Medical Hypotheses 83 (2014) 359–364
Contents lists available at ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
CCNB1 is a prognostic biomarker for ER+ breast cancer Kun Ding, Wenqing Li, Zhiqiang Zou, Xianzhi Zou, Chengru Wang ⇑ Yantai City Hospital for Infectious Diseases, Yantai, China
a r t i c l e
i n f o
Article history: Received 10 March 2014 Accepted 15 June 2014
a b s t r a c t Identification of effective prognostic biomarkers and targets are of crucial importance to the management of breast cancer. CCNB1 (also known as CyclinB1) belongs to the highly conserved cyclin family and is significantly overexpressed in various cancer types. In this study, we demonstrated that CCNB1 had significant predictive power in distant metastasis free survival, disease free survival, recurrence free survival and overall survival of ER+ breast cancer patients. We also found that CCNB1 was closely associated with hormone therapy resistance. In addition, gene set enrichment analysis (GSEA) revealed that its expression was positively associated with genes overexpressed in endocrine therapy resistant samples. Finally, using CCNB1-Drug interaction network, we demonstrated the interactions between CCNB1 and several available cancer drugs. Overall, we suggest that CCNB1 is a biomarker for the prognosis of ER+ breast cancer and monitoring of hormone therapy efficacy. It is also a promising target for developing new strategies to prevent or even reverse hormone therapy resistance. Moreover, CCNB1 expression may help to monitor hormone therapy and to direct personalized therapies. Nevertheless, in vivo and in vitro experiments and multi-center randomized controlled clinical trials are still needed before its application in clinical settings. Ó 2014 Elsevier Ltd. All rights reserved.
Introduction Breast cancer is the most common malignancy among women in the United States, among which 70% are estrogen receptor positive (ER+). Adjuvant hormone therapy greatly improves the overall outcome. The selective ER modulator tamoxifen has shown great success in the treatment of ER+ breast cancer [1]. However, over 40% ER+ patients with advanced disease fail to respond to tamoxifen effectively [2]. Studies have also demonstrated clinical responses to aromatase inhibitors in 40–70% of ER-positive tumours [3]. Approximately 25% of all women diagnosed with breast cancer die from their disease despite having been treated according to state-of-the-art clinical guidelines [4–6]. While adjuvant systemic therapy saves a significant number of lives [7–9]. However, many patients are subjected to unnecessary adjuvant therapies with the potential of causing more harm than good [10]. The lack of criteria to help individualize breast cancer treatment indicates the need for a novel way to predict prognosis and therapy response. Since about one-half of the patients with ER+
⇑ Corresponding author. Address: Department of Infectious Diseases, Yantai City Hospital for Infectious Diseases, Yantai 264001, PR China. Tel.: +86 05356221371; fax: +86 05356628542. E-mail address: [email protected] (C. Wang). http://dx.doi.org/10.1016/j.mehy.2014.06.013 0306-9877/Ó 2014 Elsevier Ltd. All rights reserved.
cancer fail on adjuvant therapy, identification of effective and reliable biomarkers that could be used to monitor hormone therapy efficacy and new targets to reverse hormone therapy resistance is of crucial importance [11,12]. CCNB1 (also known as CyclinB1) belongs to the highly conserved cyclin family and is expressed in almost all tissues in human body [13]. It complexes with p34 (cdc2) to form the maturation-promoting factor (MPF) and plays critical roles in the control of cell cycle at the G2/M transitions [14]. Data from the Human Protein Atlas show that CCNB1 is expressed in dozens of cancer types, which indicates its potential roles in cancer transformation and progression [15]. It is also reported that CCNB1 may be involved in the processes of epithelial-mesenchymal transitions (EMT) and metastasis [16]. We propose that CCNB1 is a promising biomarker for overall survival and hormone therapy efficacy in ER+ breast cancer, and that its presence may guide the clinician in a rational choice of chemotherapy for these patients. Nevertheless, the prognostic power of CCNB1 in ER+ breast cancer and its relation with hormone therapy resistance have never been reported before [17]. In this study, we explored the possibility of CCNB1 as a biomarker for the prognosis of ER+ breast cancer patients and prediction of hormone therapy efficacy. Furthermore, an interaction network was constructed to show how CCNB1 and available anti-cancer drugs could interact with each other.
