Original Paper
Urologia Internationalis
Received: April 22, 2014 Accepted after revision: July 1, 2014 Published online: August 7, 2014
Urol Int 2015;95:79–85 DOI: 10.1159/000365595
Expression of Neural Wiskott-Aldrich Syndrome Protein in Clear Cell Renal Cell Carcinoma and Its Correlation with Clinicopathological Features Guang-hua Liu Jian Chen Zhi-gang Ji Li Zhou Department of Urology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, PR China
Abstract Introduction: Neural Wiskott-Aldrich syndrome protein (NWASP) expression is associated with tumor cell invasion and migration. However, its expression status in clear cell renal cell carcinoma (CCRCC) remains unclear. We examined the level of N-WASP in CCRCC and its association with clinicopathological features characteristic. Materials and Methods: 73 CCRCC patients who underwent radical nephrectomy or partial nephrectomy were enrolled. Immunohistochemical staining for N-WASP was performed on tissue microarrays constructed from tumor and para-tumor tissue obtained from these patients. The difference in N-WASP expression between tumor tissue and adjacent normal renal tissue was examined. Correlations between N-WASP expression in the tumor and clinicopathological parameters were analyzed and the relationship between N-WASP expression and overall survival also assessed. Uni- and multivariate survival analyses were performed. Results: N-WASP expression was significantly reduced in tumor tissues and was significantly related to the histological grade of CCRCC. A higher level of N-WASP expression in the tumor was associated with relatively poor survival in CCRCC patients. The level of N-
© 2014 S. Karger AG, Basel 0042–1138/14/0951–0079$39.50/0 E-Mail [email protected] www.karger.com/uin
WASP expression, age at time of surgery, and histological grade were all responsible for clinical outcome in CCRCC patients. N-WASP was an independent predictor for overall survival. Conclusions: N-WASP was downregulated in CCRCC and could serve as a prognostic biomarker for predicting clinical outcome of CCRCC. © 2014 S. Karger AG, Basel
Introduction
Renal cell carcinoma (RCC) is the most common malignancy of the kidney, and its incidence is increasing [1]. It is estimated that approximately 37.7 men and 16.6 women per 100,000 Chinese people are diagnosed with RCC every year [2]. Clear cell renal cell carcinoma (CCRCC) is the most prevalent subtype of RCC, accounting for 75% of these renal neoplasms [3]. Despite years of research, biomarkers have yet to be found that effectively predict the likelihood of metastases or clinical outcome in this disease. Local tumor extent, regional nodal metastasis, and distant metastases remain the most important prognostic factors affecting survival in CCRCC.
G. Liu and J. Chen are co-first authors and contributed equally to this article.
Ji Zhi-gang Department of Urology, Surgical Building of Peking Union Medical College Hospital No.1 Shuaifuyuan, Wangfujing, Dong Cheng District Beijing 100730 (PR China) E-Mail 372543078 @ qq.com
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Key Words Biomarker · Clear cell renal cell carcinoma · Neural Wiskott-Aldrich syndrome protein · Tissue microarray · Tumor
Table 1. Clinicopathological features of patients with CCRCC
Age at the time of operation, years Tumor diameter, cm Gender Male Female Histological grade I II III IV Tumor stage T1a T1b T2 T3 Lymph node metastasis N0 N1 Distal metastasis M0 M1 AJCC staging 1 2 3 4
59 6.7 48 25 3 35 27 8 19 16 25 13 68 5 72 1
International Union against Cancer [14]. Clinical staging was in accordance with the seventh edition of the American Joint Committee on Cancer (AJCC) Staging Manual [14]. All human specimens used in this study were approved by the Ethics Committees of PUMCH and written informed consent was obtained from each patient. Construction of Tissue Microarrays (TMA) 73 pairs of CCRCC specimens and adjacent non-tumor renal tissue were fixed in 10% formaldehyde solution and embedded in blocks of paraffin. Following routine histopathological examination (hematoxylin-eosin staining), two cores of tumor and paratumor tissue per patient were obtained from representative regions using a 1.5-mm punch. The manual tissue arrayer (Beecher Instruments, Inc., Sun Prairie, Wisc., USA) was used for TMA construction. Immunohistochemistry Immunohistochemical (IHC) staining for N-WASP was performed on the TMA using a rabbit N-WASP polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA) and a twostep staining kit (EnVisionTM + Kit; Dako, Glostrup, Denmark). Briefly, 4-μm thick sections were mounted, deparaffinized in xylene and rehydrated through a graded series of ethanol washes. After antigen retrieval in 10 mmol/l citrate buffer (pH 6.0) at 100 ° C for 10–15 min, slides were then incubated with 3% hydrogen peroxide for 10 min to block endogenous peroxidase. Subsequently, slides were incubated overnight at 48 ° C with the primary antibody at a dilution of 1:50. Following washing in phosphate-buffered saline, horseradish peroxidase-conjugated secondary antibody was added for 30–60 min. N-WASP staining was then visualized with the chromogen 3,3-diaminobenzidine. Non-immune rabbit serum of the same dilution as the primary antibody was used as the negative control.
32 24 16 1
Neural Wiskott-Aldrich syndrome protein (N-WASP) is a key regulator of actin polymerization and cytoskeletal remodeling. Expression of this protein has previously been shown to associate with tumor cell invasion and migration in breast and esophageal carcinomas [4–12]. Until now, however, reports on the level of N-WASP expression and its association with clinicopathological features of CCRCC have been lacking. The objective of the present study was to determine the expression and prognostic value of N-WASP in CCRCC.
Evaluation of IHC Two pathologists who were unaware of the clinicopathologic and follow-up data performed the IHC analysis. Cytoplasmic staining of tissue samples was taken to denote a positive signal. The intensity of cell staining was classified according to one of three grades (50%, grade 2) using a previously reported method [15]. In addition, a simple grading for overall N-WASP expression was applied as follows: weak (grade 0), moderate (grade 1), and high expression (grade 2). Follow-Up 43 of the patients were followed up after surgery. The duration of follow-up ranged from 2 to 74 months, with a median term of 53 months.
