ITF2 prevents activation of the β-catenin-TCF4 complex in colon cancer cells and levels decrease with tumor progression.
OBJECTIVE
Immunoglobulin transcription factor 2 (ITF2) was believed to promote neoplastic transformation via activation of β-catenin. However, ITF2 recently was reported to suppress colon carcinogenesis. We investigated the roles of ITF2 in colorectal cancer cell lines and tumor formation and growth in mice.


METHODS
Levels of ITF2, β-catenin, and c-Myc were measured in 12 human colorectal tumor samples and by immunohistochemistry. ITF2 regulation of β-catenin and T-cell factor (TCF) were analyzed using luciferase reporter, reverse-transcription quantitative polymerase chain reaction, flow cytometry, and immunoblot analyses. Mice were given subcutaneous injections of human colorectal cancer cell lines that stably express ITF2, small hairpin RNAs to reduce levels of ITF2, or control plasmids; xenograft tumor growth was assessed. Human colorectal carcinoma tissue arrays were used to associate levels of ITF2 expression and clinical outcomes.


RESULTS
Levels of β-catenin, cMyc, and ITF2 were increased in areas of human colon adenomas and carcinomas, compared with nontumor areas of the same tissues. ITF2 levels were reduced and cMyc levels were increased in areas of carcinoma, compared with adenoma. In human colorectal cancer cell lines, activation of the β-catenin-TCF4 complex and expression of its target genes were regulated negatively by ITF2. ITF2 inhibited formation of the β-catenin-TCF4 complex by competing with TCF4 for β-catenin binding. Stable transgenic expression of ITF2 in human colorectal cancer cell lines reduced their proliferation and tumorigenic potential in mice, whereas small hairpin RNA knockdown of ITF2 promoted growth of xenograft tumors in mice. In an analysis of colorectal tumor tissue arrays, loss of ITF2 from colorectal tumor tissues was associated with poor outcomes of patients. A gene set enrichment analysis supported the negative correlation between the level of ITF2 and activity of the β-catenin-TCF4 complex.


CONCLUSIONS
In human colorectal cancer cell lines and tissue samples, ITF2 appears to prevent activation of the β-catenin-TCF4 complex and transcription of its gene targets. Loss of ITF2 promotes the ability of colorectal cancer cells to form xenograft tumors, and is associated with tumor progression and shorter survival times of patients.
Department of Pharmacology, Seoul National University College of Medicine, Seoul, Korea; Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul, Korea; Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea.
Hyun-Woo
HW
Shin
Department of Pharmacology, Seoul National University College of Medicine, Seoul, Korea.
Hyunsung
H
Choi
Department of Pharmacology, Seoul National University College of Medicine, Seoul, Korea; Department of Biomedical Science, Seoul National University College of Medicine, Seoul, Korea.
Daeho
D
So
Department of Pharmacology, Seoul National University College of Medicine, Seoul, Korea; Department of Biomedical Science, Seoul National University College of Medicine, Seoul, Korea.
Young-Im
YI
Kim
Department of Biomedical Science, Seoul National University College of Medicine, Seoul, Korea.
Kumsun
K
Cho
Seoul National University Biomedical Informatics, Seoul National University College of Medicine, Seoul, Korea.
Hee-Joon
HJ
Chung
Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul, Korea; Department of Physiology, Seoul National University College of Medicine, Seoul, Korea.
Kyoung-Hwa
KH
Lee
Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul, Korea; Department of Biomedical Science, Seoul National University College of Medicine, Seoul, Korea; Department of Physiology, Seoul National University College of Medicine, Seoul, Korea.
Yang-Sook
YS
Chun
Department of Pharmacology, Seoul National University College of Medicine, Seoul, Korea; Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul, Korea; Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea.
Chung-Hyun
CH
Cho
Department of Pathology, Seoul National University College of Medicine, Seoul, Korea.
Gyeong Hoon
GH
Kang
Department of Pathology, Seoul National University College of Medicine, Seoul, Korea.
Woo Ho
WH
Kim
Department of Pharmacology, Seoul National University College of Medicine, Seoul, Korea; Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul, Korea; Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea; Department of Biomedical Science, Seoul National University College of Medicine, Seoul, Korea. Electronic address: parkjw@snu.ac.kr.
Jong-Wan
JW
Park
D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D000236:Adenoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / Q000235:genetics / Q000378:metabolism*; D049109:Cell Proliferation; D015179:Colorectal Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D018450:Disease Progression; D015536:Down-Regulation; D025461:Feedback, Physiological; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D045325:HCT116 Cells; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D011379:Prognosis; D011485:Protein Binding; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D034622:RNA Interference; D015398:Signal Transduction; D013997:Time Factors; D000073940:Transcription Factor 4; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D047368:Tumor Burden; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism*
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C495270:CTNNB1 protein, human; C489427:MYC protein, human; D016271:Proto-Oncogene Proteins c-myc; C532096:TCF4 protein, human; D000073940:Transcription Factor 4; D014157:Transcription Factors; D051176:beta Catenin
Mouse Model; Signal Transduction; Tumor Suppressor; Wnt Signaling