Divisive influence of interleukin-1 receptor antagonist polymorphisms in melanoma patients

Abstract

OBJECTIVE:

The interleukin-1 receptor antagonist (IL-1RA) contributes to tumor survival and progression in multiple cancer entities. IL-1RA polymorphisms influence IL-1RA expression patterns and function. A known polymorphism was correlated with clinical outcomes in melanoma patients with particularly aggressive disease.

METHODS:

DNA of 343 controls and 97 melanoma patients with poor prognostic indicators (time from diagnosis to death, nodal status, metastasis) was analyzed for a variable number of tandem repeat polymorphisms (VNTR) of the IL-1RA gene. Five alleles containing two (allele 2), three (allele 4), four (allele 1), five (allele 3) or six (allele 5) 86-bp repeats were targeted via PCR amplification.

RESULTS:

Genotype 1/2 is less common in the melanoma patient group vs. the control (28.8% vs. 39.6%; p = 0.06). Significant was the stage of the melanoma in order to predict the survivability (p = 0.008). The 1/1 and 1/2 genotype appeared to have lower hazards ratios than the 2/2 genotype (p > 0.05).

CONCLUSIONS:

Compared to the general population, the distribution of alleles coding for IL-1RA is different in melanoma patients. This alteration and the potential impact on tumor protein function and systemic inflammatory response may warrant further investigation.

Keywords

Melanoma interleukin-1 receptor antagonist IL-1RA polymorphism inflammation

Abbreviations

IL-1RA

Interleukin-1 receptor antagonist

VNTR

Variable tandem number repeat

SNP

Single nucleotide polymorphism

VEGF

Vascular endothelial growth factor

TNF

Tumor necrosis factor

CSF

Colony-stimulating growth factors

Base pairs

Kilo base

1 Introduction

Melanoma is the most frequent cause of death from skin cancer. Despite prevention campaigns and exhaustive efforts to increase early detection, 20–25% of all melanoma patients will eventually die from the disease [1]. Once melanoma has spread to regional lymph nodes, mortality increases to 50–70%, and after dissemination to visceral organs, only few patients can be cured [2]. It is, therefore, important to enhance diagnostic tests and treatment modalities for this malignancy and to improve its prognosis by timely detection and treatment.

The tumor microenvironment consists of tumor cells, stromal and inflammatory cells, all of which produce a plethora of cytokines, chemokines, growth factors and adhesion molecules. Interestingly, approximately 15% of all cancers can be attributed to inflammatory etiologies linking chronic inflammation to tumorigenesis [3, 4]. Additionally, disturbed pH-gradients with particularly low extracellular pH seem to largely contribute to the immune escape mechanisms tumors use.

A physiologic inflammatory response normally occurs upon tissue injury due to mechanical stimuli, infectious agents or chemical irritation. Chronic pathological inflammation is mediated via the continuing presence of a stimulus, such as malignant melanoma cells. The resulting prolonged inflammatory cytokine or chemokine exposure has the potential to promote tumor growth through the induction of angiogenesis, DNA damage, and other events favorable to tumor invasion and spread [5]. Signaling molecules induced by hypoxia, a hallmark of progressive cancers, include vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF), IL-6 and, of our interest, IL-1 [4, 5].

Interleukin 1 (IL-1) is a pro-inflammatory cytokine, which is produced by monocytes, macrophages, neutrophils, and epithelial cells [6]. IL-1 has effects in both physiological and pathological states, including the stimulation of expression pattern of metastatic and angiogenic genes and growth factors [7, 8]. The interleukin 1 family comprises 11 different cytokines encoded by three genes on the long arm of chromosome 2q14.22, mapping a region of 430 kb. This region encodes IL-1 α, IL-1 ß, and the IL-1 receptor antagonist (IL-1RA) [9 –11].

