Oncogene (2013) 32, 947–960 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc ORIGINAL ARTICLE Prox1 suppresses the proliferation of neuroblastoma cells via a dual action in p27-Kip1 and Cdc25A IP Foskolou, D Stellas, I Rozani, MD Lavigne and PK Politis Neuroblastoma is a pediatric tumor that originates from precursor cells of the sympathetic nervous system with less than 40% long- term survival in children diagnosed with high-risk disease. These clinical observations underscore the need for novel insights in the mechanisms of malignant transformation and progression. Accordingly, it was recently reported that Prox1, a homeobox transcription regulator, is expressed in higher levels in human neuroblastoma with favorable prognosis. Consistently, we have recently shown that Prox1 exerts a strong antiproliferative effect on neural precursor cells during embryonic development. Thus, Prox1 is a candidate gene with a critical role in suppressing malignant neuroblastoma transformation. Here, we provide evidence that Prox1 strongly suppresses the proliferation of mouse and human neuroblastoma cell lines and blocks the growth of neuroblastoma tumors in SCID mice. Conversely, short hairpin RNA (shRNA) -mediated knockdown of basal Prox1 expression significantly induces proliferation, genomic instability and the ability of neuroblastoma cells to form tumors. Mechanistically, analysis of an inducible Prox1-overexpressing Neuro2A cell line indicates that Prox1 is sufficient to suppress CyclinD1, CyclinA and CyclinB1, consistent with a role in cell cycle arrest. Surprisingly, Prox1 strongly induces CyclinE1 expression in the same system despite its action on blocking cell cycle progression, which could account for the context dependent oncogenic function of Prox1. Most importantly, Prox1 was sufficient to decrease Cdc25A and induce p27-Kip1, but not p21-Cip1 or p53. By alleviating the Prox1 action in Cdc25A and p27-Kip1 expression, we were able to rescue its effect on cell cycle arrest. Together these data suggest that Prox1 negatively regulates neuroblastoma carcinogenesis through suppression of Cdc25A and induction of p27-Kip1 to counteract CyclinE1 overexpression and block cell cycle progression. Furthermore, these observations render Prox1 a candidate target for the treatment of neuroblastoma tumors. Oncogene (2013) 32, 947–960; doi:10.1038/onc.2012.129; published online 16 April 2012 Keywords: prospero; cyclinE1; G0/G1-arrest; cell cycle; SH-SY5Y; tet-on INTRODUCTION of proliferation and differentiation in neural precursor cells.5 In Neuroblastoma is a malignancy of early childhood that arises from agreement, recent data suggest that Notch1 expression predicts the developing autonomic nervous system.1 The clinical behavior an unfavorable prognosis for patients with neuroblastoma and is variable, ranging from spontaneous regression to highly could serve as a therapeutic target.6 Therefore, Prox1 may be aggressive metastatic disease with a poor overall survival rate. It involved in the malignant transformation, progression and/or is the most frequently diagnosed neoplasm during infancy and regression of neuroblastoma through Notch signaling regulation less than 40% of children with high-risk neuroblastoma are likely and/or additional genes and pathways. to achieve long-term cure, despite dramatic escalations in the Multiple lines of evidence suggest important roles for Prox1 in intensity of therapy provided.2 Future therapies will focus different aspects of embryonic development and morphogenesis, increasingly on the genes and biological pathways that while mouse embryos deficient in Prox1 die at E14.5. Prox1 has contribute to this malignancy. been previously shown to have essential roles during lymphatic, Towards this aim, it was recently reported that expression of hepatocyte, pancreatic, heart, lens, retinal and spinal cord Prox1, a homeobox transcription regulator, in human neuroblas- development.5,7–10 Prox1 has also been implicated in both toma tumors is correlated with the stage 4S, which has favorable progression and suppression of malignancies. A function for prognosis with high incidence of spontaneous regression. More- Prox1 as tumor suppressor has been described previously in other over, it was shown that Prox1 is highly expressed in sympathetic systems. Decreased expression levels have been found in neurons during early stages of development in chick, mouse and hepatocellular and pancreatic carcinoma,11,12 which may be due human peripheral nervous system and its expression levels are to epigenetic silencing. Hypermethylation of the Prox1 gene has reduced in highly proliferative human neuroblastoma cell lines.3,4 been found in breast cancer and lymphomas and in brain These observations suggest