Abstract P4-04-05: circHEACA promotes anti-HER2 drug resistance by HEACA-GRB7/AKT axis in HER2+ breast cancer patients

Abstract Background: Some HER2-positive (HER2+) breast cancer patient are resistant to anti-HER2 therapy, and the potential reasons of anti-HER2 drug resistance remain unclear. It has been demonstrated that circRNAs play an important role in breast cancer progression, however, the role of circRNAs in anti-Her2 drug resistance remains unknown. In our study, we found and identified a HER2-related circRNA that is associated with malignant progression of HER2 positive breast cancer and preliminary explore their potential mechanisms and clinical significance. Materials and Methods: To identify the HER2-related circRNAs, the deep circRNAs RNA sequencing was performed between the HER2 positive and HER2 negative human breast cancer tissues, and then selected the candidate circRNAs using qPCR to verify the expression level. K-M model was used to define the prognostic performance of circRNA candidates in a breast cancer patient cohort with anti-HER2 drug treatment(N=120). Divergent primers assay and actinomycin D experiments were used to identify the circular features of circRNA candidates. To detect the biological function in breast cancer cells, we knocked-down or over-expressed the circular RNA by siRNA and plasmid transfection. We conducted CHiRP (Chromatin Isolation by RNA Purification) assay to pull down protein complex using biotinylated circRNA probe, and performed mass spectrometric to find out the potential binding protein. Western blot and immunofluorescence (IF) verified that circRNA interacted the protein candidate Results: In this study, we identified a HER2 -Associated CircRNA (named HEACA), and found that divergent primers were amplified in cDNA but not gDNA. Convergent primers amplified in the both cDNA and gDNA. As expected, HEACA was more stable than its linear mRNA when both of them were treated by actinomycin D. Clinical data indicated that HER2+ breast cancer patients with high expression level of HEACA was significantly associated with larger tumour size, more much lymphatic metastasis and higher ki67 index. Kaplan-Meier survival curves showed that HER2+ breast cancer patients with high HEACA who received anti-HER2 therapy has shorter disease-free survival (DFS) and overall survival (OS) than those with low HEACA. Next, we found that know-down HEACA significantly inhibited the proliferation of HER2 positive breast cancer cells through cell variability assay and colony formation, while over-express HEACA highly increased cell proliferation. However, neither know-down nor over-express resulted in affecting the biological function in MCF-7 cells. CircRNA pull down assay and mass spectrometric indicated HEACA may bind to Growth factor receptor-bound protein 7(GRB7). Western blot and IF showed that the protein expression of GRB7 increased or decreased by knocking down or over-express HEACA. Furthermore, we demonstrated that upregulated HEACA promoted breast cancer progression via GRB7/AKT axis in HER2+ cell lines, consequently resulted in a malignant progression of breast cancer cells in vitro and in vivo. Conclusions: HEACA-GRB7/AKT axis plays a critical role in HER2+ breast cancer progression and may be a predictive biomarker for anti-her2 therapy for HER2+ BC patients. Citation Format: Yun Ling, Liang Gehao, Zihao Liu, Zhenluan Tian, Wenjing Zhong, Wanyi Lin, Erwei Song, Gong Chang. circHEACA promotes anti-HER2 drug resistance by HEACA-GRB7/AKT axis in HER2+ breast cancer patients [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 P4-04-05.