Tetsukan Woo

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.

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.

KRAS gene mutations in lung cancer: particulars established and issues unresolved.

Lung cancer, like other cancers, is considered to develop through the accumulation of genetic alterations. Mutation of the KRAS gene is one of the most important events in carcinogenesis of the lung. The KRAS gene, belonging to the RAS gene family, encodes a membrane‐bound 21‐kd guanosine triphosphate (GTP)‐binding protein. Single point mutations in this protein result in continuous activation to transmit excessive signals, promoting a variety of biological events. In lung cancers, the mutations concentrate at codon 12 and mostly affect adenocarcinomas (ADCs). They also affect atypical adenomatous hyperplasia, the precursor of ADCs. Therefore, mutation of the KRAS gene is suggested to confer a growth advantage to airway epithelial cells enabling them to expand clonally early in the development of ADCs. The mutation is also a reliable marker of an unfavorable response to certain molecular‐targeting therapies. Furthermore, patients with ADCs affected by mutations have been reported to exhibit a significantly higher risk of postoperative disease recurrence. Thus, the significance of KRAS gene mutations has been investigated extensively. However, not all the details emerged. In this review, particulars that have been established are introduced, and important issues remaining to be resolved are discussed, with special reference to carcinogenesis of the lung.

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.

Superior vena cava (SVC) reconstruction using autologous tissue in two cases of differentiated thyroid carcinoma presenting with SVC syndrome

Herein, we report two extremely rare cases of differentiated thyroid carcinoma (DTC) with extended tumor thrombus or mediastinum lymph node metastasis (LNM) involving the superior vena cava (SVC), causing SVC syndrome. Both of these patients were successfully treated with radical resection and reconstruction of the SVC using autologous tissue instead of an expanded polytetrafluoroethylene (ePTFE) graft. The left brachiocephalic vein was used to reconstruct the SVC in a papillary thyroid carcinoma patient with mediastinum LNM and a pericardial patch was used in a follicular thyroid carcinoma patient with tumor thrombus. Our search of the English-language literature found sporadic reports of SVC resection with reconstruction by vascular graft (ePTFE), interposed between the brachiocephalic vein and the right atrium. However, SVC reconstruction using autologous tissue in thyroid carcinoma has not been reported to date. To our knowledge, this is the first report describing such an unusual technique in DTC patients.

Insulin-like growth factor binding protein-4 gene silencing in lung adenocarcinomas

Gene silencing by promoter hypermethylation plays an important role in molecular pathogenesis. We previously reported that insulin‐like growth factor (IGF) binding protein‐4 (IGFBP‐4), which inhibits IGF‐dependent growth, is expressed via early growth response‐1 (EGR‐1) and is often silenced in cultivated lung cancer cells. The purpose of the present study was to clarify clinicopathological factors associated with IGFBP‐4 gene silencing in lung adenocarcinomas. Seventy‐six surgically resected adenocarcinomas (20 well‐, 35 moderately‐, and 21 poorly‐differentiated) were subjected to methylation‐specific polymerase chain reaction (PCR) analysis for EGR‐1‐binding sites located in the IGFBP‐4 promoter and immunohistochemistry for IGFBP‐4, EGR‐1, and Ki‐67. Thirty‐two adenocarcinomas (42%) revealed IGFBP‐4 promoter hypermethylation, and the severity inversely correlated with the level of IGFBP‐4 expression (P < 0.0001) and tumor differentiation (well versus poor, P= 0.0278; well/moderate versus poor, P= 0.0395). Furthermore, there was a negative correlation between Ki‐67 labeling index and IGFBP‐4 expression (P= 0.0361). These findings suggest that the expression of IGFBP‐4 in adenocarcinoma cells in vivo is downregulated by epigenetic silencing in association with tumor differentiation, resulting in disruption of the mechanism of IGFBP‐4‐mediated growth inhibition.

A case of epithelial-myoepithelial carcinoma of the bronchus - a review of reported cases and a comparison with other salivary gland-type carcinomas of the bronchus.

We present a case of a polypoid tumor located in the right middle lobe bronchus. The tumor was composed of bi-layered glandular or ductular structures consisting of inner cuboidal cells and outer multipolar cells. Immunohistochemical examinations confirmed epithelial and myoepithelial differentiation in the inner and outer components, respectively. Consequently, the tumor was diagnosed as an epithelial-myoepithelial carcinoma. Epithelial-myoepithelial carcinomas of the bronchus are very rare neoplasms with low-grade malignant potential. To date, including our case, only 27 cases have been reported in the English literature. Here, we review the reported cases and compare them with other salivary gland-type carcinomas regarding clinical, biological, and genetic features.