Overexpression of SOX 4 promotes cell migration and invasion of renal cell carcinoma by inducing epithelial-mesenchymal transition

Incomplete understanding remains in the molecular mechanisms underlying progression and metastasis of renal cancer. The transcription factor SOX4 is upregulated in various human malignancies, including renal cancer, indicating it may be involved in renal tumorigenesis. In this study, we explored this hypothesis by loss-of-function and gain-of-function assays of SOX4 in renal cancer cell lines and renal epithelial cell line. We found that specific knockdown of SOX4 in renal cancer cell lines significantly suppressed the migration and invasion of cancer cells; specific overexpression of SOX4 in renal epithelial cell line markedly promoted the migration and invasion of the cell line. Epithelial-mesenchymal transition (EMT), a fundamental morphogenesis process, is implicated in renal cancer progression and metastasis. Our results demonstrated that SOX4 positively regulated the expression of mesenchymal cell markers and negatively regulated the expression of epithelial cell marker, and was involved in signal transduction pathway of TGFβ-induced EMT. In addition, SOX4 induced EMT probably through modulating the AKT/p-AKT signaling cascade. Finally, we found that SOX4 was significantly upregulated in clinical renal cancer samples compared with corresponding normal tissues and associated with EMT process in clinical samples. Taken together, our findings confirm a crucial function of SOX4 in the metastasis of renal cancer through orchestrating EMT and establish that the function suppression of SOX4-AKT-EMT axis might be an attractive therapeutic intervention during renal cancer metastasis.


Introduction
Renal cell carcinoma (RCC) accounting for 80-90% renal malignancies derives from renal tubule epithelium, which is also called renal adenocarcinoma.It is estimated that approximately 62,700 new cases of renal cancer and 14,240 deaths from renal cancer occurred in the United States in 2016 (1).According to 2004 WHO histopathological classification criteria, RCC was classified into a variety of histologic subtypes.Clear cell RCC (ccRCC) represents the most common subtype of RCC that is characterized by high frequency of metastasis and mortality, and resistance to traditional radiotherapy and chemotherapy.Cancer metastasis is the overwhelming cause of tumor-related mortality in tumor-bearing patients (2)(3)(4).Unfortunately, approximately 30% of patients with renal cancer have developed local invasion or distant metastasis when diagnosed, resulting in poor prognosis.Currently, the underlying molecular mechanisms of metastasis of renal cell carcinoma are elusive.Therefore, it is greatly needed to make a thorough inquiry of metastatic mechanisms in RCC, aiming to provide a novel target for clinical treatment.
Epithelial -mesenchymal transition (EMT), a fundamental morphogenesis process in which the epithelial cells transform into motile mesenchymal cells, plays a pivotal role in embryogenesis (5,6), the formation of organs, the differentiation of tissues, wound healing, stem cell property, and malignant tumor progression and metastasis (7,8).During an EMT process, epithelial cells lose their characteristics, such as the loss of cell polarity, cell-cell adhesion, and downregulation of epithelial markers, substituted by mesenchymal cell

