoassays were investigated. RESULTS The developed assays exhibited sensitivity high enough to detect target haptens in serum samples. The limit of detection of the ELISA for E2 was 3.13 pg/mL, and that of the fully automated chemiluminescent enzyme immunoassay (CLEIA) system was 2.1 ng/mL for 25(OH)D. The cross-reactivity with immunoreactive derivatives was effectively improved compared with the competitive assay. The CVs for the sandwich ELISA for E2 were 4.2%-12.6% (intraassay) and 6.2%-21.8% (total imprecision). The CVs for the sandwich CLEIA for 25(OH)D were 1.0%-2.3% (intraassay) and 1.9%-3.5% (total imprecision). In particular, the sandwich CLEIA for 25(OH)D showed correlations of r = 0.99 with both LC-MS/MS and a commercially available (125)I RIA. CONCLUSIONS Our method represents a potentially simple and practical approach for routine assays of haptens, including vitamins, hormones, drugs, and toxins.

Protein kinase D2 and heat shock protein 90 beta are required for BCL6-associated zinc finger protein mRNA stabilization induced by vascular endothelial growth factor-A

Vascular endothelial growth factor (VEGF) is a major angiogenic factor that activates pro-angiogenic molecules to generate new vessels. Recently, we identified a VEGF-A-induced pro-angiogenic gene, BCL-6 associated zinc finger protein (BAZF), in endothelial cells. BAZF interacts with CBF1, a transcriptional regulator of Notch signaling, and downregulates Notch signaling by inducing the degradation of CBF1. A signal inhibition assay with a combination of chemical inhibitors and siRNA revealed that the protein kinase D (PRKD) family, mainly PRKD2, mediated BAZF gene expression by VEGF-A stimulation. A luciferase reporter assay showed that the promoter activity of the BAZF gene was unchanged by VEGF-A stimulation. However, we found that the stability of BAZF mRNA increased in a VEGF-A/PRKD2-dependent manner. In further studies to investigate the underlying mechanism, we successfully identified heat shock protein 90 beta (HSP90β) as a molecule that interacts with and stabilizes BAZF mRNA following VEGF-A/PRKD2 activation. These data suggest that HSP90β may positively regulate angiogenesis, not only as a protein chaperone, but also as an mRNA stabilizer for pro-angiogenic genes, such as BAZF, in a PRKD2 activity-dependent manner.

Novel monoclonal antibodies recognizing the active conformation of epidermal growth factor receptor.

The precise regulation of epidermal growth factor receptor (EGFR) is crucial for its function in cellular growth control. Although many antibodies against EGFR have been developed and used to analyze its regulation and function, it is not yet easy to analyze activated EGFR specifically. Activated EGFR has been mainly detected by its phosphorylation state using anti-phospho-EGFR and anti-phosphotyrosine antibodies. In the present study, we have established novel monoclonal antibodies which recognize the activated EGFR independently of its phosphorylation. Our antibodies detected active state of EGFR in immunoprecipitation and immunofluorescence, by recognizing the epitopes which are exposed through the conformational change induced by ligand-binding. Furthermore, we found that our antibodies preferentially detected the conformation of constitutively active EGFR mutants found in lung cancer cell lines. These results indicate that our antibodies may become novel research and diagnostic tools for detecting and analyzing the conformation of active EGFR in various cells and tissues.

Abstract B46: Soluble AXL is a candidate circulating biomarker for predicting acquired and intrinsic resistance to molecular-targeted agents in NSCLC cells.