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Materials and methods
Statistical analysis
Ethics statement
Clinical and gene expression profiling data were analyzed using standard statistical tests including the logrank test and unpaired t-test. In GSEA, we assess the significance of an observed enrichment score (ES) by comparing it with the set of scores ESNULL computed with randomly assigned phenotypes. The nominal p value estimates the statistical significance of the enrichment score for a single gene set. A P value of zero (0.0) indicates an actual P value of less than 1/number-of-permutations. In the present study, we performed 1000 permutations. Significance was defined as a P value of less than 0.05. Analyses were performed using R 3.0.1 (R Foundation for Statistical Computing [http://www.r-project. org/]), GraphPad Prism 5.01 (GraphPad Software, Inc. [www. graphpad.com]) and SPSS version 21 (SPSS Inc., Chicago, Illinois).
We have the right to use datasets from Gene Expression Omnibus (GEO) by complying with all requirements according to each dataset. The Research Ethics Committee of Peking University Cancer Hospital & Institute waived the requirement for ethical approval of this analysis because the registry is a de-identified database. Patients and cell lines Several publicly available datasets were downloaded and analyzed to explore the prognostic value of CCNB1 in ER+ breast cancer patients and its potential role in hormone therapy resistance. GSE47561 contains expression data from 1570 breast cancer samples, of them, 514 ER+ patients with distant metastasis free survival (DMFS) data (114 of them are known to be treated with tamoxifen), 513 ER+ patients with recurrence free survival (RFS) data, 125 ER+ patients with disease free survival (DFS) and overall survival (OS) data, 167 ER patients with DMFS data. The Van cohort contains expression data from 226 ER+ breast cancer samples that with DMFS, DFS and OS data. Gyorffy dataset (256 samples) and GSE3494 (83 samples) are expression data from ER+ breast cancer samples that with DMFS data and are known to be treated with tamoxifen. ISDB3008 dataset contains expression data from 2795 breast cancer samples and is obtained through InsilicoMerging package in R 3.0.1. Raw data (⁄.CEL) in this dataset was normalized using the FRMA method and all the probes were mapped to gene symbols [18]. Among all the datasets used in this study, GSE47561, GSE3494, GSE33366 and GSE26459 are obtained from Gene Expression Omnibus (GEO) dataset; van dataset and Gyorffy dataset were obtained from supplementary data of previous publications. For all the study subjected in this research, ER positivity was defined as greater than 10 fmol/mg tumor tissue and greater than 1% nuclear staining or immunohistochemistry (IHC) score of at least 3 for the biochemical and immunohistochemical assays, respectively.
Results CCNB1 is a prognosis biomarker in ER+ breast cancer By analyzing gene expression profiles and corresponding clinical information of breast cancer patients from publicly available datasets, we found that high expression of CCNB1 confers poor distant metastasis free survival (DMFS), disease free survival (DFS), recurrence free survival (RFS) and overall survival (OS) in ER+ breast cancer patients (Figs. 1A and 2). However, the expression levels of CCNB1 are not correlated with DMFS in ER breast cancer patients (Fig. 1B). Specifically, CCNB1 overexpression is associated with poor DMFS in ER+ breast cancer cohorts GSE47561 (Fig. 1A, P = 2.9e 05) and Van dataset (Fig. 2D, P = 0.014). Besides, overexpression of CCNB1 also confers poor DFS (Fig. 2A, P = 5.6e 05 and Fig. 2E, P = 0.0014), RFS (Fig. 2B, P = 0.00067) and OS (Fig. 2C, P = 0.028 and Fig. 2F, P = 0.038) in ER+ breast cancer cohorts as noted in each figure. However, the association between CCNB1 expression levels and DMFS in ER breast cancer patients is not significant (Fig. 1B, P = 0.23). All these data indicates that CCNB1 confers poor prognosis in ER+ breast cancer.
Genomic analysis
CCNB1 is a biomarker for hormone therapy resistance
The mRNA expression profiling of all the samples in this study were performed on the Human U133A Gene Chip or Human genome U133 plus 2.0 platforms (Affymetrix, Santa Clara, CA). GS E47561 [19], Van dataset [19], Gyorffy dataset [20], and GSE3494 [21] were used for survival analysis. Patients were classified into two groups according to the CCNB1 expression values in Kaplan– Meier plots for DMFS, DFS, RFS and OS. The classification was accorded to the upper 50% against the rest of CCNB1 expression values. GSE33366 and GSE26459 were used to show the association between CCNB1 expression and tamoxifen efficacy [22]. Gene Set Enrichment Analysis (GSEA) was performed using expression data of GSE47561 and ISDB3008 on gene sets related with hormone therapy resistance including ‘ENDOCRINE THERAPY RESISTANCE’ and ‘TAMOXIFEN RESISTANCE DN’ etc [23]. The Comparative Toxicogenomics Database (CTD) was explored to construct CCNB1Drug interaction network [24]. More specifically, CCNB1 was searched in the CTD database for drugs or chemicals that could decrease/increase the mRNA or protein expression of CCNB1. Then drugs or chemicals were selected based on their applications in breast cancer management. Finally, CCNB1-Drug interaction network was constructed through Cytoscape version 3.0.2 [25].
