ERRα promotes breast cancer cell dissemination to bone by increasing RANK expression in primary breast tumors

Abstract

Bone is the most common metastatic site for breast cancer. Estrogen-related-receptor alpha (ERRα) has been implicated in cancer cell invasiveness. Here, we established that ERRα promotes spontaneous metastatic dissemination of breast cancer cells from primary mammary tumors to the skeleton. We carried out cohort studies, pharmacological inhibition, gain-offunction analyses in vivo and cellular and molecular studies in vitro to identify new biomarkers in breast cancer metastases. Meta-analysis of human primary breast tumors revealed that high ERRα expression levels were associated with bone but not lung metastases. ERRα expression was also detected in circulating tumor cells from metastatic breast cancer patients. ERRα overexpression in murine 4T1 breast cancer cells promoted spontaneous bone micro-metastases formation when tumor cells were inoculated orthotopically, whereas lung metastases occurred irrespective of ERRα expression level. In vivo, Rank was identified as a target for ERRα. That was confirmed in vitro in Rankl stimulated tumor cell invasion, in mTOR/pS6K phosphorylation, by transactivation assay, ChIP and bioinformatics analyses. Moreover, pharmacological inhibition of ERRα reduced primary tumor growth, bone micro-metastases formation and Rank expression in vitro and in vivo. Transcriptomic studies and meta-analysis confirmed a positive association between metastases and ERRα/RANK in breast cancer patients and also revealed a positive correlation between ERRα and BRCA1 mut carriers. Taken together, our results reveal a novel ERRα/RANK axis by which ERRα in primary breast cancer promotes early dissemination of cancer cells to bone. These findings suggest that ERRα may be a useful therapeutic target to prevent bone metastases.

ERRα/VDR Axis Promotes Calcitriol Degradation and Estrogen Signaling Activation and Correlates with Poor Prognosis in Basal-like Breast Cancer Patients.

“…Through BioGRID, we built an extended network to query VDR, ESRRA, ESR1, BRCA1 and KDM1A as main interacting protein hubs. Such a choice was based on ESRRA interacting proteins emerged by our study and by recent report showing a direct interaction between ESRRA and BRCA1 in BRCA1-mutated carriers [37], which is a setting represented by the SUM149PT cell line in our experiments, while others have demonstrated a direct interaction between ESRRA and KDM1A [28,27]. The subnetwork, identi ed from the whole database (Fig.…”

ERRα Expression in Bone Metastases Leads to an Exacerbated Antitumor Immune Response

“…Cells lines were tested for mycoplasma regularly. Mouse and human ESRRA cDNA (ERRα) and the dominant-negative co-activator domain AF2 (AF2) mutant were described previously (16) (18). Briefly, pSRα-ERRαWT and pEcmv-ERRαAF2 or respective empty vectors (CT) constructs were transfected into parental 4T1 cells and cultured for 4 weeks in puromycin (2 µg/mL) (Life-Technologies).…”

“…Two independent clones were obtained from empty vectors transfection respectively pSRα-4T1-CT (4T1-CT) and pEcmv-4T1-CT (4T1-CTaf2). For MCF7 clones, a mix containing 1.5µg Retroviral pLPCX-Human-ERRαWT, pLPCX-HumanERRαAF2 or empty vector and 0.5µg pCMV-VSV-G envelope vector (Cell-Biolabs) were used previously (16). 4T1-ERRα and 4T1-ERRαAF2 (pool of 3clones each) cells were treated for 24 hours with the ERRα inverseagonists XCT790 (Sigma) or C29 (AGVdiscovery, France) at 1µM and 5µM, respectively, as described (13) (16) (19).…”

“…Indeed, two qPCR Sign Arrays: Cytokines and Inflammation Arrays (AnyGenes, CT1M1-IFM1)(CliniSciences) were used to quantify expression of cytokines, chemokines and growth factors. Total RNA was extracted from 4T1-CT and 4T1-ERRα cells and 2 µg were reverse-transcribed as previously described (16). Real-Time PCR was performed according to the manufacturer's instructions.…”

“…Notably, ERRα-positive tumors are associated with more invasive BCa and a higher risk of recurrence (9) (14). The over-expression of ERRα in BCa promotes tumor growth in the mammary gland and BCa metastatic dissemination to the bone (16). However, the role of ERRα in BM outcome once they are anchorage in the bone microenvironment remains elusive.…”