Promoter methylation of Retinol Binding Protein 7 (RBP7) predicts worse prognosis in breast cancer patients

doi:10.21203/rs.3.rs-4976990/v1

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Promoter methylation of Retinol Binding Protein 7 (RBP7) predicts worse prognosis in breast cancer patients

https://doi.org/10.21203/rs.3.rs-4976990/v1

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Breast cancer is the most common cancer among women all over the world. Genetic and epigenetic events are accumulated in breast tumorigenesis. Retinoid-binding protein7 (RBP7) is a member of the cellular retinol-binding protein (CRBP) family, which is involved in the pathogenesis of breast cancer. Previous study indicated high expression of RBP7 was significantly related to good relative percent survival in the luminal A subtype. However, promoter methylation of RBP7 and its relationship with clinicopathological features remains unclear. In order to investigate the methylation status of RBP7 in breast cancer patients. We collected and conducted MSP in a cohort of breast cancer patients. Our results showed that high expression of RBP7 correlates with better prognosis in breast cancer from TCGA database. And the MSP experiment in the breast cancer cohort indicated promoter methylation of RBP7 might be one of the reasons causing RBP7 down-regulation in breast cancer patient. Further study indicated that RBP7 methylation was found to be significantly associated with molecular status, LNM and cancer related death. Furthermore, the univariate and multivariable Cox regression analysis showed that RBP7 methylation acted as a predictor of poor survival either in the whole cohort or ER, PR, Her2 negative subtypes. Our study in conclusion indicates that promoter methylation of RBP7 may predict poor prognosis in breast cancer regardless of its molecular status.

Breast cancer

RBP7

promotor methylation

MSP

clinical outcomes

Breast cancer (BC), as a common cancer affecting women worldwide, accounts for about 30% of female cancers, and has a mortality-to-incidence ratio of 15%[1, 2]. According to Chinese Women's survey, breast cancer is now the most common malignant tumor in Chinese women, and the incidence rate is increasing yearly[3–6]. Biomarker testing is always conducted with a new diagnosis of breast cancer and these biomarkers including estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2) are performed through immunohistochemistry[7–9]. These receptors are both prognostic and treatment predictive that be widely used to determine the patient’s prognosis and can provide information that predicts for response to certain treatments such as endocrine therapy or trastuzumab[10–13]. Through the past recent years, many research have been conducted to find and validate new molecular biomarkers to serve as a prognostic and predictive biomarker for patients with breast cancer[14–17]. However, excepted for the 21 genes at the mRNA level, other novel biomarkers still need to be carefully validated before their clinical applications[18–20]. Thus, novel molecular characters and effective treatment approaches for breast cancer patients are still urgently required.

Previous studies have confirmed that cellular retinol-binding protein (CRBP) family members are involved in the pathogenesis of breast cancer[21]. CRBPs belong to the family of fatty acid-binding proteins and whose members are required for vitamin A stability and metabolism. Epigenetic silence of CRBPs is a common event in cancers[22]. For example, Kuppumbatti et al. reported that CRBPs were lowly expressed in 24% of human breast cancer. Retinoid-binding protein 7 (RBP7), also named CRBP4, which belongs to a clearly distinct CRBP subfamily, represent a relatively different mode of retinol binding for this protein[23]. It was reported that RBP7 regulates the occurrence and development of various diseases. RBP7 also plays an important role in adipose tissue during adipogenesis, cold exposure, and nutritional treatment[24–26]. From present studies, it is shown that RBP7 is a strong prognostic biomarker contributing to the malignant phenotype in colon cancer[25]. However, as a novel member of the CRBP family, and previous studies focus on the expression of RBP7 and its role in promoting malignant transformation of numerous cancers, the aims of the present study are to investigate the methylation of RBP7 in a cohort of breast cancer patients and its association with their clinicopathological features to better understand its pathogenesis role in breast cancer.

Patients.

