Publications by authors named "Jorma J Palvimo"

117 Publications

Reprogramming of glucocorticoid receptor function by hypoxia.

EMBO Rep 2022 Jan 26;23(1):e53083. Epub 2021 Oct 26.

VIB Center for Inflammation Research, Ghent, Belgium.

Here, we investigate the impact of hypoxia on the hepatic response of glucocorticoid receptor (GR) to dexamethasone (DEX) in mice via RNA-sequencing. Hypoxia causes three types of reprogramming of GR: (i) much weaker induction of classical GR-responsive genes by DEX in hypoxia, (ii) a number of genes is induced by DEX specifically in hypoxia, and (iii) hypoxia induces a group of genes via activation of the hypothalamic-pituitary-adrenal (HPA) axis. Transcriptional profiles are reflected by changed GR DNA-binding as measured by ChIP sequencing. The HPA axis is induced by hypothalamic HIF1α and HIF2α activation and leads to GR-dependent lipolysis and ketogenesis. Acute inflammation, induced by lipopolysaccharide, is prevented by DEX in normoxia but not during hypoxia, and this is attributed to HPA axis activation by hypoxia. We unfold new physiological pathways that have consequences for patients suffering from GC resistance.
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http://dx.doi.org/10.15252/embr.202153083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8728616PMC
January 2022

The androgen receptor depends on ligand-binding domain dimerization for transcriptional activation.

EMBO Rep 2021 Dec 18;22(12):e52764. Epub 2021 Oct 18.

Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium.

Whereas dimerization of the DNA-binding domain of the androgen receptor (AR) plays an evident role in recognizing bipartite response elements, the contribution of the dimerization of the ligand-binding domain (LBD) to the correct functioning of the AR remains unclear. Here, we describe a mouse model with disrupted dimerization of the AR LBD (AR ). The disruptive effect of the mutation is demonstrated by the feminized phenotype, absence of male accessory sex glands, and strongly affected spermatogenesis, despite high circulating levels of testosterone. Testosterone replacement studies in orchidectomized mice demonstrate that androgen-regulated transcriptomes in AR mice are completely lost. The mutated AR still translocates to the nucleus and binds chromatin, but does not bind to specific AR binding sites. In vitro studies reveal that the mutation in the LBD dimer interface also affects other AR functions such as DNA binding, ligand binding, and co-regulator binding. In conclusion, LBD dimerization is crucial for the development of AR-dependent tissues through its role in transcriptional regulation in vivo. Our findings identify AR LBD dimerization as a possible target for AR inhibition.
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http://dx.doi.org/10.15252/embr.202152764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8647150PMC
December 2021

Combined glucocorticoid resistance and hyperlactatemia contributes to lethal shock in sepsis.

Cell Metab 2021 Sep 23;33(9):1763-1776.e5. Epub 2021 Jul 23.

VIB Center for Inflammation Research, VIB, Ghent 9052, Belgium; Department of Biomedical Molecular Biology, Ghent University, Ghent 9052, Belgium. Electronic address:

Sepsis is a potentially lethal syndrome resulting from a maladaptive response to infection. Upon infection, glucocorticoids are produced as a part of the compensatory response to tolerate sepsis. This tolerance is, however, mitigated in sepsis due to a quickly induced glucocorticoid resistance at the level of the glucocorticoid receptor. Here, we show that defects in the glucocorticoid receptor signaling pathway aggravate sepsis pathophysiology by lowering lactate clearance and sensitizing mice to lactate-induced toxicity. The latter is exerted via an uncontrolled production of vascular endothelial growth factor, resulting in vascular leakage and collapse with severe hypotension, organ damage, and death, all being typical features of a lethal form of sepsis. In conclusion, sepsis leads to glucocorticoid receptor failure and hyperlactatemia, which collectively leads to a lethal vascular collapse.
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http://dx.doi.org/10.1016/j.cmet.2021.07.002DOI Listing
September 2021

ZFP451-mediated SUMOylation of SATB2 drives embryonic stem cell differentiation.

Genes Dev 2021 08 8;35(15-16):1142-1160. Epub 2021 Jul 8.

Department of Cellular and Molecular Immunology, Max Planck Institute of Immunobiology and Epigenetics, 79108 Freiburg, Germany.

The establishment of cell fates involves alterations of transcription factor repertoires and repurposing of transcription factors by post-translational modifications. In embryonic stem cells (ESCs), the chromatin organizers SATB2 and SATB1 balance pluripotency and differentiation by activating and repressing pluripotency genes, respectively. Here, we show that conditional gene inactivation weakens ESC pluripotency, and we identify SUMO2 modification of SATB2 by the E3 ligase ZFP451 as a potential driver of ESC differentiation. Mutations of two SUMO-acceptor lysines of ( ) or knockout of impair the ability of ESCs to silence pluripotency genes and activate differentiation-associated genes in response to retinoic acid (RA) treatment. Notably, the forced expression of a SUMO2-SATB2 fusion protein in either or ESCs rescues, in part, their impaired differentiation potential and enhances the down-regulation of The differentiation defect of ESCs correlates with altered higher-order chromatin interactions relative to ESCs. Upon RA treatment of ESCs, SATB2 interacts with ZFP451 and the LSD1/CoREST complex and gains binding at differentiation genes, which is not observed in RA-treated cells. Thus, SATB2 SUMOylation may contribute to the rewiring of transcriptional networks and the chromatin interactome of ESCs in the transition of pluripotency to differentiation.
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http://dx.doi.org/10.1101/gad.345843.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8336893PMC
August 2021

Genome-wide crosstalk between steroid receptors in breast and prostate cancers.

Endocr Relat Cancer 2021 Jul 22;28(9):R231-R250. Epub 2021 Jul 22.

