BANCR-containing extracellular vesicles enhance breast cancer resistance

Abstract

Extracellular vesicles (EVs) are nano-sized particles secreted by many cell types-including tumor cells-and play key roles in cellular communication by transporting functional RNAs. This study aims to elucidate the role of long noncoding RNA BRAF-activated nonprotein coding RNA (BANCR) in EVs derived from breast cancer (BC) cells in trastuzumab resistance. Differentially expressed long noncoding RNA and downstream targets in BC-resistant samples were identified. SKBR-3 cells were treated with trastuzumab to generate resistant cells (SKBR-3TR), and EVs from these cells (SKBR-3TR-EVs) were isolated and characterized. Functional studies of BANCR were performed in SKBR-3 and SKBR-3TR cells. Coculturing SKBR-3 cells with SKBR-3TR-EVs assessed changes in cell behavior. A xenograft model in nude mice examined in vivo tumorigenicity and trastuzumab resistance. BANCR was highly expressed in SKBR-3TR cells and EVs, linked to trastuzumab resistance. SKBR-3TR-EVs transferred BANCR to SKBR-3 cells, where BANCR inhibited miR-34a-5p, reducing its expression. High-mobility group A1 (HMGA1) was identified as a miR-34a-5p target. BANCR activated the HMGA1/Wnt/β-catenin pathway by inhibiting miR-34a-5p, promoting resistance. In vivo experiments showed that BANCR inhibition delayed tumorigenesis and reversed trastuzumab resistance. BC cell-derived EVs containing BANCR may enhance resistance to trastuzumab by regulating the miR-34a-5p/HMGA1/Wnt/β-catenin axis, presenting a potential target for BC therapy.

Keywords: HMGA1; Wnt/β-catenin pathway; breast cancer; extracellular vesicles; lncRNA BANCR; miR-34a-5p; trastuzumab resistance.

Figure 1

Significance of lncRNAs in the resistance of BC cells to trastuzumab.A, a heat map showing the differentially expressed lncRNAs in trastuzumab-resistant (n = 3) and trastuzumab-sensitive cell line samples (n = 3) in the GSE155478 dataset. B, a volcano map showing the differentially expressed lncRNAs in trastuzumab-resistant (n = 3) and trastuzumab-sensitive cell line samples (n = 3) in the GSE155478 dataset. Red indicates up-regulated lncRNAs, and green indicates downregulated lncRNAs. C, a heat map showing the differentially expressed lncRNAs in EVs (n = 3) and BC cells (n = 3) in the GSE115040 dataset. D, a volcano map showing the differentially expressed lncRNAs in EVs (n = 3) and BC cells (n = 3) in the GSE115040 dataset. Red indicates upregulated lncRNAs, and green indicates downregulated lncRNAs. E, Venn diagram of the upregulated lncRNAs from the GSE155478 and GSE155478 datasets. The blue circle represents the highly expressed lncRNAs in drug-resistant BC cells in the GSE155478 dataset, while the red circle indicates the highly expressed lncRNAs in EVs in the GSE115040 dataset. F, BANCR expression in trastuzumab-resistant (n = 3) and trastuzumab-sensitive cell line samples (n = 3) in the GSE155478 dataset. G, BANCR expression in EVs (n = 3) and BC cells (n = 3) in the GSE115040 dataset. LncRNA, long noncoding RNA; EV, extracellular vesicle; BANCR, BRAF-activated nonprotein coding RNA; BC, breast cancer.

Figure 2

BANCR facilitates the resistance of BC cells to trastuzumab.A, BANCR expression in SKBR-3TR and SKBR-3 cells determined by qRT-PCR. B, BANCR expression in SKBR-3TR transduced with lentivirus harboring sh-BANCR and SKBR-3 cells harboring oe-BANCR determined by qRT-PCR. C, IC₅₀ value of trastuzumab in SKBR-3TR treated with sh-BANCR and SKBR-3 cells treated with oe-BANCR determined by CCK-8. D, viability of SKBR-3TR treated with sh-BANCR and of SKBR-3 cells treated with oe-BANCR determined by CCK-8. E, apoptosis rate of SKBR-3TR treated with sh-BANCR and of SKBR-3 cells treated with oe-BANCR determined by flow cytometry. The apoptotic rate is calculated by summing Q2 (late apoptotic cells) and Q3 (early apoptotic cells). ∗p < 0.05 versus sh-NC or oe-NC. Cell experiments were repeated three times. BANCR, BRAF-activated nonprotein coding RNA; BC, breast cancer; Oe-BANCR, overexpression of BANCR.

