Autoregulatory Loop Research Articles

Bone Morphogenetic Protein-2 (BMP-2) Activates NFATc1 Transcription Factor via an Autoregulatory Loop Involving Smad/Akt/Ca2+ Signaling

Bone remodeling is controlled by dual actions of osteoclasts (OCs) and osteoblasts (OBs). The calcium-sensitive nuclear factor of activated T cells (NFAT) c1 transcription factor, as an OC signature gene, regulates differentiation of OCs downstream of bone morphogenetic protein-2 (BMP-2)-stimulated osteoblast-coded factors. To analyze a functional link between BMP-2 and NFATc1, we analyzed bones from OB-specific BMP-2 knock-out mice for NFATc1 expression by immunohistochemical staining and found significant reduction in NFATc1 expression. This indicated a requirement of BMP-2 for NFATc1 expression in OBs. We showed that BMP-2, via the receptor-specific Smad pathway, regulates expression of NFATc1 in OBs. Phosphatidylinositol 3-kinase/Akt signaling acting downstream of BMP-2 also drives NFATc1 expression and transcriptional activation. Under the basal condition, NFATc1 is phosphorylated. Activation of NFAT requires dephosphorylation by the calcium-dependent serine/threonine phosphatase calcineurin. We examined the role of calcium in BMP-2-stimulated regulation of NFATc1 in osteoblasts. 1,2Bis(2aminophenoxy)ethaneN,N,N',N'-tetraacetic acid acetoxymethyl ester, an inhibitor of intracellular calcium abundance, blocked BMP-2-induced transcription of NFATc1. Interestingly, BMP-2 induced calcium release from intracellular stores and increased calcineurin phosphatase activity, resulting in NFATc1 nuclear translocation. Cyclosporin A, which inhibits calcineurin upstream of NFATc1, blocked BMP-2-induced NFATc1 mRNA and protein expression. Expression of NFATc1 directly increased its transcription and VIVIT peptide, an inhibitor of NFATc1, suppressed BMP-2-stimulated NFATc1 transcription, confirming its autoregulation. Together, these data show a role of NFATc1 downstream of BMP-2 in mouse bone development and provide novel evidence for the presence of a cross-talk among Smad, phosphatidylinositol 3-kinase/Akt, and Ca(2+) signaling for BMP-2-induced NFATc1 expression through an autoregulatory loop.

Regulation of Mutant p53 Protein Expression.

For several decades, p53 has been detected in cancer biopsies by virtue of its high protein expression level which is considered indicative of mutation. Surprisingly, however, mouse genetic studies revealed that mutant p53 is inherently labile, similar to its wild type (wt) counterpart. Consistently, in response to stress conditions, both wt and mutant p53 accumulate in cells. While wt p53 returns to basal level following recovery from stress, mutant p53 remains stable. In part, this can be explained in mutant p53-expressing cells by the lack of an auto-regulatory loop with Mdm2 and other negative regulators, which are pivotal for wt p53 regulation. Further, additional protective mechanisms are acquired by mutant p53, largely mediated by the co-chaperones and their paralogs, the stress-induced heat shock proteins. Consequently, mutant p53 is accumulated in cancer cells in response to chronic stress and this accumulation is critical for its oncogenic gain of functions (GOF). Building on the extensive knowledge regarding wt p53, the regulation of mutant p53 is unraveling. In this review, we describe the current understanding on the major levels at which mutant p53 is regulated. These include the regulation of p53 protein levels by microRNA and by enzymes controlling p53 proteasomal degradation.

