Expression of regulatory factor X1 can predict the prognosis of breast cancer

  • Authors:
    • Masahiro Shibata
    • Mitsuro Kanda
    • Dai Shimizu
    • Haruyoshi Tanaka
    • Shinichi Umeda
    • Masamichi Hayashi
    • Takahiro Inaishi
    • Noriyuki Miyajima
    • Yayoi Adachi
    • Yuko Takano
    • Kenichi Nakanishi
    • Dai Takeuchi
    • Sumiyo Noda
    • Yasuhiro Kodera
    • Toyone Kikumori
  • View Affiliations

  • Published online on: April 7, 2017     https://doi.org/10.3892/ol.2017.6005
  • Pages: 4334-4340
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Abstract

Breast cancer (BC) is the most common malignancy among women. Identifying novel biomarkers to predict prognosis accurately is important in managing this disease. The regulatory factor X1 (RFX1) gene is a member of the regulatory factor X gene family. Its protein reportedly downregulates the proto‑oncogene c‑myc, but its role in BC has been unclear. In this study, expression and methylation status of RFX1 were determined in BC cell lines. We then evaluated RFX1 mRNA expression levels with regard to clinicopathological factors including postoperative prognosis in 167 patients with BC. Expression of RFX1 was heterogeneous among cell lines, and we found no DNA methylation at the RFX1 promoter region. Patients were categorized into groups with high or low RFX1 expression, based on ratio of RFX1 mRNA expression in BC and adjacent non‑cancerous tissues. The high RFX1 group was significantly associated with low T factor (P=0.028), earlier disease stage (P=0.015), positive expression of estrogen receptor (P=0.005) and progesterone receptor (P=0.011), negative expression of human epidermal growth factor receptor 2 (P=0.001). The high RFX1 group experienced more favorable disease‑free survival (P=0.007) and overall survival (P=0.013). In multivariate analysis, RFX1 expression was an independent prognostic factor for disease‑free survival. Our findings indicate that RFX1 may serve as a prognostic marker for BC.

Introduction

Breast cancer (BC) is the most commonly diagnosed cancer and the leading cause of cancer-related death in women worldwide (1). Approximately 1.7 million new cases of BC and 521,900 deaths related to BC are estimated to have occurred worldwide in 2012 (2). Recurrent BC after resection is frequently intractable and recognized as a major challenge in clinical practice. Currently, adjuvant therapies such as chemotherapy, endocrine therapy and anti-human epidermal growth factor 2 (HER2) agent are administered to BC patients with the aim of prevention of disease recurrences (3). However, many patients have suffered from adverse events and other costs caused by such treatment. Risk stratification is necessary for assessing indication for adjuvant therapy; development of biomarkers for this purpose is therefore needed.

Several commercial multigene expression assays (Oncotype Dx®, MammaPrint®, PAM-50 ROR score®, EndoPredict® and Breast Cancer Index®) are currently available to predict patients' prognoses and to evaluate whether adjuvant chemotherapy is appropriate (3). However, such assays are limited to estrogen receptor (ER)-positive, HER2-negative BC. It's still difficult to predict prognosis of all BC subtypes.

The regulatory factor X1 (RFX1) gene is a member of the regulatory factor X gene family which codes transcription factors (4). Expression and regulatory mechanisms of RFX1 transcription have been reported in connection with several malignancies, including brain tumors (5,6) and hepatocellular carcinoma (7). RFX1 is considered to work as a tumor suppressor by downregulating the proto-oncogene c-myc (8). However, the relationship between RFX1 mRNA expression and patients' clinicopathological factors in BC has not been studied. Here, we focused on RFX1 and investigated its expression in BC cell lines and patients' tumor samples to determine whether it could be a prognostic marker for BC.

