Triple-negative breast cancer (TNBC) as a poor prognosis disease, attracted more and more people’s concern and attention. Owing to lack HR, ER and HER2, TNBC still do not have available target therapy options . In clinical practice, chemotherapy still the main treatment to TNBC patients . Recently, targeted therapy has been a hot topic. And new targeted site and new targeted drugs had been proved very beneficial for tumor patients . Higher response rates were seen when targeted inhibitors are combined with chemotherapy . Therefore, it is very beneficial for us to find new targeted site and new potential ideal targeted drugs.
In this study, we combined bioinformatics with molecular biology to provide new ideas for TNBC treatments. Firstly, we analyzed GSE62931 and GSE76275 database, which contains 245 TNBC tissues and 120 normal tissues. Trough the Heat maps and Volcano plots, we got 1,212 and 353 differentially expressed genes (DEGs) form these 2 databases. And the Venn plot showed that there are 299 DEGs in both GSE62931 and GSE76275 database, including 88 up-regulated and 141 down-regulated genes. These DEGs could be regarded as potential biomarkers and targeted site for TNBC.
Then, we used metascape website to make the functional enrichment analysis, to study molecular pathways in TNBC. The figures and tables indicated that DEGs were mostly enriched in ‘developmental growth’, ‘regulation of hormone level’ and ‘epithelial cell differentiation’. Also, we respectively analyzed the up-regulated and down-regulated DEGs by functional enrichment in DAVID, and results illustrated that the enrichment of up-regulated DEGs was mainly in BP, MF, CC terms, such as ‘mitotic nuclear division’, ‘identical protein binding’ and ‘cytoplasm’. As for down-regulated DEGs, they were also enriched in BP, MF and CC terms, including ‘negative regulation of cell proliferation’, ‘heme binding’ and ‘extracellular exosome’. In addition, in KEGG pathways, the enrichments of up-regulated and down-regulated DEGs were respectively mostly in ‘cell cycle’ and ‘PPAR signaling pathway’. For example, M. Rath et al. demonstrated that mitotic nuclear division was associated with tumorigenesis, and mitotic kinesins were being validated as drug targets [35, 36]. And extracellular exosome is a vesicle released into extracellular region. Some studies had shown that tumor cells can produce more exosomes than normal cell . Therefore, in short, these pathways were all contribute to the progression of TNBC.
PPI network analysis was very important in bioinformatics research. By the STRING and cytoscape software, we got the top 20 DEGs as the hub genes based on the degrees. We also got the most significant 3 modules, which could be regarded as the most important gene clusters of TNBC. Among these, module 1 included CDC20 and other 17 genes. We also got the functional pathway enrichment analysis of these 3 modules. DEGs in module 1 were mostly enriched in the BP, CC and KEGG pathway, including ‘spindle’, ‘sister chromatid cohesion’, ‘cell division’, ‘cell cycle’ and ‘mitotic nuclear division’. CDC20 plays a great role in these 5 GO terms and pathway. Meanwhile, most of the DEGs in these 3 modules were enriched in the MF and BP of GO terms, including ‘transcription regulatory region DNA binding’ and ‘structural molecule activity’ and ‘mitotic nuclear division’. On the one hand, it is obvious that abnormal transcription and mitosis could lead to tumorigenesis [38, 39]. On the other hand, there were some studies demonstrated that inhibition of the cellular machinery required for the assembly and maintenance can inhibit the tumor growth [40, 41]. Therefore, these terms and pathways maybe new therapeutic targets for TNBC.
In addition, in Kaplan website, we found 14 hub genes were relevant to OS of TNBC patients, and 19 hub genes were relevant to RFS of TNBC patients. Higher expression of most of them contributed to shorter lifetime, including CDC20, ANLN, ASE1, ASPM, CEP55 and so on. Among these hub genes, cell-division cycle protein 20 homologue (CDC20), as the second important gene based on the degrees in cytoscape software and the significant gene in module 1, took part in cell division . CDC20, which was key to chromosome segregation and mitosis exit, plays an important role in cell cycle progressing . It can activate a ligase, the anaphase-promoting complex/cyclosome (APC/C), which starts the anaphase and mitotic exit . Cheng et al. shown that overexpression of CDC20 promotes the metastasizing of breast cancer . Meanwhile, some study confirmed that overexpression of CDC20 lead to short-term breast cancer survival again . It was also proved that CDC20 was a great target for anti-tumor drug development . Therefore, CDC20 was a potential treatment target, and we chose CDC20 as a targeted spot for further study.
It is obvious that finding effective CDC20 inhibitor was very important for breast cancer targeted therapy. There were many CDC20 targeted drugs, including tosyl-L-arginine methyl ester (TAME) and apcin, but they still have many issues to be addressed . TAME was proved that can inhibit the binding of free CDC20 and APC and promote the CDC20 removal from the APC [47, 48]. And apcin can bind to CDC20 and simultaneously disrupt the APC/C-Cdc20-substrate ternary complex by competitively inhibition to blockade the mitotic exit . It was also proved that apcin can inhibit the growth and invasion of osteosarcoma cell by targeting CDC20 . However, whether TAME and apcin were useful in clinical needs further investments. Among these CDC20 inhibitor, we chose apcin as the reference drug to screen new potential ideal compounds for TNBC patients.
We got 7,416 natural ligands by LibDock, and based on the LibDock score, we chose top 20 ligands to do the further study. Safety is one of the most important things in drug development. Therefore, after analyzing their biochemico-pharmacological properties by ADME and Toxicity Prediction module, we chose ZINC000004098930, which was non-hepatotoxicity, more solubility level and less carcinogenicity than apcin, as the safe lead compounds among the top 20 ligands.
Then we analyzed the pharmacophore and the ligand binding mechanisms of ZINC000004098930 and apcin with CDC20. The results demonstrated that the CDOCKER potential energy of ZINC000004098930 was lower than apcin, which means ZINC000004098930 could bind more firmly than apcin. Meanwhile, ZINC000004098930 had more hydrogen bonds and the π-related interactions with CDC20 than apcin. In general, ZINC000004098930 had a higher binding force with CDC20 than apcin.
In the end, we run RMSD and calculated the potential energy of ZINC000004098930-CDC20 and apcin-CDC20 complexes to study the stability of them by molecular dynamics simulation. As the results suggested, the trajectories of both ZINC000004098930-CDC20 and apcin-CDC20 complexes reached their equilibrium after 18 ps. They become gradually stabilized, which indicated these two complexes could exist stability in natural. In conclusion, ZINC000004098930 could be regarded as ideal lead compounds for drug development for TNBC patients and may give new thoughts to TNBC targeted therapy.
Recently, targeted therapy is a hot topic for tumor treatment, but we still do not have perfect drugs for TNBC treatment. In this study, we combined bioinformatics with molecular biology to screen a new ideal ligand, which targeted inhibit CDC20. Although there is a long way from clinical application, it provided a new way to treat TNBC. ZINC000004098930 as a natural ligand has unique advantages. To sum up, we did the first step of drug development for TNBC patients. And what’s more, we provided 18 else hub genes and many targeted pathways, which may be useful in future study.