Latent Epstein-Barr virus infection collaborates with Myc over-expression in normal human B cells to induce Burkitt-like Lymphomas in mice.

Epstein-Barr virus (EBV) is an important cause of human lymphomas, including Burkitt lymphoma (BL). EBV+ BLs are driven by Myc translocation and have stringent forms of viral latency that do not express either of the two major EBV oncoproteins, EBNA2 (which mimics Notch signaling) and LMP1 (which activates NF-κB signaling). Suppression of Myc-induced apoptosis, often through mutation of the TP53 (p53) gene or inhibition of pro-apoptotic BCL2L11 (BIM) gene expression, is required for development of Myc-driven BLs. EBV+ BLs contain fewer cellular mutations in apoptotic pathways compared to EBV-negative BLs, suggesting that latent EBV infection inhibits Myc-induced apoptosis. Here we use an EBNA2-deleted EBV virus (ΔEBNA2 EBV) to create the first in vivo model for EBV+ BL-like lymphomas derived from primary human B cells. We show that cord blood B cells infected with both ΔEBNA2 EBV and a Myc-expressing vector proliferate indefinitely on a CD40L/IL21 expressing feeder layer in vitro and cause rapid onset EBV+ BL-like tumors in NSG mice. These LMP1/EBNA2-negative Myc-driven lymphomas have wild type p53 and very low BIM, and express numerous germinal center B cell proteins (including TCF3, BACH2, Myb, CD10, CCDN3, and GCSAM) in the absence of BCL6 expression. Myc-induced activation of Myb mediates expression of many of these BL-associated proteins. We demonstrate that Myc blocks LMP1 expression both by inhibiting expression of cellular factors (STAT3 and Src) that activate LMP1 transcription and by increasing expression of proteins (DNMT3B and UHRF1) known to enhance DNA methylation of the LMP1 promoters in human BLs. These results show that latent EBV infection collaborates with Myc over-expression to induce BL-like human B-cell lymphomas in mice. As NF-κB signaling retards the growth of EBV-negative BLs, Myc-mediated repression of LMP1 may be essential for latent EBV infection and Myc translocation to collaboratively induce human BLs.

Infantile (congenital) anaplastic intracranial solitary fibrous tumor/hemangiopericytoma-A case report with brief literature review.

Solitary fibrous tumor/hemangiopericytoma (SFT/HPC) is a rare primary central nervous system (CNS) tumor, included in the World Health Organization (WHO) 2016 classification. Very few cases have been described in the literature so far, especially the infantile type. It is a mesenchymal tumor of the fibroblastic type, characterized by the fusion of NAB 2 and STAT 6 genes. A 10-month-old boy presented to our neurosurgery department with complaints of increasing head circumference since 1 month of age. The magnetic resonance imaging (MRI) showed a space-occupying lesion measuring 8.2 cm × 7 cm × 6.9 cm in the fronto-temporo-parietal region with a clinical diagnosis of glioma/atypical teratoid rhabdoid tumor (ATRT). The microscopy revealed a spindle cell tumor arranged in a patternless pattern with variable cellularity, increased mitosis, and areas of coagulative necrosis. The immunohistochemistry showed vimentin, CD 34, STAT6, CD99 positivity whereas Glial fibrillary acidic protein, Epithelial membrane antigen, and S-100 negativity. Hence, a diagnosis of anaplastic SFT/HPC (grade-III) was rendered. The patient improved after gross total resection (GTR). The primary intracranial congenital SFT/HPC are extremely rare, often a clinico-radiologically misdiagnosed entity. Thus, the immunohistochemistry/molecular study in addition to histology is mandatory for accurate diagnosis.

Epstein-Barr virus LMP1 protein promotes proliferation and inhibits differentiation of epithelial cells via activation of YAP and TAZ.

