CDK8 and CDK19: positive regulators of signal-induced transcription and negative regulators of Mediator complex proteins.

We have conducted a detailed transcriptomic, proteomic and phosphoproteomic analysis of CDK8 and its paralog CDK19, alternative enzymatic components of the kinase module associated with transcriptional Mediator complex and implicated in development and diseases. This analysis was performed using genetic modifications of CDK8 and CDK19, selective CDK8/19 small molecule kinase inhibitors and a potent CDK8/19 PROTAC degrader. CDK8/19 inhibition in cells exposed to serum or to agonists of NFκB or protein kinase C (PKC) reduced the induction of signal-responsive genes, indicating a pleiotropic role of Mediator kinases in signal-induced transcriptional reprogramming. CDK8/19 inhibition under basal conditions initially downregulated a small group of genes, most of which were inducible by serum or PKC stimulation. Prolonged CDK8/19 inhibition or mutagenesis upregulated a larger gene set, along with a post-transcriptional increase in the proteins comprising the core Mediator complex and its kinase module. Regulation of both RNA and protein expression required CDK8/19 kinase activities but both enzymes protected their binding partner cyclin C from proteolytic degradation in a kinase-independent manner. Analysis of isogenic cell populations expressing CDK8, CDK19 or their kinase-inactive mutants revealed that CDK8 and CDK19 have the same qualitative effects on protein phosphorylation and gene expression at the RNA and protein levels, whereas differential effects of CDK8 versus CDK19 knockouts were attributable to quantitative differences in their expression and activity rather than different functions.

CDK8 Selectively Promotes the Growth of Colon Cancer Metastases in the Liver by Regulating Gene Expression of TIMP3 and Matrix Metalloproteinases.

: Unresectable hepatic metastases of colon cancer respond poorly to existing therapies and are a major cause of colon cancer lethality. In this study, we evaluated the therapeutic viability of targeting the mediator kinase CDK8, an early clinical stage drug target, as a means to suppress metastasis of colon cancer. CDK8 was amplified or overexpressed in many colon cancers and CDK8 expression correlated with shorter patient survival. Knockdown or inhibition of CDK8 had little effect on colon cancer cell growth but suppressed metastatic growth of mouse and human colon cancer cells in the liver. This effect was due in part to inhibition of already established hepatic metastases, indicating therapeutic potential of CDK8 inhibitors in the metastatic setting. In contrast, knockdown or inhibition of CDK8 had no significant effect on the growth of tumors implanted subcutaneously, intrasplenically, or orthotopically in the cecum. CDK8 mediated colon cancer growth in the liver through downregulation of matrix metalloproteinase (MMP) inhibitor TIMP3 via TGFß/SMAD-driven expression of a TIMP3-targeting microRNA, miR-181b, along with induction of Mmp3 in murine or MMP9 in human colon cancer cells via Wnt/ß-catenin-driven transcription. These findings reveal a new mechanism for negative regulation of gene expression by CDK8 and a site-specific role for CDK8 in colon cancer hepatic metastasis. Our results indicate the utility of CDK8 inhibitors for the treatment of colon cancer metastases in the liver and suggest that CDK8 inhibitors may be considered in other therapeutic settings involving TGFß/SMAD or Wnt/ß-catenin pathway activation. SIGNIFICANCE: These findings demonstrate that inhibition of the transcription-regulating kinase CDK8 exerts a site-specific tumor-suppressive effect on colon cancer growth in the liver, representing a unique therapeutic opportunity for the treatment of advanced colon cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/23/6594/F1.large.jpg.

CDK8/19 Mediator kinases potentiate induction of transcription by NFκB.

