Medical Hypotheses 82 (2014) 398–400

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Medical Hypotheses

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IFI16 mis-localization can be a contributing factor to hepatocellularcarcinoma progression

0306-9877/$ - see front matter � 2014 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.mehy.2014.01.017

⇑ Corresponding author. Tel.: +86 028 85501267; fax: +86 028 85502243.E-mail address: lmy3985@sina.com (M. Li).

Xinli Shi a,b, Jingli Liu c, Qiujun Liu d, Mingyuan Li b,⇑a Department of Pathobiology and Immunology, Hebei University of Traditional Chinese Medicine, Shijiazhuang, Hebei 050200, Chinab Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, Chinac Department of Repairing and Servicing Technology of Medical Equipment, Bethune Medical Non-commissioned Officer Academy of PLA, Shijiazhuang, Hebei 050081, Chinad Department of Biochemistry, Luzhou Medical College, Luzhou 646000, China

a r t i c l e i n f o

Article history:Received 29 October 2013Accepted 18 January 2014

a b s t r a c t

Hepatocellular carcinoma (HCC) is a highly deadly cancer, with usually drug resistance. However themechanisms responsible for this phenomenon are poorly understood. Interferon-c inducible protein 16(IFI16), a multifunctional protein, has roles in anti-proliferation, autophagy, cell senescence, anti-inflam-mation, and DNA sensor to trigger innate immunity. IFI16 physiologically absents in adult healthy hepa-tocyte, but exists in liver cancer cells. Interestingly, increasing evidences suggest that dysregulationor/and loss of IFI16 function have a critical role in drug resistance and tumor progression. Furthermore,interaction with DNA or other protein depends on IFI16 localization. In our study, to our knowledge, wefirst showed that IFI16 is a chromatin-binding protein in four HCC cell lines with different TP53 genotype,but not in fetal liver cell line, L02 cells. However, the function of IFI16 subcellular localization has notbeen determined in HCC. Therefore, we present our study and theoretical basis and presume that chro-matin-bounding localization of IFI16 is associated with HCC progression. If we are able to acetylate or/and delete NLS of IFI16 with activated-p53 restoration, we may offer an alternative for HCC therapy.

� 2014 Elsevier Ltd. All rights reserved.

Introduction

Hepatocellular carcinoma (HCC) is the fifth most commonlydiagnosed cancer in men and the seventh in women, and remainsthird most deadly cancer in the world [1]. At present, radical sur-gery and chemotherapy are the main treatments for HCC. However,drug resistance usually limits the result of HCC chemotherapy.

Interferon-c inducible protein 16 (IFI16) physiologically ex-presses in tissues such as epithelial cells of the skin, gastrointesti-nal tract, and glands and ducts of breast tissue, but not interminally differentiated tissues [2,3]. However, modest levels ofIFI16 mRNA were found in adult healthy liver through tissue north-ern blot of 32P-labelled IFI16 cDNA and autoradiography analysis[2]. IFI16 protein expresses in fetal hepatocytes [4], but not in adulthealthy liver cells [3,5]. Meanwhile, IFI16 exists in most humanHCC tissues and cell lines [6]. Alternatively, IFI16 expression is reg-ulated at a post-transcriptional level as well as by the transcrip-tional mechanism [7]. In addition, hepatocytic expression of IFI16is mainly detected during acute liver graft rejection [8] and inthe liver infected with virus [9]. For instance, an estimated 80%of global HCC is due to HBV and/or HCV infections [10]. IFI16 up-regulates in chronic HCV liver tissue through DNA microarray

analysis [11]. IFI16 has roles in anti-proliferation [12], autophagy[13], cell senescence [14], and anti-inflammation [15]. Interest-ingly, IFI16 acts as DNA sensor to trigger IFN-b expression in thecytoplasm [16,17]. However, IFI16 primarily localizes in the nu-cleus with multipartite nuclear localization signal (NLS) [18].Therefore, sensing DNA or interaction with other protein is dueto IFI16 subcellular localization [4,19].

The hypothesis

We hypothesized that IFI16 mis-localization may be a contrib-uting factor to HCC progression, but not IFI16 expression level.

Evaluation of the hypothesis

IFI16 missing or/and dysregulation induce some tumor

IFI16 is involved in liver cancer progression. Interestingly,increasing evidences suggest that dysregulation or/and loss ofIFI16 function have a critical role in drug resistance and tumor pro-gression [20].

