- Email: [email protected]
119 EXPRESSION PROFILE OF MOLECULAR MARKERS ASSOCIATED WITH EPITHELIAL TO MESENCHYMAL TRANSITION IN RENAL CELL CARCINOMA
e50
THE JOURNAL OF UROLOGY姞
Source of Funding: None
118 EZH2 IS OVEREXPRESSED AND CONTRIBUTES TO APOPTOTIC-RESISTANCE AND METASTASIS IN RENAL CELL CARCINOMA Dong Zhang*, Yan Xue, Xiaojie Yang, Weimin Gan, Tie Chong, Ziming Wang, Dalin He, Xi’an, China, People’s Republic of INTRODUCTION AND OBJECTIVES: The enhancer of zeste homolog 2 (EZH2) gene has been recognized as a proto-oncogene and linked to human malignancies. Here, we determined EZH2 expression in primary renal cell carcinoma (RCC) specimens and explored its potential role in prosurvival and metastasis of RCC. METHODS: EZH2 protein expression in RCC specimens was determined by immunohistochemistry. siRNA targeting EZH2 were transfected into RCC CaKi-2, 786-O, and 769-P cells. Cell cycle and apoptosis were detected by flow cytometry following EZH2 knockdown. Transwell assay were used to determine invasive potential in response to EZH2 inhibition. Western blot was performed to detect the expression of cell cycle and apoptosis associated proteins. RESULTS: EZH2 was higher expressed in RCC sample than in paired adjacent nontumorous tissue. 76.5% of RCC sample showed dense staining of EZH2, whereas the neighboring unaffected cells stained at the normal level. After RCC cells were transfected with siRNA targeting to EZH2, all three cells were arrested in G1/S phase (P⬍0.05, compared to control siRNA transfected cells). In addition, significant increased apoptosis were observed in the three cell lines following EZH2 silencing (P⬍0.05, compared to control cells). Furthermore, inhibition of EZH2 resulted in decreased invasive capability of RCC cells in vitro. Mechanically, EZH2 knockdown led to upregulation of p21, cleaved caspase-9, -3, and PARP. Downregulation of PCNA and MMP-2 was also observed following EZH2 silencing. CONCLUSIONS: EZH2 is overexpressed in RCC. Moreover, it plays a critical role in both proliferation and apoptotic-resistance in RCC cell lines. In addition, EZH2 can enhance invasive potential of RCC cell in vitro. These data suggest that inhibition of EZH2 could provide an advantage in improving therapeutic effect of RCC. Source of Funding: None
119 EXPRESSION PROFILE OF MOLECULAR MARKERS ASSOCIATED WITH EPITHELIAL TO MESENCHYMAL TRANSITION IN RENAL CELL CARCINOMA Mototsugu Muramaki*, Hideaki Miyake, Tomoaki Terakawa, Yuuji Kusuda, Masato Fujisawa, Kobe, Japan INTRODUCTION AND OBJECTIVES: As a feature of aggressive tumors, epithelial to mesenchymal transition (EMT) is characterized by reduced E-cadherin and increased several protein expression including N-cadherin, MMP-2 and MMP-9. This molecular alternation is
Vol. 185, No. 4S, Supplement, Sunday, May 15, 2011
thought to be contributing to a stroma-oriented cellular adhesion profile with increased tumor cell motility and invasive properties, and it has recently reported in several tumors. However, it is not known whether the EMT phenotype is important for the progress and prognosis of renal cell carcinoma (RCC). The objective of this study was to investigate the association between EMT markers and the progression of RCC after radical nephrectomy. METHODS: Expression levels of several EMT markers, including E-cadherin, N-cadherin, MMP-2 and MMP-9, were evaluated by immunohistochemical staining in a consecutive series of 99 patients RCC, consisting of 51 with organ-confined disease and 48 with nonorgan-confined disease. All patients including who had metastatic disease at diagnosis underwent radical nephrectomy as a primary treatment. Progression-free survival rates were calculated using the Kaplan-Meier method. Various predictive factors for postoperative recurrence or progression were analyzed using multivariate analysis. RESULTS: Median follow-up period was 19.3 months (range 5–101). Among the 48 patients with non-organ-confined disease, 27 patients had metastatic disease at diagnosis. There were significant increase in the incidence of positive expression of N-cadherin, MMP-2 and -9 in non-organ-confined group (p ⫽ 0.002, 0.002 and 0.03, respectively). Patients with positive expression of N-cadherin, MMP-2 or MMP-9 had significantly shorter progression-free survival (p ⬍ 0.0001, 0.04 and 0.011, respectively). Moreover, presence of metastasis at diagnosis and positive expression of N-cadherin and MMP-9 were shown to be an independent predictor of progression-free survival evaluated by multivariate analysis (p ⫽ 0.007, ⬍ 0.0001 and ⬍ 0.0001, respectively). CONCLUSIONS: Among ENT markers investigated in this study, N-cadherin and MMP-9 were significantly associated with worse prognosis in patients with RCC, and they appeared to be independent prognostic factors after radical nephrectomy. These findings suggest that EMT may have an important role for progression of RCC. Patients with high expression levels of N-cadherin and MMP-9 should undergo intensive post-treatment follow-up. Source of Funding: None
