Association of Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4) Genetic Variants with Risk and Outcome of Cutaneous Melanoma

Abstract

This study aimed to verify whether germline single nucleotide variants (SNV) in CTLA-4 gene, c.-1765C>T, c.-1661A>G, c.-1577G>A, and c.-1478G>A, influence the risk, clinicopathological aspects, and survival of patients with CM, as well as its functional consequences. A total of 432 patients with CM and 504 controls were evaluated. CTLA-4 genotypes were identified by real-time polymerase chain reaction (RT-PCR) and expression of CTLA-4 by quantitative PCR (qPCR) and luciferase assay. Cell cycle, proliferation, apoptosis/necrosis, and migration analyses were performed in SK-MEL-28 and A-375 cell lines modified to present homozygous ancestral or variant genotypes by CRISPR technique. Individuals with the CTLA-4 c.-1577 AA genotype and the combined CTLA-4 c.-1577 and c.-1478 AA + AA genotypes were at 1.60- and 3.12-fold higher risk of developing CM, respectively. The CTLA-4 c.-1577 AA genotype was seen as an independent predictor of worse event-free survival and was also associated with higher gene expression, higher cell proliferation, lower cell apoptosis, and higher cell migration. Our data present, for the first time, evidence that CTLA-4 c.-1577G>A alters the risk and clinical aspects of CM treated with conventional procedures and may be used for selecting individuals for tumor prevention and patients for distinct treatment.

Keywords: CTLA-4; cutaneous melanoma; prognosis; risk; single nucleotide variants.

Figure 1

Signaling pathway of CTLA-4. The dendritic cell identifies antigens in the microenvironment and, with the help of the major histocompatibility complex (MHC), presents these antigens to inactive T lymphocytes, initiating the activation process (A). The first step occurs when the MHC binds to the T-cell receptor (TCR). After this binding, surface proteins of the CD80/86 family on dendritic cells bind to the CD28 receptor on T lymphocytes, promoting increased cell proliferation, enhancing cytokine production, and combating tumor melanocytes. During activation, CTLA-4, initially stored in vesicles within the cytoplasm, is released, becomes a receptor, and binds with higher affinity than CD28 to the CD80/86 family proteins (B). This binding leads to the inactivation and apoptosis of T lymphocytes, allowing tumor melanocytes to survive, as lymphocytes do not target them, blocking T-lymphocyte activation from the binding of melanocytes to antigen-presenting cells (C). By binding to tumor melanocytes, the dendritic cell prevents the antigen from being presented to T lymphocytes, thus inhibiting T-lymphocyte activation. As a result, the tumor melanocyte evades the immune system’s response.

Figure 2

Functional analyses of the ancestral and variant genotypes of the CTLA-4 c.-1577G>A. Analysis of CTLA-4 gene expression in peripheral blood of patients with cutaneous melanoma (A). Gene expression was higher in patients with AA genotype than in those with GG genotype. Relative luciferase activity in SK-MEL-28 and A-375 melanoma cell lines transfected with the ancestral plasmid (GG genotype) or with the variant plasmid (AA genotype) (B). Luciferase activity was higher in cells with AA genotype than in cells with GG genotype. Assessment of the cell cycle in strains modified to present ancestral and variant genotypes (C). Cells were identified in the G1, S, and G2 phases using flow cytometry. A higher percentage of SK-MEL-28 cells with the GG genotype was found in the G1 phase compared to those with the AA genotype *, and a higher percentage of SK-MEL-28 cells with the AA genotype was found in S phase compared to those with GG genotype **; a similar percentage of A-375 cells were seen in the G1, S, and G2 phases. Cell proliferation in SK-MEL-28 and A-375 melanoma cell lines (D). A higher percentage of SK-MEL-28 and A-375 cells with AA genotype was found in proliferation when compared to those with the GG genotype. Analysis of the assessment of apoptosis and necrosis by flow cytometry with stimulation of the immunotherapy drug ipilimumab (E). A higher percentage of SK-MEL-28 cells with GG genotype was found in necrosis compared to those with the AA genotype *; a higher percentage of cells with the AA genotype were alive compared to those with the GG genotype **; the A-375 cells with the GG genotype were in initial apoptosis when compared to cells with the AA genotype.

Figure 3

Transcription factor binding sites for the CTLA-4 1577G>A (rs11571316) single-nucleotide polymorphism (SNP). Binding of the transcription factor POUPF2 in the 3′-5′ direction of the CTLA-4 gene (A). Binding of the transcription factor HMGA1 in the 3′-5′ direction of the CTLA-4 gene (B). The gray square represents the SNP alleles.

Figure 4

Cell migration by wound healing assay in melanoma cell lines SK-MEL-28 (A) and A-375 (B). Cells with the AA genotype of the CTLA-4 c.-1577G>A single nucleotide variant showed a higher percentage of wound closure after 16 h.