Macrophage-elicited loss of estrogen receptor-α in breast cancer cells via involvement of MAPK and c-Jun at the ESR1 genomic locus

Abstract

Estrogen receptor-α (ERα, ESR1) is a pivotal transcriptional regulator of breast cancer physiology and is targeted by endocrine therapies. Loss of ERα activity or expression is an indication of endocrine resistance and is associated with increased risk of tumor recurrence and worse prognosis. In this study, we sought to investigate whether elements of the tumor microenvironment, namely macrophages, would impact on ERα and we found that macrophage-derived factors caused loss of ERα expression in breast cancer cells. Conditioned media from macrophages caused activation of several intracellular pathways in breast cancer cells of which c-Src, protein kinase c and mitogen-activated protein kinase (MAPK) were essential for loss of ERα expression. Moreover, a prolonged hyperactivation of MAPK was observed. The activation of this kinase cascade resulted in recruitment of extracellular signal regulated kinase 2 (ERK2) directly to chromatin at the ESR1 gene locus in a process that was dependent upon activation and recruitment of the c-Jun transcription factor. Thus, we identify a novel mechanism for loss of ERα expression in breast cancer cells via macrophage activation of kinase cascades in the cancer cells causing transcriptional repression of the ESR1 gene by a direct chromatin action of a c-Jun/ERK2 complex. The findings in this study support an alternative mechanism, not intrinsic to the tumor cell but derived from the cross-talk with the tumor microenvironment, that could lead to endocrine resistance and might be targeted therapeutically to prevent loss of ERα expression in breast tumors.

Fig.1. Polarization of the monocytic THP-1 cell line into M1- or M2-like macrophages

A) Schematic of experimental protocol (for details see Material and Methods) B) Comparison between q-PCR data obtained with the protocol used in panel A with data from expression cDNA microarrays in primary human macrophages (Martinez et al., 2006). Data are represented as M1/M2 ratio and are average +/− SEM of 6 independent experiments. C) q-PCR data for selected genes that are specific for M1- vs. M2- like macrophages in THP-1 cells.

Fig.2. Conditioned Media (CMs) from polarized THP-1 cells, but not cytokines alone, cause loss of ERα mRNA and protein in a time-dependent manner in MCF-7 cells

A-B) MCF-7 cells were treated with THP-1 CMs or cytokines for the times indicated and ERα mRNA and protein levels were assessed by q-PCR and Western blot. Panel B shows quantification of Western blot data for ERα protein from four independent experiments. C) ChIP assay was performed in MCF-7 cells after treatment with vehicle or CM1 for 45 min using RNA polymerase II or IgG control antibodies. Recovered DNA was used to assess the ESR1 promoter A. All experiments were performed a minimum of three times. Data are average +/− SEM. CM0: CM from vehicle treated THP-1 cells; CM1: CM from IFNG treated THP-1 cells; CM2: CM from IL4 treated THP-1 cells.

Fig.3. MAPK, c-Src and PKC pathways are required for loss of ERα elicited by polarized THP-1 cell conditioned media

A) MCF-7 cells were treated with conditioned media from IFNG-treated THP-1 cells (CM1) for 15 min and activation of various pathways was tested by Western blot using phospho-specific antibodies. B) MCF-7 cells were pre-treated for 1 h with the indicated pathway inhibitors and then treated with CM1 for 8 h. ERα mRNA was quantified by q-PCR. Data are average +/− SEM of three independent experiments. C) MCF-7 cells were pre-treated for 1 h with the indicated pathway inhibitors and then treated with CM1 for 24 h. ERα protein was then quantified by Western blot. Values for CM1 treated samples are expressed relative to control vehicle treated samples which are set at 1x. D–E) MCF-7 cells were pretreated for 1 h with PKC (Gö6976), c-Src (PP-2) or MEK1 (U0126) inhibitors prior to 15 min of CM1 or IFNG 20ng/ml treatment. Activated pathways were probed with phospho-specific antibodies via Western blot. F) Time-course of MAPK activation in MCF-7 cells after treatment with CM1.

Fig.4. ERK2 is recruited to the ERα promoter and its knock-down prevents loss of ERα after CM1 treatment

A) UCSC Genome Browser schematics of the ESR1 locus; the location of ERK2 binding sites (black boxes, 244–248) is based on (Madak-Erdogan et al., 2011). B) ChIP assay was performed in MCF-7 cells treated with vehicle or CM1 for 45 min; the isolated chromatin was probed with antibodies for ERK2 or IgG as control. C) MCF-7 cells were transfected with GL3 siRNA or ERK2 siRNA for 48 h prior to 8 h of CM1 treatment. ERα and ERK2 levels were analyzed via q-PCR and Western blot. Fold change values are expressed relative to that of vehicle control GL3 siRNA treated samples which are set at 1x. Data are average +/− SEM of three independent experiments.

Fig.5. c-Jun is activated in a PKC-, c-Src- and MAPK-dependent manner, and is recruited to the ESR1 promoter and required for ERα loss upon CM1 treatment

A) Activation of c-Jun was monitored by Western blot after 15 min of CM1 treatment in MCF-7 cells using phospho-specific antibody. B) MCF-7 cells were treated as in (A) except that they were pretreated for 1 h with the indicated pathway inhibitors. C) MCF-7 cells were treated with CM1 for 45 min and then ChIP was performed using c-Jun specific antibody. D) MCF-7 cells were transfected with GL3 or c-Jun siRNA for 48 h prior to CM1 treatment for 8 h (for mRNA) or 24 h (for protein). ERK2 was used as a loading control. Fold change values are expressed relative to that of vehicle control GL3 siRNA treated samples which are set at 1x. Data are average +/− SEM of three independent experiments.

Fig.6. c-Jun is required for ERK2 recruitment to the ESR1 locus

A) MCF-7 cells were treated with vehicle or CM1 for 45 min and then ChIP-reChIP was performed with the indicated antibodies. B) MCF-7 cells were transfected with GL3 control or cJun siRNA for 72 h prior to 45 min of vehicle or CM1 treatment. ERK2 ChIP was then performed. In both panels A and B, DNA was quantitated by q-PCR and the data are mean +/− SEM of 3 experiments. C) Proposed model for ERα down-regulation in breast cancer cells by macrophage-breast cancer cell interrelationships involving hyper-activation of MAPK and recruitment of c-Jun and ERK2 to the ESR1 genomic locus.