Institute of Biomedicine, School of Medicine, University of Eastern Finland, Kuopio, Finland.

Steroid receptors (SRs) constitute an important class of signal-dependent transcription factors (TFs). They regulate a variety of key biological processes and are crucial drug targets in many disease states. In particular, estrogen (ER) and androgen receptors (AR) drive the development and progression of breast and prostate cancer, respectively. Thus, they represent the main specific drug targets in these diseases. Recent evidence has suggested that the crosstalk between signal-dependent TFs is an important step in the reprogramming of chromatin sites; a signal-activated TF can expand or restrict the chromatin binding of another TF. This crosstalk can rewire gene programs and thus alter biological processes and influence the progression of disease. Lately, it has been postulated that there may be an important crosstalk between the AR and the ER with other SRs. Especially, progesterone (PR) and glucocorticoid receptor (GR) can reprogram chromatin binding of ER and gene programs in breast cancer cells. Furthermore, GR can take the place of AR in antiandrogen-resistant prostate cancer cells. Here, we review the current knowledge of the crosstalk between SRs in breast and prostate cancers. We emphasize how the activity of ER and AR on chromatin can be modulated by other SRs on a genome-wide scale. We also highlight the knowledge gaps in the interplay of SRs and their complex interactions with other signaling pathways and suggest how to experimentally fill in these gaps.
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http://dx.doi.org/10.1530/ERC-21-0038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8345902PMC
July 2021

Chromatin-directed proteomics-identified network of endogenous androgen receptor in prostate cancer cells.

Oncogene 2021 07 14;40(27):4567-4579. Epub 2021 Jun 14.

Institute of Biomedicine, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland.

Treatment of prostate cancer confronts resistance to androgen receptor (AR)-targeted therapies. AR-associated coregulators and chromatin proteins hold a great potential for novel therapy targets. Here, we employed a powerful chromatin-directed proteomics approach termed ChIP-SICAP to uncover the composition of chromatin protein network, the chromatome, around endogenous AR in castration resistant prostate cancer (CRPC) cells. In addition to several expected AR coregulators, the chromatome contained many nuclear proteins not previously associated with the AR. In the context of androgen signaling in CRPC cells, we further investigated the role of a known AR-associated protein, a chromatin remodeler SMARCA4 and that of SIM2, a transcription factor without a previous association with AR. To understand their role in chromatin accessibility and AR target gene expression, we integrated data from ChIP-seq, RNA-seq, ATAC-seq and functional experiments. Despite the wide co-occurrence of SMARCA4 and AR on chromatin, depletion of SMARCA4 influenced chromatin accessibility and expression of a restricted set of AR target genes, especially those involved in cell morphogenetic changes in epithelial-mesenchymal transition. The depletion also inhibited the CRPC cell growth, validating SMARCA4's functional role in CRPC cells. Although silencing of SIM2 reduced chromatin accessibility similarly, it affected the expression of a much larger group of androgen-regulated genes, including those involved in cellular responses to external stimuli and steroid hormone stimulus. The silencing also reduced proliferation of CRPC cells and tumor size in chick embryo chorioallantoic membrane assay, further emphasizing the importance of SIM2 in CRPC cells and pointing to the functional relevance of this potential prostate cancer biomarker in CRPC cells. Overall, the chromatome of AR identified in this work is an important resource for the field focusing on this important drug target.
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http://dx.doi.org/10.1038/s41388-021-01887-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8266679PMC
July 2021

BCOR modulates transcriptional activity of a subset of glucocorticoid receptor target genes involved in cell growth and mobility.

J Steroid Biochem Mol Biol 2021 06 17;210:105873. Epub 2021 Mar 17.

Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland. Electronic address:

Glucocorticoid (GC) receptor (GR) is a key transcription factor (TF) that regulates vital metabolic and anti-inflammatory processes. We have identified BCL6 corepressor (BCOR) as a dexamethasone-stimulated interaction partner of GR. BCOR is a component of non-canonical polycomb repressor complex 1.1 (ncPCR1.1) and linked to different developmental disorders and cancers, but the role of BCOR in GC signaling is poorly characterized. Here, using ChIP-seq we show that, GC induces genome-wide redistribution of BCOR chromatin binding towards GR-occupied enhancers in HEK293 cells. As assessed by RNA-seq, depletion of BCOR altered the expression of hundreds of GC-regulated genes, especially the ones linked to TNF signaling, GR signaling and cell migration pathways. Biotinylation-based proximity mapping revealed that GR and BCOR share several interacting partners, including nuclear receptor corepressor NCOR1. ChIP-seq showed that the NCOR1 co-occurs with both BCOR and GR on a subset of enhancers upon GC treatment. Simultaneous depletion of BCOR and NCOR1 influenced GR target gene expression in a combinatorial and gene-specific manner. Finally, we show using live cell imaging that the depletion of BCOR together with NCOR1 markedly enhances cell migration. Collectively, our data suggest BCOR as an important gene and pathway selective coregulator of GR transcriptional activity.
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http://dx.doi.org/10.1016/j.jsbmb.2021.105873DOI Listing
June 2021

Stress-induced nuclear condensation of NELF drives transcriptional downregulation.

Mol Cell 2021 03 5;81(5):1013-1026.e11. Epub 2021 Feb 5.

Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany; CIBSS, Centre for Integrative Biological Signaling Studies, Freiburg, Germany; MRC, University of Cambridge, Cambridge, UK. Electronic address:

In response to stress, human cells coordinately downregulate transcription and translation of housekeeping genes. To downregulate transcription, the negative elongation factor (NELF) is recruited to gene promoters impairing RNA polymerase II elongation. Here we report that NELF rapidly forms nuclear condensates upon stress in human cells. Condensate formation requires NELF dephosphorylation and SUMOylation induced by stress. The intrinsically disordered region (IDR) in NELFA is necessary for nuclear NELF condensation and can be functionally replaced by the IDR of FUS or EWSR1 protein. We find that biomolecular condensation facilitates enhanced recruitment of NELF to promoters upon stress to drive transcriptional downregulation. Importantly, NELF condensation is required for cellular viability under stressful conditions. We propose that stress-induced NELF condensates reported here are nuclear counterparts of cytosolic stress granules. These two stress-inducible condensates may drive the coordinated downregulation of transcription and translation, likely forming a critical node of the stress survival strategy.
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http://dx.doi.org/10.1016/j.molcel.2021.01.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7939545PMC
March 2021

SUMOylation regulates the protein network and chromatin accessibility at glucocorticoid receptor-binding sites.

Nucleic Acids Res 2021 02;49(4):1951-1971

Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland.

Glucocorticoid receptor (GR) is an essential transcription factor (TF), controlling metabolism, development and immune responses. SUMOylation regulates chromatin occupancy and target gene expression of GR in a locus-selective manner, but the mechanism of regulation has remained elusive. Here, we identify the protein network around chromatin-bound GR by using selective isolation of chromatin-associated proteins and show that the network is affected by receptor SUMOylation, with several nuclear receptor coregulators and chromatin modifiers preferring interaction with SUMOylation-deficient GR and proteins implicated in transcriptional repression preferring interaction with SUMOylation-competent GR. This difference is reflected in our chromatin binding, chromatin accessibility and gene expression data, showing that the SUMOylation-deficient GR is more potent in binding and opening chromatin at glucocorticoid-regulated enhancers and inducing expression of target loci. Blockage of SUMOylation by a SUMO-activating enzyme inhibitor (ML-792) phenocopied to a large extent the consequences of GR SUMOylation deficiency on chromatin binding and target gene expression. Our results thus show that SUMOylation modulates the specificity of GR by regulating its chromatin protein network and accessibility at GR-bound enhancers. We speculate that many other SUMOylated TFs utilize a similar regulatory mechanism.
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http://dx.doi.org/10.1093/nar/gkab032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7913686PMC
February 2021

New insights into the genetic basis of premature ovarian insufficiency: Novel causative variants and candidate genes revealed by genomic sequencing.

Maturitas 2020 Nov 20;141:9-19. Epub 2020 Jun 20.

Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, VIC, 3052, Australia; Department of Paediatrics, University of Melbourne, Melbourne, VIC, 3052, Australia.

Ovarian deficiency, including premature ovarian insufficiency (POI) and diminished ovarian reserve (DOR), represents one of the main causes of female infertility. POI is a genetically heterogeneous condition but current understanding of its genetic basis is far from complete, with the cause remaining unknown in the majority of patients. The genes that regulate DOR have been reported but the genetic basis of DOR has not been explored in depth. Both conditions are likely to lie along a continuum of degrees of decrease in ovarian reserve. We performed genomic analysis via whole exome sequencing (WES) followed by in silico analyses and functional experiments to investigate the genetic cause of ovarian deficiency in ten affected women. We achieved diagnoses for three of them, including the identification of novel variants in STAG3, GDF9, and FANCM. We identified potentially causative FSHR variants in another patient. This is the second report of biallelic GDF9 and FANCM variants, and, combined with functional support, validates these genes as bone fide autosomal recessive "POI genes". We also identified new candidate genes, NRIP1, XPO1, and MACF1. These genes have been linked to ovarian function in mouse, pig, and zebrafish respectively, but never in humans. In the case of NRIP1, we provide functional support for the deleterious nature of the variant via SUMOylation and luciferase/β-galactosidase reporter assays. Our study provides multiple insights into the genetic basis of POI/DOR. We have further elucidated the involvement of GDF9, FANCM, STAG3 and FSHR in POI pathogenesis, and propose new candidate genes, NRIP1, XPO1, and MACF1, which should be the focus of future studies.
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http://dx.doi.org/10.1016/j.maturitas.2020.06.004DOI Listing
November 2020

BCOR-coupled H2A monoubiquitination represses a subset of androgen receptor target genes regulating prostate cancer proliferation.

Oncogene 2020 03 10;39(11):2391-2407. Epub 2020 Jan 10.

Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland.

We have identified BCL6 corepressor (BCOR) as a hormone-dependent interaction partner of androgen receptor (AR), a key transcription factor in the development of normal and cancerous prostate. BCOR is often mutated in cancers and hematological diseases and as a component of a non-canonical polycomb repressive complex 1 (ncPRC1.1) required for arranging many facets of cellular differentiation. However, its role in androgen signaling or prostate cancer cells remains unknown. Here, our genome-wide analyses reveal that BCOR is recruited in an androgen-dependent fashion to majority of AR-binding chromatin sites in castration-resistant prostate cancer (CRPC) cells. Interestingly, depletion of BCOR has a significant effect on the expression of androgen-repressed genes linked to regulation of cell proliferation, differentiation and development. At many of these genes, such as HOX genes, the depletion leads to a decrease in H2A K119 monoubiquitination and an increase in mRNA expression. Consistently, BCOR depletion impairs the proliferation and viability of CRPC cells, inducing their apoptosis. Collectively, our data indicate a key role for the BCOR-ncPRC1.1 complex in the corepression of an important subset of AR target genes and the regulation of prostate cancer cell proliferation.
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http://dx.doi.org/10.1038/s41388-020-1153-3DOI Listing
March 2020

A long hypoxia-inducible factor 3 isoform 2 is a transcription activator that regulates erythropoietin.

Cell Mol Life Sci 2020 Sep 25;77(18):3627-3642. Epub 2019 Nov 25.

