Genome-Wide Analysis of Differentially Expressed Genes and Splicing Isoforms in Clear Cell Renal Cell Carcinoma

Alessio Valletti,Massimo Carella,Anna Maria D’Erchia,Loreto Gesualdo,Elena Ranieri,Apollonia Tullo,Margherita Gigante,Ernesto Picardi,Elisabetta Sbisà,Michele Battaglia,Chiara Divella,Orazio Palumbo,Graziano Pesole

doi:10.1371/journal.pone.0078452

Abstract

Clear cell renal cell carcinoma (ccRCC) is the most common malignant renal epithelial tumor and also the most deadly. To identify molecular changes occurring in ccRCC, in the present study we performed a genome wide analysis of its entire complement of mRNAs. Gene and exon-level analyses were carried out by means of the Affymetrix Exon Array platform. To achieve a reliable detection of differentially expressed cassette exons we implemented a novel methodology that considered contiguous combinations of exon triplets and candidate differentially expressed cassette exons were identified when the expression level was significantly different only in the central exon of the triplet. More detailed analyses were performed for selected genes using quantitative RT-PCR and confocal laser scanning microscopy. Our analysis detected over 2,000 differentially expressed genes, and about 250 genes alternatively spliced and showed differential inclusion of specific cassette exons comparing tumor and non-tumoral tissues. We demonstrated the presence in ccRCC of an altered expression of the PTP4A3, LAMA4, KCNJ1 and TCF21 genes (at both transcript and protein level). Furthermore, we confirmed, at the mRNA level, the involvement of CAV2 and SFRP genes that have previously been identified. At exon level, among potential candidates we validated a differentially included cassette exon in DAB2 gene with a significant increase of DAB2 p96 splice variant as compared to the p67 isoform. Based on the results obtained, and their robustness according to both statistical analysis and literature surveys, we believe that a combination of gene/isoform expression signature may remarkably contribute, after suitable validation, to a more effective and reliable definition of molecular biomarkers for ccRCC early diagnosis, prognosis and prediction of therapeutic response.

Highlights

Renal cell carcinoma (RCC) is the most widespread adult renal epithelial cancer, accounting for more than 90% of all renal malignancies; clear cell Renal Cell Carcinoma is the most common RCC subtype, affecting about 70% of the surgical cases [1,2]
The morphological features of the tumor tissues corresponded to well-differentiated renal cell carcinomas of the clear cell type, showing prominent cytoplasmic clearing and thin-walled vascular channels
A preliminary Analysis of Variance (ANOVA) highlighted the “Status” variable (i.e., NT and clear cell Renal Cell Carcinoma (ccRCC) tissues) as the major source of variance, with all other variables considered accounting for the residual variance, with small differences between random variables (i.e., “Patient”) and factors (i.e., “Fuhrman Grade” and “Gender”)

Summary

Introduction

Renal cell carcinoma (RCC) is the most widespread adult renal epithelial cancer, accounting for more than 90% of all renal malignancies; clear cell Renal Cell Carcinoma (ccRCC) is the most common RCC subtype, affecting about 70% of the surgical cases [1,2]. A very recent study by the Cancer Genome Atlas Research Network identified a recurrent pattern in ccRCC that correlates with tumor stage and severity and offers new views on the opportunities for disease treatment This remodelling of the cellular metabolism is characterized by the down-regulation of genes involved in the TCA cycle, decreased AMPK and PTEN protein levels, up-regulation of the pentose phosphate pathway and the glutamine transporter genes, increased acetyl-CoA carboxylase protein, and altered promoter methylation of miR-21 ( known as MIR21) and GRB10 [6]. In the last 5 years, more targeted therapies have offered a significant increase in progression-free survival of ccRCC patients; but despite their better efficacy and tolerability than chemotherapy, the majority of patients will eventually develop resistant disease and succumb [7] This lack of therapeutic efficacy highlights the need to identify new molecular markers able to discriminate RCC for an early diagnosis, prognosis and for a better prediction of the therapeutic response

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Conclusion