The scaffold protein MEK Partner 1 is required for the survival of estrogen receptor positive breast cancer cells
© Marina et al.; licensee BioMed Central Ltd. 2012
Received: 6 February 2012
Accepted: 9 July 2012
Published: 9 July 2012
MEK Partner 1 (MP1 or MAPKSP1) is a scaffold protein that has been reported to function in multiple signaling pathways, including the ERK, PAK and mTORC pathways. Several of these pathways influence the biology of breast cancer, but MP1’s functional significance in breast cancer cells has not been investigated. In this report, we demonstrate a requirement for MP1 expression in estrogen receptor (ER) positive breast cancer cells. MP1 is widely expressed in both ER-positive and negative breast cancer cell lines, and in non-tumorigenic mammary epithelial cell lines. However, inhibition of its expression using siRNA duplexes resulted in detachment and apoptosis of several ER-positive breast cancer cell lines, but not ER-negative breast cancer cells or non-tumorigenic mammary epithelial cells. Inhibition of MP1 expression in ER-positive MCF-7 cells did not affect ERK activity, but resulted in reduced Akt1 activity and reduced ER expression and activity. Inhibition of ER expression did not result in cell death, suggesting that decreased ER expression is not the cause of cell death. In contrast, pharmacological inhibition of PI3K signaling did induce cell death in MCF-7 cells, and expression of a constitutively active form of Akt1 partially rescued the cell death observed when the MP1 gene was silenced in these cells. Together, these results suggest that MP1 is required for pro-survival signaling from the PI3K/Akt pathway in ER-positive breast cancer cells.
The small protein MEK Partner 1 (MP1, also known as Map Kinase Scaffold Protein 1 and LAMTOR3) was originally identified as a scaffold protein that potentiates MAPK signaling by binding to MEK1 and ERK1 . MP1 interacts with another small protein p14, and together these two proteins are localized to endomembrane compartments as part of larger signaling complexes. For example, an MP1-p14-MEK1 complex is localized to late endosomes, and this localization is required for EGF-induced ERK1/2 signaling [2–4]. A second MP1-p14-p18 Ragulator complex is required for the recruitment of mTORC1 to the lysosomal surface, and is essential for amino acid-dependent signaling . In addition to these trimeric complexes, MP1 has been reported to bind PAK1 at the plasma membrane, and the MP1-PAK1 interaction is required for MEK phosphorylation by PAK1 in the absence of Raf [6, 7]. Thus, MP1 can regulate the function of several intracellular kinases in different subcellular locations.
Both in vitro and in vivo approaches have been taken to investigate the biological functions of MP1. Transient inhibition of its expression using RNA interference in fibroblasts resulted in decreased Rho activity and delayed cell spreading on fibronectin , and similar knockdown experiments in DU145 prostate cancer cells resulted in decreased migration on fibronectin . The effect on migration was independent of MP1’s ability to activate ERK and PAK1, since the levels of phosphorylated ERK and PAK1 were unchanged upon MP1 knockdown. However, MP1 gene silencing in prostate cancer cells was associated with both decreased expression of paxillin and decreased number and turnover of focal adhesions at the migratory edge. Taken together, these data indicate that one function of MP1 in cell culture is related to cell spreading and migration.
Studies performed in conditional p14 knockout mice and in Drosophila have addressed the in vivo functions of MP1. The endosomal p14-MP1-MEK1 complex is required for cell proliferation in the epidermis during mouse embryogenesis . In Drosophila, the MP1/ERK complex regulates cell differentiation during development of the wing, since both down-regulation and overexpression of dMAPKSP1 led to an ectopic wing vein phenotype . In summary, MP1 is a widely expressed protein that interacts with multiple protein kinases and may impact various cellular processes including proliferation, spreading, migration, and differentiation.
Many of the pathways and processes in which MP1 has been implicated play important roles in cancer biology, including breast cancer. Breast cancer is the most common type of cancer and the second most common cause of death from cancer in women in the United States . A majority of breast tumors express estrogen receptor alpha (ER) and depend on estrogen to grow . There is extensive cross-talk between ER and other cellular signaling pathways, including those in which MP1 functions [12–14]. We therefore hypothesized that MP1 might play an important role in ER-positive breast cancer cells. To test this hypothesis, we analyzed MP1 expression and function in a panel of tumorigenic and non-tumorigenic human mammary epithelial cell lines. Immunoblotting experiments demonstrated that MP1 protein is expressed in both ER-positive and ER-negative breast cancer cell lines, as well as in non-tumorigenic cells. However, the effects of inhibiting MP1 expression by transient transfection with siRNA duplexes differed between different cell types. MP1 gene silencing induced apoptosis of three ER-positive breast cancer cell lines, including one with acquired endocrine resistance. In contrast, no cell death was observed in ER-negative breast cancer or non-tumorigenic cell lines. The apoptosis observed in ER-positive cells was associated with cell detachment, and with decreased ER expression and Akt activity. The cell death phenotype could be partially reversed by overexpressing a constitutively active form of Akt1, suggesting that MP1 plays a novel role in promoting survival of ER-positive breast cancer cells at least in part via the Akt pathway.
