TNFα/Grnd mediate JNK/MMPs activation during glioma progression and neurodegeneration

Glioblastoma (GB) is a type of brain tumour that involves the transformation of glial cells. This is the most aggressive and lethal tumour of the central nervous system and currently there are not efficient treatments. In GB, glial cells display a network of membrane projections (cytonemes) which mediate cell to cell communication. Under pathological conditions like GB, cytonemes transform into ultra-long tumour microtubes (TMs) that infiltrate into the brain, enwrap neurons and deplete wingless (Wg)/WNT from the neighbouring healthy tissue. GB cells establish a positive feedback loop including Wg signalling, JNK and matrix metalloproteases (MMP) required for GB progression and neuronal synapse loss and degeneration. Frizzled1 receptor mediates Wg signalling upregulation which is required for JNK activation in GB. Consequently, MMPs are upregulated and facilitate TMs infiltration in the brain, hence GB TMs network expands and mediate further Wg depletion to close the loop. Thus, cellular signals other than primary mutations emerge as a central feature of GB which correlates with a poor prognosis in patients and animal models. Here we describe the molecular mechanisms that regulate TMs production, infiltration and maintenance, in a Drosophila model of GB. The contribution of the bi-directional signals between healthy tissue (neurons) and GB cells, mediate the progression of the disease. JNK pathway signalling mediated by Grindelwald (Grnd) receptor is activated by the ligand Eiger (Egr)/TNFα secreted by the neurons surrounding the GB. Then, MMPs are secreted to facilitate TM progression and GB dissemination. Here we show that the coordination among different signals facilitate GB progression and contribute to the complexity and versatility of these incurable tumours.


Introduction
Glioblastoma multiforme (GB) is the most frequent and aggressive primary malignant brain tumour with a 3 per 100.000 incidence per year (Gallego 2015). GB patients´ median survival is 12-15 months, with less than 5% of survival after 5 years ( The recent discovery of a network of ultra-long tumour microtubes (TMs) in GB (Osswald et al. 2015) improves our understanding of GB progression and therapy resistance (Osswald et al. 2016). TMs are actin-based filopodia which infiltrate into the brain and reach long distances within the brain (Osswald et al. 2015). TMs are required in GB cells to mediate The confocal images do not show morphological evidence of glioma and the TMs glial network does not expand ( Figure S1E compare to glioma in S1B). Moreover, no detectable Wg or Fz1 proteins were found in the glial membranes ( Figure S1E-H) and Cyt-Arm is homogeneously distributed along the brain (Figure S1K compare to glioma in S1J) similar to control samples ( Figure S1I). Taking these results together, dMyc it is not sufficient to reproduce the features of the GB even though it is a convergent point of EGFR and PI3K pathways. These results suggest that both PI3K and EGFR together are necessary to activate a pathway downstream responsible for the expansion of glial projections, Fz1 accumulation and activation of the Wg pathway in glial transformed cells, and dMyc signaling is not sufficient to cause these phenotypes.
To determine the epistatic relations behind MMPs expression, we studied single gene modifications related to GB and the effect on MMPs. We analyzed MMP1 localization in the brain which is key to trigger TMs expansion and is a target of the JNK pathway. We assessed the presence of MMP1 in glial membranes. A specific monoclonal antibody was used to visualize the MMP1 that localized homogeneously across the brain in control samples ( Figure 1A). However, in the GB brain, the TMs expand and accumulate MMP1 ( Figure 1B). Again, we expressed separately the constitutively active forms of PI3K (dp110 CAAX ) and EGFR (TOR-DER CA ) in glial cells marked with ihog-RFP. In both cases we did not observe a significant upregulation of MMP1 or the formation of TMs ( Figure 1C-D, quantified in F). Besides, we also tested dMyc upregulation in glial cells as a candidate to upregulate MMP1. However, we did not observe any significant change in MMPs expression or localization upon dMyc upregulation ( Figure 1E-F). Taking these results together, EGFR, PI3K or dMyc are not sufficient to reproduce the features of glioma. These results suggest that the combined activity of PI3K and EGFR pathways are necessary to activate a downstream pathway responsible for the expansion of TM glial projections and MMP1 accumulation in glial transformed cells, but dMyc expression is not sufficient to cause these phenotypes.

