Differential expression of neurogenes among breast cancer subtypes identifies high risk patients The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Fernández-Nogueira, Patricia, Paloma Bragado, Vanessa Almendro, Elisabet Ametller, Jose Rios, Sibgat Choudhury, Mario Mancino, and Pedro Gascón. 2016. “Differential expression of neurogenes among breast cancer subtypes identifies high risk patients.” Oncotarget 7 (5): 5313-5326. doi:10.18632/oncotarget.6543. http:// dx.doi.org/10.18632/oncotarget.6543. Published Version doi:10.18632/oncotarget.6543 Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:27320293 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 5 Differential expression of neurogenes among breast cancer subtypes identifies high risk patients Patricia Fernández-Nogueira1,3, Paloma Bragado1, Vanessa Almendro4, Elisabet Ametller1,2, Jose Rios5,6, Sibgat Choudhury4, Mario Mancino1,2,*, Pedro Gascón1,2,3,* 1Department of Medical Oncology, Hospital Clínic, Barcelona, Spain 2Institut d’Investigacions Biomediques August Pi i Sunyer Barcelona, Barcelona, Spain 3Department of Medicine, University of Barcelona, Barcelona, Spain 4 Division of Medical Oncology, Department of Medicine, Harvard Medical School, Dana-Farber Cancer Institute, Brigham and Women’s Hospital, Boston, MA, USA 5 Medical Statistics Core Facility, IDIBAPS, (Hospital Clinic) Barcelona, Barcelona, Spain 6Biostatistics Unit, Faculty of Medicine, Universitat Autònoma de Barcelona, Barcelona, Spain *These authors have contributed equally to this work Correspondence to: Mario Mancino, email: [email protected] Pedro Gascón, email: [email protected] Keywords: breast cancer, neurogenes, neuropeptides, neurotransmitters, tumor microenvironment Received: September 01, 2015 Accepted: November 22, 2015 Published: December 10, 2015 ABSTRACT The nervous system is now recognized to be a relevant component of the tumor microenvironment. Receptors for neuropeptides and neurotransmitters have been identified in breast cancer. However, very little is known about the role of neurogenes in regulating breast cancer progression. Our purpose was to identify neurogenes associated with breast cancer tumorigenesis with a potential to be used as biomarker and/or targets for treatment. We used three databases of human genes: GeneGo, GeneCards and Eugenes to generate a list of 1266 relevant neurogenes. Then we used bioinformatics tools to interrogate two published breast cancer databases SAGE and MicMa (n=96) and generated a list of 7 neurogenes that are differentially express among breast cancer subtypes. The clinical potential was further investigated using the GOBO database (n=1881). We identified 6 neurogenes that are differentially expressed among breast cancer subtypes and whose expression correlates with prognosis. Histamine receptor1 (HRH1), neuropilin2 (NRP2), ephrin-B1 (EFNB1), neural growth factor receptor (NGFR) and amyloid precursor protein (APP) were differentially overexpressed in basal and HER2-enriched tumor samples and syntaxin 1A (STX1A) was overexpressed in HER2-enriched and luminal B tumors. Analysis of HRH1, NRP2, and STX1A expression using the GOBO database showed that their expression significantly correlated with a shorter overall survival (p < 0.0001) and distant metastasis-free survival (p < 0.0001). In contrast, elevated co-expression of NGFR, EFNB1 and APP was associated with longer overall (p < 0.0001) and metastasis- free survival (p < 0.0001). We propose that HRH1, NRP2, and STX1A can be used as prognostic biomarkers and therapeutic targets for basal and HER2-enriched breast cancer subtypes. INTRODUCTION its metastases. Intratumor heterogeneity facilitates tumor progression and fosters the continuous adaptation and Intratumor heterogeneity refers to the coexistence of survival of the different tumor-propagating clones to the subpopulations of cancer cells diverging in their genetic, different microenvironments in which a tumor resides. A phenotypic, or behavioral characteristics within a given high degree of heterogeneity has been observed in many primary tumor, and between a given primary tumor and tumor types, including breast [1], prostate [2], ovarian [3], www.impactjournals.com/oncotarget 5313 Oncotarget bladder [4], and pancreatic cancers [5, 6], as well as in markers have poor prognosis [21]. Synaptophysin, a glioma [7], chronic lymphocytic leukemia [8], multiple protein found in neuroendocrine cells and in virtually myeloma [9], and acute myeloid leukemia [5]. all neurons in the central nervous system participating Insights from genomics have led to