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. 2018 Sep 4;20(1):106.
doi: 10.1186/s13058-018-1041-8.

Key Regulators of Lipid Metabolism Drive Endocrine Resistance in Invasive Lobular Breast Cancer

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Free PMC article

Key Regulators of Lipid Metabolism Drive Endocrine Resistance in Invasive Lobular Breast Cancer

Tian Du et al. Breast Cancer Res. .
Free PMC article

Abstract

Background: Invasive lobular breast carcinoma (ILC) is a histological subtype of breast cancer that is characterized by loss of E-cadherin and high expression of estrogen receptor alpha (ERα). In many cases, ILC is effectively treated with adjuvant aromatase inhibitors (AIs); however, acquired AI resistance remains a significant problem.

Methods: To identify underlying mechanisms of acquired anti-estrogen resistance in ILC, we recently developed six long-term estrogen-deprived (LTED) variant cell lines from the human ILC cell lines SUM44PE (SUM44; two lines) and MDA-MB-134VI (MM134; four lines). To better understand mechanisms of AI resistance in these models, we performed transcriptional profiling analysis by RNA-sequencing followed by candidate gene expression and functional studies.

Results: MM134 LTED cells expressed ER at a decreased level and lost growth response to estradiol, while SUM44 LTED cells retained partial ER activity. Our transcriptional profiling analysis identified shared activation of lipid metabolism across all six independent models. However, the underlying basis of this signature was distinct between models. Oxysterols were able to promote the proliferation of SUM44 LTED cells but not MM134 LTED cells. In contrast, MM134 LTED cells displayed a high expression of the sterol regulatory element-binding protein 1 (SREBP1), a regulator of fatty acid and cholesterol synthesis, and were hypersensitive to genetic or pharmacological inhibition of SREBPs. Several SREBP1 downstream targets involved in fatty acid synthesis, including FASN, were induced, and MM134 LTED cells were more sensitive to etomoxir, an inhibitor of the rate-limiting enzyme in beta-oxidation, than their respective parental control cells. Finally, in silico expression analysis in clinical specimens from a neo-adjuvant endocrine trial showed a significant association between the increase of SREBP1 expression and lack of clinical response, providing further support for a role of SREBP1 in the acquisition of endocrine resistance in breast cancer.

Conclusions: Our characterization of a unique series of AI-resistant ILC models identifies the activation of key regulators of fatty acid and cholesterol metabolism, implicating lipid-metabolic processes driving estrogen-independent growth of ILC cells. Targeting these changes may prove a strategy for prevention and treatment of endocrine resistance for patients with ILC.

Keywords: Cholesterol; Endocrine resistance; Fatty acid; Invasive lobular breast; LTED; SREBP1.

