SPOP Could Play a Potential Inhibitory Role in Human Renal Cell Carcinoma

Background: SPOP, a substrate adaptor of Cul3 ubiquitin ligase, plays crucial roles in solid neoplasms by promoting the ubiquitination and degradation of substrates. Limited studies have shown that SPOP is overexpressed in human renal cell carcinoma (RCC) tissue. However, the exact role of SPOP in RCC remains unclear and needs to be further elucidated. The present study showed that SPOP was expressed at different levels in different RCC cell lines. The purpose of this study was to explore the roles of SPOP in the biological features of RCC cells and determine the expression levels of SPOP in human tissue microarrays (TMAs) and kidney tissues. Methods: Here, SPOP was overexpressed by lentiviral vector transfection in ACHN and Caki-1 cells, and SPOP was knocked down in Caki-2 cells with similar transfection methods. The transfection eciency was evaluated by quantitative PCR and western blotting analyses. The role of SPOP in the proliferation, migration, invasion and apoptosis of cell lines was determined by the MTT, wound-healing, Transwell and ow cytometry assays. Moreover, the cells were treated with different drug concentrations in proliferation and apoptosis assays to investigate the effect of sunitinib and IFN-α2b on the proliferation and apoptosis of SPOP-overexpressing cells and SPOP-knockdown RCC cells. Finally, immunohistochemical staining of SPOP was performed in kidney tissues and TMAs, which included RCC tissues and corresponding adjacent normal tissues. Results: Overexpression of SPOP inhibited cell proliferation, migration and invasion and increased cell apoptosis. Interestingly, sunitinib and IFN-α2b at several concentrations increased the proliferation inhibitory rate and total apoptosis rate of cells overexpressing SPOP. The ndings of the present study showed that the SPOP protein was signicantly expressed at low levels in most clear cell RCC (ccRCC) tissues and at relatively high levels in the majority of adjacent normal tissues and kidney tissues. Kaplan-Meier survival analysis showed that there was no statistically signicant difference in cumulative RCC: renal cell carcinoma; ccRCC: clear cell renal cell carcinoma; SPOP (cid:0) Speckle-type POZ protein; TMAs: tissue microarrays; VEGF: Vascular endothelial growth factor;PI3K: Phosphoinositide-3 kinase; AKT: protein kinase B;mTOR:mammalian target of rapamycin; CRL3: cullin3-RING ubiquitin ligase; AR: androgen receptor; SRC: steroid receptor coactivator; PTEN: phosphatase and tensin homolog deleted on chromosome ten; VEGFR: vascular endothelial growth factor receptor; DMEM (cid:0) Dulbecco's modied Eagle's medium. HHIs:Hedgehog inhibitors.

inhibitory rate and total apoptosis rate of cells overexpressing SPOP. The ndings of the present study showed that the SPOP protein was signi cantly expressed at low levels in most clear cell RCC (ccRCC) tissues and at relatively high levels in the majority of adjacent normal tissues and kidney tissues. Kaplan-Meier survival analysis showed that there was no statistically signi cant difference in cumulative survival based on the data of different SPOP expression levels in TMA and patients.
Conclusions: In contrast to previous studies, our ndings demonstrated that overexpression of SPOP might suppress the progression of RCC cells, which was supported by cell experiments and immunohistochemical staining. SPOP could be a potential tumour inhibitor in RCC.

