Estrogen receptor α regulates the expression of syndecan-1 in human breast carcinoma cells

in Endocrine-Related Cancer
Correspondence should be addressed to J Levallet:
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Breast cancer (BC) is the primary cause of cancer-related mortality among women. Patients who express the estrogen receptor (ER), which mediates the tumorigenic effects of estrogens, respond to antihormonal therapy. Loss of ER expression or acquired resistance to E2 is associated with aggressive malignant phenotypes, which lead to relapse. These BC subtypes overexpress syndecan-1 (SDC1), a transmembrane heparan sulfate proteoglycan that mediates angiogenesis as well as the proliferation and invasiveness of cancer cells. We showed here that the activation of ER-alpha (ERα) by estrogens induces downregulation of SDC1 expression in ER(+) MCF7 cells but not in T47D cells. Loss of ERα expression, induced by RNA interference or a selective ER downregulator, led to subsequent SDC1 overexpression. E2-dependent downregulation of SDC1 expression required de novo protein synthesis and was antagonized by treatment with BAY 11-7085, an irreversible inhibitor of IκBα phosphorylation, which inhibits the activation of NFκB. Downregulation of SDC1 expression required ERα and activation of IKK, but was independent to downstream transcriptional regulators of NFκB. BAY 11-7085 prevented E2-mediated phosphorylation of ERα on Ser118, increasing its proteasomal degradation, suggesting that IKK stabilized E2-activated ERα, leading to subsequent downregulation of SDC1 expression. Our results showed that sustained ER signaling inhibits SDC1 expression. Such antagonism elucidates the inverse correlation between SDC1 and ER expression in ER(+) BC as well as the overexpression of SDC1 in hormone receptor-negative BC subtypes with the most aggressive phenotypes. These results identify SDC1 as an attractive therapeutic target for BC as well as for other endocrine-associated cancers.

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    Estradiol induces a dose- and time-dependent inhibition of SDC1 expression in MCF7 cells. (A) Expression of SDC1 (and PGR in the insert panel) mRNA in MCF-7 cells cultured in estrogen-deprived medium (MEM-10% FBSdest) and treated for 24 h with increasing concentration of estradiol (10−12–10−8 mol/L) (n = 4). (B) Expression of SDC1 mRNA measured after addition of E2 (10−8 mol/L) for increasing period of time (1–24 h). (C) Immunocytochemical observation of SDC1 expression using goat polyclonal anti-SDC1 (AF2780 from R&D) in cells treated without or with E2 (10−8 mol/L) during 24 h. In same experimental conditions, the content of SDC1 protein was estimated by Western blot (D) (C and D are representative results from three independent experiments). (E) Expression of SDC1 and PGR measured by QRT-PCR in estrogen-deprived medium MEM or in medium containing phenol red and non-charcoal-treated FBS (HD) (n = 3). (F) Immunocytochemical observation of SDC1 expression after 48 h of cell culture in MEM or HD medium (n = 3) (scale bar = 50 µm).

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    E2-dependent regulation of syndecan-1 expression in MCF7, T47D and MDA-MB-231 cells. (A) Relative expressions of SDC1, ESR1, ESR2, GPER and PGR in MCF-7, T47D and MDA-MB-231 (MDA) cells cultured in MEM-10% FBSdest (n = 3). (B) Expression of SDC1 in T47D and MDA-MB-231 cells cultured in MEM-10% FBSdest and treated for 24 h with increasing concentration of estradiol (10−12–10−8 mol/L). (C) MCF7 and T47D cells were incubated for 24 h with E2, P4 or both (10−8 mol/L) and SDC1 mRNA level measured by QRT-PCR (n = 3).