Latent Epstein-Barr virus (EBV) infection promotes undifferentiated nasopharyngeal carcinomas (NPCs) in humans, but the mechanism(s) for this effect has been difficult to study because EBV cannot transform normal epithelial cells in vitro and the EBV genome is often lost when NPC cells are grown in culture. Here we show that the latent EBV protein, LMP1 (Latent membrane protein 1), induces cellular proliferation and inhibits spontaneous differentiation of telomerase-immortalized normal oral keratinocytes (NOKs) in growth factor-deficient conditions by increasing the activity of the Hippo pathway effectors, YAP (Yes-associated protein) and TAZ (Transcriptional coactivator with PDZ-binding motif). We demonstrate that LMP1 enhances YAP and TAZ activity in NOKs both by decreasing Hippo pathway-mediated serine phosphorylation of YAP and TAZ and increasing Src kinase-mediated Y357 phosphorylation of YAP. Furthermore, knockdown of YAP and TAZ is sufficient to reduce proliferation and promote differentiation in EBV-infected NOKs. We find that YAP and TAZ are also required for LMP1-induced epithelial-to-mesenchymal transition. Importantly, we demonstrate that ibrutinib (an FDA-approved BTK inhibitor that blocks YAP and TAZ activity through an off-target effect) restores spontaneous differentiation and inhibits proliferation of EBV-infected NOKs at clinically relevant doses. These results suggest that LMP1-induced YAP and TAZ activity contributes to the development of NPC.

Type 1 and Type 2 Epstein-Barr viruses induce proliferation, and inhibit differentiation, in infected telomerase-immortalized normal oral keratinocytes.

Differentiated epithelial cells are an important source of infectious EBV virions in human saliva, and latent Epstein-Barr virus (EBV) infection is strongly associated with the epithelial cell tumor, nasopharyngeal carcinoma (NPC). However, it has been difficult to model how EBV contributes to NPC, since EBV has not been shown to enhance proliferation of epithelial cells in monolayer culture in vitro and is not stably maintained in epithelial cells without antibiotic selection. In addition, although there are two major types of EBV (type 1 (T1) and type 2 (T2)), it is currently unknown whether T1 and T2 EBV behave differently in epithelial cells. Here we inserted a G418 resistance gene into the T2 EBV strain, AG876, allowing us to compare the phenotypes of T1 Akata virus versus T2 AG876 virus in a telomerase-immortalized normal oral keratinocyte cell line (NOKs) using a variety of different methods, including RNA-seq analysis, proliferation assays, immunoblot analyses, and air-liquid interface culture. We show that both T1 Akata virus infection and T2 AG876 virus infection of NOKs induce cellular proliferation, and inhibit spontaneous differentiation, in comparison to the uninfected cells when cells are grown without supplemental growth factors in monolayer culture. T1 EBV and T2 EBV also have a similar ability to induce epithelial-to-mesenchymal (EMT) transition and activate canonical and non-canonical NF-κB signaling in infected NOKs. In contrast to our recent results in EBV-infected lymphoblastoid cells (in which T2 EBV infection is much more lytic than T1 EBV infection), we find that NOKs infected with T1 and T2 EBV respond similarly to lytic inducing agents such as TPA treatment or differentiation. These results suggest that T1 and T2 EBV have similar phenotypes in infected epithelial cells, with both EBV types enhancing cellular proliferation and inhibiting differentiation when growth factors are limiting.

Glutamate binding triggers monomerization of unliganded mGluR2 dimers.

The Metabotropic glutamate receptor 2 (mGluR2) is involved in several neurological and psychiatric disorders and is an attractive drug target. It is believed to form a strict dimer and the dimeric assembly is necessary for glutamate induced activation. Although many studies have focused on glutamate induced conformational changes, the dimerization propensity of mGluR2 with and without glutamate has never been investigated. Also, the role of the unstructured loop in dimerization of mGluR2 is not clear. Here, using Forster Resonance Energy Transfer (FRET) based assay in live cells we show that mGluR2 does not form a "strict dimer" rather it exists in a dynamic monomer-dimer equilibrium. The unstructured loop moderately destabilizes the dimers. Furthermore, binding of glutamate to mGluR2 induces conformational change that promotes monomerization of mGluR2. In the absence of an unstructured loop, mGluR2 neither undergoes conformational change nor monomerizes upon binding to glutamate.

Defining the Homo- and Heterodimerization Propensities of Metabotropic Glutamate Receptors.

The eight metabotropic glutamate receptors (mGluRs) serve critical modulatory roles throughout the nervous system. The molecular diversity of mGluRs is thought to be further expanded by the formation of heterodimers, but the co-expression of mGluR subtypes at the cellular level and the relative propensities of heterodimer formation are not well known. Here, we analyze single-cell RNA sequencing data and find that cortical pyramidal cells express multiple mGluR subtypes with distinct profiles for different receptor combinations. We then develop quantitative, fluorescence-based assays to define the relative homo- and heterodimer propensities across group-I, -II, and -III mGluRs. We find a strong preference for heterodimerization in a number of cases, including mGluR2 with mGluR3, which we confirm in frontal cortex using in situ RNA hybridization and co-immunoprecipitation. Together, our findings support the biological relevance of mGluR heterodimerization and highlight the complex landscape of mGluR populations in the brain.

