NSG-70, a new glioblastoma cell line with mixed proneural-mesenchymal features, associates NOTCH1-WNT5A signaling with stem cell maintenance and angiogenesis.

BACKGROUND: Glioblastoma initiation and progression is believed to be driven by Glioma stem cells (GSCs). Activation of NOTCH1 and WNT, and more recently, non-canonical WNT5A signaling, has been demonstrated to regulate self-renewal and differentiation of the GSCs crucially. High expression levels of NOTCH1 and WNT in GBM tumors contribute to the sustenance of GSCs and mediate characteristic phenotypic plasticity, which is reflected by the different subtypes and tremendous intra-tumor heterogeneity. However, the coregulation of NOTCH1 and WNT5A is not well understood. Here, we studied the role of these molecules in regulating the characteristics of different GSC subtypes. METHODS: We established a novel GSC-enriched cell model, referred to as NSG-70, from a patient with recurrent GBM. NSG-70 cells harbor a unique cytogenetic feature, viz. isochromosome 9q. At the same time, its expression profiles indicate that it is a mixed lineage comprising proneural and mesenchymal subtypes. We examined the relevance of NOTCH1 and WNT5A signaling and their coordinated action in GBM using these cells and other patient-derived models representing different GSC subtypes. RESULTS: Our data revealed that the downregulation of NOTCH1 resulted in the suppression of stem cell and mesenchymal markers and significantly reduced the levels of WNT5A. NOTCH1 knockdown also led to a notable reduction in the vasculogenic mimicry of GSCs. Interestingly, knockdown of WNT5A exhibited similar effects and drove quiescent GSC towards proliferation. In a complementary manner, ectopic expression of WNT5A or rhWNT5A treatment rescued the effects of NOTCH1 knockdown. CONCLUSION: The resistance of GSCs towards conventional therapies in part due to subtype interconversion demands therapies targeting specific GSC subtype. Our study suggests the need for a combinatorial approach that could effectively target the NOTCH1-WNT5A signaling axis toward eliminating GSCs.

Noncoding RNA Ginir functions as an oncogene by associating with centrosomal proteins.

Long noncoding RNAs constitute a major fraction of the eukaryotic transcriptome, and together with proteins, they intricately fine-tune various growth regulatory signals to control cellular homeostasis. Here, we describe the functional characterisation of a novel pair of long intergenic noncoding RNAs (lincRNAs) comprised of complementary, fully overlapping sense and antisense transcripts Genomic Instability Inducing RNA (Ginir) and antisense RNA of Ginir (Giniras), respectively, from mouse cells. This transcript pair is expressed in a spatiotemporal manner during embryonic development. The individual levels of the sense and antisense transcripts are finely balanced during embryonic growth and in adult tissues. Functional studies of the individual transcripts performed using overexpression and knock-down strategies in mouse cells has led to the discovery that Ginir RNA is a regulator of cellular proliferation and can act as an oncogene having a preeminent role in malignant transformation. Mechanistically, we demonstrate that the oncogenic function of Ginir is mediated by its interaction with centrosomal protein 112 (Cep112). Additionally, we establish here a specific interaction between Cep112 with breast cancer type 1 susceptibility protein (Brca1), another centrosome-associated protein. Next, we prove that the mutual interaction between Cep112 with Brca1 is significant for mitotic regulation and maintenance of genomic stability. Furthermore, we demonstrate that the Cep112 protein interaction with Brca1 protein is impaired when an elevated level of Ginir RNA is present in the cells, resulting in severe deregulation and abnormality in mitosis, leading to malignant transformation. Inhibiting the Ginir RNA function in transformed cells attenuates transformation and restores genomic stability. Together, these findings unravel, to our knowledge, a hitherto-unknown mechanism of oncogenesis mediated by a long noncoding RNA and establishes a unique role of Cep112-Brca1 interaction being modulated by Ginir RNA in maintaining mitotic fidelity.

