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Oncogenic dependence of glioma cells on kish/TMEM167A regulation of vesicular trafficking

Genetic lesions in glioblastoma (GB) include constitutive activation of PI3K and EGFR pathways to drive cellular proliferation and tumor malignancy. An RNAi genetic screen, performed in Drosophila melanogaster to discover new modulators of GB development, identified a member of the secretory pathway: kish/TMEM167A. Downregulation of kish/TMEM167A impaired fly and human glioma formation and growth, with no effect on normal glia. Glioma cells increased the number of recycling endosomes, and reduced the number of lysosomes. In addition, EGFR vesicular localization was primed toward recycling in glioma cells. kish/TMEM167A downregulation in gliomas restored endosomal system to a physiological state and altered lysosomal function, fueling EGFR toward degradation by the proteasome. These endosomal effects mirrored the endo/lysosomal response of glioma cells to Brefeldin A (BFA), but not the Golgi disruption and the ER collapse, which are associated with the undesirable toxicity of BFA in other cancers. Our results suggest that glioma growth depends on modifications of the vesicle transport system, reliant on kish/TMEM167A. Noncanonical genes in GB could be a key for future therapeutic strategies targeting EGFR‐dependent gliomas.

cells, rather multiple mutations that co-activate EGFR-Ras and PI3K/ AKT pathways are required to induce a glioma in mouse models (Holland et al., 2000;Read, Cavenee, Furnari, & Thomas, 2009). In Drosophila models, a combination of constitutively active mutant forms of dEGFR (dEGFR λ ) and dPI3K (Dp110 CAAX ) effectively causes a glioma-like condition that shows features of human tumors, including glial expansion and invasion (Kegelman et al., 2014;Read, 2011;Read et al., 2009). Moreover, this model has proved to be valuable in finding new kinase activities relevant to the glioma progression (Read et al., 2013).
Vesicle transport plays a central role in cell biology as it provides membranous platforms to assemble specific signaling complexes and to terminate signal transduction (Stasyk & Huber, 2016).
In GB, for example, members of the small GTPases involved in cytoskeletal dynamics and vesicle trafficking, are overexpressed and promote tumor progression (Kim et al., 2014;Wang & Jiang, 2013).
Several growth factor receptors and adhesion molecules are clients of the vesicular transport, both for proper membrane localization and for degradation in the lysosomes (Mattissek & Teis, 2014). In fact, defective endocytic downregulation of EGFR has been associated with cancer, in particular with gliomas (Jones & Rappoport, 2014) (Zahonero & Sanchez-Gomez, 2014). Moreover, it has been shown that NHE9 (a Na + /H + exchanger) limits luminal acidification in glial cells to circumvent EGFR turnover, and thus prolongs downstream signaling pathways that drive glioma growth and migration (Kondapalli et al., 2015). Therefore, modulators of RTK vesicular transport are promising targets in these tumors. In fact, therapeutic strategies to induce non-apoptotic cell death in glioma cells rely on endosomal "mis-trafficking" (Pasupuleti, Grodzki, & Gorin, 2015).
However, inhibitors of the endo/lysosomal system are still far from preclinical development as they target too many common cellular mechanisms with potentially high toxicity at the organism levels (Stasyk & Huber, 2016).
In this work, we have taken advantage of the Drosophila glioma model, which allows us to look for novel oncogene dependence mechanisms of tumor cells, as we can simultaneously test the effect of target genes in glioma cells and normal glia. On these premises, we have identified kish/TMEM167A, a protein associated with vesicle transport and secretion (Gershlick, Schindler, Chen, & Bonifacino, 2016;Wendler et al., 2010), which is necessary for glioma growth in human cells and Drosophila models, but not for glial cells during fly development. Mechanistically, TMEM167A/kish downregulation alters the endo-lysosomal system, resulting in a change of dEGFR localization and the induction of proteasomal dEGFR degradation, which prevents tumoral growth. Moreover, TMEM167A/kish downregulation mirrors part of the effects of Brefeldin A (BFA), a lactone antiviral that alters the morphology and function of the Golgi apparatus and endosomal compartments in different cell types (Lippincott-Schwartz et al., 1991). We propose vesicle transport as a key property of tumoral cell biology and a target for EGFRassociated GB; this novel strategy takes advantage of a general feature of GB cells, therefore, it could be relevant for a plethora of de novo and recurrent GBs. To generate a glioma in Drosophila melanogaster, the Gal4/UAS system (Brand & Perrimon, 1993) was used. The Repo enhancer (repo-Gal4), which drives the activation of the expression system specifically to glial cells, was used to induce the expression of dEGFR (UAS-dEGFRλ) and PI3K (UAS-dp110) constitutively active forms. This genetic combination is termed as glioma. To restrict the expression of this genetic combination to the adulthood, we used the thermosensitive repression system Gal80 TS . Individuals maintained at 17 C did not activate the expression of the UAS constructs, after switching the flies to 29 C, the protein Gal80 ts is degraded and the expression system UAS/Gal4 activated. Moreover, we used a UAS-GFP NLS reporter to monitor repo cells.
A list of the fly genotypes is detailed in the Supporting Information "Materials and Methods".

