Quaking-5 suppresses aggressiveness of lung cancer cells through inhibiting β-catenin signaling pathway

Quaking-5 (QKI-5) belongs to the STAR (signal transduction and activation of RNA) family of RNA binding proteins and functions as a tumor suppressor in several human malignancies. In this study, we attempt to elucidate the role of QKI-5 in the pro-metastasis processes of lung cancer (LC) cells and the underlying mechanisms. We confirmed that QKI-5 was decreased in human LC tissues and cell lines, especially in high-metastatic cells. Moreover, QKI expression was positively correlated with LC patients’ survival. Functional studies verified that QKI-5 suppressed migration, invasion and TGF-β1-induced epithelial-mesenchymal transition (EMT) of LC cells. Mechanistically, we affirmed that QKI-5 reduced β-catenin level in LC cells via suppressing its translation and promoting its degradation, whereas QKI-5 promoter hypermethylation suppressed QKI-5 expression. Our findings indicate that QKI-5 inhibits pro-metastasis processes of LC cells through interdicting β-catenin signaling pathway, and that QKI-5 promoter hypermethylation is a crucial epigenetic regulation reducing QKI-5 expression in LC cells, and reveal that QKI-5 is a potential prognostic biomarker for LC patients.


INTRODUCTION
Lung cancer (LC) currently ranks as one of the most prevalent neoplasms and the leading cause of cancer-related deaths worldwide [1]. According to the histological characters, LC can be classified into small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) which accounts for about 85% of all the LCs [2]. Confronting the facts of its increasing incidence, considerable mortality and poor prognosis, LC has become a great threat for human health [3]. Therefore, further efforts are urgently needed to comprehensively understand the internal biological mechanisms and to identify more candidate therapy targets for improving the clinical outcomes of LC patients.
As is known, metastasis occurs in about 40% of newly diagnosed LC patients [4]. For all types of LCs, treatment failure is mainly attributed to distant metastasis involving lymph node, brain and liver [5]. Epithelial-mesenchymal transition (EMT) is a biological process that enables epithelial cells to acquire mesenchymal phenotype [6]. It can be triggered by various ligand-receptor interactions, including TGF-β1, and involves extensive regulatory networks which are www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 47), pp: 82174-82184 Research Paper controlled by transcription factors and else [7]. Over the past decades, increasing evidences have demonstrated that EMT contributes to tumor progression, especially to tumor invasion and metastasis [6]. Wnt/β-catenin signaling pathway is crucial in human malignancies, including LC [8]. Notably, numerous studies have linked the metastasis and EMT induction with Wnt/β-catenin activation [9][10][11]. Hence, it is important to understand how the crosstalk between key factors and Wnt/βcatenin signaling pathway promotes the aggression of LC cells, which may provide new modalities for LC therapeutic intervention.
Quaking (QKI), a member of signal transduction and activation of RNA (STAR) protein family, is a pivotal post-transcriptional regulator [12]. The QKI gene encodes three major alternatively spliced mRNAs, i.e., QKI-5, QKI-6, and QKI-7 with different C-terminals. QKI-5 is prominently expressed in early embryogenesis, while QKI-6 and -7 are mainly expressed in the central nervous system in late development stage [13]. Through recognition and binding to the QKI responsive element (QRE, ACUAAY [N1-20] UAAY) in the 3′-UTR of mRNA, QKI impacts diverse aspects of target mRNAs including the location, stability, and translational efficiency in multiple physiological and pathological processes [14]. In LC, it has been demonstrated that QKI-5 is the dominant isoform of QKIs [15], and its downexpression is an important cause leading to cell proliferation acceleration by influencing alternative mRNA splicing of at least two genes, NUMB and MacroH2A1 [15,16]. However, the effect of QKI-5 on LC metastasis and the underlying mechanism remain poorly understood.
In the present study, we confirm that QKI-5 is decreased in human LC tissues and cell lines, especially in high-metastatic cells, and identify that QKI-5 inhibits the migration, invasion and TGF-β1-induced EMT of LC cells by directly decreasing β-catenin. Moreover, we uncover that QKI-5 reduction in LC cells results from QKI promoter hypermethylation. Our data demonstrate for the first time that QKI-5 is a key inhibitor of LC cell aggressiveness and that methylation of QKI promoter contributes to QKI-5 downexpression in LC.