Egr/Grnd in GB
A recent study using gene expression profiling identified genes that were significantly correlated with the overall survival in patients with GB and were further analysed for their involvement in pathways and possible biological roles. Eight pathways were identified as core pathways. Half of these pathways were signal transduction pathways correlated with cell survival, death, and growth. 104 genes were identified, which are common between patients with GB and those with low grade gliomas and can be used as core genes related to patient survival. Of these, 10 genes (CTSZ, EFEMP2, ITGA5, KDELR2, MDK, MICALL2, MAP 2 K3, PLAUR, SERPINE1, and SOCS3) can potentially classify patients with gliomas into different risk groups and could be used to estimate the prognosis of patients with gliomas. Among these pathways identified from the enrichment analysis of survival-related genes, the TNF-alpha signalling pathway stands out. Four genes from this 10-gene group (MAP 2 K3, PLAUR, SERPINE1, and SOCS3) are involved in TNFα signalling, and they might have potential prognostic value for patients with GB, (Hsu et al. 2019).

Egr translocates from Neuron to Glia in GB and Grnd accumulates in GB cells
The Drosophila orthologue of TNFα and its receptor are Egr and grnd respectively. We used an Egr-GFP protein fusion and monitored GFP signal in the Drosophila brain. The results from confocal microscopy show that most Egr-GFP signal is localized in the neurons (~70%) that contact with glial cells in control brains ( Figure A-B, E). However, there is a shift of Egr-GFP signal from neurons to glia (~50%) in GB samples ( Figure 2C-E). Next, we monitored the expression pattern of the JNK receptor grnd, with a specific antibody in GB and control samples ( Figure 2F-H). The results show that grnd protein accumulates in GB brains.
Taking these data together, GB cells accumulate grnd and Egr protein translocates from neurons to glia in GB brains. Thus, we propose that Egr produced in neurons mediates JNK pathway activation in GB cells.

Progressive JNK activation in glioma
JNK is upregulated in a number of tumours including GB and it is related to glioma malignancy (Hagemann et al. 2005;Zeng et al. 2018;Mu et al. 2018;Huang et al. 2003).
Moreover, JNK is a target for specific drugs in combination with temozolomide treatments as it was proven to play a central role in GB progression (Matsuda et al. 2012;Kitanaka et al. 2013;Feng et al. 2016;Okada et al. 2014). However, little is known about the molecular mechanisms underlying JNK activation in glioma cells and the functional consequences for GB progression.
We had previously confirmed JNK pathway activation in GB cells, by using the TRE-RFP reporter that confer transcriptional activation in response to JNK signalling (Chatterjee & Bohmann 2012;Jemc et al. 2012;Ruan et al. 2016). Now we decided to study the activation of the JNK pathway in a timely manner to understand when is the JNK activated in GB. We took advantage of another JNK reporter (puc-LacZ) that monitors the transcriptional activation of the downstream JNK target puckered (Martin-Blanco et al. 1998;Langen et al. 2013). To analyse JNK activity in GB at two different timepoints (48 and 96h after GB induction), we used the thermo sensitive repression system Gal80TS that restricts the expression of the GAL4/UAS system (see materials and methods). In control brains, the JNK reporter puc-LacZ is mostly activated in neurons (~78%). 48h after the induction of the GB, puc-LacZ activation in neurons is reduced (~37%) and GB cells show a progressive upregulation of puc-LacZ and a shift from neurons to GB cells from 63% puc-LacZ activation in glia 48h after the tumour induction to ~80% 96h after tumour induction ( Figure 3A-D), indicating that JNK pathway is activated in GB cells progressively.

Timeline: GB first causes neurodegeneration, then TMs infiltrate and finally proliferates
To evaluate the progressive growth of the TMs and the number of glial cells, we dissected larval brains at 24, 48 and 72h after tumour induction (referred to as day 1D, 2D or 3 days in Figure 4A-C). To visualize and quantify the TMs network volume, we expressed a membrane-bound myristoylated Red Fluorescent Protein (UAS-myrRFP) in glial cells and to quantify the glial cell number we stained the glia nuclei with a specific anti-Repo antibody and obtained confocal images from whole brains ( Figure 4A-C).
The statistical analysis of TMs volume ( Figure 4D) shows no significant increase in the volume of the TM network between day 1 and day 2 after tumour induction. Nevertheless, there is a significant increase in the volume of the TMs between day 2 and day 3 after tumour induction. Similarly, the statistical analysis of the number of glial cells ( Figure 4E) shows no significant increase in the number of glial cells between day 1 and day 2, but there is a significant increase between day 2 and day 3, and between day 1 and day 3 after tumour induction. This suggests a progressive growth and expansion of the TMs and a progressive increase in the number of glioma cells.
TMs are required in GB cells to mediate Wg signaling imbalance among neurons and GB cells. Wg signaling is upregulated in GB cells to promote proliferation, at expenses of the surrounding neurons in which the downregulation of the Wg pathway results in synapse loss, neurodegeneration and lethality.
To evaluate the impact of the progressive GB growth on the surrounding neurons, we quantified the number of synapses in the neuromuscular junction (NMJ) of third-instar larvae 1, 2 and 3 days after GB induction through immunofluorescence. NMJs were stained with anti-bruchpilot to reveal the synapses visualised by confocal microscopy ( Figure 5A-C).
The statistical analysis of synapse number ( Figure 5D) shows a progressive loss of synapse number between day 1 and day 2, and between day 2 and day 3 after tumour induction. The overall loss of synapses at the NMJ is highly significant ( Figure 5D).
To determine whether there is an association between the volume of the network and the number of synapses at the NMJ, we plotted the number of synapses against the volume of the glial network in a correlation graph ( Figure 5E). The correlation index is -0.966 (3s.f.) indicating there is a negative association between the volume of the TMs and the number of synapses at the NMJ. Therefore, larger TMs network leads to a greater synapse loss.

