Src promotes estrogen-dependent estrogen receptor α proteolysis in human breast cancer
Isabel Chu, Angel Arnaout, Sophie Loiseau, Jun Sun, Arun Seth, Chris McMahon, Kathy Chun, Bryan Hennessy, Gordon B. Mills, Zafar Nawaz, Joyce M. Slingerland
Isabel Chu, Angel Arnaout, Sophie Loiseau, Jun Sun, Arun Seth, Chris McMahon, Kathy Chun, Bryan Hennessy, Gordon B. Mills, Zafar Nawaz, Joyce M. Slingerland
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Research Article Endocrinology

Src promotes estrogen-dependent estrogen receptor α proteolysis in human breast cancer

Abstract

Estrogen drives both transcriptional activation and proteolysis of estrogen receptor α (ERα; encoded by ESR1). Here we observed variable and overlapping ESR1 mRNA levels in 200 ERα-negative and 50 ERα-positive primary breast cancers examined, which suggests important posttranscriptional ERα regulation. Our results indicate that Src cooperates with estrogen to activate ERα proteolysis. Inducible Src stimulated ligand-activated ERα transcriptional activity and reduced ERα t1/2. Src and ERα levels were inversely correlated in primary breast cancers. ERα-negative primary breast cancers and cell lines showed increased Src levels and/or activity compared with ERα-positive cancers and cells. ERα t1/2 was reduced in ERα-negative cell lines. In both ERα-positive and -negative cell lines, both proteasome and Src inhibitors increased ERα levels. Src inhibition impaired ligand-activated ERα ubiquitylation and increased ERα levels. Src siRNA impaired ligand-activated ERα loss in BT-20 cells. Pretreatment with Src increased ERα ubiquitylation and degradation in vitro. These findings provide what we believe to be a novel link between Src activation and ERα proteolysis and support a model whereby crosstalk between liganded ERα and Src drives ERα transcriptional activity and targets ERα for ubiquitin-dependent proteolysis. Oncogenic Src activation may promote not only proliferation, but also estrogen-activated ERα loss in a subset of ERα-negative breast cancers, altering prognosis and response to therapy.

Authors

Isabel Chu, Angel Arnaout, Sophie Loiseau, Jun Sun, Arun Seth, Chris McMahon, Kathy Chun, Bryan Hennessy, Gordon B. Mills, Zafar Nawaz, Joyce M. Slingerland

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Figure 5

Src stimulates ERα ubiquitylation and degradation in vivo and in vitro.

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Src stimulates ERα ubiquitylation and degradation in vivo and in vitro. ...
(A) Serum- and estrogen-deprived MCF-7 cells were treated with estradiol and 5% cFBS for 6 hours with or without immediate prior addition of MG132 or PP1, and ERα levels were assayed. Equal loading was confirmed by β-actin. ERα was precipitated, ERα complexes were resolved, and ubiquitylated ERα (Ub-ERα) was detected with anti-ubiquitin antibody. (B) For in vitro ERα ubiquitylation, recombinant ERα protein was reacted with E1 and E2, with or without E3 and with or without prior treatment of ERα with Src kinase (as described in Methods), for 60 minutes. ERα immunoprecipitates were resolved and blotted with anti-ubiquitin or anti-phosphotyrosine (pY) antibodies. The membrane was stripped and reprobed for ERα. (C) In vitro degradation of recombinant ERα used E1, E2, and E3 with or without prior incubation with Src and/or addition of 26S proteasome fraction as described in Methods.