The KDM3A-KLF2-IRF4 Axis Maintains Myeloma Cell Survival

Histone methylations are tightly regulated by a balance between methyltransferases and demethylases that mediate the addition and removal of these modifications. Importantly, dysregulation of histone methylation is implicated in pathogenesis of cancers, including multiple myeloma (MM). For example, the t(4;14) (p16;q32) is present in 15 - 20% of MM patients and results in overexpression of WHSC1, a histone H3 lysine 36 (H3K36) methyltransferase. On the other hand, approximately 10% of MM patients without the t(4;14) have inactivating mutations in KDM6A, a H3K27 demethylase.KDM3A is a Jumonji C-domain-containing histone demethylase which catalyzes removal of H3K9 mono- and dimethylation (H3K9me1 and H3K9me2). KDM3A is implicated in pathogenesis of different types of cancers. Here we investigated the biological impact of KDM3A in MM.KDM3A expression was significantly elevated in MM patient samples compared to normal plasma cells in publicly available dataset (GSE5900, GSE6691). To evaluate the functional role of KDM3A, shRNAs targeting KDM3A were transduced into MM cell lines: knockdown of KDM3A significantly inhibited MM cell growth (RPMI8226, MM.1S, U266, H929) in vitro and in xenograft model (MM.1S). Apo2.7 staining showed that apoptotic cells were significantly increased after knockdown of KDM3A.We next examined gene expression profiles after knockdown of KDM3A in RPMI8226 cells. With a cutoff of > 1.5-fold downregulation, a total of 305 probe sets were downregulated in KDM3A-knockdown cells relative to control cells. Among putative KDM3A targets, a gene of particular interest is KLF2 which plays a key role in maintenance of B cell and plasma cell phenotype, and function. Another intriguing gene is IRF4, given its known crucial role in MM cell survival. We confirmed that expression of KLF2 and IRF4 was downregulated after knockdown of KDM3A by quantitative realtime PCR and immunoblots in RPMI82226, MM.1S, and U266 cells. KDM3A binding to KLF2 and IRF4 core promoters was demonstrated by chromatin immunoprecipitation (ChIP) assay in RPMI8226 cells. Moreover, knockdown of KDM3A increased H3K9me1 and me2 levels at both promoter regions, indicating that KDM3A directly regulates KLF2 and IRF4 expression by removing H3K9 methylation marks at their promoters in MM cells.shRNAs targeting KLF2 were next transduced into MM cell lines: silencing of KLF2 significantly reduced cell growth of MM cell lines, associated with decreased IRF4. Promoter reporter assays using human IRF4 promoter showed that KLF2 significantly increased luciferase expression in a dose-dependent manner. Moreover, ChIP assay showed that KLF2 bound to IRF4 promoter in RPMI8226 cells. Since transcription factors could form an autoregulatory feedback loop, we hypothesized that IRF4 might regulate KLF2 expression. As expected, knockdown of IRF4 downregulated KLF2 expression at both the mRNA and protein levels in 3 MM cell lines. In addition, ChIP assays demonstrated that IRF4 bound to KLF2 second intron that contains tandem IRF4 motifs in RPMI8226 cells. Collectively, these results suggest that KLF2 activates IRF4 expression and vice versa, forming an autoregulatory loop in MM cells.KLF2 has been reported to control homing of plasma cells to the bone marrow; we therefore hypothesized that KDM3A-KLF2-IRF4 axis might regulate adhesion and homing of MM cells to the bone marrow. Importantly, knockdown of KDM3A, KLF2, or IRF4 decreased adhesion of 3 MM cell lines to bone marrow stromal cells. Furthermore, bone marrow homing of MM.1S cells was significantly reduced after knockdown of KDM3A, KLF2, or IRF4 in a murine xenograft MM model, indicating that KDM3A-KLF2-IRF4 axis regulates, at least in part, MM cell adhesion and homing to the bone marrow.In conclusion, our study demonstrated that KDM3A is a crucial epigenetic regulator of MM cell survival, and that inhibition of KDM3A represents a novel therapeutic strategy in MM. [Display omitted] DisclosuresRaje:Amgen: Consultancy; Takeda: Consultancy; Novartis: Consultancy; Celgene Corporation: Consultancy; BMS: Consultancy; Acetylon: Research Funding; Eli Lilly: Research Funding; Onyx: Consultancy; AstraZeneca: Research Funding; Millenium: Consultancy. Richardson:Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Harigae:Chugai Pharmaceutical Co., Ltd.: Research Funding. Anderson:Oncopep: Equity Ownership; Gilead: Consultancy; BMS: Consultancy; Millennium: Consultancy; Celgene: Consultancy; Acetylon: Equity Ownership.

Revised Morning Loops of the Arabidopsis Circadian Clock Based on Analyses of Direct Regulatory Interactions.

The network structure of the plant circadian clock is complex and direct regulatory interactions between individual components have proven particularly difficult to predict from genetic analyses. Here, we systematically investigate in vivo binding interactions between the morning-specific transcription factor, LATE ELONGATED HYPOCOTYL (LHY) and the promoters of other components of the network. We then demonstrate the functionality of these interactions by testing the responsiveness of the target gene to an ethanol-induced change in expression level of the LHY protein. We uncover novel, negative autoregulatory feedback loops from LHY and the closely related CIRCADIAN CLOCK ASSOCIATED-1 (CCA1) onto their own and each other’s expression. Furthermore we show that LHY acts as a repressor of all other clock components, including PSEUDO-RESPONSE REGULATORs (PRRs) 9 and 7, which were previously thought to be positive regulatory targets. These experimental results lead to a substantial revision of the morning loops of the clock.

