Triterpenoid saponin from Ardisia gigantifolia Stapf inhibits cancer cell metastasis behavior of TNBC via suppressing EMT and stemness through ITGB4/FAK signals

Triple-negative breast cancer (TNBC) is a subtype of breast cancer that has a relatively poor prognosis after surgery and chemotherapy. The major challenge in the treatment of TNBC is the lack of effective targets and drug resistance, therefore, the search for new and effective drugs for the treatment of TNBC remains a critical clinical need. 3β-O-α-L-rhamnopyranosyl-(1→3)-[β-D-xylopyranosyl-(1→2)]-β-D-glucopyranosyl-(1→4)-[β-D-glucopyranosyl-(1→2)]-α-L-arabinopyranosyl-16α -hydroxy-13,28-epoxy-oleanane (AG) is a kind of triterpenoid saponin from Ardisia gigantifolia Stapf and showed anti-tumor activities against several kinds of cancers especially breast cancer. However, the effects of AG on cancer metastasis, stemness of TNBC and the underlaying mechanism were not very clear. The aim of our study is to further investigate the effects of AG on metastasis and stemness of MDA-MB-231 cells and MCF-10A cells. Cell viability assay of MDA-MB-231 and MCF-10A cells was performed using MTT method. Scratch and transwell invasion assay was used to test cell motility ability. Cell apototise and breast cancer stem marker ALDH was tested by flow cytometry. RNA-seq was used to analyze the AG-related biological process and pathway. Protein levels of Vimentin, E-cadherin, MMP-2, p-FAK, FAK, ITGB4 and OCT4 were detected using western blotting. The in vivo antitumor activity of AG was tested using tumor bearing nude mice induced by MDA-MB-231 cells. AG inhibited the proliferation of MDA-MB-231 cells dose-dependently. AG suppressed the motility and invasion of MDA-MB-231 cells through influences on Vimention, E-cadherin, MMP-2 and Cancer stem cells (CSCs) marker OCT. RNA-seq results identified 2516 differentially expressed genes along with 1163 up-regulated and 1353 down-regulated genes. GO and KEGG analysis revealed that these differentially expressed genes were obviously enriched in adhesion, cell migration and pathways in cancer. Meanwhile, AG significantly suppressed p-FAK and ITGB4 does-dependently. Moreover, the effects were restored by EGFR activation. In the in vivo experiment, AG significantly decreased the tumor weight and tumor volume with no statistically significant effects on body weight. These findings indicated the inhibiting activities of AG on Epithelial-to-mesenchymal transition (EMT), stemness and metastasis in breast cancer, and suggested the possibility being used as a potent anti-metastatic agent of AG for the treatment of TNBC. Conclusion: AG inhibited EMT, stemness and subsequently suppressed cell migration and invasion of MDA-MB-231 cells through ITGB4/FAK signaling in an EGFR-dependent manner.