Reduced Expression of the Breast Cancer Metastasis Suppressor 1 mRNA Is Correlated with Poor Progress in Breast Cancer Free

Understanding the mechanism of breast cancer metastasis will benefit patients who need early aggressive therapy and improve their survival. It is known that metastasis is a multistep process involving dissociation of malignant cells in the primary tumor, local invasion, angiogenesis, intravasation of invading cells into the vasculature and lymphatic systems, survival in these channels, extravasation, and proliferation at a distant site (reviewed in refs. 1, 2). These processes are modulated by many factors, and of these the metastasis activators and suppressors are of particular importance in elucidating the underlying genetic and biochemical mechanisms. Metastasis suppressor genes, by definition, suppress metastasis without affecting primary tumor growth (3–6). Breast cancer metastasis suppressor 1 (BRMS1) was identified by differential display comparing metastasis-suppressed chromosome 11 hybrids with metastatic, parental MDA-MB-435 human breast carcinoma cells, and BRMS1 has subsequently been shown to suppress metastasis, but not tumorigenicity, of human melanoma cells in nude mice (7).

About the underlying mechanism of action, previous studies have shown that transfection with BRMS1 cDNA can restore cell-cell gap junction communications in the metastatic human breast carcinoma cell line MDA-MB-435 (8). One study suggested that BRMS1 inhibits metastasis in part through gene regulation via interaction with histone deacetylases (9). Another recent study suggested that inhibition of nuclear factor-κB activity and subsequent suppression of urokinase-type plasminogen activator expression contribute to BRMS1-dependent suppression of metastasis in tumors derived from breast cancer and melanoma cells (10). Metastasis suppression by BRMS1 also involves reduction of phosphoinositide signaling in MDA-MB-435 breast carcinoma cells (11).

Although the metastasis suppression function of BRMS1 is well established in experimental animals with tumors derived from cancer cell lines, its clinical importance remains undetermined. Previous studies showed that BRMS1 mRNA expression is reduced in breast cancer brain metastases compared with primary tumors (12) and in breast tumors compared with matched normal breast tissues (10). However, the clinical significance of its expression in breast cancer remains unclear; for example, one recent study using reverse transcription-PCR (RT-PCR) showed that expression of BRMS1 was independent of tumor size, tumor grade, metastasis to axillary lymph nodes, and hormone receptor status of the primary mammary tumor (13). In the present study, we used quantitative real-time RT-PCR to measure BRMS1 mRNA expression in samples taken from 161 female patients with invasive carcinoma of the breast and report a correlation between expression level and several clinicopathologic factors.

Patients and sample. Primary invasive breast carcinoma specimens were obtained by surgical excision from 161 female patients at the Department of Breast and Endocrine Surgery, Nagoya City University Hospital (Nagoya, Japan) between 1992 and 2000. The 161 patients were from a consecutive series, and no exclusion criteria were applied. Samples were snap frozen in liquid nitrogen and stored at −80°C until RNA extraction. Informed consent was obtained from all patients before surgery. The ethics committee of Nagoya City University Graduate School of Medicine (Nagoya, Japan) approved the study protocol. The median age of the patients was 53 years (range, 34-88 years). As postoperative adjuvant treatment, tamoxifen was given to patients whose tumors tested positive for estrogen receptor α (ERα) and/or progesterone receptor (PgR). Depending on tumor stage, the following chemotherapy regimens were given: oral 5-fluorouracil, CMF (100 mg oral cyclophosphamide, days 1-14; 40 mg i.v. methotrexate, days 1 and 8; 500 mg i.v. 5-fluorouracil, days 1 and 8), or CEF (500 mg cyclophosphamide, 60 mg epirubicin, 500 mg 5-fluorouracil; every 3 or 4 weeks). Since 1995, postoperative treatment has been done in accordance with the recommendations of St. Gallen (14). On recurrence, patients with ERα- and PgR-negative tumors were treated with CMF, CEF, and taxanes. Patients with hormone receptor–positive tumors and nonvisceral metastases were treated with endocrine therapy, such as antiestrogens, aromatase inhibitors, and medroxyprogesterone acetate. Patients were followed postoperatively every 3 months. The median follow-up period was 61 months (range, 48-144 months). Patients were graded histopathologically according to the modified Bloom and Richardson method proposed by Elston and Ellis (15).

