|Year : 2018 | Volume
| Issue : 1 | Page : 49-53
Distribution of the chemokine receptor-5 gene in Egyptian breast cancer patients
Amal I.A Youssef, Amal A Hassan, Shuzan A Mohammed, Hebat Allah E.M Ahmed
Medical Biochemistry and Head of Molecular Biology Unit, Faculty of Medicine, Benha University, Benha, Egypt
|Date of Submission||03-Oct-2017|
|Date of Acceptance||15-Oct-2017|
|Date of Web Publication||28-Feb-2018|
Hebat Allah E.M Ahmed
El-Shorouq City, Cairo, 11837
Source of Support: None, Conflict of Interest: None
Background Chemokine receptor type-5 (CCR5)-Δ32, a 32-base pair deletion of the C–C CCR5 gene, is associated with slowed human immunodeficiency virus disease progression in heterozygotes and protection against infection in homozygotes between carriers and noncarriers of each genetic variant.
Aim The present study aimed to investigate the frequency of the CCR5-Δ32 mutation in Egyptian breast cancer (BC) patients.
Materials and methods We determined the genotypic frequency of wild and mutant variants of CCR5 in 40 BC patients and 20 healthy individuals using restriction fragment length polymorphism and reverse hybridization.
Results We found the absence of heterozygous and homozygous mutant gene variants in both BC patients and controls.
Conclusion No significant difference in the frequency of CCR5 genotypes and alleles between patients and controls and no association between that gene and BC.
Keywords: breast cancer, chemokine receptor-5, polymorphism
|How to cite this article:|
Youssef AI, Hassan AA, Mohammed SA, Ahmed HE. Distribution of the chemokine receptor-5 gene in Egyptian breast cancer patients. Benha Med J 2018;35:49-53
|How to cite this URL:|
Youssef AI, Hassan AA, Mohammed SA, Ahmed HE. Distribution of the chemokine receptor-5 gene in Egyptian breast cancer patients. Benha Med J [serial online] 2018 [cited 2018 Aug 21];35:49-53. Available from: http://www.bmfj.eg.net/text.asp?2018/35/1/49/226415
| Introduction|| |
Breast cancer (BC) is one of the most common malignant diseases in the world. BC has a highest mortality worldwide among women, accounting for 522 000 deaths in 2012 . Among all malignancies, BC is the most common cancer in Egyptian women. It represents about 38% of all reported cancer cases in Egyptian women, with an average age of 49.6 per 100 000 populations . It has been reported that there is a relationship between inflammation and cancer. Leukocyte infiltration in tumor mediated by chemokine and chemokine receptors is regarded as one of the major factors in tumor proliferation, invasion, and progression . In this case, CCL5 and its chemokine receptor type-5 (CCR5) have markedly been considered in this process .
The C–C CCR5 is related to the superfamily of the seven-transmembrane G-protein-coupled receptors . It interacts with chemokines that mediate the trafficking and function of memory/effector T-lymphocytes, macrophages, and immature dendritic cells toward the sites of inflammation . After its activation with chemokine ligands, CCR5 are rapidly phosphorylated at serine and threonine residues within the C-tail and the third intracellular loop . When bound by their ligands, these receptors can be internalized, impairing the subsequent ability to bind their ligands. Once internalized, these receptors tend to recycle to the cell surface in time. Most chemokines activate more than one receptor subtype and like other chemokine receptors, CCR5 can also bind several chemokines ,. Apart from the role in HIV infection, the CCR5-Δ32 mutation seems to have a role in BC.
It was proved that CCR5 and its ligand CCL5 interaction involved in signaling pathway in BC cell proliferation, cell invasion, metastasis, and angiogenesis ,. One of the most common polymorphism in the CCR5 gene is a 32 bp deletion polymorphism (denoted as delta CCR5 or CCR5-Δ32). CCR5-Δ32 polymorphism leads to a premature stop codon and results in a nonfunctional form of the chemokine receptor that is unable to bind CC chemokine ligands such as CCL5 , and subsequently leading to major defects in the chemotaxis mediated by these ligands . The aim of this study was to determine whether the genetic variant CCR5-Δ32 has a relation to BC susceptibility.
| Materials and methods|| |
Peripheral blood samples were obtained from 20 healthy volunteers free from cancer and 40 women who were clinically and histopathologically diagnosed as BC of different stages. The study was carried out after receiving approval from the ethics committee of Benha Faculty of Medicine and after obtaining informed consent from the included participants, the participants were recruited from the General Surgery Department Faculty of Medicine, Benha University Hospital.