Discussion
Activating mutations for EGFR and PI3K pathways are the most frequent initial signals in GB. However, the attempts to treat GB reducing the activation of these pathways have so far been limited by acquired drug resistance. The current tendencies suggest that a multiple approach is required to obtain a more positive result (Taylor et al. 2012;Westphal et al. 2017;Prasad et al. 2011;Westhoff et al. 2014;Zhao et al. 2017). GB cells show a high mutation rate and usually present more than two sub-clones within the same patient and from the same primary tumour (McGranahan & Swanton 2017;Qazi et al. 2017).

Progressive tumour growth
Although there is an overall significant expansion in volume of the TMs and an increase in the number of glial cells in the Drosophila model of GB, between day 1 and day 2 of tumour development there is no significant increase for either parameters. Looking at the volume on day 2 after tumour induction there are some values above the average. There is also a large dispersion in volume and number of glial cells data in brains on day 3 after tumour induction.
A possible explanation for this is that under physiological conditions, there is a stable state that prevents cells from exiting the cell cycle, proliferating or extending a network of TMs.

Progressive neurodegeneration
There is a significant progressive decrease in the number of synapses at the NMJ.
Nevertheless, the variance in the number of synapses on day 1 after tumour induction is very large, reaching uniform values on days 2 and 3 after tumour induction. The individuals show different resistances to the changes caused by the GB, therefore, on day 1 after tumour induction, some individuals are largely affected by the tumour and suffer a great loss of synapses at the NMJ while other individuals are more resistant and maintain the number of synapses. On day 2 after tumour induction, the GB causes severe changes that affect all individuals in a similar manner regardless of their initial resistance.
We have observed a negative correlation between the volume of TMs and the number of synapses at the NMJ, suggesting that the TMs are responsible for the neurodegeneration.
Previous studies from our laboratory have proved that TMs surround neurons and relocate which leads to the degeneration of neurons (Rich & Bigner 2004). We can conclude that as the tumour progresses, it extends a network of TMs that grows progressively infiltrating in the brain and surrounding neurons depleting the ligand WNT from them and leading to their degeneration, process previously described as vampirization (Portela et al. 2019). The expansion of the TMs is slow during the first 24h of tumour development but increases after 48h of tumour development. The associated neurodegeneration is also progressive but is visible from the first 24h of tumour development.

Progressive activation of JNK pathway via Egr/Grnd
JNK signalling regulates MMPs expression in GB, which is required for TMs network formation and infiltration. Consequently, Wg pathway responds to JNK and TMs expansion, these three events conform a regulatory positive feedback loop in GB progression. Grnd is the receptor that interacts with the ligand Egr and activates JNK pathway in GB cells. GFP fused protein showed that Egr is produced in the surrounding neurons and accumulated in the membrane of GB TMs in contact with healthy neuronal tissue. The progressive activation of JNK pathway in glial cells correlate with the morphological changes (TM expansion and number of glial cells) that GB undergo after 48 of induction, and JNK pathway activation is essential for GB progression. Therefore, here is another example of neuron-glia molecular interaction which mediates the physiological status of the brain and the evolution of GB and reinforce the proposal of a bidirectional interaction between GB and surrounding healthy tissue. It is of interest to unravel the regulatory mechanisms that mediate Egr expression and secretion in neurons in response to GB induction, and the response in GB cells mediated by Grnd-Egr as a potential modulator for brain tumour advance.

Fly stocks
Flies were raised in standard fly food at 25ºC. Fly stocks from the Bloomington stock Centre: (Dominguez et al. 1998).