Total RNA isolation and reverse transcription. Total RNA was isolated from ∼500 mg specimens of frozen breast cancer tissue (which were microscopically confirmed) using Trizol reagent (Life Technologies, Inc., Tokyo, Japan) according to the manufacturer's instructions. Total RNA from a flask of HepG2 cells, kindly provided by Dr. N. Harada (Division of Molecular Genetics, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi, Japan) (16), was used as a positive control and to generate standard curves. Reverse transcription reactions were done as described previously (17).

Primers and probes. We conducted BLAST searches (Genbank) to confirm both the specificity of the nucleotide sequences chosen for the primers and probes and the absence of DNA polymorphism. To avoid detection of contaminating genomic DNA, the primers were located at exons 7 and 9. The specific oligonucleotide primers were synthesized according to published information on the BRMS1 gene (NM_015399) as follows: sense, 5′-TGGTGGGACGACAAACTG-3′ (659-676); antisense, 5′-CTGCCCTAGCCTTTTTGATG-3′ (824-805). The PCR product size is 166 bp. The donor probe 5′-CCTCTGGTTTCTGGCCCATACATCGTGT-3′ was labeled with fluorescein at its 3′ end. The acceptor probe 5′-CATGCTTCAAGAGATCGACATCCTGGAGG-3′ was labeled with LC Red 640 at its 5′ end.

To ensure the fidelity of mRNA extraction and reverse transcription, all samples were subjected to PCR amplification with oligonucleotide primers and probes specific for the constitutively expressed gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and normalized. GAPDH primers were as follows: forward, 5′-AAATCAAGTGGGGCGATGCTG-3′; reverse, 5′-GCAGAGATGATGACCCTTTTG-3′. The sequences of the GAPDH probes used for real-time LightCycler PCR were 5′-AGAAGGCTGGGGCTCATTTGCAGGG-3′ and 5′-GTCCACTGGCGTCTTCACCACCATG-3′. All primers and probes were purchased from the Japanese Gene Institute (Saitama, Japan).

Real-time RT-PCR. PCR was done using a LightCycler (Roche Molecular Biochemicals, Mannheim, Germany) as described previously (18). In brief, the PCR was carried out in a 20 μL final volume containing (a) 3.2 μL of 25 mmol/L MgCl₂, (b) 0.5 μL of 20 pmol/μL sense and antisense primers, (c) 0.4 μL of 10 pmol/μL donor and acceptor probes, (d) 2 μL PCR master mix, (e) 1.5 μL cDNA, and (f) H₂O up to 20 μL. After initial denaturation at 95°C for 60 seconds, temperature cycling was initiated. Each cycle consisted of denaturation at 95°C for 2 seconds, hybridization at 57°C for 5 seconds, and elongation at 72°C for 7 seconds. The fluorescence signal was acquired at the end of the hybridization step. A total of 55 cycles was done. Cycling conditions for GAPDH were as follows: initial denaturation at 95°C for 60 seconds followed by 50 cycles at 95°C for 2 seconds, 60°C for 5 seconds, and 72°C for 8 seconds.

Standard curves and expression of results. For each PCR run, a standard curve was constructed from serial dilutions of cDNA from the HepG2 cell line. The level of expression of BRMS1 mRNA is given as relative copy numbers normalized against GAPDH mRNA and shown as mean ± SD. Relative BRMS1 mRNA expression was calculated by the following formula: BRMS1 / GAPDH × 1,000. A nontemplate control was included in each experiment. All of the nontemplate controls, the standard cDNA dilutions from the HepG2 cell line, and the tumor samples were assayed in duplicate. All of the patients' samples with a coefficient of variation for gene mRNA copy number >10% were retested following the method of Bieche et al. (19).

