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BZW1 promotes cell proliferation in prostate cancer by regulating TGF-β1/Smad pathway

ABSTRACT Recently, basic leucine zipper and the W2 domain-containing protein 1 (BZW1) are reported to be implicated in tumor progression. However, the role of BZW1 in prostate cancer remains unknown. This study is aimed to investigate the expression of BZW1 and its influence on cell proliferation in prostate cancer. Then, the expression levels of BZW1 were measured in 136 cases of prostate cancer and matched adjacent non-cancerous prostate tissues by quantificational real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). The effect of BZW1 on cell proliferation was further explored. QRT-PCR analysis showed that the mRNA levels of BZW1 in prostate cancer were significantly greater compared with those in matched adjacent non-cancerous prostate tissues (P< 0.001). IHC results showed that the high-expression rates of BZW1 in prostate cancer and matched adjacent non-cancerous prostate tissues were 68.4% and 32.4%, and the difference was statistically significant (P< 0.001). BZW1 high expression significantly correlated with T stage, lymph node metastasis, prostate-specific antigen (PSA) and Gleason score (P< 0.05). Patients with BZW1 high expression presented unfavorable prognosis compared with those with BZW1 low expression (P= 0.002). In addition, CCK-8 and colony formation assays revealed that BZW1 overexpression significantly promoted cell proliferation in vitro. Tumor xenograft has shown that BZW1 knockdown significantly inhibited tumor growth in vivo. Moreover, BZW1 overexpression activated the TGF-β1/Smad1/Smad3 pathway. Therefore, these data indicate that BZW1 overexpression predicts poorer prognosis and promotes cell proliferation in prostate cancer by regulating TGF-β1/Smad pathway.


Introduction
Prostate cancer (PCa) is one of the most prevalent urological cancers in men worldwide [1][2][3]. The incidence of PCa is increased worldwide in recent years [4,5]. In the USA and Europe, PCa accounts for the second most leading cause of cancerrelated mortality [6,7]. Although the testing of PSA is generally used in the early diagnosis, the incidence and mortality rate of PCa are still increasing [8][9][10]. Therefore, identifying new potential markers of PCa is crucial for the early diagnosis and clinical therapy.
BZW1 is a member of the bZIP superfamily of transcription factors, which contains C-terminal W2-type HEAT domains [11,12]. BZW1 plays a key role in the cell cycle by regulating histone H4 gene transcription [13]. Moreover, BZW1 promotes cell proliferation in salivary mucoepidermoid carcinoma and lung adenocarcinoma [14][15][16]. BZW1 overexpression is an independent prognostic marker in lung adenocarcinoma [16]. However, the biological function of BZW1 in prostate cancer remains unknown.
Here, the expression of BZW1 and its clinical significance in PCa were investigated. Moreover, the effect of BZW1 expression on cell proliferation and its potential mechanism were explored.

Materials
A total of 136 PCa and matched adjacent noncancerous prostate tissues were enrolled in this study. All patients received surgical resection during 2010-2018, while no patient was treated with chemotherapy or radiotherapy before surgery. Clinicopathological characteristics were obtained from hospitalized records. The follow-up time was ranged from 8 to 64 months. This study was supported by the ethics committee of Sun Yat-sen University (No. Z81771573) and performed in accordance with the Declaration of Helsinki. The informed consent was signed by each patient.

QRT-PCR
Total RNA was extracted from fresh tissues and cells by using TRIzol Reagent (Life Technologies). According to the manufacturer's protocol, the RNA was reversed into cDNA using a PrimeScript RT kit (Takara, Dalian, China). Real-time quantitative PCR was performed by SYBR1 Premix Ex Taq (DDR041A, TaKaRa, Dalian, China). Primers for BZW1 were 5'-ACTGGTGTTCTTCTGGCTAA-3' (forward) and 5'-GTGCTCATTACACTTGACCA-3' (reverse). The primer sequences for GAPDH were 5'-CTGAACGGGAAGCTCACTGG-3' (forward) and 5'-TGAGGTCCACCACCCTGTTG-3' (reverse). GAPDH was considered as the internal control, and each test was repeated three times. The relative expression level of the target gene was calculated using the 2 −ΔΔCt method.

