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.

A trojan horse approach for silencing thymidylate synthase.

In this paper we present a new and possibly more effective way of inhibiting thymidylate synthase (TS) in cells than through the use of substrate analogue inhibitors. An inactive double mutant of TS (DM), Arg(126)Glu/Cys(146)Trp, is shown to progressively impair the reactivation of native Escherichia coli TS when the two are denatured together in vitro. The individual single mutant proteins Arg(126)Glu and Cys(146)Trp showed little or no inhibition. When the DM is introduced into E. coli and induced from an expression plasmid, the mutant subunits act as a decoy in deceiving newly formed native TS subunits to fold with them to yield inactive heterodimers. As a consequence of the depletion of TS, the cells die a "thymineless" death when grown in medium devoid of thymine. Addition of thymine to the medium enables the cells to grow normally, although only very low levels of TS activity could be detected in those cells containing induced DM. The individual single-site mutations of the DM, Arg(126)Glu and Cys(146)Trp, did not inhibit growth, as might be expected from the in vitro studies. However, when a nontoxic level of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) is added to growing DM-transformed cells, the combination is lethal to the cells. These experiments suggest that a similar dominant-negative response to the DM of TS could be affected in tumor cells, for which preliminary evidence is presented. This technique, either alone or combined with other modalities, suggest a new approach to targeting cells for chemotherapy.