Immunohistochemical staining of ERα, PgR, and HER2. Immunostaining of ERα and PgR was done as described previously (20). Briefly, the slides were incubated with anti-ERα primary antibody (ER1D5; DAKO, Kyoto, Japan) or anti-PgR primary antibody (PgR636; DAKO) at a 1:100 dilution and developed using the streptavidin-biotin system (SAB-PO kit, Nichirei, Tokyo, Japan) according to the manufacturer's instructions. The immunostaining of ERα and PgR was subjectively assessed by two independent investigators (Z.Z. and H.I.), and discordant results were resolved by consultation with a third investigator (H.Y.) as described previously (21). Expression of ERα and PgR was scored by assigning a proportion score and an intensity score according to Allred's procedure (22). In brief, the proportion of positive staining throughout the entire slide was assessed as 0 (negative), 1 (<1%), 2 (1-10%), 3 (10-33%), 4 (33-67%), and 5 (>67%), and the average staining intensity was lodged 0 (negative), 1 (weak), 2 (moderate), or 3 (strong) under light microscopy. The immunohistochemical score of each slide (0 or 2-8) was obtained as the sum of the proportion and intensity. ERα and PgR status by immunohistochemistry was then assessed as negative (scores 0 and 2) or positive (scores 3-8). Immunostaining of HER2 was done as described previously (23). Briefly, primary antibody for HER2 was rabbit anti-human c-erB2 oncoprotein antibody (DAKO) at 1:200 dilution. The DAKO EnVision System (DAKO EnVision labeled polymer, peroxidase) was used for the detection system for HER2. HER2 immunostaining was evaluated using the same method as is used by the HercepTest (DAKO). To determine the score of HER2 expression on the membrane, staining pattern was estimated and scored on a scale of 0 to 3+. Tumors with scores of ≥2 were considered to be positive for HER2 overexpression.

Statistical analysis. The nonparametric Mann-Whitney U test was adopted for statistical analysis of associations between BRMS1 mRNA expression and clinicopathologic factors. Disease-free survival and overall survival curves were generated by the Kaplan-Meier method and verified by the log-rank (Mantel-Cox) test. Cox's proportional hazards model was used for the univariate and multivariate analyses of prognostic values. Differences were considered significant when a P of <5% was obtained.

Characteristics of the patients. Clinical characteristics are summarized in Table 1. The relative copy number of BRMS1 mRNA of the 161 cases ranged from 0 to 254 with a median of 83.

The level of BRMS1 mRNA expression correlates with age of patients and tumor size. The level of BRMS1 mRNA expression was observed to be significantly higher in the group of patients >50 years of age (113 ± 82) than in those of age ≤50 (79 ± 64; P = 0.001). It was also found to be higher in the group with tumors <2 cm in diameter (113 ± 63) than in those with tumors ≥2 cm (97 ± 82; P = 0.03; Table 1).

The level of BRMS1 mRNA expression correlates marginally with ERα but significantly with PgR and HER2 expression. The level of BRMS1 mRNA expression was found to be marginally higher in the ERα-positive group (102 ± 63) than in the ERα-negative group (97 ± 103; P = 0.05). It was also found that BRMS1 mRNA was expressed at higher levels in the PgR-positive group (105 ± 68) than in the PgR-negative group (91 ± 91; P = 0.02; Table 1). We also analyzed BRMS1 mRNA expression between HER2-positive and HER2-negative group, and we found that it was higher in HER2-negative group (106 ± 78) than that in HER2-positive group (80 ± 74; P = 0.01; Table 1).

No differences were found between BRMS1 mRNA expression and axillary lymph node metastasis (P = 0.1) and histologic grade groups (P = 0.2; Table 1).

Patients whose tumors express higher levels of BRMS1 mRNA have better disease-free and overall survivals. In the analysis of disease-free survival, local recurrences and distant metastases were considered as an event. Among 38 cases of events, there were 34 cases of distant metastases events and 4 cases of local recurrent events.

