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Cell, Tumor, and Stem Cell Biology |
by Phosphatidylinositol 3-Kinase Signaling Cascade1 Department of Molecular and Cellular Biochemistry, 2 Department of Pathology, 3 Comprehensive Cancer Center, College of Medicine, Ohio State University, Columbus, Ohio; 4 Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois; and 5 Department of Molecular and Integrative Physiology and Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan
Requests for reprints: Samson T. Jacob, Department of Molecular and Cellular Biochemistry, College of Medicine, The Ohio State University, Columbus, OH 43210. Phone: 614-688-5494; Fax: 614-688-5600; E-mail: jacob.42{at}osu.edu and Kalpana Ghoshal, Department of Molecular and Cellular Biochemistry, College of Medicine, The Ohio State University, Columbus, OH 43210. Phone: 614-292-8865; Fax: 614-292-4118; E-mail: ghoshal.1{at}osu.edu.
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Abstract |
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(C/EBP), a target of GSK-3, impeded MT expression, which could not be reversed by PI3K inhibitors. DNA binding activity of C/EBP and its phosphorylation at T222 and T226 by GSK-3 are required for MT expression. MTF-1 and C/EBP act in concert to increase MT-2A expression, which probably explains the high level of MT expression in the liver. This study shows the role of PI3K/AKT signaling pathway and C/EBP in regulation of MT expression in hepatocarcinogenesis. [Cancer Res 2007;67(6):2736–46] |
Introduction |
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Generation of excessive free radicals in tissues resulting from chronic inflammation damages cellular macromolecules, including DNA, which lead to mutation, apoptosis, hyperproliferation, and ultimately cancer (7). Metallothioneins (MT), a group of stress response proteins induced at a high level by oxidative stress, are efficient scavengers of reactive oxygen species (ROS) and reactive nitrogen species (8–10). These are evolutionarily conserved, ubiquitously expressed, and cysteine-rich, heavy metal binding proteins. Four isoforms of MT are arranged in tandem on mouse chromosome 8 and on human chromosome 16 (11). Human genome contains several MT-1 variants of which some are pseudogenes. MT-2A is the major MT isoform expressed in human. Expression of MT-1 and MT-2 are coordinately regulated in all tissues. An important role of MTs is to preserve homeostasis of biologically essential metals, such as zinc and copper, and to scavenge the toxic metals, such as cadmium and mercury (8, 9). The major function of MT is to scavenge free radicals by using its unique metal-thiolate clusters that act as redox sensor and are rapidly oxidized by ROS releasing apo-MT and the metal ions (12, 13). Overproduction of MT at a high level selectively in the heart can protect mice from the cardiotoxic effects of the potent anticancer drug Adriamycin (13).
MTs are expressed at high levels in the liver and are dramatically induced by a variety of agents (8, 9), whereas these genes are not induced in rodent hepatomas following exposure to heavy metals (14). We explored the molecular mechanism of MT down-regulation in a transplanted rat hepatoma (15). This study showed that methylation of CpG islands (CGI) located on MT-1 gene promoter played a causal role in silencing this gene, which was demethylated and reactivated by treatment of animals bearing the tumor with DNA hypomethylating agent 5-azacytidine.
The primary objective of the present study was to determine whether down-regulation of MTs is a common event in primary HCCs and to elucidate the underlying mechanism. This study showed that MT is suppressed in primary HCC by transcriptional repression rather than by promoter methylation and it is mediated through the activation of phosphatidylinositol 3-kinase (PI3K)/AKT pathway inducing dephosphorylation of the transcription factor CCAAT/enhancer binding protein (C/EBP) .
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Materials and Methods |
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and ß-actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), anti-Myc (9E10) and anti-Flag (M2) antibodies were from Sigma, and anti-HA antibody was from Covance (Philadelphia, PA). Phospho-T222/T226 and phospho–S21-C/EBP were from Cell Signaling (Danvers, MA).