With the approval of our institutional review board and human ethics committee in accordance with the Declaration of Helsinki and the International ethical guidelines for medical research involving human subjects, 2016, where required, a total of 200 paraffin-embedded breast cancer tissues were randomly obtained at the First Affiliated Hospital of Xi’an Jiaotong University School of Medicine. All individuals signed informed consent to participate in the study. Tumor staging was performed according to the tumor, node, and metastasis (TNM) classification. Tumor samples were obtained following surgical resection and their pathological features were examined on macro dissection of the samples. All samples were histologically examined by a senior pathologist at Department of Pathology of the Hospital based on World Health Organization (WHO) criteria. The demographic and clinical features of the study population are summarized in Table 1.

Table 1

Clinicopathological characteristics of breast cancer patients
Characteristics	No. of patients (%)	No. of methylated RBP7 (%)
Age,years
Mean	51.7	n/a
SD	13.2	n/a
WHO grade
I	32.2	75.0
II	48.3	43.5
III + IV	19.5	73.7
Recurrence
No	79.3	55.4
Yes	20.7	73.7
ER
-	40.2	52.6
+	59.8	63.6
PR
-	46.0	61.4
+	54.0	57.1
Her²
-	77.0	53.4
+	23.0	80.0
Lymph node metastasis (LNM)
No	58.6	66.7
Yes	41.4	50.0
Radiotherapy
No	80.5	58.7
Yes	19.5	61.1
Chemotherapy
No	19.5	76.5
Yes	80.5	55.3
Survival status
Alive	80.5	54.7
Dead	19.5	77.8

DNA extraction and bisulfite modification.

All tissues sections were reviewed by boardcertified pathologists to ensure that ≥ 50% of the cells used for DNA purification were neoplastic. The tissues were first treated with xylene for 12 h at room temperature to remove the paraffin and were then subjected to digestion with 1% sodium dodecylsulfate (SDS) and proteinase K at 48°C for 72 h with the addition of several spiking aliquots of concentrated proteinase K to facilitate digestion. Genomic DNA was subsequently isolated from the digested tissues followed by a standard phenol-chloroform extraction and ethanol precipitation protocol. The samples were then stored at -80°C until use. Quantity and quality of the extracted DNA were evaluated by a spectrophotometer and 186/200 samples passed (Nanodrop 2000, Thermo Scientific, USA). For each sample, 1 µg of genomic DNA was used for sodium bisulfite modification. Methylation specific PCR. The extracted DNA was treated with bisulfite to convert unmethylated cytosine to uracil prior to a methylation-specific polymerase chain reaction (MS-PCR) using Epi Tect Bisulfite Kit (Qiagen, Germany) according to the manufacturer’s instructions.

Methylation-specific PCR (MSP) assay

The modified DNA was amplified using MS-PCR primers. Methylated RBP7 promoter forward primer sequence 5’-GCGGAGGTTGTAGTGAGTC-3’; Reverse primer sequence 5’-TAACCTTATAATCCGCCCG-3’. Unethylated RBP7 promoter forward primer sequence 5’-GAGGTGGAGGTTGTAGTGAGTT-3’; Reverse primer sequence 5’-ATAACCTTATAATCCACCCACC-3’. PCR amplification was carried out in the buffer containing 16.6 mM ammonium sulfate, 67 mM Tris base, 2.5 mM MgCl2, 10 mM 2-mercaptoethanol, 0.1% DMSO, 0.2 mM each of dATP, dCTP, dGTP and dTTP, 600 nM each of forward and reverse primers, 0.6 unit Platinum Taq polymerase. Each sample was run in triplicate. The condition for RBP7 gene amplification was 10 minutes at 95°C, followed by amplification cycles including 30 seconds at 95°C, 45 seconds at 59°C and 60 seconds at 72°C for the extension, in addition to final elongation of 10 minutes at 72°C. Normal leukocyte DNA was methylated in vitro with Sss I methylase (New England Biolabs, Beverly, MA) to generate completely methylated DNA as a positive control. After PCR, electrophoresis was performed on a 1.5% agarose gel.

Statistical analysis.

