Lysosomal Zn2+ release triggers rapid, mitochondria-mediated, non-apoptotic cell death in metastatic melanoma

Abstract

During tumor progression, lysosome function is often maladaptively upregulated to match the high energy demand required for cancer cell hyper-proliferation and invasion. Here, we report that mucolipin TRP channel 1 (TRPML1), a lysosomal Ca²⁺ and Zn²⁺ release channel that regulates multiple aspects of lysosome function, is dramatically upregulated in metastatic melanoma cells compared with normal cells. TRPML-specific synthetic agonists (ML-SAs) are sufficient to induce rapid (within hours) lysosomal Zn²⁺-dependent necrotic cell death in metastatic melanoma cells while completely sparing normal cells. ML-SA-caused mitochondria swelling and dysfunction lead to cellular ATP depletion. While pharmacological inhibition or genetic silencing of TRPML1 in metastatic melanoma cells prevents such cell death, overexpression of TRPML1 in normal cells confers ML-SA vulnerability. In the melanoma mouse models, ML-SAs exhibit potent in vivo efficacy of suppressing tumor progression. Hence, targeting maladaptively upregulated lysosome machinery can selectively eradicate metastatic tumor cells in vitro and in vivo.

Keywords: ML-SAs; ML-SIs; TRPML1; Zn(2+); cell death; lysosome; metastatic melanoma; mitochondria; small molecule.

Figure 1.. Lysosomal ML1 is upregulated in metastatic melanoma cells

(A) Representative images of human tissue sections from normal skin, nevus (benign tumor), and metastatic melanoma immunostained with an anti-ML1 antibody (brown DAB staining). Scale bar, 200 μm. (B) Quantification of ML1 immunostaining from normal skin tissue (Normal; n = 2 cases), nevus (Benign; n = 8 cases), and metastatic melanoma (Mets; n = 8 cases). Each open circle represents one case. (C–E) Whole-endolysosome ML1 currents (I_ML1) were activated by ML-SA1 (20 μM), a synthetic agonist of ML1, in an immortalized human melanocyte (C), a MeWo cell (D), and a M12 cell (E). Currents were recorded with a ramp voltage protocol from −120 to +120 mV (only partial voltage ranges are shown). Pipette (luminal) solution was a standard external (Tyrode’s) solution adjusted to pH 4.6 to mimic the acidic environment of the lysosome lumen. Bath (cytoplasmic) solution was a K⁺-based solution (140 mM K-gluconate). Note that the inward currents indicate cations flowing out of endolysosomes. (F) Mean current densities of whole-endolysosome I_ML1 in normal melanocytes (n = 4), MeWo cells (n = 7), and M12 cells (n = 12). Each open circle represents one endolysosome/patch/cell; average data are presented as mean ± SEM; **p < 0.01.

Figure 2.. Selective cytotoxic effects of ML-SAs on metastatic melanoma cells

(A–C) Dose-dependent effects of SA5 and SA8 on cell viabilities of normal melanocytes, MeWo, and M12 cells, which were measured using the CellTiter-Glo ATP assay 24 h after drug treatment. The average data represent mean ± SEM from three independent experiments, each with triplicates. (D) PI staining of MeWo and M12 cells in the presence or absence of SA5 (3 μM). Normal melanocytes and MeWo cells were drug treated for 6 h, and M12 cells were drug treated for 3 h. Overlay phase contrast (Ph) images are also shown. Scale bar, 25 μm. (E) The percentage of PI-positive cells in the presence of SA5 (3 μM, the left panels), as in (D), and SA8 (1 μM, right panels). Each open circle represents the average data from one batch of cells for each independent experiment. (F and G) Dose-dependent effects of SA5/8 on the percentages of PI-positive MeWo (6 h) and M12 (3 h) cells. (H) The effects of ML-SAs (3 μM SA5 or 1 μM SA8 for 12 h) on the cell viabilities of multiple cell types (n = 3 independent experiments). (I) Overlay phase contrast and fluorescence images of PI-stained SA5 (3 μM)-treated MeWo (for 6 h) and M12 (for 3 h) cells in the presence or absence of ML-SIs (SI3 and SI4, 20 μM). Scale bar, 25 μm. (J) The percentage of PI-positive cells under indicated conditions. Each open circle represents one batch of cells. Note that the control (Ctrl) groups of MeWo and M12 treated with SA5 or SA8 are re-plotted from (E), as they were from the same experiments. (K) Cell viability analysis of MeWo and M12 cells treated with SA5 in the presence or absence of ML-SI3 (12 h). In all panels, average data are presented as mean ± SEM; ***p < 0.001.