the in synaptic transmission, has been detected in breast identification of five molecular subtypes of breast cancer [22], colon [23], prostate [24], and brain [25] tumors as on the basis of gene expression patterns. Different well as melanoma [26], supporting the idea that nerve molecular subtypes of breast cancer have different clinical fibers infiltrate tumors. Furthermore, the release of outcomes and responses to chemotherapy [10]; therefore, neurotrophic factors such as norepinephrine, dopamine, intratumor heterogeneity represents a major challenge for and substance P appears to stimulate the growth of the design of effective therapies. Intratumor heterogeneity nerve fibers inside tumors [27–29]. Nerve endings results from the differentiation of stem-like cells and in turn release factors that stimulate the migratory along with clonal selection, enables the propagation of activity of tumor cells and promote metastasis [20]. the fittest clones for a given tumor microenvironment In addition, netrin-1 (an axonal guidance molecule) [11]. Breast cancer stem cells were initially identified and its receptor neogenin are involved in maintaining using membrane antigenic markers. In 2003, Al-Hajj et adhesion between basal and luminal cells in adjacent al.[12] first described the existence of a CD44+CD24- cap cells of the mammary gland terminal end buds subpopulation (hereinafter referred to as CD44+) in [30]. Netrin-1 regulates invasion and migration of breast cancer with properties of tumor stem cells. The breast epithelial tumor cells [31] and promotes the tumorigenic CD44+CD24−/low Lineage− population survival of tumor cells in metastatic breast cancer [32]. shares with normal stem cells the ability to proliferate In this context, we recently demonstrated that netrin-1 extensively, and to give rise to diverse cell types with negatively regulates the expression of stem cell markers reduced developmental or proliferative potential. (Nanog, Oct3/4, and CRIPTO-1) in human embryonic Moreover, this cell population is rich in cells capable carcinoma cells and mouse embryonic stem cells [33]. of initiating tumors in immunosuppressed animals [12]. Furthermore, individual studies have linked various However, a large body of evidences has demonstrated that aspects of cancer biology to certain neurotransmitter this phenotype is heterogeneous and not expressed in all receptors, such as the beta-2 adrenergic receptor [34] breast cancers [13] [14]. In addition, Honeth et al. found and the tachykinin NK1 receptor [35] as well as to CD44 expression predominantly on the cells surface soluble factors such as bradykinin [36]. Substance P, membrane along with CD24 in the cytoplasm, and, most an inflammatory neuropeptide and its receptor NK1, interesting, they showed that CD44 protein distribution are overexpressed in breast cancer [37]. Our group or its degradation during tumor initiation and metastasis, found that blocking substance P signaling promotes may favor the enrichment of CD24 on the membrane [15]. death in breast cancer cells [38]. Moreover, we have Furthermore, It has been described that cancer cells can shown that substance P promotes cancer progression acquire a CD44+ /CD24– phenotype through epithelial- and drug resistance by contributing to persistent HER2 to-mesenchymal transition (EMT) [16] Moreover, Meyer activation [35]. However, the role of neurotransmitters et al. hypothesized that an interconversion between and their receptors in breast cancer progression is still the differentiated and stem-like phenotypes occurs in unclear. It could be hypothesized that, analogous to the breast cancer and suggested that epithelial like CD44+/ proinflammatory cytokines, certain neurotransmitters CD24+ can generate CD44+/CD24− cells during tumor and neuropeptides in the microenvironment may initiation [17]. Therefore, the CD44+ is heterogeneous; promote tumor progression by selecting certain nevertheless the expression of CD44 is correlated with a specifically responsive clones. The nervous system more aggressive phenotype in breast cancer and with poor could exert direct and indirect control of tumor outcome of patients with basal-like breast cancer [15]. progression mainly through modulation of the immune Increasing evidence suggests that the nervous system [39]. system itself, as well as neurotransmitters and To characterize the role of neurotransmitters, neuropeptides present in the tumor microenvironment, neuropeptides, neurotrophic factors, and axonal guidance play a role in orchestrating tumor progression. molecules in breast cancer progression, we