Data were analyzed using SPSS 18.0 (SPSS Inc. Chicago, USA). The Mann-Whitney U test and Kruskal-Wallis test were performed for numerical data and the Chi-square test was used to analyze the relationship between parameter data. Multivariate models were then developed that adjusted for the most important covariates, including ER, PR, Her2, survival status and lymph node metastasis. Survival length was determined from the day of primary tumor surgery to the day of death or last clinical follow-up. The Kaplan–Meier method was used for survival analysis grouping with methylation status of RBP7. Differences between curves were analyzed using the log-rank test. Multivariate Cox regression analysis was used to evaluate the effect of gene methylation on survival of independently of the number of lymph node metastasis, ER, PR, Her2. Differences were considered statistically significant if p < 0.05.

High expression of RBP7 correlates with better prognosis in breast cancer

Previous studies had indicated that high expression of RBP7 was an independent biomarker of poor cancer specific survival in breast cancer, colon cancer and bladder cancer. Through analysis the mRNA level of RBP7 using TCGA database, as shown in Fig. 1A, the expression of RBP7 was downregulated in breast cancer, colon cancer and bladder cancer tissues compared with their normal samples in TCGA. What’s more, the expression level of RBP7 was not related with tumor stage in breast cancer (Fig. 1B). However, the Kaplan-Meier curve indicated that the breast cancer patients with high level of RBP7 mRNA trends to have a longer survival time especially in the Lumina A subtype (Fig. 2).

Methylation status of RBP7 gene in breast cancer tissues and its association with clinicopathological features

We then examined promoter methylation of RBP7 using MSP approach in a large cohort of breast cancer samples. As shown in Table 1, the methylation rate of RBP7 was 59.1% (110/186) in the cohort. However, no significant difference of RBP7 methylation rate was seen between WHO grade, LNM, radiotherapy, chemotherapy and either hormone receptors.

In order to further examine the relationship of RBP7 methylation status with clinicopathological characteristics in this cohort. Logistic regression analysis was then explored. The univariate analyses showed that lower RBP7 methylation rate was found to be significantly associated with patients with chemotherapy (p = 0.03) and patients with lymph node metastasis (p = 0.02). However, higher RBP7 methylation rate was associated with Her2 positive group (p = 0.004), cancer relapsed (p = 0.04), and cancer related death (p = 0.01) (Table 2). In order to assess the independent association of gene methylation with molecular status (including ER, PR and Her2), lymph node metastasis, radiotherapy, chemotherapy and cancer-related death, we conducted multiple multivariable logistic regressions. As also shown in Table 2, lower RBP7 methylation rate remained significantly associated with lymph node metastasis. However, higher RBP7 methylation rate remained associated with Her2 status and cancer related death.

Table 2

Methylation of *RBP7* in breast cancer: univariate and multivariate models with clinicopathological characteristics
Characteristics	Univariate		Multivariate
Characteristics	OR* (95% CI)	P	OR* (95% CI)	P
ER	1.58 (0.87–2.86)	0.13	6.16 (1.97–19.27)	0.02
PR	0.84 (0.47–1.51)	0.56	0.35 (0.12–1.03)	0.06
Her²	3.49 (1.06–11.44)	0.004*	5.42 (1.91–15.43)	0.002
LNM	0.50 (0.28–0.91)	0.02	0.38 (0.18–0.81)	0.01
Radiotherapy	1.11 (0.53–2.33)	0.79	2.76 (1.08–7.09)	0.04
Chemotherapy	0.38 (0.16–0.89)	0.03	0.64 (0.24–1.72)	0.37
Relapse	2.25 (1.02–4.97)	0.04	n/a	n/a
Survival status	2.90 (1.24–6.78)	0.01	4.71 (1.66–13.35)	0.04*
*OR: odds ratio with 95% confidence interval; Survival status (alive vs. dead).

