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The p38 mitogen activated protein kinase pathway (MAPK) is known to promote cell survival, endocrine therapy resistance and hormone independent breast cancer cell proliferation. Therefore, we utilized the novel p38 inhibitor RWJ67657 to investigate the relevance of targeting this pathway in the ER+ breast cancer cell line MCF-7. Our results show that RWJ67657 inhibits both basal and estrogen stimulated phosphorylation of p38α, resulting in decreased activation of the downstream p38α targets hsp27 and MAPAPK. Furthermore, inhibition of p38α by RWJ67657 blocks clonogenic survival of MCF-7 cells with little effect on non-cancerous breast epithelial cells. Even though p38α is known to phosphorylate ERα at residue within ER’s hinge region at Thr311, resulting in increased ERα transcriptional activation, our results suggest RWJ67657 inhibits the p38α-induced activation of ER by targeting both the AF-1 and AF-2 activation domains within ERα. We further show that RWJ67657 decreases the transcriptional activity of the ER coactivators SRC-1, SRC-2 and SRC-3. Taken together, our results strongly suggest that in addition to phosphorylating Thr311 within ERα, p38α indirectly activates the ER by phosphorylation and stimulation of the known ERα coactivators, SRC-1, -2 and-3. Overall, our data underscore the therapeutic potential of targeting the p38 MAPK pathway in the treatment of ER+ breast cancer.
The mitogen activated protein kinase (MAPK) cascade is a well-studied cancer signaling pathway. Five distinct MAPK signaling pathways have been identified in mammals; extracellular signal-related kinases (ERKs) 1 and 2, c-Jun N-terminal kinases (JNKs) 1, 2 and 3, p38 isoforms alpha, beta, gamma and delta, ERKs 3 and 4 and ERK 5.1 A variety of stimuli can activate MAPKs, leading to a sequential phosphorylation cascade. Activation of the MAPK pathways results in alterations in a broad variety of cell functions, including mitosis, metabolism, survival, apoptosis, differentiation and altered gene expression.1-5 In breast cancer, MAPK signaling pathways are often pathologically activated to promote both hormone dependent and hormone independent tumor growth.6-8 Furthermore, in estrogen receptor-positive (ER+) breast cancer, 17-β-estradiol acts as a stimulus to induce MAPK activation and initiates a downstream signaling cascade promoting proliferation and endocrine therapy resistance.6,9
p38 MAPK is a primary growth signaling cascade in clinical breast cancer tumors.10,11 Multiple studies have confirmed that p38α signaling can regulate ERα activity and increased p38α phosphorylation in tumors correlates with increased tamoxifen resistance.12-14 Activation of p38α is known to enhance ERα transcriptional activity, though the exact mechanisms of p38’s ER effect are not fully understood. A p38α specific phosphorylation site has been recently identified at Thr311 in the hinge region of ERα.4,12,15 p38 is also known to potentiate the recruitment and activity of ER cofactors to enhance ER-mediated gene transcript. For example, the coactivator GRIP1 is synergistically activated by p38-MAPK in thyroid hormone and the ER.13,16 In addition, we have shown that p38α phosphorylates and activates steroid receptor coactivator 2 (SRC-2), a known ERα coactivator.13 Others have demonstrated cross-talk between p38α and SRC-1, and SRC-3, both of which are known ERα coactivators.16-21 However, the effect of inhibiting p38 on ER function has not been well-studied.
Given that p38α has recently implicated in endocrine resistance in breast cancer, targeting this MAPK as a breast cancer treatment strategy has been of increasing interest. Preliminary studies have demonstrated that pharmacologically targeting p38 and inhibiting its activity results in decreased proliferation in MCF-7 cells.13 However, to date, the effect of p38α inhibition on ERα function has yet to be fully elucidated. Here, we investigate the putative p38 inhibitor, RWJ67657 as an anticancer agent in the treatment of ER(+) breast cancer. We further use this inhibitor as a pharmacological tool to determine the therapeutic potential of blocking p38α and subsequent ERα signaling, in the treatment of ER+ breast cancer.
