Growth factor stimulation induces a distinct ER(alpha) cistrome underlying breast cancer endocrine resistance - PubMed (original) (raw)

Growth factor stimulation induces a distinct ER(alpha) cistrome underlying breast cancer endocrine resistance

Mathieu Lupien et al. Genes Dev. 2010.

Abstract

Estrogen receptor α (ERα) expression in breast cancer is predictive of response to endocrine therapy; however, resistance is common in ERα-positive tumors that overexpress the growth factor receptor ERBB2. Even in the absence of estrogen, ERα can be activated by growth factors, including the epidermal growth factor (EGF). EGF induces a transcriptional program distinct from estrogen; however, the mechanism of the stimulus-specific response is unknown. Here we show that the EGF-induced ERα genomic targets, its cistromes, are distinct from those induced by estrogen in a process dependent on the transcription factor AP-1. The EGF-induced ERα cistrome specifically regulates genes found overexpressed in ERBB2-positive human breast cancers. This provides a potential molecular explanation for the endocrine therapy resistance seen in ERα-positive breast cancers that overexpress ERBB2. These results suggest a central role for ERα in hormone-refractory breast tumors dependent on growth factor pathway activation and favors the development of therapeutic strategies completely antagonizing ERα, as opposed to blocking its estrogen responsiveness alone.

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Figures

Figure 1.

Figure 1.

ERα is required for growth factor-mediated breast cancer cell proliferation. (A) Proliferation of MCF7 breast cancer cells pretreated or not with the full anti-estrogen fulvestrant (Ful) was measured following EGF stimulation. (*) P ≤ 0.05; (**) P ≤ 0.01; (***) P ≤ 0.001. (B) Comparison of estrogen-up (E2), EGF-up, and ERα-dependent EGF up-regulated as well as down-regulated transcriptional programs in MCF7 breast cancer cells. (C) mRNA levels derived from RT-qPCR of the ERα-dependent EGF target genes LIF and ACP6 are presented under EGF stimulation in MCF7 cells pretreated or not with Ful (100 nM). TFF1 is used as a negative control. (D) Oncomine Concepts Map analysis (Compendia Biosciences, Inc.;

https://www.oncomine.com

) was used to compare the EGF-induced gene signature in MCF7 breast cancer cells against all published gene signatures from primary breast tumors. This revealed significant correlations between EGF-up as well as ERα-dependent EGF-up gene signatures from MCF7 cells with gene signatures from poor-outcome (metastasis, recurrence, death, and high grade) as well as ERBB2-positive breast tumors (P ≤ 1e-2, odds ratio [O.R.] ≥2). No significant correlations were revealed between EGF-up or ERα-dependent EGF-up gene signatures and expression signatures from ERα-positive primary breast tumors. Each green circle in the left figure corresponds to the gene signature from primary breast tumors established in an independent study. The red circle corresponds to the EGF-up gene signature in MCF7 breast cancer cells. The purple circle corresponds to the ERα-dependent EGF-up gene signature from MCF7 breast cancer cells. Genes signatures significantly correlated with each other are linked to each other by a straight line. The right figure presents an example of how significant correlation between EGF-up and ERα-dependent EGF-up gene signatures with the ERBB2-positive breast cancer gene signature were established in one published study (Richardson et al. 2006). Specifically, the expression profile established in primary breast tumors from different patients (vertical axis) is presented for the genes found in the EGF-up and ERα-dependent EGF-up gene signature from MCF7 breast cancer cells (horizontal axis).

Figure 2.

Figure 2.

AP-1 is central to the growth factor-induced ERα cistromes. (A) Genome-wide ChIP-on-chip analysis following EGF stimulation in MCF7 breast cancer cells reveals 12,550 ERα-binding sites (false discovery rate [FDR] 1%), 31% overlapping with the estrogen (E2)-induced ERα cistrome. (B) Sequence analysis of EGF-unique (orange), shared (purple), or E2-unique (green) ERα-binding sites reveals the preferential enrichment of EREs in the center of the E2-unique and shared binding sites, while the FKH and AP-1 motifs are preferentially enriched in the center of the shared and EGF-unique ERα cistromes. (C) ChIP–reChIP assays directed against ERα and AP-1 were performed to reveal the corecruitment of these factors following EGF stimulation on ERα-binding sites. (*) P ≤ 0.05; (**) P ≤ 0.01; (***) P ≤ 0.001. (D) Proliferation of MCF7 breast cancer cells transfected with the mock (pcDNA3.1) or AP-1 dominant-negative (TAM67) vectors was measured following EGF stimulation (*) P ≤ 0.05; (**) P ≤ 0.01; (***) P ≤ 0.001. (E) mRNA levels of EGF target genes were measured following EGF stimulation in MCF7 breast cancer cells transfected with the mock (pcDNA3.1) or AP-1 dominant-negative (TAM67) vectors. (*) P ≤ 0.05; (**) P ≤ 0.01; (***) P ≤ 0.001.