Sample collection and DNA isolation
Peripheral blood samples were collected in a volume of 3 ml and genomic DNA was extracted using 50 ml of lysis solution and 5 ml GENxTRACT resin (ViennaLab Diagnostics, A-1120, Vienna, Austria).
Polymerase chain reaction amplification
Amplification was performed in a single reaction using biotinylated primers (HVD strip kit; HVD Life Science, HVD Vertriebs-Ges.m.b.H., Wurzbachgasse 18, Vienna, Austria). All PCR reagents and DNA templates were kept refrigerated whereas all steps were performed until start of the thermal cycling program, using a G-storm thermocycler.
After getting our target amplified, we went through two assays:
- Reverse hybridization: which was performed at 45°C; in a thermoshaker plate.
- PCR-restriction fragment length polymorphism: which is performed by PCR with appropriate PCR primers  that flank the 32 bp deletion without using restriction endonuclease. The primer set: 5′-AGG TCT TCA TTA CAC CTG CAG C-3′ and 5′-CTT CTC ATT TCG ACA CCG AAG C-3′ were used to amplify a fragment of 169 bp for wild-type and 137 bp for the mutant variant ([Figure 1]). The CCR5-Δ32 variant was detected by electrophoresis on 3% agarose gel and using ethidium bromide staining. PCR was done for 35 cycles, consisting of a 94°C for 3 min as initial denaturation, denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and further extension at 72°C for 5 min.
|Figure 1 PCR results of chemokine receptor type-5 gene on 3% agarose gel. Lane 1: DNA ladder (marker). Lane 2–10: one single 169 bp band in wild-type homozygotes, no mutant type detected.|
Click here to view
The analysis of data was determined by χ2-test and Student’s t-test was used to compare the mean for continuous variables. Microsoft excel 2016 was used. Results were considered significant at P value of less than 0.05.
| Results|| |
In this case–control study, 40 clinically confirmed breast ductal carcinoma cancer patients and 20 unrelated healthy control individuals were analyzed. The mean ages of patients and controls were 49.9±12.84 and 45.9±11.66 years, respectively. The selected characteristics of the cases and controls are presented in [Table 1].
The clinical pathological features of the IDBC group are summarized in [Table 2]. It observed that more than 90% of patients had positive hormonal receptors (estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2-neu receptors). The majority of patients had node-positive and tumor, node, metastasis stage II disease.
Genotype distribution of CCR5-Δ32 polymorphisms are shown in [Table 3]. The absence of mutant allele was observed in our study.
Gel electrophoresis confirmed the absence of a mutant variant in our study as shown in [Figure 1].
| Discussion|| |
Chemokines and chemokine receptors are expressed by many tumor cells, and these molecules can affect both tumor progression and antitumor immune response. Genetic polymorphisms of some chemokine receptors including the CCR5 receptor gene were found to be closely related to cancer development or involved in metastatic process, including BC ,.
Our data did not find any association between CCR5-Δ32 polymorphism and BC as the mutant allele was not found in our participants (neither homozygous nor heterozygous).
These results were in agreement with Salem and Batzer , Martinson et al. , and Voevodin et al.  who reported that CCR5-Δ32 allele was expected to be completely absent among Egyptians from the Sinai, Sudanese, and Yamanis. These findings might not reflect the real frequency of this deletion among the studied populations due to the small size of samples (˂100).
Moreover, this deletion was completely absent to extremely low among individuals from Venezuela, Central and Western Africa, Japanese, Filipino, Korean, Chinese, Brazilian, Indians, and African-Americans .
The distribution of the CCR5-Δ32 allele varies widely with 21.7% in North American Caucasians, 6.9% in Hispanics, 5.8% in African-Americans, 5.3% in Colombia, 2% in North Africa, and 0.6% in Asian-Americans .
In Arabic countries, the frequency of the CCR5-Δ32 polymorphism is very low in these populations. Within the Middle East region, the frequency was found to be 2.8% among Bahrainis, followed by 2.5, 2.4, 2.1, and 0.6% in Lebanese, Iranians, Saudis, and Jordanians, respectively ,,,. Low frequencies have been observed among Kuwaitis, Syrians, and Egyptians from Ismailia (1, 0.6, and 0.5%, respectively) ,.
