Overexpression of transient receptor potential mucolipin-2 ion channels in gliomas: role in tumor growth and progression

The Transient Receptor Potential (TRP) superfamily consists of cation-selective and non-selective ion channels playing an important role both in sensory physiology and in physiopathology in several complex diseases including cancers. Among TRP family, the mucolipin (TRPML1, −2, and −3) channels represent a distinct subfamily of endosome/lysosome Ca2+ channel proteins. Loss-of-function mutations in human TRPML-1 gene cause a neurodegenerative disease, Mucolipidosis Type IV, whereas at present no pathology has been associated to human TRPML-2 channels. Herein we found that human TRPML-2 is expressed both in normal astrocytes and neural stem/progenitor cells. By quantitative RT-PCR, western blot, cytofluorimetric and immunohistochemistry analysis we also demonstrated that TRPML-2 mRNA and protein are expressed at different levels in glioma tissues and high-grade glioma cell lines of astrocytic origin. TRPML-2 mRNA and protein levels increased with the pathological grade, starting from pylocitic astrocytoma (grade I) to glioblastoma (grade IV). Moreover, by RNA interference, we demonstrated a role played by TRPML-2 in survival and proliferation of glioma cell lines. In fact, knock-down of TRPML-2 inhibited the viability, altered the cell cycle, reduced the proliferation and induced apoptotic cell death in glioma cell lines. The DNA damage and apoptosis induced by TRPML-2 loss increased Ser139 H2AX phosphorylation and induced caspase-3 activation; furthermore, knock-down of TRPML-2 in T98 and U251 glioma cell lines completely abrogated Akt and Erk1/2 phosphorylation, as compared to untreated cells. Overall, the high TRPML-2 expression in glioma cells resulted in increased survival and proliferation signaling, suggesting a pro-tumorigenic role played by TRPML-2 in glioma progression.


INTRODUCTION
Malignant gliomas are the most common type of primary brain tumors [1]. Diffuse gliomas are classified histologically according to the hypothesized line of differentiation as astrocytomas, oligodendrogliomas and oligoastrocytomas. Astrocytomas are pathologically graded in accordance with the World Health Organization as low grade astrocytoma (grade II), anaplastic astrocytoma (grade III) or high grade glioblastoma multiforme (GBM, grade IV). GBM is the most aggressive and prevalent type of glioma, accounting for at least 80% of malignant gliomas [1,2]. It is one of the most lethal forms of cancer in human, with a median overall survival of 12-18 months. Although new treatments have been developed on the basis of new knowledge about the molecular nature of glioma disease, surgery in association with radiation therapy and chemotherapy remains the standard of care.
Since no data on the expression and functions of TRPML-2 channels in gliomas has been provided so far, the present work evaluated the expression of TRPML-2 channels in astrocytomas and glioblastomas compared to normal brain tissues (NB), astrocytes (NHA) and neural stem/progenitor cells (NS/PC). In addition, by RNA interference, the role of TRPML-2 in controlling cell survival and proliferation was studied in glioma cell lines.

RESULTS
Expression of TRPML-2 gene in normal human brain, astrocytes and neural stem/progenitor cells We first evaluated by quantitative real timepolymerase chain reaction (qRT-PCR) the expression of TRPML-2 gene in two different normal human brain (NB1 and NB2) samples, astrocyte cell lines (NHA1 and NHA2) and neural stem/progenitor cell lines (NS/PC1 and NS/ PC2). We demonstrated that TRPML-2 transcripts were found at higher levels in NS/PCs and NHAs as respect to NB specimens ( Figure 1).