Oulu Center for Cell-Matrix Research, University of Oulu, PO Box 5400, 90014, Oulu, Finland.

Hypoxia-inducible factor (HIF), an αβ dimer, is the master regulator of oxygen homeostasis with hundreds of hypoxia-inducible target genes. Three HIF isoforms differing in the oxygen-sensitive α subunit exist in vertebrates. While HIF-1 and HIF-2 are known transcription activators, HIF-3 has been considered a negative regulator of the hypoxia response pathway. However, the human HIF3A mRNA is subject to complex alternative splicing. It was recently shown that the long HIF-3α variants can form αβ dimers that possess transactivation capacity. Here, we show that overexpression of the long HIF-3α2 variant induces the expression of a subset of genes, including the erythropoietin (EPO) gene, while simultaneous downregulation of all HIF-3α variants by siRNA targeting a shared HIF3A region leads to downregulation of EPO and additional genes. EPO mRNA and protein levels correlated with HIF3A silencing and HIF-3α2 overexpression. Chromatin immunoprecipitation analyses showed that HIF-3α2 binding associated with canonical hypoxia response elements in the promoter regions of EPO. Luciferase reporter assays showed that the identified HIF-3α2 chromatin-binding regions were sufficient to promote transcription by all three HIF-α isoforms. Based on these data, HIF-3α2 is a transcription activator that directly regulates EPO expression.
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http://dx.doi.org/10.1007/s00018-019-03387-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7452874PMC
September 2020

TNF-α inhibits glucocorticoid receptor-induced gene expression by reshaping the GR nuclear cofactor profile.

Proc Natl Acad Sci U S A 2019 06 10;116(26):12942-12951. Epub 2019 Jun 10.

Mouse Genetics in Inflammation, VIB Center for Inflammation Research, 9052 Ghent, Belgium;

Glucocorticoid resistance (GCR) is defined as an unresponsiveness to the therapeutic effects, including the antiinflammatory ones of glucocorticoids (GCs) and their receptor, the glucocorticoid receptor (GR). It is a problem in the management of inflammatory diseases and can be congenital as well as acquired. The strong proinflammatory cytokine TNF-alpha (TNF) induces an acute form of GCR, not only in mice, but also in several cell lines: e.g., in the hepatoma cell line BWTG3, as evidenced by impaired Dexamethasone (Dex)-stimulated direct GR-dependent gene up- and down-regulation. We report that TNF has a significant and broad impact on this transcriptional performance of GR, but no impact on nuclear translocation, dimerization, or DNA binding capacity of GR. Proteome-wide proximity-mapping (BioID), however, revealed that the GR interactome was strongly modulated by TNF. One GR cofactor that interacted significantly less with the receptor under GCR conditions is p300. NFκB activation and p300 knockdown both reduced direct transcriptional output of GR whereas p300 overexpression and NFκB inhibition reverted TNF-induced GCR, which is in support of a cofactor reshuffle model. This hypothesis was supported by FRET studies. This mechanism of GCR opens avenues for therapeutic interventions in GCR diseases.
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http://dx.doi.org/10.1073/pnas.1821565116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6600915PMC
June 2019

IRF2BP2 modulates the crosstalk between glucocorticoid and TNF signaling.

J Steroid Biochem Mol Biol 2019 09 28;192:105382. Epub 2019 May 28.

Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland. Electronic address:

IRF2BP2 (interferon regulatory factor-2 binding protein-2) is an uncharacterized interaction partner of glucocorticoid (GC) receptor (GR), an anti-inflammatory and metabolic transcription factor. Here, we show that GC changes the chromatin binding of IRF2BP2 in natural chromatin milieu. The GC-induced IRF2BP2-binding sites co-occur with GR binding sites and are associated with GC-induced genes. Moreover, the depletion of IRF2BP2 modulates transcription of GC-regulated genes, represses cell proliferation and increases cell movement in HEK293 cells. In A549 cells, the depletion extensively alters the responses to GC and tumor necrosis factor α (TNF), including metabolic and inflammatory pathways. Taken together, our data support the role of IRF2BP2 as a coregulator of both GR and NF-κB, potentially modulating the crosstalk between GC and TNF signaling.
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http://dx.doi.org/10.1016/j.jsbmb.2019.105382DOI Listing
September 2019

Lack of androgen receptor SUMOylation results in male infertility due to epididymal dysfunction.

Nat Commun 2019 02 15;10(1):777. Epub 2019 Feb 15.

Institute of Biomedicine, University of Eastern Finland, FI-70211, Kuopio, Finland.

Androgen receptor (AR) is regulated by SUMOylation at its transactivation domain. In vitro, the SUMOylation is linked to transcriptional repression and/or target gene-selective regulation. Here, we generated a mouse model (ArKI) in which the conserved SUMO acceptor lysines of AR are permanently abolished (Ar). ArKI males develop normally, without apparent defects in their systemic androgen action in reproductive tissues. However, the ArKI males are infertile. Their spermatogenesis appears unaffected, but their epididymal sperm maturation is defective, shown by severely compromised motility and fertilization capacity of the sperm. Fittingly, their epididymal AR chromatin-binding and gene expression associated with sperm maturation and function are misregulated. AR is SUMOylated in the wild-type epididymis but not in the testis, which could explain the tissue-specific response to the lack of AR SUMOylation. Our studies thus indicate that epididymal AR SUMOylation is essential for the post-testicular sperm maturation and normal reproductive capability of male mice.
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http://dx.doi.org/10.1038/s41467-019-08730-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6377611PMC
February 2019

Androgen receptor SUMOylation regulates bone mass in male mice.

Mol Cell Endocrinol 2019 01 24;479:117-122. Epub 2018 Sep 24.