| Viability assays
Flies were crossed and progeny was raised at 25 C under standard conditions. The number of adult flies emerged from the pupae was counted for each genotype. The number of control flies was considered 100% viability and all genotypes are represented relative to controls. Experiments were performed in triplicates.

| Glioma model
We grew U87 cells from the ATCC (in vitro and in vivo) as previously described (Pozo et al., 2013). We obtained GB1 cells by dissociation of human GB surgical specimens from patients treated at Hospital Ramón y Cajal (Madrid, Spain). The lentiviral vectors pTRIPZ, pTRIPZ-shTMEM167A (a and b) and pTRIPZ-shTRAPPC11(c) (Open-Biosystems) were used to produce conditionally interfered cells. shRNA expression was induced by 1 μg/ml of Dox (Sigma-Aldrich, St. Louis, MO) in vitro or by adding 2 mg/ml Dox in the drinking water in vivo.

| Glioma cell culture
U-87 MG cells were obtained from the ATCC. GBM1 cells were obtained by dissociation of a human GBM surgical specimen from Hospital 12 de Octubre (Madrid, Spain), after patient's written consent and with the approval of the ethic committee. We digested fresh tissue samples enzymatically using Accumax (Millipore), and then we purified isolated cells by a Ficoll gradient (GE Healthcare, Chicago, IL).

| Mouse Xenografts
All the protocols with animals were reviewed and approved by the Research Ethics and Animal Welfare Committee at our institution (Instituto de Salud Carlos III, Madrid), in agreement with the European Union and national directives. Animals were sacrificed by cervical dislocation, and the tumors induced were removed and either fixed in 4% PFA for 24 hr before immunofluorescent staining or fresh frozen for RNA extraction.

| Proteasomal inhibitor (MG132) treatment
We resuspended MG132 (Cayman chemical company 10012628, Ann Arbor, MI) in DMSO or just the vehicle DMSO (control) was added to standard cornmeal agar food to a final concentration of 50 μM.
Crosses were set up in normal vials and transferred 24 hr later to MG132 or control DMSO vials. We collected, dissected, and processed third-instar larvae as described earlier.

| Drug studies
Flies were crossed and the progeny grew in 3 ml of standard fly food supplemented with the corresponding compounds: Brefeldin A (TOCRIS 145E #1231 14 mg/ml) at 1:200 of, 1:500.
We dissolved all compounds in DMSO (14 mg/ml) and we used equivalent amount of vehicle (DMSO) for each case as control.
For studies in heterotopic tumors, we injected wild-type U87 cells and animals started receiving daily intratumoral injections of BFA (0.24 mg/day). We dissolved BFA in Tween80-H 2 O (TOCRIS 145E #1231). For studies in U87 cells were treated with BFA at 1 μg/μl.

| qRT-PCRs
We converted RNA (from flies, mouse tumor tissue, human samples, and cultured cells) to cDNA and subjected to qPCR using SYBR Green for detection. We analyzed gene expression data by the ΔΔCt method.

| Imaging
Fluorescent labeled samples (flies, tumor sections, and fixed cells) were mounted in Vectashield mounting media (Vector Laboratories) and images were acquired by confocal microscopy (LEICA TCS SP5).
Images were processed using Leica LAS AF Lite and Fiji (Image J 1.50e) and analyzed by using Imaris 6.3.1 Bitplane Scientific Solutions software. Images were assembled using Adobe Photoshop CS5.1.

