Sustained inactivation of the Polycomb PRC1 complex induces DNA repair defects and genomic instability in epigenetic tumors.

Cancer initiation and progression are typically associated with the accumulation of driver mutations and genomic instability. However, recent studies demonstrated that cancer can also be driven purely by epigenetic alterations, without driver mutations. Specifically, a 24-h transient downregulation of polyhomeotic (ph-KD), a core component of the Polycomb complex PRC1, is sufficient to induce epigenetically initiated cancers (EICs) in Drosophila, which are proficient in DNA repair and characterized by a stable genome. Whether genomic instability eventually occurs when PRC1 downregulation is performed for extended periods of time remains unclear. Here, we show that prolonged depletion of PH, which mimics cancer initiating events, results in broad dysregulation of DNA replication and repair genes, along with the accumulation of DNA breaks, defective repair, and widespread genomic instability in the cancer tissue. A broad misregulation of H2AK118 ubiquitylation and to a lesser extent of H3K27 trimethylation also occurs and might contribute to these phenotypes. Together, this study supports a model where DNA repair and replication defects accumulate during the tumorigenic transformation epigenetically induced by PRC1 loss, resulting in genomic instability and cancer progression.

Sustained inactivation of the Polycomb PRC1 complex induces DNA repair defects and genomic instability in epigenetic tumors.

Cancer initiation and progression are typically associated with the accumulation of driver mutations and genomic instability. However, recent studies demonstrated that cancers can also be purely initiated by epigenetic alterations, without driver mutations. Specifically, a 24-hours transient down-regulation of polyhomeotic (ph-KD), a core component of the Polycomb complex PRC1, is sufficient to drive epigenetically initiated cancers (EICs) in Drosophila, which are proficient in DNA repair and are characterized by a stable genome. Whether genomic instability eventually occurs when PRC1 down-regulation is performed for extended periods of time remains unclear. Here we show that prolonged depletion of a PRC1 component, which mimics cancer initiating events, results in broad dysregulation of DNA replication and repair genes, along with the accumulation of DNA breaks, defective repair, and widespread genomic instability in the cancer tissue. A broad mis-regulation of H2AK118 ubiquitylation and to a lesser extent of H3K27 trimethylation also occurs, and might contribute to these phenotypes. Together, this study supports a model where DNA repair and replication defects amplify the tumorigenic transformation epigenetically induced by PRC1 loss, resulting in genomic instability and cancer progression.

Loss of SUMO-specific protease 2 causes isolated glucocorticoid deficiency by blocking adrenal cortex zonal transdifferentiation in mice.

SUMOylation is a dynamic posttranslational modification, that provides fine-tuning of protein function involved in the cellular response to stress, differentiation, and tissue development. In the adrenal cortex, an emblematic endocrine organ that mediates adaptation to physiological demands, the SUMOylation gradient is inversely correlated with the gradient of cellular differentiation raising important questions about its role in functional zonation and the response to stress. Considering that SUMO-specific protease 2 (SENP2), a deSUMOylating enzyme, is upregulated by Adrenocorticotropic Hormone (ACTH)/cAMP-dependent Protein Kinase (PKA) signalling within the zona fasciculata, we generated mice with adrenal-specific Senp2 loss to address these questions. Disruption of SENP2 activity in steroidogenic cells leads to specific hypoplasia of the zona fasciculata, a blunted reponse to ACTH and isolated glucocorticoid deficiency. Mechanistically, overSUMOylation resulting from SENP2 loss shifts the balance between ACTH/PKA and WNT/ß-catenin signalling leading to repression of PKA activity and ectopic activation of ß-catenin. At the cellular level, this blocks transdifferentiation of ß-catenin-positive zona glomerulosa cells into fasciculata cells and sensitises them to premature apoptosis. Our findings indicate that the SUMO pathway is critical for adrenal homeostasis and stress responsiveness.

Sexually dimorphic activation of innate antitumor immunity prevents adrenocortical carcinoma development.

