Teppei Sugano

Inhibition of ABCB1 Overcomes Cancer Stem Cell–like Properties and Acquired Resistance to MET Inhibitors in Non–Small Cell Lung Cancer

Patients with non–small cell lung cancer (NSCLC) EGFR mutations have shown a dramatic response to EGFR inhibitors (EGFR-TKI). EGFR T790M mutation and MET amplification have been recognized as major mechanisms of acquired resistance to EGFR-TKI. Therefore, MET inhibitors have recently been used in NSCLC patients in clinical trials. In this study, we tried to identify the mechanism of acquired resistance to MET inhibitors. We analyzed the antitumor effects of two MET inhibitors, PHA-665752 and crizotinib, in 10 NSCLC cell lines. EBC-1 cells with MET amplification were the only cells that were sensitive to both MET inhibitors. We established PHA-665752–resistant EBC-1 cells, namely EBC-1R cells. Activation of KRAS, EGFR, and FGFR2 signaling was observed in EBC-1R cells by FISH and receptor tyrosine kinase phosphorylation antibody arrays. EBC-1R cells also showed overexpression of ATP-binding cassette subfamily B member 1 (ABCB1) as well as phosphorylation of MET. EBC-1R cells grew as cell spheres that exhibited cancer stem cell–like (CSC) properties and epithelial–mesenchymal transition (EMT). The level of miR-138 that targeted ABCB1 was decreased in EBC-1R cells. ABCB1 siRNA and the ABCB1 inhibitor elacridar could reduce sphere numbers and suppress EMT. Elacridar could also reverse resistance to PHA-665752 in EBC-1R cells. Our study demonstrated that ABCB1 overexpression, which was associated with CSC properties and EMT, was involved in the acquired resistance to MET inhibitors. Inhibition of ABCB1 might be a novel therapeutic strategy for NSCLC patients with acquired resistance to MET inhibitors. Mol Cancer Ther; 14(11); 2433–40. ©2015 AACR.

miR-200/ZEB axis regulates sensitivity to nintedanib in non-small cell lung cancer cells

Nintedanib (BIBF1120) is a multi-targeted angiokinase inhibitor and has been evaluated in idiopathic pulmonary fibrosis and advanced non-small cell lung cancer (NSCLC) patients in clinical studies. In the present study, we evaluated the antitumor effects of nintedanib in 16 NSCLC cell lines and tried to identify microRNA (miRNA) associated with sensitivity to nintedanib. No correlations between FGFR, PDGFR and VEGFR family activation and sensitivity to nintedanib were found. The difference in miRNA expression profiles between 5 nintedanib-sensitive and 5 nintedanib-resistant cell lines was evaluated by miRNA array and quantitative RT-PCR analysis (qRT-PCR). Expression of miR-200b, miR-200a and miR-141 belonging to the miR-200 family which contributes to epithelial-mesenchymal transition (EMT), was significantly lower in 5 nintedanib-resistant than in 5 nintedanib-sensitive cell lines. We examined the protein expression of EMT markers in these 10 NSCLC cell lines. E-cadherin expression was lower, and vimentin and ZEB1 expression were higher in 5 nintedanib-resistant cell lines. PC-1 was the most sensitive of the NSCLC cell lines to nintedanib. We established nintedanib-resistant PC-1 cells (PC-1R) by the stepwise method. PC-1R cells also showed decreased expression of miR-200b, miR-141 and miR-429 and increased expression of ZEB1 and ZEB2. We confirmed that induction of miR-200b or miR-141 enhanced sensitivity to nintedanib in nintedanib-resistant A549 and PC1-R cells. In addition, we evaluated the response to gefitinib in combination with nintedanib after TGF-β1 exposure of A549 cells. Nintedanib was able to reverse TGF-β1-induced EMT and resistance to gefitinib caused by miR-200b and miR-141 upregulation and ZEB1 downregulation. These results suggested that the miR-200/ZEB axis might be predictive biomarkers for sensitivity to nintedanib in NSCLC cells. Furthermore, nintedanib combined with gefitinib might be a novel therapeutic strategy for NSCLC cells with EMT phenotype and resistance to gefitinib.