Background
Renal cell carcinoma (RCC) ranks as the sixth most frequently diagnosed cancer in men and the 10th most frequently diagnosed cancer in women in the United States. Moreover, it has been reported that RCC incidence and mortality are increasing around the world [1][2][3]. Clear cell carcinoma represents the most common tissue subtype, accounting for 70%~90% of RCCs. Approximately 25% of patients diagnosed with RCC have evidence of advanced disease or metastases, although the increasing incidence is correlated with the wide use of multiple medical techniques for RCC screening [4,5]. Primary localized renal cancers can be cured by radical nephrectomy. The treatment outcome for patients with metastatic RCC (mRCC) has improved since the introduction of VEGF inhibitors and agents targeting the PI3K/Akt/mTOR signalling pathway, including combination strategies, such as lenvatinib and everolimus; however, drug resistance is still a major problem. Unfortunately, previous systemic treatment options could not provide long-term e cacy for mRCC, which ultimately becomes resistant to rst-line drugs [6][7][8]. The molecular mechanism of resistance to targeted therapy for advanced or mRCC has become a research hotspot.
Previous studies have shown that SPOP, as a substrate adaptor of cullin3-RING ubiquitin ligase (CRL3), recruits substrates to CRL3 for ubiquitination and degradation, such as the androgen receptor (AR), steroid receptor coactivator (SRC)-3 and PTEN proteins [10][11][12]. Over the past decades, SPOP has been con rmed as a tumour suppressor in several cancers, including prostate cancer, lung cancer and gastric cancer, and studies of the differential expression levels and mutation status of SPOP have indicated that SPOP plays different roles in cancer cell development [13]. It is well known that dramatically decreased SPOP expression is negatively correlated with tumorigenesis in gastric cancer tissues [14]. In addition, SPOP gene mutation is the most common missense point mutation in prostate cancer and affects the progression of prostate tumours through coordinated regulation of the PI3K/mTOR and AR signalling pathways [15,16]. Interestingly, to date, mutation of SPOP has not been found in RCC tumours [11,16].
However, some studies have shown overexpression of SPOP in the cytoplasm of clear cell RCC (ccRCC) cells and have indicated some correlation with high pathological stages, lymph node invasion and metastasis [17,18]. However, the exact role of SPOP in the biological features of RCC and its potential molecular mechanism in RCC tumours remain unclear. Based on the results of preliminary cell experiments, SPOP might act as a protective factor in RCC.

Materials And Methods
Cell lines and cell culture ACHN and Caki-1 cell lines were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences. Caki-2 cell lines were provided by Guangzhou Cell cook Biotech Co. Ltd. ACHN, Caki-1 and Caki-2 cell lines were maintained in Dulbecco's modi ed Eagle's medium (DMEM) (Gibco) supplemented with 10% foetal bovine serum (FBS). All the cells were cultured in a humidi ed incubator containing 5% CO 2 at 37°C and were used in further experiments.

Transfections
The plasmids were transfected into cells with Lipofectamine 2000 according to the manufacturer's instructions. The following day, the cells were cultured with media containing neomycin and selected for two weeks to obtain stably transfected cells. The SPOP plasmid or shRNA plasmid was packaged and transfected into retroviral packaging cells. Retroviral supernatants were added to the cells, spun for 45 min at 1800 rpm and incubated for 4 h at 37 ℃. The cell medium was switched to medium supplemented with puromycin for one week to select stable cell lines. The overexpression and knockdown e ciency of SPOP in the cells was tested by western blot and QPCR analyses.

Western blot analysis
As previously described [19], cell protein samples were harvested using RIPA buffer, and 20 µg of the protein sample was separated on 12% SDS-polyacrylamide gels followed by wet transfer at room temperature. The blots were then blocked with non-fat milk, followed by incubation with diluted primary antibody overnight at 4°C. The membranes were washed three times with TBST for 5 min and subsequently incubated with the appropriate secondary antibody conjugated to IRDye800 at room temperature for 2 h. Protein bands were visualized with the ECL detection system and analysed using ImageJ software.

Quantitative real-time PCR analysis
Total RNA from cells was extracted and reverse transcribed using a cDNA synthesis kit (Invitrogen). Realtime PCR analysis was performed using a LightCycler 96 (Roche). The peak of the melting curve was de ned as the criterion for ampli cation speci city. The relative expression levels of mRNAs were determined by normalization to the expression levels of the internal control gene GAPDH, and the data were analysed by the ΔΔCt method.

Cell invasion assay
The capacity of cell invasion was evaluated by the Transwell assay. A total of 2×10 5 cells in serum-free medium were plated on top of the Transwell chamber, which was coated with Matrigel matrix (Corning 354230). Medium supplemented with 10% FBS as the chemoattractant was added to the bottom of the chamber. The cells were then incubated in Transwell plates at 37°C in 5% CO 2 for 48 h. The non-invading cells at the top of the chamber were carefully removed with a cotton swab. The cells on the lower surface of the Transwell chamber were stained with crystal violet for 30 min after xation with paraformaldehyde.
The inserts were washed three times with PBS, and the number of invading cells was counted under a microscope.

