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Link to original content: https://pubmed.ncbi.nlm.nih.gov/20460518
Extranuclear functions of ER impact invasive migration and metastasis by breast cancer cells - PubMed Skip to main page content
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. 2010 May 15;70(10):4092-101.
doi: 10.1158/0008-5472.CAN-09-3834. Epub 2010 May 11.

Extranuclear functions of ER impact invasive migration and metastasis by breast cancer cells

Dimple Chakravarty  1 , Sujit S Nair, Bindu Santhamma, Binoj C Nair, Long Wang, Abhik Bandyopadhyay, Joseph K Agyin, Darrell Brann, Lu-Zhe Sun, I-Tien Yeh, Francis Y Lee, Rajeshwar Rao Tekmal, Rakesh Kumar, Ratna K Vadlamudi
Affiliations

Extranuclear functions of ER impact invasive migration and metastasis by breast cancer cells

Dimple Chakravarty et al. Cancer Res. .

Abstract

The molecular basis of breast cancer progression to metastasis and the role of estrogen receptor (ER) signaling in this process remain poorly understood. Emerging evidence suggests that ER participates in extranuclear signaling in addition to genomic functions. Recent studies identified proline-, glutamic acid-, and leucine-rich protein-1 (PELP1) as one of the components of ER signalosome in the cytoplasm. PELP1 expression is deregulated in metastatic breast tumors. We examined the mechanism and significance of ER-PELP1-mediated extranuclear signals in the cytoskeletal remodeling and metastasis. Using estrogen dendrimer conjugate (EDC) that uniquely activate ER extranuclear signaling and by using model cells that stably express PELP1 short hairpin RNA (shRNA), we show that PELP1 is required for optimal activation of ER extranuclear actions. Using a yeast two-hybrid screen, we identified integrin-linked kinase 1 (ILK1) as a novel PELP1-binding protein. Activation of extranuclear signaling by EDC uniquely enhanced E2-mediated ruffles and filopodia-like structures. Using dominant-negative and dominant-active reagents, we found that estrogen-mediated extranuclear signaling promotes cytoskeleton reorganization through the ER-Src-PELP1-phosphoinositide 3-kinase-ILK1 pathway. Using in vitro Boyden chamber assays and in vivo xenograft assays, we found that ER extranuclear actions contribute to cell migration. Collectively, our results suggest that ER extranuclear actions play a role in cell motility/metastasis, establishing for the first time that endogenous PELP1 serves as a critical component of ER extranuclear actions leading to cell motility/invasion and that the ER-Src-PELP1-ILK1 pathway represents a novel therapeutic target for preventing the emergence of ER-positive metastasis.