Unlike most cancers, adrenocortical carcinomas (ACCs) are more frequent in women than in men, but the underlying mechanisms of this sexual dimorphism remain elusive. Here, we show that inactivation of Znrf3 in the mouse adrenal cortex, recapitulating the most frequent alteration in ACC patients, is associated with sexually dimorphic tumor progression. Although female knockouts develop metastatic carcinomas at 18 months, adrenal hyperplasia regresses in male knockouts. This male-specific phenotype is associated with androgen-dependent induction of senescence, recruitment, and differentiation of highly phagocytic macrophages that clear out senescent cells. In contrast, in females, macrophage recruitment is delayed and dampened, which allows for aggressive tumor progression. Consistently, analysis of TCGA-ACC data shows that phagocytic macrophages are more prominent in men and are associated with better prognosis. Together, these data show that phagocytic macrophages are key players in the sexual dimorphism of ACC that could be previously unidentified allies in the fight against this devastating cancer.

Mislocalization of protein kinase A drives pathology in Cushing's syndrome.

Mutations in the catalytic subunit of protein kinase A (PKAc) drive the stress hormone disorder adrenal Cushing's syndrome. We define mechanisms of action for the PKAc-L205R and W196R variants. Proximity proteomic techniques demonstrate that both Cushing's mutants are excluded from A kinase-anchoring protein (AKAP)-signaling islands, whereas live-cell photoactivation microscopy reveals that these kinase mutants indiscriminately diffuse throughout the cell. Only cAMP analog drugs that displace native PKAc from AKAPs enhance cortisol release. Rescue experiments that incorporate PKAc mutants into AKAP complexes abolish cortisol overproduction, indicating that kinase anchoring restores normal endocrine function. Analyses of adrenal-specific PKAc-W196R knockin mice and Cushing's syndrome patient tissue reveal defective signaling mechanisms of the disease. Surprisingly each Cushing's mutant engages a different mitogenic-signaling pathway, with upregulation of YAP/TAZ by PKAc-L205R and ERK kinase activation by PKAc-W196R. Thus, aberrant spatiotemporal regulation of each Cushing's variant promotes the transmission of distinct downstream pathogenic signals.

Steroidogenic Factor-1 Lineage Origin of Skin Lesions in Carney Complex Syndrome.

Carney complex is a rare familial multineoplastic syndrome predisposing to endocrine and nonendocrine tumors due to inactivating mutations of PRKAR1A, leading to perturbations of the cAMP‒protein kinase A signaling pathway. Skin lesions are the most common manifestation of Carney complex, including lentigines, blue nevi, and cutaneous myxomas in unusual locations such as oral and genital mucosa. Unlike endocrine disorders, the pathogenesis of skin lesions remains unexplained. In this study, we show that embryonic invalidation of the Prkar1a gene in steroidogenic factor-1‒expressing cells leads to the development of familial skin pigmentation alterations, reminiscent of those in patients with Carney complex. Immunohistological and molecular analyses, coupled with genetic monitoring of recombinant cell lineages in mouse skin, suggest that familial lentiginosis and myxomas occur in skin areas specifically enriched in dermal melanocytes. In lentigines- and blue nevi‒prone areas from mutant mice and patients, Prkar1a/PRKAR1A invalidation occurs in a subset of dermal fibroblasts capable of inducing, under the influence of protein kinase A signaling, the production of promelanogenic EDN3 and hepatocyte GF signals. Our model strongly suggests that the origin of the typical Carney complex cutaneous lesions is the result of noncell-autonomous promelanogenic activity of a dermal fibroblast population sharing a community of origin with steroidogenic factor-1 lineage.

Comprehensive characterization of the epigenetic landscape in Multiple Myeloma.

