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Inhibition of the proliferation, invasion, migration, and epithelial-mesenchymal transition of prostate cancer cells through the action of ATP1A2 on the TGF-β/Smad pathway

Background Prostate cancer (PC) is one of the major male malignancies worldwide. Because Na+-K+-ATPase is widely involved in various pathological processes, but the action of its α2 subtype (ATP1A2) in PC is unclear, we investigated the role of ATP1A2 in the invasion and migration of PC cells. Methods We measured the expression levels of ATP1A2 in human normal prostate epithelial cell line (RWPE-1) and PC cell lines (PC-3 and DU145) by quantitative real-time PCR (qRT-PCR) and western blot. Cell proliferation, apoptosis, migration, and invasion of PC-3 and DU145 cells were investigated through clone formation assay, EdU assay, flow cytometry and transwell assay, respectively. The effect of ATP1A2 on a tumor-inhibitory pathway [transforming growth factor-β (TGF-β)/Smad] was assessed using western blot. In addition, tumor formation was detected using in vivo xenograft model in male BALB/c nude mice. Results The Cancer Genome Atlas (TCGA) analysis showed that ATP1A2 expression was reduced in PC patients (P<0.05), and patients with low ATP1A2 expression had a lower survival rate (P<0.05). ATP1A2 levels were significantly reduced in PC-3 and DU145 cells, compared with RWPE-1 cells (P<0.01). We also demonstrated that overexpression of ATP1A2 significantly inhibited the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of PC-3 and DU145 cells (P<0.01) and promoted apoptosis (P<0.01). However, silencing ATP1A2 had the opposite effect (P<0.01). In addition, overexpression of ATP1A2 significantly inhibited the TGF-β/Smad pathway (P<0.01), whereas silencing ATP1A2 activated the TGF-β/Smad pathway (P<0.01). Meanwhile, the effect of ATP1A2 silencing on the proliferation, apoptosis, migration and invasion was reversed by TGF-β/Smad pathway inhibitor (LY364947). Furthermore, ATP1A2 inhibited tumor growth in vivo. Conclusions ATP1A2 inhibited proliferation, apoptosis, migration, invasion, and EMT in PC by inhibiting the TGF-β/Smad pathway.


Introduction
Prostate cancer (PC) is a relatively common malignancy of the male genitourinary system, particularly affecting middle-aged and older men, and has a high incidence in Europe and the USA (1), with an increasing incidence in Asian countries in recent years (2). PC is highly aggressive and prone to metastasis (3), which is the cause of almost all deaths of PC patients and occurs mostly in the late stage of the disease, is usually resistant to various treatment methods and has a 5-year survival rate of >30% (4,5). Therefore, it is particularly urgent to understand the mechanism of the occurrence and development of PC and to find new and effective diagnostic techniques and treatments for PC.
The well-studied sodium pump (Na + -K + -ATPase) is widely involved in various pathological processes (6). It has different subtypes, including four different subtypes of the α subunit, which all have different functions. As an example, α1, or ATP1A1, is considered to play a tumor-promoting role in malignant gliomas, small cell lung cancer, and other tumors (7,8); α3 (ATP1A3) is considered to have the ability to promote metastasis of colon cancer (9); α4 (ATP1A4) exists only in testicular tissue (10); and repeated mutations in ATP1A2 have been found to be involved in the biological processes of lung cancer cell metabolism, division, death, and immune regulation (11). Moreover, the data of The Cancer Genome Atlas (TCGA) have demonstrated that ATP1A2 expression is markedly reduced in PC tissues, and low ATP1A2 expression in patients is associated with a lower survival rate. However, there are few published studies of the relationship between α2 (ATP1A2) and PC. Therefore, the purpose of this study was to measure the expression of ATP1A2 in PC and explore its effect on the invasion and migration of PC cells in two cell lines (PC-3 and DU-145), with the goal of expanding our knowledge of the role of Na + -K+-ATPase in tumors and to provide new ideas for the treatment of PC in clinical practice.
The ligand, receptor, and intracellular signal transduction molecule Smad protein of the transforming growth factor-β (TGF-β) signal transduction pathway forms a tumorinhibitory pathway (12,13). Abnormality of any element in the pathway can cause signal transduction disorders and lead to tumorigenesis. Epithelial-mesenchymal transition (EMT) is a process in which epithelial cells lose their epithelial phenotype and acquire a stromal phenotype, which then transforms into stromal cells (14). As an important part of the deterioration and metastasis of PC, the activation of EMT is regulated by a series of gene expression changes. Previous studies have found that the TGF-β1/Smad pathway regulates the expression of Snail and Twist1 (15). An abnormally activated TGF-β1/Smad pathway mediates EMT by inducing Snail1 overexpression in most tumors (16). The binding of TGF-β1 to the TGF receptor on the cell surface leads to phosphorylation of TGF-β RII and further activates Smad3. P-Smad3 enters the nucleus to initiate the expression of Snail1 and Twist (17). Basic studies have found that the EMT of tumor cells can be reversed by inhibiting the TGF-β/Smad pathway.
In this study, we for the first time revealed the expression level and effect of ATP1A2 in PC and unraveled that ATP1A2 played significant roles in PC through the regulation of TGF-β/Smad pathway. We present the following article in accordance with the ARRIVE reporting checklist (available at https://dx.doi.org/10.21037/tau-21-1117).

