Hepatocellular carcinoma (HCC) ranks many tasks in clinical oncology due to possibly developing a general tumor in men and, usually lead to malignant to death within years.1 Research had reported major factors for being HCC was male sex and HCC associated with cirrhosis in childhood is found more common in males than females.2,3 In certain mouse strains as studied, breeding with testosterone significantly increases the chemically induced hyperplastic nodules to the development of HCC.4 Furthermore, castration of male mice diminished the frequent development of liver tumors.5 Male hepatitis B virus transgenic mice have a greater occurrence of HCC than females.6 The report describes sex hormone in animals regulated growth of HCC.7 In addition, the hormonal-additive environment at the initiation of tumor development may be based upon the linkage of the usage of anabolic steroids.5 Other possible mechanisms, including sexual status with respect to body metabolism by environmental carcinogens, have been reported.6 Such as the pathway leading to the active metabolite of aflatoxin is observed much more progressive in certain male animals compared to females.3 In humans, however, the molecular developmental mechanisms between HCC and sexual status are not clear. Steroid4 hormones initiate their functional cascade by binding to the receptors and turn on the transcriptional regulatory machinery within the cells.8 A study finds that EGF-triggered growth and invasiveness of cells expressing estrogen and androgen receptors to be correlated to the cell malignancy and carcinogenesis.9 Cofactors for steroid receptors have been confirmed to be involved in transcriptional signal which will define the difference of cell destiny.10 Based on these observations, we will try to rule out the patterns of steroid receptors and possible cofactors expressed in liver, analysis of expression and characterization of their mechanisms in hepatoma would be important. Herein, we had observed high levels of androgen receptor in liver. After screen. liver cell library by AR, we found a novel ARCAP from hepatoma cells. Analysis of expression patterns of ARCAP and characterization of their mechanisms in hepatoma would be important. Our approach is to clarify the distribution of ARCAP and verify the expression in cells specifically. AR and ARCAP are expressed in liver. Analysis of their interaction patterns and characterization of their mechanisms during malignant process in liver would be important to understand genomic signaling in modulatory transactivation and thus leading to tumorigenesis.
2.1. Liver samples
HCC were obtained from the Department of Surgery, Veterans General Hospital-Taipei Clinical information of each patient was recorded and informed consent was obtained. Samples obtained after surgery were stored in liquid nitrogen. This study was approved by review at Taipei General Veterans Hospital (IRB# 91-08-05).
2.2. Cell culture and cell proliferation assay
HepG2, HepA2, HA22T/VGH, Hep3B, SK-Hep-1, Huh7, 293T, PC3, and BNL were grown under standard conditions following ATCC protocols. Hepatoma G2, A2, HA22T/VGH, SK-Hep-1, Huh7 cells, BNL mouse normal liver cells, human embryonic kidney 293T cells, and human prostate cancer cell line PC3 were routinely subcultured in DMEM and NEAA medium (Life Technologies, Inc., Carlsbad, CA) supplemented with 10% fetal bovine serum (Life Technologies, Inc.). A Celltiter 96 (Promega, Madison, MA) was utilized to determine the number of viable cells in proliferation assays. Cells grew with different time courses and at different concentrations following the treatment with dihydrotestosterone (DHT; Sigma, St. Louis, MO) as various studies required. The proliferation assay was performed and analyzed with an ELISA reader Spectra Max 250 (Molecular Devices, Sunnyvale, CA) as the manual suggested.
2.3. RNA extraction and reverse transcription
Total RNA was extracted from tissues and cells using a Trizol Kit (Life Technologies, Inc.). cDNA was synthesized from 10 µg of total RNA. Reverse transcription was performed in a total volume of 30 µΛ containing RNA in 1x first strand buffer (Life Technologies, Inc.) with 10 µM DTT, 500 µM dNTPs, 50 ng/µΛ oligo-dT, and 100 units MMLV reverse transcriptase at 37°C for 1 hour. The samples were then denatured for 5 minutes at 95°C.
2.4. Degenerate priming
One pair of degenerate primers of TGYGARGGYTGYAARRG and CATICCIACIIIVADRCA encoding the zinc finger DNA binding domain from the p box to the second finger domain, sized around approximately a 170 bp PCR product, was utilized in the PCR reaction.
cDNA (1 µΛ) was used for a first PCR amplification in a volume of 25 µl containing 0.8 µM of primer sets, 50 µM of each dNTP (GE Pharmacia, Piscataway, NJ), 1x PCR buffer, and 1.25 units Taq Polymerase (GE Pharmacia). The primers for human transferrin gene are shown as followed. F: TGAGAACTTGGCAAACAAG R: ATTCATGATCTTGGCGATGC. The PCR consisted of a denaturation beginning at 94°C for 3 minutes and 35 cycles of 1 minute at 94°C, 1 minute at 60°C, 1 minute at 72°C, and with a final extension for 10 minutes at 72°C, using a PTC-100 programmable thermal controller (MJ Research, Inc., Watertown, MA). The PCR products were resolved on 1% ethidium bromide-stained agarose gels (FMC Bioproducts, Rockland, ME).
