TAE226, a Bis-Anilino Pyrimidine Compound, Inhibits the EGFR-Mutant Kinase Including T790M Mutant to Show Anti-Tumor Effect on EGFR-Mutant Non-Small Cell Lung Cancer Cells.

TAE226, a bis-anilino pyrimidine compound, has been developed as an inhibitor of focal adhesion kinase (FAK) and insulin-like growth factor-I receptor (IGF-IR). In this study, we investigated the effect of TAE226 on non-small-cell lung cancer (NSCLC), especially focusing on the EGFR mutational status. TAE226 was more effective against cells with mutant EGFR, including the T790M mutant, than against cells with wild-type one. TAE226 preferentially inhibited phospho-EGFR and its downstream signaling mediators in the cells with mutant EGFR than in those with wild-type one. Phosphorylation of FAK and IGF-IR was not inhibited at the concentration at which the proliferation of EGFR-mutant cells was inhibited. Results of the in vitro binding assay indicated significant differences in the affinity for TAE226 between the wild-type and L858R (or delE746_A750) mutant, and the reduced affinity of ATP to the L858R (or delE746_A750) mutant resulted in good responsiveness of the L858R (or delE746_A750) mutant cells to TAE226. Of interest, the L858R/T790M or delE746_A750/T790M mutant enhanced the binding affinity for TAE226 compared with the L858R or delE746_A750 mutant, resulting in the effectiveness of TAE226 against T790M mutant cells despite the T790M mutation restoring the ATP affinity for the mutant EGFR close to that for the wild-type. TAE226 also showed higher affinity of about 15-fold for the L858R/T790M mutant than for the wild-type one by kinetic interaction analysis. The anti-tumor effect against EGFR-mutant tumors including T790M mutation was confirmed in mouse models without any significant toxicity. In summary, we showed that TAE226 inhibited the activation of mutant EGFR and exhibited anti-proliferative activity against NSCLCs carrying EGFR mutations, including T790M mutation.

Preclinical evaluation of microRNA-34b/c delivery for malignant pleural mesothelioma.

The microRNA-34s (miR-34s) have p53 response elements in their 5'-flanking regions and demonstrate tumor-suppressive functions. In malignant pleural mesothelioma (MPM), we previously reported that expression of miR-34b and miR-34c (miR-34b/c) was frequently downregulated by methylation in MPM cell lines and primary tumors. The forced overexpression of miR-34b/c showed significant antitumor effects with the induction of apoptosis in MPM cells. In this study, we examined the in vivo antitumor effects of miR-34b/c using adenovirus vector on MPM. We subcutaneously transplanted NCI-H290, a human MPM cell line, into BALB/C mice and injected adenovirus vector expressing miR-34b/c, luciferase driven by the cytomegalovirus promoter (Ad-miR-34b/c or Ad-Luc), or PBS control into tumors over 5mm in diameter. A statistically significant growth inhibition of the tumor volume was observed in the Ad-miR-34b/c group from day 6 onward compared to the Ad-Luc group. The inhibition rate of Ad-miR-34b/c, compared to the tumor volume treated with Ad-Luc, was 58.6% on day 10 and 54.7% on day13. Our results indicate that adenovirus-mediated miR-34b/c gene therapy could be useful for the clinical treatment of MPM.

Heparin-conjugated gelatin as a growth factor immobilization scaffold.

Tissue engineering requires growth factors, cells and a scaffold to permit effective tissue regeneration. This study aimed to develop a scaffold with a focus on immobilizing growth factors within gelatin. We focused on the extracellular matrix and developed a heparin-conjugated gelatin (Hep-gela). Conjugation was confirmed using the alcian blue assay and X-ray diffraction patterns. The mechanical strength and stability of the Hep-gela gel in protease solution were improved compared with collagen gel. Hep-gela was able to immobilize vascular endothelial growth factor (VEGF) even in the presence of albumin, with an efficiency of 54.2%. Immobilized VEGF promoted proliferation of human umbilical vein endothelial cells. Hep-gela-immobilized VEGF maintained its native biological activity. In summary, Hep-gela has the potential to become an effective material in the field of regenerative medicine.

MicroRNA miR-34b/c enhances cellular radiosensitivity of malignant pleural mesothelioma cells.

