Purpose: The Mediator complex is a multiprotein assembly, which serves as a hub for diverse signaling pathways to regulate gene expression. Because gene expression is frequently altered in cancer, a systematic understanding of the Mediator complex in malignancies could foster the development of novel targeted therapeutic approaches.Experimental Design: We performed a systematic deconvolution of the Mediator subunit expression profiles across 23 cancer entities (n ¼ 8,568) using data from The Cancer Genome Atlas (TCGA). Prostate cancer-specific findings were validated in two publicly available gene expression cohorts and a large cohort of primary and advanced prostate cancer (n ¼ 622) stained by immunohistochemistry. The role of CDK19 and CDK8 was evaluated by siRNA-mediated gene knockdown and inhibitor treatment in prostate cancer cell lines with functional assays and gene expression analysis by RNAseq.Results: Cluster analysis of TCGA expression data segregated tumor entities, indicating tumor-type-specific Mediator complex compositions. Only prostate cancer was marked by high expression of CDK19. In primary prostate cancer, CDK19 was associated with increased aggressiveness and shorter disease-free survival. During cancer progression, highest levels of CDK19 and of its paralog CDK8 were present in metastases. In vitro, inhibition of CDK19 and CDK8 by knockdown or treatment with a selective CDK8/CDK19 inhibitor significantly decreased migration and invasion.Conclusions: Our analysis revealed distinct transcriptional expression profiles of the Mediator complex across cancer entities indicating differential modes of transcriptional regulation. Moreover, it identified CDK19 and CDK8 to be specifically overexpressed during prostate cancer progression, highlighting their potential as novel therapeutic targets in advanced prostate cancer.
Despite considerable progress in recent years, the overall prognosis of metastatic malignant melanoma remains poor, and curative therapeutic options are lacking. Therefore, better understanding of molecular mechanisms underlying melanoma progression and metastasis, as well as identification of novel therapeutic targets that allow inhibition of metastatic spread, are urgently required. The current study provides evidence for aberrant cyclin-dependent kinase 5 (CDK5) activation in primary and metastatic melanoma lesions by overexpression of its activator protein CDK5R1/p35. Moreover, using melanoma in vitro model systems, shRNA-mediated inducible knockdown of CDK5 was found to cause marked inhibition of cell motility, invasiveness, and anchorage-independent growth, while at the same time net cell growth was not affected. In vivo, CDK5 knockdown inhibited growth of orthotopic xenografts as well as formation of lung and liver colonies in xenogenic injection models mimicking systemic metastases. Inhibition of lung metastasis was further validated in a syngenic murine melanoma model. CDK5 knockdown was accompanied by dephosphorylation and overexpression of caldesmon, and concomitant caldesmon knockdown rescued cell motility and proinvasive phenotype. Finally, it was found that pharmacological inhibition of CDK5 activity by means of roscovitine as well as by a novel small molecule CDK5-inhibitor, N-(5-isopropylthiazol-2-yl)-3-phenylpropanamide, similarly caused marked inhibition of invasion/migration, colony formation, and anchorage-independent growth of melanoma cells. Thus, experimental data presented here provide strong evidence for a crucial role of aberrantly activated CDK5 in melanoma progression and metastasis and establish CDK5 as promising target for therapeutic intervention.
The generation and expansion of functionally competent NK cells in vitro is of great interest for their application in immunotherapy of cancer. Since CD33 constitutes a promising target for immunotherapy of myeloid malignancies, NK cells expressing a CD33-specific chimeric antigen receptor (CAR) were generated. Unexpectedly, we noted that CD33-CAR NK cells could not be efficiently expanded in vitro due to a fratricide-like process in which CD33-CAR NK cells killed other CD33-CAR NK cells that had upregulated CD33 in culture. This upregulation was dependent on the stimulation protocol and encompassed up to 50% of NK cells including CD56dim NK cells that do generally not express CD33 in vivo. RNAseq analysis revealed that upregulation of CD33+ NK cells was accompanied by a unique transcriptional signature combining features of canonical CD56bright (CD117high, CD16low) and CD56dim NK cells (high expression of granzyme B and perforin). CD33+ NK cells exhibited significantly higher mobilization of cytotoxic granula and comparable levels of cytotoxicity against different leukemic target cells compared to the CD33− subset. Moreover, CD33+ NK cells showed superior production of IFNγ and TNFα, whereas CD33− NK cells exerted increased antibody-dependent cellular cytotoxicity (ADCC). In summary, the study delineates a novel functional divergence between NK cell subsets upon in vitro stimulation that is marked by CD33 expression. By choosing suitable stimulation protocols, it is possible to preferentially generate CD33+ NK cells combining efficient target cell killing and cytokine production, or alternatively CD33− NK cells, which produce less cytokines but are more efficient in antibody-dependent applications.
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