Koji Okudela

PIK3CA mutation and amplification in human lung cancer

To explore the significance of phosphatidylinositol‐3‐kinase, catalytic, alpha (PIK3CA) in the carcinogenesis in human lung, mutations and copy number changes were investigated in 148 Japanese patients with primary cancer of the lung. For biological validation, the effects of exogenously expressed wild‐type and mutated PIK3CA were studied in an immortalized human airway epithelial cell line. Mutations in PIK3CA were found in five (3.6%) of the 139 available patients, and copy number gains were found in 21 (18.3%) of 115 patients, respectively. Overall, mutations or copy number gains were detected in 24 of the 106 patients (22.6%) for whom results in both analyses were available. The prevalence of copy number gains was higher in men, smokers, and in patients with squamous cell carcinoma than in the opposite categories. The copy number changes showed a trend toward higher prevalence in the earlier stages (P = 0.038). Interestingly, the presence of mutations and of copy number alterations were mutually exclusive in the present patients, implying that both entail equivalent oncogenic potential. Over‐expressed wild‐type PIK3CA and its two common mutants, K545E and H1047R, significantly enhanced the anchorage‐independent growth activity and migration activity of immortalized airway epithelium 16HBE14o– cells, but the effects of the K545E and H1047R mutants were more remarkable than those of the wild‐type. The present demonstrates an important role of PIK3CA in human lung carcinogenesis.

Acetylcholine from vagal stimulation protects cardiomyocytes against ischemia and hypoxia involving additive non‐hypoxic induction of HIF‐1α

Electrical stimulation of the vagal efferent nerve improves the survival of myocardial infarcted rats. However, the mechanism for this beneficial effect is unclear. We investigated the effect of acetylcholine (ACh) on hypoxia‐inducible factor (HIF)‐1α using rat cardiomyocytes under normoxia and hypoxia. ACh posttranslationally regulated HIF‐1α and increased its protein level under normoxia. ACh increased Akt phosphorylation, and wortmannin or atropine blocked this effect. Hypoxia‐induced caspase‐3 activation and mitochondrial membrane potential collapse were prevented by ACh. Dominant‐negative HIF‐1α inhibited the cell protective effect of ACh. In acute myocardial ischemia, vagal nerve stimulation increased HIF‐1α expression and reduced the infarct size. These results suggest that ACh and vagal stimulation protect cardiomyocytes through the PI3K/Akt/HIF‐1α pathway.

K-ras Gene Mutation Enhances Motility of Immortalized Airway Cells and Lung Adenocarcinoma Cells via Akt Activation: Possible Contribution to Non-Invasive Expansion of Lung Adenocarcinoma

Point mutations of the K-ras gene, which are found in 10 to 30% of lung adenocarcinomas, are regarded as being an early event during the carcinogenesis. Autonomous vigorous motility of neoplastic cells, as well as growth and survival advantages, are considered to be necessary for cancer development and progression. The present study describes the contributions of the K-ras gene mutation and its downstream pathway via phosphatidylinositol 3-OH kinase (PI3K)-Akt to the cell motility in an immortalized human peripheral airway epithelial cell (HPL1D) and lung adenocarcinoma cells (A549, H820, TKB6, and TKB14). We have also evaluated the relationship between pathological events and the K-ras-Akt pathway using surgically resected lung tumors. The HPL1D cells transfected with the mutated K-ras gene (HPL-V12) showed a significant increase in cell motility compared to those transfected with empty vector (HPL-E) or wild-type K-ras gene (HPL-K). The enhanced motility in the HPL-V12 cells was markedly reduced by either treatment with inhibitors of ras, PI3K, and/or MEK, or by transfection with the dominant-negative mutant Akt (dnAkt). The lung adenocarcinoma cells bearing the K-ras gene mutation (A549 and H820) showed consistently higher levels of cell motilities than those without the mutation (TKB6 and TKB14), and the motility of A549 and H820 cells were significantly inhibited by dnAkt transfection. These results suggest that the K-ras gene mutation could enhance the motility of neoplastic cells through a pathway involving PI3K-Akt. Actually, among the surgically resected lung tumors, the adenocarcinomas with the K-ras gene mutation tended to show a higher frequency and intensity of immunoreactivity for phosphorylated Akt (p-ser473Akt) than those without the mutation, supporting the in vitro observation that the mutated K-ras can activate the PI3K-Akt pathway. Immunoreactivity for p-ser473Akt was also seen in the pre-malignant and early lesions at a frequency similar to that in the advanced lung adenocarcinomas,. No correlation was seen between p-ser473Akt immunoreactivity and lymphatic/organ metastasis or prognosis. These results taken together suggest that the K-ras-Akt pathway might facilitate the motility of neoplastic cells during the early period of carcinogenesis in lung adenocarcinomas, and may contribute to their non-invasive expansion along the alveolar septa, rather than invasion or metastasis.