Cell counting kit-8 (CCK-8) analysis
CCK-8 assay was used to evaluate the cell proliferation. Cells were transferred and seeded in 96well plates (1.0 × 10 3 cells/well) for 5 days. Then, cells were cultured with 10 µL of CCK-8 (Dojindo, Kumamoto, Japan) for 3 h, and the optical density of each well was examined by a microplate reader (BioTek, Winooski, VT, USA) at 450 nm. Each test was repeated in triplicate under the same conditions.

Colony formation analysis
Transfected cells (1.0 × 10 3 cells/well) were seeded into six-well plates. After 2 weeks, cells were obtained and fixed with 100% methanol. Then, cells were incubated with 0.1% crystal violet and stained with a hemocytometer for counting. A colony contained more than 50 cells. Each experiment was repeated in triplicate under the same conditions.

Tumor xenograft in vivo
Balb/c nude mice (4 weeks old) were purchased from animal center and kept in specific pathogenfree conditions in accordance with institutional guidelines. To evaluate the effect of BZW1 on tumor growth, 6 × 10 6 prostate cancer cells with shRNA lentivirus vectors were subcutaneously injected into the flank regions of legs (5 mice per group). Negative controls (NC) were treated with prostate cancer cells with non-target shRNA lentivirus vectors. After 2 weeks, tumor volume, tumor weight and Ki-67 index were calculated.

Statistical analysis
All data were characterized as mean ± standard deviation and analyzed by SPSS software (version 19.0; SPSS, Chicago, IL, USA). The mRNA expression levels of BZW1 between prostate cancer and matched adjacent non-cancerous tissues were analyzed by paired t-test. The correlation between BZW1 expression and clinicopathological characteristics was evaluated by Chi-square test (χ 2 ). Survival analysis was performed by using the Kaplan-Meier method with log-rank test. Hazard ratio (HR) was evaluated using the Cox's proportional hazards model. P < 0.05 was considered as statistical significance.

BZW1 is highly expressed in PCa and correlated with clinicopathological characteristics
To evaluate the expression of BZW1, the mRNA levels of BZW1 were detected in 136 cases of PCa and matched adjacent non-cancerous prostate tissues by qRT-PCR. Results showed that the mRNA levels of BZW1 were significantly greater in PCa compared with those in matched adjacent noncancerous prostate tissues (Figure 1a, P ≤ 0.001). Then, the protein expression levels of BZW1 were detected by IHC. As shown in Figure 1b-e, positive staining of BZW1 was mainly located in the cell cytoplasm as brown and yellow. Semiquantitative analysis showed that the high-expression rates of BZW1 in PCa and non-cancerous prostate tissues were 68.4% and 32.4%, respectively, and the difference was statistically significant (Table 1, P ≤ 0.001).
To investigate the clinical significance of BZW1 in PCa, the correlation between BZW1 expression and clinicopathological characteristics was investigated. Results showed BZW1 high expression was significantly correlated with T stage, lymph node metastasis, PSA and Gleason score (P ≤ 0.05, Table  2). However, no significant correlation was observed in BZW1 expression with age and smoke history (P ≥ 0.05, Table 2). In addition, Kaplan-Meier analysis revealed that patients with BZW1 high expression presented unfavorable prognosis than those with BZW1 low expression (Figure 2, P = 0.002). T stage, lymph node metastasis, PSA and Gleason score rather than age and smoke history were significantly correlated with overall survival time (P ≤ 0.05, Table 3). Moreover, multivariate Cox regression analysis has shown that BZW1 expression, T stage, lymph node metastasis, PSA and Gleason score were independent prognostic factors for predicting overall survival in PCa (Table 4, P ≤ 0.05).

