Original Article Correlation Between LSP1 Polymorphisms and the Susceptibility to Breast Cancer

Int J Clin Exp Pathol 2015;8(5):5798-5802 www.ijcep.com /ISSN:1936-2625/IJCEP0005835

Original Article Correlation between LSP1 polymorphisms and the susceptibility to breast cancer

Hai Chen, Xiaodong Qi, Ping Qiu, Jiali Zhao

Department of Galactophore, The General Hospital of Beijing Military Command, Beijing, China Received January 12, 2015; Accepted March 16, 2015; Epub May 1, 2015; Published May 15, 2015

Abstract: Objective: The present study aimed at assessing the relationship between Leukocyte-specific protein 1 gene (LSP1) polymorphisms (rs569550 and rs592373) and the pathogenesis of breast cancer (BC). Methods: 70 BC patients and 72 healthy subjects were enrolled in the study. Rs569550 and rs592373 polymorphisms were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Odds ratio (OR) with 95% confidence interval (CI) were calculated by the chi-squared test to assess the relationship between LSP1 polymorphisms and BC risk. Linkage disequilibrium (LD) and haplotypes were also analyzed by HaploView software. Results: Genotype distribution of the control was in accordance with Hardy-Weinberg equilibrium (HWE). The homozygous genotype TT and T allele of rs569550 could significantly increase the risk of BC (TT vs. GG: OR=3.17, 95% CI=1.23-8.91; T vs. G: OR=1.63, 95% CI=1.01-2.64). For rs592373, mutation homozygous genotype CC and C allele were significantly associated with BC susceptibility (CC vs. TT: OR=4.45, 95% CI=1.38-14.8; C vs. T: OR=1.70, 95% CI=1.03-2.81). LD and haplotypes analysis of rs569550 and rs592373 polymorphisms showed that T-C haplotype was a risk factor for BC (T-C vs. G-T: OR=1.74, 95% CI=1.04-2.92). Conclusion: LSP1 rs569550 and rs592373 polymorphisms are both risk factors for BC.

Keywords: LSP1, breast cancer, polymorphisms

Introduction demonstrated that LSP1 is related with many diseases, such as HIV, inflammatory diseases, Breast cancer (BC) is the most frequency diag- hepatocellular carcinoma and so on [11-13]. So nosed cancer and the leading cause of cancer- far, the relationship between LSP1 polymor- related deaths among the women [1]. The phisms and the development of BC was hardly occurrence of BC is still clearly increasing all reported. over the world [2]. The previous studies have shown that the occurrence and development of So a case-control study was carried out to eval- BC were a complex interaction between genes uate the relevance of LSP1 rs569550 (T>G) and environment [3, 4]. Only a small number of and rs592373 (T>C) polymorphisms with the individuals who expose to the same environ- risk of BC. ment develop BC, so genes play vital roles in Material and methods the occurrence of BC. But the tumorigenesis mechanism of BC is still unclear [5]. In recent Objects of study years, a number of studies have reported that some gene polymorphisms could influence the Total 70 female cases with BC in the study were susceptibility to BC, such as CCND1 rs9344, outpatients and inpatients from The General Hif-1α rs11549465, LEP G-2548A, LEPR Q2- Hospital of Beijing Military Command. They 23R and so on [6-9]. Leukocyte-specific protein were unrelated with each other and belonged to 1 (LSP1) is an F-actin binding protein specially Chinese Han population. The cases were con- expressed in endothelial cells and hematopoi- firmed by pathological diagnosis. The BC-free etic lineage. LSP1 gene is located on 11p15.5 group was consisted of 72 healthy women from and contains 20 exons [10, 11]. It has been the examination center at the same period as LSP1 polymorphisms and breast cancer

