Neuroprotective effects of G9a inhibition through modulation of peroxisome-proliferator activator receptor gamma-dependent pathways by miR-128.

JOURNAL/nrgr/04.03/01300535-202419110-00033/figure1/v/2024-03-08T184507Z/r/image-tiff Dysregulation of G9a, a histone-lysine N-methyltransferase, has been observed in Alzheimer's disease and has been correlated with increased levels of chronic inflammation and oxidative stress. Likewise, microRNAs are involved in many biological processes and diseases playing a key role in pathogenesis, especially in multifactorial diseases such as Alzheimer's disease. Therefore, our aim has been to provide partial insights into the interconnection between G9a, microRNAs, oxidative stress, and neuroinflammation. To better understand the biology of G9a, we compared the global microRNA expression between senescence-accelerated mouse-prone 8 (SAMP8) control mice and SAMP8 treated with G9a inhibitor UNC0642. We found a downregulation of miR-128 after a G9a inhibition treatment, which interestingly binds to the 3' untranslated region (3'-UTR) of peroxisome-proliferator activator receptor γ (PPARG) mRNA. Accordingly, Pparg gene expression levels were higher in the SAMP8 group treated with G9a inhibitor than in the SAMP8 control group. We also observed modulation of oxidative stress responses might be mainly driven Pparg after G9a inhibitor. To confirm these antioxidant effects, we treated primary neuron cell cultures with hydrogen peroxide as an oxidative insult. In this setting, treatment with G9a inhibitor increases both cell survival and antioxidant enzymes. Moreover, up-regulation of PPARγ by G9a inhibitor could also increase the expression of genes involved in DNA damage responses and apoptosis. In addition, we also described that the PPARγ/AMPK axis partially explains the regulation of autophagy markers expression. Finally, PPARγ/GADD45α potentially contributes to enhancing synaptic plasticity and neurogenesis after G9a inhibition. Altogether, we propose that pharmacological inhibition of G9a leads to a neuroprotective effect that could be due, at least in part, by the modulation of PPARγ-dependent pathways by miR-128.

Loss of G9a does not phenocopy the requirement for Prdm12 in the development of the nociceptive neuron lineage.

Prdm12 is an epigenetic regulator expressed in developing and mature nociceptive neurons, playing a key role in their specification during neurogenesis and modulating pain sensation at adulthood. In vitro studies suggested that Prdm12 recruits the methyltransferase G9a through its zinc finger domains to regulate target gene expression, but how Prdm12 interacts with G9a and whether G9a plays a role in Prdm12's functional properties in sensory ganglia remain unknown. Here we report that Prdm12-G9a interaction is likely direct and that it involves the SET domain of G9a. We show that both proteins are largely co-expressed in dorsal root ganglia during early murine development, opening the possibility that G9a plays a role in DRG and may act as a mediator of Prdm12's function in the development of nociceptive sensory neurons. To test this hypothesis, we conditionally inactivated G9a in neural crest using a Wnt1-Cre transgenic mouse line. We found that the specific loss of G9a in the neural crest lineage does not lead to dorsal root ganglia hypoplasia due to the loss of somatic nociceptive neurons nor to the ectopic expression of the visceral determinant Phox2b as observed upon Prdm12 ablation. These findings suggest that Prdm12 function in the initiation of the nociceptive lineage does not critically involves its interaction with G9a.

G9a Inhibition Promotes Neuroprotection through GMFB Regulation in Alzheimer's Disease.

Epigenetic alterations are a fundamental pathological hallmark of Alzheimer's disease (AD). Herein, we show the upregulation of G9a and H3K9me2 in the brains of AD patients. Interestingly, treatment with a G9a inhibitor (G9ai) in SAMP8 mice reversed the high levels of H3K9me2 and rescued cognitive decline. A transcriptional profile analysis after G9ai treatment revealed increased gene expression of glia maturation factor ß (GMFB) in SAMP8 mice. Besides, a H3K9me2 ChIP-seq analysis after G9a inhibition treatment showed the enrichment of gene promoters associated with neural functions. We observed the induction of neuronal plasticity and a reduction of neuroinflammation after G9ai treatment, and more strikingly, these neuroprotective effects were reverted by the pharmacological inhibition of GMFB in mice and cell cultures; this was also validated by the RNAi approach generating the knockdown of GMFB/Y507A.10 in Caenorhabditis elegans. Importantly, we present evidence that GMFB activity is controlled by G9a-mediated lysine methylation as well as we identified that G9a directly bound GMFB and catalyzed the methylation at lysine (K) 20 and K25 in vitro. Furthermore, we found that the neurodegenerative role of G9a as a GMFB suppressor would mainly rely on methylation of the K25 position of GMFB, and thus G9a pharmacological inhibition removes this methylation promoting neuroprotective effects. Then, our findings confirm an undescribed mechanism by which G9a inhibition acts at two levels, increasing GMFB and regulating its function to promote neuroprotective effects in age-related cognitive decline.

