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[Cancer Research 64, 7030-7038, October 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Regulation of {alpha}-Fetoprotein by Nuclear Factor-{kappa}B Protects Hepatocytes from Tumor Necrosis Factor-{alpha} Cytotoxicity during Fetal Liver Development and Hepatic Oncogenesis

Lakita G. Cavin1, Manickam Venkatraman1, Valentina M. Factor2, Swayamjot Kaur1, Insa Schroeder2, Frank Mercurio3, Amer A. Beg4, Snorri S. Thorgeirsson2 and Marcello Arsura1

1 Department of Pharmacology, Center for Anticancer Drug Research, University of Tennessee Cancer Institute, College of Medicine, Memphis, Tennessee; 2 Laboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland; 3 Celgene Signal Research Division, San Diego, California; and 4 Department of Biosciences, Columbia University, New York, New York


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nuclear factor-{kappa}B (NF-{kappa}B) plays a critical role during fetal liver development and hepatic oncogenesis. Here, we have assessed whether NF-{kappa}B activity is required for murine hepatocellular carcinoma cell survival. We show that adenoviral-mediated inhibition of inhibitor of NF-{kappa}B kinase-ß (IKK-2) activity in hepatocellular carcinomas derived from transforming growth factor (TGF)-{alpha}/c-myc bitransgenic mice leads to inhibition of NF-{kappa}B and promotes tumor necrosis factor (TNF)-{alpha}–mediated cell death of malignant hepatocytes but not the surrounding peritumorous tissue. Induction of apoptosis is accompanied by inhibition of Bcl-XL and XIAP, two pro-survival NF-{kappa}B target genes. In addition, we have identified the {alpha}-fetoprotein (AFP) as a novel downstream target of NF-{kappa}B. We show that repression of IKK-2 activity in hepatocellular carcinomas promotes down-regulation of AFP gene expression. Likewise, genetic disruption of the RelA subunit results in reduced AFP gene expression during embryonic liver development, at a time in which fetal hepatocytes are sensitized to TNF-{alpha}–mediated cell killing. In this regard, we show that AFP inhibits TNF-{alpha}–induced cell death of murine hepatocellular carcinomas through association with TNF-{alpha} and inhibition of TNFRI signaling. Thus, NF-{kappa}B-mediated regulation of AFP gene expression during liver tumor formation and embryonic development of the liver constitutes a potential novel mechanism used by malignant and fetal hepatocytes to evade immune surveillance.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The inhibitor of nuclear factor-{kappa}B (I{kappa}B) kinase (IKK) complex plays a central role during liver cell survival through regulation of nuclear factor-{kappa}B (NF-{kappa}B) activity (1) . This protein complex is formed by the two catalytic subunits IKK-{alpha} (IKK-1) and IKK-ß (IKK-2), and by the regulatory subunit IKK-{gamma} (2) . In response to a wide variety of stimuli, the IKK complex promotes NF-{kappa}B activation through phosphorylation-induced ubiquitination of cytoplasmic I{kappa}B inhibitors (2) . Mouse embryos lacking either the IKK-2 or IKK-{gamma} subunit (3, 4, 5, 6) display enhanced liver apoptosis during gestation, which is reminiscent of that observed in RelA null mice (7) due to sensitization to TNF-{alpha} cell killing (5 , 8, 9, 10) . Recent evidence indicates that aberrant activation of NF-{kappa}B caused by constitutive activation of the IKK complex might also be involved in epithelial neoplastic progression through protection from cell death and induction of cell growth (11, 12, 13, 14, 15) . In this context, recent data suggest a bridging role of NF-{kappa}B between tumor initiation and inflammation in hepatitis B virus-infected and hepatitis C virus-infected livers (16 , 17) as well as in animal models of gastric cancer (18) .

Previously, we have reported aberrant NF-{kappa}B activity in liver tumors derived from bitransgenic mice overexpressing transforming growth factor (TGF)-{alpha} and c-myc, which was accompanied by constitutive activity of the IKK complex (11 , 19) . These bitransgenic animals develop multiple liver tumors between 4 and 8 months of age, which represents a dramatic acceleration of neoplastic development compared with the long latency of hepatocellular carcinomas derived from single c-myc or TGF-{alpha} transgenic mice (20 , 21) . This effect correlated with enhanced cell survival of bitransgenic hepatocellular carcinomas compared with that of single c-myc–derived liver tumors (22) , indicating that ectopic expression of TGF-{alpha} opposed c-myc–induced apoptosis, thereby favoring the development of a more aggressive neoplastic phenotype. Interestingly, TGF-{alpha}/c-myc–derived hepatocellular carcinomas are characterized by a pronounced up-regulation of reactive oxygen species (23) , and treatment with antioxidant scavengers such as vitamin E leads to inhibition of neoplastic development (24) . Based on these data, we have now tested the hypothesis that the constitutive NF-{kappa}B activity observed in TGF-{alpha}/c-myc–derived hepatocellular carcinomas is essential for tumor cell survival in vivo, a process that is thought to favor neoplastic growth of epithelial cells. In agreement with recent evidence implicating NF-{kappa}B/Rel factors in mammary tumor development (13) , we show that inhibition of IKK complex activity of TGF-{alpha}/c-myc–derived hepatocellular carcinomas promotes extensive cell death of malignant hepatocytes through down-regulation of pro-survival genes. Overall, our findings indicate a major role of the IKK/NF-{kappa}B axis in cell survival of oncogene/growth factor–induced hepatocellular carcinomas with possible implications for human liver cancer.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice.
Male TGF-{alpha}/c-myc double transgenic mice were generated by crossing homozygous MT/TGF-{alpha} (21) and Alb/c-myc (20) single transgenic mice and housed as described previously (23) . RelA knock-out mice have been described previously (7) . The animal study protocols were conducted according to the NIH guidelines for animal care. Mice had free access to standard rodent chow and water.

