Purpose : Associations between p160 coactivator proteins and the development of resistance to endocrine treatment have been described. We hypothesized that nuclear receptor coregulatory proteins may interact with nonsteroid receptors. We investigated the mitogen-activated protein kinase -activated transcription factors, Ets, as possible interaction proteins for the coactivators SRC-1 and AIB1 and the corepressor NCoR in human breast cancer.Experimental Design: Expression and coexpression of Ets and the coregulatory proteins was investigated using immunohistochemistry and immunofluorescence in a cohort of breast tumor patients (N = 134). Protein expression, protein-DNA interactions and protein-protein interactions were assessed using Western blot, electromobility shift, and coimmunoprecipitation analysis, respectively.Results: Ets-1 and Ets-2 associated with reduced disease-free survival (P < 0.0292, P < 0.0001, respectively), whereas NCoR was a positive prognostic indicator (P < 0.0297). Up-regulation of Ets-1 protein expression in cell cultures derived from patient tumors in the presence of growth factors associated with tumor grade (P < 0.0013; n = 28). In primary breast tumor cell cultures and in the SKBR3 breast cell line, growth factors induced interaction between Ets and their DNA response element, induced recruitment of coactivators to the transcription factor-DNA complex, and up-regulated protein expression of HER2. Ets-1 and Ets-2 interacted with the coregulators under basal conditions, and growth factors up-regulated Ets-2 interaction with SRC-1 and AIB1. Coexpression of Ets-2 and SRC-1 significantly associated with the rate of recurrence and HER expression, compared with patients who expressed Ets-2 but not SRC-1 (P < 0.0001 and P < 0.0001, respectively).Conclusions: These data describe associations and interactions between nonsteroid transcription factors and coregulatory proteins in human breast cancer.

The ADAMs (a disintegrin and metalloprotease) are membrane proteins containing both protease and adhesion domains and thus may be potentially important in cancer invasion and metastasis. The aim of our study was to investigate the distribution and potential clinical significance of ADAM-9 in breast cancer. ADAM-9 expression was measured using both reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. ADAM-9 mRNA was expressed more frequently in both breast carcinomas (72/110, 66%) and fibroadenomas (21/38, 55%) compared to normal breast tissue (6/25, 24%) (p ‫؍‬ 0.0004, p ‫؍‬ 0.028, respectively). Multiple forms of ADAM-9 protein were detected by Western blotting, i.e., at 124, 84 and 48 kDa under reducing conditions and at 115, 76, 55, 52 and 46 kDa under nonreducing conditions. The 84 and 55 kDa forms were detected more frequently in the primary cancers compared to normal breast tissue (p < 0.0001, p ‫؍‬ 0.0002, respectively). In addition, relative levels of the 84 kDa mature form were significantly higher in the primary cancers than in the fibroadenomas (p ‫؍‬ 0.003), while the reverse was found for the 124 kDa precursor form (p ‫؍‬ 0.026). In the carcinomas, the 84 kDa form of ADAM-9 protein was expressed at higher levels in node-positive than node-negative cancers (p ‫؍‬ 0.05) and correlated positively with HER-2/neu protein levels (r ‫؍‬ 0.313, p ‫؍‬ 0.016). This is the first report to describe expression of any ADAM in a large number of human carcinomas. © 2003 Wiley-Liss, Inc. Key words: breast cancer; ADAMs; ADAM-9, HER-2/neu; metastasisThe process of cancer invasion and metastasis is a multistep event that involves angiogenesis, local invasion, cell migration, intravasation, extravasation and growth at a secondary site (for review, see reference 1). Although multiple genes have been implicated in cancer dissemination, among the best characterised are those encoding matrix degrading proteases and adhesion proteins (for review, see reference 2). Proteases such as urokinase plasminogen activator (uPA) and specific matrix metalloproteinases (MMPs) degrade or remodel the extracellular matrix (ECM) allowing cancer cells to invade locally and ultimately form distant metastases. 3 Proteases may also promote metastasis by releasing or activating factors [e.g., fibroblast growth factor-2 (FGF-2), transforming growth factor-␤ (TGF-␤), vascular endothelial growth factor (VEGF)], which enhance cell growth, cell migration and angiogenesis. 4,5 Consistent with their role in metastasis, high levels of multiple proteases have been associated with adverse prognosis in different malignancies (for review, see reference 6).As with proteases, adhesion molecules are also involved at multiple stages during invasion and metastasis. 2,7 In the initial stages of the metastatic pathway, cells must detach themselves from their neighbouring cells and adhere to the basement membrane. As the invading cell migrates through the extracellular matrix (ECM), the leading edge undergoes consecutive cycles of adhesio...

The Ets family of transcription factors regulate expression of multiple genes involved in tumour progression. The aim of this study was to investigate the expression of Ets-1 in a large panel of human breast cancers and relate its levels to the parameters of tumour progression and metastasis. Using RT -PCR, Ets-1 mRNA was detected in 30 out of 42 (71%) fibroadenomas and 131 out of 179 (73%) primary breast carcinomas. Similarly, levels of Ets-1 mRNA were not significantly different in fibroadenomas and primary breast carcinomas. Using Western blotting, four forms of the Ets-1 protein were detected, that is, p33, p42, p51 and p52. Levels of both p51 and p52 but not p33 and p42 were present at significantly higher levels in the carcinomas compared to the fibroadenomas (for p51, Po0.007; for p52, Po0.02; Mann -Whitney U-test). Levels of p52, p51 and p33 correlated significantly with uPA protein levels (Po0.01), while only levels of p52 correlated significantly with HER-2/neu protein levels (Po0.01). Using immunohistochemistry, Ets-1 was found predominantly in tumour cells, but was also detected in some stromal cells surrounding tumour islands. We conclude that, while at the mRNA level, Ets-1 was found at similar levels in fibroadenomas and primary breast carcinomas, higher protein levels were detected in the cancers compared to the benign specimens. Since p52, p51 and p33 correlate with uPA levels, these forms of Ets-1 may play a role in breast cancer metastasis.