Akiteru Goto

MicroRNA Expression and Clinical Outcomes in Patients Treated with Adjuvant Chemotherapy after Complete Resection of Non–Small Cell Lung Carcinoma

This study determined whether expression levels of a panel of biologically relevant microRNAs can be used as prognostic or predictive biomarkers in patients who participated in the International Adjuvant Lung Cancer Trial (IALT), the largest randomized study conducted to date of adjuvant chemotherapy in patients with radically resected non-small cell lung carcinoma (NSCLC). Expression of miR-21, miR-29b, miR-34a/b/c, miR-155, and let-7a was determined by quantitative real-time PCR in formalin-fixed paraffin-embedded tumor specimens from 639 IALT patients. The prognostic and predictive values of microRNA expression for survival were studied using a Cox model, which included every factor used in the stratified randomization, clinicopathologic prognostic factors, and other factors statistically related to microRNA expression. Investigation of the expression pattern of microRNAs in situ was performed. We also analyzed the association of TP53 mutation status and miR-34a/b/c expression, epidermal growth factor receptor and KRAS mutation status, and miR-21 and Let-7a expression. Finally, the association of p16 and miR-29b expression was assessed. Overall, no significant association was found between any of the tested microRNAs and survival, with the exception of miR-21 for which a deleterious prognostic effect of lowered expression was suggested. Otherwise, no single or combinatorial microRNA expression profile predicted response to adjuvant cisplatin-based chemotherapy. Together, our results indicate that the microRNA expression patterns examined were neither predictive nor prognostic in a large patient cohort with radically resected NSCLC, randomized to receive adjuvant cisplatin-based chemotherapy versus follow-up only.

c-Met activation in lung adenocarcinoma tissues: An immunohistochemical analysis

c‐Met is often overexpressed in non‐small cell lung cancer, but it remains unsolved whether its overexpression leads to its activation. We used an antibody specific to phospho‐c‐Met (Tyr1235) to investigate c‐Met activation immunohistochemically in 130 surgically resected lung adenocarcinomas. The expression of c‐Met and hepatocyte growth factor (HGF) was also investigated. Phospho‐c‐Met was positive in 21.5% (28/130) of cases. c‐Met was positive in 74.6% of cases (97/130) and was expressed at high levels in 36.1% of cases (47/130). HGF was expressed at high levels in 31.5% of cases (41/130). Phospho‐c‐Met was correlated with high levels of HGF (P =0.0010) and high levels c‐Met expression (P = 0.0303), but it was also found to be positive in 12 cases with little to no HGF expression. Phospho‐c‐Met expression was significantly associated with tumor differentiation (P = 0.0023) and papillary histology (P = 0.0011), but not with pathological stage, lymph node metastasis or survival. High levels of c‐Met and HGF were also associated with papillary histology (P = 0.0056 and P = 0.0396, respectively), but not with tumor differentiation. Phospho‐c‐Met was correlated with phospho‐Akt (P = 0.0381), but not with phospho‐Erk or phospho‐Stat3. Phospho‐Akt expression was marginally correlated with the expression of phospho‐epidermal growth factor receptor (EGFR) (P = 0.0533) and, importantly, it was strongly correlated with the expression of either phospho‐c‐Met or phospho‐EGFR (P = 0.0013). The data suggest that in lung adenocarcinoma tissue, c‐Met activation may take place either ligand‐dependently or ligand‐independently via c‐Met overexpression. c‐Met activation may play special roles in the papillary subtype and in well differentiated lung adenocarcinomas. (Cancer Sci 2007; 98: 1006–1013)

Genome structure-based screening identified epigenetically silenced microRNA associated with invasiveness in non-small-cell lung cancer†

MicroRNA (miRNA) expression is frequently altered in human cancers. To search for epigenetically silenced miRNAs in non‐small‐cell lung cancer (NSCLC), we mapped human miRNAs on autosomal chromosomes and selected 55 miRNAs in silico. We treated six NSCLC cell lines with the DNA methylation inhibitor 5‐aza‐2′‐deoxycytidine (5‐aza‐CdR) and determined the expressions of the 55 miRNAs. Fourteen miRNAs were decreased in the cancer cell lines and were induced after 5‐aza‐CdR treatment. After a detailed DNA methylation analysis, we found that mir‐34b and mir‐126 were silenced by DNA methylation. Mir‐34b was silenced by the DNA methylation of its own promoter, whereas mir‐126 was silenced by the DNA methylation of its host gene, EGFL7. A chromatin immunoprecipitation assay revealed H3K9me2 and H3K9me3 in mir‐34b and EGFL7, and H3K27me3 in EGFL7. The overexpression of mir‐34b and mir‐126 decreased the expression of c‐Met and Crk, respectively. The 5‐aza‐CdR treatment of lung cancer cell line resulted in increased mir‐34b expression and decreased c‐Met protein. We next analyzed the DNA methylation status of these miRNAs using 99 primary NSCLCs. Mir‐34b and mir‐126 were methylated in 41 and 7% of all the cases, respectively. The DNA methylation of mir‐34b was not associated with c‐Met expression determined by immunohistochemistry, but both mir‐34b methylation (p = 0.007) and c‐Met expression (p = 0.005) were significantly associated with lymphatic invasion in a multivariate analysis. The DNA methylation of mir‐34b can be used as a biomarker for an invasive phenotype of lung cancer.

