Long non‐coding RNA SNHG14 induces trastuzumab resistance of breast cancer via regulating PABPC1 expression through H3K27 acetylation

Abstract Currently, resistance to trastuzumab, a human epidermal growth factor receptor 2 (HER2) inhibitor, has become one major obstacle for improving the clinical outcome of patients with advanced HER2+ breast cancer. While cell behaviour can be modulated by long non‐coding RNAs (lncRNAs), the contributions of lncRNAs in progression and trastuzumab resistance of breast cancer are largely unknown. To this end, the involvement and regulatory functions of lncRNA SNHG14 in human breast cancer were investigated. RT‐qPCR assay showed that SNHG14 was up‐regulated in breast cancer tissues and associated with trastuzumab response. Gain‐ and loss‐of‐function experiments revealed that overexpression of SNHG14 promotes cell proliferation, invasion and trastuzumab resistance, whereas knockdown of SNHG14 showed an opposite effect. PABPC1 gene was identified as a downstream target of SNHG14, and PABPC1 mediates the SNHG14‐induced oncogenic effects. More importantly, ChIP assays demonstrated that lncRNA SNHG14 may induce PABPC1 expression through modulating H3K27 acetylation in the promoter of PABPC1 gene, thus resulting in the activation of Nrf2 signalling pathway. These data suggest that lncRNA SNHG14 promotes breast cancer tumorigenesis and trastuzumab resistance through regulating PABPC1 expression through H3K27 acetylation. Therefore, SNHG14 may serve as a novel diagnostic and therapeutic target for breast cancer patients.

breast cancer patients. 5,6 Trastuzumab is designed to target HER2 and silence its function, and is mostly used for early stage or metastatic gastric and breast cancer patients with positive HER2 mutations. However, trastuzumab might be effective at initial treatments, and resistance increases substantially after a period of exposure. In addition, there is a clear need for useful therapeutic biomarkers that can be used for predicting chemo-response to trastuzumab treatment. 7 Hence, it is urgent and meaningful to reveal the mechanism of trastuzumab resistance and find useful molecular markers and therapeutic targets for breast cancer patients.
With the advanced development of whole genome and transcriptome sequencing technologies and the ENCODE project, it is more and more clear that most of the genome DNA is represented in processed transcripts without or lacking of protein-coding capacity. 8 Long non-coding RNAs (lncRNAs) are a recently discovered major class of non-coding RNAs (ncRNAs) with more than 200 nucleotides in length. 9 In recent years, emerging evidence indicates that they play important roles in regulating cellular and biological functions.
LncRNAs can regulate gene expression at post-transcriptional level via sponging microRNAs 10 and modulate transcriptional gene silencing via the chromatin regulation. 11,12 Thus, the investigation of the role of lncRNAs in breast cancer could help with the understanding of tumorigenesis and the identification of novel diagnostic and therapeutic targets.
The lncRNAs work in a complicated way as critical regulator of epigenetic modulation, transcription and translation in a spatiotemporal manner. 13,14 SNHG14, alternatively named UBE3A-ATS, is located on chromosome 15q11.2. SNHG14 can overlap with the entire UBE3A gene and promoter, thus inhibited the expression of UBE3A, causing neurogenetic disorders, such as Angelman syndrome. 15 Recently, Liu et al 16 17 showed that SNHG14 is a tumour suppressor gene by inhibiting proliferation and invasion in glioma. These contradictory conclusions lead us to identify the function of SNHG14 in breast cancer. Currently, however, the expression pattern, biological function and underlying mechanism of SNHG14 in breast cancer progression and trastuzumab resistance are largely unknown.
In this study, we has been suggested that lncRNA SNHG14 regulated breast cancer progression and resistance via regulating PABPC1 expression through H3K27 acetylation. To verify this hypothesis, we determined the expression level of SNHG14 in breast cancer tissues and cell lines. By performing in vitro and in vivo experimental assays, we further investigated the functional relevance of SNHG14 with breast cancer progression.