2.6. Multiple Tissue Northern blot
Three Multiple Tissue Northern blots Cat. # 7760-1, 7759-1, and 7767-1 were ordered from BD-Clontech for the hybridization experiments with ARCAP probe. Hybridizations are performed as the user manual.
2.7. Northern hybridization
Samples of total RNA (10 µg) were size-fractionated in 1% agarose-0.67% formaldehyde gels and then transferred to nitrocellulose membranes. Hybridization to α-32P dCTP (GE Amersham Corp., Piscataway, NJ)-labeled DNA probe was performed in 5X SSC, 50% formamide, 2X Denhart’s solution containing 250 µg/µL salmon sperm DNA and 0.1% SDS overnight at 42°C. Membranes were washed in different stringent conditions and exposed to X-ray film (Fuji, Tokyo, Japan).
2.8. Fluorescence in situ hybridization
Cells were prepared as a logarithmic phrase and then by adding 10 μL/mL Colecemid to cell flasks. Cells were broken and hybridized with Cy5 label ARCAP probe. Wash after hybridization and visualized by fluorescence microscope.
2.9. Microscopic analysis of GFP-containing proteins
Full-length ARCAP and ARA70 were cloned into the EcoR1 site in a pEGFP-C1vector (Clontech) to generate GFP-ARCAP and GFP-ARA70. Cells were seeded on chamber glass culture plates (Corning) in a volume of 2 mL at densities of 1 × 105 cells/well. After24 hours, cells were washed with PBS and then transfected with 1 µg of the GFP-containing plasmid constructs using the FuGENE 6. After 6 hours, cells were then washed twice with incubating overnight in fresh media supplemented with 10% FBS. Following PBS wash once, cells were placed in serum-free RPMI 1640 medium for a further 18 hours. Cover slips were washed with PBS, fixed in methanol for 10 minutes at120°C, dried, and inverted on a microscope finally. Slides were analyzed using an Olympus AX80 microscope with 100× magnification.
2.10. Microscopic analysis of GFP-containing proteins
Immunoprecipitation pull-down assay was applied to study the interaction of the AR and the ARCAP. Recombinant constructs of AR-pCDNA3.1 in 293T cell lysate and ARCAP-containing GST-BL21 bacteria lysate respectively incubated with Sepharose-GST beads conjugates were recovered by centrifugation at 14,000 rpm for 3 minutes, and resuspended in 1 mL of buffer A (0.2% Triton X-100 and 350 mM NaCl in PBS), and re-spun. Samples were resuspended in 1 mL of buffer B (PBS containing 0.2% Triton X-100), re-spun, and resuspended in SDS sample buffer. Samples were resolved by electrophoresis in 10% polyacrylamide gels at 200 V for 45 minutes with equal loading Western transferred and coimmunoprecipitated with anti-AR antibody.
2.11. Transient transfection of 293T cells with various ARCAP constructs and luciferase reporter assay followed
Full-length ARCAP and N- and C-terminal truncates of ARCAP GST vector were constructed into pcDNA 3.1 vectors (Invitrogen, Carlsbad, CA). The N-terminal truncate (Y1) had deletions from the position in the amino acid residues 1 to 230 of ARCAP. C-terminus truncate (Y2) abolished the amino acid residues from 710 to 860 of the ARCAP. ARE-containing Luc, a luciferase reporter construct derived from the α-fetoprotein enhancer region, was used for the AR-based transactivation experiments in 293T cells. Beta-gal pC100 vector (Pharmacia, Piscataway, NJ) was included in the transfection for normalization of the luciferase activity. After transfection for a period of 24 hours, cells were washed by PBS, and medium containing vehicle or DHT was added as indicated. Cells were harvested at 48 hours after transfection and analyzed as protocols.
3.1. Degenerated priming method to detect the expression in HCC
Because the regulation of hormonal steroid receptors and cofactors and their actions are. not completely understood in the development of hepatomas, the expression patterns of steroid hormone receptors in hepatoma cells were investigated first. Degenerate primers encoding a conserved region of the zinc finger DNA binding domain of the steroid receptor superfamily were utilized to profile the expression of these receptors in hepatoma (Fig. 1A).