Background: Human multiple myeloma (MM) cell lines (HMCLs) have been widely used to understand the molecular processes that drive MM biology. Epigenetic modifications are involved in MM development, progression, and drug resistance. A comprehensive characterization of the epigenetic landscape of MM would advance our understanding of MM pathophysiology and may attempt to identify new therapeutic targets. Methods: We performed chromatin immunoprecipitation sequencing to analyze histone mark changes (H3K4me1, H3K4me3, H3K9me3, H3K27ac, H3K27me3 and H3K36me3) on 16 HMCLs. Results: Differential analysis of histone modification profiles highlighted links between histone modifications and cytogenetic abnormalities or recurrent mutations. Using histone modifications associated to enhancer regions, we identified super-enhancers (SE) associated with genes involved in MM biology. We also identified promoters of genes enriched in H3K9me3 and H3K27me3 repressive marks associated to potential tumor suppressor functions. The prognostic value of genes associated with repressive domains and SE was used to build two distinct scores identifying high-risk MM patients in two independent cohorts (CoMMpass cohort; n = 674 and Montpellier cohort; n = 69). Finally, we explored H3K4me3 marks comparing drug-resistant and -sensitive HMCLs to identify regions involved in drug resistance. From these data, we developed epigenetic biomarkers based on the H3K4me3 modification predicting MM cell response to lenalidomide and histone deacetylase inhibitors (HDACi). Conclusions: The epigenetic landscape of MM cells represents a unique resource for future biological studies. Furthermore, risk-scores based on SE and repressive regions together with epigenetic biomarkers of drug response could represent new tools for precision medicine in MM.

Mechanisms of Polycomb group protein function in cancer.

Cancer arises from a multitude of disorders resulting in loss of differentiation and a stem cell-like phenotype characterized by uncontrolled growth. Polycomb Group (PcG) proteins are members of multiprotein complexes that are highly conserved throughout evolution. Historically, they have been described as essential for maintaining epigenetic cellular memory by locking homeotic genes in a transcriptionally repressed state. What was initially thought to be a function restricted to a few target genes, subsequently turned out to be of much broader relevance, since the main role of PcG complexes is to ensure a dynamically choregraphed spatio-temporal regulation of their numerous target genes during development. Their ability to modify chromatin landscapes and refine the expression of master genes controlling major switches in cellular decisions under physiological conditions is often misregulated in tumors. Surprisingly, their functional implication in the initiation and progression of cancer may be either dependent on Polycomb complexes, or specific for a subunit that acts independently of other PcG members. In this review, we describe how misregulated Polycomb proteins play a pleiotropic role in cancer by altering a broad spectrum of biological processes such as the proliferation-differentiation balance, metabolism and the immune response, all of which are crucial in tumor progression. We also illustrate how interfering with PcG functions can provide a powerful strategy to counter tumor progression.

Protein kinase A drives paracrine crisis and WNT4-dependent testis tumor in Carney complex.

Large-cell calcifying Sertoli cell tumors (LCCSCTs) are among the most frequent lesions occurring in male Carney complex (CNC) patients. Although they constitute a key diagnostic criterion for this rare multiple neoplasia syndrome resulting from inactivating mutations of the tumor suppressor PRKAR1A, leading to unrepressed PKA activity, LCCSCT pathogenesis and origin remain elusive. Mouse models targeting Prkar1a inactivation in all somatic populations or separately in each cell type were generated to decipher the molecular and paracrine networks involved in the induction of CNC testis lesions. We demonstrate that the Prkar1a mutation was required in both stromal and Sertoli cells for the occurrence of LCCSCTs. Integrative analyses comparing transcriptomic, immunohistological data and phenotype of mutant mouse combinations led to the understanding of human LCCSCT pathogenesis and demonstrated PKA-induced paracrine molecular circuits in which the aberrant WNT4 signal production is a limiting step in shaping intratubular lesions and tumor expansion both in a mouse model and in human CNC testes.

Co-culture of monocytes and zona fasciculata adrenal cells: An in vitro model to study the immune-adrenal cross-talk.

