[show abstract][hide abstract] ABSTRACT: To study epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) resistance mechanisms, we established a novel gefitinib-resistant lung cancer cell line derived from an EGFR-mutant non-small cell lung cancer cell line (PC-9) pretreated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (designated PC9-GR). We found that gefitinib substantially suppressed the EGFR signaling pathway, whereas ERK was reactivated after several hours in PC9-GR but not in PC-9. The combination of gefitinib with ERK inhibition (by U0126) restored gefitinib susceptibility in PC9-GR, but PI3K-Akt inhibition with LY294002 did not. Although the levels of phosphorylated Src were up-regulated simultaneously with ERK reactivation, neither ERK suppression using U0126 nor an ERK-specific siRNA induced Src phosphorylation. Furthermore, dual inhibition of EGFR and Src restored gefitinib sensitivity in PC9-GR in vitro and in vivo. In conclusion, our results indicate that Src-mediated ERK reactivation may play a role in a novel gefitinib resistance mechanism, and that the combined use of gefitinib with a Src inhibitor may be a potent strategy to overcome this resistance.
Experimental Cell Research 01/2014; · 3.56 Impact Factor
[show abstract][hide abstract] ABSTRACT: Rapid on-site evaluation (ROSE) is used widely during endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA). BIOEVALUATOR(®) is a device used for determining whether the tissues obtained by EBUS-TBNA are appropriate for a pathological diagnosis. This study describes our experience with ROSE using BIOEVALUATOR(®) during EBUS-TBNA for diagnosing pulmonary and mediastinal diseases.
We retrospectively evaluated the results of 35 patients who underwent EBUS-TBNA with BIOEVALUATOR(®) between December 2011 and February 2013. For the diagnosis, the tissue areas were appearing white and red through BIOEVALUATOR(®) are considered to be appropriate and inappropriate, respectively. We examined their medical records to obtain information concerning the examination of BIOEVALUATOR(®) results of the patient's materials (white/red), the diagnosis yield, site and size of lymph nodes and number of needle passes.
The median longest diameter of 40 lymph nodes (21 #7, 13 #4R, 4 #4L and 2 #11) from 35 patients was 27.9 (range 12.4-50.6) mm and the median number of needle passes was 2 (range 1-5). The definitive diagnosis was made by EBUS-TBNA in 28 of 35 patients, by thoracotomy in one patient and BIOEVALUATOR(®) results were white and lymphocytes were seen in the rest six patients. The BIOEVALUATOR(®) results of other patients without accurate diagnosis were left indefinitive. Finally, the six patients were judged as having benign lymphadenopathy because the lymph node size on computed tomography decreased or remained stable after for at least 8 months.
Checking aspirated samples using BIOEVALUATOR(®) appears useful for determining their adequacy for pathological diagnosis.
Annals of Thoracic Medicine 01/2014; 9(1):14-7. · 1.12 Impact Factor
[show abstract][hide abstract] ABSTRACT: We encountered a family of Japanese descent in which multiple members developed lung cancer. Using whole-exome sequencing, we identified a novel germline mutation in the transmembrane domain of the human epidermal growth factor receptor 2 (HER2) gene (G660D). A novel somatic mutation (V659E) was also detected in the transmembrane domain of HER2 in one of 253 sporadic lung adenocarcinomas. Because the transmembrane domain of HER2 is considered to be responsible for the dimerization and subsequent activation of the HER family and downstream signaling pathways, we performed functional analyses of these HER2 mutants. Mutant HER2 G660D and V659E proteins were more stable than wild-type protein. Both the G660D and V659E mutants activated Akt. In addition, they activated p38, which is thought to promote cell proliferation in lung adenocarcinoma. Our findings strongly suggest that mutations in the transmembrane domain of HER2 may be oncogenic, causing hereditary and sporadic lung adenocarcinomas.
[show abstract][hide abstract] ABSTRACT: STAT3 plays a vital role in inducing and maintaining a pro-carcinogenic inflammatory microenvironment and is reported to be a critical mediator of the oncogenic effects of EGFR mutations. STAT3 activation is mediated through JAK family kinases. We investigated the effect of the JAK1/2 inhibitor AZD1480 on lung tumors induced by an activating EGFR mutation.
Three EGFR tyrosine kinase inhibitor-resistant cell lines (RPC-9, PC-9/Van-R and PC-9/ER3) established from PC-9 harboring an EGFR exon19 deletion mutation were used. Growth inhibition was measured using an MTT assay. Effects of AZD1480 were also evaluated in the xenograft model and in the EGFR transgenic mice model. Protein expressions were assessed by immunoblotting and immunohistochemistry. Group differences were compared using Student's t-test. To evaluate the efficacy of AZD1480 on survival, AZD1480 or vehicle was administered orally from 7 weeks of age of the transgenic mice. Overall survival curves were calculated using the Kaplan-Meier method.
