Inhibition of CDK8 mediator kinase suppresses estrogen dependent transcription and the growth of estrogen receptor positive breast cancer

Hormone therapy targeting estrogen receptor (ER) is the principal treatment for ER-positive breast cancers. However, many cancers develop resistance to hormone therapy while retaining ER expression. Identifying new druggable mediators of ER function can help to increase the efficacy of ER-targeting drugs. Cyclin-dependent kinase 8 (CDK8) is a Mediator complex-associated transcriptional regulator with oncogenic activities. Expression of CDK8, its paralog CDK19 and their binding partner Cyclin C are negative prognostic markers in breast cancer. Meta-analysis of transcriptome databases revealed an inverse correlation between CDK8 and ERalpha expression, suggesting that CDK8 could be functionally associated with ER. We have found that CDK8 inhibition by CDK8/19-selective small-molecule kinase inhibitors, by shRNA knockdown or by CRISPR/CAS9 knockout suppresses estrogen-induced transcription in ER-positive breast cancer cells; this effect was exerted downstream of ER. Estrogen addition stimulated the binding of CDK8 to the ER-responsive GREB1 gene promoter and CDK8/19 inhibition reduced estrogen-stimulated association of an elongation-competent phosphorylated form of RNA Polymerase II with GREB1. CDK8/19 inhibitors abrogated the mitogenic effect of estrogen on ER-positive cells and potentiated the growth-inhibitory effects of ER antagonist fulvestrant. Treatment of estrogen-deprived ER-positive breast cancer cells with CDK8/19 inhibitors strongly impeded the development of estrogen independence. In vivo treatment with a CDK8/19 inhibitor Senexin B suppressed tumor growth and augmented the effects of fulvestrant in ER-positive breast cancer xenografts. These results identify CDK8 as a novel downstream mediator of ER and suggest the utility of CDK8 inhibitors for ER-positive breast cancer therapy

Oncogenic senescence: a multi-functional perspective

Cellular senescence is defined as an irreversible growth arrest with the acquisition of a distinctive secretome. The growth arrest is a potent anticancer mechanism whereas the secretome facilitates wound healing, tissue repair, and development. The senescence response has also become increasingly recognized as an important contributor to aging and age-related diseases, including cancer. Although oncogenic mutations are capable of inducing a beneficial senescence response that prevents the growth of premalignant cells and promotes cancer immune-surveillance, the secretome of senescent cells also includes factors with pro-tumorigenic properties. On June 23rd and 24th, 2016, the Division of Cancer Biology of the National Cancer Institute sponsored a workshop to discuss the complex role of cellular senescence in tumorigenesis with the goal to define the major challenges and opportunities within this important field of cancer research. Additionally, it was noted how the development of novel tools and technologies are required to accelerate research into a mechanistic understanding of senescent cells in carcinogenesis in order to overcome the current limitations in this exciting, yet ill-defined area

The C-terminal extension of Lsm1 is able to bind RNA by itself and the C-terminal most 8 residues are important for such binding.

<p><i>A</i>, Synthetic untagged wild type Lsm1 C-terminal extension peptide (10μM) or increasing concentrations (0.5μM, 2μM and 10μM) of synthetic 6xHis-tagged C-terminal extension peptides corresponding to <i>LSM1</i>, <i>lsm1-39</i>, <i>lsm1-40</i> or <i>lsm1-41</i> were incubated with radiolabeled <i>in vitro</i> transcript carrying the 3’-most 43 residues of the yeast <i>MFA2</i> mRNA and then subjected to pull down using the Ni-NTA matrix. After washing, the co-precipitated RNA was run alongside untreated RNA (10% of total amount used for the binding) and then visualized by denaturing PAGE and phosphorimaging. <i>B</i>, A plot of the percentage of RNA bound vs the concentration of the peptide used is shown. Plotted values are mean ± SD from three independent trials. <i>C</i>, <i>Top panel</i>, bovine serum albumin (BSA) or synthetic C-terminal segment peptides (2 nmols each) corresponding to <i>LSM1</i>, <i>lsm1-39</i> or <i>lsm1-41</i> were incubated with radiolabeled <i>in vitro</i> transcript carrying the 3’-most 43 residues of the yeast <i>PGK1</i> mRNA, UV crosslinked, treated with ribonuclease and then visualized by SDS-PAGE and phosphorimaging. <i>Bottom panel</i>, Similar UV crosslinking analysis carried out with synthetic wild type Lsm1 C-terminal extension peptide (untagged or 6xHis-tagged) or BSA is shown.</p

