Hiromu Yoshioka

Transcriptional silencing of secreted frizzled related protein 1 (SFRP1) by promoter hypermethylation in non-small-cell lung cancer

Secreted frizzled related protein 1 (SFRP1) is an antagonist of the transmembrane frizzled receptor, a component of the Wnt signaling pathway, and has been suggested to be a candidate tumor suppressor in several human malignancies. Since SFRP1 is located at chromosome 8p11, where lung cancers also exhibit frequent allelic loss, we hypothesized that the inactivation of SFRP1 is also involved in lung carcinogenesis. To substantiate this, we performed mutational analysis of SFRP1 for 29 non-small-cell lung cancer (NSCLC) and 25 small-cell lung cancer (SCLC) cell lines, and expression analysis for the same cell lines. Although somatic mutations were not detected in the coding sequence, downregulation of SFRP1 was observed in 14 (48%) NSCLC and nine (36%) SCLC cell lines. We analysed epigenetic alteration of the SFRP1 promoter region and detected hypermethylation in 15 (52%) of 29 NSCLC cell lines, two (8%) of 25 SCLC cell lines, and 44 (55%) of 80 primary lung tumors. By comparing the methylation status with SFRP1 expression, we found a significant correlation between them. We also performed loss of heterozygosity (LOH) analysis and found that 15 (38%) of 40 informative surgical specimens had LOH in the SFRP1 gene locus. Furthermore, we performed colony formation assay of two NSCLC cell lines (NCI-H460 and NCI-H2009) and found the reduction of colony formation with SFRP1 transfection. In addition, we also detected that SFRP1 inhibits the transcriptional activity of β-catenin, which is thought to be a downstream molecule of SFRP1, with luciferase reporter assay. Our current studies demonstrated that the SFRP1 gene is frequently downregulated by promoter hypermethylation and suppresses tumor growth activity of lung cancer cells, which suggests that SFRP1 is a candidate tumor suppressor gene for lung cancer.

Genetic alteration of the β-catenin gene (CTNNB1) in human lung cancer and malignant mesothelioma and identification of a new 3p21.3 homozygous deletion

The β-catenin gene (CTNNB1) has been shown to be genetically mutated in various human malignancies. To determine whether the β-catenin gene is responsible for oncogenesis in thoracic malignancies, we searched for the mutation in 166 lung cancers (90 primary tumors and 76 cell lines), one blastoma and 10 malignant mesotheliomas (two primary tumors and eight cell lines). Among the lung cancers, including 43 small cell lung cancers (SCLCs) and 123 non-small cell lung cancers (NSCLCs), we identified four alterations in exon 3, which is the target region of mutation for stabilizing β-catenin. One primary adenocarcinoma had a somatic mutation from C to G, leading to an amino acid substitution from Ser to Cys at codon 37. Among the cell lines, SCLC NCI-H1092 had a mutation from A to G, leading to an Asp to Gly substitution at codon 6, NSCLC HCC15 had a mutation from C to T, leading to a Ser to Phe substitution at codon 45, and NSCLC NCI-H358 had a mutation from A to G, leading to a Thr to Ala substitution at codon 75. One blastoma also had a somatic mutation from C to G, leading to a Ser to Cys substitution at codon 37. Among the 10 malignant mesotheliomas, we identified a homozygous deletion in the NCI-H28 cell line. Cloning of the rearranged fragment from NCI-H28 indicated that all the exons except exon 1 of the β-catenin gene are deleted and that the deletion junction is 13 kb downstream from exon 1. Furthermore, Northern blot analysis of 26 lung cancer and eight mesothelioma cell line RNAs detected ubiquitous expression of the β-catenin messages except NCI-H28, although Western blot analysis showed that relatively less amounts of protein products were expressed in some of lung cancer cell lines. Our findings suggest that the β-catenin gene is infrequently mutated in lung cancer and that the NCI-H28 homozygous deletion of the β-catenin gene might indicate the possibility of a new tumor suppressor gene residing in this region at 3p21.3, where various types of human cancers show frequent allelic loss.

p53 apoptotic pathway molecules are frequently and simultaneously altered in nonsmall cell lung carcinoma

Lung carcinomas show frequent inactivation of the p53 tumor suppressor, which regulates an apoptotic pathway. The objective of the current study was to assess how the p53 apoptotic pathway is altered in nonsmall cell lung carcinoma (NSCLC), especially in tumors without p53 alterations.

