The impact of Caspase-8 on the activity of Cyclin Dependent Kinase 9 in regulating RNA Polymerase II mediated transcription in ovarian cancer

• Applicant: Dr. Khayal Gasimli, since 6/2023
• Subject Area: Gynaecology and Obstetrics ; Cell Biology
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 456711327

Ovarian cancer is one of the most lethal gynecological cancers in the world. Over 70% of ovarian cancer cases are diagnosed at advanced stages, resulting in poor prognosis, and are largely incurable. In ovarian cancer, caspase-8 (C8) is often weakly expressed, which is associated with a significantly worse prognosis for patients (OV-TCGA, 491 patients). Our preliminary works showed that Caspase-8:1. Is a negative regulator of cell migration and invasion2. Interacts with CDK93. Negatively regulates CDK9 activation, and its activity on RNA Polymerase II (POLR2A), which mediates productive transcriptional elongation, in vitro and ex vivo4. KO cells are less sensitive to the transcription inhibitor GemcitabineIn the next steps, we will determine:1. How Caspase-8 negatively regulates CDK9 activity. 5-ethynyl uridine (EU) assay would be used to compare and quantify global transcription between Caspase-8 WT and KO cancer cell lines. Inactive CDK9 is sequestered in the cytoplasm by the Hsp90/Cdc37 complex and shuttles to the nucleus to get activated. Cell fractionation and Heterokaryon Fusion Assay would be used to determine whether Caspase-8 inhibits this shuttling. Interactome analysis would be used to identify any additional members of this complex and down-regulated to determine their effects on this shuttling.2. How Caspase-8 altered POLR2A mediated transcription of metastasis regulating proteins. Global Transcriptomics and Proteomics analysis of Caspase-8 WT and KO cells would be performed to identify genes which are significantly up- or down-regulated between the WT and KO cells. The expressions of these genes would be verified by qRT-PCRs and immunoblots, and would be subjected to gene ontology/pathway analysis using IPA and DAVID Bioinformatics Resource. Genes known to be involved in the regulation of metastasis would be confirmed through their knock-down or over-expression, migration and invasion assays.3. How these genes regulate metastasis in ovarian cancer cell lines and patient-derived tumor cells (PDCs). The expressions of the most interesting genes would be checked in low or high Caspase-8 expressing PDCs. The effects of these genes on the expressions and activities of known biomarkers of cancer metastasis would be determined.4. The effects of CDK9 inhibition in combination with chemotherapeutic agents, in a spheroid/organoid bank. A spheroid bank of several Caspase-8 WT and KO cancer cell lines and organoid bank of PDCs would be established to test the response of several CDK9 inhibitors in combination with different standard chemotherapeutics.5. The effect of CDK9 inhibition in combination with chemotherapeutic agents, in a mouse model. WT and KO cancer cells, stably expressing the Luciferase gene, would be generated to perform the non-invasive analysis of metastasis, following their orthotopic injection into the mouse ovaries. These mice would be treated with combinations of chemotherapeutics and the CDK9 inhibitor LDC000067.

• DFG Programme: Research Grants
• International Connection: China
• Cooperation Partner: Professor Shengtao Zhou, Ph.D.
• Ehemaliger Antragsteller: Dr. Ranadip Mandal, Ph.D., until 6/2023