Fig. 2: NRAS ASO treatment reduces NRAS-mRNA, protein levels, and MAPK-pathway signaling in NRAS-mutant melanoma. | Communications Medicine

Fig. 2: NRAS ASO treatment reduces NRAS-mRNA, protein levels, and MAPK-pathway signaling in NRAS-mutant melanoma.

From: Suppression of NRAS-mutant melanoma growth with NRAS-targeting Antisense Oligonucleotide treatment reveals therapeutically relevant kinase co-dependencies

Fig. 2

a Using qRT-PCR to compare RNA levels in D04 and MM415 cells that were either treated with NRAS ASO-1 or NRAS ASO-2, showed a robust reduction of NRAS-mRNA levels after 6, 24, 48, and 72 hours, when compared to treatment with non-targeting Control ASO. Final oligonucleotide concentration was 100 nM; error bars represent s.e.m. (n = 3). b, c Representative images of RNA in situ hybridization (RNA-ISH) derived from pellets of b D04 or c MM415 cells, either treated with NRAS ASO-1, or Control ASO. Fluorescent signals were produced by DAPI DNA staining to mark the nuclear regions (blue), probes that stain the NRAS-mRNA (red), and two different antibodies that stain for NRAS protein (ProteinTech 10724-1-AP – green, LsBio LS-C174539 – orange). NRAS ASO-1 treatment strongly reduced NRAS-mRNA levels in the cytoplasm and nucleus of the cells and NRAS protein expression. Final oligonucleotide concentration was 100 nM and treatment period lasted for 24 h. d Immunoblotting showing a strong decrease in NRAS protein levels 1 day after NRAS ASO-1 treatment compared to Control ASO treatment in D04 (−66%) and MM415 (−87%) cell lysates. B-ACTIN served as loading control and normalization parameter. e Immunoblotting showing a decrease in p-ERK1/2 protein levels 2 days after NRAS ASO treatment compared to Control ASO treatment in D04 (−50%) and MM415 (−50%) cell lysates, while total ERK1/2 levels were not altered significantly. GAPDH served as loading control and normalization parameter. f Immunoblotting showing a decrease in p-S6 protein levels 2 days after NRAS ASO-1 treatment compared to Control ASO treatment in D04 (−70%) and MM415 (−71%) cell lysates, while total S6 levels were not altered significantly. g Immunoblotting showing a small increase in p-AKT protein levels 2 days after NRAS ASO-1 treatment compared to Control ASO treatment in D04 (+18%) and MM415 (+12%) cell lysates. Total AKT levels were not altered significantly. Final oligonucleotide concentration was 100 nM. h A simplified illustration depicting key signaling pathways in NRAS-mutant melanoma, emphasizing the activation of crucial proteins contributing to cellular survival. Through transcription, the mutations in the NRAS gene are carried over to the NRAS-mRNA, which is translated into the constitutively active mutant NRAS protein, initiating downstream signaling cascades. This activation prompts the RAF kinase (not shown) to activate MEK, which, in turn, activates ERK. ERK signaling influences the activation of S6 ribosomal protein and translocates to the nucleus, regulating transcription and supporting cellular proliferation. S6 plays a pivotal role in translation, facilitating protein synthesis. The activation of this signaling pathways enhances cellular survival in NRAS-mutant melanoma. Phosphorylation-dependent activation steps are denoted by (P). i A simplified illustration highlighting the impact of NRAS ASO treatment: NRAS ASOs reduce NRAS-mRNA levels in both the cytoplasm and nucleus. This reduction is followed by decreased NRAS protein levels and the inhibition of MAPK-pathway signaling activity, as evidenced by diminished p-ERK and p-S6 protein levels. The pathway is essential for the NRAS-mutant cancer cells’ ability to proliferate and survive.

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