Plasmid construction. Mouse MTF-1 cDNA and rat C/EBP cDNA were PCR amplified with gene-specific primers with Accuprime polymerase (Invitrogen, Carlsbad, CA) and cloned into pcDNA-Flagx3 (Sigma) and pcDNA3.1-HA, respectively (Invitrogen).
Human HCC samples and immunohistochemical analysis. HCC and matching liver tissues were obtained from Ohio State University Tissue Bank (Institutional Review Board protocol no. 2004C0081). Formalin-fixed tissue sections from cancerous liver were stained with MT (E9) antibody (DAKO, Carpinteria, CA) and developed using avidin-biotin method (16).
Mouse primary tumors. Liver tumor was induced in C57B/6XSLJ mice by diethylnitrosamine following published protocol (17).
Real-time reverse transcription-PCR of MT-2A and MT-1. An aliquot of cDNA synthesized from total RNA was subjected to real-time SYBR Green PCR using gene-specific primers following published protocol (18, 19). Reverse transcription-PCR (RT-PCR) primers for human MT-2A, MT-1, and C/EBP are provided in the Supplementary Materials and Methods.
Cell culture, treatment with inhibitors, and transfection. Human HCC cell line (HepG2 and Hep3B) were cultured according to supplier's [American Type Culture Collection (ATCC), Manassas, VA] instruction. Immortalized human liver derived THLE-2 cells (ATCC) were cultured in the recommended medium (Cambrex, Walkersville, MD). These cells maintain properties of hepatocytes and express liver-specific markers (20). Exponentially growing cells were treated with 50 µmol/L LY290002, DMSO (vehicle), or 100 nmol/L Wortmannin for 1 h followed by treatment with ZnSO4 (50 µmol/L) for 3 h. Similarly, cells were treated with 25 mmol/L LiCl or NaCl for 1 h before zinc treatment. H293T cells were transfected with constitutively active or dominant-negative PI3K, AKT expression vector, or the empty vector using LipofectAMINE 2000 (Invitrogen) following manufacturer's protocol. After 36 h, cells were harvested and the RNA isolated was subjected to RT-PCR analysis.
Western blot analysis. The whole-cell extracts were immunoblotted with different antibodies as described earlier (21, 22). The signal was developed with enhanced chemiluminescence (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) after incubation with appropriate secondary antibodies.
Generation of Hep3B cells depleted of C/EBP by short hairpin RNA. A short hairpin RNA (shRNA) specific for C/EBP (23) cloned into pRetroSuper vector (24) and transfected Phoenix cells were used to generate infectious viral particles using calcium phosphate precipitation method. After 24 h, the cells were washed and allowed to grow. The retroviral particles collected after 24 h from the culture medium were used to infect Hep3B cells. Cells infected with C/EBP-specific shRNA or pRetroSuper were selected with puromycin (2 µg/mL) and the expression of C/EBP and MT-2A was analyzed in the selected cells.
Ectopic expression of C/EBP mutants and their effect on MT expression. The C/EBP mutants (S193A and S193A+R290A) were a generous gift of Dr. Nicholai Timchenko (Baylor College of Medicine, Houston, TX; ref. 23). The generation of S21A and T222A/T226A mutants of C/EBP has been described earlier (21, 22). The mutants along with the wild-type (WT) C/EBP or empty vector were transfected to C/EBP-depleted Hep3B cells using LipofectAMINE 2000. After 24 h, cells were split into three plates, and 12 h later, they were either treated with ZnSO4 (50 µmol/L) or left untreated for an additional 3 h. Whole-cell extracts were subjected to Western blot analysis with anti-C/EBP antibody. Real-time RT-PCR analyses were for MT-1 and MT-2A done with total RNA.
Immunoprecipitation. C/EBP-depleted Hep3B cells were transfected with pcMTF-1–Flag and pcC/EBP-HA separately or in combination. Cells transfected with the empty vector were used as control. After 48 h, cells were harvested and washed, and extracts made in TNN buffer were subjected to immunoprecipitation with anti-Flag or anti-HA antibodies (25). Immunocomplexes were pulled down with protein G-agarose and washed with TNN buffer followed by Western blot analysis.