| Image quantification
To quantify the number of glial cells GFP nls (Figures 1 and 7), the number of endosomes (Rab5 + , Rab7 + , Rab11 + , and dArl8 + puncta; Figures 3 and 5, Supporting Information Figure S4), the number of dEGFR + puncta (Figures 4, 5 and 6, Supporting Information Figure S4) and the number of Lysotracker + puncta ( Figure 5), we took advantage of the "spots tool" from the Imaris 6.3.1 Bitplane scientific solutions 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 dEGFR-Endosomal co-localization studies ( Figure 5 and Supporting Information Figure S4), we quantified the total number of an endosomal compartment (e.g., Rab + puncta) and the total number of dEGFR puncta, and then applied a co-localization filter (intensity mean of the channel of interest) using the Spots tool from the Imaris 6.3.1 Bitplane Scientific Solutions software.

| Statistics
To analyze and plot the data, we used Microsoft Excel 2013 and GraphPad Prism 6. We performed a D'Agostino & Pearson normality test and the data found to have a normal distribution were analyzed by a two-tailed t test with Welch-correction. In the case of multiple comparisons, we used a One-way ANOVA with Bonferroni post-test.
The data that did not pass the normality test were subjected to a two-

| Kish or gryzun knockdown prevent glioma progression
We used the Drosophila glioma model to search for genes expressed in glial cells, implicated in vesicle transport and necessary for tumor progression. Glioma induction in flies results in 100% lethality; thus, we established survival as the read-out to perform the screening. The screening was performed silencing target genes specifically in glial cells driven by the pan-glial driver repo-Gal4 (Casas-Tinto, Arnes, & Ferrus, 2017), most of the RNAi against vesicle transport hits (including Rab family) were lethal after activation in normal glia or enhanced glioma lethality (data not shown). However, we found that the interference of two genes, kish and gryzun, rescued this lethality in 20% and 30% of the cases, respectively (Figure 1a).
To validate our results, we marked glial cells nuclei with GFP NLS .
The number of glial/glioma cells (GFP+) was quantified in adult control and glioma brains in normal conditions and expressing kish or gryzun RNAi. The results showed an increase of glial cell number in glioma brains when compared with control brains and a prevention of glioma cell number increase upon kish or gryzun knockdown (Figure 1b-g). In addition, we observed a significant increase in total GFP fluorescence in animals developing a glioma, which upon kish or gryzun silencing was comparable to control levels in glioma brains (Figure 1h-m).
The results show that kish and gryzun are necessary for glioma progression and that detrimental effects caused by the glioma are reduced upon knockdown of either of these genes in glioma cells. In addition, our results indicate that kish or gryzun RNAi expression in wild type glial cells do not induce detrimental effects on brain development as the number of glial cells is similar to control brains ( Figure 1g) and 100% of the individuals reached adulthood (Figure 1a columns 3 and 5). Altogether, these data suggest that kish or gryzun downregulation are harmless for normal glia development but they can inhibit glioma growth.