Since high expression of CCNB1 confers poor DMFS, DFS, RFS and OS in ER+ breast cancer patients. Hormone therapy, including tamoxifen and novel aromatase inhibitor, is one of the most widely used drugs for the management of those ER+ patients, CCNB1 may play a potential role in hormone therapy resistance. To validate this hypothesis, several datasets were downloaded from publicly available datasets and analyzed. Results showed that high expression of CCNB1 was significantly associated with poor hormone therapy efficacy in ER+ breast cancer within GSE47561, Miller et al. and Pawitan et al (Figs. 1C, 3A and B, P = 3.1e 05, 0.0099 and 0.00031, respectively). Besides, Gene Set Enrichment Analysis performed on expression data from GSE47561 indicated that CCNB1 expression was positively correlated with genes overexpressed in endocrine therapy resistant samples (Fig. 1D, P = 0.026). All these data suggests that CCNB1 expression is correlated with hormone therapy resistance. CCNB1-Drug interaction network indicates drugs that could decrease CCNB1 expression Next, we sought to explore how CCNB1 and available cancer drugs could influence each other. CCNB1-Drug interaction network
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Fig. 1. CCNB1 expression is associated with poor survival and hormone therapy resistance in ER+ breast cancer patients. Kaplan–Meier plot for DMFS of ER+ (A), ER (B) or hormone therapy treated (C) breast cancer patients grouped by the CCNB1 expression levels, significance was assessed by logrank test. (D) Gene set enrichment analysis of CCNB1 mRNA expression in relation to hormone resistance gene set using breast cancer expression profiles.
was constructed in Cytoscape based on data from The Comparative Toxicogenomics Database (CTD) (Fig. 4). Please see Methods section for procedure details. This network indicates that several drugs could influence the mRNA or protein expression of CCNB1. For instance, Cisplatin and Doxorubicin could decrease CCNB1 expression while Paclitaxel could increase CCNB1 expression. Interestingly, tamoxifen was also shown to upregulate the expression of CCNB1. Each arrow in this network is supported by previous reports.
Discussion The therapy of ER+ breast cancer, which represents more than 70% of breast tumors, is based on anti-hormonal compounds [26]. The anti-estrogen is a commonly used treatment for patients with ER+ breast cancer. For adjuvant therapy of ER+ breast cancer, hormone therapy improves overall survival and reduces risk for development of breast cancer [27]. Unfortunately, a subset of patients who received adjuvant hormone therapy would
eventually experience relapse and die as a result of the disease. In National Surgical Adjuvant Breast and Bowel Project (NSABP) prevention trial (P1), 30% of ER+ tumors were not prevented by hormone therapy [28]. Numerous studies have been performed, which combined endocrine therapy with agents that could modulate these mechanisms, so as to prevent the occurrence of hormone therapy resistance [29]. Due to the pressing clinical need, several other investigators have developed gene predictors that could predict outcome in ER+ breast cancer treated with adjuvant hormone therapy. For instance, Cyclin D1, Acid ceramidase 1and p53 accumulation had been reported that could predict outcome in ER+ breast cancer treated with adjuvant anti-estrogen therapy [30–36]. Likewise, Retinoic acid receptor alpha, CD44 and deltaEF1 had been reported to be involved in the development of hormone therapy resistance in breast cancer [37–39]. It has been reported that breast stem cells and Wnt signaling activation might be the underlying mechanism of resistance to tamoxifen [40,41]. CCNB1, a key regulator of cell cycle, is overexpressed in many human cancers, including breast cancer [42–45]. However, the
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Fig. 2. Kaplan–Meier plot for DFS (A and E), RFS (B), DMFS (D) and OS (C and F) of ER+ breast cancer patients classified according to the of CCNB1 expression levels. Significance was assessed by logrank test.
