10653

SPINT2

DIAR3|HAI-2|HAI2|Kop|PB;serine peptidase inhibitor, Kunitz type, 2;SPINT2;serine peptidase inhibitor, Kunitz type, 2

hepatocyte growth factor activator inhibitor type 2|kunitz-type protease inhibitor 2|placental bikunin|serine protease inhibitor, Kunitz type, 2

19q13.1

protein-coding

Medulloblastoma (MB) is a malignant cerebellar tumor that occurs primarily in children. The hepatocyte growth factor (HGF)/MET pathway has an established rolein both normal cerebellar development as well as the development and progressionof human brain tumors, including MB. To identify novel tumor suppressor genes involved in MB pathogenesis, we performed an epigenome-wide screen in MB cell lines, using 5-aza-2'deoxycytidine to identify genes aberrantly silenced by promoter hypermethylation. Using this technique, we identified an inhibitor of HGF/MET signaling, serine protease inhibitor kunitz-type 2 (SPINT2/HAI-2), as a putative tumor suppressor silenced by promoter methylation in MB. In addition, based on single nucleotide polymorphism array analysis in primary MB samples, weidentified hemizygous deletions targeting the SPINT2 locus in addition to gains on chromosome 7 encompassing the HGF and MET loci. SPINT2 gene expression was down-regulated and MET expression was up-regulated in 73.2% and 45.5% of tumors,respectively, by quantitative real-time PCR. SPINT2 promoter methylation was detected in 34.3% of primary MBs examined by methylation-specific PCR. SPINT2 reexpression in MB cell lines reduced proliferative capacity, anchorage independent growth, cell motility in vitro, and increased overall survival timesin vivo in a xenograft model (P<0.0001). Taken together, these data support the role of SPINT2 as a putative tumor suppressor gene in MB, and further implicate dysregulation of the HGF/MET signaling pathway in the pathogenesis of MB.

The hepatocyte growth factor (HGF)/MET pathway plays a critical role in the development of the nervous system and has been implicated in medulloblastoma pathogenesis. Recent studies have shown a role for gene amplification of activators of this pathway, as well as silencing of its inhibitors in medulloblastoma pathogenesis. We analyzed exon array data from a cohort of 103 primary medulloblastomas to show that HGF/MET pathway elements are dysregulated in tumors compared to normal cerebellum. To determine if mutation of HGF/MET pathway genes is a mechanism for pathway dysregulation, we conducted a mutational analysis by exon resequencing of three key components of this pathway, includingserine protease inhibitor Kunitz-type 1 (SPINT1), serine protease inhibitor Kunitz-type 2 (SPINT2), and MET, in 32 primary human medulloblastoma specimens. From this analysis, we identified multiple coding synonymous and nonsynonymous single nucleotide polymorphisms in these genes among the 32 tumor samples. Interestingly, we also discovered two unreported sequence variants in SPINT1 andSPINT2 in two tumors that resulted in Arginine to Histidine amino acid substitutions at codons 418 and 233, respectively. However, conservation assessment and functional assays of these two variants indicate that they involve nonconserved residues and that they do not affect the function of SPINT1 and SPINT2 as tumor suppressor genes. In conclusion, our data suggest that mutation alone plays a minor role in causing aberrancies of the HGF/MET pathway in medulloblastoma in comparison with other malignancies such as breast, hepatocellular, renal, and lung carcinomas.#CI- Copyright (c) 2012 Wiley Periodicals, Inc.