Abstract P1-05-12: Norcantharidin suppressed cell migration and invasion by modulating the β-catenin/miR-301a/ZBTB4 signaling axis in triple-negative breast cancer

Purpose: Breast cancer is the leading malignant tumor in women worldwide. Since not responding to endocrine therapy and anti-human epidermal growth factor receptor 2 (HER2) therapy, triple-negative breast cancer (TNBC) has the highest recurrence rate among all subtypes of breast cancer. MicroRNAs have been proved to be involved in the progression of various tumors. It has been demonstrated that miR-301a promotes cell proliferation, migration and invasion in breast cancer. However, the functions and underlying mechanism of miR-301a in TNBC are undefined. Methods: Bioinformatics analysis was conducted to analyze the expression level of miR-301a. RT-qPCR was used to confirm the level of miR-301a in breast cancer patients. Migration and invasion assay was performed to analyze the function of miR-301a. Western blot was carried out to compare the protein level of genes. A luciferase reporter assay was used to verify the downstream target gene of miR-301a. Immunohistochemistry was conducted to evaluate the protein level of ZBTB4. Immunofluorescence was performed to assess the influence of norcantharidin (NCTD) on β-catenin. Finally, lung metastasis model was established in vivo through tail vein injection of breast cancer cells to evaluate the anti-tumor effect of NCTD. Results: In this study, the expression signatures of miRNAs were analyzed using the high-throughput sequencing data from The Cancer Genome Atlas (TCGA). The results showed that miR-301a was dysregulated in TNBC, and the expression of miR-301a was upregulated in breast cancer, especially in TNBC. In order to analyze the clinical relevance and prognostic correlation of miR-301a in breast cancer, complete data with clinicopathology features and overall survival (OS) of 897 breast cancer patients were collected from the TCGA database. The result indicated a high expression level of miR-301a is an unfavorable prognostic factor for breast cancer patients. Moreover, the migration and invasion of TNBC cells were enhanced by miR-301a mimics and weakened by miR-301a inhibitors. Furthermore, ZBTB4 was identified as a downstream target gene of miR-301a by the Western blot and luciferase reporter assay, and overexpression of ZBTB4 reduced the effect of miR-301a on cell migration and invasion. The level of miR-301a was decreased and that of ZBTB4 was elevated following knockdown of β-catenin by siRNA. The result of immunohistochemistry showed that ZBTB4 was downregulated in breast cancer tissues and the result of RT-qPCR of 77 breast cancer tissue samples indicated that the mRNA level of ZBTB4 was significantly lower in TNBC samples than in non-TNBC samples. Then the plasmid of ZBTB4 was constructed and successfully transfected into TNBC cells. However, overexpression of ZBTB4 had no effect on the level of β-catenin. Importantly, we found that NCTD, the demethylated analog of cantharidin, suppressed the migration and invasion of TNBC cells by downregulating miR-301a and upregulating ZBTB4 through the β-catenin signaling pathway. Then lung metastasis model was established by tail vein injection of cancer cells and NCTD decreased the number of lung metastatic lesions in vivo. Conclusion: Collectively, our findings illustrate that miR-301a plays a role in the carcinogenesis of TNBC by targeting ZBTB4, and NCTD may be used as a potential adjuvant treatment for TNBC. Citation Format: Qiang He, Xinrong Luo, Guosheng Ren, Hongyuan Li. Norcantharidin suppressed cell migration and invasion by modulating the β-catenin/miR-301a/ZBTB4 signaling axis in triple-negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-05-12.