| Through serial analysis of gene expression of endothelial cells isolated from breast carcinoma and normal breast tissue, we found that HEYL (hairy/enhancer-of-split related with YRPW motif-like), a basic helix-loop-helix transcription repressor, was consistently overexpressed in tumor endothelial cells. Functional assays showed that overexpression of HEYL in human umbilical vein endothelial cells (HUVEC) increased cell proliferation and conferred strong anti-apoptosis ability, suggesting that HEYL may promote tumor angiogenesis. However, wild-type and HEYL knockout mice supported the growth of xenografts of a syngeneic melanoma cell line equally well, challenging the role of HEYL in neoangiogenesis. Gene expression data provided evidence that HEYL was also overexpressed in the epithelial component of breast cancers. Immunohistochemical analysis on clinical samples showed that HEYL was frequently overexpressed in breast carcinomas. Elevated mRNA expression was also observed at the same frequency in breast carcinomas. DNA amplification of the HEYL gene was not seen, thereby discounting this mechanism for the overexpression of HEYL mRNA and protein. Rather, our work suggested that activation of the Notch pathway may lead to induction of HEYL expression in breast cancer.;For the first time, we showed a direct interaction of HEYL with Smad3. This interaction inhibited the TGF-beta signaling, repressing the effects of TGF-beta on reporter gene luciferase assays. Further, the upregulations of both PAI-1 and p15 mRNA induced by TGF-beta were repressed in HEYL-overexpressing cells, and the reduction of HEYL expression in MDA-MB-231 breast cancer cells caused the partial cell growth inhibition upon TGF-beta treatment.;Taken together, the data demonstrated that HEYL was a novel negative regulator of the TGF-beta pathway. Acquired resistance to TGF-beta is a key step in the early-stage tumorigenesis. Little is known about the development of this resistance in breast cancer. On the other hand, activation of the Notch pathway is known to play a substantial role in promoting breast cancer development. Here we have presented evidence of a crosstalk between these two pathways. HEYL, a direct target of Notch signaling, repressed TGF-beta activity by binding to TGF-beta-activated Smad proteins. |
