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Cancer cells have higher reactive oxygen species (ROS) than normal cells, due to genetic and metabolic alterations. An emerging scenario is that cancer cells increase ROS to activate protumorigenic signaling while activating antioxidant pathways to maintain redox homeostasis. Here we show that, in basal-like and BRCA1-related breast cancer (BC), ROS levels correlate with the expression and activity of the transcription factor aryl hydrocarbon receptor (AhR). Mechanistically, ROS triggers AhR nuclear accumulation and activation to promote the transcription of both antioxidant enzymes and the epidermal growth factor receptor (EGFR) ligand, amphiregulin (AREG). In a mouse model of BRCA1-related BC, cancer-associated AhR and AREG control tumor growth and production of chemokines to attract monocytes and activate proangiogenic function of macrophages in the tumor microenvironment. Interestingly, the expression of these chemokines as well as infiltration of monocyte-lineage cells (monocyte and macrophages) positively correlated with ROS levels in basal-like BC. These data support the existence of a coordinated link between cancer-intrinsic ROS regulation and the features of tumor microenvironment. Therapeutically, chemical inhibition of AhR activity sensitizes human BC models to Erlotinib, a selective EGFR tyrosine kinase inhibitor, suggesting a promising combinatorial anticancer effect of AhR and EGFR pathway inhibition. Thus, AhR represents an attractive target to inhibit redox homeostasis and modulate the tumor promoting microenvironment of basal-like and BRCA1-associated BC.

The arginine methyltransferase PRMT6 is shown to methylate histone H3 on Arg2 in mammalian cells, and this modification is mutually antagonistic with methylation of Lys4 on H3 by the methyl transferase complex MLL. Eukaryotic genomes are organized into active (euchromatic) and inactive (heterochromatic) chromatin domains. Post-translational modifications of histones (or ‘marks’) are key in defining these functional states, particularly in promoter regions 1 , 2 . Mutual regulatory interactions between these marks—and the enzymes that catalyse them—contribute to the shaping of this epigenetic landscape, in a manner that remains to be fully elucidated 1 , 2 . We previously observed that asymmetric di-methylation of histone H3 arginine 2 (H3R2me2a) counter-correlates with di- and tri- methylation of H3 lysine 4 (H3K4me2, H3K4me3) on human promoters 3 . Here we show that the arginine methyltransferase PRMT6 catalyses H3R2 di-methylation in vitro and controls global levels of H3R2me2a in vivo . H3R2 methylation by PRMT6 was prevented by the presence of H3K4me3 on the H3 tail. Conversely, the H3R2me2a mark prevented methylation of H3K4 as well as binding to the H3 tail by an ASH2/WDR5/MLL-family methyltransferase complex 4 , 5 , 6 , 7 . Chromatin immunoprecipitation showed that H3R2me2a was distributed within the body and at the 3′ end of human genes, regardless of their transcriptional state, whereas it was selectively and locally depleted from active promoters, coincident with the presence of H3K4me3. Hence, the mutual antagonism between H3R2 and H3K4 methylation, together with the association of MLL-family complexes with the basal transcription machinery 8 , may contribute to the localized patterns of H3K4 tri-methylation characteristic of transcriptionally poised or active promoters in mammalian genomes 1 , 2 , 3 , 9 , 10 .

Mutations in the tumor suppressor BRCA1 predispose women to breast and ovarian cancers. The mechanism underlying the tissue-specific nature of BRCA1's tumor suppression is obscure. We previously showed that the antioxidant pathway regulated by the transcription factor NRF2 is defective in BRCA1-deficient cells. Reactivation of NRF2 through silencing of its negative regulator KEAP1 permitted the survival of BRCA1-null cells. Here we show that estrogen (E2) increases the expression of NRF2-dependent antioxidant genes in various E2-responsive cell types. Like NRF2 accumulation triggered by oxidative stress, E2-induced NRF2 accumulation depends on phosphatidylinositol 3-kinase–AKT activation. Pretreatment of mammary epithelial cells (MECs) with the phosphatidylinositol 3-kinase inhibitor BKM120 abolishes the capacity of E2 to increase NRF2 protein and transcriptional activity. In vivo the survival defect of BRCA1-deficient MECs is rescued by the rise in E2 levels associated with pregnancy. Furthermore, exogenous E2 administration stimulates the growth of BRCA1-deficient mammary tumors in the fat pads of male mice. Our work elucidates the basis of the tissue specificity of BRCA1-related tumor predisposition, and explains why oophorectomy significantly reduces breast cancer risk and recurrence in women carrying BRCA1 mutations.

