Insulin-like growth factor 1 attenuates antiestrogen- and antiprogestin-induced apoptosis in ER⁺ breast cancer cells by MEK1 regulation of the BH3-only pro-apoptotic protein Bim

MCF-7 and T-47D ER⁺ breast adenocarcinoma cells (early passage) were procured from the American Type Culture Collection (Rockville, MD, USA) and cultured, as previously described [17, 30]. Before hormonal treatments, cells were placed in DMEM-F12 medium (Invitrogen, Carlsbad, CA, USA), supplemented with 5% dextran-coated charcoal stripped fetal bovine serum (DCC FBS; Hyclone, Logan UT, USA), 2% antibiotics-antimycotics (Invitrogen), 1% sodium pyruvate (Invitrogen) and 10 μg/ml insulin (Sigma-Aldrich, St Louis, MO, USA). For hormonal treatments, cells were seeded either in the absence or presence of insulin, allowed to adhere to the culture vessel for 16 to 24 hours, and then treated with one of the following: 10 nM estradiol (E2; Sigma), 10 nM E2 plus 1 μM 4-OHT in the presence or absence of 10 μM MIF (Sigma Aldrich). For experiments in which cells were seeded in medium containing insulin, cells were washed with HBSS to remove insulin, before administration of hormonal treatment. As indicated in the text and figure legends, hormonal treatments also were conducted in the presence of the following agents alone or in combination: 10 μ;g/ml Insulin (Sigma),1-20 ng/ml IGF-1 (Novozymes/Gropep, North Rocks, NSW, Australia), 5 μM U0126 (EMD Biosciences, Billerica, MA, USA), 25 or 50 μ;M PD 98059 (Calbiochem), and/or 500 μ;M vitamin E (Sigma Aldrich).

Cells were evenly seeded in triplicate at a density to attain 50% to 70% confluence within 24 hours and treated with drugs and/or hormones, as described in the figure legends. For cell counts of the detached cell population, detached cells were collected, concentrated by centrifugation, and counted by using a hemacytometer. Adherent cells were washed twice with cold 1× PBS, trypsinized, diluted in Isoton II, and counted by using a Coulter Counter. For total cell counts, the adherent, monolayer cells were released from the culture dish by trypsinization and pooled with the detached cells collected from the medium. Before all cell counts, the cells were syringed 3 times with a 25 7/8-gauge needle to obtain single-cell suspension. Where indicated in the figure legends, trypan blue (0.08%; Sigma Aldrich) was added to the cell suspension for the identification of dead cells; trypan blue-positive cells demonstrate compromised plasma membrane integrity in dying or dead cells. Cell counts are graphed as the mean ± SD values, and statistically significant differences between treatment groups are described in the figure legends.

The mitochondrial depolarization assay was conducted by using the compound 5,5',6,6- tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine, also referred to as JC-1, according to the manufacturer's protocol (Biotium, Inc., Hayward, CA, USA) and as previously described by our laboratory [31]. All experiments were performed in triplicate. The results are expressed as the mean ± SD values, and statistically significant differences between treatment groups are described in the figure legends.

Cell lysates were harvested as described in our previous studies [13, 17, 30, 31]. Immunoblotting was conducted according to the manufacturer's protocol by using primary antibodies to: LC3 (ab48394), p62 (ab56416), cleaved lamin A (ab52300) [Abcam]; cleaved PARP (9541), phospho-p44/42 MAP kinase (Thr202/Tyr204, 9106), total MAPK (9102), Akt (9272), phospho-Akt (Ser473, 9271), MEK1 (9124), pBim (4581), and Bim (2819) [Cell Signaling]; pERK1/2 (SC-7383), ERα (SC-8002), and IGF-1Rβ (SC-713) [Santa Cruz Biotechnology]; and β-actin (A5441) [Sigma]. Secondary antibodies included antimouse IgG (715-035-150) and anti-rabbit IgG (711-035-152) [Jackson ImmunoResearch]. Immunodetection was performed by using the ECL detection system (34080; Thermo Scientific Pierce) and HyBlot CL autoradiography film (E3012, Denville Scientific Inc., Parsippany, NJ, USA). Densitometry was used to compare signal intensity among samples by using β-actin as the loading control.

