The lncRNA AATBC is highly expressed in breast cancer and associated with advanced tumor stages and poor prognosis
To screen for differentially expressed lncRNAs between breast cancer samples and adjacent breast epithelial tissues, three breast cancer microarray datasets were downloaded from the GEO database (Table S1). The GSE42568 and GSE65194 datasets were used for differential expression analysis between breast cancer samples and adjacent breast epithelial tissues[11]. After SAM, the differential expression probes from the two datasets were integrated and analyzed. There were 1,751 common differentially expressed probes including 1,642 that were highly representing 1,434 genes, and 109 that were relatively lowly expressed, representing 106 genes. The analysis process is shown in Fig.S1a. Based on NetAffx, RefSeq, and Ensembl annotations of noncoding RNAs, seven lncRNAs including AATBC, HOTAIR, LOC642852, taurine up-regulated 1 (TUG1), deleted in lymphocytic leukemia 2 (DLEU2), myocardial infarction associated transcript (MIAT), and uncharacterized LOC730101 were overexpressed, and uncharacterized LOC730101 were overexpressed and one lncRNA [LOC100133920, methylenetetrahydrofolate dehydrogenase (NADP+dependent) 1 like pseudogene] was downregulated in breast cancer tissues, compared to in healthy breast epithelial tissues, in both datasets (Fig.S1b-c).
Among these lncRNAs, AATBC was highly expressed in breast cancer samples according to the GSE42568 and GSE65194 datasets (Fig.1a). Further analysis showed that AATBC overexpression was positively associated with poor overall survival and poor recurrent-free survival of breast cancer patients, according to the GSE20711 and GSE42568 datasets (Fig.1b). In situ hybridization based on 21 pairs of paraffin-embedded breast cancer and adjacent breast epithelial tissue samples confirmed the high expression of AATBC in breast cancer sample (Fig.1c-d).
AATBC promoted breast cancer cell migration and invasion.
First, we verified the overexpression and knockdown effects of an AATBC overexpression vector and two siRNA sequences, respectively, in the MDA-MB-231 and MCF7 cell lines (Fig. S2a). Next, we performed wound healing and transwell experiments and our results showed that AATBC overexpression could promote the migration and invasion of MDA-MB-231 and MCF7 breast cancer cells (Fig. 2a-b; Fig. S2b). A Matrigel-based 3D cell culture model was also used to explore the role of AATBC in MDA-MB-231 cell adhesion and invasion. AATBC overexpression showed a significantly increased number of pseudopodia, whereas expression of the two AATBC siRNAs led to the opposite phenotype (Fig. 2c). We also detected a change in the metastasis-associated molecules in MDA-MB-231 and MCF7 cells after AATBC siRNA treatment or overexpression. AATBC was found to promote the expression of MMP2, MMP9, N-Cadherin, ZEB1, ZEB2, Slug, Snail, and Claudin-1 (Fig. 2d). These results suggested that AATBC promoted breast cancer cell migration and invasion in vitro. In addition, we also confirmed that AATBC affected breast cancer cell apoptosis (Fig. S2c).
AATBC enhanced breast cancer lung metastasis in nude mice
We next examined the in vivo effects of AATBC expression in breast cancer cells. Four-week-old nude mice were randomly divided into three groups (n = 7 mice/group), and were injected with 1 × 107 MDA-MB-231 cells transfected with siAATBC siRNA or the AATBC overexpression plasmid. After 6 weeks, mice were sacrificed. Their lung tissues were removed, fixed, and imaged, and the number of metastatic nodules was counted (Fig. 3a-c). Next, mouse lungs were paraffin-embedded for H&E staining (Fig. 3d). Nodules and cancer nests in nude mice injected with siAATBC-treated cells were smaller and significantly reduced in number than in the negative control mice. In contrast, nude mice injected with the AATBC overexpressing MDA-MB-231 cells had larger nodule numbers and cancer nests than the control mice.
AATBC promoted breast cancer cell migration and invasion by inhibiting the YAP1/Hippo signaling pathway
To determine the mechanism behind AATBC-driven breast cancer metastasis, we identified AATBC-regulated proteins in MDA-MB-231 cells transfected with siAATBC or scrambled siRNA by LC-MS/MS (Fig. S3a). We identified 690 differentially expressed proteins, including 298 that were downregulated by AATBC and 392 that were upregulated by AATBC (Table S4). Enrichment analysis in DAVID revealed that several components of the Hippo signaling pathway were altered upon AATBC knockdown (Fig. S3b). Western blotting was used to confirm the expression of the main components in this pathway among different groups, and p-YAP1 (Ser127) was found to be altered after AATBC knockdown or overexpression (Fig. 4a). Despite the changes observed at the protein level, qRT-PCR analysis indicated that AATBC did not affect the mRNA levels of the main components of the Hippo signaling pathway in MDA-MB-231 cells (Fig. S4). It is well known that YAP1 acts as a transcriptional regulator in the Hippo signaling pathway and activates transcription of genes involved in cell proliferation; further, it suppresses apoptotic genes. Analysis of cytoplasmic and nuclear fractions revealed that the overexpression of AATBC increased YAP1 protein translocation to the nucleus in MDA-MB-231 and MCF7 cells, while siAATBC had the opposite effect (Fig. 4b).
