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Cell and Tumor Biology |
Departments of 1 Molecular and Cellular Oncology and 2 Pathology, University of Texas M.D. Anderson Cancer Center, Houston, Texas and 3 Department of Genetics, Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, New Orleans, Louisiana
Requests for reprints: Rakesh Kumar, Department of Molecular and Cellular Oncology, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 108, Houston, TX 77030. Phone: 713-745-3558; Fax: 713-745-3792; E-mail: rkumar{at}mdanderson.org or Ratna K. Vadlamudi, Department of Genetics, Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, 533 Bolivar Street, New Orleans, LA 70112; E-mail: rvadla{at}lsuhsc.edu.
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Introduction |
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60% to 70% of breast tumors are ER-positive at presentation (1, 2). Although antiestrogens and selective ER modulators (SERM) are effective in curbing the progression of ER-positive breast tumors to more invasive phenotypes (3), many patients with metastatic breast tumors eventually become resistant to this treatment (4). Several mechanisms have been proposed to explain this resistance to hormonal therapy, including the expression of variant or mutant ER, the ligand-independent activation of ER, the adaptation of tumors to lower concentrations of estrogen, and pharmacologic alterations (4, 5). Emerging data also suggest that ER coregulators play a role in hormonal responsiveness and tumor progression (6, 7). However, the causes of ER coregulator–linked resistance to hormonal therapy and ways to interfere with this phenomenon remain elusive. The answer may be in the fact that, in addition to its well-studied nuclear functions, ER also participates in cytoplasmic and membrane-mediated signaling events (nongenomic signaling; refs. 8, 9). Such nongenomic signaling has been linked to rapid responses to estrogen and generally involves the stimulation of the Src kinase, mitogen-activated protein kinase (MAPK), phosphatidylinositol-3-kinase (PI3K), and protein kinase C pathways in the cytosol (10, 11). Further, ER-activated nongenomic pathways have been shown to modify ER or its coactivators by phosphorylation, resulting in the altered topology of ER and its coregulator proteins and eventually leading to ligand-independent activation or differential responses to SERMs (9, 12).
Other factors implicated in the development and progression of breast cancer are deregulated epidermal growth factor receptor (EGFR) signaling and the constitutive activation of cytosolic pathways (MAPK, PI3K, and AKT; ref. 13). Now, there is also emerging evidence that resistance to endocrine therapies may stem from complex interactions between ER and EGFR signaling components (14). The phosphorylation of ER and its associated coregulatory proteins has been suggested as one such mechanism by which growth factor signaling contributes to hormonal resistance (15). However, very little is known about the molecular mechanisms that lead to EGFR-ER crosstalk and the molecular adaptors that facilitate EGFR-ER signaling crosstalk.
Proline-, glutamic acid–, and leucine-rich protein-1 (PELP1)/modulator of nongenomic activity of estrogen receptor (MNAR) is a novel ER coactivator (16) that plays an important role in the genomic (17) and nongenomic actions of ER (18), and promotes cell proliferation by sensitizing cells to G1-to-S progression (19). PELP1 expression is up-regulated by estradiol (E2)-ER signaling and differentially modulated by SERMs (20). PELP1 is widely deregulated in hormone-responsive cancers, including breast and endometrial cancers (16, 21). Although PELP1 is predominantly localized in the nucleus in hormonally responsive tissues (16, 17), recent studies have suggested that under certain conditions, PELP1/MNAR could be exclusively localized in the cytoplasm of cancer cells (21). However, the functional implications of the localization of PELP1 are unknown. Therefore, in the present study, we investigated the localization and functional consequences of PELP1 in human breast tumor specimens. We found that PELP1 was localized in the cytoplasm only in 58% of PELP1-positive tumors. To mimic this situation to gain further insight into the functional consequences of this event, we generated a novel MCF-7 model cell line to specifically express PELP1 in the cytoplasm. We found that the cytoplasmic localization of PELP1 made these cells hypersensitive to estrogen but resistant to SERMs and promoted tumorigenesis in nude mice. Our findings suggest that the altered localization of the ER coactivator PELP1 might lead to the excessive stimulation of cytosolic signaling pathways, thereby causing hormonal independence and increasing tumorigenic potential of breast cancer cells.