We first determined the ability E2 to activate p38α in our MCF-7 cell model system. A time-course analysis of phosphorylation of p38α in MCF-7 cells treated with E2 was performed at various time intervals from 15–120 min. Cells lysates were then analyzed for total and phosphorylated protein levels of p38α. Treatment with E2 significantly increased phosphorylation of p38α in a time-dependent manner, without altering total protein levels p38 (Fig. 1A).
We next determined the ability RWJ67657 to inhibit activation of p38α in ER-positive breast cancer cells. MCF-7 cells were treated with RWJ67657 in the presence and absence of E2, followed by western blot analysis of phosphorylated and total p38α. Treatment with RWJ67657 resulted in a marked decrease in both basal and E2 stimulated p38α protein phosphorylation (Fig. 1B). In order to validate these findings, we analyzed the effect of RWJ67657 on downstream protein targets of p38α. Activated p38α is known to phosphorylate hsp27 and MAPAPK to mediate its biological effects.22-25 MCF-7 cells were treated with RWJ67657, with and without E2, and analyzed for total and phosphorylated levels of p38α, hsp27 and MAPAPK (Fig. 1B). The decrease in p38α phosphorylation with inhibitor treatment was associated with decreased phosphorylated levels of both hsp27 and MPAPK, suggesting that the RWJ67657-mediated decrease in activated p38α translates to diminished downstream p38α signaling. Taken together, these results provide proof of principle that RWJ67657 blocks p38 signal transduction in ER+ breast cancer.
The p38-signaling pathway is known to cross talk with that of the ER.10,26 Our laboratory has previously shown that p38α promotes ERα signaling and increases ER transcription.13 We next determined the effect of RWJ67657 on ERα transcriptional activity using an estrogen response element (ERE) reporter construct. The endogenously ER-negative HEK293 cell line was transiently transfected with both ERα and an ERE-luciferase promoter construct followed by treatment with increasing concentrations of RWJ67657. Treatment with RWJ67657 resulted in a dose-dependent decrease in ERE-transcriptional activity (Fig. 2A). We next validated that these results in an ER+ system. MCF-7 cells were transfected with the same ERE-luciferase construct and treated with RWJ67657. Exposure to RWJ67657 also decreased ERE activity in the MCF-7 cell system, suggesting that this inhibitor can decrease ER activity in vitro (Fig. 2B). We utilized a dominant negative-p38α (DN-p38) as a molecular tool to confirm that RWJ67657 inhibits p38 in an on-target fashion. As seen in Figure 2C, molecular inhibition of p38 using DN-p38α greatly reduced ERE-transcriptional activity. These results correlate with those using pharmacological inhibition of p38 with RWJ67657.
p38α is known to phosphorylate ERα at Thr311, resulting in enhanced ER genomic activity.12,15 Therefore, we determined whether the RWJ67657 induced decrease in ERα transcriptional activity was the result of decreased phosphorylation of Thr311. HEK293 cells were transiently transfected with a mutant ERα lacking the Thr311 site and treated with estrogen, RWJ6765, or estrogen plus RWJ67657. Interestingly, treatment with RWJ67657 alone decreased the transcriptional activity of ERα (Thr311Ala) by approximately 70% (Fig. 3A). These findings suggest p38α is targeting ERα not only by direct phosphorylation of Thr311 but also by phosphorylation of other proteins within the cell that affect ERα activity.
We explored this hypothesis further using individual activation domain mutants of ERα. There are two activation regions within ERα, AF-1 and AF-2. Normally, these two domains act in a synergistic manner, however, they can also be activated independently of each other.27,28 AF-1 is the dominant activator of ER transcription in certain tissue types, while in others AF-2 is the primary modulator of ERα function. E2 is a pure ERα agonist and activates both domains in all organ systems. Tamoxifen blocks only the AF-2 domain, which accounts for the tissue-type specific effects of tamoxifen.29 To determine which ERα domain RWJ67657 affects, we utilized two ERα mutants that preferentially inactivate or knockout either AF-1 or AF-2. Treatment with RWJ67657 inhibited the activity of both the AF-1 and AF-2 mutants (Fig. 3B and C). These findings demonstrate that RWJ67657 targets both AF-1 and AF-2 domains within ERα, and further suggests the phosphorylation site at Thr311 is not the only functional target of p38α.