IFI16 expression levels negatively associate with head and necksquamous cell carcinomas (HNSCC) tumor histopathologic gradeand aggressive activity [21]. Consistently, one study showed thatIFI16 anti-proliferative activity is related to the expression level

X. Shi et al. / Medical Hypotheses 82 (2014) 398–400 399

in mice HNSCC cell xenograft models with restored IFI16 expres-sion in HNO136 (wild-type TP53) cells [22]. In addition, functionalp53 is one of the main regulators of IFI16 activity [23]. However,liver cancer is usually with inactivated- or null-p53 [24]. For in-stance, transfection IFI16 retards cell growth in oral squamous cellcarcinoma (OSCC) cells with wild-type p53, but not with inactived-p53 [25]. Similarly, loss of IFI16 function results in deregulation ofp53-mediated apoptosis leading to breast cancer development[26]. Adenovirus expression of IFI16 in MCF7 cells (null-IFI16 andwild-type p53) or co-expressed with p53 in EJ cells (null-p53 andwild-type IFI16) induces cell apoptosis in response to DNA-damag-ing agents [26].

In addition, one study proposes that male predominance inhepatocellular carcinoma [27]. Consistently, IFI16 binds anddown-regulates androgen receptor (AR) expression by a negativefeedback loop in prostate cancer cells [28]. About 60% of HCC isassociated with HBV in Africa and Asia, where HBV is endemic[10]. Genomic analysis of liver biopsy from three chimpanzeesshowed that elevated IFI16 blocks HBV replication and promotesHBV clearance [29]. Therefore, we proposed that increased IFI16expression in the HCC is similar to acute liver graft rejection [8]and virus-infected liver cells [9], which only might reflect hepaticreversion to fetal status after liver injury.

IFI16 is a chromatin-associated protein in HCC cells

IFI16 interacts with certain length dsDNA, which is required forIFI16 oligomerization to activate IFN-b expression and eliminateviral infection in the cytoplasm [16]. However, IFI16 was predom-inantly in the nucleus [18]. IFI16 containing an intact NLS is able torecognize dsDNA [18]. Interestingly, NLS deletion mutants aremore responsive to transfected viral dsDNA compared to wild-typeIFI16 [18]. In other words, subcellular localization may affect IFI16function as an innate immune sensor.

NLS acetylation on lysine residues suppresses IFI16 nuclear im-port to promote IFI16 cytoplasm accumulation and maximize im-mune system sensitivity in many cancer chemotherapies [18].Histone deacetylases (HDACs) and p300 regulate IFI16 subcellularlocalization through deacetylation and acetylation of the lysineresidues on NLS, respectively [18]. For instance, HDAC-dependentsilence of IFI16 gene in prostate epithelial cells contributes to pros-tate cancer development [30]. Some prostate cancer cell lines lackIFI16, in which missing IFI16 causes AR expression increased, lead-ing to cell proliferation and survival [30]. HDAC inhibitors, such asTrichostatin A (TSA) or CGK1026, upregulate the expression levelsof IFI16 mRNA and protein in human prostate cancer cells [30]. TSAleads also to cytoplasmic accumulation of nucleus protein IFI16[18].

Fig. 1. The subcelluar localization of IFI16 in HCC cell lines and L02 cells. (A) Representatcontrol. (B) Chromatin fractions were analyzed via Western blot. H2b and b-actin servedcytoplasmic proteins; (S2) soluble nuclear proteins; (P2) chromatin-enriched sediment;

In our study, we used a fetal liver cell line, L02 cells, and fourHCC cell lines with different TP53 genotype, including SMMC-7721 (wild-type TP53), HepG2 (wild-type TP53), Hep3B (nullTP53), and Huh-7 (mutant TP53) cells to investigate the expressionand subcellular localization of IFI16. First, total RNA was isolatedand reverse transcribed. Specific human primers were used as fol-lows (forward and reverse): IFI16: 50-GAA GTG CCA GCG TAA CTCCTA-30 and 50-TAC CTC AAA CAC CCC ATT CAC-30; TP53: 50-TAGTGT GGT GGT GCC CTA TGA G-30 and 50-AGT GTG ATG ATG GTGAGG ATG G-30; ACTB: 50-GGC ATC CAC GAA ACT ACC TTC A-30

and 50-GTG ATC TCC TTC TGC ATC CTG TC-30. ACTB was used asthe internal control. The RT-PCR was performed as follows: 40 cy-cles of 95 �C for 5 s and 60 �C for 30 s. All samples were analyzed intriplicate on the same plate. We found that the expression level ofIFI16 mRNA was lower in HCC cells compared with L02 cells by realtime RT-PCR (Fig. 1A). Then, we extracted the chromatin fractions[31] and used Western blot analysis to investigate the associationof IFI16 with chromatin by a special monoclonal anti-IFI16 anti-body (ab50004, Abcam, diluted 1:1000). Interestingly, IFI16 wasdetected in the chromatin-binding fraction of four HCC cells, butnot of L02 cells (Fig. 1B). These results suggest that IFI16 is a chro-matin-bound protein in four HCC cell lines and a nucleoplasmicprotein in L02 cells.

