Basic research on the growth factor and cell adhesion factor in a mechanism of invasion and metastasis of pancreatic cancer

[TGF-β信号与RhoB蛋白的功能]我们通过TGF-β刺激培养细胞诱导上皮间充质转化（epithelial mesenchymal transition， EMT）。此外，我们还发现了EMT与TGF-β刺激细胞的侵袭性之间的关系。我们还发现RhoGTPases的蛋白和mRNA表达在TGF-β信号传导中受到抑制。利用野生型和突变型RhoB表达载体建立稳定克隆，分析RhoB蛋白的功能。我们在这些细胞中检测了EMT的诱导、细胞增殖、不依赖于锚定的细胞增殖、迁移和侵袭。TGF-β刺激信号通路被认为主要由Smad信号通路和MAPKs级联（ERK1/2、JNK/SAPK和p38MAPK）组成。我们发现与侵袭和转移相关的信号通路是MAPKs级联。【胰腺癌癌基因的改变与侵袭转移】我们选用K-Ras、DPC4和TGF-βRII基因突变状态不同的胰腺癌细胞系。建立了具有DNRas的稳定克隆。还建立了RhoB、MEK1/2和SMAD4的DN和CA型稳定克隆。利用这些细胞检测TGF-β刺激的侵袭转移能力。观察TGF-β刺激下EMT状态及RhoB蛋白、Cdc42、cadherin的表达。我们发现TGF-β信号通路的改变和Ras蛋白的强制激活增加了侵袭和转移。我们最终证实了K-Ras和DPC4在胰腺癌组织中的改变。

[TGF-β signal and Function of RhoB protein] We induced epithelial mesenchymal transition (EMT) in the cultured cell by TGF-β stimulation. In addition, we found the relation between EMT and invasiveness of the cells stimulated by TGF-β. We also found that the protein and mRNA expression of RhoGTPases was suppressed in TGF-β signaling. Stable clones were established by using wild-type and mutant-type of RhoB expression vectors in order to analyze RhoB protein function. We examined, in these cells, the induction of EMT, cell proliferation, anchorage-independent cell proliferation, migration and invasion. The TGF-β stimulation signal pathway is thought to be chiefly composed of Smad signal pathway and MAPKs cascade (ERK1/2, JNK/SAPK and p38MAPK). We showed that the signal pathway which related to invasion and metastasis is MAPKs cascade. [Alteration of oncogene and invasion and metastasis in pancreatic cancer] We used pancreatic cancer cell lines with the different status of K-Ras, DPC4 and to TGF-βRII gene mutation. Stable clone with DNRas was established. Stable clones with DN and CA form of RhoB, MEK1/2 and SMAD4 were also established. The ability of invasion and metastasis stimulated by TGF-β was examined by using these cells. Moreover, the state of EMT, and the expression of RhoB protein, Cdc42 and cadherin were examined in response to the TGF-β stimulation. We showed alteration of TGF-β signaling and increased invasion and metastasis by enforced activation of Ras protein. We finally confirmed alteration of K-Ras and DPC4 in pancreatic cancer tissues.