Cell culture and treatment
Human PC cell lines (PC-3, VCaP, LNCaP and DU145) and human normal prostate epithelial cell line (RWPE-1) were acquired from the American Type Culture Collection (ATCC, USA). The cells were cultured in complete culture medium containing 10% fetal bovine serum (Dulbecco's Modified Eagle Medium) at a constant temperature of 37 ℃ in a 5% CO 2 incubator. Cells in the logarithmic growth phase and in good condition were selected for the experiment.

Quantitative real-time PCR (qRT-PCR)
qRT-PCR was performed to analyze ATP1A2 expression as previously described (18,19). Total RNA from cells was extracted using TRIzol reagent. After concentration and purity detection, cDNA was synthesized with FirstStrand cDNA Synthesis Supermix (Transgen Biotech, Beijing, China) according to the manufacturer's instructions. The expression of ATP1A2 was determined through qRT-PCR using an ABI 7500 system (Applied Biosystems, USA), with Green qPCR Supermix (Transgen Biotech, Beijing, China). The gene expression levels were calculated using the 2 -ΔΔCt method. GAPDH was used as an internal reference. The primers were supplied by GenePharma as follows: ATP1A2 (

Colony formation
Cells in the logarithmic growth phase were inoculated into 6-well plates according to appropriate cell density, and were evenly dispersed and cultured in a cell incubator for 2 weeks. When cell colonies were observable in the well plate with the naked eye, the cells were fixed with methanol, stained with crystal violet, and counted directly.

EdU detection
Cells were seeded in a 96-well plate at a density of 5×10 3 cells/well and 200 μL/well. After the cells fused, they were incubated with 50 mmol/L EdU solution (Beyotime, Nantong, China) for 2 h, fixed with 4% formaldehyde and treated with 0.5% Triton for 20 min to permeabilize at room temperature. Staining of the cell nuclei was performed using 4',6-diamidino-2-phenylindole (DAPI; Sigma, USA). The prepared samples were photographed under a fluorescence microscope.

Cell apoptosis
The apoptotic ability of PC-3 and DU145 cells was analyzed using the Annexin-V-FITC cell apoptosis assay kit (Beyotime) following the manufacturer's protocols. After being resuspended in the binding buffer, the transfected PC-3 and DU145 cells were stained with Annexin V-FITC and propidium iodide in the dark for 15 min. The cells were then analyzed with a flow cytometer (FACScan, BD Bioscience, USA).