EGFR point mutation in non‐small cell lung cancer is occasionally accompanied by a second mutation or amplification

Activating mutations of EGFR are found frequently in a subgroup of patients with non‐small cell lung cancer (NSCLC) and are highly correlated with the response to gefitinib and erlotinib. In the present study, we searched for mutations of EGFR, HER2 and KRAS in 264 resected primary NSCLC from Japanese patients and determined whether there is a correlation between genetic alterations of these genes and clinicopathological factors, together with 85 tumors that we reported previously. EGFR mutations were found in 102 of the total 349 tumors, and seven tumors had two missense mutations. Reverse transcription–polymerase chain reaction of EGFR and subsequent subcloning analyses identified that the double mutations occurred in the same allele. Furthermore, in 202 NSCLC analyzed by Southern blotting, we identified 11 tumors with gene amplification of EGFR, with eight tumors containing a mutation in EGFR. Sequence analysis detected only weak or no signals of the wild‐type allele in the eight tumors, strongly suggesting that the mutated allele was amplified selectively. These findings indicate that a dual genetic change of EGFR can occur in the same allele either with a possible second‐hit mutation or with amplification, which may imply a more selective growth advantage in a cancer cell. Meanwhile, HER2 mutations and amplifications were found in six of 349 tumors and three of 202 tumors, respectively, and KRAS mutations in 21 of 349 tumors. Mutations of the EGFR and HER2 genes were more frequently found in female never or light‐smoking patients with adenocarcinoma, and there were no tumors that had two or more mutations simultaneously among EGFR, HER2 and KRAS. The current study further demonstrates that a double genetic event in EGFR can occasionally occur in lung cancer, thus providing new clues for understanding the involvement of epidermal growth factor receptor signaling cascades in the pathogenesis of NSCLC. (Cancer Sci 2006; 97: 753–759)

Loss of heterozygosity of chromosome 12p does not correlate with KRAS mutation in non‐small cell lung cancer

Activating mutations of RAS gene families have been found in a variety of human malignancies, including lung cancer, suggesting their dominant role in tumorigenesis. However, several studies have shown a frequent loss of the wild‐type KRAS allele in the tumors of murine models and an inhibition of oncogenic phenotype in tumor cell lines by transfection of wild‐type RAS, indicating that wild‐type RAS may have oncosuppressive properties. To determine whether loss of wild‐type KRAS is involved in the development of human lung cancer, we investigated the mutations of KRAS, NRAS and BRAF in 154 primary non‐small cell lung cancers (NSCLCs) as well as 10 NSCLC cell lines that have been shown to have KRAS mutations. We also determined the loss of heterozygosity status of KRAS alleles in these tumors. We detected point mutations of KRAS in 11 (7%) of 154 NSCLCs, with 10 cases at codon 12 and 1 at codon 61, but no mutations of NRAS or BRAF were found. Using the laser capture microdissection technique, we confirmed that 9 of the 11 tumors and 7 of the 10 NSCLC cell lines retained the wild‐type KRAS allele. Among the cell lines with heterozygosity of mutant and wild‐type KRAS, all of the cell lines tested for expression were shown to express more mutated KRAS than wild‐type mRNA, with higher amounts of KRAS protein also being expressed compared to the cell lines with a loss of wild‐type KRAS allele. In addition, among 148 specimens available for immunohistochemical analysis, 113 (76%) showed positive staining of KRAS, indicating that the vast majority of NSCLCs continue to express wild‐type KRAS. Our findings indicate that the wild‐type KRAS allele is occasionally lost in human lung cancer, and that the oncogenic activation of mutant KRAS is more frequently associated with an overexpression of the mutant allele than with a loss of the wild‐type allele in human NSCLC development.

β-catenin inhibits cell growth of a malignant mesothelioma cell line, NCI-H28, with a 3p21.3 homozygous deletion

We have found that a malignant mesothelioma cell line, NCI-H28, had a chromosome 3p21.3 homozygous deletion containing the β-catenin gene (CTNNB1), which suggested that the deletion of β-catenin might have a growth advantage in the development of this tumor. To determine whether β-catenin has a growth-inhibitory activity, we transfected wild-type β-catenin, Ser37Cys mutant β-catenin as an activated type, and C-terminus deletion mutant β-catenin that lacks the transcription activity, into the NCI-H28 cells. A non-small cell lung cancer cell line, NCI-H1299, which expressed endogenous β-catenin, was also studied. We tested the localization of exogenous β-catenin in the NCI-H28 cells with immunofluorescence, and found that the wild-type β-catenin and the C-terminus deletion mutant were more strongly expressed in the plasma membrane and cytoplasm than in the nucleus, while the Ser37Cys mutant was more in the nucleus than in the cytoplasm. By using luciferase-reporter assay, the β-catenin/T-cell factor 4-mediated transactivity of the Ser37Cys mutant was shown to be higher than that of the wild-type β-catenin in both cell lines. However, the transactivity of the C-terminus deletion mutant was strongly reduced in both. Colony formation of the NCI-H28 cells was reduced by 50% after transfection with the wild-type β-catenin, and 60% with the Ser37Cys mutant, but only 20% with the C-terminus deletion mutant compared to the vector control. Inhibition of colony formation in NCI-H28 cells was because of apoptosis, manifested by positive staining of Annexin V and TUNEL assays in transfected cells. In contrast, when transfected with the wild-type β-catenin, no significant reduction in colony formation was seen in β-catenin wild-type NCI-H1299 cells. In conclusion, our data indicate that inactivation of β-catenin by a 3p21.3 homozygous deletion might be a crucial event in the development of the mesothelioma NCI-H28 cells. Thus, while β-catenin is well known to be a positive growth-stimulating factor for many human cancers, it can also act as a potential growth suppressor in some types of human cancer cells.