120 E2EPF AS AN UBIQUITIN CARRIER PROTEIN PLAYS A ROLE IN THE PATHOGENESIS OF PAPILLARY RENAL CELL CARCINOMA Frederik Roos*, Mainz, Germany; Andrew Evans, Toronto, Canada; Walburgis Brenner, Mainz, Germany; Christian Thomas, Vancouver, Canada; Pardeep Heir, Olga Roche, Toronto, Canada; Kyle Furge, Grand Rapids, MI; Christian Hampel, Joachim Thu¨roff, Mainz, Germany; Michael Ohh, Toronto, Canada INTRODUCTION AND OBJECTIVES: Molecular pathways associated with pathogenesis of sporadic papillary renal cell carcinoma (PRCC), the second most common form of kidney cancer, are poorly understood. There is no effective therapy for metastatic PRCC, and patients are often excluded from clinical cancer trials. METHODS: We analyzed primary tumour specimens from 35 PRCC patients treated by nephrectomy via gene expression analysis and tissue microarrays (TMAs) constructed from additional 57 paraffinembedded PRCC samples via immunohistochemistry. Gene products were validated and further studied by Western blot analyses using primary PRCC tumour samples and established RCC cell lines, and potential associations with pathological variables and survival in 27 patients with follow-up information were determined. RESULTS: The expression of E2-EPF ubiquitin carrier protein, which targets the principal negative regulator of hypoxia-inducible factor (HIF), von Hippel-Lindau (VHL) protein, for proteasome-dependent degradation, is markedly elevated in the majority of PRCC tumors exhibiting increased HIF1␣ expression and associated with poor prognosis. In addition, we identified multiple hypoxia-responsive elements (HREs) within the E2-EPF promoter and demonstrate for the first time that E2-EPF is a hypoxia inducible gene directly regulated via HIF1.
THE JOURNAL OF UROLOGY姞
Source of Funding: None
118 EZH2 IS OVEREXPRESSED AND CONTRIBUTES TO APOPTOTIC-RESISTANCE AND METASTASIS IN RENAL CELL CARCINOMA Dong Zhang*, Yan Xue, Xiaojie Yang, Weimin Gan, Tie Chong, Ziming Wang, Dalin He, Xi’an, China, People’s Republic of INTRODUCTION AND OBJECTIVES: The enhancer of zeste homolog 2 (EZH2) gene has been recognized as a proto-oncogene and linked to human malignancies. Here, we determined EZH2 expression in primary renal cell carcinoma (RCC) specimens and explored its potential role in prosurvival and metastasis of RCC. METHODS: EZH2 protein expression in RCC specimens was determined by immunohistochemistry. siRNA targeting EZH2 were transfected into RCC CaKi-2, 786-O, and 769-P cells. Cell cycle and apoptosis were detected by flow cytometry following EZH2 knockdown. Transwell assay were used to determine invasive potential in response to EZH2 inhibition. Western blot was performed to detect the expression of cell cycle and apoptosis associated proteins. RESULTS: EZH2 was higher expressed in RCC sample than in paired adjacent nontumorous tissue. 76.5% of RCC sample showed dense staining of EZH2, whereas the neighboring unaffected cells stained at the normal level. After RCC cells were transfected with siRNA targeting to EZH2, all three cells were arrested in G1/S phase (P⬍0.05, compared to control siRNA transfected cells). In addition, significant increased apoptosis were observed in the three cell lines following EZH2 silencing (P⬍0.05, compared to control cells). Furthermore, inhibition of EZH2 resulted in decreased invasive capability of RCC cells in vitro. Mechanically, EZH2 knockdown led to upregulation of p21, cleaved caspase-9, -3, and PARP. Downregulation of PCNA and MMP-2 was also observed following EZH2 silencing. CONCLUSIONS: EZH2 is overexpressed in RCC. Moreover, it plays a critical role in both proliferation and apoptotic-resistance in RCC cell lines. In addition, EZH2 can enhance invasive potential of RCC cell in vitro. These data suggest that inhibition of EZH2 could provide an advantage in improving therapeutic effect of RCC. Source of Funding: None