| kish/TMEM167A interference inhibits human glioma growth
The human ortholog of kish is TMEM167A and its five-exon gene is located in chromosome 5. It encodes for a small trans-membrane protein (72 amino acids) with unknown function. The human ortholog of gryzun is transport protein particle complex 11 (TRAPCC11), which gene is located in chromosome 4 and has been associated with membrane trafficking and Golgi apparatus architecture (Bogershausen et al., 2013). To determine the relevance of these two genes in glioma cells, we used shRNA lentiviral plasmids directed toward TMEM167A or TRAPCC11 or a control shRNA (all Dox-inducible), in the well-known U87 glioma cell line. We grew these cells in the absence of serum as floating neurospheres. In this defined media, cells re-express stem cell markers, display high tumorogenic potential and depend on glioma oncogenic signals, like EGFR (Pozo et al., 2013). We injected U87 infected cells into the flank of immunodeficient mice and once tumors were visible, we induced RNA interference, and we measured tumor size twice a week. The results show that shTRAPCC11 RNAi silenced the expression of the target gene and reduced the growth of the tumors, although the reduction in the final tumor volume was not significant after TRAPCC11 downregulation (Supporting Information Figure S1). However, we observed a strong reduction in xenograft growth upon shTMEM167A knockdown with two different shRNA constructs (Figure 2a-d). Final tumor size was significantly smaller after shTMEM167A induction (Figure 6c) and xenograft growth inhibition correlated well with the level of reduction in TMEM167A expression (Figure 6d). Based on these results, we decided to continue our studies on the function of kish/TMEM167A on glioma progression. The viability assay shows that the lethality induced by the glioma is partially rescued upon inhibition of kish or gryzun expression. We quantified a minimum of 50 animals for each genotype). (b) Diagram of adult Drosophila brain anatomy, each brain is composed of two symmetrical optic lobes attached to the central brain. (cf ) confocal images from Drosophila adult brains displayed at the same scale. Glia are labeled with nuclear GFP (green) driven by repo-Gal4. The quantification of the number of glial cells is shown in (g) N = 9 for each genotype. Scale bar size = 50 μm. (h) Diagram of larval nervous system composed by the central nervous system (brain) and the peripheral nervous system. Anatomy of the larval brain composed by the central brain, optic lobes and ventral nerve cord. (i-m) Pictures of whole live larvae with GFP labeled glia (green) driven by repo-Gal4 where the GFP pixel intensity was measured. (i-l) In repo > dEGFR; dp110CAAX (glioma) brains (j), there is an increased in GFP pixel intensity relative to wt control (i). This is restored to control levels upon kish RNAi or gryzun RNAi in glioma brains (k-l). The quantification of the pixel intensity is shown in (m) N = 5 for each genotype [Color figure can be viewed at wileyonlinelibrary.com] To determine glioma proliferation, we analyzed the number of mitosis in the dissected U87-glioma cells. The quantification of these results shows a significant reduction in BrdU incorporation, which suggests a decrease in proliferation after TMEM167A downregulation (Supporting Information Figure S2A). This correlates with the observed decrease in BrdU incorporation when U87 infected cells were incubated with Dox in vitro (Supporting Information Figure S2B).
In addition, we performed intracranial injection of U87 cells (shControl and shTMEM167Aa) in the brain of immunodeficient mice.
One week after implantation, animals received Dox in the drinking water. We monitored tumor growth by the expression of the RFP reporter in an IVIS equipment (e.g., see the picture taken at Day 30 post-injection in Figure 6e). The resulting data shows that there is a reduction in tumor growth after TMEM167A downregulation, which correlates with a significant reduction in tumor burden ( Figure 6f ). In addition, these results were corroborated in a primary glioma cell line (GB1), the results show that the expression of shTMEM167Aa also reduced significantly the growth of the GB1 tumors in situ (Figure 6g).

| kish/TMEM167A is upregulated in human GB
To determine the contribution of TMEM167A to GB, we analyzed the data from The Cancer Genome Atlas database (TCGA). In silico analysis showed that TMEM167A gene is highly expressed in several tumors of the nervous system, including gliomas (Supporting Information Figure S3A). Although no mutations or alterations of this gene have been described in these tumors, we observed a significant upregulation of TMEM167A expression levels in tumor tissue obtained from glioma patients compared with normal brain samples (Figure 6h). Regarding its function, kish/TMEM167A is a resident protein from endoplasmic reticulum (ER) and Golgi apparatus related to secretory pathways (Wendler et al., 2010). In accordance with its vesicular trafficking role in invertebrates, the DAVID gene analysis of the pathways coupregulated with TMEM167A in tumoral cell lines, showed an association with ER functions, protein transport and extracellular matrix (Supporting Information Figure S3B). Altogether, these results suggest that TMEM167A has a pro-oncogenic function in GB that could affect vesicular trafficking. Membrane localization and EGFR signaling activation depends on kish/TMEM167A. (a) The viability assay shows that the lethality induced by the glioma is partially rescued upon inhibition of kish. UAS-dp110 CAAX and UAS-Ras V12 expressing glioma is also lethal but the kish inhibition is not efficient to rescue viability (a minimum of 50 animals were quantified for each genotype). (b) We stained control, glioma and glioma kish-RNAi brains with anti-dEGFR (green) and the graph shows the total number of dEGFR puncta quantification. (c) Confocal images and quantification of heterotopic U87 xenografts sections stained with EGFR. (d) U-87 neurospheres were disaggregated and labeled with a specific antibody against the extracellular domain of EGFR and analyzed by flow cytometry (N = 3). (e) Growth factor-starved U87 cells (control or shTMEM167Aa) were incubated in the presence of Dox (12 h). Cycloheximide was added to the cells and 1 hr later, EGF (100 ng/ml) was added for the times indicated. The cells were chilled on ice and cell pellets were processed for WB analysis to measure EGFR signaling pathway. WB densitometry analysis of the levels of pEGFR, AKT and pAKT and GAPDH are shown on (f ) [Color figure can be viewed at wileyonlinelibrary.com]

