cell biochemistry and function Cell Biochem Funct 2015; 33: 241–248. Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cbf.3110
Tumour promotion versus tumour suppression in chronic hepatic iron overload Steven A. Bloomer1 and Kyle E. Brown2,3,4* 1
Division of Science and Engineering, Penn State Abington College, Abington, PA, USA Iowa City Veterans Administration Medical Center, Iowa City, IA, USA 3 Division of Gastroenterology-Hepatology, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA, USA 4 Program in Free Radical and Radiation Biology, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA, USA 2
Although iron-catalysed oxidative damage is presumed to be a major mechanism of injury leading to cirrhosis and hepatocellular carcinoma in hemochromatosis, these events have been difficult to recapitulate in an animal model. In this study, we evaluated regulators of hepatocarcinogenesis in a rodent model of chronic iron overload. Sprague–Dawley rats were iron loaded with iron dextran over 6 months. Livers were harvested and analysed for markers of oxidative stress, as well as the following proteins: p53, murine double minute 2, the Shc proteins p66, p52, p46; β-catenin, CHOP, C/EBPα and Yes-associated protein. In this model, iron loading is associated with hepatocyte proliferation, and indices of oxidative damage are mildly increased in tandem with augmented antioxidant defenses. Alterations potentially favouring carcinogenesis included a modest but significant decrease in p53 levels and increases in p52, p46 and β-catenin levels compared with control livers. Countering these factors, the iron-loaded livers demonstrated a significant decrease in CHOP, which has recently been implicated in the development of hepatocellular carcinoma, as well as a reciprocal increase in C/EBPα and decrease in Yes-associated protein. Our results suggest that chronic iron overload elicits both tumour suppressive as well as tumour-promoting mechanisms in rodent liver. Copyright © 2015 John Wiley & Sons, Ltd. key words—β-catenin; C/EBPα; CHOP; hemochromatosis; liver; MDM2; oxidative stress; Yes-associated protein
INTRODUCTION Iron-catalysed oxidative damage to cellular components is presumed to be a major mechanism of tissue damage caused by iron overload disorders such as hemochromatosis. As a primary site of iron storage under physiologic conditions, the liver is a target of iron-related damage, which can progress to cirrhosis if untreated. Once cirrhosis is present, there is a substantial risk of hepatocellular carcinoma (HCC) developing within the iron-loaded liver.1 Although less common, HCC can also occur in noncirrhotic livers of hemochromatosis patients,2 and multiple studies have linked excess iron with liver cancer risk in various forms of chronic liver disease.3–7 Taken together, these data support the concept that oxidative damage driven by excess iron may be directly involved in hepatocarcinogenesis. These clinical observations notwithstanding, it has proven difficult to confirm a causative role for iron-catalysed oxidative damage in the genesis of HCC in an animal model. HCC has not been reported in rodents iron-loaded with either dietary carbonyl iron or parenteral iron dextran, which
*Correspondence to: Kyle E. Brown, Division of GastroenterologyHepatology, 4553 JCP, 200 Hawkins Drive, Iowa City, IA 52242, USA. E-mail: [email protected]
Copyright © 2015 John Wiley & Sons, Ltd.
are two of the most commonly used models. Furthermore, the combination of either of these two methods of iron administration with other hepatocarcinogenesis induction protocols has yielded variable results, with iron enhancing the number of preneoplastic foci and/or HCCs in some models, while having no effect or even decreasing numbers of preneoplastic foci in others.8–10 The failure of exogenous iron to promote hepatocarcinogenesis in animal models is particularly surprising in view of the fact that hepatic iron overload stimulates hepatocyte proliferation and commonly results in liver enlargement.11 Hepatomegaly is a frequent physical finding in hemochromatosis and is a consistent consequence of iron loading in rodent models, irrespective of the chemical form of iron used or the route of its administration. Although the mechanisms responsible for liver enlargement have not been characterized in all models of iron overload, we previously reported that iron dextran appears to act as a direct mitogen.11 In that work, we observed upregulation of cyclin D1 protein and messenger RNA in the livers of rats exposed to iron dextran over a period of 6 months. Like other key regulators of hepatocyte proliferation such as transforming growth factor-α and epidermal growth factor, chronic overexpression of cyclin D1 in transgenic mice is sufficient to drive hepatocarcinogenesis.12–14 Thus, the lack of HCC in Received 1 December 2014 Revised 25 February 2015 Accepted 26 March 2015
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rodent models despite chronic proliferative stimulation as well as the potential for iron-induced oxidative damage is intriguing and suggested to us that iron may induce tumour suppressive mechanisms.
expression of which is sometimes affected by experimental variables (Figure S1). After normalization to Ponceau, the mean protein expression within the control group was determined. Samples in the iron-loaded group were then normalized to the control group, which was given a value of 1. All immunoblots were performed in duplicate or triplicate.