3.2. AR strongly expressed in HCC
A stronger DNA signal was observed in hepatoma tissue than in normal liver tissue after degenerate priming PCR. This PCR-amplified DNA fragment was isolated from the gel and ligated to a pGEM-T vector. Then, the DNAs were transformed into DH5α bacteria cells. To clarify the distribution of steroid receptor expression, we sequenced 100 clones from DNA-transformed DH5α bacteria. After data analysis, the AR ranked 24% of the steroid receptors expressed within hepatoma tissues (Fig. 1B).
3.3. A novel ARCAP cloned by yeast-two hybrid system
Since the AR was found to be expressed in hepatoma tissues and since no coregulators for the AR have been reported in liver, a yeast two-hybrid screening assay using the AR ligand-binding domain as the bait was performed. One candidate protein ARCAP (Androgen Receptor Complex Associated Protein) was found in the liver matchmaker library (BD Clontech). Originally, a short cDNA fragment was obtained. A longer cDNA fragment containing the original sequence was searched for by the screening of a HepG2 hepatoma cDNA library (StrateGene). Subsequently, the full-length ARCAP was obtained using 5’ and 3’ RACE (BD Clontech) with the RNA template from HepG2 cells. The human ARCAP has an open reading frame of 2583 bp that encodes 860 amino acids with a calculated molecular weight of 95 Kd (GenBank: DQ768089.1, GenBank: ABG76793.1).11,12 Analysis of the coding sequence of the human ARCAP revealed that the genomic structure of ARCAP spanned 19 exons of 135 kb of genomic DNA at chromosome1q23.2-q24.3 region. (Fig. 2). Fluorescence in situ hybridization in hepatoma G2 cell indicates the mapping of ARCAP was located at Chromosome I which was shown as yellow spot within Chromosome 1 region (Fig. 3).
3.4. The expression of ARCAP
ARCAP is a novel gene. The tissue distribution of its transcriptional expression was determined by Northern and PCR techniques. ARCAP mRNA was not expressed in most normal human tissues, except it was weakly expressed in heart and skeletal muscle (Fig. 4A). There was a barely detectable level of ARCAP in normal liver, but it was highly expressed in all human hepatoma cell lines examined (Fig. 4B). ARCAP was more highly expressed in 13 HCC tissues with in the adjacent liver tissue from 15 pairs of the tumor and adjacent tissue samples studied (Fig. 4C). It’s interesting that ARCAP is most expressed in liver tumor cells and hepatoma.
3.5. Localization of ARCAP
Ligand-bound AR is present in the nucleus.8 The intracellular location of ARCAP was determined with an ARCAP-containing GFP fusion construct after it was transiently transfected into hepatoma G2 cells. Fluorescence microscopic observation, ARCAP is primarily localized in the nucleus of liver tumor cells (Fig. 5) with less located in the cytoplasm.
3.6. ARCAP interacts with AR to modulate AR transactivation in liver cells
To further examine the interaction between these two proteins, an ARCAP-containing GST expression BL21 bacteria lysate with an GST-express epitope and an AR subsequently fuse pcDNA3.1-containing 293T cell lysate are incubated with GST resin beads. Interaction complexes were purified and Western blotting to recognize the signal with the AR antibody against an immunoprecipitated. Our data demonstrate the in vitro pulldown can identify the interaction between ARCAP and AR, but not with the control protein (Fig. 6).
3.7. Partial localization of the ARCAP binding regions with AR
Since there is no AR and ARCAP expressing in 293T cells, thus vector only, AR, and wild type of, truncates of the N-terminus (Y1) and the C-terminus (Y2) of ARCAP-containing DNA and an ARE-containing luciferase vector were transiently transfected into 293T cells. After 48 hours, cells were treated with different agents as 1% alcohol, or 10nM DHT and cells lysed after 24 hours, and then luciferase activity was determined. We demonstrate AR only can transactivate ARE in a DHT-dependent function. Meanwhile, data indicate a full-length of ARCAP DNA can co-transactivate with AR in a hormone-dependent manner as shown in Fig. 7.
3.8. Map ARCAP regions in regulating cell growth
There is no ARCAP expressing in mouse normal liver cells (BNL), thus vector only, and wild type of, truncates of the N-terminus (Y1) and the C-terminus (Y2) of ARCAP-containing DNA are transfected into BNL cells. After 48 hours, cells were treated with different agents as 1% alcohol in Fig. 8A; 100nM estrogen (E2) in Fig. 8B, or 100nM DHT in Fig. 8C. We observe overexpression of ARCAP stimulating BNL cell growth but truncates of ARCAP lose partial ability to increase BNL cell growth. Meanwhile, data indicate a full length of ARCAP can modulate liver cell proliferation independent of hormone treatment as shown in Fig. 8.