Methods
80
Urol Int 2015;95:79–85 DOI: 10.1159/000365595
Statistical Analysis McNemar and Mann-Whitney U tests were applied to analyze the difference in N-WASP expression between tumor tissue and adjacent normal renal tissue. Correlations between N-WASP expression within the tumor and clinicopathological parameters were analyzed using a χ2 test. Overall survival was assessed by the Kaplan-Meier method, and a log-rank test was used to analyze survival curves. Uni- and multivariate survival analysis was performed using the Cox proportional hazards regression model. Statistical significance was set at p < 0.05. All statistical analysis was carried out using the SPSS statistical software package (standard version 13.0; SPSS, Inc., Chicago, Ill., USA).
Liu/Chen/Ji/Zhou
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Patient Samples Tissue samples were obtained from 73 patients who underwent radical nephrectomy or partial nephrectomy for renal neoplasm at the Peking Union Medical College Hospital (PUMCH) between August 2006 and November 2008. Postoperative pathology examinations confirmed that all renal neoplasms were CCRCC. Information regarding patient age, gender, tumor diameter, histological grade, and clinical stage are listed in table 1. Histological grade was determined with reference to the World Health Organization (WHO) classification of tumors [13]. Tumor, Node, Metastasis (TNM) staging was according to the fifth edition of the TNM classification of the
Expression Profiling of N-WASP in CCRCC and Normal Renal Tissue by IHC The expression level of N-WASP was determined by IHC for 73 pairs of CCRCC and adjacent normal renal tissue. N-WASP staining of normal tissue was intense and was observed primarily in the nuclear region and cytoplasm. In contrast, CCRCC tissue was poorly stained with little, if any, staining of the nuclear region (fig. 1). The level of N-WASP expression in CCRCC was significantly lower than that of normal tissue as determined using both McNemar and Mann-Whitney U tests (p < 0.0005; table 2).
Discussion
Association of N-WASP Expression in CCRCC with Patient Survival The association between N-WASP expression in CCRCC and patient survival was analyzed using the Kaplan-Meier method. Among the 43 patients who were followed up during a mean period of 53 months, 14 died of CCRCC. 27 of these 43 patients exhibited weak-to-moderate N-WASP expression in tumor tissue, and 4 of these 27 patients died. The remaining 16 patients exhibited strong N-WASP expression and 10 deaths were recorded among this group. The group of patients with weak-to-moderate N-WASP expression tended to have a longer overall survival than those in the group associated with a high tumor N-WASP level (p = 0.002; fig. 2). To estimate the prognostic value of N-WASP expression in CCRCC, we applied univariate analysis to control for other prognostic factors. Results indicated that NWASP (p = 0.002), age at time of operation (p = 0.032), and histological grade (p = 0.013) were all responsible for clinical outcome in CCRCC patients (table 4). The above three statistically significant parameters were further subjected to Cox proportional hazards regression analysis to evaluate the significance of N-WASP expression in CCRCC prognosis. Results suggested that N-WASP was also an independent predictor for overall survival (p = 0.032; table 5).
Wiskott-Aldrich syndrome (WAS) is an X-linked hereditary disease that is characterized by thrombocytopenia, eczema, and immunodeficiency. The gene that is mutated in WAS patients was identified by Derry et al. [4] in 1994. The product of the WAS gene is WASP. In 1996, a novel protein with approximately 50% amino acid sequence identity to WASP was purified. Unlike WASP, which is exclusively expressed in hematopoietic cells, this novel protein is expressed ubiquitously, although strongest expression is observed in nerve cells, and thus it was named Neural-WASP (N-WASP) [4, 16]. WASP and N-WASP proteins contain three characteristic functional domains, a pleckstrin homology domain, which binds phosphatidyl-inositol bisphosphate; a Cdc42-binding domain, and a 70-amino-acid conserved verprolin-homology domain, which constitutes the actin-binding region and is critical for the regulation of the actin cytoskeleton. Both WASP and N-WASP are downstream effectors of the small GTPase Cdc42, and participate in the regulation of actin polymerization and cytoskeletal organization. Cdc42-dependent actin assembly requires the interaction of WASP/N-WASP with the Arp2/3 complex [5, 6]. The invasion and metastasis of a malignant tumor is a multistep and complex process involving tumor cell local amplification and extension, tumor vascularization, detachment of tumor cells from primary foci, remodeling of extracellular matrix (ECM), the migration of tumor cells across basement membrane barriers, intravasation, extravasation, and ultimately cell growth at secondary sites. The whole process of invasion is driven by the migration of tumor cells. N-WASP activates Arp2/3-complex-mediated actin polymerization, and is therefore important for regulating changes in cell morphology and motility [7–9]. Also, to migrate through barriers, carcinoma cells need to remodel ECM, which is a process controlled by proteases, most notably matrix metalloproteases (MMPs). N-WASP activity is required for the MMP-dependent invasion of carcinoma cells in vivo [13]. Although N-WASP plays a crucial role in the process of tumor invasion and metastasis, the expression pattern of this protein among different carcinoma types varies. Studies of breast cancer cell invasion and migration revealed that N-WASP is involved in the formation of invadopodia in tumor cells in vivo. Furthermore, these studies demonstrated a direct link between invadopodium formation and remodeling of stroma by cancer cells during invasion, intravasation and lung metastasis
N-WASP in Clear Cell Renal Cell Carcinoma
Urol Int 2015;95:79–85 DOI: 10.1159/000365595
Correlation between Clinicopathological Features and N-WASP Expression in CCRCC Correlations between N-WASP expression in the tumor and clinicopathological parameters were analyzed using a χ2 test (table 3). The histological grade of CCRCC was shown to be significantly related to the degree of NWASP staining of tumor tissue (p = 0.03).
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Results
Color version available online
a
b
c
d
Fig. 1. N-WASP expression in normal renal tissues and CCRCC.
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Urol Int 2015;95:79–85 DOI: 10.1159/000365595
e
Liu/Chen/Ji/Zhou
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Representative image of N-WASP staining in normal kidney tissue (a), grade I (b), grade II (c), grade III (d), and grade IV (e). NWASP was observed primarily and intensively stained in the nuclear region and cytoplasm of normal tissue. In contrast, CCRCC was poorly stained, if any in the nuclear. Also, a tendency of significantly increased of N-WASP expression in upgoing histological grade of CCRCC was shown.