Figure 1: QKI-5 downexpression in human LC tissues and cell lines with metastasis is accompanied by EMT. (A)
IHC representative images of QKI-5 expression in bronchial epithelial (a), alveolar epithelium (b), lung adenocarcinoma (c, d) and lung squamous cancer (e, f). Scale bar, 50 μm. (B) Comparisons among groups of QKI-5 mRNA level in 20 non-cancerous lung tissues, 20 LC tissues without (No LNM) and 20 LC tissues with (LNM) lymph node metastasis. The QKI-5 mRNA level was analyzed by qRT-PCR and normalized against GAPDH. The ratio of QKI-5/GAPDH in LNM group was arbitrarily set to 1.0. The data are presented as the mean ± SD. ***P<0.001. (C) Western blotting analyses of QKI-5 and EMT markers (E-cadherin, N-cadherin, Vimentin) in two pairs of differential metastatic LC cell lines (AGZY vs ANIP, 95C vs 95D). α-tubulin was used as the loading control. www.impactjournals.com/oncotarget

QKI-5 expression is decreased in high-metastatic LC tissues and cell lines
To investigate the potential roles of QKI in LC progression, we firstly characterized the expressions of several QKI isoforms generated by alternative splicing, and found that QKI-5 was the dominant isoform expressed in LC cells (Supplementary Figure 1A-1C). By utilizing the specific antibody as determined in Supplementary Figure 1C, we analyzed QKI-5 expression in LC tissues by IHC. The results showed that QKI-5 was homogeneously presented in the nucleus of bronchial epithelium and pneumocytes in non-cancerous lung tissues, but was nearly absent in lung adenocarcinoma and squamous cell carcinoma ( Figure 1A). Significantly, the survival analysis of 1926 LC patients based on Kaplan Meier plotter demonstrated that lower QKI mRNA expression predicted a shorter overall survival (P<0.001; Supplementary Figure 2). The result reveals that QKI-5 is a potential prognostic biomarker for LC patients. We next detected QKI-5 mRNA expression in 60 tissue samples (20 non-cancerous lung tissues, 20 LC samples without (No LNM) and 20 LC samples with lymph node metastasis (LNM)). In comparison with non-cancerous lung tissues, QKI-5 mRNA reduction was more obvious in metastatic tumors than those of non-metastatic tumors (P<0.001; Figure 1B). We then examined the expressions of QKI-5 mRNA and protein in two pairs of low and high metastatic LC cell lines (AGZY vs ANIP, 95C vs 95D). As shown in Supplementary Figure 3A, QKI-5 mRNA was significantly reduced in high-metastatic ANIP and 95D cells (P<0.05~0.01), which was accompanied by the decrease of QKI-5 protein and E-cadherin as an epithelial marker, and increase of N-cadherin and Vimentin as mesenchymal markers ( Figure 1C). These data indicate that QKI-5 is an inhibitor of LC metastasis and that its downexpression is an important cause inducing EMT and metastasis of LC.

QKI-5 inhibits the migration and invasion of LC cells
To elucidate the possible roles of QKI-5 in the pro-metastasis processes of LC cells, we examined its expression level in multiple LC cell lines. The results showed that QKI-5 was decreased in these LC cell lines compared with a bronchial epithelial cell (Beas2B), accompanied by the increase of N-cadherin and Vimentin (Supplementary Figure 3B). As shown in Supplementary Figure 3B, QKI-5 level was the lowest in H1299 cells and modest in A549 cells. Therefore, gain-of-function studies were performed in H1299 cells, whereas loss-of-function studies in A549 cells. Stable QKI-5-overexpressing H1299 cells and QKI-5-silenced A549 cells were constructed by respective retroviruses or plasmids (Figure 2A and 2B).
Wound healing and transwell invasion assays showed that QKI-5 overexpression significantly suppressed the migration and invasion of H1299 cells (P<0.001; Figure 2C). In contrast, QKI-5 knockdown facilitated the migration and invasion of A549 cells (P<0.01~0.001; Figure 2D). Identical effects were also observed in the SPC-A1 cells (P<0.05~0.001; Supplementary Figure 4A-4C). Since migration and invasion are the key procedures of LC metastasis, these findings further identify the inhibitory effect of QKI-5 on the pro-metastatic behaviors of LC cells.