To identify a clinically meaningful cutoff point for BRMS1 mRNA expression in prognosis analysis, various levels of BRMS1 mRNA expression were tested by the Kaplan-Meier method and verified by the log-rank (Mantel-Cox) test. When the cutoff point for BRMS1 mRNA level was set at 100, patients with a high level of expression (171 ± 85, n = 60) had significantly better disease-free survival than those with low expression [59 ± 26, n = 101; log-rank (Mantel-Cox) test; P < 0.0004; Fig. 1A]. When analyzing overall survival, the same method was adopted. When the cutoff point was set at 100, patients with high level of BRMS1 mRNA expression (171 ± 85, n = 60) had a better prognosis than those with low expression [59 ± 26, n = 101; log-rank (Cox-Mantel) test; P = 0.0177; Fig. 1B].

Univariate and multivariate prognostic analysis of BRMS1 mRNA expression in breast cancer for disease-free and overall survivals. From the limited 161 cases of breast cancer patients, it was found that BRMS1 mRNA expression is an independent prognostic factor for disease-free survival (Table 2) but not for overall survival (Table 3).

BRMS1 was discovered by differential display comparing metastasis-suppressed chromosome 11-MDA-MB-435 hybrids with metastatic parental cells (7). Fluorescence in situ hybridization placed BRMS1 on chromosome11q13.1-11q13.2 (7). Genomic BRMS1 consists of 10 exons and 9 introns, spanning ∼7 kb, with the first exon being untranslated. The predicted protein is 246 amino acids long (M_r, ∼27 kDa), and an epitope-tagged BRMS1 migrates as expected on SDS-PAGE (7). There are two putative nuclear localization sequences at amino acids 198 to 205 and amino acids 239 to 245, and BRMS1 has been localized to the nucleus by both fractionation studies and immunofluorescence (24).

Despite the thorough studies of its metastasis suppression in cell line–derived tumors in nude mice, its clinical importance still remains to be studied. Previous studies showed that BRMS1 mRNA was down-regulated in breast cancer brain metastasis (12) and breast tumor tissue (10); however, its importance in lymph node metastasis and its correlation with hormone receptor status remain unclear. One recent study using RT-PCR failed to show any relationships between BRMS1 mRNA expression and tumor size, tumor grade, axillary nodes, or hormone receptor status, which does not suggest a role for BRMS1 in suppressing breast cancer metastasis to local lymph nodes (13).

In the present study, we used quantitative RT-PCR to investigate the level of BRMS1 gene expression in 161 clinical cases of breast cancer. We found that BRMS1 mRNA was expressed at significantly higher levels in patients >50 years of age, with tumor size <2 cm, and with PgR-positive and HER2-negative tumors. Patients with high levels of BRMS1 mRNA expression have a better prognosis than those with low expression. We have previously (25) used real-time RT-PCR to quantitate HDAC1 mRNA expression in the same tumor samples used in the present study. Comparing these results with our present data shows a highly significant positive correlation between mRNA expression levels of HDAC1 and BRMS1 (P < 0.0001; data not shown), which provides support for the proposal that BRMS1 suppresses metastasis by interacting with the mSin3-histone deacetylase complex, thus altering local chromatin structure (9). In the present study, the difference in BRMS1 expression between the lymph node–positive and lymph node–negative groups did not show significance; however, decreased expression was significantly correlated with poor survival, which supports the conclusions of functional studies in experimental cell line–derived tumors. The correlation of expression and survival also provides clinical evidence that BRMS1 is a breast carcinoma metastasis suppressor gene, suggesting that during the progression of breast cancer metastasis the suppressive function of BRMS1 may be decreased or lost. Further functional studies are needed to elucidate the mechanism of metastasis suppression of BRMS1 and to confirm its metastasis suppression function in other tumor types and models. In the present study, RNA was isolated from nonmicrodissected samples and that interpretation could be affected by this technical issue. Further study using microdissected breast cancer tissues is needed. In addition, the clinical significance of BRMS1 mRNA expression in breast cancer warrants study of BRMS1 protein levels in a large cohort of patients.

In conclusion, these results provide clinical evidence to support the notion that BRMS1 is a breast carcinoma metastasis suppressor gene. Our results also suggest that measuring BRMS1 expression will help to identify those breast cancer patients with worse disease-free survival.

We thank Mariko Nishio for her excellent technical support.