NCOA5 is correlated with progression and prognosis in luminal breast cancer

Accepted Manuscript NCOA5 is correlated with progression and prognosis in luminal breast cancer Xiao-He Ye, Du-Ping Huang, Rong-Cheng Luo PII:

S0006-291X(16)31905-2

DOI:

10.1016/j.bbrc.2016.11.051

Reference:

YBBRC 36753

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 29 October 2016 Accepted Date: 10 November 2016

Please cite this article as: X.-H. Ye, D.-P. Huang, R.-C. Luo, NCOA5 is correlated with progression and prognosis in luminal breast cancer, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.11.051. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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NCOA5 is correlated with progression and prognosis in luminal breast cancer Xiao-He Ye1, Du-Ping Huang2*, Rong-Cheng Luo3

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The email address of each author Xiao-He Ye:[email protected] Du-Ping Huang: [email protected] Rong-Cheng Luo: [email protected]

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1 Department of Radiology, The First Affiliated Hospital of Wenzhou Medical University,Zhejian, Wenzhou, P.R. China. 2 Department of Oncology, The First Affiliated Hospital of Wenzhou Medical University,Zhejian, Wenzhou, P.R. China. 3 Cancer Centre, TCM-Integrated Hospital of Southern Medical University, Guangzhou, Guangdong, P.R. China.

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*Correspondence to: Du-Ping Huang, Department of Oncology, The First Affiliated Hospital of Wenzhou Medical University, Nan Bai Xiang Street, Ouhai District, Wenzhou, 325000, Zhejiang, China. Tel: +86-577-55789462; Fax: +86-557-8999618767; E-mail: [email protected].

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Abstract Nuclear receptor coactivator 5 (NCOA5) is known to modulate ERα-mediated transcription and has been found to be involved in the

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progression of several malignancies. However, the potential correlation between NCOA5 and clinical outcome in patients with luminal breast cancer remains unknown. In the present study, we demonstrated that

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NCOA5 was significantly up-regulated in luminal breast cancer tissues

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compared with adjacent non-cancerous tissues both in validated cohort and TCGA cohort. Moreover, Kaplan-Meier analysis indicated that patients with high NOCA5 expression had significantly lower overall survival (P = 0.021). Cox regression analysis indicated that the high

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NOCA5 expression was independent high risk factor as well as old age (> 60) and HER-2 expression (P=0.039; P=0.003; P=0.005; respectively). This study provides new insights and evidences that NOCA5

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over-expression was significantly correlated with progression and

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prognosis in luminal breast cancer. However, the precise cellular mechanisms for NOCA5 in luminal breast cancer need to be further explored.

Keywords: NCOA5; Luminal breast cancer; Prognosis

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Introduction Breast cancer is the most common cancer in the world and the leading cause of cancer death among females[1,2]. Luminal or estrogen receptor

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(ER)-positive tumours represent approximately two-thirds of all breast cancers[3,4], and most of these tumors show a high overall response rate to endocrine treatments[5]. However, the development of primary and

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secondary resistance to endocrine treatments still remains a major clinical

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problem[6,7]. Considering that luminal breast cancer is a heterogeneous disease, with varying biological behaviors, biomarker profiles, and prognoses[4,8], highlighting the importance of identifying novel prognostic biomarkers that would allow the application of more effective

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therapeutic strategies.

The nuclear receptor coactivator 5(NCOA5, also known as coactivator independent of AF-2, CIA, or KIAA1637) gene encodes a novel

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coregulator that would function independently of AF-2[9]. The protein

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has both coactivator and corepressor functions, and is known to modulate ERα-mediated transcription[9,10,11]. Recently, NCOA5 has been shown to be involved in tumorigenesis in several types of cancer including hepatocellular

carcinoma

and

esophageal

squamous

cell

carcinoma[12,13]. However, the potential correlation between NCOA5 and clinical outcome in patients with breast cancer remains unknown. In this study, the expression of NCOA5 in luminal breast cancer tissues and

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adjacent non-cancerous tissues was analyzed in a large scale samples. Moreover, the predictive value of NCOA5 expression for prognosis was also explored.

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Materials and methods Patients and samples

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The study protocol and acquisition of tissue specimens were approved by the clinical ethical committee of First Affiliated Hospital of Wenzhou

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Medical University. Twenty luminal breast cancer tissues and twenty paired adjacent non-cancerous tissues were resected during surgery and snap-frozen in liquid nitrogen immediately for subsequent total RNA extraction.