For phosphatase experiments, cell lysates were prepared and analyzed as recently described [32] in a triton-based lysis buffer with protease inhibitors, but not NaF or Na₃VO₄. The lysates (50 μ;g protein) were incubated for 20 minutes or 1 hour with lambda phosphatase (λ-PPase (15 μ;g/200 U) (NEB; PO753S) or calf alkaline phosphatase (CIP, 50 U) (NEB, M02909), according to the manufacturer's recommendations.

Evenly seeded adherent cells were treated with the drugs and/or hormones for 48, 72, and 96 hours. Detached and adherent cells were collected and lysed in ice-cold lysis buffer (10 mM Tris-HCl, pH 7.4/10 mM MgCl₂/150 mM NaCl/0.5% NP-40), and the cleavage of cytokeratin 18 was measured in the cell extracts by using Peviva M30-Apoptosense ELISA, according to the manufacturer's protocol (DiaPharma, West Chester, OH, USA). Three independent experiments were performed for each treatment group. Values expressed as the mean ± SD and statistically significant differences between treatment groups are described in the figure legends.

MCF-7 cells (5 × 10⁴ cells in 200 μl per well of a 96-well dish) were seeded. After 24 hours to allow cell attachment, cells were treated with the drugs and/or hormones for various times. At the end of the experimental period, the cells were washed with HBSS and loaded with 25 μM 5(6)-carboxy-2',7'-dichlorofluorescein diacetate (CM-H₂DCFDA, C6827; Invitrogen) for 30 minutes. This nonfluorescent ester CM-H₂DCFDA enters cells and is deacetylated to nonfluorescent 5-(and -6)-chloromethyl- 2',7'-dichlorodihydrofluorescein (CM-H₂DCF) by cellular esterases. ROS rapidly oxidizes CM-H₂DCF to the highly fluorescent 5- (and 6-) chloromethyl-2',7'-DCF (CM-DCF). After 30 minutes of incubation, intracellular ROS levels are directly proportional to CM-DCF generation. To quantify the level of intracellular CM-DCF, the cells were washed with HBSS to remove extracellular CM-DCF, treatment medium was replaced, and cells were incubated at 37°C for a short recovery period (0 to 5 minutes). CM-DCF fluorescence was measured at an excitation wavelength of 485 nm and emission at 520 nm on a fluorescent plate reader (Tecan Spectraflour Plus). Values are expressed as mean ± SD of three independent experiments, and statistically significant differences between treatment groups are described in the figure legends.

RNA interference (RNAi) targeted to MEK1 or Bim (M-003571-00 and M004383-02-0020, respectively, Dharmacon) was carried out according to the manufacturer's protocol. Cells were seeded in DMEM-F12 medium containing 5% DCC FBS (antibiotic free). Twenty-four hours after seeding, cells were washed and treated with either MEK- or Bim-targeting RNAi by using Oligofectamine (Invitrogen; 12252-011). For controls, cells were treated with scrambled RNAi. Twenty-four or forty-eight hours after RNAi treatment, the cells were treated with drugs and/or hormones for various times, and harvested for either protein analysis or cell counts.

A MEK1-GFP plasmid expression vector [33] was purchased from Addgene (Plasmid 147461; Billerica, MA, USA), and the pEGFP-N1 parent vector, from Clonetech, 6085-1. Twenty-four hours before transfection, MCF-7 cells were seeded in DMEM-F12 medium containing 5% DCC FBS (antibiotic free) to yield approximately 50% confluence. Cells were then transfected with plasmids (4.0 μg) by using lipofectamine LTX (Invitrogen) according to the manufacturer's protocol. The transfected cell population was maintained in culture medium for 24 hours, treated with drugs for various times, and harvested for either ROS determinations, mitochondrial membrane permeabilization, or protein analysis.

Recombinant adenovirus [34] expressing dominant-negative MEK1 (Ad-CMV-MEK1DN, Cat 1165; Vector Biolabs) or the Ad-CMV-Null control vector (Ad-CMV, Cat 1300; Vector Biolabs) [35] was used to infect cells at an estimated multiplicity of infection of 100, which results in > 80% infection of ER⁺ breast cancer cells. Twenty-four hours after infection, cells were treated with hormones and/or drugs for various times, and harvested for either ROS determinations, mitochondrial membrane permeabilization, or protein analysis.