Cell migration assay
Cell migration was determined by the wound-healing assay. A total of 1.2×10 5 cells were plated in a 12well plate at 37°C and 5% CO 2 overnight. A horizontal scratch was then made in the plate using a sterile pipette tip, followed by washing with PBS three times to remove the oating cells. Finally, the cells were incubated in serum-containing medium at 37°C in 5% CO 2 for 24 h. The scratch migration area was calculated using ImageJ software after 0 h and 24 h.

Cell proliferation and apoptosis assays
For the proliferation assay, 1.5×10 5 cells were cultured in 96-well plates with regular medium at 37°C and 5% CO 2 for 24 h. The next day, the culture medium was replaced with medium supplemented with IFN-α2b (20, 80, 4000 and 5000 IU/ml) or sunitinib (2.50, 5.01, 7.00 and 10.05 µmol/L) and incubated for 48 h. During culture, 10 ml CCK-8 chromogenic agent and 100 ml DMEM without FBS were added to the wells and incubated for 1 h. The absorbance (A) at 450 nm was analysed using a microplate reader. The inhibitory rate of cell growth (%) was quanti ed as follows: (1-(A treated )/(A control )) × 100%. For the apoptosis assay, 1x10 5 cells were seeded into 6-well plates, allowed to attach overnight and were treated with 10% FBS (control), IFN-α2b (20, 40 and 80 IU/ml), or sunitinib (5.0, 5.5 and 6.0 µmol/L) for 48 h. The cells were collected by centrifugation at 1200 rpm for 5 min at 37°C. The collected cells were washed with PBS and 50 µl 1x binding buffer. Then, the cells were stained with Annexin V-APC and 7-AAD and incubated at room temperature in the dark for 15 min. Subsequently, 50 µl 1x binding buffer was added, and the samples were tested using an Accuri TM C6 PLUS ow cytometer (BD Biosciences). The sum of early apoptosis and late apoptosis was de ned as total apoptosis. Tissue microarrays (TMA) and normal kidney tissues Immunohistochemical staining Immunohistochemical staining of the SPOP protein in the cytoplasm of TMA tissues and kidney tissues was performed with appropriate antibodies according to the methods of a previous study [20]. Brie y, para n-embedded sections were subjected to depara nization, rehydration, and heat-induced antigen retrieval. The sections were incubated with primary SPOP antibody overnight at 4°C after blocking endogenous peroxidase activity with 3% hydrogen peroxide. Rabbit IgG antibody was used for the isotype control. 3,3'-Diaminobenzidine (DAB) was added as a chromogen followed by counterstaining with haematoxylin. The staining intensity and positive staining rate were assessed by two independent pathologists according to the histologic scoring system (H-score). SPOP expression was scored comprehensively based on the positive staining rate and staining intensity. The positive staining rate was scored as follows: 0 (negative), 1+ (1-25%), 2+ (26-50%), 3+ (51-75%), and 4+ (76-100%). The intensity of cytoplasmic staining was classi ed as follows: 0 (negative), 1+ (weak), 2+ (moderate), and 3+ (strong). The above two scores were multiplied to obtain the nal score. A total score of SPOP immunohistochemical staining ≥ 6 was de ned as high expression; otherwise, it was considered as low expression.

Statistical analysis
Statistical analyses and gure preparation were performed using SPSS 24.0 (SPSS, Inc, Chicago, IL, USA) and GraphPad Prism 7.0 (San Diego, California, USA) software. Values of the in vitro cell experiments are presented as the mean ± standard deviation based on results obtained from at least three independent experiments. Comparisons were made between homogeneous experimental groups using the t-test or ANOVA, as appropriate. Mann-Whitney U test were used to analyse the differences of SPOP expression between tumor tissues and adjacent normal tissues. Survival analysis was determined by the Kaplan-Meier method and compared by the log rank test. A p-value less than 0.05 was considered to be statistically signi cant.

