IJC
International Journal of Cancer
Aryl hydrocarbon receptor nuclear translocator is
associated with tumor growth and progression
of hepatocellular carcinoma
Ying Liang1*, Wei-Wei Li1*, Bi-Wei Yang1, Zhong-Hua Tao1, Hui-Chuan Sun1, Lu Wang1, Jing-Lin Xia1, Lun-Xiu Qin1,2,
Zhao-You Tang1,2, Jia Fan1,2 and Wei-Zhong Wu1
1
bHLH/PAS proteins play important roles in tumor progression. Lost or reduced expression of single-minded homolog 2 (SIM)
as well as aryl hydrocarbon receptor repressor (AHRR) has been observed in cancerous human tissues. Here, we investigated
the role of aryl hydrocarbon receptor nuclear translocator (ARNT), another bHLH/PAS protein, in hepatocellular carcinoma
(HCC). Using tissue microarray and immunohistochemistry, we found that intratumoral ARNT was inversely correlated with
time to recurrence and overall survival of HCC patients after resection. Knockdown of ARNT in HepG2, HCCLM3 and HCCLM6
cells significantly shortened cell doubling time, increased S-phase cell populations and accelerated in vivo HCCLM6 growth
and metastasis. After ARNT expression was rescued, prolonged cell doubling time and decreased S-phase cell populations
were observed in HepG2, HCCLM3 and HCCLM6 cells. And, HCCLM6 growth and metastasis in vivo were remarkably inhibited.
Screening by quantitative reverse-transcription PCR and PCR arrays revealed that cyclin E1, CDK2, Fos and Jun were negatively
regulated by ARNT, whereas CDKN1C, CNKN2A, CDKN2B, MAPK11 and MAPK14 were positively regulated in HCC. According to
the results of immunoprecipitation assay, both ARNT/ARNT and ARNT/AHRR complexes were clearly formed in HCCLM6
xenograft with increased ARNT expression. In summary, ARNT is an important regulator of HCC growth and metastasis and
could be a promising prognostic candidate in HCC patients.
Hepatocellular carcinoma (HCC) is the third leading cause of
cancer death in the world and the second in China.1,2 The
long-term prognosis of HCC patients after hepatectomy still
remains a challenge, largely due to its high recurrence rate
Key words: hepatocellular carcinoma, ARNT, cell cycle, proliferation,
prognosis
Abbreviations: AHR: aryl hydrocarbon receptor; ARNT: aryl
hydrocarbon receptor nuclear translocator; CDK: cyclin-dependent
kinase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HCC:
hepatocellular carcinoma; HIF-1a: hypoxia-inducible factor-1 alpha;
OS: overall survival; TTR: time to recurrence
Additional Supporting Information may be found in the online
version of this article.
*Ying Liang and Wei-Wei Li, as first co-authors.
Grant sponsor: National Natural Science Foundation of China;
Grant numbers: 30670953 and 81071904; Grant sponsor: China
National Key Projects for Infectious Disease; Grant number:
2008ZX10002-019, 021
DOI: 10.1002/ijc.26166
History: Received 7 Jan 2011; Accepted 21 Apr 2011; Online 4 May
2011
Correspondence to: Wei-Zhong Wu, Liver Cancer Institute &
Zhongshan Hospital of Fudan University, 180 Fenglin Road,
Shanghai 200032, China, Tel. & Fax: þ86-21-5423 7181,
E-mail: [email protected]
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Int. J. Cancer: 130, 1745–1754 (2012) V
and metastasis.3 The molecular mechanisms of HCC progression need be further investigated for new insights and interventions against metastatic recurrence.
Aryl hydrocarbon receptor nuclear translocator (ARNT),
also known as hypoxia-induced factor-1b (HIF-1b), is one of
the most important nuclear transcription factors within the
basic helix-loop-helix/Per-ARNT-SIM (bHLH/PAS) superfamily. It is widely expressed in human cells, including hepatocytes. When responding to different extracellular stimuli,
ARNT can form a heterodimeric complex with aryl hydrocarbon receptor (AHR), hypoxia-inducible factor-1a (HIF-1a)
and its homologous factors (HIF-2a, HIF-3a), or with singleminded homolog 2 (SIM) to mediate various biological
actions, such as hypoxia reaction, xenobiotic metabolism, teratogenesis, immunosuppression4 and embryonic development.5 However, its onco-biological function is still unclear.