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Public data of NCOA5 gene expression for breast cancer patients and their clinical information were obtained from the Cancer Genome Atlas

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(TCGA) database. Totally 745 patients with luminal breast cancer were enrolled from 2001–2013, and 63 paired patients’ expression data were

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analyzed, after excluding male patients and female patients with incomplete information or non-luminal types. Quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA of 20 paired samples was isolated by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) . RNA purity and concentration were detected by NanoDrop 2000 (Thermo Fisher Scientific). The samples were then reversely transcribed using a ReverTra Ace qPCR RT Kit

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(Toyobo, Osaka, Japan) according to the manufacturer’s instructions. qRT-PCR analysis of NOCA5 expression and data collection was performed on the Roche 480 System (Roche, USA). The relative

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quantitation value for NOCA5 gene expression was calculated by using the comparative Ct method, and β-actin was used as an internal control. primers

sequences

of

NCOA5

5’-CAGTCAGCAGCAGTTACC-3’,

were and

as

follows:

antisense

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The

sense 5’-

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CAGAGTCTCAGAACCAACTT-3’, and primers sequences of β-actin were as follows: 5’-GGGAAATCGTGCGTGACATTAAGG-3’, and antisense 5’- CAGGAAGGAAGGCTGGAAGAGTG -3’. Statistical analysis

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SPSS version 22.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism version 6.01 (GraphPad Software Inc., La Jolla, CA, USA) were performed to analyze data. Expression differences were evaluated by

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Paired t-test. Clinicopathological characteristics were compared by

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Chi-square test. Kaplan-Meier survival curves with log-rank test and Cox regression analyses were used to assess the overall survival with different NCOA5 expression and other clinical features. Two-tailed P values less than 0.05 were considered statistically significant.

Results

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The expression of NOCA5 mRNA was assessed in 20 paired breast cancer tissues and adjacent non-cancerous tissues by qRT-PCR analysis. As shown in Fig 1, NOCA5 mRNA expression was significantly

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up-regulated in breast cancer tissues compared with the adjacent non-cancerous tissues (P<0.001). To further validate dysregulated expression of NOCA5, RNA sequencing data of luminal breast cancer

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was extracted from TCGA database which contained 63 paired breast

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cancer tissues and non-cancerous tissues. Consistently, the expression of NOCA5 mRNA was significantly up-regulated in luminal breast cancer tissues (Fig 2, P < 0.001).

To investigate the relationship between NOCA5 expression and the

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clinical features of luminal breast cancer, total 745 luminal patients were divided into low and high NOCA5 expression groups according to the median value. As showed in Table 1, there were no differences between

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the two groups in terms of ages, tumor size, lymph-node metastasis,

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distant metastasis, clinical stage, hormone receptor, and human epidermal growth factor receptor-2 receptor(HER-2). In order to assessed the effects of NOCA5 expression on the prognosis

of luminal breast cancer, we analyzed follow-up data of TCGA database. As showed in Fig 3, those with high NOCA5 expression had significantly lower overall survival (P=0.021). Moreover, Cox regression analysis indicated that the high NOCA5 expression was independent high risk

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factor as well as old age (> 60) and HER-2 expression (HR=4.740, 95%Confidence Interval[CI] 1.079-20.820, P=0.039; HR=5.921, 95%CI 1.867-18.770, P=0.003; HR=4.459, 95%CI 1.568-12.676, P=0.005;

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respectively) (Table 2). Discussion

In the present study, we reported and validated that NOCA5

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over-expression was significantly correlated with progression and

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prognosis in luminal breast cancer. This study provides new insights and evidences that NCOA5 is a predictor for survival of patients with luminal breast cancer.

Luminal breast cancer represents approximately 60% to 75% of breast

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cancer around the world[4]. Despite blockade of the E2 signal through either antiestrogens or aromatase inhibitors is highly effective and appropriate for nearly all women with luminal breast cancer, resistance to

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endocrine therapy still remains a significant clinical problem[1,3,8]. As

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luminal breast cancer is a highly heterogeneous disease with different biological behaviors, biomarker profiles, and prognoses, identification of novel molecular biomarkers is critical for providing individualized therapies and efficient treatment in luminal breast cancer[8,14,15]. NCOA5 is a coregulator of estrogen receptor α (ERα)-mediated transcription[9]. Previous study indicated that NCOA5 cooperate with TIP30 to repress ERα-mediated c-myc transcription, which is implicated

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in the tumorigenesis[11]. Moreover, it has been reported that NOCA5 is involved in tumorigenesis in hepatocellular carcinoma and esophageal squamous cell carcinoma[2,4]. Besides, previous studies indicated that

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NOCA5 was associated with inflammation[16,17], which is known as a common pathogenic condition leading to breast cancer[18]. However, the expression status of NOCA5 and the its potential role in luminal breast

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cacner remains unknown. Herein, we reported that NOCA5 was

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significantly up-regulated in luminal breast cancer tissue when compared with normal tissues. Interestingly, NOCA5 was reported to be significantly reduced in tumors compared to the adjacent non-tumor area in both hepatocellular carcinoma and esophageal squamous cell

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carcinoma[4,13]. Considering the majority of breast cancer originates from the milk duct and the lobe, both of which highly express ERα[19,20], the various expression status of NOCA5 in human cancer

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tissues may due to different process of embryonic development and

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tumorigenesis[21]. Moreover, survival analysis showed that patients with high NOCA5 expression had significantly worse overall survival than those with low NOCA5 expression, suggesting that NOCA5 is a predictor for survival of patients with luminal breast cancer. To the best of our knowledge, this is the first presentation that NOCA5 was involved in the progression and prognosis of luminal breast cancer. However, the precise cellular mechanisms for NOCA5 in luminal breast cancer need to be

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further explored. And future studies should also address the biological effects of NOCA5 in vitro and in vivo experiments. In summary, this study provides the first evidence that NOCA5 is

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up-regulated in luminal breast cancer tissues when compared with adjacent non-cancerous tissues. And NOCA5 is an independent biological indicator of poor prognosis in luminal breast cancer. However, the precise

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cellular mechanisms for NOCA5 in luminal breast cancer deserve further

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exploration.

