SOX4 induces tumor invasion by targeting EMT-related pathway in prostate cancer

Abstract

SOX4 (sex-determining region Y-related high-mobility group box 4) is associated with tumor progression and poor clinical outcome in several cancers. This study aims to evaluate whether SOX4 affects the biological behaviors of prostate cancer and further elucidate whether this effect works through the epithelial–mesenchymal transition pathway. We investigated the expression of SOX4 in a series of prostate cancer tissues and adjacent noncancerous tissues, as well as in a panel of prostate cancer cell lines. Cell proliferation, migration, and invasion were evaluated in SOX4 knockdown prostate cancer cell lines by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Transwell assay. Our results showed that the expression of SOX4 was remarkably upregulated both in prostate cancer tissues and in cell lines. Knockdown of SOX4 repressed the ability of cell proliferation and migration of DU145 cells. Moreover, inhibition of SOX4 could reverse the epithelial–mesenchymal transition processes through upregulation of E-cadherin and downregulation of vimentin. This study provided evidence that SOX4 could serve as a potential therapeutic target in prostate cancer.

Introduction

Prostate cancer (PCa) remains the second most common cause of cancer-related deaths in the United States and the leading noncutaneous malignancy in older men. ¹ Epidemiological studies have suggested that multiple risk factors are involved in prostate carcinogenesis. Although radical prostatectomy and radiation therapy are commonly used to improving outcomes of the PCa patients, the postoperative 5-year survival rate is low. ² Thus, there is an urgent need for a better understanding of the factors related to the development of PCa.

Sex-determining region Y-related high-mobility group box 4 (SOX4), one of group-C SOX genes, plays an important role in the regulation of transcription during developmental processes.^3–6 Recently, multiple studies reported that SOX4 plays essential roles in various cancers, often correlating with tumor angiogenesis and resistance to chemotherapy.^7,8 These findings provide evidence to indicate that SOX4 functions as a key mediator of metastatic progression. To date, epithelial–mesenchymal transition (EMT) is an attractive target for therapeutic interventions and provides a new basis of the progression of carcinoma toward dedifferentiated and more malignant states. The aim of this study was to determine whether SOX4 affects the biological behaviors of PCa and further elucidate whether this effect works through EMT.

Materials and methods

Human tissue samples

A total of 98 pairs of PCa tissue samples and matched normal prostate tissues were obtained from patients who undergone surgical resection at Fifth Affiliated Hospital of Guangzhou Medical University between December 2010 and November 2014. Consent was obtained from all patients, and the experimental protocols were approved by the local ethics committee of Fifth Affiliated Hospital of Guangzhou Medical University. Clinical and pathological information were collected from a retrospective review of well-documented medical records, and characteristics of patients were detailed.

Cell lines and culture conditions

The PC3, LNCaP, and 22Rv1 cell lines (ATCC, Manassas, VA, USA) were cultured in RPMI 1640, supplemented with 10% fetal bovine serum (FBS) and antibiotics. The DU145 cell line was cultured in minimum essential Eagle’s medium supplemented with 10% FBS and 2 mM l-glutamine with antibiotics. The human nontumorigenic prostate epithelial cell line RWPE-1 was cultured in keratinocyte serum-free medium supplemented with 5 ng/mL human recombinant epidermal growth factor and 30 mg/mL bovine pituitary extract (Invitrogen, Carlsbad, CA, USA). Cultures were maintained in a 5% CO₂ humidified atmosphere at 37°C.

Immunohistochemistry

For immunohistochemical analysis of SOX4 expression, 4 µm tissue microarray (TMA) sections were automatically pre-treated using the PT Link System and then stained in an Autostainer Plus (DAKO; Glostrup, Copenhagen, Denmark) with primary antibody anti-SOX4 (1:100; Abcam Inc., Cambridge, MA, USA). The immunohistochemistry (IHC) sections were scored by three pathologists independently to ensure interobserver agreement. Each section was scored according to the intensity and percentage of positive cells. Staining intensity was scored as follows: 0 (negative), 1 (weakly positive), 2 (moderately positive), and 3 (strongly positive). The percentage of positive cells was also graded according to four categories: 1 point for 0%–25% positive cells, 2 for 26%–50% positive cells, 3 for 51%–75% positive cells, and 4 for more than 75% positive cells. Overall scores ⩽3 were defined as negative; scores >3 were defined as positive.