Can active components of licorice, glycyrrhizin and glycyrrhetinic acid, lick rheumatoid arthritis?

This review stated the possible application of the active components of licorice, glycyrrhizin (GL) and glycyrrhetinic acid (GA), in rheumatoid arthritis (RA) treatment based on the cyclooxygenase (COX)-2/thromboxane A2 (TxA2) pathway. The extensive literature from inception to July 2015 was searched in PubMed central, and relevant reports were identified according to the purpose of this study. The active components of licorice GL and GA exert the potential anti-inflammatory effects through, at least in part, suppressing COX-2 and its downstream product TxA2. Additionally, the COX-2/TxA2 pathway, an auto-regulatory feedback loop, has been recently found to be a crucial mechanism underlying the pathogenesis of RA. However, TxA2 is neither the pharmacological target of non-steroidal anti-inflammatory drugs (NSAIDs) nor the target of disease modifying anti-rheumatic drugs (DMARDs), and the limitations and side effects of those drugs may be, at least in part, attributable to lack of the effects on the COX-2/TxA2 pathway. Therefore, GL and GA capable of targeting this pathway hold the potential as a novel add-on therapy in therapeutic strategy, which is supported by several bench experiments. The active components of licorice, GL and GA, could not only potentiate the therapeutic effects but also decrease the adverse effects of NSAIDs or DMARDs through suppressing the COX-2/TxA2 pathway during treatment course of RA.

Gene-Regulatory Logic to Induce and Maintain a Developmental Compartment.

Gene-Regulatory Logic to Induce and Maintain a Developmental Compartment.

CBX7 and miR-9 are part of an autoregulatory loop controlling p16(INK) (4a).

Polycomb repressive complexes (PRC1 and PRC2) are epigenetic regulators that act in coordination to influence multiple cellular processes including pluripotency, differentiation, cancer and senescence. The role of PRCs in senescence can be mostly explained by their ability to repress the INK4/ARF locus. CBX7 is one of five mammalian orthologues of Drosophila Polycomb that forms part of PRC1. Despite the relevance of CBX7 for regulating senescence and pluripotency, we have a limited understanding of how the expression of CBX7 is regulated. Here we report that the miR-9 family of microRNAs (miRNAS) downregulates the expression of CBX7. In turn, CBX7 represses miR-9-1 and miR-9-2 as part of a regulatory negative feedback loop. The miR-9/CBX7 feedback loop is a regulatory module contributing to induction of the cyclin-dependent kinase inhibitor (CDKI) p16(INK4a) during senescence. The ability of the miR-9 family to regulate senescence could have implications for understanding the role of miR-9 in cancer and aging.

Tgif1 Counterbalances the Activity of Core Pluripotency Factors in Mouse Embryonic Stem Cells

Core pluripotency factors, such as Oct4, Sox2, and Nanog, play important roles in maintaining embryonic stem cell (ESC) identity by autoregulatory feedforward loops. Nevertheless, the mechanism that provides precise control of the levels of the ESC core factors without indefinite amplification has remained elusive. Here, we report the direct repression of core pluripotency factors by Tgif1, a previously known terminal repressor of TGFβ/activin/nodal signaling. Overexpression of Tgif1 reduces the levels ofESC core factors, whereas its depletion leads to the induction of the pluripotency factors. We confirm the existence of physical associations between Tgif1 and Oct4, Nanog, and HDAC1/2 and further show the level of Tgif1 is not significantly altered by treatment with an activator/inhibitor of the TGFβ/activin/nodal signaling. Collectively, our findings establish Tgif1 as an integral member of the core regulatory circuitry of mouse ESCs that counterbalances the levels of the core pluripotency factors in a TGFβ/activin/nodal-independent manner.

Circadian molecular clock in lung pathophysiology.

