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. 2016 Sep 20;18(1):92.
doi: 10.1186/s13058-016-0748-7.

WNT4 Mediates Estrogen Receptor Signaling and Endocrine Resistance in Invasive Lobular Carcinoma Cell Lines

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Free PMC article

WNT4 Mediates Estrogen Receptor Signaling and Endocrine Resistance in Invasive Lobular Carcinoma Cell Lines

Matthew J Sikora et al. Breast Cancer Res. .
Free PMC article

Abstract

Background: Invasive lobular carcinoma (ILC) of the breast typically presents with clinical biomarkers consistent with a favorable response to endocrine therapies, and over 90 % of ILC cases express the estrogen receptor (ER). However, a subset of ILC cases may be resistant to endocrine therapies, suggesting that ER biology is unique in ILC. Using ILC cell lines, we previously demonstrated that ER regulates a distinct gene expression program in ILC cells, and we hypothesized that these ER-driven pathways modulate the endocrine response in ILC. One potential novel pathway is via the Wnt ligand WNT4, a critical signaling molecule in mammary gland development regulated by the progesterone receptor.

Methods: The ILC cell lines MDA-MB-134-VI, SUM44PE, and BCK4 were used to assess WNT4 gene expression and regulation, as well as the role of WNT4 in estrogen-regulated proliferation. To assess these mechanisms in the context of endocrine resistance, we developed novel ILC endocrine-resistant long-term estrogen-deprived (ILC-LTED) models. ILC and ILC-LTED cell lines were used to identify upstream regulators and downstream signaling effectors of WNT4 signaling.

Results: ILC cells co-opted WNT4 signaling by placing it under direct ER control. We observed that ER regulation of WNT4 correlated with use of an ER binding site at the WNT4 locus, specifically in ILC cells. Further, WNT4 was required for endocrine response in ILC cells, as WNT4 knockdown blocked estrogen-induced proliferation. ILC-LTED cells remained dependent on WNT4 for proliferation, by either maintaining ER function and WNT4 regulation or uncoupling WNT4 from ER and upregulating WNT4 expression. In the latter case, WNT4 expression was driven by activated nuclear factor kappa-B signaling in ILC-LTED cells. In ILC and ILC-LTED cells, WNT4 led to suppression of CDKN1A/p21, which is critical for ILC cell proliferation. CDKN1A knockdown partially reversed the effects of WNT4 knockdown.

Conclusions: WNT4 drives a novel signaling pathway in ILC cells, with a critical role in estrogen-induced growth that may also mediate endocrine resistance. WNT4 signaling may represent a novel target to modulate endocrine response specifically for patients with ILC.

Keywords: Breast cancer; Endocrine resistance; Endocrine therapy; Estradiol; Estrogen receptor; Lobular carcinoma; Wnt signaling.