| Kish modulates vesicular trafficking in gliomas
As kish has been associated with the control of vesicular trafficking in cell culture, we sought to determine if it could have a similar role in Drosophila glioma models. We used specific antibodies to visualize and detect the total number of early, late, recycling endosomes and lysosomes (Rab5, Rab7, Rab11 and dArl8, respectively). We quantified the images automatically (Imaris). Not surprisingly, gliomas showed a significant increase in the amount of Rab11 (recycling) endosomes and a strong reduction in dArl8 positive lysosomes in comparison with control brains. However, the number of Rab5 (early) or Rab7 (late) endosomes did not show any significant difference (Figure 3a-

| EGFR signaling require kish/TMEM167A expression
Interference of kish expression rescued glioma cells proliferation and lethality when dEGFR and dPI3K were affected in glial cells (Figure 1).
To determine which mechanisms involved in glioma growth depend on kish, we induced gliomas by overexpressing a constitutively active form of Ras (downstream effector of EGFR) and PI3K. This Ras-induced glioma is independent of dEGFR activity. Figure 4a shows that Rasinduced gliomas are lethal during development, similar to dEGFRinduced gliomas. However, kish depletion in Ras-induced gliomas could FIGURE 5 dEGFR stability and distribution in glioma depends on kish. (a-b) control, glioma and glioma kish-RNAi brains were stained with anti-dEGFR (green) and vesicle markers (red) for early endosomes (Rab5), late endosomes (Rab7), recycling endosomes (Rab11) and lysosomes (dArl8). (a) We assessed the co-localization of dEGFR with vesicle markers in control, glioma and glioma kish-RNAi brains. Statistical significance of dEGFR co-localization in endosomal vesicles is shown in (b). (c-f ) Confocal images of larval brain sections from third-instar larvae. Control, glioma, kish-RNAi and glioma; kish-RNAi brains were stained with Lysotracker to assess vesicle acidification. (g) Total number of Lysotracker+ puncta quantification in all genotypes. N = 9 for each genotype. Scale bar size = 10 μm [Color figure can be viewed at wileyonlinelibrary.com] not rescue this lethality (Figure 4a). These results suggest that Kish is required for dEGFR protein biology in glioma but is no longer necessary when the activated components of this pathway are downstream.
To determine if the effects of kish downregulation on vesicular trafficking (Figure 3) affect to dEGFR, we quantified the total amount of the receptor (measured as dEGFR positive puncta) in glioma samples expressing kish RNAi. The results showed a reduction of total dEGFR now comparable to control levels ( Figure 4b). In addition, we determined dEGFR mRNA levels by quantitative RT-PCR and we observed no significant differences between glioma and glioma expressing kish RNAi (Supporting Information Figure S4A

| dEGFR localization in glioma depends on kish
To confirm the relevance of kish in dEGFR vesicular trafficking, we quantified the co-localization of dEGFR protein with each of the endosomal or lysosomal markers in Drosophila brains. The results indicate that glioma cells accumulate dEGFR protein in early endosomes, where dEGFR is active. In addition, there is an increase of dEGFR protein in the recycling endosomes (Figure 5a

