Epigenetic inactivation of ID4 in colorectal carcinomas correlates with poor differentiation and unfavorable prognosis.
OBJECTIVE
ID4 gene is a member of the inhibitor of DNA binding (ID) family proteins that inhibit DNA binding of basic helix-loop-helix transcription factors. The epigenetic inactivation of ID4 gene on colorectal cancer (CRC) development and its clinical significance was assessed.


METHODS
In CRC cell lines, ID4 methylation status of the promoter region was assessed by methylation-specific PCR and bisulfite sequencing. The mRNA expression level was assessed by quantitative real-time reverse transcription-PCR. The methylation status of 9 normal epithelia, 13 adenomas, 92 primary CRCs, and 26 liver metastases was assessed by methylation-specific PCR. ID4 protein expression was assessed by immunohistochemistry analysis of tissue specimen.


RESULTS
CRC cell lines were shown to be hypermethylated, and mRNA expression was suppressed and could be restored by 5-aza-cytidine treatment. In clinical specimens from normal epithelia, adenomas, primary CRCs, and liver metastases, the frequency of ID4 hypermethylation was 0 of 9 (0%), 0 of 13 (0%), 49 of 92 (53%), and 19 of 26 (73%), respectively, with a significant elevation according to CRC pathological progression. Methylation status of primary CRCs significantly correlated with histopathological tumor grade (P = 0.028). Immunohistochemistry analysis showed ID4 expression of normal colon epithelia, adenomas, and unmethylated primary CRCs but not hypermethylated CRC specimens. Among 76 American Joint Committee on Cancer stage I to IV patients who had undergone curative surgical resection, overall survival was significantly poorer in patients with hypermethylated ID4 bearing tumors (P = 0.0066).


CONCLUSIONS
ID4 gene is a potential tumor suppressor gene for which methylation status may play an important role in the CRC progression.
Umetani
Naoyuki
N
Department of Molecular Oncology, and Division of Gastrointestinal Oncology, John Wayne Cancer Institute, Santa Monica, California 90404, USA.
Takeuchi
Hiroya
H
Fujimoto
Akihide
A
Shinozaki
Masaru
M
Bilchik
Anton J
AJ
Hoon
Dave S B
DS
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001374:Azacitidine / Q000494:pharmacology; D002277:Carcinoma / Q000175:diagnosis / Q000235:genetics*; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D003106:Colon / Q000473:pathology; D015179:Colorectal Neoplasms / Q000175:diagnosis / Q000235:genetics*; D019175:DNA Methylation; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000235:genetics*; D018450:Disease Progression; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D051797:Inhibitor of Differentiation Protein 2; D008297:Male; D008875:Middle Aged; D008957:Models, Genetic; D009369:Neoplasms / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011379:Prognosis; D012333:RNA, Messenger / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics*; D012306:Risk; D017422:Sequence Analysis, DNA; D013447:Sulfites / Q000494:pharmacology; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics*
D017931:DNA Primers; D004268:DNA-Binding Proteins; C497450:ID2 protein, human; D051797:Inhibitor of Differentiation Protein 2; D012333:RNA, Messenger; D012097:Repressor Proteins; D013447:Sulfites; D014157:Transcription Factors; D001374:Azacitidine; C009279:sodium bisulfite