Study on inhibition of Britannin on triple-negative breast carcinoma through degrading ZEB1 proteins

Background: Triple-negative breast carcinomas (TNBCs) are a breast carcinoma with the most aggressive form, which is demonstrated as enhanced invasion and recurrence. Britannin is extracted mainly from the traditional Chinese herb Inula japonica Thunb, and few studies have focused on its effect on TNBC. Moreover, there is still no report concerning the role of Britannin in degrading the transcripts of Zinc finger E-box-binding homeobox 1 (ZEB1) proteins. Purpose: To explore the potential effect of Britannin on invasion and stemness of TNBCs and its underlying mechanism. Methods: Cellular activity was measured using MTT, and cell cycle was measured using flow cytometry (FCM). The effect of Britannin on the migrating and invading abilities of MDA-MB-231 and 4T1 cells were measured using the wound healing and transwell assays. The sizes and number of breast carcinoma cells were measured by tumor formation assay and in vitro limiting-dilution assay. CD44 expression in tumor spheroids was tested by immunofluorescence assay. Nextly, the expressions of epithelial-mesenchymal transition (EMT) markers and ZEB1 protein expressional level were detected by western blot . ZEB1 mRNA expressional level was analyzed using RT-qPCR. Drug affinity-responsive target stability (DARTS) method was used to detect the binding activity between Britannin and ZEB1. Co-immunoprecipitation (Co-IP) analysis was applied to test the ubiquitination of ZEB1. The mouse models for experimental lung metastasis of 4T1 cells were established to detect the anti-metastasis effect of Britannin in vivo, and the expressional levels of EMT markers in lung metastases were detected by immunohistochemistry. Results: Britannin could inhibit cell growth and G2/M arrest in TNBC cells. Britannin could inhibit the migrating and invading ability without inducing severe apoptosis of MDA-MB-231 and 4T1 cells. Meanwhile, Britannin reduced the size and number of spheroids formed in these two cells, and decreased the expressional level of stem cells biomarker CD44 in tumor spheroids. Mechanism research showed that Britannin specifically bound to ZEB1 and induced its ubiquitination in MDA-MB-231 cells. Afterwards, Britannin disturbed protein stability and promoted ZEB1 protein degradation. Importantly, Britannin could not inhibit cell invasion and spheroid formation after ZEB1 expression was knocked down. Finally, Britannin inhibition of 4T1 cell metastasis was confirmed through establishing mouse models for the experimental lung metastasis. It was proved that both Britannin and paclitaxel could decrease the lung metastases, and Britannin could also down-regulate the protein expressional levels of ZEB1, MMP9 and CD44. Conclusion: This study reveals that Britannin suppresses the invasion and metastasis of TNBC cells through degrading ZEB1, which suggests that Britannin can be used to prevent tumor metastasis and recurrence via degrading ZEB1 proteins.