Functional Dissection of HOXD Cluster Genes in Regulation of Neuroblastoma Cell Proliferation and Differentiation Yunhong Zha1,4., Emily Ding3., Liqun Yang1, Ling Mao1,5, Xiangwei Wang1,6, Brian A. McCarthy1, 2 1 Shuang Huang , Han-Fei Ding * 1 Cancer Center and Department of Pathology, Medical College of Georgia, Georgia Health Sciences University, Augusta, Georgia, United States of America, 2 Department of Biochemistry and Molecular Biology, Medical College of Georgia, Georgia Health Sciences University, Augusta, Georgia, United States of America, 3 Lakeside High School, Evans, Georgia, United States of America, 4 Department of Neurology, the First People’s Hospital of Yichang, Three Gorges University College of Medicine, Yichang, Hubei, China, 5 Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China, 6 Department of Urology, Second Affiliated Hospital, Third Military Medical University, Chongqing, China Abstract Retinoic acid (RA) can induce growth arrest and neuronal differentiation of neuroblastoma cells and has been used in clinic for treatment of neuroblastoma. It has been reported that RA induces the expression of several HOXD genes in human neuroblastoma cell lines, but their roles in RA action are largely unknown. The HOXD cluster contains nine genes (HOXD1, HOXD3, HOXD4, and HOXD8-13) that are positioned sequentially from 39 to 59, with HOXD1 at the 39 end and HOXD13 the 59 end. Here we show that all HOXD genes are induced by RA in the human neuroblastoma BE(2)-C cells, with the genes located at the 39 end being activated generally earlier than those positioned more 59 within the cluster. Individual induction of HOXD8, HOXD9, HOXD10 or HOXD12 is sufficient to induce both growth arrest and neuronal differentiation, which is associated with downregulation of cell cycle-promoting genes and upregulation of neuronal differentiation genes. However, induction of other HOXD genes either has no effect (HOXD1) or has partial effects (HOXD3, HOXD4, HOXD11 and HOXD13) on BE(2)-C cell proliferation or differentiation. We further show that knockdown of HOXD8 expression, but not that of HOXD9 expression, significantly inhibits the differentiation-inducing activity of RA. HOXD8 directly activates the transcription of HOXC9, a key effector of RA action in neuroblastoma cells. These findings highlight the distinct functions of HOXD genes in RA induction of neuroblastoma cell differentiation. Citation: Zha Y, Ding E, Yang L, Mao L, Wang X, et al. (2012) Functional Dissection of HOXD Cluster Genes in Regulation of Neuroblastoma Cell Proliferation and Differentiation. PLoS ONE 7(8): e40728. doi:10.1371/journal.pone.0040728 Editor: Neil A. Hotchin, University of Birmingham, United Kingdom Received December 2, 2011; Accepted June 13, 2012; Published August 7, 2012 Copyright: 2012 Zha et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Institutes of Health grant CA124982 and a Georgia Cancer Coalition Distinguished Scholar Award to HD. LM was supported in part by the National Science Foundation of China (NSFC 81101905) and XW by the National Natural Science Foundation of China (No. 81172443). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: hding@georgiahealth.edu . These authors contributed equally to this work. Introduction Retinoic acid (RA), a derivative of vitamin A, has a key role in vertebrate morphogenesis, cellular differentiation, and tissue homeostasis. In embryonic development of the nervous system, RA is essential for the organization of the posterior hindbrain and anterior spinal cord, and for the differentiation and specification of motor neurons [1,2]. This function of RA depends critically on its ability to regulate the expression of HOX genes that encode a family of transcriptional factors with the ability to specify positional identities of tissues along the anteroposterior axis [3,4,5]. In vitro, RA can induce neuronal differentiation of embryonal carcinoma stem cells [6,7], embryonic stem cells [8] and neuroblastoma cells [9], which is associated with induction of HOX genes [10,11,12,13,14,15]. However, the precise roles of these upregulated HOX genes in RA-induced neuronal differen- tiation are not well understood. Neuroblastoma is a common childhood malignant tumor of the sympathetic nervous system that arises either in the adrenal medulla or in paravertebral sympathetic ganglia [16,17]. It has long been recognized that neuroblastoma differentiation states strongly affect clinical outcomes: patients with neuroblastomas of differentiating histology have a significantly better chance of survival than those with undifferentiated or poorly differentiated neuroblastomas [18,19,20,21,22]. Because of its ability to induce differentiation of neuroblastoma cells, RA has been used in clinic as a therapeutic agent for high-risk neuroblastomas [23,24]. Thus, identification of the downstream effectors of RA signaling and elucidation of the modes of their action may identify new drug targets for differentiation-based neuroblastoma therapy. We have recently reported that high-level HOXC9 expression is associated with neuroblastoma differentiation and is prognostic for better survival in neuroblastoma patients. HOXC9 induces the growth arrest and neuronal differentiation of neuroblastoma cells by directly targeting both cell cycle-promoting and neuronal PLoS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e40728