The sensitivities of resistant and parent cells to AZD1480 were similar in vitro. AZD1480 (30 or 50mg/kg/day, per os) reduced angiogenesis and revealed significant tumor regression in a mouse xenograft model. Subsequently, the transgenic mice were treated with AZD1480 (30mg/kg/day) or vehicle alone. The numbers of lung tumors (long axis exceeding 1mm) in the AZD1480-treated group and control group were 0.37±0.18 and 2.25±0.53 (p<0.001), respectively. AZD1480 treatment suppressed pSTAT3, pJAK1, pJAK2 and angiogenesis. The median survival time in the AZD1480-treated group (217 days) was significantly greater than that in the control group (106 days) (log-rank test, p<0.0001).
AZD1480 may be effective against lung tumors driven by an activating EGFR mutation.
Lung cancer (Amsterdam, Netherlands) 10/2013; · 3.14 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cisplatin can induce severe renal toxicity. However, the degree and pattern of hydration that is most efficient at preventing it have scarcely been formally evaluated. We here performed a prospective feasibility study of cisplatin-based chemotherapy with short-term low-volume hydration in advanced lung cancer.
Chemo-naïve patients with advanced lung cancer and reserving renal function who were suitable for cisplatin use (≥60 mg/m(2) on Day 1) were eligible for this study. Two-and-a-half-liter hydration within ∼4.5 h was investigated. The primary end point was the proportion of patients who underwent cisplatin-based chemotherapy without any Grade 2 or more renal toxicity in the first cycle.
A total of 46 patients were registered, all of whom were evaluable for renal toxicity. The median baseline creatinine score was 0.70 mg/dl and the median cisplatin dose on Day 1 was 80 mg/m(2). In the first cycle, none of the patients developed Grade 2 or more creatinine toxicity, which met the primary endpoint. Four patients (9%) had Grade 1 toxicity, with a median worst creatinine score of 1.19 mg/dl, but it disappeared rapidly. Creatinine toxicity was influenced by several clinical factors, including the performance status. Ten patients (22%) needed extra hydration during the first cycle, mainly due to gastrointestinal toxicity. However, all 10 were able to undergo further cycles of treatment. Thirty-two (86%) of the 37 patients who were assumed to be able to undergo further treatment at our institute received it in an outpatient setting.
This study demonstrated prospectively the feasibility of short-term low-volume hydration.
Japanese Journal of Clinical Oncology 09/2013; · 1.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: Non-small cell lung cancers (NSCLCs) with epidermal growth factor receptor (EGFR) mutations are sensitive to EGFR tyrosine kinase inhibitors (TKIs); however, unlike cytotoxic agents, it is generally accepted that minimal doses of drugs inhibiting target molecules are sufficient when molecular-targeted agents, including EGFR-TKIs, are administered. Thus, any utility of higher doses remains unclear. We compared low-dose (15 mg/kg) gefitinib therapy with high-dose (50 mg/kg) therapy using an EGFR-mutated lung cancer xenograft model. Both gefitinib doses induced tumor shrinkage, but tumors regrew in the low-dose group within 1 month, whereas tumors in the high-dose group did not. Neither the T790M mutation nor MET amplification was apparent in regrown tumors. We also compared outcomes after administration of two doses of gefitinib (5 and 25 mg/kg) in a transgenic EGFR-mutated lung cancer mouse model. In line with the results obtained using the xenograft model, both gefitinib doses completely inhibited tumor growth, but tumors treated with the lower dose of gefitinib developed earlier drug resistance. In conclusion, administration of a low gefitinib dose caused tumors to become drug-resistant prior to acquisition of the T790M mutation or MET amplification in EGFR-mutated models of lung cancer. This suggests that it is important to optimize the EGFR-TKI dose for treatment of EGFR mutation-associated lung cancer. Gefitinib may need to be administered at a dose greater than the minimum required for inhibition of target molecules. This article is protected by copyright. All rights reserved.
[show abstract][hide abstract] ABSTRACT: Endobronchial ultrasound-guided transbronchial needle aspiration is a new minimally invasive test for investigating mediastinal and hilar lymphadenopathy. It is sometimes difficult to distinguish between a recurrent malignant lymph node and lymphadenopathy due to sarcoidosis in patients who develop lymphadenopathy after surgery for a malignant tumor.
Between December 2009 and October 2012, we performed endobronchial ultrasound-guided transbronchial needle aspiration in 13 selected patients with a suspected recurrence in the mediastinum and/or hilum of the lung after surgical resection of a malignant tumor. We examined their medical records to obtain information on the diagnosis, the sizes of lymph nodes, the number of needle passes and other complications.