Several prototypes of bHLH/PAS factors have been identified as being involved in tumor progression. HIF-1a promotes tumor progression and metastasis via its regulation of
cancerous glycolysis,6 proliferation, apoptosis7 and angiogenesis.8 AHR appears to be a regulator of cell proliferation, albeit
growth-inhibitory and promoting roles in MCF-7 and HepG2
cells, respectively, are observed.9 SIM is frequently lost or
reduced in primary breast tumors, promoting malignant
transformation of cells and tumor invasion.10 More recently,
aryl hydrocarbon receptor repressor (AHRR) has been
Cancer Cell Biology
Liver Cancer Institute and Zhongshan Hospital, Fudan University, Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai,
People’s Republic of China
2
Institute of Biomedical Sciences of Fudan University, Shanghai, People’s Republic of China
Inhibitory role of ARNT on HCC progression
1746
identified as a tumor suppressor in multiple human cancers.11 These findings collectively suggest that nuclear transcription factors of bHLH/PAS play critical roles in tumor
progression. Although ARNT has been widely studied in tumor angiogenesis and tumorigenesis in the past 2 decades, its
role in tumor progression, especially in HCC, has not been
explored. In our study, the expression levels of ARNT in
HCC surgical specimens were investigated and their prognostic significance was then analyzed. Furthermore, the effects of
ARNT on HCC growth and metastasis were experimentally
tested to confirm clinical observations.
Material and Methods
Cancer Cell Biology
Patients, specimens and follow-up
One hundred five patients were randomly enrolled in this
study. All patients underwent curative liver resection between
January 1999 and March 2006 and had histological confirmation of HCC.12 None of them received any preoperative anticancer treatment. Of the enrolled patients, 90 had a history
of hepatitis B. Preoperative liver functions of all patients were
classified as Child-Pugh A. Tumor stages were determined by
TNM classification according to the 2002 International Union
Against Cancer guidelines.13 Tumor differentiation was graded
by the Edmondson grading system. The Scheuer system was
applied in 100 patients for grading (necroinflammatory activity) and staging (fibrosis and cirrhosis) of the nontumor liver
tissue (Supporting Information Table 1; the surrounding liver
tissue was not adequate for scoring in five patients)14,15 The
study was approved by the Zhongshan Hospital Research
Ethics Committee. Informed consent was obtained from each
patient according to the committee’s regulations.
Patients were followed until March 2008, with a median
follow-up time of 31.2 months. Briefly, all patients were evaluated every other month during the first year and thereafter
at least every 3–4 months. At each visit, alpha fetoprotein
measurement and liver ultrasonography were performed. A
computed tomography scan was performed on the abdomen
every 6 months. A bone scan or magnetic resonance imaging
was conducted if localized bone pain was reported.12,16 Treatment modalities following a relapse were administered based
on uniform guidelines as previously described.12,17,18 Overall
survival (OS) was defined as the interval between surgery and
death, and time to recurrence (TTR) as the interval between
surgery and recurrence. Patients in whom recurrence was not
detected were censored on the date of death or the last follow-up.
Tissue microarray, immunohistochemistry and evaluation
Immunohistochemical staining for molecules of interest was
performed on tissue microarrays made of formalin-fixed, paraffin-embedded tumor resection specimens. Two cores were
taken from the margin of the tumor and from normal liver tissue within 10 mm of the tumor (Shanghai Biochip Company
Ltd, Shanghai, China).16 Rabbit primary antibodies against
human ARNT, HIF-1a, CD31 and CD34 (1:200; Santa Cruz
Biotechnology, Santa Cruz, CA) were used with the components of the Envision-plus detection system (EnVision þ
HRP/Mo; Dako, Carpinteria, CA). The reaction products were
visualized via incubation with 3,30 -diaminobenzidine. The
negative controls were treated identically but without primary
antibody. The expression of ARNT and HIF-1a was analyzed
comprehensively by the extent of staining and the range
according to the Fromowitz standard.19,20 In brief, five visual
fields were randomly observed and 100 cells in each field were
counted. The range scores were graded as 0, 1, 2, 3 and 4
when 0–5%, 6–25%, 26–50%, 51–75% and >75% of the examined cells were positively stained, respectively. The extent
scores were graded as 0 or 1, 2, or 3 when the examined cells
were unstained or stained light yellow, brown or dark brown,
respectively. The expression status of the target protein was
judged jointly by its range and extent score. If the total scores
were 3, the protein was regarded as having low expression;
otherwise, it was considered as highly expressed. CD31 as well
as CD34 levels in cancerous and liver tissues were detected as
previously described.21
Cell lines
Human hepatoma HepG2, HCCLM3 and HCCLM6 cell lines
and their derivates were used in the study; HCCLM3 and
HCCLM6 are hepatitis B virus (HBV) positive. All cell lines
were cultured in Dulbecco’s modified Eagle’s medium (Gibco
BRL, Grand Island, NY) supplemented with 10% fetal bovine
serum and maintained at 37 C in a humidified incubator
containing 5% CO2.
