Glioblastoma (GB) is the most lethal brain tumor, and Wingless (Wg)-related integration site (WNT) pathway activation in these tumors is associated with a poor prognosis. Clinically, the disease is characterized by progressive neurological deficits. However, whether these symptoms result from direct or indirect damage to neurons is still unresolved. Using Drosophila and primary xenografts as models of human GB, we describe, here, a mechanism that leads to activation of WNT signaling (Wg in Drosophila) in tumor cells. GB cells display a network of tumor microtubes (TMs) that enwrap neurons, accumulate Wg receptor Frizzled1 (Fz1), and, thereby, deplete Wg from neurons, causing neurodegeneration. We have defined this process as “vampirization.” Furthermore, GB cells establish a positive feedback loop to promote their expansion, in which the Wg pathway activates cJun N-terminal kinase (JNK) in GB cells, and, in turn, JNK signaling leads to the post-transcriptional up-regulation and accumulation of matrix metalloproteinases (MMPs), which facilitate TMs’ infiltration throughout the brain, TMs’ network expansion, and further Wg depletion from neurons. Consequently, GB cells proliferate because of the activation of the Wg signaling target, β-catenin, and neurons degenerate because of Wg signaling extinction. Our findings reveal a molecular mechanism for TM production, infiltration, and maintenance that can explain both neuron-dependent tumor progression and also the neural decay associated with GB.

Abbreviations: APC, adenomatous polyposis coli; Arm, Armadillo; bGal, beta-galactosidase; Bsk DN , dominant negative form of the effector Basket; Cyt-Arm, Cytoplasmic-Armadillo; dEGFRλ, constitutively active form of the Epidermal Growth Factor Receptor; dPI3K92E caax , activated membrane-localized version of the PI3K catalytic subunit p110α/PI3K92E; ECM, extracellular matrix; EGFR, Epidermal Growth Factor Receptor; egr, eiger; ELAV, embryonic lethal abnormal vision; EST, Expression Sequence Tags; Fz, Frizzled; Fz1, Frizzled1; FZD, Frizzled; GAP43, Growth-Associated Protein-43; GB, Glioblastoma; GBSC, glioblastoma multiforme stem-like cell; GFP, green fluorescent protein; GFP-MLC, GFP tagged version of Myosin Light Chain protein; GMA-GFP, GFP tagged version of Moesin; GPI-YFP, glycosylphosphatidylinositol-anchored Yellow Fluorescent Protein; GRASP, GFP Reconstitution Across Synaptic Partners; Grnd, Grindelwald; grnd-extra, extracellular domain of Grnd; Hh, hedgehog; Hrp, horseradish peroxidase; HS, hybridization solution; HSP90B, Heat Shock Protein 90B; igl, igloo; Ihog, Interference hedgehog; JNK, cJun N-terminal kinase; LRP, lipoprotein-related protein; MMP, matrix metalloproteinase; myr, myristoilated; nkd-lacZ, transcriptional beta-galactosidase reporter of naked gene NLGN3, Neuroligin-3; NLS, nuclear localization signal; NMJ, neuromuscular junction; NPC, neural precursor cell; PI3K, phosphatidylinositol-3 kinase; PLA, proximity ligation assay; PP2A, protein phosphatase 2A; PTEN, Phosphatase and tensin homolog; qPCR, quantitative Polimerase Chain Reaction; RFP, Red Fluorescent Protein; Rho/ROCK, rhomboid/Rho associated kinase; RTK, receptor tyrosine kinase; SPARC/SPARCL1, Secreted Protein Acidic And Cysteine Rich; Sqh-GFP, GFP tagged form of spaghetti squash; SVZ, subventricular zone; TEM, transmission electron miscroscopy; TM, tumor microtube; TOR, Target of Rapamycin; TRE-RFP, RFP fluorescent protein regulated by a transcriptional response element of JNK pathway; TTHY1, Tweety homologue-1; Wg, Wingless; WNT, wingless-related integration site; WT, wild type

Funding: MP holds a fellowship from the Juan de la Cierva program IJCI-2014-19272 from the Spanish MICINN. Research has been funded by grant BFU2015-65685P. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2019 Portela et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

All in all, both the rapid growth of malignant gliomas and the neurological sequelae of affected patients are considerable challenges in neurooncology until today. A positive correlation between neuronal activity and glioma progression has recently been suggested [ 42 , 43 ]. The neuron-glioma interactions might, however, be manifold. Thus, the major aim of this study is to better characterize the complex world of interactions between neurons of the brain on one side and tumor cells on the other. The results can provide a framework of a novel understanding of the disease.