TRPML-2 mRNA expression increases during glioma progression
The expression of TRPML-2 was also evaluated at mRNA levels in human glioma tissues of both sexes, with different pathological grade (Table 1) and was compared to the mean values of NHA samples ( Figure  2). Messenger RNA from estrogen receptor positive (ER + ) breast cancer tissues was used as positive control [20]. Starting from low-grade pylocytic astrocytoma, grade I and astrocytomas, grade II towards the more invasive anaplastic astrocytomas, grade III and GBM, grade IV, a progressive up-regulation of TRPML-2 mRNA expression was detected by qRT-PCR compared with NHA samples (Figure 2), with a strong increase (about 45-fold) of TRPML-2 expression in GBM biopsies, compared to pilocytic astrocytomas. The results obtained in glioma tissues prompted us to evaluate the expression of TRPML-2 protein by immunohistochemistry. In agree with qRT-PCR data, astrocytoma, anaplastic astrocytoma and GBM were more immunoreactive, compared to grade I astrocytomas (Figure 3), suggesting an inverse correlation between differentiation status and TRPML-2 protein expression. In addition, no significative difference in TRPML-2 protein expression was found between grade II and III astrocytomas. High TRPML-2 mRNA and protein levels were detected in ER-positive, epidermal growth factor receptor 2 positive (HER2 + ) breast cancer tissues, used as positive controls (Figure 2 and 3). No reactivity was found in tissue sections used as negative control incubated with the omission of primary Ab ( Figure 3). www.impactjournals.com/oncotarget TRPML-2 mRNA and protein expression levels in human glioma cell lines We further analyzed both at mRNA and protein levels, the TRPML-2 expression in human high-grade T98, U251 and U87 glioma cell lines. By qRT-PCR we observed high expression of TRPML-2 mRNA in T98 and U251 GBM cell lines as well as in MCF-7 cell line, used as positive control, compared to the mean value of NHA samples ( Figure 4A). U87 cells showed low TRPML-2 mRNA level compared to T98 and U251 cell lines ( Figure  4A). Immunocytochemistry demonstrated a positive immunoreaction for TRPML-2 in all glioma cell lines and in MCF-7 cells ( Figure 4B). We further confirmed the protein expression by western blot analysis. In parallel to qRT-PCR data, immunoblots showed that T98 and U251 glioma cell lines displayed a higher TRPML-2 protein level, corresponding to a band of 68 kDa, respect to U87 cells ( Figure 4C). Moreover, blots of plasma membrane (ME) and cytoplasm (CE) fraction ( Figure 4D) and cytofluorimetric analysis of unpermeabilized and permeabilized glioma cells demonstrated that TRPML-2 localization was both intracytoplasmic and in the plasma membrane, with highest expression in the cytosol (Supplementary Figure 1).

Silencing of TRPML-2 mRNA inhibits viability and proliferation in glioma cell lines
To demonstrate the involvement of TRPML-2 channels in the modulation of glioma cell viability and proliferation, we performed a TRPML-2 gene knockdown experiment in T98 and U251 glioma cell lines. Transfection with siTRPML-2 reduced the TRPML-2 mRNA and protein levels, as evaluated by qRT-PCR ( Figure 5A) and western blot analysis in both cell lines ( Figure 5B).
The effect of TRPML-2 knock-down in glioma cells viability and proliferation was evaluated by MTT and BrdU assay, respectively. A reduced viability was evidenced in siTRPML-2 U251 and siTRPML-2 T98 glioma cell lines, respectively, compared with siGLOtransfected cell lines ( Figure 6A). Moreover, evaluation of DNA synthesis through BrdU/PI assay showed that TRPML-2 silencing significantly reduced DNA synthesis rate of about 25% in both cell lines after 72 h of transfection ( Figure 6B).
The ERK and Akt/PKB pathways regulate the survival and proliferation in gliomas [21]. Therefore, the activation status of Akt/PKB and Erk1/2 proteins was analyzed by western blot analysis. Constitutive Akt and Erk1/2 phosphorylation was observed in T98 and U251 cells. Knock-down of TRPML-2 gene markedly reduced the activation in both cell lines, compared to siGLO cells ( Figure 6C).
Overall the reduction in survival and proliferation induced by the loss of TRPML-2 expression is associated with Akt/Erk1/2 pathway inhibition, suggesting a pro-tumorigenic role played by TRPML-2 in glioma progression.  astrocyte (NHA 1,2), glioma tissues (n = 63) at different tumour grades and ER + breast cancers (positive control) were evaluated by qRT-PCR. TRPML-2 mRNA levels were normalized for GAPDH expression and were expressed as relative fold with respect to the mean value of NHA samples, used as control. Data are expressed as mean ± SD. *p<0.05 vs NHA; # p<0.01 vs pilocytic astrocytoma and astrocytoma; § p<0.01 vs anaplastic astrocytoma (Anova with Bonferroni's post-test). Clinicopathological characteristics of the 63 glioma patients analyzed for TRPML-2 mRNA and protein expression.