Adenoviruses and Mouse Tail Vein Injection.
The adenoviral vector SPAV-2 expressing the dominant-negative forms of IKK-2 (IKK-2 K>M), was constructed by blunt ligation of the respective IKK cDNA into the replication-deficient vector pAxCA. The adenoviral vector expressing the green fluorescent protein (adGFP) was similarly constructed and used as negative control. Virus stocks were amplified to high titer (Quantum Biotechnologies, Montreal, Canada). The concentration of viral particles was determined by A260 measurement. Plaque assay to determine infectious virus units gave a viral particle to infectious virus unit ratio of less than 100:1. Adenoviral preparations were re-titered using human umbilical vein endothelial cells to determine the optimum multiplicity of infection. SPAV-2 and adGFP were administered into tumor-bearing TGF-{alpha}/c-myc mice via the tail vein at doses of 1 x 1011 viral particle per animal. Vectors were diluted in a physiologic saline in a total volume of 200 µL.

Cell Culture.
The 223ma2 cell line was isolated from a hepatocellular carcinoma of a TGF-{alpha}/c-myc transgenic mouse and maintained in Dulbecco’s modified Eagle’s medium/Ham’s F-12 high glucose medium (Gibco/BRL, Rockville, MD) containing 1 mg/mL D-galactose, 30 µg/mL proline, 2 mmol/L glutamine, 5 mmol/L sodium pyruvate, 18 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 5 ng/mL insulin, 5 µg/mL transferrin, 5 ng/mL selenium, 100 µg/mL streptomycin, and 10% fetal bovine serum (Gibco/BRL). Where indicated, cells were incubated with 100 µg/mL purified human {alpha}-fetoprotein (The Binding Site Inc., San Diego, CA), which was extensively dialyzed to remove sodium azide, with or without 0.2, 2, or 20 ng/mL TNF-{alpha} (Sigma, St. Louis, MO). The human HepG2 cell line was a kind gift of Dr. Edwards Park (University of Tennessee Health Sciences Center, Memphis, TN). Cells were cultivated in Dulbecco’s modified Eagle’s medium/F-12 medium (Gibco/BRL) supplemented with 10% fetal bovine serum.

RNA Interference and Transfection Conditions.
The SMARTpool siRNAs specific for murine {alpha}-fetoprotein (AFP) was purchased from Upstate Technologies (Lake Placid, NY) and was transfected (100 nmol/L) into 223ma2 cells according to the manufacturer’s specifications using LipofectAMINE 2000 (Invitrogen, Carlsbad, CA). 223ma2 cells were transiently transfected with a solution of DNA and LipofectAMINE reagent according to manufacturer’s instructions (Invitrogen). Sixteen hours after transfection, cells were treated with 20 ng/mL TNF-{alpha} or bovine serum albumin carrier solution for 6 hours. For 5-bromo-4-chloro-3-indoyl-ß-D-galactoside (X-Gal) staining, transfected cells were rinsed twice with 1 x PBS (pH 7.4) and fixed in 2% formaldehyde (Sigma) and 0.2% glutaraldehyde (Sigma) for 5 minutes at room temperature. Cells were then rinsed twice with 1 x PBS and stained for 12 hours in 1 x PBS containing 0.1% X-Gal (Invitrogen), 0.5 mmol/L potassium ferricyanide, 0.5 mmol/L potassium ferrocyanide, and 2 mmol/L MgCl2. Viable blue-stained cells were visually counted at the microscope. The XIAP expression vector was a kind gift of Dr. R. Korneluk (Children’s Hospital of Eastern Ontario, Ottawa, Ontario, Canada; ref. 25 ).

RNA Isolation and Analysis.
Total cellular RNA was isolated using the RNeasy kit following the manufacturer’s instructions (Qiagen, Valencia, CA) and samples (1 µg) subjected to reverse transcription-PCR using the Omniscript and the TaqPCR core kits all from Qiagen. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were obtained from Maxim Biotech. Inc. (San Francisco, CA). The sequences of the murine AFP- and GAPDH-specific primers are as follows: AFP(+), 5'-GCTGCGTCCAAAGCATTGCA-3'; AFP(–), 5'-GGCCAGCTTCTGAATCTCAG -3'; GAPDH(+), 5'-GGGTGGAGCCAAACGGGTC-3'; and GAPDH(–), 5'-GGAGTTGCTGTTGAAGTCGCA-3'.

For PCR, samples were subjected to 25 cycles each consisting of a denaturation step at 94°C for 1', annealing at 52°C for the AFP or at 58°C for GAPDH for 1', and elongation at 72°C for 1' in a Mastercycler Gradient thermocycler (Eppendorf Scientific, Westbury, NY). DNA was visualized in a 0.5x Tris-borate EDTA-agarose gel stained with ethidium bromide.

Immunohistochemistry and TUNEL Assay.
Immunohistochemitry and terminal deoxynucleotidyltransferase-mediated nick end labeling (TUNEL) staining were performed on formalin-fixed, paraffin-embedded 5 µmol/L sections from at least four different hepatocellular carcinomas and peritumorous tissues each obtained from a total of six adGFP- and six adIKK-2 K>M-infected TGF-{alpha}/c-myc bitransgenic mice. Upon antigen unmasking, immunostaining was performed using the rabbit, goat, or M.O.M. Vectastain ABC Elite kit followed by Vector 3,3'-diaminobenzidine peroxidase substrate (Vector Laboratories, Burlingame, CA). Sections were then dehydrated and stained with Vector Hematoxylin QS nuclear counterstain (Vector Laboratories). The antibody preparations for IKK1/2 (sc-7607), GFP (sc-9996), RelA (sc-372-G), and AFP (sc-8108) were all purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The antibody against TNF-{alpha} (ab6671-200) was purchased from Abcam (Cambridge, MA). TUNEL assay was performed either on paraffin-embedded tissue sections or cultures of live cells using the DeadEnd Colorimetric TUNEL System (Promega, Madison, WI) following the manufacturer’s instructions.