Chromatin immunoprecipitation. Formaldehyde cross-linked chromatin was prepared from THLE-2 cells following published protocol (26) and subjected to immunoprecipitation with anti-C/EBP, MTF-1 IgG, or normal rabbit IgG. The immunocomplexes were pulled down by protein A-agarose and DNA was purified (26). Input and pulled-down DNA were subjected to PCR with primers specific for MT-2A, albumin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoters. The primer sequences are provided in the Supplementary Data.
Methylation analysis. Genomic DNA from liver and HCCs were subjected to bisulfite conversion as described (26–28) followed by amplification of the promoter region of each MT isoform using strand-specific primers (see Supplementary Data). MT-2A PCR product was also subjected to sequencing using dideoxy termination kit (26).
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To examine whether down-regulation of MT is a common molecular event in the etiology of HCCs, we used diethylnitrosamine-induced hepatocarcinogenesis in mice as a model system. These mice develop liver tumors after 30 weeks of diethylnitrosamine injection (see Materials and Methods for details). Real-time RT-PCR analysis showed significant suppression (92%) of MT-1 expression in all four tumors compared with the three age-matched control livers (Fig. 1C), which is consistent with the data obtained with primary human HCCs (Fig. 1B).
Expression of the key transcription factor, MTF-1, or one of its target genes, Zn-T1, is not significantly altered in human primary HCCs. MTF-1 is the transcription factor required for both basal and heavy metal-induced expression of MT genes (8, 29). This ubiquitously expressed transcription factor binds to the metal response elements (MRE) on the MT promoters and activates expression of these genes. To examine whether decreased MT levels in HCCs are due to reduced expression of MTF-1 in primary HCCs, we did real-time RT-PCR analysis. The results showed that MTF-1 level was not significantly altered (P = 0.075) in HCCs relative to the matching normal tissues (Fig. 1D).
To rule out the possibility that the transactivation potential of MTF-1 is compromised in HCCs, we measured mRNA level of zinc transporter-1 (Zn-T1), another MTF-1 target gene (30). Real-time RT-PCR analysis showed that the expression of Zn-T1 in tumors was not significantly different from the liver tissues (Fig. 1D). Thus, it is unlikely that MT down-regulation in HCCs is due to inactivation of MTF-1.
Inhibitors of PI3K and AKT up-regulate both basal and zinc-induced expression of MT-2A and MT-1 in HCC. PI3K/AKT signaling pathway, which regulates cell growth and cell survival, is activated in different forms of cancer, including HCC (31). We tested the possibility that the activation of PI3K/AKT pathway in HCCs may play a key role in down-regulation of MT expression. To address this issue, we compared the effect of LY294002, a specific inhibitor of PI3K, on MT expression in human liver-derived epithelial cell line THLE-2 and HCC cell line Hep3B. Real-time RT-PCR analysis showed 12- to 13-fold increase in basal expression of MT-2A and MT-1 genes in the inhibitor-treated Hep3B cells (Fig. 2A
), suggesting that PI3K negatively regulates their expression. As anticipated, zinc induced expression of both MT-2A (
53-fold) and MT-1 (50-fold), which was further up-regulated (6-fold and 4-fold, respectively) in cells pretreated with LY294002 (Fig. 2A). In contrast, LY294002 exerted minimal effect on both basal and zinc-induced MT-2A and MT-1 expression in a human liver-derived epithelial cell line THLE-2 (Fig. 2A), supporting the notion that higher activity of PI3K indeed down-regulates MT expression in HCC cells. The higher level of MT-2A and MT-1 mRNA in THLE-2 cells (liver epithelial) compared with Hep3B (HCC) cells concurs with the observation in primary liver tumors (Fig. 1B and C). Treatment of THLE-2 cells with zinc further stimulated expression of MT-2A and MT-1 2.4- and 3.8-fold, respectively (Fig. 2A). The relatively lower level of stimulation of MT expression in these cells compared with that in Hep3B cells is probably due to their higher basal expression in THLE-2 cells. LY294002 exerted similar effect on the expression of MT genes in other HCC cell lines, such as HepG2 (human), Hepa (mouse), and H4 (rat; data not shown). We also tested another potent PI3K inhibitor, Wortmannin, on MT expression in HCC and liver epithelial cells. Like LY294002, Wortmannin treatment increased MT-2A and MT-1 levels 7.8- and 2.6-fold, respectively, in Hep3B cells without any significant effect on THLE-2 cells (Fig. 2B). These results suggest that relatively high PI3K activity suppresses MT expression specifically in hepatocarcinoma cells.