METHODS Chronic iron administration Male Sprague–Dawley rats weighing 200–250 g (Harlan Laboratories, Indianapolis, IN) were individually housed in polyethylene cages with stainless steel tops and were fed a standard rat diet (Dyets, Inc., Bethlehem, PA) and allowed water ad libitum. Iron dextran (50 mg iron per rat for the first four injections, 100 mg per rat thereafter) was administered by intraperitoneal injection every 2 weeks for 6 months as previously described.15 Iron-loaded animals received a total of 900 mg of iron during the course of this experiment. Control animals received intraperitoneal injections of an equivalent quantity of dextran on an identical schedule. The animals were cared for in accordance with criteria from the National Research Council, and the protocol was approved by the Animal Research Committee of the John Cochran Veterans Administration Medical Center. Rats (n = 5 per group) were euthanized by exsanguination while anesthetized with pentobarbital sodium (65 mg kg 1). At the time of death, the livers were quickly excised, weighed and divided for analysis as described in the succeeding text. Biochemical assays Frozen liver tissue was assayed for nonheme iron using the spectrophotometric method of Torrance and Bothwell.16 Thiobarbituric acid reactive species (TBARS) were measured in liver samples as described previously.15 Protein carbonyls were determined by the dinitrophenyl hydrazine method as described previously.15 Immunoblotting Liver tissue was homogenized in radioimmunoprecipitation assay buffer (50 mM Tris, 150 mM NaCl, 0.25% sodium deoxycholate, 1% triton-X, 1 mM EDTA and 1 mM Na3VO4), protein concentrations were determined by the Bradford protein assay (BioRad, Hercules, CA) and immunoblotting was performed as previously described.15 Antibody information and conditions are given in Table S1. Images were taken with the ChemiDoc XRS+ system (BioRad, Hercules, CA), and brightness of bands was quantified with Image Lab software (BioRad). To confirm equal loading and transfer, membranes were subsequently stained with Ponceau S staining solution, and all protein bands within each sample lane were quantified using Image Lab software. Band densities of the protein of interest were normalized to the density of a consistent band on the Ponceau-stained membrane for each sample (shown in Figure S1). Ponceau staining has been shown by us and others17 to quantify protein loading more accurately than routinely used loading controls such as β-actin, the Copyright © 2015 John Wiley & Sons, Ltd.
Statistics Biochemical measures and protein values were compared between groups by t-test for independent samples, controlling for variance as appropriate. Results were considered significant at a p-value of less than 0.05. RESULTS Iron loading resulted in a highly significant increase in hepatic nonheme iron concentration (10 706 vs. 189 μg g 1, p < 0.001). As previously reported, liver sections from rats iron-loaded in this manner show heavy iron deposition in both hepatocytes and sinusoidal lining cells with no conspicuous inflammatory reaction and no histologically evident fibrosis.15,18 No tumours were observed in control or iron-loaded livers. To assess for evidence of iron-induced oxidative damage, we measured TBARS and protein carbonyls. Iron loading was associated with significant (p < 0.05), but rather modest increases in both TBARS (60% increase, Figure 1A) and protein carbonyls (33% increase; Figure 1B). In contrast, levels of the redox-sensitive protein, thioredoxin-1 (Trx-1) were elevated nearly fourfold with iron loading compared with controls (Figure 1C). To further examine cellular pathways controlling hepatocyte proliferation in this model, we first determined the effects of iron loading on the tumour suppressor protein, p53. In the iron-loaded rat livers, there was a subtle but significant decrease in p53 compared with control animals (Figure 2). Because murine double minute 2 (MDM2) is a negative regulator of p53 that has been reported to be responsive to iron,19 we evaluated its expression in this model. Protein levels of MDM2 were increased twofold by iron loading (Figure 2), consistent with the role of MDM2 in stimulating degradation of p53. The oncogenic protein β-catenin is a mediator of the Wnt pathway, and iron has been implicated in Wnt signalling;20 therefore, we evaluated the expression of β-catenin in this model. Iron overload was associated with a twofold increase in β-catenin protein abundance (Figure 3). However, immunohistochemical staining showed no nuclear localization of β-catenin in either the iron-loaded livers or the controls; only membranous staining was observed with no appreciable differences between the groups (not shown). Consistent with this result, levels of c-myc, another β-catenin target, were similar in iron-loaded and control livers (Figure 3). The Src-homology and collagen homolog (Shc) family of adaptor proteins contains three related redox-sensitive proteins (p66, p52 and p46) transcribed from the same gene, which contain overlapping amino acid sequences. Initially, these proteins were shown to transform NIH 3T3 cells and Cell Biochem Funct 2015; 33: 241–248.