TRPML-2 knock-down induces DNA damage and cell cycle alteration in glioma cell lines
Inhibition of glioma cell viability and proliferation in siTRPML-2 glioma cells may depend on DNA damage-induced cell cycle arrest. Thus, the effect of TRPML-2 gene silencing on histone γ-H2AX (H2AX) phosphorylation and cell cycle arrest was evaluated by western blot analysis and cell cycle assay. An increased level of Ser-139 γ-H2AX phosphorylation was observed in T98 and U251 siTRPML-2 cells, compared to siGLO cells ( Figure 7A). Moreover, knock-down of TRPML-2 increased the percentage of cells arrested at subG0 phase, indicating an elevated percentage of hypodiploid cells with fragmented DNA in U251 and T98 siTRPML-2 cell lines, compared with siGLO cells ( Figure 7B).

The siTRPML-2-mediated DNA damage response triggers apoptosis in glioma cell lines
Autophagy regulates both cell survival and death during stress conditions in different cell types [22]. Therefore, to evaluate the role of TRPML-2 in autophagy, rapamycin, a potent autophagic inducer was used. In particular, U251 and T98 siTRPML-2 cells were treated with rapamycin 50 nM for 72h and modulation of LC3 and p62 proteins was assessed by western blot analysis ( Figure  8A and 8B). During autophagy induction, LC3-I isoform is converted into LC3-II to allow the insertion of this protein into autophagosome membrane. Therefore, the amount of LC3-II correlates with the extent of autophagosome formation. Immunoblots revealed that the transfection condition increased the LC3-I levels in both cell lines; moreover, in siTRPML-2 U251 cells increased levels of LC3-II were detected under basal conditions ( Figure 8A). When U251 and T98 siTRPML-2 cells were treated with rapamycin to stimulate autophagy, an increased LC3-II and a decreased p62 levels, as respect to basal condition, was observed ( Figure 8A, 8B). Overall, these results indicate that TRPML-2 doesn't seem to be involved in the autophagy of glioma cells.
The response to DNA damage results in repair of DNA lesions or caspase cascade activation and apoptosis. Therefore, the triggering of apoptosis in siTRPML-2 glioma cells as well as in siGLO control cells was investigated by Annexin V/PI staining and biparametric cytofluorimetric analysis. Concordantly with the increase of subG0 cell phase populations, the number of early, Annexin V + and late, PI + /Annexin V + , apoptotic cells was enhanced in siTRPML-2 T98 and U251 cells, as compared to siGLO cells ( Figure 9A).
To elucidate the molecular mechanism by which the silencing of TRPML-2 induced apoptotic cell death, the mitochondrial transmembrane potential (ΔΨm) was analyzed in glioma cells. Mitochondrial depolarization was evident after 72 h of transfection in siTRPML-2 cells, as respect to siGLO cells ( Figure 9B). In addition, by western blot analysis a reduced level of procaspase-3 protein, as result of caspase-3 activation, was observed in siTRPML-2 T98 and U251 cells, compared to siGLO cells ( Figure 9C).
Taken together, loss of TRPML-2 in glioma cell lines induces DNA damage, reduces cell proliferation, and activates caspase-dependent apoptosis.