Disrupted daily or circadian rhythms of lung function and inflammatory responses are common features of chronic airway diseases. At the molecular level these circadian rhythms depend on the activity of an autoregulatory feedback loop oscillator of clock gene transcription factors, including the BMAL1:CLOCK activator complex and the repressors PERIOD and CRYPTOCHROME. The key nuclear receptors and transcription factors REV-ERBα and RORα regulate Bmal1 expression and provide stability to the oscillator. Circadian clock dysfunction is implicated in both immune and inflammatory responses to environmental, inflammatory, and infectious agents. Molecular clock function is altered by exposomes, tobacco smoke, lipopolysaccharide, hyperoxia, allergens, bleomycin, as well as bacterial and viral infections. The deacetylase Sirtuin 1 (SIRT1) regulates the timing of the clock through acetylation of BMAL1 and PER2 and controls the clock-dependent functions, which can also be affected by environmental stressors. Environmental agents and redox modulation may alter the levels of REV-ERBα and RORα in lung tissue in association with a heightened DNA damage response, cellular senescence, and inflammation. A reciprocal relationship exists between the molecular clock and immune/inflammatory responses in the lungs. Molecular clock function in lung cells may be used as a biomarker of disease severity and exacerbations or for assessing the efficacy of chronotherapy for disease management. Here, we provide a comprehensive overview of clock-controlled cellular and molecular functions in the lungs and highlight the repercussions of clock disruption on the pathophysiology of chronic airway diseases and their exacerbations. Furthermore, we highlight the potential for the molecular clock as a novel chronopharmacological target for the management of lung pathophysiology.

Abstract 068: Role for the Rho-GAP Graf3 in the Pathogenesis of Human Hypertension

Activation of RhoA in vascular smooth muscle cells (SMC) has been linked to vasoconstrictor-induced hypertension (HTN), but the relevance of this pathway to human disease was undetermined. We identified GRAF3 as a RhoA-GAP expressed specifically in SMC in mice and humans and reported that global GRAF3-deficient mice exhibited significant basal HTN (+ 25 mm Hg) that was fully reversed by treatment with a Rho-kinase inhibitor (Nature Comm. 2013;4:2910). Importantly, we recently created a tamoxifen inducible SMC-GRAF3 re-expression model which resulted in a near complete reversal of MAP (from 123 mmHg to 95 mm Hg), indicating that GRAF3's ability to limit RhoA activity in SMC is required for the maintenance of normal BP. Given that a BP-associated locus within the GRAF3 gene was identified by Genome Wide Association, we next sought to characterize the mechanisms that control GRAF3 expression. Through the use of a series of siRNA-dependent approaches in cultured SMC, we found that SMC-specific expression of GRAF3 is mediated by the RhoA/MRTF-A/SRF-signaling axis. Interestingly, RhoA is known to be activated by mechanical forces and we found that GRAF3 expression was significantly increased (8-fold) in vessels subjected to pathological stress. The finding that GRAF3 expression was significantly increased in two separate hypertensive animal models relative to their littermate controls provides further evidence that GRAF3 expression is regulated as part of an auto-regulatory negative feedback loop to inhibit RhoA activity and SMC tone. Interestingly, the BP associated locus maps to the 80Kb first intron of the GRAF3 gene and we found that the minor GRAF3 allele that decreases BP was associated with a significant increase in GRAF3 mRNA in human tibial artery samples (3-fold increase; N=123; p=1e-10 ). We have identified regulatory elements within the hypertensive locus that exhibit SMC-selective transcriptional activity and have shown that a minor allele variation within one of these elements significantly increased transcriptional activity. Our studies add significantly to our understanding of the development and pathophysiologic consequences of hypertension and may lead to novel and perhaps individualized approaches to its treatment.

Circadian clock-coupled lung cellular and molecular functions in chronic airway diseases.

Airway diseases are associated with abnormal circadian rhythms of lung function, reflected in daily changes of airway caliber, airway resistance, respiratory symptoms, and abnormal immune-inflammatory responses. Circadian rhythms are generated at the cellular level by an autoregulatory feedback loop of interlocked transcription factors collectively referred to as clock genes. The molecular clock is altered by cigarette smoke, LPS, and bacterial and viral infections in mouse and human lungs and in patients with chronic airway diseases. Stress-mediated post-translational modification of molecular clock proteins, brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1 (BMAL1) and PERIOD 2, is associated with a reduction in the activity/level of the deacetylase sirtuin 1 (SIRT1). Similarly, the levels of the nuclear receptor REV-ERBα and retinoic acid receptor-related orphan receptor α (ROR α), critical regulators of Bmal1 expression, are altered by environmental stresses. Molecular clock dysfunction is implicated in immune and inflammatory responses, DNA damage response, and cellular senescence. The molecular clock in the lung also regulates the timing of glucocorticoid sensitivity and phasic responsiveness to inflammation. Herein, we review our current understanding of clock-controlled cellular and molecular functions in the lungs, the impact of clock dysfunction in chronic airway disease, and the response of the pulmonary clock to different environmental perturbations. Furthermore, we discuss the evidence for candidate signaling pathways, such as the SIRT1-BMAL1-REV-ERBα axis, as novel targets for chronopharmacological management of chronic airway diseases.

Coupling circadian rhythms of metabolism and chromatin remodelling.