Construction and infection of lentiviral vectors for
modulating ARNT expression
ARNT v1 (NM_001668) and ARNT v2 (NM_178426), two
transcripts of approximately 2.37 kb and 1.0 kb, respectively,
were constitutively expressed in liver tissues. Four sequencespecific shRNAs against human ARNT v1 and ARNT v2
were designed and evaluated in 293T cells by Western blot
and immunofluorescence assays. The stem-loop DNA oligonucleotides with the highest knockdown efficiency were (1)
sense: 50 -CTC AGA TGA AAT TGA GTA C-30 and antisense: 50 -GTA CTC AAT TTC ATC TGA G-30 against
ARNT v1; (2) sense: 50 -ATG ACC CAG CCT GAG GTC T30 and antisense: 50 -AGA CCT CAG GCT GGG TCA T-30
against ARNT v2 (Supporting Information Fig. 1A). A mock
oligonucleotide (sense: 50 -TTC TCC GAA CGT GTC ACG
T-30 and antisense: 50 -AAG AGG CTT GCA CAG TGC A30 ) was used against a scrambled human gene. One mutant
of ARNT v1, starting at oligonucleotide position 1,348 and
ending at position 1,368 was successfully changed from TAC
TCA GAT GAA ATT GAG TAC to TAT TCT GAC GAG
ATA GAA TAT. The mutant together with a FLAG tag
encoding sequence was cloned into a pLVTHM vector
(Shanghai Genechem Co., China; Supporting Information
Fig. 2 and Supporting Information Table 2) and was able to
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Figure 1. Expression levels of ARNT on HCC prognosis. Representative photographs of the immunostaining with (a, c) high and (b, d) low
levels of ARNT in intratumoral and peritumoral tissues, detected using HCC tissue microarrays. (e) The OS of HCC patients with high
intratumoral ARNT level was significantly higher than that of the patients with low levels (p < 0.001). (f) The TTR was lower in HCC patients
with high intratumoral ARNT level than the patients with low levels (p ¼ 0.016).
be translated into the same amino acids as the wild-type
gene. All lentiviral particles were prepared as previously
described.22 Wild-type (WT) HepG2, HCCLM3 and
HCCLM6 were infected with either Lenti-ARNTi-v1 virus,
Lenti-ARNTi-v2 virus or Lenti-Mock virus. In addition,
HCCLM6 cells were co-infected with Lenti-ARNTi-v1 and
Lenti-ARNT-v1 viruses to rescue ARNT expression. All vectors except Lenti-ARNT-v1 expressed the green fluorescent
protein (GFP) signal.
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Detection of ARNT-regulated genes by quantitative
reverse-transcription PCR (qRT-PCR) and qRT-PCR array
Total RNA was extracted by the RNeasyV Mini kit (Qiagen,
Valencia, CA). The quality of RNA from HCCLM6 and its
derivates (Supporting Information Fig. 1B) was assessed via
A260/280 absorbance, and 1.0 lg of total RNA was used to
synthesize the first-strand cDNA with MuLV reverse transcriptase (Applied Biosystems, Foster City, CA) at 42 C for
60 min and then at 95 C for 5 min. The forward and reverse
R
Inhibitory role of ARNT on HCC progression
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1748
Figure 2. The roles of ARNT on HepG2, HCCLM3 and HCCLM6 proliferation. (a) The relative ARNT protein levels and (b) their doubling time
were analyzed using HCCLM6-WT, HCCLM6-Mock, HCCLM6-ARNTi-v1, HCCLM6-ARNTi-v2 and HCCLM6-ARNT-v1 cells. The S-phase cell
populations were significantly increased in (c) ARNT knockdown cells, while decreased in (d) cells with rescued ARNT expression. (e) Downregulated ARNT expression in HepG2, HCCLM3 and HCCLM6 cells significantly accelerated in vitro cell proliferation and vice versa.