The hypothalamic-pituitary-adrenal axis is the primary neuroendocrine system activated to re-establish homeostasis during periods of stress, including critical illness and major surgery. During critical illness, evidence suggests that locally induced inflammation of the adrenal gland could facilitate immune-adrenal cross-talk and, in turn, modulate cortisol secretion. It has been hypothesized that immune cells are necessary to mediate the effect of inflammatory stimuli on the steroidogenic pathway that has been observed in vivo. To test this hypothesis, we developed and characterized a trans-well co-culture model of THP1 (human monocytic cell)-derived macrophages and ATC7 murine zona fasciculata adrenocortical cells. We found that co-culture of ATC7 and THP1 cells results in a significant increase in the basal levels of IL-6 mRNA in ATC7 cells, and this effect was potentiated by treatment with LPS. Addition of LPS to co-cultures of ATC7 and THP1 significantly decreased the expression of key adrenal steroidogenic enzymes (including StAR and DAX-1), and this was also found in ATC7 cells treated with pro-inflammatory cytokines. Moreover, 24-h treatment with the synthetic glucocorticoid dexamethasone prevented the effects of LPS stimulation on IL-6, StAR and DAX-1 mRNA in ATC7 cells co-cultured with THP1 cells. Our data suggest that the expression of IL-6 and steroidogenic genes in response to LPS depends on the activation of intra-adrenal immune cells. Moreover, we also show that the effects of LPS can be modulated by glucocorticoids in a time- and dose-dependent manner with potential implications for clinical practice.

Having Therapeutic Conversations With Fathers Grieving the Death of a Child.

A child's death is a traumatic life experience for parents. Health-care professionals (HCPs) have sought guidance on how to intervene with grieving parents, particularly with fathers. Having therapeutic conversations is an effective way for HCPs to support grieving fathers. In our previous study, fathers identified core beliefs that influenced their experience of grief and coping. In this article, the Illness Beliefs Model was integrated with the findings to provide a framework for interventions to create open conversations, ease fathers' suffering, and thereby help their spouse and family suffering as well. This article will guide HCPs to engage in therapeutic conversations to support bereaved fathers.

HOX genes promote cell proliferation and are potential therapeutic targets in adrenocortical tumours.

BACKGROUND: Understanding the pathways that drive adrenocortical carcinoma (ACC) is essential to the development of more effective therapies. This study investigates the role of the transcription factor HOXB9 and other HOX factors in ACC and its treatment. METHODS: We used transgenic mouse models to determine the role of Hoxb9 in adrenal tumour development. Patient transcriptomic data was analysed for the expression of HOX genes and their association with disease. Drug response studies on various adrenocortical models were done to establish novel therapeutic options. RESULTS: Our human ACC dataset analyses showed high expression of HOXB9, and other HOX factors, are associated with poorer prognosis. Transgenic overexpression of Hoxb9 in the adrenal cortex of mice with activated Ctnnb1 led to larger adrenal tumours. This phenotype was preferentially observed in male mice and was characterised by more proliferating cells and an increase in the expression of cell cycle genes, including Ccne1. Adrenal tumour cells were found to be dependent on HOX function for survival and were sensitive to a specific peptide inhibitor. CONCLUSIONS: These studies show Hoxb9 can promote adrenal tumour progression in a sex-dependent manner and have identified HOX factors as potential drug targets, leading to novel therapeutic approaches in ACC.

Role of Polycomb Complexes in Normal and Malignant Plasma Cells.

Plasma cells (PC) are the main effectors of adaptive immunity, responsible for producing antibodies to defend the body against pathogens. They are the result of a complex highly regulated cell differentiation process, taking place in several anatomical locations and involving unique genetic events. Pathologically, PC can undergo tumorigenesis and cause a group of diseases known as plasma cell dyscrasias, including multiple myeloma (MM). MM is a severe disease with poor prognosis that is characterized by the accumulation of malignant PC within the bone marrow, as well as high clinical and molecular heterogeneity. MM patients frequently develop resistance to treatment, leading to relapse. Polycomb group (PcG) proteins are epigenetic regulators involved in cell fate and carcinogenesis. The emerging roles of PcG in PC differentiation and myelomagenesis position them as potential therapeutic targets in MM. Here, we focus on the roles of PcG proteins in normal and malignant plasma cells, as well as their therapeutic implications.

Involvement of CREB-regulated transcription coactivators (CRTC) in transcriptional activation of steroidogenic acute regulatory protein (Star) by ACTH.