The circadian clock controls a large variety of neuronal, endocrine, behavioural and physiological responses in mammals. This control is exerted in large part at the transcriptional level on genes expressed in a cyclic manner. A highly specialized transcriptional machinery based on clock regulatory factors organized in feedback autoregulatory loops governs a significant portion of the genome. These oscillations in gene expression are paralleled by critical events of chromatin remodelling that appear to provide plasticity to circadian regulation. Specifically, the nicotinamide adenine dinucleotide (NAD)(+) -dependent deacetylases SIRT1 and SIRT6 have been linked to circadian control of gene expression. This, and additional accumulating evidence, shows that the circadian epigenome appears to share intimate links with cellular metabolic processes and has remarkable plasticity showing reprogramming in response to nutritional challenges. In addition to SIRT1 and SIRT6, a number of chromatin remodellers have been implicated in clock control, including the histone H3K4 tri-methyltransferase MLL1. Deciphering the molecular mechanisms that link metabolism, epigenetic control and circadian responses will provide valuable insights towards innovative strategies of therapeutic intervention.

Energy Homeostasis Control in Drosophila Adipokinetic Hormone Mutants.

Maintenance of biological functions under negative energy balance depends on mobilization of storage lipids and carbohydrates in animals. In mammals, glucagon and glucocorticoid signaling mobilizes energy reserves, whereas adipokinetic hormones (AKHs) play a homologous role in insects. Numerous studies based on AKH injections and correlative studies in a broad range of insect species established the view that AKH acts as master regulator of energy mobilization during development, reproduction, and stress. In contrast to AKH, the second peptide, which is processed from the Akh encoded prohormone [termed "adipokinetic hormone precursor-related peptide" (APRP)] is functionally orphan. APRP is discussed as ecdysiotropic hormone or as scaffold peptide during AKH prohormone processing. However, as in the case of AKH, final evidence for APRP functions requires genetic mutant analysis. Here we employed CRISPR/Cas9-mediated genome engineering to create AKH and AKH plus APRP-specific mutants in the model insect Drosophila melanogaster. Lack of APRP did not affect any of the tested steroid-dependent processes. Similarly, Drosophila AKH signaling is dispensable for ontogenesis, locomotion, oogenesis, and homeostasis of lipid or carbohydrate storage until up to the end of metamorphosis. During adulthood, however, AKH regulates body fat content and the hemolymph sugar level as well as nutritional and oxidative stress responses. Finally, we provide evidence for a negative autoregulatory loop in Akh gene regulation.

HSF2 autoregulates its own transcription.

Heat shock factor 2 (HSF2) is one of the most important regulators affecting stress mechanisms, and is frequently amplified in the ubiquitin proteasome pathway. Despite its significance, the mechanisms which regulate HSF2 expression remain unclear. In the present study, we describe the existence of a negative autoregulatory mechanism of HSF2. Transfection assays demonstrated that HSF2 decreased endogenous HSF2 mRNA expression in human K562 erythroleukemia cells. Luciferase reporter assays revealed that HSF2 inhibited the activity of its own promoter in a dose-dependent manner and that the downstream region (-1.5kb) relative to the transcription start site was responsible for this inhibitory effect. Furthermore, chromatin immunoprecipitation(ChIP) assat indicated that HSF2 is directly recruited onto its own promoter, which contains a putative heat shock element(HSE). Collectively, the findings of our studys suggest that HSF2 contributes to its own expression by forming a negative autoregulatory loop.

Abstract 1785: Chemical inhibition or transient knockdown of wild-type p53 induced phosphatase 1 (WIP1/PPM1D) potentiates the response to MDM2 inhibitors in a p53-dependent manner