primers of qRT-PCR were 50 -AGT GGC GTT TAA GTC
CCC TGA-30 and 50 -GGG ATA CTG CGG CAG TAG CA30 for cyclin E and 50 -CAA GCC AGT ACC CCA TCT
TCG-30 and 50 -CAA ATA GCC CAA GGC CAA GC-30 for
CDK2, respectively. The reactions were performed on a DNA
Engine Opticon system (MJ Research, Reno, NV) using
R Green PCR Master Mix (Applied Biosystems). FolSYBRV
lowing each cycle, SYBR green fluorescence was monitored
and the melting curve was analyzed to ensure that a single
PCR product was obtained. Afterwards, the size and specificity of amplicons were confirmed by 2.5% agarose gel electrophoresis. All reactions were repeated in three separate runs
and evaluated with the Opticon Monitor software (Version
1.02). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
was employed to normalize the samples. RNase-free water
(Qiagen) was included as a negative control in RNA
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Liang et al.
Detection of ARNT protein by Western blotting
ARNT protein in HCCLM6 cells and their derivates was evaluated by Western blotting. About 20 lg of total protein was
extracted and separated by 10% SDS-PAGE, transferred onto
polyvinylidene fluoride membranes, and then reacted with
primary antibodies against ARNT (1:200) and GAPDH. After
being extensively washed with phosphate-buffered saline
(PBS) containing 0.1% Triton X-100, the membranes were
incubated with alkaline phosphatase-conjugated goat antirabbit antibody for 30 min at room temperature. The bands
were visualized using 1-stepTM NBT/BCIP reagents (Thermo
Fisher Scientific, Rockford, IL) and detected by an Alpha
Imager (Alpha Innotech, San Leandro, CA).
Cell proliferation and cell doubling time
HCCLM6 cells and their derivates in exponential growth
phase were trypsinized to yield a single-cell suspension. A
total of 2 104 cells in 1 ml of medium was added to each
well of 24-well plates and incubated at 37 C in 5% CO2. Cell
numbers in three replicate wells were counted by Coulter
Counter after 72 h incubation. Cell doubling time was calculated by the following formula: TD ¼ T(log 2)/log(N/N0)
(TD, doubling time; T: time interval; N0, initial cell number;
N, endpoint cell number).23 Viable cells were also determined
by Cell Count Kit-8 (CCK-8) assay (Dojin Laboratories,
Kumamoto, Japan).24 Briefly, WT HepG2, HCCLM3
HCCLM6 and their derivates with ARNT knockdown or rescued expression were seeded into 96-well plates at an initial
density of 2 103 cells/well. After 24-, 48- or 72-h incubation, 10 ll of the kit reagent was added to each well and the
plates were incubated for another 3 h. All plates were then
scanned by a microplate reader at 450 nm. Cell proliferation
was calculated on the basis of absorbency.
Cell cycle analysis
About 1 106 HCCLM6 cells and their derivates were harvested, washed with cold PBS twice and fixed with 70% ethanol solution. The pellets were then resuspended in PBS and
incubated with RNase A solution at a final concentration of
100 lg/ml at 37 C for 30 min. After staining with 1 mg/ml
of propidium iodide, the cell cycle was measured by flow
cytometry (BD Biosciences, San Jose, CA).
Tumor growth assays in vivo
Ten nude mice (Institute of Materia Medica, CAS, Shanghai,
China) were divided into groups of two mice apiece. Both
mice in each group were injected in the upper right flank
region with 1 107/0.2 ml of HCCLM6-WT, HCCLM6Mock,
HCCLM6-ARNTi-v1,
HCCLM6-ARNTi-v2
or
HCCLM6-ARNT-v1, respectively, to establish subcutaneous
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xenograft models. Four weeks later, subcutaneous tumors 1
cm in diameter were removed, cut into 1-mm3 pieces and
implanted into the livers of other 30 mice to establish orthotopic xenograft models.25 In vivo green fluorescence imaging
of HCCLM6-Mock, HCCLM6-ARNTi-v1, HCCLM6-ARNTiv2 and HCCLM6-ARNT-v1 xenografts was done once a
week (stereomicroscope: Leica MZ6; illumination: Leica L5
FL; C-mount: 0.63/1.25; CCD: DFC 300FX) and quantified
with Image Pro Plus software. On day 42, all mice were sacrificed and spontaneous metastasis to the lung was observed
by the presence of GFP signals. All procedures were
approved by the Animal Care and Use Committee of Shanghai, China.