Transwell assay
After cells were digested and centrifuged, 800 μL of culture medium containing 10% serum was added to the lower chamber of transwell chambers (Corning, Corning, NY, USA), 100 μL cell suspension was added to the upper chamber, and the mixtures were cultured for 24 h in an incubator at 37 ℃. The chamber was then removed to allow the fluid in the upper chamber to dry out, the cells was fixed with 4% paraformaldehyde at room temperature for 30 min, and finally stained with crystal violet at room temperature for 30 min. The chamber was then gently rinsed with clean water and photographed under a microscope.
Cell invasion assay was firstly carried out using a Corning Polycarbonate Membrane Insert transwell chamber (8-μm pore size, Corning, USA) coated with Matrigel (BD Bioscience). The subsequent steps were as described above.

In vivo xenograft model
Ten male BALB/c nude mice (4 weeks old), obtained from Beijing Vital River, were injected subcutaneously in their right dorsal flanks with 1×10 7 DU145 cells transfected with either pcDNA-NC or pcDNA-ATP1A2. Animal experiments were performed under a project license (No. KYKT2018-043-A1) granted by the Animal Ethics Committee of Affiliated Dongguan People's Hospital, Southern Medical University, in compliance with Affiliated Dongguan People's Hospital, Southern Medical University guidelines for the care and use of animals. Subcutaneous tumor volumes were measured every 6 days and calculated as: volume = length × width 2 × 1/2. On the 30th day after injection, the mice were killed humanely and the subcutaneous tumors were removed and weighed.

Bioinformatic analysis
The differential expression of ATP1A2 in PC and normal tissues, and the prognostic analysis of PC patients were performed by Gene Expression Profiling Interactive Analysis (GEPIA; http://gepia2.cancer-pku.cn) based on TCGA (https://cancergenome.nih.gov/).

Statistical analysis
The data were analyzed using SPSS 21.0, and the measurement data were expressed as mean ± standard deviation (SD). Statistical analysis was performed using Student's t-test or one-way analysis of variance (ANOVA) test. The difference was considered statistically significant at P<0.05.

ATP1A2 expression in PC tissues and cells
The results of GEPIA based on TCGA database revealed that ATP1A2 expression is markedly reduced in PC tissues ( Figure 1A), and low ATP1A2 expression in patients is associated with a lower survival rate ( Figure 1B). Moreover in our study, the mRNA and protein expressions of ATP1A2 were significantly reduced in PC cells compared with normal human prostate epithelial cells ( Figure 1C,1D). Compared with the other PC cells, PC-3 and DU145 cells showed the lowest ATP1A2 expression, which is why we selected them for the subsequent experiments.

Effect of ATP1A2 on PC cell proliferation
We transfected PC-3 and DU145 cells with pcDNA-ATP1A2 and si-ATP1A2 to evaluate the role of ATP1A2 in cell proliferation. The mRNA and protein expressions of ATP1A2 increased after transfection with pcDNA-ATP1A2, but decreased after transfection with si-ATP1A2 ( Figure  2A,2B). As shown in Figure 2C, ATP1A2 overexpression reduced the number of PC-3 and DU145 cell clones. In contrast, ATP1A2 silencing increased the colony number ( Figure 2C). EdU staining also showed that proliferation of PC-3 and DU145 cells was inhibited by ATP1A2 overexpression and promoted by ATP1A2 silencing ( Figure  2D). These data suggested that ATP1A2 markedly inhibited the proliferation of PC cells.

Effect of ATP1A2 on apoptosis of PC-3 and DU145 cells
Overexpression and silencing of ATP1A2 promoted or inhibited apoptosis in PC-3 and DU145 cells, respectively ( Figure 3A). Western blot results revealed that overexpression of ATP1A2 elevated the expression of Bax and cleaved caspase-3, and reduced the expression of Bcl-2 in PC-3 and DU145 cells, whereas ATP1A2 silencing had the opposite effect ( Figure 3B). These data demonstrated that ATP1A2 promoted apoptosis of PC cells.