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10-fold) and zinc-induced (9.6-fold) MT-2A mRNA level in Hep3B cells (Fig. 2C). Similarly, AKT inhibitor induced basal and zinc-stimulated expression of MT-1 5- and 5.7-fold, respectively (Fig. 2C). Comparatively lesser stimulation of MT-1 by PI3K and AKT inhibitors is probably due to their differential effects on MT-1 variants. AKT inhibitor did not have any significant effect on MT induction in THLE-2 cells (Fig. 2D). Taken together, these results indicate that the activation of PI3K/AKT signaling cascade leads to down-regulation of MT expression in HCC cells.
Constitutively active and dominant-negative mutants of PI3K and AKT reciprocally regulate MT gene expression. To confirm the role of PI3K/AKT in MT gene regulation, we expressed constitutively active (P110* and AKT*) and dominant-negative (kinase dead) mutants (P110*KR and AKTKR; ref. 32) separately in H293T cells. We used H293T cells, as detectable expression of the ectopic kinases was achieved in these cells. Western blot analysis showed that the levels of both PI3K and AKT variants were comparable (Fig. 3A, top
). The size of the constitutively active AKT protein was higher than the dominant-negative mutant due to its myristoylation (32). Because the basal expression of MT is very low in H293T cells, their zinc-induced expression was analyzed. The results showed that both constitutively active PI3K and AKT impeded MT-2A expression by 54% and 60%, respectively, compared with the vector-transfected cells (Fig. 3A). The inhibitory effects of P110* and AKT* on MT-1 mRNA level were comparatively less prominent (28% and 21%, respectively) than those observed on MT-2A expression. In contrast, MT-2A mRNA level increased 83- and 43-fold in cells expressing dominant-negative mutants of PI3K and AKT, respectively (Fig. 3A). MT-1 expression also increased 4- and 7-fold, respectively, when KR mutants of P13K and AKT were ectopically expressed (Fig. 3A, bottom). Relatively low level of MT-1 induction might be due to differential effect of PI3K/AKT on different MT isoforms. These results further confirm that MT-2A and MT-1 genes are subjected to negative regulation by PI3K/AKT signaling pathway.
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, a downstream target of PI3K/AKT, reduces MT expression. We entertained the possibility that C/EBP may be one of the transactivators involved in MT expression in the liver for the following reasons. First, it is a major transcription factor that negatively regulates hepatocyte proliferation by controlling cell cycle progression and maintains terminal differentiation state (for review, see ref. 33). Second, its function is compromised in different rodent and human malignancies due to down-regulation of its expression (34, 35). Third, its activity is negatively regulated in HCC cells by PI3K/AKT signaling (23). Fourth, both MT-2A and MT-1 promoters harbor multiple C/EBP cognate sites (analyzed by TRANSFAC database).