Figures

Fig. 1
Fig. 1
WNT4 is necessary for estrogen-induced growth in invasive lobular carcinoma (ILC) cells. a Breast cancer cell lines (BCCLs) were reverse-transfected with 10 nM siWNT4 or siSCR (Scrambled siRNA control) pools. WNT4 expression was assessed by quantitative polymerase chain reaction. Bars represent mean of biological triplicate ± SD. p < 0.05 for each siSCR vs siWNT4 (t test). b BCCLs were transfected as in (a) with increasing concentrations of small interfering RNA (siRNA), and proliferation was assessed 6 days posttransfection. siWNT4-treated cell proliferation was normalized to siSCR of equivalent concentration. *p < 0.01 by analysis of variance (ANOVA) of siRNA effect (siSCR vs siWNT4). c MDA-MB-134-VI (MM134) cells were hormone-deprived and reverse-transfected with siSCR or individual siWNT4 constructs. Cells were then treated with 100 pM 17β-estradiol (E2) or 0.01 % EtOH approximately 16 h posttransfection, and proliferation was assessed at the indicated time posttreatment. *,p < 0.0001 by ANOVA of E2 effect (siSCR without E2 vs with E2). x p = n.s. by ANOVA of E2 effect (siSCR without E2 vs either siWNT4). d Cells were treated as in (c), and proliferation was assessed 6 days posttreatment. *p < 0.05 for condition with E2 siSCR vs siWNT4 (t test). n.s. Not significant. e BCCLs were reverse-transfected with 10 nM siWNT4 or siSCR. The following day (after approximately 16 h), cells were treated with CellTox Green dye and 1 μM ICI 182,780 (fulvestrant; ICI) or staurosporine (STS) as indicated. Increased fluorescence represents accumulation of nonviable cells. Time points represent repeated measures of the same initial cell populations. *p < 0.05 by two-way ANOVA vs control, treatment effect. IDC Invasive ductal carcinoma, 44PE SUM44PE cell line
Fig. 2
Fig. 2
Estrogen regulation of WNT4 correlates with estrogen receptor (ER) binding at the WNT4 estrogen receptor binding site (ERBS). a Breast cancer cell lines (BCCLs) were hormone-deprived and treated in biological triplicate with vehicle (0.2 % EtOH), 1 nM 17β-estradiol (E2), 100 nM progesterone (P4), 1 μM ICI 182,780 (fulvestrant; ICI), or 1 μM RU486 (RU), as indicated. RNA was harvested 24 h posttreatment. Bars represent mean ± SD as fold change vs vehicle control; red error bars indicate analysis of variance (Dunnett’s multiple comparisons test) vs vehicle control (p < 0.05). b Data from (a) were normalized to a pan-average of all samples across BCCLs tested. c BCCLs were hormone-deprived and treated as indicated for 45 minutes. Chromatin immunoprecipitation (ChIP) was performed as described in the Methods section. Red values indicate fold enrichment for E2 vs vehicle in ER ChIP. The data were derived from a single experiment but are representative of two or three experiments. SUM44 SUM44PE cell line, HR Hormone receptor, IDC Invasive ductal carcinoma, IgG Immunoglobulin G, ILC Invasive lobular carcinoma, PR Progesterone receptor
Fig. 3
Fig. 3
Expression and regulation of WNT4 in invasive lobular carcinoma endocrine-resistant long-term estrogen-deprived (ILC-LTED) cells correlates with use of the WNT4 estrogen receptor binding site (ERBS). a Breast cancer cell lines (BCCLs) in their respective standard conditions were treated in biological triplicate with 0.1 % EtOH or 1 μM ICI 182,780 (fulvestrant; ICI). RNA was harvested 24 h posttreatment. Bars represent mean ± SD. *p < 0.05 for vehicle control vs ICI (t test). b BCCLs were treated as in (a) for 60 minutes. Chromatin immunoprecipitation (ChIP) was performed as described in the Methods section. Data derived from single experiment, but are representative of duplicate experiments. “E2 status” denotes the hormone status of the experimental culture medium. +FBS-containing medium; Charcoal-stripped FBS-containing medium. qPCR Quantitative polymerase chain reaction, E2 17β-Estradiol, MM134 MDA-MB-134-VI, SUM44 SUM44PE cell line
Fig. 4
Fig. 4
WNT4 dependence in invasive lobular carcinoma endocrine-resistant long-term estrogen-deprived cells (ILC-LTED) cells is linked to endocrine response context. a Breast cancer cell lines were reverse-transfected with increasing concentrations of siWNT4 or 12.5 nM siSCR, or were mock-transfected (Ctrl). Growth was assessed 6 days posttransfection. siSCR was toxic in 134:L/A and 44:L/B, and these lines were not included in analyses or future small interfering RNA (siRNA) experiments. b Cells were reverse transfected with 35 nM siRNA and treated with increasing concentrations of ICI 182,780 (fulvestrant; ICI) approximately 16 h posttransfection. Growth was assessed 6 days posttreatment. *p < 0.05; bottom of nonlinear regression for siSCR vs siWNT4. 44PE SUM44PE cell line, MM134 MDA-MB-134-VI
Fig. 5
Fig. 5