Overexpression of SOX4 promotes cell migration and invasion of renal cell carcinoma by inducing epithelial-mesenchymal transition
traits involving acquisition of migration and invasiveness, resistance to apoptosis and senescence, stemness and immunosuppression, and upregulation of mesenchymal markers (7).SOX4, belonging to the sex-determining region Y (SRY)related high-mobility group (HMG)-box family, is a crucial transcriptional factor associated with a series of development procedures, such as retinal differentiation, central nervous system, heart development, lymphocyte development, via its transcriptional activation (9)(10)(11)(12)(13)(14).Recently, SOX4, as an oncogenic gene, has aroused great interest of researchers, and has been implicated in various human malignancies, including leukemia (15), cancers of lung (16), prostate (17), liver (18), colon (19), breast (3), and ovarian (20).In breast cancer and prostate cancer, SOX4 can induce epithelial-mesenchymal transition and promote metastasis (21,22).In a few human malignancies, however, SOX4 has been classified as a tumor suppressor gene, including gallbladder cancer (23), bladder cancer (24), and glioblastoma multiforme (25).However, few studies have been carried out to investigate SOX4 expression in renal cell carcinoma and its biological behavior in renal cancer cells.TGFβ signaling transduction pathway has been reported to be involved in EMT and malignancies progression (26).Moreover, TGFβ signaling has been demonstrated to induce SOX4 expression in breast cancer and prostate cancer (21,22).Thus, we conjectured that SOX4 might play a catalytic role in the progression and metastasis of RCC via induction of EMT.
In this study, we verified SOX4 overexpression in renal cancer tissues and cell lines.Downregulation of SOX4 greatly inhibited the migratory and invasive abilities of renal cancer cells 786-O and SN12-pM6, while overexpression of SOX4 strongly increased the migratory and invasive properties of the immortalized non-transformed renal HK-2 cells.besides, the gain or loss of SOX4 expression could partly induce or reverse the EMT process, respectively, and upregulate or downregulate the expression of phospho-AKT (p-AKT) in renal cancer cells.Therefore, we speculated that SOX4 might play a role in renal cancer cell migration and invasiveness by inducing EMT through AKT/p-AKT signaling cascade.These data indicated that SOX4 might be an oncogenic gene through orchestrating EMT in RCC tumor microenvironment and provided a firm theoretical principle for consideration of SOX4 as a target for therapy of RCC.

Materials and methods
Human renal cancer samples.All samples (paired tumor and normal tissues) were surgically resected from patients with RCC between 2012 and 2014, at Union Hospital of Huazhong University of Science and Technology (Wuhan, China).Resected tissues were immediately frozen in liquid nitrogen for subsequent experiments.None of the patients had received any antitumor therapy before surgery.Informed written consent was obtained from each of the RCC patients.This study and experiment procedures were approved by the Institutional Review board of Huazhong University of Science and Technology.
Immunohistochemistry assay.Immunohistochemistry (IHC) assay was performed as previously described (27).Briefly, RCC tissues and corresponding normal tissues were fixed in 10% formalin, dehydrated, and embedded in paraffin sequentially.The sections were incubated with rabbit polyclonal antibody SOX4 (1:100, ab80261, Abcam, Cambridge, MA, USA) overnight at 4˚C.After washing three times with PBS for 10 min each time, the sections were incubated with anti-rabbit secondary antibodies conjugated to horseradish peroxidaselabeled polymers.Finally, the sections were counterstained with hematoxylin.
Immunofluorescence.Appropriate amounts of cells were seeded on glass coverslips in 12-well plates, washed with pbS three times, fixed in 4% paraformaldehyde and permeabilized with 0.3% TritonX-100 for 10 min.Cells were blocked with 3% bSA for 1 h.Grass coverslips were incubated with primary antibodies overnight at 4˚C, followed by incubation with CY3-conjugated secondary antibodies for 1 h at room temperature, and then stained with DApI.After washing with PBS, glass coverslips were photographed under a fluorescence microscope.
Transwell migration and invasion assays and wound healing assays.In vitro cell migration and invasion assays were performed using 24-well Transwell chambers with 8.0-µm pore polycarbonate membrane inserts (Corning).For the migration assay, 1x10 4 (786-O, SN12-pM6) or 2.5x10 4 (HK-2) cells were added to the top chambers.For the invasion assay, 2x10 4 (786-O, SN12-pM6) or 5x10 4 (HK-2) cells were added to the top chambers precoated with Matrigel (bD biosciences, Franklin Lakes, NJ, USA).As a chemoattractant, complete medium supplemented with 10% FbS was added to the bottom chambers to stimulate migration or invasion.After cells were incubated for 24 h, cells on the top surface of the membrane were gently scraped with a cotton swab and cells on the lower surface of the membrane were fixed with 100% methanol and then stained with 0.1% crystal violet.The cells migrating or invading through the membrane were counted in 5 random fields.For wound healing assays, cells transfected with si-RNA were seeded in 6-well plates.When cells reached 70-80% confluence, a scratch was made using 10-µl pipette tip, washed, then images were at 0 and 24 h.
Statistical analysis.Data were analyzed using Graphpad prism 5.0 (Graphpad Software, Inc., La Jolla, CA, USA).Comparisons of different groups were performed using Student's two-tailed t-test.Data of each group were expressed as mean ± SEM and p<0.05 was considered to indicate a statistically significant difference.

