Recruitment Of Receptors Research Articles

Abstract B42: Role of TRAIL signaling in tumor killing

Abstract TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is an endogenous ligand, which plays role in immune surveillance and anti-tumor immunity. Binding of TRAIL to its death receptor (DR4 or DR5) leads to receptor oligomerization and recruitment of the death-inducing signaling complex (DISC), activates the caspase-8 and its downstream caspase cascade and ultimately leads to cell death. Although TRAIL induces apoptosis in many tumor cell lines, almost all normal cells are insensitive to TRAIL-induced cell death. The physiological role of TRAIL remains to be elucidated. Recently, scientists have turned their attention to the emerging role of TRAIL in modulating immune responses. TRAIL administration induced anti-inflammatory effects in several autoimmune animal models. All these results suggest an apoptosis-independent role of TRAIL in the immune system. In this study, we studied the role of TRAIL signaling in immune resistance to tumor growth by using the TRAILR-R knockout mice. We demonstrated TRAIL suppressed T cell activation stimulated by anti-CD3/CD28. T cells from TRAIL-R knockout mice resisted to these effects and were with higher proliferation and cytokine production capability to anti-CD3/CD28 stimulation. However, when the melanoma cell lines B16-F10 were inoculated, there was enhanced tumor growth in TRAIL-R knockout mice compared to the wild type control. This finding suggests TRAIL/TRAIL-R interaction may play a role in host against tumor growth. The immunophenotype of the tumor infiltrating lymphocytes and cytokine production are analyzed. Apoptosis and activation of caspases of tumor cells and infiltrating immune cells are also characterized. This study will help to elucidate the role of TRAIL signaling in tumor killing and immune surveillance. Citation Format: Shih-Hong Siao, Ping-Ning Hsu. Role of TRAIL signaling in tumor killing. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B42.

RIP3 Mediates Necroptotic Cell Death in Response to Acid Stimulation of ASIC1a

Acidosis occurs commonly under pathological conditions when body homeostasis is disrupted due to inflammation, hypoxia, cancer and other illnesses. However, the effects of acidosis on cell fates differ dramatically from one cell type to another. On one hand, acidosis is usually harmful to brain neurons, which are particularly sensitive to tissue acidosis that occurs very often among neurological disorders, including ischemic stroke. Because of this, acidotoxicity represents one of the major causes of neurodegeneration and brain injury. On the other hand, cancer cells thrive in acidity as the microenvironment of tumor is often acidic. Previously, we have shown that extracellular acid induces neuronal cell death through activation of acid-sensing ion channel isoform 1a (ASIC1a) in a manner that involves recruitment and phosphorylation of receptor interacting protein 1 (RIP1), but independent of the ion channel function of ASIC1a. Here, we demonstrate that receptor interacting protein 3 (RIP3) is also critically required for acid-induced cell death and the presence of ASIC1a and/or RIP3 determines the nature of cellular responses to acidosis. Whereas both ASIC1a and RIP3 are expressed in neurons, at least one of them tends to be absent in many cancer cell lines. In the absence of RIP3, acidosis activated NF-κB signaling to promote survival of the tumor through ASIC1a. Re-introduction of RIP3 then converted the cancer cells to undergo necrosis in response to acidosis. Conversely, inhibiting RIP3 reduced both acid-induced death of mouse cortical neurons in vitro and brain infarction in mice elicited by middle cerebral artery occlusion in vivo. Our data also implicate the formation of necrosome downstream of ASIC1a/RIP1 in response to acidosis in the presence of RIP3. Thus, ASIC1a works similarly as conventional death receptors that regulate both cell survival and demise. Differently controlling RIP3 expression and function may be of therapeutic values in treating different diseases.

Acetylated tau in Alzheimer's disease: An instigator of synaptic dysfunction underlying memory loss: Increased levels of acetylated tau blocks the postsynaptic signaling required for plasticity and promotes memory deficits associated with tauopathy.

Pathogenesis in tauopathies involves the accumulation of tau in the brain and progressive synapse loss accompanied by cognitive decline. Pathological tau is found at synapses, and it promotes synaptic dysfunction and memory deficits. The specific role of toxic tau in disrupting the molecular networks that regulate synaptic strength has been elusive. A novel mechanistic link between tau toxicity and synaptic plasticity involves the acetylation of two lysines on tau, K274, and K281, which are associated with dementia in Alzheimer's disease (AD). We propose that an increase in tau acetylated on these lysines blocks the expression of long-term potentiation at hippocampal synapses leading to impaired memory in AD. Acetylated tau could inhibit the activity-dependent recruitment of postsynaptic AMPA-type glutamate receptors required for plasticity by interfering with the postsynaptic localization of KIBRA, a memory-associated protein. Strategies that reduce the acetylation of tau may lead to effective treatments for cognitive decline in AD.