Mutagenic Analysis of the C-Terminal Extension of Lsm1 - Fig 1

<p><i>A</i>, Alignment of the C-terminal most 55 residues of <i>S</i>. <i>cerevisiae</i> and <i>P</i>. <i>stipitis</i> Lsm1 proteins. Numbers of the first and last residues are indicated on the left and right of the sequence. <i>B</i>, Three dimensional structure of Lsm1 subunit in the Lsm1-7 complex (PDB ID: 4C92; Sharif and Conti, 2013) is shown as ribbon diagram. Parts of the ribbon corresponding to some of the C-terminal extension residues targeted in the <i>lsm1</i> mutants are shown in red. For these residues the orientation of the side chains is also shown. Rest of the C-terminal extension is shown in green. The N-terminal extension and the Sm domain are shown in gray.</p

Residue changes in the various <i>lsm1</i> alleles generated in this study.

<p>Residue changes in the various <i>lsm1</i> alleles generated in this study.</p

Effect of the C-terminal extension mutations of the <i>lsm1-39</i>, <i>lsm1-40</i> and <i>lsm1-41</i> alleles on mRNA decay.

<p>RNA isolated from <i>lsm1Δ</i> cells expressing wild type or various mutant alleles of <i>LSM1</i> (left panel) or from <i>lsm1-27</i> cells expressing wild type or various mutant versions of the C-terminal extension peptide of Lsm1 from multi copy 2μ vectors (right panel) were subjected to Northern analysis to reveal the <i>MFA2pG</i> mRNA and the poly(G) fragment. Poly(G) fragment levels were approximated and presented as described in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158876#pone.0158876.g002" target="_blank">Fig 2</a>.</p

Suppression of the mRNA decay phenotype of the <i>lsm1-27</i> mutant (C-terminal truncation mutant of <i>LSM1</i>) in <i>trans</i> by the various mutant versions of the C-terminal extension peptide, upon over expression.

<p>RNA isolated from <i>lsm1-27</i> cells expressing wild type or various mutant versions of the C-terminal extension peptide of Lsm1 from multi copy <i>2μ</i> vectors were subjected to Northern analysis to reveal the <i>MFA2pG</i> mRNA and the poly(G) fragments. The fractional contribution of the poly(G) fragments to the total signal (total = full-length mRNA + trimmed and normal poly(G) fragments) was approximated for each sample via quantitation using the phosphorimager and normalized to the value obtained for <i>lsm1-27</i> cells expressing wild type C-terminal extension peptide. Samples with approximate poly(G) fragment levels that are ≥ 80%, 60% to 80% and <60% of the value for cells expressing wild type peptide are marked with +++, ++ and + below the corresponding lanes in the figure.</p

Cyclin-dependent kinase 8 mediates chemotherapy-induced tumor-promoting paracrine activities

Conventional chemotherapy not only kills tumor cells but also changes gene expression in treatment-damaged tissues, inducing production of multiple tumor-supporting secreted factors. This secretory phenotype was found here to be mediated in part by a damage-inducible cell-cycle inhibitor p21 (CDKN1A). We developed small-molecule compounds that inhibit damage-induced transcription downstream of p21. These compounds were identified as selective inhibitors of a transcription-regulating kinase CDK8 and its isoform CDK19. Remarkably, p21 was found to bind to CDK8 and stimulate its kinase activity. p21 and CDK8 also cooperate in the formation of internucleolar bodies, where both proteins accumulate. A CDK8 inhibitor suppresses damage-induced tumor-promoting paracrine activities of tumor cells and normal fibroblasts and reverses the increase in tumor engraftment and serum mitogenic activity in mice pretreated with a chemotherapeutic drug. The inhibitor also increases the efficacy of chemotherapy against xenografts formed by tumor cell/fibroblast mixtures. Microarray data analysis revealed striking correlations between CDK8 expression and poor survival in breast and ovarian cancers. CDK8 inhibition offers a promising approach to increasing the efficacy of cancer chemotherapy