Significance of Suppressor of Cytokine Signaling-3 Expression in Bladder Urothelial Carcinoma in Relation to Proinflammatory Cytokines and Tumor Histopathological Grading

Bladder cancer is the second most common malignancy affecting the urinary system, with the incidence being four times higher in men than in women (Fajkovic et al., 2011). Urothelial carcinoma originates from an epithelial origin and represents approximately 90% of bladder cancers. Cigarette smoking, occupational exposures to carcinogens and chronic infection with Schistosoma haematobium have been established as risk factors for bladder cancer (Kobeissi et al., 2013). The mammalian signal transducers and activators of transcription (STATs) family comprises seven intracytoplasmic proteins; STAT1, 2, 3, 4, 5A, 5B, and 6 , whose biological activities control critical aspects of cell growth, survival and differentiation (Santos et al., 2011). STATs activation depends on the phosphorylation of a conserved tyrosine residue (Y705) by Janus kinases


Introduction
Bladder cancer is the second most common malignancy affecting the urinary system, with the incidence being four times higher in men than in women (Fajkovic et al., 2011).Urothelial carcinoma originates from an epithelial origin and represents approximately 90% of bladder cancers.Cigarette smoking, occupational exposures to carcinogens and chronic infection with Schistosoma haematobium have been established as risk factors for bladder cancer (Kobeissi et al., 2013).
The mammalian signal transducers and activators of transcription (STATs) family comprises seven intracytoplasmic proteins; STAT1, 2, 3, 4, 5A, 5B, and 6 , whose biological activities control critical aspects of cell growth, survival and differentiation (Santos et al., 2011).STATs activation depends on the phosphorylation of a conserved tyrosine residue (Y705) by Janus kinases

Significance of Suppressor of Cytokine Signaling-3 Expression in Bladder Urothelial Carcinoma in Relation to Proinflammatory Cytokines and Tumor Histopathological Grading
Hanaa Hibishy Gaballah 1 *, Noha Mohamed Shafik 1 , Rania Elsayed Wasfy 2 , Mohamed Osama Abou Farha 3 (JAKs) in response to specific ligands such as cytokines (IL-6), growth factors, and extracellular signals (Quintás-Cardama et al., 2013).Dysregulation of the JAK-STAT pathway is observed in many primary human tumours (Furqan et al., 2013).Due to the disastrous biologic consequences of uncontrolled cytokine-driven STAT activation, the JAK/STAT pathway is tightly controlled by the suppressors of cytokine signaling (SOCS) proteins, namely, SOCS1-SOCS7 and the SH2 domain-containing.
As a regulator of STAT-3 activation, SOCS-3 was thought to serve as multifunctional molecular switches facilitating or suppressing neoplastic transformation depending on cellular context (Culig 2013).Altered expression of SOCS-3 protein were found to be involved in the development and progression of several malignancies such as hepatocellular (Wu et al., 2011) and renal cell carcinoma (Stofas et al., 2014).However, the existing information about the role of SOC-3 protein in bladder urothelial carcinoma remains limited and inconclusive.
Growing body of evidences highlights the pivotal role of Th2 cytokines particularly  and  in controlling growth and differentiation of normal and malignant cells and in contributing to the evasion of immune surveillance (Töpfer et al., 2011).IL-6 is a multifunctional cytokine associated with a number of biological functions in bladder cancer (Ebadi N et al., 2014), including cell proliferation, cell transformation, inflammation, and detrusor smooth muscle contractility (Tsui et al., 2013).IL-8 is a proinflammatory CXC chemokine that modulates both the cellular transcriptome and proteome within the tumor microenvironment.It also affects the organization of the cell cytoskeleton through posttranslational regulation of regulatory proteins (Zarogoulidis et al., 2014).
Cytokeratins provide mechanical stability to tissues; their synthesis is usually maintained during malignant transformation representing one of the hallmarks of epithelium-derived tumors (Karantza, 2011).Cytokeratin 18 (CK-18), a member of the intermediate-filament gene family, is important for cellular processes such as apoptosis, mitosis, cell cycle progression, and cell signaling (Weng et al., 2012).Recently, CK-18 is attracting considerable interest as measure of cell death for monitoring of disease progression in cancer patients (Benedict et al., 2014).
Deciphering the well-orchestrated signaling pathways coordinating the bladder carcinogesis is crucial to optimizing diagnosis and treatment strategies.Therefore, we herein aimed to investigate the m RNA expression pattern of SOCS3 in normal and cancerous bladder tissue and to explore its relation to urinary levels of some proinflammatory cytokines, CK -18 and with histopathological grading, in order to evaluate their role as potential diagnostic and prognostic markers for bladder urothelial carcinoma.

