Activated Notch signaling augments cell growth in hepatocellular carcinoma via up-regulating the nuclear receptor NR4A2

// Bo Zhu 1, * , Lichun Sun 1, 2, 3, 4, * , Wei Luo 3 , Min Li 4, 5 , David H. Coy 4 , Long Yu 1 , Wenbo Yu 1 1 State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Fudan University, Shanghai 200433, China 2 Hunan Province Cooperative Innovation Center for Molecular Target New Drug Study, Institute of Pharmacy and Pharmacology, University of South China, Hengyang 421001, China 3 Department of Orthopedics, Xiangya Hospital, Central South University, Changsha, Hunan 410008, China 4 Department of Medicine, School of Medicine, Tulane Health Sciences Center, New Orleans, LA 70112-2699, USA 5 Department of Radiation Oncology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China * These authors contributed equally to this work Correspondence to: Lichun Sun, email: peptide612@gmail.com , lsun@tulane.edu Wenbo Yu, email: 12111010012@fudan.edu.cn Keywords: hepatocellular carcinoma, Notch signaling, Notch1, nuclear receptor, NR4A2 Received: November 02, 2016      Accepted: February 13, 2017      Published: February 21, 2017 ABSTRACT Hepatocellular carcinoma (HCC) is one of the most malignant cancers. Conventional therapies are limited due to the human liver being such a unique organ and easily showing side-effects. The unclear molecular mechanisms are tough challenges for scientists searching for new and effective anti-HCC targeting drugs. We identified that the nuclear receptor NR4A2 is a novel oncogene in HCC progression. In this study, we show that NR4A2 and the notch recceptor Notch1 were expressed highly in primary HCC tissues and immortal HCC cells by using qPCR, western blot and immuno-histochemistry assays. Both genes were observed to stimulate HCC cell proliferation, anti-apoptosis and cell cycle arrest by using cell proliferation assays and FACS assays. We also observed that the four notch receptor subtypes (Notch1-4) displayed different effects on HCC cell growth. The over-expression of Notch1 by transiently transfecting the intracellular domain of Notch1 (ICN1, Notch1 active form) increased the expression of NR4A2, with the knockdown of Notch1 decreasing NR4A2. This indicates that NR4A2 is one of the Notch-mediated downstream genes. Moreover, both NR4A2 and Notch1 suppressed the expression of tumor suppressors p21 and p63. These findings support that Notch1/NR4A2 co-regulate HCC cell functions by playing oncogenic roles and regulating the associated downstream signaling pathways. Novel Notch1/NR4A2-mediated oncogenic signaling may provide us a great opportunity for anti-HCC drug development.