Materials and methods

Sample collection

We obtained 13 BC cell lines (BT-20, BT-474, BT-549, HCC1419, HCC1954, Hs578T, MCF7, MDA-MB-231, MDA-MB-361, MDA-MB-415, MDA-MB-468, SK-BR-3 and ZR-75-1) and two non-cancerous mammary gland epithelial cell lines (MCF-10A and MCF-12A). BT-549, HCC1419, HCC1954 and Hs578T were purchased from Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan), BT-474, MCF-7 and MCF-12A were provided to our research team by Johns Hopkins University and Dr. David Sidransky, the director of the Department of Otolaryngology-Head and Neck Surgery of Johns Hopkins University (Baltimore, MD, USA), and others were from the American Type Culture Collection (Manassas, VA, USA). Cells were stored at −80°C using cell preservative solution (Cell Banker; Mitsubishi Chemical Medience Corporation, Tokyo, Japan) and cultured in RPMI-1640 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum and in an atmosphere containing 5% CO2 at 37°C (9,10). We acquired 167 primary BC tissues from patients who underwent breast surgery at Nagoya University Hospital between March 2002 and November 2009. All tissue samples were diagnosed histologically as BC, frozen immediately after resection, and stored at −80°C. Adjacent non-cancerous specimens were resected >3 cm away from the edge of the tumor. BC specimens were classified histologically using the 7th edition of the Union for International Cancer Control (UICC) staging system for BC. Adjuvant chemotherapy, endocrine therapy and anti-HER2 therapy were administered to selected patients according to the patient's condition, pathological factors, subtype and the physician's discretion.

This study conforms to the ethical guidelines of the World Medical Association Declaration of Helsinki: Ethical Principles for Medical Research Involving Human Subjects. Written informed consent for use of clinical samples and data was required by the Institutional Review Board at Nagoya University (Nagoya, Japan) and was obtained from all patients.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Levels of RFX1 mRNA were determined using RT-qPCR. We extracted RNA from cell lines (8.0×106 cells per each cell line), 167 primary BCs, and adjacent normal tissues using the RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacture's protocol. cDNA was synthesized from total RNAs (1 µg) by M-MLV Reverse Transcriptase (Invitrogen Life Technologies, Frederick, MD, USA) and Primer ‘random’ (Sigma-Aldrich). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were quantified to normalize expression levels. RT-qPCR was performed using the SYBR-Green PCR Core Reagents kit (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) as follows: 1 cycle at 95°C for 10 min, 40 cycles at 95°C for 5 sec, and 60°C for 60 sec. All samples were tested in triplicate, and samples without templates were included in each PCR plate as negative controls. Real-time detection of SYBR Green fluorescence was conducted using an ABI StepOnePlus Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The expression level of each sample was taken as the value of the RFX1 amplicon divided by that of GAPDH (11,12). Primers specific for RFX1 and GAPDH are listed in Table I.

Table I.

Primers.

Table I.

Primers.

GeneExperimentTypeSequence (5′-3′)Product sizeAnnealing temperature
RFX1RT-qPCRForward GCAGACAGAAGTGGGGAGAA241 bp60°C
Reverse CAGTATACGCCTGTGTTGCC
Bisulfite sequencing in promoter regionForward TGTTTTTGAGGGTTTAGTTTTTTTT207 bp56°C
Reverse ACAACTATTACTACCCACCCTAATTAC
Bisulfite sequencing in intron 7Forward GGTGGAGGTTTGGAGTTT273 bp60°C
Reverse ACAAAAACAAATATAAAAACAACA
MSPForward TTTTTCGTTTTTATTTAATTTCGAC167 bp56°C
Reverse ATTTCTAACTTCTTACGCTAACGTC
U-MSPForward GGAGGTGTTATAGTTATGGTAGTTGT164 bp56°C
Reverse CTTTTCAAAAATCTCAAAATCAAT
GAPDHRT-qPCRForward GAAGGTGAAGGTCGGAGTC226 bp60°C
Probe CAAGCTTCCCGTTCTCAGCC
Reverse GAAGATGGTGATGGGATTTC

[i] RFX1, regulatory factor X1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; MSP, methylation specific PCR; U-MSP, un-methylation specific PCR.