All three (IL-1 α, IL-1 ß, and IL-1RA) molecules bind to IL-1 receptors [12 –14]. While IL-1 α and IL-1 ß are potent pro-inflammatory cytokines, IL-1RA is an anti-inflammatory cytokine that competes with IL-1 α and IL-1ß in binding to IL-1 receptors, without exerting an intrinsic effect [15, 16]. Within the tumor microenvironment, high IL-1 concentrations are associated with more aggressive tumor phenotypes. The exact mechanism through which IL-1 exerts its proliferative and angiogenic effects remains unknown.

Several studies have shown that certain polymorphic genes possessing specific alleles are associated with the prevalence and severity of a number of diseases [17]. It is estimated that there are approximately 200,000 single-nucleotide polymorphisms within the coding regions of the greater than 80,000 distinct protein-coding human gene isoforms with a population allele frequency of at least 1% [17, 18]. Another type of common polymorphisms are variable number tandem repeats (VNTRs) [19]. Such variations may affect the rate of gene transcription, mRNA-stability as well as the quantity and activity of the resulting protein. Polymorphisms have been reported in all three IL-1 genes. The polymorphisms of IL-1, IL-1ß, and IL-1RA produce alterations of IL-1, IL-1ß, and IL-1RA protein expression, and may have crucial effects on oncogenic processes [12 , 20–23].

Based on these reports, we hypothesized that secondary to a genetic predisposition, “hypersecreting” alleles may lead to overproduction of the protein and thereby decrease the inflammatory response and malignant potential of melanoma [24]. Certain allelic polymorphisms would therefore be protective of and underrepresented in metastatic melanoma patients compared to patients with less aggressive disease, while other polymorphisms may predispose the patient to a more virulent course of disease.

2 Methods

The COPATH system of the Yale pathology department was used to identify frozen tissue samples of patients. The patient cohort included 432 primary melanoma cases with clinical and pathological analysis and seven-year average follow-up (Yale Tissue Microarray YTMA59). The samples and data contained in the repository were coded with a separate password protected file for the linking code. All tissue arrays are stored in the Yale Cancer Center/Pathology Tissue Microarray Facility (http://tissuearray.org/yale). Information about the subject-donors was maintained in a password-protected computer and password-protected data files. Yale University Human Investigation Committee (HIC) approved the study.

To perform DNA extraction, a DNeasy ^TM Kit (Qiagen, U.S.A.) was used following the manufacturer’s recommendation. The sequence of the forward primer to amplify the region of interest was 5’-CTCAGCAACACTCCTAT-3’ and the reverse primer sequence was 5’-TCCTGGTCTGCAGGTAA-3’ [25]. The amplification products were visualized using gel-electrophoresis on a 3% agarose gel and stained with SYBR Green I (FMC B Bio Products Europe Vallensbaek Strand, Denmark) (Figs. 1 and 2). Sequencing of the primer region was performed to assure that precisely the area coding for IL-1RA was amplified, not including unwanted nucleotide areas. Allele distributions were compared to a healthy control group of 343 patients. These patients had no past medical history or family history of cancer or skin cancers including melanoma, and were seen by their physician for routine medical examinations.

Fig.1

Illustration of gel-electrophoresis product with base pair (bp) ladder and bands corresponding to the different alleles/bp-repetitions.

Fig.2

Photograph of actual gel representing different alleles.

Out of the 432 melanoma patients, genomic DNA from 97 patients was extracted and then analyzed for the VNTR polymorphism in the IL-1RA gene using PCR single strand-conformation polymorphism analysis (SSCP). The 97 patients were chosen based on apparently aggressive disease courses, including either stage III or higher at initial presentation, age <30 years, or survival <6 months.