Effect of ATP1A2 on the migration and invasion of PC cells
Inhibition of tumor cell migration and invasion can effectively inhibit tumor metastasis. Overexpression and silencing of ATP1A2 inhibited or promoted the migration and invasion of PC cells, respectively ( Figure 4A,4B). Overexpression of ATP1A2 significantly inhibited the expression of MMP-2 and MMP-9, whereas silencing of ATP1A2 significantly promoted their expression ( Figure 4C).

Effect of ATP1A2 on EMT in PC cells
Subsequently, we detected the expression of EMT-related marker proteins. Overexpression of ATP1A2 significantly inhibited Snail, α-SMA, vimentin, N-cadherin, and β-catenin expression and promoted E-cadherin expression, whereas silencing of ATP1A2 had the opposite result ( Figure 5A,5B). These data revealed that ATP1A2 could significantly inhibit EMT in PC cells.

Effect of ATP1A2 on the TGF-β/Smad pathway in PC cells
The TGF-β-Smad signaling pathway plays a key role in cellular EMT, which can reduce the adhesion between © Translational Andrology and Urology. All rights reserved.
Transl  cells, allowing tumor cells to penetrate the basement membrane, extensively invade adjacent tissues, and develop distant metastases. Therefore, we examined the effect of ATP1A2 on the TGF-β/Smad pathway. As shown in Figure  6A,6B, overexpression of ATP1A2 markedly inhibited the expression of TGF-β, p-Smad2, and p-Smad3 in PC-3 and DU145 cells, whereas ATP1A2 silencing significantly promoted their expression. These data confirmed that ATP1A2 inhibited the activation of the TGF-β/Smad pathway.

Effect of ATP1A2 inhibiting the TGF-β/Smad pathway
As seen in Figure 7A, the facilitating effect of ATP1A2 silencing on the expression of TGF-β, p-Smad2, and p-Smad3 in PC-3 and DU145 cells was significantly weakened by LY364947 (an inhibitor of the TGF-β/Smad pathway). The effect of ATP1A2 silencing on DU145 cell proliferation was reversed by LY364947 ( Figure 7B), whereas the inhibitory effect of ATP1A2 silencing on the apoptosis of DU145 cells was reversed by LY364947 ( Figure 7C). Moreover, LY364947 reversed the effect of ATP1A2 silencing on the migration and invasion of DU145 cells ( Figure 7D). These results suggested that ATP1A2 can suppress the proliferation, migration and invasion of PC cells, and promote their apoptosis by inhibiting the TGF-β/Smad pathway.

Effect of ATP1A2 on tumor growth in vivo
To explore the function of ATP1A2 on tumor growth in vivo, the xenograft assay was performed ( Figure 8A). Tumor volume and weight were significantly decreased in the pcDNA-ATP1A2 group when compared with the pcDNA-NC group ( Figure 8B,8C). These results revealed that ATP1A2 overexpression can inhibit tumor growth in vivo.