4.1. Novel AR CAP
AR as known plays an important role in sexual development.13 The distribution of AR in hepatoma has been reported by several laboratories.14 A report described the presence of androgen receptors within liver tumor was significantly related to smaller tumor size.14 As many reports about hepatomas, there is an elevation of both androgen receptors and estrogen receptors activities, and the progression from hyperplasia to cancer parallels suppresses of ER expression, but maintain of AR expression.15 The signal pathways involving androgen signaling may affect the risk of HBV-related HCC among men.16 The case of CAG polymorphism in exon 1 region within AR gene has demonstrated to be associated with a human male. HCC cases with shorter AR alleles found to confer a higher risk as reported.17 Since the molecular mechanisms of AR seem to be important in liver tumor pathogenesis, but are still ambiguous, the understanding of these mechanisms should provide new avenues leading toward successful treatments for hepatoma. As reported, several studies have identified and characterized cofactors (coactivators or corepressors) for steroid receptors.17,18 The interactions of the steroid receptor and the cofactors are essential for the regulation of target gene transcription.18,19 AR complexes containing acetyltransferase and other coactivators have been reported.20 Herein, we present ARCAP express a higher level in malignant liver cells and tumors. Our findings indicate a novel ARCAP cloned and mapped to Chromosome 1q23.2 may initiate the cascade pathway.
4.2. Expression and localization of ARCAP
It has been speculated that differential levels of steroid receptor-coregulators among tissues contribute, at least partly, lead to the different responses elicited by these hormones.20 The present findings indicate that the ARCAP is a cofactor with possible activation capability for AR. ARCAP is mostly expressed in liver tumor cell lines and liver tumor tissues. Although less is known about the functional domains of ARCAP yet, little input let us define the physiological roles in normal development and homeostasis, hormone regulation, and for the progression of cancer. Some of the characteristics of the ARCAP are studied further for understanding ARCAP might play a key point in possible liver tumorigenesis.
4.3. Interaction of ARCAP and AR
Our findings indicate that the ARCAP is a cofactor with activation capability for the AR. ARCAP not only stimulates the transactivating ability of AR, but stimulates proliferative growth of liver cells. ARCAP is the first coactivator that specifically acts on AR in liver tumors. Whereas the ARCAP is highly expressed in liver tumors and hepatoma cell lines, in normal tissues, it is only expressed at very low heart and skeletal muscle,
The ability of ARCAP to affect AR transcriptional activation depends on the ARCAP and AR interacting regions, due to AR C-terminal ligand-containing interaction domain (C-terminal ligand-containing AR utilized as a “bait” dependent transcription) is enhanced by ARCAP coexpression. Thus, the ARCAP represents a novel AR ligand domain-associated coactivator. Interestingly, ARCAP and AR are present in only a subset of hepatomas, suggesting that ARCAP may play a role in regulating AR-dependent liver function. Although we have searched and mapped the possible regions of ARCAP that might interact with the ligand-binding domain of AR, sequence analysis of the ARCAP has not revealed any classical interaction motifs found for other nuclear receptors. Several WD40 containing motifs21,22 were found in both the N- and C-termini of the ARCAP which might contribute to the interaction of the ARCAP and the AR. These will be studied further. Deletion analysis of ARCAP failed to define a distinct interface within the ARCAP that mediates the association with AR.
One plausible explanation is that ARCAP may associate with AR ligand-binding domain through multiple weak interactions and that removal of any one of these contacts might make the ARCAP incapable of binding. ARCAP molecule may be involved in and the whole completeness of ARCAP may be necessary for the requirement of any region interacting with AR.
In conclusion, hepatocytes have a remarkable proliferative capacity that is little noted in normal liver. During tumorigenesis or following injury, the activation of the hepatocyte cell cycle can be directly stimulated by overexpression or combinations of genes. The critical steps required for the transition in liver from manipulating ARCAP-regulated to constitutive hepatocyte proliferation to malignancy are being studied. Verification of ARCAP performance may lead us to understand liver tumorigenesis.
We thank Dr. Ming-Jer Tsai (Baylor College of Medicine, Houston) for providing the AR plasmid construct, and Dr. William Scher (Mount. Sinai Medical Center, New York) for previous suggestions and for reviewing this manuscript. Taipei Veterans General Hospital research grant supported the project process.
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