Calcium-permeable AMPA receptors and silentsynapses in cocaine-conditioned place preference.

Exposure to cocaine generates silent synapses in the nucleus accumbens (NAc), whose eventual unsilencing/maturation by recruitment of calcium-permeable AMPA-type glutamate receptors (CP-AMPARs) after drug withdrawal results in profound remodeling of NAc neuro-circuits. Silent synapse-based NAc remodeling was shown to be critical for several drug-induced behaviors, but its role in acquisition and retention of the association between drug rewarding effects and drug-associated contexts has remained unclear. Here, we find that the postsynaptic proteins PSD-93, PSD-95, and SAP102 differentially regulate excitatory synapse properties in the NAc. Mice deficient for either of these scaffold proteins exhibit distinct maturation patterns of silent synapses and thus provided instructive animal models to examine the role of NAc silent synapse maturation in cocaine-conditioned place preference (CPP). Wild-type and knockout mice alike all acquired cocaine-CPP and exhibited increased levels of silent synapses after drug-context conditioning. However, the mice differed in CPP retention and CP-AMPAR incorporation. Collectively, our results indicate that CP-AMPAR-mediated maturation of silent synapses in the NAc is a signature of drug-context association, but this maturation is not required for establishing or retaining cocaine-CPP.

Endosomal Phosphatidylinositol 3-Phosphate Promotes Gephyrin Clustering and GABAergic Neurotransmission at Inhibitory Postsynapses

The formation of neuronal synapses and the dynamic regulation of their efficacy depend on the proper assembly of the postsynaptic neurotransmitter receptor apparatus. Receptor recruitment to inhibitory GABAergic postsynapses requires the scaffold protein gephyrin and the guanine nucleotide exchange factor collybistin (Cb). In vitro, the pleckstrin homology domain of Cb binds phosphoinositides, specifically phosphatidylinositol 3-phosphate (PI3P). However, whether PI3P is required for inhibitory postsynapse formation is currently unknown. Here, we investigated the role of PI3P at developing GABAergic postsynapses by using a membrane-permeant PI3P derivative, time-lapse confocal imaging, electrophysiology, as well as knockdown and overexpression of PI3P-metabolizing enzymes. Our results provide the first in cellula evidence that PI3P located at early/sorting endosomes regulates the postsynaptic clustering of gephyrin and GABAA receptors and the strength of inhibitory, but not excitatory, postsynapses in cultured hippocampal neurons. In human embryonic kidney 293 cells, stimulation of gephyrin cluster formation by PI3P depends on Cb. We therefore conclude that the endosomal pool of PI3P, generated by the class III phosphatidylinositol 3-kinase, is important for the Cb-mediated recruitment of gephyrin and GABAA receptors to developing inhibitory postsynapses and thus the formation of postsynaptic membrane specializations.

Structure of the Toll/Interleukin-1 Receptor (TIR) Domain of the B-cell Adaptor That Links Phosphoinositide Metabolism with the Negative Regulation of the Toll-like Receptor (TLR) Signalosome

Ligand binding to Toll-like receptors (TLRs) results in dimerization of their cytosolic Toll/interleukin-1 receptor (TIR) domains and recruitment of post-receptor signal transducers into a complex signalosome. TLR activation leads to the production of transcription factors and pro-inflammatory molecules and the activation of phosphoinositide 3-kinases (PI3K) in a process that requires the multimodular B-cell adaptor for phosphoinositide 3-kinase (BCAP). BCAP has a sequence previously proposed as a “cryptic” TIR domain. Here, we present the structure of the N-terminal region of human BCAP and show that it possesses a canonical TIR fold. Dimeric BCAP associates with the TIR domains of TLR2/4 and MAL/TIRAP, suggesting that it is recruited to the TLR signalosome by multitypic TIR-TIR interactions. BCAP also interacts with the p85 subunit of PI3K and phospholipase Cγ, enzymes that deplete plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2), and these interactions provide a molecular explanation for BCAP-mediated down-regulation of inflammatory signaling.