Recent studies show p38α phosphorylates SRC-1, -2 and -3 (Frigo, 2006; Proia DA, 2006; Chai Z, 2009) and we know that all three are coactivaters of ERα (Onate SA, 1995; McInerney EM, 1996; Norris JD, 1998; Suen CS, 1998). Therefore, we hypothesized that RWJ67657 may target ERα cofactors to exert its effect outside of direct inhibition of ERα phosphorylation. The ERα mutant Tyr537Ser is a constitutively active mutant found in nature that confers resistance to tamoxifen.30 This mutation results in a conformational change within ERα which mimics a receptor that is bound by coactivators. We transiently transfected ER (Tyr537Ser) into HEK293 cells and treated the cells with RWJ67657. As seen in Figure 4A, RWJ67657 blocks both basal and E2-induced ER (Tyr537Se). Altogether, our results suggest that while it has a consensus phosphorylation site within ERα, p38α also affects ERα activity by indirectly modulating ER-associated coregulatory proteins.
We next investigated the ability of RWJ67657 to alter the function of three ERα coregulators, SRC-1, -2 and -3, utilizing a mammalian one-hybrid approach. HEK293 cells were transfected with a Gal-4-luciferase reporter and Gal-4-SRC-1, -2, -3 constructs. Cells were then exposed to increasing concentrations of RWJ67657. We observed a dose-dependent decrease in all three SRC activities following treatment with RWJ67657, with SRC-1 being the most affected (Fig. 4B). These findings suggest that RWJ67657 blocks p38α’s ability to both directly phosphorylate ERα at Thr311 and alter the activity of ERα associated coactivators.
Selective toxicity for cancer, as opposed to normal cells, is an important characteristic of any potential anticancer agent. Because p38α activity is increased in cancer cells compared with normal cells, we hypothesized that pharmacological inhibition of p38α by RWJ67657 would selectively target cancer cells. We utilized the MCF10A cell system as a model of normal breast tissue. MCF10A cells are non-transformed, noncancerous breast epithelial cells and are commonly used as controls for MCF-7 ductal carcinoma cells.31-33 We determined the effect of RWJ67657on MCF-7 and MCF10A viability using MTT assays. As seen in Figure 5A, treatment with RWJ67657 resulted in a statistically significant decrease in MCF-7 cell viability. Interestingly, while RWJ67657 diminished breast cancer viability, there was little effect on MCF10A breast epithelial cell viability (Fig. 5B). These data suggest that RWJ67657 targets cancerous breast cells and exhibits little effect on noncancerous breast epithelial cell viability.
A major hallmark of breast cancer is unregulated cell proliferation and survival.34 Therefore, we determined whether inhibition of p38α and ERα signaling by RWJ67657 translated into decreased downstream biological effects of these pathways. MCF-7 cells were treated with RWJ67657, ICI, or tamoxifen, followed by measurement for long-term clonogenic survival (Fig. 5). Exposure to RWJ67657 resulted in a dose dependent decrease of breast cancer clonogenic survival, suggesting that inhibition of p38α by RWJ67657 abrogates downstream biological effects of p38α signaling.
To further validate these findings, we investigated the effects of p38α inhibition of breast cancer tumor growth. Utilizing well-established murine xenograft models, we injected MCF-7 cells into the mammary fat pads of immunocompromised mice. Upon tumor formation on day 18, mice were treated with either vehicle control or RWJ67657 and measured for tumor growth for 31 d. As seen in Figure 6B, treatment with RWJ65657 resulted in markedly decreased breast cancer tumor growth compared with vehicle treated tumors. These results demonstrate the therapeutic potential of inhibiting p38-MAPK signaling in the treatment of ER+ breast cancer (Fig. 7).