The nuclear factor-κB (NFκB) family of transcription factors has been implicated in inflammatory disorders, viral infections, and cancer. Most of the drugs that inhibit NFκB show significant side effects, possibly due to sustained NFκB suppression. Drugs affecting induced, but not basal, NFκB activity may have the potential to provide therapeutic benefit without associated toxicity. NFκB activation by stress-inducible cell cycle inhibitor p21 was shown to be mediated by a p21-stimulated transcription-regulating kinase CDK8. CDK8 and its paralog CDK19, associated with the transcriptional Mediator complex, act as coregulators of several transcription factors implicated in cancer; CDK8/19 inhibitors are entering clinical development. Here we show that CDK8/19 inhibition by different small-molecule kinase inhibitors or shRNAs suppresses the elongation of NFκB-induced transcription when such transcription is activated by p21-independent canonical inducers, such as TNFα. On NFκB activation, CDK8/19 are corecruited with NFκB to the promoters of the responsive genes. Inhibition of CDK8/19 kinase activity suppresses the RNA polymerase II C-terminal domain phosphorylation required for transcriptional elongation, in a gene-specific manner. Genes coregulated by CDK8/19 and NFκB include IL8, CXCL1, and CXCL2, which encode tumor-promoting proinflammatory cytokines. Although it suppressed newly induced NFκB-driven transcription, CDK8/19 inhibition in most cases had no effect on the basal expression of NFκB-regulated genes or promoters; the same selective regulation of newly induced transcription was observed with other transcription signals potentiated by CDK8/19. This selective role of CDK8/19 identifies these kinases as mediators of transcriptional reprogramming, a key aspect of development and differentiation as well as pathological processes.

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 ERα 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.

The adapter protein CD2AP binds to p53 protein in the cytoplasm and can discriminate its polymorphic variants P72R.

Proline-rich motifs are widely distributed in eukaryotic proteomes and are usually involved in the assembly of functional complexes through interaction with specific binding modules. The tumour-suppressor p53 protein presents a proline-rich region that is crucial for regulating apoptosis by connecting the p53 with a complex protein network. In humans, a common polymorphism determines the identity of residue 72, either proline or arginine, and affects the features of the motifs present in the polyproline domain. The two isoforms have different biochemical properties and markedly influence cancer onset and progression. In this article, we analyse the binding of the p53 proline-rich region with a pool of selected polyproline binding domains (i.e. SH3 and WW), and we present the first demonstration that the purified SH3 domains of the CD2AP/Cin85 protein family are able to directly bind the p53 protein, and to discriminate between the two polymorphic variants P72R.

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.

TP53 codon 72 polymorphism affects accumulation of mtDNA damage in human cells.

Human TP53 gene is characterised by a polymorphism at codon 72 leading to an Arginine-to-Proline (R/P) substitution. The two resulting p53 isoforms have a different subcellular localisation after stress (more nuclear or more mitochondrial for the P or R isoform, respectively). p53P72 variant is more efficient than p53R72 in inducing the expression of genes involved in nuclear DNA repair. Since p53 is involved also in mitochondrial DNA (mtDNA) maintenance, we wondered whether these p53 isoforms are associated with different accumulation of mtDNA damage. We observed that cells bearing p53R72 accumulate lower amount of mtDNA damage upon rotenone stress with respect to cells bearing p53P72, and that p53R72 co-localises with polymerase gamma more than p53P72. We also analysed the in vivo accumulation of heteroplasmy in a 300 bp fragment of mtDNA D-loop of 425 aged subjects. We observed that subjects with heteroplasmy higher than 5% are significantly less than expected in the p53R72/R72 group. On the whole, these data suggest that the polymorphism of TP53 at codon 72 affects the accumulation of mtDNA mutations, likely through the different ability of the two p53 isoforms to bind to polymerase gamma, and may contribute to in vivo accumulation of mtDNA mutations.

Molecular remodeling of potassium channels in fibroblasts from centenarians: a marker of longevity?

Aging is a complex process resulting from, among other, dynamic non-linear interactions between genetics and environment. Centenarians are the best example of successful aging in humans, as they escaped from, or largely postponed, major age-related diseases. Ionic fluxes changes play a key role in several patho-physiological cellular processes, but their relation to human aging is largely unexplored. In the present study we have compared patch-clamp potassium (K(+)) current recordings from dermal fibroblasts (DF) obtained from young, elderly and centenarian donors. We found that in DF from elderly donors, but not from centenarians, K(+) current amplitude is significantly smaller with respect to DF from young donors. Moreover, cell membrane capacitance of DF from elderly donors is smaller with respect to young donors and centenarians. We also observed that the voltage-gated Shaker Kv1.1 channel is expressed in higher percentage of elderly's and centenarian's DF than young's, whereas the large-conductance calcium-activated K(+) (BK(Ca)) channel ß1 subunit is expressed in lower percentage of centenarian's DF than in elderly's and young's. The maintenance of "young" K(+) currents and the peculiar age-related remodeling of K(+) channel subtypes in centenarian's DF is likely associated with successful aging and might provide a predictive marker of longevity.

