Estrogen promotes breast cancer cell survival in an inhibitor of apoptosis (IAP)-dependent manner

Abstract

The estrogen receptor (ER) is a major prognostic and therapeutic marker that is expressed in nearly 75% of breast tumors. We have previously shown that the presence of inflammatory mediators can alter the genomic function of the estrogen receptor (ER) in a gene specific manner. In particular, 17β-estradiol (E2) works in combination with the pro-inflammatory cytokines to enhance the expression of a number of pro-survival factors, including the Inhibitor of Apoptosis (IAP) family member, cIAP2. Here we confirm that mRNA and protein levels for cIAP2, but not the related family members cIAP1 and XIAP, are highly up-regulated in MCF-7 breast cancer cells by E2 and cytokines. Similar regulation of cIAP2 is evident in other ER positive but not ER negative cell lines. In agreement with its role as a pro-survival factor, cIAP2 is highly expressed in a subset of invasive breast carcinomas but not in normal breast tissue or ductal carcinoma in situ. Antagonizing IAPs with mimetics of SMAC, which is a known endogenous IAP antagonist, or knockdown of IAPs by siRNA led to greater cell death by TNFα and prevented E2 from promoting cell survival. In addition, a SMAC mimetic reversed TNFα resistance in ER positive breast cancer cells that express high levels of endogenous IAPs. In summary, our findings indicate a new mechanism by which E2 allows breast cancer cells to evade cell death and suggest that an antagonist of IAPs may be a potential therapeutic option for a subset of ER positive breast tumors.

Keywords: Breast Cancer; Cell Survival; Cytokine; Estrogen; IAPs.

Fig. 1

Regulation of IAP mRNA by E2 and TNFα in MCF-7 Cells. a Time course analysis of IAP mRNA expression following treatment of MCF-7 cells with E2 (10 nM), TNFα (10 ng/ml), or both was carried out by QPCR. Data shown are fold change relative to untreated controls using the ΔΔCt method with 36B4 as an internal control. b cIAP2 mRNA was examined by QPCR in ER positive (BT-474, T47D) and ER negative (MDA-MB-231) breast cancer cell lines following treatment with E2, TNFα or both for 24 h.

Fig. 2

Regulation of IAP protein by E2 and TNFα in MCF-7 cells. MCF-7 cells were treated with the combination of E2 (10 nM) and TNFα (10 ng/ml) for up to 24 h (left) or individually with E2 (10 nM) or TNFα (10 ng/ml) or the combination of both for 24 h (right). Western Blot for cIAP1, cIAP2 and XIAP were carried out with β-actin serving as a loading control.

Fig. 3

Expression of cIAP2 in invasive breast carcinoma. A TMA containing paraffin embedded breast tissue was examined for cIAP2 expression by IHC. Positive staining for cIAP2 (a at 10×, b at 40×) was observed in ~13% of invasive breast tumors while ~87% of tumors were negative (c at 10×, d at 40×).

Fig. 4

Smac mimetics sensitize MCF-7 cells to TNFα-induced cell death and block E2-mediated cell survival. MCF-7 cells were treated for 24 h with E2 (10 nM), TNFα (50 ng/ml), or both in the absence or presence of increasing doses of three different SMs, Cmpd3 (a), MV1 (b), and BV6 (c), and cell viability was assessed by a MTS assay. **P < 0.001 for E2 + TNFα compared to E2 alone at the same dose of SM, ^# P < 0.01 for TNFα with a SM compared to TNFα alone in the absence of SMs.

Fig. 5

Smac mimetics down-regulate cIAP1 and cIAP2 protein levels. WCE were prepared from MCF-7 cells treated with or without the combination of E2 (10 nM) and TNFα (10 ng/ml) for 24 h followed by a 1-h treatment with vehicle or BV6 (5 μM). Western Blot for cIAP1, cIAP2, XIAP, and Bcl-2 were carried out with β-actin as the loading control.

Fig. 6

Inhibition of IAPs sensitizes BT-474 Cells to TNFα. a BT-474 cells were treated for 24 h with E2, TNFα (50 ng/ml), or both in the presence of Cmpd 3 (10 nM) and cell viability was determined by MTS assay. b The basal levels of IAP mRNA in untreated BT-474 were compared to that found in untreated MCF-7 cells by QPCR.

Fig. 7

siRNA for IAPs Prevent E2-mediated Cell Survival. a MCF-7 cells were transfected with siRNA for each IAP or a negative control (siNeg) for 48 h. Cells were then treated with the combination of E2 (10 nM) and TNFα (10 ng/ml) for an additional 24 h for cIAP2 detection whereas cIAP1, XIAP and Bcl-2 detection was carried out in untreated cells. Protein expression was examined by Western Blot. Percent knockdown for each siRNA relative to siNeg was determined by densitometry and normalized to β-actin. b MCF-7 cells were transfected with siRNA for single, double, and triple knockdowns of IAPs for 48 h before cells were treated for an additional 24 h with the combination of E2 (10 nM) and TNFα (10 ng/mL). Viability was measured by methylene blue assay. Survival is reported as percent of control (siNeg) for the mean and standard error of three biological replicates, with four technical replicates for each sample. *P < 0.05 compared to siNeg.