The p38 MAPK pathway is known to promote mitogenesis, survival, tumorigenesis and endocrine therapy resistance in cancer cells.11,35 p38 mediates these biological effects through regulation of downstream kinases and gene transcription.10,11 Several studies have shown increased p38α activity as a mechanism of hormone independent tumor growth. Furthermore, phosphorylation of ERα by p38 enhances ERα mediated growth and signaling.5,12,36,37 We show here, similar to other reports, that E2 is also a potent activator of the p38α MAPK pathway. Therefore, targeting the p38 MAPK pathway represents a potential therapeutic treatment option in the treatment of ER+ breast cancer.
In this study we utilized the novel p38α inhibitor RWJ67657 as a pharmacological tool to further determine the role of p38α signaling in breast cancer. We demonstrated that RWJ67657 blocks both basal and E2 stimulated phosphorylation of p38α. This decrease of activated p38 resulted in decreased activation of the downstream effectors hsp27 and MAPAPK. This RWJ67657-induced decrease in p38α activation abrogated downstream biological effects of p38α. Treatment with RWJ67657 blocked both viability and survival of breast cancer cells. Interestingly, exposure of RWJ67657 to normal breast epithelial cells had no effect on cell viability, suggesting that RWJ67657 selectively targets cancerous cells. Selective inhibition of cancer cells, thereby minimizing general toxicity, is an important characteristic of an anticancer agent.
We further investigated the role of RWJ67657 in ERα signaling in the ER+ MCF-7 breast cancer cells. We found that RWJ67657 dose dependently decreased ERα transcriptional activity, both in artificial and endogenous ER cell models. The mechanism of RWJ67657’s effect on ERα was further investigated. p38α is known to phosphorylate and activate ERα at Thr311.4,12,15 Utilizing the endogenously ER- HEK293 cell line, we used a Thr311Ala ERα mutant to determine whether RWJ67657 was acting through direct inhibition of ER phosphorylation at this site. Interestingly, treatment with RWJ67657 partially blocked ERα (Thr311Ala) activity in this system, suggesting RWJ67657 utilizes an alternative mechanism to antagonize ER activity. Further investigation revealed that RWJ67657 targeted both the AF-1 and AF-2 functional domains of the ER and was able to block ERα transcriptional activity in a constitutively active ERα mutant. This led us to hypothesize that RWJ67657 may affect ERα coactivators as a means of decreasing ERα signaling. We found that RWJ67657 inhibited the transcriptional activation of SRC-1, SRC-2 and SRC-3.
Our results show that inhibiting p38α not only blocks the direct downstream mitogenic effects of this MAPK, but also decreases activation of ERα signaling. Dual inhibition of these growth pathways with a single agent is an attractive therapeutic option. Taken together, our findings demonstrate the therapeutic potential of targeting the p38 MAPK pathway as an ER+ breast cancer treatment.
RWJ67657 was obtained from Johnson and Johnson Pharmaceutical Research and Development, L.L.C. ICI 182,780 and 4-hydroxytamoxifen (tamoxifen) were purchased from Tocris Bioscience. Dimethyl- sulfoxide (DMSO) and estradiol were purchased from Fisher Scientific.
MCF-7, MCF10A and HEK293 cells were cultured as previously described.38,39 Briefly, the MCF-7 cell line used is a subclone of MCF-7 cells obtained from the American Type Culture Collection (ATCC) generously provided by Louise Nutter (University of Minnesota, MN).40 The culture flasks were maintained in a tissue culture incubator in a humidified atmosphere of 5% CO2 and 95% air at 37°C. For estrogen studies, cells were grown in phenol red-free Dulbecco's Modified Eagle Medium (DMEM) supplemented with 5% dextran-coated charcoal-treated fetal bovine serum (5% CS-FBS) for 48 h before the addition of additional hormones.