BACKGROUND: We previously reported that epigenetic silencing of microRNA-34b/c (miR-34b/c) plays an important role in the pathogenesis of malignant pleural mesothelioma (MPM). We examined the impact of miR-34b/c restoration on the radiosensitivity of MPM cells. MATERIALS AND METHODS: We established stable miR-34b/c and scramble transfectants of two MPM cell lines, H2052 and H28. We examined these transfectants by clonogenic survival assay, phosphorylated histone H2AX (γH2AX) foci assay, cell-cycle analysis, and western blotting. RESULTS: The clonogenic survival assay revealed that miR-34b/c radiosensitized MPM cells. γH2AX foci assay showed that DNA double-strand break repair was delayed in miR-34b/c transfectants. The proportion of sub-G(1) phase cells was increased in miR-34b/c transfectants after irradiation. miR-34b/c inhibited expression of cyclin-D1, cyclin-dependent kinase 4/6, B-cell lymphoma-2 (BCL-2) and increased cleaved poly (ADP-ribose) polymerase (cPARP) and cleaved caspase-3 after irradiation. CONCLUSION: Our results indicate that miR-34b/c enhances radiosensitivity by promoting radiation-induced apoptosis and suggested that miR-34b/c might be a useful therapeutic molecule to enhance radiotherapy in MPM.

Knockdown of the epidermal growth factor receptor gene to investigate its therapeutic potential for the treatment of non-small-cell lung cancers.

BACKGROUND: Epidermal growth factor receptor (EGFR) is often overexpressed in non-small-cell lung cancer (NSCLC). Anti-EGFR agents, including EGFR-tyrosine kinase inhibitors are considered to be effective when a drug-sensitive EGFR mutation is present. However, inherent and acquired resistances are major problems of EGFR-targeting therapies. In this study, we performed EGFR knockdown by using small interfering RNAs in NSCLC cell lines to examine the significance of targeting EGFR for NSCLC therapy. METHODS: We treated 13 NSCLC cell lines, including 8 EGFR mutant and 5 EGFR wild type by using gefitinib or small interfering RNAs against EGFR (siEGFR). Three cell lines (PC-9-GR1, RPC-9, and HCC827-ER) were experimentally established with acquired resistance to EGFR-tyrosine kinase inhibitors. The antitumor effect was determined by using an 3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt (MTS) or colony formation assay. The protein expression was evaluated by using Western blotting. RESULTS: All 13 cell lines expressed EGFR protein, and siEGFR downregulated EGFR protein expression in all. The cell viability was suppressed by siEGFR in 6 of 8 EGFR-mutant cell lines (suppressed 57%-92% of control cells), including PC-9-GR1 and RPC-9. The NCI-H1650 and HCC827-ER harbored EGFR mutations but were not suppressed. Of note, PTEN (phosphatase and tensin homolog) was deleted in NCI-H1650, and c-MET was amplified in HCC827-ER. It was not suppressed in any of the EGFR wild-type cells except in the NCI-H411, in which EGFR is phosphorylated, which indicates its activation. CONCLUSIONS: Analysis of the results indicated that EGFR can be a therapeutic target in NSCLCs with EGFR activation. In contrast, targeting EGFR is not appropriate for tumors in which EGFR is not activated, even if EGFR is expressed.

Frequent methylation and oncogenic role of microRNA-34b/c in small-cell lung cancer.

Small-cell lung cancer (SCLC) is an aggressive tumor with a dismal prognosis among primary lung cancers. MicroRNAs (miRNAs) can act as oncogenes or tumor-suppressor genes in human malignancy. The miR-34 family is comprised of tumor-suppressive miRNAs, and its reduced expression by methylation has been reported in various cancers, including non-small cell lung cancer (NSCLC). In this study, we investigated the alteration and tumor-suppressive impact of miR-34s in SCLC. The methylation of miR-34a and miR-34b/c was observed in 4 (36%) and 7 (64%) of 11 SCLC cell lines, respectively. Among the 27 SCLC clinical specimens, miR-34a and miR-34b/c were methylated in 4 (15%) and 18 (67%), respectively. In contrast, 13 (28%) miR-34a methylated cases and 12 (26%) miR-34b/c methylated cases were found in 47 NSCLC primary tumors. The frequency of miR-34b/c methylation was significantly higher in SCLC than in NSCLC (p<0.001). The expressions of miR-34s were reduced in methylated cell lines and tumors and restored after 5-aza-2'-deoxycytidine treatment, indicating that methylation was responsible for the reduced expression of miR-34s. Because the frequency of methylation was higher in miR-34b/c, we focused on miR-34b/c for a functional analysis. We examined the effect of miR-34b/c introduction on cell proliferation, migration and invasion. The transfection of miR-34b/c to two SCLC cell lines (H1048 and SBC5) resulted in the significant inhibition of cell growth, migration, and invasion, compared with control transfectants. Our results indicate that the aberrant methylation of miR-34b/c plays an important role in the pathogenesis of SCLC, implying that miR-34b/c may be a useful therapeutic target for SCLC.