MP1 protein expression in human mammary epithelial cells
Since the number of cell lines investigated was limited, we also queried publicly available breast tumor databases for MP1 mRNA expression as described in the methods section. Briefly, eight independent datasets were combined, resulting in a single dataset containing 1459 samples. Batch effects were removed using the BFRM algorithm, and MP1 expression levels were examined. In agreement with the protein results, MP1 mRNA was expressed in both ER positive and negative tumors, but showed a statistically significant elevation in samples that were scored either ER+/PR+ or ER+/PR- by immunohistochemistry (p < 0.001 by t-test for both). Since not all of the samples in the combined dataset were scored for ER and PR, we predicted ER/PR status in the entire dataset using genomic signatures as previously described , and compared MP1 levels in ER/PR positive vs. negative tumors. This analysis also indicated that MP1 mRNA is expressed in both groups, but is present at higher levels in ER/PR positive tumors (p < 0.001). One clinical study identified MP1 as a gene associated with a poor prognosis signature in sporadic lymph-node negative breast cancer patients , but in our analysis high MP1 expression was not correlated with either time to distant metastasis or disease free survival.
Inhibition of MP1 expression induces cell death and detachment of ER-positive breast cancer cells
Inhibition of MP1 expression does not induce death of ER-negative breast cancer cells or non-tumorigenic cells
Inhibition of MP1 expression results in apoptosis of MCF-7 cells
MP1 silencing reduces ER protein expression and decreases Akt activity but does not impact ERK expression or activity in MCF-7 cells
Differential requirement for PI3K/Akt pathway for survival of MCF-7 and MDA-MB-231 cells
Constitutively active Akt1 partially rescues MP1 siRNA induced apoptosis of MCF-7 cells
The results presented here reveal a novel role for the small scaffold protein MP1 in ER-positive breast cancer cells. Although MP1 is expressed in both ER-positive and ER-negative breast cancer cells, its depletion using RNAi-mediated gene silencing leads to detachment and death of several ER-positive cell lines, including one (LCC9) with acquired estrogen independence and antiestrogen resistance. In contrast, MP1 gene silencing had no detectable effect in three ER-negative breast cancer cell lines or a non-tumorigenic mammary epithelial cell line. Although this is a limited sample, MP1 has also been depleted in rat fibroblasts and human prostate cancer cells, and cell detachment or death was not reported in either case [7, 8]. Therefore, MP1 expression seems to be required for survival in a subset of cell types, including ER-positive breast cancer cells. The mechanism of cell death that occurs as a result of inhibiting MP1 expression in MCF-7 cells was shown to be apoptosis, as demonstrated by decreased Bcl-2 expression, increased PARP cleavage, Annexin V staining, and rescue of the death phenotype by treatment with the pan-caspase inhibitor z-VAD-FMK.
Several interesting questions are raised by these results. One is what pro-survival pathways are affected by loss of MP1 expression in MCF-7 cells. Depletion of MP1 did not result in decreased ERK activation, indicating that its pro-survival functions are not mediated by the ERK pathway. The lack of an effect on ERK activity was somewhat surprising, since MP1 was originally identified as a scaffold protein that increases ERK signaling , but is consistent with results obtained in prostate cancer cells . In contrast, inhibition of MP1 expression resulted in a greater than two fold decrease in Akt phosphorylation. The extent of Akt inhibition may be an underestimate, since by 48 h a majority of cells were dead, and the remaining live cells might represent ones with the lowest extent of MP1 knockdown. Akt plays a known pro-survival function in breast cancer cells, where it relays signals from upstream molecules including integrins, growth factor receptors, PI3K and mTORC1 to downstream molecules such as Bcl-2 and NF-κB [21–26]. In MCF-7 cells, the activity of Akt1 can be modulated by estradiol and IGF [27–29]. The fact that this kinase likely also plays a role in MP1 mediated survival is supported by the fact that expression of a constitutively active Akt1 partially rescued the cell death phenotype observed upon MP1 knockdown. However, the partial effect observed indicates that there are likely to be additional survival pathways involved as well.