As a first step to determine whether the PI3K-mediated inactivation of C/EBP results in down-regulation of MT genes in HCCs, we depleted C/EBP from Hep3B cells by RNA interference. Hep3B cells expressing C/EBP-specific shRNA were generated using a retroviral vector (23). C/EBP RNA was reduced by 88% in puromycin-resistant pool of cells infected with pRetroSuper harboring C/EBP-shRNA compared with the cells infected with the vector alone (Fig. 3B, top). Two isoforms of C/EBP (42 and 30 kDa) that arise due to usage of alternate ATG codon are expressed in Hep3B cells (Fig. 3B). The level of both polypeptides was significantly reduced (70%) in depleted cells (sh) compared with the vector-infected cells or parental Hep3B cells. Protein levels of other C/EBP isoforms (ß and ) and MTF-1 were unaltered in C/EBP-depleted cells (Fig. 3B), showing its specific depletion by shRNA. Real-time RT-PCR analysis showed that the basal MT-2A and MT-1 RNA levels were reduced by 52% and 55%, respectively, in C/EBP-depleted cells compared with the control cells (Fig. 3B, bottom). Zinc-induced expression of MT-2A and MT-1 was also decreased by 48% and 74%, respectively, in C/EBP-depleted cells (Fig. 3B, bottom). These results were reproducible in different batches of cells. These results indicate that C/EBP is one of the key transcription factors involved in regulating MT expression in human HCC cells.
C/EBP is the target of PI3K/AKT signaling to the MT promoter. Next, we investigated whether the observed decrease in MT level in HCCs was due to depletion or inactivation of C/EBP. Because C/EBP mRNA level in 12 HCC samples (where MT expression was substantially reduced) was not significantly altered (Fig. 3C), we explored the possibility that inactivation of this transcription factor by PI3K/AKT suppresses MT expression. We hypothesized that if C/EBP mediates the regulatory effect of PI3K on MT expression, depletion of the factor will abrogate the stimulatory effect of the PI3K inhibitor LY294002. To test this possibility, MT expression was measured in C/EBP-depleted cells treated with this inhibitor. As expected, MT-2A and MT-1 mRNA levels increased 13- and 15-fold, respectively, in control (vector infected) cells treated with LY294002 (Fig. 3D). On the contrary, the inhibitor failed to stimulate MT expression in C/EBP-depleted cells. These results support the notion that C/EBP is the key target of PI3K/AKT signaling ensuing inhibition of MT expression in HCC cells.
Phosphorylation of C/EBP by GSK-3 is critical to MT gene expression. Phosphorylation of C/EBP at different serine/threonine residues located within different domains (Fig. 4A
) regulates its function (for review, see ref. 33). It is phosphorylated at S21 by Erk1 (22), at T222/T226 by GSK-3 (21), at S193 by cyclin-dependent kinase (Cdk) 2/Cdk4, and at S248 by an unidentified kinase (36). Among these, phosphorylation at S193 and T222/T226 is negatively regulated by PI3K/AKT pathway. To delineate the phospho-serine/threonine residue critical for MT expression, we measured zinc-induced expression of endogenous MT genes following ectopic expression of the WT and mutants of mouse C/EBP in Hep3B cells depleted of endogenous C/EBP. The results showed that the WT C/EBP elevated MT-2A mRNA 4-fold, whereas T222A/T226A double mutant failed to increase its expression (Fig. 4B). In contrast, MT-2A expression was not significantly inhibited in cells expressing S21A, which corroborated with the inability of Erk1 inhibitors to affect MT expression in Hep3B cells (data not shown). S193A mutant that inhibits its interaction with Cdk2 and Brm (23, 36) did not alter MT-2A expression (Fig. 4C). It is, therefore, unlikely that recruitment of these proteins by CEBP is involved in MT expression. T222A/T226A mutant also failed to stimulate basal MT-2A expression (data not shown). Western blot analysis showed that the expression of the WT and mutant proteins was comparable (Fig. 4D). These results show that phosphorylation at T222/T226 by GSK-3 is required for MT gene activation.
