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First published online March 12, 2004
doi: 10.1242/10.1242/jcs.01025

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p130Cas interacts with estrogen receptor and modulates non-genomic estrogen signaling in breast cancer cells

Sara Cabodi^1,*, Laura Moro^2,*, Germano Baj^3,*, Monica Smeriglio¹, Paola Di Stefano¹, Silvana Gippone³, Nicola Surico³, Lorenzo Silengo^1,4, Emilia Turco¹, Guido Tarone^1,4 and Paola Defilippi^1,4,

¹ Dipartimento di Genetica, Biologia e Biochimica, Università di Torino, Via Santena 5 bis, 10126 Torino, Italy
² Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Università del Piemonte Orientale, V.le Ferrucci 33, 28100 Novara, Italy
³ Dipartimento di Scienze Mediche, Università del Piemonte Orientale, Via Solaroli 17, 28100 Novara, Italy
⁴ Centro Ricerche di Medicina Sperimentale, CeRMS, C. so Bramate 88, 10126 Torino, Italy

View larger version (36K): [in a new window]   Fig. 1. p130Cas transiently associates with the estrogen receptor , c-Src and the p85 subunit of PI 3-kinase (PI3K). T47D cells grown to confluence were made quiescent for rapid estrogen signaling by incubating them in phenol red-free RPMI-1640 medium supplemented with charcoal-treated serum for 24 hours. Cells were then left untreated or treated with 10 nM E2 for the indicated times (in hours) and detergent extracted. Cell lysates were immunoprecipitated with antibody to estrogen receptor . Material co-immunoprecipitated with estrogen receptor were run on an 8% SDS-PAGE gel and immunoblotted with antibodies to p130Cas, c-Src, estrogen receptor and p85 subunit of PI 3-kinase. The data reported here are a representative experiment out of five independent experiments.

View larger version (39K): [in a new window]   Fig. 2. Estrogen-dependent c-Src activation is required for complex formation. (A) T47D cells grown to confluence and made quiescent as indicated (in Fig. 1), were left untreated or treated with 10 nM E2 for the indicated times (in seconds and minutes) and then detergent extracted. Cell extracts were immunoprecipitated with c-Src antibodies and immunoprecipitates subjected to kinase assay. Immunoprecipitates were run on 6% SDS-PAGE gel, dried and exposed for 3 hours at –80°C. Half of the immunoprecipitates was run in a parallel gel, transferred and blotted with antibodies to c-Src. (B) T47D cells in the same conditions as in A were treated with 10 nM estrogen in the presence of 5 µM PP1 for the indicated minutes. Cell lysates were immunoprecipitated with antibodies to estrogen receptor . Material co-immunoprecipitated with estrogen receptor were run on an 8% SDS-PAGE gel and immunoblotted with antibodies to p130Cas, c-Src and estrogen receptor . The data reported here are of a representative experiment out of three separate experiments. (C) T47D were treated for 3 minutes with 10 nM estrogen and cell extracts were immunoprecipitated either with antibodies to estrogen receptor or first with antibodies to Src and then to estrogen receptor . The immunoprecipitates were processed as described in B.

View larger version (38K): [in a new window]   Fig. 3. p130Cas over-expression modulates estrogen-dependent c-Src activation. T47D cells were transiently transfected with empty vector or the p130Cas cDNA by the Lipofectamine 2000 method. 24 hours post-transfection, cells grown to confluence and made quiescent (as indicated in Fig. 1) for an additional 24 hours, were left untreated or treated with 10 nM E2 for the indicated times and then detergent extracted. Cell extracts were immunoprecipitated with c-Src antibodies and immunoprecipitates subjected to kinase assay. Immunoprecipitates were run on a 6% SDS-PAGE gel, dried and exposed at –80°C. Half of the immunoprecipitates was run in a parallel gel, transferred and blotted to antibodies with c-Src and re-blotted with polyclonal antibodies to myc epitope (lower panel). The relative amount of c-Src autophosphorylation was determined by densitometric analysis (on the right) of the autoradiographs. In each case, the autophosphorylation signal was normalized for the corresponding amounts of c-Src immunoprecipitated and expressed as arbitrary units relative to the untransfected and untreated control. The statistical significance of the different values was calculated using Student's t-test (*) (P<0.05). Similar results were obtained in two other experiments.