Given that RBP7 methylation was found to be associated with the key molecular status including ER, PR and Her2 in our cohort. We then divided the cohort based on this molecular status and examine the relationship of methylated RBP7 with clinicopathological characteristics again using logistic regression analysis. In the ER genitive group, as shown in Table S1, the univariate and multivariable logistic regressions showed that higher RBP7 methylation rate was significantly associated with Her2 positive group and cancer related death. Besides, in the PR genitive group, as shown in Table S2, we also found that higher RBP7 methylation rate was significantly associated with Her2 positive group and cancer related death. What’s more, in the Her2 genitive group, as shown in Table S3, we found that higher RBP7 methylation rate was significantly associated with ER positive group and cancer related death both using univariate and multivariable logistic regressions. The above results indicated that patients with high level of promoter methylation of RBP7 might have a worse survival time especially in those patients with some negative hormone receptor.

RBP7 gene promoter methylation predicted worse prognosis in breast cancer

Whether methylation of RBP7 is indeed associated with a worse survival in breast cancers, as suggested by its association with clinicopathological characteristics of breast cancer patients, was subsequently investigated in unvariate and multivariable survival analysis. As shown in Table 3, univariate Cox regression showed RBP7 methylation (p = 0.001), ER negative status (p = 0.02), Her2 positive status (p = 0.001), patients without radiotherapy (p = 0.04), patients without chemotherapy (p = 0.003), and cancer relapse (p < 0.001) acted as a predictor of poor survival for breast cancer patients. Cox multivariate regression showed that RBP7 methylation (p < 0.001) and lymph node metastasis (p < 0.001) is also a predictor of poor survival as an independently variable with respect to the ER, PR, Her2. And radiotherapy act as a predictor of better survival factor (Table 4).

Table 3

Prognostic value of clinicopathological factors and methylation of *RBP7* using univariate Cox regression analysis
Characteristics	HR* (95% CI)	P
RBP7 methylation	5.67 (1.98–16.20)	0.001
ER	0.44 (0.22–0.86)	0.02
PR	0.55 (0.28–1.08)	0.08
Her²	3.04 (1.55–5.98)	0.001
LNM	2.62 (1.31–5.24)	0.07
Radiotherapy	0.23 (0.05–0.94)	0.04
Chemotherapy	0.35 (0.17–0.70)	0.003
Relapse	127.132 (30.31-533.25)	< 0.001
*HR: hazard ratio with 95% confidence interval (CI)

Table 4

Prognostic value of clinicopathological factors and methylation of *RBP7* using multivariate Cox regression analysis
Characteristics	HR* (95% CI)	P
RBP7 methylation	9.52 (3.05–29.72)	< 0.001
ER	0.39 (0.14–1.12)	0.08
PR	1.22 (0.46–3.26)	0.69
Her²	1.06 (0.42–2.72)	0.91
LNM	4.27 (2.05–8.91)	< 0.001
Radiotherapy	0.12 (0.03–0.54)	0.006
*HR: hazard ratio with 95% confidence interval (CI)

In order to understand the relationship of RBP7 methylation with the key molecular status including ER, PR and Her2 in the survival status of these breast cancer patients. We then again divided the cohort based on these molecular status and explored unvariate and multivariable survival analysis using Cox regression. As show in Table S4, in the ER genitive group, the univariate and multivariable Cox regression analysis showed that RBP7 methylation and lymph node metastasis acted as a predictor of poor survival. Besides, as show in Tables 5 and 6, in the PR and Her2 genitive group, we also found that RBP7 methylation acted as a predictor of poor survival.

The Kaplan-Meier estimator of the survivorship function was used to evaluate the impact of aberrant gene methylation on the survival of breast cancer patients. The survival of cancer patients with and without gene methylation was compared using the log-rank test. As shown in Fig. 3, methylation of RBP7 was significantly associated with worse survival of breast cancer patients (median survival time: 71.4 vs 90 months; p = 0.019). What’s more, we also found that methylation of RBP7 was significantly associated with worse survival of breast cancer patients both in the Her2 negative and ER negative subtypes (Fig. 3). Since lymph node metastasis, cancer relapse, radiotherapy and chemotherapy play an important role of survival time. We then analyze the survival times after surgery under those conditions. As also shown in Fig. 3, methylation of RBP7 was significantly associated with worse survival both in the patients with lymph node metastasis and patients without radiotherapy subtype.