RNA binding motif 47 (RBM47): emerging roles in vertebrate development, RNA editing and cancer.

RNA-binding proteins (RBPs) are critical players in the post-transcriptional regulation of gene expression and are associated with each event in RNA metabolism. The term 'RNA-binding motif' (RBM) is assigned to novel RBPs with one or more RNA recognition motif (RRM) domains that are mainly involved in the nuclear processing of RNAs. RBM47 is a novel RBP conserved in vertebrates with three RRM domains whose contributions to various aspects of cellular functions are as yet emerging. Loss of RBM47 function affects head morphogenesis in zebrafish embryos and leads to perinatal lethality in mouse embryos, thereby assigning it to be an essential gene in early development of vertebrates. Its function as an essential cofactor for APOBEC1 in C to U RNA editing of several targets through substitution for A1CF in the A1CF-APOBEC1 editosome, established a new paradigm in the field. Recent advances in the understanding of its involvement in cancer progression assigned RBM47 to be a tumor suppressor that acts by inhibiting EMT and Wnt/[Formula: see text]-catenin signaling through post-transcriptional regulation. RBM47 is also required to maintain immune homeostasis, which adds another facet to its regulatory role in cellular functions. Here, we review the emerging roles of RBM47 in various biological contexts and discuss the current gaps in our knowledge alongside future perspectives for the field.

Effective Visualization and Easy Tracking of Extracellular Vesicles in Glioma Cells.

Extracellular vesicles (EVs) are nano-sized, membrane-bound structures secreted by cells and play critical roles in mediating intercellular signaling. EVs based on their size as well as mechanisms of biosynthesis are categorized as either microvesicles (200-1000 nm) or exosomes (30-200 nm). The EVs carry several biomolecules like proteins, DNAs, RNAs, and lipids into other cells and modulate several cellular functions. Being of very small sizes, it is very challenging to analyze them using conventional microscopes. Here, we report a new method developed by us for visualizing EVs using simple immune-fluorescence based technique, wherein the isolated EVs can be stained with fluorescently tagged antibodies to proteins present in EVs. The stained EVs can then be analyzed by using either confocal or super-resolution microscopes. Our method detailed here is equally effective in staining proteins that are present inside the EVs as well as those localized to the membranes of vesicles. By employing unique staining strategies, we have minimized the background noise and thereby improved the signal strength in confocal microscope. Using electron microscopy, we have ascertained that the structural integrity of the labeled EVs is intact. More importantly, the labeling of EVs does not affect their functionality and their localization can be tracked after its uptake by recipient cells without resorting to any conventional reporter-based strategies or lipophilic dyes. In conclusion, the method described here is a simple, sensitive and efficient immune-fluorescence based method for visualization of molecules within the EVs.

Mitochondria Targeting Non-Isocyanate-Based Polyurethane Nanocapsules for Enzyme-Triggered Drug Release.

Surface engineering of nanocarriers allows fine-tuning of their interactions with biological organisms, potentially forming the basis of devices for the monitoring of intracellular events or for intracellular drug delivery. In this context, biodegradable nanocarriers or nanocapsules capable of carrying bioactive molecules or drugs into the mitochondrial matrix could offer new capabilities in treating mitochondrial diseases. Nanocapsules with a polymeric backbone that undergoes programmed rupture in response to a specific chemical or enzymatic stimulus with subsequent release of the bioactive molecule or drug at mitochondria would be particularly attractive for this function. With this goal in mind, we have developed biologically benign nanocapsules using polyurethane-based, polymeric backbone that incorporates repetitive ester functionalities. The resulting nanocapsules are found to be highly stable and monodispersed in size. Importantly, a new non-isocyanate route is adapted for the synthesis of these non-isocyanate polyurethane nanocapsules (NIPU). The embedded ester linkages of these capsules' shells have facilitated complete degradation of the polymeric backbone in response to a stimulus provided by an esterase enzyme. Hydrophilic payloads like rhodamine or doxorubicin can be loaded inside these nanocarriers during their synthesis by an interfacial polymerization reaction. The postgrafting of the nanocapsules with phosphonium ion, a mitochondria-targeting receptor functionality, has helped us achieve the site-specific release of the drug. Co-localization experiments with commercial mitotracker green as well as mitotracker deep red confirmed localization of the cargo in mitochondria. Our in vitro studies confirm that specific release of doxorubicin within mitochondria causes higher cytotoxicity and cell death compared to free doxorubicin. Endogenous enzyme triggered nanocapsule rupture and release of the encapsulated dye is also demonstrated in a zebrafish model. The results of this proof-of-concept study illustrate that NIPU nanocarriers can provide a site-specific delivery vehicle and improve the therapeutic efficacy of a drug or be used to produce organelle-specific imaging studies.

