Yeast two-hybrid screening was used to explore novel
proteins that interact with a
breast tumor or
metastasis suppressor, SYK (
spleen tyrosine kinase). The screening yielded
NHERF (
Na+/H+ exchanger regulatory factor, also known as NHERF1 or EBP-50) that binds to the interdomain B of SYK.
NHERF is an
estrogen-responsive gene that encodes an inhibitory factor for epithelial Na+/H+ exchanger
isoform 3 (NHE3). We found intragenic mutation of the
NHERF gene accompanied by loss of heterzygosity (LOH) in approximately 3% (3/85) of
breast cancer cell lines and primary
breast tumors. Mutations occurred at the conserved PDZ domains at
NHERF NH2-terminus that bound to SYK, or at its COOH-terminus motif that binds to
MERLIN, the product of
Neurofibromatosis 2 (NF2) tumor suppressor gene.
NHERF tumorigenic mutations decreased or abolished its interaction with SYK or
MERLIN, suggesting a pathway link among these three molecules that may play a critical role in mammary neoplastic progression. Primary
breast tumors with LOH at the
NHERF locus had clinical presentations of higher aggressiveness, indicating that deregulated
NHERF signaling may be associated with
disease progression. Moreover, the LOH was inversely correlated with SYK promoter methylation, suggesting that
NHERF and SYK may transduce a common suppressive signal. Taken together, the results indicated
NHERF to be a candidate tumor suppressor gene in human
breast carcinoma that may be interconnected to the SYK and
MERLIN suppressors.