Coexpression of TRPML1 and TRPML2 Mucolipin Channels Affects the Survival of Glioblastoma Patients

Abstract

Among brain cancers, glioblastoma (GBM) is the most malignant glioma with an extremely poor prognosis. It is characterized by high cell heterogeneity, which can be linked to its high malignancy. We have previously demonstrated that TRPML1 channels affect the OS of GBM patients. Herein, by RT-PCR, FACS and Western blot, we demonstrated that TRPML1 and TRPML2 channels are differently expressed in GBM patients and cell lines. Moreover, these channels partially colocalized in ER and lysosomal compartments in GBM cell lines, as evaluated by confocal analysis. Interestingly, the silencing of TRPML1 or TRPML2 by RNA interference results in the decrease in the other receptor at protein level. Moreover, the double knockdown of TRPML1 and TRPML2 leads to increased GBM cell survival with respect to single-channel-silenced cells, and improves migration and invasion ability of U251 cells. Finally, the Kaplan-Meier survival analysis demonstrated that patients with high TRPML2 expression in absence of TRPML1 expression strongly correlates with short OS, whereas high TRPML1 associated with low TRPML2 mRNA expression correlates with longer OS in GBM patients. The worst OS in GBM patients is associated with the loss of both TRPML1 and TRPML2 channels.

Keywords: glioblastoma; heterogeneity; mucolipin channels; overall survival.

Figure 1

Survival analysis by Kaplan–Meier curves and log-rank (Mantel–Cox) test according to different TRPML1/TRPML2 phenotypes of GBM patients. (A) Stratification of GBM patients based on TRPML2 mRNA expression. (B) p-values related to Kaplan–Meier analysis. * p < 0.05 was considered statistically significant. (C) OS of patients stratified by negative, low- or high-expressing TRPML1 or TRPML2. Percentages represent the number of patients in each subgroup. (D) Stratification of GBM patients based on negative, low or high mRNA expression of both TRPML1 and TRPML2. (E) OS of patients stratified in the six subgroups. Percentages represent the number of patients in each subgroup. Neg, negative; pos, positive; high, high-expressing; low, low-expressing.

Figure 2

TRPML1 and TRPML2 expression in cancer cell lines. (A) Representative immunoblots reflecting TRPML1 and TRPML2 protein levels in U251 and T98 (glioma), DU145 (prostate cancer), 5637 and T24 (bladder cancers), and SKBR3 and BT549 (breast cancers) cell lines, and PBMCs used as positive control. Blots are representative of one of three separate experiments. (B) Representative FACS dot plots of TRPML1 and TRPML2 expression in U251, T98, T24, 5637, DU145, SKBR3 and BT549 cell lines and PBMCs. Plots with FITC and PE as labels represent the staining of cells with secondary Abs alone. Percentages refer to the totality (100%) of cells.

Figure 3

Subcellular distribution of TRPML1 and TRPML2 in glioma cell lines. Confocal microscopy analysis of TRPML1 and TRPML2 in U251 and T98 cell lines. 40,6-diamidino-2-phenylindole (DAPI) was used to counterstain nuclei. Magnification 60×.

Figure 4

Subcellular distribution of TRPML1 and TRPML2 in glioma cell lines. Total membrane fraction (Mem), cytoplasm (Cyto), and nuclear (Nuc) extracts from T98, U251 and PMBCs, used as positive control, were immunoblotted with anti-TRPML1, anti-TRPML2, anti-LAMP1, anti-GAPDH and anti-histone H3 Ab. Whole-cell lysate (WCL) was used as control. The purity of subcellular fractions was assessed by blotting against specific markers. Cytosolic and membrane marker: GAPDH; membrane-bound organelles: LAMP1; nuclear marker: histone H3. Blots are representative of three separate experiments.

Figure 5

Confocal microscopy analysis to evaluate colocalization of TRPML1 or TRPML2 with different cellular organelles in T98 (A) and U251 (B) cells using specific Abs. Endoplasmic reticulum (ER): anti-calreticulin Ab; plasma membrane: anti-caveolin-1 Ab; lysosome: anti-LAMP1; mitochondria: anti-COX IV. 40,6-diamidino-2-phenylindole (DAPI) was used to counterstain nuclei. Magnification 60×.

Figure 6

TRPML1 and TRPML2 silencing in glioma cell lines. (A,C,E) TRPML1 and TRPML2 mRNA levels were evaluated by qRT-PCR after 72 h of transfection in T98 and U251 cells silenced for TRPML1 (A), TRPML2 (C) or double-silenced (E). Relative TRPML1 and TRPML2 expression, normalized to GAPDH mRNA levels, was calculated using siGLO as calibrator. * p < 0.05 vs. siGLO transfected cells. (B,D,F) TRPML1 and TRPML2 protein levels were evaluated after 72 h of transfection in T98 and U251 cells silenced for TRPML1 (B), TRPML2 (D) or double-silenced (F). Blots are representative of one of three separate experiments. TRPML1 and TRPML2 densitometric values were normalized to GAPDH, used as loading control.

Figure 7

Effects of double silencing on growth of GBM cell lines. A) Cell growth was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in DN, siTRPML1 and siTRPML2 T98 and U251 GBM cells for up to 48 h after silencing. Data shown are expressed as mean ± SE of three separate experiments. * p < 0.05 vs. siTRPML2 or siTRPML1 cells.

Figure 8

Double silencing increases the migration/invasion capability in U251 cell line. (A) The extent of wound closure in wound healing assays of siTRPML1 siTRPML2 (DN) T98 and U251cells at 24 h in cultures with 1% FBS. Data shown are the means ± SE. * p < 0.01. Representative images are shown of the progression of wound closure at 24 h. Original magnification ×10. (B) Representative images showing DAPI fluorescence after 24 h culture in transwell chambers (×10 magnification). The siGLO and siTRPML1 siTRPML2 T98- and U251-invading cells were counted in 10 randomly chosen microscopic fields per transwell chamber. Each sample was run in triplicate, and three independent experiments were performed. Bars represent the quantification of invaded cells in each field. Error bars represent ± SE. * p< 0.05.

Figure 9

Schematic representation of TRPML1 and TRPML2 modification in GBM.