Backbone Engineered γ-Peptide Amphitropic Gels for Immobilization of Semiconductor Quantum Dots and 2D Cell Culture.

We are reporting a spontaneous supramolecular assembly of backbone engineered γ-peptide scaffold and its utility in the immobilization of semiconductor quantum dots and in cell culture. The stimulating feature of this γ-peptide scaffold is that it efficiently gelates both aqueous phosphate buffers and aromatic organic solvents. A comparative and systematic investigation reveals that the greater spontaneous self-aggregation property of γ-peptide over the α- and ß-peptide analogues is mainly due to the backbone flexibility, increased hydrophobicity, and π-π stacking of γ-phenylalanine residues. The hydrogels and organogels obtained from the γ-peptide scaffold have been characterized through field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), FT-IR, circular dichroism (CD), wide-angle X-ray diffraction, and rheometric study. Additionally, the peptide hydrogel has displayed a stimuli-responsive and thixotropic signature, which leads to the injectable hydrogels. 2D cell culture studies using normal and cancer cell lines reveal the biocompatibility of γ-peptide hydrogels. Further, the immobilization of semiconductor core-shell quantum dots in the transparent γ-peptide organogels showed ordered arrangement of quantum dots along the peptide fibrillar network with retaining photophysical property. Overall, γ-peptide scaffolds may serve as potential templates for the design of new functional biomaterials.

Cancer stem cell-vascular endothelial cell interactions in glioblastoma.

Glioblastoma (GBM), a higher grade glial tumor, is highly aggressive, therapy resistant and often shows poor patient prognosis due to frequent recurrence. These features of GBM are attributed to presence of a significantly smaller proportion of glioma stem cells (GSCs) that are endowed with self-renewal ability, multi-potent nature and show resistance to therapy in patients. GSCs preferably take shelter close to tumor vasculature due to paracrine need of soluble factors secreted by endothelial cells (ECs) of vasculature. The physical proximity of GSCs to ECs creates a localized perivascular niche where mutual GSC-EC interactions regulate GSC stemness, migration, therapy resistance, and cellular kinetics during tumor growth. Together, perivascular niche presents a therapeutically targetable tumor structure for clinical management of GBM. Thus, understanding cellular and non-cellular components in perivascular niche is vital for designing in vitro and in vivo GBM tumor models. Here, we discuss the components and structure of tumor vascular niche and its impact on tumor progression.

Wnt3a mediated activation of Wnt/ß-catenin signaling promotes tumor progression in glioblastoma.

Presence of a distinct population of cells that drives tumor progression supports the hierarchical model of tumor development in Glioblastoma (GBM) and substantiates the cancer stem cell hypothesis. Amongst the various developmental signaling pathways that are aberrantly activated, we here show that activated Wnt/ß-catenin signaling pathway plays a critical role in malignant transformation and tumor progression in gliomas. We demonstrate that Wnt ligands - Wnt1 and Wnt3a are expressed in a graded manner in these tumors as well as over-expressed in glioma stem cell-lines. A selective inhibition of Wnt signaling pathway by selective knock-down of its ligands Wnt1 and Wnt3a in glioma-derived stem-like cells led to decreased cell proliferation, cell migration and chemo-resistance. Furthermore, Wnt silencing in glioma cells reduced the capacity to form intra-cranial tumors in vivo. Taken together, our study indicates Wnt/ß-catenin signaling pathway as an essential driver of glioma tumorigenesis, recognizing role of Wnt3a as an oncogene and thereby offering novel therapeutic strategies for management of these tumors.

