Tanshinone IIA suppresses the progression of lung adenocarcinoma through regulating CCNA2-CDK2 complex and AURKA/PLK1 pathway

Lung adenocarcinoma (LUAD) belongs to a subgroup of non-small cell lung cancer (NSCLC) with an increasing incidence all over the world. Tanshinone IIA (TSA), an active compound of Salvia miltiorrhiza Bunge., has been found to have anti-tumor effects on many tumors, but its anti-LUAD effect and its mechanism have not been reported yet. In this study, bio-information analysis was applied to characterize the potential mechanism of TSA on LUA, biological experiments were used to verify the mechanisms involved. TCGA, Pubchem, SwissTargetPrediction, Venny2.1.0, STRING, DAVID, Cytoscape 3.7.2, Omicshare, GEPIA, RSCBPDB, Chem Draw, AutoDockTools, and PyMOL were utilized for analysis in the bio-information analysis and network pharmacology. Our experiments in vitro focused on the anti-LUAD effects and mechanisms of TSA on LUAD cells (A549 and NCI-H1975 cells) via MTT, plate cloning, Annexin V-FITC and PI dual staining, flow cytometry, and western blot assays. A total of 64 differentially expressed genes (DEGs) of TSA for treatment of LUAD were screened out. Gene ontology and pathway analysis revealed characteristic of the DEGs network. After GEPIA-based DEGs confirmation, 46 genes were considered having significant differences. Further, 10 key DEGs (BTK, HSD11B1, ADAM33, TNNC1, THRA, CCNA2, AURKA, MIF, PLK1, and SORD) were identified as the most likely relevant genes from overall survival analysis. Molecular Docking results showed that CCNA2, CDK2 and PLK1 had the lowest docking energy. MTT and plate cloning assays results showed that TSA inhibited the proliferation of LUAD cells in a concentration-dependent manner. Annexin V-FITC and PI dual staining and flow cytometry assays results told that TSA promoted the apoptosis of the two LUAD cells in different degrees, and induced cycle arrest in the G1/S phase. Western blot results showed that TSA significantly down-regulated the expression of CCNA2, CDK2, AURKA, PLK1, and p-ERK. In summary, TSA could suppress the progression of LUAD by inducing cell apoptosis and arresting cell cycle, and these were done by regulating CCNA2-CDK2 complex and AURKA/PLK1 pathway. These findings are the first to demonstrate the molecular mechanism of TSA in treatment of LUAD combination of network bio-information analysis and biological experiments in vitro.

Cyclin E/CDK2: DNA Replication, Replication Stress and Genomic Instability

DNA replication must be precisely controlled in order to maintain genome stability. Transition through cell cycle phases is regulated by a family of Cyclin-Dependent Kinases (CDKs) in association with respective cyclin regulatory subunits. In normal cell cycles, E-type cyclins (Cyclin E1 and Cyclin E2, CCNE1 and CCNE2 genes) associate with CDK2 to promote G1/S transition. Cyclin E/CDK2 complex mostly controls cell cycle progression and DNA replication through phosphorylation of specific substrates. Oncogenic activation of Cyclin E/CDK2 complex impairs normal DNA replication, causing replication stress and DNA damage. As a consequence, Cyclin E/CDK2-induced replication stress leads to genomic instability and contributes to human carcinogenesis. In this review, we focus on the main functions of Cyclin E/CDK2 complex in normal DNA replication and the molecular mechanisms by which oncogenic activation of Cyclin E/CDK2 causes replication stress and genomic instability in human cancer.

Identification of novel cyclin A2 binding site and nanomolar inhibitors of cyclin A2-CDK2 complex

Aims: In this study we aim to simultaneously target cyclin A2 and CDK2 to increase the chance of finding modulators of DNA damage response mechanism. Objective: Based on cyclin A2’s recently discovered role in DNA repair, we hypothesized that small molecule inhibitors that were predicted to bind to both cyclin A2 and CDK2 will be useful as a radiosensitizer of cancer cells. In this study, we used structure-based drug discovery to find inhibitors that target both cyclin A2 and CDK2. Background: Given the diverse roles of cyclin A2 both in cell cycle regulation and in DNA damage response, identifying small molecule regulators of cyclin A2 activity carries significant potential to regulate diverse cellular processes in both ageing/neurodegeneration and in cancer. Method: Molecular dynamics simulations were used to generate diverse binding pocket conformations for application of the relaxed complex scheme. We then used structure-based virtual screening to find potential dual cyclin A2 and CDK2 inhibitors. Based on a consensus score of docked poses from Glide and AutoDock Vina, we identified about 40 promising hit compounds, where all PAINS scaffolds were removed from consideration. A biochemical luminescence assay of cyclin A2-CDK2 function was used for experimental verification. Result: Four lead inhibitors of cyclin A2-CDK2 complex have been identified using a relaxed complex scheme virtual screen have been verified in a biochemical luminescence assay of cyclin A2-CDK2 function. The most potent leads were all ribofuranosyl-pyrrolo[2,3-d]pyrimidines with inhibitory concentrations in the nanomolar range. Conclusion: The four cyclin A2-CDK2 complex inhibitors are the first reported inhibitors that were specifically designed not to target the cyclin A2-CDK2 protein-protein interface. Overall, our results highlight the potential of combined advanced computational tools and biochemical verification to discover novel binding scaffolds.

The CIC-DUX4 fusion oncoprotein drives metastasis and tumor growth via distinct downstream regulatory programs and therapeutic targets in sarcoma

Transcription factor fusion genes create oncoproteins that drive oncogenesis, and represent challenging therapeutic targets. Understanding the molecular targets by which such fusion oncoproteins promote malignancy offers an approach to develop rational treatment strategies to improve clinical outcomes. CIC-DUX4 is a transcription factor fusion that defines certain undifferentiated round cell sarcomas with high metastatic propensity and poor clinical outcomes. The molecular targets regulated by the CIC-DUX4 oncoprotein that promote this aggressive malignancy remain largely unknown. We show that increased expression of ETV4 and CCNE1 occurs via neo-morphic, direct effects of CIC-DUX4 and drives tumor metastasis and survival, respectively. We demonstrate a molecular dependence on the CCNE-CDK2 cell cycle complex that renders CIC-DUX4 tumors sensitive to inhibition of the CCNE-CDK2 complex, highlighting a therapeutic strategy for CIC-DUX4 tumors. Our findings highlight a paradigm of functional diversification of transcriptional repertoires controlled by a genetically-aberrant transcriptional regulator, with therapeutic implications.