Tyrosine Phosphorylation of the Pioneer Transcription Factor FoxA1 Promotes Activation of Estrogen Signaling

The pioneer transcription factor FoxA1 plays an important role in estrogen signaling by opening closed chromatin and promoting recruitment of the estrogen receptor to its target regions in DNA. In this study, we analyzed tyrosine phosphorylation of FoxA1 by the non-receptor-type tyrosine kinase c-Abl. c-Abl was shown to phosphorylate FoxA1 at multiple sites, especially in the N- and C-terminal regions. Tyr429 and Tyr464 were identified as the major phosphorylation sites in the FoxA1 C-terminal region. The phosphomimetic and nonphosphorylatable FoxA1 mutants were generated by glutamic acid and phenylalanine substitutions at these tyrosine residues, respectively. The phosphomimetic FoxA1 promoted the activation of estrogen signaling, whereas the nonphosphorylatable FoxA1 suppressed its activation. Stimulation with the epidermal growth factor, which activates c-Abl, enhanced the activation of estrogen signaling. In contrast, the c-Abl inhibitor imatinib reduced its activation. The phosphomimetic FoxA1 mutant showed a higher affinity toward histone H3 than the wild-type. These results suggest that c-Abl-mediated phosphorylation of FoxA1 promotes the activation of estrogen signaling by inducing its binding to histones. J. Cell. Biochem. 118: 1453-1461, 2017. © 2016 Wiley Periodicals, Inc.

BNN27, a 17-Spiroepoxy Steroid Derivative, Interacts With and Activates p75 Neurotrophin Receptor, Rescuing Cerebellar Granule Neurons from Apoptosis.

Neurotrophin receptors mediate a plethora of signals affecting neuronal survival. The p75 pan-neurotrophin receptor controls neuronal cell fate after its selective activation by immature and mature isoforms of all neurotrophins. It also exerts pleiotropic effects interacting with a variety of ligands in different neuronal or non-neuronal cells. In the present study, we explored the biophysical and functional interactions of a blood-brain-barrier (BBB) permeable, C17-spiroepoxy steroid derivative, BNN27, with p75NTR receptor. BNN27 was recently shown to bind to NGF high-affinity receptor, TrkA. We now tested the p75NTR-mediated effects of BNN27 in mouse Cerebellar Granule Neurons (CGNs), expressing p75NTR, but not TrkA receptors. Our findings show that BNN27 physically interacts with p75NTR receptors in specific amino-residues of its extracellular domain, inducing the recruitment of p75NTR receptor to its effector protein RIP2 and the simultaneous release of RhoGDI in primary neuronal cells. Activation of the p75NTR receptor by BNN27 reverses serum deprivation-induced apoptosis of CGNs resulting in the decrease of the phosphorylation of pro-apoptotic JNK kinase and of the cleavage of Caspase-3, effects completely abolished in CGNs, isolated from p75NTR null mice. In conclusion, BNN27 represents a lead molecule for the development of novel p75NTR ligands, controlling specific p75NTR-mediated signaling of neuronal cell fate, with potential applications in therapeutics of neurodegenerative diseases and brain trauma.

HRG/HER2/HER3 signaling promotes AhR-mediated Memo-1 expression and migration in colorectal cancer.

Colorectal cancer (CRC) is a complex disease with still unsatisfactory prognosis even in western societies, although substantial progress has been made in pre-screening programs, surgical techniques and targeted therapy options. Mediator of motility-1 (Memo-1) was previously recognized as an important effector of cell migration downstream of receptor tyrosine kinase signaling in breast cancer. This study identified Memo-1 as frequently overexpressed in CRC and established a close link between extracellular HER2 activation, AhR/ARNT transcriptional activity and Memo-1 expression. Dissection of the hMemo-1 gene promoter using reporter assays and chromatin IP techniques revealed recruitment of Aryl hydrocarbon receptor (AhR)/Aryl hydrocarbon receptor nuclear-translocator (ARNT) complex, which positively influenced Memo-1 expression in cancer cells. We found that Memo-1 depletion negatively influenced the cellular actin network and that its expression is required for HER2-mediated cell migration and invasion. Moreover, analyses of Memo-1 expression in primary CRC revealed correlation with clinical parameters that point to Memo-1 as a new prognostic factor of aggressive disease in CRC patients. Altogether, these observations demonstrate that Memo-1 is an important downstream regulator of HER2-driven CRC cell migration and invasion through connecting extracellular signals from membrane to the cytoskeletal actin network.