However, other studies reported an association between the CCR5-Δ32 allele and BC. Degerli et al.  studied Δ32 allele of CCR5 gene in BC and other tumors. They demonstrated that the heterozygote genotype is an independent risk factor for the development of BC.
Guleria et al.  and Aoki et al.  have also found no association for Δ32 allele in relation to BC increased susceptibility in Indian and Turkish populations, respectively.Studies have shown that chemokine receptor systems play an important role in tumor progression . Tumor cells secrete chemotactic factors in a variety of processes, which mediate tumor cell invasion and metastasis through binding with corresponding receptors .
Moreover, Azenshtein et al.  have found that Regulated on Activation, Normal T Expressed and Secreted (RANTES) expression in the CCR5 ligand occurs in breast tissue and BC cell lines T47D and MCF-7. It is also associated with the degree of malignancy of BC and disease course. The higher the degree of malignancy and the later the course of the disease, the higher is the expression of RANTES. These results indicate that RANTES is involved in BC.
Using immunohistochemical methods Tan  has found that high CCR5 mRNA expression in human BC stem cells accompanied stronger invasion and metastasis capabilities than those of BC cells, which may be a key factor in BC metastasis.
In another study Mañes et al.  have found that CCR5 expression is negatively correlated with p53 wild-type gene expression in the progression of breast tumors. CCR5 may be involved in the progression of BC in a p53 gene mutation-mediated process.
| Conclusion|| |
According to the CCR5 gene, the involvement of CCR5 gene polymorphism in human immunodeficiency virus pathogenesis and treatment is currently a focus of attention; however, research on the association of CCR5 gene polymorphism and BC is relatively limited. More studies with a bigger sample size are needed to show the effect of the CCR5 gene in BC.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Bray F, Ren J-S, Masuyer E, Ferlay J. Global estimates of cancer prevalence for 27 sites in the adult population in 2008. Int J Cancer 2013; 132:1133–1145.
Dey S, Soliman AS, Hablas A, Seifeldein IA, Ismail K, Ramadan M et al.
Urban–rural differences in breast cancer incidence in Egypt (1999–2006). Breast 2010; 19:417–423.
Lee S, Margolin K. Cytokines in cancer immunotherapy. Cancers (Basel) 2011; 3:3856–3893.
Balkwill F. Cancer and the chemokine network. Nat Rev Cancer 2004; 4:540–550.
Allen SJ, Crown SE, Handel TM. Chemokine: receptor structure, interactions, and antagonism. Annu Rev Immunol 2007; 25:787–820.
De Oliveira CEC, Oda JMM, Losi Guembarovski R, Oliveira KBD, Ariza CB, Neto JS et al.
CC chemokine receptor 5: the interface of host immunity and cancer. Dis Markers 2014; 2014:126954.
Yang Z, Yang F, Zhang D, Liu Z, Lin A, Liu C et al.
Phosphorylation of G protein-coupled receptors: from the barcode hypothesis to the flute model. Mol Pharmacol 2017; 92:201–210.
Hütter G, Neumann M, Nowak D, Klein S, Klüter H, Hofmann W-K. The effect of the CCR5-delta32 deletion on global gene expression considering immune response and inflammation. J Inflamm (Lond) 2011; 8:29.
Scholten DJ, Canals M, Maussang D, Roumen L, Smit MJ, Wijtmans M et al.
Pharmacological modulation of chemokine receptor function. Br J Pharmacol 2012; 165:1617–1643.
Velasco-Velázquez M, Pestell RG. The CCL5/CCR5 axis promotes metastasis in basal breast cancer. Oncoimmunology 2013; 2:e23660.
Gao D, Rahbar R, Fish EN. CCL5 activation of CCR5 regulates cell metabolism to enhance proliferation of breast cancer cells. Open Biol 2016; 6:6.
Hoppin JA, Tolbert PE, Holly EA, Brock JW, Korrick SA, Altshul LM et al.
Pancreatic cancer and serum organochlorine levels. Cancer Epidemiol Biomarkers Prev 2000; 9:199–205.
Khademi B, Razmkhah M, Erfani N, Gharagozloo M, Ghaderi A. SDF-1
genes polymorphism in patients with head and neck cancer. Pathol Oncol Res 2008; 14:45–50.
Guleria K, Sharma S, Manjari M, Uppal MS, Singh NR, Sambyal V. p.R72P, PIN3 Ins16bp polymorphisms of TP53 and CCR5-Δ32 in north Indian breast cancer patients. Asian Pac J Cancer Prev 2012; 13:3305–3311.