Quantitative real-time polymerase chain reaction

Total RNA was extracted from specimens or cell lines using TRIzol reagent (Invitrogen). For quantitative real-time polymerase chain reaction (qRT-PCR), RNA reverse transcribed to complementary DNA (cDNA) from 1 µg of total RNA was reverse transcribed in a final volume of 20 µL using random primers and a Reverse Transcription Kit (TaKaRa, Dalian, China). According to the manufacturer’s instructions, the reverse transcription was performed at 37°C for 15 min and then at 85°C for 5 s. qRT-PCR analyses were performed using a standard protocol from Power SYBR Green (TaKaRa, Dalian, China). All protocols were performed according to the manufacturer’s instructions. The Δct values were normalized to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primers were synthesized by Shanghai Sangon Biological Engineering Technology Services (Shanghai, China). The sequences of human SOX4 primers were 5′-GGTCTCTAGTTCTTGCACGCTC-3′ (forward) and 5′-CGGAATCGGCACTAAGGAG-3′ (reverse). The primers for human GAPDH were 5′-GCAGTGGCAAAGTGGAGATT-3′ (forward) and 5′-TGAAGTCGCAGGAGACAACC-3′ (reverse).

Western blot analyses

Cells were lysed using the mammalian protein extraction reagent radioimmunoprecipitation assay (RIPA; Beyotime, Beijing, China) supplemented with a protease inhibitor cocktail (Roche, West Sussex, UK) and phenylmethylsulfonyl fluoride (PMSF; Roche, West Sussex, UK). Protein was separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to 0.22 mm nitrocellulose (NC) or polyvinylidene difluoride membranes (Sigma, St. Louis, MO, USA). The membranes were washed, blocked, and incubated with specific primary antihuman antibodies anti-SOX4, anti-E-cadherin, anti-N-cadherin, anti-Vimentin, and anti-GAPDH (Abcam, Cambridge, MA, USA). The secondary antibody was horseradish peroxidase–conjugated goat anti-rabbit IgG. An enhanced chemiluminescence (ECL) chromogenic substrate was used to visualize the bands, and the intensity of the bands was quantified by densitometry (Quantity One Software; Bio-Rad, Hercules, CA, USA).

Knockdown of SOX4 by siRNA transfection

Sequence-specific siRNA against SOX4 was purchased from Thermo Scientific Dharmacon (Lafayette, CO, USA). Cells were seeded into six-well plates at a density of 3 × 10⁴ to 5 × 10⁴ cells per well and transfected with siRNA using Lipofectamine 2000 reagent (Invitrogen). After 48 h, SOX4 expression levels were detected by RT-PCR and/or western blot.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

After transfection, cells were seeded in a 96-well plate at a density of 10⁴ cells/well. Thereafter, the media was changed and the cells were incubated with 20 µL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 5 mg/mL; Sigma-Aldrich, St. Louis, MO, USA) for 4 h. The mixture was centrifuged at 12,000×g at 4°C for 10 min. The supernatant was removed, 50 µL dimethyl sulfoxide (DMSO) was added to each well, and the solutions were agitated on a decolorization shaker for 10 min. The optical density (OD) of each well was measured using an enzyme immunoassay analyzer (Power Wave XS; BioTek Instruments Inc., Winooski, VT, USA) at 490 nm.

Migration and invasion assays

After transfection, 1 × 10⁵ cells in serum-free medium were seeded into the Boyden chamber without Matrigel (8-µm pore; BD Falcon, San Jose, CA, USA) for migration or the chamber with Matrigel (8-µm pore; BD Falcon) for invasion. Then, the chambers were put in 24-well plates with medium with 10% FBS. The chambers were incubated for 24 h at 37°C with 5% CO₂. The cells on the underside of filter membrane were fixed in ethanol and stained with crystal violet. The cells were counted under a microscope.