RBM47 is a Critical Regulator of Mouse Embryonic Stem Cell Differentiation.

RNA-binding proteins (RBPs) are pivotal for regulating gene expression as they are involved in each step of RNA metabolism. Several RBPs are essential for viable growth and development in mammals. RNA-binding motif 47 (RBM47) is an RRM-containing RBP whose role in mammalian embryonic development is poorly understood yet deemed to be essential since its loss in mouse embryos leads to perinatal lethality. In this study, we attempted to elucidate the significance of RBM47 in cell-fate decisions of mouse embryonic stem cells (mESCs). Downregulation of Rbm47 did not affect mESC maintenance and the cell cycle but perturbed the expression of primitive endoderm (PrE) markers and increased GATA4 + PrE-like cells. However, the PrE misregulation could be reversed by either overexpressing Rbm47 or treating the knockdown mESCs with the inhibitors of FGFR or MEK, suggesting an implication of RBM47 in regulating FGF-ERK signaling. Rbm47 knockdown affected the multi-lineage differentiation potential of mESCs as it regressed teratoma in NSG mice and led to a skewed expression of differentiation markers in serum-induced monolayer differentiation. Further, lineage-specific differentiation revealed that Rbm47 is essential for proper differentiation of mESCs towards neuroectodermal and endodermal fate. Taken together, we assign a hitherto unknown role(s) to RBM47 in a subtle regulation of mESC differentiation.

Rictor regulates MMP-9 activity and invasion through Raf-1-MEK-ERK signaling pathway in glioma cells.

Glioblastoma multiforme (GBM) is the most common and highly aggressive type of primary brain tumor. Tumor-associated macrophages (TAMs) secrete TNF-α that activates important survival pathways including Akt (PKB)/mTOR network. The mammalian target of rapamycin (mTOR) network functions downstream of PI3K/Akt pathway to regulate cell growth, proliferation and survival. mTOR exists in two distinct complexes-mTORC1 and mTORC2 that differ in their components and sensitivity to rapamycin. The rapamycin-insensitive complex (mTORC2) consists of mTOR, mLST8, Rictor, mSin1 and Protor and regulates the actin cytoskeleton in addition to activating Akt (protein kinase B). The present study aimed to investigate the role of Rictor-a core component of mTORC2 in regulating proliferation, survival, and invasion in gliomas. siRNA-mediated loss of Rictor function in human glioma cell lines, LN18 and LN229 and in primary GBM cells resulted in elevated expression and activity of MMP-9 and significant increase in the invasive potential of these cells. Mechanistic studies revealed that the activation of Raf-1-MEK-ERK pathway was essential for induction of MMP-9 activity and enhanced invasion. Interestingly, ablation of Rictor did not affect TNF-α-induced MMP-9 activity and invasiveness suggesting that TNF-α in the microenvironment of tumor might overrule the function of Rictor as a negative regulator of MMP-9 and invasion. Silencing Rictor had no effect on the survival or proliferation in the cell lines in the presence or absence of TNF-α. Our findings identify a role for Rictor in bridging two major pathways-Akt (PKB)/mTOR and Raf-1-MEK-ERK in regulating MMP-9 activity and invasion of glioma tumor cells.

Epigenetic regulation of DNA methyltransferases: DNMT1 and DNMT3B in gliomas.