Immunoprecipitation assay
Approximately 1 mg of tissue from HCCLM6-WT,
HCCLM6-Mock, HCCLM6-ARNTi-v1 and HCCLM6-ARNTv1 xenografts was lysed with 3 ml of RIPA buffer at 4 C for
30 min. One ml of lysate was incubated with 0.25 lg of rabbit IgG together with 20 ll of protein A-agarose at 4 C for
30 min and centrifuged at 10,000 rpm for 10 min. The supernatant was incubated with 2 lg of rabbit anti-human ARNT
(Santa Cruz Biotechnology) or anti-FLAG antibody (SigmaAldrich, St. Louis, MO) at 4 C for 2 h, and then with 20 ll
of protein A-agarose at 4 C overnight. Pellets were washed
three times with RIPA buffer before analysis for HIF-1a,
p300, AHR, ARNT (1:200; Santa Cruz Biotechnology, Santa
Cruz, CA), AHRR (1:1,000; Abcam, Cambridge, MA) and
FLAG levels (1:5,000; Sigma-Aldrich, St. Louis, MO) by the
method described in Detection of ARNT protein by Western
blotting.
Statistical analysis
Statistical analysis was performed with SPSS 16.0 for Windows (SPSS, Chicago, IL). Pearson chi-square test or Fisher
exact test was applied for the comparison of qualitative variables, and quantitative variables were analyzed by ANOVA
and Pearson’s correlation test. Kaplan-Meier analysis was
performed to determine survival probability; Log-rank test, to
compare patients’ survival probability between subgroups;
and Cox regression model, to conduct a multivariate analysis.
Results
Expression levels of ARNT and HIF-1a on HCC prognosis
To understand ARNT expression on HCC prognosis, we first
investigated the protein level in resection specimens from
105 HCC patients using a tissue microarray and immunohistochemical staining (Supporting Information Fig. 1C). Our
results showed that ARNT protein was primarily found in
the nucleus and occasionally in the cytoplasm of hepatocytes
and tumor cells and strongly stained to be a brown or a dark
brown mass. The expression level of ARNT was significantly
higher in normal liver tissues compared with HCC tissues (p
¼ 0.007). Specifically, 75 of 100 peritumoral tissues (75.0%)
and 56 of 105 intratumoral tissues (53.3%) had a high level
Cancer Cell Biology
extraction and in each run. More cell cycle-control genes
were screened by RT Profiler PCR Arrays (PAHS-061A,
SABiosciences, Frederick, MD) and performed by Kangchen
Bio-tech Co. (Shanghai).
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1750
of ARNT (Figs. 1a–1d). Overall survival of patients with a
high intratumoral ARNT level was significantly longer than
survival of those with a low ARNT level, and recurrence incidence was lower in patients with a high intratumoral ARNT
level than in those with a low ARNT level (p < 0.001 and p
¼ 0.016, respectively; Figs. 1e and 1f). Median survival time
of patients with high and low ARNT levels was 84.0 and 26.3
months, respectively (p < 0.001). Thirty-five patients with a
low intratumoral ARNT level experienced tumor recurrence
within 2 years, whereas 11 patients with a high ARNT level
experienced tumor recurrence during the same period of
time. The median survival and recurrence time for patients
with low and high ARNT expression were significantly different (p < 0.001 for both). Intratumoral ARNT level was a
prognostic factor both for OS and TTR in univariate analysis,
but only for OS in multivariate analysis (Supporting Information Table 3). HIF-1a was mainly found in the cytoplasm of
hepatocytes and tumor cells appeared as weakly stained light
yellow or yellow granules. CD31þ and CD34þ blood vessels
were detected both in tumor tissues and in normal liver tissues. Staining hotspots were observed at the interface of tumor and liver tissues. Neither HIF-1a, CD31 nor CD34 levels
had any relationship with OS or TTR in postoperative
patients (Supporting Information Fig. 3).