Another signaling pathway that has been associated with glial proliferation is the cJun-N-terminal Kinase (JNK) pathway. GB cells normally activate the JNK pathway to maintain the stem-like state, and it has become a pharmacological target for the treatment of GB [ 29 ]. Moreover, the JNK pathway is the main regulator of several factors that regulate cell motility in different organisms and tissues [ 30 – 34 ]. Cell motility demands the remodeling of the extracellular matrix (ECM), which is mainly performed by a family of endopeptidases known as Matrix metalloproteinases (MMPs). There are more than 20 members, including collagenases, gelatinases, stromelysins, some elastases, and aggrecanases [ 35 ]. The vertebrate MMPs have overlapping substrates, they exhibit functional redundancy and compensation, and pharmacological inhibitors are nonspecific. In contrast, there are only 2 MMP genes in Drosophila, MMP1 and MMP2, categorized by their pericellular localization, with MMP1 being secreted and MMP2 being plasma membrane anchored. However, the products of both genes are found at the cell surface and released into media, the 2 Drosophila MMPs cleave different substrates, and GPI-anchored MMPs promote cell adhesion when they become inactive [ 36 , 37 ]. MMPs are up-regulated in a number of tumors, including gliomas [ 38 ]. MMP up-regulation in GB is associated with the diffuse infiltrative growth, and they have been proposed to play a role in glioma cell migration and infiltration ([ 39 , 40 ] reviewed in the work by Nakada and colleagues [ 38 ]). Consequently, MMP up-regulation in GB is an indicator of poor prognosis [ 41 ], and therefore the study of the mechanisms mediated by MMPs is relevant for the biology of GB and cancer in general.

Among signaling pathways, the most prominent candidate to play a role in GB progression is the WNT canonical pathway, which is activated upon the ligand “Wingless-related integration site” (WNT) binding to the specific family of receptors, low-density lipoprotein-related protein (LRPs) or Frizzled (FZD) in the plasma membrane. As a consequence of WNT binding, the “destruction complex” that includes the tumor suppressors Axin and adenomatous polyposis coli (APC), the Ser/Thr kinases GSK-3 and CK1, protein phosphatase 2A (PP2A), and the E3-ubiquitin ligase β-TrCP [ 10 ], is inactivated, and β-catenin (armadillo in Drosophila) accumulates. β-catenin translocates to the cell nucleus where it promotes the expression of target genes (i.e., Cyclin D1 and Myc) that are important for cell proliferation [ 11 , 12 ]. The WNT pathway is evolutionarily conserved in all metazoans, and it plays a central role in brain development [ 13 ], adult neuronal physiology [ 14 ], and synaptogenesis [ 15 , 16 ]. Perturbations in WNT signaling are associated with neural deficits, Alzheimer disease [ 16 – 19 ], and, most notably, GB [ 20 – 22 ]. WNT/FZD signaling is frequently up-regulated in GB [ 23 , 24 ] (reviewed in the work by Suwala and colleagues [ 25 ]). In particular, one of the hallmarks of bad prognosis is the accumulation of ß-catenin in tumor cells [ 26 , 27 ], indicating an activation of WNT/FZD pathway [ 28 ].

GB cells extend ultra-long membrane protrusions that interconnect tumor cells [ 7 ]. These TMs are associated with the worst prognosis in human gliomas. TMs contribute to invasion and proliferation, resulting in effective brain colonization by GB cells. Moreover, TMs constitute a multicellular network that connects GB cells over long distances (up to 500 μm in length), a feature that likely provides resistance against radiotherapy, chemotherapy, and surgery [ 7 , 8 ]. In addition, TMs seem akin to a basic mechanism of cell−cell connection and molecular communication called "cytonemes" in Drosophila [ 9 ]. Growth-Associated Protein-43 (GAP43) is essential for the development of TMs and, thus, the tumor cell network associated with GB progression [ 7 ]. However, many aspects of this paradigmatic finding in glioma biology are still unexplored, including the molecular mechanisms underlying TM expansion and its impact on neighboring neurons.

The progression of glioblastoma (GB) is accompanied by broad neurological dysfunctions, including neurocognitive disturbances that compromise quality of life during the short life span of affected patients, which is still around 1 year [ 1 ]. These tumors are often resistant to standard treatments, which include resection, radiotherapy, and chemotherapy with temozolomide [ 2 ]. Numerous studies are focused on new molecular targets to treat GBs [ 3 – 6 ]; however, none of them has yet proven effective, which is in stark contrast to the considerable progress made in many other tumor types. It is therefore necessary to better understand the key glioma-specific features of tumor biology that can lead to additional therapeutic strategies against GBs.

