HRAS-driven cancer cells are vulnerable to TRPML1 inhibition (2019)
Figure 1

Fig 1. BLCA, HNSC, and THCA tumors bearing oncogenic HRAS mutations exhibit upregulation of the CLEAR endolysosomal gene network

(A) Plot showing the average Z-scores of 210 endolysosomal genes plotted against the negative log10 of the P values from the one-sample t-test of whether average Z-score ≠ 0. Green circles represent significantly upregulated genes (Z > 0 and P < 0.05). Magenta circles represent significantly downregulated genes (Z < 0 and P < 0.05). Grey circles represent genes whose expression is not significantly changed (P > 0.05). All comparisons were between cancers and normal cells from the same tissue. Dashed line shows position on Y-axis that corresponds to P = 0.05. Positions of MCOLN1 (red circle), VAC14 (blue circle), and MTM1 (yellow circle) are indicated. Inset, schematic showing that Mtm1 and Vac14 regulate the levels of PI(3,5)P2, and thereby, influence TRPML1 activity.

(B) Unsupervised hierarchical clustering of Pearson's coefficients of pairwise correlation of gene expression reveal 4 indicated clusters.

(C) Violin plots of average Z-scores of genes that belong to the four clusters derived from hierarchical clustering. The thick horizontal dashed lines in each sample set represent median; thin dashed lines represent the quartiles; polygons represent density estimates of data and extend to extreme values. Statistical tests employed were 1-sample t-test or Wilcoxon signed rank test to examine significance of differences from a hypothetical value of 0.

(D) GSEA conducted on endolysosomal genes ranked on the basis of increasing Z-scores. Positions of the CLEAR target genes are indicated.

(E) Bar graph showing the relative expression of the indicated genes in the indicated cell types. Values were normalized to respective 'HN31+HRAS shRNA' average and represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.

(F) Western blots performed on extracts isolated from the indicated cells probed with antibodies against Tfeb and tubulin. Molecular weights are shown on the left. Bar graph shows relative Tfeb levels in the indicated samples. Values were normalized to 'HN31+HRAS shRNA' and represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.

(G) Bar graph showing the relative expression of the indicated genes in the indicated cell types. Values were normalized to respective 'HN31+HRAS shRNA' average and represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.

(H) 'Attribute circle layout' generated from Pearson's coefficients of pairwise correlation of gene expression using CytoScape. Decreasing node size (clockwise from MCOLN1) reflects decreasing closeness centrality; node color represents degree (number of directed edges); edge color represents Pearson's correlations.

(I) Bar graph showing the relative expression of MCOLN1 in the indicated cell types. Values were normalized to HT1197 average and represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.