Differential gene expression screening uncovers downregulation of the cell cycle regulator BTG2 as an important step in renal tumor biology

Abstract

Renal cell cancer (RCC) is the most common malignancy affecting the adult kidney. The most prevalent RCC subtypes is clear cell RCC (cRCC), which accounts for 75% of all malignant lesions. RCC is characterized by an unpredictable clinical course and a poor prognosis. Very little is known about molecular alterations involved in initiation and progression of RCC. Identification of molecular markers might facilitate diagnosis and outcome predictions and help to develop innovative and novel treatment options. For this reason, it was the aim of this thesis to identify RCC associated genes. In a first part, potentially renal tumor relevant genes were uncovered using a combination of cDNA and tissue microarray technology. In a second part, one gene was selected to further evaluate its impact on RCC biology using Northern blot analysis, quantitative RT-PCR, and functional studies in cRCC cell lines. To uncover renal cancer associated genes, gene expression profiles of four cRCC cell lines and normal renal tissue were compared using BD AtlasTM Human Cancer 1.2 cDNA microarrays. Twenty-five genes were found significantly differentially expressed. To evaluate the relevance of those genes for RCC, mRNA expression levels were further studied by RNA in situ hybridization on a tissue microarray generated from 61 snap frozen primary RCC and 12 normal renal tissues. Five genes (VIM, CD74, CHES1, LITAF, and BTG2) appeared to be highly interesting renal carcinogenesis relevant genes. Of those genes, three (CHES1, LITAF, and BTG2) have never been associated with renal cancer before. BTG2, a negative cell cycle regulator, which was expressed in normal renal tissue but downregulated in cRCC cell lines and primary cRCCs, was chosen for further experiments. Northern blot analysis confirmed the results obtained by cDNA and tissue microarray analysis. Quantitative RT-PCR analysis in 42 primary cRCCs and 17 normal renal tissues revealed up to 44-fold reduced BTG2 mRNA expression in the tumor tissues. Decrease of BTG2 mRNA expression was not associated with advanced disease indicating that reduction of BTG2 mRNA expression is rather an early event in renal carcinogenesis. In cRCC cell lines, BTG2 mRNA expression was weakly inducible by the phorbolester TPA in one of four cultures. In contrast, increasing cell densities lead to slightly elevated BTG2 mRNA expression in three of four cRCC cell lines. Importantly, in both experiments BTG2 mRNA levels did not reach values observed in normal renal tissue by far. The results obtained in the second part of this thesis strongly suggest that downregulation of BTG2 is an important step in renal cancer development. Further experiments (allelic deletion, mutation and methylation analysis and also re-expression of BTG2 in cRCC cell lines) are necessary to show whether BTG2 is a new renal tumor suppressor gene. In summary, application of high throughput cDNA microarray analysis in combination with tissue microarray technology allowed the identification of five genes, which might play a role in renal tumor biology. Further studies on BTG2, the most interesting candidate gene, indicate that downregulation of BTG2 mRNA expression is an early and important step in renal carcinogenesis. More experiments will show whether BTG2 is a new renal tumor suppressor gene.