Results
Overexpression of SPOP inhibits the invasion and migration of RCC cells in vitro ACHN, Caki-1 and Caki-2 cell lines are commonly used in RCC studies. Our preliminary western blotting results showed that SPOP expression was signi cantly downregulated in ACHN and Caki-1 cell lines and upregulated in the Caki-2 cell line ( Fig. 1A and B). To explore the effect of SPOP on the biological features of RCC cells, ACHN and Caki-1 cells were transfected using a lentiviral vector overexpressing SPOP, and SPOP in Caki-2 cells was knocked down with similar transfections using a small hairpin RNA (shRNA) lentiviral vector. The overexpression and knockdown e ciency of SPOP were veri ed by western blot (Fig. 1C and D) and QPCR analyses. By Transwell assays, we found that overexpression of SPOP in ACHN and Caki-1 cells signi cantly inhibited the invasive ability of the cells after incubation for 48 h compared with the negative control (NC). Similar results were observed in the si-NC group of Caki-2 cells compared with the SPOP-silenced group (P < 0.0001, Fig. 2A). Moreover, cell migration is another malignant behaviours of cancer cells. The effect of SPOP on cell migration was measured by the woundhealing assay. High expression of SPOP in Caki-1 and Caki-2 cells signi cantly decreased the migration capacity of RCC cells after 24 h compared to that of cells with low expression of SPOP (P < 0.01 Fig. 2B).
A similar phenomenon was observed in ACHN cells; however, no signi cant difference was noted (P = 0.0649). The above ndings suggested that SPOP may play an important role in suppressing the malignant biological behaviour of RCC cells.

SPOP suppresses RCC cell proliferation and induces cellular apoptosis
The data showed that SPOP plays a key role in the invasion and migration of RCC cells. Next, we performed experiments to investigate the effect of SPOP on the proliferation and apoptosis of RCC cells by using the MTT assay and ow cytometry. Previous studies suggested that the motility of Caki-2 cells was stronger than that of ACHN and Caki-1 cells, which could be suppressed by IFN or sorafenib, especially when the two drugs were combined, which indicated that Caki-2 cells were more aggressive than ACHN and Caki-1 cells [21]. Advanced or mRCC cannot be cured due to drug resistance, which remains one of the most challenging issues. In the present study, we investigated the effects of rst-line drugs (sunitinib and IFN-α2b) on the above cell lines with different SPOP expression levels. The results showed that overexpression of SPOP decreased RCC cell proliferation (Fig. 3A and B) and induced cellular apoptosis under several drug concentrations (Fig. 3C and D) compared to low SPOP expression.
All of these ndings suggested that the SPOP protein may improve the susceptibility of RCC cells to drug treatments.

SPOP is mainly expressed at low levels in the cytoplasm of ccRCC tissue
Cell experiments indicated that SPOP expression signi cantly inhibited the growth and progression of RCC cells. To determine SPOP expression in human RCC tissue and normal kidney samples, immunohistochemical staining was performed on TMA tissues consisting of ccRCC tissues (n = 88), papillary RCC tissues (n = 2) and corresponding adjacent normal tissues. We found that the expression of SPOP protein in the cytoplasm was signi cantly downregulated in 83% of ccRCC tumour tissues and upregulated in 88% of adjacent nontumour tissues (P < 0.0001, Fig. 4). To further con rm these preliminary results, we continued to analyse SPOP expression in the normal kidney samples. Consistent ndings were observed in the immunohistochemical staining of normal kidney tissues (Fig. 4), in which the SPOP protein was mainly overexpressed in the cytoplasm of kidney tissues. This is quite different from previous studies, which showed that the SPOP protein was overexpressed in over 80% of RCC tissues, even in nearly 100% of primary ccRCCs showing SPOP accumulation, and negative in 82% of normal kidney tissues [17,18,22].

