Hedgehog-interacting
protein (HHIP) was identified as a putative antagonist of the Hh pathway and as a target of Hh signalling. Our aim was to clarify the expression profiles and epigenetic alterations of the HHIP gene in
gastrointestinal cancer. The expression and promoter epigenetic status of HHIP in
cancer cell lines and freshly resected
gastrointestinal cancer tissues were examined using RT-PCR, tissue microarray analysis, methylation-specific PCR, and
chromatin immunoprecipitation assay. Cells were treated with the demethylating agent
5-aza-2'-deoxycytidine and/or
histone deacetylase inhibitor trichostatin A.
WST-8 assays and in vitro invasion assays
after treatment with HHIP-specific
siRNA were performed. HHIP expression levels were reduced in most of the
gastrointestinal cancer cell lines and in a certain subset of
cancer tissues, and these were correlated with promoter hypermethylation. A heterochromatic structure characterized by neither acetylated H3 nor acetylated H4, and
histone H3 lysine 9 hypermethylation and
histone H3 lysine 4 hypomethylation was observed in
cancer cells in which the HHIP gene was aberrantly silenced. On the other hand, overexpression of the HHIP gene was also found in some
cancer tissues and there were significant correlations between
protein expression levels of HHIP and those of Sonic hedgehog (Shh), Indian hedgehog, Patched, and
glioma-associated oncogene homologue-1. An association was found between
lymph node metastasis and HHIP silencing in
colorectal cancer tissues with strong Shh expression and between advanced TNM stage and HHIP silencing in diffuse-type
gastric cancer tissues with strong Shh expression. Down-regulation of HHIP expression by
siRNA resulted in a significant increase in
colon cancer cell growth and invasion in vitro. Silencing of the HHIP gene due to hypermethylation and
chromatin remodelling appears to be frequently involved in gastrointestinal tumourigenesis.