So far, breast cancer remains the most common cancer among women worldwide[1, 27]. Due to the increasing in breast cancer incidence, strategies to combat the breast cancer pandemic are geared toward prevention, early detection, and treatment[28, 29]. With the development of gene sequencing, targeted therapies, and molecular diagnostics, breast cancer treatment has the potential to become directed toward each patient’s specific tumor characteristics[30]. Estrogen and progesterone receptors are already used to predict a tumor’s response to hormone therapy and ERBB2 expression predicts response to treatment with trastuzumab[31–34]. The Oncotype DX assay measures expression of 21 genes and predicts which patients with node-negative disease are less likely to benefit from chemotherapy. Biomarkers are also being developed to predict severe drug-related toxicity[35]. The improvement of tumor biomarkers ready for clinical use is a long process. A good biomarker should be a predictor of not only prognosis but also the response to therapies. For HR-positive/HER2-negative subtype, only two biomarkers, the 21-gene recurrence score and the 70-gene signature, have been tested in prospective randomized clinical trials[36, 37]. There are no diagnostic multigene assays for HER2-positive and TN breast cancers. Robust predictive biomarkers to accelerate clinical progress are needed. RBP7 is a member of the CRBP family which involved in the pathogenesis of breast cancer. Previous study indicated that RBP7 involved in regulating the occurrence and development of ER⁺ breast cancer through the PPAR and PI3K/AKT pathways[38]. In this study, we investigated promoter methylation of RBP7 in a cohort of breast cancers using methylation-specific PCR approach. Firstly, we found that in TCGA database, RBP7 was downregulated in breast cancer compared to the normal tissues. Consider that previous reaches indicated that RBP7 as a novel biomarker that is helpful for the prognosis of ER + breast cancer patients. Promoter methylation of RBP7 is involved in its gene silencing in breast cancer, thus regulating the occurrence and development of ER + breast cancer through the PPAR and PI3K/AKT pathways. We then examined promoter methylation of RBP7 using MSP approach in our cohort. We found the methylation rate of RBP7 was 59.1% in our cohort which indicated promoter methylation might be one mechanism causing gene downregulated in breast cancer. We further examine the relationship of RBP7 methylation with clinicopathological characteristics and found that RBP7 methylation was found to be significantly associated with molecular subtype and cancer related death. In addition, univariate and multivariate Cox regression showed RBP7 methylation act as a predictor of better survival for breast cancer patients. The Kaplan-Meier estimator of the survivorship function also indicated that methylation of RBP7 was significantly associated with longer survival time of breast cancer patients after surgery.

In conclusion, this is the first study which investigates the methylation status of the RBP7 gene in breast cancers providing evidence for a possible pathogenetic role of RBP7 promoter methylation in the development of breast cancer.

Ethics approval and consent to participate

200 paraffin-embedded breast cancer tissues were randomly obtained with the approval of our institutional review board and human ethics committee in accordance with the Declaration of Helsinki and the International ethical guidelines for medical research involving human subjects, 2016.

Clinical trial number: not applicable.

Consent for publication

The authors declare no conflict of interest to publication.

Availability of data and material

All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author.

Competing interests

The authors declare no conflict of interest.

Funding

This work was supported by the Nature Science Foundation of Shaanxi (No. 2020JQ-503) and Fundamental Research Funds for the Central Universities (No. xzy012019092)

Authors' contributions

Peng Hou and Yiping Qu designed of the work and wrote the main manuscript text. Xumin Zhao, Rongrong Cui and Pu Chen explore the experiment and prepared figures 1-5. Yuanyuan Wang and Rong Lu analysis the data and prepared the tables. All authors reviewed the manuscript. The authors declare no conflict of interest.

Acknowledgements

Not applicable.

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Promoter methylation of Retinol Binding Protein 7 (RBP7) predicts worse prognosis in breast cancer patients

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Abstract

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Introduction

Patients and methods

Methylation-specific PCR (MSP) assay

Statistical analysis.

Results

RBP7 gene promoter methylation predicted worse prognosis in breast cancer

Discussion

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References

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