DISCUSSION
Changes in TRP channel expression were found in gliomas, and a significant relationship between overexpression of TRP genes and survival of patients has been recently reported [23][24][25].
Here, we evaluated the expression of TRPML-2 at mRNA and protein levels in glioma tissues with different pathological grades. We observed that TRPML-2 mRNA expression is increased in all glioma specimens analyzed, as compared to NHA. In particular, the expression of TRPML-2 mRNA increased with the pathological grade, from the pylocytic astrocytoma, grade I to the GBM grade IV, with the last showing the highest TRPML-2 mRNA levels. Similarly, we also found that TRPML-2 protein is expressed in all glioma specimens, with the high expression in GBM and low expression in astrocytoma grade I. Concordingly, high level of TRPML-2 mRNA and proteins were observed in high-grade U87 astrocytoma as well as in T98 and U251 GBM cell lines. TRPML-2 protein localizes both in the plasma membrane and in the cytoplasm. In line with our findings, the transcriptional activator of the TRPML-2 gene [26], Paired box 5 (PAX5), also known as B-cell lineage specific activator protein, has been found to be overexpressed in human astrocytoma tissues, and its expression correlates with increasing malignancy and pathological grade [27]. The PAX proteins promote tumor cell survival, proliferation and apoptosis resistance and a reduction in PAX gene expression induces apoptosis in tumor cells [28,29]. Taken together, these results demonstrate for the first time the expression of TRPML-2 mRNA and proteins in gliomas, suggesting that these channels play an important role and can interfere with gliomas growth.
At present limited data in the physiologic and pathologic role played by TRPML-2 has been provided. So, we evaluated the role of TRPML-2 in glioma cell survival, proliferation and death, by silencing the TRPML-2 gene in T98 and U251 GBM cell lines. We found that siTRPML-2 U251 and T98 cells show a reduced viability, compared to siGLO cells, correlated with a decreased cell proliferation. Furthermore, silencing of TRPML-2 resulted in a strong reduction in G1 phase cell populations, with a marked increase of subG0 DNA fragmentation, a sign of an enhanced cell death. Thus, an increased percentage of Ann V + and PI + /Ann V + apoptotic cells was evidenced in siTRPML-2 U251 and T98MG cells, respect to relative controls. Finally, the cellular response to DNA damage induced by TRPML-2 loss, leading to cell cycle arrest involves the γ-H2AX phosphorylation, caspase-3 activation and mitochondriadependent apoptosis as evaluated by increased Ser-139 γ-H2AX, and reduced ΔΨm and procaspase-3 levels in siTRPML-2 T98 and U251 cells, compared to siGLO cells. In agreement with our results, knock-down of TRPML-2 expression in HEK-293 cells causes severe cell degeneration, characterized by lysosomal inclusions and mitochondrial fragmentation, suggesting that the presence of functional TRPML-2 channel is necessary to ensure cell viability [7]. Finally, since the role for TRPML-1  and -3 channels in autophagy [30,31], the involvement of TRPML-2 in this pathway was evaluated. In agree with previous findings obtained in TRPML-2 silenced HEK-293 and Hela cells [32], the silencing of TRPML-2 gene in glioma cell lines does not affect the autophagic process.
The findings of the dependence of TRPML-2mediated survival and proliferation of glioma cells to the PI3K/Akt pathway may be relevant in the view of a role of phosphoinositides (PIPs) in the regulation of TRPMLs expression and functional activity [19,38] and on the ability of PI(3,5)P2 to activate TRPML-2 [19]. The PI(3,5)P2 levels are tightly regulated and involved in signaling response to hyperosmotic stress and growth factors in mammalian cells [39,40]. It has been suggested that different PIP isoforms determine the trafficking and the functional activity of TRPML ion channel between specific cell compartments in the endocytic pathway (endosome/lysosome) and plasma membrane [41]. Thus it is tempting to speculate that tuning of Akt/PKB and Erk1/2 pathways by TRPML-2 in the glioma microenvironment may regulate the survival and proliferation of GBM cells.
Finally, our findings evidenced that the level of TRPML-2 mRNA and proteins was high in neural stem/ progenitor cells; since, the glioblastoma stem-like cells, suspected to support the gliomagenesis, derive from neural stem and/or progenitor cells or differentiated cells such as astrocytes or oligodendrocytes [42], the peculiar expression of TRPML-2 channels in neural stem/progenitor cells and astrocytes may be particularly www.impactjournals.com/oncotarget interesting to stimulate additional studies on the expression and functions of these channels in glioma stem-like cells.
The low TRPML-2 expression in normal brain and the progressive overexpression in low vs high grade gliomas may give TRPML-2 channels as predictive and specific biomarker in brain tumors of astrocyte origin [43] and may point to novel approaches in glioma therapy. Understanding the role of TRPML-2 in the regulation of survival and proliferative pathways could shed light on the mechanisms of resistance of these cancers to apoptotic signals. Further in depth studies in the physio-pathologic role of TRPML-2 channels in normal cells should be required to improve understanding of the molecular mechanisms that regulate their expression and functions in brain tumors.