Depletion of MP1 in MCF-7 cells also resulted in decreased levels of ERα protein, mRNA (data not shown) and transcriptional activity on ERE-containing promoters. The apoptosis observed is unlikely to be due solely to a loss of ER signaling, since we and others have found that inhibition of ER expression using siRNA does not result in MCF-7 cell death . The fact that LCC9 cells, which are estrogen independent and antiestrogen resistant, die in response to MP1 gene silencing also supports a model in which loss of ER expression is not the sole cause of cell death. Several studies indicate that ER may be implicated in breast cancer cell survival via cross-talk with the PI3K/Akt pathway , or by regulating the activity of NF-κB [32, 33], Bcl-2 [34, 35], or IAP family members . We therefore cannot rule out the possibility that decreased ER expression may contribute in some way to the apoptosis observed in MCF-7 cells.
A second question raised by these results is the molecular basis for the differential requirement for MP1 for survival of ER-positive vs. ER-negative breast cancer cells. One possibility is that activation of pro-survival proteins such as Akt is not dependent on MP1 in ER-negative cells, and a second is that the ER-negative cells are less dependent on these pro-survival signaling pathways. The fact that LY294002 caused a concentration-dependent apoptotic response in MCF-7 cells, but did not affect MDA-MB-231 cells supports the latter hypothesis. This is in agreement with previous reports describing a differential sensitivity to this compound between the two breast cancer cell lines [19, 20].
A final question is whether the cell death that we have observed is related to the previously identified roles of MP1 in cell spreading and motility. Since the phenotype involves cell rounding and detachment, inhibition of MP1expression may disrupt cell adhesion signals, which could then trigger cell death. Preliminary PCR array experiments indicated that inhibiting MP1 expression leads to decreased expression of molecules involved in cell adhesion in MCF-7 cells, including several integrins (data not shown). Immunoblotting analysis indicated a small but reproducible decrease in beta 1 integrin protein levels upon MP1 silencing (Additional file 2: Figure S2). Since integrins can initiate pro-survival signaling , future experiments will investigate if a loss of integrin expression plays a role in the decreased Akt activation and/or apoptosis observed as a result of MP1 knockdown.
This is the first report investigating the role of the small scaffold protein MP1 in breast cancer cells. We have demonstrated that inhibiting MP1 protein expression results in apoptosis in ER-positive breast cancer cells, but not ER-negative breast cancer or non-tumorigenic mammary epithelial cells. Furthermore, MP1 gene silencing led to decreased Akt activity in ER-positive MCF-7 cells, these cells are highly dependent upon the Akt pathway for survival, and expression of a constitutively active form of Akt partially rescued cells from apoptosis. We therefore hypothesize that MP1 is required for pro-survival signaling mediated by Akt, and that it may provide a novel target for the treatment of ER-positive breast cancers, including those (such as LCC9) with acquired endocrine resistance.
Cell lines and culture conditions
MCF-7 and LCC9 cells were obtained from the Lombardi Cancer Center. T47D, ZR-75-1, MDA-MB-231, BT-549, and Sk-Br-3 cells were purchased from the American Type Culture Collection. Cells were maintained in Improved Modified Eagle’s Medium (IMEM) containing phenol red (GIBCO-Invitrogen-Applied Biosystems), supplemented with 5% fetal bovine serum (HyClone), and 100 Units/ml Penicillin/100 μg/ml Streptomycin (Invitrogen) and incubated at 37°C with 5% CO2.
All siRNA transfection reagents were purchased from Dharmacon-Thermo Scientific. Two independent MP1 siRNA duplexes (ON-TARGETplus), a non-targeting siRNA (ON-TARGETplus siCONTROL) and an ER siRNA (ON-TARGETplus) were used. Cells were plated in six-well plates at 105 to 3 x 105 cells per well in FBS containing medium. After 24 h, cells were transfected with 30–150 nM of either control or MP1 siRNA using DharmaFECT 1 transfection reagent. For MP1 siRNA and ER siRNA cotransfection cells were treated with a 30 nM mix of two duplexes. Cells were harvested after 24 or 48 h, then lysed in CelLytic M lysis buffer (Sigma), supplemented with cocktail tablets of protease (Roche - Complete Mini EDTA-free) and phosphatase inhibitors (Roche – PhosSTOP).
Determination of cell death
Cell death was assessed at 48 h post transfection using Trypan blue exclusion assays. Briefly, floating cells were collected, centrifuged, and resuspended in PBS, while attached cells were trypsinized, centrifuged, and resuspended in PBS. For each cell suspension, 18 μl were incubated with 2 μl trypan blue for 15 min and both total number and the number of dead cells were counted with a hemacytometer. The remaining harvested cells were processed for protein determination and immunoblotting.
