Oncogene (2011) 30, 1892–1900 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE LITAF and TNFSF15, two downstream targets of AMPK, exert inhibitory effects on tumor growth J Zhou1, Z Yang2, T Tsuji3, J Gong1, J Xie1, C Chen3,WLi1, S Amar4 and Z Luo1 1Department of Biochemistry, Boston University School of Medicine, Boston, MA, USA; 2Department of Biochemistry and Molecular Biology, College of Life Sciences, Shaanxi Normal University, Xi’an, PR China; 3Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA and 4Department of Periodontology and Oral Biology, Boston University School of Dental Medicine, Boston, MA, USA Lipopolysaccharide (LPS)-induced tumor necrosis factor Keywords: AMPK; LITAF; TNFSF15; p53; tumor (TNF) a factor (LITAF) is a multiple functional molecule suppressor; tumorigenesis whose sequence is identical to the small integral membrane protein of the lysosome/late endosome. LITAF was initially identified as a transcription factor that activates transcription of proinflammatory cytokine in Introduction macrophages in response to LPS. Mutations of the LITAF gene are associated with a genetic disease, called Lipopolysaccharide (LPS)-induced tumor necrosis fac- Charcot–Marie–Tooth syndrome. Recently, we have tor (TNF) a factor (LITAF) was initially characterized reported that mRNA levels of LITAF and TNF super- as a transcription factor that activates transcription of family member 15 (TNFSF15) are upregulated by 50 cytokines such as TNFa, interleukin 6, soluble TNF- adenosine monophosphate (AMP)-activated protein ki- receptor II (sTNF-RII) and chemokine interleukin-8 in nase (AMPK). The present study further assesses their macrophages in response to LPS (Myokai et al., 1999; biological functions. Thus, we show that 5-aminoimidazole- Tang et al., 2006). It was also described as a p53- 4-carboxamide ribonucleoside (AICAR), a pharmacological inducible gene (Polyak et al., 1997). Upon stimulation, activator of AMPK, increases the abundance of LITAF LITAF translocates to the nucleus and binds to a and TNFSF15 in LNCaP and C4-2 prostate cancer cells, specific sequence of the TNFa promoter to cooperate which is abrogated by small hairpin RNA (shRNA) or the with other transcription factors such as signal transdu- dominant-negative mutant of AMPK a1 subunit. Our cer and activator of transcription 6(B) to upregulate the data further demonstrate that AMPK activation upregu- transcription (Tang et al., 2005). Therefore, LITAF has lates the transcription of LITAF. Intriguingly, silencing an important role in inflammatory response. Interestingly, LITAF by shRNA enhances proliferation, anchorage- the sequence of LITAF is identical to the Small Integral independent growth of these cancer cells and tumor Membrane Protein of the Lysosome/late Endosome growth in the xenograft model. In addition, our study (SIMPLE), mutations of which are associated with a reveals that LITAF mediates the effect of AMPK by genetic disease called Charcot–Marie–Tooth disease, binding to a specific sequence in the promoter region. characterized by demyelinating disorders of the peripheral Furthermore, we show that TNFSF15 remarkably inhibits nervous system (Niemann et al., 2006). It is not clear the growth of prostate cancer cells and bovine aortic whether the mutations disable the transcriptional activity endothelial cells in vitro, with a more potent effect toward of LITAF or other functions. the latter. In conjuncture, intratumoral injection of TNF superfamily member 15 (TNFSF15) was first TNFSF15 significantly reduces the size of tumors and identified as an inhibitor of vascular endothelial cell number of blood vessels and induces changes that are growth factor, thus also named VEGI (Sethi et al., characteristic of tumor cell differentiation. Therefore, our 2009). This TNF subfamily contains three isoforms studies for the first time establish the regulatory axis of resulting from different splicing; the first one consists of AMPK–LITAF–TNFSF15 and also suggest that LITAF 174 amino acids, which shares 20–30% homology to may function as a tumor suppressor. other TNF family members, and two additional iso- Oncogene (2011) 30, 1892–1900; doi:10.1038/onc.2010.575; forms comprise 192 and 251 amino acids, respectively published online 10 January 2011 (Sethi et al., 2009). All the three isoforms are identical in the carboxy-terminal 151 amino acids. TNFSF15 has been shown to potently inhibit the growth of vascular endothelial cells (Yue et al., 1999; Zhai et al., 1999a) as Correspondence: Dr Z Luo, Department of Biochemistry, Boston well as tumor cells (Haridas et al., 1999). Several tumor University School of Medicine, 715 Albany Street, Evans 645, Boston, xenograft studies have revealed that TNFSF15 sup- MA 02118, USA E-mail: [email protected] presses tumor growth (Zhai et al., 1999b; Pan et al., Received 13 May 2010; revised 15 November 2010; accepted 16 2004; Hou et al., 2005). In addition, the longer form, November 2010; published online 10 January 2011 VEGI-251, also called TNF-like ligand 1, has been LITAF inhibition of tumorigenesis J Zhou et al 1893 implicated in autoimmunity and inflammation-induced immunity (Sethi et al., 2009; Bayry, 2010). 