Endocrine Abstracts

Mutations of the transcription factor GATA3, which is important for maintaining human breast luminal epithelial cell differentiation and quiescence, have been reported in 17 oestrogen receptor (ER) positive breast cancers, although the functional effects of these mutations have not been studied. We therefore investigated 56 ER-positive breast cancers for GATA3 mutations. The tumours were macrodissected, and immunohistochemistry for GATA3 revealed a strong correlation between ER and GATA3 immunostaining with 65% of tumours expressing both proteins (P<0.001). DNA was extracted from tumours, and GATA3 mutational analysis performed. Heterozygous GATA3 mutations were identified in 4 of 56 (7%) ER-positive tumours and consisted of an inframe deletion (330delArg), a seven nucleotide insertion (330InsTGGAGGA) leading to a frameshift and premature termination at codon 353, and two single nucleotide insertions (408InsG and 408InsA) that lead to a frameshift and extension of the predicted protein by 62 amino acids. Functional studies using western blot, immunofluorescence analysis, electrophoretic mobility shift assays and luciferase reporter assay, revealed that: the GATA3 mutant proteins 408InsG, 408InsA and 330InsTGGAGGA were unstable and did not accumulate in the nucleus; only the mutants 408InsG and 408InsA retained DNA binding to the GATA3 consensus sequence; and that the transactivational activities of the GATA3 mutants 330InsTGGAGGA and 330delArg were reduced by 100 and 75%, respectively, and that of the 408InsG and 408InsA by 50% each, when compared to wild-type GATA3. There was a lack of correlation between the presence of GATA3 mutations and GATA3 immunostaining, and three of the four mutations occurred in tumours that had GATA3 immunostaining. The GATA3 immunostaining observed in such tumours may be due to detection of the expressed normal and mutant GATA3 proteins. However, a heterozygous mutation is nevertheless sufficient for tumourigenesis, and thus mutational analysis is necessary for identifying abnormalities of GATA3 in breast cancers.