Project description:RNA sequencing of HCT116 colon cancer cells following single and combined depletions of Mediator kinase module subunits CDK8, CDK19, MED12, MED13 and MED13L and the BET family protein BRD4. The results provide insight into the shared and specific functions of kinase module subunits and BRD4 in regulation of expression of transcriptional programs including genes associated with cancer acquired super-enhancers.
Project description:CDK8 Mediator kinase is amplified and overexpressed in colon cancers; elevated CDK8 expression is associated with shorter patient survival. Nevertheless, CDK8 kinase inhibitors do not generally suppress colon cancer growth. We addressed this paradox by investigating the effects of CDK8 knockdown or a CDK8 kinase inhibitor on tumor growth at primary and metastatic sites. CDK8 knockdown or inhibition had no significant effect on primary tumors but suppressed the growth of hepatic metastases in murine and human colon cancer models. The effect of CDK8 inhibition on liver metastasis is mediated by upregulation of matrix metalloproteinase (MMP) inhibitor TIMP3 and downregulation of several MMPs.
Project description:CDK8 Mediator kinase is amplified and overexpressed in colon cancers; elevated CDK8 expression is associated with shorter patient survival. Nevertheless, CDK8 kinase inhibitors do not generally suppress colon cancer growth. We addressed this paradox by investigating the effects of CDK8 knockdown or a CDK8 kinase inhibitor on tumor growth at primary and metastatic sites. CDK8 knockdown or inhibition had no significant effect on primary tumors but suppressed the growth of hepatic metastases in murine and human colon cancer models. The effect of CDK8 inhibition on liver metastasis is mediated by upregulation of matrix metalloproteinase (MMP) inhibitor TIMP3 and downregulation of several MMPs.
Project description:The NFM-NM-:B transcription factor is constitutively active in a number of hematologic and solid tumors, and many signaling pathways implicated in cancer are likely connected to NFM-NM-:B activation. A critical mediator of NFM-NM-:B activity is TGFM-NM-2-activated kinase 1 (TAK1). Here, we identify TAK1 as a novel interacting protein and direct target of fibroblast growth factor receptor 3 (FGFR3) tyrosine kinase activity. We further demonstrate that activating mutations in FGFR3 associated with both multiple myeloma and bladder cancer can modulate expression of genes which regulate NFM-NM-:B signaling, and promote both NFM-NM-:B transcriptional activity and cell adhesion in a manner dependent on TAK1 expression in both cancer cell types. Our findings suggest TAK1 as a potential therapeutic target for FGFR3-associated cancers, and other malignancies in which TAK1 contributes to constitutive NFM-NM-:B activation. A total of 12 samples of MGHU3 (Y375C) mutant FGFR3 bladder cancer cells (a kind gift from Dr. Margaret Knowles (University of Leeds, Leeds, UK)) were used for array-based gene expression analysis. 3 replicates of each condition: Control siRNA, Control siRNA + PD173074, TAK1 siRNA, and TAK1 siRNA + PD173074.
Project description:Med26 is a subunit of the Human Mediator complex. The Mediator complex is an evolutionarily conserved coregulatory complex that interacts with RNA polymerase II to regulate gene expression. In metazoa, Mediator is composed of some 30 distinct subunits. Mediator exists in multiple, functionally distinct forms that share a common core of subunits and can be distinguished by the presence or absence of a kinase module composed of Med12, Med13, Cdk8, and Cyclin C. In higher eukaryotes, a subset of Mediator complexes is associated with an additional subunit, Med26. This Med26-containing Mediator copurifies from cells with little or no kinase module, but near-stoichiometric Pol II. Evidence suggests that Med26-containing Mediator plays a key role in transcriptional activation however, the mechanism(s) by which Med26 contributes to this process are not known. To identify Med26 target genes, we used Affymetrix U133A plus 2.0 expression arrays to analyze mRNA expression in 293T cells from which Med26 had been depleted by transient transfection by each of three different siRNAs. Three different independent Med26 siRNA knockdowns were compared to a non-targeting control in triplicate, for a total of 12 samples.
Project description:The Mediator kinase module regulates eukaryotic transcription by phosphorylating transcription-related targets and by modulating the association of Mediator and RNA Polymerase II. The activity of its catalytic core, cyclin-dependent kinase 8 (CDK8), is controlled by Cyclin C and MED12, with its deregulation contributing to numerous malignancies. Here, we combine in vitro biochemistry, crosslinking coupled to mass spectrometry and in vivo studies to describe the binding location of the N-terminal segment of MED12 on the CDK8/Cyclin C complex and to gain mechanistic insights into the activation of CDK8 by MED12. Our data demonstrate that the N-terminal portion of MED12 wraps around CDK8, whereby it positions an "activation helix" close to the T-loop of CDK8 for its activation. Intriguingly, mutations in the activation helix that are frequently found in cancers do not diminish the affinity of MED12 for CDK8, yet likely alter the exact positioning of the activation helix. Furthermore, we find the transcriptome-wide gene expression changes in human cells that result from a mutation in the MED12 activation helix to correlate with deregulated genes in breast and colon cancer. Last, functional assays in presence of kinase inhibitors reveal that binding of MED12 remodels the active site of CDK8 and thereby precludes the inhibition of ternary CDK8 complexes by type-II kinase inhibitors. Taken together, our results not only allow us to propose a revised model of how CDK8 activity is regulated by MED12, but they also offer a path forward in developing small molecules that target CDK8 in its MED12-bound form.
Project description:The NFκB transcription factor is constitutively active in a number of hematologic and solid tumors, and many signaling pathways implicated in cancer are likely connected to NFκB activation. A critical mediator of NFκB activity is TGFβ-activated kinase 1 (TAK1). Here, we identify TAK1 as a novel interacting protein and direct target of fibroblast growth factor receptor 3 (FGFR3) tyrosine kinase activity. We further demonstrate that activating mutations in FGFR3 associated with both multiple myeloma and bladder cancer can modulate expression of genes which regulate NFκB signaling, and promote both NFκB transcriptional activity and cell adhesion in a manner dependent on TAK1 expression in both cancer cell types. Our findings suggest TAK1 as a potential therapeutic target for FGFR3-associated cancers, and other malignancies in which TAK1 contributes to constitutive NFκB activation.
Project description:Dysregulation of Wnt/TCF signaling is closely associated with cancers arising from the gastrointestinal tract, inlcluding colon cancer and liver cancer. The goal of this study is to understand the transcriptional programs underlying Wnt/TCF activation in gastrointestinal cancers. We examined the transcriptional responses to TCF inhibition in cultured human colon cancer cells and liver cancer cells that are characteristic of Wnt pathway activation. Human liver cancer cell line HepG2 and colon cancer cell line LS174T with or without expression of a dominant negative form of TCF4