Surveillance of CpG island around the RFX1 gene

Nucleotide sequence analysis was conducted to determine the presence of CpG island around the promoter region of RFX1. CpG island was defined using the following criteria: At least 500-bp region of DNA with a high GC content (>50%) and an observed CpG/expected CpG ratio of ≥0.6 (13). We used the MethPrime (http://www.urogene.org/methprimer/) to determine the location of CpG island (14).

Bisulfite sequence analysis

Genomic DNA samples from 15 cell lines were subjected to bisulfite treatment using the BisulFlash DNA Modification Kit (EPIGENTEK, Farmingdale, NY, USA); PCR was conducted to detect hypermethylation of the RFX1 promoter region and intron 7 (5), using specific primers (Table I). The PCR amplification protocols were, for the promoter region: 50 cycles at 94°C for 20 sec, 56°C for 20 sec, and 72°C for 25 sec; and for intron 7: 50 cycles at 94°C for 20 sec, 60°C for 20 sec, and 72°C for 25 sec; both amplifications used an initial denaturation step at 94°C for 2 min. PCR products were purified directly using the MinElute PCR Purification Kit (Qiagen). Sequence analysis was carried out by Eurofins Genomics Co. (Tokyo, Japan) (15).

Methylation-specific polymerase chain reaction (MSP)

DNA samples from BC cell lines, clinical BC specimens, and corresponding non-cancerous specimens were subjected to bisulfite treatment. MSP was conducted using specific primers to evaluate the presence of hypermethylation of RFX1 promoter region (Table I). The PCR amplification protocols for MSP and un-MSP (U-MSP) were as follows: 50 cycles at 94°C for 15 sec, 56°C for 15 sec, and 72°C for 30 sec; both amplifications used an initial denaturation step at 94°C for 2 min. Each PCR product was loaded directly on to 2% agarose gels, stained with ethidium bromide and visualized under UV illumination.

Statistical analysis

Differences in levels of RFX1 mRNA between BC and adjacent normal tissues were analyzed using the Mann-Whitney U test. The χ2 test was used to analyze the significance of the association between RFX1 expression and clinicopathological parameters. Overall survival (OS) and disease-free survival (DFS) rates were calculated using the Kaplan-Meier method; difference in survival curves was analyzed using the log-rank test. We performed multivariate regression analyses using the Cox proportional hazards model to identify prognostic factors, and variables for which P<0.05 were entered into the final model. Statistical analyses were performed using JMP 11 software (SAS Institute, Inc., Cary, NC, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

RFX1 mRNA expression and DNA methylation status of BC cell lines

RFX1 mRNA expression levels were heterogeneous among the 13 BC cell lines and two non-cancerous mammary cell lines (Fig. 1). We accepted the reports of each cell line's immunostaining status from previous studies (16,17).

A CpG island was found at upstream of the transcription initiation site of RFX1 (length 1,519 bases; Fig. 2A). We examined the methylation status of the 15 cell lines using bisulfite sequence analysis, which detected no CpG methylation in the RFX1 promoter regions of all cell lines, whereas CpG hypermethylation in intron 7 was detected in all cell lines including the non-cancerous mammary cell lines (Fig. 2B). BT-474, MCF-10A, and three representative clinical BC and corresponding non-cancerous tissues were subjected to MSP. We found absence of methylation in the promoter region of RFX1 gene both in cell lines and clinical samples (Fig. 2C).

Patient characteristics

All 167 patients were women. Their mean age was 54.4±11.6 years (mean ± standard deviation; range, 26–78 years). Their disease stage (by UICC staging system) were stage 0, 7; stage I, 47; stage II, 78; stage III, 34; and stage IV, 1. The median duration of patient follow-up was 100.0 months (range, 8–155 months) or until death. The immunostaining status of primary tumors were ER positive, n=127; negative, n=40; progesterone receptor (PgR) positive, n=115; negative, n=52; and HER2 positive, n=39; negative, n=119.