Proliferation inhibition of ARNT in HCC cells in vitro
To confirm clinical observations, we successfully constructed
four lentiviral vectors to modulate ARNT expression in HCC
cells. Among them, Lenti-ARNTi-v1 and Lenti-ARNTi-v2
were used to suppress ARNT expression (Supporting Information Figs. 1A and 1B), Lenti-ARNT-v1 for rescued expression and mock vector for the control (Supporting Information Figs. 1B and 2, Supporting Information Table 2). After
the vectors were introduced into HCCLM6 cells, the relative
ARNT levels were significantly down-regulated in both
HCCLM6-ARNTi-v1 and HCCLM6-ARNTi-v2 cells, especially in the former, and up-regulated in HCCLM6-ARNT-v1
cells (p < 0.001, Fig. 2a). There was no significant difference
in ARNT levels between HCCLM6-Mock and HCCLM6-WT
cells.
Using these stable infected cell lines, we next evaluated
the roles of ARNT on HCC proliferation and cell cycle progression. HCCLM6-ARNTi-v1 cells grew the fastest, while
HCCLM6-ARNT-v1 cells were the slowest. The doubling
times
of
HCCLM6-ARNTi-v1,
HCCLM6-ARNTi-v2,
HCCLM6-Mock, HCCLM6-WT and HCCLM6-ARNT-v1
were 29.54 6 0.98 h, 40.81 6 1.05 h, 48.06 6 0.71 h, 47.74
6 0.98 h and 72.93 6 3.12 h, respectively (Fig. 2b). To further examine the effect of ARNT on cell cycle progression,
HCCLM6 cells and their derivates were analyzed on the third
day after subculture. The S-phase cell populations of
HCCLM6-ARNTi-v1 and HCCLM6-ARNTi-v2 cells were
conspicuously increased, especially for the former (p < 0.01),
and the S-phase cell populations of HCCLM6-ARNT-v1 cells
were dramatically decreased (p < 0.01, Figs. 2c and 2d).
Inhibitory role of ARNT on HCC progression
To explore the universal significance of ARNT, cell proliferations in ARNT-modulated HepG2, HCCLM3 and
HCCLM6 cells were tested again by a cell counting kit. As
expected, cell proliferation was significantly accelerated in
ARNT-decreased HepG2, HCCLM3 and HCCLM6 cells and
slowed in ARNT-increased cells (Fig. 2e).
Anti-tumor effects of ARNT on HCCLM6 xenograft in vivo
The average tumor size of HCC patients with high intratumoral ARNT expression was statistically smaller than that of
patients with low expression (Fig. 3a, p ¼ 0.001). To elucidate
the effects of ARNT on HCC progression, we compared tumor
metastatic foci in lung in parallel by observing GFP signals. On
day 42 after orthotopic implantation, the numbers of lung metastatic foci of HCCLM6-ARNTi-v1 and HCCLM6-ARNTi-v2
xenografts, especially of the former, were much more numerous than those of HCCLM6-Mock xenografts, while the numbers of lung metastatic foci of HCCLM6-ARNT-v1 were also
fewer than those of HCCLM6-Mock (Figs. 3b and 3c). To
dynamically measure in vivo tumor growth, the fluorescence
area of HCCLM6-Mock, HCCLM6-ARNTi-v1, HCCLM6ARNTi-v2 and HCCLM6-ARNT-v1 xenografts were monitored in parallel once a week for six successive weeks. Again,
HCCLM6-ARNTi-v1 xenografts grew the fastest, whereas
HCCLM6-ARNT-v1 xenografts were the slowest (Figs. 3d and
3e). Our in vivo experiments indicated once more that ARNT
played an inhibitory role on HCCLM6 growth and metastasis.
Heterodimeric and homodimeric complexes formed by
ARNT in HCCLM6 xenografts
Previous studies have shown that bHLH-PAS proteins usually
exert their biological functions as heterodimers or homodimers. To elucidate this issue, the total level of ARNT in
ARNT-modulated HCCLM6 xenografts was first analyzed by
immunoprecipitation assays. Similar to the results in Western
blotting, a relatively low level of the ARNT complex was
pulled down from the HCCLM6-ARNTi-v1 xenograft and a
relatively high level was from HCCLM6-ARNT-v1 xenograft,
as compared with the HCCLM6-WT and HCCLM6-Mock
xenografts. Although a tiny amount of HIF-1 a/ARNT and
AHR/ARNT heterodimer was identified, its level as well as
the cofactor P300 were not markedly changed. However, a
significant decease of AHRR level in the HCCLM6-ARNTiv1 xenograft and an increase in the HCCLM6-ARNT-v1 xenograft were found in ARNT pull-down complexes, as compared with the HCCLM6-WT and HCCLM6-Mock xenografts. In addition, the homodimer of ARNT in the
HCCLM6-ARNT-v1 xenograft was clearly detected by use of
the antibody against the FLAG tag (Fig. 4a).
