Tumor Suppressor Activity and Epigenetic Inactivation of Hepatocyte Growth Factor Activator Inhibitor Type 2/SPINT2 in Papillary and Clear Cell Renal Cell Carcinoma

Mark R Morris,Michael S Wiesener,Esther N Maina,Farida Latif,Mahera Abdulrahman,Takeshi Kishida,Masahiro Yao,Eamonn R Maher,Bin Teh,Rosamonde E Banks,Kunal Gupta,Dean Gentle

doi:10.1158/0008-5472.can-04-3371

Abstract

Following treatment with a demethylating agent, 5 of 11 renal cell carcinoma (RCC) cell lines showed increased expression of hepatocyte growth factor (HGF) activator inhibitor type 2 (HAI-2/SPINT2/Bikunin), a Kunitz-type protease inhibitor that regulates HGF activity. As activating mutations in the MET proto-oncogene (the HGF receptor) cause familial RCC, we investigated whether HAI-2/SPINT2 might act as a RCC tumor suppressor gene. We found that transcriptional silencing of HAI-2 in RCC cell lines was associated with promoter region methylation and HAI-2/SPINT2 protein expression was down-regulated in 30% of sporadic RCC. Furthermore, methylation-specific PCR analysis revealed promoter region methylation in 30% (19 of 64) of clear cell RCC and 40% (15 of 38) of papillary RCC, whereas mutation analysis (in 39 RCC cell lines and primary tumors) revealed a missense substitution (P111S) in one RCC cell line. Restoration of HAI-2/SPINT2 expression in a RCC cell line reduced in vitro colony formation, but the P111S mutant had no significant effect. Increased cell motility associated with HAI-2/SPINT2 inactivation was abrogated by treatment with extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and phospholipase C-gamma inhibitors, but not by an inhibitor of atypical protein kinase C. These findings are consistent with frequent epigenetic inactivation of HAI-2/SPINT2, causing loss of RCC tumor suppressor activity and implicate abnormalities of the MET pathway in clear cell and papillary sporadic RCC. This information provides opportunities to develop novel targeted approaches to the treatment of RCC.

Highlights

Renal cell carcinoma (RCC) is a heterogeneous disorder with the majority (f75%) of cases classified as clear cell and the most frequent subtype is papillary renal cell carcinoma (RCC) (f15% of all cases; ref. 1)
Despite reports of increased expression of hepatocyte growth factor (HGF) and MET in RCC and synergy between the effect of VHL inactivation and increased MET signaling [12], direct genetic evidence for importance of the HGF/ MET signaling in the pathogenesis of RCC is only present in a small minority of cases
We analyzed the expression of HAI-2/SPINT2 by RT-PCR pretreatment and posttreatment with 5-azacytidine in the original four RCC cell lines and in a further seven RCC cell lines

Summary

Introduction

Renal cell carcinoma (RCC) is a heterogeneous disorder with the majority (f75%) of cases classified as clear cell (conventional) and the most frequent subtype is papillary RCC (f15% of all cases; ref. 1). Germline activating mutations in the MET proto-oncogene and inactivating mutations in the VHL tumor suppressor gene. HPRC1-associated MET mutations activate MET signaling to promote cell growth and motility [4,5,6,7]. The VHL tumor suppressor gene is inactivated by somatic mutation or promoter methylation in most sporadic clear cell RCC [8,9,10,11]. Despite reports of increased expression of hepatocyte growth factor (HGF) and MET in RCC and synergy between the effect of VHL inactivation and increased MET signaling [12], direct genetic evidence for importance of the HGF/ MET signaling in the pathogenesis of RCC is only present in a small minority of cases

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Conclusion