Functional Significance of Wnt Inhibitory Factor-1 Gene in Kidney Cancer

Kazumori Kawakami,Shahana Majid,Yuichiro Tanaka,Rajvir Dahiya,Sharanjot Saini,Nobuyuki Kikuno,Hiroshi Hirata,Soichiro Yamamura,Hideki Enokida,Ken Kawamoto,Masayuki Nakagawa

doi:10.1158/0008-5472.can-09-2534

Kazumori Kawakami, Shahana Majid + Show 9 more

Open Access

https://doi.org/10.1158/0008-5472.can-09-2534

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Abstract

Wnt inhibitory factor-1 (WIF-1) has been identified as one of the secreted antagonists that bind Wnt protein. WIF-1 has been described as a tumor suppressor in various types of cancer. However, the molecular function of WIF-1 gene has never been examined in human renal cell carcinoma (RCC). Therefore, we hypothesized that WIF-1 functions as a tumor suppressor gene and overexpression of this gene may induce apoptosis and inhibit tumor growth in RCC cells. Immunohistochemistry and real-time reverse transcription-PCR revealed that WIF-1 was significantly downregulated in RCC samples and RCC cell lines, respectively. Bisulfite sequencing of the WIF-1 promoter region in RCC cell lines showed it to be densely methylated, whereas there was no methylation of WIF-1 promoter in normal kidney. Significant inhibition of cell growth and colony formation in WIF-1-transfected cells compared with controls were observed. WIF-1 transfection significantly induced apoptosis and suppressed in vivo tumor growth. Also, Wnt signaling activity and beta-catenin expression were reduced by WIF-1 transfection. In conclusion, this is the first report documenting that the WIF-1 is downregulated by promoter methylation and functions as a tumor suppressor gene by inducing apoptosis in RCC cells.

Highlights

Renal cell carcinoma (RCC) accounts for 2% to 3% of human malignancies and is the seventh most frequent cause of tumor-dependent death among men [1]
The methylation status at 28 CpG sites of the Wnt inhibitory factor-1 (WIF-1) CpG island was characterized in three RCC cell lines and normal kidney DNA by bisulfite genomic sequencing (Fig. 2A-C)
The WIF-1 gene has been recently identified as a secreted protein that binds to Wnt proteins and inhibits their interaction with the frizzled receptor; this leads to the termination of transcription of genes activated by the β-catenin/TCF/LEF transcriptional complex [18]

Summary

Introduction

Renal cell carcinoma (RCC) accounts for 2% to 3% of human malignancies and is the seventh most frequent cause of tumor-dependent death among men [1]. The most common histologic subtypes of sporadic kidney tumors are clear cell RCC The wingless-type (Wnt) proteins constitute a 19-member family of secreted glycoproteins that are involved in development and oncogenesis [3]. Aberrant expression of Wnt signaling has been reported in various human cancers, including colorectal cancer [4], melanoma [5], non–small cell lung cancer [6], leukemia [7], and bladder cancer [8]. Constitutive activation of the Wnt signaling pathway can be induced by deregulation of Wnt pathway members, such as overexpression of β-catenin and disheveled [4, 6].

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