Definitive diagnoses were made using endobronchial ultrasound-guided transbronchial needle aspiration in 10 patients (three lung adenocarcinomas, one prostate carcinoma, one renal cell carcinoma, one neuroendocrine tumor and four sarcoidosis). Pathological specimens showing non-caseating granulomas led to the diagnosis of sarcoidosis in four patients; their previous malignancies had been papillary adenocarcinoma of the thyroid, carcinoma of the gingiva, thymoma and bladder cancer, but no recurrences were observed. The median of the longest diameter in 15 lymph nodes was 22 mm (range 13-35), and the median number of needle passes was two times (range 1-5) without severe complications.
Endobronchial ultrasound-guided transbronchial needle aspiration might be useful in differentiating between benign lymphadenopathy, including sarcoidosis, and cancer recurrence in patients with mediastinal or hilar lymphadenopathy after surgical resection of a malignant tumor.
Japanese Journal of Clinical Oncology 08/2013; · 1.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: Gefitinib is an essential drug for the treatment of non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) gene mutations. The approved dosage is 250mg/body/day without adjustment for physical size such as body surface area (BSA), and the impact of physical size on the efficacy of gefitinib has not been evaluated. Here, we sought to clarify this issue using a retrospective cohort. We reviewed the medical records of patients with consecutive advanced NSCLC harboring EGFR mutations who underwent gefitinib monotherapy at Okayama University Hospital. In total, 101 patients were included in this study, and the median BSA in this cohort was 1.5m(2). The median progression-free survival (PFS) of the patients with higher BSA (≥1.5m(2)) was significantly worse than that of those with lower BSA (<1.5m(2)) (10.4 vs. 18.0 months; p=0.019, log-rank test). Multivariate analysis also showed a significant impact of BSA on PFS (hazards ratio, 2.34; 95% confidence interval, 1.78-2.89; p=0.002). By contrast, no significant association between BSA and PFS was observed in those undergoing cytotoxic chemotherapy (4.0 vs. 5.1 months; p=0.989, log-rank test), suggesting that BSA is a predictive, rather than a prognostic, marker for gefitinib therapy in EGFR-mutated NSCLC. In conclusion, BSA affected PFS in patients with EGFR-mutated NSCLC who underwent gefitinib monotherapy, suggesting the need for appraisal of BSA-based dose adjustment, even for this molecular target-based therapy.
Lung cancer (Amsterdam, Netherlands) 06/2013; · 3.14 Impact Factor
[show abstract][hide abstract] ABSTRACT: Activation of the epidermal growth factor receptor (EGFR) has been observed in many malignant tumors and its constitutive signal transduction facilitates the proliferation of tumors. EGFR-tyrosine kinase inhibitors, such as gefitinib, are widely used as a molecular-targeting agent for the inactivation of EGFR signaling and show considerable therapeutic effect in non-small cell lung cancers harboring activating EGFR mutations. However, prolonged treatment inevitably produces tumors with additional gefitinib-resistant mutations in EGFR, which is a critical issue for current therapeutics. We aimed to characterize the distinct molecular response to gefitinib between the drug-resistant and drug-sensitive lung adenocarcinoma cells in order to learn about therapeutics based on the molecular information. From the quantitative PCR analysis, we found a specific increase in p14ARF expression in gefitinib-sensitive lung adenocarcinoma clones, which was absent in gefitinib-resistant clones. Moreover, mitochondria-targeted p14ARF triggered the most apoptosis in both clones. We identified the amino acid residues spanning from 38 to 65 as a functional core of mitochondrial p14ARF (p14 38-65 a.a.), which reduced the mitochondrial membrane potential and caused caspase-9 activation. The synthesized peptide covering the p14 38-65 a.a. induced growth suppression of the gefitinib-resistant clones without affecting non-neoplastic cells. Notably, transduction of the minimized dose of the p14 38-65 peptide restored the response to gefitinib like that in the sensitive clones. These findings suggest that the region of p14ARF 38-65 a.a. is critical in the pharmacological action of gefitinib against EGFR-mutated lung adenocarcinoma cells and has potential utility in the therapeutics of gefitinib-resistant cancers.
