Identifying Cancers Impacted by CDK8/19.

CDK8 and CDK19 Mediator kinases are transcriptional co-regulators implicated in several types of cancer. Small-molecule CDK8/19 inhibitors have recently entered or are entering clinical trials, starting with breast cancer and acute myeloid leukemia (AML). To identify other cancers where these novel drugs may provide benefit, we queried genomic and transcriptomic databases for potential impact of CDK8, CDK19, or their binding partner CCNC. sgRNA analysis of a panel of tumor cell lines showed that most tumor types represented in the panel, except for some central nervous system tumors, were not dependent on these genes. In contrast, analysis of clinical samples for alterations in these genes revealed a high frequency of gene amplification in two highly aggressive subtypes of prostate cancer and in some cancers of the GI tract, breast, bladder, and sarcomas. Analysis of survival correlations identified a group of cancers where CDK8 expression correlated with shorter survival (notably breast, prostate, cervical cancers, and esophageal adenocarcinoma). In some cancers (AML, melanoma, ovarian, and others), such correlations were limited to samples with a below-median tumor mutation burden. These results suggest that Mediator kinases are especially important in cancers that are driven primarily by transcriptional rather than mutational changes and warrant an investigation of their role in additional cancer types.

CDK8 mediates the dietary effects on developmental transition in Drosophila.

The complex interplay between genetic and environmental factors, such as diet and lifestyle, defines the initiation and progression of multifactorial diseases, including cancer, cardiovascular and metabolic diseases, and neurological disorders. Given that most of the studies have been performed in controlled experimental settings to ensure the consistency and reproducibility, the impacts of environmental factors, such as dietary perturbation, on the development of animals with different genotypes and the pathogenesis of these diseases remain poorly understood. By analyzing the cdk8 and cyclin C (cycC) mutant larvae in Drosophila, we have previously reported that the CDK8-CycC complex coordinately regulates lipogenesis by repressing dSREBP (sterol regulatory element-binding protein)-activated transcription and developmental timing by activating EcR (ecdysone receptor)-dependent gene expression. Here we report that dietary nutrients, particularly proteins and carbohydrates, modulate the developmental timing through the CDK8/CycC/EcR pathway. We observed that cdk8 and cycC mutants are sensitive to the levels of dietary proteins and seven amino acids (arginine, glutamine, isoleucine, leucine, methionine, threonine, and valine). Those mutants are also sensitive to dietary carbohydrates, and they are more sensitive to monosaccharides than disaccharides. These results suggest that CDK8-CycC mediates the dietary effects on lipid metabolism and developmental timing in Drosophila larvae.

Cyclin C stimulates ß-cell proliferation in rat and human pancreatic ß-cells.

Activation of pancreatic ß-cell proliferation has been proposed as an approach to replace reduced functional ß-cell mass in diabetes. Quiescent fibroblasts exit from G0 (quiescence) to G1 through pRb phosphorylation mediated by cyclin C/cdk3 complexes. Overexpression of cyclin D1, D2, D3, or cyclin E induces pancreatic ß-cell proliferation. We hypothesized that cyclin C overexpression would induce ß-cell proliferation through G0 exit, thus being a potential therapeutic target to recover functional ß-cell mass. We used isolated rat and human islets transduced with adenovirus expressing cyclin C. We measured multiple markers of proliferation: [(3)H]thymidine incorporation, BrdU incorporation and staining, and Ki67 staining. Furthermore, we detected ß-cell death by TUNEL, ß-cell differentiation by RT-PCR, and ß-cell function by glucose-stimulated insulin secretion. Interestingly, we have found that cyclin C increases rat and human ß-cell proliferation. This augmented proliferation did not induce ß-cell death, dedifferentiation, or dysfunction in rat or human islets. Our results indicate that cyclin C is a potential target for inducing ß-cell regeneration.

Cyclin C mediates stress-induced mitochondrial fission and apoptosis.

Mitochondria are dynamic organelles that undergo constant fission and fusion cycles. In response to cellular damage, this balance is shifted dramatically toward fission. Cyclin C-Cdk8 kinase regulates transcription of diverse gene sets. Using knockout mouse embryonic fibroblasts (MEFs), we demonstrate that cyclin C directs the extensive mitochondrial scission induced by the anticancer drug cisplatin or oxidative stress. This activity is independent of transcriptional regulation, as Cdk8 is not required for this activity. Furthermore, adding purified cyclin C to unstressed permeabilized MEF cultures induced complete mitochondrial fragmentation that was dependent on the fission factors Drp1 and Mff. To regulate fission, a portion of cyclin C translocates from the nucleus to the cytoplasm, where it associates with Drp1 and is required for its enhanced mitochondrial activity in oxidatively stressed cells. In addition, although HeLa cells regulate cyclin C in a manner similar to MEF cells, U2OS osteosarcoma cultures display constitutively cytoplasmic cyclin C and semifragmented mitochondria. Finally, cyclin C, but not Cdk8, is required for loss of mitochondrial outer membrane permeability and apoptosis in cells treated with cisplatin. In conclusion, this study suggests that cyclin C connects stress-induced mitochondrial hyperfission and programmed cell death in mammalian cells.

