Exosome-mediated transfer of lncRNA‑SNHG14 promotes trastuzumab chemoresistance in breast cancer

Abstract

Currently, resistance to trastuzumab, a human epidermal growth factor receptor 2 (HER2) inhibitor, has become an important obstacle to improving the clinical outcome of patients with advanced HER2+ breast cancer. While cell behavior may be modulated by long non‑coding RNAs (lncRNAs), the contributions of lncRNAs within extracellular vesicles (exosomes) are largely unknown. To this end, the involvement and regulatory functions of potential lncRNAs contained within exosomes during the formation of chemoresistance in human breast cancer were investigated. Trastuzumab-resistant cell lines were established by continuously grafting HER2+ SKBR-3 and BT474 cells into trastuzumab-containing culture medium. An lncRNA microarray assay followed by reverse transcription‑quantitative polymerase chain reaction analysis identified that lncRNA-small nucleolar RNA host gene 14 (SNHG14) was upregulated in trastuzumab-resistant cells when compared with parental breast cancer cells. Functional experimentation demonstrated that knockdown of lncRNA‑SNHG14 potently promoted trastuzumab-induced cytotoxicity. Furthermore, extracellular lncRNA‑SNHG14 was able to be incorporated into exosomes and transmitted to sensitive cells, thus disseminating trastuzumab resistance. Treatment of sensitive cells with exosomes highly expressing lncRNA‑SNHG14 induced trastuzumab resistance, while knockdown of lncRNA‑SNHG14 abrogated this effect. The Signal Transduction Reporter Array indicated that lncRNA‑SNHG14 may promote the effect of trastuzumab by targeting the apoptosis regulator Bcl‑2 (Bcl‑2)/apoptosis regulator BAX (Bax) signaling pathway. Furthermore, the expression level of serum exosomal lncRNA‑SNHG14 was upregulated in patients who exhibited resistance to trastuzumab, compared with patients exhibiting a response. Therefore, lncRNA‑SNHG14 may be a promising therapeutic target for patients with HER2+ breast cancer.

Figure 1

Establishment and characterization of trastuzumab-resistant cells. (A) Trastuzumab-resistant cell lines, SKBR-3/Tr and BT474/Tr, presented with specific morphological alterations compared with parental cells (×20 magnification). The arrows indicate the specific alterations, including loss of cell polarity, and increased intercellular separation and formation of pseudopodia. (B) Western blot analysis of epithelial-mesenchymal transition-associated protein expression in trastuzumab-resistant cells compared with parental cells. (C) The cell viability of trastuzumab-resistant and -sensitive cells was detected during treatment with trastuzumab. (D) The half-maximal inhibitory concentration value of trastuzumab was detected for sensitive and resistant cells by cell viability assay. (E) Cell invasion ability was detected by Matrigel Transwell assay for trastuzumab-resistant and -sensitive cells (×20 magnification). ^*P<0.05; ^**P<0.01.

Figure 2

Characterization of exosomes released from trastuzumab-resistant and -sensitive SKBR-3 cells. (A) Transmission electron microscopy images of the exosomes released by SKBR-3/Pr and SKBR-3/Tr cells. (B) Nanoparticle tracking analysis on an LM10 Nanosight unit demonstrating a mean size of 100 nm for SKBR-3/Tr and 120 nm for SKBR-3/Pr exosomes. The size distribution and relative concentration were calculated using the Nano-sight software. (C) Exosomal protein marker (CD63 and CD81) detection by western blotting from purified exosomes and cell extracts. (D) Flow cytometric analysis of the MFI for a panel of exosomal markers: CD9, CD63, CD81 and Alix. Data are presented as the median ± interquartile range of triplicate experiments. MFI, mean fluorescence intensity; CD, cluster of differentiation; Alix, programmed cell death 6-interacting protein.

