Fig 2: Goblet cells of intestine display dominant expression of Rab7.(A, B) Confocal imaging of healthy and dextran sulfate sodium (DSS)-treated mice (7 d) colon sections showing Rab7 (red) expression within goblet cells (UEA1-FITC) marked with asterisk. Inset shows zoomed areas of the image (scale bar = 20 µm). Graph shows Rab7 fluorescence intensity within UEA1 positive goblet cells measured within region of interest (ROI). 20 goblet cells were selected randomly from three different fields of each mouse (n = 3). (C, D) Representative co-immunofluorescence images of human ulcerative colitis (UC) and control colon biopsy sections stained for Rab7 (red) and Muc2 (green) for goblet cells (marked with asterisk) (scale bar = 50 µm). Inset shows zoomed areas of the image (scale bar = 20 µm). Graph shows Rab7 fluorescence intensity within Muc2-positive goblet cells measured within ROI. 20 goblet cells were selected randomly from five different fields of each sample (n = 3). (E) Co-immunofluorescence of Rab7 (green) and Muc2 (red) in undifferentiated and differentiated HT-29 cells (scale bar = 50 µm). Graphs show Rab7 and Muc2 fluorescence intensity measured in 10 different fields of three independent experiments each. (F) Rab7 protein visualized in HT29-MTX-E12 cells by immune-EM. Arrows indicate presence of Rab7 on vacuoles of cells (scale bar = 1 µm). Inset showed zoomed area of image (scale bar = 200 nm). Error bars represent mean + SEM. Statistical analysis by Student’s t-test.

Fig 3: Expression of MITF and GSK3β in different melanoma lines and effects of Rab7a or TPC2 KO or small molecule blockers.a Representative Western blots for MITF and Rab7 protein expression in different melanoma lines and in the breast cancer line MDA-MB-231, normalized to Vinculin. b, c Correlation plot for MITF/Rab7 expression (b) and statistical analysis of experiments as shown in (a) (mean values ± SEM, n = 5–8). Data points represent biological replicates (c). d Representative images of sections from healthy lymphnode (male, abdomen) and melanoma lymphnode metastasis (male, iliacal) samples stained with hMITF antibody (IHC). Scale bars = 5 mm. e IHC evaluation was carried out considering the percentage of stained tumor cells. Statistical significance was assessed by two-tailed unpaired t-test, *p = 0.0125 (mean ± SD). One dot corresponds to one independent human donor (n = 10 for each condition). Genetic knockout of either Rab7a (f, g) or TPC2 (h, i) in SK-MEL-5 cells shows reduction in the protein levels of MITF and β-Catenin but increased expression of GSK3β. Statistical analysis for the expression levels of MITF, GSK3β, and β-Catenin, WT vs. Rab7a KO or TPC2 KO is shown in (g, i), respectively; significance determined by two-tailed unpaired t-test (g) or by one-way ANOVA (i). Shown are mean values ± SEM, n = 8–14 in (g) and 3–9 in (i). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. j, k Representative blots for MITF protein expression in different melanoma lines after treatment with the TPC2 inhibitor SG-094 (7 µM) or DMSO control for 24 h. Statistical significance was determined by one-way ANOVA. Shown are mean values ± SEM, n = 5–7. *p < 0.05, ***p < 0.001, ****p < 0.0001. Proliferation experiments showing effect of SG094 treatment (7 µM) in SK-MEL-5 and other SK-MEL melanoma lines, significance determined by one-way ANOVA (l) or two-tailed unpaired t-test (m). Shown are mean values ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All n numbers represent biological replicates. Source data are provided as Source Data file.

Fig 4: Rescue experiments in SK-MEL-5 TPC2 and Rab7a KO.Proliferation of Rab7a KO (a) or TPC2 KO (b) cells expressing vector alone, Rab7WT- or Rab7Q67L-mCherry, assessed for 24-, 48-, and 72-h and normalized to vector. Proliferation assessed for 24-, 48-, and 72-h for Rab7a KO (c) or TPC2 KO (d) SK-MEL-5 cells expressing TPC2WT-mCherry normalized to TPC2L265P-mCherry, TPC2M484L-YFP normalized to TPC2WT-YFP, and treatment with the agonist TPC2-A1P normalized to DMSO control. e–h Representative images of the invasive phenotype determined by OE of mCherry vector, Rab7WT- or Rab7Q67L-mCherry in Rab7a KO and TPC2 KO, and statistical analysis shown in (e) and (g), respectively. Statistical significance was determined using one-way ANOVA, mean values ± SEM. i–l Representative images and statistical analysis of the invasive phenotype determined by OE of TPC2WT-mCherry, TPC2M484L-YFP, and/or treatment with TPC2-A1P in Rab7a and TPC2 KO, normalized to resp. control. Statistical significance in a–e, g, i, k was determined using one-way ANOVA followed by Bonferroni multiple comparisons test, mean values ± SEM (a–d: n = 9–15; e, g: n = 4; i: n = 3–9, k: n = 5–8). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. m–p In vivo experiments showing tumor growth/weight after subcutaneous injection of B16F10luc WT, TPC2 KO or Rab7a KO cells into 5–6 week old C57Bl/6-Tyr mice. Treatment with vehicle control, ML-098 (0.04 mg/g) or TPC2-A1-P (0.02 mg/g) was performed daily (m, “Created in BioRender”). BioRender.com/n88t710.”). Bioluminescence images (d7 after injection) and bioluminescence signal intensities of tumors, mean ± SEM (n = 15 for Rab7a KO + TPC2-A1-P, n = 17 for Rab7a KO). Statistical significance was assessed by two-tailed unpaired t-test, **p < 0.0001 (mean ± SEM) (n). Tumor weights and representative tumors at the endpoint (day 14) are shown in (o, p). Statistical significance was determined using one-way ANOVA, mean values ± SEM (WT n = 16, TPC2 KO ± ML-098 n = 14 each, Rab7a KO n = 17, Rab7a KO + TPC2-A1-P n = 15), *p < 0.05, **p < 0.01. q In vivo experiments showing tumor cell dissemination after intravenous injection of 2 × 105 B16F10-luc WT, TPC2 KO or Rab7a KO cells. Data from day 14 (endpoint) are shown. Statistical significance was determined using ordinary one-way ANOVA, mean ± SD (WT n = 7, TPC2 KO and Rab7a KO n = 9), *p < 0.05, **p < 0.01. Source data are provided as Source Data file.

Fig 5: Proposed mechanism of β-Catenin and MITF regulation in SK-MEL-5 WT and Rab7/TPC2 KO cells.In WT cells, Rab7a upregulates activity of TPC2 which induces endolysosomal degradation of the GSK3β complex. Downregulation of GSK3β leads to decreased phosphorylation of β-Catenin, which then translocates in its dephosphorylated state to the nucleus and activates MITF transcription. The transcription factor MITF induces the transcription of various genes upregulating cell migration, invasion, proliferation and tumor growth. Furthermore, decreased GSK3β levels lead to decreased phosphorylation of MITF, hence less proteosomal degradation of MITF. When Rab7a or TPC2 is dysfunctional or knocked out, GSK3β phosphorylates β-Catenin and MITF promoting their proteosomal degradation, and blocking β-Catenin from translocation to the nucleus. Less MITF transcription leads to a decrease in migration, invasion, proliferation and tumor growth, suggesting that TPC2 GOF SNPs and high Rab7a expression may be risk factors for melanoma. “Created in BioRender. BioRender.com/c52w590”.