Protein analysis was performed as described in.32,41 Briefly, Cells were plated at 50–60% confluency in 10-cm2 dishes in 5% DMEM for 48 h. Cells were then treated with vehicle, E2, RWJ67657 ± E2, ICI ± E2, or Tam ± E2 for 0, 15, 30, 60 or 120 min. Following treatment, cells were scraped into PBS-EDTA and centrifuged. The cells were lysed in 60–100 µL lysis buffer (Mammalian Protein Extraction Reagent and Halt protease inhibitor, Pierce; and PhoSTOP phosphatase inhibitor). Lysed cells were centrifuged for 10 min. at 12,000 x g at 4°C. The supernatants were combined with loading buffer (5% 2-mercaptoethanol in 4x LDS Loading Buffer, Invitrogen), boiled for 5 min, and loaded onto 4–12% Bis Tris Polyacrilamide Gels (Invitrogen) followed by PAGE at 150V for 1.5 h. Protein was transferred to nitrocellulose membranes using the iBlot (Invitrogen) transfer unit. Nitrocellulose membranes were blocked in 5% milk (BioRad Lab) in Tris Buffered Saline-Tween 20 (TBS-T) for 1 h. at room temperature. Blots were washed briefly with 1X TBS-T (USB) and primary antibodies were diluted in 5% BSA (Bovine Serum Albumin, Sigma-Aldrich) TBS-T according to manufacturer’s recommended dilutions. Antibodies for Rho-GDI, phospho-p38, total-p38, phospho-hsp27, total-hsp27 and phospho-MAPAPK, total-MAPAPK, were purchased from Cell Signaling Technology, Inc. Membranes were incubated in primary antibody overnight at 4°C with gentle agitation. Secondary infrared conjugated antibodies (Li-Cor Biosciences) were diluted in 5% BSA-TBST solution at 1:10,000 and membranes were incubated for 1 h under gentle agitation at room temperature. Membranes were washed and scanned using the LI-COR Odyssey imager and software to detect total and phosphorylated protein levels in cell lysates. Protein bands were quantified using the Li-Cor software and normalized for protein load using the Rho-GDI band.
As previously described,31,41,42 the cells were seeded in 24-well plates at a density of 5 x 105 cells/well in media containing charcoal stripped FBS and allowed to attach overnight. The cells were then transfected for 5 h. with 300 μg pGL2-ERE2X-TK-luciferase plasmid (Panomics) using the Effectene kit according to the manufacturer’s protocol (Qiagen). After 5 h. vehicle, E2, RWJ67657, or E2 + RWJ67657 were added to the cells and cells were incubated at 37°C overnight. The following morning, the media was removed, and 100 μl of lysis buffer was added per well and then incubated for 5 min at room temperature. Cell debris was pelleted by centrifugation at 15,000 x g for 5 min. Luciferase activity of 100 μL of cell extracts was determined an equal volume of Bright Glo luciferase assay reagent (Promega Corp.) in an Autoluminat Plus luminometer (Berthhold Technologies).
Colony assays were performed as previously described.32,38 MDA-MB-361 cells were plated in 6-well plates at a density of 500 cells/well in 3 mL DMEM with 5% DCC-FBS. Twenty-four hours later vehicle (DMSO), E2 (1.0 nM) alone, or E2 + increasing concentrations of RWJ67657 were added and then monitored for colony growth. Ten days later the cells were fixed with 3% glutaraldehyde for 15 min. Following fixation, the plates were washed and stained with a solution of 0.4% crystal violet in 20% methanol for 30 min., washed with PBS, and dried. Colonies of ≥ 30 cells were counted as positive. Results were normalized to DMSO vehicle treated control cells.
Proliferation assays were performed as previously described.43,44 Briefly, cells were plated at a density of 7.5 x 105 cells per well in a 96-well plate in phenol-free DMEM supplemented with 5% FBS and allowed to attach overnight. Cells were then treated with RWJ67657 for 24 h. Following treatment, 20 μL of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg/ml) (Promega) reagent was incubated in each well for 4hr. Cells were lysed with 20% SDS in 50% dimethylformamide. The pH (is this a colorimetric pH assay) and absorbances were read on an ELx808 Microtek plate reader (Bio-Tek Instruments) at 550 nm, with a reference wavelength of 630 nm.
Data was analyzed using Student’s unpaired t-test with p < 0.05 as the limit of statistical significance. Experiments comparing multiple concentrations to the control were tested with one-way ANOVA with Bonferroni post-test to compare individual concentrations. All statistical analyses were done using GraphPad Prism 5.0 (GraphPad Software).
No potential conflicts of interest were disclosed.
Previously published online: www.landesbioscience.com/journals/cbt/article/20992