that Prox1 might have a tumor metastases of breast cancer.13,14 Moreover, mutations of Prox1 suppressor function in neuroblastoma cancer. Accordingly, we and loss of heterozygosity have been reported in various have recently reported that Prox1 negatively regulates the cancers.13,15,16 Although most studies point to a tumor proliferation of neural precursor cells during embryonic suppressor function of Prox1, a recent study reports that it development. Furthermore, we showed that a cross-inhibitory enhances colorectal cancer progression.17 These data suggest that interaction between Prox1 and Notch1 is involved in the regulation Prox1 may function in a context dependent manner, being a Center for Basic Research, Biomedical Research Foundation of the Academy of Athens, Athens, Greece. Correspondence: Dr PK Politis, Center for Basic Research, Biomedical Research Foundation of the Academy of Athens, 4 Soranou Efesiou, Athens 11527, Greece. E-mail: [email protected] Received 1 November 2011; revised 28 February 2012; accepted 7 March 2012; published online 16 April 2012 Prox1 blocks cell cycle progression in neuroblastoma cells IP Foskolou et al 948 tumor suppressor in certain cells, while in other cell types could be RESULTS related to tumor progression. This dual function may reside in the Prox1 blocks proliferation of neuroblastoma cancer cells fact that Prox1 affects differentially the expression of genes that To evaluate whether the observed correlation between Prox1 promote or inhibit proliferation and cell cycle progression. expression and suppression of neuroblastoma progression has Here, we provide functional evidence that Prox1 is indeed functional importance, we used neuroblastoma cell lines as a involved in the suppression of neuroblastoma cell growth both model system to analyze proliferation. We first examined the in vitro and in vivo. Mechanistically, activated Notch1 signaling effect of mouse or human Prox1 overexpression in Neuro2A (N2A) cannot rescue the negative effect of Prox1 on neuroblastoma mouse neuroblastoma cell line. BrdU incorporation assays proliferation, suggesting an alternative mode of action. Accord- revealed a strong block in proliferation after mouse or human ingly, we showed that Prox1 acts in basic components of cell cycle Prox1 overexpression (Figures 1a and b and Supplementary Figure machinery to regulate cellular proliferation in neuroblastoma cells. S1a). In addition, immunostainings with phosphorylated-histo- Collectively, these observations indicate that Prox1 negatively neH3 (pH3) revealed a strong reduction in Prox1 þ N2A cells regulates tumorigenic properties of neuroblastoma cells, may be undergoing mitosis (Figures 1d and e and Supplementary Figure implicated in suppressing neuroblastoma carcinogenesis, and S1b). To exclude the possibility of non-cell autonomous effects, we could be utilized for its treatment. also measured the proliferation rate in the nontransfected cells. In Figure 1. Prox1 has a strong antiproliferative effect on mouse and human neuroblastoma cell lines. (a) Prox1 or GFP transfected N2A cells were pulsed for 2 h with BrdU and then labeled for BrdU and 4,6-diamidino-2-phenylindole (DAPI). Scale bar: 25 mm. (b) Quantification of the transgene-positive/BrdU-positive cells. (GFP: 43.6±4.26%, Prox1: 1.86±0.59%; Po0.01) (c) Quantification of the transgene negative/BrdU- positive cells (GFP: 40.56±3.01%, Prox1: 39.13±1.66%; P40.1). (d) Prox1- or GFP-transfected N2A cells were labeled for pH3 and DAPI. Scale bar: 25 mm. (e) Quantification of the transgene-positive/pH3-positive cells (GFP: 11.52±1.2%, Prox1: 1.09±0.25%; Po0.05). (f) Quantification of the transgene-negative/pH3-positive cells (GFP: 11.66±0.47%, Prox1: 11.47±1.2%; P40.1). (g, h) Quantification of the transgene-positive/ BrdU-positive cells in SH-SY5Y and Kelly human neuroblastoma cell lines, respectively, (GFP: 23.78±1.68%, Prox1: 3.89±0.59%; Po0.01 in SH-SY5Y cells; GFP: 51.37±5.29%; Prox1: 20.5±0.86%; Po0.05 in Kelly cells). Oncogene (2013) 947 – 960 & 2013 Macmillan Publishers Limited Prox1 blocks cell cycle progression in neuroblastoma cells IP Foskolou et al 949 all cases, the untransfected cells in Prox1 experiments showed observations with MTT assay and PCNA immunostainings (Figures proliferation rates similar to green fluorescent protein (GFP) 3k–m). Collectively, these observations show that Prox1 negatively experiments (Figures 1c and f), further confirming that the defect affects proliferative and tumor forming abilities of neuroblastoma in proliferation is because of Prox1 overexpression. Prox1 was also cells in vitro with no indication of increased cell death. sufficient to suppress proliferation in human neuroblastoma cell lines, including SH-SY5Y and Kelly (Figures