Systems pharmacology of mifepristone (RU486) reveals its 47 hub targets and network: comprehensive analysis and pharmacological focus on FAK-Src-Paxillin complex

Abstract

Mifepristone (RU486), a synthetic steroid compound used as an abortifacient drug, has received considerable attention to its anticancer activity recently. To explore the possibility of using mifepristone as a cancer metastasis chemopreventive, we performed a systems pharmacology analysis of mifepristone-related molecules in the present study. Data were collected by using Natural Language Processing (NLP) and 513 mifepristone-related genes were dug out and classified functionally using a gene ontology (GO) hierarchy, followed by KEGG pathway enrichment analysis. Potential signal pathways and targets involved in cancer were obtained by integrative network analysis. Total thirty-three proteins were involved in focal adhesion-the key signaling pathway associated with cancer metastasis. Molecular and cellular assays further demonstrated that mifepristone had the ability to prevent breast cancer cells from migration and interfere with their adhesion to endothelial cells. Moreover, mifepristone inhibited the expression of focal adhesion kinase (FAK), paxillin, and the formation of FAK/Src/Paxillin complex, which are correlated with cell adhesion and migration. This study set a good example to identify chemotherapeutic potential seamlessly from systems pharmacology to cellular pharmacology, and the revealed hub genes may be the promising targets for cancer metastasis chemoprevention.

Figure 1. Processing flow chart of the NLP analysis of mifepristone.

Figure 2. Genes involved in cancer-related pathways discovered by KEGG analysis.

The red indicates mifepristone-related.

Figure 3. Network analysis of mifepristone-related therapeutic targets involved in cancer-related pathways.

The connectivity of CCND1 or EGFR is the highest one that has a total of 12-related genes (z-test, P < 0.05).

Figure 4. Genes involved in focal adhesion pathway.

A, focal adhesion-related pathways generated by KEGG analysis. Red indicates mifepristone-related. B, network and connectivity analysis of mifepristone-related therapeutic targets in focal adhesion pathways. The connectivity of FAK (PTK2) is the highest one that has a total of 10-related genes (z-test, P < 0.05).

Figure 5. Cellular pharmacology analysis of mifepristone.

A, in vitro activity of mifepristone against MDA-MB-231 cell line. B, dose-dependent inhibition by mifepristone on cell migration. A Corning transwell system was used to assay cell migration as described in methods. The amount of MDA-MB-231 cells migrated through polycarbonate membranes was counted by microscopic observation (10×). Each experiment was carried out at least three times. *, P < 0.05, **, P < 0.01. C, inhibition by mifepristone of MDA-MB-231 cells adhesion to HUVECs. Representative microscopic observation of the inhibition by mifepristone at 0, 50, and 100 μM. DMSO (0.1%) was used as vehicle control (average of 10 independent microscope fields for each of 3 independent experiments).

Figure 6. Concentration-dependent effects of mifepristone on FAK activity.

A. Western blot assay showing that mifepristone inhibits the expression of FAK; B. Real-time PCR assay showing that mifepristone inhibited mRNA expression. Data are the means ± SEM of 3 repeat experiments. N = 3. *, p < 0.05; **, p < 0.01.

Figure 7. Mifepristone-mediated formation of FAK/Src/Paxillin complex.

Cells were pretreated in the absence and presence of mifepristone (50 μM) for 1 hour. Cell lysates were analyzed by western blot with antibodies that recognize FAK, Src, and Paxillin. Immunoprecipitation (IP) with the anti-FAK antibody or control IgG antibody followed by blotting with the anti-Src or anti-Paxillin antibody was performed. Each of the examples is representation of three independent experiments. The lower part depicting the bars denotes the mean ± SE of three independent experiments for each condition determined from densitometry relative to control. *, P < 0.05.