Neuroblastoma RAS viral oncogene homolog mRNA is differentially spliced to give five distinct isoforms: implications for melanoma therapy

Abstract

Neuroblastoma RAS viral oncogene homolog is a commonly mutated oncogene in melanoma, and therapeutic targeting of neuroblastoma RAS viral oncogene homolog has proven difficult. We characterized the expression and phenotypic functions of five recently discovered splice isoforms of neuroblastoma RAS viral oncogene homolog in melanoma. Canonical neuroblastoma RAS viral oncogene homolog (isoform-1) was expressed to the highest degree and its expression was significantly increased in melanoma metastases compared to primary lesions. Isoform-5 expression in metastases showed a significant, positive correlation with survival and tumours over-expressing isoform-5 had significantly decreased growth in a xenograft model. In contrast, over-expression of any isoform resulted in enhanced proliferation, and invasiveness was increased with over-expression of isoform-1 or isoform-2. Downstream signalling analysis indicated that the isoforms signalled differentially through the mitogen-activated protein kinase and PI3K pathways and A375 cells over-expressing isoform-2 or isoform-5 showed resistance to vemurafenib treatment in vitro. The neuroblastoma RAS viral oncogene homolog isoforms appear to play varying roles in melanoma phenotype and could potentially serve as biomarkers for therapeutic response and disease prognosis.

Figure 1:. Structure of the NRAS isoforms.

Isoform 1 is the canonical NRAS isoform with an open reading frame spanning exons 2 through 5. Isoform 2 contains an additional exon: 3b. Isoforms 3 and 4 are lacking exon 3 or exons 3 and 4, respectively. Isoform 5 contains a fusion of the first 17 codons of exon 2 with 3 codons near the end of exon 5. Depicted are cDNA, mRNA splicing products, and protein lengths of the 5 isoforms. Numbered boxes represent the exons (Adapted from ref. 14).

Figure 2:. NRAS isoforms are expressed in melanoma tumor tissues.

(A) Expression analysis by qRT-PCR of NRAS isoforms 1–5 mRNA in 21 melanoma tumor tissues. (B) Transcript abundance in transcripts per million of NRAS isoforms 1–5 from publicly available RNAseq data of 472 skin cutaneous melanoma tumor tissues. (C) Expression analysis by qRT-PCR of NRAS isoforms 1–5 mRNA in 23 additional melanoma tumor tissues representing primary lesions and metastases to various locations. (D) Transcript abundance in transcripts per million of NRAS isoforms 1–5 from publicly available RNAseq data of 472 skin cutaneous melanoma tumor tissues separated into primary or metastatic samples. Bars represent mean ± s.e.m. *** p<0.001.

Figure 3:. Relationship of NRAS isoform levels to survival in melanoma.

(A) Overall survival of the skin cutaneous melanoma patient cohort from the TCGA database separated by top quartile (blue) or bottom quartile (red) isoform expression as measured by RNAseq.

Figure 4:. Phenotypic characterization of NRAS isoform over-expressing cell lines.

(A) MTS proliferation assay measuring cell proliferation after 48 or 72 hours of A375 cell line transduced with an empty vector control (A375 EV) or A375 transduced with vectors specific for each NRAS isoform (A375 iso 1–5). (B) Quantification of matrigel invasion assay, (C) Visualization of soft agar colony formation assay at day 4, 4x magnification of 96 well plate and quantification of cell growth by fluorescent staining, and (D) Immunoblot probing for p-ERK, p-AKT, total ERK and AKT and β-actin of same panel of A375 cell lines (left), and densitometry analysis for n=3 immunoblots (right). Bars represent three biological replicates, displayed as mean ± s.e.m. * p <0.05, ** p<0.01, *** p< 0.001.

Figure 5:. NRAS Isoform over-expression alters tumor growth in vivo.

(A) A375 cells that over-express either NRAS isoforms 1–5 or an empty vector control (A375 EV) were injected s.c. into athymic nude mice (1×10⁶ cells). and tumor growth was followed. On day 27, mice were sacrificed and tumors were removed and measured for volume (B) and weight (C). Bars depict mean ± s.e.m.

Figure 6:. NRAS Isoform expression alters response to vemurafenib treatment.

(A) Proliferation of NRAS isoform over-expressing cell lines in the presence of vemurafenib doses from 0–20 μM was measured by MTS assay after 48 hours of drug exposure, displayed as mean ± s.e.m of three replicate experiments. (B) Proportion of NRAS isoforms in BRAF/NRAS-mutant vs. wild-type (wt) metastatic melanoma patient tumors. The top and bottom quartile of NRAS isoform mRNA expression (RNAseq TCGA data in transcripts per million (TPM)). P values were determined via Fisher’s exact test.