Revisiting a controversy: The effect of EGF on EGFR dimer stability.

EGFR is a receptor tyrosine kinase that plays a critical role in cell proliferation, differentiation, survival and migration. Its activating ligand, EGF, has long been believed to stabilize the EGFR dimer. Two research studies aimed at quantitative measurements of EGFR dimerization, however, have led to contradicting conclusions and have questioned this view. Given the controversy, here we sought to measure the dimerization of EGFR in the absence and in the presence of saturating EGF concentrations, and to tease out the effect of ligand on dimer stability, using a FRET-based quantitative method. Our measurements show that the dissociation constant is decreased ~150 times due to ligand binding, indicative of significant dimer stabilization. In addition, our measurements demonstrate that EGF binding induces a conformational change in the EGFR dimer.

Unstructured loop is essential for the activation of mGluR2.

Metabotropic Glutamate Receptors (mGluRs) are Class C G-protein coupled receptors (GPCRs) that are expressed throughout the central nervous system and are involved in several neurological and psychiatric disorders. Although, many studies focused on Glutamate induced activation of mGluR2, however, the role of unstructured loop (or "BC loop") in activation of metabotropic Glutamate receptors is currently unknown. Here, using Förster Resonance Energy Transfer (FRET) based assay in live cells we show that unstructured loop is required for Glutamate induced conformation and hence the activation of the receptor.

Newly Discovered Micropeptide Regulators of SERCA Form Oligomers but Bind to the Pump as Monomers.

The recently-discovered single-span transmembrane proteins endoregulin (ELN), dwarf open reading frame (DWORF), myoregulin (MLN), and another-regulin (ALN) are reported to bind to the SERCA calcium pump in a manner similar to that of known regulators of SERCA activity, phospholamban (PLB) and sarcolipin (SLN). To determine how micropeptide assembly into oligomers affects the availability of the micropeptide to bind to SERCA in a regulatory complex, we used co-immunoprecipitation and fluorescence resonance energy transfer (FRET) to quantify micropeptide oligomerization and SERCA-binding. Micropeptides formed avid homo-oligomers with high-order stoichiometry (n > 2 protomers per homo-oligomer), but it was the monomeric form of all micropeptides that interacted with SERCA. In view of these two alternative binding interactions, we evaluated the possibility that oligomerization occurs at the expense of SERCA-binding. However, even the most avidly oligomeric micropeptide species still showed robust FRET with SERCA, and there was a surprising positive correlation between oligomerization affinity and SERCA-binding. This comparison of micropeptide family members suggests that the same structural determinants that support oligomerization are also important for binding to SERCA. Moreover, the unique oligomerization/SERCA-binding profile of DWORF is in harmony with its distinct role as a PLB-competing SERCA activator, in contrast to the inhibitory function of the other SERCA-binding micropeptides.

Erratum: Author Correction: The EphA2 receptor is activated through induction of distinct, ligand-dependent oligomeric structures.

[This corrects the article DOI: 10.1038/s42003-018-0017-7.].

The EphA2 receptor is activated through induction of distinct, ligand-dependent oligomeric structures.

The EphA2 receptor tyrosine kinase is capable of activating multiple diverse signaling pathways with roles in processes such as tissue homeostasis and cancer. EphA2 is known to form activated oligomers in the presence of ephrin-A ligands. Here, we characterize the lateral interactions between full-length EphA2 molecules in the plasma membrane in the presence of three types of ligands (dimeric ephrinA1-Fc, monomeric ephrinA1, and an engineered peptide ligand) as well as in the absence of ligand, using a quantitative FRET technique. The data show that EphA2 forms higher-order oligomers and two different types of dimers that all lead to increased EphA2 tyrosine phosphorylation, which is indicative of increased kinase-dependent signaling. We find that different ligands stabilize conformationally distinct oligomers that are assembled through two different interfaces. Our results suggest that these different oligomeric assemblies could have distinct signaling properties, contributing to the diverse activities of the EphA2 receptor.

Outcome of compartment resection of locally advanced oral cancers extending to infratemporal fossa: a tertiary rural hospital experience.

