Src promotes estrogen-dependent estrogen receptor α proteolysis in human breast cancer
Isabel Chu, … , Zafar Nawaz, Joyce M. Slingerland
Isabel Chu, … , Zafar Nawaz, Joyce M. Slingerland
Published August 1, 2007
Citation Information: J Clin Invest. 2007;117(8):2205-2215. https://doi.org/10.1172/JCI21739.
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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 2

Src promotes estrogen-dependent ERα degradation.

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Src promotes estrogen-dependent ERα degradation. (A) ERα before and 6 ho...
(A) ERα before and 6 hours after addition of estradiol (Est) with or without the proteasome inhibitor n-acetyl-Leu-Leu-norleucinal (LLnL) to estrogen-depleted MCF-7 cells. Equal loading was confirmed by β-actin. (B) ERα t1/2 was assayed by CHX chase in estrogen-depleted cells and at 2, 4, and 6 hours after addition of estradiol. Graph shows results of densitometric analysis of 3 CHX chase experiments (mean ± SEM). (C) Cells were grown in 0.1% cFBS for 48 hours and then treated with estradiol alone, 5% cFBS plus estradiol, or 5% cFBS alone. ERα and β-actin were assayed 6 hours later. (D) Serum- and estrogen-deprived MCF-7 cells were transferred to 5% FBS plus estradiol with or without added Src inhibitor PP1, and ERα was assayed 6 hours later. (E and F) MCF-7 cells were transfected with PCI-Src Y530F (Src) or empty vector (Mock). After 24 hours, (E) ERα and Src levels were assayed and (F) ERα t1/2 was assayed by CHX chase (mean ± SEM). (G and H) The MCFpINDSrc2 line was estrogen depleted for 72 hours, and Src was induced or not for 24 hours with PA prior to addition of estradiol. (G) Src at time of estradiol addition (left), and ERα and β-actin before and 6 hours after estradiol (Est + or –) was added (right). (H) CHX pulse chase, starting 6 hours after estradiol addition with (+Src) or without (–Src) prior induction of Src by PA.