Centre for Bone and Arthritis Research, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden. Electronic address:

The crucial effects of androgens on the male skeleton are at least partly mediated via the androgen receptor (AR). In addition to hormone binding, the AR activity is regulated by post-translational modifications, including SUMOylation. SUMOylation is a reversible modification in which Small Ubiquitin-related MOdifier proteins (SUMOs) are attached to the AR and thereby regulate the activity of the AR and change its interactions with other proteins. To elucidate the importance of SUMOylation of AR for male bone metabolism, we used a mouse model devoid of the two AR SUMOylation sites (AR; K381R and K500R are substituted). Six-month-old male AR mice displayed significantly reduced trabecular bone volume fraction in the distal metaphyseal region of femur compared with wild type (WT) mice (BV/TV, -19.1 ± 4.9%, P < 0.05). The number of osteoblasts per bone perimeter was substantially reduced (-60.5 ± 7.2%, P < 0.001) while no significant effect was observed on the number of osteoclasts in the trabecular bone of male AR mice. Dynamic histomorphometric analysis of trabecular bone revealed a reduced bone formation rate (-32.6 ± 7.4%, P < 0.05) as a result of reduced mineralizing surface per bone surface in AR mice compared with WT mice (-24.3 ± 3.6%, P < 0.001). Furthermore, cortical bone thickness in the diaphyseal region of femur was reduced in male AR mice compared with WT mice (-7.3 ± 2.0%, P < 0.05). In conclusion, mice devoid of AR SUMOylation have reduced trabecular bone mass as a result of reduced bone formation. We propose that therapies enhancing AR SUMOylation might result in bone-specific anabolic effects with minimal adverse effects in other tissues.
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http://dx.doi.org/10.1016/j.mce.2018.09.008DOI Listing
January 2019

Response: Commentary: Analysis of SUMO1-conjugation at synapses.

Front Cell Neurosci 2018 1;12:117. Epub 2018 May 1.

Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany.

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http://dx.doi.org/10.3389/fncel.2018.00117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5938361PMC
May 2018

Agonist-specific Protein Interactomes of Glucocorticoid and Androgen Receptor as Revealed by Proximity Mapping.

Mol Cell Proteomics 2017 08 13;16(8):1462-1474. Epub 2017 Jun 13.

From the ‡Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland;

Glucocorticoid receptor (GR) and androgen receptor (AR) are steroid-inducible transcription factors (TFs). The GR and the AR are central regulators of various metabolic, homeostatic and differentiation processes and hence important therapeutic targets, especially in inflammation and prostate cancer, respectively. Hormone binding to these steroid receptors (SRs) leads to DNA binding and activation or repression of their target genes with the aid of interacting proteins, coregulators. However, protein interactomes of these important drug targets have remained poorly defined. We used proximity-dependent biotin identification to map the protein interaction landscapes of GR and AR in the presence and absence of their cognate agonist (dexamethasone, 5α-dihydrotestosterone) and antagonist (RU486, enzalutamide) in intact human cells. We reproducibly identified more than 30 proteins that interacted with the GR in an agonist-specific manner and whose interactions were significantly influenced by the DNA-binding function of the receptor. Interestingly, the agonist-dependent interactome of the GR overlapped considerably with that of the AR. In addition to known coactivators, corepressors and components of BAF (SWI/SNF) chromatin-remodeling complex, we identified a number of proteins, including lysine methyltransferases and demethylases that have not been previously linked to glucocorticoid or androgen signaling. A substantial number of these novel agonist-dependent GR/AR-interacting proteins, BCOR, IRF2BP2, RCOR1, and TLE3, have previously been implicated in transcription repression. This together with our data on the effect of BCOR, IRF2BP2, and RCOR1 on GR target gene expression suggests multifaceted functions and roles for SR coregulators. These first high confidence SR interactomes will aid in therapeutic targeting of the GR and the AR.
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http://dx.doi.org/10.1074/mcp.M117.067488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5546198PMC
August 2017

Analysis of SUMO1-conjugation at synapses.

Elife 2017 06 9;6. Epub 2017 Jun 9.

Max Planck Institute of Experimental Medicine, Molecular Neurobiology, Göttingen, Germany.

SUMO1-conjugation of proteins at neuronal synapses is considered to be a major post-translational regulatory process in nerve cell and synapse function, but the published evidence for SUMO1-conjugation at synapses is contradictory. We employed multiple genetic mouse models for stringently controlled biochemical and immunostaining analyses of synaptic SUMO1-conjugation. By using a knock-in reporter mouse line expressing tagged SUMO1, we could not detect SUMO1-conjugation of seven previously proposed synaptic SUMO1-targets in the brain. Further, immunostaining of cultured neurons from wild-type and SUMO1 knock-out mice showed that anti-SUMO1 immunolabelling at synapses is non-specific. Our findings indicate that SUMO1-conjugation of synaptic proteins does not occur or is extremely rare and hence not detectable using current methodology. Based on our data, we discuss a set of experimental strategies and minimal consensus criteria for the validation of SUMOylation that can be applied to any SUMOylation substrate and SUMO isoform.
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http://dx.doi.org/10.7554/eLife.26338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5493437PMC
June 2017

Chromatin SUMOylation in heat stress: To protect, pause and organise?: SUMO stress response on chromatin.

Bioessays 2017 06 25;39(6). Epub 2017 Apr 25.

University of Eastern Finland, Institute of Biomedicine, Kuopio, Finland.