Molecular Cancer Therapeutics 06/2013; · 5.60 Impact Factor
[show abstract][hide abstract] ABSTRACT: Acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) is a critical problem in the treatment of lung cancer. While several mechanisms have been demonstrated to be responsible for acquired resistance, all mechanisms have not been uncovered. In this study, we investigated the molecular and cellular profiles of the acquired resistant cells to EGFR-TKI in EGFR mutant lung cancers. Four EGFR-mutant cell lines were exposed to gefitinib by stepwise escalation and high-concentration exposure methods, and resistant sublines to gefitinib were established. The molecular profiles and cellular phenotypes of these resistant sublines were characterized. Although previously reported alterations including secondary EGFR T790M mutation, MET amplification, and appearance of epithelial to mesenchymal transition (EMT) features were observed, these 2 drug-exposure methods revealed different resistance mechanisms. The resistant cells with EMT features exhibited down-regulation of microRNA-200c by DNA methylation. Furthermore, the HCC827-derived subline characterized by the high-concentration exposure method exhibited not only EMT features but also stem cell-like properties, including aldehyde dehydrogenase isoform 1 (ALDH1A1) overexpression, increase of side-population, and self-renewal capability. Resistant sublines with stem cell-like properties were resistance to conventional chemotherapeutic agents but equally sensitive to histone deacetylase and proteasome inhibitors, compared with their parental cells. ALDH1A1 was up-regulated in clinical samples with acquired resistance to gefitinib. In conclusion, our study indicates that the manner of EGFR-TKI exposure influences the mechanism of acquired resistance and the appearance of stem cell-like property with EGFR-TKI treatment.
[show abstract][hide abstract] ABSTRACT: A 72-year-old woman was referred to our hospital with Stage IV non-small-cell lung cancer (NSCLC). Chest computed tomography revealed a mass in the upper lobe of the right lung, with pleural effusion. Cytologic examination identified adenocarcinoma cells in the right pleural effusion. Furthermore, both a deletion mutation in exon 19 and a threonine–methionine substitution mutation at position 790 in exon 20 (T790M) were detected in the epidermal growth factor receptors (EGFR) in the malignant cells. As systemic chemotherapy consisting of car-boplatin and pemetrexed or erlotinib proved ineffective, docetaxel monotherapy was initiated as a third-line treatment. Following salvage chemotherapy, her Eastern Cooperative Oncology Group performance status improved from 3 to 1, with tumor regression over 5 months. To the best of our knowledge, this is the first report of successful docetaxel treatment for a patient with NSCLC harboring the T790M EGFR-activating mutation identified before treatment with EGFR tyrosine kinase inhibitors.
[show abstract][hide abstract] ABSTRACT: An irreversible ErbB family blocker is expected to inhibit tumors with activating epidermal growth factor receptor (EGFR) mutations more strongly than reversible EGFR tyrosine kinase inhibitors and to overcome acquired resistance to the T790M secondary mutation. Eleven-week-old transgenic mice with Egfr exon 19 deletion mutation were treated with afatinib, gefitinib, or vehicle for 4 weeks. All mice were sacrificed at 15 weeks of age, and the number of superficial left lung tumors with a long axis exceeding 1 mm was counted. The afatinib-treated group had significantly fewer tumors than the vehicle group (P < 0.01) and tended to have fewer tumors than the gefitinib-treated group (P = 0.06). Pathologically, gefitinib-treated mice had clearer, more nodular tumors than afatinib-treated mice. Immunoblotting showed that afatinib suppressed not only pEGFR but also pHER2, and induced apoptosis for longer periods than gefitinib. Subsequently, when each drug was administered 5 days per week until death, afatinib significantly enhanced mouse survival compared with gefitinib (median survival time: 456 days versus 376.5 days; logrank test, P < 0.01). Finally, the combination of afatinib with bevacizumab was found to be superior to either drug alone in exon 19 deletion/T790M and L858R/T790M xenograft tumors. Overall, afatinib was more potent than gefitinib in tumors harboring an exon 19 deletion mutation, and the combination of afatinib with bevacizumab efficiently suppressed tumors harboring the T790M secondary mutation.
Molecular Cancer Therapeutics 02/2013; · 5.60 Impact Factor
[show abstract][hide abstract] ABSTRACT: Midkine (MDK) is a heparin-binding growth factor that is highly expressed in many malignant tumors, including lung cancers. MDK activates the PI3K pathway and induces anti-apoptotic activity, in turn enhancing the survival of tumors. Therefore, the inhibition of MDK is considered a potential strategy for cancer therapy. In the present study, we demonstrate a novel small molecule compound (iMDK) that targets MDK. iMDK inhibited the cell growth of MDK-positive H441 lung adenocarcinoma cells that harbor an oncogenic KRAS mutation and H520 squamous cell lung cancer cells, both of which are types of untreatable lung cancer. However, iMDK did not reduce the cell viability of MDK-negative A549 lung adenocarcinoma cells or normal human lung fibroblast (NHLF) cells indicating its specificity. iMDK suppressed the endogenous expression of MDK but not that of other growth factors such as PTN or VEGF. iMDK suppressed the growth of H441 cells by inhibiting the PI3K pathway and inducing apoptosis. Systemic administration of iMDK significantly inhibited tumor growth in a xenograft mouse model in vivo. Inhibition of MDK with iMDK provides a potential therapeutic approach for the treatment of lung cancers that are driven by MDK.
PLoS ONE 01/2013; 8(8):e71093. · 3.73 Impact Factor