[show abstract][hide abstract] ABSTRACT: 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.
American Journal Of Pathology 09/2009; 175(2):867-81. · 4.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Small cell lung cancer (SCLC) exhibits insulin-like growth factor-dependent growth. SCLC is the most aggressive among known in vivo lung cancers, whereas in vitro growth of SCLC is paradoxically slow as compared with that of non-SCLC (NSCLC). In this study, we demonstrate that SCLC cells overexpress insulin-like growth factor binding protein (IGFBP)-2 via NeuroD, a neuroendocrine cell-specific transcription factor. Chromatin immunoprecipitation, electrophoretic mobility shift, and IGFBP-2 promoter assays all revealed that NeuroD binds to the E-box in the 5'-untranslated region of IGFBP-2. A NeuroD transgene in both airway epithelial and NSCLC cells up-regulated the transcription of IGFBP-2 and retarded cell growth. Recombinant IGFBP-2 repressed the growth of both airway epithelial and NSCLC cells in a dose-dependent manner. A NeuroD-specific small interfering RNA repressed IGFBP-2 expression in SCLC, and neutralization of IGFBP-2 and an IGFBP-2-specific small interfering RNA increased SCLC cell growth. Pathological samples of SCLC also expressed IGFBP-2 abundantly, as compared with NSCLC, and showed only rare (8%) IGFBP-2 promoter methylation, whereas the IGFBP-2 promoter was methylated in 71% of adenocarcinomas and 29% of squamous cell carcinomas. These findings suggest that 1) SCLC has an IGFBP-2 overexpression mechanism distinct from NSCLC, 2) secreted IGFBP-2 contributes to the slow growth of SCLC in vitro, and 3) the epigenetic alterations in the IGFBP-2 promoter contribute to the striking differences in IGFBP-2 expression between SCLC and NSCLC in vivo.
American Journal Of Pathology 09/2009; 175(3):976-87. · 4.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Gain-of-function point mutations in K-ras affect early events in pulmonary bronchioloalveolar carcinoma. We investigated altered mRNA expression on K-Ras activation in human peripheral lung epithelial cells (HPL1A) using oligonucleotide microarrays. Mutated K-Ras stably expressed in HPL1A accelerated cell growth and induced the expression of insulin-like growth factor (IGF)-binding protein (IGFBP)-4 and IGFBP-2, which modulate cell growth via IGF. Other lung epithelial cell lines (NHBE and HPL1D) revealed the same phenomena as HPL1A by mutated K-ras transgene. Lung cancer cell growth was also accelerated by mutated K-ras gene transduction, whereas IGFBP-4/2 induction was weaker compared with mutated K-Ras-expressing lung epithelial cells. To understand the differences in IGFBP-4/2 inducibility via K-Ras-activated signaling between nonneoplastic lung epithelia and lung carcinoma, we addressed the mechanisms of IGFBP-4/2 transcriptional activation. Our results revealed that Egr-1, which is induced on activation of Ras-mitogen-activated protein kinase signaling, is crucial for transactivation of IGFBP-4/2. Furthermore, IGFBP-4 and IGFBP-2 promoters were often hypermethylated in lung carcinoma, yielding low basal expression/weak induction of IGFBP-4/2. These findings suggest that continuous K-Ras activation accelerates cell growth and evokes a feedback system through IGFBP-4/2 to prevent excessive growth. Moreover, this growth regulation is disrupted in lung cancers because of promoter hypermethylation of IGFBP-4/2 genes.
American Journal Of Pathology 12/2006; 169(5):1550-66. · 4.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
American Journal Of Pathology 02/2004; 164(1):91-100. · 4.52 Impact Factor