Cell cycle-control genes regulated by ARNT
To address the underlying mechanisms, mRNA levels of cell
cycle-control genes were then screened by qRT-PCR. Both
cyclin E1 and CDK2 were found to be up-regulated in
HCCLM6-ARNTi-v1
and
HCCLM6-ARNTi-v2
cells,
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Figure 3. The effects of ARNT on in vivo HCC growth in clinical samples and HCC progression in xenograft models. (a) The average tumor
sizes of HCC patients with high intratumoral ARNT levels were statistically smaller than those with low levels (p ¼ 0.001). (b, c) The
numbers of lung metastatic foci of HCCLM6-ARNTi-v1 and HCCLM6-ARNTi-v2 xenografts, especially in the former, were much more numerous
than those of HCCLM6-Mock xenografts, while the numbers of lung metastatic foci of HCCLM6-ARNT-v1 were also fewer than those of
HCCLM6-Mock on day 42 after orthotopic implantation. (d, e) HCCLM6-Lenti-ARNTi-v1 xenograft grew the fastest (p <0.01, vs. HCCLM6Mock, on day 21 and thereafter) and HCCLM6-Lenti-ARNT-v1 xenograft the slowest (p < 0.05, vs. HCCLM6-Mock, on day 42) as evaluated
by GFP fluorescent area.
especially in the former, and to be down-regulated in
HCCLM6-ARNT-v1 cells (Fig. 4b, Supporting Information
Fig. 1D). To gain a comprehensive understanding, more genes
involved in cell cycle progression were then screened by using
qRT-PCR assays. Our results showed that the expression of
CDKN1C, CNKN2A, CDKN2B, MAPK11 and MAPK14 was
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positively regulated by ARNT, while the expression of
CDKN2C, Fos and Jun was negatively regulated (Fig. 4c).
Discussion
Several members of the bHLH/PAS superfamily have been
shown to be involved in tumor progression; however, little is
Inhibitory role of ARNT on HCC progression
Cancer Cell Biology
1752
Figure 4. ARNT complexes and the regulated genes in HCCLM6 xenografts. The levels of HIF-1a, P300, AHR AHRR and ARNT with FLAG tag in
total ARNT complexes were analyzed by immunoprecipitation assays. (a) Heterodimers of HIF-1a/ARNT and AHR/ARNT were not significantly
changed in the HCCLM6 xenograft and its derivates, while the ARNT homodimer and the ARNT/AHRR heterodimer were clearly formed in the
HCCLM6-Lenti-ARNT-v1 xenograft. (b) Cyclin E1 and CDK2 were negatively regulated by ARNT in the qRT-PCR assay. (c) CDKN1C, CNKN2A,
CDKN2B, MAPK11 and MAPK14 were positively regulated, but CDKN2C, Fos and Jun were negatively regulated by ARNT in the qRT-PCR array
assay.
known about the role of ARNT protein per se on clinical
HCC progression. The preliminary clinical results from our
immunohistochemical study suggest for the first time that
ARNT might be gradually lost during HCC progression and
could serve as a prognostic factor for postoperative HCC
patients.
To confirm clinical observation, loss- and gain-of-function
studies were performed by using HCC cell lines and xenograft models in nude mice. We found attenuated ARNT
expression in HepG2, HCCLM3 and HCCLM6 cells with
specific siRNA, especially with Lenti-ARNTi-v1 vector,
greatly shortened in vitro cell doubling time, increased Sphase cell populations and promoted in vivo tumor growth
and lung metastasis in a HCCLM6 xenograft. After ARNT
expression was rescued, prolonged cell doubling time and
decreased S-phase cell populations were observed in HepG2,
HCCLM3 and HCCLM6 cells. And HCCLM6 growth and
metastasis in vivo were remarkably inhibited. Therefore, we
believe that ARNT is a robust and negative regulator of HCC
progression.