Results
The expression of SOX4 is upregulated in RCC tissues and cell lines.Our previous findings demonstrated low miR-129-3p levels were associated with short disease-free and overall survival of RCC patients.miR-129-3p impaired RCC cell migratory and invasive properties by decreased multiple metastasis-related genes, including SOX4 (29).based on these observations, we first aimed to examine the levels of SOX4 expression in RCC cell lines and tissues by qRT-pCR and western blot analysis.Of note, the levels of SOX4 mRNA and protein expression were dramatically upregulated in RCC cell lines (786-O, SN12-pM6, ACHN and A498) compared with the normal renal epithelial cell line HK-2 (Fig. 1A and b).Unexpectedly, no remarkable difference of SOX4 mRNA was observed between ccRCC and normal tissues.However, RCC tissues exhibited significantly higher SOX4 protein expression in 17 of 20 pairs RCC compared with corresponding normal tissues by western blotting and IHC (Fig. 1C and D).Moreover, immunofluorescence revealed that SOX4 was overexpressed in RCC cells compared with the normal renal cells (Fig. 1E).Collectively, these results indicate SOX4 expression is up regulated in RCC.

The effects of SOX4 on migration and invasion in 786-O and SN12-PM6 and HK-2 cell lines.
To explore the roles of SOX4 upregulation in RCC cell lines, we then performed a loss-of-function assay by using small interfering RNA specifically targeting SOX4 (Fig. 2A and D).The effects of SOX4 knockdown on migration and invasion of these two cell lines were examined using wound healing and Transwell chamber assays.The results revealed that SOX4 knockdown markedly undermined the migration and invasion of these two RCC cell lines (Fig. 2B and C, and E and F), however, it did not influence cell proliferation and apoptosis (data not shown).To further confirm the involvement of SOX4 in migration and invasion, we then carried out a gain-of-function assay in HK-2 cell line by constructing plasmid vector expressing SOX4 (Fig. 2G).The results indicated that SOX4 overexpression prominently promoted the migration and invasion of HK2 cell line (Fig. 2H), but it did not affect cell proliferation and apoptosis (data not shown).In addition, downregulation or upregulation of SOX4 did not cause significant changes in cell morphology.Therefore, our loss-of-function and gain-of-function assays demonstrated that SOX4 might promote migration and invasion in RCC cell lines in vitro.

EMT process exists in RCC tissues and cell lines.
Accumulating evidence demonstrates that EMT has been implicated in cancer metastasis (2,30,31).Therefore, in order to investigate the potential metastatic mechanisms in RCC, we examined the EMT marker expression in RCC cell lines and tissues by qRT-pCR and western blotting.As shown in Fig. 3A and B, the RCC cell lines exhibited a significant downregulation of E-cadherin compared with the HK-2 cell line; the mesenchymal cell markers N-cadherin, Twist, and Vimentin were markedly upregulated.Moreover, RCC tissues exhibited a significant downregulation of E-cadherin compared with the corresponding normal tissues; whereas, the mesenchymal markers Vimentin, ZEB1, MMP-9, and Twist were prominently upregulated (Fig. 3C and D).Data above indicated that EMT process might have occurred in the formation and progression of RCC.