Figure 3.. ML1 is required for ML-SA-induced cell death

(A) Mean current densities of whole-endolysosome I_ML1 in Ctrl or ML1-specific siRNA (siML1)-transfected MeWo and M12 cells. Each open circle represents one endolysosome/cell/patch. (B) PI-staining of Ctrl or siML1-transfected MeWo and M12 cells in the presence of SA5 (3 μM) for 3 to 6 h. The overlay phase contrast and PI images are also shown. Scale bar, 25 μm. (C and D) The percentages of PI-positive control or siML1-transfected cells treated with SA5 (C) or SA8 (D). (E) Dose-dependent effects of SA5 (left panel) and SA8 (right panel) (12 h) on cell viability of Tet-On HEK293-GCaMP7-ML1 cells with (Dox⁺) or without doxycycline (Dox⁻) induction. (F) PI staining of HEK293 and HEK293 transiently expressing EGFP-ML1 (HEK ML1 OE) cells under indicated drug treatments for 6 h. The overlay phase contrast images are shown together with the green (EGFP-ML1) and red (PI staining) fluorescence channels. White arrows point to the EGFP-positive cells (i.e., with successful expression of EGFP-ML1) that are also PI positive. Scale bar, 25 μm. (G) The percentages of PI-positive ML1-overexpressing HEK293 cells (HEK293 ML1 OE) upon SA5 treatment (3 μM) in the presence and absence of ML-SI3/4 (20 μM), as shown in (F). Average data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4.. Lysosomal Zn²⁺ release is required for ML-SA-induced cell death

(A) The effects of SA5 (3 μM) treatment on PI staining of MeWo (for 6 h) and M12 (for 3 h) cells that were pretreated with BAPTA-AM (10 μM) or TPEN (2.5 μM). (B) The percentages of PI-positive cells as shown in (A). Each open circle represents one batch of cells. Note that the control groups of MeWo and M12 cells treated with ML-SA5 are re-plotted from Figure 2E, as they were from the same experiments. (C and D) Cell viability analysis of MeWo (C) and M12 (D) cells treated with indicated chemicals for 12 h. (E) PI staining of HEK293 and HEK293 transient expressing of EGFP-ML1 (HEK ML1 OE) cells under indicated drug treatments for 6 h. Phase contrast overlay images are shown together with the green (EGFP-ML1) and red (PI staining) fluorescence channels. White arrows point to the EGFP-positive cells that are also PI positive. Scale bar, 25 μm. (F) The percentages of PI-positive ML1-overexpressing HEK293 cells (HEK293 ML1 OE) upon SA5 treatment (3 μM) in the presence and absence of BAPTA-AM (10 μM) or TPEN (2.5 μM), as shown in (E). Note that the left two columns are re-plotted from Figure 3G, as they were from the same experiments. (G) Cell viability analysis of HEK293-GCaMP7-ML1 cells (with Dox induction) in the presence of SA5, BATPA-AM, and TPEN for 12 h. (H) Fluorescence images of MeWo and M12 cells stained with both FluoZin-3 (−AM) and LysoTracker. Scale bar, 5 μm. (I) FluoZin-3 staining of M12 cells treated with DMSO (Ctrl) and SA5 (3 μM) for 30 min. Scale bar, 5 μm. (J) Statistical analysis of relative FluoZin-3 intensity in control (n = 55 cells) and SA5 treatment (n = 56 cells) experiments, as shown in (I). (K) The effects of SA1 on GZnP3 imaging of MeWo cells transfected with GZnP3-ML1 or GZnP3-Rab7 in the presence of 100 μM Zn²⁺. (L) The rates of SA1-induced Zn²⁺ flux in GZnP3 (ML1- or Rab7-) transfected HEK293 and MeWo cells (calculated as in K). (M) FluoZin-3 imaging of M12 cells that were incubated with a normal (Ctrl) or high Zn²⁺ (100 μM) growth medium for 12 h. (N) Cell viability analysis of M12 (left panel) and MeWo (right panel) cells that were treated with indicated chemicals for 12 h. All data are mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5.. ML-SAs cause mitochondrial swelling in metastatic melanoma cells