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Materials and Methods |
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and the phosphotyrosine (pY20) were purchased from Lab Vision (Fremont, CA). Anti–T7-epitope antibody was purchased from Novagen (Milwaukee, WI). Antibodies against AKT, phospho-AKT, MAPK, phospho-MAPK, phospho-Src Tyr416, and phospho-ER-Ser167, ER-Ser118 were purchased from Cell Signaling (Beverly, MA). Antibody for Src and specific Src kinase inhibitor PP2 was purchased from Calbiochem (La Jolla, CA). Antibodies for EGFR were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Charcoal-stripped serum (DCC serum), ICI 182,780, and LY294002 were purchased from Sigma. Human samples and tumor array staining. Tissue sample from patients who had undergone routine surgery for breast cancer were snap frozen in liquid nitrogen and stored at –80°C in the M. D. Anderson Breast Core Pathology Laboratory. Paraffin-embedded archival breast tissue specimen array was obtained from the pathology core at University of Texas M. D. Anderson Cancer Center. The utilization of human tissue samples and array used in this study was approved by M. D. Anderson Cancer Center institutional human research committee. All tissue classifications were verified by light microscopic examination of H&E-stained slides by a breast pathologist (A.A. Sahin). For immunohistochemical detection of PELP1, sections were deparaffinized with xylene and rehydrated using graded ethanol. The tumor array section was boiled for 10 minutes in 0.01 mol/L citrate buffer and cooled for 30 minutes at room temperature to expose antigenic epitopes. The tumor array was blocked with 2% normal goat serum in 1% bovine serum albumin and PBS for 30 minutes. The tumor array section was incubated with polyclonal rabbit antihuman-PELP1 antiserum at a dilution of 1:500 and incubated overnight at room temperature. PELP1 antibody was developed in our laboratory, well characterized, and used in immunohistochemistry in earlier published studies (16, 21). The sections were washed thrice with 0.05% Tween in PBS for 10 minutes, incubated with secondary antibody for 1 hour, washed thrice with 0.05% Tween in PBS for 10 minutes, developed with 3,3'-diaminobenzidine-H2O2, and then counterstained with Mayer's hematoxylin. Negative controls were done by replacing the primary antibody with control rabbit IgG or peptide-absorbed PELP1 antibody. The finding that no cells or <10% of cells were immunoreactive was considered as a negative, and the finding that >10% of the cells were immunoreactive was considered positive.
Generation of PELP1 cytoplasmic mutant cells. PELP1 cytoplasmic mutant (PELP1-cyto) lacking nuclear localization signal was generated using a Quick Change kit (Stratagene, La Jolla, CA) by mutating PELP1 amino acid 495KKLK498 to EELE using T7-tagged wild-type PELP1 as a template. The sequence of the primer used for mutagenesis was 5'-CCTAGCGCCCCCGAGGAGCTAGAGCTGGATGTG-3'. MCF-7 cells stably expressing PELP1-cyto were generated by transfecting PELP1-cyto using FuGENE-6 transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN). Stable clones were selected using G418 selection (1 mg/mL).