Studies in vivo have suggested the involvement of CREB-regulated transcription coactivator (CRTC)2 on ACTH-induced transcription of the key steroidogenic protein, Steroidogenic Acute Regulatory (StAR). The present study uses two ACTH-responsive adrenocortical cell lines, to examine the role of CRTC on Star transcription. Here we show that ACTH-induced Star primary transcript, or heteronuclear RNA (hnRNA), parallels rapid increases in nuclear levels of the 3 isoforms of CRTC; CRTC1, CRTC2 and CRTC3. Furthermore, ACTH promotes recruitment of CRTC2 and CRTC3 by the Star promoter and siRNA knockdown of either CRTC3 or CRTC2 attenuates the increases in ACTH-induced Star hnRNA. Using pharmacological inhibitors of PKA, MAP kinase and calcineurin, we show that the effects of ACTH on Star transcription and CRTC nuclear translocation depend predominantly on the PKA pathway. The data provides evidence that CRTC2 and CRTC3, contribute to activation of Star transcription by ACTH, and that PKA/CRTC-dependent pathways are part of the multifactorial mechanisms regulating Star transcription.

EZH2 cooperates with E2F1 to stimulate expression of genes involved in adrenocortical carcinoma aggressiveness.

BACKGROUND: EZH2 is overexpressed and associated with poor prognosis in adrenocortical carcinoma (ACC) and its inhibition reduces growth and aggressiveness of ACC cells in culture. Although EZH2 was identified as the methyltransferase that deposits the repressive H3K27me3 histone mark, it can cooperate with transcription factors to stimulate gene transcription. METHODS: We used bioinformatics approaches on gene expression data from three cohorts of patients and a mouse model of EZH2 ablation, to identify targets and mode of action of EZH2 in ACC. This was followed by ChIP and functional assays to evaluate contribution of identified targets to ACC pathogenesis. RESULTS: We show that EZH2 mostly works as a transcriptional inducer in ACC, through cooperation with the transcription factor E2F1 and identify three positive targets involved in cell cycle regulation and mitosis i.e., RRM2, PTTG1 and ASE1/PRC1. Overexpression of these genes is associated with poor prognosis, suggesting a potential role in acquisition of aggressive ACC features. Pharmacological and siRNA-mediated inhibition of RRM2 blocks cell proliferation, induces apoptosis and inhibits cell migration, suggesting that it may be an interesting target in ACC. CONCLUSIONS: Altogether, these data show an unexpected role of EZH2 and E2F1 in stimulating expression of genes associated with ACC aggressiveness.

Hormonal and spatial control of SUMOylation in the human and mouse adrenal cortex.

SUMOylation is a highly conserved and dynamic post-translational mechanism primarily affecting nuclear programs for adapting organisms to stressful challenges. Alteration of SUMOylation cycles leads to severe developmental and homeostatic defects and malignancy, but signals coordinating SUMOylation are still unidentified. The adrenal cortex is a zonated endocrine gland that controls body homeostasis and stress response. Here, we show that in human and in mouse adrenals, SUMOylation follows a decreasing centripetal gradient that mirrors cortical differentiation flow and delimits highly and weakly SUMOylated steroidogenic compartments, overlapping glomerulosa, and fasciculata zones. Activation of PKA signaling by acute hormonal treatment, mouse genetic engineering, or in Carney complex results in repression of small ubiquitin-like modifier (SUMO) conjugation in the inner cortex by coordinating expression of SUMO pathway inducers and repressors. Conversely, genetic activation of canonical wingless-related integration site signaling maintains high SUMOylation potential in the outer neoplastic cortex. Thus, SUMOylation is tightly regulated by signaling pathways that orchestrate adrenal zonation and diseases.-Dumontet, T., Sahut-Barnola, I., Dufour, D., Lefrançois-Martinez, A.-M., Berthon, A., Montanier, N., Ragazzon, B., Djari, C., Pointud, J.-C., Roucher-Boulez, F., Batisse-Lignier, M., Tauveron, I., Bertherat, J., Val, P., Martinez, A. Hormonal and spatial control of SUMOylation in the human and mouse adrenal cortex.

Cell Fate and Developmental Regulation Dynamics by Polycomb Proteins and 3D Genome Architecture.

