TGF-β-activated Kinase-1 : A Potential Prognostic Marker for Clear Cell Renal Cell Carcinoma

Clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, initially has an asymptomatic clinical course, 25-30% of patients present with metastatic disease at time of diagnosis. Worldwide incidence and mortality rates are rising at a rate of approximately 2-3% per decade (Jemal et al., 2011). Surgery and chemotherapy have limited or no effect, leaving ccRCC patients underserved in the realm of cancer treatment (Lam et al., 2005). About 70% of patients were present with localized or locally advanced renal cell carcinoma (LARCC), unfortunately, 20 -40% of these patients would experience recurrence and metastasis (Lam et al., 2005), suggesting that surgical excision is insufficient. Once metastasis occurred, the patients with metastatic ccRCC would face a poor prognosis with a median survival of 6-10 months and a 10-20% 2-year survival rate (Janzen et al., 2003). In recent years, some prognostic markers for ccRCC, such as RALYL (Cui et al., 2012), CXCR4 (Li et al., 2011), Cdc25B (Yu et al., 2012), have appeared, but large-scale clinical application is impossible. Despite the continual progress in medical technology, the clinical characteristic of ccRCC is still difficult to predict (Wei et al., 2012). Therefore, novel diagnostic and prognostic markers of ccRCC would be very valuable in high-risk people and in those with existing


Introduction
Clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, initially has an asymptomatic clinical course, 25-30% of patients present with metastatic disease at time of diagnosis.Worldwide incidence and mortality rates are rising at a rate of approximately 2-3% per decade (Jemal et al., 2011).Surgery and chemotherapy have limited or no effect, leaving ccRCC patients underserved in the realm of cancer treatment (Lam et al., 2005).About 70% of patients were present with localized or locally advanced renal cell carcinoma (LARCC), unfortunately, 20 -40% of these patients would experience recurrence and metastasis (Lam et al., 2005), suggesting that surgical excision is insufficient.Once metastasis occurred, the patients with metastatic ccRCC would face a poor prognosis with a median survival of 6-10 months and a 10-20% 2-year survival rate (Janzen et al., 2003).In recent years, some prognostic markers for ccRCC, such as RALYL (Cui et al., 2012), CXCR4 (Li et al., 2011), Cdc25B (Yu et al., 2012), have appeared, but large-scale clinical application is impossible.Despite the continual progress in medical technology, the clinical characteristic of ccRCC is still difficult to predict (Wei et al., 2012).Therefore, novel diagnostic and prognostic markers of ccRCC would be very valuable in high-risk people and in those with existing RESEARCH ARTICLE
TGF-β-activated kinase-1 (TAK1) is a member of mitogen-activated protein kinase (MAPK) kinase kinase (MAPKKK), and is an indispensable signaling intermediate in several MAPK and NF-κB pathways (Ninomiya et al., 1999;Takaesu et al., 2001;Shim et al., 2005).TAK1 activity has been related to a greater aggressiveness in several types of cancer (Kondo et al., 1998;Edlund et al., 2003;Kaur et al., 2005), but the underlying mechanisms is not clear until now.Moreover, the relationship between TAK1 expression and prognosis status is still ambiguous.In this study, we examined the expression and clinical significance of TAK1 in ccRCC and explored the association between TAK1 expression level and prognosis status.

Patients and tissue specimens collection
51 fresh ccRCC and matched normal tissue, surgically resected between 2011 and 2012 at the Department of Urology, Peking University Shenzhen Hospital, were used for real-time polymerase chain reaction (RT-PCR).All of these specimens were immediately immersed in RNAlater (Qiagen, Germany) after resection and subsequently stored at -80℃.Additionally, 177 paraffinembedded RCC samples and 91 adjacent normal tissues from patients operated in our department between 2002 and 2012 were used for immunohistochemical assay to detect the expression level of TAK1 in ccRCC and normal tissue.All of these tumors were diagnosed and classified or reclassified according to the American Joint Committee on Cancer (AJCC) staging system.Clinical and pathological characteristics of these 177 patients are listed in Table 1.The study was approved by the institutional review board and ethical committee of Peking University Shenzhen hospital and all patients provided written informed consent.

Real-time polymerase chain reaction (RT-PCR) assay for mRNA of TAK1
After extracted from the fresh tissue with Trizol solution (Invitrogen; USA) and purified using the RNAeasy Maxi Kit (Qiagen, Germany) according to the manufacturer's protocol, 1μg mRNA of each sample was used for reverse transcription with Omniscript RT kit (Qiagen, Germany).The reactions of qRT-PCR were performed and analyzed with the ABI PRISM 7000 Fluorescent Quantitative PCR System.Reaction mixture was set up in a total volume of 20μl, consisting of 1μl of cDNA template synthesized previously, 10μl SYBR Green master mix (Invitrogen; USA), 1μl of each primer (sense and antisense primer) and RNase-free water.Primers against TAK1 (p1: GGGGCCACCGTAAAACCGCT; glyceraldehydes-3-phosphate dehydrogenase (GAPDH) (p1: AACGGGAAGCCCATCACCATCTT; p2: CAGCCTTGGCAGCACCAGTGG) .Cycling parameters were set as 95°C for 2 min, followed by 40 cycles of 95°C (15sec), 55°C (30 sec), and 72°C (40 sec).Relative expression of TAK1 was normalized and the data was analyzed with the comparative threshold cycle (2 -∆CT, ∆CT= CTTAK1-CTGAPDH) method (Livak et al., 2001).