Tectal-derived interneurons contribute to phasic and tonic inhibition in the visual thalamus.

The release of GABA from local interneurons in the dorsal lateral geniculate nucleus (dLGN-INs) provides inhibitory control during visual processing within the thalamus. It is commonly assumed that this important class of interneurons originates from within the thalamic complex, but we now show that during early postnatal development Sox14/Otx2-expressing precursor cells migrate from the dorsal midbrain to generate dLGN-INs. The unexpected extra-diencephalic origin of dLGN-INs sets them apart from GABAergic neurons of the reticular thalamic nucleus. Using optogenetics we show that at increased firing rates tectal-derived dLGN-INs generate a powerful form of tonic inhibition that regulates the gain of thalamic relay neurons through recruitment of extrasynaptic high-affinity GABAA receptors. Therefore, by revising the conventional view of thalamic interneuron ontogeny we demonstrate how a previously unappreciated mesencephalic population controls thalamic relay neuron excitability.

Molecular Determinants of Scaffold-induced Linear Ubiquitinylation of B Cell Lymphoma/Leukemia 10 (Bcl10) during T Cell Receptor and Oncogenic Caspase Recruitment Domain-containing Protein 11 (CARD11) Signaling

The activation of NF-κB downstream of T cell receptor (TCR) engagement is a key signaling step required for normal lymphocyte function during the adaptive immune response. During TCR signaling, the adaptor protein Bcl10 is inducibly recruited to the CARD11 scaffold protein as part of a multicomponent complex that induces IκB kinase (IKK) activity and NF-κB activation. Here, we show that a consequence of this recruitment is the TCR-induced conjugation of Bcl10 with linear-linked polyubiquitin chains to generate the signaling intermediate Lin(Ub)n-Bcl10, which is required for the association of Bcl10 with the NEMO subunit of the IKK complex. The TCR-induced generation of Lin(Ub)n-Bcl10 requires Bcl10 lysines 17, 31, and 63, CARD11, MALT1, and the HOIP subunit of the linear ubiquitin chain assembly complex (LUBAC) but not the HOIP accessory protein SHARPIN. CARD11 promotes signal-induced Lin(Ub)n-Bcl10 generation by co-recruiting Bcl10 with HOIP, thereby bringing substrate to enzyme. The CARD11-HOIP interaction is rendered TCR-inducible by the four autoinhibitory repressive elements in the CARD11 inhibitory domain and involves the CARD11 coiled-coil domain and two independent regions of HOIP. Interestingly, oncogenic CARD11 variants associated with diffuse large B cell lymphoma spontaneously induce Lin(Ub)n-Bcl10 production to extents that correlate with their abilities to activate NF-κB and with their enhanced abilities to bind HOIP and Bcl10. Our results define molecular determinants that control the production of Lin(Ub)n-Bcl10, an important signaling intermediate in TCR and oncogenic CARD11 signaling.

A phenomenological model of cell-cell adhesion mediated by cadherins.

We present a phenomenological model intended to describe at the protein population level the formation of cell-cell junctions by the local recruitment of homophilic cadherin adhesion receptors. This modeling may have a much wider implication in biological processes since many adhesion receptors, channel proteins and other membrane-born proteins associate in clusters or oligomers at the cell surface. Mathematically, it consists in a degenerate reaction-diffusion system of two partial differential equations modeling the time-space evolution of two cadherin populations over a surface: the first one represents the diffusing cadherins and the second one concerns the fixed ones. After discussing the stability of the solutions of the model, we perform numerical simulations and show relevant analogies with experimental results. In particular, we show patterns or aggregates formation for a certain set of parameters. Moreover, perturbing the stationary solution, both density populations converge in large times to some saturation level. Finally, an exponential rate of convergence is numerically obtained and is shown to be in agreement, for a suitable set of parameters, with the one obtained in some in vitro experiments.

Ribosome-stalk biogenesis is coupled with recruitment of nuclear-export factor to the nascent 60S subunit.