50 Adenosine monophosphate (AMP)-activated pro- tein kinase (AMPK) is a highly conserved serine/ threonine protein kinase, consisting of three subunits, a catalytic subunit (a) and two regulatory subunits (b and g) (Hardie, 2007). In mammals, each subunit of AMPK contains two to three isoforms (a1, a2; b1, b2; and g1, g2, g3). AMPK serves as a fuel gauge in maintaining energy homeostasis. Thus, it is activated under metabolic stress that increases the intracellular levels of AMP, which serves as an allosteric activator by binding to the g regulatory subunit. In addition, AMPK is phosphorylated and activated by upstream kinases, such as the tumor suppressor liver kinase B 1/serine/ threonine kinase 11 (LKB1) (Hardie, 2007; Luo et al., 2010). Under physiological conditions, AMPK is acti- vated by hormones and cytokines, including leptin (for example, in skeletal muscle) and adiponectin secreted from adipocytes, interleukin 6, and ciliary neurotrophic Figure 1 AMPK-dependent induction of LITAF by AICAR. factor (Zhang et al., 2009). In addition, AMPK can (a) Promoter activity assay. LNCaP cells were transfected with be activated by a variety of pharmacological agents. The a promoter-less firefly luciferase reporter plasmid (PGL3) or the prototypical activator is 5-aminoimidazole-4-carboxamide plasmid with insertion of LITAF promoter region and Renilla 1-D-ribonucleoside (AICAR), a cell-permeable agent that luciferase plasmid in the presence or absence of a plasmid encoding wild-type AMPK a1 subunit. Two days later, the cells were treated is phosphorylated after entering the cell and converted with AICAR for 8 h and luciferase assay was conducted as to ZMP (AICAR monophosphate), an AMP analog. described in Materials and methods. The luciferase activity was Importantly, two clinically used anti-diabetic drugs, normalized with Renilla luciferase activity and expressed as ratios metformin and thiazolidinediones, have been known to (mean±s.d., n ¼ 3). *Po0.01 as compared with basal LITAF activate AMPK (Zhang et al., 2009). promoter activity. (b) LNCaP cells were stably infected with a1 shRNA or scrambled shRNA retroviruses were treated with Recently, we have demonstrated that inhibition of AICAR (AI, 1 mM) for the indicated times. Cell lysates were AMPK exacerbates the malignant behavior of prostate blotted with antibodies, as indicated. cancer cells (Zhou et al., 2009). In exploring the mechanism underlying the inhibitory effects of AMPK, activity of LITAF. Thus, we placed the promoter region we have identified several interesting targets, among of LITAF upstream of the firefly luciferase report which are LITAF and TNFSF15. In the present gene (Tang et al., 2007). We then transfected LNCaP study, we aim to explore the relationship of these cells with this plasmid together with a plasmid encod- molecules with AMPK and assess their biological effects ing Renilla luciferase in the presence or absence of on tumor cell growth and tumor development. Our the plasmid for the wild-type a1 subunit of AMPK. results show that AMPK activation by AICAR up- After 48 h, the cells were treated with AICAR for 8 h. regulates LITAF, which in turn increases TNFSF15 Firefly luciferase activity was assayed and normalized expression. All these regulations occur at the level of with Renilla luciferase activity. As shown in Figure 1a, transcription. We further show that the knockdown of AICAR enhanced the luciferase activity by about two- LITAF expression by shRNA enhances cell prolifera- fold, while transfection of AMPK a1 subunit caused a tion, anchorage-independent growth of prostate cancer 1.5-fold increase, which was further increased by the cells and tumor development in xenograft model. treatment with AICAR. To ascertain if the change holds Finally, we show that TNFSF15 inhibits the growth of true at protein levels, we treated a prostate cancer cell prostate cancer cells in vitro and in vivo, with a more line, LNCaP cells, with AICAR for different periods of potent effect on endothelial cell growth and tumor time and examined the expression of LITAF. As shown angiogenesis. in Figure 1b, the treatment progressively increased the abundance of LITAF, whereas the induction was diminished in the cells expressing shRNA. A similar result was obtained