Oncogene (2015) 34, 237–245 & 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc ORIGINAL ARTICLE MIF4G domain containing protein regulates cell cycle and hepatic carcinogenesis by antagonizing CDK2-dependent p27 stability CWan1,4, S Hou2,4,RNi2,LLv2, Z Ding3, X Huang2, Q Hang3,SHe3, Y Wang3, C Cheng3,XXGu3,GXu1 and A Shen3 The CDK inhibitor p27kip1 plays crucial roles in cell cycle regulation and cancer progression. Through yeast two-hybrid screening, we identified MIF4G domain containing protein (MIF4GD) as a novel binding partner for p27. The association of MIF4GD and p27 was verified using immunoprecipitation and glutathione S-transferase (GST) pull-down assays. Interaction with MIF4GD led to the stabilization of p27 both in the nucleus and in the cytoplasm in hepatocellular carcinoma (HCC) cells as a result of suppressed phosphorylation of p27 by CDK2 at threonine187. Serum stimulation decreased the levels of MIF4GD and p27 simultaneously. In addition, MIF4GD overexpression resulted in increased p27 levels and reduced cell proliferation, while knockdown of MIF4GD promoted cell cycle progression with decreased p27 levels in cells. Furthermore, overexpression of MIF4GD reduced colony formation and inhibited xenograft tumor growth in nude mice. Finally, we found that both MIF4GD and p27 were expressed at low levels in HCC tissues compared to non-cancerous tissues, and that low expression levels of MIF4GD and p27 were associated with significantly worse prognosis in HCC patients. Our results suggest that MIF4GD is a potential regulator of p27-dependent cell proliferation in HCC. These findings provide a rational framework for the development of potential HCC therapy by targeting the MIF4GD–p27 interaction. Oncogene (2015) 34, 237–245; doi:10.1038/onc.2013.536; published online 16 December 2013 Keywords: p27; hepatocellular carcinoma; cell cycle regulation; MIF4GD INTRODUCTION stage and poorer disease-free survival in patients with HCC.8,11,14 Hepatocellular carcinoma (HCC) represents the fifth most common Previous studies have shown that phosphorylation of p27 protein malignancy and the third leading cause of cancer-related deaths (T187) by the cyclin E/CDK2 complex in mid-to-late G1 phase 15,16 in the world, especially in Asia and Africa. The high frequency of targets p27 to ubiquitin-mediated degradation, allowing G1–S HCC is mainly attributed to an increasing incidence of alcoholic transition to occur. Notably, hyperactive cyclin E/CDK2 can liver disease, along with hepatitis B or hepatitis C viral infections.1 promote nuclear p27 degradation in cancer cells, partly account- Despite vigorous efforts by both basic scientists and clinical ing for disrupted cell cycling and consequently malignant 9,17 practitioners, the underlying mechanisms of the pathogenesis of proliferation. The molecular switch that modulates p27 HCC remain poorly understood.2 Disruption of cell cycle control is activity in hepatocarcinogenesis is not clearly defined. one of the most frequent alterations in tumor cells, which often Our previous investigations have revealed a correlation results in uncontrolled cell proliferation.3 Therefore, many studies between p27 protein level and hepatocarcinogenesis with the have sought to elucidate the mechanism(s) underlying the underpinning that a ubiquitin–proteasome system is responsible 18,19 uncontrolled cell growth during hepatocarcinogenesis.4,5 These for p27 protein stability during that process. This observation studies have provided key evidence that cell cycle regulators, prompted us to search for proteins that could potentially regulate including pRb, p16INK4A, p21WAF1/CIP1 and p27Kip1, when aberrantly p27 stability and as such influence its activity in hepatocarcino- expressed or malfunctioning, may contribute to the pathogenesis genesis. Using yeast two-hybrid screening, we identified MIF4G of HCC.6–8 domain containing protein (MIF4GD) as a novel binding partner p27Kip1 belongs to the family of cyclin-dependent kinase for p27. MIF4GD, also known as stem-loop interacting protein 1, is inhibitors that encompass several structurally and functionally involved in replication-dependent translation of histone related proteins. Cyclin-dependent kinase inhibitors ensure the mRNAs.20,21 Interaction with MIF4GD stabilized p27 by correct timing of CDK activation in different phases of cell cycle antagonizing CDK2-mediated phosphorylation and subsequently and are thus considered tumor suppressors.9,10 p27 directly proteasomal degradation, rendering the cyclin E/CDK2 complex inhibits the cyclin E/CDK2 complex, thereby inducing G1-phase inactive and stalling cell cycle progression in HCC, but not normal arrest and halting cell cycle progression.9,11 Downregulation of cells. Importantly, high MIF4GD expression levels were found to p27 has been detected in various human cancers,8,9 including correlate with suppressed cell proliferation and improved HCC, suggesting that p27 may contribute to liver oncogenesis.12,13 prognosis in patients with HCC. Therefore, our finding suggests In addition, it has been reported that reduced p27 expression is that MIF4GD may function as a suppressor of HCC by linking p27 associated with a higher tumor grade, more advanced tumor stabilization to decelerated cell cycle progression. 