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Figure 2. Effect of iron loading on the murine double minute 2 (MDM2)– p53 axis. Top panel: representative immunoblots for MDM2 (115 kDa) and p53 (53 kDa). Bottom panel: quantitation of MDM2 and p53 expression. Band densities were normalized to the Ponceau stain, and results are pre* sented as this ratio. Significant difference between control and iron-loaded animals, n = 5 animals per group
Figure 1. Assessment of the redox environment with iron loading. Assays for thiobarbituric acid reactive species (TBARS; Panel A), and protein carbonyls (Panel B) were performed as described in the Methods section. Panel C: immunoblot analysis of thioredoxin-1 (Trx-1; 12 kDa) in control and iron-loaded livers. Top panel: representative immunoblot. Bottom panel: quantitation of Trx-1 expression. Band densities of Trx-1 were normalized * to the Ponceau stain, and results are presented as this ratio. Significant difference between control and iron-loaded animals, n = 5 animals per group
induce tumourigenesis in mice.21 More recently, p66 was shown to have a stimulatory role on p53 expression22 and to enhance mitochondrial H2O2 generation.23 Additionally, in human and animal models of HCC, p46 and p52 are overexpressed.24,25 In agreement with reports in various types of cultured cells, the abundance of p66 was low compared with that of p46 and p52 in both control and ironloaded livers.26 We found no significant difference in p66 expression between the control and iron groups; however, Copyright © 2015 John Wiley & Sons, Ltd.
iron loading was associated with robust increases in both p46 and p52 proteins (Figure 4). The absence of HCC despite the decrease in the tumour suppressor p53 and the increase of several proteins associated with oncogenesis prompted us to investigate mechanisms that may oppose cancer development. In this regard, CCAAT/enhancer binding protein α (C/EBPα) is of particular interest because it is an inhibitor of cellular proliferation27 and it is induced by iron overload.28 In addition, C/EBPα is a negative regulator of YAP. Overexpression of YAP induces hepatomegaly and strongly stimulates development of HCC in murine liver.29,30 YAP expression has also been described in human HCC, in which it is suggested to be a prognostic marker.31,32 We first confirmed the responsiveness of C/EBPα to iron in our model, showing an approximate 50% increase in C/EBPα protein levels in the iron-loaded livers (Figure 5). Consistent with the increase in C/EBPα, YAP expression was decreased nearly 50% in the iron-loaded livers compared with controls (Figure 5). To examine an upstream mechanism for the iron-induced increase in C/EBPα, we analysed C/EBP homologous protein (CHOP), which is a negative regulator of C/EBPα.29 CHOP has recently been implicated in liver injury and in Cell Biochem Funct 2015; 33: 241–248.
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Figure 3. Iron loading increases hepatic β-catenin levels. Top panels: representative immunoblots for β-catenin (82 kDa) and c-myc (50 kDa). Bottom panel: quantitation of β-catenin and c-myc levels, normalized to the * Ponceau stain. Significant difference between control and iron-loaded animals, n = 5 animals per group
the development of HCC.33,34 Interestingly, iron loading was associated with a ~30% decrease in CHOP protein expression (Figure 5).