SPOP protein expression and clinical correlations in RCC
Based on the immunohistochemical results, we showed that the SPOP protein was expressed at lower levels in most RCC tissues. Next, we investigated whether SPOP expression was associated with overall survival. Survival analysis was performed using the Kaplan-Meier method based on SPOP expression levels and follow-up time. We found that there was no signi cant correlation between SPOP expression and patient cumulative survival (P > 0.05), as shown in Fig. 5. Analysis of Kaplan-Meier curves suggested that the role of SPOP in RCC remains controversial, and the mechanism of action of SPOP needs to be explored further. However, all of our ndings suggest that the SPOP protein could act as a protective factor in RCC.

Discussion
SPOP, a CRL3 substrate adaptor protein, plays an important role in the development of some cancers [10][11][12]. Over the past decade, the potential functions of SPOP in urologic cancers have gradually attracted much attention from investigators. SPOP was found to be the most common missense mutated gene in human prostate cancers and has been shown to be associated with the pathogenesis of primary prostate tumours, but SPOP mutations in RCC tumours have not yet been reported [15,16,23,24]. Recent studies have shown that SPOP is an oncoprotein that is overexpressed in RCC [17,18,25]. However, the results of our preliminary cell experiments showed that overexpression of SPOP inhibited RCC cell proliferation, migration and invasion and increased cellular apoptosis rates. Similar to our cell experiment results, some studies also found that high expression of SPOP suppressed the malignant biological behaviour of cancer cells in vitro via ubiquitin-dependent proteolysis of the signalling pathway [14,[26][27][28]. Therefore, SPOP may be associated with inhibition of the aggressiveness of RCC cells.
SPOP plays key roles in cancer development by promoting ubiquitination and degradation of the substrate protein of speci c signalling pathways. For example, SPOP has a de nitive tumour suppressing role in gastric cancer by promoting the degradation of the transcription factor Gli2 of the Hedgehog (Hh)/Gli2 signalling pathway [14]. In an in vitro drug sensitivity experiment, we found that the proliferation inhibitory rates of cells were signi cantly increased and cellular apoptosis was induced when SPOP was overexpressed in RCC cells that were treated with sunitinib or IFN-α2b. Sunitinib, a tyrosine kinase inhibitor targeting the VEGF receptor, has been the rst-line targeted therapy for patients with mRCC who have been classi ed as having MSKCC intermediate-risk or poor-risk disease and has shown an improvement in survival [6,29]. Cellular migration, proliferation and survival of cancer cells as well as endothelial cell differentiation are driven mainly by VEGF/VEGFR activation, which in turn activates the PI3K/Akt/mTOR signalling pathway [30,31]. One study showed that 4-chloro fascaplysin, a marine sponge alkaloid derivative, inhibited tumour growth and VEGF-mediated angiogenesis by disrupting the PI3K/Akt/mTOR signalling cascade [32]. The PI3K/Akt/mTOR axis, which is involved in cancer cell proliferation, differentiation and cellular metabolism, is frequently activated in many cancers and is one of the most signi cant molecular pathways in mRCC [33,34]. Activation of the PI3K/Akt/mTOR pathway is correlated with aggressive behaviour and poor prognosis of RCC tumours and is more signi cantly altered in ccRCC, high TNM stage tumours, and tumours with poor prognostic features [35,36]. SPOP binding to the substrate is a crucial event for E3 ligase-mediated ubiquitination and subsequent proteasome degradation. Levels of the PI3K/Akt pathway have been found to be correlated with SPOP expression, which could inhibit colorectal cancer and osteosarcoma invasion by signi cantly reducing the levels of PI3K and p-Akt [37,38]. In the present study, there was a signi cant difference in the sensitivity of different cell lines overexpressing SPOP to several concentrations of sunitinib. It could be a promising potential molecular mechanism that may provide an effective therapeutic strategy for patients with advanced kidney cancer by exploring the relationships among the SPOP, VEGF and PI3K/Akt/mTOR pathways.