1School of Public Health, Nantong University, Nantong, People’s Republic of China; 2Department of Gastroenterology, Nantong University, Nantong, People’s Republic of China and 3Department of Immunology, Nantong University, Nantong, People’s Republic of China. Correspondence: Professor A Shen, Department of Immunity, Nantong University, 19 Qixiu Road, Nantong 226001, People’s Republic of China. E-mail: [email protected] 4These authors contributed equally to this work. Received 14 April 2013; revised 9 November 2013; accepted 11 November 2013; published online 16 December 2013 MIF4GD stabilizes p27 to regulate HCC progression C Wan et al 238 RESULTS p27 in two different HCC cell lines without altering the abundance MIF4GD interacts with p27 of cyclin A. In contrast, depletion of endogenous MIF4GD by short To tackle how p27 activity is regulated during cell cycling, we hairpin RNA (shRNA) accelerated degradation of p27 in Huh7 and performed a yeast two-hybrid screening using p27 as a bait. HepG2 HCC cells (Figures 3c and d). MIF4GD was identified in this process as a potential binding p27 protein is present both in the nucleus and in the cytoplasm partner for p27 (data not shown). The interaction between p27 with distinct functionalities. In the nucleus, p27 serves as a CDK and MIF4GD was verified by reciprocal immunoprecipitations as inhibitor to impede cell proliferation, while cytoplasmic p27 binds p27 was brought down by an anti-MIF4GD antibody from 293T with RhoA to promote cell migration. We then investigated cell lysate and vice versa (Figure 1a). Similarly, we observed that whether MIF4GD could influence p27 subcellular distribution. To MIF4GD interacted with p27 in normal liver tissue and HCC cells, this end, Huh7 cells transfected with MIF4GD-myc were subjected while the interactions of MIF4GD and p27 with another important to cell fractionation followed by western blot analysis. As shown in cell cycle regulator, p53, were not detected (Figure 1b). To Figure 3e, both nuclear p27 and cytoplasmic p27 were increased evaluate whether the association of p27 with MIF4GD was direct, in the presence of ectopic MIF4GD. However, the ratio of nuclear GST pull-down assay was performed. Indeed, GST-p27, but not and cytoplasmic p27 did not alter significantly. Collectively, these GST, precipitated with HA-tagged MIF4GD (Figure 1c). These data suggest that MIF4GD stabilizes p27 protein in HCC cells. observations were corroborated by immunofluorescence assay that showed partial co-localization of p27 and MIF4GD in HCC cells MIF4GD modulates the phosphorylation status of p27 (Figure 1d). The degradation of p27 is mainly regulated through phosphoryla- To further delineate the p27–MIF4GD interaction, we performed tion-dependent ubiquitin–proteasome proteolysis. Three major immunoprecipitation assays employing various p27 and MIF4GD phosphorylation sites have been reported to regulate the stability mutants. As shown in Figure 2, p27 interacted with MIF4GD via a of p27, namely S10, T187 and T198.22 Thus, we examined whether region flanked by the CDK binding domain and the nucleus MIF4GD regulates p27 stability by virtue of modulating the localization signal motif, while MIF4GD interacted with p27 via its phosphorylation status of p27. As shown in Figure 4a, while the MIF4G domain. Taken together, these data suggest that p27 phosphorylation of S10 and T198 sites seemed to be elevated, interacts with MIF4GD in vivo and in vitro, indicating that MIF4GD T187 phosphorylation was reduced. In order to confirm the could potentially influence p27 function. hypothesis that MIF4GD impacts p27 stability in a T187 phosphorylation-dependent manner, we assessed the stability of both wild-type (WT) and phosphorylation-defective (S10A, T187A MIF4GD stabilizes p27 in HCC cells and T198A) p27 proteins either in the absence or in the presence Of great intrigue, the level of endogenous p27 increased of MIF4GD. Indeed, MIF4GD prolonged the half-life of the wild dramatically in MIF4GD-transfected cells (Figure 1d, white type (WT), S10A and T198A p27, but failed to impact the T187A arrowheads). Thus, we investigated the detailed mechanism mutant (Figure 4b). Importantly, MIF4GD suppressed ubiquitina- for how MIF4GD regulates