Background: Drug resistance is an important factor that compromise therapeutic effect of most anti-cancer drugs including molecular-targeted agents. Non-invasive assessment of drug resistance by circulating biomarkers would therefore be useful for determining therapeutic options for resistant patients. Recently, a receptor tyrosine kinase AXL has been implicated in acquired resistance to EGFR inhibitors in NSCLC cells. In this study, we examined the potential utility of AXL as a circulating biomarker to predict acquired or intrinsic drug resistance using NSCLC cells. Materials and methods: Drug resistant NSCLC cell lines were developed by exposing NSCLC cells with either EGFR inhibitor gefitinib or MET inhibitor PHA-665752. AXL-positive cells in parent and established resistant cell lines were isolated by anti-AXL antibody-immobilized magnetic beads. Drug resistance was assessed by MTS growth inhibition assay. Immunoblot analysis was performed on cell lysate and conditioned medium to evaluate expression and release of AXL. The role of AXL in cell growth and motility was examined by MTS growth inhibition assay and transwell migration assay. Results: Drug-resistant NSCLC cell lines were successfully established, and increased release of fragmented soluble form of AXL was observed in these cell lines. They showed increased cell motility, which was effectively inhibited by AXL knockdown using siRNA. In contrast, their cell growth was not affected by AXL knockdown, even when these cells were simultaneously treated with molecular-targeted agents. Furthermore, cells highly expressing AXL were immunologically isolated from a gefitinib-sensitive parent cell line. These cells showed partial resistance to gefitinib, indicating that such cells intrinsically exist in NSCLC cell populations. Conclusions: Soluble form of AXL is a candidate circulating biomarker for predicting acquired and intrinsic resistance to molecular-targeted agents in NSCLC cells. It may also be used to predict AXL-mediated increased motility of resistant cells. Patients harboring such resistant cells may have increased chance of responding to AXL-targeted anti-metastasis therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B46. Citation Format: Nobuyuki Ise, Nobuaki Takemori, Kazuya Omi, Katsutoshi Goishi, Shigeki Higashiyama. Soluble AXL is a candidate circulating biomarker for predicting acquired and intrinsic resistance to molecular-targeted agents in NSCLC cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B46.

Abstract A139: Development of EML4-ALK addicted cell models for evaluating sensitivity to ALK inhibitors.

Background: EML4-ALK fusion tyrosine kinase has been implicated in the pathogenesis of a subset of non-small-cell lung cancers (NSCLCs). The fusion kinase is a rational therapeutic target for addicted cancer cells whose sustained proliferation and survival depend on it. Cancer cell lines harboring the fusion kinase gene are useful for screening effective therapeutic agents. However, few cell lines have been identified so far, and these cell lines do not represent all the variants discovered in clinical specimens. Moreover, drug-resistant mutations in the fusion gene have recently been identified in patients treated with ALK inhibitors. Techniques to establish cell models addicted to certain genes will be invaluable for developing effective drugs for molecular targeted therapy. We have developed a new method to easily and efficiently establish cell models exhibiting addiction to introduced oncogenes. We evaluated the method for variants of EML4-ALK fusion oncogene including drug-resistant mutants. Material and methods: EGFR-addicted NSCLC cell lines were transfected with each variant of EML4-ALK fusion gene, and the cells addicted to the introduced fusion gene for their growth and survival were selected. Expression of EML4-ALKs in the obtained cells was confirmed by immunoblotting. Their dependence on the introduced fusion gene was assessed by MTS assay following ALK inhibitors treatment. Direct inhibitory effect of the drugs was analyzed by monitoring the autophosphorylation levels of EML4-ALKs. Results: EML4-ALK addicted cells were efficiently established from all the transfectants. They expressed the introduced variant of EML4-ALK. Each of the ALK inhibitors tested in this study exerted distinct anti-growth activities toward the established cell models. PF-2341066 had the strongest, dose-dependent inhibitory effect on the growth of the addicted cells. This agent also inhibited autophosphorylation of EML4-ALKs in parallel with the growth inhibition activity, but failed to inhibit cells expressing drug-resistant EML-4-ALK. Conclusions: We demonstrated that cancer cell models addicted to EML4-ALK can be easily established from EGFR-addicted cell lines. Since the established cells depended on the introduced EML4-ALK genes for their growth and survival, the introduced genes likely preserved their oncogenic functions in these cells. Our method will provide useful cell models to evaluate the efficacy of various inhibitors in vitro and elucidate functions and signaling pathway of EML4-ALKs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A139.