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90%) the level of phospho-T222/T226 C/EBP (both p42 and p30) without altering C/EBP protein level (Fig. 5B). A 2-fold increase in phospho-T222/T226 level in cells treated with PI3K inhibitor (Fig. 5B) substantiates the conclusion that PI3K/AKT negatively regulates activity of C/EBP by inhibiting GSK-3 (31). However, zinc treatment did not significantly change C/EBP protein level or its phosphorylation status at either site (Fig. 5B). As expected, phosphorylation of C/EBP at S21, catalyzed by Erk1 (22), was not significantly affected by inhibitors of GSK-3 or PI3K (Fig. 5B).
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is essential for MT gene activation. C/EBP is a unique transcription factor that regulates gene expression by directly binding to cognate cis elements as well as indirectly by interacting with other transcription factors or chromatin-modifying factors (different domains of C/EBP are depicted in Fig. 4A; ref. 37). To identify the underlying mechanism of regulation of MT genes by C/EBP, we ectopically expressed a mutant (S193A and R290A) devoid of DNA binding activity due to mutation of R290 in the basic region (23). Real-time RT-PCR analysis showed that MT-2A mRNA levels increased in cells expressing the WT or S193A mutant compared with the control cells transfected with the vector alone (Fig. 4C). In contrast, MT-2A did not increase in cells expressing the double mutant (S193A/R290A), indicating that the DNA binding activity of C/EBP is essential for MT gene transactivation. Western blot analysis showed that expression of the WT and mutant proteins was comparable (Fig. 4D).
To confirm that C/EBP indeed binds to MT promoters in vivo, we did chromatin immunoprecipitation assay. Formaldehyde cross-linked chromatin was immunoprecipitated with specific antibody or normal rabbit IgG (negative control). Both the precipitated DNA and the input DNA were subjected to PCR with MT-2A promoter-specific primers. The results showed that C/EBP indeed interacts with MT-2A promoter although at a reduced level compared with MTF-1 (Fig. 5C). Inability of rabbit IgG to pull down the promoter suggests specific interaction of these transcription factors to the promoter. Lack of amplification of GAPDH promoter from DNA pulled down by C/EBP or MTF-1 antibody further confirms specific association of these transcription factors with MT-2A promoter. However, MTF-1 was not associated with ALBUMIN, another C/EBP target gene, showing specificity of the antibodies. Real-time PCR analysis showed that associations of C/EBP and MTF-1 with MT-2A promoter (normalized to input) were 20- and 45-fold, respectively, compared with the negative control (rabbit IgG; Fig. 5D). Altogether, these results show that C/EBP activates MT-2A gene by directly binding to its promoter.
C/EBP and MTF-1 cooperatively activate MT genes. C/EBP interacts with many transcription factors through its transregulatory domains, basic region, and leucine zipper (Fig. 4A) to modulate gene expression (33, 37). Because its depletion reduced both basal and heavy metal-induced expression of MT genes, we tested whether it modulates the transactivation potential of MTF-1, a transcription factor essential for the basal and metal-induced expression of MT (8). For this purpose, we overexpressed these two factors alone or in combination in C/EBP-depleted Hep3B cells and measured endogenous MT-2A and MT-1 levels by real-time RT-PCR. As expected, MTF-1 increased endogenous MT-2A level (5.5- and 8-fold by 3 and 6 µg MTF-1, respectively; Fig. 6A, left
). Similarly, C/EBP also elevated MT-2A mRNA 3.6- and 5.1-fold, at 3 and 6 µg, respectively. Basal MT-2A expression was synergistically activated (16-fold) when both transcription factors were coexpressed (Fig. 6A). Zinc-stimulated expression of MT-2A also increased 3.7- and 2.2-fold by 6 µg MTF-1 and C/EBP, respectively (Fig. 6A, right). On the other hand, these transcription factors exerted only additive effect on zinc-stimulated expression of MT-2A (5.7-fold). Western blot analysis showed that the expression of ectopic MTF-1 and C/EBP was proportional to the amount of plasmids transfected (Fig. 6B, compare lanes 2 and 3, lanes 4 and 5, and lanes 6 and 7, respectively).
