The LncRNA LENOX Interacts with RAP2C to Regulate Metabolism and Promote Resistance to MAPK Inhibition in Melanoma

Abstract

Tumor heterogeneity is a key feature of melanomas that hinders development of effective treatments. Aiming to overcome this, we identified LINC00518 (LENOX; lincRNA-enhancer of oxidative phosphorylation) as a melanoma-specific lncRNA expressed in all known melanoma cell states and essential for melanoma survival in vitro and in vivo. Mechanistically, LENOX promoted association of the RAP2C GTPase with mitochondrial fission regulator DRP1, increasing DRP1 S637 phosphorylation, mitochondrial fusion, and oxidative phosphorylation. LENOX expression was upregulated following treatment with MAPK inhibitors, facilitating a metabolic switch from glycolysis to oxidative phosphorylation and conferring resistance to MAPK inhibition. Consequently, combined silencing of LENOX and RAP2C synergized with MAPK inhibitors to eradicate melanoma cells. Melanomas are thus addicted to the lncRNA LENOX, which acts to optimize mitochondrial function during melanoma development and progression.

Significance: The lncRNA LENOX is a novel regulator of melanoma metabolism, which can be targeted in conjunction with MAPK inhibitors to eradicate melanoma cells.

Graphical abstract

Figure 1.

LENOX is expressed in normal skin melanocytes and melanoma. A, Top 50 protein-coding and noncoding transcripts based on expression across melanoma states (GSE116237) and in GTEx and SKCM TCGA. LENOX is highlighted by dark bars. B, Expression of LENOX, SAMMSON, and TFAP2A in melanoma subpopulations. Only cells where the RNAs were captured were included in the analysis. The percentage of cells analyzed for each population is indicated. C, RNAscope detection of LENOX and MITF in normal skin and melanoma FFPE sections. D, LENOX expression in melanocytic (blue) and undifferentiated (orange) cell lines. E, LENOX expression in 501Mel cells expressing the dCas9-KAP1 protein with control or LENOX promoter-targeting sgRNAs. F–H, Proliferation, apoptosis, and colony formation following dCas9-KAP1-mediated LENOX silencing compared with negative control by one-way ANOVA (Dunnett test). I and J, Proliferation and apoptosis of LENOX or control GapmeR-transfected cells compared by one-way ANOVA (Dunnett test). K, Tumor growth of A375 cells expressing shCTRL or shLENOX (N = 5). L, Bioluminescence emission was measured on day 7 and 14 using the IVIS Spectrum Image and compared by Mann–Whitney test. M, Growth of MEL006res PDX treated with control or LENOX targeting ASO. Tumor size was measured daily for 20 days. Statistical analysis were calculated using two-way ANOVA. *, P < 0.033; **, P < 0.0021; ***, P < 0.0002; ****, P < 0.0001.

Figure 2.

LENOX interacts with the RAP2 GTPases. A, Enrichment of LENOX or TINCR after oligonucleotide-mediated pulldown from MM011 cells. B, Top, proteins detected by mass spectrometry only in the LENOX pulldown from MM011 cells. Bottom left, selective enrichment of LENOX following RAP2 IP. Bottom right, IP of RAP2 with 1% input showing short and long exposures. C, RAP2A, B, and C protein sequences illustrating amino acid homology. D, RAP2A/B/C expression in indicated cells normalized over housekeeping genes. E and F, RAP2A/B/C expression in the above cells after transfection with indicated siRNAs compared with siCtrl by one-way ANOVA (Dunnett test). VINCULIN was used as a loading control in right panel. G, Proliferation and apoptosis following silencing with indicated siRNA and comparison with control by one-way ANOVA (Dunnett test). *, P < 0.033; **, P < 0.0021; ***, P < 0.0002; ****, P < 0.0001.

Figure 3.