The effects of SOX4 on EMT profile in 786-O and SN12-PM6
and HK-2 cell lines.Recent studies have identified SOX4 as a master regulator of EMT (32).To explore the potential correlation between SOX4 and EMT in RCC, we successfully knocked down the expression of SOX4 in 786-O and SN12-pM6 cell lines that overexpressed endogenous SOX4, by using small interfering RNA, as confirmed by qRT-PCR and western blotting (Fig. 4A and b).We then examined by western blotting the expression of several EMT markers in the 786-O and SN12-pM6 cells that were subjected to SOX4 knockdown.The results indicated that 786-O and SN12-PM6 cell lines that were knocked down by SOX4 had a significant higher E-cadherin expression compared with corresponding control cells; whereas, the mesenchymal cell markers N-cadherin, Vimentin, and Twist expression were prominently downregulated (Fig. 4A and b).To further corroborate the correlation between SOX4 and EMT, we then overexpressed SOX4 in HK-2 by using plasmid vector expressing SOX4, as confirmed by qRT-PCR and western blotting (Fig. 4C).Next, we examined the EMT markers expression in HK-2 cells that overexpressed the SOX4 by western blotting.As shown in Fig. 4C, the HK-2 of SOX4 overexpression exhibited a significant lower E-cadherin expression compared with the corresponding control cells; whereas, N-cadherin, Vimentin, and Twist expression were significantly upregulated.These results suggested that SOX4 could induce EMT in RCC cell lines, and SOX4 knockdown could partially reverse EMT.

The effects of SOX4 knockdown and rescue on EMT expression profile in the 786-O and SN12-PM6 and HK-2 cell lines.
To further substantiate the findings, we next performed SOX4 knockdown and rescue tests in the 786-O and SN12-PM6 and HK-2 cell lines, respectively.As shown, in all the three cell lines tested, downregulation of SOX4 upregulated epithelial marker and downregulated mesenchymal markers in 786-O and SN12-pM6 and HK-2 cell lines; while overexpression of SOX4 yielded opposite effects (Fig. 5A-C).These results further solidified our findings that SOX4 could induce the EMT program.

SOX4 is an important component involving in TGFβinduced EMT.
Multiple lines of evidence have confirmed the involvement of TGFβ signaling in EMT program.To further investigate whether SOX4 was involved in the process of TGFβ signaling-induced EMT in HK-2 cells, we stimulated HK-2 cells by using 2.5 ng/ml concentration TGFβ1 for 0, 3, 7, 10 day(s).Compared with the parental HK-2 cells, the TGFβ1-treated HK-2 cells represented morphologic changes from cobblestone-like to fibroblast-like appearances (Fig. 6A).SOX4 expression was upregulated in HK-2 cells at mRNA and protein level, in a time-dependent manner following the addition of TGFβ1 into the cell culture medium (Fig. 6b and C).Moreover, TGFβ1 modulated the expression of EMT markers and p-AKT at protein level (Fig. 6C).These data indicated SOX4 might be an important component involving TGFβinduced EMT.
SOX4 induces EMT possibly via modulating expression of p-AKT.AKT signaling cascade has been reported involved in metastasis in a variety of cancers (33)(34)(35); previous studies also showed that AKT pathway could induce EMT (36)(37)(38)(39).To further explore the molecular mechanisms how SOX4 induces EMT and metastasis in RCC, we expanded our study to the metastasis-related protein p-AKT, which may be the potential downstream target of SOX4.As shown in Fig. 7A, SOX4 knockdown in 786-O and SN12-PM6 cell lines downregulated the expression of p-AKT, while overexpression of SOX4 in HK-2 cell line upregulated the expression of p-AKT (Fig. 7b).Furthermore, downregulation of p-AKT was accompanied by the upregulation of E-cadherin, while upregulation of p-AKT reversed this result.based on these results, we preliminarily established a pathway diagram that SOX4 modulated EMT (Fig. 7C).Collectively, these data indicated that SOX4 might contribute to RCC metastasis via modulating the AKT/EMT signaling cascade.

