KU-0060648 inhibits hepatocellular carcinoma cells through DNA-PKcs-dependent and DNA-PKcs-independent mechanisms

// Min-Bin Chen 1, * , Zhen-Tao Zhou 2, * , Lan Yang 3, * , Mu-Xin Wei 4 , Min Tang 1 , Ting-Yan Ruan 5 , Jun-Ying Xu 5 , Xiao-zhong Zhou 2 , Gang Chen 6 , Pei-Hua Lu 5 1 Department of Oncology, Kunshan First People’s Hospital Affiliated to Jiangsu University, Kunshan 215300, China 2 Department of Orthopedics, the Second Affiliated Hospital of Soochow University, Suzhou 215000, China 3 Department of Breast Surgery, the Third Affiliated Hospital of Soochow University, Changzhou 213003, China 4 Department of Traditional Chinese Medicine, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China 5 Department of Medical Oncology, Wuxi People’s Hospital Affiliated to Nanjing Medical University, Wuxi 214023, China 6 Department of Neurosurgery, the First Affiliated Hospital of Soochow University, Suzhou 215006, China * These authors have contributed equally to this work Correspondence to: Pei-Hua Lu, e-mail: lphty1_1@163.com Xiao-zhong Zhou, e-mail: zhouxz@suda.edu.cn Gang Chen, e-mail: nju_neurosurgery@163.com Keywords: hepatocellular carcinoma (HCC), DNA-PKcs, KU-0060648, PI3K-AKT-mTOR signaling, miRNA Received: October 22, 2015     Accepted: February 05, 2016     Published: February 26, 2016 ABSTRACT Here we tested anti-tumor activity of KU-0060648 in preclinical hepatocellular carcinoma (HCC) models. Our results demonstrated that KU-0060648 was anti-proliferative and pro-apoptotic in established (HepG2, Huh-7 and KYN-2 lines) and primary human HCC cells, but was non-cytotoxic to non-cancerous HL-7702 hepatocytes. DNA-PKcs (DNA-activated protein kinase catalytic subunit) is an important but not exclusive target of KU-0060648. DNA-PKcs knockdown or dominant negative mutation inhibited HCC cell proliferation. On the other hand, overexpression of wild-type DNA-PKcs enhanced HepG2 cell proliferation. Importantly, KU-0060648 was still cytotoxic to DNA-PKcs-silenced or -mutated HepG2 cells, although its activity in these cells was relatively weak. Further studies showed that KU-0060648 inhibited PI3K-AKT-mTOR activation, independent of DNA-PKcs. Introduction of constitutively-active AKT1 (CA-AKT1) restored AKT-mTOR activation after KU-0060648 treatment in HepG2 cells, and alleviated subsequent cytotoxicity. In vivo , intraperitoneal ( i.p. ) injection of KU-0060648 significantly inhibited HepG2 xenograft growth in nude mice. AKT-mTOR activation was also inhibited in xenografted tumors. Finally, we showed that DNA-PKcs expression was significantly upregulated in human HCC tissues. Yet miRNA-101, an anti-DNA-PKcs miRNA, was downregulated. Over-expression of miR-101 in HepG2 cells inhibited DNA-PKcs expression and cell proliferation. Together, these results indicate that KU-0060648 inhibits HCC cells through DNA-PKcs-dependent and -independent mechanisms.