Color version available online
Table 2. Expression profiling of N-WASP in CCRCC and normal
renal tissue Adjacent normal renal tissue IHC weak, moderate
strong
total
Weak, moderate Strong
4 1
46 22
50 23
Total
5
68
73
Table 3. Relation between clinicopathological features and N-
WASP expression level in CCRCC N-WASP expression total Age ≥65 years 6.7 cm ≤6.7 cm Histological grade I, II III, IV AJCC staging 1 2, 3, 4 T stage T1 T2 T3 N stage N0 N1 M stage M0 M1
weak, moderate
0.6
0.4
0.2
N-WASP low, moderate N-WASP strong
p value 0
strong 0.509
24 49
16 34
8 15
48 25
32 18
16 7
0.425
0
20
40
60
80
Overall survival time (months)
Fig. 2. Survival analysis of N-WASP expression in CCRCC. N-
WASP protein level showed a prognostic role in overall survival. 0.221
38 35
24 26
14 9
38 35
32 18
6 17
32 41
24 26
8 15
35 25 13
25 16 8
10 9 5
68 5
48 3
20 2
72 1
50 0
22 1
[5–7]. Gligorijevic et al. [17] disturbed endogenous NWASP activity in MTLn3 breast cancer cells either by overexpressing a dominant-negative form of N-WASP defective in its ability to activate the Arp2/3 complex, or by silencing N-WASP expression with small hairpin RNA, and demonstrated a decline in invadopodia formation, ECM remodeling, and tumor cell migration in vivo.
However, Martin et al. [18] reported that N-WASP was significantly downregulated in human breast cancer, particularly in aggressive and node-positive tumors from patients with a poor prognosis. These authors also demonstrated that overexpression of N-WASP in the invasive breast cancer cell line MDA-MB-231 resulted in reduced cell motility and invasion and increased cell adhesion. Moreover, these cells produced tumors of significantly reduced growth and volume in vivo. Wang et al. [19] demonstrated that N-WASP mRNA and protein expression is increased in esophageal squamous cell carcinoma. However, a correlation was not observed between N-WASP expression and overall patient survival. Research by Jin et al. [20] also showed that NWASP was highly expressed in hepatocellular carcinoma and demonstrated its prognostic importance in this disease. Our study is the first study to demonstrate the expression pattern of N-WASP in CCRCC. We provide compelling evidence for the downregulation of N-WASP in tumor tissue when compared with matched, normal renal tissue. Also, our findings demonstrate that N-WASP expression within the tumor correlates with the histological grade of the cancer. Despite this downregulation in NWASP expression, higher levels of N-WASP expression
N-WASP in Clear Cell Renal Cell Carcinoma
Urol Int 2015;95:79–85 DOI: 10.1159/000365595
0.03 0.212 0.751
0.661 0.138
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Parameters
0.8
Cumulative survival
CCRCC IHC
1.0
expression and clinicopathologic variables in CCRCC patients and overall survival (log-rank test) Variables Age, years ≤65 >65 Gender Male Female Tumor diameter, cm >6.7 ≤6.7 Histological grade I, II III, IV AJCC staging 1 2, 3, 4 N-WASP expression Weak, moderate Strong T stage T1 T2 T3 N stage N0 N1 ASA score I II III
All cases
Mean survival, months
31 12
62 50
25 18
58 60
26 17
60 56
20 23
67 51
19 24
63 56
27 16
66 46
20 18 5
55 53 40
41 2
53 40
23 17 3
57 55 47
p value 0.032 0.915 0.89 0.013 0.193 0.002 0.355
0.07 0.201
Table 5. COX multivariate analysis of clinicopathological parameters and overall survival
Variables
Hazard ratio (95% CI)
p value
Age Histological grade N-WASP expression
1.695 (0.536–5.361) 2.418 (0.574–10.182) 3.800 (1.123–12.862)
0.369 0.229 0.032
within the tumor are indicative of poor survival among CCRCC patients. Furthermore, uni- and multivariate analysis suggests that N-WASP is a potential prognostic factor of overall survival. However, the results of this study should be interpreted with caution. First of all, N-WASP 84
Urol Int 2015;95:79–85 DOI: 10.1159/000365595
expression within the tumor correlates with the histological grade of the cancer instead of the TNM stage. Usually, stage has more prognostic value than grade. However, in our study the univariate analysis did not suggest that TNM stage indicated the clinical outcome of the patients (table 4). We believe it was due to the relatively early stage cancer features of our patients. Most of the patients enrolled in the study had localized CCRCC with stage T1– T2, which were resectable by curative radical nephrectomy or partial nephrectomy. The prognostic value of TNM stage might be shielded by the relatively optimistic overall survival of the patient’s attitude to the early cancer stage and sophisticated surgery approach. On the other hand, histological grading of the cancer was subject to interobserver variability. Therefore, the value of N-WASP expression to assess the prognosis of renal cancer might not be as ideal as the results of the study suggested. Secondly, the prognosis of the patients studied was evaluated by the overall survival instead of progression-free survival or cancer-specific survival. Even though the American Society of Anesthesiologists (ASA) scores of the patients showed no influence on overall survival (table 4), overall survival was influenced by many other factors. Lastly, the follow-up rate in our study is relatively low (59%), this also compromises our findings on the value of N-WASP expression in CCRCC prognosis evaluation. In the present study, we used IHC to compare the level of N-WASP expression in CCRCC tissue with that of normal renal tissue. The intensity of N-WASP staining was found to be significantly reduced in tumor tissues. These findings are in agreement with those of Martin et al. [18]. However, our finding that N-WASP expression in tumor tissues correlates with the histological grade of the cancer is more similar to that of Gligorijevic et al. [17]. Variations in the expression pattern of N-WASP among different carcinoma types suggest that the contribution of this protein to carcinogenesis may be tissue-specific. Findings from our study also demonstrate that N-WASP expression is not constant during the process of cancer development: we observe high levels of expression in normal renal tissue, a downregulation of this expression in the early stage of CCRCC, and then elevated levels of expression associated with the progression of the cancer. This phenomenon suggests that N-WASP expression demonstrates temporal specificity. Interestingly, another biomarker for CCRCC demonstrates a similar pattern of expression to that of N-WASP. Cai et al. [21] showed that the expression of ZBP-89, a Krüppel-type zinc-finger transcription factor, was decreased in CCRCC when compared with normal renal tisLiu/Chen/Ji/Zhou
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Table 4. Univariate analysis of the correlation between N-WASP
sue. The authors also found that ZBP-89 expression in tumor tissue significantly correlated with two clinicopathological parameters – TNM stage and distal metastasis. Furthermore, high ZBP-89 expression within the tumor was associated with poor survival of CCRCC patients. Uniand multivariate analysis also showed that ZBP-89 was a potential prognostic factor for overall survival. In conclusion, we have shown that while N-WASP expression is reduced in CCRCC, the expression level of this protein within the tumor correlates with the histological grade of the cancer. Furthermore, higher levels of NWASP expression within the tumor are associated with poorer survival in CCRCC patients. Importantly, NWASP is a potential prognostic factor for overall survival
in CCRCC. Further studies are now required to further verify the value of N-WASP expression in CCRCC prognosis evaluation, and elucidate the mechanisms by which N-WASP contributes to the progression of CCRCC. Acknowledgment The study was supported by a grant from Peking Union Medical College Hospital.