Figure 3

Exosomal lncRNA-SNHG14 is upregulated in trastuzumab-resistant breast cancer cells. (A) The heat map presents the significantly increased and decreased exosomal lncRNAs in SKBR-3/Tr and BT474/Tr cells when compared with their respective parental cells, as analyzed by Agilent human lncRNA microarray. (B) Extracellular (exosomal) lncRNA-SNHG14 was detected in the respective cells by RT-qPCR. (C) Intracellular (total) lncRNA-SNHG14 was detected in the respective cells by RT-qPCR. ^*P<0.05; ^**P<0.01. lncRNA, long non-coding RNA; SNGH14, small nucleolar RNA host gene 14; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Figure 4

Knockdown of long non-coding RNA-SNHG14 in exosomes partially reverses trastuzumab resistance. (A) The silencing efficacy was evaluated via transfection of three siRNAs targeting SNHG14. (B) Knockdown of exosomal SNHG14 increased the proportion of cell death induced by treatment with trastuzumab in the two trastuzumab-resistant cell lines. (C) Western blotting was used to evaluate the effect of SNHG14-knockdown on c-PARP and caspase-3. (D) Flow cytometry assay of cellular apoptosis caused by knockdown of SNHG14. (E) si-SNHG14 #1 abrogated the improved invasive ability due to trastuzumab in the two cell lines (×20 magnification). ^*P<0.05; ^**P<0.01; ^***P<0.001 vs. respective si-NC group. si, small interfering; SNGH14, small nucleolar RNA host gene 14, c, cleaved; NC, negative control; PARP, poly(ADP-ribose) polymerase.

Figure 5

Exosome-mediated transfer of SNHG14 induces trastuzumab resistance. (A) Intercellular trafficking of exosomes among the different cell lines, as analyzed via isolated exosomes labeled with PKH26 dye. Images are presented at ×40 magnification. (B) Reverse transcription-quantitative polymerase chain reaction assay for the detection of exosomal SNHG14 in cells treated with extracted exosome, or the PBS control, for 48 h. (C) The Cell Counting Kit-8 assay was used for the detection of cell viability in the two cell lines following treatment with extracted exosomes or PBS control for 48 h. (D) Cell viability exhibited little difference when recipient cells were transfected with si-SNHG14 #1 prior to exosomal treatment. ^**P<0.01 vs. respective PBS control. si, small interfering; SNGH14, small nucleolar RNA host gene 14; lncRNA, long non-coding RNA; NC, negative control.

Figure 6

Exosomal long non-coding RNA-SNHG14 promotes trastuzumab resistance by activating Bcl-2/Bax signaling. (A) The histogram illustrates the fold changes in the activities of different signaling pathways, as indicated by reporter activity. (B) Western blotting illustrated that knockdown of SNHG14 prior to exosomal treatment markedly reversed the effect of exosomal treatment on the Bcl-2/Bax pathway. (C) The TUNEL assay demonstrated that exosomal treatment inhibited cellular apoptosis levels, although this effect was abrogated following treatment with the Bcl-2 inhibitor venetoclax (images are presented at ×20 magnification). Bcl-2, apoptosis regulator Bcl-2; Bax, apoptosis regulator BAX; TUNEL, terminal deoxynucleotidyl-transferase-mediated dUTP nick end labelling; si, small interfering; SNGH14, small nucleolar RNA host gene 14.

Figure 7

Serum exosomal SNHG14 levels are upregulated in trastuzumab-resistant patients. (A and B) Reverse transcription-quantitative polymerase chain reaction analysis of serum exosomal lncRNA-SNHG14 (A) and serum total lncRNA-SNHG14 (B) in patients responding or not responding to treatment with trastuzumab. (C) Receiver operating characteristic curve (ROC) analysis of the diagnostic value of exosomal SNHG14 in breast cancer patients receiving treatment with trastuzumab. (D) The proportion of patients that exhibited resistance to trastuzumab therapy was significantly increased in the high exosomal SNHG14-expressing group compared with the low expression group. SNGH14, small nucleolar RNA host gene 14; lncRNA, long non-coding RNA; AUC, area under the curve.

Figure 8

Schematic diagram of exosomal lncRNA-SNHG14 in breast cancer trastuzumab resistance. In trastuzumab-resistant breast cancer cells, lncRNA-SNHG14 promotes trastuzumab resistance by targeting Bcl-2/Bax signaling, inducing the suppression of apoptotic proteins expression and inhibition of cell apoptosis. Moreover, lncRNA-SNHG14 can be packaged into exosomes and secreted from trastuzumab-resistant breast cancer cells, transferring resistance to recipient-sensitive cells. SNGH14, small nucleolar RNA host gene 14; lncRNA, long non-coding RNA; Bcl-2, apoptosis regulator Bcl-2; Bax, apoptosis regulator BAX; HER-2, human epidermal growth factor receptor 2; Casp 3, caspase-3; c-PARP, cleaved poly(ADP-ribose) polymerase.