| Patient samples
Primary cancer tissue and adjacent non-cancerous tissue samples were collected from one cohort of 36 patients with breast cancer (male/female: 0/36, range of age (median): 35-62 (46)), and another independent cohort of 62 breast cancer patients that received trastuzumab treatment (male/female: 0/62, range of age (median): 49-81 (55)). All the patients were pathologically confirmed and the clinical tissue samples were collected before chemotherapy was started at Hainan General Hospital and The Second Affiliated Hospital of Chongqing Medical University. They were obtained during operation and immediately frozen at −80°C until RNA extraction. Written informed consents obtained from all patients were approved according to the guidelines revised by the Hainan General Hospital and The Second Affiliated Hospital of Chongqing Medical University.

| Cell lines and reagents
The human breast cancer cell lines SKBR-3 and BT474, which harbour HER2 activating mutations, were purchased from Chinese Type Culture Collection, Chinese Academy of Sciences (Shanghai, China).

| RNA oligoribonucleotides and cell transfection
The full-length of lncRNA SNHG14 and the coding sequence of PABPC1 were amplified, cloned into the lentivirus vector for retrovirus production with BT474 cells (Lv-SNHG14 and Lv-PABPC1) by GeneChem (Shanghai, China). Negative control vectors were also generated (Lv-NC). The lentivirus vector containing shRNA sequence targeting PABPC1 (sh-PABPC1), SNHG14 (Lv-SNHG14) or negative control vector (sh-NC) was also amplified and cloned by GeneChem.
All the vectors were labelled with green fluorescence protein (GFP).

| Cell viability assay
The altered cell viability after transfection was assayed using the

| Chromatin immunoprecipitation (ChIP)
ChIP was performed with the EZ ChIP ™ Chromatin Immunoprecipi-

| TUNEL assay
TUNEL staining was performed to evaluate cell apoptosis. In brief, cells were fixed using 4% formaldehyde followed by staining with

| Immunohistochemistry analysis
Immunohistochemical staining was performed on 4-μm-thick TMA slides. Briefly, the slides were deparaffinized and antigen retrieval was then performed in a steam cooker for 1.5 minutes in 1 mmol/L EDTA. Rabbit anti-PABPC1 antibody (#4992, Cell Signaling Technology, Beverly, MA, USA) at 1:150 dilution was used for culture over night at 4 □. Universal secondary antibody (DAKO) was applied for 15 minutes at room temperature. Diaminobenzidine or 3-amino-9ethylcarbazole was used as chromogens and slides were counterstained with haematoxylin before mounting.

| In vivo tumorigenesis assay
Male BALB/C nude mice (6 weeks of age) were purchased from

| Western blots and antibodies
Cell lysates were prepared with RIPA buffer containing protease inhibitors (Sigma-Aldrich). Membranes were incubated overnight at 4°C with a 1:1000 solution of antibodies. A secondary antibody was then used for immunostaining for one hour at room temperature.

| lncRNA SNHG14 is up-regulated in breast cancer and can serve as a diagnostic indicator
To investigate whether the expression of SNHG14 is altered in breast cancer, we detected the expression of SNHG14 in 36 breast cancer tissues and paired adjacent non-tumour tissues. As shown in Figure 1A To determine the expression of SNHG14 in trastuzumab-treated patients, we collected 62 cancer tissues from advanced HER2 + breast cancer patients who received single trastuzumab treatment.
Patients were divided into responding (CR + PR, 33 patients) and non-responding (SD + PD, 29 patients) groups according to the Response Evaluation Criteria In Solid Tumors (RECIST, version 1.1). 18 RT-qPCR showed that SNHG14 was up-regulated in patients who did not respond to trastuzumab treatment than those who showed response to trastuzumab therapy ( Figure 1E). We then investigated the diagnostic potential of SNHG14 by establishing a ROC curve. As shown in Figure 1F, the area under the curve (AUC), diagnostic sensitivity and specificity for differentiating responding and nonresponding patients reached 0.776, 67.65% and 70.59% with the established cut-offs (0.033 at highest AUC), respectively. Under these stratification criteria (0.033), patients were divided into a low and a high SNHG14 expression groups, and the proportion of patients not responding to chemotherapy was significantly higher in the high SNHG14 expression group than in the low expression group ( Figure 1G). Taken together, our clinical data indicate that SNHG14 may be a promising diagnostic marker for breast cancer patients.