Post-translational modifications, e.g. SUMO modifications (SUMOylation), provide a mechanism for swiftly changing a protein's activity. Various stress conditions trigger a SUMO stress response (SSR) - a stress-induced rapid change in the conjugation of SUMO to multiple proteins, which predominantly targets nuclear proteins. The SSR has been postulated to protect stressed cells by preserving the functionality of crucial proteins. However, it is unclear how it exerts its protective functions. Interestingly, heat stress (HS) increases SUMOylation of proteins at active promoters and enhancers. In promoters, HS-induced SUMOylation correlates with gene transcription and stress-induced RNA polymerase II (Pol2) pausing. Conversely, a disappearance of SUMOylation in HS occurs at chromatin anchor points that maintain chromatin-looping structures and the spatial organisation of chromatin. In reviewing the literature, we hypothesise that the SSR regulates Pol2 pausing by modulating the interactions of pausing-regulating proteins, whereas deSUMOylation alters the function of chromatin anchors.
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http://dx.doi.org/10.1002/bies.201600263DOI Listing
June 2017

ZNHIT3 is defective in PEHO syndrome, a severe encephalopathy with cerebellar granule neuron loss.

Brain 2017 05;140(5):1267-1279

The Folkhälsan Institute of Genetics, Haartmaninkatu 8, 00290 Helsinki, Finland.

Progressive encephalopathy with oedema, hypsarrhythmia, and optic atrophy (PEHO) syndrome is an early childhood onset, severe autosomal recessive encephalopathy characterized by extreme cerebellar atrophy due to almost total granule neuron loss. By combining homozygosity mapping in Finnish families with Sanger sequencing of positional candidate genes and with exome sequencing a homozygous missense substitution of leucine for serine at codon 31 in ZNHIT3 was identified as the primary cause of PEHO syndrome. ZNHIT3 encodes a nuclear zinc finger protein previously implicated in transcriptional regulation and in small nucleolar ribonucleoprotein particle assembly and thus possibly to pre-ribosomal RNA processing. The identified mutation affects a highly conserved amino acid residue in the zinc finger domain of ZNHIT3. Both knockdown and genome editing of znhit3 in zebrafish embryos recapitulate the patients' cerebellar defects, microcephaly and oedema. These phenotypes are rescued by wild-type, but not mutant human ZNHIT3 mRNA, suggesting that the patient missense substitution causes disease through a loss-of-function mechanism. Transfection of cell lines with ZNHIT3 expression vectors showed that the PEHO syndrome mutant protein is unstable. Immunohistochemical analysis of mouse cerebellar tissue demonstrated ZNHIT3 to be expressed in proliferating granule cell precursors, in proliferating and post-mitotic granule cells, and in Purkinje cells. Knockdown of Znhit3 in cultured mouse granule neurons and ex vivo cerebellar slices indicate that ZNHIT3 is indispensable for granule neuron survival and migration, consistent with the zebrafish findings and patient neuropathology. These results suggest that loss-of-function of a nuclear regulator protein underlies PEHO syndrome and imply that establishment of its spatiotemporal interaction targets will be the basis for developing therapeutic approaches and for improved understanding of cerebellar development.
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http://dx.doi.org/10.1093/brain/awx040DOI Listing
May 2017

Crosstalk between androgen and pro-inflammatory signaling remodels androgen receptor and NF-κB cistrome to reprogram the prostate cancer cell transcriptome.

Nucleic Acids Res 2017 01 26;45(2):619-630. Epub 2016 Sep 26.

Institute of Biomedicine, University of Eastern Finland, 70211 Kuopio, Finland

Inflammatory processes and androgen signaling are critical for the growth of prostate cancer (PC), the most common cancer among males in Western countries. To understand the importance of potential interplay between pro-inflammatory and androgen signaling for gene regulation, we have interrogated the crosstalk between androgen receptor (AR) and NF-κB, a key transcriptional mediator of inflammatory responses, by utilizing genome-wide chromatin immunoprecipitation sequencing and global run-on sequencing in PC cells. Co-stimulation of LNCaP cells with androgen and pro-inflammatory cytokine TNFα invoked a transcriptome which was very distinct from that induced by either stimulation alone. The altered transcriptome that included gene programs linked to cell migration and invasiveness was orchestrated by significant remodeling of NF-κB and AR cistrome and enhancer landscape. Although androgen multiplied the NF-κB cistrome and TNFα restrained the AR cistrome, there was no general reciprocal tethering of the AR to the NF-κB on chromatin. Instead, redistribution of FOXA1, PIAS1 and PIAS2 contributed to the exposure of latent NF-κB chromatin-binding sites and masking of AR chromatin-binding sites. Taken together, concomitant androgen and pro-inflammatory signaling significantly remodels especially the NF-κB cistrome, reprogramming the PC cell transcriptome in fashion that may contribute to the progression of PC.
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http://dx.doi.org/10.1093/nar/gkw855DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5314794PMC
January 2017

Global analysis of transcription in castration-resistant prostate cancer cells uncovers active enhancers and direct androgen receptor targets.

Sci Rep 2016 09 19;6:33510. Epub 2016 Sep 19.

Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland.

Androgen receptor (AR) is a male sex steroid-activated transcription factor (TF) that plays a critical role in prostate cancers, including castration-resistant prostate cancers (CRPC) that typically express amplified levels of the AR. CRPC-derived VCaP cells display an excessive number of chromatin AR-binding sites (ARBs) most of which localize to distal inter- or intragenic regions. Here, we analyzed direct transcription programs of the AR in VCaP cells using global nuclear run-on sequencing (GRO-seq) and integrated the GRO-seq data with the ARB and VCaP cell-specific TF-binding data. Androgen immediately activated transcription of hundreds of protein-coding genes, including IGF-1 receptor and EGF receptor. Androgen also simultaneously repressed transcription of a large number of genes, including MYC. As functional enhancers have been postulated to produce enhancer-templated non-coding RNAs (eRNAs), we also analyzed the eRNAs, which revealed that only a fraction of the ARBs reside at functional enhancers. Activation of these enhancers was most pronounced at the sites that also bound PIAS1, ERG and HDAC3, whereas binding of HDAC3 and PIAS1 decreased at androgen-repressed enhancers. In summary, our genome-wide data of androgen-regulated enhancers and primary target genes provide new insights how the AR can directly regulate cellular growth and control signaling pathways in CPRC cells.
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http://dx.doi.org/10.1038/srep33510DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5027586PMC
September 2016

Complete androgen insensitivity syndrome caused by a deep intronic pseudoexon-activating mutation in the androgen receptor gene.