DISCUSSION Clinical observations suggest a role for iron in the genesis of HCC in patients with cirrhosis due to hemochromatosis and other etiologies.3,4,35,5–7 Iron excess may promote hepatocarcinogenesis by several routes, including (1) enhancement of oxidative damage to DNA, leading to mutagenesis; (2) acceleration of the development of cirrhosis, which is the primary risk factor for HCC in humans; and (3) stimulation of proliferation. Despite these potentially pro-carcinogenic actions of iron, rodent livers have proven to be quite resistant to the development of HCC in the context of exogenous iron overload. One major difference between rodent models and human liver disease is that iron overload models in rodents fail to induce cirrhosis or even severe fibrosis, thus eliminating this important risk factor for HCC. Although oxidative damage and growth promotion may be sufficient to induce the development of HCC in the absence of cirrhosis, this appears to be a rare phenomenon, based on the observation that the occurrence of Copyright © 2015 John Wiley & Sons, Ltd.
Figure 4. Abundance of p66, p52 and p46 Shc was detected via immunoblot. Top panels: representative immunoblots for p66, p52 and p46 (66, 52 and 46 kDa, respectively). The p66 and p52/p46 bands are shown separately because the conditions required to detect p66 caused the p52 and p46 bands to be indistinguishable. Because p52 was undetectable in control livers, * band densities for this protein were not quantified. Significant difference between control and iron-loaded animals, n = 5 animals per group
HCC in noncirrhotic patients with hemochromatosis remains the subject of case reports. Thus, the objective of this work was to examine specific mechanisms that may account for resistance of rodent liver iron-induced hepatocarcinogenesis. The present results demonstrate that although potentially pro-carcinogenic pathways are activated by iron excess, there is also upregulation of mechanisms that suppress tumour development. Given the nearly 60-fold increase in iron concentration achieved in our model, the observation that Cell Biochem Funct 2015; 33: 241–248.
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Figure 5. Reciprocal regulation of C/EBP homologous protein (CHOP), CCAAT/enhancer binding protein α (C/EBPα) and Yes-associated protein (YAP) with iron administration. Top panel: representative immunoblots for CHOP (37 kDa), C/EBPα (45 kDa) and YAP (75 kDa). Bottom panel: quanti* tation of protein expression, normalized to the Ponceau stain. Significant difference between control and iron-loaded animals, n = 5 animals per group
levels of the tumour suppressor protein p53 were decreased may seem surprising. p53 is stabilized by DNA damage and thus would be expected to increase in the context of widespread oxidative damage. However, in keeping with previous findings in this model,15 indices of oxidative damage were only modestly increased in the iron-loaded livers. We also found that iron loading was associated with an increase in Trx-1; Trx-1 has been shown to have a protective role in other models of liver damage.36,37 We and others have previously shown induction of additional protective responses by iron loading including increased glutathione and cysteine concentrations and enhanced activity of the glutathione Copyright © 2015 John Wiley & Sons, Ltd.
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synthetic machinery, as well as increased expression of metallothionein and heme oxygenase-1.15,38,18 Iron loading of rats with carbonyl iron and ferrocene for as long as 32 months has proven to be the exception to the rule that iron overload fails to induce liver cancer. In those studies, the development of preneoplastic nodules and a single HCC was accompanied by robust increases in multiple indices of oxidative damage, including oxidatively modified DNA.39 Taken together, we postulate that an adaptive response leading to enhanced cellular defenses in our model mitigates oxidative damage, which likely plays an important role in inhibiting carcinogenesis. It is worth pointing out that the decrease in p53 levels in the iron-loaded livers in the present study, while statistically significant, is both of modest magnitude and associated with increased proliferation as previously reported.11 Diverse models, which result in induction of benign hepatocyte proliferation [partial hepatectomy (PH), acute treatment with carbon tetrachloride or the nongenotoxic direct mitogen 1,4 bis [2-(3,5-dichloropyridyloxy)] benzene], have consistently shown an inverse relationship between proliferation and p53 expression.40–42 Furthermore, in support of the general finding that decreases in p53 do not invariably cause carcinogenesis, conditional knockdown of p53 in the liver has also been shown to increase proliferation without tumourigenesis. Other studies have demonstrated that ablation of p53 alone is insufficient to cause hepatocellular carcinoma; however, cancers developed when p53 ablation was combined with knockout of retinoblastoma protein