Impairing hydrolase transport machinery prevents human melanoma metastasis

Metastases are the major cause of cancer-related death, yet, molecular weaknesses that could be exploited to prevent tumor cells spreading are poorly known. Here, we found that perturbing hydrolase transport to lysosomes by blocking either the expression of IGF2R, the main receptor responsible for their trafficking, or GNPT, a transferase involved in the addition of the specific tag recognized by IGF2R, reduces melanoma invasiveness potential. Mechanistically, we demonstrate that the perturbation of this traffic, leads to a compensatory lysosome neo-biogenesis devoided of degradative enzymes. This regulatory loop relies on the stimulation of TFEB transcription factor expression. Interestingly, the inhibition of this transcription factor playing a key role of lysosome production, restores melanomas’ invasive potential in the absence of hydrolase transport. These data implicate that targeting hydrolase transport in melanoma could serve to develop new therapies aiming to prevent metastasis by triggering a physiological response stimulating TFEB expression in melanoma.


Gel zymography
Melanoma cells were cultured in 150 mm 2 dishes before to be washed twice with cold PBS prior to be incubated 15 min in 2 ml lysis buffer (Tris/HCl 0.025M pH 7.5, NaCl 0.1M, NP-40 1%, Aprotinin 10 μg/mL, Leupeptin, EDTA 0.004M).Then, protein concentrations were determined by BCA method (Pierce).Equal amount of protein was subjected to 0.1 % gelatin 10 % SDS-PAGE.After migration, the gel was renatured in a 2,5% Triton X-100 solution for 30 min prior to be washed twice with distillated water.Subsequently, the gel was incubated for 30 min shaking at room temperature in developing buffer (Tris/HCl 0.5M pH 7.8, NaCl 2M, CaCl2 0,05M and Brij-35 0,2%) and finally the gel was kept in fresh developing buffer for overnight at 37 °C.After 23 h incubation gels were stained with 0.1 % Coomassie blue for 1 h and destained in 5 % methanol and 10 % acetic acid.Zones of gelatinolytic activity were detected as clear bands against a blue background.

Supplementary Figure 2 :
Validation of IGF2R, GNPTAB and NAGPA depletion by siRNA.(a) Each indicated cell line were transfected with either siRNA specific for IGF2R or GNPTAB prior to be analysed by qRT-PCR.Each bar represent the relative expression of the mRNA targeted by the siRNA.The results are the average of at least three independent experiments.Error bars represent ± SD.(b) WM1716 cell line, transfected with indicated siRNA were analysed by qRT-PCR.Each bar represent the relative expression of the mRNA targeted by the siRNA.Error bars represent ± SD.
a) Immunofluorescence confocal microscopy images of WM1745 cells transfected with indicated siRNA and stained with Red LysoTracker (red) and DAPI (blue).The quantifications of lysosomes numbers are shown on the right panel.Central bars indicate the average number of lysosomes per cell.One-way anova tests were used for statistical analysis.Error bars represent ± SD, ** indicates p<0.01, *** p < 0.001, **** indicates p < 0.0001.Scale bar = 10µm.(b) WM1745 cells were transfected with either control siRNA or specific for GNPTAB prior to be analysed by qRT-PCR.Each bar represent the relative expression of the mRNA targeted by the siRNA.The results are the average of at least three independent experiments.Error bars represent ± SD.
TFEB is present inside the nucleus under phosphorylated and unphosphorylated forms in basal conditions in WM1716 cells.(a) WM1716 cells under basal conditions were lysed and fractioned using NE-PER fractionation kit (ThermoFisher).Cytoplasmic and nuclear fractions were analyzed by immunoblotting against TFEB.Lamin A/C served as a nuclear marker and vinculin served as a cytoplasmic marker.(b) Protein extract from WM1716 cells exposed to indicated siRNA were subjected to western blot analysis using antibody raised against total or S65 phosphorylated from of 4E-BP1.

5 :
Modulation of TFEB expression regulates lysosome biogenesis of melanoma cells.WM1716 cells were transfected with indicated siRNA and stained with Red LysoTracker and DAPI (upper panel).The number of lysosomes per cell was quantified using Volocity software.Central bars indicate the average number of lysosomes per cell (lower panel).Kruskal and Wallis tests were used for statistical analysis.Error bars represent ± SD, ** indicates p<0.01, *** p < 0.001, **** indicates p < 0.0001.Scale bar = 10µm.Characterisation of WM1716 cell line ectopically expressing TFEB (a) TFEB mRNA expression comparison between WM1716 transduced with empty vector (FUGW) or with a lentivirus coding for TFEB (WM1716-TFEB).Results are shown as fold over control.Error bars represent ± SD.(b) Western blot analysis of protein extracts from WM1716-FUGW and WM1716-TFEB cell lines using TFEB and actin antibodies.(c) Number of WM1716-FUGW and WM1716-TFEB invasive cells assessed by matrigel coated transwell assay.Unpaired t-test was used for statistical analysis.Error bars represent ± SD.**** indicates p < 0.0001