Materials and Methods
This study was carried out on fifty individuals presented to the Urology department, Tanta University Hospital and its Outpatient Clinics.They included 36 male patients with age range of (46-55) and with histopathologically confirmed urothelial bladder carcinoma after transurethral resection of suspicious bladder mass.The tumors were graded according to the World Health Organization 2004 criteria (Montironi and Lopez-Beltran, 2005); pathological grades were classified to low-grade (group I) and high-grade uro¬thelial carcinoma (group II).Patients with other malignant disease, or urinary tract infection, renal insufficiency or prior chemotherapeutic or radiation therapy were excluded from the study.Also, 14 individuals with no evidence of urothelial carcinoma, gross h,ematuria, active urinary tract infection or urolithiasis who were scheduled for some endourological procedures that involved bladder visualization were representing the control group ( group III).They were matched to the patients group with respect to the age, sex and smoking status.
Informed written consent was obtained from all participants.The study protocol was approved by the Local Research Ethics Committee, Tanta University and was in accordance with the principles of the Declaration of Helsinki II.All of the participants were subjected to detailed history taking, clinical examination, abdominopelvic ultrasonography, and routine laboratory investigations.Besides, a cystoscopic examination was performed for each patient, by which transurethral resection biopsies were taken from the apparent lesion, processed by standard oncological procedures, studied and graded by a specialized pathologist.
For tissue sampling, the bladder biopsies were divided into 2 portions: one portion was kept on liquid nitrogen for studying of SOCS-3 mRNA gene expression and the other one for histopathological and immunohistochemistry studies.
For histopathological study bladder tissue biopsies were kept in 10% formalin solution and embedded in paraffin.Five-µm slices were stained with hematoxylin & eosin (H.&E.) and photomicrographs were taken at 200x and 400x.All cases were histopathologically diagnosed as urothelial carcinoma.

Immunohistochemistry
Five-micrometer sections from selected tumor blocks were mounted on 3-aminopropyltriethoxysilane coated (Sigma, St. Louis, MO USA) slides and were deparaffinized in xylene, rehydrated in graded alcohols, plus rinsed in 0.05 m Tris-buffered saline.Sections were boiled in 10 mm citrate buffer for antigen retrieval, at pH 6.0.Endogenous peroxidase was blocked with aqueous 0.3% H 2 O 2 for 15 min.An avidin-biotin-peroxidase method was employed as described in manufacturers' kit manual.The sections were incubated in 5% normal rabbit serum followed by one hour incubation in CK 18 (1:100, Dako).As negative control, the primary antibody was replaced by TBS, and the appropriate normal areas in the sections served as positive controls.Positive immunereactivity was defined as more than 20% of cells staining with the proper pattern of reactivity.Immunopositivity to CK18 appeared as brown cytoplasmic staining of tumor cells.