In view of the above findings and our research, we consideredthat mis-localization in chromatin-bounding fraction may associ-ate with HCC progression, while higher expression level of IFI16protein in HCC might reflect hepatic reversion to fetal status afterliver injury.

Consequences of the hypothesis

In summary, the aforementioned data suggested that chroma-tin-bounding localization of IFI16 is related to HCC progressionand chemotherapy effect. These results indicate that NLS acetyla-tion or/and NLS deletion of IFI16 with activated-p53 restorationmight offer an alternative for HCC therapy.

Conflict of interest

We declare that we have no conflict of interest.

Acknowledgments

This work was financially supported by the National ScienceFoundation of China (Grant no. 81071362) and the Science andTechnology Project of Hebei Province of China (Grant no.132777189). We thank Professor Liu Cong (West China Second

ive gels for real time RT-PCR analysis of IFI16 mRNA. The ACTB gene was used as theas the quality control for nuclear fraction and cytoplasm fraction, respectively. (S1)(WCE) whole-cell extracts.

400 X. Shi et al. / Medical Hypotheses 82 (2014) 398–400

University Hospital/West China Women’s and Children’s Hospital)for kindly supplying us with the liver cell lines.

References

[1] Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin2013;63(1):11–30.

[2] Wei W, Clarke CJP, Somers GR, Cresswell KS, Loveland KA, Trapani JA, et al.Expression of IFI 16 in epithelial cells and lymphoid tissues. Histochem CellBiol 2003;119(1):45–54.

[3] Gariglio M, Azzimonti B, Pagano M, Palestro G, de Andrea M, Valente G, et al.Immunohistochemical expression analysis of the human interferon-induciblegene IFI16, a member of the HIN200 family, not restricted to hematopoieticcells. J Interferon Cytokine Res 2002;22(7):815–21.

[4] Ludlow LEA, Johnstone RW, Clarke CJP. The HIN-200 family: more thaninterferon-inducible genes? Exp Cell Res 2005;308(1):1–17.

[5] Xin H, Curry J, Johnstone RW, Nickoloff BJ, Choubey D. Role of IFI 16, a memberof the interferon-inducible p200-protein family, in prostate epithelial cellularsenescence. Oncogene 2003;22(31):4831–40.

[6] Boyault S, Rickman DS, De Reyniès A, Balabaud C, Rebouissou S, Jeannot E, et al.Transcriptome classification of HCC is related to gene alterations and to newtherapeutic targets. Hepatology 2007;45(1):42–52.

[7] Clarke C, Apostolidis V, Hii L, Gough D, Trapani J, Johnstone R. Critical role ofthe transcription factor AP-1 for the constitutive and interferon-inducedexpression of IFI 16. J Cell Biochem 2003;89(1):80–93.

[8] Borgogna C, Toniutto P, Smirne C, Azzimonti B, Rittà M, Avellini C, et al.Expression of the interferon-inducible proteins MxA and IFI16 in liverallografts. Histopathology 2009;54(7):837–46.

[9] Jenner RG, Young RA. Insights into host responses against pathogens fromtranscriptional profiling. Nat Rev Microbiol 2005;3(4):281–94.

[10] Arzumanyan A, Reis HM, Feitelson MA. Pathogenic mechanisms in HBV-andHCV-associated hepatocellular carcinoma. Nat Rev Cancer 2013;13(2):123–35.

[11] Chen L, Borozan I, Sun J, Guindi M, Fischer S, Feld J, et al. Cell-type specific geneexpression signature in liver underlies response to interferon therapy inchronic hepatitis C infection. Gastroenterology 2010;138(3):1123–33. e1123.

[12] Song LL, Ponomareva L, Shen H, Duan X, Alimirah F, Choubey D. Interferon-inducible IFI16, a negative regulator of cell growth, down-regulates expressionof human telomerase reverse transcriptase (hTERT) gene. PLoS ONE2010;5(1):e8569.

