Michihiko Tajiri

Down-Regulation of DUSP6 Expression in Lung Cancer —Its Mechanism and Potential Role in Carcinogenesis

Our preliminary studies revealed that oncogenic KRAS (KRAS/V12) dramatically suppressed the growth of immortalized airway epithelial cells (NHBE-T, with viral antigen-inactivated p53 and RB proteins). This process appeared to be a novel event, different from the so-called premature senescence that is induced by either p53 or RB, suggesting the existence of a novel tumor suppressor that functions downstream of oncogenic KRAS. After a comprehensive search for genes whose expression levels were modulated by KRAS/V12, we focused on DUSP6, a pivotal negative feedback regulator of the RAS-ERK pathway. A dominant-negative DUSP6 mutant, however, failed to rescue KRAS/V12-induced growth suppression, but conferred a stronger anchorage-independent growth activity to the surviving subpopulation of cells generated from KRAS/V12-transduced NHBE-T. DUSP6 expression levels were found to be weaker in most lung cancer cell lines than in NHBE-T, and DUSP6 restoration suppressed cellular growth. In primary lung cancers, DUSP6 expression levels decreased as both growth activity and histological grade of the tumor increased. Loss of heterozygosity of the DUSP6 locus was found in 17.7% of cases and was associated with reduced expression levels. These results suggest that DUSP6 is a growth suppressor whose inactivation could promote the progression of lung cancer. We have here identified an important factor involved in carcinogenesis through a comprehensive search for downstream targets of oncogenic KRAS.

Prognostic value of the IASLC/ATS/ERS classification of lung adenocarcinoma in stage I disease of Japanese cases

A new classification of adenocarcinoma (ADC) was proposed by the International Association for the Study of Lung Cancer, the American Thoracic Society, and the European Respiratory Society (IASLC/ATS/ERS) in 2011. The present study evaluates its prognostic value in stage I disease of Japanese cases. One‐hundred‐and‐seventy‐nine cases with pathological stage I ADC were classified according to the new classification system and their association with disease recurrence was analyzed. Eighteen (10.1%) and 24 (13.4%) cases were adenocarcinoma in situ (AIS) and minimally invasive adenocarcinoma (MIA), respectively. One‐hundred‐and‐thirty‐seven cases (76.5%) were invasive adenocarcinoma (IVA), in which 43 (24.0%) were lepidic (LEP), 59 (33.0%) were acinar (ACN), 16 (8.9%) were papillary (PAP), 1 (0.6%) was micropapillary (MPAP), 12 (6.7%) were solid predominant subtypes (SOL), and 6 (3.4%) were invasive mucinous adenocarcinoma (MUC). The5‐year disease‐free survivals (DFS) of patients with AIS and MIA were 100%. Those of LEP, ACN, PAP, SOL and MUC were 93.5%, 83.7%, 75.0%, 44.4% and 62.5%, respectively. Multivariate analysis showed that high‐histological grade (SOL, MPAP, MUC) had an independent prognostic value to predict post‐operative recurrence (HR 3.661, 95% CI 1.421–9.437, P = 0.007). In conclusion, the present study demonstrates a prognostic value of the 2011 IASLC/ATS/ERS classification of ADC in Japanese cases.

Expression of the potential cancer stem cell markers, CD133, CD44, ALDH1, and β-catenin, in primary lung adenocarcinoma—their prognostic significance

The present study investigated expression profiles of the potential CSC markers including CD133, CD44, ALDH1, and β‐catenin, and evaluated their prognostic value in lung adenocarcinomas. One‐hundred‐and‐seventy‐seven tumors (stage I) were immunohistochemically examined for the expression of these markers, and thresholds to subdivide expression levels were determined using receiver operating characteristics curves. Tumors with high levels of CD133 (adjusted hazard ratio (HR) 4.55 (95% confidence interval (CI) 1.26–16.40, P = 0.021), CD44 (HR 3.73, 95% CI 1.20–11.58, P = 0.023) or ALDH1 (HR 3.61, 95% CI 1.09–12.3, P = 0.036), but not β‐catenin (HR 2.43, 95% CI 0.59–10.8, P = 0.220), showed a significantly higher risk of recurrence than the corresponding low expressers. In conclusion, levels of CD133, CD44, and ALDH1 had independent prognostic value to predict the recurrence of lung adenocarcinoma.