The role of epigenetics and significance of aberrant gene regulation in etiology of cancer is a well-established phenomenon. The hallmark of cancer epigenetics is aberrant DNA methylation consisting of global hypomethylation and regional hypermethylation of tumor suppressor genes (TSGs) by DNA methyltransferases (DNMTs). In mammals, DNA methylation is catalyzed by DNMTs encoded by DNMT1, DNMT3A, and DNMT3B. Interestingly, little is known about variation in the methylation status of epigenetic regulators themselves in gliomas. Here, we report significant overexpression of DNMT1 and DNMT3B. A study of the methylation status and histone modifications at the promoter region of DNA methyltransferase I (DNMT1) gene revealed an unmethylated DNA promoter, similar to that detected in normal brain tissues. However, a differential histone code with distinct euchromatin marks--AcH3, AcH4, and H3k4me2--was specifically detected in tumors, unlike in normal brain tissues, which were found predominantly enriched with heterochromatin marks such as H3K9me2 and H3K27me3. In contrast, a differential methylation pattern of DNMT3B gene promoter occurred in glioma tumors, wherein it was found hypomethylated. Transcriptional silencing by CpG island methylation is a prevalent mechanism for inactivation of TSGs. Inhibiting DNMTs by 5-azacytidine (DNMT inhibitor) treatment led to significant inhibition of expression of DNMT1 and DNMT3B and enhanced expression of TSGs such as PTEN and p21 analyzed in this study. Our studies have identified effects of increased presence of DNMTs on inhibition of tumor suppressors that are epigenetically silenced in gliomas, thereby leading to aberrant regulation of cell cycle progression and failure to maintain genomic stability.

Dlxin-1, a MAGE family protein, induces accelerated neurite outgrowth and cell survival by enhanced and early activation of MEK and Akt signalling pathways in PC12 cells.

Dlxin-1 (also known as NRAGE or MAGED1) is a member of Type II melanoma-associated antigen (MAGE) family of proteins characterized by presence of a unique region of about 200 amino acids known as the MAGE homology domain (MHD). Dlxin-1 is associated with a large number of diverse cellular functions ranging from transcriptional regulation, cell cycle progression and differentiation to developmental apoptosis. While there are numerous studies reporting the role of NRAGE in facilitating cell death by interaction with p75NTR, we found varied effects of Dlxin-1 over-expression on PC12 cells grown in presence of NGF. These include induction of increased cell survival in presence of NGF and accelerated neuronal differentiation. We here categorically demonstrate that the effects on neuritogenesis are promoted through interactions of Dlxin-1 with the neurotrophin receptor TrkA. Further, using pharmacological inhibitors to specific pathways, we delineate the effects on enhanced neuritogenesis to the early and sustained activation of MEK pathway whereas the effects on cell survival to the early activation of Akt pathway. Next, we demonstrate a physical interaction of necdin with Dlxin-1 in PC12 cells. Our results establish that Dlxin-1 is an enhancer of neuronal differentiation and suggests that its possible interaction with NGF and necdin is critical in mediating pathways involved in neuronal survival and differentiation. Further in-depth analyses of the activation of various signalling pathways mediated through interaction with Dlxin-1 may provide valuable insight on the mechanisms that govern decisions regarding neuronal survival, growth, differentiation or apoptosis.

Biocompatible gellan gum-reduced gold nanoparticles: cellular uptake and subacute oral toxicity studies.

Currently gold nanoparticles are being explored for drug delivery and other biomedical applications; therefore it is necessary to study the fate of such nanoparticles inside the body. The objective of the present study was to investigate the cellular uptake and toxicity of the gold nanoparticles synthesized using a microbial polysaccharide, gellan gum, as a capping and reducing agent. The cellular uptake of gold nanoparticles was studied on mouse embryonic fibroblast cells, NIH3T3 and human glioma cell line, LN-229. The cellular uptake study indicated that the gellan gum-reduced gold nanoparticles were located in cancer cells (LN-229) while no uptake was observed in normal mouse embryonic fibroblast cells (NIH3T3). The toxicity of the gold nanoparticles was evaluated by carrying out subacute 28 day oral toxicity studies in rats. Subacute administration of gum-reduced gold nanoparticles to the rats did not show any hematological or biochemical abnormalities. The weight and normal architecture of various organs did not change compared with control. The current findings, while establishing the specific uptake of nanoparticles into cancerous cells, also demonstrates that the gellan gum-reduced gold nanoparticles are devoid of toxicity in animals following oral administration.