For all experiments in which data are graphed as the mean ± SD values, a minimum of three independent experiments was performed. Comparisons were made between treatment groups, and statistically significant differences were determined by one-way ANOVA by using Sigma Plot 11 for Windows, as identified in the figure legends.

In past studies, we reported that 4-OHT and/or MIF treatment induces MCF-7 cell detachment from the monolayer and demonstrated that the detached cells were undergoing caspase-dependent apoptosis with cleavage of PARP, lamin A, and high-molecular-weight DNA as measurable apoptotic markers [13, 14, 17]. These past studies were conducted in phenol red-free DMEM/F12 medium supplemented with 5% FBS that was depleted of endogenous steroid hormones through charcoal stripping. However, we now show that if hormonal treatments are conducted in this medium plus 10 μ;g/ml insulin, as recommended by the ATCC for optimal growth of MCF-7 cells, cell detachment and cell death are not detected (see Additional file 1). This concentration of insulin is supraphysiologic, and insulin at high doses can bind to and activate the IGF-1R [36]. Thus, we hypothesized that the prosurvival effects of insulin were mediated through the IGF-1R and predicted that IGF-1, at physiologic doses, would similarly attenuate the cytotoxic action of 4-OHT and/or MIF. To test this prediction, experiments were conducted in varying concentrations of IGF-1, used in combination with the hormonal treatments. These studies showed that IGF-1 at 10 and 20 ng/ml also attenuated 4-OHT- and/or MIF-induced cell death, as evidenced by a reduction in the number of detached, dying cells, while enhancing E2-mediated cell growth by approximately 50% (Figure 1a). However, cell counts did show that 4-OHT, MIF, and 4-OHT plus MIF treatments conducted in the presence of IGF-1 effectively reduced overall cell number, with the combination of 4-OHT plus MIF most effectively inhibiting cell proliferation (Figure 1a). Western blot analysis further showed predominantly the hypophosphorylated form of Rb110 in the cells treated with 4-OHT plus MIF in IGF-1-supplemented medium, in contrast to significantly higher levels of the hyperphosphorylated, inactive Rb110 present in the cells treated with either 4-OHT or MIF (Figure 1b; compare lane 8 with lanes 5 to 7). Active, dephosphorylated Rb is known to play a key role in 4-OHT- and/or MIF-induced growth arrest of ER⁺ breast cancer cells in the G₁ phase of the cell cycle [13, 17]. So, in the presence of IGF-1, the combined treatment of 4-OHT plus MIF was able to induce cytostasis effectively, but did not appear to affect a significant death response.

Once we established that 20 ng/ml IGF-1 maximally induced cell proliferation, while blocking cell detachment (Figure 1 and data not shown), this concentration was used in all subsequent experiments, including the experiments shown in Figure 1 (b through d), in which we further characterized the IGF-1 prosurvival action. Specifically, IGF-1-treated cells showed very low levels of cleaved PARP and lamin A (Figure 1b). Further IGF-1 blocked the ability of 4-OHT and/or MIF treatment to affect the cleavage of cytokeratin 18 (Figure 1c) and depolarize the mitochondrial membrane (Figure 1d). Cytokeratin 18 cleavage, in particular, occurs during caspase-dependent apoptosis in epithelial cells and tumors derived from epithelial cells [37]. Thus, IGF-1, at physiologically relevant concentrations, blocks 4-OHT-induced and/or MIF- induced apoptotic cell death, which has been partially characterized in our previous studies and involves the activation of caspase-9, -8, and -6 [17, 30].

Because oxidative stress can be an upstream effector of caspase activation [38], and is suppressed by IGF-1 in breast cancer cells [39], we further determined the levels of ROS in cells undergoing the hormonal treatments in medium supplemented with or devoid of IGF-1. ROS levels were determined at various times after treatment and in multiple independent experiments. These experiments showed that ROS levels were significantly higher in cells treated with 4-OHT and/or MIF compared with E2-treated cells, but significantly reduced if IGF-1 was in the treatment medium. Figure 2a shows representative levels of ROS in cells treated with hormones for 24 hours in the presence and absence of IGF-1. The determination of ROS levels in cells harvested at earlier time points (that is, 1 hour) showed that 4-OHT plus MIF treatment induced higher levels of ROS than did either 4-OHT or MIF used as a single agent (data not shown). An essential role of ROS in mediating cell death was demonstrated by using the antioxidant vitamin E. When vitamin E was added to the treatment medium, no significant increase in ROS levels was noted in cells treated with 4-OHT and/or MIF at any time point analyzed (Figure 2b and data not shown). Further, mitochondrial membrane permeability (Figure 2c) and the cleavage of PARP and lamin A were minimally affected (Figure 2d) by hormonal therapy conducted in the presence of vitamin E. Thus, the proapoptotic actions of both 4-OHT and MIF require ROS, and the IGF-1-mediated antiapoptotic action involves a mechanism that, in large part, reduces ROS in hormonally treated breast cancer cells.

