Globally, renal cell carcinoma (RCC) is the fifth common malignancy and it formed about 2–3% of all cancers in adults [1][2]. Patients that are having early stages RCC is increasing, but 30% of RCC are liable to develop metastatic and incurable disease [3]. Identification of novel predictive and prognostic bio-markers for RCC is a must, which could help to discover recent beneficial therapeutic agents for patients with variable clinicopathological parameters. TNM stage, cancer size and Fuhrman grade are the usual factors that could predict patients’ outcome [4]. But it was found that RCC patients that had the same stage can have different outcomes and that have not been understood yet. The studying of epigenetic-changes could explain cancer biological criteria and gives chances for discovering novel therapeutic agents for RCC [5]. Enhancer of zeste homolog 2 (EZH2) that have been mapped on chromosome 7q-35, is a polycomb group (PcG) genes family member which have allowed the gene expression patterns transmission to daughter cells with a stability. It is as a histone methyl-transferase that can silence many different tumor suppressor genes. The EZH2 have recently found to play essential roles in RCC oncogenesis [6]. But, the exact mechanism of its action is still uncertain.

Signal-transducer and activator of transcription (STAT) proteins are large family of transcription factors and STAT3 which is one member of such family have controlled many normal cellular processes as cell proliferation, survival, differentiation, and even inflammation [7], also it can be expressed in many malignant human tumors [8]. STAT3 activation stimulated cancer cells proliferation, invasion, carcinogenesis and also maintenance of tumor promoting inflammatory microenvironment [9]. Many cancer stimulatory STAT3 functions were discovered, e.g., drugs resistance [10], role in epigenetic regulations [11] and cancer stem cells [12]. All previously discovered data showed that STAT3 formed an important novel therapeutic target for patients with RCC [13]. But its exact prognostic role in such type of cancer is still controversial.

Our objective in this study was to assess the EZH2 and p-STAT3 as tissue protein markers expressions in RCC patients, exploring their significance in disease progression and patient survival.

In our prospective cohort study, formalin-fixed, paraffin embedded tissue samples retrieved from 60 RCC patients were processed and diagnosed in Pathology Department, Faculty of Medicine, Zagazig University. We have followed patients for five years, between December 2011 and December 2016. Biopsies have been taken by radical nephrectomy and pelvic lymphadenectomy which have been done in Urology Department, Zagazig University-hospitals. We recorded the detailed clinico-pathological data for all cases. We used the TNM classification for pathologic RCC staging [14] and Fuhrman classification for histologic-grading [15][16]. Our study followed local-ethics-committee-guidelines. We followed patients for five years from December 2011 and December 2016 in medical-oncology, clinical-oncology and nuclear medicine departments, faculty of medicine, Zagazig University. The follow-up deadline was December 2016.

We used streptavidine-biotin technique of immunohistochemical staining [17], where we incubated tissue sections with primary mouse monoclonal anti-EZH2 (1:100; cell signaling technology, MA, Danvers, USA) and primary rabbit polyclonal antibody p-STAT3 (1:50; cell signaling technology, MA, Beverly), which detected only STAT3-phosphorylated-form. We have used sections from carcinomas of the breast and heart tissues as positive controls for EZH2 and p-STAT3 respectively. For negative controls we have removed the primary antibodies then replaced them with phosphate buffered saline. All slides were read by two pathologists.

We calculated the intensity of stain as zero that equals negative, one equals weak, two equals moderate and three equals strong stain intensity, the extent of immune-positive cells have been calculated as one (0–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%) [18].

Nuclear p-STAT3 immuno-expression was scored as:

• no expression equals
• 0 weak-positive expression when, <10% of nuclei were positive;
• moderate-positive, 10–50% was positive;
• strong-positive, more than 90% were positive [19].

We have multiplied intensity and extent of both markers expression to reach scores from 0–12, the final cut-off stain-score was four, scores less than four have been considered low expressions and a scores equal to or more than four have been considered as high expression.

We expressed continuous and categorical variables by the mean±SD and median (range) and by number (percentage) respectively, checked continuous variables normality by Shapiro. Wilk test, used Pearson’s chi-square test or Fisher’s exact test for comparison between categorical variables percent, calculated progression free survival (PFS) as the time from diagnosis date to time at which any kind of progressions (local, or regional or distant metastasis) were detected, calculated overall survival (OS) as time from diagnosis date to date of death. Overall survival and progression free survival rates stratifications were done according to EZH2 and STAT3 expressions, estimated these rates by using the method of Kaplan Meier curve, and compared them by using two-sided log-rank test. We considered the p-value <0.05 as significant. We used SPSS 22.0 for windows (SPSS Inc., USA Chicago, IL) and MedCalc-windows (MedCalc-Software 13, Ostend, Belgium) in performing all statistics.