Urine sample collection and storage
Before any therapeutic intervention, 100 mL of voided urine was obtained from each subject.Fifty milliliters of urine was used for clinical laboratory analyses as urinary cytology and urinalysis, according to standard procedures.The remaining 50 ml of urine was centrifuged at 600 ×g 4°C for 5 min.The supernatant was decanted and aliquoted, and the urinary pellet was snap frozen.Both the supernatant and pellet were stored at -80 °C prior to analysis.
Quantitative determination of total CK-18 in urine: The M65 ELISA kit (#Cat 10020, Peviva AB, Bromma, Sweden) was used according to the manufacturer's instructions.The M65 ELISA consists of two monoclonal antibodies (clones M5 and M6) specific for conventional epitopes on CK-18, present on both intact/uncleaved and cleaved CK-18.In brief, urine samples were first placed into 96-well plates which had been coated with a mouse M6 monoclonal antibody directed against CK-18 as solid phase catcher antibody.Following incubation for 4 h at room temperature, the plate was washed five times with phosphate buffered saline.A horseradish peroxidase conjugated M5 antibody directed against another epitope on CK-18 was then used to detect the presence and concentration of M65 (Roth et al., 2011).The absorbance at 450 nm was determined in a microplate reader within 30 min of setting up the assay.

Estimation of of SOCS-3 mRNA expression levels by real-time PCR
i) RNA extraction: Total RNA from bladder tissue samples was prepared using Qiagen RNeasy Mini Kit according to the protocol supplied by the manufacturer.RNA was eluted, its concentration was measured spectrophotometrically (280) and then stored at -80°C.
ii) cDNA synthesis was performed using the RevertAid H Minus First Strand cDNA Synthesis kit (#K1632,Thermo Scientific Fermentas, St. Leon-Ro, Germany) according to the manufacturer's instructions.Ten μl of random hexamer primers (Roche, Mannheim, Germany) were added to 21 μl of RNA which was denatured for 5 minutes in the thermal cycler (Biometra, USA).The RNA-primer mixture was cooled to 4°C.The cDNA master mix was prepared (5 μl of first strand buffer, 10 mM of dNTPs,1 μl of RNase inhibitor, 1 μl of reverse transcriptase Superscript™ II-RT enzyme and 10 μl of DEPC treated water ) according to the kit protocol and was added to each sample.The total volume of the cDNA master mix was 19 μl for each sample.This was added to 31 μl RNA-primer mixture resulting in a reaction volume of 50 μl, which was then incubated in the programmed thermal cycler one hour at 37 °C, followed by inactivation of enzymes at 95 °C for 10 minutes, and finally cooled at 4°C.The RNA was reverse transcribed into cDNA which was then stored at -20°C.
iii) Real-time quantitative PCR: One μl of the cDNA was added to a 20 μl reaction mixture of the QuantiTect SYBR-Green PCR kit (Qiagen) and 0.5 μM from the specific primer pair for human SOCS-3.This cDNA was then amplified using the Step One instrument (Applied Biosystems, USA) as follows: Initial denaturation at 95˚C for 15 minutes was followed by 40 cycles with denaturation at 95˚C for 15 sec, annealing at 60˚C for 30 sec and extension at 72˚C for 20 sec.A control reaction without a DNA template was performed in parallel to detect genomic DNA contamination.Primer sequences specific for SOCS-3 were designed according to Walsh.et al., 2006; as follows: forward primer: 5'-CCCTCGCCACCTACTGAA-3' and reverse primer: 5'-TCCGACAGAGATGCTGAAGA-3. Primers for β-actin were included as an internal control: forward primer: 5'-TGGCATTGCCGACAGGATGCAGAA-3, and reverse primer: 5'CTCGTCATACTCCTGCTTGCTGAT -3'.The determination of the relative levels of gene expression was performed using the cycle threshold) rrCt (method and normalized to the reference gene β-actin, which was not altered by the experimental conditions. Statistical Analysis: The data were analyzed using statistical package for the social science (SPSS) version 20.0 software (SPSS Inc., Chicago, IL, USA).Quantitative data expressed as mean and standard deviation.Categorical variables were compared using Chi-square test.Multiple comparisons were performed by one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test.Correlations were analyzed using the Pearson test.Multiple logistic regression analysis was used to estimate the odds ratios (ORs) and 95% confidence intervals (CI) for the studied parameters.Receiver operating characteristics (ROC) analysis was used to identify the optimal threshold values of the studied parameters.