and exposure to the carcinogen diethylnitrosamine.43 Thus, the observation that small decreases in p53 levels are not associated with tumourigenesis in the absence of a strong carcinogenic stimulus is consistent with prior data. At a mechanistic level, the decrease in p53 protein in our model appears to be because, at least in part, of ironmediated upregulation of MDM2. It is interesting to note that our data regarding the effects of iron on MDM2 and p53 conflict with those reported by Dongiovanni et al.19 Those authors found that treatment with the iron chelator deferoxamine increased MDM2 and diminished p53 levels, both in immortalized foetal mouse hepatocytes and in rat liver. Conversely, iron in the form of ferric ammonium citrate suppressed MDM2 and elevated p53 levels in immortalized hepatocytes—the inverse of our findings. There are important experimental differences that may explain the discrepant results. First, Dongiovanni et al. did not examine the effects on MDM2 and p53 of chronic iron overload in a whole animal model, limiting their whole animal study to a relatively short-term (2 weeks) chelation experiment. The current paper is the first to examine the effects of chronic iron excess on MDM2 and p53 in a whole animal. Second, in the study of Dongiovanni and colleagues, the treatment of cultured hepatocytes with ferric ammonium citrate was of short duration (24 h). The acute nature of the exposure is important in view of their observation that iron-related changes in p53 expression were blunted by co-treatment with antioxidants.19 Given prior and current data showing that long-term iron overload upregulates multiple Cell Biochem Funct 2015; 33: 241–248.
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antioxidants,15,19,18 this suggests that adaption to chronic iron-induced oxidative stress may modulate its effects of excess iron on MDM2 and p53. Previous studies have shown that overexpression of β-catenin is associated with hepatomegaly.44,45 For this reason and given the important role of β-catenin in hepatocellular carcinoma,46,35 as well as the observation that iron might be involved in Wnt signalling,47 we were interested in characterizing β-catenin in our model. While we noted a significant increase in immunoreactive β-catenin with iron loading, we did not observe nuclear localization of β-catenin nor did the iron-loaded livers demonstrate alterations in levels of c-myc, a β-catenin target (Figure 3). Cyclin D1 is also a β-catenin target, and while we and others have previously shown that cyclin D1 is upregulated with iron loading,11,48 our data suggest that this may involve mechanisms other than activation of β-catenin. The increase in β-catenin levels in the iron-loaded livers in the absence of nuclear translocation suggests that the protein may be sequestered at the cell membrane. In murine liver, prolonged proliferative stimulation induced by hepatocyte growth factor results in an increase in membrane-bound β-catenin that has been proposed to serve as a tumour suppressive mechanism by limiting availability of β-catenin.49 Although the mechanism and significance of the increase in β-catenin in this model will require additional study, the current data provide further evidence for the concept that β-catenin is an iron-responsive protein. The Shc family of proteins is involved in diverse functions, including mitogenic signalling,21 cell migration and adhesion, and generation of oxidative stress and apoptosis.23 Members of this family are also implicated in various cancers including HCC.24,25 We observed that iron induced robust increases in p46 and p52 with no consistent change in levels of p66. Prior studies have shown that expression of p66 is induced by oxidative stress.50,51 We speculate that the lack of an increase in p66 levels in response to chronic iron overload may reflect the relatively low level of oxidative stress in this model. Furthermore, although previous studies have indicated that p66 antagonizes the growth stimulatory effects of p52,26 relatively little is known about the biology of these proteins in benign proliferation in the liver. One study in which proliferation was induced by PH reported a marked increase in p46 levels in regenerating rat livers.52 Conversely, p66 appears to act as a ‘brake’ for proliferative processes, and impaired regeneration after PH in aged mice has been linked to enhanced oxidative stress and increased apoptosis mediated by p66.53 Further studies will be required to confirm this relationship, but taken together with our data, these observations suggest that p46 and p52 may be involved in benign proliferation of hepatocytes. Finally, our results suggest that an additional important factor mitigating the risk of HCC in our model is the increase in C/EBPα, which is a powerful tumour suppressor.54,55 The elevation in C/EBPα likely occurs via iron-mediated downregulation of CHOP, which negatively regulates both protein levels of C/EBPα as well as its transcriptional activity.56 Copyright © 2015 John Wiley & Sons, Ltd.