Figure 3.

Figure 3.

Growth factor-induced transcriptional response relates to stimuli-specific ERα cistromes. (A) Correlation between E2, EGF-up, or ERα-dependent EGF-up target genes with EGF-unique, shared, or E2-unique ERα-binding sites from MCF7 breast cancer cells. The occurrence of ERα-binding sites within 20 kb of the TSS of regulated genes was compared with that on nonregulated genes. (*) P ≤ 0.05; (**) P ≤ 0.01; (***) P ≤ 0.001. (B) ChIP-qPCR results against ERα performed under EGF or E2 stimulation in MCF7 cells on the regulatory element associated with the TNFRSF21 and LIF genes. (C) RT-qPCR results measuring expression of the EGF-specific responsive genes TNFRSF21 and LIF following 3 h of E2 or EGF stimulation.

Figure 4.

Figure 4.

Selective coactivation of ERα-binding sites under growth factor stimulation. (A) Coactivation as measured by H3K18ac levels established following either EGF or estrogen (E2) stimulation by ChIP-on-chip of chromosomes 8, 11, and 12 in MCF7 breast cancer cells. The MAT score for H3K18ac (EGF/control and E2/control) was established on all ERα-binding sites shared under E2 and EGF stimulation found on chromosomes 8, 11, and 12. K-means cluster analysis was used to identify ERα-binding sites preferentially associated with induced H3K18ac under E2 or EGF treatment. (B) Correlation between gene expression and the three subsets of shared ERα-binding sites based on H3K18ac levels was performed as described for Figure 2D. (C) Specific examples validating the concept of stimuli-specific coactivation at ERα-binding sites common to estrogen and EGF stimulation. RT-qPCR studies validate the stimuli-specific expression of ACP6, LIF, TFF1, and XBP1 in MCF7 breast cancer cells following E2 or EGF treatment in MCF7 cells. (D) ChIP-qPCR analysis demonstrates E2 and EGF equivalent induction of ERα recruitment to regulatory regions associated with ACP6, LIF, TFF1, and XBP1. Coactivation measured through acetylation of H4K12ac or H3K18ac (Supplemental Material) is specific to ERα-binding sites near genes induced under the same treatment in MCF7 cells.

Figure 5.

Figure 5.

Growth factor ERα cistrome relates to poor-outcome expression signatures in breast tumors. (A) Oncomine Concepts Map analysis reveals significant association between genes specifically associated with an EGF-specific ERα-binding site within 20 kb of their TSS and gene expression signatures from ERBB2-positive, poor-outcome (metastasis, recurrence, death, and high grade), or ERα-positive breast tumors characterized in eight independent studies (each represented by individual circles). Only significantly associated gene lists are linked by a straight line (P ≤ 1e-4, O.R. ≥ 1.35). The gene list associated with estrogen (E2)-induced ERα-binding sites within 20 kb of their TSS does not significantly associate with any of the gene expression signatures linked to EGF–ERα-associated genes. The right panel presents an example of the expression profile for EGF–ERα-associated genes differentially expressed in ERBB2-positive breast tumors according to one independent study. Only genes significantly differentially expressed in ERBB2-positive versus -negative breast tumors are presented (P ≤ 5e-2). (B) Schematic representation of mechanisms involved in stimuli-specific transcriptional response acting through a shared transcription factor. E2 and the growth factor pathway (GF) induce a unique transcriptional response dependent on the ERα. This involves stimuli-specific ERα cistromes and coactivation. Current therapies for ERα-positive primary breast tumors, including aromatase inhibitors (AI) or selective ERα modulators such as tamoxifen (T), block only E2-mediated activation of the receptor. Full antiestrogens (FA) such as fulvestrant lead to ERα degradation and can therefore block E2- as well as GF-mediated activation of ERα. Growth factor inhibitors (GFI) should also block ERα activation following growth factor pathway activation.

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