Involvement of Mediator complex in malignancy.

Mediator complex (MED) is an evolutionarily conserved multiprotein, fundamental for growth and survival of all cells. In eukaryotes, the mRNA transcription is dependent on RNA polymerase II that is associated to various molecules like general transcription factors, MED subunits and chromatin regulators. To date, transcriptional machinery dysfunction has been shown to elicit broad effects on cell proliferation, development, differentiation, and pathologic disease induction, including cancer. Indeed, in malignant cells, the improper activation of specific genes is usually ascribed to aberrant transcription machinery. Here, we focus our attention on the correlation of MED subunits with carcinogenesis. To date, many subunits are mutated or display altered expression in human cancers. Particularly, the role of MED1, MED28, MED12, CDK8 and Cyclin C in cancer is well documented, although several studies have recently reported a possible association of other subunits with malignancy. Definitely, a major comprehension of the involvement of the whole complex in cancer may lead to the identification of MED subunits as novel diagnostic/prognostic tumour markers to be used in combination with imaging technique in clinical oncology, and to develop novel anti-cancer targets for molecular-targeted therapy.

Cryptococcus neoformans mediator protein Ssn8 negatively regulates diverse physiological processes and is required for virulence.

Cryptococcus neoformans is a ubiquitously distributed human pathogen. It is also a model system for studying fungal virulence, physiology and differentiation. Light is known to inhibit sexual development via the evolutionarily conserved white collar proteins in C. neoformans. To dissect molecular mechanisms regulating this process, we have identified the SSN8 gene whose mutation suppresses the light-dependent CWC1 overexpression phenotype. Characterization of sex-related phenotypes revealed that Ssn8 functions as a negative regulator in both heterothallic a-α mating and same-sex mating processes. In addition, Ssn8 is involved in the suppression of other physiological processes including invasive growth, and production of capsule and melanin. Interestingly, Ssn8 is also required for the maintenance of cell wall integrity and virulence. Our gene expression studies confirmed that deletion of SSN8 results in de-repression of genes involved in sexual development and melanization. Epistatic and yeast two hybrid studies suggest that C. neoformans Ssn8 plays critical roles downstream of the Cpk1 MAPK cascade and Ste12 and possibly resides at one of the major branches downstream of the Cwc complex in the light-mediated sexual development pathway. Taken together, our studies demonstrate that the conserved Mediator protein Ssn8 functions as a global regulator which negatively regulates diverse physiological and developmental processes and is required for virulence in C. neoformans.

Gene encoding a c-type cyclin in Mycosphaerella graminicola is involved in aerial mycelium formation, filamentous growth, hyphal swelling, melanin biosynthesis, stress response, and pathogenicity.

Mycosphaerella graminicola is an important wheat pathogen causing Septoria tritici blotch. To date, an efficient strategy to control M. graminicola has not been developed. More significantly, we have a limited understanding of the molecular mechanisms of M. graminicola pathogenicity. In this study, we attempted to characterize an MCC1-encoding c-type cyclin, a gene homologous to FCC1 in Fusarium verticillioides. Four independent MCC1 knock-out mutants were generated via Agrobacterium tumefaciens-mediated transformation. All of the MCC1 mutants showed consistent multiple phenotypes. Significant reductions in radial growth on potato dextrose agar (PDA) were observed in all of the MCC1 mutants. In addition, MCC1 gene-deletion mutants produced less aerial mycelium on PDA, showed delayed filamentous growth, had unusual hyphal swellings, produced more melanin, showed an increase in their stress tolerance response, and were reduced significantly in pathogenicity. These results indicate that the MCC1 gene is involved in multiple signaling pathways, including those involved in pathogenicity in M. graminicola.

Cyclin C regulates human hematopoietic stem/progenitor cell quiescence.

Hematopoietic stem cells (HSCs) can remain quiescent or they can enter the cell cycle, and either self-renew or differentiate. Although cyclin C and cyclin dependent kinase (cdk3) are essential for the transition from the G(0) to the G(1) phase of the cell cycle in human fibroblasts, the role of cyclin C in hematopoietic stem/progenitor cells (HSPCs) is not clear. We have identified an important role of cyclin C (CCNC) in regulating human HSPC quiescence, as knocking down CCNC expression in human cord blood CD34(+) cells resulted in a significant increase in quiescent cells that maintain CD34 expression. CCNC knockdown also promotes in vitro HSPC expansion and enhances their engraftment potential in sublethally irradiated immunodeficient mice. Our studies establish cyclin C as a critical regulator of the G(0)/G(1) transition of human HSPCs and suggest that modulating cyclin C levels may be useful for HSC expansion and more efficient engraftment.