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 hyper-methylation. 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, hyper-methylation, 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.

Overexpression of the YAP1 oncogene in clear cell renal cell carcinoma is associated with poor outcome

“…Samples were obtained according to our previous reports (26,27). In short, dissected tissue samples of primary ccRCC tumors (n=86, named T), normal kidney (n=86, named C as control) and adrenal gland or the whole lymph node (n=12, named M), were collected in the operating theatre (by J.K.) and placed immediately in approximately five volumes of RNAlater (Ambion Inc., Austin, Tx, uSA).…”

“…RNA isolation and cDNA synthesis were performed as previously described (26,27). Briefly, ExtractMe RNA kit (DNAGdansk, Gdansk, Poland) was used for RNA extraction.…”

Decreased expression of p73 in colorectal cancer

“…The collection of neoplastically changed (tumor) and normal colon tissue samples was performed as described previously [12]. For immunohistochemistry, the tissue samples were fixed in 4% buffered formalin for 3-4 days and standard histologic preparation followed [13]. For the RNA and protein isolation, part of each sample was immediately stored in RNA--later buffer (Ambion Inc., Austin, TX, USA) at 4°C overnight or immersed and kept in liquid nitrogen overnight; eventually all samples were stored at -80°C until further use.…”

“…6.05 program (GraphPad Software, San Diego, CA, USA) as described previously [13]. Mann-Whitney U and Kruskal-Wallis ANOVA tests were applied to check the statistical significance between two or more groups of samples/patients, respectively.…”

“…Primers were designed with the use of PrimerBlast online software: p73a: 5'-CAC-CACGTTTGAGCACCTCTGG; 5'-TGCTCAG-CAGATTGAACTGGGC, PGK1 (normalization gene): 5'-GTTGACCGAATCACCGACCTCTC; 5'-AGAACAGAACATCCTTGCCCAGC. The qPCR time-temperature protocol was applied as previously described [13].…”