Tissue and cell extracts, immunoblotting, immunoprecipitation, and kinase assays. Tissue and cell lysates were prepared using Triton X-100 buffer [50 mmol/L Tris-HCl (pH 7.5), 100 mmol/L NaCl, 0.5% Triton X-100, 1x protease inhibitor mixture, 1 mmol/L sodium vanadate] for 15 minutes on ice. The lysates were centrifuged in an Eppendorf centrifuge at 4°C for 15 minutes. Cell lysates containing equal amounts of protein (200 µg) were resolved on SDS-polyacrylamide gels (8% acrylamide), transferred to nitrocellulose membranes, probed with the appropriate antibodies, and developed using either the enhanced chemiluminescence method or the alkaline phosphatase–based color reaction method. Immunoprecipitation was done for 2 hours at 4°C using 1 µg of antibody per milligram of protein. For PI3K assay, cell lysates (1 mg) were immunoprecipitated with antiphosphotyrosine monoclonal antibody (mAb) PY20 (Labvision, Fremont, CA) and subjected to in vitro kinase reaction in 50 µL of kinase buffer containing 0.2 mg/mL phosphatidylinositol (Sigma) and 20 µCi of [
-32P]ATP and 20 mmol/L MgCl2. The reaction products were separated on TLC plates using chloroform/methanol/ammonium hydroxide/water (87:76:10:14) buffer and visualized by autoradiography.
Cell proliferation, soft agar, and tumorigenesis assays. For the cell proliferation assays, cells were grown in phenol red–free medium supplemented with 5% DCC serum for 48 hours, and then estrogen or tamoxifen was added. The proliferation rate of the cells was measured by counting them in a Beckman Coulter Counter as previously described (19). Soft-agar colony-growth assays were done as previously described (22). Briefly, 1 mL of 0.6% Difco agar in DMEM supplemented with 5% DCC serum and insulin was layered onto tissue culture plates. Test cells (1 x 104) mixed with 1 mL of 0.36% bactoagar solution in DMEM were layered on top of the 0.6% bactoagar layer. The plates were incubated at 37°C in 5% CO2 for 21 days. For tumorigenesis studies, 5 x 107 cells were implanted s.c. into the mammary fat pads of eight nude mice as previously described (23) and allowed to grow for 8 weeks. Tumor size was then measured.
Reporter gene assay. For reporter gene transient transfections, COS-1 cells were cultured for 48 hours in minimal essential medium without phenol red–containing 5% DCC serum. Estrogen response element (ERE)-luciferase reporter constructs were cotransfected with ER in the presence or absence of pcDNA vector, wild-type PELP1 (PELP1-WT), or PELP1-cyto expression plasmids using FuGENE-6 according to the instructions of the manufacturer. Twenty-four hours later, cells were treated with EGF for 12 hours. Cells were then lysed with passive lysis buffer, and the luciferase assay was done using a luciferase reporter assay kit (Promega, Madison, WI). The total amount of DNA used in the transfections was kept constant by adding a parental vector. Each transfection was carried out in six-well plates in triplicate wells.
Immunofluorescence and confocal microscopy. The cellular localization of PELP1 was determined using indirect immunofluorescence as previously described (17).
Statistical analysis. Statistical analysis was done using Student's t test and values with P < 0.05 were considered statistically significant.
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Cytoplasmic retention enhances PELP1 interactions with Src kinase. Recent studies have shown that in addition to its suggested nuclear functions (17), PELP1 participates in nongenomic signaling activities, such as activation of Src (18, 24). Therefore, we examined whether the localization of PELP1 to the cytoplasmic compartment, a physiologic situation existing in a subset of breast tumors, promotes PELP1 interaction with Src. Results from confocal microscopy showed that a substantial amount of PELP1 was localized in the nuclear compartment of PELP1-WT–expressing cells and that E2 treatment stimulated colocalization of PELP1 with Src at the membrane in some cells (Fig. 2A, top). In contrast, in PELP1-cyto–expressing cells, E2 treatment promoted substantial colocalization of PELP1-cyto with Src both at the membrane and in the cytoplasm (Fig. 2A, bottom). Another study found that PELP1 interaction with Src promotes Src activation (24); this raised the possibility that the cytoplasmic localization of PELP1 might provide an opportunity for PELP1 to interact with Src, thereby leading to enhanced Src kinase activation. To test whether PELP1-cyto interacts with Src kinase, immunoprecipitation was done by using total cellular lysates from pcDNA- and PELP1-cyto–expressing cells. Results showed that PELP1-cyto interacts with Src kinase (Fig. 2B, left). To confirm that overexpression of PELP1-cyto activates Src kinase, we did Western blotting with a phosphospecific antibody (phospho-Src Tyr416) that recognizes activated Src kinase. This showed that PELP1-cyto–expressing cells increased the activation of Src kinase compared with pcDNA-expressing clones (Fig. 2B, right). These findings suggest that PELP1 cytolocalization promotes PELP1 interaction with Src kinase. Because PELP1-WT functions as a scaffolding protein for the coupling of ER with Src kinase, we then investigated whether PELP1-cyto also interacts with ER. The immunoprecipitation of PELP1-cyto showed that PELP1-cyto could interact with ER (Fig. 2C).