Figure 3

Transfer BANCR via BC cell–derived EVs regulates BC trastuzumab resistance.A, subcellular localization of BANCR determined by FISH. Red indicates BANCR, and blue indicates DAPI. B, BANCR expression in the nucleus and cytoplasm measured by qRT-PCR. C, extracellular BANCR expression in cell medium added with RNase A or combined with Triton X-100 measured by qRT-PCR (∗p < 0.05 versus RNase A). D, SKBR-3TR-EVs and SKBR-3-EVs observed under a TEM. E, size distribution of SKBR-3TR-EVs and SKBR-3-EVs measured by NTA. F, Western blot of EV marker proteins in the SKBR-3TR-EVs and SKBR-3-EVs and cell extracts. G, the expression of BANCR in cell culture medium, EVs, and EV-free medium measured by qRT-PCR (∗p < 0.05 versus culture medium). H, The expression of BANCR in SKBR-3TR-EVs and SKBR-3-EVs measured by qRT-PCR (∗p < 0.05 verss SKBR-3 EVs). All data are presented as mean ± SD. Cell experiments were repeated three times. EV, extracellular vesicle; BANCR, BRAF-activated nonprotein c oding RNA; BC, breast cancer; NTA, nanoparticle tracking analysis; TEM, transmission electron microscope.

Figure 4

BC cell–derived EVs transfer BANCR to enhance the trastuzumab resistance of BC cells.A, uptake of the SKBR-3TR-EVs labeled with PKH26 by SKBR-3 cells. Red indicates EVs, and blue indicates DAPI. B, BANCR expression in SKBR-3 cells cocultured with SKBR-3TR-EVs or (SKBR-3TR + sh-BANCR) EVs determined by qRT-PCR. C, IC₅₀ value of trastuzumab in SKBR-3 cells cocultured with SKBR-3TR-EVs or (SKBR-3TR + sh-BANCR) EVs determined by CCK-8. D, viability of SKBR-3 cells cocultured with SKBR-3TR-EVs or (SKBR-3TR + sh-BANCR) EVs determined by CCK-8. E, size distribution and number of SKBR-3TR-EVs upon treatment with GW4869 analyzed by NTA. F, IC₅₀ value of trastuzumab in SKBR-3 cells cocultured with SKBR-3TR-EVs or (SKBR-3 TR + GW4869) EVs determined by CCK-8. G, viability of SKBR-3 cells cocultured with SKBR-3TR-EVs or (SKBR-3TR + GW4869) EVs determined by CCK-8. H, apoptosis rate of SKBR-3 cells cocultured with SKBR-3TR-EVs, (SKBR-3TR + sh-BANCR) EVs or (SKBR-3TR + GW4869) EVs determined by flow cytometry. ∗p < 0.05 versus PBS, #p < 0.05 versus SKBR-3TR EVs. All data are presented as mean ± SD. Cell experiments were repeated three times. EV, extracellular vesicle; BANCR, BRAF-activated nonprotein coding RNA; BC, breast cancer; NTA, nanoparticle tracking analysis.

Figure 5

BANCR facilitates trastuzumab resistance of BC cells by acting as ceRNA to sponge miR-34a-5p.A, binding sites of BANCR to the miR-34a-5p sequence predicted by RNA22 website. B, the binding of BANCR to miR-34a-5p verified by dual luciferase reporter assay (∗p < 0.05 versus mimic-NC). C, the binding of BANCR to miR-34a-5p analyzed by RIP assay (∗p < 0.05 versus IgG). D, the expression of miR-34a-5p in SKBR-3TR after BANCR downregulation measured by qRT-PCR (∗p < 0.05 versus sh-NC). E, miR-34a-5p inhibition efficiency measured by qRT-PCR. SKBR-3TR were treated with sh-BANCR or combined with miR-34a-5p inhibitor (∗p < 0.05 versus inhibitor-NC). F, IC₅₀ value of trastuzumab in SKBR-3TR determined by CCK-8. G, viability of SKBR-3TR determined by CCK-8. H, apoptosis rate of SKBR-3TR determined by flow cytometry. In panel F−H, ∗p < 0.05 versus sh-NC or inhibitor-NC, #p < 0.05 versus sh-BANCR. Cell experiments were repeated three times. BANCR, BRAF-activated nonprotein coding RNA; BC, breast cancer.