MEK1 signaling has been shown to protect against breast cancer cell death more effectively than PI3K/Akt signaling under certain cell contexts (that is, nutrient deprivation) [40]. Thus, we sought to determine whether MEK1 was a key downstream effector of the IGF-1/IGF-1R prosurvival signaling that protected MCF-7 cells from 4-OHT and/or MIF-induced death. Our read-out of MEK1 activity was the phosphorylation/activation of the mitogen-activated protein kinases MAPK1/2 that are activated by MEK1-mediated phosphorylation [41]. Under our treatment conditions and at multiple time points analyzed, cells treated with IGF-1 and E2 showed higher levels of MAPK1/2 phosphorylation (designated pMAPK) than did E2-treated or IGF-1-treated cells (Figure 3a, b; compare lane 5 with lane 1, and data not shown). Further, 4-OHT or MIF used as a single agent or in combination did lead to a significant reduction in pMAPK1/2 phosphorylation in these cells (Figure 3a). Nonetheless, at multiple time points analyzed, pMAPK1/2 in the 4-OHT- and/or MIF-treated cells was detectable by Western blot (Figure 3b, compare lanes 6 to 8 with lane 5 and data not shown), indicating that MEK1 action is only moderately suppressed by these hormonal treatments. Even when treatments were conducted in the absence of IGF-1, active, phosphorylated MAPK1/2 was detected in 4-OHT- and/or MIF-treated cells at multiple time points, including 6 hours (Figure 3b, compare lanes 2 to 4 with lane 1). Thus, MEK1 appears to be active in a large percentage of cells undergoing 4-OHT and/or MIF treatments.

To determine whether the apparent MEK1 activity imparted a growth or survival advantage to 4-OHT- and/or MIF- treated cells, we combined the small-molecule MEK1 inhibitors PD 98059 [42] or U0126 [43] with the hormonal treatments. In initial experiments, we determined that PD 98059 and U0126 effectively blocked IGF-1-and E2-induced MEK1 activity, as evidenced by the lack of detectable MAPK1/2 phosphorylation, with minimal affect on Akt phosphorylation (Figure 3b; compare lanes 9 to 12 with lanes 5 to 9 and data not shown). Importantly, PD 98059 treatment restored the basal and induced level of cell detachment (Figure 3c) and cleavage of PARP in the (Figure 3d) cell populations undergoing 4-OHT and/or MIF treatment, in addition to reducing cell proliferation in all treatment groups (Figure 3c). In a similar fashion, treatment with U0126 also blocked the proliferative (data not shown) and prosurvival effects of IGF-1 and restored the cytotoxic action of 4-OHT and MIF, which included enhancing ROS levels and increasing the percentage of mitochondrial membrane depolarization (see Additional files 2a and 2b). Treatment with vitamin E again reduced the levels of ROS, mitochondrial membrane depolarization, and cleavage of PARP and lamin A resulting from MEK1 blockade (see Additional files 2c through 2e). Considered together, these data show that small-molecule inhibitors of MEK1 effectively block the proliferative and antiapoptotic action of IGF-1 and enhance the ability of 4-OHT and MIF to induce an ROS-dependent apoptosis in ER⁺ MCF-7 breast cancer cells.