We have included 60 patients with RCC which are reevaluated and diagnosed as 46 (76.7%) cases with clear cell renal cell carcinoma, 9 (15%) papillary renal cell carcinoma 5 (8.3%) chromophope renal cell carcinoma with age ranged from (40–77) years (Mean: 59.65±10.12 years). 48 (80%) patients were males and 12 (20%) were females.

EZH2 expression (Figure 1), (Figure 2), (Table 2) and (Table 3)

• High expression of EZH2 was detected in 30 out of 60 (50%) cases of RCC and was significantly positively correlated with increased age of the patient (p = 0.037), higher grade, T stage of the tumor (p < 0.001). Advanced AJCC stage (p = 0.004), presence of lymph nodes metastases (p = 0.002)
• There were no significant correlations between EZH2 expression with sex of the patient, histopathological subtypes or presence of distant metastases.

p-STAT3 expression (Figure 3), (Figure 4), (Table 2) and (Table 3)

• High expression of p-STAT3 was detected in 31 out of 60 (51.7%) cases of RCC and was significantly positively correlated with higher grade, T stage of the tumor, advanced AJCC stage of the tumor, presence of lymph nodes (p<0.001) and distant metastases (p = 0.004).
• There were no significant correlations between p-STAT3 expression, age, sex of the patient or histopathological subtypes.

Progression and survival analysis in relation to EZH2 expression (Figure 5), (Table 4) and (Table 5)

All cases with high EZH2 expression showed progression of the cancer while cases with low expression only three cases (10%) of them showed progression of the diseases (p<0.001).

• Progression free survival rate of all cases was 40 months and was 26 months only for cases with high EZH2 expression and 56 months for cases with low expressions (p<0.001).
• months and was 30 months only for cases with high EZH2 expression and 57 months for cases with low expressions (p<0.001).

Progression and survival analysis in relation to p-STAT3 expression (Figure 5), (Table 4) and (Table 5)

• Twenty nine (93.5%) cases with high STAT3 expression showed cancer progression, while only four cases (13.8%) with low expression showed progression of the diseases (p<0.001).
• Progression free survival rate of all cases was 40 months and was 28 months only for cases with high p-STAT3 expression and 54 months for cases with low expressions (p<0.001).
• Five years overall survival rate of all cases was 44 months and was 31 months only for cases with high p-STAT3 expression and 57 months for cases with negative expressions (p<0.001).

We found a significant positive correlations between EZH2 and p-STAT3 expressions in RCC (p<0.001).

Pathologic stages have been considered the most important RCC prognostic factor. But, patients that had the same stage were still having variable outcomes. Cancer is a genetic disease which is initiated, promoted and controlled by alterations in many oncogenes and tumor suppressor genes. Epigenetic mutations which included histone-modifications and DNA-methylations were recently found to be significant as cancer promoting factors [20][21][22]. In genetic changes and mutations, the DNA sequence is altered by mutation which is difficult to be restored. On the contrary, epigenetic mutations could be restored by their inhibitors.

In the present study, we found that high expression of EZH2 have been significantly correlated with older age of the patient, higher tumor grade, advanced AJCC stage, presence of lymph node metastases, high incidence of disease progression and shortened overall survival and progression free survival rates.

Our results were similar to Xu et al., who found that EZH2 expression levels were increased with RCC progression, in high grade RCC, advanced TNM stage and presence of distant metastasis that suggested EZH2 as an important prognostic biomarker for RCC [23].

Our results were also similar to that of Liu et al., who found that EZH2 expression is a predictive of RCC patients’ survival [24]. The prognostic values of TNM-stage in RCC are improved when combined with EZH2 expression levels. EZH2 expressions can be clinically applicable procedures for discrimination of patients with variable outcomes. It might lead to more individualized RCC patients’ managements and improved patients suitable for systemic therapies. Wagener et al. proved that EZH2 is a novel poor prognostic bio-marker in RCC-patients [25] and it had been applied according to the REMARK criteria [26]. These included large samples size, long prospective follow-up periods of patients and predictive value description of marker.

Results similar to our study were found by other researchers that EZH2 expression have been correlated with poor clincopathological parameters and poor outcomes of patients with malignancies of different organs [27][28].

Hinz and colleagues found different results from us and showed that EZH2 high levels of expression related to less aggressive criteria and associated with RCC patients’ favorable prognosis when assessed by real-time PCR [29]. These conflicting results may be related to different detection methods of EZH2 expression levels.

Xu et al. explained how EZH2 over-expression allowed RCC progression by that it increased angiogenesis that lead to increased RCC in size, invasion and spread [23].

