Downregulation of CPA4 inhibits non small–cell lung cancer growth by suppressing the AKT/c‐MYC pathway

Abstract Carboxypeptidase A4 (CPA4) is a member of the metallocarboxypeptidase family. A previous study indicated that CPA4 may participate in the modulation of peptide hormone activity and hormone‐regulated tissue growth and differentiation. However, the role of CPA4 in lung tumorigenesis remains unclear. Our study revealed that CPA4 expression was higher in both lung cancer cells and tumor tissues. We performed 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide assays, colony‐formation assays, and Cellomics ArrayScan Infinity analysis to demonstrate that CPA4 knockdown inhibited non small–cell lung cancer (NSCLC) cell proliferation. Conversely, ectopic expression of CPA4 enhanced lung cancer cell proliferation. Consistent with these observations, we generated xenograft tumor models to confirm that CPA4 downregulation suppressed NSCLC cell growth. Mechanistically, we revealed that CPA4 downregulation may induce apoptosis and G1‐S arrest by suppressing the protein kinase B/c‐MYC pathway. These results suggest that CPA4 has an oncogenic effect on lung cancer growth. Taken together, we identified a novel gene in lung cancer that might provide a basis for new therapeutic targets.

and differentiation by modulating or inactivating peptide hormone activity resulting from the cleavage of the COOH-terminus. 4 Meanwhile, CPA4 is a maternally imprinted gene that is located in a region of chromosome 7q32 that is speculated to be associated with prostate cancer aggressiveness. [7][8][9] Ross et al 10 demonstrated that a nonsynonymous coding single-nucleotide polymorphism in CPA4 correlates with an increased risk of aggressive prostate cancer among younger men (<66 years). Moreover, several studies have shown that CPA4 expression is increased in multiple cancer tissues, including tissues from patients with pancreatic cancer, gastric cancer, esophageal squamous cell carcinoma, and lung cancer, and may serve as a potential diagnostic and prognostic marker. [11][12][13][14] These findings suggest that CPA4 may play a crucial role in cancers; however, the role of CPA4 in lung tumorigenesis remains poorly understood.
In this study, we investigated the function of CPA4 in lung tumorigenesis. Our work revealed that CPA4 was overexpressed in most human lung cancer tissues, and decreasing CPA4 inhibited lung cancer growth by inducing apoptosis and G1-S arrest. Mechanistically, we showed that CPA4 activated the protein kinase B(AKT)/c-MYC pathway to promote lung tumorigenesis.

| Quantitative real-time polymerase chain reaction
The total RNA was extracted from the tumor tissues and lung cancer cells using TRIzol reagent (Invitrogen, Carlsbad, CA). Complementary DNA was synthesized with murine leukemia virus-reverse transcriptase (Invitrogen), and each reaction was performed in triplicate using SYBR Green (Tiangen, Beijing, China) according to the manufac-turerʼs instructions using an ABI7300 real-time detection system (Applied Biosystems). Glyceraldehyde  After deparaffinization and rehydration, the sections were stained with hematoxylin and eosin and analyzed by immunohistochemistry.
For the immunohistochemistry, the sections were antigen-retrieved, washed, and treated with peroxidase (Santa Cruz Biotechnology) for 5 minutes. Each slide was blocked with normal donkey serum for 20 minutes. Then, the slides were incubated with an anti-CPA4 antibody (1:100; Abcam), and mouse tumor tissues were incubated with an anti-Ki67 antibody (1:100; Abcam). After washing twice with phosphate-buffered saline (PBS), the slides were hybridized with a secondary antibody, followed by incubation with a peroxidasestreptavidin reagent. Then, the color was developed using a diaminobenzidine substrate, and the sections were counterstained with hematoxylin. The results were assessed by two independent pathologists. The following criteria were used to quantify the protein expression level of CPA4: the CPA4 staining intensity was classified as no (score = 0), weak (score = 1), moderate (score = 2), or strong (score = 3) staining; and the positive percentage of CPA4 cells was scaled as 0% (score = 0), 1% to 25% (score = 1), 26% to 50% (score = 2), 51% to 75% (score = 3), or 76% to 100% (score = 4). The values of the staining intensity were multiplied by the percentage to obtain a single CPA4 score for each sample. The mean score of tumors vs adjacent tissues was compared by the Student t test (two-tailed).