Control of the MYC-eIF4E axis plus mTOR inhibitor treatment in small cell lung cancer

BackgroundMammalian target of rapamycin (mTOR) inhibitors have anti-tumor effects against renal cell carcinoma, pancreatic neuroendocrine cancer and breast cancer. In this study, we analyzed the antitumor effects of mTOR inhibitors in small cell lung cancer (SCLC) cells and sought to clarify the mechanism of resistance to mTOR inhibitors.MethodsWe analyzed the antitumor effects of three mTOR inhibitors including everolimus in 7 SCLC cell lines by MTS assay. Gene-chip analysis, receptor tyrosine kinases (RTK) array and Western blotting analysis were performed to identify molecules associated with resistance to everolimus.ResultsOnly SBC5 cells showed sensitivity to everolimus by MTS assay. We established two everolimus resistant-SBC5 cell lines (SBC5 R1 and SBC5 R10) by continuous exposure to increasing concentrations of everolimus stepwise. SPP1 and MYC were overexpressed in both SBC5 R1 and SBC5 R10 by gene-chip analysis. High expression levels of eukaryotic translation initiation factor 4E (eIF4E) were observed in 5 everolimus-resistant SCLC cells and SBC5 R10 cells by Western blotting. MYC siRNA reduced eIF4E phosphorylation in SBC5 cells, suggesting that MYC directly activates eIF4E by an mTOR-independent bypass pathway. Importantly, after reduction of MYC or eIF4E by siRNAs, the SBC5 parent and two SBC5-resistant cells displayed increased sensitivity to everolimus relative to the siRNA controls.ConclusionThese findings suggest that eIF4E has been shown to be an important factor in the resistance to everolimus in SCLC cells. Furthermore, a link between MYC and mTOR-independent eIF4E contribute to the resistance to everolimus in SCLC cells. Control of the MYC-eIF4E axis may be a novel therapeutic strategy for everolimus action in SCLC.

Significance of osteopontin in the sensitivity of malignant pleural mesothelioma to pemetrexed

Pemetrexed (PEM) is currently recommended as one of the standard anticancer drugs for malignant pleural mesothelioma (MPM). However, the mechanism of the sensitivity of MPM to PEM remains unclear. We analyzed the antitumor effects of PEM in six MPM cell lines by MTS assay. To identify genes associated with drug sensitivity, we conducted gene expression profiling on the same set of cell lines using GeneChips and pathway analysis. Three cell lines were sensitive to PEM. A total fo 18 transcripts and 14 genes identified by GeneChips were significantly correlated with sensitivity to PEM. Pathway analysis revealed that osteopontin (SPP1/OPN) was an important target in PEM sensitivity. Overexpression of SPP1/OPN was observed in the sensitive cells by quantitative PCR and western blot analysis. Introduction of SPP1/OPN by lentiviral vector significantly enhanced the invasion activities of MPM cells. PEM treatment with SPP1/OPN knockdown inhibited the PEM-induced cell growth-inhibitory effect in PEM-sensitive cells. Expression of SPP1/OPN and AKT phosphorylation significantly decreased after PEM treatment of the PEM-sensitive cells. High immunohistochemical expression of SPP1/OPN was observed in two of three MPM patients who had a partial response to PEM-based chemotherapy. PEM has antitumor effects in MPM cells dependent on SPP1/OPN overexpression resulting in AKT activation. Our results suggest that SPP1 may be used as a single predictive biomarker of the effectiveness of PEM treatment in MPM.

Pembrolizumab-induced agranulocytosis in a pulmonary pleomorphic carcinoma patient who developed interstitial lung disease and ocular myasthenia gravis

Abstract An 82-year-old man with a recurrence of pulmonary pleomorphic carcinoma was treated with pembrolizumab. He achieved partial response after three cycles of pembrolizumab. However, he developed febrile neutropenia. A bone marrow aspiration sample revealed a decrease of mature neutrophils, and anti-neutrophil antibody was detected in blood. Computed tomography scans revealed consolidation in the right lung. Pathological findings in lung biopsy tissue revealed organizing pneumonia. Pembrolizumab-induced agranulocytosis and interstitial lung disease (ILD) were diagnosed. We initiated antibacterial therapy and granulocyte colony-stimulating factor (G-CSF). The neutrophil count immediately increased, and the fever decreased. The improvement of ILD was achieved without using systemic steroids. Moreover, the patient developed ocular myasthenia gravis induced by pembrolizumab. This is the first case report of pembrolizumab-induced agranulocytosis. Agranulocytosis was improved by administration of G-CSF without using systemic steroids. However, further studies are needed to determine the optimal treatment for patients with anti-neutrophil antibody whose tumor has progressed.