Prognostic value of KRAS mutations and Ki-67 expression in stage I lung adenocarcinomas.

The purpose of the present study was to establish accurate prognostic markers to predict the post-operative recurrence of stage I lung adenocarcinomas (ADC). One-hundred and ninety cases of stage I ADC were examined for KRAS mutations and Ki-67 expression, and their associations with disease recurrence were analyzed. KRAS-mutated cases showed a significantly higher risk of recurrence than cases without mutations (5-year disease-free survival (DFS) 61.0% vs. 85.8%, P=0.017: adjusted Hazard ratio (HR) 4.55, 95% Confidence Interval (CI) 1.61-12.82, P=0.004). Ki-67 high-expressers (labeling index >10%) also showed a higher risk of recurrence than low-expressers (5-year DFS 68.7% vs. 93.2%, P<0.001: adjusted HR 3.84, 95% CI 1.18-12.45, P=0.025). Ki-67 high-expressers with KRAS mutations showed an additional higher risk of recurrence compared to low-expressers without mutations (5-year DFS 37.5% vs. 93.3%, P<0.001: adjusted HR 16.82, 95% CI 3.77-74.98, P<0.001) and their 5-year DFS was nearly equivalent to that of stage II non-small cell lung cancer (NSCLC) in our facility (37.5% vs. 37.2% for stage II NSCLC, p=0.577). The combined use of KRAS status and Ki-67 expression level could be an excellent prognostic marker to predict the post-operative recurrence of stage I ADC.

Decreased invasiveness via two methods of thoracoscopic lobectomy for lung cancer, compared with open thoracotomy.

Background and objective:  Video‐assisted thoracic surgery (VATS) lobectomy for primary lung cancer is considered minimally invasive. However, different procedures may be used for the VATS lobectomy, from complete videoscopic (CV) surgery to a technique similar to the muscle‐sparing thoracotomy. We divided patients into two groups based on the surgical approach and analysed the outcomes.

Early Growth Response-1 Induces and Enhances Vascular Endothelial Growth Factor-A Expression in Lung Cancer Cells

Vascular endothelial growth factor-A (VEGF-A) is crucial for angiogenesis, vascular permeability, and metastasis during tumor development. We demonstrate here that early growth response-1 (EGR-1), which is induced by the extracellular signal-regulated kinase (ERK) pathway activation, activates VEGF-A in lung cancer cells. Increased EGR-1 expression was found in adenocarcinoma cells carrying mutant K-RAS or EGFR genes. Hypoxic culture, siRNA experiment, luciferase assays, chromatin immunoprecipitation, electrophoretic mobility shift assays, and quantitative RT-PCR using EGR-1-inducible lung cancer cells demonstrated that EGR-1 binds to the proximal region of the VEGF-A promoter, activates VEGF-A expression, and enhances hypoxia inducible factor 1alpha (HIF-1alpha)-mediated VEGF-A expression. The EGR-1 modulator, NAB-2, was rapidly induced by increased levels of EGR-1. Pathology samples of human lung adenocarcinomas revealed correlations between EGR-1/HIF-1alpha and VEGF-A expressions and relative elevation of EGR-1 and VEGF-A expression in mutant K-RAS- or EGFR-carrying adenocarcinomas. Both EGR-1 and VEGF-A expression increased as tumors dedifferentiated, whereas HIF-1alpha expression did not. Although weak correlation was found between EGR-1 and NAB-2 expressions on the whole, NAB-2 expression decreased as tumors dedifferentiated, and inhibition of DNA methyltransferase/histone deacetylase increased NAB-2 expression in lung cancer cells despite no epigenetic alteration in the NAB-2 promoter. These findings suggest that EGR-1 plays important roles on VEGF-A expression in lung cancer cells, and epigenetic silencing of transactivator(s) associated with NAB-2 expression might also contribute to upregulate VEGF-A expression.

Morphometric profiling of lung cancers-its association with clinicopathologic, biologic, and molecular genetic features.

