Tumor suppressor, AT motif binding factor 1 (ATBF1), translocates to the nucleus with runt domain transcription factor 3 (RUNX3) in response to TGF-beta signal transduction.
OBJECTIVE
AT motif binding factor 1 (ATBF1), a homeotic transcription factor, was identified as a tumor suppressor, and loss of heterozygosity at ATBF1 locus occurs frequently in gastric cancers. We previously showed that ATBF1 expression inversely correlated with the malignant character of gastric cancer and that ATBF1 enhanced the promoter activity of p21Waf1/Cip1. We also found that ATBF1 moves between cytoplasm and nucleus, but the precise mechanism of translocation is unknown. In this study, we investigated the mechanism of ATBF1 translocation to the nucleus with the runt domain transcription factor 3 (RUNX3) in cooperation with TGF-beta signal transduction.


METHODS
To analyze the expression of ATBF1 and RUNX3 in gastric cancer cells, we performed immunohistochemistry on 98 resected gastric cancer tissue samples and scored the nuclear staining intensity as grade 0 to grade 5. Co-immunoprecipitation (co-IP) of ATBF1 and RUNX3 was performed. Dual luciferase assays were performed by transfecting ATBF1 and RUNX3 with a p21Waf1/Cip1 reporter vector. To investigate the nuclear translocation of endogenous ATBF1 and RUNX3 in response to TGF-beta signal, we examined the subcellular localization of ATBF1 and RUNX3 in gastric cancer cells treated with recombinant TGF-beta1 using confocal laser scanning microscopy.


RESULTS
Strong immunohistochemical nuclear staining of ATBF1 was observed in 37 (37.8%) of the gastric cancer tissue samples, and RUNX3 nuclear staining was observed in 15 (15.3%). There was a statistically significant correlation between ATBF1 and RUNX3 nuclear localization (rs=0.433, p<0.001). Co-IP revealed a physical association between ATBF1 and RUNX3. ATBF1 and RUNX3 up-regulated p21Waf1/Cip1 promoter activity synergistically. In SNU16 gastric cancer cells, ATBF1 and RUNX3 were cytoplasmic before TGF-beta1 stimulation, but after 24h of TGF-beta1 stimulation, endogenous ATBF1 and RUNX3 translocated to the nucleus.


CONCLUSIONS
ATBF1 associates with RUNX3 and translocates to the nucleus in response to TGF-beta signal transduction and might function in the nucleus as tumor suppressor and transcriptional regulator.
Mabuchi
Motoshi
M
Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.
Kataoka
Hiromi
H
Miura
Yutaka
Y
Kim
Tae-Sun
TS
Kawaguchi
Makoto
M
Ebi
Masahide
M
Tanaka
Mamoru
M
Mori
Yoshinori
Y
Kubota
Eiji
E
Mizushima
Takashi
T
Shimura
Takaya
T
Mizoshita
Tsutomu
T
Tanida
Satoshi
S
Kamiya
Takeshi
T
Asai
Kiyofumi
K
Joh
Takashi
T
D021581:Active Transport, Cell Nucleus; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D050677:Core Binding Factor Alpha 3 Subunit / Q000378:metabolism*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism; D018398:Homeodomain Proteins / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D011401:Promoter Regions, Genetic; D015398:Signal Transduction; D013274:Stomach Neoplasms / Q000378:metabolism* / Q000473:pathology; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
C493956:CDKN1A protein, human; D050677:Core Binding Factor Alpha 3 Subunit; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018398:Homeodomain Proteins; C493729:Runx3 protein, human; D016212:Transforming Growth Factor beta; D025521:Tumor Suppressor Proteins; C071660:ZFHX3 protein, human