A case of interstitial lung disease with alveolar hemorrhage induced by pembrolizumab

We herein describe the case of a 67-year-old woman with advanced lung adenocarcinoma who developed interstitial lung disease (ILD) with alveolar hemorrhage induced by pembrolizumab. She received four courses of pembrolizumab therapy and achieved a partial response. She had no respiratory symptoms; however, chest radiography and computed tomography (CT) revealed ground-glass opacities (GGOs) and crazy-paving pattern. Based on findings of bloody bronchoalveolar lavage fluid and transbronchial lung biopsy samples, pembrolizumab-induced ILD with alveolar hemorrhage was diagnosed. Corticosteroid therapy rapidly improved alveolar hemorrhage and regressed GGOs on CT scan. This is the first report on ILD with alveolar hemorrhage induced by pembrolizumab.

Pembrolizumab and salvage chemotherapy in EGFR T790M-positive non-small-cell lung cancer with high PD-L1 expression

Immuno-checkpoint inhibitors (ICI) have become an effective treatment option for non-small-cell lung cancer patients. However, ICI therapy was reported to be less effective in patients with epidermal growth factor receptor (EGFR) mutations than in those with wild-type EGFR. We report here that an non-small-cell lung cancer patient with the EGFR mutant T790M showed a programmed cell death ligand 1 (PD-L1) expression level that increased from <25% to >90% after eighth-line osimertinib therapy. He was treated with pembrolizumab as a ninth-line treatment, and attained stable disease. After the pembrolizumab therapy, he was treated with gemcitabine, which produced a good response despite being the 10th-line treatment. We should consider administering ICI and chemotherapy even to EGFR mutant patients after failure of EGFR tyrosine kinase inhibitor, especially in cases with high PD-LI expression.

Abstract 756: Inhibition of ABCB1 overcomes cancer stem cell-like properties and acquired resistance to MET inhibitor in non-small cell lung cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Non-small cell lung cancer (NSCLC) patients with EGFR mutations have shown a dramatic response to EGFR inhibitors (EGFR-TKI). EGFR T790M mutation and MET amplification have been recognized as mechanisms of acquired resistance to EGFR-TKI. Recently, MET inhibitors have been used in NSCLC patients for clinical trials. In this study, we tried to identify the mechanisms of acquired resistant to MET inhibitor. We analyzed the antitumor effects of two MET inhibitors; PHA-665752 and crizotinib, in 10 NSCLC cell lines. EBC1 cells with MET amplification were only sensitive cells to both of MET inhibitors. We have established PHA-665752 resistant cells; namely EBC1-R cells. EBC1-R cells showed high expression levels of ATP-binding Cassette Sub-family B Member 1 (ABCB1) as wells as phosphorylation MET (p-MET). EBC1-R cells grew as cell sphere that exhibited cancer stem cell-like (CSC) properties and epithelial mesenchymal transition (EMT) feature. Two miRNAs, miR-374a and miR-138 which targeted ABCB1, were decreased in EBC1-R cells. ABCB1 inhibitor elacridar could suppress sphere numbers and EMT phenomenon and restore to the resistance to PHA-665752 in EBC1-R cells. Our study demonstrated that ABCB1 overexpression associated with CSC properties and EMT may be critical for acquire resistance for MET inhibitor. ABCB1 inhibition might be a novel therapeutic strategy for resistance to MET inhibitor in NSCLC cells. Citation Format: Teppei Sugano, Masahiro Seike, Rintaro Noro, Chie Soeno, Shinji Nakamichi, Nobuhiko Nishijima, Masaru Matsumoto, Susumu Takeuchi, Akihiko Miyanaga, Kaoru Kubota, Akihiko Gemma. Inhibition of ABCB1 overcomes cancer stem cell-like properties and acquired resistance to MET inhibitor in non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 756. doi:10.1158/1538-7445.AM2015-756