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Figures

Figure 1
Figure 1
Activation of ER-extranuclear signaling promotes actin reorganization. A, MCF7 control or MCF7-PELP1-shRNA cells were lysed and expression of PELP1 was analyzed by Western blotting. B, MCF7 vector control and MCF7-PELP1-shRNA cells were cultured in 5% DCC serum containing medium treated with or without E2. The activation of signaling pathways was analyzed by Western blotting of total protein lysates with phospho-specific antibodies. Densitometric analysis of the western blots of phospho bands from triplicate samples were performed and corrected with the values of respective total bands. Each column is an average of triple determinations. Bars, SEM. *, p<0.05. **, P<0.001. C, MCF7 cells were treated with FITC-labeled EDC for 45 min and localization of EDC was analyzed by confocal microscopy (left panel). MCF7 and MCF7-PELP1-shRNA cells were treated with EDC and activation of signaling pathways was analyzed by Western blotting (C, middle panel). Quantitation of the bands was as described in Fig. B legend (C, right panel). D, MCF7 or MCF7-PELP1-shRNA cells were treated either with E2 or EDC and the F-actin status was analyzed by phalloidin staining and visualized by confocal microscopy.
Figure 2
Figure 2
Src kinase is needed for optimal activation of PELP1-mediated E2 extranuclear actions. A, Schematic representation of PELP1 mutant that cannot bind or be phosphorylated by Src kinase. B, MCF7 cells were transfected with PELP1WT or PELP1SrcMT and treated with or without E2. The ability of the expressed proteins to interact with Src kinase was analyzed by immunoprecipitation (left panel). MCF7 control and MCF7 cells were transiently transfected with either PELP1WT or PELP1SrcMT were treated with EDC for 5 min. The status of F-actin was analyzed by confocal microscopy (right panel). C, MCF7 cells were transfected with PELP1WT or PELP1SrcMT using the Amaxa nucleofection method and treated with or without E2. Activation of extranuclear signaling was measured by Western blotting. D, MCF7 or MCF7 cells that stably expressed PELP1 were treated with or without dasatinib and with or without E2. Activation of extranuclear signaling was measured by Western blotting.
Figure 3
Figure 3
Integrin linked kinase 1 (ILK1) is a novel PELP1 binding protein A, Confirmation of PELP1 interaction with ILK1 is shown in a yeast-based growth assay (left panel). Identification of the domain of interaction between PELP1 and ILK1 using a yeast-based growth assay (right panel). B, MCF7 cells that express T7-tagged PELP1WT (left panel) or T7-PELPcyto mutant (right panel) were treated with EDC and the PELP1 and ILK1 interaction was confirmed by immunoprecipitation assay. C, MCF7 cells were treated with or without EDC for 5 min and the colocalization of PELP1 and ILK1 were analyzed by confocal microscopy. D, MCF7- T7-PELPcyto cells were treated with or without EDC and total protein lysates were immunoprecipitated with T7-tagged antibody. The presence of PELP1, Src, ILK1, ER, and p85 in the immunoprecipitates was analyzed by Western blotting.
Figure 4
Figure 4
PELP1–ILK1 axis plays a productive role in E2-mediated cytoskeleton reorganization. Only one representative image for each experimental condition is shown and the results are representative of three independent replicates. A, MCF7 breast cancer cells were transfected with dominant negative (DN)-ILK1 (red). After 72 h, the cells were treated with EDC for 10 min and F-actin (green) status was analyzed by confocal microscopy (A, top panel). MCF7-PELP1 shRNA cells were transiently transfected with dominant active (DA)-ILK1. F-actin status was verified by confocal microscopy (A, Bottom panel). B, MCF7-PELP1 shRNA cells were transfected with constitutively active RFP-P110α subunit of PI3K (red) without (B, top panel) or with DN-ILK1 (B, bottom panel, blue color) and F-actin (green) changes were analyzed by confocal microscopy. C, MCF7-PELP1 shRNA cells were transiently transfected with DA-CDC42 (green) without (C, top panels) or with DN-ILK1 (C, bottom panels, blue) and F-actin status was verified by confocal microscopy. D, The ability of PELP1 to enhance ILK1 activity was measured by an in vitro kinase assay by incubating immunoprecipitated GFP-ILK1 with increasing of amounts of GST-PELP1 (D, left panel). MCF7 cells were transfected with ILK1 expression vector with or without PELP1 expression vector and the ability of PELP1 to enhance ILK1 downstream signaling was analyzed by Western blotting (D, left panel). MCF7 and MCF7-PELP1 shRNA cells were treated with or without EDC. ILK1 was immunoprecipitated and kinase activity was measured using an in vitro kinase assay (D, middle panel). Cells were transfected with ILK1 expression vector with or without PELP1 expression vector. After 72 hours, cells were treated with PI3K inhibitor (LY294002, 50 uM) or Src kinase inhibitor (dasatinib, 100 nM). ILK1 was immunopreciptated and the ILK1 activity was measured by an in vitro kinase assay (D, right panel).
Figure 5
Figure 5
E2-mediated extranuclear actions promote cell migration and metastasis. A, MCF7 cells, MCF7 cells transfected with dominant negative ILK1 (DN-ILK1), and MCF7 cells stably transfected with PELP1-shRNA were treated with or without EDC and the migratory potential was analyzed by using Boyden chamber assay. Photomicrographs of migrated cells in various treatments (right panel). Data shown are the means of ± SEM from three independent experiments performed in triplicate wells. **, p <0.001. B, MCF7 cells were treated with EDC in the presence or absence of Src inhibitor dasatinib (100 nM). The cell migratory potential was analyzed by using Boyden chamber assay. Data shown are the means of ± SEM from three independent experiments performed in triplicate wells. *, p <0.05. C, Wound healing assay was performed in the presence or absence of E2 and in presence or absence of dasatinib. D, ZR75 cells expressing GFPvector or GFP-PELP1WT were injected into nude mice either via tail vein (D, Left panel) or cardiac route (D, middle panel) and metastases were recorded after 8 weeks. MCF7 cells expressing control GFP-vector or GFP+PELP1cyto were injected into nude mice (n=5) via tail vein (D, right panel). Representative images of metastatic nodules as observed by fluorescence microscope are shown. The average number of tumor nodules is shown in the graph. Bars, SEM, **, P<0.0001.
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