Drosophila glioblastoma model
The most frequent genetic lesions in human gliomas include mutation or amplification of the genetic combination and control it in a temporal manner, we used the thermo sensitive repression system Gal80 TS . Individuals maintained at 17ºC did not activate the expression of the UAS constructs, but when the larvae were switched to 29ºC, the protein Gal80 TS changed conformation and was not longer able to bind to Gal4 to prevent its interaction with UAS sequences, and the expression system was activated and therefore the GB was induced.

Imaging
Fluorescent labeled samples were mounted in Vectashield mounting media with DAPI (Vector Laboratories) and analyzed by Confocal microscopy (LEICA TCS SP5). Images were processed using Leica LAS AF Lite and Fiji (Image J 1.50e). Images were assembled using Adobe Photoshop CS5.1.

Quantifications
Relative MMP1 and grnd staining within brains was determined from images taken at the same confocal settings. Average pixel intensity was measured using measurement log tool from Fiji 1.51g and Adobe Photoshop CS5.1. Average pixel intensity was measured in the glial tissue and in the adjacent neuronal tissue (N<10 for each sample) and expressed as a Glia/Neuron ratio. Glial network volume was quantified using Imaris surface tool (Imaris 6.3.1 Bitplane Scientific Solutions software).
The number of Repo + cells, the number of synaptic active sites and the number of puc-lacZ positive cells was quantified by using the spots tool Imaris 6.3.1 software, we selected a minimum size and threshold for the puncta in the control samples of each experiment. Then we applied these conditions to the analysis of each corresponding experimental sample. For the puc-lacZ glia or neuron co-localization studies we quantified the total number of puc-lacZ + cells and then applied a co-localization filter (intensity center of the channel of interest) using the Spots tool from the Imaris 6.3.1 software.
For the co-localization of Egr-GFP in glial cells, GFP channel volume was quantified using Imaris surface tool. We selected a specific threshold for the total volume in the control samples and then we applied these conditions to the analysis of the corresponding experimental sample. Then we applied a co-localization filter (intensity mean of the red channel)

Statistical Analysis
To analyze and plot the data, we used Microsoft Excel 2013 and GraphPad Prism 6. We    Genotypes: Genotypes:  , A d v C a n c e r R e s 1 2 1 ,  a  t  r  i  x  m  e  t  a  l  l  o  p  r  o  t  e  i  n  a  s  e  s  (  M  M  P  s  )  i  n  h  e  a  l  t  h  a  n  d  d  i  s  e  a  s  e  :  a  n  o  v  e  r  v  i  e  w  .   F  r  o  n  t  B  i  o  s  c  i  1  1  ,   1  6  9  6  -1  7  0  1  .   M  a  r  t  i  n  -B  l  a  n  c  o  ,  E  .  ,  G  a  m  p  e  l  ,  A  .  ,  R  i  n  g  ,  J  .  ,  V  i  r  d  e  e  ,  K  .  ,  K  i  r  o  v  ,  N  .  ,  T  o  l  k  o  v  s  k  y  ,  A  .  M  .  a  n  d  M  a  r  t  i  n  e  z  -A  r  i  a  s  ,  A  . ( 1  9  9  8  )  p  u  c  k  e  r  e  d  e  n  c  o  d  e  s  a  p  h  o  s  p  h  a  t  a  s  e  t  h  a  t  m  e  d  i  a  t  e  s  a  f  e  e  d  b  a  c  k  l  o  o  p  r  e  g  u  l  a  t  i  n  g  J  N  K   a  c  t  i  v  i  t  y  d  u  r  i  n  g  d  o  r  s  a  l  c  l  o  s  u  r  e  i  n  D  r  o  s  o  p  h  i  l  a  .   G  e  n  e  s  D  e  v  1  2  ,   5  5  7  -5  7 T  u  m  o  u  r  s  :  r  e  t  r  o  s  p  e  c  t  i  v  e  a  p  p  l  i  c  a  t  i  o  n  t  o  a  c  o  h  o  r  t  o  f  d  i  f  f  u  s  e  g  l  i  o  m  a  s  .   J  N  e  u  r  o  o  n  c  o  l  1  3  7  ,   1  8  1  -1  8  9  .   R  o  m  e  ,  C  .  ,  A  r  s  a  u  t  ,  J  .  ,  T  a  r  i  s  ,  C  .  ,  C  o  u  i  l  l  a  u  d  ,  F  .  a  n  d  L  o  i  s  e  a  u  ,  H  .  (  2  0  0  7  )  M  M  P  -7  (  m  a  t  r  i  l  y  s  i  n  )  e  x  p  r  e  s  s  i  o  n  i  n   h  u  m  a  n  b  r  a  i  n  t  u  m  o  r  M o l C a n c e r 1 6 , 1 0 0 .