Results

Glioma depletes Wg from neuronal membranes The mechanisms of Wg delivery are currently under debate. This protein was initially described as a secreted protein [69]. Recent studies have proved that Wg secretion is not necessary for Drosophila development [70]. A membrane-tethered version of Wg protein [71] (WgNRT) can substitute the endogenous gene, in mimicking Wg’s normal functions and produce viable organisms [70]. We took advantage of this tool to determine the cellular mechanisms mediating glioma Wg retrieval from neurons. We created a genetic combination to substitute 1 copy of endogenous wg with 1 copy of wgNRT exclusively in neurons (to avoid crossed expression, we used a different genetic driver system, the LexA-LexAop system [72]), while inducing a glioma marked with Ihog-RFP (by using the Gal4/UAS system). In addition, upon LexA system activation, neurons are marked with membranous GFP (CD8-GFP), whereas the rest of the cells are WT. We first analyzed the interaction of glioma cells with wgNRT-expressing neurons (Fig 4G–4I). The results show that heterozygous wgNRT/wg in neurons, prevented glioma network expansion (Fig 4I compared to Fig 4H, quantified in Fig 4J). WgNRT is anchored to neuronal membranes; thus it would be expected to reduce the total Wg signaling in glioma cells thereby decreasing cell proliferation/survival and, consequently, resulting in a normal sized brain. In conclusion, glioma cells produce a network of TMs that reach neighboring neurons, increasing intimate membrane contact that facilitates neuronal-Wg sequestering (which we refer to as vampirization) mediated by the Fz1 receptor in glioma TMs. Moreover, Wg/Fz1 signaling in glioma mediates glioma cell proliferation and tumor progression.

Wg/Fz1 pathway disruption causes neurodegeneration Neuronal development and physiology are dependent on Wg/Fz1 signaling, and disruptions in this signaling pathway lead to synapse loss, an early symptom of neurodegeneration (reviewed in the work by Libro and colleagues, the work by Kahn, the work by Arrazola and colleagues, and the work by Garcia and Arias [17,74–76]). To determine whether an imbalance in Wg distribution caused by glioma cells can affect the neighboring neurons, we determined the contribution of Fz1/Wg pathway in neural cell function. To inhibit Wg/Fz1 signaling, we expressed UAS-fz1-RNAi or UAS-wg-RNAi in motor neurons under the control of a D42-Gal4 driver [46] and quantified the number of active zones (synapses) in the NMJ. The results showed a significant reduction in the number of synapses (Fig 5G–5J). These data show that Wg/Fz1 signaling in neurons is necessary for synaptogenesis. Because the glioma cells vampirize Wg from the neural cells and the neural cells require Fz1-Wg signaling for synaptogenesis, we attempted to restore this signaling equilibrium by overexpressing Fz1 receptor in neurons surrounded by glioma cells. To avoid crossed expression, we generated LexAop-Fz1 transgenic flies based on the LexA-LexAop genetic driver system [72] to target neurons. Fz1 was ectopically overexpressed specifically in neurons by using the ELAV-LexA driver, whereas the Gal4-UAS system was used to generate the glioma. Oversized glioma brains showed the expected glioma network compartmentalizing nests of neurons in the brain (S8A and S8B Fig). However, Fz1 overexpression in neurons restored the homogeneous distribution of Fz1 protein in the brain (S8C and S8D Fig) and prevented brain oversize (S8C and S8D Fig) and neuron nests (S8A’–S8D’ Fig). In addition, Wg/Fz1 signaling equilibrium restoration partially rescued lethality, and most animals reached adulthood. To verify Fz1 activation of the pathway, we stained for Wg and Cyt-Arm (S8E–S8H Fig). As previously shown, glioma brains showed a heterogeneous distribution for Wg protein (S8E Fig), and, as a consequence, an imbalance in pathway activation reported by Cyt-Arm accumulation (S8G Fig). As expected, neuronal Fz1 overexpression in glioma brains restored Wg distribution and Cyt-Arm signal toward that of control brains (S8F and S8H Fig). To further determine the effect of Wg/Fz1 signal restoration in neurons, we quantified the number of synapses in the NMJs. Synapse number reduction by the glioma is prevented by Fz1 overexpression in neurons (Fig 5K–5N). Altogether, these results indicate that the Wg/Fz1 pathway disruption caused by glioma is responsible for the synapse loss, and restoration of the signaling equilibrium between glia and neurons prevents synapse loss and therefore neurodegeneration.