Extrachromosomal DNA: Redefining the pathogenesis of glioblastoma.

Glioblastoma is an incurable most prevalent primary malignant brain tumor in adults. Surgery followed by radiotherapy with concomitant chemotherapy is the standard of care in patients with glioblastoma. Although, prognosis remains poor with a median survival in the range of 12-15 months. Over the decades of research has identified the gene mutation, angiogenesis, cell signaling for the development novel therapeutics. However, recent understanding on extrachromosomal DNA (ecDNA) put extra-layer of complexity in glioblastoma pathogenesis. These ecDNAs are present in significantly higher copy number in the nucleus of the cancer cells and contains several oncogenes which are instrumental for intra-tumoral genetic heterogeneity, accelerated tumor evolution and therapy resistance. In this review, we will discuss the current understanding on biogenesis, disease progression and potential therapeutic implications of ecDNAs in glioblastoma.

Laminin-1 induces neurite outgrowth in human mesenchymal stem cells in serum/differentiation factors-free conditions through activation of FAK-MEK/ERK signaling pathways.

Mesenchymal stem cells (MSCs) can be differentiated into cell types derived from all three germ layers by manipulating culture conditions in vitro. A multitude of growth and differentiation factors have been employed for driving MSCs towards a neuronal phenotype. In the present study, we investigated the potential of extracellular matrix (ECM) proteins-fibronectin, collagen-1, collagen-IV, laminin-1, and laminin-10/11, to induce a neuronal phenotype in bone marrow derived human MSCs in the absence of growth factors/differentiating agents. All of the ECM proteins tested were found to support adhesion of MSCs to different extents. However, direct interaction only with laminin-1 triggered sprouting of neurite-like processes. Cells plated on laminin-1 exhibited neurite out growth as early as 3h, and by 24h, the cells developed elaborate neurites with contracted cell bodies and neuronal-like morphology. Function-blocking antibodies directed against alpha6 and beta1 integrin subunits inhibited neurite formation on laminin-1 which confirmed the involvement of integrin alpha6beta1 in neurite outgrowth. Mechanistic studies revealed that cell adhesion to laminin-1 activated focal adhesion kinase (FAK), and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathways. Abrogation of FAK phosphorylation by herbimycin-A inhibited neurite formation and also decreased activities of MEK and ERK. Pharmacological inhibitors of MEK (U0126) and ERK (PD98059) also blocked neurite outgrowth in cells plated on laminin-1. Our study demonstrates the involvement of integrin alpha6beta1 and FAK-MEK/ERK signaling pathways in laminin-1-induced neurite outgrowth in MSCs in the absence of serum and differentiation factors.

Generation of two control iPSC clones NCCSi008-A and NCCSi008-B from an individual of Indian ethnicity.

Two iPSC clones, NCCSi008-A and NCCSi008-B, were generated from a healthy male individual of Indian origin by reprogramming his CD4+ T cells with an integration free Sendai viral vector. The established iPSC clones showed high alkaline phosphatase (ALP) activity, expression of pluripotency markers, a normal male karyotype consistent with the donor gender (46, XY) and has potential for multi-lineage differentiation. These iPSC lines of Indian origin would serve as valuable resources for disease modeling, drug development and screening.

Development and characterization of two independent integration free iPSC clones NCCSi010-A and NCCSi010-B from a patient with alcoholic liver cirrhosis of Indian ethnicity.