Knowledge of protein domains that function as the biological effectors for diverse post-translational modifications of histones is critical for understanding how nuclear and epigenetic programs are established. Indeed, mutations of chromatin effector domains found within several proteins are associated with multiple human pathologies, including cancer and immunodeficiency syndromes. To date, relatively few effector domains have been identified in comparison to the number of modifications present on histone and non-histone proteins. Here we describe the generation and application of human modified peptide microarrays as a platform for high-throughput discovery of chromatin effectors and for epitope-specificity analysis of antibodies commonly utilized in chromatin research. Screening with a library containing a majority of the Royal Family domains present in the human proteome led to the discovery of TDRD7, JMJ2C, and MPP8 as three new modified histone-binding proteins. Thus, we propose that peptide microarray methodologies are a powerful new tool for elucidating molecular interactions at chromatin.

Although the asymmetric dimethylation of histone H3R2 acts as a repressive mark, new studies reveal that symmetrically dimethylated H3R2 (H3R2me2s) is a functional histone mark in vivo . The RBBP7 co-repressor is excluded from binding H3R2me2s in favor of the coactivator WDR5, which poises euchromatic genes for transcription activation upon cell-cycle exit and differentiation. The asymmetric dimethylation of histone H3 arginine 2 (H3R2me2a) acts as a repressive mark that antagonizes trimethylation of H3 lysine 4. Here we report that H3R2 is also symmetrically dimethylated (H3R2me2s) by PRMT5 and PRMT7 and present in euchromatic regions. Profiling of H3-tail interactors by SILAC MS revealed that H3R2me2s excludes binding of RBBP7, a central component of co-repressor complexes Sin3a, NURD and PRC2. Conversely H3R2me2s enhances binding of WDR5, a common component of the coactivator complexes MLL, SET1A, SET1B, NLS1 and ATAC. The interaction of histone H3 with WDR5 distinguishes H3R2me2s from H3R2me2a, which impedes the recruitment of WDR5 to chromatin. The crystallographic structure of WDR5 and the H3R2me2s peptide elucidates the molecular determinants of this high affinity interaction. Our findings identify H3R2me2s as a previously unknown mark that keeps genes poised in euchromatin for transcriptional activation upon cell-cycle withdrawal and differentiation in human cells.

By the late 1990s, a quest for the major tumor suppressor gene within the frequently deleted region of chromosome 10 found in advanced cancers culminated in the discovery of PTEN, named based on amino acid sequence homology with the protein tyrosine phosphatases and tensin (1 ). In cells, PIP3 concentrations in the plasma membrane increased after cellular stimulation with growth factors, leading to activation of the PI3K-PKB pathway, a signaling cascade that regulates diverse cellular processes, including cell metabolism, survival, proliferation, apoptosis, growth, and migration.

The tumor suppressor PTEN is disrupted in a large proportion of cancers, including in HER2-positive breast cancer, where its loss is associated with resistance to therapy. Upon genotoxic stress, ataxia telangiectasia mutated (ATM) is activated and phosphorylates PTEN on residue 398. To elucidate the physiological role of this molecular event, we generated and analyzed knock-in mice expressing a mutant form of PTEN that cannot be phosphorylated by ATM (PTEN-398A). This mutation accelerated tumorigenesis in a model of HER2-positive breast cancer. Mammary tumors in bi-transgenic mice carrying MMTV-neu and Pten398A were characterized by DNA damage accumulation but reduced apoptosis. Mechanistically, phosphorylation of PTEN at position 398 is essential for the proper activation of the S phase checkpoint controlled by the PI3K–p27Kip1–CDK2 axis. Moreover, we linked these defects to the impaired ability of the PTEN-398A protein to relocalize to the plasma membrane in response to genotoxic stress. Altogether, our results uncover a novel role for ATM-dependent PTEN phosphorylation in the control of genomic stability, cell cycle progression, and tumorigenesis.

