암유전자인 Ras의 암유발기작규명

Abstract

Ras oncogenes는 human cancers의 많은 부부에서 돌연변이 되어 있고, 다른 cancer에서는 다양한 메커니즘을 통해 Ras pathway는 활성화 되어 있다. Ras가 유도하는 종양의 진행 메커니즘은 충분히 ?P혀지지 않았다. 이번 연구에서 NLEF level은 human fibroblast GM00637 cells에서 oncogenic H-Ras발현에 의해 상당히 증가되었다. NLEF protein level는 Ras-dominant active cancer cells인 A549, H1299, H460, Sw480, DLD1, Capan-1, and Panc-1 cells에서 감소되어 있고 H-RasN17는 이러한 cell들의 NLEF발현은 억제시킨다. 게다가 ERK and PI3K 억제제의 처리는 Ras-dominant active cancer cells에서 NLEF발현은 상당히 억제시컸다. 이러한 결과는 NLEF는 oncogenic H-Ras의 새로운 downstream target molecule이고 H-Ras-mediated 에 의해 증가되는 ERK and PI3K 활성는 NLEF발현이 필요하였다. Oncogenic-H-Ras-induced NLEF 발현의 생물학적 기능을 연구하기 위해서 우리는oncogenic Ras-mediated 종양발생의 증가에 NLEF가 관련되어 있는지를 조사하였다. NLEF의 이소성 발현이 GM00637 cells 에서 ERK1/2 and Akt의 serum-stimulated phosphorylation이 증가 시킴을 찾아냈다. Human colon cancer cells인 HCT116, DLD1, and SW480 cells에서 NLEF shRNA 처리는 몇 개의 cell cycle progression proteins인 p-histone-H3, p-Rb, and p-cdc2의 phosphorylation의 억제의 원인이 되었다. 나는 NLEF 발현이 부드러운 배지에서 집단형성을 증가 시킴을 이끌었다. 더욱이, NLEF shRNA를 형질 도입 시킨 A569, H460, H1299, HCT116, and DLD1 cells은 anchorage-independent 성장이 상당히 감소함을 보여주었다. 본 연구에서는 huamn cancer cells의 세포 증식 능력은 NLEF shRNA를 형질 도입 시킴으로서 현저히 억제됨을 보여주고 있다. 이러한 결과들로 NLEF의 upregulation은 Ras에 의해 유도된 매개종양변환발생의 중요한 역할을 수행할 것으로 생각한다.|Ras oncogenes are mutated in a large proportion of human cancers, and Ras pathway are activated by a variety of other mechanisms in may other cancers. The mechanisms by which Ras induces tumor progression is , however, not fully elucidated. In this report, I found that the levels of nucleotide exchange factor (NLEF) protein are significantly increased in oncogenic H-Ras expression in human fibroblast GM00637 cells. The levels of NLEF protein was decreased in Ras-dominant active cancer cells, including A549, H1299, H460, Sw480, DLD1, Capan-1, and Panc-1 cells, and dominant negative form H-RasN17 suppressed NLEF expression in these cells. In addition, treatment of ERK and PI3K inhibitors led to significant suppression of NLEF expression in those Ras-dominant active cancer cells. These results suggest that NLEF is a novel downstream target molecule of oncogenic H-Ras, and H-Ras-mediated increase in ERK and PI3K activity is required for NLEF expression. To investigate the biological function of oncogenic-H-Ras-induced NLEF expression, I examined whether NLEF is contributed to oncogenic Ras-mediated increase in cancer progression. I found that ectopic expression of NLEF increased serum-stimulated phosphorylation of ERK1/2 and Akt in HGM00637 cells. NLEF shRNA treatment caused suppression of phosphorylation of several cell cycle progression proteins, such as p-histone-H3, p-Rb, and p-cdc2 in human colon cancer cells, HCT116, DLD1, and SW480 cells. I further showed that expression of NLEF led to increase in colony formation in soft agar. Furthermore, NLEF shRNA-transfected A569, H460, H1299, HCT116, and DLD1 cells exhibited significant reduction of anchorage-independent growth rate. Finally, our results showed that the ability of cellular proliferation of huamn cancer ccells was significantly suppressed by transfection of NLEF shRNA. These results suggest that the upregulation of NLEF induced by oncogenic Ras may be an important role for oncogenic Ras-mediated neoplastic transformation.