As a nuclear co-transcription regulator, YAP1 can exert biological functions upon entering the nucleus. Connective tissue growth factor (CTGF) is a classical target gene of YAP1, and the transcriptional activity of YAP1 can be detected by the luciferase reporter gene vector harboring the CTGF promoter[12]. The regulatory effect of AATBC on YAP1 prompted us to study whether AATBC regulated the transcriptional activity of YAP1. Thus, we measured the luciferase activity of the CTGF promoter in MDA-MB-231 and MCF7 cells after overexpression or knockdown of AATBC. As shown in Fig. 4C, downregulation of AATBC impaired the transcriptional activity of YAP1 in the MDA-MB-231 and MCF7 cells, whereas forced expression of AATBC enhanced the transcriptional activity of YAP1 in these cells.
Next, we checked whether YAP1 regulated the effects of AATBC on migration and invasion in breast cancer through overexpression or knockdown of YAP1 (two siRNAs) (Supplementary Fig.5a). The results showed that the two siYAP1 and the YAP1 overexpression vector could modulate the function of AATBC with respect to migration and invasion in vitro (Supplementary Fig.5b-g). The wound healing and transwell experiments showed that the knockdown of AATBC or YAP1 inhibited the migration and invasion of MDA-MB-231 and MCF7 cells, whereas the overexpression of AATBC or YAP1 enhanced the migratory and invasive properties of MDA-MB-231 and MCF7 cells. Notably, overexpression of YAP1 reversed the inhibitory effect of siAATBC when the YAP1 overexpression vector and AATBC siRNAs were co-expressed in MDA-MB-231 and MCF7 cells. Moreover, knockdown of YAP1 also reversed the enhancing functions of AATBC when the AATBC overexpression vector and siYAP1 were co-expressed in MDA-MB-231 and MCF7 cells. These data suggested that YAP1 participated in the AATBC-modulated migration and invasion of MDA-MB-231 and MCF7 cells.
AATBC promoted breast cancer cells migration and invasion by interacting with YBX1
To explore the mechanism through which AATBC regulates YAP1/Hippo signaling in breast cancer, RNA pulldown experiments were performed. Biotinylated sense and antisense AATBC strands were transcribed in vitro and incubated with MDA-MB-231 lysates. The RNA-protein complexes were then captured on streptavidin affinity magnetic beads and subjected to mass spectrometry (Fig. 5a). However, the YAP1 protein was not identified, indicating that AATBC did not directly interact with YAP1.
We next screened the mass spectra data and found that YBX1, a Y-box protein, was among the identified potential interacting proteins (Supplementary Table 5); YBX1 was also identified as a potential AATBC-binding protein by the catRAPID algorithm [13](Fig.S6; Table S6). Therefore, we next sought to verify the interaction between YBX1 and AATBC. Western blot experiments showed that the AATBC sense strand could bind to YBX1, whereas the antisense strand displayed very weak binding (Fig. 5b). To confirm this interaction between AATBC and YBX1, RIP experiment were conducted. MDA-MB-231 and MCF7 cell lysates were incubated with YBX1 antibody, and co-precipitated RNA was analyzed by qRT-PCR using primers targeting AATBC, LOC284454 (another lncRNA; used as a negative control), and U1 small nuclear ribonucleoprotein subunit 70 (SNRP70; used as a positive control). The enrichment of AATBC, but not LOC284454 lncRNA, was observed in both cell lines, indicating a specific interaction between AATBC and YBX1 (Fig. 5c). MST1 was selected as a negative control, and it did not interact with AATBC. We next performed a deletion-mapping assay to determine the region of AATBC that interacts with YBX1. The AATBC sense strand was divided into three fragments. The data showed that the Del 1 fragment (nucleotides 1–2270) bound to YBX1 with higher affinity than the Del 2 (nucleotides 2271–3767) and Del 3 (nucleotides 3768–4622) fragments (Fig. 5d). Taken together, these observations indicate that AATBC binds YBX1 via the fragment containing nucleotides 1-2270. Fortunately, we have also proved AATBC mainly interacting with C-terminal fragment (Fig. 5e).
Next, we measured whether AATBC Del 1 promoted migration and invasion through the YAP1/Hippo pathway. The data of the wound healing assay and transwell assay with or without Matrigel showed that overexpression of AATBC Del 1 could induce migration and invasion of MDA-MB-231 and MCF7 cells through the Hippo signaling pathway (Fig. 5f-h).