Disclosure Statement The authors have no conflicts of interest to disclose.
References
N-WASP in Clear Cell Renal Cell Carcinoma
8 Yamaguchi H, Lorenz M, Kempiak S, Sarmiento C, Coniglio S, Symons M, Segall J, Eddy R, Miki H, Takenawa T, Condeelis J: Molecular mechanisms of invadopodium formation: the role of the N-WASP-Arp2/3 complex pathway and cofilin. J. Cell Biol 2005;168:441–452. 9 Xue C, Wyckoff J, Liang F, Sidani M, Violini S, Tsai KL, Zhang ZY, Sahai E, Condeelis J, Segall JE: Epidermal growth factor receptor overexpression results in increased tumor cell motility in vivo coordinately with enhanced intravasation and metastasis. Cancer Res 2006;66:192–197. 10 Oser M, Mader CC, Gil-Henn H, Magalhaes M, Bravo-Cordero JJ, Koleske AJ, Condeelis J: Specific tyrosine phosphorylation sites on cortactin regulate Nck1-dependent actin polymerization in invadopodia. J Cell Sci 2010; 123:3662–3673. 11 Egeblad M, Ewald AJ, Askautrud HA, Truitt ML, Welm BE, Bainbridge E, Peeters G, Krummel MF, Werb Z: Visualizing stromal cell dynamics in different tumor microenvironments by spinning disk confocal microscopy. Dis Model Mech 2008;1:155–167. 12 Koblinski JE, Ahram M, Sloane BF: Unraveling the role of proteases in cancer. Clin Chim Acta 2000;291:113–135. 13 Eple JN, Sauter G, Epstein JI, Sesterhenn IA: Pathology and Genetics: Tumours of the Urinary System and Male Genital Organs; Lyon, IARC Press, 2004. 14 Edge S, Byrd DR, Compton CC, Fritz AG, Greene FL, Trotti A: AJCC Cancer Staging Manual. New York, Springer, 2009.
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15 Zhang H, Liu J, Cagle PT, Allen TC, Laga AC, Zander DS: Distinction of pulmonary small cell carcinoma from poorly differentiated squamous cell carcinoma: an immunohistochemical approach. Mod Pathol 2005;18:111–118. 16 Miki H, Takenawa T: Regulation of actin dynamics by WASP family proteins. J Biochem 2003;134:309–313. 17 Gligorijevic B, Wyckoff J, Yamaguchi H, Wang Y, Roussos ET, Condeelis J: N-WASP-mediated invadopodium formation is involved in intravasation and lung metastasis of mammary tumors. J Cell Sci 2005;125:724–734. 18 Martin TA, Pereira G, Watkins G, Mansel RE, Jiang WG: N-WASP is a putative tumour suppressor in breast cancer cells, in vitro and in vivo, and is associated with clinical outcome in patients with breast cancer. Clin Exp Metastasis 2008;25:97–108. 19 Wang WS, Zhong HJ, Xiao DW, Huang X, Liao LD, Xie ZF, Xu XE, Shen ZY, Xu LY, Li EM: The expression of CFL1 and N-WASP in esophageal squamous cell carcinoma and its correlation with clinicopathological features. Dis Esophagus 2010;23:512–521. 20 Jin KM, Lu M, Liu FF, Gu J, Du XJ, Xing BC: N-WASP is highly expressed in hepatocellular carcinoma and associated with poor prognosis. Surgery 2013;153:518–525. 21 Cai MY, Luo RZ, Li YH, Dong P, Zhang ZL, Zhou FJ, Chen JW, Yun JP, Zhang CZ, Cao Y: High-expression of ZBP-89 correlates with distal metastasis and poor prognosis of patients in clear cell renal cell carcinoma. Biochem Biophys Res Commun 2012;426:636–642.
85
Downloaded by: Stockholms Universitet 198.143.54.1 - 8/14/2015 4:21:16 AM
1 Qin C, Sun LJ, Cui L, Cao Q, Zhu J, Li P, Zhang GM, Mao X, Shao PF, Wang ML, Zhang ZD, Gu M, Zhang W, Yin CJ: Application of the revised Tumour Node Metastasis (TNM) staging system of clear cell renal cell carcinoma in eastern China: advantages and limitations. Asian J Androl 2013; 15: 550– 557. 2 Yang L, Parkin DM, Ferlay J, Li L, Chen Y: Estimates of cancer incidence in China for 2000 and projections for 2005. Cancer Epidemiol Biomarkers Prev 2005;14:243–250. 3 Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, Thun MJ: Cancer statistics. CA Cancer J Clin 2008;58:71–96. 4 Derry JM, Ochs HD, Francke U: Isolation of a novel gene mutated in Wiskott-Aldrich syndrome. Cell 1994;78:635–644. 5 Miki H, Miura K, Takenawa T: N-WASP, a novel actin-depolymerizing protein, regulates the cortical cytoskeletal rearrangement in a PIP2-dependent manner downstream of tyrosine kinases. EMBO J 1996;15:5326–5335. 6 Kowalski JR, Egile C, Gil S, Snapper SB, Li R, Thomas SM: Cortactin regulates cell migration through activation of N-WASP. J Cell Sci 2005;118:79–87. 7 Parsons M, Monypenny J, Ameer-Beg SM, Millard TH, Machesky LM, Peter M, Keppler MD, Schiavo G, Watson R, Chernoff J, Zicha D, Vojnovic B, Ng T: Spatially distinct binding of Cdc42 to PAK1 and N-WASP in breast carcinoma cells. Mol Cell Biol 2005;25:1680– 1695.