Oncofetal protein glypican-3 in testicular germ-cell tumor

The expression of an oncofetal protein, the glypican-3 (GPC3), was immunohistochemically evaluated in a wide variety of primary testicular germ-cell tumors (GCTs) in comparison with other markers, alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG)-beta, and OCT3/4. Eighty-nine cases of GCT including 22 cases of mixed GCT were evaluated with reference to each tumor component. GPC3 expression was observed in neoplastic cells of yolk-sac tumor (YST) (25/25), teratoma (2/10), components of syncytiotrophoblastic giant cells (STGCs) (10/14), and choriocarcinoma (1/3), but none in intratubular germ-cell neoplasias, unclassified type (0/33), seminomas (0/61), or embryonal carcinoma (0/19). All cases of YST showed diffuse labeling of neoplastic cells in cytoplasmic and membranous patterns, and the positive area of GPC3 was much larger than that of AFP. Glandular structures in teratomas showed GPC3 immunostaining as well as AFP. Although the number of GPC3-positive cells was smaller in STGC components and choriocarcinoma, there was no diffusion artifact in GPC3 immunostaining, as was frequently encountered in hCG-beta staining. Thus, GPC3 is a unique oncofetal protein, which is useful as an immunohistochemical marker for GCT differentiated to extraembryonic tissue, especially YST.

Differential expression of S100A2 and S100A4 in lung adenocarcinomas: Clinicopathological significance, relationship to p53 and identification of their target genes

Previous studies suggest that some S100 proteins are involved in the progression of certain types of cancer. However, no comprehensive data is currently available on the expression of S100 family genes in lung adenocarcinomas. Oligonucleotide array, quantitative reverse transcription–polymerase chain reaction and western blot analyses of lung adenocarcinoma cell lines and bronchiolar epithelial cells (SAEC and NHBE) revealed that S100A2 and S100A4 were the most strikingly downregulated and upregulated members of the S100 family, respectively. Immunohistochemical analyses of 94 primary lung adenocarcinomas showed that positive S100A2 expression (33/94, 35.1%) was significantly associated with lymphatic invasion (P = 0.0233) and positive S100A4 expression (19/94, 20.2%) with vascular invasion (P = 0.0454). Interestingly, a strong inverse relationship was found between S100A4 and p53 expression (P = 0.0008). Survival analyses showed that S100A4 positivity was associated with poor patient prognosis (P = 0.042). S100A2 positivity was not associated with patient survival when the whole patient group was analyzed; however, S100A2 positivity was a favorable prognostic indicator in patients with p53‐negative tumors (P = 0.0448). Finally, we used oligonucleotide array analyses and identified potential S100A2 and S100A4 target genes involved in cancer progression: S100A2 induced RUNX3 and REPRIMO; S100A4 induced EZRIN, RUNX1 and WISP1; S100A2 repressed EGFR, NFKB2 and RELA2; and S100A4 repressed ANXA10 and IL1RN. Thus, the present study demonstrates involvement of S100A2 and S100A4 in the progression of lung adenocarcinomas and an inverse association between S100A4 and p53 expression, and provides a list of targets regulated by S100A2 and S100A4. (Cancer Sci 2005; 96: 844–857)

Overexpression of cdk4/cyclin D1, a possible mediator of apoptosis and an indicator of prognosis in human primary lung carcinoma

The relation between expression of cell cycle–regulator molecules and apoptosis was examined in surgical specimens and cultured human lung carcinoma cell lines. Immunohistochemical analysis for 133 cases revealed 2 types of staining pattern. The first group consisted of 95 cases (71.4%) characterized by apoptotic cells showing intensely positive staining for cdk4 and cyclin D1 but negative for other proteins (type A). In the second group (type B), comprising 38 cases (28.6%), apoptotic cells exhibited intense positive staining for any cyclins and cdks. Most of the latter cases had lost expression of Rb protein. When tumor cells retrieved from paraffin‐embedded tissue were examined by flow cytometry, higher proportions of cells expressing only cdk4 or cyclin D1 in type A cases and of cells expressing any cyclin or cdk in type B cases showed a subdiploid DNA content. In survival analysis using the LI of apoptotic cells and cyclin/cdk‐positive cells, the high‐apoptosis/high‐cyclin D1 group showed the poorest prognosis. Furthermore, forced overexpression of only cdk4 or cyclin D1 induced apoptosis in cultured cells with normal Rb protein, whereas overexpression of any cyclin or cdk induced apoptosis in cells defective for Rb protein. In conclusion, upregulation of cdk4/cyclin D1 may be a primary and critical factor in induction of apoptosis in human lung carcinomas in vivo. Moreover, inactivation of Rb protein renders cells more prone to apoptosis by abnormal expression of any cell‐cycle protein.

