Association of SIPA1 545 C>T polymorphism with survival in Chinese women with metastatic breast cancer

Abstract

It has been demonstrated that single nucleotide polymorphisms (SNPs) of SIPA1 (signal-induced proliferation associated gene 1) are associated with metastatic efficiency in both human and rodents. The purpose of this study was to determine whether SIPA1 545 C>T polymorphism was associated with overall survival in patients with metastatic breast cancer. In this study, SIPA1 545 C>T polymorphism was detected in 185 metastatic breast cancer patients using polymerase chain reaction-restriction fragment length polymorphism assay (PCR-RFLP). Survival curves for patients with SIPA1 545 C>T polymorphism was compared using the Kaplan-Meier method with log-rank tests. We found that SIPA1 545 C>T polymorphism was significantly associated with survival in 185 patients with metastatic breast cancer. Patients with SIPA1 545 T/T genotype had a significantly worse overall survival (OS) than did patients with C/T or C/C genotype (50.0% vs. 62.9%, P = 0.042). Moreover, in multivariate analysis, as compared with the C/C or C/T genotype, the T/T genotype remained an independent unfavorable prognostic marker of OS in this cohort (hazard ratio [HR] = 2.16; 95% CI= 1.12–4.15; P = 0.022). Our findings indicate that metastatic breast cancer patients with SIPA1 545 T/T genotype have a poorer survival compared to patients with C/C or C/T genotype.

Keywords

SIPA1 / polymorphism / metastatic breast cancer / survival

Introduction

Breast cancer is one of the most common cancers in women worldwide. Although the prognosis is good for patients with early-stage breast cancer, it is dramatically poor for patients with metastatic breast cancer [1]. Metastatic breast cancer is a biologically heterogeneous disease, and some metastatic breast cancer patients may have relatively longer survival than others [2]. Therefore, it is of the great importance to predict the prognosis of metastatic breast cancer patients in the clinical setting.

SIPA1 (signal-induced proliferation-associated gene 1), located on human chromosome 11q13.3 [3], was cloned by Hattari in 1995 [4]. It was the first identified as a potential cancer metastasis modulator in the mouse model. The N-terminal domain of SIPA1 has GTPase activating protein (GAP) activity, which is specific for RapGTPases (RapGTP). Rap is a member of the superfamily of Ras-related proteins, and involves in cell proliferation, morphology and adhesion. SIPA1 negatively regulates Rap via its RapGAP activity, which catalyzes the hydrolysis of active RapGTP into inactive RapGDP. Recently, it has been demonstrated that the expression of SIPA1 is correlated with cellular metastatic capacity [5-10].

One polymorphism in exon 1 of SIPA1 generates a 545 C to T transition, leading to phenylalanine (Phe, F) 182 to serine (Ser, S) amino acid substitution [11]. In the current study, SIPA1 545 C>T polymorphism was determined in 185 metastatic breast cancer patients using PCR-restriction fragment length polymorphism assay (PCR-RFLP). We aimed to investigate the association between this polymorphism and survival in this cohort.

Materials and methods

Study subjects

192 metastatic breast cancer patients were recruited at the Breast Center, Peking University Cancer Hospital, from March 2003 to December 2007. Of these, four patients lost the follow-up; and three specimens failed to obtain the PCR products because of poor-quality DNA when detecting SIPA1 545 C>T polymorphism. Thus, a total of 185 patients were available for SIPA1 545 C>T polymorphism. The age of patients ranged from 23 to 76 years, with a median of 52 years, and the median follow-up is 25 months (range: 4 to 85 months). This study was approved by the Research and Ethical Committee of Peking University Cancer Hospital.

DNA extraction and genotyping

Genomic DNA was obtained from peripheral blood lymphocytes of each patient using phenol-chloroform extraction. SIPA1 polymorphism was detected by using a PCR-RFLP technique. Forward primer 5′-CCAGCTCGGACCTGCTGCAT-3′ and reverse primer 5′-GATGGACACGGCCGCGTT-3′ were used for detection of SIPA1 545 C>T, as previously described by Crawford et al. [11]. PCR was performed in 20 μl reaction mixture containing 100 ng of genomic DNA template, 2 μl 10 × PCR buffer, 0.8 mM dNTP, 2.5 mM MgCl₂, 0.5 μM primers, and 1 unit AmpliTaq DNA polymerase (Promega, USA). The reaction condition employed was initial denaturation at 94°C for 2 min, followed by 35 step cycles of denaturation at 94°C for 30 s, annealing 57°C for 45 s, and extension at 72°C for 30 s followed by a terminal extension time of 10 min. 15 μl of PCR product was digested with Bsm I restriction endonuclease for SIPA1 545 C>T (New England Biolabs Inc.) for 2 h at 37°C. The digestion products were then resolved on a 2.5% agarose gel containing ethidium bromide. The homozygous SIPA1 545 C/C genotype was identified by one band (128 bp), the homozygous SIPA1 545 T/T genotype produced two bands (78 bp and 50 bp), and heterozygous SIPA1 545 C/T genotype displayed three bands (128 bp, 78 bp and 50 bp).