Nuclear export of preribosomal subunits is a key step during eukaryotic ribosome formation. To efficiently pass through the FG-repeat meshwork of the nuclear pore complex, the large pre-60S subunit requires several export factors. Here we describe the mechanism of recruitment of the Saccharomyces cerevisiae RNA-export receptor Mex67-Mtr2 to the pre-60S subunit at the proper time. Mex67-Mtr2 binds at the premature ribosomal-stalk region, which later during translation serves as a binding platform for translational GTPases on the mature ribosome. The assembly factor Mrt4, a structural homolog of cytoplasmic-stalk protein P0, masks this site, thus preventing untimely recruitment of Mex67-Mtr2 to nuclear pre-60S particles. Subsequently, Yvh1 triggers Mrt4 release in the nucleus, thereby creating a narrow time window for Mex67-Mtr2 association at this site and facilitating nuclear export of the large subunit. Thus, a spatiotemporal mark on the ribosomal stalk controls the recruitment of an RNA-export receptor to the nascent 60S subunit.

The Neurotrophic Factor Receptor p75 in the Rat Dorsolateral Striatum Drives Excessive Alcohol Drinking.

Brain-derived neurotrophic factor (BDNF) signaling in the dorsolateral striatum (DLS) keeps alcohol intake in moderation. For example, activation of the BDNF receptor tropomyosin receptor kinase B (TrkB) in the DLS reduces intake in rats that consume moderate amounts of alcohol. Here, we tested whether long-term excessive consumption of alcohol produces neuroadaptations in BDNF signaling in the rat DLS. We found that BDNF was no longer able to gate alcohol self-administration after a history of repeated cycles of binge alcohol drinking and withdrawal. We then elucidated the possible neuroadaptations that could block the ability of BDNF to keep consumption of alcohol in moderation. We report that intermittent access to 20% alcohol in a two-bottle choice paradigm that models excessive alcohol drinking produces a mobilization of DLS p75 neurotrophin receptor (p75NTR), whose activities oppose those of the Trk receptors, including TrkB. These neuroadaptations were not observed in the DLS of rats exposed to continuous access to 10% alcohol or in rats consuming sucrose. Furthermore, short hairpin RNA (shRNA)-mediated knockdown of the p75NTR gene in the DLS, as well as intra-DLS infusion or systemic administration of the p75NTR modulator, LM11A-31, significantly reduced binge drinking of alcohol. Together, our results suggest that excessive alcohol consumption produces a change in BDNF signaling in the DLS, which is mediated by the recruitment of p75NTR. Our data also imply that modulators of p75NTR signaling could be developed as medications for alcohol abuse disorders. Neuroadaptations gate or drive excessive, compulsive alcohol drinking. We previously showed that brain-derived neurotrophic factor and its receptor, TrkB, in the dorsolateral striatum (DLS), are part of an endogenous system that keeps alcohol drinking in moderation. Here, we show that a history of excessive alcohol intake produces neuroadaptations in the DLS that preclude BDNF's ability to gate alcohol self-administration in rats by the recruitment of the low-affinity neurotrophin receptor, p75NTR, whose activities opposes those of the Trk receptors. Finally, we show that the administration of the p75NTR modulator, LM11A-31, significantly reduces excessive alcohol intake suggesting that the drug may be developed as a new treatment for alcohol abuse disorders.

AT2 Receptors: Potential Therapeutic Targets for Hypertension.

The renin-angiotensin system (RAS) is arguably the most important and best studied hormonal system in the control of blood pressure (BP) and the pathogenesis of hypertension. The RAS features its main effector angiotensin II (Ang II) acting via its 2 major receptors, angiotensin type-1(AT1R) and type-2 (AT2R). In general, AT2Rs oppose the detrimental actions of Ang II via AT1Rs. AT2R activation induces vasodilation and natriuresis, but its effects to lower BP in hypertension have not been as clear as anticipated. Recent studies, however, have demonstrated that acute and chronic AT2R stimulation can induce natriuresis and lower BP in the Ang II infusion model of experimental hypertension. AT2R activation induces receptor recruitment from intracellular sites to the apical plasma membranes of renal proximal tubule cells via a bradykinin, nitric oxide, and cyclic guanosine 3',5' monophosphate signaling pathway that results in internalization and inactivation of sodium (Na+) transporters Na+-H+ exchanger-3 and Na+/K+ATPase. These responses do not require the presence of concurrent AT1R blockade and are effective both in the prevention and reversal of hypertension. This review will address the role of AT2Rs in the control of BP and Na+ excretion and the case for these receptors as potential therapeutic targets for hypertension in humans.

ATP from synaptic terminals and astrocytes regulates NMDA receptors and synaptic plasticity through PSD-95 multi-protein complex.

