Polymorphisms in the CCR5 promoter associated with cervical intraepithelial neoplasia in a Chinese Han population

C-C motif chemokine receptor type 5 (CCR5), a transmembrane G-coupled cell-surface chemokine receptor, binds to five kinds of CC-chemokines: human macrophage inflammatory protein-1α (MIP-1α), MIP-1β, RANTES (regulated on activation, normal T cell expressed and secreted), monocyte chemotactic protein 2 (MCP-2) and MCP-4 [1, 2]. CCR5 is known to be the principal coreceptor of macrophage-tropic (R5) strains of human immunodeficiency virus-type 1 (HIV-1), which is highly variably expressed on the cell surface of the memory T cells, macrophages, dendritic cells, hematopoietic stem cells, and microglial cells [1, 3].

The human CCR5 gene located on 3p21.31, is composed of four exons and two introns. The promoter region has been described previously [4, 5], and the differences in the promoter region may regulate the expression of CCR5 in monocyte/macrophages and T lymphocytes [5]. CCR5 expression in tumor cells and various host cells plays a very important role in tumor progression [6]. Studies have demonstrated that CCR5 and CCR5 ligands CCL5 might stimulate angiogenesis as growth factors, modulate the recruitment of inflammatory cells, and induce the tumor immune evasion [6, 7]. It has been reported that men with a loss of functional CCR5 and carrying the CCR5 Δ32 mutant are resistant to the development of prostate cancer. Additionally, the expression of the CCR5 ligand CCL5 promotes the migration and invasiveness of pancreatic cancer [8].

Cervical carcinoma is the second most common cancer affecting women worldwide [9]. Approximately 75,000 women develop cervical cancer, and 40,000 women die from this disease in China each year [10]. The development of cervical carcinoma is strongly linked to persistent infection of high-risk human papillomaviruses (HPV), such as HPV16 and HPV18 [11]. Most HPV infections do not cause any symptoms or disease and clear up spontaneously; however, a minority of women exhibit persistent HPV infection, which might lead to cervical intraepithelial neoplasia (CIN) or cervical cancer (CC) [12, 13], which means that the host immune system and genetic background play important roles in the progression of cervical cancer [14]. In recent years, Che et al [15] and Sales et al [16] found that CCR5 expression is extremely higher in cervical cancer tissue than in matched normal control tissue and that downregulation of CCR5 suppresses cervical cancer cell growth and proliferation in vivo [15]. Variability of the promoter region of CCR5 gene might be the reason for differing CCR5 expression levels.

In the present study, we detected the genetic polymorphisms in the promoter region of CCR5 of a Chinese Han population and investigated the association between genetic polymorphisms of CCR5 gene promoter and cervical cancer in Han Chinese.

Tumor development and progression depend heavily on the presence of angiogenesis factors including chemokines and their receptors [22]. There has been evidence that chemokines and their receptors expressed on tumor cells contribute to recruitment and activation of tumor-suppressor cells [6, 23] and recruitment of inflammatory cells to the tumor microenvironment [24], which may induce the process of tumor cell migration, invasion, and metastasis [22, 24‐26]. As one of the key chemokines involved in many pathological processes, including inflammation, angiogenesis, tumor cell growth and infiltration, CCR5 may influence the cervical cancer progression. In the current study, we performed the association study of CCR5 promoter (P₁) polymorphisms with cervical carcinoma and found that the promoter variations of CCR5 might be associated with development of cervical lesions, cervical intraepithelial neoplasia.

Two CCR5 promoters have been previously described: a weak upstream promoter called P_U or P₂, and a stronger downstream promoter called P_D or P₁ [4, 27]. The expression of CCR5 on cell surface is highly variable, and CCR5 promoter polymorphisms may alter the CCR5 cell surface expression, which might consequently influence the disease progression. Previously, several SNPs in the CCR5 promoter have been shown to affect CCR5 expression. Salkowitz et al found that rs1799987 (−2459G > A) located in the P₁ was related to a different expression level of CCR5 on CD14⁺ monocytes, and individuals carrying homozygous rs1799987-G exhibited lower CCR5 density on CD14⁺ monocytes [28]. Mummidi et al [29] reported that rs1799987 was associated with the rate of AIDS progression. CCR5 promoter polymorphisms correlate with HIV disease progression possibly by regulating CCR5 cell surface expression and CD4⁺ T cell apoptosis in HIV patients. In this study, rs1799987-AA significantly increased the risk of susceptibility to CIN, which indicated that rs1799987-AA might be a risk factor for cervical lesions development, possibly by influencing the expression level of CCR5.

CCR5 promoter haplogroups constructed by -2733A/G, −2554G/T, −2459G/A, − 2135 T/C, −2132C/T, −2086A/G, −1835C/T and + 554(Δ32), have been characterized and described before, and CCR5 haplogroups have been named as CCR5-HHA (A-G-G-T-C-A-C-Δ32wild), −HHB (A-T-G-T-C-A-C-Δ32wild), −HHC (A-T-G-T-C-G-C-Δ32wild), −HHD (A-T-G-T-T-A-C-Δ32wild), −HHE (A-G-A-C-C-A-C-Δ32wild), −HHF (A-G-A-C-C-A-T-Δ32wild), −HHG*1 (G-G-A-C-C-A-C-Δ32wild) and -HHG*2 (G-G-A-C-C-A-C-Δ32mutant) [27, 30, 31]. A specific CCR5 promoter haplogroup was demonstrated to correlate with transcriptional activity [27] and affect the rate of AIDS progression [30]. CCR5-HHA haplotype is associated with lower CCR5 expression and protective phenotype in humans, whereas CCR5-HHF or CCR5-HHG is related to higher CCR5 expression [32]. We performed haplotype analysis with 6 polymorphism loci in CCR5 promoter, and only SNP rs2227010 was not comprised in the defined CCR5 haplogroups. Our results showed that haplotype H1: A-T-G-T-G-C, which was similar to CCR5-HHC, was the most-common haplotype in our participants and consisted of the major alleles of these 6 SNPs revealing a protective effect against CIN; and a tendency of susceptibility to CIN appeared in H2 (similar to CCR5-HHE), H3 (similar to CCR5-HHF) and H4 (similar to CCR5-HHA), although these haplotypes did not show statistical association with CIN. The haplotype analysis results coincided with the results of our allelic and genotypic analysis and indicated that the CCR5 promoter haplotype was associated with cervical lesions development. However the correlation between haplotype of 6 SNPs in this study and expression of CCR5 need to test and verify.