View larger version (31K): [in a new window]   Fig. 4. p130Cas over-expression modulates estrogen-dependent Erk1/2 MAPK activation. (A) T47D cells were grown to confluence and made quiescent (as indicated in Fig. 1) for 24 hours, left untreated or treated with 10 nM E2 for the indicated times and detergent extracted. Erk1/2 MAPK activation was measured and the relative amount of MAPK activation was calculated by densitometric analysis (on the right) as explained in Fig. 3. (B) T47D cells in the same conditions as in A were treated with 10 nM estrogen in the presence of 5 µM PP1 for the indicated times. (C) Alternatively, T47D cells were transiently transfected either with empty vector or the p130Cas cDNA using Lipofectamine 2000. 24 hours post transfection, cells were made quiescent in phenol red-free RPMI-1640 medium supplemented with charcoal-treated serum for an additional 24 hours, left untreated or treated with 10 nM E2 for the indicated times and detergent extracted. Densitometric analysis (right) was performed as previously described and the statistical significance was calculated using Student's t-test (*) P<0.05. (A-C) Cell extracts were run on 10% SDS-PAGE, transferred to nitrocellulose, blotted with anti-phospho Erk1/2 antibodies (upper panels) and re-blotted with polyclonal antibodies to Erk1/2 (lower panels in A and B) and c-myc (lower panel in C). Similar results were obtained in four independent experiments.

View larger version (37K): [in a new window]   Fig. 5. p130Cas silencing by siRNAs inhibits estrogen-dependent Erk1/2 MAPK activation. T47D cells were transfected with p130Cas siRNAs (+) or with control siRNAs (–) as described in Materials and Methods. 48 hours after transfection cells were stimulated with 10 nM estrogen for different times and detergent extracted. Cell extracts were run on 8% SDS-PAGE and blotted with the indicated antibodies. The same results were obtained in two independent experiments.

View larger version (31K): [in a new window]   Fig. 6. p130Cas modulates estrogen-dependent cyclin D1 expression. (A) T47D cells were transiently transfected with empty vector or the myc-tagged p130Cas cDNA by the Lipofectamine 2000 method. 24 hours post transfection, cells made quiescent (as indicated in Fig. 1) for an additional 24 hours, were left untreated or treated with 10 nM E2 for the indicated times and detergent extracted. Cell extracts were run on 10% SDS-PAGE, transferred to nitrocellulose, blotted with cyclin D1 antibodies (upper panels) and re-blotted with polyclonal antibodies to myc epitope (lower right panel). (B) T47D cells were treated with 10 nM E2 for the indicated times in the presence of either 5 µM PP1 or 25 µM PD98059 and detergent extracted. Cell extracts were run on 10% SDS-PAGE, transferred to nitrocellulose and blotted with anti-cyclin D1 antibodies. (C) T47D were depleted of p130Cas by siRNA transfection as indicated in Fig. 5. After 48 hours, cells were treated with 10 nM estrogen for the indicated times and detergent extracted. Cell extracts were run on 10% SDS-PAGE, transferred to nitrocellulose, blotted with p130Cas antibodies (upper panel) or with cyclin D1 (lower panel). Similar results were obtained in three independent experiments.

View larger version (19K): [in a new window]   Fig. 7. Model of the assembly and function of the ER macromolecular complex. In the absence of hormone, ER does not interact with p85 or p130Cas (1). Estradiol triggers association of ER , c-Src, the p85 subunit of PI 3-kinase (PI3K) and p130Cas in a macromolecular complex (2) and activates the c-Src kinase leading to p130Cas-dependent Erk1/2 phosphorylation (see Fig. 5). Increased amount of cellular p130Cas (3) induces a further activation of the c-Src kinase and of the downstream Erk1/2 MAPK pathways.

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