The traditional histologic classification of lung cancer is not satisfactory to describe the morphologic characteristics of individual tumors, because it does not fully cover cytologic features. This paper describes a novel typing system using morphometric profiling that covers a variety of morphologic features including histologic architecture, cell type, cytoplasmic color and internal structure, nuclear outline, chromatin pattern, nucleoli count and remarkableness, and average and deviation of nuclear size and circularity. In all, 201 cases of lung tumors (whose sizes are <20 mm) were examined. Results of a hierarchical clustering analysis were used to draw a dendrogram. We here tentatively focused on 8 morphometric clusters and analyzed their potential association with a variety of clinicopathologic and molecular genetic features. Significant differences in postoperative recurrent risk, growth activity, oncogenic mutation (EGFR or KRAS), impairments of tumor suppressors (p53 and p16), sex predisposition, and smoking status were found among the 8 clusters. The system has the potential to improve histopathologic diagnosis and our understanding of carcinogenesis in the lung.

Thoracoscopic lung biopsy in 285 patients with diffuse pulmonary disease

Background Surgical lung biopsy is generally considered the most appropriate method for diagnosing diffuse lung disease. However, there are few reports focusing on only one thoracoscopic technique. This study was designed to determine the morbidity and mortality related to video-assisted thoracoscopic lung biopsy in a single center, thereby providing data on the severity of morbidity and clarifying the risk factors. Methods We analyzed 285 patients with undiagnosed diffuse lung disease who underwent video-assisted thoracoscopic lung biopsy at Kanagawa Cardiovascular and Respiratory Center from February 2007 to April 2012. We recorded the severity of postoperative complications using the Clavien-Dindo classification. Results The surgical morbidity was 7.0% (20/285), including delayed pulmonary fistulas in 11 patients, acute exacerbation in 3, prolonged air leakage (>7 days) in 2, hypoxemia in 2, atrial fibrillation in 1, and premature ventricular contraction in 1. Based on the Clavien-Dindo classification, grade I, II, IIIa, IIIb, and IVa complications accounted for 20%, 10%, 50%, 5%, and 15%, respectively. The 30-day mortality was 0%. The diagnostic yield was 100%. Although acute exacerbation occurred in 2 patients with idiopathic pulmonary fibrosis and 1 with fibrotic nonspecific interstitial pneumonia, there were no distinctive features that allowed preoperative prediction of acute exacerbation. Conclusions Our findings indicate that video-assisted thoracoscopic lung biopsy is a feasible procedure. We hope to clarify risk factors in future research.

Allelic Imbalance in the miR-31 Host Gene Locus in Lung Cancer - Its Potential Role in Carcinogenesis

Small non-protein coding RNA, microRNA (miR), which regulate messenger RNA levels, have recently been identified, and may play important roles in the pathogenesis of various diseases. The present study focused on miR-31 and investigated its potential involvement in lung carcinogenesis. The expression of miR-31 was altered in lung cancer cells through either the amplification or loss of the host gene locus. The strong expression of miR-31 in large cell carcinomas was attributed to the gene amplification. Meanwhile, the loss of miR-31 expression was more frequently observed in aggressive adenocarcinomas. Thus, miR-31 may play a pleiotropic role in the development of lung cancers among different histological types. To the best of our knowledge, this is the first study to show the potential causative mechanism of the altered expression of miR-31 and suggest its potentially diverse significance in the different histological types of lung cancers.

Proteome Analysis for Downstream Targets of Oncogenic KRAS - the Potential Participation of CLIC4 in Carcinogenesis in the Lung

This study investigated the proteome modulated by oncogenic KRAS in immortalized airway epithelial cells. Chloride intracellular channel protein 4 (CLIC4), S100 proteins (S100A2 and S100A11), tropomyosin 2, cathepsin L1, integrinsα3, eukaryotic elongation factor 1, vimentin, and others were discriminated. We here focused on CLIC4 to investigate its potential involvement in carcinogenesis in the lung because previous studies suggested that some chloride channels and chloride channel regulators could function as tumor suppressors. CILC4 protein levels were reduced in some lung cancer cell lines. The restoration of CLIC4 in lung cancer cell lines in which CLIC4 expression was reduced attenuated their growth activity. The immunohistochemical expression of the CLIC4 protein was weaker in primary lung cancer cells than in non-tumorous airway epithelial cells and was occasionally undetectable in some tumors. CLIC4 protein levels were significantly lower in a subtype of mucinous ADC than in others, and were also significantly lower in KRAS-mutated ADC than in EGFR-mutated ADC. These results suggest that the alteration in CLIC4 could be involved in restrictedly the development of a specific fraction of lung adenocarcinomas. The potential benefit of the proteome modulated by oncogenic KRAS to lung cancer research has been demonstrated.