Electrophoretic Mobility Shift Analysis.
Nuclear extracts were prepared from hepatocellular carcinomas or normal surrounding livers as described previously (11) . The sequences of the URE-{kappa}B– and Octamer-1–containing oligonucleotides are as follows: URE-{kappa}B, 5'-AAGTCCGGGTTTTCCCCAACC-3'; and Oct-1, 5'-TGTCGAATGCAAATCACTAGAA-3'. Oligonucleotides were end-labeled with Klenow and [{alpha}-32P]dNTPs, and electrophoretic mobility shift analysis was performed as described previously (11) .

Immunoblot Analysis, Immunoprecipitation, and Antibodies.
For isolation of whole-cell extracts, pulverized tissues were resuspended in cold extraction buffer [40 mmol/L Tris (pH 8), 500 mmol/L NaCl, 6 mmol/L EDTA, 6 mmol/L EGTA, 10 mmol/L glycerophosphate, 10 mmol/L NaF, 10 mmol/L p-nitrophenyl phosphate, 300 µmol/L Na3VO4, 1 mmol/L benzamidine, 2 µmol/L phenylmethylsulfonyl fluoride, 1 mmol/L dithiothreitol, 1 µg/mL leupeptin, 10 µg/mL aprotinin, 1 µg/mL pepstatin, and 0.5% NP40] and sonicated for 30 seconds using a Microson Ultrasonic Cell Disruptor XL (Misonix Incorporated, Farmingdale, NY). Extracts were then cleared by centrifugation at 40,000 rpm for 30 minutes at 4°C. For immunoblotting, samples (20–40 µg) were subjected to electrophoresis on a 10% polyacrylamide-SDS gel, transferred to nitocellulose membrane (Millipore, Bedford, MA), and immunoblotted, as described previously (26) . Chemiluminescence was acquired and quantified using the Chemi-Doc XRS and Quantity One software from Bio-Rad (Hercules, CA).

The polyclonal antibody against human AFP used in immunoblotting was purchased from United States Biologicals (Swampscott, MA). Despite several technical efforts, we were unable to detect murine AFP protein expression in whole-cell extracts of either the hepatocellular carcinomas or 223ma2 cells using the only commercially available antibody targeted against murine AFP (sc-8108; data not shown). The monoclonal antibodies against Bcl-XL (Bcl-XL-Mab) and XIAP (XIAP-Mab) were purchased from BD-Transduction Laboratories (Lexington, KY) and from Stressgen (San Diego, CA), respectively. The monoclonal antibody against phospho-c-Jun (sc-822) and the polyclonal antibody anti-c-Jun (sc-45) were purchased from Santa Cruz Biotechnology, Inc.. The monoclonal antibody specific for ß-actin (AC-15) was purchased from Sigma.

Immunoprecipitation was carried out using protein A-Sepharose CL-4B beads coated with a monoclonal antibody against human AFP (ab3980) that was purchased from Abcam. After low-stringency washings, the beads were loaded on a 15% polyacrylamide-SDS gel and subjected to immunoblotting using an antibody against human TNF-{alpha} (AF-210-NA) purchased from R&D Systems (Minneapolis, MN).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Mouse Tail Vein Injection of Adenoviral IKK-2 K>M Down-regulates Constitutive Nuclear Factor-{kappa}B Activity in Transforming Growth Factor-{alpha}/c-Myc-Derived Hepatocellular Carcinomas.
To determine whether the IKK complex mediates the aberrant NF-{kappa}B activation seen in hepatocellular carcinomas derived from TGF-{alpha}/c-myc double transgenic mice, we subjected bitransgenic TGF-{alpha}/c-myc 30- to 48-week-old mice bearing hepatocellular carcinomas to tail vein injections of an adenoviral construct directing expression of the IKK-2 K>M dominant-negative mutant (adIKK-2 K>M). As negative control, we injected an adenovirus expressing green fluorescent protein (adGFP) into tail veins. Hepatocellular carcinomas and normal surrounding livers were isolated at 24 and 72 hours post injection, and whole-tissue extracts were subjected to immunoblot analysis using an antibody raised against IKK-2. We observed increased expression levels of an immunoreactive band in both T and SL of mice infected with the adIKK-2 K>M (K) at 24 or 72 hours post injection compared with those of mice that received injections of adGFP (G; Fig. 1ACitation ). This band comigrated with that seen in extracts of adIKK-2 K>M-infected HEK 293 cells (Fig. 1ACitation , Lane 1). Furthermore, upon prolonged exposure, we did not detect significant expression levels of IKK-2 in the tissue samples of adGFP-injected mice (data not shown). Thus, we concluded that the band seen in adIKK2 K>M-injected tissues was specific to the IKK-2 K>M transgene. In addition, we detected IKK-2 K>M and GFP protein staining in both peritumorous tissues and neoplastic lesions of livers infected with adIKK2 K>M or adGFP, respectively (see Supplementary Discussion 1 and Supplementary Fig. 1A–Bi).