Fig. 3. (A and B) Kaplan–Meier plot for DMFS of hormone treated breast cancer patients classified according to the CCNB1 expression level, significance was assessed by logrank test.
association between CCNB1 overexpression and hormone therapy resistance in ER+ breast cancer remains unclear. Our results show the significant prognostic power of CCNB1 in ER+ breast cancer progression and hormone therapy resistance. Importantly, high level of CCNB1 is correlated with hormone treatment failure and poor DMFS. We might be able to stratify ER+ breast cancer patients
by testing the mRNA expression level of CCNB1 and decide when and how to use hormone therapy treatment in combination with appropriate therapeutic drug in the future. There are many studies that connect CCNB1 and poor prognosis. It is easy to ascribe the poor outcome to the CCNB1’s critical role in regulating cell cycle at G2/M phase. High expression of CCNB1 indicates active
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Fig. 4. Interaction network of CCNB1 and chemotherapeutic drugs. The gene–drug interaction network shows us how available chemotherapeutic drugs could decrease the expression of CCNB1. For example, Doxorubicin could decrease the expression of CCNB1, while Paclitaxcel could increase the expression of CCNB1.
proliferation and uncontrolled tumor growth. Alternatively, high level of CCNB1 indicated the failure of hormone therapy including tamoxifen and aromatase inhibitor [3]. However, there are several other ways. For instance, Song et al. have showed that CCNB1 promoted cancer invasion and metastasis through enhancing epithelial-mesenchymal transition [16]. Lu et al. found that CCNB1/ CDK1 inhibition could restore p53 function, which implies that CCNB1 participate in impairing p53 function and promote tumorigenesis [46]. Here, we also showed evidence that CCNB1 played a role in hormone therapy resistance in breast cancer, in vivo/ in vitro experiments are still needed to confirm the finding. Nevertheless, whether CCNB1 is a driver gene in hormone therapy resistance still requires experimental validation. Provided the high expression of CCNB1 is involved in the development of hormone therapy resistance, how to manage cancer patients with CCNB1 overexpression remains a great challenge. Here we show several drugs that could influence the expression of CCNB1 (Fig. 2). For instance, Doxorubicin and Cisplatin could decrease the expression of CCNB1, while Paclitaxel could increase the expression of CCNB1. In addition, tamoxifen seems to upregulate the expression of CCNB1. Since CCNB1 participate in the hormone therapy resistance, it is interesting to know whether ER+ breast cancer patient with CCNB1 overexpression could benefit from the repression of CCNB1, or in other words, whether CCNB1 is a promising target for preventing or could reverse hormone therapy resistance. More clinical data is needed to solve these issues. For example, the effects of CCNB1 overexpression could be verified in a clinical trial of early ER+ breast cancer (without distant metastasis), we estimate 120 patients (60 cases with high CCNB1 expression and 60 with low expression) would need to be included to demonstrate the effect based on our retrospective data. If this could be demonstrated, a further step would be to investigate whether early addition of platinum/etoposide-based chemotherapy improved outcomes in these patients. Although much information about ER and cancer has been provided in the past three decades since the arrival of hormone therapy in the clinic, a lot more needs to be elucidated for favorable therapeutic outcomes. More concrete research outcomes will warrant the translational research that may lead to more efficient and safer treatment for breast cancer patients as well as women at high risk of advanced breast cancer.
Taken together, this study indicates that CCNB1 expression may help to monitor hormone therapy efficacy. In addition, it suggests the relevance of CCNB1 in the development of hormone therapy resistance. Furthermore, it could provide guidance to personalized therapies. Nevertheless, multi-center randomized controlled clinical trials and in vivo/in vitro experiments are still needed before its application in clinical settings. Conflict of interest statement None. References [1] Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwide burden of cancer in 2008: GLOBOCAN. Int J Cancer 2008;127(2893– 2917):2010. [2] Vendrell JA, Ghayad S, Ben-Larbi S, Dumontet C, Mechti N, Cohen PA. A20/ TNFAIP3, a new estrogen-regulated gene that confers tamoxifen resistance in breast cancer cells. Oncogene 2007;26:4656–67. Epub 2007 Feb 4612. [3] Miller WR. Clinical, pathological, proliferative and molecular responses associated with neoadjuvant aromatase inhibitor treatment in breast cancer. J Steroid Biochem Mol Biol 2010;118:273–6. [4] Brenner H. Long-term