Takayuki Muraoka

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 G1 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. Clin Cancer Res; 17(15); 4965–74. ©2011 AACR.

Prognostic impact of cancer stem cell-related markers in non-small cell lung cancer patients treated with induction chemoradiotherapy

The expression of several cancer stem cell (CSC)-related markers has been confirmed in non-small cell lung cancer (NSCLC). The aim of this study was to clarify the clinical role of CSC-related markers in patients with NSCLC undergoing induction chemoradiotherapy (CRT). Fifty patients with clinically diagnosed N2 or N3 NSCLC who underwent induction CRT with docetaxel and cisplatin concurrently with thoracic radiation followed by surgery were examined in this study. The expressions of CSC related markers (CD133, ALDH1, ABCG2, and Bmi-1) were examined using immunohistochemical staining in surgically resected specimens. Among the 50 patients, 20 patients had no residual tumor cells in the resected specimen when examined pathologically; CSC-related marker expressions and their correlation to survival were evaluated in the other 30 patients. After a median follow-up period of 72 months, the 5-year overall survival rate of the patients with CD133-positive or ALDH1-positive specimens was significantly worse than that of the patients with both CD133-negative and ALDH1-negative expressions (44.9% vs. 90.0%, respectively; P = 0.042). In a multivariate analysis, CD133 and ALDH1 negativity (P = 0.047) and cN2-3 single station metastasis (P = 0.03) were significant independent prognostic factors for prolonged survival. The expressions of CSC-related markers after CRT were significantly correlated with a poor prognosis in patients with NSCLC. The development of therapeutic strategies including adjuvant therapy that take CSC-related marker positivity into consideration is likely to be a key factor in further improvements of the prognosis of patients undergoing trimodality therapy.

Strong anti-tumor effect of NVP-AUY922, a novel Hsp90 inhibitor, on non-small cell lung cancer

The anti-tumor activity of a newly developed Hsp90 inhibitor, NVP-AUY922 (AUY922), against non-small cell lung cancer (NSCLC) was examined. Twenty-one NSCLC cell lines were used, the somatic alterations of which were characterized. Cell proliferation was analyzed using a modified MTS assay. Expression of the client proteins was assessed using Western blotting. The cell cycle was analyzed using flow cytometry. The IC50 value of AUY922 for the NSCLC cell lines ranged from 5.2 to 860 nM (median, 20.4 nM). Based on previous data, cells with an IC50 of less than 50 nM were classified as sensitive cells and 19 of the 21 NSCLC cell lines were judged to be sensitive. The IC50 of five malignant pleural mesothelioma (MPM) cell lines revealed that the MPM cells had a significantly higher IC50 value (median, 89.2 nM; range, 22.2-24,100 nM) than the NSCLC cells (p=0.015). There was significant depletion of both the total and phosphorylated client proteins--EGFR, MET, HER2 and AKT--at low drug concentrations (50-100 nM) in drug-sensitive cell lines. Cell-cycle analysis was performed for two sensitive cell lines, H1975 and H838. Following AUY922 treatment, an increase in the sub-G0-G1 cell population, as well as appearance of cleaved PARP expression, indicated the induction of apoptosis. In conclusion, AUY922 was effective against most NSCLC cell lines, independent of the type of known molecular alteration, and appears to be a promising new drug for the treatment of NSCLC.

The degree of microRNA-34b/c methylation in serum-circulating DNA is associated with malignant pleural mesothelioma