(A) Live imaging of MitoTracker-loaded normal melanocytes (left panels, 60 min), MeWo (middle panels, 60 min), and M12 (right panels, 30 min) cells under DMSO (Ctrl) or SA5 (3 μM) treatment. Scale bar, 10 μm. (B and C) Statistical analysis of mitochondrial swelling in melanocytes, MeWo, and M12 cells that were treated with SA5 (3 μM) or SA8 (1 μM) for 30 or 60 min. Each open circle represents the average result from one batch of cells. (D and E) The effects of ML-SIs and TPEN on mitochondrial swelling in SA5 (D) or SA8 (E)-treated MeWo and M12 cells. Note that the control groups of MeWo and M12 treated with SA5 or SA8 are re-plotted from (B) and (C), as they were from the same experiments. (F) Representative transmission electron microscopy (TEM) images of M12 cells that were treated with DMSO (Ctrl) or SA5 (3 μM) for 30 min. M, mitochondria. Scale bar, 200 nm. (G) Statistical analysis of mitochondrial size in control (n = 14 mitochondria from 3 cells) and SA5-treated cells (n = 22 mitochondria from 5 cells). All data are mean ± SEM; **p < 0.01.

Figure 6.. In vivo efficacy of ML-SAs on suppressing tumor growth in advanced melanoma mouse models

(A) Representative bioluminescence images (BLI) of MeWo-FmC-tumor bearing mice treated with vehicle or SA5 (5 mg/kg mouse weight). (B) Statistical analysis of in vivo MeWo-FmC tumor growth in vehicle versus SA5-treated mice (n = 5 mice per group). (C) Representative mCherry fluorescence imaging of MeWo-FmC tumor cells (red) in skin sections from vesicle or SA5-treated tumor bearing mice (sample collection at day 42). Scale bar, 50 μm. (D) The effects of drug treatment on mouse body weight. (E) Representative bioluminescence images of M12-FmC-tumor bearing mice treated with vehicle or SA8 on day 7 after tumor implantation (before drug administration at day 8) and day 14 (1 week after drug administration); single intracranial injection with vehicle (DMSO) or SA8 (10 mM/5 μl) was conducted at day 8. (F) Statistical analysis of in vivo M12-FmC brain tumor growth in vehicle versus SA8-treated mice (n = 8 mice per group). Each open circle/square represents one mouse. (G) Kaplan-Meier survival analysis of M12-FmC tumor-bearing mice treated with vehicle or SA8 (n = 8 mice per group). Log-rank test, p < 0.001. For all other panels, data are mean ± SEM; **p < 0.01.

Figure 7.. Activation of upregulated ML1 channels in the lysosomal Zn²⁺ stores in duces Zn²⁺ release and selective necrotic cell death in metastatic melanoma cells

Lysosome number and machinery, including lysosomal ML1 channels, are maladaptively upregulated in metastatic melanoma cells compared with normal melanocytes. In metastatic melanoma cells, but not in normal melanocytes, ML-SA administration results in excessive ML1 activation and lysosomal Zn²⁺ release, causing mitochondrial swelling/damage, cellular ATP depletion, and necrotic cell death (which we termed “Lysozincrosis”).