BZW1 promotes cell proliferation in PCa by regulating TGF-β1/Smad pathway
To explore the biological functions of BZW1 in PCa, the influence of BZW1 expression on cell proliferation was investigated. As shown in Figure 3a,BZW1 was highly expressed in PC3 cells but was relatively lower in DU145 and LnCap cells. Thus, BZW1 expression was knocked down in PC3 cells and overexpressed in LnCap cells. QRT-PCR and Western blot analysis indicated that BZW1 downregulation and upregulation were successfully performed (Figure 3b and 3c, P ≤ 0.05). Then, cell proliferation was evaluated by CCK-8 and colony formation assays in vitro. CCK-8 assay showed that BZW1 knockdown significantly inhibited the proliferation in PC3 cells (Figure 3d, P ≤ 0.05), while BZW1 overexpression significantly promoted the proliferation in LnCap cells (Figure 3d, P ≤ 0.05). The colony number in    cells with BZW1 downregulation was significantly decreased, while it was significantly increased in cells with BZW1 overexpression (Figure 4a, P ≤ 0.05). To further investigate the effect of BZW1 expression on cell proliferation, xenografts in vivo were performed. Results showed that tumor volume and tumor weight were significantly decreased in the BZW1-knockdown groups and increased in the BZW1-overexpression groups compared with those in NC groups (Figure 4b and 4c, P ≤ 0.05). Moreover, the positive staining of Ki-67 was obviously decreased in the tumor tissues with BZW1 knockdown (Figure 4d). TGF-β/Smad pathway is reported to be involved in cell proliferation, but its role in PCa remains unclear [17][18][19]. Thus, to explore the potential mechanism of BZW1, TGF-β1/Smad pathway was investigated. As shown in Figure 4e, the expression of TGF-β1 was significantly decreased with the knockdown of BZW1, while it was significantly upregulated by BZW1 overexpression. The protein expression levels of Smad1 and Smad3 were unchanged regardless of whether the BZW1 expression interference, while BZW1 downregulation resulted in the decrease of p-Smad1 and p-Smad3.

Discussion
BZW1, as one of the transcription factors, encodes an N-terminal bZIP domain and a C-terminal nucleotide-binding domain [11,12]. The proteins and transcription factors of bZIP domain play crucial roles in cancer development [20,21]. Recently, BZW1 overexpression was observed in mucoepidermoid carcinoma [14] and lung    adenocarcinoma [16]. However, the role of BZW1 in PCa remains unknown.
Here, the expression and clinical significance of BZW1 were investigated in PCa. Results showed that the mRNA and protein levels of BZW1 in PCa were significantly higher compared with those in non-cancerous prostate tissues. These data indicated that BZW1 overexpression was associated with the formation of PCa, which was consistent with the observations in mucoepidermoid carcinoma [14] and lung adenocarcinoma [16]. BZW1 overexpression might be helpful for the diagnosis of PCa. In addition, BZW1 overexpression significantly correlated with T stage, lymph node metastasis, PSA and Gleason score, suggesting that BZW1 overexpression was connected with the progression of PCa. Furthermore, survival analysis shown that BZW1 as well as T stage, lymph node metastasis, PSA and Gleason score were associated with patients' survival, which could be served as independent prognostic factors in PCa. Moreover, Chiou et al. [16] reported that BZW1 overexpression was an independent poor prognosis marker in lung adenocarcinoma.
Subsequently, the influence of BZW1 expression on cell proliferation and its potential mechanism were explored. Results have shown that BZW1 downregulation significantly suppressed cell proliferation, while BZW1 overexpression significantly promoted cell proliferation in vitro. Xenograft assays further validated that BZW1 overexpression significantly promoted cell proliferation in vivo. To explore the potential mechanism of BZW1, TGF-β1/ Smad pathway was investigated. It is well known that TGF-β/Smad pathway is involved in cell proliferation [17][18][19]. So the impact of BZW1 expression on the TGF-β1/Smad pathway was further explored. The results showed that BZW1 overexpression activated the TGF-β1/ Smad pathway. Therefore, these data indicated that BZW1 overexpression significantly promoted cell proliferation, which was connected with the TGF-β1/Smad pathway.
In conclusion, these data indicate that BZW1 is highly expressed in PCa and correlates with tumor progression and prognosis. Moreover, BZW1 overexpression promotes cell proliferation by regulating TGF-β1/Smad pathway.