survival rates of cancer patients achieved by the end of the 20th century: a period analysis. Lancet 2002;360:1131–5. [5] Goldhirsch A, Glick JH, Gelber RD, Coates AS, Senn HJ. Meeting highlights: international consensus panel on the treatment of primary breast cancer. Primary breast cancer. J Clin Oncol 2001;19:3817–27. [6] Eifel P, Axelson JA, Costa J, et al. National Institutes of Health Consensus Development Conference Statement: adjuvant therapy for breast cancer, November 1–3, 2000. J Natl Cancer Inst 2001;93:979–89. [7] Group EBCTC. Tamoxifen for early breast cancer: an overview of the randomised trials. Early Breast Cancer Trialists’ Collaborative Group. Lancet 1998;351:1451–67. [8] Group EBCTC. Polychemotherapy for early breast cancer: an overview of the randomised trials. Early Breast Cancer Trialists’ Collaborative Group. Lancet 1998;352:930–42. [9] Bergh J. Where next with stem-cell-supported high-dose therapy for breast cancer? Lancet 2000;355:944–5. [10] Group EBCTC. Favourable and unfavourable effects on long-term survival of radiotherapy for early breast cancer: an overview of the randomised trials. Early Breast Cancer Trialists’ Collaborative Group. Lancet 2000;355:1757–70. [11] Clarke R, Liu MC, Bouker KB, et al. Antiestrogen resistance in breast cancer and the role of estrogen receptor signaling. Oncogene 2003;22:7316–39. [12] Osborne CK, Schiff R. Growth factor receptor cross-talk with estrogen receptor as a mechanism for tamoxifen resistance in breast cancer. Breast 2003;12: 362–7. [13] Miyazaki T, Arai S. Two distinct controls of mitotic cdk1/cyclin B1 activity requisite for cell growth prior to cell division. Cell Cycle 2007;6:1419–25.
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Contents lists available at ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
CCNB1 is a prognostic biomarker for ER+ breast cancer Kun Ding, Wenqing Li, Zhiqiang Zou, Xianzhi Zou, Chengru Wang ⇑ Yantai City Hospital for Infectious Diseases, Yantai, China
a r t i c l e
i n f o
Article history: Received 10 March 2014 Accepted 15 June 2014
a b s t r a c t Identification of effective prognostic biomarkers and targets are of crucial importance to the management of breast cancer. CCNB1 (also known as CyclinB1) belongs to the highly conserved cyclin family and is significantly overexpressed in various cancer types. In this study, we demonstrated that CCNB1 had significant predictive power in distant metastasis free survival, disease free survival, recurrence free survival and overall survival of ER+ breast cancer patients. We also found that CCNB1 was closely associated with hormone therapy resistance. In addition, gene set enrichment analysis (GSEA) revealed that its expression was positively associated with genes overexpressed in endocrine therapy resistant samples. Finally, using CCNB1-Drug interaction network, we demonstrated the interactions between CCNB1 and several available cancer drugs. Overall, we suggest that CCNB1 is a biomarker for the prognosis of ER+ breast cancer and monitoring of hormone therapy efficacy. It is also a promising target for developing new strategies to prevent or even reverse hormone therapy resistance. Moreover, CCNB1 expression may help to monitor hormone therapy and to direct personalized therapies. Nevertheless, in vivo and in vitro experiments and multi-center randomized controlled clinical trials are still needed before its application in clinical settings. Ó 2014 Elsevier Ltd. All rights reserved.
Introduction Breast cancer is the most common malignancy among women in the United States, among which 70% are estrogen receptor positive (ER+). Adjuvant hormone therapy greatly improves the overall outcome. The selective ER modulator tamoxifen has shown great success in the treatment of ER+ breast cancer [1]. However, over 40% ER+ patients with advanced disease fail to respond to tamoxifen effectively [2]. Studies have also demonstrated clinical responses to aromatase inhibitors in 40–70% of ER-positive tumours [3]. Approximately 25% of all women diagnosed with breast cancer die from their disease despite having been treated according to state-of-the-art clinical guidelines [4–6]. While adjuvant systemic therapy saves a significant number of lives [7–9]. However, many patients are subjected to unnecessary adjuvant therapies with the potential of causing more harm than good [10]. The lack of criteria to help individualize breast cancer treatment indicates the need for a novel way to predict prognosis and therapy response. Since about one-half of the patients with ER+
⇑ Corresponding author. Address: Department of Infectious Diseases, Yantai City Hospital for Infectious Diseases, Yantai 264001, PR China. Tel.: +86 05356221371; fax: +86 05356628542. E-mail address: [email protected] (C. Wang). http://dx.doi.org/10.1016/j.mehy.2014.06.013 0306-9877/Ó 2014 Elsevier Ltd. All rights reserved.