Structural characterization of p53 isoforms due to the polymorphism at codon 72 by mass spectrometry and circular dichroism.

A common polymorphism at codon 72 of human TP53 gene determines a proline to arginine aminoacidic substitution within the proline-rich domain of p53 protein. The two resulting isoforms (p53P(72) and p53R(72)) are different from a biochemical and biological point of view and many reports suggest that they can modulate individual cancer susceptibility and overall survival. In the attempt to explain the observed biological differences, we characterized the two isoforms by mass spectrometry and circular dichroism (CD) to evaluate the possible alteration in the secondary structure of p53 introduced by this polymorphism. Recombinant human p53R(72) and p53P(72) were produced by using E. coli expression system then purified by chromatography (affinity chromatography and RP-HPLC), and the whole proteins identified by HPLC-ESI-IT and MALDI-TOF analysis. A bottom-up approach, using both MALDI-TOF and HPLC-ESI-QTOF analysis, was then adopted to obtain the sequence information on the two p53 isoforms. To this purpose, peptide maps were obtained by trypsin proteolysis on the two p53 isoforms. The two isoforms proteolytic digests were separated by LC and subsequent mass spectrometry analysis of both entire and fragmented peptides was performed. In particular, precursor peptide ions obtained by ESI were subjected to collision by the triple quadrupole and TOF separation, allowing us to determine the isoforms aminoacidic peptide sequence by peptide ladder sequencing. Because of the presence of arginine, a selective trypsin proteolytic cleavage at R(72), giving rise to two selective shorter peptides, occurred in p53R(72), but was missing in the case of p53P(72) trypsin digest, in which an uncleaved longer peptide was instead identified. Upon primary structure confirmation, the two p53 isoforms were studied by CD in order to investigate the experimental variables, which affect ordered secondary structure adoption. CD analysis indicated that the two isoforms are not structurally different, thus allowing us to exclude that the observed biological differences can be due to a different conformation of the two isoforms introduced by this polymorphism. Furthermore, these studies establish a mass spectrometry method to identify the two isoforms that can be useful for future interactome studies and cancer drug discovery.

The frequency of Klotho KL-VS polymorphism in a large Italian population, from young subjects to centenarians, suggests the presence of specific time windows for its effect.

In mice a defect of Klotho gene expression results in multiple aging-like phenotypes including short lifespan, osteoporosis and atherosclerosis, while its over-expression suppresses aging and extends lifespan. Contrasting data have been reported as far as the importance of the functional variant of Klotho termed "KL-VS" on human longevity, depending on the average age of the old subjects that were compared with young controls. We therefore performed a study on a large Italian population sample including people from very young to very old age (centenarians). A total of 1,089 (669 women and 420 men) unrelated individuals from 19 to 109 years, born and residing in northern and central Italy, were subdivided into three age classes defined on the basis of the survival curve constructed using Italian demographic mortality data, and genotyped for the KL-VS allele. We found a significant increase of the heterozygous Klotho genotype in the class of elderly people compared to young controls. On the contrary, no difference was present between centenarians and young controls. Such a non monotonic trajectory is evident only when a large, comprehensive age range is investigated, and is compatible with the hypothesis that this KL-VS heterozygous genotype is favorable for survival in old people, its beneficial effect decreasing thereafter, and becoming no more evident at the extreme ages. Such unusual age-related changes in the Klotho KL-VS genotype frequency is compatible with the hypothesis that alleles and genotypes involved in aging and longevity may exert their biological effect at specific time windows.

Different types of cell death in organismal aging and longevity: state of the art and possible systems biology approach.

Cell death is as important as cell proliferation for cell turn-over, and susceptibility to cell death is affected by a number of parameters that change with time. A time-dependent derangement of such a crucial process, or even the simple cell loss mediated by cell death impinges upon aging and longevity. In this review we will discuss how cell death phenomena are modulated during aging and what is their possible role in the aging process. We will focus on apoptosis and autophagy, which affect mostly proliferating and post-mitotic cells, respectively, and on mitochondrial degradation in long living cells. Since the "decisional process" that leads the cell to death is very complex, we will also discuss the possibility to address this topic with a systems biology approach.

Association studies on human mitochondrial DNA: methodological aspects and results in the most common age-related diseases.

