CDK8 is a colorectal cancer oncogene that regulates β-catenin activity

Abstract

Aberrant activation of the canonical Wnt/β-catenin pathway occurs in almost all colorectal cancers and contributes to their growth, invasion and survival. Although dysregulated β-catenin activity drives colon tumorigenesis, additional genetic perturbations are required to elaborate fully malignant disease. To identify genes that both modulate β-catenin activity and are essential for colon cancer cell proliferation, we conducted two loss-of-function screens in human colon cancer cells and compared genes identified in these screens with an analysis of copy-number alterations in colon cancer specimens. One of these genes, CDK8, which encodes a member of the mediator complex, is located at 13q12.13, a region of recurrent copy number gain in a substantial fraction of colon cancers. Suppression of CDK8 expression inhibited proliferation in colon cancer cells characterized by high levels of CDK8 and β-catenin hyperactivity. CDK8 kinase activity was necessary for β-catenin driven transformation and expression of several β-catenin transcriptional targets. Together these observations suggest that therapeutic interventions targeting CDK8 may confer clinical benefit in β-catenin-driven malignancies.Correspondence and Requests for materials should be addressed to W.C.H. (Email: william_hahn@dfci.harvard.edu).. The Wnt/β-catenin pathway is implicated in over 90% of colon cancers and in a fraction of other human malignancies. Loss of the tumor suppressor APC or activating CTNNB1 (β-catenin) mutations results in constitutive activity of the β-catenin-T cell factor (TCF) transcriptional complex, which drives adenoma formation 1,2 . Although mutations in TP53 or K-RAS cooperate with dysregulated β-catenin signaling to program a fully malignant phenotype 3 , these mutations are found in less than half of β-catenin-driven colon cancers 4 . NIH Public AccessTo identify oncogenes that modulate β-catenin-dependent transcription and regulate colon cancer cell proliferation, we conducted two RNAi-based loss-of-function screens. We engineered DLD1 colon cancer cells, which harbor APC deletions and depend on β-catenin for proliferation 5 , to stably express "TOPFLASH" β-catenin-luciferase and "FOPFLASH" mutant-Renilla reporter constructs 6,7 (DLD1 Rep ). Suppression of β-catenin expression in DLD1 Rep cells by three β-catenin-specific short hairpin RNAs (shRNA) markedly reduced the TOPFLASH/FOPFLASH ratio (Fig. 1a), confirming that reporter activity requires β-catenin expression. We then screened DLD1 Rep cells with a shRNA library containing 4849 shRNAs that target 1000 genes, including 95% of the human kinome 6 . We found 34 genes whose expression was necessary for β-catenin activity, including two known β-catenin regulators, CSNK1G3 8 and CSNK1E 9 ( Fig. 1b and Supplementary Table 1).In parallel, we performed an arrayed, kinase-enriched shRNA screen in another β-catenindependent colon cancer cell line, HCT116, to identify genes essential for cancer cell proliferation. We identified 166 candidate genes necessary for proliferatio...

Tumor-suppressive effects of CDK8 in endometrial cancer cells

“…Keywords: cyclin-dependent kinae 8 (CDK8), endometrial cancer, tumorigenesis, cell growth, cell migration identified as an oncoprotein, 6,7 yet the underlying mechanisms of how elevated CDK8 promotes tumorigenesis remain unclear. Second, because CDK8 is a kinase, there is considerable interest in developing cancer drugs by targeting CDK8.…”

“…Thus to further examine the notion that CDK8 inhibits the growth of endometrial cancer cells, we depleted CDK8 in AN3 CA cells by transducing the cells with pLKO.1-shRNAs, which were previously shown to specifically target CDK8. 6,7,31,36,38 Similar construct targeting GFP (sh-GFP) was used as the negative control. Transduced cells were selected by puromycin for over 2 wk, and the efficiency of CDK8 knockdown was verified by qRT-PCR and western blot.…”

“…In addition, we tested the effect of CDK8 reduction on cell cycle distribution by flow cytometry analyses, and we observed that knockdown of CDK8 evidence that links dysregulated CDK8 to a variety of human cancers in recent years. 17 For example, CDK8 gene is amplified or overexpressed in colorectal and gastric cancers, 6,[17][18][19][20][21][22][23] and overexpression of wild-type CDK8 in untransformed 3T3 cells led to anchorage-independent growth. 6 In addition, loss of the histone variant macroH2A increases the expression of CDK8 in melanoma.…”

Frequent genomic loss at chr16p13.2 is associated with poor prognosis in colorectal cancer

“…However, in the carcinomas several other regions on several chromosomes including 1, 3 and 10 were found to be significantly associated with chr16p-loss. To reduce the possibility that the associations identified were false positives, an independent sample set consisting of 128 colorectal carcinomas, copy-number profiled by 250K Sty SNP arrays in a recent study, 15 was also analyzed. In accordance with our study, we also found chr16p-loss to be a frequent alteration, 41% (52/128), in this sample set and comparison of the alterations associated with chr16p-loss in this set and our carcinoma set pointed to only a single CNA, loss of a region at chr3p22.3 (34.3-36.3 Mb).…”

“…Genomic analyses of cancer samples, by cytogenetic studies and more recently by array-based profiling, have identified recurrent alterations associated with all investigated cancer types. [7][8][9][10][11] With respect to CRC, a few CNAs have been associated with outcome, [12][13][14] some have led to the identification of cancercausing genes, 15 and others have suggested potential therapeutic strategies. 16 Over the recent years, advances in genome characterization technologies have enabled increasingly systematic efforts to characterize genomic alterations in human cancer samples.…”