Table 1 reflects both the individual patient’s allele distribution as well as the overall allele frequencies. SSCP is the most suitable method to detect mutations in short stretches of DNA, the conformations of which is based on the primary nucleotide sequence. Given the ability of single-stranded DNA molecules to fold into unique secondary structures, changes in the nucleotide sequence, secondary to a polymorphism or a mutation, will alter the secondary structure of the molecule resulting and as such the mobility through a non-denaturing polyacrylamide gel. This aberrant migration pattern can visualize the presence of DNA sequence alterations. SSCP has been very successful and widely used to detect disease-causing mutations and polymorphisms [26]. The VNTR polymorphism in the IL-1RA gene shows five alleles containing two (allele 2), three (allele 4), four (allele 1), five (allele 3) or six (allele 5) 86-bp repeats, located in the intron 2 region of the IL-1RA gene.

Table 1

Overall frequencies of each allel

Allel	Melanoma patients #	Melanoma patients %	Control population	Control population
l	142	73.20%	492	71.72% NS
2	46	23.71%	194	28.28% NS
3	2	1.03%	0	0.00% NS
4	4	2.06%	0	0.00% NS

The study was performed according to the ethical guidelines of Clinical Hemorheology and Microcirculation [27]. Groups were compared using chi-squared analysis. Length of survival using a Cox proportional hazards model (Fig. 4).

Fig.3

Graphic depiction of expression of different genotypes.

Fig.4

Survival curves evaluating allele 1/2 vs. all other alleles, showing no significant difference.

3 Results

For both melanoma patients and the control group, genotypes were recorded and allele frequencies calculated for each of the genotypes (Fig. 3), as well as for each individual allele (Table 1). The level of statistical significant was defined as p < 0.05.

A statistical difference was found only when comparing genotype 1/2 to the control population with 28.8% vs. 39.6%, (Pearson Chi-Square p = 0.06) (Fig. 3). No statistical significant difference was found for either other genotypes or for the overall allele distribution.

Survival curves were generated to evaluate for correlation of certain genotypes with patient demographics, prognostic factors (sun exposure, family history, age at time of diagnosis, Breslow depth, stage, metastasis). Controlling for age and sex, only stage was a significant predictor of survival times (p = 0.008). The 1/1 and 1/2 genotypes appeared to have lower hazards ratios than the 2/2 genotype (p > 0.05). The genotypes were not significantly associated with increased or decreased survival.

4 Discussion

The effects of IL-1 and IL-1RA on various cancers including melanoma have been proven in the past. However, these data mainly stem from animal and in-vitro models. Voronov et al. showed that IL-1 knock-out mice failed to develop solid tumors post injection of melanoma cells and exhibited significantly improved survival compared with wild type animals [28]. Saijo and Sawai et al. proofed that overexpression of IL-1 is associated with an aggressive tumor behavior, while other studies have shown that IL-1RA may in fact be protective of tumor growth or may at least help to slow down its aggressive growth pattern [6, 29]. Weinreich et al. transduced xenografts with IL-1RA and showed decreased tumor growth and metastases in murine models, and Elaraj et al. where able to decrease tumor proliferation rate, metastases and IL-8/VEGF mRNA pattern using the same model [30, 31]. Wu T. et al. demonstrated that targeting of IL-1 could interrupt oral carcinogenesis by reprogramming the TME [31].