[13] Duan X, Ponomareva L, Veeranki S, Choubey D. IFI16 Induction by glucoserestriction in human fibroblasts contributes to autophagy through activationof the ATM/AMPK/p53 pathway. PLoS ONE 2011;6(5):e19532.

[14] Xin H, Pereira-Smith OM, Choubey D. Role of IFI 16 in cellular senescence ofhuman fibroblasts. Oncogene 2004;23(37):6209–17.

[15] Veeranki S, Duan X, Panchanathan R, Liu H, Choubey D. IFI16 protein mediatesthe anti-inflammatory actions of the type-I interferons through suppression ofactivation of caspase-1 by inflammasomes. PLoS ONE 2011;6(10):e27040.

[16] Unterholzner L, Keating SE, Baran M, Horan KA, Jensen SB, Sharma S, et al. IFI16is an innate immune sensor for intracellular DNA. Nat Immunol2010;11(11):997–1004.

[17] O’Neill LA. Sensing the dark side of DNA. Science 2013;339(6121):763–4.[18] Li T, Diner BA, Chen J, Cristea IM. Acetylation modulates cellular distribution

and DNA sensing ability of interferon-inducible protein IFI16. Proc Natl AcadSci 2012;109(26):10558–63.

[19] Choubey D, Duan X, Dickerson E, Ponomareva L, Panchanathan R, Shen H, et al.Interferon-inducible p200-family proteins as novel sensors of cytoplasmicDNA: role in inflammation and autoimmunity. J Interferon Cytokine Res2010;30(6):371–80.

[20] Choubey D, Deka R, Ho SM. Interferon-inducible IFI16 protein in humancancers and autoimmune diseases. Front biosci 2008;13:598–608.

[21] Azzimonti B, Pagano M, Mondini M, De Andrea M, Valente G, Monga G, et al.Altered patterns of the interferon-inducible gene IFI16 expression in head andneck squamous cell carcinoma: immunohistochemical study includingcorrelation with retinoblastoma protein, human papillomavirus infectionand proliferation index. Histopathology 2004;45(6):560–72.

[22] Mazibrada J, Andrea MD, Rittà M, Borgogna C, Pfeffer U, Chiusa L, et al. In vivogrowth inhibition of head and neck squamous cell carcinoma by theinterferon-inducible gene IFI16. Cancer Lett 2010;287(1):33–43.

[23] Liao JCC, Lam R, Brazda V, Duan S, Ravichandran M, Ma J, et al. Interferon-inducible protein 16: insight into the interaction with tumor suppressor p53.Structure 2011;19(3):418–29.

[24] Petitjean A, Achatz M, Borresen-Dale A, Hainaut P, Olivier M. TP53 mutationsin human cancers: functional selection and impact on cancer prognosis andoutcomes. Oncogene 2007;26(15):2157–65.

[25] Kondo Y, Nagai K, Nakahata S, Saito Y, Ichikawa T, Suekane A, et al.Overexpression of the DNA sensor proteins, absent in melanoma 2 andinterferon-inducible 16, contributes to tumorigenesis of oral squamous cellcarcinoma with p53 inactivation. Cancer Sci 2012;103(4):782–90.

[26] Fujiuchi N, Aglipay JA, Ohtsuka T, Maehara N, Sahin F, Su GH, et al.Requirement of IFI16 for the maximal activation of p53 induced by ionizingradiation. J Biol Chem 2004;279(19):20339–44.

[27] Lui W-Y, Lin H-L, Chau G-Y, Liu T-Y, Chi C-W. Male predominance inhepatocellular carcinoma: new insight and a possible therapeuticalternative. Med Hypotheses 2000;55(4):348–50.

[28] Alimirah F, Chen J, Xin H, Choubey D. Androgen receptor auto-regulates itsexpression by a negative feedback loop through upregulation of IFI16 protein.FEBS Lett 2006;580(6):1659–64.

[29] Wieland S, Thimme R, Purcell RH, Chisari FV. Genomic analysis of the hostresponse to hepatitis B virus infection. Proc Natl Acad Sci U S A2004;101(17):6669–74.

[30] Alimirah F, Chen J, Davis FJ, Choubey D. IFI16 in human prostate cancer. MolCancer Res 2007;5(3):251–9.

[31] Zou L, Cortez D, Elledge SJ. Regulation of ATR substrate selection by Rad17-dependent loading of Rad9 complexes onto chromatin. Genes Dev2002;16(2):198–208.