Mitochondrial DNA (mtDNA) follows direct maternal inheritance and, as such, can be used in phylogenetic studies to determine a human lineage tree. The presence of common polymorphisms allows a classification of mtDNA in haplogroups and sub-haplogroups, according to the branch they belong to. Thanks to the rapidly growing number of mtDNA sequences available, this classification is being corrected and redefined to be more accurate. In parallel with this process, several studies are trying to identify an association between common mtDNA polymorphisms and common complex traits, as hypothesized by the common disease-common variant theory. Here we review the associations already reported with the main age-related complex diseases and we identify the critical points (sample size, size of the recruiting area, careful matching between cases and controls regarding geographical origin and ethnicity, data quality checking) to be taken in account in planning such studies. On the whole, this research area is opening a new perspective as an important component of "mitochondrial medicine", capable of identifying new molecular targets for the diagnosis, prevention and treatment of common complex diseases.

Complexity of anti-immunosenescence strategies in humans.

Immunosenescence is characterized by three main aspects: (i) the shrinkage of the T cell repertoire and the accumulation of oligoclonal expansions (megaclones) of memory/effector cells directed toward ubiquitary infectious agents; (ii) the involution of the thymus and the exhaustion of naïve T cells; and (iii) a chronic inflammatory status called inflamm-aging. We present here possible strategies to counteract these main aspects of immunosenescence in humans with particular attention to the reduction of antigenic load by pathogens, such as CMV, and the normalization of intestinal microflora, the possible utilization of IL-7 to reverse thymic involution, the purging of megaclones, the forced expression of CD28 on T lymphocytes, the reduction of inflamm-aging and the administration of nutrients such as vitamin D. Possible drawbacks of all these strategies are discussed. Finally, the complexity of a rejuvenation approach is stressed, with particular attention to the inhibitory role played by the "old microenvironment" on the performance of progenitor cells, the best candidate to counteract the decline in regenerative potential characteristic of organs and tissues from old organisms.

Age-dependent effects of in vitro radiofrequency exposure (mobile phone) on CD95+ T helper human lymphocytes.

Recent studies on "nonthermal" effects of mobile phone radiofrequency (RF) suggest that RF can interact with cellular functions and molecular pathways. To study the possible RF effects on human lymphocyte activation, we analyzed CD25, CD95, CD28 molecules in unstimulated and stimulated CD4+ e CD8+ T cells in vitro. Peripheral blood mononuclear cells (PBMCs) from young and elderly donors were exposed or sham-exposed to RF (1,800 MHz, Specific Absorption Rate 2 W/kg) with or without mitogenic stimulation. No significant changes in the percentage of these cell subsets were found between exposed and sham-exposed lymphocytes in both young and elderly donors. Nevertheless, after RF exposure we observed a slight, but significant, downregulation of CD95 expression in stimulated CD4+ T lymphocytes from elderly, but not from young donors. This age-related result is noteworthy given the importance of a such molecule in regulation of the immune response.

Abnormal splicing of ABCA1 pre-mRNA in Tangier disease due to a IVS2 +5G>C mutation in ABCA1 gene.

Two point mutations of ABCA1 gene were found in a patient with Tangier disease (TD): i) G>C in intron 2 (IVS2 +5G>C) and ii) c.844 C>T in exon 9 (R282X). The IVS2 +5G>C mutation was also found in the brother of another deceased TD patient, but not in 78 controls and 33 subjects with low HDL. The IVS2 +5G>C mutation disrupts ABCA1 pre-mRNA splicing in fibroblasts, leading to three abnormal mRNAs: devoid of exon 2 (Ex2-/mRNA), exon 4 (Ex4-/mRNA), or both these exons (Ex2-/Ex4-/mRNA), each containing a translation initiation site. These mRNAs are expected either not to be translated or generate short peptides. To investigate the in vitro effect of IVS2 +5G>C mutation, we constructed two ABCA1 minigenes encompassing Ex1-Ex3 region, one with wild-type (WTgene) and the other with mutant (MTgene) intron 2. These minigenes were transfected into COS1 and NIH3T3, two cell lines with a different ABCA1 gene expression. In COS1 cells, WTgene pre-mRNA was spliced correctly, while the splicing of MTgene pre-mRNA resulted in Ex2-/mRNA. In NIH3T3, no splicing of MTgene pre-mRNA was observed, whereas WTgene pre-mRNA was spliced correctly. These results stress the complexity of ABCA1 pre-mRNA splicing in the presence of splice site mutations.