Deciphering the molecular mode of action of histone deacetylase inhibitors in human germ cell cancer cell lines in vitro and in vivo

• Applicant: Professor Dr. Daniel Nettersheim
• Subject Area: Pathology ; Hematology, Oncology
• Term: from 2015 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 270478324

Our previous analyses have shown that ISTODAX causes hyperacetylation of histones H3 and H4, apoptosis and G2/M-arrest at very low concentrations in gem cell cancer (GCC) cell lines. In human fibroblasts, only a G2/M-arrest, but no apoptosis was detectable. Additionally, we identifed putative key factors driving the cellular response to ISTODAX. We postulate that downregulation of the SWI/SNF-regulator ARID1A as well as upregulation of stress-sensors GADD45B, ATF3, ID2, ZFP36 and DUSP1 induced apoptosis in GCC cell lines. Furthermore, we found 5 genes to be commonly expressed between GCC cell lines and fibroblasts (DHRS2, RHOB, CRISPLD2, BAIPA2 and p21). We assume that one of these factors induces p21 expression, causing G2/M-arrest.In this study, we would like to shed light on the hierarchical structure of the ISTODAX-cascade and the functional role of each key player by cDNA overexpression or CRISPR/Cas-mediated knock down of these factors in GCC cells and fibroblasts, followed by analyses of the molecular effetcs. We will utilize qRT-PCR- and western blot- as well as chromatin-immunoprecipitation- and co-immunoprecipitation-analyses to screen for changes in gene expression and detect protein-DNA- / protein-protein-interactions, respectively. Induction of apoptosis or G2/M-arrest will be measured by FACS-based methods.Next, Luciferase-expressing, cDNA-overexpressing and CRISPR/Cas-knock out cell lines will be transplanted into the seminiferous tubule of nude mice. During a two month ISTODAX application, tumor growth will be measured by live imaging technology. Additionally, tumors will be isolated and analyzed for the molecular effects of an ISTODAX in vivo treatment as described before. Finally, gathered data will be correlated to the results of the in vitro analyses.These experiments will decipher the mode of action of ISTODAX in vitro and in vivo and provide insight into the response mechanisms of GCC cells towards ISTODAX that might influence the success of a therapy. The seminiferous tubule injections will demonstrate that ISTODAX is able to pass the blood-testis-barrier, highlighting ISTODAX as a valuable GCC in vivo therapy option.

• DFG Programme: Research Grants