Increased Expression of Exosomal AGAP2-AS1 (AGAP2 Antisense RNA 1) In Breast Cancer Cells Inhibits Trastuzumab-Induced Cell Cytotoxicity

Abstract

BACKGROUND Trastuzumab therapy is important for patients with HER2-positive breast cancer, but more and more patients have experienced trastuzumab resistance during recent years. Accumulating evidence from recent studies showed that long non-coding RNAs (lncRNAs) play essential roles in chemoresistance of various cancer types, but the precise role of lncRNAs in trastuzumab resistance is unclear. In the present study, we aimed to identify the biofunction of lncRNA APAP2-AS1 in tranastuzumab resistance and to reveal the underlying regulatory mechanism. MATERIAL AND METHODS By culturing HER2-positive SKBR-3 and BT474 cells with transtuzumab-containing medium, we built trastuzumab-resistant cells. Quantitative real-time PCR was used to test the expression of AGAP2-AS1 in the built trastuzumab-resistant cells. Cell viability assay and TUNEL assay were used to test the cell viability and apoptosis in each group. Exosomes were purified from cells cultured in exosomes-depleted FBS and identified by transmission electron microscopy. RESULTS qRT-PCR assay suggested that AGAP2-AS1 was upregulated in the built trastuzumab-resistant cells when compared with parental sensitive cells. Cell viability assay showed that silencing of AGAP2-AS1 enhanced the cytotoxicity induced by trastuzumab treatment. Mechanistically, we revealed that AGAP2-AS1 was secreted outside cells by incorporation into exosomes in an hnRNPA2B1-dependent manner. More importantly, co-culture AGAP2-AS1-containing exosomes with sensitive cells reduced the trastuzumab-induced cell death, and silencing of AGAP2-AS1 from exosomes reversed this effect. In summary, AGAP2-AS1 promotes trastuzumab resistance of breast cancer cells through packaging into exosomes. CONCLUSIONS Knockdown of AGAP2-AS1 may be helpful for improving the clinical outcome for HER2+ breast cancer patients and could serve as a therapeutic target.

Figure 1

Trastuzumab resistance induces high expression of AGAP2-AS1 in breast cancer. (A) The established trastuzumab-resistant cell lines showed specific morphologic changes, including decreased cell polarity and cell interaction, and increased pseudopodia formation (as indicated by arrows). (B) The cell viability was measured by using CCK8 (cells treated with trastuzumab for 48 h, ** P<0.01). (C) The cell survival rate was determined by CCK8 assay in cells cultured with trastuzumab at different concentrations. (D) The expression levels of AGAP2-AS1 in indicated cell lines were measured with qRT-PCR assay, P<0.05, ** P<0.01 and *** P<0.001. (E) qRT-PCR determination of AGAP2-AS1 expression in trastuzumab-resistant cells and parental cells, ** P<0.01 compared to parental cells.

Figure 2

AGAP2-AS1 promoted trastuzumab resistance of breast cancer cells. (A) Three siRNAs against AGAP2-AS1 were generated and transfected accordingly, * P<0.05; ** P<0.01; *** P<0.001. (B) The transfection efficiency was shown by labeling cells with GFP. (C) Cell viability was evaluated by performing CCK8 assay in cells silenced with AGAP2-AS1 or not. (D, E) Cell apoptosis induced by trastuzumab was determined by using flow cytometry (D) and TUNEL assay (E), * P<0.05, ** P<0.01 compared to si-NC.

Figure 3

AGAP2-AS1 is excreted through incorporating into exosomes. (A) LncLocator (http://www.csbio.sjtu.edu.cn/bioinf/lncLocator/) online software was used to predict the relative scoring of AGAP2-AS1 in different subcellular locations. (B) lncRNA AGAP2-AS1 expression was measured with qRT-PCR, * P<0.05; ** P<0.01 compared to control. (C) The exosomes released from breast cancer cells were imaged and presented by TEM scanning. (D) A Zetasizer device was used to determine the size distribution of purified exosomes. (E) Western blot analysis was performed to verify the existence of HSP70 and CD9 protein. (F) The expression of AGAP2-AS1 contained in exosomes was measured by qRT-PCR, ** P<0.01.

Figure 4

hnRNPA2B1 mediates the incorporation of AGAP2-AS1 into exosomes, which disseminates trastuzumab resistance. (A) The enrichment of AGAP2-AS1 by hnRNPA2B1 was measured by performing RNA immunoprecipitation (RIP). (B) Specific vectors were used to dysregulate the expression of hnRNPA2B1 in breast cancer cells. (C) qRT-PCR was used to detect the influence of hnRNPA2B1 on expression of exosomal AGAP2-AS1, * P<0.05 compared to control vector. (D) The existence of exosomes in recipient cells was tracked by labeling with PKH26 dye. (E) qRT-PCR was performed to determine the expression level of intracellular AGAP2-AS1 in cells co-cultured with exosome or not, ** P<0.01. (F) Cell viability of cells in indicated groups was detected by performing CCK8 assay.