Cells and tissues
Formalin-fixed paraffin-embedded brain tissues from human tumor biopsies surgically removed from patients who gave informed consent to the study. (n = 63), were kindly provided by Prof. Felice Giangaspero (I.N.M., Neuromed, Pozzilli, Isernia, Italy), Glioma tissues, histologically graded according to the World Health Organization classification criteria [2], were grouped on the basis of malignancy, in pylocitic astrocytomas, grade I (n = 11), astrocytomas, grade II (n = 16), anaplastic astrocytomas, grade III (n = 17) and GBM, grade IV (n = 19). Breast cancer samples (n = 3) from invasive ER and HER2-positive, high-grade (G3) breast cancers, were collected during surgery and formalin-fixed by the Pathology Unit, AU3, Macerata, from patients giving their informed written consent, that covered the use of their tissues for research purposes. All procedures were conducted in accordance with the Declaration of Helsinki [44,45].

Western blot
Total lysates from T98, U251, U87 and MCF-7 cell lines were lysed in a lysis-buffer containing protease inhibitor cocktail (Sigma Aldrich). Plasma membrane and cytosol fractions from glioma cell lines were isolated using the Subcellular Protein Fractionation kit (Thermo Scientific, Rockford, IL, USA), according to the manufacturer's directions.
Proteins were separated on 8-14% SDS polyacrylamide gel, transferred onto Hybond-C extra membranes (GE Healthcare) and blotted with the specific Abs. Non-specific binding sites were blocked with 5% low-fat dry milk and 2% bovine serum albumin (BSA) in phosphate-buffered saline 0.1% Tween 20 for 1 h at room temperature. Blots were incubated with the anti-TRPML-2 primary Ab for 25 min at 37 °C followed by HRPconjugated anti-rabbit Ab for 1h at room temperature. Membrane were incubated overnight at 4°C in primary Abs (anti-caspase 3; anti-H2AX, anti-pAKT, anti-AKT, anti-pERK, anti-ERK, anti-p62, anti-LC3, anti-GAPDH), followed by the incubation for 1 h at room temperature with HRP-conjugated anti-rabbit or anti-mouse secondary Abs. The detection was performed using the LiteAblot PLUS kit (EuroClone, Milan, Italy), and densitometric analysis was carried out by a Chemidoc using the Quantity One software (Bio-Rad, Hercules, CA, USA). For quantification, GAPDH was used as loading control. One representative out of three independent experiments is shown.

Cell viability
Cell viability was assessed by the MTT assay. Three x 10 4 untransfected, siTRPML-2 and siGLO cells/ml were seeded in 96-well plates. After 72 h of incubation, 0.8 mg/ml of MTT was added to the samples and incubated for 3 h. After the removal of medium from the wells, the formazan crystals were dissolved with 100 μl per well of DMSO and the coloured solutions were read by microtiter plate spectrophometer (BioTek Instruments, Winooski, VT, USA). Four replicates were used for each treatment.

Cell cycle analysis
Cell cycle analysis was performed in untransfected, siTRPML-2 and siGLO cells. After 72 h of transfection cells were fixed in ice-cold 70% ethanol, treated for 30 min at 37° C with 100 μg/ml ribonuclease A solution, stained for 30 min at room temperature with PI 20 μg/ml and analyzed by flow cytometry using linear amplification.

BrdU cell proliferation assay
BrdU incorporation was determined in untransfected, siTRPML-2 and siGLO cells after 72 h of transfection. Cells were labelled by adding 20 μM BrdU for 24 h and fixed in cold ethanol 70%. After this, cells were washed with PBS and incubated with HCl 2N for 30 min at room temperature. Thereafter, cells were washed twice with PBS and incubated with the anti-BrdU FITCconjugated Ab, diluted 1:50 in a PBS-T solution (1% BSA and 0.5% Tween 20), for 1 h, in the dark, under agitation. Finally, cells were washed once with PBS-T and incubated with the same solution used for cell cycle analysis, followed by flow cytometry.

Cell death analysis
Cell death was evaluated using FITC-conjugated Annexin V (Ann V) and PI staining followed by single and biparametric FACS analysis. Briefly, untreated, siTRPML-2 and siGLO cells were incubated with 5 μl Annexin V-FITC (Enzo Life Sciences, Farmingdale, NY, USA) and 20 μg/ml PI for 10 min at room temperature. The cells were then analyzed by flow cytometry using CellQuest software.