cancer fail on adjuvant therapy, identification of effective and reliable biomarkers that could be used to monitor hormone therapy efficacy and new targets to reverse hormone therapy resistance is of crucial importance [11,12]. CCNB1 (also known as CyclinB1) belongs to the highly conserved cyclin family and is expressed in almost all tissues in human body [13]. It complexes with p34 (cdc2) to form the maturation-promoting factor (MPF) and plays critical roles in the control of cell cycle at the G2/M transitions [14]. Data from the Human Protein Atlas show that CCNB1 is expressed in dozens of cancer types, which indicates its potential roles in cancer transformation and progression [15]. It is also reported that CCNB1 may be involved in the processes of epithelial-mesenchymal transitions (EMT) and metastasis [16]. We propose that CCNB1 is a promising biomarker for overall survival and hormone therapy efficacy in ER+ breast cancer, and that its presence may guide the clinician in a rational choice of chemotherapy for these patients. Nevertheless, the prognostic power of CCNB1 in ER+ breast cancer and its relation with hormone therapy resistance have never been reported before [17]. In this study, we explored the possibility of CCNB1 as a biomarker for the prognosis of ER+ breast cancer patients and prediction of hormone therapy efficacy. Furthermore, an interaction network was constructed to show how CCNB1 and available anti-cancer drugs could interact with each other.
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K. Ding et al. / Medical Hypotheses 83 (2014) 359–364
Materials and methods
Statistical analysis
Ethics statement
Clinical and gene expression profiling data were analyzed using standard statistical tests including the logrank test and unpaired t-test. In GSEA, we assess the significance of an observed enrichment score (ES) by comparing it with the set of scores ESNULL computed with randomly assigned phenotypes. The nominal p value estimates the statistical significance of the enrichment score for a single gene set. A P value of zero (0.0) indicates an actual P value of less than 1/number-of-permutations. In the present study, we performed 1000 permutations. Significance was defined as a P value of less than 0.05. Analyses were performed using R 3.0.1 (R Foundation for Statistical Computing [http://www.r-project. org/]), GraphPad Prism 5.01 (GraphPad Software, Inc. [www. graphpad.com]) and SPSS version 21 (SPSS Inc., Chicago, Illinois).
We have the right to use datasets from Gene Expression Omnibus (GEO) by complying with all requirements according to each dataset. The Research Ethics Committee of Peking University Cancer Hospital & Institute waived the requirement for ethical approval of this analysis because the registry is a de-identified database. Patients and cell lines Several publicly available datasets were downloaded and analyzed to explore the prognostic value of CCNB1 in ER+ breast cancer patients and its potential role in hormone therapy resistance. GSE47561 contains expression data from 1570 breast cancer samples, of them, 514 ER+ patients with distant metastasis free survival (DMFS) data (114 of them are known to be treated with tamoxifen), 513 ER+ patients with recurrence free survival (RFS) data, 125 ER+ patients with disease free survival (DFS) and overall survival (OS) data, 167 ER patients with DMFS data. The Van cohort contains expression data from 226 ER+ breast cancer samples that with DMFS, DFS and OS data. Gyorffy dataset (256 samples) and GSE3494 (83 samples) are expression data from ER+ breast cancer samples that with DMFS data and are known to be treated with tamoxifen. ISDB3008 dataset contains expression data from 2795 breast cancer samples and is obtained through InsilicoMerging package in R 3.0.1. Raw data (⁄.CEL) in this dataset was normalized using the FRMA method and all the probes were mapped to gene symbols [18]. Among all the datasets used in this study, GSE47561, GSE3494, GSE33366 and GSE26459 are obtained from Gene Expression Omnibus (GEO) dataset; van dataset and Gyorffy dataset were obtained from supplementary data of previous publications. For all the study subjected in this research, ER positivity was defined as greater than 10 fmol/mg tumor tissue and greater than 1% nuclear staining or immunohistochemistry (IHC) score of at least 3 for the biochemical and immunohistochemical assays, respectively.
Results CCNB1 is a prognosis biomarker in ER+ breast cancer By analyzing gene expression profiles and corresponding clinical information of breast cancer patients from publicly available datasets, we found that high expression of CCNB1 confers poor distant metastasis free survival (DMFS), disease free survival (DFS), recurrence free survival (RFS) and overall survival (OS) in ER+ breast cancer patients (Figs. 1A and 2). However, the expression levels of CCNB1 are not correlated with DMFS in ER breast cancer patients (Fig. 1B). Specifically, CCNB1 overexpression is associated with poor DMFS in ER+ breast cancer cohorts GSE47561 (Fig. 1A, P = 2.9e 05) and Van dataset (Fig. 2D, P = 0.014). Besides, overexpression of CCNB1 also confers poor DFS (Fig. 2A, P = 5.6e 05 and Fig. 2E, P = 0.0014), RFS (Fig. 2B, P = 0.00067) and OS (Fig. 2C, P = 0.028 and Fig. 2F, P = 0.038) in ER+ breast cancer cohorts as noted in each figure. However, the association between CCNB1 expression levels and DMFS in ER breast cancer patients is not significant (Fig. 1B, P = 0.23). All these data indicates that CCNB1 confers poor prognosis in ER+ breast cancer.
