Amplification of AKT2 in human pancreatic cells and inhibition of AKT2 expression and tumorigenicity by antisense RNA.

We previously demonstrated that the putative oncogene AKT2 is amplified and overexpressed in some human ovarian carcinomas. We have now identified amplification of AKT2 in approximately 10% of pancreatic carcinomas (2 of 18 cell lines and 1 of 10 primary tumor specimens). The two cell lines with altered AKT2 (PANC1 and ASPC1) exhibited 30-fold and 50-fold amplification of AKT2, respectively, and highly elevated levels of AKT2 RNA and protein. PANC1 cells were transfected with antisense AKT2, and several clones were established after G418 selection. The expression of AKT2 protein in these clones was greatly decreased by the antisense RNA. Furthermore, tumorigenicity in nude mice was markedly reduced in PANC1 cells expressing antisense AKT2 RNA. To examine further whether overexpression of AKT2 plays a significant role in pancreatic tumorigenesis, PANC1 cells and ASPC1 cells, as well as pancreatic carcinoma cells that do not overexpress AKT2 (COLO 357), were transfected with antisense AKT2, and their growth and invasiveness were characterized by a rat tracheal xenotransplant assay. ASPC1 and PANC1 cells expressing antisense AKT2 RNA remained confined to the tracheal lumen, whereas the respective parental cells invaded the tracheal wall. In contrast, no difference was seen in the growth pattern between parental and antisense-treated COLO 357 cells. These data suggest that overexpression of AKT2 contributes to the malignant phenotype of a subset of human ductal pancreatic cancers. IMAGES:

我们此前的研究已证实，推定致癌基因AKT2在部分人类卵巢癌中发生扩增并过度表达。本研究进一步在约10%的胰腺癌样本中检测到AKT2扩增：18株细胞系与10例原发性肿瘤标本中，分别有2株和1例出现该扩增。AKT2异常的两株细胞系（PANC1与ASPC1）的AKT2扩增倍数分别为30倍和50倍，其AKT2 RNA与蛋白水平均显著升高。我们将反义AKT2转染至PANC1细胞中，经G418筛选后成功构建了多个克隆株，上述克隆株中的AKT2蛋白表达水平因反义RNA（antisense RNA）的作用大幅下调。此外，表达反义RNA的PANC1细胞在裸鼠体内的致瘤性显著降低。为进一步明确AKT2过度表达在胰腺癌发生过程中的关键作用，我们将反义AKT2分别转染至PANC1细胞、ASPC1细胞以及不发生AKT2过度表达的胰腺癌细胞（COLO 357）中，并通过大鼠气管异种移植实验（rat tracheal xenotransplant assay）对各组细胞的生长与侵袭能力进行了表征。结果显示，表达反义RNA的ASPC1与PANC1细胞仍被限制于气管腔内，而对应的亲本细胞则可侵袭气管管壁；与之相反，亲本与反义RNA处理后的COLO 357细胞的生长模式未出现明显差异。上述实验数据表明，AKT2过度表达可促进部分人类导管型胰腺癌的恶性表型形成。图像：