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-HA were coexpressed in C/EBP-depleted cells and the extracts were immunoprecipitated with anti-Flag or anti-HA antibody. Western blot analysis of the precipitated proteins confirmed that each antibody specifically pulled down only the respective polypeptide (Fig. 6C, compare lanes 6 and 7 and lanes 10 and 11, respectively). As expected, neither antibody precipitated any protein from cells transfected with the vectors (Fig. 6C, lanes 1, 5, and 9). Anti-HA antibody precipitated both C/EBP and MTF-1 when these proteins were coexpressed (Fig. 6C, lane 8), implicating their interaction in vivo. Similarly, anti-Flag antibody pulled down both MTF-1 and C/EBP from this extract (Fig. 6C, lane 12). Detection of the ectopic proteins (in extracts and in immunoprecipitates) by anti–MTF-1 and C/EBP antibodies show authenticity of the recombinant proteins. The endogenous MTF-1 in 50 µg whole-cell extracts was too little to be detected by MTF-1 antibody (Fig. 6C, lanes 1 and 2), whereas C/EBP was not detected in vector-transfected cells because we used C/EBP-depleted cells to maximize the effect of ectopic protein on MT expression.
Based on the results generated in the present study, we propose a model (Fig. 6D) that depicts regulation of MT expression by PI3K and its downstream effectors. To our knowledge, the data have revealed for the first time that phosphorylation of C/EBP at threonines by GSK-3 is essential for MT expression and PI3K/AKT negatively regulates MT expression by inactivating GSK-3.
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We have shown previously that MT expression is suppressed in mouse and rat cell lines (27, 43) and in a transplanted rat hepatoma (15) due to promoter methylation. One of the goals of the present study was to determine whether similar mechanism is operative for MT suppression in primary human HCCs. Interestingly, combined bisulfite restriction analysis and bisulfite genomic sequencing of MT-2A and MT-1 promoters did not, however, reveal tumor-specific methylation of CGIs in their promoters (see Supplementary Fig. S2 and Supplementary Data). The results presented here show that transcriptional repression is the primary mechanism of down-regulation of MT expression in HCCs, which may be permanently silenced by promoter methylation during subsequent rounds of replication in the host or in cell culture. This notion is further supported by the observation that MT-1 suppression in primary hepatomas developed in rat livers in response to folate/methyl-deficient diet was also caused by transcriptional mechanism rather than methylation of its promoter region (data not shown).
An important finding here is the potential role of PI3K/AKT signaling in the regulation of MT expression. Studies to date have emphasized the role of the metal transcription factor MTF-1in the basal and induced expression of MT. The lack of significant change in the level and activity of MTF-1 led us to search for other factor(s) that mediates suppression of MT in the liver tumors. C/EBP was considered a potential candidate for the following reasons. First, C/EBP is an abundant transcription factor in the liver whose growth-inhibitory property is compromised in primary HCCs by PI3K signaling cascade (23). Second, C/EBP is essential for the maintenance of liver energy metabolism and regulation of hepatocyte proliferation (33). Third, a recent report indicates significantly diminished C/EBP level in diethylnitrosamine-induced hepatic tumors (44), which is correlated with drastic reduction of MT level in these tumors (Fig. 1D). Further, ectopic C/EBP has been shown to inhibit HCC formation in a knockin mouse model where C/EBP is expressed from -fetoprotein promoter, a functional promoter in liver tumors (44). It is, therefore, conceivable that down-regulation of MT, a C/EBP target and abundantly expressed protein in the liver, may play a causal role in hepatocarcinogenesis. Abundance of C/EBP in the liver may explain relatively high MT level in this tissue.
The present study also established that inhibition of MT expression in HCC cells is mediated by activation of the PI3K/AKT pathway as a result of inhibition of GSK-3 activity and subsequent phosphorylation of C/EBP at T222/T226. In contrast, phosphorylation at Ser193 by cyclin D3-Cdk4/Cdk6 complex (36) that is also negatively regulated by PI3K independent of GSK-3 activity is not essential for MT activation. These findings reveal a specific role of GSK-3 in suppression of MT in HCC.