LENOX and RAP2 modulate oxidative phosphorylation. A, Co-imaging of LENOX, p32, and RAP2 in 501Mel cells transfected with the indicated GapmeRs. B, RAP2-p32 colocalization in a human melanoma FFPE section. C, Levels of 16S rRNA, SAMMSON, and LENOX in mitochondrial and cytoplasmic fractions (top) and levels of RAP2, P32, and HSP60 proteins (bottom) D. Mitostress test of 501Mel cells 48 hours after transfection with indicated GapmeRs or siRAP2C. Experimental groups were compared by one-way ANOVA (Dunnett test). E, Mito Stress Test on cells with ectopic LENOX isoform expression. Experimental groups were compared as in D. *, P < 0.033; **, P < 0.0021; ***, P < 0.0002.

Figure 4.

LENOX promotes RAP2 interaction with DRP1 regulating its phosphorylation on serine 637 and mitochondrial fusion. A, MitoTracker CMXROS Red staining of 501Mel cells transfected with the indicated GapmeR or siRAP2C. MitoTracker signal is shown in grayscale and Hoescht in blue. Total mitochondrial count, area, perimeter, and mean form factor were calculated and compared by one-way ANOVA (Dunnett test). B, MitoTracker CMXROS Red staining of 501Mel cells with ectopic LENOX isoform expression. Mitochondrial parameters were calculated and compared as above. C–E, Phospho-DRP1 S637 and total DRP1 levels in 501Mel and A375 cell extracts, with H3 and ACTB as loading controls. F, Left, RAP2 IP from 501Mel cells expressing shRNAs revealed with antibodies to the indicated proteins. Right, LENOX and SAMMSON levels in the indicated IP fractions. G, IP of DRP1 from 501Mel cells (left) and LENOX and SAMMSON levels in the IP (right). H, PLA-mediated detection RAP2–DRP1 interaction in 501Mel cells. Appropriate negative controls using secondary antibodies only are shown. *, P < 0.033; **, P < 0.0021; ****, P < 0.0001.

Figure 5.

LENOX and SAMMSON cooperatively promote melanoma cell survival. A and B, Proliferation and apoptosis after transfection with suboptimal doses of LENOX or SAMMSON GapmeRs as single or pairwise combinations compared between groups by one-way ANOVA. C, Crystal violet staining of cells transfected as indicated and cultured for 10 days. Percentages of area occupied in each condition were compared by one-way ANOVA. *, P < 0.033; **, P < 0.0021; ***, P < 0.0002; ****, P < 0.0001.

Figure 6.

LENOX and RAP2C promote metabolic switch upon BRAF inhibition. A and B, Mito Stress Test of 501Mel cells grown for 3 days with DMSO or vemurafenib (1 μmol/L) and transfected as indicated. Experimental groups were compared by one-way ANOVA (Tukey test). C, MitoTracker CMXROS Red staining of cells treated with DMSO or vemurafenib, transfected as indicated and analyzed by confocal microscopy as in Fig. 4A. D, Phospho-DRP1 S637 and total-DRP1 levels in cells treated as above. H3 was used as loading control. E and F, Proliferation and apoptosis of cells treated as indicated. Experimental groups were compared by one-way ANOVA. G and H, Crystal violet staining of GapmeR-transfected cells. Percentages of occupied areas were compared by one-way ANOVA. I, Growth of MEL006res PDX in mice treated with DT with or without nontargeting ASO (DT+GAP-CTR) or LENOX-targeting ASO (DT+GAP-LENOX). Tumor size was measured daily for 20 days. Statistics were calculated using two-way ANOVA (Šidák multiple comparisons test). J, Kaplan–Meier plot showing overall survival (OS) of mice described in I. DT, n = 5; DT+GAP-CTR, n = 3; DT+GAP-LENOX ASO, n = 4. Statistical analysis were calculated by log-rank (Mantel–Cox) test. *, P < 0.033; **, P < 0.0021; ***, P < 0.0002; ****, P < 0.0001.