Decreased expression of RASSF1A tumor suppressor gene is associated with worse prognosis in clear cell renal cell carcinoma

Abstract

Clear-cell renal cell carcinoma (ccRCC) is the most common subtype of RCC (70-80%) and is associated with poor prognosis in 40% of cases mainly due to metastasis in the course of the disease. RASSF1, with its isoforms RASSF1A and RASSF1C, is a tumor suppressor gene which has not been fully analyzed in ccRCC yet. The epigenetic downregulation of RASSF1A is commonly associated with promoter hypermethylation. The aim of the present study was to compare the ccRCC outcomes with the expression of RASSF1A and RASSF1C. Tissues were obtained from 86 ccRCC patients. RASSF1A and RASSF1C mRNA levels were assessed in tumor and matched normal kidney tissue, and in 12 samples of local metastases by quantitative PCR (qPCR). RASSF1A and RASSF1C proteins levels were semi-quantified in 58 samples by western blot analysis and their tissue localization was assessed by immunohistochemistry. Hypermethylation of RASSF1A promoter was measured by high-resolution-melting methylation-specific qPCR. RASSF1A mRNA levels were 4 and 5 times lower in 66% of tumor and 75% metastasized samples. RASSF1A hypermethylation was found in 40% of analyzed T cases. RASSF1A protein expression was 5 or 20 times decreased in 70% tumor and 75% metastatic samples, respectively. RASSF1A hypermethylation, mRNA and protein levels were associated with TNM progression and higher Fuhrman's grading. Decreased RASSF1A expression, hypermethylation, TNM and Fuhrman's grading were associated with poorer overall survival (OS). Cox hazard ratio (HR) analysis revealed predictor role of RASSF1A mRNA levels on OS and progression-free survival (PFS) in relation to Fuhrman's grading (OS HR=2.25, PFS HR=2.93). RASSF1C levels were increased in ccRCC; no correlations with clinicopathological variables were found. We conclude that RASSF1C gene is not involved in ccRCC progression and we propose that the measurements of RASSF1A mRNA levels in paired tumor-normal kidney tissue could serve as a new prognostic factor in ccRCC.

Figure 1

RASSF1A and RASSF1C gene expression in ccRCC. (A) RASSF1A and (B) RASSF1C mRNA levels in tissue samples of ccRCC patients were assessed by qPCR. (C) Plots and (D) show gene expression in tumor samples related to TNM and Fuhrman's grading. (E and F) RASSF1A and RASSF1C protein levels assessed by western blot analysis. Bars and whiskers represent mean ± SEM normalized to control kidney samples. ^*P<0.05; ^**P<0.01; ^***P<0.001; ^****P< 0.0001 between groups (Mann-Whitney U test).

Figure 2

Quantitative comparison between RASSF1A mRNA levels in tumor and metastasized samples divided by methylation of the RASSF1A gene promoter. Impact of RASSF1A promoter methylation on the gene expression. mRNA results of kidney tumor and metastasized samples (T, M) were divided according to DNA methylation; 25% DNA methylation was treated as threshold. Mann-Whitney U test was applied: ^*P<0.05; ^**P<0.01 between groups.

Figure 3

Correlation plots between either RASSF1A promother methylation, methylation and mRNA (A) or mRNA and protein levels of (B) RASSF1A and (C) RASSF1C. Details in the text.

Figure 4

Analysis of RASSF1A and RASSF1C proteins in ccRCC by western blot analysis. Semi-quantitative analysis of RASSF1A and RASSF1C proteins in tumor (T), control kidney (C ) and metastasized (M) samples normalized to GAPDH protein level. Lines 1 and 2 represent biopsies from patient characterized by TNM 3 and Fuhrman's 2 grade, whereas lines 3–5 represent biopsies from patient with TNM 4, Fuhrman's 3 grade.

Figure 5

Association between RASSF1A promoter methylation and expression at mRNA and protein levels in ccRCC tumor samples. Graphic presentation of multivariate regression analysis of RASSF1A expression pattern; promoter methylation, mRNA level and protein level. XYZ plot represent results of 58 T, C and 12 M biopsies; single results are shown by empty dots. Darkening area represents increasing association between variables (white area for <0.1 association to black area for >0.7 association). Regression analysis with methylation as an independent variable: b=−0.63, P<0.001.

Figure 6

Immuhistochemical localization of RASSF1A and RASSF1C proteins in ccRCC. Immuhistochemical localization of (A–D) RASSF1A and RASSF1C (F–I) proteins in ccRCC. Normal kidney (A and F), tumor kidney of TNM 3 and Fuhrman's grade 2 (B and G) or TNM 4, Fuhrman's grade 4 (C and H) or metastasized lymph node (D and I) of two ccRCC patients (according to Fig. 5) are shown. Strong reaction was observed for RASSF1C in all samples, RASSF1A is characterized by strong presence in epithelial cells of control kidney; weak expression was observed in tumor and mestastized samples as compared to negative control (primary antibody was omitted) of (J) either tumor or (E) metastasized lymph node samples. Magnification, ×20.

Figure 7

Kaplan-Meier's survival analysis for ccRCC patients related to clinicopathological and molecular data. Progression-free survival plots for (A and B) 86 or 58 (C) ccRCC patients. Overall survival plots in 86 (D–F) or 58 (G and H) ccRCC patients.