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Cytoplasmic retention of PELP1 promotes constitutive activation of the AKT pathway. Because the activation of PI3K is another recognized nongenomic signaling activator of ER (13), we examined the functionality of PI3K signaling in PELP1-cyto clones by using phospho-AKT as a downstream marker of this pathway. Interestingly, PELP1-cyto clones showed excessive constitutive activation of the AKT pathway that could not be further induced by E2 treatment (Fig. 3A). Because the AKT pathway has also been shown to promote tamoxifen resistance (25), we examined the status of the AKT pathway in PELP1-cyto clones after treating the clones with tamoxifen. PELP1-cyto–expressing cells showed increased AKT activity compared with pcDNA-expressing clones (Fig. 3B), and the status of already elevated levels of AKT in PELP1-cyto clones was not affected by tamoxifen. Our finding that PELP1-cyto clones exhibit significant constitutive stimulation of the AKT pathway even in the absence of E2 treatment raised the possibility that PELP1 activation of the AKT pathway might be independent of the ability of PELP1 to interact with ER. To examine this possibility, we treated pcDNA and PELP1-cyto clones with the antiestrogen ICI 182,780 for 3 days to down regulate ER expression and activity. The ICI 182,780 did not affect the levels of constitutive AKT stimulation observed in the PELP1-cyto clones (Fig. 3C), suggesting that the increased activation seen in the PELP1-cyto clones is independent of ER. We then examined the possibility that increased AKT was due to the activation of PI3K, an upstream activator of AKT. Treatment of PELP1-cyto clones with LY294002, a widely used inhibitor of PI3K, blocked the constitutive activation of AKT (Fig. 3D). These findings suggested that the cytoplasmic localization of PELP1 might activate PI3K, thereby contributing to enhanced AKT signaling.
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PELP1 associates with epidermal growth factor receptor. Previous studies have suggested bidirectional signaling between ER and EGFR pathways, and growth factors promote ER phosphorylation through the PI3K-AKT pathway (13). Because we observed that PELP1 interacts with PI3K, whose activity is modulated by growth factor signaling, we examined whether PELP1 interacts with growth factor receptors as well. To determine whether there is physiologic evidence of endogenous PELP1-EGFR interactions, we initially examined this in HeLa cells, which abundantly express EGFR and PELP1. HeLa cells were treated with EGF, and the colocalization of PELP1 and EGFR was analyzed by confocal microscopy. Our results showed that EGF stimulation caused a small portion of PELP1 to associate with membranous regions, and, in some areas, the colocalization of PELP1 with EGFR was observed (Fig. 4A, left). We next examined whether PELP1-cyto also interacts with EGFR. Confocal microscopy examination of EGF-treated PELP1-cyto clones revealed colocalization of PELP1-cyto with EGFR (Fig. 4A, right). To confirm these results, we did immunoprecipitation experiments in COS-1 cells, which were treated with or without EGF. Immunoprecipitation of EGFR followed by Western blot analysis with PELP1 confirmed that PELP1 indeed associates with EGFR upon EGF stimulation (Fig. 4B).