PRMT5 triggers glucocorticoid-induced cell migration in triple-negative breast cancer.

Triple-negative breast cancers (TNBCs) are the most aggressive breast cancers, and therapeutic options mainly rely on chemotherapy and immunotherapy. Although synthetic glucocorticoids (GCs) are given to alleviate the side effects of these treatments, GCs and their receptor, the glucocorticoid receptor (GR), were recently associated with detrimental effects, albeit the mechanisms involved remain elusive. Here, we identified the arginine methyltransferase PRMT5 as a master coregulator of GR, serving as a scaffold protein to recruit phospho-HP1γ and subsequently RNA polymerase II, independently of its methyltransferase activity. Moreover, the GR/PRMT5/HP1γ complex regulated the transcription of GC-target genes involved in cell motility and triggering cell migration of human TNBC cells in vitro and in a zebrafish model. Of note, we observed that GR/PRMT5 interaction was low in primary tumors but significantly increased in residual tumors treated with chemotherapy and GCs in neoadjuvant setting. These data suggest that the routine premedication prescription of GCs for early TNBC patients should be further assessed and that this complex could potentially be modulated to specifically target deleterious GR effects.

Nuclear PRMT5 is a biomarker of sensitivity to tamoxifen in ERα+ breast cancer.

Endocrine therapies targeting estrogen signaling, such as tamoxifen, have significantly improved management of estrogen receptor alpha (ERα)-positive breast cancers. However, their efficacy is limited by intrinsic and acquired resistance to treatment, and there is currently no predictive marker of response to these anti-estrogens to guide treatment decision. Here, using two independent cohorts of breast cancer patients, we identified nuclear PRMT5 expression as an independent predictive marker of sensitivity to tamoxifen. Mechanistically, we discovered that tamoxifen stimulates ERα methylation by PRMT5, a key event for its binding to corepressors such as SMRT and HDAC1, participating in the inhibition of the transcriptional activity of ERα. Although PRMT5 is mainly localized in the cytoplasm of tumor cells, our analyses show that tamoxifen triggers its nuclear translocation in tamoxifen-sensitive tumors but not in resistant ones. Hence, we unveil a biomarker of sensitivity to tamoxifen in ERα-positive breast tumors that could be used to enhance the response of breast cancer patients to endocrine therapy, by fostering its nuclear expression.

Transcriptional coactivation by EHMT2 restricts glucocorticoid-induced insulin resistance in a study with male mice.

The classical dogma of glucocorticoid-induced insulin resistance is that it is caused by the transcriptional activation of hepatic gluconeogenic and insulin resistance genes by the glucocorticoid receptor (GR). Here, we find that glucocorticoids also stimulate the expression of insulin-sensitizing genes, such as Irs2. The transcriptional coregulator EHMT2 can serve as a transcriptional coactivator or a corepressor. Using male mice that have a defective EHMT2 coactivation function specifically, we show that glucocorticoid-induced Irs2 transcription is dependent on liver EHMT2's coactivation function and that IRS2 play a key role in mediating the limitation of glucocorticoid-induced insulin resistance by EHMT2's coactivation. Overall, we propose a model in which glucocorticoid-regulated insulin sensitivity is determined by the balance between glucocorticoid-modulated insulin resistance and insulin sensitizing genes, in which EHMT2 coactivation is specifically involved in the latter process.

A hotspot for posttranslational modifications on the androgen receptor dimer interface drives pathology and anti-androgen resistance.

