Strict regulation of CAIXG250/MN by HIF-1α in clear cell renal cell carcinoma

Abstract

Renal cell carcinoma of the clear cell type (ccRCC) is associated with loss of functional von Hippel-Lindau (VHL) protein and high, homogeneous expression of the G250 MN protein, an isoenzyme of the carbonic anhydrase family. High expression of G250MN is found in all ccRCCs, but not in most normal tissues, including normal human kidney. We specifically studied the mechanism of transcriptional regulation of the CAIX G250 gene in RCC. Previous studies identified Sp1 and hypoxia-inducible factor (HIF) as main regulatory transcription factors of G250 MN in various non-RCC backgrounds. However, G250MN regulation in RCC has not been studied and may be differently regulated in view of the HIF accumulation under normoxic conditions due to VHL mutations. Transient transfection of different G250 MN promoter constructs revealed strong promoter activity in G250 MN -positive RCC cell lines, but no activity in G250 MN -negative cell lines. DNase-I footprint and bandshift analysis demonstrated that Sp1 and HIF-1a proteins in nuclear extracts of RCC cells bind to the CAIX promoter and mutations in the most proximal Sp1 binding element or HIF binding element completely abolished CAIX promoter activity, indicating their critical importance for the activation of G250 expression in RCC. A close correlation between HIF-1a expression and G250 MN expression was observed. In contrast, no relationship between HIF-2a expression and G250 MN was seen. The participation of cofactor CBP/p300 in the regulation of G250 transcription was shown. In conclusion, HIF-1a and Sp1, in combination with CBP/p300, are crucial elements for G250 MN expression in ccRCC, and CAIX G250 can be regarded as a unique HIF-1a target gene in ccRCC.

Mutations of the von Hippel–Lindau gene confer increased susceptibility to natural killer cells of clear-cell renal cell carcinoma

“…Our evidence that NK cells are activated differently by targets that bear or not VHL mutations are consistent with earlier findings that suggest that somatic VHL alteration is associated with better cancer-specific survival among those patients presenting with early stage (pT1 and pT2) clear-cell RCC (Parker et al, 2005). Strong expression of the cancer antigen, carbonic anhydrase IX, associated with VHL loss of function (Grabmaier et al, 2004) is correlated with a greater spontaneous immune response Mutated VHL RCC cells and NK cell activation A Perier et al leading to a better outcome in some cases. Furthermore, carbonic anhydrase IX expression strongly predicts the response to IL2-based immunotherapy in metastatic clear-cell RCC (Atkins et al, 2009).…”

Target gene selectivity of hypoxia-inducible factor-α in renal cancer cells is conveyed by post-DNA-binding mechanisms

“…We performed a detailed analysis of two genes displaying highly contrasting HIF-a isoform-specific regulation that is typical of previously analysed patterns of response for two distinct groups of genes. The gene encoding CA9 conforms to a pattern first described among genes encoding glycolytic enzymes (Hu et al, 2003), and is specifically responsive to HIF-1a, showing no response to HIF-2a in any cell type, whether non-RCC or RCC (Grabmaier et al, 2004;Raval et al, 2005). The gene encoding PHD3 conforms to a pattern previously reported for genes encoding a variety of proteins including VEGF and GLUT1 (Raval et al, 2005); it responds dominantly or exclusively to HIF-2a in RCC cells, but to both HIF-1a and HIF-2a in non-RCC cells.…”

“…To simplify the analysis of mechanisms underlying these patterns of transcriptional selectivity, we selected two genes, CA9 and PHD3, that manifest particularly strong hypoxia-inducible responses from low basal levels of expression in normoxic non-RCC (VHL competent) cells. CA9 has previously been reported to be induced by HIF-1a alone in all cell types (Grabmaier et al, 2004;Raval et al, 2005), whereas PHD3 had been demonstrated to be regulated by both HIF-1a and HIF-2a in non-RCC cells, but had not been studied in this way in RCC cells (Aprelikova et al, 2004). First, we showed the (D) and (F) Quantitation of changes in PHD3 protein levels, measured by densitometry, from at least three independent repetitions of this experiment (error bars corresponding to one standard deviation).…”

Clear-cell papillary renal cell carcinoma: molecular and immunohistochemical analysis with emphasis on the von Hippel–Lindau gene and hypoxia-inducible factor pathway-related proteins

“…18 Silencing of VHL expression ultimately results in overexpression of a number of proteins, including HIF-1a, glucose transporter-1 (GLUT-1), and CA9. [19][20][21] We evaluated the morphological, immunohistochemical, and molecular features of nine cases of clear-cell papillary RCC with emphasis on the VHL gene and HIF pathway-related markers, given the importance of this gene and pathway in clear-cell RCC-the main differential diagnostic consideration. Mutational analysis of the entire VHL gene was performed in each case to exclude such mutations as a possible mechanism of gene silencing.…”