Locally advanced oral cancers extending to infratemporal fossa (ITF) are a challenge to head and neck surgeons. These tumors are classified as T4b whenever the masticator space (MS), pterygoid muscles (PM), and pterygoid plates (PP) are involved according to AJCC classification. Until recently, these tumors were considered inoperable and treated only with palliative intent. However, a few studies in the last decade showed that many of these tumors could be resected with a reasonably favorable prognosis by compartment resection of ITF, particularly when the tumor was below sigmoid notch of mandible. A few studies attempted to downstage these tumors by neo-adjuvant chemotherapy before attempting resection. Oral Squamous cell carcinoma has a high prevalence in South India. Majority of these patients are females addicted to tobacco quid chewing and present with locally advanced disease. In this retrospective analysis, we evaluated the outcome of treatment of oral squamous cell carcinoma extending to ITF and staged T4b in 52 patients. All patients underwent Composite resection including compartment resection of ITF followed by adjuvant treatment. 20 patients had received neo-adjuvant chemotherapy. Pectoralis major myocutaneous flap was the mainstay of reconstruction. After mean follow-up of 2 years, 31 patients are alive and disease free. 14 patients had local recurrence in ITF and 2 patients had recurred in cervical nodes. 8 patients died due to disease and 6 are on palliative care. Neo-adjuvant chemotherapy did not benefit the outcome. Close margins of resection, extra nodal spread from lymph nodes and supra notch and involvement of posterior part of ITF were factors which predisposed to recurrence.

Intracellular Domain Contacts Contribute to Ecadherin Constitutive Dimerization in the Plasma Membrane.

Epithelial cadherin (Ecadherin) is responsible for the intercellular cohesion of epithelial tissues. It forms lateral clusters within adherens cell-cell junctions, but its association state outside these clusters is unknown. Here, we use a quantitative Forster resonance energy transfer (FRET) approach to show that Ecadherin forms constitutive dimers and that these dimers exist independently of the actin cytoskeleton or cytoplasmic proteins. The dimers are stabilized by intermolecular contacts that occur along the entire length of Ecadherin, with the intracellular domains having a surprisingly strong favorable contribution. We further show that Ecadherin mutations and calcium depletion induce structural alterations that propagate from the N terminus all the way to the C terminus, without destabilizing the dimeric state. These findings provide context for the interpretation of Ecadherin adhesion experiments. They also suggest that early events of adherens junction assembly involve interactions between from preformed Ecadherin dimers.

The SAM domain inhibits EphA2 interactions in the plasma membrane.

All members of the Eph receptor family of tyrosine kinases contain a SAM domain near the C terminus, which has been proposed to play a role in receptor homotypic interactions and/or interactions with binding partners. The SAM domain of EphA2 is known to be important for receptor function, but its contribution to EphA2 lateral interactions in the plasma membrane has not been determined. Here we use a FRET-based approach to directly measure the effect of the SAM domain on the stability of EphA2 dimers on the cell surface in the absence of ligand binding. We also investigate the functional consequences of EphA2 SAM domain deletion. Surprisingly, we find that the EphA2 SAM domain inhibits receptor dimerization and decreases EphA2 tyrosine phosphorylation. This role is dramatically different from the role of the SAM domain of the related EphA3 receptor, which we previously found to stabilize EphA3 dimers and increase EphA3 tyrosine phosphorylation in cells in the absence of ligand. Thus, the EphA2 SAM domain likely contributes to a unique mode of EphA2 interaction that leads to distinct signaling outputs.

A small peptide promotes EphA2 kinase-dependent signaling by stabilizing EphA2 dimers.

BACKGROUND: The EphA2 receptor tyrosine kinase is known to promote cancer cell malignancy in the absence of activation by ephrin ligands. This behavior depends on high EphA2 phosphorylation on Ser897 and low tyrosine phosphorylation, resulting in increased cell migration and invasiveness. We have previously shown that EphA2 forms dimers in the absence of ephrin ligand binding, and that dimerization of unliganded EphA2 can decrease EphA2 Ser897 phosphorylation. We have also identified a small peptide called YSA, which binds EphA2 and competes with the naturally occurring ephrin ligands. METHODS: Here, we investigate the effect of YSA on EphA2 dimer stability and EphA2 function using quantitative FRET techniques, Western blotting, and cell motility assays. RESULTS: We find that the YSA peptide stabilizes the EphA2 dimer, increases EphA2 Tyr phosphorylation, and decreases both Ser897 phosphorylation and cell migration. CONCLUSIONS: The experiments demonstrate that the small peptide ligand YSA reduces EphA2 Ser897 pro-tumorigenic signaling by stabilizing the EphA2 dimer. GENERAL SIGNIFICANCE: This work is a proof-of-principle demonstration that EphA2 homointeractions in the plasma membrane can be pharmacologically modulated to decrease the pro-tumorigenic signaling of the receptor.