Results
A statistical comparison between the studied groups with respect to the age and laboratory biochemical findings using ANOVA test followed by Tukey's test is demonstrated in Table (1) and Figure (1 Regarding the correlations of the studied parameters with each other in different grades of urothelial carcinoma, the mRNA relative expression levels of SOC-3 showed significant negative correlations with the urinary levels of IL-6 (r= -0.56, p<0.01),IL-8 (r= -0.68 and total CK-18 (r= -0.49, p<0.01) .Moreover urinary levels of IL-6 and IL-8 exhibited a significant positive correlation with each other(r= -0.61, p<0.01) and with urinary total CK-18 (r=0.59, and r= 0.47, respectively) p<0.01.None of the studied parameters showed significant correlations with age.These data are summarized in Table (2).
Receiver operating characteristics (ROC) analysis was used to assess the diagnostic and prognostic value of the urinary cytokines as well as to identify their optimal cut off values.The area under the curve can range from 0.5 to 1 and diagnostic tests that approach 1 indicate a perfect discriminator.The optimal cut off value of urinary IL-8 was 10 pg/L, the sensitivity at this cut off point was 90%, the specificity was 100%, the positive predictive value was 100% and the negative predictive value was 83.3%   and the area covered was 0. 966.Urinary IL-6 monitoring revealed a sensitivity of 70%, specificity of 85%, positive predictive value of 90.3 %, negative predictive value of 58.6%, and an accuracy of 0.815 at an optimal cut off value of 66.5pg/ml.Likewise, the cut off value of urinary total CK-18 was 150 U/L , at this point the sensitivity was 87.5%, the specificity was 85.0%, the positive predictive value was 92.1% and the negative predictive value was 77.3% and the accuracy was 0. 925.Data are summarized in Table (4) and Figure (2

).
During Immunohistochemical analysis; normal areas in the sections served as positive controls showed superficially diffuse cytoplasmic staining of CK-18 in the umbrella cells.CK-18 positivity was detected by diffuse cytoplasmic staining in all tumor cells.Ten cases of low grade urothelial carcinoma were CK-18 positive and eight cases were negative.Sixteen cases of high grade urothelial carcinoma were CK-18 positive and two cases were negative.Using Chi-square test, the expression of CK-18 was significantly higher in high grade compared to low grade urothelial carcinoma (χ²=4.15,p <0.05).These data are demonstrated in Table (5) and Figures (3-6).