Urologia Internationalis
Received: April 22, 2014 Accepted after revision: July 1, 2014 Published online: August 7, 2014
Urol Int 2015;95:79–85 DOI: 10.1159/000365595
Expression of Neural Wiskott-Aldrich Syndrome Protein in Clear Cell Renal Cell Carcinoma and Its Correlation with Clinicopathological Features Guang-hua Liu Jian Chen Zhi-gang Ji Li Zhou Department of Urology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, PR China
Abstract Introduction: Neural Wiskott-Aldrich syndrome protein (NWASP) expression is associated with tumor cell invasion and migration. However, its expression status in clear cell renal cell carcinoma (CCRCC) remains unclear. We examined the level of N-WASP in CCRCC and its association with clinicopathological features characteristic. Materials and Methods: 73 CCRCC patients who underwent radical nephrectomy or partial nephrectomy were enrolled. Immunohistochemical staining for N-WASP was performed on tissue microarrays constructed from tumor and para-tumor tissue obtained from these patients. The difference in N-WASP expression between tumor tissue and adjacent normal renal tissue was examined. Correlations between N-WASP expression in the tumor and clinicopathological parameters were analyzed and the relationship between N-WASP expression and overall survival also assessed. Uni- and multivariate survival analyses were performed. Results: N-WASP expression was significantly reduced in tumor tissues and was significantly related to the histological grade of CCRCC. A higher level of N-WASP expression in the tumor was associated with relatively poor survival in CCRCC patients. The level of N-
© 2014 S. Karger AG, Basel 0042–1138/14/0951–0079$39.50/0 E-Mail [email protected] www.karger.com/uin
WASP expression, age at time of surgery, and histological grade were all responsible for clinical outcome in CCRCC patients. N-WASP was an independent predictor for overall survival. Conclusions: N-WASP was downregulated in CCRCC and could serve as a prognostic biomarker for predicting clinical outcome of CCRCC. © 2014 S. Karger AG, Basel
Introduction
Renal cell carcinoma (RCC) is the most common malignancy of the kidney, and its incidence is increasing [1]. It is estimated that approximately 37.7 men and 16.6 women per 100,000 Chinese people are diagnosed with RCC every year [2]. Clear cell renal cell carcinoma (CCRCC) is the most prevalent subtype of RCC, accounting for 75% of these renal neoplasms [3]. Despite years of research, biomarkers have yet to be found that effectively predict the likelihood of metastases or clinical outcome in this disease. Local tumor extent, regional nodal metastasis, and distant metastases remain the most important prognostic factors affecting survival in CCRCC.
G. Liu and J. Chen are co-first authors and contributed equally to this article.
Ji Zhi-gang Department of Urology, Surgical Building of Peking Union Medical College Hospital No.1 Shuaifuyuan, Wangfujing, Dong Cheng District Beijing 100730 (PR China) E-Mail 372543078 @ qq.com
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Key Words Biomarker · Clear cell renal cell carcinoma · Neural Wiskott-Aldrich syndrome protein · Tissue microarray · Tumor
Table 1. Clinicopathological features of patients with CCRCC
Age at the time of operation, years Tumor diameter, cm Gender Male Female Histological grade I II III IV Tumor stage T1a T1b T2 T3 Lymph node metastasis N0 N1 Distal metastasis M0 M1 AJCC staging 1 2 3 4
59 6.7 48 25 3 35 27 8 19 16 25 13 68 5 72 1
International Union against Cancer [14]. Clinical staging was in accordance with the seventh edition of the American Joint Committee on Cancer (AJCC) Staging Manual [14]. All human specimens used in this study were approved by the Ethics Committees of PUMCH and written informed consent was obtained from each patient. Construction of Tissue Microarrays (TMA) 73 pairs of CCRCC specimens and adjacent non-tumor renal tissue were fixed in 10% formaldehyde solution and embedded in blocks of paraffin. Following routine histopathological examination (hematoxylin-eosin staining), two cores of tumor and paratumor tissue per patient were obtained from representative regions using a 1.5-mm punch. The manual tissue arrayer (Beecher Instruments, Inc., Sun Prairie, Wisc., USA) was used for TMA construction. Immunohistochemistry Immunohistochemical (IHC) staining for N-WASP was performed on the TMA using a rabbit N-WASP polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA) and a twostep staining kit (EnVisionTM + Kit; Dako, Glostrup, Denmark). Briefly, 4-μm thick sections were mounted, deparaffinized in xylene and rehydrated through a graded series of ethanol washes. After antigen retrieval in 10 mmol/l citrate buffer (pH 6.0) at 100 ° C for 10–15 min, slides were then incubated with 3% hydrogen peroxide for 10 min to block endogenous peroxidase. Subsequently, slides were incubated overnight at 48 ° C with the primary antibody at a dilution of 1:50. Following washing in phosphate-buffered saline, horseradish peroxidase-conjugated secondary antibody was added for 30–60 min. N-WASP staining was then visualized with the chromogen 3,3-diaminobenzidine. Non-immune rabbit serum of the same dilution as the primary antibody was used as the negative control.
32 24 16 1
Neural Wiskott-Aldrich syndrome protein (N-WASP) is a key regulator of actin polymerization and cytoskeletal remodeling. Expression of this protein has previously been shown to associate with tumor cell invasion and migration in breast and esophageal carcinomas [4–12]. Until now, however, reports on the level of N-WASP expression and its association with clinicopathological features of CCRCC have been lacking. The objective of the present study was to determine the expression and prognostic value of N-WASP in CCRCC.
Evaluation of IHC Two pathologists who were unaware of the clinicopathologic and follow-up data performed the IHC analysis. Cytoplasmic staining of tissue samples was taken to denote a positive signal. The intensity of cell staining was classified according to one of three grades (50%, grade 2) using a previously reported method [15]. In addition, a simple grading for overall N-WASP expression was applied as follows: weak (grade 0), moderate (grade 1), and high expression (grade 2). Follow-Up 43 of the patients were followed up after surgery. The duration of follow-up ranged from 2 to 74 months, with a median term of 53 months.