Establishment of a large cell lung cancer cell line (Y-ML-1B) producing granulocyte colony-stimulating factor

We established a new lung cancer cell line, designated Y-ML-1B, from a lung cancer of a 70-year-old Japanese man with leukocytosis and thrombocytosis. Before surgical resection, the white blood cell and platelet counts were elevated to 34,400/mm3 and 668,000/mm3, respectively, and the granulocyte colony-stimulating factor (G-CSF) level in the serum was increased at 141 pg/mL. The primary tumor showed an undifferentiated morphology with large cells and induced extensive thickening of the pleura in the right hemithorax. The Y-ML-1B cells grow as a monolayer, with a doubling time of 19 hours, and are tumorigenic in nude mice, which showed a morphology similar to the primary tumor in xenografts. Analysis of the supernatant of cell culture medium of Y-ML-1B showed elevated levels of G-CSF and other cytokines such as interleukin (IL)-6, IL-8, and granulocyte-macrophage colony-stimulating factor (GM-CSF), consistent with the high levels detected in the patients serum. Cytogenetic analysis revealed aneuploidy of greater than 56 in metaphases with many structural abnormalities. Mutation analysis of the tumor suppressor genes showed that Y-ML-1B is inactivated in TP53 and RASSF1A, but not in p14(ARF), p16(INK4A), or RB. Neither activating mutations of KRAS or NRAS nor amplification of MYC or MDM2 were detected. Y-ML-1B expressed N-cadherin but not E-cadherin. This newly established cell line might serve as a useful model for studying the molecular pathogenesis for large cell cancers of the lung which express high levels of cytokines.

Pulmonary Neoplasms in Patients with Birt-Hogg-Dubé Syndrome: Histopathological Features and Genetic and Somatic Events

Birt-Hogg-Dubé syndrome (BHD) is an inherited disorder caused by genetic mutations in the folliculin (FLCN) gene. Individuals with BHD have multiple pulmonary cysts and are at a high risk for developing renal cell carcinomas (RCCs). Currently, little information is available about whether pulmonary cysts are absolutely benign or if the lungs are at an increased risk for developing neoplasms. Herein, we describe 14 pulmonary neoplastic lesions in 7 patients with BHD. All patients were confirmed to have germline FLCN mutations. Neoplasm histologies included adenocarcinoma in situ (n = 2), minimally invasive adenocarcinoma (n = 1), papillary adenocarcinoma (n = 1), micropapillary adenocarcinoma (n = 1), atypical adenomatous hyperplasia (n = 8), and micronodular pneumocyte hyperplasia (MPH)-like lesion (n = 1). Five of the six adenocarcinoma/MPH-like lesions (83.3%) demonstrated a loss of heterozygosity (LOH) of FLCN. All of these lesions lacked mutant alleles and preserved wild-type alleles. Three invasive adenocarcinomas possessed additional somatic events: 2 had a somatic mutation in the epidermal growth factor receptor gene (EGFR) and another had a somatic mutation in KRAS. Immunohistochemical analysis revealed that most of the lesions were immunostained for phospho-mammalian target of rapamycin (p-mTOR) and phospho-S6. Collective data indicated that pulmonary neoplasms of peripheral adenocarcinomatous lineage in BHD patients frequently exhibit LOH of FLCN with mTOR pathway signaling. Additional driver gene mutations were detected only in invasive cases, suggesting that FLCN LOH may be an underlying abnormality that cooperates with major driver gene mutations in the progression of pulmonary adenocarcinomas in BHD patients.

Transfer of Cefaclor and Ceftibuten into Lung Tissue and Dosing Schedules for Patients with Respiratory Tract Infections

Cefaclor (CCL) and ceftibuten (CETB) are cephem antibiotics synthesized in Japan for oral use. CCL has a broad antibacterial spectrum and CETB is specific for gram-negative bacteria. In our studies, serum and lung tissue CCL and CETB levels were determined from samples obtained at the time of lung surgery after administration of a single fasting dose of 500 mg of CCL or 200 mg of CETB. This study was undertaken to investigate the transfer of the drugs into lung tissue and to determine the appropriate dosing schedules of these drugs for patients with respiratory tract infections. The mean peak serum CCL level was 4.04 μg/mL at 1.5 hours after administration, and the mean alveolar tissue CCL level was 0.30 μg/g tissue 5 hours after administration. The mean peak serum CETB level was 8.47 μg/mL 2 hours after administration, and the mean alveolar, subsegmental bronchial, and bronchiolar tissue CETB levels were 2.65,1.64 and 1.75 μg/g tissue, respectively, at 5 hours after administration, while the CETB level in cancerous lung tissue was 1.41 μg/g 5 hours after administration. From our results, both CCL and CETB showed favorable uptake into lung tissue, suggesting that both compounds may be useful for respiratory tract infections. CCL should be selected for patients with respiratory tract infections in which the pathogens are unknown, with a dose of 500 mg administered every 8 hours. When the pathogen has been identified as a gram-negative organism in patients with pulmonary or bronchiolar infections, 200 mg of CETB should be administered every 12 hours.