The anti-proliferative effect of heat shock protein 90 inhibitor, 17-DMAG, on non-small-cell lung cancers being resistant to EGFR tyrosine kinase inhibitor.

Acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), gefitinib and erlotinib, is frequently observed after initiation of TKIs therapy. Non-small-cell lung cancers (NSCLC) with activating EGFR mutations were reported to be sensitive to heat shock protein 90 (Hsp90) inhibitors regardless of the secondary TKI-resistant T790M mutation. We established EGFR-TKI resistant clones for PC-9 cell lines, harboring EGFR exon 19 deletions, with or without the secondary T790M mutation. We examined the anti-proliferative effect of 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), an orally active Hsp90 inhibitor, on the growth of NSCLC cell lines in vitro and in vivo. In MTS assay, the IC(50) values of 17-DMAG for 13 EGFR-mutant cell lines including eight EGFR-TKI resistant cell lines ranged from 0.04 to 0.16 µM while those for seven EGFR-wild type cell lines ranged from 1.6 to 27.4 µM. Western blot analysis revealed that phospho-EGFR, phospho-Akt, phospho-MAPK, cdk4, and cyclin D1 were more readily depleted by 17-DMAG treatment in EGFR-mutant cell lines than in EGFR-wild type cell lines. Cleaved PARP expression confirmed apoptosis in response to 17-DMAG treatment in EGFR-mutant cell lines but not in EGFR-wild type cell lines. In mice xenograft models, 17-DMAG significantly reduced the growth of EGFR-mutant lines irrespective of T790M mutation. These results suggested that 17-DMAG is a potential novel therapeutic agent for NSCLC patients with EGFR mutations with or without EGFR-TKI resistance.

Epigenetic silencing of microRNA-34b/c plays an important role in the pathogenesis of malignant pleural mesothelioma.

PURPOSE: Malignant pleural mesothelioma (MPM) is an aggressive tumor with a dismal prognosis. Unlike other malignancies, TP53 mutations are rare in MPM. Recent studies have showed that altered expression of microRNA (miRNA) is observed in human malignant tumors. In this study, we investigated the alterations of miR-34s, a direct transcriptional target of TP53, and the role of miR-34s on the pathogenesis of MPM. EXPERIMENTAL DESIGN: Aberrant methylation and expression of miR-34s were examined in MPM cell lines and tumors. miR-34b/c was transfected to MPM cells to estimate the protein expression, cell proliferation, invasion, and cell cycle. RESULTS: Aberrant methylation was present in 2 (33.3%) of 6 MPM cell lines and 13 (27.7%) of 47 tumors in miR-34a and in all 6 MPM cell lines (100%) and 40 (85.1%) of 47 tumors in miR-34b/c. Expression of miR-34a and 34b/c in all methylated cell lines was reduced and restored with 5-aza-2'-deoxycytidine treatment. Because epigenetic silencing was the major event in miR-34b/c, we investigated the functional role of miR-34b/c in MPM. miR-34b/c-transfected MPM cells with physiologic miR-34b/c expression exhibited antiproliferation with G(1) cell cycle arrest and suppression of migration, invasion, and motility. The forced overexpression of miR-34b/c, but not p53, showed a significant antitumor effect with the induction of apoptosis in MPM cells. CONCLUSIONS: We show that the epigenetic silencing of miR-34b/c by methylation is a crucial alteration and plays an important role in the tumorigenesis of MPM, suggesting potential therapeutic options for MPM.

Aberrant methylation of p21 gene in lung cancer and malignant pleural mesothelioma.