Table 1. Primers sequences and amplified lengths of µL primers (1 µL upstream and 1 µL down- rs569550 and rs592373 stream), 25 µL Master Mix, and 21 µL of Primer Primer sequence ddH2O. The reaction program was as fol- rs569550 Former: 5’-CTGTCACCTGCTCACACCTC-3’ lows: initial degeneration at 95°C for 3 minutes, followed by 36 cycles with degenera- Reverse: 5’-GGAGAGGTGTGAGCAGGTGA-3’ tion at 94°C for 30 seconds, annealing (at rs592373 Former: 5’-GTGCTCCAAAGACGGGCGGT-3’ 57-60°C ) for 30 seconds, and extension at Reverse: 5’-CTCCTACCGCCCGTCTTTGG-3’ 72°C for 40 seconds, the final extension at 72°C for 5 minutes. Then PCR products Table 2. Essential features of cases and controls were digested by NcoI restriction enzyme. All digested products were detected by 2% Essential feature Cases n (%) Controls n (%) P value agarose gel electrophoresis. Age (mean age) 53.8±10.8 53.4±12.2 0.86 ≤50 22 (31.4) 21 (29.2) Statistical methods >50 48 (68.6) 51 (70.8) Smoking 0.87 SPSS18.0 software was used for statistical yes 31 (44.3) 30 (41.7) analysis. Genotype distribution of the con- no 39 (55.7) 42 (58.3) trol group was tested by Hardy-Weinberg Family history 0.00 equilibrium (HWE). The odds ratio (ORs) and 95% confidence intervals (CIs) were yes 45 (64.3) 16 (22.2) calculated by chi-squared test to evaluate no 25 (35.7) 56 (77.8) the association of LSP1 rs569550 and Abortion history 0.43 rs592373 polymorphisms with BC suscep- yes 19 (27.1) 15 (20.8) tibility. Linkage disequilibrium (LD) was no 51 (72.9) 57 (79.2) analyzed to assess the combined effects of Menopause 0.02 the two polymorphisms on the occurrence yes 43 (61.4) 30 (42.9) of BC in the HaploView software. P<0.05 no 27 (38.6) 42 (57.1) represents the difference with statistical significance. controls. The controls were frequency-matched Results with cases by age and race and they all had no family history of breast diseases. The study Essential features of study objects was approved by the Research Ethics Com- mittee of the hospital. There were no significant differences between cases and controls in such factors as age, DNA extraction smoking and abortion history (P>0.05 for all). While, the differences were statistically sig Genome DNA of peripheral venous blood was nificant between two groups in family history extracted from all the objects using traditional and menopause history (P<0.05 for both) (Table 2). phenol chloroform method and stored in -20°C refrigerator. Association between LSP1 rs569550 and rs592373 polymorphisms and the risk of BC Genotyping of LSP1 polymorphisms The feasible association of LSP1 polymor- Polymerase chain reaction-restriction fragment phisms with BC susceptibility was showed length of polymorphism (PCR-RFLP) was used in Table 3. The homozygous genotype TT and to detect LSP1 polymorphisms (rs569550 and allele T of LSP1 rs569550 significantly inc- rs592373). PCR primers were showed in Table reased the risk of BC (TT vs. GG: OR=3.17, 95% 1. The primers were synthesized by Shanghai CI=1.23-8.91; T vs. G: OR=1.63, 95% CI=1.01- SANGON Biological Engineering Technology 2.64). For rs592373 polymorphism, CC geno- Co., Ltd. PCR reaction system was a volume of type and C allele were both genetic-susceptibil- 50 µL solution, including 2 µL DNA template, 2 ity factors for BC (CC vs. TT: OR=4.45, 95%

5799 Int J Clin Exp Pathol 2015;8(5):5798-5802 LSP1 polymorphisms and breast cancer

Table 3. Genotypes and alleles distribution of LSP1 rs569550 and ing was related with high rs592373 polymorphisms risk of BC [19]. While, sev- Genotype Cases n (% ) Controls n (% ) P OR (95% CI) eral studies suggested that isoflavone could reduce the rs569550 occurrence of BC [20]. Until GG 23 (32.9) 30 (41.7) - 1.00 (Ref.) now, the pathogenesis of GT 30 (42.9) 35 (48.6) 0.77 1.12 (0.54-2.32) BC is still unclear. TT 17 (24.2) 7 (9.7) 0.03 3.17 (1.23-8.91) G 76 (54.3) 95 (66.0) - 1.00 (Ref.) In recent years, many stud- T 64 (45.7) 49 (34.0) 0.04 1.63 (1.01-2.64) ies found important roles of rs592373 genetic factors in the occur- TT 32 (45.7) 38 (52.8) - 1.00 (Ref.) rence of BC. The study by Sun X et al. showed that TC 23 (31.9) 30 (41.7) 0.80 0.91 (0.44-1.87) miR-21 and miR-200c were CC 15 (22.4) 4 (5.5) 0.01 4.45 (1.34-14.8) related with the onset of BC T 87 (55) 106 (73.6) - 1.00 (Ref.) [21]. In addition, MTHFR C 53 (45) 38 (26.4) 0.04 1.70 (1.03-2.81) rs1801133 polymorphism also could increase the risk for BC [22]. Moreover, the Table 4. Relationship between haplotypes of LSP1 rs569550 and study of Wang J et al. sh- rs592373 polymorphisms and BC susceptibility owed that Pin1 might serve Haplotype Cases 2n=140 (%) Controls 2n=144(%) P OR (95% CI) as a positive diagnosis bio- G-T 76 (15.0) 95 (6.9) - 1.00 (Ref.) marker for breast cancer T-C 53 (45.0) 38 (18.1) 0.03 1.74 (1.04-2.92) [23]. Meanwhile, the func- T-T 11 (40.0) 11 (75.0) 0.62 1.25 (0.51-3.04) tion of LSP1 gene on the Total 140 (100.0) 144 (100.0) - - occurrence of BC has attracted a lot of attention.

