Identification of HRK as a target of epigenetic inactivation in colorectal and gastric cancer.
OBJECTIVE
Aberrant methylation of CpG islands can be a good molecular marker for identifying genes inactivated in cancer. We found the proapoptotic gene HRK to be a target for hypermethylation in human cancers and examined the role of such methylation in silencing the gene's expression.


METHODS
Methylation of HRK was evaluated by bisulfite-PCR and bisulfite sequencing in a group of colorectal and gastric cancer cell lines and primary cancers. Gene expression and histone acetylation were examined by reverse transcription-PCR and chromatin immunoprecipitation analyses, respectively. Apoptosis of cancer cells after treatment with a DNA methyltransferase inhibitor and/or histone deacetylase inhibitor was examined with fluorescence-activated cell-sorting analysis.


RESULTS
The region around the HRK transcription start site was methylated in 36% of colorectal and 32% of gastric cancer cell lines and was closely associated with loss of expression in those cell types. HRK expression was restored by treatment with a methyltransferase inhibitor, 5-aza-deoxycytidine, and enhanced further by addition of histone deacetylase inhibitor trichostatin A or depsipeptide. Such restoration of HRK expression was well correlated with induction of apoptosis and enhancement of Adriamycin-induced apoptosis. Expression of other proapoptotic genes, including BAX, BAD, BID, and PUMA, was unaffected by treatment with 5-aza-deoxycytidine. Aberrant methylation of HRK was also frequently detected in primary colorectal cancers that showed methylation of multiple genes, including p16INK4A and hMLH1, and was associated with wild-type p53.


CONCLUSIONS
HRK methylation can be a useful molecular target for cancer therapy in a subset of colorectal and gastric cancers.
Obata
Toshiro
T
First Department of Internal Medicine, Cancer Research Institute, Sapporo Medical University, Sapporo, Japan.
Toyota
Minoru
M
Satoh
Ayumi
A
Sasaki
Yasushi
Y
Ogi
Kazuhiro
K
Akino
Kimishige
K
Suzuki
Hiromu
H
Murai
Masafumi
M
Kikuchi
Takefumi
T
Mita
Hiroaki
H
Itoh
Fumio
F
Issa
Jean-Pierre J
JP
Tokino
Takashi
T
Imai
Kohzoh
K
D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D014408:Biomarkers, Tumor; D045744:Cell Line, Tumor; D002469:Cell Separation; D002843:Chromatin / Q000378:metabolism; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000378:metabolism; D018899:CpG Islands; D019175:DNA Methylation; D000077209:Decitabine; D047630:Depsipeptides; D004317:Doxorubicin / Q000494:pharmacology; D005434:Flow Cytometry; D056572:Histone Deacetylase Inhibitors; D006657:Histones / Q000378:metabolism; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D008957:Models, Genetic; D009479:Neuropeptides / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013274:Stomach Neoplasms / Q000235:genetics* / Q000378:metabolism; D013447:Sulfites / Q000737:chemistry
D051017:Apoptosis Regulatory Proteins; D014408:Biomarkers, Tumor; D002843:Chromatin; D047630:Depsipeptides; C484586:HRK protein, human; D056572:Histone Deacetylase Inhibitors; D006657:Histones; D006877:Hydroxamic Acids; D009479:Neuropeptides; D009842:Oligopeptides; D013447:Sulfites; C403512:lysyl-lysyl-valyl-valyl--phenylalanyl-lysyl-valyl-lysyl-phenylalanyl-lysyl-lysinamide; C012589:trichostatin A; D000077209:Decitabine; D004317:Doxorubicin; D001374:Azacitidine