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  		Fig. 1. Ectopic expression of IKK-2 K>M inhibits constitutive NF-{kappa}B activity in malignant hepatocytes. A. Thirty- to 48-week-old TGF-{alpha}/c-myc bitransgenic mice bearing well-differentiated hepatocellular carcinomas were subjected to two independent tail vein injections with 1 x 1011 viral particle adGFP (G) or adIKK-2 K>M (K). At 24 (1) or 72 hours (3) post injection, surrounding liver (SL) and liver tumor (T) samples (40 µg) were subjected to immunoblotting (IB) using an antibody against IKK-1/2 (top panels). As a positive control, whole-cell extracts (20 µg) of HEK 293 cells infected with adIKK-2 K>M (+ control) were similarly analyzed. For equal loading, the same filters were hybridized with an antibody against actin (bottom panels). B. IKK-2 K>M inhibits NF-{kappa}B DNA-binding activity. Nuclear extracts were prepared from adGFP or adIKK-2 K>M-injected hepatocellular carcinomas at 24 or 72 hours post injection as described in Materials and Methods. To measure the levels of NF-{kappa}B DNA-binding activity, nuclear extracts (5 µg) were subjected to electrophoretic mobility shift analysis using the upstream NF-{kappa}B element from the c-myc gene (URE-{kappa}B) as a probe (27) . The top band represents classical NF-{kappa}B (p65/p50), the bottom band contains p50 homodimers. As control for equal loading, electrophoretic mobility shift analysis was also performed with an Octamer-1 probe (Oct-1).

 
To determine whether the IKK-2 K>M transgene was functionally active, nuclear extracts from SL and T samples of adIKK-2 K>M- or adGFP-injected livers were subjected to electrophoretic mobility shift analysis using as probe the upstream NF-{kappa}B element (URE-{kappa}B) described previously (27) . The nuclear extracts from SL infected with adIKK-2 K>M or adGFP for 24 and 72 hours displayed low levels of a single band containing the p65 (RelA) and p50 (NF-{kappa}B1) subunits, as judged by supershift analysis (Fig. 1BCitation ; data not shown). Consistent with NF-{kappa}B being constitutively active in murine hepatocellular carcinomas (11) , we observed enhanced levels of NF-{kappa}B DNA-binding activity in hepatocellular carcinomas of adGFP-infected livers (Fig. 1B)Citation . In contrast, IKK-2 K>M-expressing tumors displayed reduced levels of NF-{kappa}B DNA-binding activity (Fig. 1B)Citation . In addition, adenoviral-mediated delivery of IKK-2 K>M, but not GFP, inhibited constitutive NF-{kappa}B DNA-binding activity through cytoplasmic sequestration of RelA (see Supplementary Discussion 1 and Supplementary Fig. 2A–Bi), presumably, via inhibition of I{kappa}B-{alpha} degradation.

Ectopic Expression of IKK-2 K>M Promotes Apoptosis of Bitransgenic Hepatocellular Carcinomas.
Previously, we reported a significant reduction in the apoptotic indices of TGF-{alpha}/c-myc-derived hepatocellular carcinomas compared with those of single c-myc hepatocellular carcinomas (11) that correlated with a dramatic acceleration of liver neoplastic development of the bitransgenic mice compared with that of single transgenic animals (20) . Because we have shown that TGF-{alpha}/c-myc hepatocellular carcinomas expressed a more pronounced NF-{kappa}B activity than c-myc-derived hepatocellular carcinomas (11) , we sought to determine whether NF-{kappa}B activity is providing a survival advantage to malignant hepatocytes. To examine the effect of IKK-2 K>M on cell death, we compared levels of TUNEL staining in hepatocellular carcinomas specimens derived from adIKK-2 K>M-injected mice with those of adGFP-infected animals. We detected an average of 6 ± 3 TUNEL-positive cells per representative field of six GFP expressing hepatocellular carcinomas of mice at 24 (Fig. 2A and Ai)Citation or 72 hours post injection (Fig. 2B and Bi)Citation . In three hepatocellular carcinomas expressing IKK-2 K>M at 24 hours post injection, we saw the appearance of several small areas of TUNEL-positive hepatocytes (40 ± 10 per representative field; Fig. 2C and CiCitation ). These TUNEL-positive areas became more evident in adIKK-2 K>M-injected mice at 72 hours post injection, in which we could detect several apoptotic stained nuclei within the characteristic trabecular and pseudoglandular pattern of the hepatocellular carcinomas (Fig. 2DCitation ; Supplementary Fig. 3A–Bii). Importantly, we did not observe TUNEL staining above background levels in several matching peritumorous tissues (Supplementary Fig. 3A–Bii), indicating that the apoptosis induced by IKK-2 K>M was restricted solely to neoplastic lesions.



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  		Fig. 2. Ectopic expression of IKK-2 K>M induces apoptosis of neoplastic lesions. A and B, GFP-expressing hepatocellular carcinomas display low levels of apoptotic hepatocytes. Tissue sections of adGFP-infected livers at 24 (A) or 72 hours (B) post injection were subjected to colorimetric TUNEL assay using 3,3'-diaminobenzidine as substrate (brown). Arrows, brown apoptotic nuclei. Inset, a higher magnification of the area indicated by * in B. Matching serial tissue sections were stained with hematoxylin (H QS; blue/violet; Ai and Bi). C and D, IKK-2 K>M-injected livers display enhanced levels of apoptotic malignant hepatocytes. Tissue sections of adIKK-2 K>M-injected livers at 24 (C) or 72 hours (D) post injection were subjected to TUNEL assay as above. Arrows, brown apoptotic nuclei. Insets, higher magnifications of the areas indicated by * in C and D. Matching serial tissue sections were stained with hematoxylin as above (Ci and Di).