Statistical analysis

The data are presented as mean ± standard error of the mean (SEM). Three replicate wells were tested per assay, and each experiment was performed in triplicate. Statistical comparisons between groups were determined by the student’s t-test. All data were analyzed using SPSS 16.0 (SPSS Inc., Chicago, IL, USA). All statistical tests were two-tailed and statistical significance was assumed for p < 0.05.

Results

SOX4 is overexpressed in PCa tissues and cell lines

We first examined the SOX4 expression levels in 98 PCa tissue samples and matched normal tissue samples by IHC, qRT-PCR, and western blotting analyses. The expression of SOX4 was found to be significantly upregulated in the PCa tissues compared with the corresponding adjacent non-tumorous tissues (Figure 1). Our IHC results showed that the positive staining of SOX4 was observed in 77 of 98 (78.57%) of PCa cases and in 18 of 98 (18.36%) of adjacent non-neoplastic specimens, with statistical significance (p < 0.001). We also found that there were remarkable changes in the expression levels of SOX4 messenger RNA (mRNA) and protein between PCa and adjacent non-neoplastic tissues, which revealed that tumor tissues showed higher levels of SOX4 than that in adjacent nontumor tissues (Figure 2; p < 0.05).

Figure 1.

(a) Positive expression of SOX4 in the PCa tissues (magnification 200×) and (b) negative expression of SOX4 in the adjacent normal prostate tissues (magnification 200×).

Figure 2.

(a) qRT-PCR showing expression level of SOX4 mRNA in PCa tissues and adjacent normal prostate tissues and (b) western blot assay showing expression level of SOX4 protein in PCa tissues and adjacent normal prostate tissues.

Moreover, we examined the expression of SOX4 in a panel of PCa cell lines as well as an immortalized nontumorigenic human prostate epithelial cell line RWPE-1. Both mRNA and protein levels of SOX4 were greatly elevated in all human PCa cell lines in comparison to RWPE-1 cells, but expression levels of SOX4 varied among PCa cell lines (Figure 3(a) and (b)).

Figure 3.

(a) qRT-PCR showing expression level of SOX4 mRNA in PCa cell lines and (b) western blots showing the expression of SOX4 protein in PCa cell lines.

SOX4 regulates PCa cell proliferation, migration, and invasion

In order to test the oncogenic activity of SOX4 in PCa, DU145 cells were used to knockdown the SOX4 expression by siRNA. The levels of silenced SOX4 expression were verified by qRT-PCR and western blot (Figure 4(a) and (b)). Therefore, we investigated whether SOX4 would affect human DU145 cell proliferation. MTT assay showed that DU145-siSOX4 cell line had lower rates of cell proliferation compared with control (Figure 5(a)), suggesting that SOX4 may act as an oncogene involved in the promotion of PCa cell proliferation. Migration and invasion assays were performed to investigate the effects of SOX4 on the migratory and invasive behaviors of PCa cells in vitro by Transwell assay. The results demonstrated that the number of DU145-siSOX4 cells that transited the artificial basement membrane or/and Matrigel Matrix was significantly reduced than control cells (Figure 5(b)). These data confirmed that inhibition of SOX4 in DU145 cells could suppress their capability of migration and invasion in PCa in vitro.

Figure 4.

(a) qRT-PCR showing expression level of SOX4 mRNA in DU145-si SOX4 cells was significantly decreased compared with control cells and (b) western blots showing the expression of SOX4 protein in DU145-si SOX4 cells was significantly decreased compared with control cells.

Figure 5.

(a) Silencing of SOX4 inhibits the cell growth of DU145 cells and (b) inhibition of SOX4 suppresses the ability of migration and invasion of DU145 cells.