Mutations of the androgen receptor (AR) associated with prostate cancer and androgen insensitivity syndrome may profoundly influence its structure, protein interaction network, and binding to chromatin, resulting in altered transcription signatures and drug responses. Current structural information fails to explain the effect of pathological mutations on AR structure-function relationship. Here, we have thoroughly studied the effects of selected mutations that span the complete dimer interface of AR ligand-binding domain (AR-LBD) using x-ray crystallography in combination with in vitro, in silico, and cell-based assays. We show that these variants alter AR-dependent transcription and responses to anti-androgens by inducing a previously undescribed allosteric switch in the AR-LBD that increases exposure of a major methylation target, Arg761. We also corroborate the relevance of residues Arg761 and Tyr764 for AR dimerization and function. Together, our results reveal allosteric coupling of AR dimerization and posttranslational modifications as a disease mechanism with implications for precision medicine.

PRMT1, a Key Modulator of Unliganded Progesterone Receptor Signaling in Breast Cancer.

The progesterone receptor (PR) is a key player in major physiological and pathological responses in women, and the signaling pathways triggered following hormone binding have been extensively studied, particularly with respect to breast cancer development and progression. Interestingly, growing evidence suggests a fundamental role for PR on breast cancer cell homeostasis in hormone-depleted conditions, with hormone-free or unliganded PR (uPR) involved in the silencing of relevant genes prior to hormonal stimulation. We herein identify the protein arginine methyltransferase PRMT1 as a novel actor in uPR signaling. In unstimulated T47D breast cancer cells, PRMT1 interacts and functions alongside uPR and its partners to target endogenous progesterone-responsive promoters. PRMT1 helps to finely tune the silencing of responsive genes, likely by promoting a proper BRCA1-mediated degradation and turnover of unliganded PR. As such, PRMT1 emerges as a key transcriptional coregulator of PR for a subset of relevant progestin-dependent genes before hormonal treatment. Since women experience periods of hormonal fluctuation throughout their lifetime, understanding how steroid receptor pathways in breast cancer cells are regulated when hormones decline may help to determine how to override treatment failure to hormonal therapy and improve patient outcome.

Proximity Ligation Assay Allows the Detection, Localization, and Quantification of Protein Arginine Methylation in Fixed Tissue.

Arginine methylation is emerging as a key post-translational modification involved in a large range of biological processes. Its study in tissue is often limited by the lack of a specific antibody recognizing the target arginine residue. Proximity ligation assay (PLA) was originally developed to study protein/protein interactions. Here, we describe in detail a PLA protocol dedicated to the detection of arginine methylation that we applied to the glucocorticoid receptor (GR). Having previously shown that PRMT5 dimethylates GRs in cells, we used PLA with a pan symmetrical dimethyl antibody and an anti-GR antibody to measure GR methylation in breast tumors. We demonstrate that PLA offers a unique approach to measure arginine methylation of a target protein, even when the site of methylation has not been identified. This technique could be extended to other post-translational modifications where effective pan antibodies are available. Hence, we detail the PLA technology used to detect arginine methylation in fixed tissue using GR as an example.

MEN1 silencing triggers the dysregulation of mTORC1 and MYC pathways in ER+ breast cancer cells.

Menin, encoded by the MEN1 gene, has been identified as a critical factor regulating ESR1 transcription, playing an oncogenic role in ER+ breast cancer (BC) cells. Here, we further dissected the consequences of menin inactivation in ER+ BC cells by focusing on factors within two major pathways involved in BC, mTOR and MYC. MEN1 silencing in MCF7 and T-47D resulted in an increase in phosphor-p70S6K1, phosphor-p85S6K1 and phosphor-4EBP1 expression. The use of an AKT inhibitor inhibited the activation of S6K1 and S6RP triggered by MEN1 knockdown (KD). Moreover, MEN1 silencing in ER+ BC cells led to increased formation of the eIF4E and 4G complex. Clinical studies showed that patients with menin-low breast cancer receiving tamoxifen plus everolimus displayed a trend toward better overall survival. Importantly, MEN1 KD in MCF7 and T-47D cells led to reduced MYC expression. ChIP analysis demonstrated that menin bound not only to the MYC promoter but also to its 5' enhancer. Furthermore, E2-treated MEN1 KD MCF7 cells displayed a decrease in MYC activation, suggesting its role in estrogen-mediated MYC transcription. Finally, expression data mining in tumors revealed a correlation between the expression of MEN1 mRNA and that of several mTORC1 components and targets and a significant inverse correlation between MEN1 and two MYC inhibitory factors, MYCBP2 and MYCT1, in ER+ BC. The current work thus highlights altered mTORC1 and MYC pathways after menin inactivation in ER+ BC cells, providing insight into the crosstalk between menin, mTORC1 and MYC in ER+ BC.