Recent studies highlighted the importance of astrocyte-secreted molecules, such as ATP, for the slow modulation of synaptic transmission in central neurones. Biophysical mechanisms underlying the impact of gliotransmitters on the strength of individual synapse remain, however, unclear. Here we show that purinergic P2X receptors can bring significant contribution to the signalling in the individual synaptic boutons. ATP released from astrocytes facilitates a recruitment of P2X receptors into excitatory synapses by Ca2+-dependent mechanism. P2X receptors, co-localized with NMDA receptors in the excitatory synapses, can be activated by ATP co-released with glutamate from pre-synaptic terminals and by glia-derived ATP. An activation of P2X receptors in turn leads to down-regulation of postsynaptic NMDA receptors via Ca2+-dependent de-phosphorylation and interaction with PSD-95 multi-protein complex. Genetic deletion of the PSD-95 or P2X4 receptors obliterated ATP-mediated down-regulation of NMDA receptors. Impairment of purinergic modulation of NMDA receptors in the PSD-95 mutants dramatically decreased the threshold of LTP induction and increased the net magnitude of LTP. Our findings show that synergistic action of glia- and neurone-derived ATP can pre-modulate efficacy of excitatory synapses and thereby can have an important role in the glia-neuron communications and brain meta-plasticity.

The Arp2/3 Complex Is Essential for Distinct Stages of Spine Synapse Maturation, Including Synapse Unsilencing.

Dendritic filopodia are actin-rich structures that are thought to contribute to early spine synapse formation; however, the actin regulatory proteins important for early synaptogenesis are poorly defined. Using organotypic hippocampal slice cultures and primary neuron hippocampal cultures from Arp2/3 conditional knock-out mice, we analyze the roles of the Arp2/3 complex, an actin regulator that creates branched actin networks, and demonstrate it is essential for distinct stages of both structural and functional maturation of excitatory spine synapses. Our data show that initially the Arp2/3 complex inhibits the formation of dendritic filopodia but that later during development, the Arp2/3 complex drives the morphological maturation from filopodia to typical spine morphology. Furthermore, we demonstrate that although the Arp2/3 complex is not required for key spine maturation steps, such as presynaptic contact and recruitment of MAGUK (membrane-associated guanylate kinase) scaffolding proteins or NMDA receptors, it is necessary for the recruitment of AMPA receptors. This latter process, also known as synapse unsilencing, is a final and essential step in the neurodevelopment of excitatory postsynaptic synaptogenesis, setting the stage for neuronal interconnectivity. These findings provide the first evidence that the Arp2/3 complex is directly involved in functional maturation of dendritic spines during the developmental period of spinogenesis. Excitatory spine synapse formation (spinogenesis) is a poorly understood yet pivotal period of neurodevelopment that occurs within 2-3 weeks after birth. Neurodevelopmental disorders such as intellectual disability and autism are characterized by abnormal spine structure, which may arise from abnormal excitatory synaptogenesis. The initial stage of spinogenesis is thought to begin with the emergence of actin-rich dendritic filopodia that initiate contact with presynaptic axonal boutons. However, it remains enigmatic how actin cytoskeletal regulation directs dendritic filopodial emergence or their subsequent maturation into dendritic spines during development and on into adulthood. In this study, we provide the first evidence that the Arp2/3 complex, a key actin nucleator, is involved in distinct stages of spine formation and is required for synapse unsilencing.

Epidermal Mineralocorticoid Receptor Plays Beneficial and Adverse Effects in Skin and Mediates Glucocorticoid Responses

Glucocorticoids (GCs) regulate skin homeostasis and combat cutaneous inflammatory diseases; however, adverse effects of chronic GC treatments limit their therapeutic use. GCs bind and activate the GC receptor and the mineralocorticoid receptor (MR), transcription factors that recognize identical hormone responsive elements. Whether epidermal MR mediates beneficial or deleterious GC effects is of great interest for improving GC-based skin therapies. MR epidermal knockout mice exhibited increased keratinocyte proliferation and differentiation and showed resistance to GC-induced epidermal thinning. However, crucially, loss of epidermal MR rendered mice more sensitive to inflammatory stimuli and skin damage. MR epidermal knockout mice showed increased susceptibility to phorbol 12-myristate 13-acetate-induced inflammation with higher cytokine induction. Likewise, cultured MR epidermal knockout keratinocytes had increased phorbol 12-myristate 13-acetate-induced NF-κB activation, highlighting an anti-inflammatory function for MR. GC-induced transcription was reduced in MR epidermal knockout keratinocytes, at least partially due to decreased recruitment of GC receptor to hormone responsive element-containing sequences. Our results support a role for epidermal MR in adult skin homeostasis and demonstrate nonredundant roles for MR and GC receptor in mediating GC actions.