Downregulation of SOX4 could regulate EMT

To study whether SOX4 could regulate EMT, western blotting was performed to show expression of EMT-relevant markers, and the results indicated that E-cadherin expression was significantly increased in DU145-siSOX4 cells, indicating that SOX4 is involved in the EMT process in PCa (Figure 4).

Discussion

As the previous description, the high mortality rate of PCa has created a great need in search of factors affecting cancer progression as well as a need to understand their molecular events. The SOX4 signal pathway is currently an important topic in cancer research. Among the many SOX members, SOX4 belongs to the C subgroup of the family. SOX4 is a 47-kDa protein member of SOX family encoded by a single exon gene, and it preferentially binds the ^A/_T^A/^TCAAAG sequence motif through heavy-metal-binding (HMB) domain and regulates transcription of target genes.^7,8 The abnormal expression of SOX4 has been reported in various types of cancer, and considerable attention is focused on understanding of the physiological and pathophysiological mechanisms of SOX4 in cancer development. However, few studies have detected the expression of SOX4 in PCa patients. In this study, we aimed to detect the expression of SOX4 in human PCa tissues and cell lines. In vitro, we also downregulated SOX4 in DU145 cell line using siRNA technology and evaluated the cell proliferation, migration, and invasion after SOX4 deficiency. We provided evidence that SOX4 is highly expressed in human PCa tissues and cell lines. SOX4 knockdown inhibited PCa cell proliferation and invasion. Our results also suggested that there are potentially certain correlations between SOX4 expression and EMT markers in PCa.

SOX4 was found to be overexpressed in metastatic breast cancer cell lines and in human tissue specimens from advanced stages of breast carcinomas with metastasis.^9–11 In this study, we investigated the expression of SOX4 in a series of clinical PCa tissues. The IHC analysis showed that SOX4 staining was stronger in PCa tissues than in adjacent tissue. Our results of qRT-PCR and western blot showed that the expression level of SOX4 mRNA and protein were also significantly increased in PCa tissues compared with adjacent tissuess. Consistent with our findings, previous studies of SOX4 expression in other cancers have also revealed correlations between SOX4 expression and cancer progression. We next examined the expression of SOX4 in four human PCa cell lines (PC3, LNCaP, 22Rv1, and DU145) and a normal prostate epithelial cell (RWPE-1). Figure 3 showed that the SOX4 expression levels were indeed higher in all of the PCa cell lines examined compared with the normal epithelial cells at both the mRNA and protein levels.

In attempting to determine the possibility of SOX4 as a therapeutic target of PCa, we employed the siRNA technique to knockdown of the SOX4 expression in DU145 cells in which SOX4 expression is relatively high. Our results found that the depletion of SOX4 expression had an antitumorigenic effect in DU145 cells, indicating SOX4 is a critical factor for the proliferation of PCa cancer cells. Tumor metastasis is a multi-step process in tumor progression and is the major cause of death in the vast majority of cancer patients. ¹² Inhibition of the cancer cell invasion and migration can improve the survival rate of cancer patients. ¹³ Recent study demonstrated that the SOX4 plays an important role in the process of invasion and migration. ¹⁴ Consistent with the previous study, the findings presented in this study demonstrate that knockdown of endogenous SOX4 in PCa cell lines greatly reduces cell migration and invasion capacity in vitro, suggesting that SOX4 has an important role in PCa invasiveness. EMT was identified as a special regulator of tumor invasion and metastasis through downregulation of E-cadherin.^15–17 In our study, knockdown of SOX4 reverses the EMT process through upregulation of E-cadherin in PCa cells, suggesting that SOX4 promotes PCa aggressiveness through EMT.

In conclusion, we reported that SOX4 was upregulated in PCa tissues and cell lines. SOX4 also promoted cell proliferation, migration, and increased invasive capability. Moreover, inhibition of SOX4 could reverse EMT through upregulation of epithelial markers E-cadherin in an established human PCa cell line. SOX4 was shown to play a crucial role in the progression of PCa and might function as a therapeutic target for PCa patients.