Mean expression levels of RFX1 mRNA in BC tissues was significantly lower than those in corresponding non-cancerous tissues (P<0.001; Fig. 3A). In 43 (26%) of the 167 patients, RFX1 mRNA expression levels in BC tissues were higher than in adjacent normal tissues. We calculated the ratio of RFX1 mRNA expression levels between BC and adjacent non-cancerous tissues. Patients whose RFX1 mRNA levels were lower in BC tissue than in adjacent tissues were designated as ‘the low RFX1 group’ (n=124), and patients with higher RFX1 expression were designated as ‘the high RFX1 group’ (n=43) in the following analyses.

Clinical significance of RFX1 mRNA levels

The high RFX1 group was significantly associated with less advanced UICC T factor (P=0.028) and lower pathological stage (P=0.015). The high RFX1 group correlated significantly with ER positive (P=0.005), PgR positive (P=0.011), HER2 negative status (P=0.001; Table II).

Table II.

Associations between expression of RFX1 mRNA and clinicopathological parameters in 167 patients with breast cancer.

Table II.

Associations between expression of RFX1 mRNA and clinicopathological parameters in 167 patients with breast cancer.

Clinicopathological parameterThe low RFX1 group (n=124)The high RFX1 group (n=43)P-value
Age
  ≤60 year7830
  >60 year46130.413
Histology
  DCIS43
  IDC11038
  ILC52
  Others500.265
UICC T factor
  Tis/T15126
  T2/T3/T47317   0.028a
Node status
  Negative6223
  Positive62200.693
UICC pathological stage
  0/I/II9239
  III/IV3240.015a
ER status
  Positive8839
  Negative364   0.005a
PgR status
  Positive7936
  Negative457   0.011a
HER2 status
  Positive372
  Negative8237
  Unknown54   0.001a
Triple-negative
  True153
  False10840
  Unknown100.323
Adjuvant therapy
  Endocrine therapy alone3522
  Chemotherapy alone273
  Endocrine and chemotherapy4816
  None142   0.012a

{ label (or @symbol) needed for fn[@id='tfn2-ol-0-0-6005'] } χ2 test.

a Statistically significant (P<0.05). DCIS, ductal carcinoma in situ; IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma; UICC, union for international cancer control; Tis, carcinoma in situ; ER, estrogen receptor; PgR, progesterone receptor; HER2, human epidermal growth factor 2.

Mean RFX1 mRNA expression in T2/T3/T4 specimens (n=90) was significantly lower than in the Tis (carcinoma in situ)/T1 group specimens (n=77; P=0.004; Fig. 3B). RFX1 mRNA levels did not significantly differ between patients with negative and positive lymph node metastases (n=85 and 82 respectively, P=0.181), or between UICC stage 0/I/II specimens (n=131) and stage III/IV (n=36; P=0.069).

With regard to relevance to the conventional biomarkers (Fig. 4), RFX1 mRNA levels in ER positive specimens (n=127) were expressed significantly higher than in ER negative specimens (n=40; P=0.017); PgR positive specimens' RFX1 mRNA level (n=115) was also significantly higher than that of PgR negative specimens (n=52; P=0.010); HER2 negative specimens (n=119) expressed significantly more RFX1 mRNA than HER2 positive specimens (n=39; P=0.006; missing HER2 data for 1 patient). RFX1 mRNA levels did not significantly differ between triple-negative (n=18) and other BC subtypes (n=148; P=0.363; missing data for 1 patient).

Prognostic impact of RFX1 mRNA expression

The high RFX1 group experienced significantly longer DFS than the low RFX1 group (5-year DFS rates, high RFX1 group: 95%; low RFX1 group: 77%; P=0.007; Fig. 5A). Among 43 patients of the high RFX1 group, only two suffered recurrences. The OS in the high RFX1 group was also significantly longer than in the low RFX1 group (5-year OS rates, high RFX1 group: 98%; low RFX1 group: 90%; P=0.013; Fig. 5B). Multivariate analysis of DFS identified UICC pathological stage III/IV [hazard ratio (HR): 4.80; 95% confidence interval (CI): 1.80–14.9, P=0.001] and the low RFX1 group (HR, 4.77; 95% CI: 1.32–30.9, P=0.014) as independent prognostic factors (Table III).