Numerous studies have demonstrated that the HIF-1 signal pathway is one of the most important pathways in solid
tumor growth and metastasis.26–28 HBx protein was reported
to cross-talk with HIF-1a and promote its nuclear translocation, phosphorylation, stability and transactivation function
in HBx-inducible Chang liver cells as well as in nonhepatic
cells. This kind of cross-talk may lead to transcriptional activation of HIF-1a target genes [e.g., an increased expression
of vascular endothelial growth factor (VEGF) in HBxþ cells]
and thus stimulate endothelial cell proliferation and tumor
angiogenesis.29,30 Because about 90% of the patients enrolled
in this study had a history of HBV infection, we first evaluated the expression level of HIF-1a. Given that CD31þ and
CD34þ endothelial cells are two major target cells in VEGF
response and their tissue densities can be used to evaluate
the biological functions of VEGF and HIF-1 signaling
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Int. J. Cancer: 130, 1745–1754 (2012) V
Liang et al.
pathway, we observed the prognostic roles of CD31 and
CD34 molecules in the same patient cohort. However, neither
was found to have any prognostic values when evaluated by
TTR and OS. Our findings were consistent with observations
of epithelial ovarian tumors,31 but different from those in
HCV-related HCC,32 oropharyngeal cancer33 and esophageal
cancer.34 Such differences in the expression level of HIF-1a
might arise from the types of tumor studied, the tumor tissue
regions, or the specific pretreatments received. To avoid as
far as possible tissue necrosis and a secondary hypoxia
response caused by radiotherapy and chemotherapy, all tissues analyzed in this study were taken from the margins of
HCC tumors that had not received any pretreatment. In such
a circumstance, HIF-1a was predominantly localized in the
cytoplasm, but not in the nucleus as reported by Wada
et al.,32 suggesting that the periphery of HCC in our patients
may not have been hypoxic. Although a low basal level
expression of VEGF was found in HBx-bearing HCCLM3
and HCCLM6 cells in normoxic culture conditions, no significant changes of this factor were detected after ARNT was
down-regulated or up-regulated (data not shown). In addition, little protein interaction between HIF-1a and ARNT
was found in vivo by using HCCLM6 xenograft tissues.
Therefore, we concluded that the regulation of ARNT on
HCC growth and metastasis was not primarily mediated by
hypoxia- or HBx-induced HIF-1 signal pathway.
Recently, endogenous AHR was identified as a regulator
of cancerous cell proliferation in a ligand-independent pattern.9 Further, AHRR was reported to repress transcription
activity of AHR by competing with this transcription factor
for heterodimer formation with ARNT.11 It is very likely that
the AHRR/ARNT heterodimer plays an antagonistic role to
the AHR/ARNT heterodimer as well as the ARNT homodimer in HCC growth and metastasis. To test this hypothesis,
we analyzed AHR and AHRR levels in ARNT pulled-down
complexes. Although no obvious changes of AHR levels were
found, a marked increase in AHRR levels was detected from
HCCLM6-ARNT-v1 xenografts as compared with those from
HCCLM6-Mock and HCCLM6-ARNTi-v1 xenografts. Thus,
we supposed that the transcriptional activities of ARNT
homodimer-driven gene expression with the E box core
sequence could be reversed, at least partly, in the presence of
the AHRR/ARNT heterodimer.35,36 The relative balance
between the ARNT homodimer and the ARNT/AHRR heterodimer will be important in determining HCC progression.
Cell cycle is strictly controlled by sequential activation of
cyclin/Cdk complexes, which are negatively regulated by
Cdk-inhibitors (CKI).37–39 Two families of CKI, Cip/Kip and
INK4, have been found in human cells. CDKN1C (p57kip2), a
member of the Cip/Kip family, can strongly inhibit the activities of several G1 cyclin/Cdk complexes. CDKN2A
(p16INK4a) and CDKN2B (p15INK4b), two members of the
INK4 family, can function as inhibitors of CDK4 kinase.
Because cell cycle arrest is an obligate step in cell differentiation,40 the activity of the CyclinE/cdk2 complex is usually
inhibited by the members of the Cip/Kip family at this juncture.41,42 Therefore, up-regulation of CDKN1C, CNKN2A
and CDKN2B together with down-regulation of cyclin E1
(CCNE1) and Cdk2 by enhanced ARNT expression may
cause cell cycle arrest from G1 to S phase, resulting in
HCC stagnancy and deceasing HCC metastasis. In addition,
up-regulations of MAPK11 and MAPK14 together with
down-regulations of Fos and Jun by enhanced ARNT expression may inhibit cell proliferation and promote cell differentiation in a mechanism as reported by Johnstone et al.43 and
Hui et al.44
In conclusion, ARNT is an important regulator of the
bHLH/PAS superfamily in HCC progression and a useful
biomarker for predicting HCC prognosis after curative
resection.
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