Discussion
Accumulating evidence indicates that the SOX4 transcription factor is significantly overexpressed in various human malignancies, including melanoma (40), leukemia (15), medulloblastoma (41), and caners of colon (19), prostate (17), breast (3), liver (18), and lung (16), indicating that it may play a crucial role in cancer initiation and progression.previous study has reported that miR-335 suppresses breast cancer metastasis by directly targeting SOX4 (3).Moreover, SOX4 contributes to breast cancer progression through inducing EMT (21).In endometrial cancer, SOX4 knockdown results in attenuation of endometrial cells growth; however, it has no effects on cell migration or invasion (42).In oral squamous cell carcinoma, SOX4 is involved in miR-204-modulated cancer stemness and EMT and promotes lymph node metastasis (43).In addition, knockdown of SOX4 expression induces apoptosis in adenoid cystic carcinoma (44).Conversely, SOX4 may significantly impair cell viability and promote cell apoptosis in the bladder cell line HU609 overexpressing SOX4 (24).Moreover, SOX4 may induce cell cycle arrest and promote cell apoptosis, and inhibit tumorigenesis in a p53-dependent manner in HCC116 cells (45).These studies suggest that SOX4 may play different roles in different malignancies.Different physiological function of SOX4 in different malignancies may be attributed to distinct tumor microenvironment and genetic background.However, the role of SOX4 in RCC is still unclear.
In our studies, we demonstrated that SOX4 expression was frequently upregulated in RCC tissues and cell lines, consistent with many of previous reports in other cancer types.
Our results indicate that SOX4 may promote migration and invasion, which is consistent with the roles of SOX4 in breast cancer and oral squamous cell carcinoma.However, it has no effects on proliferation, apoptosis and colony formation in RCC.To understand the potential molecular mechanisms that SOX4 promoted RCC metastasis, we examined the EMT marker expression in RCC tissues and cell lines by qRT-PCR and western blotting.The results suggested that epithelial cell marker E-cadherin was downregulated at mRNA level and protein level in RCC tissues and cell lines; whereas, the mesenchymal cell markers N-cadherin, Vimentin, and Twist were upregulated, indicating that EMT process happened in RCC progression.
based on these results, SOX4 might promote migration and invasion of RCC through inducing EMT.previous studies have confirmed that AKT signaling pathway could induce EMT through downregulating E-cadherin (36,39).SOX4 was found to be elevated in prostate cancer and implicated in the activation of the pI3K/AKT pathway (46).Furthermore, the expression of SOX4 induced by pTEN loss could activate the pI3K-AKT-mTOR signaling in prostate cancer (17).Therefore, we investigated the expression of AKT and p-AKT in gain-of-function and loss-of-function assays of SOX4.The results indicated that the expression of p-AKT was positively correlated with SOX4 expression.Thus, we speculated that SOX4 might modulate AKT/p-AKT expression directly or indirectly.However, there is a limitation in our study that the roles of SOX4 in vivo are not clear and what regulates SOX4 expression in RCC remains unknown.Therefore, future studies that explore the roles of SOX4 in vivo will further demonstrate the function of SOX4 in RCC.
In conclusion, the current study indicates that SOX4 is overexpressed in RCC tissues and cell lines.Moreover, our results demonstrate that SOX4 promotes migration and invasion of RCC by inducing EMT through AKT signaling cascade, which provides new insight into molecular mechanisms involvement in metastasis of RCC.Therefore, our study suggests that the function suppression of SOX4-AKT-EMT axis could be an attractive therapeutic intervention in RCC metastasis.to X.Z., and the Natural Science Foundation of Zhejiang province (grant no.LY15H160052) to X.C.