| lncRNA SNHG14 promotes proliferation and invasion of breast cancer cells in vitro
We then investigated the functional role of SNHG14 in breast cancer progression and trastuzumab resistance using two HER2+ cell lines, SKBR-3 and BT474. According to the expression of SNHG14 in breast cancer cells, among which SKBR-3 showed the highest level while BT474 indicating a lowest endogenous expression, we constructed SNHG14 overexpression model using BT474 cells and SNHG14 knockdown model using SKBR-3 cells (Figure 2A,B). According to the results from CCK8 assay, we found that BT474 cells overexpressed with SNHG14 showed significantly elevated cell proliferation compared to negative controls, while knockdown of SNHG14 in SKBR-3 cells caused decreased cell growth ( Figure 2C). Further in colony formation assay, the number of formed colonies was much higher in Lv-SNHG14-BT474 cells than Lv-NC-BT474 cells; however, a suppressed colony formation ability was identified in sh-SNHG14-SKBR-3 cells when compared to sh-NC-SKBR-3 cells ( Figure 2D).
To confirm the effect of SNHG14 on cell proliferation, we detected the Ki-67 expression level by immunofluorescence assay. As expected, enhanced SNHG14 promoted Ki-67 expression, whereas SNHG14 knockdown dramatically silenced the level of Ki-67 ( Figure 2E). Next, FACS cell cycle assay showed an increased population in G0/G1 phase and a decreased population in G2/M phase in SNHG14-overexpressed BT474 cells, whereas knockdown of SNHG14 suggested an opposite effect in SKBR-3 cells ( Figure 2F).
Subsequently, we determined whether lncRNA SNHG14 had effects on cell migration and invasion. Wound healing assay indicated that overexpression of SNHG14 promoted cell migration, while knockdown of SNHG14 suppressed the migratory ability ( Figure 2G). Next, transwell assay also showed that SNHG14 promoted breast cancer cell invasion ( Figure 2H). To this end, we demonstrated that SNHG14 played an oncogenic role in breast cancer progression.
F I G U R E 1 lncRNA SNHG14 is up-regulated in breast cancer and can serve as a diagnostic indicator. A, RT-qPCR was used to detect the expression of lncRNA SNHG14 in 36 paired breast cancer tissues and adjacent non-tumour tissues. B, SNHG14 expression level was analysed in 36 primary breast cancer tissues and expressed as log 2 fold change (cancer/normal), and the log 2 fold changes were presented as follows: >1, overexpression (22 cases); <1, underexpression (4 cases); the remainder were defined as unchanged (10 cases). C, The expression level of SNHG14 was detected via RT-qPCR in breast cancer cells. D, ROC curve was drawn to show the ability of SNHG14 in differentiating breast cancer patients from healthy individuals. E, RT-qPCR was used to detect the expression of SNHG14 in 33 responding and 29 non-responding patients who received trastuzumab therapy. F, ROC curve was drawn to show the ability of SNHG14 in differentiating responding patients from non-responding patients. G, The proportion of patients that showed resistance to trastuzumab therapy was significantly higher in high SNHG14 expressing groups than in low expression group. *P < .05, **P < .01

| lncRNA SNHG14 induces trastuzumab resistance of breast cancer cells in vitro
Next, we investigated the functional relevance with trastuzumab in breast cancer cells. Two trastuzumab-resistant sublines derived from HER2 + parental cell lines SKBR-3 and BT474 were established (SKBR-3/Tr and BT474/Tr, respectively). As shown in

SNHG14 function in breast cancer
Based on the understanding of the pathologic role of SNHG14, we continued to determine the underlying functional mechanisms. RNA-pull down experiments were performed followed by mass spectrometry to search for the SNHG14-associated proteins in breast cancer cells. As shown in Table S1, a list of cor-