119 EXPRESSION PROFILE OF MOLECULAR MARKERS ASSOCIATED WITH EPITHELIAL TO MESENCHYMAL TRANSITION IN RENAL CELL CARCINOMA Mototsugu Muramaki*, Hideaki Miyake, Tomoaki Terakawa, Yuuji Kusuda, Masato Fujisawa, Kobe, Japan INTRODUCTION AND OBJECTIVES: As a feature of aggressive tumors, epithelial to mesenchymal transition (EMT) is characterized by reduced E-cadherin and increased several protein expression including N-cadherin, MMP-2 and MMP-9. This molecular alternation is
Vol. 185, No. 4S, Supplement, Sunday, May 15, 2011
thought to be contributing to a stroma-oriented cellular adhesion profile with increased tumor cell motility and invasive properties, and it has recently reported in several tumors. However, it is not known whether the EMT phenotype is important for the progress and prognosis of renal cell carcinoma (RCC). The objective of this study was to investigate the association between EMT markers and the progression of RCC after radical nephrectomy. METHODS: Expression levels of several EMT markers, including E-cadherin, N-cadherin, MMP-2 and MMP-9, were evaluated by immunohistochemical staining in a consecutive series of 99 patients RCC, consisting of 51 with organ-confined disease and 48 with nonorgan-confined disease. All patients including who had metastatic disease at diagnosis underwent radical nephrectomy as a primary treatment. Progression-free survival rates were calculated using the Kaplan-Meier method. Various predictive factors for postoperative recurrence or progression were analyzed using multivariate analysis. RESULTS: Median follow-up period was 19.3 months (range 5–101). Among the 48 patients with non-organ-confined disease, 27 patients had metastatic disease at diagnosis. There were significant increase in the incidence of positive expression of N-cadherin, MMP-2 and -9 in non-organ-confined group (p ⫽ 0.002, 0.002 and 0.03, respectively). Patients with positive expression of N-cadherin, MMP-2 or MMP-9 had significantly shorter progression-free survival (p ⬍ 0.0001, 0.04 and 0.011, respectively). Moreover, presence of metastasis at diagnosis and positive expression of N-cadherin and MMP-9 were shown to be an independent predictor of progression-free survival evaluated by multivariate analysis (p ⫽ 0.007, ⬍ 0.0001 and ⬍ 0.0001, respectively). CONCLUSIONS: Among ENT markers investigated in this study, N-cadherin and MMP-9 were significantly associated with worse prognosis in patients with RCC, and they appeared to be independent prognostic factors after radical nephrectomy. These findings suggest that EMT may have an important role for progression of RCC. Patients with high expression levels of N-cadherin and MMP-9 should undergo intensive post-treatment follow-up. Source of Funding: None
120 E2EPF AS AN UBIQUITIN CARRIER PROTEIN PLAYS A ROLE IN THE PATHOGENESIS OF PAPILLARY RENAL CELL CARCINOMA Frederik Roos*, Mainz, Germany; Andrew Evans, Toronto, Canada; Walburgis Brenner, Mainz, Germany; Christian Thomas, Vancouver, Canada; Pardeep Heir, Olga Roche, Toronto, Canada; Kyle Furge, Grand Rapids, MI; Christian Hampel, Joachim Thu¨roff, Mainz, Germany; Michael Ohh, Toronto, Canada INTRODUCTION AND OBJECTIVES: Molecular pathways associated with pathogenesis of sporadic papillary renal cell carcinoma (PRCC), the second most common form of kidney cancer, are poorly understood. There is no effective therapy for metastatic PRCC, and patients are often excluded from clinical cancer trials. METHODS: We analyzed primary tumour specimens from 35 PRCC patients treated by nephrectomy via gene expression analysis and tissue microarrays (TMAs) constructed from additional 57 paraffinembedded PRCC samples via immunohistochemistry. Gene products were validated and further studied by Western blot analyses using primary PRCC tumour samples and established RCC cell lines, and potential associations with pathological variables and survival in 27 patients with follow-up information were determined. RESULTS: The expression of E2-EPF ubiquitin carrier protein, which targets the principal negative regulator of hypoxia-inducible factor (HIF), von Hippel-Lindau (VHL) protein, for proteasome-dependent degradation, is markedly elevated in the majority of PRCC tumors exhibiting increased HIF1␣ expression and associated with poor prognosis. In addition, we identified multiple hypoxia-responsive elements (HREs) within the E2-EPF promoter and demonstrate for the first time that E2-EPF is a hypoxia inducible gene directly regulated via HIF1.