Ablation of the tumor suppressor phosophatase and tensin homlog (PTEN) unexpectedly suppresses the development of pre-B acute lymphoblastic leukemia (ALL).

In order to sustain proficient life-long hematopoiesis, hematopoietic stem cells (HSCs) must possess robust mechanisms to preserve their quiescence and genome integrity. DNA-damaging stress can perturb HSC homeostasis by affecting their survival, self-renewal, and differentiation. Ablation of the kinase ataxia telangiectasia mutated (ATM), a master regulator of the DNA damage response, impairs HSC fitness. Paradoxically, we show here that loss of a single allele of Atm enhances HSC functionality in mice. To explain this observation, we explored a possible link between ATM and the tumor suppressor phosphatase and tensin homolog (PTEN), which also regulates HSC function. We generated and analyzed a knockin mouse line (PtenS398A/S398A), in which PTEN cannot be phosphorylated by ATM. Similar to Atm+/-, PtenS398A/S398A HSCs have enhanced hematopoietic reconstitution ability, accompanied by resistance to apoptosis induced by genotoxic stress. Single-cell transcriptomic analyses and functional assays revealed that dormant PtenS398A/S398A HSCs aberrantly tolerate elevated mitochondrial activity and the accumulation of reactive oxygen species, which are normally associated with HSC priming for self-renewal or differentiation. Our results unveil a molecular connection between ATM and PTEN, which couples the response to genotoxic stress and dormancy in HSCs.

Loss of function of the phosphatase and tensin homolog (PTEN) tumor suppressor is frequently found in many human malignancies. PTEN antagonizes the Phosphatidylinositide 3-kinase (PI3K) pathway (1) by dephosphorylating the 3 position of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] (PIP3). PIP3 serves as a second messenger whose levels in the plasma membrane are elevated following cell stimulation with growth factors, mediated by the activity of PI3Ks. Proteins containing pleckstrin homology (PH) domains physically interact with PIP3, bringing them into close proximity with other PH domain-containing proteins and facilitating further functional interactions that serve to propagate the membranous signal. Although the cytoplasmic role of PTEN in antagonizing PI3K signaling has been well studied, PTEN also resides in the nucleus, where its function remains poorly understood. Here, I demonstrate that SUMOylation (SUMO) of PTEN controls its nuclear localization. Consistent with its restricted cytoplasmic localization, a SUMO-deficient PTEN mutant is fully competent for regulating PI3K signaling. Upon exposure to genotoxic stress, SUMO-PTEN is rapidly excluded from the nucleus in an ATM-dependent manner, identifying a connection between these two major tumor suppressors. Further, ATM phosphorylated PTEN on threonine 398, and a PTEN mutant that cannot be phosphorylated at this position (PTEN T398A) resist nuclear exclusion following genotoxic stress. Judging by various readouts of the DNA damage response, cells lacking nuclear PTEN are hypersensitive to DNA damage and display impaired homologous recombination-mediated repair of double-strand DNA breaks. Moreover, unlike cells with Wt PTEN, PTEN-deficient cells are susceptible to killing by a combination of genotoxic stress and a small molecule inhibitor of PI3K both in vitro and in vivo. The synergistc effect of genotoxic stress and PI3K inhibition on PTEN-null cells is dependent on the simultaneous inhibition of both p110α and p110β isoform. Further in vivo studies revealed that effective inhibition of PI3K pathway can be achieved with a discontinuous administration schedule in order to reduce the adverse effects associated with this treatment. My findings have considerable implications for individualizing therapy for patients with PTEN-deficient tumors.