Both high levels of IGF-1R, as seen in MCF-7 cells, and low levels of IGF-1R are associated with a higher risk and a less-favorable clinical prognosis [44]. Thus, we wanted to determine whether IGF-1 showed similar, MEK1-dependent prosurvival effects under conditions of low-level IGF-1R expression. We analyzed a subclone of MCF-7, designated SX13, that expresses low-level IGF-1R. SX13 cells harbor the stable integration of an expression vector containing antisense to IGF-1R, whereas the parent MCF-7 cells (designated NEO) harbor the expression vector lacking the antisense [19]. IGF-1R levels in SX13 and NEO cells differ by at least twofold (Figure 4a). However, the reduction in IGF-1R does not sensitize cells to 4-OHT- and/or MIF-induced cell death. The levels of PARP cleavage in SX13 and NEO cells in response to 4-OHT and/or MIF treatment were similar (Figure 4a). Although SX13 cells were not growth-stimulated by IGF-1 above E2-stimulated growth, even under conditions of limiting serum concentration (Figure 4b) [19], IGF-1 did block the growth-inhibitory effects of 4-OHT on SX13 cells. Importantly, MEK1 blockade restored 4-OHT sensitivity in IGF-1-supplemented medium (Figure 4c). Further, IGF-1 reduced the cytotoxic action of the 4-OHT-plus-MIF combination treatment, with detectable reductions in the numbers of dead cells (trypan blue cells) (Figure 4d). When PD 98059 inhibitor was used to block MEK1 action, however, a significant increase in the numbers of trypan blue cells was seen in all the treatment groups (Figure 4d). Microscopic evaluation of SX13 and the NEO cells after 4-OHT and/or MIF treatment, in the presence and absence of IGF-1, clearly showed that PD 98059 treatment resulted in a robust reduction in cell number (data not shown, and Figure 4e, compare c and d with a and b, respectively). These studies establish that blockade of MEK1 with small-molecule inhibitors can circumvent the protective effects of IGF-1 and increase the cytotoxic, proapoptotic action of 4-OHT and/or MIF on ER⁺ breast cancer cells with low and high levels of IGF-1R.

To confirm the role of MEK1 in regulating hormonally induced ROS and apoptosis, we used RNAi to downregulate MEK1 mRNA, a dominant negative, mutant MEK1 cDNA to block the action of MEK1, and a wild-type MEK1 cDNA to force MEK1 overexpression. In these experiments, targeting MEK1 expression with siRNA effectively reduced MEK1 protein levels in all treatment groups (Figure 5a, compare lanes 4 to 6 with 1 to 3). This reduction in MEK 1 expression significantly increased both the ROS levels (Figure 5b) and mitochondrial membrane depolarization (Figure 5c) in cells subjected to 4-OHT and/or MIF treatment in IGF-1-supplemented medium. Similar results were obtained when MEK1 action in MCF-7 cells was blocked by overexpression of a mutant, MEKDN (see Additional files 3a to 3c). In stark contrast, the overexpression of MEK1 wild-type cDNA, which led to detectable increases in MEK1 protein in the transfected cells (Figure 5d), reduced both the levels of ROS and mitochondrial membrane depolarization in cells undergoing 4-OHT and/or MIF treatment (Figure 5e and 5f, respectively). Thus, MEK1 overexpression in 4-OHT- and/or MIF-treated cells mimicked the prosurvival effects of IGF-1. Further, these MEK1 expression studies were consistent with the results obtained with the small-molecule inhibitors of MEK1 and confirmed a key antiapoptotic role of a MEK1-dependent pathway in MCF-7 breast cancer cells undergoing 4-OHT and/or MIF treatments.

The proapoptotic protein Bim/BOD, a member of the BH3-only group of Bcl-2 family members, is an effector of cell death on growth-factor withdrawal in many cell types, including epithelial cells [45]. Further, MEK1/MAPK1/2 signaling regulates BimEL expression via phosphorylation that facilitates BimEL degradation by the proteasome [46]. Thus, we considered Bim to be a strong candidate for the death effector mediating the cytotoxicity in hormonally treated MCF-7 cells with compromised MEK1 activity. Bim has three isoforms because of alternative splicing: BimEL, BimL, and BimS [47], with BimEL being the most abundant form expressed in MCF-7 cells under our treatment conditions (Figure 6a, top panel). BimS, which is the most cytotoxic Bim isoform and transiently expressed during apoptosis in other cell types [48], was the most difficult to detect. The BimL isoform was seen at higher levels in cells treated with MIF in comparison with E2- or 4-OHT-treated cells (Figure 6a, top panel: compare lanes 3 and 4 with 1 and lanes 7 to 8 with 5). MIF, but not 4-OHT, appeared to be inducing BimL.