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Growth factor signaling has been shown to modulate coactivator functions by posttranslation modifications (14). Because PELP1 interacted with EGFR and promoted EGF-mediated ERE transactivation functions, we next examined whether EGF stimulation promotes the phosphorylation of PELP1. COS-1 cells were transfected with T7-tagged PELP1, and after 48 hours cells were treated with or without EGF. Total lysates were immunoprecipitated with T7-epitope antibody (Fig. 4D, left), and immunoprecipitates were blotted with T7 and phosphotyrosine antibodies. Results showed that EGF promotes tyrosine phosphorylation of PELP1. Similarly, immunoprecipitation of PELP1-cyto from PELP1 stable clones also showed that EGF promotes tyrosine phosphorylation of PELP1-cyto (Fig. 4D, right).
Because PELP1 interacts with ER and promotes E2-mediated nongenomic signaling pathways, we examined whether E2 can promote the tyrosine phosphorylation of PELP1. MCF-7 cells stably expressing PELP1-WT were treated with or without E2. Immunoprecipitation experiments showed that E2 also promoted the tyrosine phosphorylation of PELP1 (Fig. 4E). We confirmed these results with confocal microscopic analysis. As shown by phosphotyrosine antibody staining, E2 promoted the membrane localization of PELP1 and its colocalization with phosphotyrosine (Fig. 4F). These results suggest that both EGF and E2 signaling modulate PELP1 functions and that the cytoplasmic retention of PELP1 excessively promotes the interactions of PELP1 with components of the growth factor signaling axis.
PELP1 cytoplasm retention promotes hormonal independence. To examine the effect of the cytoplasmic localization of PELP1 on biological functions, we did cell proliferation and anchorage independence assays. Results showed that PELP1-cyto–expressing clones were hypersensitive to E2 compared with parental MCF-7– or pcDNA-vector–expressing clones (Fig. 5A). Further, PELP1-cyto clones showed an increased ability to form colonies in an anchorage-independent manner compared with pcDNA-expressing cells (Fig. 5B). Because AKT activation has been shown to promote tamoxifen partial agonist action in breast cancer cells, we repeated these biological assays in the presence or absence of tamoxifen for 7 days. MCF-7 parental and pcDNA-expressing clones showed a 50% reduction in cell number. Interestingly, PELP1-cyto–expressing clones showed resistance to tamoxifen in that the cell number was not altered (Fig. 5C). In anchorage-independence assays, PELP1-cyto–expressing cells exhibited an increased ability to form colonies in the presence of tamoxifen compared with parental or pcDNA clones (Fig. 5D). Consistent with these results, PELP1-cyto clones exhibited tumorigenic potential in nude mice, and these tumors also exhibited constitutive activation of the AKT pathway (Fig. 5E). These findings suggest that a close relationship exists between PELP1 cytoplasmic localization, AKT activation, and tumorigenesis.
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Discussion |
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The altered localization of the ER coactivator PELP1, which has the potential to stimulate nongenomic ER functions (18, 24), may alter the ratio of genomic and nongenomic signaling in breast cancer cells and thus might promote hormonal independence by modulating ER-transactivating functions and SERM actions. As our study and other studies indicate, PELP1 expression is deregulated in breast and endometrial cancers (16, 21); therefore, the altered localization of PELP1 is expected to contribute toward the excessive activation of the MAPK and PI3K-AKT pathways, leading to follow-up modifications of ER. Such modifications of the ER pathway may lead to the activation of ER target genes in a ligand-independent manner. Alternatively, the modified ER may allow the recruitment of a different set of coregulator proteins, and thus may exhibit resistance to hormonal therapy. In our study, the cytoplasmic localization of PELP1 promoted increased activation of MAPK, leading to enhanced ER-Ser118 phosphorylation. Similarly, these clones also exhibited constitutive activation of the AKT pathway, which was not further induced by E2. Therefore, the enhanced E2-mediated growth and anchorage independence seen in these clones is likely to be caused by excessive activation of the MAPK pathway, whereas tamoxifen-mediated resistance could be due to the cumulative effect of the activation of the MAPK and AKT pathways, which leads to the modification of ER and its associated proteins as well as the activation of downstream pathways by activated MAPK and AKT.