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Author contributions

Du-ping Huang and Rong-Cheng Luo conceived and designed the study; Xiao-he Ye performed the majority of experiments and wrote the manuscript.

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None.

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Conflict of interest

Acknowledgments

We thank Dr Rui-min Ma for his assistance with the RT-PCR analysis. This work was supported by the Natural Science Foundation of Zhejiang poince, China(Grant No.LY16H160050).

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ACCEPTED MANUSCRIPT European population: a case-control study, Hum Immunol 72 (2011) 761-765. [18] D.P. Huang, R.M. Ma, Y.Q. Xiang, Utility of Red Cell Distribution Width as a Prognostic Factor in Young Breast Cancer Patients, Medicine (Baltimore) 95 (2016) e3430. [19] E.A. Musgrove, R.L. Sutherland, Biological determinants of endocrine resistance in breast cancer, Nat Rev Cancer 9 (2009) 631-643. [20] Y. Diao, A. Azatyan, M.F. Rahman, C. Zhao, J. Zhu, K. Dahlman-Wright, P.G. Zaphiropoulos, breast cancer cells, Oncotarget (2016).

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Blockade of the Hedgehog pathway downregulates estrogen receptor alpha signaling in [21] C.Y. Liu, G.S. Feng, NCOA5, a molecular link between type 2 diabetes and liver cancer,

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Hepatobiliary Surg Nutr 3 (2014) 106-108.

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Figure legends Figure1. RT-PCR results of NOCA5 mRNA expression levels in human samples. Relative mRNA levels of NOCA5 between twenty breast cancer tissues (Tumor) and twenty paired adjacent non-cancerous tissues (Control) were presented. (P<0.001).

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Figure2. NOCA5 expression in luminal breast cancer in TCGA dataset. Totally 63 luminal breast cancer patients’ paired expression data in the TCGA dataset were analyze.

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Figure3. Kaplan-Meier analysis for the overall survival of luminal breast cancer patients with different expression of MLF1IP in NOCA5 dataset. Patients with high NOCA5 expression had a worse overall survival than low NOCA5 expression(P=0.021).

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Table legends Table1. The relationship between NOCA5 expression and clinicopathologic characteristics in luminal breast cancer patients of TCGA dataset.

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Table2. Multivariable Cox regression analysis for NOCA5 and clinical features.

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TABLE.1 The relationship between NCOA5 expression and clinicopathologic characteristics in luminal breast cancer patients of TCGA cohort

Positive Human epidermal growth factor

225(59.7%) 213(57.9%) 317(58.3%) 120(60.0%)

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227(41.7%) 80(40.0%) 137(47.5%) 166(42.2%)

228 (41.5%) 72(40.4%) 8(53.3%) 299(41.0%)

0.618

0.180

0.671

0.359

0.549

0.000

1.000

0.056

0.813

0.929

0.335

1.855

0.173

0.479

0.489

322(58.5%) 106(59.6%) 7(46.7%) 431(59.0%)

54(47.0%)

61(53.0%)

253(40.2%)

377(59.8%)

165(39.2%)

0.249

369(60.7%) 7(58.3%)

receptor-2 receptor

Negative

P

205(52.5%) 227(57.8%)

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239(39.3%) 5(41.7%)

X2

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152(40.3%) 155(42.1%)

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High NCOA5 (%)

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Age < 60 ≥ 60 Tumor size ≤ 2 cm > 2 cm Lymph-node metastasis None Present Distant metastasis No Yes Clinical stage I-II III-IV Estrogen receptor Negative Positive Progesterone receptor Negative

Low NCOA5 (%)

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Characteristic

256(60.8%)

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67(57.3%)

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50 (42.7%)

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Positive

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HR

95%CI

P

Age ≥ 60 vs < 60

5.921

1.867-18.770

0.003

HER-2 Positive vs HER-2 Negative

4.459

1.568-12.676

0.005

High NCOA5 vs Low NCOA5

4.740

1.079-20.820

0.039

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HER-2 = Human epidermal growth factor receptor-2 receptor; HR = Hazard ratio; CI = confidence intervals

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Highlights

NCOA5 is significantly over-expressed in human luminal breast cancer tissues.

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NOCA5 was involved in the progression of luminal breast cancer.

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NCOA5 can predict the progression of luminal breast cancer.