Abstract 4430: Implication of the primary cilium in the Hedgehog pathway in colorectal cancer cells

Abstract Background: The Hedgehog (HH) pathway is involved in the maintenance of numerous cell types both during development and in the adult. Often deregulated in cancers, its involvement in colorectal cancer has come into view during the last few years, although its role remains poorly defined. In most tissues, the HH pathway is highly connected to the primary cilium (PC), an organelle not expressed in the normal colonic epithelium which recruits the functional components and regulates the pathway. Aims: Since the intestinal epithelium is known to be a non-ciliated tissue, the HH pathway as related to the PC has not been explored. We investigated the presence of PC in colon cancer tumors and cell lines. Our hypothesis is that PC formation controls the HH activation that has been reported in numerous colon cancer studies. Materials and methods: We used cellular models to look at HH activation, focusing on the final effector Gli1. The link between PC and the HH pathway was shown by the recruitment of the Smo receptor in the PC. We looked for PC in a subset of 63 tumors from a Tissue Micro Array and in 4 colorectal cell lines by immunofluorescence using two well-known markers, acetylated α-tubulin and polyglutamylated tubulin. 3D deconvoluted pictures were obtained to characterize the shape and size of PC. Using cellular models we investigated HH pathway expression by qPCR. We assessed the functional link between HH pathway and PC through localization of the Smo receptor in the PC using immunofluorescence. Results: We observed the presence of the PC in the epithelium of primary colorectal tumors at all stages but not in their normal counterparts. Using human colorectal cancer cell lines we found a clear correlation between the presence of the PC and the expression of the final HH effector, GLI1, and provide evidence of a functional link between the two by demonstrating the recruitment of the SMO receptor to the PC membrane. Conclusion: We conclude that the PC directly participates in the HH pathway in colorectal cancer cells which is the first observation of PC expression in tumors arising from a non-ciliated tissue. Functional studies are required to determine the mode of action of this organelle in carcinogenesis. Citation Format: Blanche Senicourt. Implication of the primary cilium in the Hedgehog pathway in colorectal cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4430.

Abstract 5139: Adjuvant immunotherapy targeting CSF1R to limit metastatic progression

Abstract Metastasis is often considered a late stage event, although the first changes in the metastatic site occur very early during localized primary tumor development. Although understanding of these initiating events is limited, immune suppression plays an essential role in allowing for the outgrowth of disseminating tumor cells. Targeting this immune suppressive milieu can hold promise to effectively inhibit metastatic progression. Colony stimulating factor-one (CSF-1) is overexpressed by many diverse tumor types and can induce expansion and recruitment of CSF-1 receptor (CSF-1R) expressing cells. CSF-1R is expressed on a multitude of myeloid cells including inflammatory monocytes, myeloid derived suppressor cells and macrophages, which are key immune suppressive cells in a primary tumor site. Using a metastatic rhabdomyosarcoma model, we have identified expansion of immune suppressive myeloid cells in the early metastatic microenvironment. PLX3397, which is small molecular inhibitor that selectively targets CSF-1R, Kit and Oncogenic FLT3, was used to determine the impact of targeting this immune suppressive microenvironment in limiting metastatic progression. PLX3397 and GW2580, a selective CSF1R inhibitor, reduce the immune suppressive capacity of bone marrow-derived monocytes on activated T cells. Adjuvant therapy with PLX3397 in resected rhabdomyosarcoma reduces metastatic spread. Targeting CSF-1R is associated with an expansion of CD11C expressing antigen presenting cells in the metastatic tissue and in combination with PD-1 blockade improves anti-tumor immunity. Continued investigation of combination immunotherapy with metastatic microenvironment targeting in the adjuvant setting holds promise to limit metastatic spread. Citation Format: Justin Evans, Amber J. Giles, Meera Murgai, Miki Kasai, Caitlin Reid, Rosandra Natasha Kaplan. Adjuvant immunotherapy targeting CSF1R to limit metastatic progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5139.