Abstract The mechanisms that determine p53 cell fate decision after its non-genotoxic activation by MDM2 inhibitors are poorly understood. Wild-type p53-inducible phosphatase-1 (WIP1/PPM1D) forms an autoregulatory loop with p53 and is involved in the homeostasis of the p53 stress response network. WIP1 is activated, gained/amplified and overexpressed in a range of p53 wild-type malignancies. We investigated cellular growth/proliferation of p53 wild-type (wt) and matched p53 mutant (mut)/null cell line pairs, differing in WIP1 genetic status, in response to Nutlin-3 + GSK2830371 (WIP1i)/WIP1 siRNA-mediated knockdown. We also assessed the effects of WIP1i or knockdown in combination with Nutlin-3 on p53 post-translational modifications, p53 downstream transcriptional targets and apoptosis. Phosphorylation of p53S15 (pp53S15) was used as a marker of WIP1 catalytic activity. WIP1-amplified MCF-7p53wt cells were very sensitive to the WIP1i, with a 50% growth inhibitory concentration (GI50) of 2.65μM + 0.54 (SEM). The three p53 wild-type and mutant/null cell line pairs showed relatively little sensitivity to single agent WIP1i (GI50>10μM). However the WIP1i, at a non-growth inhibitory dose (2.5μM) in vitro, potentiated their response to Nutlin-3 in a p53-dependent manner. HCT116p53+/+; WIP1L450fs*1/+ (L450fs*1 is a WIP1 activating mutation), NGPp53wt and SJSA-1p53wt cell lines showed a 2.4-fold (p = 0.007), 2.1-fold (p = 0.04) and 1.3-fold (p = 0.02) decrease in their Nutlin-3 GI50 values respectively in the presence of WIP1i at 2.5μM. In contrast Nutlin-3 GI50 did not change in HCT116p53-/−, N20R1p53Mut and SN40R2p53Mut, NGP and SJSA-1 Nutlin-3 resistant daughter cell lines respectively. WIP1 transient siRNA knockdown also resulted in potentiation of cells to Nutlin-3. In WIP1-non-amplified (WNA) cells treated with either Nutlin-3 at the GI50 dose or 2.5μM WIP1i, pp53S15 was not detected, however in combination, these drugs resulted in dramatic levels of pp53S15. Interestingly protein expression of p53 targets MDM2 and p21 were not affected in WNA cells by the increased p53S15 phosphorylation; whereas BAX expression was elevated. The Nutlin-3 + WIP1i/siRNA knockdown combination also resulted in a significant increase in cell death markers between 24-48Hrs post-treatment in a p53-dependent manner. Although the strongest WIP1i potentiation of Nutlin-3 was observed in HCT116p53+/+; WIP1L450fs*1/+ cells which possess an activating WIP1 mutation, the NGPp53wt and SJSA-1p53wt cell lines with wild-type WIP1 also showed potentiation. These findings highlight the mechanistic importance of the p53-WIP1 autoregulatory loop in cell fate determination after non-genotoxic p53 activation by Nutlin-3. Potent inhibitors of WIP1 may potentiate the response to other therapeutics aiming to activate p53 mediated tumour suppression. Citation Format: Arman Esfandiari, Nicola J. Curtin, John Lunec. Chemical inhibition or transient knockdown of wild-type p53 induced phosphatase 1 (WIP1/PPM1D) potentiates the response to MDM2 inhibitors in a p53-dependent manner. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1785. doi:10.1158/1538-7445.AM2015-1785

Autoregulatory loop between TGF-β1/miR-411-5p/SPRY4 and MAPK pathway in rhabdomyosarcoma modulates proliferation and differentiation.

The origin of rhabdomyosarcoma (RMS) remains controversial. However, specific microRNAs (miRNAs) are downregulated in RMS and it is possible that re-expression of these miRNAs may lead to differentiation. Transforming growth factor-β1 (TGF-β1) is known to block differentiation of RMS. We therefore analyzed miRNA microarrays of RMS cell lines with or without TGF-β1 knockdown and identified a novel anti-oncogene miR-411-5p. Re-expression of miR-411-5p inhibited RMS cell proliferation in vitro and tumorigenicity in vivo. Using a luciferase reporting system and sequence analysis, the potential target of miR-411-5p was identified as sprouty homolog 4 (SPRY4), which inhibits protein kinase Cα-mediated activation of mitogen-activated protein kinases (MAPKs), especially p38MAPK phosphorylation. These results revealed an inverse correlation between TGF-β1/SPRY4 and miR-411-5p levels. SPRY4 small interfering RNA and miR-411-5p both activated p38MAPK phosphorylation and also promoted apoptosis and myogenic differentiation, indicated by increased caspase-3, myosin heavy chain, and myosin expression. SPRY4 and miR-411 mRNA levels correlated with TGF-β1 expression levels in RMS tissues, which was confirmed by immunohistochemical staining for TGF-β1, SPRY4, and phosphorylated p38MAPK proteins. Overall, these results indicate that miR-411-5p acts as an RMS differentiation-inducing miRNA prompting p38MAPK activation via directly downregulating SPRY4. These results establish an autoregulatory loop between TGF-β1/miR-411-5p/SPRY4 and MAPK in RMS, which governs the switch between proliferation and differentiation.