Figure 6

BANCR activates the HMGA1/Wnt/β-catenin axis by competitively binding to miR-34a-5p, thereby inducing trastuzumab resistance of BC cells.A, a heat map showing the differentially expressed mRNAs in BC-resistant (n = 2) and BC-sensitive cell line samples (n = 2) in the GSE24460 dataset. B, a volcano map showing the differentially expressed mRNAs in BC-resistant (n = 2) and BC-sensitive cell line samples (n = 2) in the GSE24460 dataset. Red indicates upregulated mRNAs, and green indicates downregulated mRNAs. C, a heat map showing the differentially expressed mRNAs in BC-resistant (n = 4) and BC-sensitive cell line samples (n = 4) in the GSE89216 dataset. D, a volcano map showing the differentially expressed mRNAs in BC-resistant (n = 4) and BC-sensitive cell line samples (n = 4) in the GSE89216 dataset. Red indicates upregulated mRNAs, and green indicates downregulated mRNAs. E, Venn diagram of the downstream targets of miR-34a-5p and the upregulated mRNAs in BC-resistant samples. The blue circle represents the prediction results of starBase website, the red circle indicates prediction results of miRWalk website, and the green circle indicates the highly expressed mRNAs in BC-resistant samples in the GSE24460 dataset, while the yellow circle indicates the highly expressed mRNAs in BC-resistant samples in the GSE89216 dataset. F, HMGA1 expression in BC-resistant (n = 2) and BC-sensitive cell line samples (n = 2) in the GSE24460 dataset. G, HMGA1 expression in BC-resistant (n = 4) and BC-sensitive cell line samples (n = 4) in the GSE89216 dataset. H, binding sites of miR-34a-5p in the sequence of HMGA1 predicted by starBase website. I, binding of miR-34a-5p to HMGA1 verified by dual-luciferase reporter experiment. J, correlation of BANCR expression and HMGA1 expression in BC samples (n = 1104) analyzed by starBase website. K, overexpression efficiency of HMGA1 determined by qRT-PCR (∗p < 0.05). SKBR-3TR were treated with miR-34a-5p mimic, oe-HMGA1 alone or in combination. L, viability of SKBR-3TR determined by CCK-8. M, IC₅₀ value of trastuzumab in SKBR-3TR determined by CCK-8. N, apoptosis rate of SKBR-3TR determined by flow cytometry. O, Western blot of the Wnt/β-catenin–related proteins. In panel L−O, ∗p < 0.05 versus mimic-NC, #p < 0.05 versus Oe-BANCR, miR-34a-5p mimic, or oe-HMGA1, and p < 0.05 versus oe-BANCR + oe-miR-34a-5p or oe-miR-34a-5p + oe-HMGA1. Cell experiments were repeated three times. BANCR, BRAF-activated nonprotein coding RNA; BC, breast cancer; HMGA1, high-mobility group A1; Oe-BANCR, overexpression of BANCR.

Figure 7

CBP elevates BANCR expression by mediating acetylation of H3K27.A, H3K27ac enrichment in the BANCR promoter region through the UCSC website. B, H3K27ac enrichment in the BANCR promoter region analyzed by ChIP. C, BANCR expression in SKBR-3TR treated with C646 for 48 h determined by qRT-PCR. D, CBP enrichment in the BANCR promoter region in SKBR-3TR analyzed by ChIP. E, CBP knockdown efficiency measured by Western blot. F, H3K27ac enrichment in the BANCR promoter region in SKBR-3TR after CBP knockdown. G, BANCR expression in SKBR-3TR after CBP knockdown measured by qRT-PCR. ∗p < 0.05. Cell experiments were repeated three times. BANCR, BRAF-activated nonprotein coding RNA; ChIP, chromatin immunoprecipitation.

Figure 8

The effectof BANCR on the resistance and metastasis of breast cancer cells to trastuzumabin vivo.A, qRT-PCR detection of the downregulation efficiency of BANCR after lentivirus infection; B, line graph of tumor volume changes in nude mice from each group after trastuzumab treatment; C, images of tumors from nude mice in each group after trastuzumab treatment; D, comparison of tumor weights in nude mice from each group after trastuzumab treatment; E, immunohistochemical detection of relevant protein expression in tumor tissues from each group of nude mice after trastuzumab treatment; the horizontal line in the bottom right corner represents 25 μm (∗ indicates a significant difference between the two groups with p < 0.05; n = 10). LncRNA, long noncoding RNA; EV, extracellular vesicle; BANCR, BRAF-activated nonprotein coding RNA; BC, breast cancer.

Figure 9

Molecular mechanism graph of BANCR in trastuzumab resistance of BC cells. BC cell–derived EVs transfer BANCR into BC cells, where BANCR competitively binds to miR-34a-5p and activates the HMGA1/Wnt/β-catenin pathway, thus promoting trastuzumab resistance of BC cells. HMGA1, high-mobility group A1; EV, extracellular vesicle; BANCR, BRAF-activated nonprotein coding RNA; BC, breast cancer.

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