There is one report that suggests positive regulation of mouse MT promoter activity by PI3K (45). This conclusion was based on decreased reporter gene activity driven by several copies of MRE-d (the MTF-1 binding site) in cells treated with PI3K inhibitor. We also observed inhibitory effect of PI3K inhibitor, LY294002, on luciferase activity driven by five copies of MRE-d in tandem [(MRE-d)5-Luc] when transfected into HepG2 cells (data not shown). In contrast, luciferase expression transcribed by the native MT-1 promoter (–457 to +67) was stimulated by LY294002 (data not shown), mimicking the endogenous gene expression in the chromatin context. These results show that the artificial promoter (MRE-d–Luc) behaves very differently from the endogenous promoter at least with respect to PI3K signaling. It is, therefore, critical to study the effect of any inhibitors in the context of the native promoter.
A common characteristic of the liver cancer–causing agents, such as HBV and HCV infection and alcohol, is the induction of oxidative stress by inflammatory cells, which results in chronic hepatic injury and eventually transformation of regenerating hepatocytes to HCC (46, 47). Mice overexpressing MT are relatively resistant to agents that cause oxidative stress and hepatic hyperplasia (48), whereas MT-1 and MT-2 knockout mice are markedly more sensitive to chemical carcinogenesis (49, 50). Future studies with MT-1 and MT-2 null or MT-1 overexpressor mice in different animal models of hepatocarcinogenesis will further elucidate the role of C/EBP and P13K/AKT signalling in the protective function of MTs against malignant transformation of hepatocytes.
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Acknowledgments |
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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.
We thank Drs. Nicholai Timchenko, Anke Klippel (Merck Research Laboratories, Boston, MA), Rene Bernards (The Netherlands Cancer Institute, Plesmanlann, Amsterdam, The Netherlands), and Glen Andrews (University of Kansas Medical Center, Kansas City, KS) for providing us C/EBP mutants, PI3K and AKT mutants, pRetroSuper, and anti–MTF-1 antibody, respectively.
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Footnotes |
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This work is dedicated to the memory of Dr. Robert Costa.
Received 12/ 1/06. Revised 12/27/06. Accepted 1/10/07.
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 B. Ramaswamy, S. Majumder, S. Roy, K. Ghoshal, H. Kutay, J. Datta, M. Younes, C. L. Shapiro, T. Motiwala, and S. T. Jacob Estrogen-Mediated Suppression of the Gene Encoding Protein Tyrosine Phosphatase PTPRO in Human Breast Cancer: Mechanism and Role in Tamoxifen Sensitivity Mol. Endocrinol., February 1, 2009; 23(2): 176 - 187. [Abstract] [Full Text] [PDF]
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T. E. Miller, K. Ghoshal, B. Ramaswamy, S. Roy, J. Datta, C. L. Shapiro, S. Jacob, and S. Majumder MicroRNA-221/222 Confers Tamoxifen Resistance in Breast Cancer by Targeting p27Kip1 J. Biol. Chem., October 31, 2008; 283(44): 29897 - 29903. [Abstract] [Full Text] [PDF]
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 J. Datta, H. Kutay, M. W. Nasser, G. J. Nuovo, B. Wang, S. Majumder, C.-G. Liu, S. Volinia, C. M. Croce, T. D. Schmittgen, et al. Methylation Mediated Silencing of MicroRNA-1 Gene and Its Role in Hepatocellular Carcinogenesis Cancer Res., July 1, 2008; 68(13): 5049 - 5058. [Abstract] [Full Text] [PDF]
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 B. J. Murphy, T. Kimura, B. G. Sato, Y. Shi, and G. K. Andrews Metallothionein Induction by Hypoxia Involves Cooperative Interactions between Metal-Responsive Transcription Factor-1 and Hypoxia-Inducible Transcription Factor-1{alpha} Mol. Cancer Res., March 1, 2008; 6(3): 483 - 490. [Abstract] [Full Text] [PDF]
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