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    ERα mediates ligand-dependent and -independent downregulation of SDC1 expression in MCF7 cells. (A) Expression of SDC1 estimated by QRT-PCR in MCF7 cells cultured in MEM before addition of E2 (10−8 mol/L) for 24 h or specific agonist of ERα (PPT), ERβ (DPN) or GPER (G1) used at 10−8 mol/L. GPER antagonist G15 was added 30 min before E2 stimulation (n = 3). (B) MCF7 cells were incubated for 24 h with E2, tamoxifen (Tam) at 10−6 mol/L or both and SDC1 mRNA level measured by QRT-PCR (n = 3). (C) Immunocytochemical observation of SDC1 after 24 h of treatment with E2 (10−8 mol/L) and Tam (10−6 mol/L) (Representative experiments from n = 3). (D) SDC1 mRNA expression in MCF-7 cells cultured in MEM and treated with E2, ICI (10−8 mol/L) or both for 24 h. (E) Representative Western blot showing ERα and SDC1 protein expression after ICI treatment for increasing period of time. Expression levels were normalized with actin as loading control. (F) Immunocytochemical observation of SDC1 and ERα in MCF-7 treated for 24 h with or without ICI (10−8 mol/L). (G) Expression of SDC1 mRNA estimated by QRT-PCR 24 h after transfection of siRNA (10 nM) followed by an additional incubation 24 h without or with E2 (n = 3–5). (H) ERα and SDC1 expression were estimated by Western blot 72 h after transfection with increasing concentration of siRNA. (I) Immunocytochemical observation of SDC1 and ERα in MCF7 transfected with 10 nM of siNeg or siERα (n = 3) (scale bar = 50 µm).

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    E2-dependent transcriptional regulation of SDC1 expression requires a de novo protein synthesis and is independent to ERK/AKT/JNK pathways. (A) Cycloheximide (CHX at 10 µg/mL) and actinomycin D (ACT at 2 × 10−6 mol/L) or both were first added for 30 min before subsequent E2 treatment (10−8 mol/L). Expression of SDC1 was evaluated by QRT-PCR after 24 h of incubation (n = 3). (B) Cells were treated with ACT for 30 min and stimulated for additional 1, 3 or 6 h with E2. (C) Activation/phosphorylation of ERK, AKT and JNK pathways evaluated by Western blot 20 min after E2 (10−10 mol/L) or EGF (10 ng/mL) treatment (representative experiments). (E) MCF-7 cells were first treated with MEK, AKT and JNK inhibitors (UO126, Ly294,002 and Sp600,125, respectively) for 30 min before treatment for 24 h with E2 (10−10 mol/L) or EGF (10 ng/mL). SDC1 mRNA expression was quantified by QRT-PCR. Data were means ± s.e.m. from four independent experiments (n = 4). ##P < 0.01 compared to untreated control cells.

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    The inhibitor of IKK, Bay-11-7085 reduces the ER-dependent inhibition of SDC1 expression. (A) Cells were first treated for 30 min with the NFΚB inhibitor, BAY 11-7085 (Bay) (10−5 mol/L) and E2 or TNFα (10 ng/mL) was added for additional 24 h before SDC1 mRNA quantification by qRT-PCR. (B) Representative immunocytochemical observation of SDC1 in MCF7 after Bay pre-treatment and E2 or TNF stimulation. (Scale bar = 50 µm). (C) Expression of RELA mRNA 48 h after RelA-silencing (n = 3). (D) MCF7 was transfected with siRelA for 48 h and stimulated with E2, TNFα or both for additional 24 h. SDC1 mRNA expression was measured by QRT-PCR (n = 3). (E) Expression of ESR1 mRNA 48 h after transfection with siESR1 (n = 3). (F) SDC1 mRNA expression was measured in MCF7 transfected 48 h with siESR1 and stimulated with E2, Bay or both for an additional period of 24 h (n = 3). For D and F ##P < 0.01; ###P < 0.001 compared to respective transfected control cells by one-way ANOVA with Tukey multiple comparison tests. *P < 0.05 using Student’s t-test.

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    Bay regulates ESR1 expression and E2-dependent phosphorylation of ERα on Ser118. Expression of ESR1 (A), PGR (B) mRNA in MCF7 pre-treated or not with Bay (30 min) before 24 h of E2 treatment (n = 3). (C) Expression of ESR1 and PGR mRNA in MCF-7 cells treated with E2 for 1–24 h of incubation. (D) ERα content in MCF-7 cells was determined by Western blot 6 h after incubation with Bay or E2 or after pre-treatment with Bay (30 min) before the E2 treatment. Upper panel is a representative experiment and lower panel a densitometric quantification of ERα/Actin ratio (n = 3). (E) Same experiments than in D were performed but with a first treatment of 30 min with 10 mM of the proteasome inhibitor, MG-132. (F) Phosphorylation of ERα on Ser118, Ser167 and Ser104/106 observed by Western blot in MCF7 cells treated for 30 min with Bay or E2 or first treated with Bay for 30 min prior to E2 stimulation (representative experiment). Quantitative analysis of Ser118/ERα and Ser167/ERα ratio (n = 3).


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