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

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Abstract

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/β-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/β-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.

Introduction

Wnt proteins form a family of highly conserved secreted signaling molecules that regulate a myriad of complex developmental cellular processes that include proliferation, tissue homeostasis, stem cell maintenance, cell fate decisions and tissue polarity. The Wnt proteins are about 40 kDa in size with many conserved cysteines (Tanaka et al., 2002) and essentially function as morphogens (Nusse and Varmus, 2012). One of the predominant pathways employed by members of Wnt family to arbitrate these diverse cellular processes is the canonical Wnt/β-catenin signaling pathway regulated through stability and sub-cellular localization of its key effector β-catenin. In the absence of Wnt ligands, cytoplasmic β-catenin protein is constantly degraded by action of the Axin complex, composed of scaffolding protein Axin, tumor suppressor adenomatous polyposis coli (APC), casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3β) (Cadigan, 2008a, Cadigan, 2008b, Kanwar et al., 2010, Su et al., 2008, Zheng et al., 2008). CK1 and GSK3β sequentially phosphorylate β-catenin, leading to its ubiquitination and subsequent proteasome mediated degradation. The binding of Wnt to two types of cell surface receptors, seven trans-membrane protein Frizzled (Fz) and single-pass membrane protein low-density lipoprotein receptor related protein 5 and 6 (LRP5/6) is required for transducing Wnt signal. LRP5/6 is homo and hetero-oligomerized with Fz through binding to Wnt proteins leading to phosphorylation of LRP5/6. This results in inactivation of the destruction complex, inducing stabilization of β-catenin and its entry into the nucleus (Cadigan, 2008a, Clevers and Nusse, 2012). Once inside, β-catenin displaces the repressor Groucho from TCF, forming a multi-protein activator complex of Pygopus and Legless/Bcl9 culminating in activation of Wnt target genes like cyclin D1 and c-Myc that promote proliferation and self-renewal (Angers and Moon, 2009, MacDonald et al., 2009, Morin, 1999).

Imbalance in the structural and signaling properties of β-catenin often results in disease and deregulated growth connected to cancer and metastasis. Abnormal activation is linked to cancers of prostrate (Yamamoto et al., 2010), breast (Howard and Ashworth, 2006), and hepatocellular carcinomas (Hu et al., 2009) and more importantly to colorectal carcinogenesis wherein high Wnt signaling activity is identified as a marker for colon cancer stem cells (Vermeulen et al., 2010). Constitutively activated β-catenin signaling due to APC deficiency or mutations in APC, AXIN1, or CTNNB1 (which encodes β-catenin) enhances β-catenin stability leading to excessive stem cell renewal and proliferation that predisposes cells to tumorigenesis (Burgess et al., 2011, Minde et al., 2011). Higher expression of β-catenin is often associated with increased grades of astrocytomas wherein β-catenin is found to be either membrane-bound or cytoplasmic with few cases of glioblastomas reporting nuclear expression (Utsuki et al., 2002). The protein expression levels of Wnt1, β-catenin and cyclin D1 correlate with glioma grades (Liu et al., 2011). Studies have shown that GBM cells over-express Wnt ligands and receptors, such as Wnt3, Wnt6, and Fz9 and Wnt3a (Zheng et al., 2010). Over-expression of Wnt3a is known to promote nuclear translocation of FoxM1 forming a complex with β-catenin/TCF on promoters of Wnt target genes and thereby enhancing its transcriptional activity (Abla et al., 2012, Bowman and Nusse, 2011). In glioblastomas, the activity of FoxM1 leads to elevated β-catenin signaling activity as reduction in FoxM1 levels impede tumor formation, in a β-catenin-dependent manner (Zhang et al., 2011). Recent studies also indicate involvement of non-canonical Wnt pathway driven by Wnt5a in GBM tumor progression (Augustin et al., 2012, Kamino et al., 2011).

Like several other solid tumors, gliomas are considered to be driven by a small sub-population of cells known as glioma stem cells (GSC) (Singh et al., 2004). These GSCs exhibit many properties of stem cells that include their self-renewal potential, extended proliferation, multi-potency and more importantly are pheno-typically and geno-typically similar to normal neural stem cells (NSC). Pathways regulating normal stem cell self-renewal, proliferation and survival like Notch, Hedgehog and bone morphogenetic protein (BMP) signaling operative in normal neural stem cells are re-activated in these tumors and this significantly contributes to their aggressive phenotype (Alexson et al., 2006, Faigle and Song, 2013, Sanson, 2008).

Here, we present evidence of over-expression of Wnt ligands, Wnt1 and Wnt3a in higher grade gliomas and glioma stem cells and thereby demonstrate role of aberrantly activated canonical Wnt/β-catenin pathway in malignant transformation and tumor progression. Inhibition of this pathway by RNA interference in glioma stem cells led to decreased cell proliferation, migration, tumorigenicity and chemo-resistance suggesting that targeting WNT/β-catenin pathway may represent a potential therapeutic target in GBM.

Section snippets

Results

One of the principal objectives of this study was to determine whether aberrantly activated Wnt signaling contributed to malignant tumor progression and invasiveness in gliomas. For this, we began by investigating status of expression of Wnt ligands, Wnt1 and Wnt3a involved in canonical Wnt signaling, in glioma tumors of different grades and in glioma stem cell cultures developed by us. Further, we developed another brain tumor stem cell-line (BTSC), NSG-K16 from GBM tumor tissue and

Discussion

Various mutational and epigenetic changes in genes crucial to normal physiological development drive processes that confer extensive self-renewal and invasive properties to cells causing phenotypic heterogeneity leading to cancer. Human glioblastoma is one of the most common and aggressive malignancies of the Central Nervous System (CNS). The tumors display high level of heterogeneity due to the presence of diverse population of cells termed as brain tumor stem cells demonstrating hierarchical

Experimental methods

The present study was approved by Institutional Ethics Committee of National Centre for Cell Science (NCCS), Pune, India. The consent to use tissues for research purposes was signed by patients prior to surgery. Tumor tissue samples were collected from KEM Hospital, Mumbai and assigned specific tumor stages and pathological grades according to WHO criteria for gliomas with help of a neuro-pathologist (Nakazato, 2008). A total of forty-two glioma tumor tissue samples of various grades and two

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Role of funding source

Grant support provided by Department of Biotechnology (DBT), Government of India, New Delhi, India and National Centre for Cell Science (NCCS), Pune, India. The fellowships of S.C., N.K. and P.R. were supported by Council of Scientific and Industrial Research (CSIR), New Delhi, India. Fellowship support for S.R. was provided by DBT, India.

Acknowledgments

We are thankful to Ms. Shweta Srinivasan for providing help in TCGA analyses. Histopathogical sections of diagnosed glioma tumors were kindly provided by Dr. Sridhar Epari, ACTREC, Tata Memorial Centre, Navi Mumbai, India. We acknowledge the expert guidance from Dr. Avinash Pradhan, KEM Hospital, Pune in histopathological analyses of glioma tumor tissues. The lenti-viral vectors pLU-EF1a-Wnt3a-iTuRFP and pLU-EF1a-iTuRFP were a kind gift from Dr. Meenhard Herlyn, Wistar Institute, Philadelphia,

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    Author contributions: N.K and S.C. contributed equally to this article. N.K. and S.C.: Experimental design, data collection, data analysis and interpretation. S.R. and P.R.: Experimental design and data analysis and interpretation. D.M. GBM tissues provision and data interpretation MS: Animal experimentation and AS: conception and design, data analysis and interpretation, manuscript writing, and final approval of manuscript.

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    Equal contribution.

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