Discussion
The symptoms of early PC are subtle, and the disease is difficult to diagnosis until it has progressed (22). Although some patients improve after treatment, many others progress to resistant PC, which leads to difficulties in treatment and poor prognosis; consequently the patient's quality of life is seriously affected (23,24). Therefore, finding more effective therapeutic targets is urgent. As the target protein of cardiotonic glycosides, Na + - K + -ATPase has been widely studied in the context of heart failure, arrhythmia, and other diseases; its signal transduction function has been gradually discovered and recognized by researchers, and its role in tumorigenesis is now a major focus (25). ATP1A2 is mainly expressed in the adult heart, vascular smooth muscle, skeletal muscle, brain, adipocytes, cartilage, and bone, but less attention has been given to ATP1A2 in tumors because of its tissue specificity (26,27). In this study, we revealed that ATP1A2 is expressed at low levels in PC tissues and cells, and that PC patients with low ATP1A2 expression have a higher mortality rate. Therefore, we speculate that ATP1A2 plays a regulatory role in PC. The expression of ATP1A2 was lowest in PC-3 and DU145 cells compared with other PC cell lines, so we used them for further analyses. Previous studies have revealed that the proliferation, apoptosis, invasion and metastasis of tumor cells are the pathological basis of the development of cancers (28,29). In this study, the results showed that ATP1A2 significantly inhibited the proliferation, migration and invasion of PC-3 and DU145 cells and promoted the apoptosis of PC-3 and DU145 cells.
EMT is a necessary process for the distant metastasis of PC (28). MMPs such as MMP-2 and MMP-9 degrade the extracellular matrix (ECM) and enhance cell invasion (30). Our results confirmed that ATP1A2 overexpression inhibited MMP-2 and MMP-9 expression. Snail significantly regulates the EMT process, which is closely related to the loss of E-cadherin and upregulation of vimentin and fibronectin © Translational Andrology and Urology. All rights reserved.
Transl Androl Urol 2022 | https://dx.doi.org/10.21037/tau-21-1117 TGF, transforming growth factor. (31)(32)(33). Cytological studies have confirmed that reduced intracellular stability of the Snail protein can inhibit the EMT process and the malignant phenotype of cancer cells (34). In this study, we confirmed that ATP1A2 can significantly inhibit EMT-associated proteins and Snail in PC cells. TGF-β1 is highly expressed in a variety of tumors, and its role in tumor development has been explored both in vitro and in transgenic mice (35). However, the role of TGF-β1 in carcinogenesis is complex. Many tumor cells become insensitive to TGF-β1-mediated growth inhibition, suggesting that increased TGF-β1 expression may alter the tumor microenvironment, such as inducing local angiogenesis, ECM generation, and inhibiting immune surveillance, which provides a favorable environment for the proliferation of tumor cells (36). In the process of tumor development, the increase in TGF-β1 synthesis plays a role in promoting tumor development (37). Malignant proliferation of cancer cells originating from the prostate epithelium lose cell polarity after EMT, resulting in decreased intercellular adhesion and enhanced potential for invasion and migration. At the same time, the expression of epithelial markers E-cadherin and tight junction protein ZO-1 is downregulated, and the expression of stromal cell markers N-cadherin and vimentin is upregulated (38,39). TGF-β is an effective inducer of EMT, which is characterized by decreased expression of E-cadherin, and upregulation of interstitial proteins (40). During cancer progression, classical Smad is a key influential factor for TGF-β-induced EMT, which is abnormally reactivated during cancer progression and can be used to regulate EMT through the interaction of related transcription factors (41). TGF-β can induce EMT by activating Smad3 and Smad2 signaling (42). In this study, we confirmed that ATP1A2 significantly inhibited the activation of the TGF-β/Smad pathway in PC. Moreover, the rescue experiments further directly verified that the TGF-β/Smad pathway was a target of ATP1A2. All these results confirmed that the promotion of ATP1A2 could be choosed for PC treatment in the future.
However, there are also shortcomings in our research. There is not only one signaling pathway that has a potential targeting relationship with ATP1A2. Therefore, whether ATP1A2 affects other pathways in PC will be further investigated in our future experiments. More work is required to explore how ATP1A2 can be used as a novel therapeutic target for the treatment of PC.

Conclusions
In summary, we first confirmed that the expression of ATP1A2 was decreased in PC tissues and cells. We also revealed that ATP1A2 significantly inhibits the proliferation, migration, invasion and EMT of PC cells, and may be regulated through the TGF-β/Smad pathway. ATP1A2 may be an effective therapeutic target for the treatment of PC. Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the noncommercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.