CI=1.38-14.8; C vs. T: OR=1.70, 95% CI= LSP1 is mainly expressed in lymphocytes, neu- 1.03-2.81). trophils, macrophages and endothelium. It also regulates neutrophil movements, the adhesion LD test and haplotype analysis of fibrinogen matrix proteins and the traverse of endothelial cell layers. In other words, LSP1 The analysis of pairwise LD between rs569550 plays a regulatory role in the activation and che- and rs592373 suggested that there was nota- motaxis of neutrophils. It has been reported 2 ble linkage disequilibrium (D’=0.86, r =0.67) that LSP1 rs3817198 polymorphism could and two loci formed three haplotypes of T-T, T-C affect the susceptibility to breast cancer [24, and G-T. The heterozygous haplotype T-C signifi- 25]. But rs592373 and rs569550 polymor- cantly increased the risk of BC (OR=1.74, 95% phisms are hardly reported. CI=1.04-2.92) (Table 4). So the T-C haplotype might be a risk factor for BC. The present study investigated the association Discussion of LSP1 polymorphisms with BC risk and the results showed that there were significant dif- BC is the most common cancer with highest ferences in the distribution of TT genotype and morbidity and mortality, cases in China account- T allele in rs569550 polymorphism as well as ing for 12.2% and 9.6% of all new diagnosed BC CC genotype and C allele in rs592373 between and deaths from BC in the world, respectively cases and controls, which were significantly [14]. In Asia, the morbidity shows a significant associated with BC risk. The haplotype analysis rising trend over the years [15]. Moreover, its showed that T-C haplotype could increase the incidence rate varies due to the difference of risk for BC. races and geographical conditions [16]. Studies also found that long-term exposure to insecti- In conclusion, LSP1 rs569550 and rs592373 cides or pesticides increased the risk of BC [17, polymorphisms may be closely correlated with 18]. Izano M et al. reported that current smok- the occurrence of BC. Further study with well-