Abstract 784: Significance of osteopontin in the sensitivity of malignant pleural mesothelioma to pemetrexed

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Pemetrexed (PEM) is currently recommended as one of the standard anticancer drugs for malignant pleural mesothelioma (MPM). However, the mechanism of the sensitivity of MPM to PEM remains unclear. Materials and Methods: We analyzed the antitumor effects of PEM in six MPM cell lines by MTS assay. To identify genes associated with drug sensitivity, we conducted gene expression profiling on the same set of cell lines using GeneChips and pathway analysis. Results: Three cell lines were sensitive to PEM. Eighteen transcripts and fourteen genes identified by Gene-chips were significantly correlated with sensitivity to PEM. Pathway analysis revealed that osteopontin (SPP1/OPN) was an important target in PEM sensitivity. Overexpression of SPP1/OPN was observed in the sensitive cells by quantitative RT-PCR analysis and Western blotting. Introduction of SPP1/OPN by lentiviral vector significantly enhanced the invasion activities of MPM cells. PEM treatment with SPP1/OPN knockdown inhibited the PEM-induced cell growth-inhibitory effect in PEM-sensitive cells. Expression of SPP1/OPN and AKT phosphorylation significantly decreased after PEM treatment of the PEM sensitive cells. High immunohistochemical expression of SPP1/OPN was observed in two of three MPM patients who had a partial response to PEM-based chemotherapy. Conclusion: PEM has anti-tumor effects in MPM cells dependent on SPP1/OPN overexpression resulting in AKT activation. Our results suggest that SPP1 may be used as a single predictive biomarker of the effectiveness of PEM treatment in MPM. Citation Format: Susumu Takeuchi, Masahiro Seike, Rintaro Noro, Chie Soeno, Teppei Sugano, Fenfei Zou, Masaru Matsumoto, Akihiko Miyanaga, Yuji Minegishi, Kaoru Kubota, Akihiko Gemma. Significance of osteopontin in the sensitivity of malignant pleural mesothelioma to pemetrexed. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 784. doi:10.1158/1538-7445.AM2014-784

Abstract 5548: High copy number of the MET gene predicts resistance to EGFR-TKI in non-small cell lung cancer patients.

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background Non-small cell lung cancer (NSCLC) patients with EGFR gene mutations have shown a dramatic responses to EGFR tyrosine kinase inhibitor (EGFR-TKI). However, it is recognized that, clinically, drug resistance eventually emerges and this limits the mean duration of response. Although mechanisms of acquired resistance to EGFR-TKI, such as T790M secondary mutation and MET amplification, have recently been described, other mechanisms of resistant to EGFR-TKI should be identified to broaden the therapeutic strategy for NSCLC in patients with EGFR gene mutations. Patients and Methods We evaluated 26 lung tumor samples from NSCLC patients who had received EGFR-TKI treatment from 2009 to 2011 at Nippon Medical School Hospital. Status of copy numbers and amplification of the MET gene were examined by fluorescence in situ hybridization (FISH) and estimated by high throughout automated image analysis. Results High copy number (>4 copies/cell) was observed in 6 patients (27%) and amplification (MET/CEP7 ≥2.0) was seen in 0 of 26 NSCLC patients. MET gene copy number status was not correlated with EGFR gene mutation, gender, histology, or smoking history. In 2 of the 6 cases with high copy number, high copy numbers were found at both the pre- and post-treatment stages. Among the 6 patients with high copy number, 5 patients achieved partial response (PR) and one patient showed stable disease (SD) with EGFR-TKI therapy. However, the 6 patients with high copy number had statistically significantly shorter progression-free survival (PFS) than the 20 patients with low copy numbers. (p=0.048, log-rank test). The median PFS of patients with High copy number and low copy numbers was 9.5 months and 24.4 months, respectively. Conclusion Detection of high copy number of the MET gene by FISH may be useful for predicting response to EGFR-TKI. The correlation between MET gene copy number and response to EGFR-TKI should be further evaluated using larger sample sizes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5548. doi:1538-7445.AM2012-5548