Table III.

Prognostic factors for disease-free survival in 167 patients with breast cancer.

Table III.

Prognostic factors for disease-free survival in 167 patients with breast cancer.

VariablesnUnivariate Hazard ratio95% CIPMultivariate Hazard ratio95% CIP
Age (>60)591.060.49–2.170.879
UICC T factor (T2-4)903.961.73–10.7 <0.001a1.850.67–5.650.239
Lymph node metastasis (positive)824.982.18–13.4 <0.001a2.840.79–10.00.107
UICC pathological stage (III/IV)367.733.78–16.4 <0.001a4.801.80–14.9   0.001a
ER status (negative)402.020.93–4.140.073
PgR status (negative)521.810.87–3.670.112
HER2 status (positive)392.311.09–4.76   0.031a0.940.42–2.080.883
Triple-negative (yes)182.370.88–5.400.084
Adjuvant chemotherapy (yes)942.371.10–5.65   0.026a0.320.09–1.070.064
The low RFX1 group1245.691.72–35.2   0.002a4.771.32–30.9   0.014a

a Statistically significant in multivariate analysis (P<0.05). CI, confidence interval; UICC, Union for International Cancer Control; ER, estrogen receptor; PgR, progesterone receptor; HER2, human epidermal growth factor 2. Univariate analysis was performed using the log-rank test. Multivariate analysis was performed using the Cox proportional hazards model.

Discussion

Our data showed that higher RFX1 expression was associated with less advanced T factor and lower disease stage, and patients with higher RFX1 expression had excellent prognoses.

The RFX1 gene is cytogenetically located at 19p13.1, and belongs to the RFX family genes which share the conserved DNA-binding domain named ‘winged helix’ (4,18). The RFX family codes transcription factors that contain a highly conserved 76-amino acid DNA binding domain (4). RFX1 forms a homodimer or heterodimers with the RFX2 or RFX3 proteins through a dimerization domain (19,20). RFX1-4 can bind methylated DNA sequences preferentially within a sequence-specific, 14-bp consensus sequence (21). As RFX1 downregulates the proto-oncogene c-myc (8), it is considered to be a tumor suppressor. In hepatocellular carcinoma (HCC), RFX1 regulates SC-2001-mediated SHP1 transcription in HCC cell lines, and SC-2001 inhibits tumor growth (7). RFX1 downregulation in tumors has been also verified in glioma (5), neuroblastoma (6), and esophageal adenocarcinoma (22).

We investigated the mRNA expression level and methylation status of RFX1 using BC cell lines and non-cancerous mammary gland epithelial cell lines. The RFX1 mRNA expression levels were heterogeneous across cell lines. There were no reports on hypermethylation of the promoter region of RFX1 gene in non-neoplastic tissues (5). In this study, we found no CpG methylation within the promoter region in cell lines and some clinical tissues. Reportedly, CpGs in intron 7 were hypermethylated in glioma cell lines and tissue samples, although CpG methylation in the promoter region was not seen (5). BC cells were also reported to have hypermethylated CpGs in intron 7 (23). CpGs in gene bodies are sometimes methylated in a tissue-specific manner. Gene body methylation has been shown to increase gene expression contrary to methylation in the promoter region (24). In the current study, we found CpG hypermethylation in intron 7 of all BC cell lines and the non-cancerous mammary cell lines. These results suggest that methylation status is not associated with downregulation of RFX1 mRNA in BC cells.