PRMT1 Regulates EGFR and Wnt Signaling Pathways and Is a Promising Target for Combinatorial Treatment of Breast Cancer.

Identifying new therapeutic strategies for triple-negative breast cancer (TNBC) patients is a priority as these patients are highly prone to relapse after chemotherapy. Here, we found that protein arginine methyltransferase 1 (PRMT1) is highly expressed in all breast cancer subtypes. PRMT1 depletion decreases cell survival by inducing DNA damage and apoptosis in various breast cancer cell lines. Transcriptomic analysis and chromatin immunoprecipitation revealed that PRMT1 regulates the epidermal growth factor receptor (EGFR) and the Wnt signaling pathways, reported to be activated in TNBC. PRMT1 enzymatic activity is also required to stimulate the canonical Wnt pathway. Type I PRMT inhibitors decrease breast cancer cell proliferation and show anti-tumor activity in a TNBC xenograft model. These inhibitors display synergistic interactions with some chemotherapies used to treat TNBC patients as well as erlotinib, an EGFR inhibitor. Therefore, targeting PRMT1 in combination with these chemotherapies may improve existing treatments for TNBC patients.

How Protein Methylation Regulates Steroid Receptor Function.

Steroid receptors (SRs) are members of the nuclear hormonal receptor family, many of which are transcription factors regulated by ligand binding. SRs regulate various human physiological functions essential for maintenance of vital biological pathways, including development, reproduction, and metabolic homeostasis. In addition, aberrant expression of SRs or dysregulation of their signaling has been observed in a wide variety of pathologies. SR activity is tightly and finely controlled by post-translational modifications (PTMs) targeting the receptors and/or their coregulators. Whereas major attention has been focused on phosphorylation, growing evidence shows that methylation is also an important regulator of SRs. Interestingly, the protein methyltransferases depositing methyl marks are involved in many functions, from development to adult life. They have also been associated with pathologies such as inflammation, as well as cardiovascular and neuronal disorders, and cancer. This article provides an overview of SR methylation/demethylation events, along with their functional effects and biological consequences. An in-depth understanding of the landscape of these methylation events could provide new information on SR regulation in physiology, as well as promising perspectives for the development of new therapeutic strategies, illustrated by the specific inhibitors of protein methyltransferases that are currently available.

Targeting AKT in ER-Positive HER2-Negative Metastatic Breast Cancer: From Molecular Promises to Real Life Pitfalls?

The AKT protein kinase plays a central role in several interconnected molecular pathways involved in growth, apoptosis, angiogenesis, and cell metabolism. It thereby represents a therapeutic target, especially in hormone receptor-positive (HR) breast cancers, where the PI3K/AKT signaling pathway is largely hyperactivated. Moreover, resistance to therapeutic classes, including endocrine therapy, is associated with the constitutive activation of the PI3K/AKT pathway. Improved knowledge on the molecular mechanisms underlying resistance to endocrine therapy has led to the diversification of the therapeutic arsenal, notably with the development of PI3K and mTOR inhibitors, which are currently approved for the treatment of advanced HR-positive breast cancer patients. AKT itself constitutes a novel pharmacological target for which AKT inhibitors have been developed and tested in clinical trials. However, despite its pivotal role in cell survival and anti-apoptotic mechanisms, as well as in endocrine therapy resistance, few drugs have been developed and are available for clinical practice. The scope of the present review is to focus on the pivotal role of AKT in metastatic breast cancer through the analysis of its molecular features and to discuss clinical implications and remaining challenges in the treatment of HR-positive metastatic breast cancer.

Structure, Activity, and Function of PRMT1.