| Colony-formation assay
Cells were seed into six-well plates at 1000 cells/well and cultured for 10 days. At the assay endpoint, the cells were washed gently with PBS, fixed with prechilled methanol for 10 minutes and stained with crystal violet for 15 minutes. Stained colonies with more than 50 cells were considered colony-forming units. The experiment was performed in triplicate wells in three independent experiments.

| Cell cycle analysis
Flow cytometry was used to analyze the cell cycle according to the manufacturerʼs instructions. After a 96-hour transduction with shRNAs, the cells were subjected to cell cycle analysis. Briefly, cells (1 × 10 6 ) were harvested by centrifugation at 700 rpm for 5 minutes.
Then, cells were washed twice with PBS and fixed with ice-cold 70% ethanol overnight at 4°C. The cell pellets were washed with PBS, followed by resuspension in 500 mL of PBS containing 50 mg/mL propidium iodide, 0.1 mg/mL RNase A, and 0.05% Triton X-100, and incubated for 15 minutes in the dark at 4°C. Cell cycle distribution was determined using a flow cytometer (Millipore, Boston, MA). The experiment was performed in triplicate.

| Cellomics ArrayScan infinity
Cell proliferation was evaluated using a Cellomics ArrayScan Infinity system (Nexcelom, MA) to quantify the number of fluorescent cells.
Cells were infected with the lentiviral particles for 72 hours. Then, cells were seeded into 96-well plates at 2000 cells/well in triplicates and assessed for the indicated durations.

| Statistical analysis
All data are shown as the mean ± SD. Unless stated otherwise, statistical analyses were conducted using the Student t test.
Significance was defined as P < .05.

| CPA4 is upregulated in human lung cancer cells and primary tumors, and its expression is associated with poor prognosis
We first examined CPA4 expression in lung cancer cells and lung normal epithelial cells. The result showed that CPA4 protein level was significantly overexpressed in lung cancer cells compared with that in lung normal epithelial cell Beas-2B ( Figure 1A). Then, we performed qRT-PCR to evaluate CPA4 gene expression in 20 tumor tissues and corresponding nontumor tissues. We found that CPA4 mRNA was significantly upregulated in 14 tumor tissues ( Figure 1B).
To confirm this result, we expanded the number of lung cancer tissues to evaluate CPA4 protein expression by immunohistochemistry. We multiplied the staining intensity by the percentage to obtain a score for the quantification of CPA4 protein expression. The results showed that CPA4 was increased in 93 of the 131 (71%) tumor tissues compared with that in the adjacent tissues ( Figure 1C and 1D). Meanwhile, we analyzed TCGA datasets for CPA4 expression.
The results indicated that CPA4 expression was higher in NSCLC tissues compared with that in normal tissues ( Figure 1E). Further, the Kaplan-Meier survival curve showed that high CPA4 expression was correlated with poor prognosis ( Figure 1F). These results indicate that CPA4 may serve as a novel diagnostic marker of NSCLC.

| CPA4 promotes lung cancer cell growth
To explore the function of CPA4 in lung cancer, we generated CPA4 knockdown cells by transducing CPA4 shRNAs into H1299 and A549 cells. The effects of CPA4 knockdown were confirmed by both qRT-PCR and Western blot analysis (Figure 2A and 2B).
Colony-formation assays revealed that CPA4 depletion (shCPA4) dramatically reduced the clonogenicity of lung cancer cells compared with nontargeted (shCtrl) cells ( Figure 2C). In contrast, CPA4 overexpression in A549 cells markedly enhanced the clonogenic ability compared with vector control cells ( Figure 2D and 2E). Moreover, MTT assays showed that CPA4 downregulation resulted in a significant decrease in cell proliferation compared with that in nontargeted cells in both H1299 and A549 cells ( Figure 3A). We further performed proliferation analysis with Cellomics ArrayScan Infinity in H1299 and A549 cells. Similarly, the results indicated that decreased CPA4 led to an inhibition of lung cancer cell growth ( Figure 3B). Taken together, these results indicate that CPA4 promotes lung cancer cells growth.

| Knockdown of CPA4 induces G1-S arrest and apoptosis
To investigate the mechanism by which CPA4 enhances cell growth,   Table S2). Then, we performed functional classification based on 820 genes whose expression levels were altered upon CPA4 knockdown using ingenuity pathway analysis. The results showed that these differentially expressed genes were enriched in various biological functions, including cellular growth, cell cycle, cell death, and cellular development ( Figure 4B and 4C and an increase of proapoptosis protein cleaved-caspase 9 and cleaved-caspase 3 compared with shCtrl cells ( Figure 5F). Together, these results indicate that CPA4 knockdown inhibits lung cancer cells growth by inducing G1-S arrest and cell apoptosis.