OBJECTIVES Malignant pleural mesothelioma (MPM) is an aggressive tumor with a poor prognosis. microRNA-34b/c (miR-34b/c), which plays an important role in the pathogenesis of MPM, is frequently downregulated by DNA methylation in approximately 90% of MPM cases. In this study, we estimated the degree of miR-34b/c methylation in serum-circulating DNA using a digital methylation specific PCR assay (MSP). MATERIALS AND METHODS A real-time MSP assay was performed using the SYBR Green method. The melting temperature (Tm) of each PCR product was examined using a melting curve analysis. For a digital MSP assay, 40 wells were analyzed per sample. A total of 110 serum samples from 48 MPM cases, 21 benign asbestos pleurisy (BAP) cases, and 41 healthy volunteers (HVs) were examined. RESULTS Positive range of Tm value for miR-34b/c methylation was defined as 77.71-78.79 °C which was the mean ± 3 standard deviations of 40 wells of a positive control. The number of miR-34b/c methylated wells was counted per sample according to this criterion. The number of miR-34b/c methylated wells in MPM cases was significantly higher than that in BAP cases (P=0.03) or HVs (P<0.001). Advanced MPM cases tended to have higher number of miR-34b/c methylated wells than early MPM cases. Receiver-operating characteristic (ROC) curve analysis revealed that three number of miR-34b/c methylated wells per sample was the best cut-off of positivity of MPM with a 67% of sensitivity and a 77% specificity for prediction. The area under the ROC curve was 0.77. CONCLUSIONS Our digital MSP assay can quantify miR-34b/c methylation in serum-circulating DNA. The degree of miR-34b/c methylation in serum-circulating DNA is associated with MPM, suggesting that this approach might be useful for the establishment of a new detection system for 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.

The proliferative effects of asbestos-exposed peripheral blood mononuclear cells on mesothelial cells

Malignant mesothelioma (MM) is thought to arise from the direct effect of asbestos on mesothelial cells. However, MM takes a long time to develop following exposure to asbestos, which suggests that the effects of asbestos are complex. The present study examined the effects of asbestos exposure on the cell growth of MeT-5A human mesothelial cells via cytokines produced by immune cells. Peripheral blood mononuclear cells (PBMCs) were stimulated with antibodies against cluster of differentiation (CD)3 and CD28 upon exposure to the asbestos chrysotile A (CA) or crocidolite (CR); the growth of MeT-5A cells in media supplemented with PBMC culture supernatants was subsequently examined. MeT-5A cells exhibited an increase in proliferation when grown in supernatant from the 7-day PBMC culture exposed to CA or CR. Analysis of cytokine production demonstrated increased levels of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-1α, IL-1β, IL-3, IL-5, IL-13 and IL-17A in supernatants. Individual administration of these cytokines, excluding G-CSF and GM-CSF, led to an increase in cell growth of MeT-5A, whereas this effect was not observed following the combined administration of these cytokines. The results indicate that cytokines secreted by immune cells upon exposure to asbestos cause an increase in the growth activity of mesothelial cells, suggesting that alterations in the production of cytokines by immune cells may contribute to tumorigenesis in individuals exposed to asbestos.

Abstract 7: Knockdown of EGFR to investigate its therapeutic potential for treatment of non-small cell lung cancers

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Epidermal growth factor receptor (EGFR) is a transmembrane protein consisting of an extracellular ligand-binding domain, and its activation leads to a multitude of effects including cell proliferation, cell differentiation, angiogenesis, metastasis, and antiapoptosis. EGFR can be a partner of heterodimer of other EGFR family such as HER2 and HER3. EGFR is often overexpressed in non-small cell lung cancer (NSCLC). Anti-EGFR agents including EGFR-tyrosine kinase inhibitors (EGFR-TKIs) have considered to be effective for NSCLC when drug sensitive EGFR mutation is present. However, inherent and acquired resistances are major problems of EGFR targeting therapies. In this study, we knock-downed EGFR using small interfering RNAs (siRNAs) in NSCLC cell lines to examine the significance of targeting EGFR for NSCLC therapy. Materials and methods: We treated 14 NSCLC cell lines including nine EGFR mutant and five EGFR wild-type cell lines by geftinib or siRNAs for EGFR knock-down (siR-EGFR). Three cell line, PC-9-GR-N1, RPC-9, and HCC827-Met, were experimentally established as acquired resistant cells to gefitinib. The anti-tumor effect was determined by MTS or colony formation assay. The protein expression was evaluated using Western blotting. Results: All cell lines showed the expression of EGFR protein and siR-EGFR treatment down-regulated EGFR protein in all 14 cell lines. siR-EGFR suppressed cell viability in 7 of 9 EGFR mutant cells ranged from 8.0% to 73%. PC-9-GR-N1 and RPC-9 also showed inhibition. NCI-H1670 and HCC827-Met harboured EGFR mutation but were not inhibited. Of note, PTEN was deleted in NCI-H1670 and c-MET was amplified in HCC827-Met. Cell viability of all EGFR wild-type cells was not inhibited except NCI-H411. Conclusion: Our results indicated that EGFR can be the therapeutic target of NSCLC with EGFR activation. By contrast, targeting EGFR is not appropriate strategy for tumor of which EGFR is not activated even though EGFR is expressed. 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 7. doi:1538-7445.AM2012-7