 
AdIKK-2 K>M Infection Sensitizes Transforming Growth Factor-{alpha}/c-Myc–Derived Hepatocellular Carcinomas to Tumor Necrosis Factor-{alpha} Cytotoxicity through Inhibition of XIAP and Bcl-XL Protein Expression.
To analyze the molecular events leading to enhanced cell death in IKK-2 K>M expressing bitransgenic livers, we determined the levels of protein expression of XIAP and Bcl-XL, two antiapoptotic targets of NF-{kappa}B (1) that have been found to be highly expressed in hepatocellular carcinomas (28, 29, 30) . In two independent experiments, we observed up-regulation of both Bcl-XL and XIAP expression levels in adGFP-infected tumors compared with the adjacent normal tissues (Fig. 3A and B)Citation . In contrast, adIKK-2 K>M tumors displayed low levels of protein expression of either Bcl-XL or XIAP, comparable with those seen in normal surrounding livers (Fig. 3A and B)Citation . Because we found enhanced TNF-{alpha} protein expression levels in both GFP- and IKK-2 K>M-expressing hepatocellular carcinomas (Supplementary Fig. 4A–Bi) compared with normal livers (data not shown), we asked whether inhibition of NF-{kappa}B was sensitizing malignant hepatocytes to TNF-{alpha} cytotoxicity. For this purpose, we established a cell line from a TGF-{alpha}/c-myc–derived hepatocellular carcinoma and assessed its sensitivity to TNF-{alpha} cytotoxicity after inhibition of NF-{kappa}B activity. Indeed, we detected a dramatic enhancement of TUNEL staining after treatment with TNF-{alpha} of adIKK-2 K>M-infected cells compared with adGFP-expressing cells (Fig. 3C)Citation . Furthermore, ectopic expression of XIAP rescued IKK-2 K>M-expressing cells from TNF-{alpha} cell killing (Fig. 3D)Citation . Thus, our data are consistent with a model in which NF-{kappa}B-mediated up-regulation of antiapoptotic genes rescues malignant hepatocytes, presumably from TNF-{alpha} cytotoxicity.



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  		Fig. 3. adIKK-2K>M-mediated down-regulation of Bcl-XL and XIAP in murine hepatocellular carcinomas. A and B, Whole-cell extracts of the surrounding liver (SL) and liver tumor (T) samples (30 µg) infected with either adGFP or adIKK-2 K>M for 24 or 72 hours were subjected to immunoblotting using antibodies against Bcl-XL, XIAP, and actin. C, Ectopic expression of IKK-2 K>M sensitizes 223ma2 hepatocytes to TNF-{alpha} cytotoxicity. TGF-{alpha}/c-myc bitransgenic 223ma2 cells were infected with 10 plaque forming units (pfu)/mL adenoviral construct directing expression of IKK-2 K>M or GFP for 24 hours. Subsequently, cells were treated for 6 hours with 20 ng/mL TNF-{alpha} and subjected to TUNEL assay. Means and SDs of TUNEL-positive cells relative to the total cell number (% cell death) are representative of three independent experiments performed in duplicate. For each condition, a minimum of 100 cells was counted. D, Ectopic expression of XIAP rescues hepatocellular carcinomas from TNF-{alpha} cell killing. 223ma2 cells were plated in 96-well plates and transfected by lipofection with 25 ng of pON407 ß-Gal expression vector in the presence or absence of 50 ng of XIAP expression vector. The final concentration of DNA was adjusted to 150 ng with the XIAP backbone vector. Six hours after transfection, 10 pfu/mL adGFP or adIKK-2 K>M were added to the cultures. After 18 hours of incubation, cells were treated for 6 hours with 0.2, 2, or 20 ng/mL TNF-{alpha}. Cell death was monitored by X-Gal staining as described in Materials and Methods and expressed as the percentage of blue viable cells relative to total cell number. Means and SDs are representative of three independent experiments carried out in triplicate.

 
AdIKK-2 K>M Infection Causes Diminished Expression of {alpha}-Fetoprotein.
To further elucidate the mechanism of IKK-2 K>M-induced apoptosis, we subjected mRNA of infected livers to gene array analysis using murine whole-genome Affymetrix microchips. Upon cluster data analysis, we identified several genes and expressed sequence tags that appeared to be differentially regulated in adGFP- and adIKK-2 K>M-infected hepatocellular carcinomas (data not shown). We focused on one of them, the AFP, because of its relevance during liver development and neoplastic transformation (31 , 32) . We observed down-regulation of AFP mRNA in IKK-2 K>M-expressing hepatocellular carcinomas compared with the adGFP expressing tumors, as judged by reverse transcription-PCR analysis (Fig. 4A)Citation . Furthermore, ectopic expression of IKK-2 K>M in HepG2 cells resulted in down-regulation of AFP protein expression levels (Fig. 4B)Citation . Consistent with the gene array and reverse transcription-PCR data, we detected a marked immunostaining for AFP in adGFP-infected hepatocellular carcinomas (data not shown), which was significantly reduced in adIKK-2 K>M-expressing hepatocellular carcinomas at 24, 48, and 72 hours post injection (Fig. 4Ci and DiCitation ; data not shown). Interestingly, when we analyzed serial tissue sections of two different adIKK-2 K>M-injected hepatocellular carcinomas at 24 hours post injection, we observed that the immunostaining for IKK-2 K>M and AFP was mutually exclusive (Fig. 4C–Di)Citation , again indicating that inhibition of NF-{kappa}B activity down-regulates AFP protein expression. In addition, we noticed that the appearance of TUNEL-positive cells was restricted exclusively to the AFP-negative/IKK-2 K>M-positive areas (Fig. 4Cii and Dii)Citation , suggesting that down-regulation of AFP coincided with induction of apoptosis.