QKI-5 overexpression attenuates the TGF-β1induced EMT of LC cells
It has been previously reported that NSCLC H1299 cells are converted to fibroblastic phenotype in response to TGF-β1. To ascertain the role of QKI-5 in LC cell EMT, we conducted the experiment inducing EMT with TGF-β1 (5 ng/ml). At 48 h after induction, H1299-vector control cells showed spindle fibroblastlike morphology with reduced cell-cell contact and appeared more significant at 72 h, whereas QKI-5overexpressing H1299-QKI-5 cells still remained epithelial shape at 48 h after induction and only The mRNAs were detected by qRT-PCR and normalized against GAPDH. The ratios of the above mRNAs to GAPDH mRNA in control cells (vector) were arbitrarily set to 1.0. The data are presented as the mean ± SD. **P<0.01, ***P<0.001. (C) Protein levels of QKI-5, E-cadherin and N-cadherin measured by Western blotting in the indicated H1299 cells. www.impactjournals.com/oncotarget exhibited minor morphological alteration at 72 h ( Figure  3A). qRT-PCR and Western blotting analyses verified that TGF-β1-induced morphological transformation of H1299-vector cells was accompanied by significant decrease of E-cadherin as an epithelial marker and increase of N-cadherin, Vimentin, Snail as mesenchymal markers (P<0.001; Figure 3B and 3C). Moreover, QKI-5 overexpression in H1299-QKI-5 cells not only increased E-cadherin but also significantly reversed the suppressive or stimulative effect of TGF-β1 on the expression of E-cadherin, N-cadherin, Vimentin or Snail (P<0.01~0.001; Figure 3B and 3C), which was consistent with their epithelial phenotype with enhanced cell-cell contact ( Figure 3A). QKI-5 overexpression in A549 cells also induced similar resistance to TGF-β1induced EMT (P<0.01~0.001; Supplementary Figure 5A and 5B). These results verify that QKI-5 overexpression can indeed interdict TGF-β1-induced EMT of LC cells.
As shown in Figure 5C, β-catenin accumulation in H1299-QKI-5 cells was still lower than that in H1299-vector cells when its degradation was blocked by GSK3β inhibitor (SB415286) or proteasome inhibitor (MG132), but both the overexpression and knockdown of QKI-5 did not change β-catenin mRNA transcription in two LC cells (Supplementary Figure 5D), suggesting that QKI-5 might also suppress β-catenin expression through inhibiting its translation. It is known that the 3'-UTR of β-catenin mRNA contains two potential QREs. RNA immunoprecipitation (RIP) confirmed that flag-tagged QKI-5 was co-precipitated with β-catenin mRNA in H1299 cells ( Figure 5D). We then constructed the luciferase reporter containing the two potential QREs of β-catenin 3'-UTR, and verified that QKI-5 overexpression obviously inhibited the activity of β-catenin 3'-UTR in H1299 and A549 cells (P<0.01~0.001; Figure 5E). The above results reveal that QKI-5 not only expedites β-catenin degradation, but also suppresses β-catenin translation by binding with the QREs in its mRNA 3'-UTR, thereby decreasing β-catenin and directly blocking its signaling pathway in LC cells.

QKI promoter hypermethylation inhibits its expression in LC cells
To further clarify the underlying mechanism resulting in QKI-5 downexpression in LC, we focused on QKI promoter methylation. The online promoter analysis found that QKI promoter region, especially from -700bp to transcription initiation site (TIS), had abundant CpG islands ( Figure 6A). Methylation specific PCR (MSP) verified that QKI promoter methylation was very frequent in LC and LC cell lines, and more obvious in LC with LNM and high-metastatic ANIP and 95-D cells ( Figure  6B), which was in accord with the QKI-5 expression level in Figure 1B and 1C. Furthermore, the treatment of 5-aza-2′-deoxycytidine (DAC), a demethylation agent, significantly increased QKI-5 mRNA level in LC cells ( Figure 6C). The results indicate that QKI-5 promoter hypermethylation is an important mechanism leading to QKI-5 downexpression in LC cells.