YBX1 regulated the YAP1/Hippo signaling pathway by interacting with MST1.
Further, transwell experiments with or without Matrigel and wound healing experiments showed that YBX1 reversed the migrative and invasive phenotype of MDA-MB-231 and MCF7 cells mediated by AATBC (Fig.S7). These results suggested that AATBC promoted the migration and invasion of breast cancer cells by directly interacting with YBX1.
Immunofluorescence showed that YBX1 could colocalize with MST1, an upstream kinase of the YAP1/Hippo signaling pathway (Fig. 6a). This indicated further interaction between YBX1 and MST1. Accordingly, this interaction was confirmed by immunoprecipitation (Fig. 6b). Co-immunoprecipitation experiment was used to detect the interaction between MST1 and YBX1 mutants. We transfect YBX1 full length or YBX1 deletion mutants (C-terminal and △C) into MDA-MB-231 cells respectively. 48h later, collect cells for Co- IP experiment. WB detects the specific structural region of MST1 binding YBX1, through experimental results, we determined that the C-terminal of YBX1 is the structural basis for interaction with MST1(Fig. 6c).Moreover, as we predicted, there was an interaction between YBX1 and MST1 in MDA-MB-231 and MCF7 cells, and YBX1 was found to regulate the expression of many components of the YAP1/Hippo signaling pathway (Fig. 6d).
Further, we checked whether YBX1 participated in the regulatory effect of AATBC on the YAP1/Hippo signaling pathway. When AATBC was knocked down, the expression of MST1, MST2, LATS1, and pYAP1 proteins was markedly increased, and overexpression of YBX1 rescued this increase. In contrast, the overexpression of AATBC decreased the expression of MST1, MST2, LATS1, and pYAP1 proteins, and YBX1 depletion could rescue these changes (Fig. 6e and Fig.S7). These data suggested that AATBC regulated the YAP1/Hippo signaling pathway by binding to YBX1.
MST1, a serine/threonine kinase and core component of the mammalian YAP1/Hippo pathway, has two isoforms, a 59-kDa full-length protein and a truncated 36-kDa amino-terminal fragment, both of which have full catalytic activity [14]. To determine whether AATBC contributed to MST1 stability, AATBC was overexpressed in MDA-MB-231 or MCF7 cells, which were then treated with bortezomib to inhibit proteasome-mediated degradation (Fig. 6f; dimethyl sulfoxide [DMSO] was used as a control). In AATBC-overexpressing cells, compared to the negative control cells, levels of 36-kDa MST1 were increased and that of 58-kDa MST1 were decreased, suggesting that AATBC overexpression promoted MST1 cleavage and degradation. AATBC-modulated cleavage and degradation of MST1 could be rescued when YBX1 was knocked down. These results suggested that AATBC regulated MST1 expression by decreasing its physical stability and YBX1 acted as a bridge connecting AATBC and the YAP1/Hippo signaling pathway.
The expression and correlation between AATBC and YAP1, YBX1, and MST1 in clinical samples and mice tissues.
Next, we utilized 21 pairs of paraffin-embedded breast cancer and adjacent breast epithelial tissue samples to assess the potential clinical relationships among AATBC, YBX1, MST1, and YAP1 (Fig. S8a). Immunohistochemical staining showed that MST1 was mainly expressed in the cytoplasm of only 19.04% (4/21) of the breast cancer samples, in contrast to 71.43% (15/21) of the adjacent breast epithelial samples. Conversely, both YAP1 and YBX1 were highly expressed in breast cancer tissues, compared with in adjacent breast epithelial tissues (high expression rate: breast cancer, 100% [21/21] vs. adjacent breast epithelium, 9.5% [2/21]). As shown in Fig.S8b, we defined the patients who showed higher AATBC, YBX1, and YAP1 expression and lower MST1 expression in tumor tissues than in normal tissues as relevant patients. Conversely, we defined the patients who showed the opposite findings as irrelevant patients. We found that 95.24% of patients were relevant patients. We also found that 60% of these patients showed high AATBC, YBX1, and YAP1 expression and low MST1 expression in tumor tissues and low AATBC, YXB1, and YAP1 expression and high MST1 expression in normal tissues. We defined these patients as highly relevant patients. Both types of patients showed that AATBC expression was positively correlated with YAP1 expression and negatively correlated with MST1 expression in breast cancer tissues. Meanwhile, we also examined the expression of AATBC, YBX1, MST1, and YAP1 in the lung tissues of nude mice (Fig.S8c and Table S7). Compared to the negative control group, the AATBC group showed high YBX1 and YAP1 expression and low MST1 expression, whereas the siAATBC group showed the opposite findings. These data suggested that AATBC could negatively regulate MST1 expression and positively regulate YBX1 and YAP1 in vivo.