In 2016, Che et al indicated that CCR5 mRNA and protein expression levels were extremely higher in cervical cancer tissue than in matched normal control tissue, and downregulation of CCR5 inhibited cervical cancer growth and invasion [15]. Sales et al also presented similar results that the level of CCR5 was high in cervical cancer tissue and the expression of CCR5 could be regulated by inflammatory pathways [16]. However, no previous reports on the CCR5 expression level in CIN tissues were identified. So we performed the transcription factor binding site prediction in the CCR5 promoter and try to find the possible mechanism of association of SNPs to development of cervical lesions. The computer algorithms suggested that there are many binding sites of distinct nuclear and transcription factors in the CCR5 promoter, and the 6 SNPs studied were located in the binding region of specific transcription factors (Fig. 1). Nucleotide substitutions in the cis-regulatory region of a gene may cause a loss of binding of some nuclear proteins, or binding of some novel nuclear factors, or alteration in nuclear factor binding affinity. A previous study found that SNPs in human CCR5 cis-regulatory sequences were associated with alteration in DNA/nuclear factor binding. Mummidi et al found both G and T in − 2554 site (rs2734648) could bind to four nuclear factor complexes (NF4-NF7), but the affinities for NF5-NF4 complexes bound to the -2554 T were significantly greater than the binding affinities to -2554G [27]. Bream et al [33] also found that -2554 T bound p65, c-Rel, and p50 with greater affinity than -2554G. We predicted that rs2734648-T was located in the binding region of FOXP3, TFII-I, C/EBP-beta, NF-AT1 and PR–B. Therefore, the transcriptional status of rs2734648-G, especially rs2734648-GG, might be different. So we infer that rs2734648-GG is significantly associated with the susceptibility of CIN by affecting the transcription factors binding. Mummidi et al also found the wild-type C allele in − 1835 site (rs1800024) specifically bound to two novel nuclear factors NF2 and NF3, but the mutant-T allele lost the ability to bind nuclear factor NF3 [27]. The binding region of transcription factor GR-α contains two polymorphic loci rs1799988 (− 2135 T > C) and rs41469351 (−2132C > T). These two polymorphisms are located so closely, only at a 2-base-pair interval. For there is no polymorphism in rs41469351, so nucleotide substitution in rs1799988 might affect the transcription factor GR-α binding. In addition, Mummidi et al [27] found that CCR5 haplotypes conferred the haplotype-specific differences in the promoter transcriptional efficiency, of which the haplotype HHA showed the least promoter activity, whereas the transcriptional activity of HHF haplotype was the highest. That is to say the haplotype H1, similar to HHC, have a medium promoter activity. And haplotype H1 showed a significant resistance to CIN. So we infer that nucleotide substitutions such as rs2734648-GG, rs1799987-AA, rs1799988-CC and rs1800023 could associated with susceptibility to CIN by alternate nuclear factors binding,, then subsequently affect the efficiency of CCR5 translation and CCR5 expression level on cell surface and, finally, affect the development of cervical lesions. By the way, our results revealed that the susceptibility of SNPs in CCR5 promoter arising more in recessive inheritance model, such as rs2734648, rs1799987, rs1799988 and rs1800023. That is to say individuals carried two mutations in both alleles raise the susceptibility to CIN. This can be understood as the efficiency of nuclear factors binding, CCR5 translation and expression could be affected more when two mutations in both alleles of SNP.

In this study, we found the frequencies of the minor alleles of rs2734648 was significantly higher in the CIN group than that in the control group and the frequencies of rs2734648-GG, rs1799987-AA, rs1799988-CC and rs1800023-AA were distinctly increase the susceptibility of CIN. However, rare associations of susceptible to CC or from CIN progressing to CC were found. It is known that persisting high-risk HPV infection, which usually related to the lack of HPV-specific T-cell immunity and some immune-response pathway [11, 13], is associated with the development of vassal cell layer infection and hyperplastic lesions called cervical intraepithelial neoplasia (CIN). CIN lesions may regress, persist or progress to invasive cervical carcinogenesis [14]. Carcinoma development depends on different strategies to escape recognition by immune cells. So we think the mechanisms of normal cervical tissue progressing to CIN and CIN progressing to CC might be different, and the host immunogenetic factors played in development and progression of cervical cancer need to further study.

In conclusion, we demonstrated that SNPs in the CCR5 promoter are significantly associated with development of cervical intraepithelial neoplasia. These polymorphisms could potentially influence not only the CCR5 gene expression but also the CCR5 mRNA stability and the translation efficiency, which subsequently influence the density of CCR5 on cell-surface and, thus, have an impact on virus infection or disease pathogenesis. However, there might be some other mechanism of cervical cancer development, because our results indicated that polymorphisms in the CCR5 promoter are not associated with progression from CIN to CC. Thus, sample size expansion for association study and the functional study on correlation of CCR5 polymorphisms with cervical intraepithelial neoplasia development, and finding other immunogenetic factors associated with cervical carcinoma will be performed in the future.