Sci Rep 2016 09 9;6:32819. Epub 2016 Sep 9.

Physiology, Faculty of Medicine, University of Helsinki, Helsinki, Finland.

Mutations in the X-linked androgen receptor (AR) gene underlie complete androgen insensitivity syndrome (CAIS), the most common cause of 46,XY sex reversal. Molecular genetic diagnosis of CAIS, however, remains uncertain in patients who show normal coding region of AR. Here, we describe a novel mechanism of AR disruption leading to CAIS in two 46,XY sisters. We analyzed whole-genome sequencing data of the patients for pathogenic variants outside the AR coding region. Patient fibroblasts from the genital area were used for AR cDNA analysis and protein quantification. Analysis of the cDNA revealed aberrant splicing of the mRNA caused by a deep intronic mutation (c.2450-118A>G) in the intron 6 of AR. The mutation creates a de novo 5' splice site and a putative exonic splicing enhancer motif, which leads to the preferential formation of two aberrantly spliced mRNAs (predicted to include a premature stop codon). Patient fibroblasts contained no detectable AR protein. Our results show that patients with CAIS and normal AR coding region need to be examined for deep intronic mutations that can lead to pseudoexon activation.
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http://dx.doi.org/10.1038/srep32819DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5016895PMC
September 2016

SUMOylation of AMPKα1 by PIAS4 specifically regulates mTORC1 signalling.

Nat Commun 2015 Nov 30;6:8979. Epub 2015 Nov 30.

Research Programs Unit, Faculty of Medicine, University of Helsinki, Biomedicum Helsinki, Haartmaninkatu 8, PO Box 63, Helsinki FI-00014, Finland.

AMP-activated protein kinase (AMPK) inhibits several anabolic pathways such as fatty acid and protein synthesis, and identification of AMPK substrate specificity would be useful to understand its role in particular cellular processes and develop strategies to modulate AMPK activity in a substrate-specific manner. Here we show that SUMOylation of AMPKα1 attenuates AMPK activation specifically towards mTORC1 signalling. SUMOylation is also important for rapid inactivation of AMPK, to allow prompt restoration of mTORC1 signalling. PIAS4 and its SUMO E3 ligase activity are specifically required for the AMPKα1 SUMOylation and the inhibition of AMPKα1 activity towards mTORC1 signalling. The activity of a SUMOylation-deficient AMPKα1 mutant is higher than the wild type towards mTORC1 signalling when reconstituted in AMPKα-deficient cells. PIAS4 depletion reduced growth of breast cancer cells, specifically when combined with direct AMPK activator A769662, suggesting that inhibiting AMPKα1 SUMOylation can be explored to modulate AMPK activation and thereby suppress cancer cell growth.
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http://dx.doi.org/10.1038/ncomms9979DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4674823PMC
November 2015

A new vertebrate SUMO enzyme family reveals insights into SUMO-chain assembly.

Nat Struct Mol Biol 2015 Dec 2;22(12):959-67. Epub 2015 Nov 2.

Department of Epigenetics, Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany.

SUMO chains act as stress-induced degradation tags or repair factor-recruiting signals at DNA lesions. Although E1 activating, E2 conjugating and E3 ligating enzymes efficiently assemble SUMO chains, specific chain-elongation mechanisms are unknown. E4 elongases are specialized E3 ligases that extend a chain but are inefficient in the initial conjugation of the modifier. We identified ZNF451, a representative member of a new class of SUMO2 and SUMO3 (SUMO2/3)-specific enzymes that execute catalysis via a tandem SUMO-interaction motif (SIM) region. One SIM positions the donor SUMO while a second SIM binds SUMO on the back side of the E2 enzyme. This tandem-SIM region is sufficient to extend a back side-anchored SUMO chain (E4 elongase activity), whereas efficient chain initiation also requires a zinc-finger region to recruit the initial acceptor SUMO (E3 ligase activity). Finally, we describe four human proteins sharing E4 elongase activities and their function in stress-induced SUMO2/3 conjugation.
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http://dx.doi.org/10.1038/nsmb.3114DOI Listing
December 2015

Global SUMOylation on active chromatin is an acute heat stress response restricting transcription.

Genome Biol 2015 Jul 28;16:153. Epub 2015 Jul 28.

Institute of Biomedicine, University of Eastern Finland, P.O. Box 1627, FI-70211, Kuopio, Finland.

Background: Cells have developed many ways to cope with external stress. One distinctive feature in acute proteotoxic stresses, such as heat shock (HS), is rapid post-translational modification of proteins by SUMOs (small ubiquitin-like modifier proteins; SUMOylation). While many of the SUMO targets are chromatin proteins, there is scarce information on chromatin binding of SUMOylated proteins in HS and the role of chromatin SUMOylation in the regulation of transcription.

