Pharmacological inhibition of DOT1L blocks estrogen receptor signaling in breast cancer.

BackgroundThe RNA-binding protein Argonaute 2 (AGO2) is a key effector of RNA-silencing pathways It exerts a pivotal role in microRNA maturation and activity and can modulate chromatin remodeling, transcriptional gene regulation and RNA splicing. Estrogen receptor beta (ERβ) is endowed with oncosuppressive activities, antagonizing hormone-induced carcinogenesis and inhibiting growth and oncogenic functions in luminal-like breast cancers (BCs), where its expression correlates with a better prognosis of the disease.ResultsApplying interaction proteomics coupled to mass spectrometry to characterize nuclear factors cooperating with ERβ in gene regulation, we identify AGO2 as a novel partner of ERβ in human BC cells. ERβ–AGO2 association was confirmed in vitro and in vivo in both the nucleus and cytoplasm and is shown to be RNA-mediated. ChIP-Seq demonstrates AGO2 association with a large number of ERβ binding sites, and total and nascent RNA-Seq in ERβ + vs ERβ − cells, and before and after AGO2 knock-down in ERβ + cells, reveals a widespread involvement of this factor in ERβ-mediated regulation of gene transcription rate and RNA splicing. Moreover, isolation and sequencing by RIP-Seq of ERβ-associated long and small RNAs in the cytoplasm suggests involvement of the nuclear receptor in RISC loading, indicating that it may also be able to directly control mRNA translation efficiency and stability.ConclusionsThese results demonstrate that AGO2 can act as a pleiotropic functional partner of ERβ, indicating that both factors are endowed with multiple roles in the control of key cellular functions.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-017-1321-0) contains supplementary material, which is available to authorized users.

Although a large fraction of high-grade serous epithelial ovarian cancers (OCs) expresses Estrogen Receptor alpha (ERα), anti-estrogen-based therapies are still not widely used against these tumors due to a lack of sufficient evidence. The histone methyltransferase Disruptor of telomeric silencing-1-like (DOT1L), which is a modulator of ERα transcriptional activity in breast cancer, controls chromatin functions involved in tumor initiation and progression and has been proposed as a prognostic OC biomarker. As molecular and clinico-pathological data from TCGA suggest a correlation between ERα and DOT1L expression and OC prognosis, the presence and significance of ERα/DOT1L association was investigated in chemotherapy-sensitive and chemotherapy-resistant ER+ OC cells. RNA sequencing before and after inhibition of these factors showed that their activity is implicated in OC cell proliferation and that they functionally cooperate with each other to control the transcription of genes involved in key cancer cell features, such as the cell cycle, epithelial-mesenchymal transition (EMT), drug metabolism, and cell-to-cell signaling, as well as expression of the ERα gene itself. Together with evidence from loss-of-function genetic screens showing that ERα and DOT1L behave as core fitness factors in OC cells, these results suggest that combined inhibition of their activity might be effective against ERα-expressing, chemotherapy-resistant ovarian tumors.

Breast cancer (BC) is a heterogeneous disease characterized by different biopathological features, differential response to therapy and substantial variability in long-term-survival. BC heterogeneity recapitulates genetic and epigenetic alterations affecting transformed cell behavior. The estrogen receptor alpha positive (ERα+) is the most common BC subtype, generally associated with a better prognosis and improved long-term survival, when compared to ERα-tumors. This is mainly due to the efficacy of endocrine therapy, that interfering with estrogen biosynthesis and actions blocks ER-mediated cell proliferation and tumor spread. Acquired resistance to endocrine therapy, however, represents a great challenge in the clinical management of ERα+ BC, causing tumor growth and recurrence irrespective of estrogen blockade. Improving overall survival in such cases requires new and effective anticancer drugs, allowing adjuvant treatments able to overcome resistance to first-line endocrine therapy. To date, several studies focus on the application of loss-of-function genome-wide screenings to identify key (hub) “fitness” genes essential for BC progression and representing candidate drug targets to overcome lack of response, or acquired resistance, to current therapies. Here, we review the biological significance of essential genes and relative functional pathways affected in ERα+ BC, most of which are strictly interconnected with each other and represent potential effective targets for novel molecular therapies.

Estrogen Receptor alpha (ERα) is a ligand-inducible transcription factor that mediates estrogen signaling in hormone-responsive cells, where it controls key cellular functions by assembling in gene-regulatory multiprotein complexes. For this reason, interaction proteomics has been shown to represent a useful tool to investigate the molecular mechanisms underlying ERα action in target cells. RNAs have emerged as bridging molecules, involved in both assembly and activity of transcription regulatory protein complexes. By applying Tandem Affinity Purification (TAP) coupled to mass spectrometry (MS) before and after RNase digestion in vitro, we generated a dataset of nuclear ERα molecular partners whose association with the receptor involves RNAs. These data provide a useful resource to elucidate the combined role of nuclear RNAs and the proteins identified here in ERα signaling to the genome in breast cancer and other cell types.