Suppression of p21 has been implicated in the genesis and progression of many human malignancies. DNA methylation is an important mechanism of gene silencing in human malignancies. In this study, we examined the expression status and aberrant methylaion of p21 in lung cancers and malignant pleural mesotheliomas (MPM). We used 12 small cell lung cancer (SCLC) cell lines, 13 non-small cell lung cancer (NSCLC) cell lines, 50 primary NSCLCs, 6 MPM cell lines and 10 primary MPMs. The expression and methylation of p21 was examined by reverse transcription-PCR (RT-PCR), Western blotting and methylation-specific PCR (MSP) assay. Loss of p21 protein expression was observed in 7 SCLC cell lines (58.3%), 5 NSCLC cell lines (38.5%) and 3 MPM cell lines (50%) while mRNA expression was lost in 2 SCLC cell lines (16.7%), 2 NSCLC cell lines (15.4%) and none of the MPM cell lines. Aberrant methylation of p21 was found in 8.3% of SCLC cell lines, 30.2% of NSCLCs and 6.3% of MPMs. Among primary NSCLCs, methylation in adenocarcinomas was significantly more frequent than in squamous cell carcinomas. Loss of p21 expression was frequently observed in lung cancers and MPMs and aberrant methylation was one of the mechanisms of suppression of p21, especially in NSCLCs.

Usefulness of cumulative smoking dose for identifying the EGFR mutation and patients with non-small-cell lung cancer for gefitinib treatment.

We examined the diagnostic accuracy of the cumulative smoking dose for identifying the epidermal growth factor receptor (EGFR) exon 19 deletion and L858R mutation among Japanese patients with non-small-cell lung cancer (NSCLC). EGFR mutations in exon 19 and exon 21 were determined in 1001 NSCLC patients. A receiver-operating characteristic (ROC) curve methodology was applied to estimate the diagnostic accuracy. EGFR mutations were detected in 314 patients (31.4%). A cumulative smoking dose of less than 13 pack-years (PY) was the optimal cut-off point for predicting a positive EGFR mutation status, producing a balance between the sensitivity (73.5%) and the specificity (77%). The area under the ROC curve was 0.77, indicating that the smoking dose had a moderate diagnostic accuracy. The median survival time or the median progression-free survival time of patients who had smoked less than 13 pack-years (PY) were 18.6 and 6.3 months, respectively, while those of patients with equal to or more than 13 PY were 9.6 and 2.4 months, respectively. The overall survival (OS) and progression-free survival (PFS) rates were significantly different between the two groups (OS: hazard ratio [HR] = 0.64, 95% confidence interval [CI] = 0.51-0.80, P = 0.0001) (PFS: HR = 0.58, 95% CI = 0.47-0.71, P < 0.0001). Our study indicated that the smoking dose predicted EGFR mutations with a moderate diagnostic accuracy. Thus, patients who have smoked less than 13 PY might be candidates for gefitinib treatment when EGFR mutation status cannot be determined.

DNA methylation in small lung adenocarcinoma with bronchioloalveolar carcinoma components.

We examined the methylation status in 100 specimens of lung adenocarcinomas measuring 2cm or less and with bronchioloalveolar carcinoma (BAC) components (Noguchi types A-C) and then compared the methylation status between noninvasive tumors (Noguchi type A or B) and invasive tumors (Noguchi type C). Methylation-specific PCR was used to determine the methylation statuses of p16(INK4a), RASSF1A, CDH13, RARbeta, and Cyclin D2. The methylation index that was regarded as representing the degree of methylation was calculated. We also determined the mutational statuses of EGFR exons 19 and 21 using a PCR-based method. A multivariate analysis showed that the aberrant methylation of p16(INK4a), RASSF1A, and CDH13 was significantly more frequent in invasive tumors than in noninvasive tumors [p16(INK4a), 36.5% versus (vs.) 8.3%, P=0.0023; RASSF1A, 46.2% vs. 14.6%, P=0.0012; CDH13, 42.3% vs. 10.4%, P=0.0006]. The methylation index was significantly higher in invasive tumors than in noninvasive tumors (P=0.004). The methylation of p16(INK4a) was significantly more frequent in EGFR wild-type tumors than in EGFR mutant tumors (P=0.021). Our results indicate the involvement of epigenetic alterations in the progression of adenocarcinoma with BAC components.

MET gene amplification or EGFR mutation activate MET in lung cancers untreated with EGFR tyrosine kinase inhibitors.