Mitochondrial transmembrane potential (ΔΨm)
Mitochondrial transmembrane potential was evaluated by JC-I staining in untransfected, siTRPML-2 and siGLO cells. After 72 h of transfection cells were incubated for l0 min at room temperature with JC-1. JC-I was excited by an argon laser (488 nm) and green (530 nrn)/red (>570 nrn) emission fluorescence was collected simultaneously. Carbonyl cyanide chlorophenylhydrazone protonophore, a mitochondrial uncoupler that collapses ΔΨm was used as positive control (data not shown). Samples were analyzed by a FACScan cytofluorimeter using the CellQuest software; fluorescence intensity was expressed in arbitrary units on logarithmic scale.

RNA extraction and reverse transcription
Total RNA from fixed paraffin-embedded tissue slices (5-7 μm-thick) was extracted by RNeasy® FFPE Mini Kit (Qiagen, Milan, Italy). RNA from U87, U251, T98 and MCF-7 cell lines was extracted by RNeasy Mini Kit (Qiagen). All RNA samples were eluted in the appropriate buffer and their concentration and purity were evaluated by A260/280 nm measurement. Five hundred nanograms of RNA extracted were subjected to reverse transcription in a total volume of 25 μl using the highcapacity cDNA archive kit (PE Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. Five microlitres of the cDNA derived by fixed paraffin-embedded tissue was pre-amplified for 15 cycles using RT 2 PreAMP cDNA Synthesis kit (Qiagen). Two microliters of the resulting cDNA products were used as template for polymerase chain reaction (PCR).

Immunohistochemistry
For immunohistochemistry, after re-hydration, sections were incubated with Tris-HCl 20 mM, EDTA 0,65 mM, Tween-20 0,0005% pH 9, in a microwave for 5 min (two times) for antigen retrieval. Sections were treated with H 2 O 2 for 20 min, washed, incubated for 1 h at room temperature with 5% bovine serum albumin (BSA) and 0.3% Triton X-100 in PBS, and then overnight at 4°C with anti-TRPML-2 Ab. Thereafter, slides were incubated for 30 min at room temperature with a biotinylated antirabbit secondary antibody (1:200) rinsed, and exposed for 30 min to the streptavidin-biotin complex (VECTASTAIN ABC Kit, Vector laboratories, Burlingame, CA, USA). Immunoreactivity was detected by the addition of diaminobenzidine (Peroxidase Substrate Kit, Vector laboratories). Four random fields of each tissue specimen were analyzed under x20 magnification using the Leica DMR Microscope collected by TV camera with an IAS 2000 system (Delta Sistemi, Rome, Italy). The density of immunoreaction was measured by image analysis in different sample portion. The intensity of immunostaining was evaluated microdensitometrically calibrating the image analyzer, taking as "zero" the background developed in sections incubated with a non-immune serum and "250" as the conventional value of maximum intensity of staining.
U87, U251, T98 and MCF-7 cell lines were maintained on cover slips of 6-well plates in fresh medium. After 24 h cells on cover slips were fixed with 4% paraformaldehyde for 15 min at room temperature, and were then counterstained with haematoxylin. Immunohistochemistry in the cells was performed as above described, without antigen-retrieval. www.impactjournals.com/oncotarget

Immunofluorescence and FACS analysis
The membranous and intracellular TRPML-2 expression was determined by flow cytometry. Cells were fixed with 4% paraformaldehyde and then each sample was divided into two groups, one to be stained with anti-TRPML-2 Ab (1:100) in staining solution (phosphatebuffered saline [PBS], 1% FBS and 0.1% NaN 3 ) and one to be stained with anti-TRPML-2 Ab (1:100) in permeabilization buffer (PBS, 1% FBS, 0.1% NaN3 and 1% saponin). After an incubation of 1 h at 4°C, cells were then incubated with FITC-conjugated anti-rabbit Ab and analysed using a FACScan cytofluorimeter with CellQuest software.

Statistical analysis
The statistical significance was determined by Student's t-test and by Anova with Bonferroni's post-test. No statistically significant difference was found between untransfected and siGLO transfected U251 and T98 cells (data not shown).