This study demonstrated that RFX1 mRNA expression has a significant relationship with patients' clinicopathological factors and prognosis. The most striking finding was that the high RFX1 group experienced excellent prognosis (5-year DFS rates, high RFX1 group: 95%; low RFX1 group: 77%). Notably, only two of 43 patients suffered recurrences in the high RFX1 group. Multivariate analysis of DFS identified UICC pathological stage and the low RFX1 group as independent prognostic factors. These results emphasize the usefulness of RFX1 as a prognostic marker. RFX1 mRNA level was indicated as a potent prognostic factor which was independent of ER, PgR or HER2 status. Considering these findings, if a patient's BC specimen's RFX1 mRNA expression is high, adjuvant chemotherapy could be tailored, thus reducing the adverse events and expense associated with chemotherapy.

Breast cancers are commonly treated according to their subtypes (3), which are determined by investigating their immunostaining status for ER, PgR and HER2. The subtype reflects each tumor's character, prognosis and sensitivity to medication. In this study, the high RFX1 group correlated significantly with ER positive, PgR positive and HER2 negative. This result corresponds to our understanding that BC with such phenotypes are less aggressive. Although triple-negative BC is thought to be a more malignant phenotype with a poorer prognosis, our results showed no significant differences between triple-negative BC and other types. This might be because of the small sample size of the triple-negative group.

This is the first study to show the relationship between RFX1 mRNA expression and BC prognosis; and our findings may have several possible clinical applications. For example, RFX1 levels in resected samples might help evaluate each patient's prognosis and need for adjuvant chemotherapy. Multigene expression assays might be refined by adding RFX1 expression. In addition, RFX1 expression can be analyzed by needle biopsy samples before operation, thus aiding in treatment decisions for neoadjuvant therapy.

This study has some limitations. First, the function of RFX1 in BC cells is unknown. Basic functional analyses in which RFX1 expression is inhibited are needed. Second, the mechanism of RFX1 downregulation is still unclear. The methylation status of two regions (the promoter and intron 7) in the RFX1 gene was determined in the present study. Therefore, it remains formally possible that additional intragenic or intergenic regions may have undergone methylation in breast cancer tissues. Mechanisms other than methylation (such as histone modification and micro RNAs) should also be investigated for this gene and its product. Further, this study was conducted retrospectively. Therefore, therapeutic intervention might affect significance of RFX1 mRNA level. Thus study for larger series of patients or prospective study is warranted.

In conclusion, our data indicate that higher expression of RFX1 mRNA level in BC tissue than adjacent normal site predicts better prognosis. RFX1 could become a useful prognostic marker for BC.

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June-2017
Volume 13 Issue 6

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Shibata M, Kanda M, Shimizu D, Tanaka H, Umeda S, Hayashi M, Inaishi T, Miyajima N, Adachi Y, Takano Y, Takano Y, et al: Expression of regulatory factor X1 can predict the prognosis of breast cancer. Oncol Lett 13: 4334-4340, 2017
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Shibata, M., Kanda, M., Shimizu, D., Tanaka, H., Umeda, S., Hayashi, M. ... Kikumori, T. (2017). Expression of regulatory factor X1 can predict the prognosis of breast cancer. Oncology Letters, 13, 4334-4340. https://doi.org/10.3892/ol.2017.6005
MLA
Shibata, M., Kanda, M., Shimizu, D., Tanaka, H., Umeda, S., Hayashi, M., Inaishi, T., Miyajima, N., Adachi, Y., Takano, Y., Nakanishi, K., Takeuchi, D., Noda, S., Kodera, Y., Kikumori, T."Expression of regulatory factor X1 can predict the prognosis of breast cancer". Oncology Letters 13.6 (2017): 4334-4340.
Chicago
Shibata, M., Kanda, M., Shimizu, D., Tanaka, H., Umeda, S., Hayashi, M., Inaishi, T., Miyajima, N., Adachi, Y., Takano, Y., Nakanishi, K., Takeuchi, D., Noda, S., Kodera, Y., Kikumori, T."Expression of regulatory factor X1 can predict the prognosis of breast cancer". Oncology Letters 13, no. 6 (2017): 4334-4340. https://doi.org/10.3892/ol.2017.6005