Targeting Transcription Regulation In Pancreatic Cancer With A Covalent CDK7 Inhibitor

BackgroundPancreatic cancer is a leading lethal disease with limited treatment choice and poor prognosis.Currently,only one targeted drug-Erlotinib has been approved by FDA.Limited"driver genes" have been found for pancreatic cancer,thus there is an urgent need for discovering an effective treatment strategy.Studies have shown that many tumors have aberrantly activated transcriptional genes.The CDK7 inhibitor,THZ1,could bind CDK7 covalently and inhibit the transcription of tumor cells effectively.THZ1 could inhibit tumor proliferation in triple negative breast cancer,small cell lung cancer,ovarian cancer and other refractory tumors.There is no study of THZ1 for pancreatic cancer,therefore,we intended to test the efficacy of CDK7 inhibitors in pancreatic cancer.MethodsThere were three main sections of the experiment.The first section was to examine the efficacy of CDK7 inhibitors in pancreatic cancer cell lines.The second section was to explore possible downstream pathway of CDK7.The third section was to examine the association between CDK7 expression and prognosis in pancreatic cancer patients.In the first section,pancreatic cell lines were exposed to CDK7 inhibitor-THZl to test the proliferation,apoptosis,cell cycle,and levels of cell-related transcription proteins.To further corroborate these results,the CDK7 gene was edited by CRISPR/Cas9 technique to test the proliferation of pancreatic cancer cell lines.In the second section,using ChIP assay in Canpan2 cell line with H3k27ac antibody,we plan to study downsteam pathways that may be regulated by THZ1.Furthermore,we tested the RNA and protein levels of certain pathway in cells lines exposed to THZ1.In the third section,the expression of CDK7 in pancreatic cancer patients was divided into high and low groups using immunohistochemistry(IHC).Kaplan-Meier and multivariate Cox model were used to detect the relationship between CDK7 and survival.Furthermore,we use the public TCGA transcription data to analyze the relationship of CDK7 transcription level and pancreatic cancer prognosis.ResultsIn the first section,we observed that pancreatic cancer cells were highly sensitive to THZ1,with cell proliferation effectively suppressed by low nanomolar concentrations of THZ1(IC50<200 nM).We further found that CDK7 inhibition efficiently induced apoptosis cell death in CAPAN2 and PANC03.27 cell lines.However,unlike small cell lung cancer and ovarian cancer,THZ1 has no significant effect on the cell cycle in pancreatic cancer cell lines,which is the same as triple negative breast cancer.Furthermore,we found that the phosphorylation level of Ser5,7 and 2 of RNA polymerase ? CTD(carboxy-terminal domain)decreased with the increasing of THZ1 concentration or prolonged drug treatment time,which inhibited the transcription.Treating cells with constructs encoding two independent small guiding RNA targeting CDK7(sg_CDK7)led to a substantial reduction of CDK7 protein and suppression of cell growth.The level of apoptosis related protein Cleaved-PARP was significantly increased and the anti-apoptotic protein MCL1 was significantly decreased.The results confirmed that proliferation of pancreatic cell lines largely depend on CDK7 protein.In the second section,we performed ChIP experiment in CAPAN2 cell line.Using H3K27ac as the target antibody,we found that THZ1 reduced the H3K27ac-conjugated PIK3CA gene.Furthermore,the RNA level of PIK3CA was decreased in CAPAN2 exposed to THZ1.The protein level of PI3K was also decreased,together with phosphorylation if AKT and phosphorylation if mTOR.Thus,we speculated that THZ1 inhibit the PI3K/AKT/mTOR pathway.In the third section,we evaluated the CDK7 protein expression level by IHC and devided pancreatic cancer patients into high/low groups.Multivariate Cox analysis showed that patients with higher expression of CDK7 had shorter overall survival(HR:1.25,95%CI:1.00-1.57)compared with low expression of CDK7.The results of TCGA transcription data also showed that high expression of CDK7 was associated with inferior survival.ConclusionIn summary,our study demonstrates that THZ1 could efficiently bind to CDK7 protein,significantly inhibit pancreatic cancer cell proliferation,induce apoptosis and reduce gene transcription.In the study of downstream target,THZ1 may inhibit the PI3K/AKT/mTOR pathway,thus inhibitinh cell proliferation.Further clinical trials of CDK7 for patients with pancreatic cancer are warrented.