Methods
80
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Statistical Analysis McNemar and Mann-Whitney U tests were applied to analyze the difference in N-WASP expression between tumor tissue and adjacent normal renal tissue. Correlations between N-WASP expression within the tumor and clinicopathological parameters were analyzed using a χ2 test. Overall survival was assessed by the Kaplan-Meier method, and a log-rank test was used to analyze survival curves. Uni- and multivariate survival analysis was performed using the Cox proportional hazards regression model. Statistical significance was set at p < 0.05. All statistical analysis was carried out using the SPSS statistical software package (standard version 13.0; SPSS, Inc., Chicago, Ill., USA).
Liu/Chen/Ji/Zhou
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Patient Samples Tissue samples were obtained from 73 patients who underwent radical nephrectomy or partial nephrectomy for renal neoplasm at the Peking Union Medical College Hospital (PUMCH) between August 2006 and November 2008. Postoperative pathology examinations confirmed that all renal neoplasms were CCRCC. Information regarding patient age, gender, tumor diameter, histological grade, and clinical stage are listed in table 1. Histological grade was determined with reference to the World Health Organization (WHO) classification of tumors [13]. Tumor, Node, Metastasis (TNM) staging was according to the fifth edition of the TNM classification of the
Expression Profiling of N-WASP in CCRCC and Normal Renal Tissue by IHC The expression level of N-WASP was determined by IHC for 73 pairs of CCRCC and adjacent normal renal tissue. N-WASP staining of normal tissue was intense and was observed primarily in the nuclear region and cytoplasm. In contrast, CCRCC tissue was poorly stained with little, if any, staining of the nuclear region (fig. 1). The level of N-WASP expression in CCRCC was significantly lower than that of normal tissue as determined using both McNemar and Mann-Whitney U tests (p < 0.0005; table 2).
Discussion
Association of N-WASP Expression in CCRCC with Patient Survival The association between N-WASP expression in CCRCC and patient survival was analyzed using the Kaplan-Meier method. Among the 43 patients who were followed up during a mean period of 53 months, 14 died of CCRCC. 27 of these 43 patients exhibited weak-to-moderate N-WASP expression in tumor tissue, and 4 of these 27 patients died. The remaining 16 patients exhibited strong N-WASP expression and 10 deaths were recorded among this group. The group of patients with weak-to-moderate N-WASP expression tended to have a longer overall survival than those in the group associated with a high tumor N-WASP level (p = 0.002; fig. 2). To estimate the prognostic value of N-WASP expression in CCRCC, we applied univariate analysis to control for other prognostic factors. Results indicated that NWASP (p = 0.002), age at time of operation (p = 0.032), and histological grade (p = 0.013) were all responsible for clinical outcome in CCRCC patients (table 4). The above three statistically significant parameters were further subjected to Cox proportional hazards regression analysis to evaluate the significance of N-WASP expression in CCRCC prognosis. Results suggested that N-WASP was also an independent predictor for overall survival (p = 0.032; table 5).
Wiskott-Aldrich syndrome (WAS) is an X-linked hereditary disease that is characterized by thrombocytopenia, eczema, and immunodeficiency. The gene that is mutated in WAS patients was identified by Derry et al. [4] in 1994. The product of the WAS gene is WASP. In 1996, a novel protein with approximately 50% amino acid sequence identity to WASP was purified. Unlike WASP, which is exclusively expressed in hematopoietic cells, this novel protein is expressed ubiquitously, although strongest expression is observed in nerve cells, and thus it was named Neural-WASP (N-WASP) [4, 16]. WASP and N-WASP proteins contain three characteristic functional domains, a pleckstrin homology domain, which binds phosphatidyl-inositol bisphosphate; a Cdc42-binding domain, and a 70-amino-acid conserved verprolin-homology domain, which constitutes the actin-binding region and is critical for the regulation of the actin cytoskeleton. Both WASP and N-WASP are downstream effectors of the small GTPase Cdc42, and participate in the regulation of actin polymerization and cytoskeletal organization. Cdc42-dependent actin assembly requires the interaction of WASP/N-WASP with the Arp2/3 complex [5, 6]. The invasion and metastasis of a malignant tumor is a multistep and complex process involving tumor cell local amplification and extension, tumor vascularization, detachment of tumor cells from primary foci, remodeling of extracellular matrix (ECM), the migration of tumor cells across basement membrane barriers, intravasation, extravasation, and ultimately cell growth at secondary sites. The whole process of invasion is driven by the migration of tumor cells. N-WASP activates Arp2/3-complex-mediated actin polymerization, and is therefore important for regulating changes in cell morphology and motility [7–9]. Also, to migrate through barriers, carcinoma cells need to remodel ECM, which is a process controlled by proteases, most notably matrix metalloproteases (MMPs). N-WASP activity is required for the MMP-dependent invasion of carcinoma cells in vivo [13]. Although N-WASP plays a crucial role in the process of tumor invasion and metastasis, the expression pattern of this protein among different carcinoma types varies. Studies of breast cancer cell invasion and migration revealed that N-WASP is involved in the formation of invadopodia in tumor cells in vivo. Furthermore, these studies demonstrated a direct link between invadopodium formation and remodeling of stroma by cancer cells during invasion, intravasation and lung metastasis
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Correlation between Clinicopathological Features and N-WASP Expression in CCRCC Correlations between N-WASP expression in the tumor and clinicopathological parameters were analyzed using a χ2 test (table 3). The histological grade of CCRCC was shown to be significantly related to the degree of NWASP staining of tumor tissue (p = 0.03).
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Results
Color version available online
a
b
c
d
Fig. 1. N-WASP expression in normal renal tissues and CCRCC.
82
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e
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Representative image of N-WASP staining in normal kidney tissue (a), grade I (b), grade II (c), grade III (d), and grade IV (e). NWASP was observed primarily and intensively stained in the nuclear region and cytoplasm of normal tissue. In contrast, CCRCC was poorly stained, if any in the nuclear. Also, a tendency of significantly increased of N-WASP expression in upgoing histological grade of CCRCC was shown.
