Bommi-Reddy Archana, Almeciga Ingrid, Sawyer Jacqueline, Geisen Christoph, Li Wenliang, Harlow Ed, Kaelin William G, Grueneberg Dorre A
Department of Medical Oncology, The Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02115, USA.
Proc Natl Acad Sci U S A. 2008 Oct 28;105(43):16484-9. doi: 10.1073/pnas.0806574105. Epub 2008 Oct 23.
Clear cell renal carcinomas are the most common form of kidney cancer and frequently are linked to biallelic inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. The VHL gene product, pVHL, has multiple functions including directing the polyubiquitylation of the HIF transcription factor. We screened 100 shRNA vectors, directed against 88 kinases, for their ability to inhibit the viability of VHL-/- renal carcinoma cells preferentially compared with isogenic cells in which pVHL function was restored. shRNAs for "hits" identified in the primary screen were interrogated in secondary screens that included shRNA titration studies. Multiple shRNAs against CDK6, MET, and MAP2K1 (also known as MEK1) preferentially inhibited the viability of 786-O and RCC4 VHL-/- cells compared with their wild-type pVHL-reconstituted counterparts. The sensitivity of pVHL-proficient cells to these shRNAs was not restored upon HIF activation, suggesting that loss of an hypoxia-inducible factor (HIF)-independent pVHL function formed the basis for selectivity. A small-molecule Cdk4/6 inhibitor displayed enhanced activity against VHL-/- renal carcinoma cells, suggesting that in some cases hits from shRNA screens such as described here might translate into therapeutic targets.
透明细胞肾细胞癌是最常见的肾癌形式，并且经常与冯·希佩尔-林道（VHL）肿瘤抑制基因的双等位基因失活有关。VHL基因产物pVHL具有多种功能，包括指导HIF转录因子的多聚泛素化。我们筛选了针对88种激酶的100个shRNA载体，以评估它们优先抑制VHL基因敲除肾癌细胞活力的能力，并与恢复了pVHL功能的同基因细胞进行比较。在包括shRNA滴定研究的二次筛选中，对在初次筛选中鉴定出的“命中”shRNA进行了检测。与野生型pVHL重构的对应细胞相比，多种针对CDK6、MET和MAP2K1（也称为MEK1）的shRNA优先抑制了786-O和RCC4 VHL基因敲除细胞的活力。在HIF激活后，pVHL功能正常的细胞对这些shRNA的敏感性并未恢复，这表明缺氧诱导因子（HIF）非依赖性pVHL功能的丧失构成了选择性的基础。一种小分子Cdk4/6抑制剂对VHL基因敲除肾癌细胞显示出增强的活性，这表明在某些情况下，此处所述的shRNA筛选中的“命中”可能转化为治疗靶点。