Abstract 2450: Identifying novel substrates of PLK2 using a chemical genetics approach

Abstract Polo like kinase 2 (PLK2/SNK) is a Ser/Thr kinase with roles identified in G1-S phase transition and in centriole duplication prior to entry into mitosis. PLK2 null embryonic fibroblasts have a slower proliferative rate and delayed entry into S-phase than their wild-type counterparts validating its role in cell cycle. Furthermore, wild-type p53, in response to DNA damage, induces PLK2 expression and checkpoint arrest. While these studies suggest that PLK2 is a tumor suppressor, studies have also shown that PLK2 binds to and phosphorylates mutant p53, but not wild-type p53, thereby promoting an oncogenic, auto-regulatory feedback loop, suggesting a potential role for PLK2 in proliferation. These observations suggest that PLK2 can function as an oncogene as well as a tumor suppressor. It is likely that cellular context and the nature of substrates are key to our understanding of the role that PLK2 plays in tumorigenesis. Although these reports highlight the importance of PLK2 in a variety of cellular processes, there is limited knowledge on the various proteins that are regulated by PLK2. We have optimized a chemical genetics system to identify novel substrates of PLK2. To that end, we mutated the conserved gatekeeper residue in the kinase domain of PLK2 (Leu159) to a smaller amino acid (Gly) to enlarge the ATP binding pocket. This engineered kinase, L159G-PLK2 (Plk2-as) is now able to utilize ATP analogs that carry a N6 modified bulky substituent of nucleoside residue such as N6-Phenylethyl ATP to phosphorylate substrates. We took advantage of this system to tag and isolate PLK2 substrates in vitro. Wild type and analog sensitive-PLK2 kinase domains were purified from bacteria and conditions were optimized for specific thio-phosphorylation of cell lysates by PLK2-as kinase. Labeling of cell lysates and subsequent mass spectrometry identified Signal transducer and activator of transcription 1 (STAT1) as a novel substrate of PLK2. In vitro PLK2 kinase assays in the presence or absence of specific PLK2 inhibitor, ON1231320, validated STAT1 to be a novel substrate of PLK2. Since mass spectrometry identified Ser708 as the potential phosphorylation site, we designed WT and mutant S708A peptides that span 16 amino acids of the phosphorylation site and used them as substrates for PLK1 and PLK2 in vitro kinase assays. While PLK2 readily phosphorylated the WT peptide, phosphorylation of the S708A mutant peptide was significantly reduced suggesting that PLK2 phosphorylates STAT1 at Ser708. This was further confirmed by our observation that ON1231320, a PLK2- specific inhibitor, inhibited PLK2-mediated phosphorylation of WT peptide. In addition, neither the WT nor the S708A peptide was phosphorylated by PLK1, indicating that Ser708 is a PLK2-specific phosphorylation site. Together, these studies validate that STAT1 is a bona fide target of PLK2. Citation Format: Poornima Ramkumar, Rebecca S. Levin, Miriam Sansó, Shashidhar Jatiani, Arvin C. Dar, Robert P. Fisher, Kevan M. Shokat, E Premkumar Reddy. Identifying novel substrates of PLK2 using a chemical genetics approach. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2450. doi:10.1158/1538-7445.AM2015-2450

The inhibitory effects of AR/miR-190a/YB-1 negative feedback loop on prostate cancer and underlying mechanism.

Prostate cancer at advanced stages including metastatic and castration-resistant cancer remains incurable due to the lack of effective therapies. MiR-190a belongs to the small noncoding RNA family and has an important role in breast cancer metastasis. However, it is still unknown whether miR-190a plays a role in prostate cancer development. Herein, we first observed AR/miR-190a/YB-1 forms an auto-regulatory negative feedback loop in prostate cancer: miR-190a expression was down-regulated by AR activation; YB-1 functions are as an AR activator; miR-190a inhibited AR expression and transactivation through direct binding to 3′UTR of YB-1 gene. MiR-190a contributes the human prostate cancer cell growth through AR-dependent signaling. Moreover, we examined the expression of miR-190a and observed a significant decrease in human prostate cancers. Reduced expression of miR-190a was inversely correlated to AR levels of prostate cancer patients, and patients with higher miR-190a expression in their tumor have improved tumor-free survival. Taken together, our findings identified a biochemical and functional link between miR-190a with reduced expression in advanced prostate cancer, YB-1 and AR signaling in prostate cancer.