Results: We mapped HS-induced genome-wide changes in chromatin occupancy of SUMO-2/3-modified proteins in K562 and VCaP cells using ChIP-seq. Chromatin SUMOylation was further correlated with HS-induced global changes in transcription using GRO-seq and RNA polymerase II (Pol2) ChIP-seq along with ENCODE data for K562 cells. HS induced a rapid and massive rearrangement of chromatin SUMOylation pattern: SUMOylation was gained at active promoters and enhancers associated with multiple transcription factors, including heat shock factor 1. Concomitant loss of SUMOylation occurred at inactive intergenic chromatin regions that were associated with CTCF-cohesin complex and SETDB1 methyltransferase complex. In addition, HS triggered a dynamic chromatin binding of SUMO ligase PIAS1, especially onto promoters. The HS-induced SUMOylation on chromatin was most notable at promoters of transcribed genes where it positively correlated with active transcription and Pol2 promoter-proximal pausing. Furthermore, silencing of SUMOylation machinery either by depletion of UBC9 or PIAS1 enhanced expression of HS-induced genes.

Conclusions: HS-triggered SUMOylation targets promoters and enhancers of actively transcribed genes where it restricts the transcriptional activity of the HS-induced genes. PIAS1-mediated promoter SUMOylation is likely to regulate Pol2-associated factors in HS.
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http://dx.doi.org/10.1186/s13059-015-0717-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4531811PMC
July 2015

Androgen receptor- and PIAS1-regulated gene programs in molecular apocrine breast cancer cells.

Mol Cell Endocrinol 2015 Oct 26;414:91-8. Epub 2015 Jul 26.

Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland. Electronic address:

We have analyzed androgen receptor (AR) chromatin binding sites (ARBs) and androgen-regulated transcriptome in estrogen receptor negative molecular apocrine breast cancer cells. These analyses revealed that 42% of ARBs and 39% androgen-regulated transcripts in MDA-MB453 cells have counterparts in VCaP prostate cancer cells. Pathway analyses showed a similar enrichment of molecular and cellular functions among AR targets in both breast and prostate cancer cells, with cellular growth and proliferation being among the most enriched functions. Silencing of the coregulator SUMO ligase PIAS1 in MDA-MB453 cells influenced AR function in a target-selective fashion. An anti-apoptotic effect of the silencing suggests involvement of the PIAS1 in the regulation of cell death and survival pathways. In sum, apocrine breast cancer and prostate cancer cells share a core AR cistrome and target gene signature linked to cancer cell growth, and PIAS1 plays a similar coregulatory role for AR in both cancer cell types.
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http://dx.doi.org/10.1016/j.mce.2015.07.024DOI Listing
October 2015

Discovery of ODM-201, a new-generation androgen receptor inhibitor targeting resistance mechanisms to androgen signaling-directed prostate cancer therapies.

Sci Rep 2015 Jul 3;5:12007. Epub 2015 Jul 3.

Orion Corporation, Orion Pharma, Finland.

Activation of androgen receptor (AR) is crucial for prostate cancer growth. Remarkably, also castration-resistant prostate cancer (CRPC) is dependent on functional AR, and several mechanisms have been proposed to explain the addiction. Known causes of CRPC include gene amplification and overexpression as well as point mutations of AR. We report here the pharmacological profile of ODM-201, a novel AR inhibitor that showed significant antitumor activity and a favorable safety profile in phase 1/2 studies in men with CRPC. ODM-201 is a full and high-affinity AR antagonist that, similar to second-generation antiandrogens enzalutamide and ARN-509, inhibits testosterone-induced nuclear translocation of AR. Importantly, ODM-201 also blocks the activity of the tested mutant ARs arising in response to antiandrogen therapies, including the F876L mutation that confers resistance to enzalutamide and ARN-509. In addition, ODM-201 reduces the growth of AR-overexpressing VCaP prostate cancer cells both in vitro and in a castration-resistant VCaP xenograft model. In contrast to other antiandrogens, ODM-201 shows negligible brain penetrance and does not increase serum testosterone levels in mice. In conclusion, ODM-201 is a potent AR inhibitor that overcomes resistance to AR-targeted therapies by antagonizing both overexpressed and mutated ARs. ODM-201 is currently in a phase 3 trial in CRPC.
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http://dx.doi.org/10.1038/srep12007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4490394PMC
July 2015

Cyclin-dependent kinase 5 acts as a critical determinant of AKT-dependent proliferation and regulates differential gene expression by the androgen receptor in prostate cancer cells.

Mol Biol Cell 2015 Jun 7;26(11):1971-84. Epub 2015 Apr 7.

Department of Biosciences, Faculty of Science and Engineering, Åbo Akademi University, FI-20520 Turku, Finland Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, FI-20520 Turku, Finland

Contrary to cell cycle-associated cyclin-dependent kinases, CDK5 is best known for its regulation of signaling processes in differentiated cells and its destructive activation in Alzheimer's disease. Recently, CDK5 has been implicated in a number of different cancers, but how it is able to stimulate cancer-related signaling pathways remains enigmatic. Our goal was to study the cancer-promoting mechanisms of CDK5 in prostate cancer. We observed that CDK5 is necessary for proliferation of several prostate cancer cell lines. Correspondingly, there was considerable growth promotion when CDK5 was overexpressed. When examining the reasons for the altered proliferation effects, we observed that CDK5 phosphorylates S308 on the androgen receptor (AR), resulting in its stabilization and differential expression of AR target genes including several growth-priming transcription factors. However, the amplified cell growth was found to be separated from AR signaling, further corroborated by CDK5-dependent proliferation of AR null cells. Instead, we found that the key growth-promoting effect was due to specific CDK5-mediated AKT activation. Down-regulation of CDK5 repressed AKT phosphorylation by altering its intracellular localization, immediately followed by prominent cell cycle inhibition. Taken together, these results suggest that CDK5 acts as a crucial signaling hub in prostate cancer cells by controlling androgen responses through AR, maintaining and accelerating cell proliferation through AKT activation, and releasing cell cycle breaks.
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http://dx.doi.org/10.1091/mbc.E14-12-1634DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4472009PMC
June 2015
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