Discussion
The suppressor of cytokine signaling SOCS family is characterized as endogenous negative regulators to the JAK/STAT signaling cascade which modulates various fundamental biological processes and cancer pathogenesis (Linossi et al., 2013).Altered expression of suppressor of cytokine signaling -3 (SOCS-3) has been implicated in various types of human cancers (Sasi et al., 2014); however its role in bladder urothelial carcinoma as well as its relevance to tumor histopathological grade remains controversial.
The present study revealed that SOC-3 mRNA expression levels were significantly lower in cancerous bladder tissue compared to normal bladder tissue samples.Moreover, high grade urothelial carcinoma was associated with lower SOC-3 expression than low grade carcinoma, suggesting a role for SOC-3 impaired expression in tumor   progression and cancer cell differentiation.In harmony with these findings, reduced expression of SOCS-3 mRNA had been reported in other types of cancer such as lung cancer and head and neck squamous cell carcinoma (Baltayiannis et al., 2008, Ying et al., 2010).Moreover, in vitro forced expression of SOCS-3 in mesothelioma and lung adenocarcinoma cell lines decreased tumor invasion by modulating expression of genes that are essential for cell proliferation, migration and invasion (Yu et al., 2009).Well in line, Sasi et al., 2010 andWu et al., 2011 reported an inverse relationship between SOCS-3 expression and tumor stage in breast and hepatocellular carcinoma respectively, where higher expression was associated with earlier tumour stage and better clinical outcome.
Given that the constitutive JAK/STAT activation plays a pivotal role in the pathogenesis of urothelial carcinoma through regulating apoptosis, proliferation and invasion-promoting genes (Chen et al., 2008), it can be anticipated that once SOCS-3 expression is impaired and its negative regulatory effect on JAK-STAT pathway is lost, the bladder cancer cells become more sensitive to aberrant growth stimulating signals, thus promoting tumor progression.Further supporting the tumor suppressor role of SOC-3, Stahl et al., 2012 reported that SOCS-3 is an endogenous feedback inhibitor of pathologic angiogenesis acting at the converging crossroads of growth factor-and cytokine-induced vessel growth.
Noteworthy, the reported decreased SOC-3 expression in urothelial carcinoma as well as in other types of cancer can be explained by the epigenetic gene silencing through aberrant gene promoter hypermethylation which causes transcriptional downregulation of SOC-3 gene and subsequent impaired its expression (Li et al., 2012).Epigenetic dysregulation of the SOCS-3 gene may interfere with the cellular response to the complex cytokine network thus contributing to oncogenesis (Al-Jamal et al., 2014) .Well in line, demethylating agents which can restore SOC-3 expression remarkably suppressed cell proliferation and induced apoptosis of pancreatic cancer cells (Wang et al., 2014).Since SOCS-3 cytoplasmic localization is critical for mediating suppression of cytokine signaling, it seems plausible that a predominant nuclear localization might cause an impairment of its function in cancer cells that still possess detecTable SOCS-3 expression (Rossa C et al., 2012).
In contrast to these findings, Huang et al., 2009 failed to establish any significant correlations between SOCS-3 expression and invasiveness or histological grade of urothelial carcinoma.Furthermore, SOC-3 overexpression has been found to be correlated with advanced disease and poor response to treatment in other types of cancer such as lymphoma and some leukemias, representing a consequence of persistent STAT3 activation and cytokine production by tumor cells (Attia et al., 2011).This sustained STAT3 activation might be attribuTable to the failure of other negative regulatory pathways of JAK-STAT signaling as well as development of strategies to by-pass negative regulation by SOCS-3 protein (Li et al., 2011).Taken together, it is tempting to speculate that SOCS-3 plays a complex role in different types of malignancies as it may facilitate or suppress oncogenic transformation depending on cellular context which underline the need for further investigations.
On the other hand, it has been hypothesized that immune mediators in urine may serve as potential noninvasive diagnostic biomarkers for bladder cancer (Wang et al., 2012);owing to the fact that the urine of bladder cancer patients is in intimate contact with tumor cells and adjacent inflamed urothelium (Ghafouri-Fard et al., 2014).In this context, we further investigated the urinary levels of Th2 cytokines in the same cohort, where IL-6 levels were significantly increased in bladder cancer patients compared to control subjects with being significantly higher in high grade than low grade urothelial carcinoma.Concurring with this finding, Chen et al., 2013 revealed that IL-6 was overexpressed in the bladder cancer specimens compared with non-malignant tissues.In addition, they postulated that IL-6 might be related to a more malignant phenotype, as its expression was significantly increased in patients with locally advanced compared to noninvasive urothelial bladder cancer.Moreover, they provided evidences that blocking IL-6 expression resulted in significant reduction of bladder tumor growth and its invasive capability.