Abstract 1614: Inhibition of MDM2 by RG7388 confers hypersensitivity to X-radiation in xenograft models of childhood sarcoma

Abstract Curative therapy for childhood sarcoma presents challenges when complete resection is not possible. Ionizing radiation (XRT) is used as a standard modality at diagnosis or recurrence for childhood sarcoma, however local recurrence is still problematic. Most childhood sarcomas are TP53 wild type at diagnosis, and approximately 5-10 per cent have MDM2 amplification or overexpression. Thus, reconstitution of a functional TP53 pathway, through inhibition of MDM2-mediated TP53 degradation, is an attractive anticancer strategy to enhance the activity of cytotoxic therapies that induce apoptosis through TP53-dependent pathways. RG7388, is a second generation MDM2 inhibitor. However, combining MDM2 inhibitors with myelosuppressive therapies may be problematic as the p53-MDM2 auto-regulatory loop in normal megakaryocytopoiesis suggests that thrombocytopenia may be an on-target toxicity. Here we have evaluated the antitumor activity of RG7388 alone or in combination with XRT in two rhabdomyosarcoma xenografts (Rh18 [embryonal], and Rh30 [alveolar]). RG7388 was administered at 80 mg/kg daily x 5 (schedule 1) or 100 mg/kg BID q7 days x 2 (schedule 2). XRT (2 Gy daily fractions) was given to a cumulative dose of 20 Gy (Rh18) or 30 Gy (Rh30). XRT (20 Gy) induced complete regressions of Rh18 xenografts followed by regrowth of all tumors with the median event time of 89.6 days compared 9.5 days for control or RG7388 treated tumors (P = 0.0287). The combination of XRT with RG7388 on either schedule induced complete regressions with no tumor regrowth (19 weeks observation). Thus, RG7388 given on either schedule significantly potentiated XRT (P<0.0003) for both schedules). RG7388 slightly potentiated XRT against Rh30 xenografts where the cumulative tumor volumes at the end of the period of observation (day 134) was significantly smaller for the combination group in Schedule 1 (P = 0.0005) and Schedule 2 (P = 0.0029) when compared with tumors receiving 30 Gy alone. Local XRT-induced skin toxicity was not enhanced by RG7388. Tumor samples were derived from untreated tumors, or tumors following 2, 4 or 6 Gy XRT and 24 and 48 Hr after the last XRT fraction with or without RG7388, or at the same time points from mice treated for 3 days with RG7388 alone (80 mg/kg). RG7388/XRT increased p21 levels over the 3 days of treatment, to a greater extent than XRT alone whereas p21 induction in tumors treated with RG7388 alone was lowest. Both XRT and combination treatments induced PARP cleavage over the first 48 hr. Although additional models should be examined to see whether the synergistic activity of XRT combined with RG7388 occurs frequently, these initial results suggest that the combination may provide more effective local control for pediatric soft tissue sarcoma. Citation Format: Peter J. Houghton, Doris A. Phelps, Kathryn Bondra, Star Seum, Christopher Chronowski, Justin Leasure, Raushan T. Kurmasheva, Stephen Middleton, Dian Wang, Xiaokui Mo. Inhibition of MDM2 by RG7388 confers hypersensitivity to X-radiation in xenograft models of childhood sarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1614. doi:10.1158/1538-7445.AM2015-1614

Identifying the ERAD ubiquitin E3 ligases for viral and cellular targeting of MHC class I.

The human cytomegalovirus (HCMV) US2 and US11 gene products hijack mammalian ER-associated degradation (ERAD) to induce rapid degradation of major histocompatibility class I (MHC-I) molecules. The rate-limiting step in this pathway is thought to be the polyubiquitination of MHC-I by distinct host ERAD E3 ubiquitin ligases. TRC8 was identified as the ligase responsible for US2-mediated MHC-I degradation and shown to be required for the cleavage-dependent degradation of some tail-anchored proteins. In addition to MHC-I, plasma membrane profiling identified further immune receptors, which are also substrates for the US2/TRC8 complex. These include at least six α integrins, the coagulation factor thrombomodulin and the NK cell ligand CD112. US2's use of specific HCMV-encoded adaptors makes it an adaptable viral degradation hub. US11-mediated degradation is MHC-I-specific and genetic screens have identified TMEM129, an uncharacterised RING-C2 E3 ligase, as responsible for US11-mediated degradation. In a unique auto-regulatory loop, US11 readily responds to changes in cellular expression of MHC-I. Free US11 either rebinds more MHC-I or is itself degraded by the HRD1/SEL1L E3 ligase complex. While virally encoded US2 and US11 appropriate mammalian ERAD, the MHC-I complex also undergoes stringent cellular quality control and misfolded MHC-I is degraded by the HRD1/SEL1L complex. We discuss the identification and central role of E3 ubiquitin ligases in ER quality control and viral degradation of the MHC-I chain.