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.

Cancer stem cell: Implications in cancer biology and therapy with special reference to lung cancer

The cancer stem cell (CSC) theory is currently central to the field of cancer research, because it is not only a matter of academic interest but also crucial in cancer therapy. CSCs share a variety of biological properties with normal somatic stem cells in terms of self-renewal, the propagation of differentiated progeny, the expression of specific cell markers and stem cell genes, and the utilization of common signaling pathways and the stem cell niche. However, CSCs differ from normal stem cells in their tumorigenic activity. Thus, CSCs are also termed cancer initiating cells. In this paper, we briefly review hitherto described study results and refer to some excellent review articles to understand the basic properties of CSCs. In addition, we focus upon CSCs of lung cancers, since lung cancer is still increasing in incidence worldwide and remains the leading cause of cancer deaths. Understanding the properties of, and exploring cell markers and signaling pathways specific to, CSCs of lung cancers, will lead to progress in therapy, intervention, and improvement of the prognosis of patients with lung cancer. In the near future, the evaluation of CSCs may be a routine part of practical diagnostic pathology.

Dimethylarsinic acid, a main metabolite of inorganic arsenics, has tumorigenicity and progression effects in the pulmonary tumors of A/J mice

The pulmonary tumorigenicity of dimethylarsinic acid (DMAA), a main metabolite of inorganic arsenics, was examined in A/J mice fed with drinking water containing DMAA for 25 and 50 weeks. Mice fed with 400 ppm DMAA for 50 weeks produced more pulmonary tumors than untreated mice (mean number per animal 1.36 versus 0.50; P < 0.05). Histological examination revealed that the number of mice which bore adenocarcinomas or papillary adenomas correlated with the concentration of DMAA given (untreated versus 400 ppm; P = 0.002), suggesting that DMAA could promote tumorigenic processes. These results are consistent with the epidemiological studies on the pulmonary carcinogenesis of arsenics and suggest that DMAA alone can act as a carcinogen in mice.

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.

Distinct characteristics of pleuroparenchymal fibroelastosis with usual interstitial pneumonia compared with idiopathic pulmonary fibrosis.

BACKGROUND Pleuroparenchymal fibroelastosis (PPFE) is a rare form of interstitial pneumonia and sometimes coexists with a histologic usual interstitial pneumonia (UIP) pattern. This study aimed to describe the distinct clinical features of PPFE with UIP pattern compared with idiopathic pulmonary fibrosis (IPF). METHODS We conducted a retrospective review of the medical records of 110 consecutive patients with IPF with a histologic UIP pattern on surgical lung biopsy specimen. Patients meeting radiologic criteria for the diagnosis of PPFE based on high-resolution CT scan and with a histologic UIP pattern were included. RESULTS Nine of eleven patients meeting radiologic criteria for the diagnosis of PPFE were histologically confirmed as having PPFE with UIP pattern. The PPFE with UIP pattern group showed a significantly higher residual volume (1.8 L vs 1.3 L, P < .01), higher Paco2 (44.6 mm Hg vs 41.7 mm Hg, P = .04), and higher complication rate of pneumothorax and pneumomediastinum than the 99 patients with IPF/UIP. The ratio of anteroposterior to transthoracic diameter in patients with PPFE with UIP pattern was significantly lower than that in patients with IPF/UIP (P = .04). Survival time tended to be shorter in patients with PPFE with UIP pattern. CONCLUSIONS The results support the view that PPFE with UIP pattern is a disease entity distinct from IPF/UIP and may well be classified as PPFE.