RAMP1 as a novel prognostic biomarker in pan-cancer and osteosarcoma

Abstract

Receptor activity modifying protein 1 (RAMP1) facilitates the localization of the calcitonin-like receptor (CLR) to the plasma membrane, but its role in osteosarcoma (OS) remains unclear. We evaluated the RAMP1 expression and prognostic value across different cancers, studying tumor immune infiltration. The prognostic value was analyzed using the GSE39058 and TARGET datasets. Differential gene expression was evaluated. a protein-protein interaction network was constructed, and gene set enrichment analysis was performed. The function of RAMP1 in the tumor microenvironment was analyzed, and its expression in OS cell lines was validated using quantitative real-time PCR. High RAMP1 expression correlated with poor prognosis relative to low RAMP1 expression (p < 0.05). Low RAMP1 expression correlated with an abundance of CD4+ memory-activated T cells. whereas a high expression level correlated with a high proportion of gamma-delta T cells (γδ T cells). Differentially expressed genes from TARGET was enriched in olfactory transduction pathways (normalized enrichment scores [NES] = 1.6998, p < 0.0001). RAMP1 expression negatively correlated with CD44 expression but positively correlated with TNFSF9 expression. The RAMP1 gene is substantially expressed in OS cells compared to the normal osteoblast cell line hFOB1.19. Thus, RAMP1 may be a prognostic biomarker and potential therapeutic target in OS.

Fig 1. Receptor activity modifying protein 1 (RAMP1) expression and prognostic implications in pan-cancer.

There was an upregulation of RAMP1 in breast invasive carcinoma (BRCA), prostate adenocarcinoma (PRAD) and liver hepatocellular carcinoma (LIHC). Conversely, downregulation of RAMP1 was observed in bladder urothelial carcinoma (BLCA) and uterine corpus endometrial carcinoma (UCEC) (1A and 1B). High RAMP1 expression levels were associated with poor clinical outcomes in rectal adenocarcinoma (READ) (1C-1E).

Fig 2

Correlation between RAMP1 gene expression and immune checkpoint molecules in pan-cancer studies (2A); various immune cell populations infiltrating the tumor microenvironment (2B); and immune scores, including ESTIMATE Score, Immune Score, and Stromal Score (2C-2E).

Fig 3. Prognostic value of RAMP1 in the GSE39058 dataset and Gene Ontology (GO) analysis.

A heatmap of differentially expressed genes (DEGs) showing the top 10 upregulated and downregulated genes (3A). A higher RAMP1 gene expression is substantially related to a poorer prognosis in the GSE39058 dataset (p = 0.021) (3B). GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to determine the GO terms and pathways (3C) and KEGG terms (3D) associated with the DEGs. A protein-protein interaction (PPI) network of RAMP1 was generated using GeneMANIA (3E). Gene Set Enrichment Analysis (GSEA) identified significantly enriched signaling pathways (3F).

Fig 4. Prognostic value of RAMP1.

High expression of RAMP1 correlates with poor prognosis (p < 0.001) (4A). The AUC value for 1‐, 3‐, and 5‐year survival were 0.824, 0.762, and 0.747, respectively (4B). The univariate (95% confidence interval [CI]  =  1.256–2.067, p < 0.001) (4C) and multivariable (95% CI  =  1.233–2.079, p < 0.001) (4D) analyses showed that RAMP1 was a significant independent prognostic risk factor. A nomogram (4E) and the calibration curve of the nomogram (4G) were constructed to predict the 1-, 3-, and 5-year survival of each patient. The prognosis of OS patients with metastasis was substantially unfavorable in the group with high RAMP1 gene expression compared to the group with low expression (4F).

Fig 5. Differential gene expression, GO, KEGG, and GSEA analyses in Therapeutically Applicable Research to Generate (TARGET).

A heatmap depicting the top 10 upregulated and downregulated genes (5A). The KEGG analysis showed that protein digestion and absorption, the Hippo signaling pathway, and the Wnt signaling pathway were significantly enriched (5B). Genes involved significantly in biological process (BP): extracellular matrix organization (GO:0030198), molecular function (MF): histone deacetylase binding (GO:0042826), and cellular component (CC): collagen-containing extracellular matrix (GO:0062023) are shown in a circle graph (5C). The GSEA analysis revealed that these DEGs were predominantly enriched in olfactory transduction pathways (normalized enrichment scores [NES] = 1.6998, p < 0.0001) (5D).

Fig 6. Correlation between RAMP1 and its co-expressed genes.

The top five co-expressed genes that were upregulated or downregulated by the RAMP1 gene were identified using Pearson correlation analysis (6A). Upregulation and downregulation are shown by the red and green curves, respectively. The top five upregulated genes (CGREF1, LTK, RARRES1, RHBDL2, and WIF1) (6B - 6F) and downregulated genes (ACTN1, ARL4C, ITGA11, DNM1, and POSTN) (6G - 6K).

Fig 7. Correlation between RAMP1 expression and tumor-infiltrating immune cells.

CD4+ memory-activated T cells and gamma-delta T cells varied considerably between the high and low RAMP1 expression groups (7A). Gamma-delta T cells, activated mast cells, and RAMP1 RNA levels were positively correlated (r > 0, p < 0.001) (7B and 7C), whereas memory B cells were negatively correlated with RAMP1 mRNA levels (r = -0.26, p = 0.017) (7D). The low RAMP1 expression group had significantly higher stromal scores than the high RAMP1 expression group (**p < 0.01) (7E). The correlation between the RAMP1 gene and immune checkpoint genes was presented (7F). As determined by real-time RT-PCR, the expression of the RAMP1 gene was higher in OS cell lines than in hFOB1.19 osteoblast cell lines (7G). RAMP1 expression was significantly associated with immunological markers in several immune cells, including CD163, VSIG4 (M2 macrophage), and ITGAM (neutrophil) (p < 0.05) (7H - 7J).