Characterization of {gamma}-aminobutyric acid type A receptor-associated protein, a novel tumor suppressor, showing reduced expression in breast cancer.
Frequent allelic loss of the chromosomal region 17p13 in breast cancer has suggested that more tumor suppressor genes, besides p53, are located in this region. By doing suppression subtractive hybridization to detect differentially expressed genes between the breast cancer cell line CAL51 and a nontumorigenic microcell hybrid CAL/17-1, we identified the gene for the gamma-aminobutyric acid type A (GABA(A)) receptor associated protein (GABARAP), located on 17p13.1. GABARAP displayed high expression levels in the microcell hybrid CAL/17-1 but only weak expression in CAL51 and other breast cancer cell lines tested. Furthermore, we observed large vesicles in CAL/17-1 by immunofluorescence staining, whereas no signal could be detected in the tumor cell line. GABARAP mRNA expression and protein expression were significantly down-regulated in invasive ductal and invasive lobular carcinomas compared with normal breast tissue measured by semiquantitative reverse transcription-PCR and immunohistochemistry, respectively. We assessed that neither mutations in the coding region of the gene nor hypermethylation of CpG islands in the promoter region are responsible for loss of gene expression in CAL51; however, 5-aza-2'-deoxycytidine treatment was effective in gene up-regulation, suggesting a methylation-dependent upstream effect. Stable transfection of GABARAP into CAL51 resulted in an increase of gene expression and remarkably influenced the ability of colony formation in soft agar and the growth rate in vitro and, moreover, suppressed the tumorigenicity of the cells in nude mice. In summary, our data suggest that GABARAP acts via a vesicle transport mechanism as a tumor suppressor in breast cancer.
Klebig
Christiane
C
Institute of Pathology, University Hospital Charité, Berlin, Germany.
Seitz
Susanne
S
Arnold
Wolfgang
W
Deutschmann
Nicole
N
Pacyna-Gengelbach
Manuela
M
Scherneck
Siegfried
S
Petersen
Iver
I
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism*; D048708:Cell Growth Processes / Q000235:genetics; D045744:Cell Line, Tumor; D019175:DNA Methylation / Q000187:drug effects; D004252:DNA Mutational Analysis; D000077209:Decitabine; D015536:Down-Regulation; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D016147:Genes, Tumor Suppressor; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection; D015854:Up-Regulation / Q000187:drug effects
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; C116741:GABARAP protein, human; D008869:Microtubule-Associated Proteins; D012333:RNA, Messenger; D000077209:Decitabine; D001374:Azacitidine