| CPA4 promotes lung cancer cells growth via the AKT/c-MYC pathway
To explore the molecular mechanism involved in the regulation of pathway. 17,18 In addition, MCP subfamily carboxypeptidase E (CPE) has been shown to increase the phosphorylation of AKT. 19 Thus, we examined whether CPA4 affected the phosphorylation of AKT. The result showed that CPA4 depletion impaired AKT phosphorylation ( Figure 6B). Conversely, CPA4 overexpression increased the protein levels of phosphorylated-AKT ( Figure 6C).
Taken together, these results suggest that CPA4 enhances lung cancer growth via the AKT/c-MYC pathway. Differentially expressed genes were grouped and statistically analyzed using IPA. C, Heatmap showing the functional classification of CPA4-regulated genes. Differentially expressed genes were grouped and statistically analyzed using IPA. Orange represents a Z score >2; blue represents a Z score <−2; white represents a Z score = 0; gray represents no Z score; Z score >2 indicates that differentially expressed genes activate this function; Z score <−2 indicates that differentially expressed genes inhibit this function. CPA4, carboxypeptidase A4; IPA, ingenuity pathway analysis [Color figure can be viewed at wileyonlinelibrary.com]  Figure 7D). In addition, fewer proliferative cells were detected in the tumors derived from H1299-shCPA4 1# cells than that derived form H1299-shCtrl cells by Ki67 staining ( Figure 7E). TUNEL assay results showed a higher level of cell apoptosis in CPA4 knockdown tumor tissues ( Figure 7E). Overall, these data indicated that downregulation of CPA4 inhibits lung cancer growth in vivo.   These results suggest that CPA4 promotes lung cancer growth though promoting cell proliferation and inhibiting cell apoptosis.

| DISCUSSION
We also explored how CPA4 regulates lung cancer cell proliferation and apoptosis. The gene expression profiles suggested that c-MYC was a potential candidate target controlled by CPA4. c-MYC is a transcription factor that is overexpressed in many malignancies.
The aberrant expression of c-MYC promotes tumorigenesis by accelerating cell cycle progression and inhibiting cell apoptosis. [23][24][25] Our results showed that CPA4 downregulation reduced the expres- indicating that c-MYC may be the cause of CPA4 promoting lung cancer growth. In accordance with our findings, N-terminally truncated CPE has been reported to induce c-MYC expression to promote osteosarcoma cells migration and invasion. 22 Thus, these results indicated that CPA4 promotes NSCLC growth via c-MYC.
We further found that CPA4 knockdown impaired AKT phosphorylation. The AKT pathway participates in multiple cellular processes, including cell proliferation, apoptosis, and cell cycle. Studies have reported that activation of AKT pathway promotes tumorigenicity of breast cancer cells and survival of lymphoid cells via the upregulation of c-MYC. 17,18 In addition, the cooperation of activated AKT and c-MYC induces cell proliferation and transformation. 26 Our study found that CPA4 promoted cell cycle progression and inhibited cell apoptosis though the AKT/c-MYC pathway. The activation of AKT pathway is generally considered to be controlled by phosphatidylinositol 3-kinase (PI3K) and epidermal growth factor receptor (EGFR) activation. 27 We first performed Western blot analysis to examine whether CPA4 can alter the phosphorylation of EGFR. The result indicated that CPA4 did not affect EGFR activity (data not shown). PI3K is a lipid kinase that comprises of a p85 regulatory subunit and a p110 catalytic subunit.
Activation of p85 relieves the inhibition of p110 by p85 and activates the AKT pathway. 28  Collectively, we found a novel gene in lung cancer that was overexpressed in tumor tissues. We demonstrated that CPA4 knockdown suppressed lung cancer growth in vitro and in vivo.
Suppressing CPA4 expression induced G1-S arrest and cell apoptosis by inhibiting the AKT/c-MYC pathway. These results indicate that CPA4 may be a potential target for the treatment of NSCLC.

| CONCLUSION
In summary, our work showed that CPA4 downregulation inhibits lung tumorigenesis by regulating the AKT/c-MYC pathway. This finding might serve as a basis for the treatment of NSCLC.

ACKNOWLEDGMENTS
We thank the Cardiothoracic Surgery Department of the First Affiliated Hospital of Wenzhou Medical University for kindly providing the tumor tissues.