Color version available online
Table 2. Expression profiling of N-WASP in CCRCC and normal
renal tissue Adjacent normal renal tissue IHC weak, moderate
strong
total
Weak, moderate Strong
4 1
46 22
50 23
Total
5
68
73
Table 3. Relation between clinicopathological features and N-
WASP expression level in CCRCC N-WASP expression total Age ≥65 years 6.7 cm ≤6.7 cm Histological grade I, II III, IV AJCC staging 1 2, 3, 4 T stage T1 T2 T3 N stage N0 N1 M stage M0 M1
weak, moderate
0.6
0.4
0.2
N-WASP low, moderate N-WASP strong
p value 0
strong 0.509
24 49
16 34
8 15
48 25
32 18
16 7
0.425
0
20
40
60
80
Overall survival time (months)
Fig. 2. Survival analysis of N-WASP expression in CCRCC. N-
WASP protein level showed a prognostic role in overall survival. 0.221
38 35
24 26
14 9
38 35
32 18
6 17
32 41
24 26
8 15
35 25 13
25 16 8
10 9 5
68 5
48 3
20 2
72 1
50 0
22 1
[5–7]. Gligorijevic et al. [17] disturbed endogenous NWASP activity in MTLn3 breast cancer cells either by overexpressing a dominant-negative form of N-WASP defective in its ability to activate the Arp2/3 complex, or by silencing N-WASP expression with small hairpin RNA, and demonstrated a decline in invadopodia formation, ECM remodeling, and tumor cell migration in vivo.
However, Martin et al. [18] reported that N-WASP was significantly downregulated in human breast cancer, particularly in aggressive and node-positive tumors from patients with a poor prognosis. These authors also demonstrated that overexpression of N-WASP in the invasive breast cancer cell line MDA-MB-231 resulted in reduced cell motility and invasion and increased cell adhesion. Moreover, these cells produced tumors of significantly reduced growth and volume in vivo. Wang et al. [19] demonstrated that N-WASP mRNA and protein expression is increased in esophageal squamous cell carcinoma. However, a correlation was not observed between N-WASP expression and overall patient survival. Research by Jin et al. [20] also showed that NWASP was highly expressed in hepatocellular carcinoma and demonstrated its prognostic importance in this disease. Our study is the first study to demonstrate the expression pattern of N-WASP in CCRCC. We provide compelling evidence for the downregulation of N-WASP in tumor tissue when compared with matched, normal renal tissue. Also, our findings demonstrate that N-WASP expression within the tumor correlates with the histological grade of the cancer. Despite this downregulation in NWASP expression, higher levels of N-WASP expression
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0.03 0.212 0.751
0.661 0.138
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Parameters
0.8
Cumulative survival
CCRCC IHC
1.0
expression and clinicopathologic variables in CCRCC patients and overall survival (log-rank test) Variables Age, years ≤65 >65 Gender Male Female Tumor diameter, cm >6.7 ≤6.7 Histological grade I, II III, IV AJCC staging 1 2, 3, 4 N-WASP expression Weak, moderate Strong T stage T1 T2 T3 N stage N0 N1 ASA score I II III
All cases
Mean survival, months
31 12
62 50
25 18
58 60
26 17
60 56
20 23
67 51
19 24
63 56
27 16
66 46
20 18 5
55 53 40
41 2
53 40
23 17 3
57 55 47
p value 0.032 0.915 0.89 0.013 0.193 0.002 0.355
0.07 0.201
Table 5. COX multivariate analysis of clinicopathological parameters and overall survival
Variables
Hazard ratio (95% CI)
p value
Age Histological grade N-WASP expression
1.695 (0.536–5.361) 2.418 (0.574–10.182) 3.800 (1.123–12.862)
0.369 0.229 0.032
within the tumor are indicative of poor survival among CCRCC patients. Furthermore, uni- and multivariate analysis suggests that N-WASP is a potential prognostic factor of overall survival. However, the results of this study should be interpreted with caution. First of all, N-WASP 84
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expression within the tumor correlates with the histological grade of the cancer instead of the TNM stage. Usually, stage has more prognostic value than grade. However, in our study the univariate analysis did not suggest that TNM stage indicated the clinical outcome of the patients (table 4). We believe it was due to the relatively early stage cancer features of our patients. Most of the patients enrolled in the study had localized CCRCC with stage T1– T2, which were resectable by curative radical nephrectomy or partial nephrectomy. The prognostic value of TNM stage might be shielded by the relatively optimistic overall survival of the patient’s attitude to the early cancer stage and sophisticated surgery approach. On the other hand, histological grading of the cancer was subject to interobserver variability. Therefore, the value of N-WASP expression to assess the prognosis of renal cancer might not be as ideal as the results of the study suggested. Secondly, the prognosis of the patients studied was evaluated by the overall survival instead of progression-free survival or cancer-specific survival. Even though the American Society of Anesthesiologists (ASA) scores of the patients showed no influence on overall survival (table 4), overall survival was influenced by many other factors. Lastly, the follow-up rate in our study is relatively low (59%), this also compromises our findings on the value of N-WASP expression in CCRCC prognosis evaluation. In the present study, we used IHC to compare the level of N-WASP expression in CCRCC tissue with that of normal renal tissue. The intensity of N-WASP staining was found to be significantly reduced in tumor tissues. These findings are in agreement with those of Martin et al. [18]. However, our finding that N-WASP expression in tumor tissues correlates with the histological grade of the cancer is more similar to that of Gligorijevic et al. [17]. Variations in the expression pattern of N-WASP among different carcinoma types suggest that the contribution of this protein to carcinogenesis may be tissue-specific. Findings from our study also demonstrate that N-WASP expression is not constant during the process of cancer development: we observe high levels of expression in normal renal tissue, a downregulation of this expression in the early stage of CCRCC, and then elevated levels of expression associated with the progression of the cancer. This phenomenon suggests that N-WASP expression demonstrates temporal specificity. Interestingly, another biomarker for CCRCC demonstrates a similar pattern of expression to that of N-WASP. Cai et al. [21] showed that the expression of ZBP-89, a Krüppel-type zinc-finger transcription factor, was decreased in CCRCC when compared with normal renal tisLiu/Chen/Ji/Zhou
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Table 4. Univariate analysis of the correlation between N-WASP
sue. The authors also found that ZBP-89 expression in tumor tissue significantly correlated with two clinicopathological parameters – TNM stage and distal metastasis. Furthermore, high ZBP-89 expression within the tumor was associated with poor survival of CCRCC patients. Uniand multivariate analysis also showed that ZBP-89 was a potential prognostic factor for overall survival. In conclusion, we have shown that while N-WASP expression is reduced in CCRCC, the expression level of this protein within the tumor correlates with the histological grade of the cancer. Furthermore, higher levels of NWASP expression within the tumor are associated with poorer survival in CCRCC patients. Importantly, NWASP is a potential prognostic factor for overall survival
in CCRCC. Further studies are now required to further verify the value of N-WASP expression in CCRCC prognosis evaluation, and elucidate the mechanisms by which N-WASP contributes to the progression of CCRCC. Acknowledgment The study was supported by a grant from Peking Union Medical College Hospital.