Close inspection of a lighter exposure of the BimEL signal (Figure 6a, middle panel) identified a doublet band, with the upper band being the predominant form in cells treated with E2 plus IGF-1 (Figure 6a, lane 5). The lower BimEL band, which is a faster-migrating BimEL protein, was consistently detected at higher levels in cells treated with U0126 as a single agent or in combination with 4-OHT (Figure 6b; compare lanes 3 and 4 with lanes 1 and 2 and lanes 7 and 8 with lanes 5 and6), and/or MIF treatment (Figure 6c and data not shown). In addition, this lower band was often detected at higher levels than the upper BimEL band in cells treated with 4-OHT and/or MIF for 60 hours or longer in medium devoid of IGF-1 (data not shown). Overall, the relative increase in the levels of the lower BimEL band correlated to the timing of 4-OHT- and/or MIF-induced cytotoxicity in MCF-7 populations. We predicted that the lower BimEL band was the dephosphorylated form of BimEL known to be more stably maintained in cells. To determine whether phosphorylation was regulating the levels of either BimEL form, we treated cells with MG132, a commonly used proteasome inhibitor, which blocks the degradation of ubiquitinated proteins by the proteasome [49]. In these experiments, the upper BimEL protein accumulated in cells treated with MG132 plus E2, 4-OHT, and/or MIF (Figure 6i; compare lanes 5 and 6 with lanes 1 and 2 and data not shown). In contrast, in the cell populations treated with PD 98059 or U0126, the phosphorylation of BimEL was impaired and did not significantly increase after MG132 treatment (Figure 6i, compare lanes 7 and 8 with lanes 5 and 6). Further, the dephosphorylated status of the lower Bim EL band was established when protein lysates isolated from cells exposed to the different hormones were subjected to calf-intestinal-phosphatase (data not shown) or λ-phosphatase. Figure 6d shows representative results of λ-phosphatase treatments conducted for 20 minutes and 1 hour that resulted in increased levels of the lower BimEL band (Figure 6d; compare lane 1 with lanes 2 and 3, respectively). The increase in the lower BimEL band occurred with a concomitant loss of the upper BimEL band (Figure 6e; compare lane 1 with lanes 2 and 3, respectively) and was similar in size to the BimEL form generated by treatment of cells with U0126 (Figure 6e; compare lanes 2 through 4). In comparison, the λ-phosphatase treatment of protein isolated from cells treated with U0126 only modestly increased the levels of the lower BimEL band (Figure 6e; compare lane 5 with lane 4). As an internal control, the loss of pMAPK signal as a result of CIP (data not shown) and λ-phosphatase treatment was apparent in all experiments (Figure 6e; lanes 2 and 3 compared with lane 1, and lane 5 compared with lane 4). Thus, these experiments identify the lower BimEL band as the dephosphorylated from of BimEL.

Because studies of CYP2D6 polymorphisms do not clearly show that 4-OHT is a key metabolite involved in the antitumor effects of TAM treatment in patients [50], we also performed similar experiments with TAM at a dose of 5.0 μ;m, commonly used for preclinical in vitro studies [51]. The results of these experiments showed that IGF-1 also reduced the ability of TAM, used as a single agent or in combination with MIF, to induce cell death (Figure 6f, lane 3 compared with lane 7, and data not shown); targeting MEK1 with inhibitors led to a robust increase in the levels of dephosphorylated BimEL, with a concomitant decrease in the levels of phosphorylated BimEL; and the levels of dephosphorylated Bim EL correlated directly to the cytotoxicity of MEK1 blockade, as evidenced by increased PARP cleavage in cells by 72 hours of treatment (Figure 6f; lanes 8 and 9 compared with lanes 6 and 7).