Activated nuclear factor kB (NF-kB) in invasive lobular carcinoma endocrine-resistant long-term estrogen-deprived (ILC-LTED) cells drives WNT4 expression. a Transcription factor reporter arrays were performed as described in the Methods section. Data are expressed as relative luciferase units (RLU) normalized to RLU from a negative control (luciferase without a response element). *p < 0.05; n.s., not significant (t test). Statistical tests were not corrected for multiple comparisons in this experiment, owing to the hypothesis-generating nature of this semibiased screen. b Breast cancer cell lines (BCCLs) were maintained in their respective standard conditions. Immunoblotting was performed as described in the Methods section. c BCCLs were reverse transfected with 10 nM small interfering (siRNA), and RNA was collected 60 h posttransfection. Bars represent the mean of biological triplicate ± SD. *p < 0.05 by analysis of variance of expression vs siSCR (Dunnett’s multiple comparisons test). d and e BCCLs were reverse-transfected with 10 nM siRNA, and lysates and/or RNA were collected 48 h posttransfection. d Immunoblotting was performed as described in the Methods section. Images are representative of duplicate experiments. e Gene expression data are shown as means of biological triplicate ± SD. p = not significant for siSCR vs siWNT4 (t test). ER Estrogen receptor, 44PE SUM44PE cell line, MM134 MDA-MB-134-VI
Fig. 6
Fig. 6
siWNT4-mediated growth suppression is mediated by increased CDKN1A/p21. a Breast cancer cell lines (BCCLs) were reverse-transfected with 10 nM small interfering RNA (siRNA), and RNA was collected 48 h posttransfection. Gene expression data are shown as the mean of biological triplicate ± SD. p < 0.05 for each SCR vs WNT4 (t test). b Left, SUM44PE (44PE) cells were reverse-transfected with 10 nM siRNA, and lysates were collected 48 h posttransfection. The samples presented were also used in Fig. 5d, and the loading control is replicated for clarity. Data are representative of duplicate experiments. Right, MDA-MB-134-VI (MM134) cells were reverse-transfected with 10 nM siRNA (20 nM total for combinations), and lysates were collected 48 h posttransfection. 1, GE Dharmacon siGENOME control pool 2; 2, Sigma-Aldrich MISSION control pool 1. Reduction in p21 protein levels with scrambled control siRNA was a class effect across all commercial scrambled siRNA pools tested, observed only in MM134 cells. c Left, 44PE cells were reverse-transfected with increasing concentrations of siWNT4 in the presence of 10 nM siSCR or siCDKN1A. Proliferation was assessed 7 days posttransfection. Data are shown as fold change vs siSCR or siCDKN1A control (no siWNT4). p < 0.01 by analysis of variance (ANOVA) for interaction (siSCR vs siCDKN1A on siWNT4 effect). Right, MM134 cells were reverse-transfected with 10 nM siRNA as indicated. Proliferation was assessed 7 days posttransfection. Data are shown as fold change vs mock transfection. *p < 0.05 by ANOVA (Dunnett’s multiple comparisons test) vs siWNT4 alone. n.s. Not significant. d Schematic of WNT4 regulation and signaling in invasive lobular carcinoma (ILC) and ILC endocrine-resistant long-term estrogen-deprived (ILC-LTED) cells. Red stars, WNT4 estrogen receptor binding site. Left, in parental ILC cells, 17β-estradiol (E2) activates the estrogen receptor (ER), which binds at WNT4 and drives WNT4 expression. Center, in 134:LTED, the ER no longer binds at WNT4, and activated nuclear factor kB (NF-kB) drives increased WNT4 expression. Right, In 44:LTED, the ER binds at WNT4 despite the absence of exogenous ligands and, potentially in coordination with Oct-4, maintains weaker WNT4 expression. Bottom, WNT4 initiates a Wnt signaling pathway that is likely β-catenin-independent. This leads to suppression of CDKN1A expression and a decrease in p21 protein, which relieves p21-mediated growth inhibition and permits cell growth.
Fig. 7
Fig. 7
WNT4 expression is increased in hormone receptor-positive and luminal breast tumors. Source data are described in the Methods section. a Left, p value by Mann-Whitney U test for estrogen receptor (ER)-negative vs ER-positive breast tumors. Right, Categories represent PAM50 molecular subtypes. AdjNor Adjacent normal breast tissue. p Values by Tukey’s test for pairwise comparisons; n.s. Not significant (p > 0.05). b and (c) p Values by Mann-Whitney U tests for invasive ductal carcinoma (IDC) vs invasive lobular carcinoma (ILC) in each subset of breast tumors. d WNT4 expression groups were determined on the basis of median expression for all ER-positive breast tumors. p Value represents uncorrected log-rank test for low vs high WNT4 expression. METABRIC Molecular Taxonomy of Breast Cancer International Consortium, TCGA The Cancer Genome Atlas

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