Background
Resistance to trastuzumab has become a leading cause of mortality in breast cancer patients and is one of the major obstacles for improving the clinical outcome. Cell behavior can be modulated by long non-coding RNAs (lncRNAs), but the contribution of lncRNAs in trastuzumab resistance to breast cancer is largely unknown. To this end, the involvement and regulatory function of lncRNA AGAP2-AS1 in human breast cancer are yet to be investigated.
Conclusion
AGAP2-AS1 could promote breast cancer growth and trastuzumab resistance by activating the NF-κB signaling pathway and upregulating MyD88 expression. Therefore, AGAP2-AS1 may serve as a novel biomarker for prognosis and act as a therapeutic target for the trastuzumab treatment.
Methods
Trastuzumab-resistant SKBR-3 and BT474 cells were obtained by continuous culture with 5 mg/mL trastuzumab for 6 months. RT-qPCR assay was used to determine the expression of AGAP2-AS1 in tissues and cells. RNA fluorescence in situ hybridization was used to investigate the subcellular location of AGAP2-AS1 in breast cancer cells. Bioinformatic analysis, chromatin immunoprecipitation (ChIP), RNA immunoprecipitation (RIP), western blotting, and immunofluorescence were carried out to verify the regulatory interaction of AGAP2-AS1, CREB-binding protein (CBP), and MyD88. In addition, a series of in vitro assays and a xenograft tumor model were used to analyze the functions of AGAP2-AS1 in breast cancer cells.
Results
AGAP2-AS1 was upregulated and transcriptionally induced by SP1 in breast cancer. Overexpression of AGAP2-AS1 promoted cell growth, suppressed apoptosis, and caused trastuzumab resistance, whereas knockdown of AGAP2-AS1 showed an opposite effect. MyD88 was identified as a downstream target of AGAP2-AS1 and mediated the AGAP2-AS1-induced oncogenic effects. Mechanistically, the RIP assay revealed that AGAP2-AS1 could bind to CBP, a transcriptional co-activator. ChIP assays showed that AGAP2-AS1-bound CBP increased the enrichment of H3K27ac at the promoter region of MyD88, thus resulting in the upregulation of MyD88. Gain- and loss-of-function assays confirmed that the NF-κB pathway was activated by MyD88 and AGAP2-AS1. Furthermore, high AGAP2-AS1 expression was associated with poor clinical response to trastuzumab therapy in breast cancer patients.