| Kish knockdown stimulates dEGFR degradation in the proteasome
The data indicate that glioma samples expressing kish-RNAi have a reduction of total dEGFR protein. These cells accumulate dEGFR in the lysosomes but these are not degradative so we can still detect it. We wondered where were the rest of the receptor from glioma; kish-RNAi brains (see Figure 4b) if they were not degraded in the lysosome. It has been previously shown that kish is required for secretion and it is localized at the ER-Golgi in Drosophila tissues (Wendler et al., 2010). Disruption of protein processing or trafficking through the ER-Golgi leads to unfolded proteins, which stimulate protein degradation via proteasome (Ellgaard, Molinari, & Helenius, 1999;Schroder & Kaufman, 2005;Shen, Zhang, & Kaufman, 2004). At this point, we hypothesized that dEGFR could be targeted to degradation in the proteasome due to kish knockdown in glioma brains.
To validate this hypothesis, we blocked the proteasome with MG132, a specific, potent, reversible, and cell-permeable proteasomal inhibitor (Griciuc et al., 2010). Quantification of confocal images shows that, kish RNAi stimulates dEGFR degradation, which is reverted upon proteasomal blockage (Figure 6a-d, g). In addition, we blocked the proteasome genetically through the expression of a FIGURE 6 Kish knockdown stimulates dEGFR proteasomal degradation. (a-f ) Confocal images of larval brain sections from thirdinstar larvae. We stained glioma and glioma kish-RNAi brains with dEGFR (grey). (a-b) Brain samples exposed to the vehicle DMSO. (cd) Brain samples exposed to the proteasomal inhibitor (MG132 at a concentration of 50 μM).  (Figure 6a,b, e-g). These data confirm that most of the reduction of dEGFR after kish downregulation is mediated by the proteasome.

| Active compounds modulate vesicle transport in glioma cells
Our results with kish/TMEM167A interference show that targeting certain components of the vesicular trafficking machinery can be detrimental for EGFR-dependent gliomas, but not for normal glia. This suggests an oncogenic dependence mechanism that could be therapeutically exploited. To explore this possibility, we decided to evaluate the impact of a collection of compounds that affect exocytosis and/or endocytosis at different levels (Supporting Information Figure S5). We performed a biased drug screening by feeding the drugs or the vehicle (DMSO) during the whole development to control larvae and larvae bearing a glioma.
The results showed that 20% of glioma flies reached adulthood after BFA treatment as compared with 0% survival in the corresponding glioma larvae exposed to the vehicle control, whereas the rest of the compounds did not have a protective effect (Supporting Information Figure S5). We dissected adult brains from the survivors to determine if BFA was preventing glioma progression. Quantifications of glial cell number from confocal images showed that the gliomas grown in the presence of BFA have a number of glial cells similar to a wt control brain (Figure 7a-c), indicating that this treatment prevents glioma progression and, as a consequence, rescues viability of the animals.
The lactone antibiotic BFA reversibly blocks traffic between the Golgi and ER and within the Golgi stacks, although it also affects the endolysosomal compartment (Lippincott-Schwartz et al., 1991) (Supporting Information Figure S5). To determine the cellular effect of BFA on glioma cells, we performed an analysis of the vesicle trafficking system in glioma third-instar larval brains after DMSO or BFA feeding during the development. The results show that the BFA does not cause an effect on early endosomes (Rab5) but it does increase the number of late endosomes (Rab7) (Figure 7d). In addition, BFA provokes a reduction of recycling endosomes (Rab11) and a significant increase of lysosomes (dArl8) as compared with glioma cells exposed to DMSO.