Immunohistochemical study of Skp2 and Jab1, two key molecules in the degradation of P27, in lung adenocarcinoma

To clarify the association of the P27 degradation pathway proteins, Skp2 and Jab1, with the development and progression of lung adenocarcinoma (AD), we immunohistochemically investigated Skp2 and Jab1 expression together with P27‐ and Ki‐67‐labeling in 110 lung AD and 11 atypical adenomatous hyperplasia (AAH) and analyzed the relationship between the expression of these proteins and the clinicopathological factors. High Skp2 or Jab1 expression was frequent in lung AD (52/110, 47%, and 59/110, 54%, respectively), and high expression of Jab1 was also frequent in AAH (4/11, 36%), while it was not observed in normal bronchiolar epithelium. The P27 labeling index (LI) was reciprocally correlated with high Skp2 and Jab1 expression, and a higher Ki‐67 LI was significantly correlated with high Skp2 and Jab1 expression. However, low P27 expression did not correlate with a higher Ki‐67 LI. High Skp2 lung AD showed significant correlation with blood and lymphatic vessel invasion, which low P27 expression did not correlate with. Furthermore, high Skp2 expression in lung AD was significantly correlated with a poor outcome for patients. Thus, Skp2 and Jab1 regulate P27 degradation, and might contribute to the development and progression of lung AD through P27‐mediated and –unmediated mechanisms.

Prevalence of CD99 protein expression in pancreatic endocrine tumours (PETs)

Aims : To determine the prevalence of CD99 expression in pancreatic endocrine tumours (PETs). We evaluated CD99 expression and analysed Ki67 labelling by immunohistochemistry in PETs.

Constitutive activation of c-Met is correlated with c-Met overexpression and dependent on cell–matrix adhesion in lung adenocarcinoma cell lines

In this study we explored the mechanisms of constitutive activation of c‐Met in lung adenocarcinoma cell lines. First, we examined levels of c‐Met and phospho‐c‐Met (Y1234/Y1235) in a panel of lung adenocarcinoma cell lines by Western blot analysis. c‐Met expression was found in 12 of 14 cell lines and an overall correlation between the expressions of c‐Met and phospho‐c‐Met was noted. c‐Met was constitutively activated particularly at high levels in five cell lines (PC3, LC‐2/ad, L27, H1648, and H2009). c‐Met amplification was identified in L27 and H1648 by single nucleotide polymorphism array analysis, but no mutations were identified in the Sema domain or in any part of the cytoplasmic domain of c‐Met. Experiments with neutralizing anti‐hepatocyte growth factor (HGF) antibody, scatter assay using Madin–Darby canine kidney cells, and Western blotting on conditioned media of the cell lines revealed that the constitutive phosphorylation of c‐Met was largely ligand‐independent. The inhibition of cell–matrix adhesion induced the dephosphorylation of c‐Met in the five cell lines tested. This was accompanied by downregulation of c‐Met in three of the five cell lines. In contrast, the inhibition of cell–cell adhesion by neutralizing E‐cadherin antibody had a minimal effect on the expression and phosphorylation of c‐Met. These results reveal three features of the constitutive activation of c‐Met in our panel of lung adenocarcinoma cell lines: (i) it correlates with c‐Met overexpression, either with or without gene amplification; (ii) it is largely ligand‐independent; and (iii) it depends on cell–matrix adhesion. (Cancer Sci 2008; 99: 14–22)

Human Papillomavirus Infection in Lung and Esophageal Cancers: Analysis of 485 Asian Cases

The role of human papillomavirus (HPV) in the development of lung and esophageal cancer remains inconclusive, which is in contrast to the established role HPV plays in the development of uterine cervical cancer. One of the reasons for this is the difference among reported HPV infection rates in these cancers. An analysis of 485 lung and esophageal cancers (176 lung squamous cell carcinoma, 128 lung adenocarcinoma, 181 esophageal carcinoma) in eight institutions in Asia (Tokyo, Kochi, Kagoshima, and Okinawa, Japan; Seoul and Daegu, Korea; Changhua, Republic of China (Taiwan); Singapore, Singapore) was carried out in order to clarify infection rates with HPV. Samples were examined in one laboratory of the Department of Pathology, the University of Tokyo, Japan in order to avoid inter‐laboratory variation using a combination of polymerase chain reaction and in situ hybridization (ISH). HPV was found in 6.3%, 7%, and 9.4% of patients with lung squamous cell carcinoma, lung adenocarcinoma, and esophageal cancer, respectively. Among the geographic areas surveyed, Kagoshima exhibited a significantly higher prevalence of HPV infection in cases of esophageal carcinoma (24.1%). There was no geographical difference in the infection rates of HPV in lung carcinomas. Subtype‐specific ISH was also performed, which identified the high‐risk HPV types 16/18 in the majority (75.7%) of the patients with lung and esophageal cancer positive for HPV. J. Med. Virol. 83:1383–1390, 2011.