The estrogen receptor-a (ERa) determines the phenotype of breast cancers where it serves as a positive prognostic indicator. ERa is a well-established target for breast cancer therapy, but strategies to target its function remain of interest to address therapeutic resistance and further improve treatment. Recent findings indicate that proteasome inhibition can regulate estrogen-induced transcription, but how ERa function might be regulated was uncertain. In this study, we investigated the transcriptome-wide effects of the proteasome inhibitor bortezomib on estrogen-regulated transcription in MCF7 human breast cancer cells and showed that bortezomib caused a specific global decrease in estrogen-induced gene expression. This effect was specific because gene expression induced by the glucocorticoid receptor was unaffected by bortezomib. Surprisingly, we observed no changes in ERa recruitment or assembly of its transcriptional activation complex on ERa target genes. Instead, we found that proteasome inhibition caused a global decrease in histone H2B monoubiquitination (H2Bub1), leading to transcriptional elongation defects on estrogen target genes and to decreased chromatin dynamics overall. In confirming the functional significance of this link, we showed that RNA interference-mediated knockdown of the H2B ubiquitin ligase RNF40 decreased ERa-induced gene transcription. Surprisingly, RNF40 knockdown also supported estrogen-independent cell proliferation and activation of cell survival signaling pathways. Most importantly, we found that H2Bub1 levels decrease during tumor progression. H2Bub1 was abundant in normal mammary epithelium and benign breast tumors but absent in most malignant and metastatic breast cancers. Taken together, our findings show how ERa activity is blunted by bortezomib treatment as a result of reducing the downstream ubiquitin-dependent function of H2Bub1. In supporting a tumor suppressor role for H2Bub1 in breast cancer, our findings offer a rational basis to pursue H2Bub1-based therapies for future management of breast cancer. Cancer Res; 71(17); 5739-53. Ó2011 AACR.

Extensive changes in posttranslational histone modifications accompany the rewiring of the transcriptional program during stem cell differentiation. However, the mechanisms controlling the changes in specific chromatin modifications and their function during differentiation remain only poorly understood. We show that histone H2B monoubiquitination (H2Bub1) significantly increases during differentiation of human mesenchymal stem cells (hMSCs) and various lineage-committed precursor cells and in diverse organisms. Furthermore, the H2B ubiquitin ligase RNF40 is required for the induction of differentiation markers and transcriptional reprogramming of hMSCs. This function is dependent upon CDK9 and the WAC adaptor protein, which are required for H2B monoubiquitination. Finally, we show that RNF40 is required for the resolution of the H3K4me3/H3K27me3 bivalent poised state on lineage-specific genes during the transition from an inactive to an active chromatin conformation. Thus, these data indicate that H2Bub1 is required for maintaining multipotency of hMSCs and plays a central role in controlling stem cell differentiation.

ABSTRACT:Residual dipolar couplings have recently found a wide range of applications in high-resolution NMR of biomolecules in the liquid state. A nonisotropic orientational distribution of a molecule of interest results in nonzero average dipolar coupling constants. Here, we present an intuitive introduction to the alignment tensor and an elementary derivation of key equations.

A role of WNT signaling for primary breast cancers of the basal-like subtype and as a predictor of brain metastasis has been described. However, a responsible WNT ligand has not been identified. To further clarify this question, we comparatively investigated 22 human breast cancer brain metastases as well as the highly invasive human breast cancer cell line MDA-MB-231 and the weakly motile MCF-7 as models for the basal-like and the luminal A subtype. WNT5A and B were found overexpressed in MDA-MB-231 cells as compared with MCF-7. This corresponded to reduction of MDA-MB-231 invasiveness by WNT inhibitors, whereas MCF-7 invasion was enhanced by recombinant WNT5B and abolished by WNT and Jun-N-terminal kinase antagonists. Expression and subcellular distribution of β-catenin remained uninfluenced. Consistently, β-catenin was not localized in the nuclei of brain metastases while there was strong nuclear c-Jun staining. Similar to MDA-MB-231, metastases showed expression of WNT5A/B and the alternative WNT receptors ROR1 and 2. These findings were validated using external gene expression datasets (Gene Expression Omnibus) of different breast cancer subtypes and brain metastases. Hierarchical cluster analysis yielded a close relation between basal-like cancers and brain metastases. Gene set enrichment analyses confirmed WNT pathway enrichment not only in basal-like primaries but also in cerebral metastases of all subtypes. In conclusion, WNT signaling seems highly relevant for basal-like and other subtypes of breast cancers metastasizing into the brain. β-catenin-independent WNT signaling, presumably via ROR1-2, plays a major role in this context.

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