Statistical analysis

The correlation between the genotype variants and clinicopathological characteristics was determined using Pearson’s χ² test. Overall survival (OS) was defined as the time from date of metastasis to the last point of follow-up, or to the date of death. Survival curves were derived from Kaplan-Meier estimates and the curves were compared by log-rank tests. A Cox regression model was applied to determine whether a factor was independent predictor of overall survival in multivariate analysis. All statistical tests were two-sided, and P values less than 0.05 were considered as statistically significant. The statistical analyses were performed using SPSS 16.0 software.

Results

Association between SIPA1 545C>T polymorphism and clinicopathological charateristics

The frequency of SIPA1 545C>T polymorphism was as follows: 44.9% (83 of 185) of the patients were homozygous for C/C genotype, 44.3% (82 of 185) were heterozygous for C/T and 10.8% (20 of 185) were homozygous for T/T genotype. This polymorphism did not deviate from the Hardy-Weinberg equilibrium (χ² = 0.001, P = 0.97).

SIPA1 545 C>T polymorphism was not statistically significantly associated with the age, menopausal status, estrogen receptor (ER), progesterone receptor (PgR), human epidermal growth factor receptor 2 (HER2) status and triple negative status (absence of the ER, PgR, HER2) (P>0.05, Table 1).

The association between the SIPA1 545C>T polymorphism and overall survival

SIPA1 545C>T polymorphism was significantly associated with the overall survival in 185 metastatic breast cancer patients. Patients with T/T genotype had a significantly poorer overall survival compared to patients with C/C or C/T genotypes (2-year overall survival rate, 50.0% vs. 62.9%, P = 0.042) (Table 2, Fig. 1).

In multivariate analysis, as compared with the C/C or C/T genotype, the T/T genotype remained an independent unfavorable prognostic marker of overall survival after adjusting for age, menopausal status, HER2 status, ER or PgR status and metastatic site in this cohort of 185 patients (hazard ratio [HR] = 2.16; 95% CI= 1.12–4.15; P = 0.022) (Table 3).

Discussion

In the present study, we showed that SIPA1 545C>T polymorphism was significantly associated with survival in Chinese women with metastatic breast cancer. Patients with T/T genotype had a poorer prognosis compared to patients with C/C or C/T genotype.

SIPA1 is a mitogen induced GTPase activating protein that may enhance or hinder cell cycle progression and plays a role in regulating cell adhesion [9,12]. Strong evidence implicates SIPA1 as a metastases modifier gene in the mouse model [13]. Previous studies showed that germline polymorphisms in the SIPA1 gene were correlated with the risk and several major clinical characteristics of breast cancer [11,14]. Hsieh et al. [15] reported that SIPA1 545C>T polymorphism was associated with breast cancer risk in case-control Caucasian cohorts, suggesting that the variants of SIPA1 gene may play a potential role in breast cancer development. In an analysis of 300 cases [11], the SIPA1 545C>T polymorphism was associated with lymph node involvement and hormone receptor negative tumors after age-adjustment in Caucasian breast cancer patients. Our previous study also showed that patients with SIPA1 545T/T genotype were more likely to have HER2 positive tumors than patients with C/T or C/C genotype in 571 Chinese women with breast cancer [16]. These results supported the hypothesis that SIPA1 may represent genetic predisposition to aggressive breast cancer behavior, such as lymph node involvement, lack of hormone receptor and over expression of HER2, which predict tumor metastasis and have poor prognosis. Moreover, in rodent models, genetic variation in SIPA1 is suspected to be responsible for the underlying Mtes1 locus found to be related to metastasis potential of mammary tumors [13]. Metastases at distant sites are the main cause of death among women with breast cancer diagnosis and are suspected to be a heritable trait [17]. In the current study, patients with SIPA1 545 T/T genotype had a poor survival and remained as an independent unfavorable factor of OS in this cohort. However, the underlying mechanism of SIPA1 545T/T genotype associated with poor outcome is currently unclear. It is possible that this exonic missense SNP which has a Ser (S) to Phe (F) change, alters a hydrophilic residue (S) to a hydrophobic residue (F), and in turn significantly changes the protein functions of SIPA1 [11].

Our study showed that SIPA1 545C>T polymorphism may act as a prognosis marker for metastasis breast cancer. However, the sample size is relatively small in this study. Further studies with larger sample size are warranted to confirm our findings and to explore the underlying mechanism(s) of this polymorphism.

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