Targeted transitions in chromatin states at thousands of genes are essential drivers of eukaryotic development. Therefore, understanding the in vivo dynamics of epigenetic regulators is crucial for deciphering the mechanisms underpinning cell fate decisions. This review illustrates how, in addition to its cell memory function, the Polycomb group of transcriptional regulators orchestrates temporal, cell and tissue-specific expression of master genes during development. These highly sophisticated developmental transitions are dependent on the context- and tissue-specific assembly of the different types of Polycomb Group (PcG) complexes, which regulates their targeting and/or activities on chromatin. Here, an overview is provided of how PcG complexes function at multiple scales to regulate transcription, local chromatin environment, and higher order structures that support normal differentiation and are perturbed in tumorigenesis.

EZH2 is overexpressed in transitional preplasmablasts and is involved in human plasma cell differentiation.

Plasma cells (PCs) play a major role in the defense of the host organism against pathogens. We have shown that PC generation can be modeled using multi-step culture systems that reproduce the sequential cell differentiation occurring in vivo. Using this unique model, we investigated the role of EZH2 during PC differentiation (PCD) using H3K27me3 and EZH2 ChIP-binding profiles. We then studied the effect of the inhibition of EZH2 enzymatic activity to understand how EZH2 regulates the key functions involved in PCD. EZH2 expression significantly increases in preplasmablasts with H3K27me3 mediated repression of genes involved in B cell and plasma cell identity. EZH2 was also found to be recruited to H3K27me3-free promoters of transcriptionally active genes known to regulate cell proliferation. Inhibition the catalytic activity of EZH2 resulted in B to PC transcriptional changes associated with PC maturation induction, as well as higher immunoglobulin secretion. Altogether, our data suggest that EZH2 is involved in the maintenance of preplasmablast transitory immature proliferative state that supports their amplification.

Steroidogenic differentiation and PKA signaling are programmed by histone methyltransferase EZH2 in the adrenal cortex.

Adrenal cortex steroids are essential for body homeostasis, and adrenal insufficiency is a life-threatening condition. Adrenal endocrine activity is maintained through recruitment of subcapsular progenitor cells that follow a unidirectional differentiation path from zona glomerulosa to zona fasciculata (zF). Here, we show that this unidirectionality is ensured by the histone methyltransferase EZH2. Indeed, we demonstrate that EZH2 maintains adrenal steroidogenic cell differentiation by preventing expression of GATA4 and WT1 that cause abnormal dedifferentiation to a progenitor-like state in Ezh2 KO adrenals. EZH2 further ensures normal cortical differentiation by programming cells for optimal response to adrenocorticotrophic hormone (ACTH)/PKA signaling. This is achieved by repression of phosphodiesterases PDE1B, 3A, and 7A and of PRKAR1B. Consequently, EZH2 ablation results in blunted zF differentiation and primary glucocorticoid insufficiency. These data demonstrate an all-encompassing role for EZH2 in programming steroidogenic cells for optimal response to differentiation signals and in maintaining their differentiated state.

Adrenocortical development: Lessons from mouse models.

The adrenocortical gland undergoes structural and functional remodelling in the fetal and postnatal periods. After birth, the fetal zone of the gland undergoes rapid involution in favor of the definitive cortex, which reaches maturity with the emergence of the zona reticularis(zR) at the adrenarche. The mechanisms underlying the adrenarche, the process leading to pre-puberty elevation of plasma androgens in higher primates, remain unknown, largely due to lack of any experimental model. By following up fetal and definitive cortex cell lines in mice, we showed that activation of protein kinase A (PKA) signaling mainly impacts the adult cortex by stimulating centripetal regeneration, with differentiation and then conversion of the zona fasciculata into a functional zR. Animals developed Cushing syndrome associated with primary hyperaldosteronism, suggesting possible coexistence of these hypersecretions in certain patients. Remarkably, all of these traits were sex-dependent: testicular androgens promoted WNT signaling antagonism on PKA, slowing cortical renewal and delaying onset of Cushing syndrome and the establishment of the zR in male mice, this being corrected by orchidectomy. In conclusion, zR derives from centripetal conversion of the zona fasciculata under cellular renewal induced by PKA signaling, determining the size of the adult cortex. Finally, we demonstrated that this PKA-dependent mobilization of cortical progenitors is sexually dimorphic and could, if confirmed in humans, account for female preponderance in adrenocortical pathologies.