Abstract 3988: Aberrant NFKBIA expression in lung adenocarcinomas arising from never smokers

[Background] Lung cancer in never-smoker (NS) is known as a seventh cause of cancer-related death. Activating mutation of epidermal growth factor receptor (EGFR) gene is frequently observed in non-small-cell lung cancers (NSCLCs) with never smoking history and it is significantly associated with the sensitivity of EGFR tyrosine kinase inhibitor (EGFR-TKI). Recently, NFKBIA gene, a tumor suppressor gene, has been reported to be silenced by deletion in glioblastomas, and in NSCLCs, its expression level is associated with clinical outcome of EGFR-TKI therapy. In this study, we examined NFKBIA expression and the genetic alterations of EGFR, K-ras, EML4-ALK to investigate the inter-relationship between these genetic alterations and clinicopathological factors among lung adenocarcinomas in NS. [Material and Method] We obtained ninety seven lung adenocarcinomas samples in NS which were resected at our institution. Four NSCLC cell lines (NCI-H1299, NCI-H1650, HCC827, NCI-H1819) were also investigated. We examined NFKBIA expression using immunohistochemistory (IHC) in tumor samples and real time reverse transcriptional PCR (RT-PCR) in cell lines, respectively. Mutational statuses of EGFR and K-ras genes were determined using mutant-enriched PCR assay and direct sequencing, respectively. EML4-ALK fusion was screened by ALK IHC and confirmed using RT-PCR assay. The methylation status of NFKBIA gene is investigated by methylation specific PCR assay and bisulfite sequencing. [Result] NFKBIA expression was silenced in 36 out of 97 samples (37.1%). EGFR mutation, K-ras mutation, and EML4-ALK fusion were detected in 61.8%, 2.1%, and 1.0% of 97 samples, respectively. Of note, the silencing of NFKBIA expression was significantly frequent in EGFR wild-type patients than in EGFR mutant patients (p=0.0024), while there was no correlation between NFKBIA expression level and K-ras mutation or EML4-ALK fusion. Although NFKBIA expression was silenced in NCI-H1299 and NCI-H1650, NFKBIA expression was not restored after 5-Aza treatment in these cell lines. In addition, NFKBIA methylation was found in these two cell lines. [Conclusion] Our findings suggest that the degradation of NFKBIA expression is a frequent event in lung adenocarcinomas of NS and may mediate an important non-tobacco carcinogenic pathway especially among lung adenocarcinoma with wild-type EGFR. 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 3988. doi:1538-7445.AM2012-3988