To confirm that Bim played a key role in apoptotic cell death induced by antiestrogen and antiprogestin treatments when conducted in the presence and absence of MEK1 blockade, we used siRNA to downregulate Bim expression in MCF-7 cells. These experiments were performed in medium supplemented with or devoid of IGF1. The siRNA targeting of Bim was very effective in reducing BimEL protein expression when conducted in cells growing in medium devoid of IGF-1 (Figure 7a; compare lanes 4 to 6 with lanes 1 to 3). Bim downregulation under these growth conditions reproducibly attenuated the ability of 4-OHT and/or MIF, in the presence or absence of U0126, to induce the cleavage of PARP and lamin A (Figure 7a; compare lanes 4 to 6 with lanes 1 to 3). Bim downregulation also significantly reduced ROS levels in the cells treated with 4-OHT and/or MIF (Figure 7b). The ROS levels in cells treated with 4-OHT, MIF, and/or U1026 were reduced to levels present in the control E2-treated cells (Figure 7b). When IGF-1 was in the treatment medium, siRNA targeting also effectively reduced Bim levels in MCF-7 cells (Figure 7c and data not shown). The reduction in Bim expression robustly reduced the proapoptotic action of U0126 in cells of all treatment groups, but most effectively in the E2- and 4-OHT-treated cells (Figure 7d; compare lane 5 with lane 2). The siRNA data shown in Figure 7a and 7b are representative of at least three independent experiments in which cells were treated in medium devoid of IGF-1 or supplemented with IGF-1, respectively. These data provide strong evidence that Bim is a key death effector for 4-OHT- and/or MIF-induced cell death, as well as the increased cytotoxicity provided by treatment with MEK1 inhibitors

T-47D is an independent breast cancer cell model that expresses ER and PR and is commonly used for studies analyzing the effects of antiestrogen blockade of ER function [17, 30, 31]. In comparison to MCF-7 cells, T-47D cells show lower basal levels of BimEL, and this reduced level of BimEL expression has been correlated to a reduced level of paclitaxel-induced apoptosis [52]. Thus, we characterized the level and phosphorylation status of BimEL relative to the induction of cytostasis and cytotoxicity in T-47D cells by 4-OHT and/or MIF treatment in the presence and absence of IGF-1 and under conditions of MEK1 blockade. Cell counts showed that IGF-1 stimulated T-47D cell growth above proliferation levels seen in the E2-treated population and that PD 98059 effectively reduced the IGF-1-mediated proliferation (Figure 8a). However, no detectable increase was seen in the number of trypan blue cells within T-47D cell populations as a result of any of the treatments, even after extended periods of treatment (that is, 144 hours) (Figure 8b and data not shown). By 72 hours of treatment, cleavage of PARP could be detected in T-47D cells treated with 4-OHT plus MIF plus U0126 (Figure 8c; compare lane 6 with lanes 1 to 5), concomitant with a reduction in the levels of procaspase 3, indicative of its activation. Cleavage of PARP and lamin A was detected in cells treated with MIF, 4-OHT plus MIF, and 4-OHT plus U0126, but only at later time points and at modest levels (data not shown). The 4-OHT-treated T-47D cells never showed evidence of apoptotic cell death; neither cleavage of PARP nor that of lamin A was detected in 4-OHT-treated cells, even after 216 hours of treatment. In comparison with the cytotoxic effect on MCF-7 cells, T-47D cells appeared essentially resistant to apoptosis, with absence of cleavage of PARP and lamin A in T-47D cells treated with 4-OHT and/or MIF in the absence or presence of U0126 for 48 hours (Figure 8d; compare lanes 1 to 4 with lanes 5 to 8, respectively). The reduced ability of T-47D cells to undergo apoptotic cell death correlated to an approximate twofold lower level of basal BimEL expression in T-47D cells compared with MCF-7 cells. This difference can readily be seen in Figure 8d, in which equal loading of lysates shows no detectable level of Bim EL in T-47D cells compared with readily detectable Bim EL expression in MCF-7 cells (Figure 8d; compare lanes 5 to 8 with lanes 1 to 4). ROS levels in 4-OHT- and/or MIF-treated T-47D also were less than those induced in MCF-7 cells (Figure 8e and data not shown).

Although apoptotic death was minimally induced in T-47D cells, treatment with U0126 effectively reduced the levels of pMAPK1/2 in T-47D cells, which were inherently higher than pMAPK1/2 levels in MCF-7 cells (Figure 8d). Because pMAPK1/2 levels were at least twofold higher in T-47D cells than in MCF-7 cells, we performed experiments with MG132 to determine whether the intrinsic turnover rate of BimEL was higher in T-47D cells than in MCF-7 cells. MG132 treatment did not increase the intracellular levels of BimEL in T-47D cells (Figure 8f; compare lanes 7 and 8 with lanes 3 and 4). These data show that the basal level of BimEL expression can vary between ER⁺ breast cancer cell models by mechanisms independent of MEK1/MAPK12-mediated phosphorylation and proteasomal turnover. Thus, MEK1 targeting may be effective only in ER⁺ breast cancer cells with high intrinsic levels of BimEL (schematically summarized in Figure 9).