PABPC1 by modulating H3K27 acetylation of the promoter region of PABPC1
To further understand the regulation of PABPC1 by SNHG14, we explored the probable mechanisms by analysis of ENCODE database (http://genome.ucsc.edu/). As shown in Figure 5A, there was high level of enrichment of H3K27ac in the promoter region of PABPC1, indicating that histone acetylation might participate in the expression of PABPC1 in transcriptional regulation. To test this hypothesis, we performed ChIP assay using SKBR-3 and BT474 cells. As shown in Figure 5B, the enrichment of H3K27ac at promoter of PABPC1 gene was identified in both cell lines. To further investigate whether lncRNA SNHG14 regulates the H3K27 acetylation at PABPC1 promoter, we sublocated the expression of SNHG14 in breast cancer cells. RT-qPCR analysis of nuclear and cytoplasmic lncRNA showed that lncRNA SNHG14 was enriched in nuclear section of both SKBR-3 and BT474 cells ( Figure 5C). FISH assay with specific probe of SNHG14 further confirmed that SNHG14 was mainly distributed in the nuclear section of both cells ( Figure 5D). Then, we detected the enrichment of H3K27ac in the promoter of PABPC1 by ChIP assay.
As expected, up-regulation of SNHG14 in BT474 cells increased the enrichment of H3K27ac in PABPC1, whereas knockdown of SNHG14 resulted in a decreased enrichment in SKBR-3 cells ( Figure 5E).
Next, we investigated whether acetylation of H3K27 is critical for trastuzumab resistance. ChIP assay showed that there was an elevated enrichment of H3K27ac of PABPC1 in trastuzumab-resistant cells when compared to the respective parental cells ( Figure 5F). As  Figure 5G). Collectively, we draw a conclusion that lncRNA SNHG14 regulates PABPC1 expression in breast cancer via modulation of H3K27 acetylation at promoter of PABPC1.  Figure 6B). In addition, immunohistochemistry (IHC) analysis was conducted to determine whether SNHG14 affects the expression of PABPC1 in xenograft tumour tissues. As shown in Figure 6C, overexpression of SNHG14 promoted the level of PABPC1 in either 1% Tween-80 or trastuzumab treatment group (Group II vs Group I or Group III vs Group IV, respectively), indicating that SNHG14 regulates breast cancer carcinogenesis and trastuzumab resistance via targeting PABPC1.

| Nrf2 pathway is promoted by the upregulation of PABPC1 induced by lncRNA SNHG14
To determine the signalling pathway that directly involved in breast cancer progression and chemo-resistance, we used Signal Reporter Array to simultaneously investigate the activity changes of canonical signalling pathways in BT474 cells upon overexpression of SNHG14.
F I G U R E 4 PABPC1 is a downstream target of lncRNA SNHG14 function in breast cancer. A, Schematic diagram of network of PABPC1 based on ENCODE database (http://genome.ucsc.edu). B, PABPC1 was positively regulated by SNHG14 at both transcript and protein levels in breast cancer cells. C, PABPC1 expression was detected via Western blot after transfection of sh-PABPC1 or Lv-PABPC1. D, Knockdown of PABPC1 dramatically abrogated the effects of Lv-SNHG14 on cell proliferation, whereas overexpression of PABPC1 reversed the effect induced by sh-SNHG14. E, PABPC1 knockdown or overexpression vector reversed the Lv-SNHG14-or sh-SNHG14-indued effects of cell invasion, respectively. F, Cells of different groups were treated with trastuzumab (7.0 mg/mL) for 48 h, then the cell viability was determined using CCK8 assay. *P < .05, **P < .01 F I G U R E 5 lncRNA SNHG14 induces an up-regulation of PABPC1 by modulating H3K27 acetylation of the promoter region of PABPC1. A, The genome bioinformatics analysis showed that the promoter of PABPC1 had a high enrichment of H3K27ac. B, ChIP assay demonstrated that H3K27 acetylation occurred in the promoter of PABPC1 in breast cancer cell lines using two primers. C, The expression level of SNHG14 in nuclear and cytoplasm of breast cancer cells. U1 (nuclear retained) and GAPDH (exported to cytoplasm) were used as controls. D, FISH analysis of the subcellular location of SNHG14 with specific probe in breast cancer cells. E, ChIP assay showed that SNHG14 positively regulated the enrichment of H3K27ac at PABPC1 promoter. F, ChIP assay demonstrated that the enrichment of H3K27ac was higher in trastuzumab-resistant cells than in parental cells. G, PABPC1 was up-regulated by Lv-SNHG14 in BT474/Tr cells and down-regulated by sh-SNHG14 in SKBR-3/Tr cells compared with the controls. *P < .05 The five most activated and five most silenced downstream pathways are shown in Table S2, among which we identified the Nrf2 pathway as the mostly activated one. Previous literatures suggested that Nrf2 pathway is one of the major signalling cascades involved in cell defense and survival against endogenous and exogenous stress, such as chemotherapy drugs. 20 Therefore, we has been suggested that SNHG14 may participate in breast cancer tumorigenesis via regulating Nrf2 pathway depending on PABPC1. Western blot assay was performed to test this hypothesis, and we found that