Along this line, the role of IL-6 in promoting bladder carcinogenesis is further underscored by the finding that IL-6, as a major activator of JAK/STAT3 signaling, enhances cell proliferation, epithelial to mesenchymal transition as well as increased angiogenesis by transcriptional activation of vascular endothelial growth factor, matrix metalloproteinase-9 and DNA methyltransferase 1 expressions (Taniguchi and Karin 2014), thus targeting IL-6 may be a promising strategy for treating bladder cancer.
Nevertheless, our data reported significantly increased urinary IL-8 levels in bladder cancer patients than controls, with being higher in high grade than low grade urothelial carcinoma.These findings are in agreement with that of Margel et al., 2011 andUrquidi et al 2012 who observed that urinary concentrations of IL-8 were significantly elevated in patients with urothelial carcinoma.Relevant to this notion, Reis et al., 2012 demonstrated that IL-8 has both mitogenic and angiogenic properties and its elevated expression was associated with enhanced bladder carcinogenicity , poor prognosis, disease recurrence and resistance to chemotherapy .Moreover, in vitro IL-8 signaling blockade significantly abrogated its promoting effects on cancer growth and metastasis, inhibited the expressions of matrix metalloproteinases, and decreased bladder cancer invasion (Lippitz, 2013).Collectively, these findings might provide a rationale for the therapeutic role of agents targeting Th2 cytokines in urothelial carcinoma microenvironment.
Furthermore, our data revealed a significant negative correlation between SOC-3 expression and urinary levels of IL-6 and IL-8 in the same cohort.This is in accordance with da Silva et al., 2013 who postulated that down regulation of SOC-3 expression enhances IL6/STAT proliferative signaling.Conceivably, loss of the negative feedback regulation of SOC-3 on these proinflammatory cytokines results in their pronounced secretion and pathological enhancement of carcinogenesis (Li al. DOI:http://dx.doi.org/10.7314/APJCP.2015.16.1.307 SOC3 in Bladder Carcinoma in Relation to Proinflammatory Cytokines andTumor Histopathological Grading 2011).Alternatively, it has been reported that IL-6 could induce the DNA methyltransferase (DMNT1) activity and mediate SOCS3 promoter hypermethylation, so maybe the reduced SOCS expression might be caused by the elevated IL-6 (Li et al., 2012).
Dysfunctional regulation of CK-18 is thought to be involved in bladder carcinogenesis by modulating intracellular signaling and operating in conjunction with various related proteins (McConkey et al., 2010).Immunohistochemical analysis of bladder tissue samples which was further validated by urinary measurement of total CK-18 demonstrated that total CK -18 expression and urinary levels were significantly were higher in high grade compared to low grade urothelial carcinoma and control subjects .This finding is harmony with that of Song et al., 2009 who concluded that CK -18 might serve as a useful marker for diagnosis of urothelial carcinoma of the bladder and for monitoring the state of illness .Well in line, Ramazan Sekeroğlu et al., 2002 reported that expression of CK-18 increases in parallel with tumor grade and cell deoxyribonucleic acid content, thus it could be recognized as a marker of aggressiveness in bladder tumors.This observed correlation between CK -18 level and the grade of malignancy may represent a general loss of urothelial differentiation features in high grade tumors (Paner et al., 2014).
Recently, changes in urinary levels of total and caspase cleaved CK-18 have been used to assess the tumor cell response to Adenoviral-mediated interferon α treatment and to predict disease outcome in bladder cancer patients (Benedict et al., 2013).Of note, CK-18 expression pattern was correlated with tumor aggressiveness, progression and poor prognosis in several types of human cancer such as renal cell (Messai et al., 2010), ovarian, colorectal and esophageal carcinomas, which might be attributed to its role in regulation of cell cycle, cell growth and apoptosis (Weng et al., 2012).
Collectively, urinary levels of IL-8, total CK-18 and IL-6 were significantly associated with urothelial bladder carcinoma; however, the diagnostic efficacy of IL-8 was superior to that of total CK-18 and IL-6 in terms of accuracy, sensitivity and specificity.Moreover, multiple logistic regression analysis underscored the significant association of the studied parameter with urothelial carcinoma development.
In conclusion, the present study demonstrated that advanced grade of urothelial bladder carcinoma was significantly associated with lowered mRNA expression of SOC-3 as well as elevated urinary levels of proinflammatory cytokines and CK-18.These findings extend our understanding of the molecular mechanisms involved in bladder carcinogenesis and lay the ground for their targeting as a therapeutic strategy for urothelial carcinoma.Furthermore, our data suggested that urinary IL-8, IL-6 and CK-18 might benefit as noninvasive biomarkers for early detection as well as histopathological subtyping of urothelial carcinoma.

Table 1 . Demographic and Laboratory Findings of the Studied Groups
).No statistically significant difference was detected between the studied groups regarding age (p>0.05).SOC-3 m RNA relative expression levels were significantly lower in high