We analyzed MET protein and copy number in NSCLC with or without EGFR mutations untreated with EGFR tyrosine kinase inhibitors (TKIs). MET copy number was examined in 28 NSCLC and 4 human bronchial epithelial cell lines (HBEC) and 100 primary tumors using quantitative real-time PCR. Positive results were confirmed by array comparative genomic hybridization and fluorescence in-situ hybridization. Total and phospho-MET protein expression was determined in 24 NSCLC and 2 HBEC cell lines using Western blot. EGFR mutations were examined for exon 19 deletions, T790M, and L858R. Knockdown of EGFR with siRNA was performed to examine the relation between EGFR and MET activation. High-level MET amplification was observed in 3 of 28 NSCLC cell lines and in 2 of 100 primary lung tumors that had not been treated with EGFR-TKIs. MET protein was highly expressed and phosphorylated in all the 3 cell lines with high MET amplification. In contrast, 6 NSCLC cell lines showed phospho-MET among 21 NSCLC cell lines without MET amplification (p = 0.042). Furthermore, those 6 cell lines harboring phospho-MET expression without MET amplification were all EGFR mutant (p = 0.0039). siRNA-mediated knockdown of EGFR abolished phospho-MET expression in examined 3 EGFR mutant cell lines of which MET gene copy number was not amplified. By contrast, phospho-MET expression in 2 cell lines with amplified MET gene was not down-regulated by knockdown of EGFR. Our results indicated that MET amplification was present in untreated NSCLC and EGFR mutation or MET amplification activated MET protein in NSCLC.

The aberrant promoter methylation of BMP3b and BMP6 in malignant pleural mesotheliomas.

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor-beta superfamily. Recent studies have showed that aberrant methylation of BMP genes is present in several types of human cancer. We examined the expression and methylation status of BMP3b and BMP6 in malignant pleural mesotheliomas (MPMs). The expression status of BMP3b, and BMP6 mRNAs were examined in seven MPM cell lines by RT-PCR assay. The expression of BMP3b was completely suppressed in 2 and partially suppressed in 2 of 7 cell lines and expression of BMP6 was partially suppressed in 2 cell lines. Methylation status of BMP3b in cell lines was determined by methylation-specific assay to find aberrant methylation in 6 cell lines which include 4 cell lines with suppressed BMP3b expression. Partial methylation of BMP6 was found in 2 cell lines whose expression was partially suppressed. Treatment with 5-Aza-dC restored BMP3b expression in methylated cell lines. Next, we examined the methylation status in 57 surgically resected MPM cases and found aberrant methylation of BMP3b in 9 (53%) out of 17 cases from Japan and 3 (8%) of 40 cases from USA and that of BMP6 in 4 (24%) cases from Japan and 12 (30%) cases from USA, showing significant difference in frequency of BMP3b methylation between MPMs of the two countries (P=0.0004). Our study indicated that BMP3b and BMP6 genes were suppressed by DNA methylation and methylation of BMP3b is significantly frequent in Japanese MPMs, suggesting its pathogenic role and the ethnic difference in MPMs.

Non-BAC component but not epidermal growth factor receptor gene mutation is associated with poor outcomes in small adenocarcinoma of the lung.

OBJECTIVE: The purpose of this study was to identify risk factors for poor clinical outcome after surgical resection of small lung adenocarcinoma. MATERIALS AND METHODS: Clinical records of 127 patients who had pathologic stage IA lung adenocarcinoma 20 mm or less and who had undergone a lobectomy with mediastinal lymph node dissection were reviewed. The percentage of non-bronchioloalveolar carcinoma (non-BAC) components quantified objectively, and epidermal growth factor receptor gene (EGFR) mutation determined by polymerase chain reaction-based assay were retrospectively linked with clinical data. RESULTS: Based on the percentage of non-BAC component, 127 patients were classified as follows: 26 in group I, BAC, 46 in group II mixed subtype with >or= 50% BAC, 18 in group III, mixed subtype with under 50% BAC, and 37 in group IV, mixed subtype with all non-BAC components or a pure pattern of one of the non-BAC components. Groups I and II were considered to be a "low non-BAC component type" and groups III and IV were considered to be a "high non-BAC component type." EGFR mutations in exon19 and exon21 were observed in 64 patients (50.4%). In terms of recurrence, the high non-BAC component type was the only independent factor for recurrence (p = 0.029). Regarding survival, the high age (p = 0.028) and high non-BAC component type (p = 0.046) were independent risk factors for poor overall survival. They were also independent risk factors for poor disease-free survival (p = 0.025 and p = 0.027, respectively). CONCLUSION: The high non-BAC component but not EGFR mutation status, is an independent risk factor for both recurrence and poor prognosis in patients with stage IA lung adenocarcinoma