Sandford AJ, Zhu S, Bai TR, FitzGerald JM, Paré PD. The role of the C-C chemokine receptor-5 Δ32 polymorphism in asthma and in the production of regulated on activation, normal T cells expressed and secreted☆. J Allergy Clin Immunol 2001; 108:69–73.
Banin-Hirata BK, Losi-Guembarovski R, Oda JMM, de Oliveira CEC, Campos CZ, Mazzuco TL et al.
CCR2-V64I genetic polymorphism: a possible involvement in HER2+
breast cancer. Clin Exp Med 2016; 16:139–145.
Salem A-H., Batzer MA. Distribution of the HIV resistance CCR5-Δ32 allele among Egyptians and Syrians. Mutat Res 2007; 616:175–180.
Martinson JJ, Chapman NH, Rees DC, Liu Y-T, Clegg JB. Global distribution of the CCR5
gene 32-basepair deletion. Nat Genet 1997; 16:100–103.
Voevodin A, Samilchuk E, Dashti S. Frequencies of SDF-1 chemokine, CCR-5, and CCR-2 chemokine receptor gene alleles conferring resistance to human immunodeficiency virus type 1 and AIDS in Kuwaitis. J Med Virol 1999; 58:54–58.
Al-Jaberi SA, Ben-Salem S, Messedi M, Ayadi F, Al-Gazali L, Ali BR. Determination of the CCR5Δ32 frequency in Emiratis and Tunisians and the screening of the CCR5
gene for novel alleles in Emiratis. Gene 2013; 529:113–118.
Su B, Sun G, Lu D, Xiao J, Hu F, Chakraborty R et al.
Distribution of three HIV-1 resistance-conferring polymorphisms (SDF1-3’A, CCR2-641, and CCR5-delta32) in global populations. Eur J Hum Genet 2000; 8:975–979.
Gharagozloo M, Doroudchi M, Farjadian S, Pezeshki AM, Ghaderi A. The frequency of CCR5Δ32 and CCR2-64I in southern Iranian normal population. Immunol Lett 2005; 96:277–281.
Karam W, Jurjus R, Khoury N, Khansa H, Assad C, Zalloua P, Jurjus A. Frequency of the CCR5-delta 32 chemokine receptor gene mutation in the Lebanese population. East Mediterr Health J 2004; 10:671–675.
Khabour OF, Abu-Haweleh LJ, Alzoubi KH. Distribution of CCR-5 Δ 32, CCR2-64I, and SDF-1-3′A alleles among Jordanians. AIDS Res Hum Retroviruses 2013; 29:151–155.
Salem AH, Farid E, Fadel R, Abu-Hijleh M, Almawi W, Han K, Batzer MA. Distribution of four HIV type 1-resistance polymorphisms (CCR5-Δ 32, CCR5-m303, CCR2-64I, and SDF1-3′A) in the Bahraini population. AIDS Res Hum Retroviruses 2009; 25:973–977.
Degerli N, Yılmaz E, Bardakci F. The Δ32 allele distribution of the CCR5
gene and its relationship with certain cancers in a Turkish population. Clin Biochem 2005; 38:248–252.
Aoki MN, da Silva do Amaral Herrera AC, Amarante MK, do Val Carneiro JL, Fungaro MHP, Watanabe MAE. CCR5 and p53 codon 72 gene polymorphisms: implications in breast cancer development. Int J Mol Med 2009; 23:429–435.
Karnoub AE, Dash AB, Vo AP, Sullivan A, Brooks MW, Bell GW et al.
Mesenchymal stem cells within tumour stroma promote breast cancer metastasis. Nature 2007; 449:557–563.
Azenshtein E, Luboshits G, Shina S, Neumark E, Shahbazian D, Weil M et al.
The CC chemokine RANTES in breast carcinoma progression: regulation of expression and potential mechanisms of promalignant activity. Cancer Res 2002; 62:1093–1102.
Tan AI. Researchers discover new hormone receptors to target when treating breast cancer. Department of Pathology, University of Miami Miller School of Medicine; February 4, 2014.
Mañes S, Mira E, Colomer R, Montero S, Real LM, Gómez-Moutón C et al.
CCR5 expression influences the progression of human breast cancer in a p53-dependent manner. J Exp Med 2003; 198:1381–1389.
[Table 1], [Table 2], [Table 3]