| DISCUSSION
Extensive efforts in the past have contributed to the understanding of both molecular and cellular mechanisms of action of chemo-resistance, one of the major causes for the failure of treatment with advanced cancer types. However, little progress has been made ever since. 21 Thus, novel molecular signatures seem to hold great promise in tumour characterization and could be used as potential prognostic markers and treatment target. To identify potential molecular therapeutic markers for trastuzumab treatment, the functional relevance between lncRNA SNHG14 and breast cancer progression and trastuzumab resistance were investigated.
Our own date showed that SNHG14 promoted breast cancer tumorigenesis and chemo-resistance via activating PABPC1 through H3K27 acetylation ( Figure 7C).  32 In addition, PABPC1 also directly participate in gastric cancer tumorigenesis by influencing cell proliferation. 33 These studies strongly suggest that PABPC1 may play an oncogenic role in cancer progression under the regulation of SNHG14. As expected, we F I G U R E 6 lncRNA SNHG14 promotes trastuzumab resistance in vivo. A, Photographs of tumours that developed in xenograft transplanted nude mouse tumour models treated orally with once daily 50 mg/kg trastuzumab or 1% Tween-80 as control for 4 wk in different groups. B, Weights of tumours that developed in xenografts from different groups are shown. C, IHC analysis of expression levels of PABPC1 in respective groups. *P < .05 confirmed that PABPC1 was essential for SNHG14-induced breast cancer tumorigenesis and trastuzumab resistance.
We further explored the underlying regulation model of increased PABPC1 by SNHG14. Mechanisms that generate transcript diversity are of fundamental importance in cancers. In recent years, regulatory factors such as histone modifications, suggesting that epigenetic features may have the ability not only to determine when and in which tissues certain genes are expressed, but also to influence how these transcripts are processed. 34 Histone acetylation is a major histone modification involved in the regulation of chromatin structure and transcription. It neutralizes the positive charge on the lysine side chain, relaxing the chromatin structure and enhancing transcriptional activity. 35 Histone acetylation named H3K27Ac was first discovered in yeast 36 and recent advancements in DNA sequencing technology have enabled the analysis of histone acetylation distribution patterns through whole genome. 37 In this study, we investigated the H3K27ac concentration at the promoter of PABPC1 by analysis of ENCODE database, followed by a serious of experimental verifications including RT-qPCR, ChIP and Western blots using breast cancer tissues and cells. We identified that PABPC1 was highly enriched with H3K27ac at the promoter of PABPC1 in breast cancer tissues. Moreover, this histone acetylation was mediated by SNHG14 as evidenced by the changed enrichment induced by overexpression or knockdown of SNHG14 in breast cancer cells.
We should address some points which need a more comprehensive investigation in future studies. First, it is still unclear whether SNHG14 directly mediates the histone acetylation or by binding with the RNA-binding proteins (RBPs), such as CREB-binding protein (CBP), which is a transcriptional co-activator with histone acetyltransferase (HAT) activity. Second, this study initially identified PABPC1 as a target protein through screening for SNHG14-interacting proteins using RNA-pulldown assay. However, we eventually verified that SNHG14 regulated the expression of PABPC1 at transcript level through mediating H3K27 acetylation. In the future, we need to reveal the underlying regulatory mechanism by which PABPC1 regulates SNHG14 expression at both transcript and protein levels.
Finally, we performed in vivo experiments to verify the in vitro results. As expected, SNHG14 abrogated the trastuzumab-induced growth suppression of xenograft tumour, indicating that SNHG14 promotes trastuzumab resistance in our mice models. Moreover, SNHG14 promotes breast cancer tumorigenesis via regulating Nfr2 pathway depending on PABPC1. In summary, our study revealed that lncRNA SNHG14 promotes breast cancer progression and chemoresistance to trastuzumab treatment both in vitro and in vivo. Therefore, SNHG14 could be considered as a promising diagnostic biomarker and therapeutic target for breast cancer patients, enhancing the clinical benefits of trastuzumab therapy.

CONFLI CT OF INTEREST
The authors declare that they have no competing interests.