IL-1RA competitively blocks the IL-1 receptor on T-lymphocytes and fibroblasts. Due to its ability to inhibit prostaglandin synthesis within chondrocytes and synovial cells, the IL-1RA drug (Anakinra) is already approved for the treatment of rheumatoid arthritis and has been shown to reverse IL-1 mediated effects in several pathological settings [12]. It is also able to block the IL-1 induced production of colony-stimulating growth factors (CSF) by fibroblasts, lymphocytes, and monocytes in acute and chronic myelogenous leukemias. Using animal models, it was found that IL-1RA inhibited both in vitro and in vivo Vascular Endothelial Growth Factor (VEGF) production in colon-adenocarcinoma. Lower ratios of IL-1RA:IL-1 correlated with higher concentrations of VEGF protein and increased tumor angiogenesis [28]. Al Toub et al. observed high E-cadherin and low IL1-Beta expression by breast cancer cells promoted reorganization of hMSCs into a niche-like formation [33]. The specific effects of IL-1RA on tumor proliferation and metastases via the transduction of human IL-1RA into two human melanoma cell lines (PMEL and SMEL) with differential IL-1 pattern (low vs. high) were illustrated. Wild-type SMEL melanoma cells and SMEL lines transduced with IL-1RA (SMEL/IL-1RA) were mixed in varying ratios ex vivo and then injected into athymic mice. All tested SMEL:SMEL/IL-1RA ratios resulted in significantly decreased tumor cross-sectional areas, thus demonstrating the paracrine effects of constitutively produced IL-1RA. Also, pulmonary metastases in the SMEL/IL-1RA group were significantly fewer than in the null-transduced and wild-type groups, demonstrating the anti-neoplastic properties of IL-1RA [30].

Different studies reported an association of IL-1 gene polymorphisms with gastric, pancreatic, cervix and breast cancer [34 –38]. Specifically, the IL-1RA homozygous mutant 2/2 was associated with shortened disease free and decreased overall survival in breast and vulvar cancer [22, 39].

Given these promising anti-tumor effects of IL-1 blocking agents and IL-1RA, as well as the fact that polymorphisms lead to increased serum levels of IL-1RA, our theory was that certain polymorphisms may exert a protective effect in melanoma patients. We therefore focused on the role of IL-1RA in respect to its gene polymorphism and its potential effects on the pathogenesis and prognosis of melanoma.

In our analysis, the frequency of homozygous 2/2 (9.3%) showed no statistically significant difference from healthy control subjects (8.5%). Unlike hypothesized, we could therefore not link a potential protective effect of the homozygous allele 2/2. However, a difference was found in the 1/2 allele group, which was present in 28.9% of our patients, compared to 39.7% in the control group (p = 0.06). While not statistically significant, to our knowledge, the allele distribution 1/2 has not been reported to differ this much from a control population in any of the evaluated disease entities.

Currently, there exists debate in the literature of how IL-1RA polymorphisms influence protein pattern and serum level of the protein. According to Denis et al., patients with IL-1RA homozygous for allele 2 have an increased production of IL-1RA in the serum and decreased production in saliva. While there exist several reports regarding the link of homozygous pattern of allele 2/2 and its effect on serum levels of the protein, we could not find any such evidence regarding allele 1/2. Since polymorphisms in the IL-1RA gene may have several intrinsic effects, it seems far-fetched to draw any conclusions regarding the potential in-vivo effects and relevance of the lower frequency of allele 1/2 in melanoma patients. However, given the difference in respect to the allele distribution (p = 0.06), we plan to prospectively obtain serum samples of patients pre- as well as postoperatively in respect to serum levels of IL-1, IL-1ß, and IL-1RA to correlate each polymorphism with the actual in-vivo effects on protein pattern.

This evaluation will substantiate the role of IL-1RA in the oncogenesis of melanoma, hence validating the IL-1RA pathway as a potential target of therapeutic intervention. Further, it may possibly impact treatment regimens by identifying patients with biologically more aggressive disease. Correlation in respect to the actual in-vivo effects may help to differentiate high risk from low risk patients based upon genetic testing and potentially alter current treatment regimens.

The influence of IL-1RA polymorphisms on the prevalence of malignant melanoma and its correlation with established clinical prognostic factors was analyzed. Compared to the general population, no statistically significant difference in the distribution of alleles coding for IL-1RA was found. However, given its potential impact on protein function and inflammatory response, it may be warranted to further investigate the systemic effects of the specific alterations.

Conflicts of interest and source of funding

There are no financial or other relationships that might lead to a conflict of interest. This investigation was supported by the Yale University OHSE Grant (“Interleukin-1 Receptor Antagonist Gen-Polymorphism and Melanoma”, P.N. Broer, PI).

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