EZH2 gene silencing could suppress cancer cell growth and have induced cell cycle arrest and also apoptosis, but EZH2 overexpression could promote cancer cell growth and decreased apoptosis. That were in agreement with the previous results in which EZH2 knocking down would inhibit cancer cell proliferation and promote apoptosis [30][31].

EZH2 plays an essential role in histone-methylations that could be reversed by its inhibitors. So, EZH2 represented a powerful novel bio-marker for prognosis prediction in RCC and would be included in well proved prognostic parameters to allow better expectation of patients’ diagnosis, management and follow-up. EZH2 expression might have therapeutic role as it contributed to RCC growth and its silencing would have anti-proliferative and therapeutic effects, so EZH2 inhibitors could repress cancer growth [32].

STAT3 is an oncogene which played an important role in cancer progression and is activated in cancers of many organs. Special tyrosine residues phosphorylation of STAT3 is an essential step for its activation. When activated, p-STAT3 can induce many genes expression that are involved in cancer cell survival and proliferation. There are many studies investigated pSTAT3 expression in cancer, but the published results are conflicting especially in relation to patient prognosis, even in the same cancer type [33]. In that study we evaluated the prognostic significance of p-STAT3 expression in RCC patients, found that high expression of p-STAT3 was significantly positively correlated with, high grade, advanced AJCC stage, presence of L.N and distant metastases, cancer progression, shorter Progression free survival and overall survival rates.

These findings indicated that p-STAT3 high expression predicted a poorer clinical prognosis than in cases with low p-STAT3 expression. Similar results found by Charles Guo et al., who proved that in RCC A high level of p-STAT3 was associated with a poor prognosis [19], and Xu YH and Lu S, have demonstrated that high p-STAT3 expression is a predictor of non-small-cell lung cancer patients poor prognosis [34], also Kun Ji, et al., findings indicated that the increased p-STAT3 expression predicted poor prognosis, high cancer grade, presence of lymph node and distant metastasis in patients having gastric cancer [33]. So, p-STAT3 has been considered a useful biomarker for expecting RCC patient prognosis. Also Li et al., found similar results esophageal carcinoma that high p-STAT3 expression have found to be correlated with advanced cancer stage, poor prognosis and shorten OS rate of patients [35].

Different results from us were found by Woo et al., that patients with p-STAT3 expression had better survival rates than those with low expression [36], and such discrepancy in the study of Woo et al, may be due to different cases number, fewer positive expression rates of p-STAT3 in cancer cases, or different clones of the antibody used in IHC [33]. Aberrant STAT3 activation had been found in a wide range of cancers [37]. p-Stat-3 is a downstream target of Janus-Kinase 2 (JAK2) [38]. JAK-STAT pathways are important oncogenic signaling cas-cades which included Janus kinase (JAK) non-receptor tyrosine kinase family and STAT transcription factors family [39], that were activated by tyrosine and serine residues phosphorylation by up-stream kinases [40]. Our results are in line with that activation of p-STAT3 is a promising RCC prognostic maker and is considered an essential novel therapeutic target for discovering novel anticancer treatments. In RCC, inhibition of STAT3 activity by protein tyrosine kinase inhibitors demonstrates a suppressive effect on cancer cells [41]. So, further studies are needed to evaluate to which degree STAT3 represented a recent prognostic bio-marker and provided targeted and effective approaches in RCC management.

Many studies had found that STAT3 and EZH2 are markedly related to cancer growth, invasion, and spread [42]. It was found that IL-6/STAT3 signaling pathway played an essential role in epigenetic changes regulation during carcinogenesis, by controlling epigenetic enzymes expression like EZH2 [43].

Our findings demonstrate that there is a significant direct relation between EZH2 and STAT3 expressions in RCC and over-expression of both markershave been found to be related to worsened clinicopathological parameters and poor patient prognosis, suggesting that a combination of STAT3 and EZH2 expression could predict disease outcome.

That was similar to Pan et al., which found similar results in gastric carcinoma, and that inhibition of STAT3 have down-regulated EZH2 at tissue protein expression levels, also this study demonstrated that STAT3 regulated EZH2 protein expression by binding to its promoter, that was in line with the results of Qiu et al., in colorectal carcinoma cells [44]. Also, STAT3 inhibition stimulated apoptosis by EZH2 suppression, through caspase-3/9 activation. Our results proved the results of previous studies which found that EZH2 and STAT3 over-expression had apoptosis antagonizing effects by activation of the Akt/Bad/Bcl-xL apoptotic pathway [43].

High levels of EZH2 and p-STAT3 expression have been found to be correlated with poor prognosis in renal cell carcinoma patients, so that the panel of both markers served as molecular prognostic biomarkers and therapeutic targets for such type of cancer. Further studies are required to explore the functional roles of these molecules as new therapeutic targets.

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