EphA2 Receptor Unliganded Dimers Suppress EphA2 Pro-tumorigenic Signaling.

The EphA2 receptor tyrosine kinase promotes cell migration and cancer malignancy through a ligand- and kinase-independent distinctive mechanism that has been linked to high Ser-897 phosphorylation and low tyrosine phosphorylation. Here, we demonstrate that EphA2 forms dimers in the plasma membrane of HEK293T cells in the absence of ephrin ligand binding, suggesting that the current seeding mechanism model of EphA2 activation is incomplete. We also characterize a dimerization-deficient EphA2 mutant that shows enhanced ability to promote cell migration, concomitant with increased Ser-897 phosphorylation and decreased tyrosine phosphorylation compared with EphA2 wild type. Our data reveal a correlation between unliganded dimerization and tumorigenic signaling and suggest that EphA2 pro-tumorigenic activity is mediated by the EphA2 monomer. Thus, a therapeutic strategy that aims at the stabilization of EphA2 dimers may be beneficial for the treatment of cancers linked to EphA2 overexpression.

Unliganded EphA3 dimerization promoted by the SAM domain.

The erythropoietin-producing hepatocellular carcinoma A3 (EphA3) receptor tyrosine kinase (RTK) regulates morphogenesis during development and is overexpressed and mutated in a variety of cancers. EphA3 activation is believed to follow a 'seeding mechanism' model, in which ligand binding to the monomeric receptor acts as a trigger for signal-productive receptor clustering. We study EphA3 lateral interactions on the surface of live cells and we demonstrate that EphA3 forms dimers in the absence of ligand binding. We further show that these dimers are stabilized by interactions involving the EphA3 sterile α-motif (SAM) domain. The discovery of unliganded EphA3 dimers challenges the current understanding of the chain of EphA3 activation events and suggests that EphA3 may follow the 'pre-formed dimer' model of activation known to be relevant for other receptor tyrosine kinases. The present work also establishes a new role for the SAM domain in promoting Eph receptor lateral interactions and signalling on the cell surface.

The sigma-1 receptors are present in monomeric and oligomeric forms in living cells in the presence and absence of ligands.

The sigma-1 receptor (S1R) is a 223-amino-acid membrane protein that resides in the endoplasmic reticulum and the plasma membrane of some mammalian cells. The S1R is regulated by various synthetic molecules including (+)-pentazocine, cocaine and haloperidol and endogenous molecules such as sphingosine, dimethyltryptamine and dehydroepiandrosterone. Ligand-regulated protein chaperone functions linked to oxidative stress and neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) and neuropathic pain have been attributed to the S1R. Several client proteins that interact with S1R have been identified including various types of ion channels and G-protein coupled receptors (GPCRs). When S1R constructs containing C-terminal monomeric GFP2 and YFP fusions were co-expressed in COS-7 cells and subjected to FRET spectrometry analysis, monomers, dimers and higher oligomeric forms of S1R were identified under non-liganded conditions. In the presence of the prototypic S1R agonist, (+)-pentazocine, however, monomers and dimers were the prevailing forms of S1R. The prototypic antagonist, haloperidol, on the other hand, favoured higher order S1R oligomers. These data, in sum, indicate that heterologously expressed S1Rs occur in vivo in COS-7 cells in multiple oligomeric forms and that S1R ligands alter these oligomeric structures. We suggest that the S1R oligomerization states may regulate its function(s).

Cervical chondrosarcoma- rare malignancy: a case report.

To highlight an uncommon bone malignancy, which presented to our institute, as a neck swelling in the supraclavicular region. A 30 year old man presented with history of swelling on the left side of neck since 1 year and numbness of left upper limb since 6 months. Magnetic Resonance Imaging of the Cervical spine & MR Angiography showed a 7.4 × 4.6 cm expansile lesion involving transverse process of C5-C7 vertebrae. As the tumour was found to be deep to the phrenic nerve & brachial plexus, a dual approach was used, anteriorly via neck incision and posteriorly via the spine. The tumour was resected & iliac crest grafted along with stabilization of the cervical spine. Patient is disease free and the cervical spine stabilized with normal movements at two and half years follow up. We need to consider tumour arising from the vertebra as a differential diagnosis for any deep seated hard neck swelling in the supraclavicular region. Even low grade malignancy of this region when resected en-bloc will have a good prognosis.