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Figures

Fig. 1
Fig. 1
MM134 and SUM44 long-term estrogen deprivation (LTED) cells have different estrogen receptor (ER) activity. a Growth curve of LTED and their parental cells. Parental cells were cultured in either their normal growth media (FBS, 1:1 DMEM:L-15 + 10% FBS) or the hormone-deprived media (CSS, IMEM + 10% CSS). Parental cells in the CSS group were hormone-deprived before seeding. “(FBS)” and “(CSS)” were used to represent the normal growth media and hormone-deprived media, respectively, throughout the article. SUM44F served as the parental cell line for SUM44 LTED cells in the article. Plots are representative of three independent experiments. Data are mean ± standard deviation (SD) of six replicates. b Two-dimensional (2D) colony formation of parental (grown in FBS) and LTED (grown in CSS) cells. Pictures of MM134 LTED-A and LTED-D colonies were selected as representative pictures for MM134 LTED cell lines. Five thousand cells were seeded per well in six-well plates, and cells were stained with crystal violet after 15 days. The colonies with radium of more than 25 μm were counted for the colony numbers. Scale bar, 1 mm. Plots are representative of two independent experiments. Data are mean ± SD of three replicates. One-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test for multiple comparisons, **P <0.01, ***P <0.001. (c, d) Dose response ICI 182, 780 (ICI) in MM134 (c) and SUM44 (d) LTED cells. Cells were treated with vehicle (Ctrl) or drugs for 5 days before collection. Plots are representative of at least two independent experiments. Data are mean ± SD of six replicates. e ERα (Western blot; top) and ESR1 (quantitative reverse transcription-polymerase chain reaction, or qRT-PCR; bottom) expression in MM134 and SUM44 LTED and parental cells. Data in the lower panel are mean ± SD of three replicates. One-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons, ***P <0.001. Abbreviations: CSS charcoal-stripped fetal bovine serum, DMEM Dulbecco’s modified Eagle’s medium, FBS fetal bovine serum, IMEM improved minimal essential medium.
Fig. 2
Fig. 2
MM134 and SUM44 long-term estrogen deprivation (LTED) cells show different top enriched pathways. (a, b) Heatmaps (a) and principal component analysis (PCA) plots (b) of the top 1000 most variable genes. The top 1000 most variable genes were selected using interquartile range (IQR) in MM134 or SUM44 cells independently. The clustering of genes in heatmaps was based on complete-linkage, Euclidean distance hierarchical clustering. c Top 10 upregulated pathways in MM134 or SUM44 LTED cells. Ranked by –log10(P value). –log10(0.05) is marked with a red line. Cholesterol- and fatty acid-related pathways are labeled in bold. Pathway analyses were performed with Ingenuity Pathway Analysis (IPA) with the commonly upregulated differential expression (DE) genes in at least three MM134 LTED variants (n = 1653) or in the two SUM44 LTED variants (n = 1448). P values were corrected with the Benjamini-Hochberg method
Fig. 3
Fig. 3
Cholesterol synthesis is predicted to be upregulated in long-term estrogen deprivation (LTED) cells. (a, b) Gene set enrichment analysis (GSEA) of (a) E2F activation signature and (b) cholesterol biosynthesis signature (Reactome Cholesterol Synthesis) in LTED variants. Differential expression (DE) genes used in GSEA were ranked by log2(fold change). c Growth of parental and LTED cells with treatment of 25-hydroxycholesterol (25-HC) and 27-hydroxycholesterol (27-HC). Parental cells were hormone-deprived before being seeded in hormone-deprived media (charcoal-stripped fetal bovine serum, or CSS). Cells were collected after 5-day treatment. Fold growth was compared with control group (data not shown), which was treated with vehicle (ethanol). Plots are representative of at least two independent experiments. Data are mean ± standard deviation (SD) of six replicates. One-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test for multiple comparisons was used to test the significance between 25-HC/27-HC–treated groups to the control groups (data not shown), *P <0.05, **P <0.01, ***P <0.001. d The activation z-score of sterol regulatory element-binding proteins (SREBPs) in LTED cells. The upstream regulator analysis was performed in individual LTED cell variants separately with Ingenuity Pathway Analysis (IPA) software. DE genes (absolute log2(fold change) > log2(1.5) and adjusted P value of less than 0.001) and their log2(fold change) were used as input. Upstream regulators with z-score of more than 2 were defined as “activated” (labeled in red). Abbreviation: ES enrichment score.
Fig. 4
Fig. 4
Cholesterol synthesis regulator, sterol regulatory element-binding protein 1 (SREBP1) is upregulated in long-term estrogen deprivation (LTED) cells. a Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) of SREBP1a in LTED and parental cells. Parental cells were cultured in their normal growth media (fetal bovine serum, or FBS). Plots are the combination of data from three independent experiments. Data are mean ± standard deviation (SD) of nine replicates. One-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test for multiple comparisons, *P <0.05, **P <0.01, ***P <0.001. b The expression of precursor SREBP1 (pre-SREBP1) and mature SREBP1 (mSREBP1) in parental and LTED cells. Because of their similar molecular weight (54 amino acid difference in length), SREBP1a and SREBP1c were not separated in the gel. β-actin and PCNA were used as internal control in the whole cell lysis and nuclear lysis, respectively. The band of mSRBP1 is labeled with an arrow. Blots of whole cell lysis are representative of three independent experiments, and the blot of nuclear lysis is a single experiment. c RNA and protein expression levels of fatty acid synthase (FASN). Plots are representative of two independent experiments. Data in the left panel are mean ± SD of three replicates. One-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons, *P <0.05, **P <0.01, ***P <0.001. d The growth inhibition of MM134 parental and LTED cells by etomoxir. MM134 LTED-D was selected as the representative variant of MM134 LTED cells. This figure is the same experiment as the dose response curve of etomoxir in Additional file 1: Figure S8B. Plot is representative of two independent experiments. Data are mean ± SD of six replicates. One-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons, ***P <0.001
Fig. 5
Fig. 5
Abrogation of sterol regulatory element-binding proteins (SREBPs) inhibits the growth of long-term estrogen deprivation (LTED) cells. a Knockdown efficiency of SREBP1 and SREBP2 in MM134 parental and LTED-D cells. mRNA was collected 72 h after reverse transfection with small interfering RNA (siRNA). Data are mean ± standard deviation (SD) of six replicates collected from two independent experiments. Two-tailed Welch’s unequal variances t test, *P ≤0.05, **P <0.01, ***P <0.001. b Growth curve of MM134 parental and LTED cells with SREBP knockdown. Parental and LTED cells in 96-well plates were reverse-transfected with 1 nM siSREBP1 and 1 nM siSREBP2 (SREBP siRNA) or 2 nM non-target siRNA. Parental cells were cultured in their normal growth media (fetal bovine serum, or FBS). Two-way analysis of variance (ANOVA), ***P <0.001. (c, d) Dose response of PF429242, an inhibitor of SREBP1 and SREBP2, in MM134 (c) and SUM44 (d) parental and LTED cells. Parental cells were grown in normal growth media (FBS) and hormone-deprived media (charcoal-stripped fetal bovine serum, or CSS) to control the effect of media on the drug. Plots are representative of two independent experiments. Data are mean ± SD of six replicates. Two-way ANOVA (LTED versus parental in FBS), ***P <0.001. e Dose response of Fatostatin in MM134 parental and LTED cells. Parental cells were grown in normal growth media (FBS) or hormone-deprived media (CSS) to control the effect of media on the drug. Plots are representative of two independent experiments. Data are mean ± SD of six replicates. Two-way ANOVA was used to compare the dose response curves (LTED versus parental in FBS). Two-tailed t tests were performed to compare the inhibition rates of Fatostatin on LTED and parental (FBS) at 10 μm, 35 μm, and 100 μm independently. **P <0.01, ***P <0.001. f The expression change of SREBP1 with 3-month letrozole treatment in letrozole responders (n = 36) and non-responders (n = 14). Black solid lines, increased expression of SREBP1 with 3-month treatment; gray dashed lines, decreased expression of SREBP1 with 3-month treatment. Gene expression data for letrozole-treated patients were downloaded from Gene Expression Omnibus [GSE20181]. Pearson’s chi-squared test. Abbreviation: NS not significant.
Fig. 6
Fig. 6
Proposed working model for role of sterol regulatory element-binding protein 1 (SREBP1) signaling in invasive lobular carcinoma (ILC) long-term estrogen deprivation (LTED) cells. Genes: the upregulated genes in MM134 or SUM44 LTED cells compared with parental cells, which were validated in the in vitro experiments or based on the RNA-sequencing (RNA-Seq) data or both. Black arrow, the potential pathway of SREBP1 promoting the survival of LTED cells

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