ER coregulatory proteins have been suggested to play a role in the generally observed tissue-specific effects of tamoxifen (28, 29). However, these ER coregulators are targeted by excessive ER-HER2 crosstalk leading to hormonal resistance in a subset of breast tumors (15). High levels of the ER coactivator AIB1 and HER2 in breast cancer contribute to tamoxifen resistance (30). Our findings that PELP1 interacts with EGFR and that PELP1 has the ability to enhance EGF-mediated ER transactivation functions suggest that PELP1 potentiates growth factor receptor–mediated hormonal resistance in a subset of tumors that have deregulated growth factor signaling.
Growth factor signaling promotes ER phosphorylation (both serine and tyrosine; refs. 14, 31). Recent evidence suggests that ER coactivators, in addition to ER, are targets of growth factor signaling (32). Growth factor–mediated activation of nongenomic pathways and phosphorylation of ER and ER-coregulatory proteins have been shown to have a role in tamoxifen resistance (33, 34). Our findings that PELP1 enhances tamoxifen resistance and that EGF promotes the phosphorylation of PELP1 suggest a possibility that growth factor–mediated posttranslational modification of PELP1 may play an important role in PELP1-mediated hormonal resistance functions.
Activation of the PI3K-AKT pathway has been shown to be an essential step in the estrogenic action of growth factors (35). Previous studies also showed that forced expression of constitutively active AKT in MCF-7 cells promotes E2-independent growth as well as tamoxifen response (25). A recent study found that overexpression of the ER coactivator AIB1 promoted high tumor incidence, which is associated with the activation of the PI3K-AKT pathway (36). Our findings suggest that cytoplasmic localization of PELP1 plays a role in tamoxifen resistance and that the ability of PELP1 to modulate the PI3K-AKT pathways may represent one mechanism by which PELP1-cyto cells develop resistance to hormonal treatment. Further, our analysis of human breast tissues provided the proof-of-principle that PELP1 localization is altered in a subset of human breast tumors.
Our results suggest that the cytoplasmic localization of PELP1 may play a role in the constitutive activation of AKT in tumor cells. Our findings also suggest the existence of a close relationship between cytoplasmic PELP1 localization and increased nongenomic signaling. Additionally, our findings suggest that the cytoplasmic localization of PELP1 might be sufficient to promote tumorigenic phenotypes and hormonal independence. Further, the ability of PELP1-cyto cells to form tumors in nude mice and the presence of increased AKT signaling in these tumors also supports the potential role of PELP1 localization in tumorigenesis. Because these tumors were formed in the absence of estrogen pellet implantation, PELP1-mediated constitutive activation of the AKT pathway rather than the MAPK pathway might play a role in PELP1-cyto–mediated in vivo tumorigenic functions. It will be interesting to discover the mechanisms by which PELP1 localization is altered in pathologic conditions. Because cytoplasmic PELP1 observed in the tumors migrated in a similar fashion as PELP-WT, it is likely that it represents a full-length protein and that its deregulation in localization could be because of posttranslational modifications or mutations that affect its localization to the nuclear compartment. Our future studies will be directed at identifying the mechanisms of PELP1 deregulation, including possible posttranslational modifications/mutations in tumors.
In summary, our results suggest that the localization of ER coactivators, such as PELP1, could activate nongenomic signals and may play an important role in the hormonal responses of ER-positive cancer cells (Fig. 5F). Furthermore, the ability of PELP1 to activate MAPK and AKT signaling pathways, its potential to enhance tamoxifen resistance, and its distinct localization in a subset of breast tumors suggest that PELP1 plays a role in the biology of a subset of breast cancers and that its deregulation might contribute to hormonal independence.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 2/22/05. Revised 5/20/05. Accepted 6/27/05.
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after PKA activation in breast cancer. Cancer Cell 2004;5:597–605.[CrossRef][Medline]
. J Biol Chem 2001;276:38272–9.This article has been cited by other articles:
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