Figure 1 .
Figure 1.The expression of SOX4 in RCC cell lines and tissues.(A and b) Real-time pCR analysis and western blot assessment of SOX4 expression in 4 human RCC cell lines (786-O, SN12-pM6, ACHN, and A498) and 1 human normal renal tubule epithelial cell line (HK-2).The SOX4 expression was normalized to GApDH.(C) Immunoblotting analysis of SOX4 expression in 20 paired RCC tissues and corresponding adjacent normal tissues.Upregulation of SOX4 expression was confirmed in 17 of 20 tissue specimens, except 3 pairs of ccRCCs (italics).GAPDH was used as a loading control.(D) Immunohistochemistry (IHC) analysis of SOX4 expression in RCC tissues and adjacent normal tissues.Representative images are displayed.(E) Immunofluorescence analysis of SOX4 expression in 786-O cells and HK-2 cells.Except IHC assay, all experiments were repeated at least three times.Error bars represent mean ± SEM from three independent experiments ( *** p<0.001, ** p<0.01, * p<0.05, compared with HK-2).

Figure 2 .
Figure 2. The effects of SOX4 on migration and invasion in 786-O and SN12-pM6 and HK-2 cell lines in vitro.(A-C) Wound healing and migration and invasion assays of 786-O transiently transfected with si-SOX4 or si-NC.(D-F) Wound healing and migration and invasion assays of SN12-pM6 transiently transfected with si-SOX4 or si-NC.(G and H) Migration and invasion assays of HK-2 transiently transfected with SOX4 or NC.The mean number of cells/field was derived from 5 random fields ( *** p<0.001, ** p<0.01, compared with the corresponding control).Each experiment was repeated at least three times.Representative images are displayed.

Figure 3 .
Figure 3.The EMT marker expression in RCC cell lines and tissues.(A and B) The mRNA levels and protein levels of EMT markers in RCC cell lines and normal renal tubule epithelial cell line.GAPDH was used as an internal control.(C and D) The mRNA levels and protein levels of EMT markers in RCC tissues and corresponding adjacent normal tissues.GApDH was used as an internal control.The data were derived from three independent experiments at least.Error bars represent mean ± SEM from three independent experiments ( *** p<0.001, ** p<0.01, * p<0.05, compared with the corresponding control).

Figure 4 .
Figure 4.The effect of SOX4 on EMT profile in 786-O and SN12-PM6 and HK-2 cell lines in vitro.(A) The expression change of EMT markers in SOX4knockdown 786-O cells and corresponding control cells.(B) The expression change of EMT markers in SOX4-knockdown SN12-PM6 cells and corresponding control cells.(C) The expression change of EMT markers in SOX4-introduced HK-2 cells and corresponding control cells.Error bars represent mean ± SEM from three independent experiments ( *** p<0.001, ** p<0.01, * p<0.05, compared with the corresponding control).

Figure 5 .
Figure 5.The effects of SOX4 knockdown and rescue on EMT expression profiles in the 786-O and SN12-PM6 and HK-2 cell lines.(A and B) The expression change of EMT markers after SOX4 upregulation in SOX4-knockdown 786-O and SN12-PM6 compared with corresponding control.(C) The expression change of EMT markers after SOX4 downregulation in SOX4-introduced HK-2 compared with corresponding control.Error bars represent mean ± SEM from three independent experiments ( *** p<0.001, ** p<0.01, * p<0.05, compared with the corresponding control).

Figure 6 .
Figure 6.SOX4 is implicated in TGFβ-induced EMT.(A) The morphology change of HK2 treated with control and TGFβ1 at indicated time.(b) The SOX4 mRNA expression change of HK-2 cells treated with TGFβ1 at indicated time.GAPDH was used as an internal control.(C) The EMT markers, SOX4, AKT and p-AKT protein levels of HK2 cells treated with TGFβ1 at indicated time.GApDH was used as a loading control.Error bars represent mean ± SEM from three independent experiments ( *** p<0.001, ** p<0.01, * p<0.05, compared with the corresponding control).