MCF-7 cells were cultured in six-well plates at 3 x 105 cells per well. The following day they were co-transfected with 0.5 μg of ERE2-tk109-luc and 0.06 μg of pβgal-Basic, using Superfect transfection reagent from Qiagen. After 3 h, the medium was changed to transfection mixes containing either control or MP1 siRNA and cells were incubated overnight. The transfection medium was then replaced with phenol red-free IMEM supplemented with 5% charcoal stripped serum (CSS) for 24 h, then cells were stimulated with 10 nM 17β-estradiol (Sigma) for 8 h. Cells were lysed and assayed for luciferase (Promega) and β-galactosidase (Clonetech) activity as suggested by each manufacturer.
Protein concentrations were determined using the Bradford protein assay (Bio-Rad). Total protein (10–20 μg) was subjected to 4-20% Tris–HCl SDS-PAGE (Bio-Rad), transferred to Immobilon-FL polyvinylidene difluoride membranes (Millipore), blocked with Odyssey Blocking Buffer and then incubated with the appropriate primary antibodies. Alexa Fluor 680 anti-goat and anti-rabbit (Invitrogen) and IRDye 800CW anti-mouse (LI-COR) secondary antibodies were used for two-color detection of proteins. Membranes were scanned and analyzed using the LI-COR Odyssey system.
Antibodies and reagents
The following primary antibodies were used for Western blotting: MP1 (A-19, Santa Cruz), actin (AC-40, SIGMA), estrogen receptor alpha (AB-17, Lab Vision-Thermo Scientific, or F-10, Santa Cruz), PARP (Cell Signaling), p-AKT (T308, Cell Signaling), AKT1 (BDI111, Santa Cruz), ERK (C-16, Santa Cruz), p-ERK (Cell Signaling), Flag M2 (Sigma), or Bcl-2 (BD Biosciences). Pan caspase inhibitor z-VAD-FMK was obtained from BD Biosciences and PI3K inhibitor LY294002 was purchased from Sigma.
Retroviral infection of MCF-7 cells
pBabe-puro (Addgene plasmid 1764) or pBabe-puro-Myr-Flag-AKT1 (Addgene plasmid 15294, ) were transfected into 293GPG packaging cells and retroviral stocks were prepared as previously described . These virus stocks were used to infect MCF-7 cells (1 ml per 10 cm dish), in the presence of polybrene (8 μg/ml), and stable colonies were selected with 0.5 μg/ml puromycin. Both single colonies and pools of 50–100 colonies were selected and propagated. Stable cell lines/pools were routinely maintained in medium supplemented with 0.25 μg/ml puromycin and plated in puromycin-free conditions for siRNA transfections.
Annexin V staining
Forty eight hours post transfection, floating and attached cells were collected, pooled, washed with PBS, and then prepared for annexin V-FITC and propidium iodide staining according to the manufacturer’s instructions (556547, BD Biosciences). Cells resuspended in 500 μl annexin V binding buffer were analyzed by flow cytometry using a FACSVantage SE system (BD Biosciences) and analyzed using the FlowJo software.
Gene expression analysis
To examine gene expression across human breast cancer samples, the following 8 breast cancer datasets were downloaded from GEO: GSE2034, GSE3494, GSE6532, GSE4922, GSE11121, GSE7390, GSE2603 and GSE14020. Data was normalized using RMA in the Affymetrix Expression console. The eight datasets were then combined, and batch effects were removed using the BFRM algorithm (Yuwanita and Andrechek, Unpublished). MP1 expression was examined in the resulting combined dataset within the various clinical parameters associated with the datasets, including ER and PR status, time to distant metastasis, and disease free survival. In addition, ER / PR status was predicted using genomic signatures as previously described , and MP1 expression levels were compared in hormone receptor positive vs. negative cells. In all cases, p values were calculated using an unpaired two tailed t test.
Data are expressed as the mean ± S.D. Experiments were performed three times unless otherwise indicated. Paired evaluations were made for experimental and control conditions within each set of experiments. For comparing groups of cell lines, an unpaired two-tailed evaluation was done. Significance was determined by Student’s t test. Significance level was set at p < 0.05.
The authors would like to thank Dr. Eran Andrechek and Ms. Inez Yuwanita for assistance with the analysis of gene expression datasets. We also thank Ms. Chotirat Rattanasinchai for assistance with experiments. Finally, we thank Dr. Robert Clarke for providing LCC9 cells. This research was supported by IDEA Grant W81XWH-07-1-0500 from the Department of Defense Breast Cancer Research Program, and by the Jean P. Schultz Endowed Oncology Research Fund in the College of Human Medicine.
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