PRMT1, the major protein arginine methyltransferase in mammals, catalyzes monomethylation and asymmetric dimethylation of arginine side chains in proteins. Initially described as a regulator of chromatin dynamics through the methylation of histone H4 at arginine 3 (H4R3), numerous non-histone substrates have since been identified. The variety of these substrates underlines the essential role played by PRMT1 in a large number of biological processes such as transcriptional regulation, signal transduction or DNA repair. This review will provide an overview of the structural, biochemical and cellular features of PRMT1. After a description of the genomic organization and protein structure of PRMT1, special consideration was given to the regulation of PRMT1 enzymatic activity. Finally, we discuss the involvement of PRMT1 in embryonic development, DNA damage repair, as well as its participation in the initiation and progression of several types of cancers.

Structure, Activity, and Function of the Protein Lysine Methyltransferase G9a.

G9a is a lysine methyltransferase catalyzing the majority of histone H3 mono- and dimethylation at Lys-9 (H3K9), responsible for transcriptional repression events in euchromatin. G9a has been shown to methylate various lysine residues of non-histone proteins and acts as a coactivator for several transcription factors. This review will provide an overview of the structural features of G9a and its paralog called G9a-like protein (GLP), explore the biochemical features of G9a, and describe its post-translational modifications and the specific inhibitors available to target its catalytic activity. Aside from its role on histone substrates, the review will highlight some non-histone targets of G9a, in order gain insight into their role in specific cellular mechanisms. Indeed, G9a was largely described to be involved in embryonic development, hypoxia, and DNA repair. Finally, the involvement of G9a in cancer biology will be presented.

Non-genomic signaling of steroid receptors in cancer.

Steroid receptors (SRs) are members of the nuclear receptor family, which are ligand-activated transcription factors. SRs regulate many physiological functions including development and reproduction, though they can also be involved in several pathologies, especially cancer. Highly controlled cellular responses to steroids involve transcriptional regulation (genomic activity) combined with direct activation of signaling cascades (non-genomic activity). Non-genomic signaling has been extensively studied in cancer, mainly in breast cancer for ER and PR, and prostate cancer for AR. Even though most of the studies have been conducted in cells, some of them have been confirmed in vivo, highlighting the relevance of this pathway in cancer. This review provides an overview of the current and emerging knowledge on non-genomic signaling with a focus on breast and prostate cancers and its clinical relevance. A thorough understanding of ER, PR, AR and GR non-genomic pathways may open new perspectives for the development of therapeutic strategies.

Analysis of genomic and non-genomic signaling of estrogen receptor in PDX models of breast cancer treated with a combination of the PI3K inhibitor alpelisib (BYL719) and fulvestrant.

BACKGROUND: Endocrine therapies targeting estrogen signaling have significantly improved breast cancer (BC) patient survival, although 40% of ERα-positive BCs do not respond to those therapies. Aside from genomic signaling, estrogen triggers non-genomic pathways by forming a complex containing methylERα/Src/PI3K, a hallmark of aggressiveness and resistance to tamoxifen. We aimed to confirm the prognostic value of this complex and investigated whether its targeting could improve tumor response in vivo. METHODS: The interaction of ERα/Src and ERα/PI3K was studied by proximity ligation assay (PLA) in a cohort of 440 BC patients. We then treated patient-derived BC xenografts (PDXs) with fulvestrant or the PI3K inhibitor alpelisib (BYL719) alone or in combination. We analyzed their anti-proliferative effects on 6 ERα+ and 3 ERα- PDX models. Genomic and non-genomic estrogen signaling were assessed by measuring ERα/PI3K interaction by PLA and the expression of estrogen target genes by RT-QPCR, respectively. RESULTS: We confirmed that ERα/Src and ERα/PI3K interactions were associated with a trend to poorer survival, the latter displaying the most significant effects. In ERα+ tumors, the combination of BYL719 and fulvestrant was more effective than fulvestrant alone in 3 models, irrespective of PI3K, PTEN status, or ERα/PI3K targeting. Remarkably, resistance to fulvestrant was associated with non-genomic ERα signaling, since genomic degradation of ERα was unaltered in these tumors, whereas the treatment did not diminish the level of ERα/PI3K interaction. Interestingly, in 2 ERα- models, fulvestrant alone impacted tumor growth, and this was associated with a decrease in ERα/PI3K interaction. CONCLUSIONS: Our results demonstrate that ERα/PI3K may constitute a new prognostic marker, as well as a new target in BC. Indeed, resistance to fulvestrant in ERα+ tumors was associated with a lack of impairment of ERα/PI3K interaction in the cytoplasm. In addition, an efficient targeting of ERα/PI3K in ERα- tumors could constitute a promising therapeutic option.