5800 Int J Clin Exp Pathol 2015;8(5):5798-5802 LSP1 polymorphisms and breast cancer design and enough large sample size is needed Morrison J, Field H, Luben R, Wareham N, to confirm the correlation and some environ- Ahmed S, Healey CS, Bowman R, Meyer KB, mental factors should be considered at the Haiman CA, Kolonel LK, Henderson BE, Le same time. Marchand L, Brennan P, Sangrajrang S, Gaborieau V, Odefrey F, Shen CY, Wu PE, Wang HC, Eccles D, Evans DG, Peto J, Fletcher O, Disclosure of conflict of interest Johnson N, Seal S, Stratton MR, Rahman N, Chenevix-Trench G, Bojesen SE, Nordestgaard None. BG, Axelsson CK, Garcia-Closas M, Brinton L, Chanock S, Lissowska J, Peplonska B, Address correspondence to: Dr. Hai Chen, De- Nevanlinna H, Fagerholm R, Eerola H, Kang D, partment of Galactophore, The General Hospital of Yoo KY, Noh DY, Ahn SH, Hunter DJ, Hankinson Beijing Military Command, Beijing, China. E-mail: SE, Cox DG, Hall P, Wedren S, Liu J, Low YL, [email protected] Bogdanova N, Schurmann P, Dork T, Tollenaar RA, Jacobi CE, Devilee P, Klijn JG, Sigurdson AJ, References Doody MM, Alexander BH, Zhang J, Cox A, Brock IW, MacPherson G, Reed MW, Couch FJ, [1] Jemal A, Bray F, Center MM, Ferlay J, Ward E Goode EL, Olson JE, Meijers-Heijboer H, van and Forman D. Global cancer statistics. CA den Ouweland A, Uitterlinden A, Rivadeneira F, Cancer J Clin 2011; 61: 69-90. Milne RL, Ribas G, Gonzalez-Neira A, Benitez J, [2] Porter P. “Westernizing” women’s risks? Breast Hopper JL, McCredie M, Southey M, Giles GG, cancer in lower-income countries. N Engl J Schroen C, Justenhoven C, Brauch H, Hamann Med 2008; 358: 213-216. U, Ko YD, Spurdle AB, Beesley J, Chen X, [3] Lichtenstein P, Holm NV, Verkasalo PK, Iliadou Mannermaa A, Kosma VM, Kataja V, A, Kaprio J, Koskenvuo M, Pukkala E, Skytthe A Hartikainen J, Day NE, Cox DR and Ponder BA. and Hemminki K. Environmental and heritable Genome-wide association study identifies nov- factors in the causation of cancer--analyses of el breast cancer susceptibility loci. Nature cohorts of twins from Sweden, Denmark, and 2007; 447: 1087-1093. Finland. N Engl J Med 2000; 343: 78-85. [11] Jongstra-Bilen J and Jongstra J. Leukocyte- [4] Xi J, Su Y, Fadiel AB, Lin Y, Su FX, Jia WH, Tang specific protein 1 (LSP1): a regulator of leuko- LY and Ren ZF. Association of physical activity cyte emigration in inflammation. Immunol Res and polymorphisms in FGFR2 and DNA meth- 2006; 35: 65-74. ylation related genes with breast cancer risk. [12] Smith AL, Ganesh L, Leung K, Jongstra-Bilen J, Cancer Epidemiol 2014; 38: 708-714. Jongstra J and Nabel GJ. Leukocyte-specific [5] Njiaju UO and Olopade OI. Genetic determi- protein 1 interacts with DC-SIGN and mediates nants of breast cancer risk: a review of current transport of HIV to the proteasome in dendritic literature and issues pertaining to clinical ap- cells. J Exp Med 2007; 204: 421-430. plication. Breast J 2012; 18: 436-442. [13] Koral K, Paranjpe S, Bowen WC, Mars W, Luo J [6] Liu LC, Su CH, Wang HC, Chang WS, Tsai CW, and Michalopoulos GK. Leukocyte-specific pro- Maa MC, Tsai CH, Tsai FJ and Bau DT. tein 1: a novel regulator of hepatocellular pro- Contribution of personalized Cyclin D1 geno- liferation and migration deleted in human he- type to triple negative breast cancer risk. Biomedicine (Taipei) 2014; 4: 3. patocellular carcinoma. Hepatology 2015; 61: [7] Bhushann Meka P, Cingeetham A, Nanchari 537-47. SR, Damineni S, Tipirisetti N, Gorre M, Jarjapu [14] Fan L, Strasser-Weippl K, Li JJ, St Louis J, S, Annamaneni S, Digumarthi R and Satti V. Finkelstein DM, Yu KD, Chen WQ, Shao ZM and HIF-1alpha (1772C>T) polymorphism as mark- Goss PE. Breast cancer in China. Lancet Oncol er for breast cancer development. Tumour Biol 2014; 15: e279-289. 2014; [Epub ahead of print]. [15] Donepudi MS, Kondapalli K, Amos SJ and [8] Rostami S, Kohan L and Mohammadianpanah Venkanteshan P. Breast cancer statistics and M. The LEP G-2548A gene polymorphism is as- markers. J Cancer Res Ther 2014; 10: 506- sociated with age at menarche and breast can- 511. cer susceptibility. Gene 2015; 557: 154-7. [16] Roshandel G, Boreiri M, Sadjadi A and [9] Mohammadzadeh G, Ghaffari MA, Bafandeh A Malekzadeh R. A Diversity of Cancer Incidence and Hosseini SM. Effect of leptin receptor and Mortality in West Asian Populations. Ann Q223R polymorphism on breast cancer risk. Glob Health 2014; 80: 346-357. Iran J Basic Med Sci 2014; 17: 588-594. [17] Holmes AK, Koller KR, Kieszak SM, Sjodin A, [10] Easton DF, Pooley KA, Dunning AM, Pharoah Calafat AM, Sacco FD, Varner DW, Lanier AP PD, Thompson D, Ballinger DG, Struewing JP, and Rubin CH. Case-control study of breast

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