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  		Fig. 4. Ectopic expression of IKK-2K>M reduces AFP mRNA and protein expression in murine and human hepatocellular carcinomas. A, AdIKK-2 K>M-infected hepatocellular carcinomas display reduced AFP mRNA expression. Total RNA was isolated from adGFP-infected (TG1.1 and TG3.1) or IKK-2 K>M-infected (TK1.1 and TK3.1) tumors at 24 (TG1.1 and TK1.1) or 72 hours (TG3.1 and TK3.1) post injection and subjected to semiquantitative reverse transcription-PCR using primers specific for AFP or GAPDH as described in Materials and Methods. As negative control, a PCR reaction was carried out in the absence of primers (no primers). DNA was visualized on a Tris-borate EDTA-agarose gel stained with ethidium bromide. The amplified AFP-specific band is 710 bp long. Bands were quantified by densitometric analysis. The ratios of the absorbance of AFP to that of the GAPDH are expressed in arbitrary units. B, Human hepatocellular carcinoma HepG2 cells were infected for 48 or 72 hours with 10 pfu/mL of adIKK-2 K>M or adGFP. Whole-cell extracts (40 µg) were subjected to immunoblotting (IB) using an antibody specific for human AFP. For equal loading, the same filters were hybridized with an antibody against actin. The ratios of the absorbance of AFP to that of the actin are expressed in arbitrary units. C and D, expression patterns of IKK-2 K>M and AFP in TGF-{alpha}/c-myc–derived neoplastic lesions. Serial tissue sections of two hepatocellular carcinomas (TK1.1 and TK1.2) isolated from an adIKK-2 K>M-infected liver at 24 hours post injection were immunostained with either an IKK1/2 antibody (C and D) or AFP antibody (Ci and Di). Proteins were visualized using 3,3'-diaminobenzidine as substrate (brown) and nuclei were lightly counterstained using hematoxylin (blue/violet). Cii–Dii. AFP-negative areas display enhanced apoptosis. Serial tissue sections of the same livers described in C through Di were subjected to TUNEL assay (Cii and Dii) as described in the legend of Fig. 3Citation . Arrows, brown apoptotic nuclei. Insets, higher magnifications of the areas indicated by * in Cii and Dii.

 
RelA Null Mice Display Reduced {alpha}-Fetoprotein Gene Expression during Fetal Liver Development.
Genetic disruption of the RelA subunit leads to embryonic lethality at 15 to 16 days of gestation due to massive degeneration of the liver by apoptosis (Fig. 5GCitation ; ref. 7 ). Because AFP gene is expressed and secreted predominantly in the fetal liver during embryogenesis, we sought to determine whether the RelA subunit plays a role in AFP gene regulation also during development. We observed a marked immunostaining for both RelA and AFP in livers of four wild-type embryos at day E14.5 (Fig. 5A and BCitation ; data not shown). In contrast, the livers of four RelA null embryos at the same day of gestation displayed only background levels of RelA and AFP staining (Fig. 5C and DCitation ; data not shown). Furthermore, we observed equal levels of actin staining in serial tissue sections of wild-type and RelA null livers, indicating that the embryos were viable (Fig. 5F and H)Citation . Thus, RelA is involved in AFP gene regulation during embryogenesis, and down-regulation of AFP gene product precedes the induction of hepatic apoptosis by TNF-{alpha} in RelA null embryos.



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  		Fig. 5. AFP protein expression is reduced in RelA null murine embryos. Serial tissue sections from livers of wild-type (WT; A, B, E, and F) or RelA null (C, D, G, and H) murine embryos at day 14.5 of gestation were immunostained with antibodies against RelA, AFP, and actin as indicated. Proteins were visualized using 3,3'-diaminobenzidine as substrate (brown), and nuclei were lightly counterstained using hematoxylin (blue/violet). Insets, higher magnifications of the areas indicated by *. Serial tissue sections were also subjected to TUNEL assay (E and G) as described in the legend of Fig. 2Citation . Arrows, brown apoptotic nuclei in the liver of the RelA null embryo.

 
{alpha}-Fetoprotein Counteracts Tumor Necrosis Factor-{alpha}–Mediated Cytotoxicity of Murine Hepatocellular Carcinomas.
Previous studies reported conflicting evidence on the role of AFP during apoptosis. High nonphysiologic concentration of AFP or AFP-derived peptides promoted growth arrest and apoptosis of hepatocellular carcinoma cell lines (31) , whereas moderate concentrations of AFP opposed TNF-{alpha}–mediated cell killing of HepG2 cells (33) . Because we detected TNF-{alpha} immunostaining in the bitransgenic hepatocellular carcinomas (Supplementary Fig. 4A–Bi), we sought to determine whether physiologically relevant concentrations of AFP (100 µg) would protect malignant hepatocytes against TNF-{alpha} cell killing. To ensure that the bovine AFP contained in the fetal calf serum would not interfere with our experimental conditions, we performed our experiments in either the presence or absence of serum in the culture medium. Furthermore, upon 1 hour of incubation in serum-free medium and before TNF-{alpha} stimulation, we removed the endogenous murine AFP, which is secreted abundantly by malignant hepatocytes (31) , through two additional washes with serum-free culture medium. Again, ectopic expression of IKK-2 K>M but not GFP-sensitized 223ma2 cells cultured with serum to TNF-{alpha} cytotoxicity (Fig. 6A)Citation . Interestingly, we observed a more pronounced cell death by TNF-{alpha} in IKK-2 K>M cells incubated with serum-free medium (Fig. 6A)Citation . In contrast, neither serum-deprived nor serum-exposed adGFP-infected 223ma2 cells displayed significant cell death in response to TNF-{alpha} treatment (Fig. 6A)Citation . Furthermore, addition of affinity-purified human AFP protein, but not bovine serum albumin, to the serum-free culture medium rescued IKK-2 K>M-infected 223ma2 from TNF-{alpha} cell killing (Fig. 6B)Citation . This effect was selective to TNF-{alpha}–mediated cytotoxicity because AFP was ineffective at rescuing hepatocellular carcinomas from staurosporine-induced apoptosis (Fig. 6B)Citation .