DISCUSSION
QKI-5 is a vital regulator of RNA processing and cell signal transduction, and the change of alternative mRNA splicing induced by its downexpression is an important cause accelerating cell proliferation in multiple cancers including LC [15][16][17]. However, whether QKI-5 suppresses LC metastasis and the underlying mechanism remain unknown. In the present study, we identified that QKI-5 expression was decreased because of its promoter hypermethylation in LC, particularly in metastatic LC. Moreover, we uncovered for the first time its new functions suppressing migration, invasion and TGF-β1-induced EMT in LC through a novel mechanism decreasing β-catenin level in LC cells. These findings facilitated our understanding of LC metastatic mechanisms.
The relations between RNA-binding proteins including QKIs and cancers have attracted much attention [16,18,19]. Our results demonstrated that QKI-5 was the dominant isoform of QKIs in LC cells and that its expression in metastatic LC tissues and high-metastatic LC cell lines was lower than that in non-metastatic LC tissues and low-metastatic LC cell lines. Moreover, patients' overall survival was positively correlated with QKI level. Furthermore, QKI-5 downexpression was accompanied the reduction of E-cadherin as an epithelial marker and the increase of N-cadherin and Vimentin as mesenchymal markers in high-metastatic LC cell lines. The functional assays confirmed that QKI-5 overexpression or knockdown could significantly suppress or facilitate the migration and invasion of LC cells, and that QKI-5 overexpression effectively inhibited the TGF-β1-induced EMT of LC cells. These facts indicated that QKI-5 was an important inhibitor of migration, invasion and EMT of LC cells, highlighting the accelerative effects of QKI-5 downexpression on LC pro-metastasis processes and revealing QKI-5 is a potential prognostic biomarker for LC patients.
It has been established that β-catenin, a key relay in Wnt signaling pathway, promotes the genesis and progression of LC via activating downstream gene transcription [20,21]. Our results showed that QKI-5 overexpression or knockdown not only decreased or increased the luciferase activity of TOP flash receptor, and β-catenin accumulation and its downstream gene expressions (c-myc, cyclin D1, MMP2) in LC cells, but also suppressed or facilitated the invasion and TGF-β1-induced EMT of LC cells. Moreover, β-catenin overexpression or knockdown could effectively reverse the above effects of QKI-5 overexpression or knockdown. These data indicated that QKI-5 inhibited the migration, invasion and EMT of LC cells through blocking β-catenin signaling pathway, highlighting the potential values of QKI-5 and β-catenin in LC therapy.
Previous studies have demonstrated that QKI-5 may suppress β-catenin translation by directly binding with QREs in β-catenin mRNA 3'-UTR [14,23], and inhibits the proliferation of colon cancer cells by decreasing β-catenin [23]. In LC cells, we verified that QKI-5 could directly bind to QREs in β-catenin mRNA 3'-UTR and attenuate its translation efficiency by RNA immunoprecipitation and the 3'-UTR luciferase reporter, but did not reduce β-catenin mRNA. Our findings revealed that QKI-5 decreased β-catenin in LC cells by not only accelerating degradation but also repressing translation, thus blocking β-catenin signaling pathway in LC cells.
QKI-5 promoter hypermethylation results in QKI-5 downexpression in colon cancer cells [23]. In the current study, we identified that QKI promoter region was rich in CpG islands, especially from -700bp to transcription initiation site. Moreover, QKI promoter was methylated in LC tissues and cell lines, and the methylation level was higher in metastatic LC tissues and high-metastatic LC cell lines. Furthermore, demethylation agent DAC significantly increased QKI-5 expression in LC cells. These results indicate that QKI-5 promoter hypermethylation is an important mechanism leading to QKI-5 downexpression in LC cells.
In summary, our study demonstrated that QKI-5 repressed the migration, invasion and TGF-β1-induced EMT of LC cells by directly decreasing β-catenin, and predicted better prognosis in LC patients. More importantly, we revealed a novel mechanism accelerating LC pro-metastatic processes, and QKI-5 and β-catenin might be the therapeutic candidates for this lethal disease.