Disclosure Statement The authors have no conflicts of interest to disclose.
References
N-WASP in Clear Cell Renal Cell Carcinoma
8 Yamaguchi H, Lorenz M, Kempiak S, Sarmiento C, Coniglio S, Symons M, Segall J, Eddy R, Miki H, Takenawa T, Condeelis J: Molecular mechanisms of invadopodium formation: the role of the N-WASP-Arp2/3 complex pathway and cofilin. J. Cell Biol 2005;168:441–452. 9 Xue C, Wyckoff J, Liang F, Sidani M, Violini S, Tsai KL, Zhang ZY, Sahai E, Condeelis J, Segall JE: Epidermal growth factor receptor overexpression results in increased tumor cell motility in vivo coordinately with enhanced intravasation and metastasis. Cancer Res 2006;66:192–197. 10 Oser M, Mader CC, Gil-Henn H, Magalhaes M, Bravo-Cordero JJ, Koleske AJ, Condeelis J: Specific tyrosine phosphorylation sites on cortactin regulate Nck1-dependent actin polymerization in invadopodia. J Cell Sci 2010; 123:3662–3673. 11 Egeblad M, Ewald AJ, Askautrud HA, Truitt ML, Welm BE, Bainbridge E, Peeters G, Krummel MF, Werb Z: Visualizing stromal cell dynamics in different tumor microenvironments by spinning disk confocal microscopy. Dis Model Mech 2008;1:155–167. 12 Koblinski JE, Ahram M, Sloane BF: Unraveling the role of proteases in cancer. Clin Chim Acta 2000;291:113–135. 13 Eple JN, Sauter G, Epstein JI, Sesterhenn IA: Pathology and Genetics: Tumours of the Urinary System and Male Genital Organs; Lyon, IARC Press, 2004. 14 Edge S, Byrd DR, Compton CC, Fritz AG, Greene FL, Trotti A: AJCC Cancer Staging Manual. New York, Springer, 2009.
Urol Int 2015;95:79–85 DOI: 10.1159/000365595
15 Zhang H, Liu J, Cagle PT, Allen TC, Laga AC, Zander DS: Distinction of pulmonary small cell carcinoma from poorly differentiated squamous cell carcinoma: an immunohistochemical approach. Mod Pathol 2005;18:111–118. 16 Miki H, Takenawa T: Regulation of actin dynamics by WASP family proteins. J Biochem 2003;134:309–313. 17 Gligorijevic B, Wyckoff J, Yamaguchi H, Wang Y, Roussos ET, Condeelis J: N-WASP-mediated invadopodium formation is involved in intravasation and lung metastasis of mammary tumors. J Cell Sci 2005;125:724–734. 18 Martin TA, Pereira G, Watkins G, Mansel RE, Jiang WG: N-WASP is a putative tumour suppressor in breast cancer cells, in vitro and in vivo, and is associated with clinical outcome in patients with breast cancer. Clin Exp Metastasis 2008;25:97–108. 19 Wang WS, Zhong HJ, Xiao DW, Huang X, Liao LD, Xie ZF, Xu XE, Shen ZY, Xu LY, Li EM: The expression of CFL1 and N-WASP in esophageal squamous cell carcinoma and its correlation with clinicopathological features. Dis Esophagus 2010;23:512–521. 20 Jin KM, Lu M, Liu FF, Gu J, Du XJ, Xing BC: N-WASP is highly expressed in hepatocellular carcinoma and associated with poor prognosis. Surgery 2013;153:518–525. 21 Cai MY, Luo RZ, Li YH, Dong P, Zhang ZL, Zhou FJ, Chen JW, Yun JP, Zhang CZ, Cao Y: High-expression of ZBP-89 correlates with distal metastasis and poor prognosis of patients in clear cell renal cell carcinoma. Biochem Biophys Res Commun 2012;426:636–642.
85
Downloaded by: Stockholms Universitet 198.143.54.1 - 8/14/2015 4:21:16 AM
1 Qin C, Sun LJ, Cui L, Cao Q, Zhu J, Li P, Zhang GM, Mao X, Shao PF, Wang ML, Zhang ZD, Gu M, Zhang W, Yin CJ: Application of the revised Tumour Node Metastasis (TNM) staging system of clear cell renal cell carcinoma in eastern China: advantages and limitations. Asian J Androl 2013; 15: 550– 557. 2 Yang L, Parkin DM, Ferlay J, Li L, Chen Y: Estimates of cancer incidence in China for 2000 and projections for 2005. Cancer Epidemiol Biomarkers Prev 2005;14:243–250. 3 Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, Thun MJ: Cancer statistics. CA Cancer J Clin 2008;58:71–96. 4 Derry JM, Ochs HD, Francke U: Isolation of a novel gene mutated in Wiskott-Aldrich syndrome. Cell 1994;78:635–644. 5 Miki H, Miura K, Takenawa T: N-WASP, a novel actin-depolymerizing protein, regulates the cortical cytoskeletal rearrangement in a PIP2-dependent manner downstream of tyrosine kinases. EMBO J 1996;15:5326–5335. 6 Kowalski JR, Egile C, Gil S, Snapper SB, Li R, Thomas SM: Cortactin regulates cell migration through activation of N-WASP. J Cell Sci 2005;118:79–87. 7 Parsons M, Monypenny J, Ameer-Beg SM, Millard TH, Machesky LM, Peter M, Keppler MD, Schiavo G, Watson R, Chernoff J, Zicha D, Vojnovic B, Ng T: Spatially distinct binding of Cdc42 to PAK1 and N-WASP in breast carcinoma cells. Mol Cell Biol 2005;25:1680– 1695.
