Wang et al. Journal of Experimental & Clinical Cancer Research 2010, 29:140 http://www.jeccr.com/content/29/1/140 Page 5 of 7 Figure 2 E2 induces HBO1 expression in breast cancer Cells. (A) T47 D Cells were treated with E2 (10-9, 10-8, 10-7 M) for 24 hours and mRNA level was quantified by real-time PCR analysis. (B) T47 D cell were treated with E2 (10-9, 10-8, 10-7 M) for 24 hours and western blot. Results quantitated by densitometric analysis were representative of three repeated experiments. (C) MCF-7 cells were treated with E2 (10-9, 10-8, 10-7 M) for 24 hours and western blot. Results quantitated by densitometric analysis were representative of three repeated experiments. by treatment with inhibitor of MEK1/2 (U0126) in T47 D (Figure 4A) and MCF-7 (Figure 4B) cells as analyzed by western blot. These results indicated that ERK1/2 signaling pathway was involved in the E2-induced HBO1 upregulation in breast cancer cells. Discussion HBO1 is a potential oncogene which maps to17q21.3, a region where frequent allelic gains are found in breast cancers and this amplification is associated with a poor prognosis of clinical outcome [14-16]. Previous studies demonstrated over-expression of HBO1 dramatically enhanced the anchorage-independent growth of both MCF7 and SKBR3 breast cancer cells while depletion of HBO1 reduced the rate of DNA synthesis, the amount of MCM complex bound to chromatin, and progression through S phase. HBO1 has also been shown to enhance transcription mediated by steroid receptors including ERa and PR [9]. However, little is known about the role of HBO1 in breast cancer and the underlying molecular mechanism. In this study, we first investigated the HBO1 protein expression in large numbers of tumor samples of Figure 3 E2-induced HBO1 expression is inhibited by ICI 182,780 or ERa RNAi. (A) T47 D cells were treated with E2 (10-8 M), ICI 182,780 (1 uM), or E2 (10-8 M) plus ICI 182,780 (1 uM) for 24 hours. Then total cell lysates were processed for western blot analysis as described in Methods. GAPDH was used as an internal control. (B) MCF-7 cells were treated with E2 (10-8 M), ICI 182,780 (1 uM), or E2 (10-8 M) plus ICI 182,780 (1 uM) for 24 hours. Then total cell lysates were processed for western blot analysis as described in Methods. GAPDH was used as an internal control. (C) T47 D cells were transiently transfected with scrambled siRNA or ERa siRNA. 48 h later, total cell lysates were processed for western blot analysis as described in Methods. (D) MCF-7 cells were transiently transfected with scrambled siRNA or ERa siRNA. 48 h later, total cell lysates were processed for western blot analysis as described in Methods. Figure 4 E2 enhances HBO1 expression through ERK1/2 signaling pathways. (A) Serum-starved T47 D cells were treated with E2 (10-8 M), or U0126 (10 uM) plus E2 (10-8 M) for 24 hours. Then equal amounts of protein (lysates) were subjected to SDS- PAGE. Western blot was performed using the Anti-phospho-ERK1/2 (Thr202/Tyr204), anti-HBO1 and anti-ERK1/2 antibodies. GAPDH was used as an internal control. (B) Serum-starved MCF-7 cells were treated with E2 (10-8 M), or U0126 (10 uM) plus E2 (10-8 M) for 24 hours. Western blot was performed as described in (A).