Genomic analysis
CCNB1 is a biomarker for hormone therapy resistance
The mRNA expression profiling of all the samples in this study were performed on the Human U133A Gene Chip or Human genome U133 plus 2.0 platforms (Affymetrix, Santa Clara, CA). GS E47561 [19], Van dataset [19], Gyorffy dataset [20], and GSE3494 [21] were used for survival analysis. Patients were classified into two groups according to the CCNB1 expression values in Kaplan– Meier plots for DMFS, DFS, RFS and OS. The classification was accorded to the upper 50% against the rest of CCNB1 expression values. GSE33366 and GSE26459 were used to show the association between CCNB1 expression and tamoxifen efficacy [22]. Gene Set Enrichment Analysis (GSEA) was performed using expression data of GSE47561 and ISDB3008 on gene sets related with hormone therapy resistance including ‘ENDOCRINE THERAPY RESISTANCE’ and ‘TAMOXIFEN RESISTANCE DN’ etc [23]. The Comparative Toxicogenomics Database (CTD) was explored to construct CCNB1Drug interaction network [24]. More specifically, CCNB1 was searched in the CTD database for drugs or chemicals that could decrease/increase the mRNA or protein expression of CCNB1. Then drugs or chemicals were selected based on their applications in breast cancer management. Finally, CCNB1-Drug interaction network was constructed through Cytoscape version 3.0.2 [25].
Since high expression of CCNB1 confers poor DMFS, DFS, RFS and OS in ER+ breast cancer patients. Hormone therapy, including tamoxifen and novel aromatase inhibitor, is one of the most widely used drugs for the management of those ER+ patients, CCNB1 may play a potential role in hormone therapy resistance. To validate this hypothesis, several datasets were downloaded from publicly available datasets and analyzed. Results showed that high expression of CCNB1 was significantly associated with poor hormone therapy efficacy in ER+ breast cancer within GSE47561, Miller et al. and Pawitan et al (Figs. 1C, 3A and B, P = 3.1e 05, 0.0099 and 0.00031, respectively). Besides, Gene Set Enrichment Analysis performed on expression data from GSE47561 indicated that CCNB1 expression was positively correlated with genes overexpressed in endocrine therapy resistant samples (Fig. 1D, P = 0.026). All these data suggests that CCNB1 expression is correlated with hormone therapy resistance. CCNB1-Drug interaction network indicates drugs that could decrease CCNB1 expression Next, we sought to explore how CCNB1 and available cancer drugs could influence each other. CCNB1-Drug interaction network
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Fig. 1. CCNB1 expression is associated with poor survival and hormone therapy resistance in ER+ breast cancer patients. Kaplan–Meier plot for DMFS of ER+ (A), ER (B) or hormone therapy treated (C) breast cancer patients grouped by the CCNB1 expression levels, significance was assessed by logrank test. (D) Gene set enrichment analysis of CCNB1 mRNA expression in relation to hormone resistance gene set using breast cancer expression profiles.
was constructed in Cytoscape based on data from The Comparative Toxicogenomics Database (CTD) (Fig. 4). Please see Methods section for procedure details. This network indicates that several drugs could influence the mRNA or protein expression of CCNB1. For instance, Cisplatin and Doxorubicin could decrease CCNB1 expression while Paclitaxel could increase CCNB1 expression. Interestingly, tamoxifen was also shown to upregulate the expression of CCNB1. Each arrow in this network is supported by previous reports.