Glucocorticoid Receptor: A Multifaceted Actor in Breast Cancer.

Breast cancer (BC) is one of the most common cancers in women worldwide. Even though the role of estrogen receptor alpha (ERα) is extensively documented in the development of breast tumors, other members of the nuclear receptor family have emerged as important players. Synthetic glucocorticoids (GCs) such as dexamethasone (dex) are commonly used in BC for their antiemetic, anti-inflammatory, as well as energy and appetite stimulating properties, and to manage the side effects of chemotherapy. However, dex triggers different effects depending on the BC subtype. The glucocorticoid receptor (GR) is also an important marker in BC, as high GR expression is correlated with a poor and good prognosis in ERα-negative and ERα-positive BCs, respectively. Indeed, though it drives the expression of pro-tumorigenic genes in ERα-negative BCs and is involved in resistance to chemotherapy and metastasis formation, dex inhibits estrogen-mediated cell proliferation in ERα-positive BCs. Recently, a new natural ligand for GR called OCDO was identified. OCDO is a cholesterol metabolite with oncogenic properties, triggering mammary cell proliferation in vitro and in vivo. In this review, we summarize recent data on GR signaling and its involvement in tumoral breast tissue, via its different ligands.

PRMT1 Is Critical for the Transcriptional Activity and the Stability of the Progesterone Receptor.

The progesterone receptor (PR) is an inducible transcription factor that plays critical roles in female reproductive processes and in several aspects of breast cancer tumorigenesis. Our report describes the type I protein arginine methyltransferase 1 (PRMT1) as a cofactor controlling progesterone pathway, through the direct methylation of PR. Mechanistic assays in breast cancer cells indicate that PRMT1 methylates PR at the arginine 637 and reduces the stability of the receptor, thereby accelerating its recycling and finally its transcriptional activity. Depletion of PRMT1 decreases the expression of a subset of progesterone-inducible genes, controlling breast cancer cells proliferation and migration. Consistently, Kaplan-Meier analysis revealed that low expression of PRMT1 predicts a longer survival among the subgroup with high PR. Our study highlights PR methylation as a molecular switch adapting the transcription requirement of breast cells during tumorigenesis.

ERα-36 regulates progesterone receptor activity in breast cancer.

BACKGROUND: Alterations in estrogen and progesterone signaling, via their respective receptors, estrogen receptor alpha (ERα) and progesterone receptor (PR), respectively, are largely involved in the development of breast cancer (BC). The recent identification of ERα-36, a splice variant of ERα, has uncovered a new facet of this pathology. Although ERα-36 expression is associated with poor prognosis, metastasis development, and resistance to treatment, its predictive value has so far not been associated with a BC subtype and its mechanisms of action remain understudied. METHODS: To study ERα-36 expression in BC specimens, we performed immunochemical experiments. Next, the role of ERα-36 in progesterone signaling was investigated by generating KO clones using the CRISPR/CAS9 technology. PR signaling was also assessed by proximity ligation assay, Western blotting, RT-QPCR, and ChIP experiments. Finally, proliferation assays were performed with the IncuCyte technology and migration experiments using scratch assays. RESULTS: Here, we demonstrate that ERα-36 expression at the plasma membrane is correlated with a reduced disease-free survival in a cohort of 160 BC patients, particularly in PR-positive tumors, suggesting a crosstalk between ERα-36 and PR. Indeed, we show that ERα-36 interacts constitutively with PR in the nucleus of tumor cells. Moreover, it regulates PR expression and phosphorylation on key residues, impacting the biological effects of progesterone. CONCLUSIONS: ERα-36 is thus a regulator of PR signaling, interfering with its transcriptional activity and progesterone-induced anti-proliferative effects as well as migratory capacity. Hence, ERα-36 may constitute a new prognostic marker as well as a potential target in PR-positive BC.