Immunohistochemical assay for TAK1
The immunohistochemical assay for TAK1 was performed according to standard procedures.Paraffinembedded samples were cut into 5μm sections and baked at 65°C for 1h, then deparaffinized in xylene and rehydrated in descending ethanol series, followed by antigen retrieval.This step was done by heating the sections in a microwave oven for 30 min in 0.01M citrate buffer (pH 6.0) antigen retrieval buffer.Then the slides were immersed in 3% hydrogen peroxide solution for 20 minutes, washed in phosphate buffered saline (PBS) triple for 5 minutes, treated in 10% bovine serum albumin for 30min in 37℃ to block non-specific protein binding.For the immunostaining of TAK1, the specimens were treated with rabbit monoclonal antibody anti-TAK1 (Epitomics, California, USA) at 1:200 dilution and overnight at 4°C.Rinsed with PBS for 3 times, the samples were incubated with anti-Rabbit IHC Kit (Maixin Bio; Fujian, China) at 37°C for 30 min.Finally, the slides were stained with 3'3-diaminobenzidine tetrahydrochloride (DAB) for 4 min, counterstained with hematoxylin, dehydrated, and mounted.Negative controls were performed with omission of the primary antibodies.
Staining evaluation of each sample was carried out by two independent observers blinded to clinicopathologic variables.Intensity of staining was graded: 0, no staining; 1, weakly stained; 2, moderately stained; 3, highly stained.Percentage of cells showing positive staining was graded: 1, 0-5%; 2, 6-25%; 3, 26-50%; 4, 51-75% and 5, 75%.All of these paraffin-embedded sections were given final scores based on the multiplication of staining intensity and percent of positive cells.In case of any discrepancy, specimens were reviewed by the two observers together and a score was agreed upon.The optimal cut-off value was calculated with log-rank test on the basis of a measure of heterogeneity in overall survival rates and final score of 4 was considered as high expression of TAK1 and 4 as low expression.

Statistical analysis
To analyze significance of differences in mRNA and protein expression of TAK1 between tumors and adjacent normal tissues, paired -sample t test was used.The relationships between expression of TAK1 and clinicopathologic variables were calculated using χ 2 test.Kaplan-Meier method and log-rank test were used to plot survival curves and test the statistical significance between stratified survival groups.To assess which covariates affect overall survival, we used univariate and multivariate Cox proportional hazards models.In all tests, p<0.05 was considered statistically significant and all statistical analyses were performed with the SPSS 17.0 software package.

RT-PCR analysis of TAK1 mRNA in 51 ccRCC tumor samples
Real-time PCR was performed to measure the expression of TAK1 mRNA in 51 ccRCC tumor tissues and ADTs.Compared with normal tissues, 45 ccRCC tumor tissues were significantly higher expression at mRNA levels (p<0.001,paired-sample t test, Figure 1).

Immunohistochemistry analysis of TAK1 expression in 91 ccRCC samples and the paired ADTs
Immunohistochemistry method was applied to assess expression and subcellular localization of TAK1 protein    in 91 paraffin-embedded ccRCC tissues and paired ADTs.

Immunohistochemistry analysis of the relationships between TAK1 protein expression and clinical features in 177 ccRCC tumor samples
Immunohistochemisstry analysis was performed in 177 paraffin-embedded ccRCC tumor samples to further assess the correlation between TAK1 expression and various clinic-pathological parameters.As shown in Table 1, low expression of TAK1 (score ≤ 4) was showed in 75 of the 177 tumor samples, high expression (score ≥ 5) was showed in another 102 samples.Increased expression of TAK1 in tumor samples was correlated with T stage, N classification, metastasis, recurrence and Fuhrman grade, while associations with age and gender were not found.

Survival analysis
To further investigate the prognostic value of TAK1 expression in ccRCC, Kaplan-Meier analysis and the logrank test were applyed to assess the relationships between TAK1 expression level in ccRCC and prognosis status.We found that the level of TAK1 expression was correlated with the overall survival of ccRCC patients.People with higher level of TAK1 expression had poorer survival rates than those with lower level.The group of high expression TAK1 patients' means survival time was 69.910 months and the medians survival time was 79 months, but the low expression group's means and medians survival time was 86.547 months and 96 months.The log-rank test showed the survival rates were significantly different between these two groups (log-rank, p=0.005, Figure 4A).Furthermore, patients without regional lymph node metastasis (N0) had a high cumulative survival rates when compared with patients with regional lymph node invasion (N+) (log-rank, p=0.037, Figure 4B).
The multivariate Cox regression analysis indicated that TAK1 expression (p=0.031) and M stage (p=0.009)were independent prognosis factors for the overall survival of ccRCC patients (Table 2).