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  		Fig. 6. AFP physically interacts with TNF-{alpha} and protects murine hepatocellular carcinomas from TNF-{alpha}-mediated apoptosis. A, enhancement of TNF-{alpha} cytotoxicity after serum deprivation of IKK-2 K>M expressing hepatocellular carcinomas. 223ma2 cells were infected with 10 pfu/mL adGFP or adIKK-2 K>M for 24 hours. Subsequently, cells were incubated in medium with or without fetal calf serum (+/–FCS) for 1 hour and treated in the absence or presence of 0.2, 2, and 20 ng/mL TNF-{alpha} for 4 hours. Cell death was determined by TUNEL assay and expressed as percent dead cells relative to total cell number. Means and SDs represent at least three independent experiments, each carried out in duplicate. B, 223ma2 cells were infected with adIKK-2 K>M as described above. After incubation in serum-free medium for 1 hour, cells were stimulated for 4 hours with 0.2 or 20 ng/mL TNF-{alpha} in the presence of either 100 µg/mL bovine serum albumin (BSA) or 100 µg/mL affinity-purified human AFP. To determine the specificity of AFP-mediated rescue, cells were treated with 100 µmol/L staurosporine (STP) for 4 hours. Cell death was measured by TUNEL assay, and results are expressed as above. Means and SDs represent at least three independent experiments, each carried out in triplicate. C, AFP gene knockdown is not sufficient to sensitize 223ma2 cells to TNF-{alpha}-induced apoptosis. Cells were transfected with 100 nmol/L siRNA specific for murine AFP (siAFP) or 100 nmol/L nonspecific siRNA control (siC). After 6 hours of transfection, cells were infected for 24 hours with adIKK-2 K>M or adGFP as described above without removing the LipofectAMINE from the culture medium. After incubation in serum-free medium for 1 hour, cells were stimulated for 3 hours with 0.2, 2, or 20 ng/mL TNF-{alpha} and cell death measured by TUNEL staining. To determine the levels of AFP gene expression, total RNA was isolated from 223ma2 cells transfected or not (none) with siC or siAFP, the latter in duplicate. Subsequently, RNA samples were subjected to semiquantitative reverse transcription-PCR using primers specific for AFP or GAPDH as described in the legend of Fig. 4Citation . Inset. DNA was visualized on Tris-borate EDTA-agarose gel stained with ethidium bromide. D, AFP associates with TNF-{alpha} in vitro. Human affinity-purified AFP (100 ng) and human recombinant TNF-{alpha} (100 ng) were incubated for 15 minutes in 1x PBS at 37°C. After immunoprecipitation (IP) with an antibody against AFP, immunoblotting (IB) was performed with an anti-TNF-{alpha} antibody. Samples were also immunoprecipitated with an anti-IgG antibody as negative control. As positive control, an aliquot of TNF-{alpha} (10 ng) was loaded onto the gel without immunoprecipitation (none). E, TNF-{alpha} associates with AFP in vivo. Culture medium of HepG2 was collected, concentrated, and immunoprecipitated with an antibody against TNF-{alpha} or interleukin-6. Immunoprecipitants were subjected to immunoblotting using an anti-AFP antibody. Purified human AFP was included as positive control. F, AFP inhibits TNF-{alpha}–mediated phosphorylation of c-Jun. HepG2 cells were infected for 24 hours with adIKK-2 K>M. After incubation in serum-free medium for 1 hour, cells were stimulated for 30 minutes with 20 ng/mL TNF-{alpha} in the presence of either 100 µg/mL bovine serum albumin (BSA) or 100 µg/mL affinity purified human AFP. Whole-cell extracts were then subjected to immunoblotting (IB) using an antibody specific for phosphorylated c-Jun or total c-Jun.

 
To determine whether inhibition of AFP gene expression was sufficient to sensitize malignant hepatocytes to TNF-{alpha}–mediated cell death, we silenced the AFP gene (Fig. 6CCitation , inset) and assessed apoptosis in serum-deprived 223ma2 cells either expressing GFP or IKK-2 K>M. We did not detect a significant induction of TNF-{alpha}–mediated apoptosis in either adGFP-infected cells treated with siRNA control or siRNA against AFP (Fig. 6C)Citation . In contrast, we observed enhanced cell death in siRNA control-treated cells after ectopic expression of IKK-2 K>M, which was more pronounced in siRNA against AFP-treated cells similarly infected (Fig. 6C)Citation . Thus, inhibition of NF-{kappa}B by ectopic expression of the IKK-2 K>M mutant promotes down-regulation of AFP gene and protein expression, thereby favoring TNF-{alpha}–induced apoptosis of malignant hepatocytes. Furthermore, silencing of AFP gene expression alone is not sufficient to sensitize cells to TNF-{alpha} cytotoxicity.

Secreted {alpha}-Fetoprotein Associates with the Circulating Tumor Necrosis Factor-{alpha}.
Because both TNF-{alpha} and AFP are secreted in the extracellular environment, we assessed whether AFP was able to physically interact with TNF-{alpha}. For this purpose, we coincubated human recombinant TNF-{alpha} with purified human AFP and subjected samples to immunoprecipitation using an antibody against AFP. Immunoprecipitation of AFP using the anti-AFP but not the IgG control resulted in coprecipitation of TNF-{alpha} (Fig. 6D)Citation . Furthermore, immunoprecipitation of secreted TNF-{alpha} but not interleukin-6 from culture medium of HepG2 resulted in coprecipitation of endogenous AFP (Fig. 6E)Citation .