| DISCUSSION
The results presented here show that kish/TMEM167A, a protein previously associated with vesicle transport and secretion (Gershlick et al., 2016;Wendler et al., 2010), is necessary for glioma growth, both in human cell xenografts and Drosophila models. Mechanistically, kish/TMEM167A downregulation alters the endo-lysosomal system. Glia are labeled with GFP (green) driven by repo-Gal4 (a-c). In repo > dEGFR λ ; dp110 CAAX (glioma) brains exposed to BFA (b), the number of glial cells is similar to wt control (a). The quantification of the number of glial cells is shown in (c), N = 6 for each genotype. Scale bar size = 50 μm. (d) Vehicle (DMSO) and BFA-treated glioma larval brains were stained with vesicle markers for early endosomes (Rab5), late endosomes (Rab7), recycling endosomes (Rab11) and lysosomes (dArl8). Quantification is shown in the graph. We analyzed an average of N = 5 brains for each genotype in each condition. (e) U87 cells were implanted into the flank of nude mice. We treated one group of mice with BFA every day during 7 days (0.240 mg/day). We measured tumor size once every 2 days and represented as fold change. (N = 6 for each). This results in a change in EGFR localization toward its degradation by the proteasome.
The quantification of the different vesicles in control and interfered cells suggest that kish/TMEM167A participates in different steps of vesicle trafficking, affecting endo-lysosomal acidification and function. Others have reported that excessive luminal alkalization by NHE9 gain-of-function circumvent EGFR turnover and prolongs downstream signaling pathways (Kondapalli et al., 2015). Moreover, the human papillomavirus type 16 E5 oncoprotein activates EGFR and PDGFR (platelet-derived growth factor receptor) with concomitant alkalization of Golgi and endosomes (Di Domenico et al., 2009). Our results suggest that TMEM167A could modulate the transport of newly synthesized proteins from the ER-Golgi to the membrane or to other organelles. Moreover, the alterations in acidic vesicles produced by TMEM167A knockdown may account for the malfunctioning of ER-Golgi trafficking. In that case, the remaining EGFR not degraded by the proteasome, would accumulate in lysosomes due to the lack of degradative capacity of these vesicles.
The screening performed in flies with exo and endocytosis regulators, shed some light into the function of kish/TMEM167A. The rescue effects of kish downregulation could only be mimicked by BFA treatment. In contrast, monensin (which blocks receptor recycling) or phenothiazine (which affects lysosomal function), did not inhibit glioma formation, suggesting that the main oncogenic function of kish/ TMEM167A is not simply mediated by a blockade of receptor turnover or by altering lysosomal function. BFA is an inhibitor of the Arf1-guanine nucleotide exchange factor (GEF) interaction. It reversibly blocks traffic between the Golgi and ER and within the Golgi stacks, disrupting Golgi morphology (Lippincott-Schwartz, Yuan, Bonifacino, & Klausner, 1989). However, the whole endosomal compartment shows morphological changes in response to BFA treatment, with normal cycling between plasma membrane and endosomes, but with impaired traffic between endosomes and lysosomes (Lippincott-Schwartz et al., 1991). The results presented here indicate that TMEM167A downregulation does not induce Golgi disruption although it has a profound effect in the endo-lysosomal system. This suggests that kish/TMEM167A downregulation could be parallel to the vacuolar effect of BFA, without its ER/Golgi effect. Interestingly, nontumoral cells can be made resistant to the cytotoxic effect of BFA if the Golgi appearance is preserved, even if the non-Golgi effects are still present (Yan, Colon, Beebe, & Melancon, 1994). The results presented here suggest that kish/TMEM167A downregulation mimic this vesicular effect of BFA, being toxic for glioma cells but not for normal glial cells. This is in line with previous reports indicating that EGFR ligands differentially affect endocytic receptors in neoplastic versus non-neoplastic astrocytes (Hussaini et al., 1999). Altogether, our data suggest that there is a window of opportunity for modulators of vesicular trafficking in gliomas, which would not have a deleterious effect in normal astrocytes although they would be able to inhibit tumor growth, at least for the EGFR-dependent gliomas, which account for more than 50% of them. BFA has an antitumor effect in certain cancer cell lines (Sausville et al., 1996) but it has not passed the preclinical stage of drug development due, in part, to its high toxicity. New BFA derivatives are being tested (Ohashi et al., 2012) but the results indicate that a more effective and less cytotoxic strategy would be to silence TMEM167A (Golan et al., 2015;Zimmermann et al., 2017).
There are no studies on the relevance of the biosynthetic pathway for growth factor receptors in gliomas yet, but it would be interesting to discern if TMEM167A has a general role in the membrane exposure of other receptors, or even in the secretion of relevant extracellular proteins. Future experiments will allow us to distinguish autocrine from paracrine effects of kish/TMEM167A downregulation and to reach a comprehensive understanding of the oncogenic functions of this protein. The data presented here indicate that in fly models, kish is expendable when downstream targets of the receptors (Ras) are active. However, in human glioma cells, we cannot discard that the effects of the conditional depletion of TMEM167A could depend as well on other pathways affected by changes in the endosomal system. PDGFRA, for example, is another key pathogenic receptor in gliomas whose stability and signaling depend on the vesicular trafficking regulation (Chen et al., 2014). Besides, MET receptor mutants, also present in gliomas, require endocytic trafficking to generate oncogenic signaling (Joffre et al., 2011). In any case, the novel approach presented here would take advantage of a general feature that is independent of the acquisition of secondary oncogenic mutations, and therefore potentially relevant for a plethora of de novo and recurrent GBs.