Tissue samples and clinical data
The surgical specimens of 40 LCs and 20 noncancerous lung tissues (control) were collected from Tianjin Medical University General Hospital (TMUGH) with written consent. The metastatic status of patients was recorded on the basis of pathological and clinical examinations at the time of resection. The 40 LC samples were divided into two groups without or with LNM. The patients' clinical features were summarized in Supplementary Table 1. For histological analysis, specimens were immediately fixed in 3.7% buffered formaldehyde solution after surgical excision and embedded in paraffin afterwards. Another repeats of tissues were stored in liquid nitrogen within 30 min after resection for detecting the mRNA and promoter methylation of QKI-5. This study was carried out in accordance with the principles of the Helsinki Declaration and approved by the Ethics Committee of TMUGH.
An independent cohort of 1926 LC patients from the Kaplan Meier plotter (http://www.kmplot.com) was used to verify the relevance of QKI mRNA level to overall survival of LC patients.

RNA isolation, RT-PCR and qRT-PCR
Total RNA was isolated using the TRIzol reagent (Invitrogen, USA) as the standard protocol. To determine the mRNA expressions of QKI transcripts, RT-PCR was performed as previously described [18], with M-MLV reverse transcriptase (Promega, USA). The specific primers were synthesized by BGI (Beijing Genomics Institute, China) and listed in Supplementary Table 2. qRT-PCR with the SYBR Green PCR kit (Takara Bio, China) was used to quantify the mRNAs involved in this study and GAPDH mRNA was used as the internal control. The specific primers were synthesized by BGI (China) and listed in Supplementary Table 3. The fold changes of mRNA levels were calculated by the 2 -ΔΔCt method.

RNAi-mediated gene silencing
The small interfering RNA (siRNA) targeting β-catenin was synthesized by Gene Pharma (China), and the sequence was listed in Supplementary Table 6. The cells were transfected with 50 nM siRNA dissolved in DEPC water using Lipofectamine RNAiMAX (Invitrogen, USA) according to the manufacturer's protocol.

Wound healing and transwell invasion assays
Uniform artificial wounds were made at 2 d after transfection and the cells were cultured for another 24 h. Cell migration ability was represented by the wound gap distance in microscopic field (×40) at the time points of 0 and 24 h. For transwell invasion assay, at 24 h after transfection, the cells (3×10 4 /well) suspended in medium containing 1% FBS were seeded into the upper well of the transwell chamber pre-coated with 50 μl 1:4 diluted Matrigel (BD Bioscience, USA) and allowed to invade towards the medium containing 10% FBS for 24 h. The cells that reached the lower surface were fixed with methanol and stained with 0.1% crystal violet. The cells were counted in 5 randomly selected microscopic fields (×400) from each chamber. www.impactjournals.com/oncotarget

RNA immunoprecipitation (RIP)
RIP assay was performed using Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) according to manufacturer's instruction. Flag antibody was used in RIP assays. The RNAs in immunoprecipitated complex and in the previously saved input fraction were extracted for further RT-PCR. Specific primers were applied for detecting the target mRNA (Supplementary Table 2).

Methylation specific PCR (MSP)
Genomic DNA was extracted from LC tissues and cell lines using a DNA extraction kit (Promega, USA). After quantification, 2 μg of DNA was treated with sodium bisulfate as previously reported [28], and used as the template DNA for PCR. The MSP primers were designed with the MethPrimer (http://www.urogene.org/cgi-bin/ methprimer/methprimer.cgi) [29] and sequences were listed in Supplementary Table 2. Then, the PCR products were electrophoresed on a 1.2% agarose gel.

Statistical analyses
Statistical analyses were performed using SPSS 21.0 software (IBM, USA). One-way ANOVA test, Student t test, Pearson correlation analysis, Kaplan-Meier analysis and log-rank test were used to analyze corresponding data in this study. Results were presented as the mean ± standard deviation (SD). Statistical significance was assigned at P<0.05 (*), P<0.01 (**) or P<0.001 (***). All the experiments of cell lines were performed at least three times with triplicate samples.