Discussion The therapy of ER+ breast cancer, which represents more than 70% of breast tumors, is based on anti-hormonal compounds [26]. The anti-estrogen is a commonly used treatment for patients with ER+ breast cancer. For adjuvant therapy of ER+ breast cancer, hormone therapy improves overall survival and reduces risk for development of breast cancer [27]. Unfortunately, a subset of patients who received adjuvant hormone therapy would
eventually experience relapse and die as a result of the disease. In National Surgical Adjuvant Breast and Bowel Project (NSABP) prevention trial (P1), 30% of ER+ tumors were not prevented by hormone therapy [28]. Numerous studies have been performed, which combined endocrine therapy with agents that could modulate these mechanisms, so as to prevent the occurrence of hormone therapy resistance [29]. Due to the pressing clinical need, several other investigators have developed gene predictors that could predict outcome in ER+ breast cancer treated with adjuvant hormone therapy. For instance, Cyclin D1, Acid ceramidase 1and p53 accumulation had been reported that could predict outcome in ER+ breast cancer treated with adjuvant anti-estrogen therapy [30–36]. Likewise, Retinoic acid receptor alpha, CD44 and deltaEF1 had been reported to be involved in the development of hormone therapy resistance in breast cancer [37–39]. It has been reported that breast stem cells and Wnt signaling activation might be the underlying mechanism of resistance to tamoxifen [40,41]. CCNB1, a key regulator of cell cycle, is overexpressed in many human cancers, including breast cancer [42–45]. However, the
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Fig. 2. Kaplan–Meier plot for DFS (A and E), RFS (B), DMFS (D) and OS (C and F) of ER+ breast cancer patients classified according to the of CCNB1 expression levels. Significance was assessed by logrank test.
Fig. 3. (A and B) Kaplan–Meier plot for DMFS of hormone treated breast cancer patients classified according to the CCNB1 expression level, significance was assessed by logrank test.
association between CCNB1 overexpression and hormone therapy resistance in ER+ breast cancer remains unclear. Our results show the significant prognostic power of CCNB1 in ER+ breast cancer progression and hormone therapy resistance. Importantly, high level of CCNB1 is correlated with hormone treatment failure and poor DMFS. We might be able to stratify ER+ breast cancer patients
by testing the mRNA expression level of CCNB1 and decide when and how to use hormone therapy treatment in combination with appropriate therapeutic drug in the future. There are many studies that connect CCNB1 and poor prognosis. It is easy to ascribe the poor outcome to the CCNB1’s critical role in regulating cell cycle at G2/M phase. High expression of CCNB1 indicates active
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Fig. 4. Interaction network of CCNB1 and chemotherapeutic drugs. The gene–drug interaction network shows us how available chemotherapeutic drugs could decrease the expression of CCNB1. For example, Doxorubicin could decrease the expression of CCNB1, while Paclitaxcel could increase the expression of CCNB1.
proliferation and uncontrolled tumor growth. Alternatively, high level of CCNB1 indicated the failure of hormone therapy including tamoxifen and aromatase inhibitor [3]. However, there are several other ways. For instance, Song et al. have showed that CCNB1 promoted cancer invasion and metastasis through enhancing epithelial-mesenchymal transition [16]. Lu et al. found that CCNB1/ CDK1 inhibition could restore p53 function, which implies that CCNB1 participate in impairing p53 function and promote tumorigenesis [46]. Here, we also showed evidence that CCNB1 played a role in hormone therapy resistance in breast cancer, in vivo/ in vitro experiments are still needed to confirm the finding. Nevertheless, whether CCNB1 is a driver gene in hormone therapy resistance still requires experimental validation. Provided the high expression of CCNB1 is involved in the development of hormone therapy resistance, how to manage cancer patients with CCNB1 overexpression remains a great challenge. Here we show several drugs that could influence the expression of CCNB1 (Fig. 2). For instance, Doxorubicin and Cisplatin could decrease the expression of CCNB1, while Paclitaxel could increase the expression of CCNB1. In addition, tamoxifen seems to upregulate the expression of CCNB1. Since CCNB1 participate in the hormone therapy resistance, it is interesting to know whether ER+ breast cancer patient with CCNB1 overexpression could benefit from the repression of CCNB1, or in other words, whether CCNB1 is a promising target for preventing or could reverse hormone therapy resistance. More clinical data is needed to solve these issues. For example, the effects of CCNB1 overexpression could be verified in a clinical trial of early ER+ breast cancer (without distant metastasis), we estimate 120 patients (60 cases with high CCNB1 expression and 60 with low expression) would need to be included to demonstrate the effect based on our retrospective data. If this could be demonstrated, a further step would be to investigate whether early addition of platinum/etoposide-based chemotherapy improved outcomes in these patients. Although much information about ER and cancer has been provided in the past three decades since the arrival of hormone therapy in the clinic, a lot more needs to be elucidated for favorable therapeutic outcomes. More concrete research outcomes will warrant the translational research that may lead to more efficient and safer treatment for breast cancer patients as well as women at high risk of advanced breast cancer.
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