To elucidate the effect of AFP during TNF-{alpha} signaling, we examined the phosphorylation levels of c-Jun, which mediates TNF-{alpha} cytotoxicity. We observed a marked reduction of c-Jun phosphorylation in AFP-treated but not bovine serum albumin-treated HepG2 cells in response to TNF-{alpha} treatment (Fig. 6F)Citation , indicating that AFP inhibited TNF-RI signaling in vivo. Thus, AFP is able to associate with TNF-{alpha} in vitro and in vivo and inhibits TNF-{alpha} signaling.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results provide evidence that NF-{kappa}B is required for cell survival of hepatocellular carcinomas derived from TGF-{alpha}/c-myc bitransgenic mice. Inhibition of aberrant NF-{kappa}B activity, which typifies these hepatocellular carcinomas (11) , through adenoviral-mediated delivery of a dominant-negative form of IKK-2 results in a dramatic induction of apoptosis, which is restricted to neoplastic lesions but not normal surrounding hepatocytes. In our model, inhibition of NF-{kappa}B and induction of apoptosis correlated with down-regulation of XIAP and Bcl-XL protein expression, two well-known pro-survival NF-{kappa}B target genes (1) that have been found to be highly expressed in murine and human hepatocellular carcinomas (28, 29, 30 ; this report). Given the high levels of TNF-{alpha} protein expression seen in the neoplastic lesions of injected and noninjected neoplastic livers, down-regulation of Bcl-XL and XIAP is likely to be responsible for enhanced sensitization of malignant hepatocytes to TNF-{alpha} cell killing. In support of this hypothesis, inhibition of NF-{kappa}B through ectopic expression of IKK-2 K>M potentiated TNF-{alpha} cell killing of a TGF-{alpha}/c-myc–derived cell line, which could be reverted by ectopic expression of XIAP. The observation that normal hepatocytes do not undergo apoptosis after infection with adIKK-2 K>M might be in part explained by a preferential distribution of the adenovirus and/or circulating TNF-{alpha} in the more richly vascularized neoplastic lesions than normal tissues. Alternatively, it is possible that malignant hepatocytes have suffered genetic changes that predisposed them to apoptotic cell death. In this context, enhanced NF-{kappa}B activity would represent a way to counteract this apoptotic tendency. Consequently, our observation raises the possibility that IKK-2 inhibitors might serve as future tools for reducing the tumor mass of nonresectable human hepatocellular carcinomas.

Another major effect of NF-{kappa}B inhibition observed in our study is the down-regulation of the tumor-associated {alpha}-fetoprotein. AFP is expressed and secreted early in development predominantly in the liver but it is repressed in the adult liver (31 , 34) . However, a reversion to early developmental levels is observed during liver regeneration and in hepatocellular carcinomas (35) . Here, we show that ectopic expression of IKK-2 K>M inhibits AFP mRNA and protein expression level in TGF-{alpha}/c-myc–derived hepatocellular carcinomas and in the human hepatoblastoma cell line HepG2, respectively. Intriguingly, we found that the immunostaining of AFP in tissue sections of bitransgenic hepatocellular carcinomas was reduced predominantly in the IKK-2 K>M-positive areas, in which we noticed the appearance of apoptotic hepatocytes. This prompted us to test whether down-regulation of AFP was required for enhanced sensitization of IKK-2 K>M-expressing tumor cells to TNF-{alpha} cytotoxicity. Indeed, addition of purified human AFP to the serum-free culture medium rescued adIKK-2 K>M-infected 223ma2 cells from TNF-{alpha} cell killing, whereas silencing of AFP further enhanced TNF-{alpha}–mediated cytotoxicity. Although high nonphysiologic concentrations of AFP or AFP-derived peptides have displayed some growth inhibitory properties (31) , our findings together with a previous report (33) support a model in which physiologically relevant concentrations of AFP (10–100 µg/mL) exert a protective role against TNF-{alpha}–induced apoptosis. In this context, the findings that AFP promotes immunosuppression of primary B splenic lymphocytes and T cells and increases the tolerance of the developing fetus against the maternal immune system (36) could be, in part, explained by the ability of AFP to act as an anti-inflammatory agent through inhibition of TNF-{alpha} signaling. Consequently, regulation of AFP protein expression levels by NF-{kappa}B could be interpreted as a potential mechanism used by malignant hepatocytes to evade immune surveillance.

The exact mechanism of AFP-mediated inhibition of TNF-{alpha} signaling still remains to be determined. We show that phosphorylation of c-Jun after TNF-{alpha} stimulation is inhibited by coincubation with affinity-purified AFP. The observation that AFP can physically associate with TNF-{alpha} suggests that AFP might function as a decoy for TNF-{alpha} in the extracellular microenvironment of neoplastic lesions. This property is reminiscent of the ability of AFP to bind to estrogen, thereby causing anovulation in AFP null mice through impairment of the hypothalamic/pituitary system (37) . In this regard, the observation that the silencing of AFP does not sensitize hepatocellular carcinoma cells bearing a functional NF-{kappa}B to TNF-{alpha} cytotoxicity might help, in part, to explain the absence of developmental abnormalities in mice lacking AFP (37) .

Given that we observed reduced levels of AFP gene product in murine embryos lacking RelA at a time in which hepatocytes undergo massive apoptosis due to sensitization to TNF-{alpha} cytotoxicity, it is tempting to speculate that the regulation of AFP protein expression by NF-{kappa}B during liver development might also contribute to protection of fetal hepatocytes against TNF-{alpha}–mediated apoptosis.

Overall, our data indicate that NF-{kappa}B is essential for cell survival of malignant hepatocytes and support a model whereby cancer cells reactivate developmental pathways to acquire a survival advantage.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge Drs. Robert Korneluk, Kuni Matsumoto, and Edwards Park for kindly providing cloned DNAs and cell lines. We thank Drs. Steve Tavalin, Kafait U. Malik, Patricia Farrar, and Suleiman Bahouth for helpful comments on the manuscript.


   FOOTNOTES
 
Grant support: ACS grant RSG-02-255-01-TBE (M. Arsura), NIH grants CA78616 (M. Arsura) and CA074892 (A. Beg), and Research Supplement for Under Represented Minorities Program CA78616-S1 (L. Cavin).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Supplementary data for this article can be found at Cancer Research Online (http://cancerres.aacrjournals.org). L. Cavin and M. Venkatraman contributed equally to this work.

Requests for reprints: Marcello Arsura, Department of Pharmacology, University of Tennessee College of Medicine, 874 Union Avenue, Memphis, TN 38163. Phone: 901-448-1733; Fax: 901-448-7206; E-mail: marsura{at}utmem.edu

Received 5/11/04. Revised 7/22/04. Accepted 7/23/04.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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