Abstract 187: Down-regulation of microRNA34 induces cell proliferation and invasion of human mesothelial cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Purpose: In the recent half-decade, many studies have shown that microRNAs (miRNAs) can act as oncogenes or tumor suppressors and that the widespread alteration of miRNA expression patterns is highly relevant to various human malignancies. We previously reported that microRNA-34s (miR-34s) are methylated and down-regulated in malignant pleural mesothelioma and might play an important role in the carcinogenesis of mesothelioma. In this study, in contrast to our previous study, we down-regulated miR-34s in the human mesothelial cells to investigate cellular effect of miR-34s knockdown. Material and methods: Two human mesothelial cell lines, MeT-5A derived from human pleura and LP-9 derived from human peritoneum, and three human primary cultured mesothelial cells from pleura were used in this study. Antisense mimics of miR-34a, b, and c (Anti-miR miRNA InhibitorR, Ambion) were transfected to these cells and effect of proliferation and invasion was estimated. Control scramble transfected cells were used as control. The protein expression status was estimated using Western Blotting. Results: Antisense miR-34a, b, and c down-regulated each miR-34s in introduced human mesothelial cells. Cell proliferation significantly increased in each antisense of miR-34s transfected human mesothelial cells than in control for all examined cells (p < 0.0001 for miR-34s with all cells). Invasion ability also increased in antisense transfected cells than in control for MeT-5A and LP-9 (P < 0.0001 for 2 cell lines). Human primary cultured mesothelial cells did not obtain invasion ability in this experiment. Western blotting confirmed up-regulation of c-Met and p-c-Met protein. Conclusion: We confirmed that down-regulation of miR-34s up-regulated oncogenic phenotype of mesothelial cells. The present study together with our previous report strongly suggests miR-34s play an important role in early carcinogenic process of human mesothelial cells to malignant mesothelioma. 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 187. doi:1538-7445.AM2012-187

Abstract 4153: Usefulness of sensitive digital PCR assay to quantify microRNA-34b/c methylation in the circulating serum DNA of malignant mesothelioma patients

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL . Background: Malignant mesothelioma (MM) is an aggressive tumor but it can sometimes be cured if its clinical stage is not advanced. It is very expected to establish a useful screening test for early detection of MM. Recently we have found that DNA methylation of microRNA-34bc (miR34b/c) plays a dismal role on pathogenesis of MM and is frequently observed in MM. Circulating DNA from malignant tumors is known to be found in the serum of the patient. Digital polymerase chain reaction (PCR) is a high sensitive quantitative assay using real time PCR assay. In order to establish a new early detection system for MM, we quantify the extent of DNA methylation of miR-34b/c using a digital PCR assay in the circulating serum DNA from MM. Material and methods: We collected serum samples from 30 MM patients, 22 patients with benign asbestos pleurisy (BAP), and 10 healthy volunteers (HV) at Okayama Rosai Hospital, NHO Yamaguchi Ube Medical Center and our institute during January 2006 to August 2009. The circulating serum DNA was extracted using QIAamp Circulating Nucleic Acid Kit (Qiagen) followed by bisulfite conversion using EpiTect Bisulfite Kits (Qiagen) according to manufactural protocols. The real time PCR assay of methylation-specific PCR (MSP) for miR-34b/c was performed using SYBR Green method. In addition, the digital PCR of real time MSP assay was explored for 40 wells per each sample with a suitable dilution to aim the increase of sensitivity of PCR reaction, and we quantified the extent of methylation in each sample by counting the number of positive well of PCR reaction per sample. As positive and negative controls, the supernatant cultured medium of NCI-H290 harboring heavy methylation of miR-34b/c and water blank was used, respectively. Results: By melting curve analysis, we distinguished the miR-34b/c methylated wells from unmethylated wells based on the temperatures of PCR products of them (78.2 degree on methylated samples vs. 76.2 degree on un-methylated samples). Using the criteria above, each 40 wells per sample was defined as positive or negative for miR-34b/c methylation. The miR-34b/c methylated samples were defined as having more than three positive wells by ROC curve analyses. Finally, we found that the number of positive wells was significantly higher in MMs than that in other groups, and the miR-34b/c methylated samples was observed in 87% of MM patients, 45% of the patients with BAP, and 10% of HV, respectively, indicating that the miR-34b/c methylation in MM patients were significantly frequent than that in others (P <0.0001). Conclusion: The digital PCR assay can detect miR-34b/c methylation in the circulation serum DNA of MM patients to find that miR-34b/c methylation is more frequent in MM than in BAP, suggesting that it may become a useful high-sensitive screening test to distinguish MM patients from the patients with BAP. 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 4153. doi:1538-7445.AM2012-4153