We generated two human induced pluripotent stem cell-line (iPSC) clones, NCCSi010-A and NCCSi010-B, from a 32-year-old alcoholic cirrhosis patient with minimal hepatic encephalopathy of Indian origin by reprogramming his CD4+ T cells with integration free Sendai viral vector system. The generated iPSC clones showed high alkaline phosphatase activity, expressed pluripotency markers, possessed potential for multi-lineage differentiation and exhibited a normal karyotype (46, XY). These two-patient specific iPSC clones of alcoholic liver cirrhosis can potentially serve as models for disease modeling, drug development and organoid generation (Shah and Bataller, 2016).

Establishment of integration free iPSC clones, NCCSi011-A and NCCSi011-B from a liver cirrhosis patient of Indian origin with hepatic encephalopathy.

Liver cirrhosis accompanied with hepatic encephalopathy commonly causes cognitive impairment in patients. To model this disease, two independent patient specific induced pluripotent stem cell-line (iPSC) clones, NCCSi011-A and NCCSi011-B were generated by reprogramming the CD4+ T cells of an Indian male patient suffering from this chronic condition. Both clones expressed the stemness markers, formed embryoid bodies (EBs) with potential for spontaneous differentiation in to all the three lineages, exhibited normal karyotype (46, XY) and demonstrated alkaline phosphatase activity. These generated iPSC lines have potential for use in understanding biology of the disease and evaluation of drugs.

Generation and characterization of three clones (NCCSi007A, NCCSi007B and NCCSi007C) of an integration free induced Pluripotent Stem Cell line from a patient with alcoholic liver cirrhosis of Indian ethnicity.

Three induced pluripotent stem cells (iPSC) clones NCCSi007-A, NCCSi007-B and NCCSi007-C were generated from CD4+T cells of a 38 years old male patient suffering from liver cirrhosis- alcoholic and minimal hepatic encephalopathy of Indian origin. The CD4+T cells of the patient were reprogrammed using integration free, Sendai viral vector system. Each of the three iPSC clones showed high alkaline phosphatase (ALP) activity, expressed pluripotency markers OCT4, SOX2, NANOG, KLF4, SSEA-4, TRA-1-60, showed normal male karyotype (46, XY) and exhibited multi-lineage differentiation.

Attenuation of Tumor Suppressive Function of FBXO16 Ubiquitin Ligase Activates Wnt Signaling In Glioblastoma.

Glioblastoma (GBM) is one of the most aggressive and lethal types of brain tumor. Despite the advancements in conventional or targeted therapies, median survival of GBM patients is less than 12 months. Amongst various signaling pathways aberrantly activated in glioma, active Wnt/ß-catenin signaling pathway is one of the crucial oncogenic players. ß-catenin, an important mediator of Wnt signaling pathway, gets phosphorylated by GSK3ß complex. Phosphorylated ß-catenin is specifically recognized by ß-Trcp1, a F-box/WD40-repeat protein and with the help of Skp1 it plays a central role in recruiting phosphorylated ß-catenin for degradation. In GBM, expression of ß-TrCP1 and its affinity for ß catenin is reported to be very low. Hence, we investigated whether any other members of the E3 ubiquitin ligase family could be involved in degradation of nuclear ß-catenin. We here report that FBXO16, a component of SCF E3 ubiquitin ligase complex, is an interacting protein partner for ß-catenin and mediates its degradation. Next, we show that FBXO16 functions as a tumor suppressor in GBM. Under normal growth conditions, FBXO16 proteasomally degrades ß-catenin in a GSK-3ß independent manner. Specifically, the C-terminal region of FBXO16 targets the nuclear ß-catenin for degradation and inhibits TCF4/LEF1 dependent Wnt signaling pathway. The nuclear fraction of ß-catenin undergoes K-48 linked poly-ubiquitination in presence of FBXO16. In summary, we show that due to low expression of FBXO16, the ß-catenin is not targeted in glioma cells leading to its nuclear accumulation resulting in active Wnt signaling. Activated Wnt signaling potentiates the glioma cells toward a highly proliferative and malignant state.