In addition, the Hedgehog signalling pathway, which increases tumour invasion and metastatic potential, is another important molecular mechanism that is worth investigating in the future. Aberrant activation of the Hedgehog pathway is associated with tumorigenesis in some cancers, including RCC, and plays an important role in RCC development [39][40][41]. Limited studies suggest that SPOP suppresses tumour development by negatively regulating the Hedgehog/Gli2 signalling pathway in gastric cancer [14]. In addition, the expression levels of the Hedgehog signalling pathway component genes Gli1 and Gli2, which are activated by the PI3K/Akt signalling pathway in RCC, are signi cantly elevated in ccRCC and provide a promising therapeutic strategy for RCC [42]. Currently, the Hedgehog inhibitors (HHIs) vismodegib and sonidegib are approved for use in advanced BCC, and other potential uses for the treatment of solid tumours beyond BCC are under development or in clinical trials [43]. Given the above, SPOP, as a tumour suppressor protein, plays an important role in inhibiting tumorigenesis by regulating different signalling pathways. However, studies on the molecular mechanism of the SPOP protein in RCC are still limited. Exploring the underlying mechanisms of signalling pathways in kidney cancer in detail is the best approach to provide a theoretical basis for the development of novel therapeutic strategies for mRCC patients in the future.
Differential expression levels or mutation pro les of SPOP in tumours play different roles in tumorigenesis and cancer progression [10,11,13]. Several studies have shown that SPOP expression is downregulated in some primary tumours, including gastric cancer, liver cancer, colorectal cancer, pancreatic cancer and non-small cell lung cancer, and low expression of SPOP is associated with poor prognosis in patients [14,[26][27][28]44]. In the current study, immunohistochemical staining demonstrated that the SPOP protein was mainly expressed at low levels in the cytoplasm of ccRCC tissues and was relatively highly expressed in most adjacent nontumour tissues and normal kidney tissues. The inhibitory role of SPOP was con rmed by an earlier study that showed that downregulation of SPOP expression in cancers might inhibit its functions as a tumour suppressor gene and might promote cancer development [45]. On the basis of the results of immunohistochemical staining and cell culture experiments in the present study, SPOP may act as a potential tumour suppressor protein in the tumorigenesis of RCC. However, the ndings of the survival analysis did not provide supportive evidence showing a correlation of SPOP expression and overall survival, suggesting that high expression of SPOP could not be regarded as a hallmark of RCC and could not yet predict the prognosis of patients. The exact role of the SPOP protein in RCC is controversial and still needs to be con rmed by further research based on a large cohort of samples.
Although there are some important discoveries in the present study, some limitations need to be discussed. First, the concentration gradient of drug experiments was too large to accurately re ect the signi cant concentration. Second, it is widely accepted that RCC is a heterogeneous tumour with distinct pathological tissue subtypes, including clear cell, papillary, and chromophobe subtypes. The TMA tissues used in this study consisted of a single pathological tissue subtype and could not be used to explore the expression of SPOP in the different subtypes of RCC. More pathological tissue subtypes should be included to analyse the expression of SPOP in RCC tissue in the future, especially fresh frozen tissue from RCC radical nephrectomy.

Conclusions
In brief, we report that SPOP reduces tumorigenesis features in RCC cell lines and induces cell apoptosis in vitro. In human RCC samples, SPOP is expressed at low levels in the majority of ccRCC samples and at higher levels in most adjacent nontumour samples. All of these ndings suggest that SPOP may act as a potential tumour suppressor protein in the tumorigenesis of human RCC. Further studies with a larger patient cohort and analysis of molecular mechanisms are needed to con rm our ndings.

Consent for publication
All authors have given their consent for the publication of this article.

Availability of data and materials
All data generated or analyzed during this study are included either in this article. The datasets used and/oranalysed during the current study are available from the corresponding author on reasonable request.

Competing interests
No potential con icts of interest are disclosed.   Immunohistochemical staining of SPOP in the cytoplasm of RCC tissues and adjacent normal tissue. SPOP expression in the TMA was assessed by Aperio image software, and the image was captured at 4X magni cation. In the TMA, SPOP was signi cantly expressed at low levels in the cytoplasm of ccRCC tissues and at high levels in adjacent normal tissue(****, P<0.0001). Similar results were observed in normal kidney tissue overexpressing the SPOP protein under a 4X eld microscope.