Invasive lobular carcinoma cell lines are characterized by unique estrogen-mediated gene expression patterns and altered tamoxifen response

Abstract

Invasive lobular carcinoma (ILC) is a histologic subtype of breast cancer that is frequently associated with favorable outcomes, as approximately 90% of ILC express the estrogen receptor (ER). However, recent retrospective analyses suggest that patients with ILC receiving adjuvant endocrine therapy may not benefit as much as patients with invasive ductal carcinoma. On the basis of these observations, we characterized ER function and endocrine response in ILC models. The ER-positive ILC cell lines MDA MB 134VI (MM134) and SUM44PE were used to examine the ER-regulated transcriptome via gene expression microarray analyses and ER ChIP-Seq, and to examine response to endocrine therapy. In parallel, estrogen response was assessed in vivo in the patient-derived ILC xenograft HCI-013. We identified 915 genes that were uniquely E2 regulated in ILC cell lines versus other breast cancer cell lines, and a subset of these genes were also E2 regulated in vivo in HCI-013. MM134 cells were de novo tamoxifen resistant and were induced to grow by 4-hydroxytamoxifen, as well as other antiestrogens, as partial agonists. Growth was accompanied by agonist activity of tamoxifen on ER-mediated gene expression. Though tamoxifen induced cell growth, MM134 cells required fibroblast growth factor receptor (FGFR)-1 signaling to maintain viability and were sensitive to combined endocrine therapy and FGFR1 inhibition. Our observation that ER drives a unique program of gene expression in ILC cells correlates with the ability of tamoxifen to induce growth in these cells. Targeting growth factors using FGFR1 inhibitors may block survival pathways required by ILC and reverse tamoxifen resistance.

Figure 1. E2 induces growth and gene expression in ILC cells

(A), Proliferation at indicated time post-treatment. (B), Zoomed scale for SUM44. (C), (D), qPCR-based gene expression; mean log2 fold-vs-vehicle of 3 biological replicates. Red, increased expression; Green, decreased expression. (C), Genes similarly regulated, (D), genes differentially regulated in ILC cells versus MCF-7.

Figure 2. E2 regulates unique genes in ILC cells

(A–D), Venn analysis of indicated gene sets; genes regulated at 3 and/or 24 hours included. (A), Experimental data for ILC cell lines. (B), Public data for IDC cell lines. (C), Union of IDC versus ILC. (D), Intersect of IDC versus ILC. (E), Validating ILC-E2 geneset in MM134 cells (Nanostring). Cells were hormone deprived and treated (0.1% EtOH or 1nM E2 ± 1μM ICI) for 3, 24, or 72 hours. Expression shown as log2 fold versus time-matched vehicle. ‘+’, common E2-regulated genes.

Figure 3. E2 induces unique distal ER binding in MM134 cells

(A), Localization of ER binding sites in MM134. Bolded/asterisk indicate q-value versus genome < 0.05. (B), ChIP-qPCR of ER binding sites in independent samples. NFERE, non-functional estrogen-response element. P-value for vehicle vs. E2 <0.05 for all sites except NFERE. Represents repeat experiments, ± SD of technical replicates. (C), MM134 binding sites versus consensus MCF-7 sites. (D), Localization of ER binding sites for specific/shared sites between MCF-7/MM134. Bolded/asterisk indicate q-value versus genome < 0.05.

Figure 4. Novel PDX HCI-013 is estrogen-responsive

(A), 5μM sections of FFPE blocks from HCI-013 and HCI-005 stained as indicated. E-cadherin/p120 staining was previously described (40). (B), PDX growth in defined estrogen conditions. Lines represent individual tumors. Growth rates ±SD shown. (C), Ki67 under indicated estrogen conditions. (D), Differential gene expression; d5 sham surgery (+E2) vs d10 pellet removal (−E2) (q < 0.05) shown as log2 fold change (−E2 vs +E2). (E), Example genes identified in (D). Points represent mean ± SD.

Figure 5. MM134 cells are de novo tamoxifen resistant

(A–C), Hormone-deprived cells were treated as indicated. Proliferation was assessed 5 (MCF-7) or 6 days (MM134, SUM44) post-treatment. Dashed line indicates growth -E2. Anti-estrogen treatment was +100pM E2 where indicated. (D–E), Hormone-deprived cells were treated with 100pM E2 or 1μM anti-estrogen ± 1μM ICI (D), or 100pM E2 ± 1μM anti-estrogen (E). Proliferation assessed 6 days post-treatment.

Figure 6. Tamoxifen induces gene expression as an ER agonist

(A), MM134 cells were hormone-deprived and treated with 100pM E2, 1μM 4OHT, or 1μM ICI for 3, 24, or 72hr. Heatmap represents log2 fold versus time-matched vehicle control. Genes clustered by Pearson complete correlation. ‘+’, commonly E2-regulated genes. Dashed white boxes highlight genes regulated by 4OHT as an agonist. (B), Gene expression (log2 fold versus control) per treatment and time point for all genes in indicated clusters. Points represent mean of n genes ± SD. P-values represent treatment effect by two-way ANOVA.

Figure 7. FGFR1 inhibitor-induced death in MM134 is dependent on estrogen signaling

(A–B), MM134 cells were hormone-deprived and treated with 0.1% EtOH vehicle, 1μM 4OHT, or 100pM E2 ± kinase inhibitor. Proliferation assessed 6 days post-treatment. Dashed line represents growth -E2. (C–D), MM134 treated as above ± 10μM kinase inhibitor, or 1μM STS. Timecourse represents repeated measures.