Discussion
The annual mortality of ccRCC is significantly higher than other in the genitourinary tract (Schrade et al., 2008) and the clinical outcome remains frustrating despite of advances in clinical technologies (Lane et al., 2007).It is extremely significant to seek specific molecular biomarkers of ccRCC for early diagnosis and evaluation of prognosis.
Recently, more and more molecular markers emerged with their potential values in predicting patients' prognoses and molecular-targeted therapy.For instance, Cui et al. (2012) found that RALY RNA binding proteinlike (RALYL) was independent prognosis factors for the overall survival of ccRCC patients, people with lower level of RALYL expression had a poorer survival rate than those with a higher level of RALYL.Li et al. (2011) found that high expression of CXC receptor 4 (CXCR4) was associated with not only increased risk for disease progression, but also worse OS of high-risk LARCC patients.Yu et al. (2012) proved that knockdown of cdc25B in ccRCC is associated with decreased malignant features and cdc25B was an attractive prognostic marker for this tumor.Some other biomarkers were also found to promising molecular predictor of tumor recurrence, such as RNA-binding protein IMP3 (Jiang et al., 2006), carbonic anhydrase 9 (Tostain et al., 2010), and P53 (Shvarts et al., 2005).However, few utility of these molecular markers for exist till now, probably because of lack of knowledge at the molecular level regarding the biology of renal cell carcinogenesis and progression (Wood et al., 2006;Nogueira et al., 2008).
TAK1, a mitogen-activated protein kinase kinase kinase (MAP3K7), was identified as a kinase that mediates TGF-β signaling to MAP kinases p38 and JNK (Yamaguchi et al., 1995).TAK1 participates not only in TGF-β signaling but also in many other molecular pathways (Delaney et al., 2006).It responds to a variety of upstream signals, including inflammatory molecules and developmental cues, therefore acting as a common effector in regulating cellular responses to stress signals coming from different molecular sensors.
Et al demonstrate that the TAK1-TAB2/TAB3 signaling axis is critical for carcinoma-induced bone lesions, mediating expression of pro-invasive and osteolytic factors.These findings identify the TAK1-TAB2 axis as a potential therapeutic target in bone metastasis (Safina et al., 2011).TAK1 might be responsible for the resistance of pancreatic cancer to the proapoptotic effect of chemotherapeutic agents by increasing the NF-κB and activator protein-1 (AP-1)-mediated transcription of cIAP-2.Targeting the expression or the kinase activity of TAK1 can reverse the intrinsic resistance of pancreatic cancer to chemotherapy (Melis et al., 2011).TAK1 inhibitors may provide significant clinical benefit for the most recalcitrant form of colon cancer.Beyond tool compounds such as 5Z-7-oxozeaenol, synthetic TAK1 inhibitors have been tested in preclinical models (Melis et al., 2011).
Our study indicated the clinical significance of TAK1 in ccRCC.Real-time PCR in 51 ccRCC tumor tissues and the paired ADT samples showed a significant increase of TAK1 mRNA in ccRCC samples.Further immunohistochemistry analysis in 91 paired samples of ccRCCs and ADTs confirmed overexpression of TAK1 protein in tumor tissue.These results indicate TAK1 may play important roles in the initiation and progression of malignancies.
To further investigate the prognostic value of TAK1, immunohistochemical analysis was performed to evaluate the correlation between TAK1 expression and various clinic-pathological parameters.In this study, we showed that the increased TAK1 expression was significantly correlated with Fuhrman grade, stage, N classification, metastasis and recurrence.According to Kaplan-Meier analysis, TAK1 protein expression in ccRCC was significantly correlated with overall survival.Patients with high TAK1 expression level had a shorter survival time than those with a low level.The log-rank test revealed that the group with lower expression of TAK1 had a favorable prognosis than higher expression group.The TNM stage of ccRCC was closely related to prognosis (Levi et al., 2008).Consistent with this, in this study, TAK1 expression and N classification were independent prognosis factors for the overall survival of ccRCC patients by multivariate Cox regression analysis.Therefore, this study reveal that there were significant correlations between the TAK1 expression level and clinic-pathological parameters and may be a potential prognostic marker and therapeutic target for ccRCC.
Fuhrman's nuclear grading system is considered to be a reliable prognostic indicator for ccRCC (Fuhrman et al., 1982).But the multivariate Cox regression analysis in our DOI:http://dx.doi.org/10.7314/APJCP.2013.14.1.315 TGF-β-activated kinase-1: A Potential Marker for Prognosis in Clear Cell Renal Cell study did not show any relations between Fuhrman grade and prognosis, this may due to our limited sample volume and the observation error pathologists made.
As far we know, this is the first study to evaluate the value of TAK1 as a clinically potential indicator for ccRCC progression, as well as a prognostic marker for patient survival.However, it should admitted that our study was a single hospital-based, retrospective study, and multi-centres or community-based prospective studies are required.

Figure 1 .
Figure 1.Real-time Quantitative RT-PCR Analysis of TAK1 Expression

Figure
Figure 2. Immunohistochemical Analysis of the Expression of TAK1 Protein