Induction of CHOP expression appears to involve the unfolded protein response and/or the integrated stress response; less is known regarding mechanisms leading to downregulation of CHOP.57 In view of the lack of significant fibrosis in our model, it is interesting to note that CHOP-deficient mice are protected from fibrosis in other models of hepatic injury.33 Also interesting and of potential direct relevance to the inhibition of fibrogenesis, two studies have shown that CHOP knockout mice are protected from tumourigenesis, thus assigning a causative role for CHOP in HCC.33,34 Combined with the observations that C/EBPα causes both apoptosis and downregulation of the pro-oncogenic YAP in cancer cell lines,58 a reciprocal relation between the roles of CHOP and C/EBPα in carcinogenesis has been established. Overexpression of YAP causes massive HCC in animal models, increases the resistance of cancer cell lines to doxorubicin and is a putative prognostic marker of HCC in humans.30,59,32 Thus, the decrease in YAP expression in our model is likely protective against iron-mediated HCC. Research to identify the mechanism by which iron overload suppresses CHOP levels and to test whether forced expression of CHOP enhances fibrogenesis and tumourigenesis in iron overload would be of great interest, as would direct assessment of the protective role of C/EBPα in this model. Future studies should also address whether similar alterations in tumour promoters versus tumour suppressors occur in iron-loaded human livers.
ACKNOWLEDGEMENTS The authors thank Kimberly Broadhurst for expert technical assistance. The c-myc antibody was a kind gift of Dr. Lori Rink at Fox Chase Cancer Center. S. A. B was supported by start-up funds from Penn State Abington. K. E. B. was supported by the Veterans’ Administration.
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31. Zender L, Spector MS, Xue W, et al. Identification and validation of oncogenes in liver cancer using an integrated oncogenomic approach. Cell 2006; 125: 1253–1267. DOI: 10.1016/j.cell.2006.05.030 32. Xu MZ, Yao TJ, Lee NP, et al. Yes-associated protein is an independent prognostic marker in hepatocellular carcinoma. Cancer 2009; 115: 4576–4585. doi:10.1002/cncr.24495. 33. DeZwaan-McCabe D, Riordan JD, Arensdorf AM, et al. The stressregulated transcription factor chop promotes hepatic inflammatory gene expression, fibrosis, and oncogenesis. PLoS Genet 2013; 9: e1003937. doi:10.1371/journal.pgen.1003937. 34. Scaiewicz V, Nahmias A, Chung RT, et al. CCAAT/enhancer-binding protein homologous (chop) protein promotes carcinogenesis in the DEN-induced hepatocellular carcinoma model. PLoS One 2013; 8: e81065. doi:10.1371/journal.pone.0081065. 35. Li P, Cao Y, Li Y, et al. Expression of wnt-5a and β-catenin in primary hepatocellular carcinoma. Int J Exp Pathol 2014; 7: 3190–3195. 36. Okuyama H, Nakamura H, Shimahara Y, et al. Overexpression of thioredoxin prevents acute hepatitis caused by thioacetamide or lipopolysaccharide in mice. Hepatology 2003; 37: 1015–1025. doi:10.1053/jhep.2003.50203. 37. Pérez VI, Cortez LA, Lew CM, et al. Thioredoxin 1 overexpression extends mainly the earlier part of life span in mice. J Gerontol A Biol Sci Med Sci 2011; 66A: 1286–1299. doi:10.1093/gerona/glr125. 38. Brown KE, Mathahs MM, Broadhurst KA, et al. Increased hepatic telomerase activity in a rat model of iron overload: a role for altered thiol redox state? Free Radic Biol Med 2007; 42: 228–235. doi:10.1016/j. freeradbiomed.2006.10.039. 39. Asare GA, Mossanda KS, Kew MC, et al. Hepatocellular carcinoma caused by iron overload: a possible mechanism of direct hepatotoxicity. Toxicology 2006; 219: 41–52. doi:10.1016/j.tox.2005.11.006. 40. Arora V, Iversen PL. Antisense oligonucleotides targeted to the p53 gene modulate liver regeneration in vivo. Drug Metab Dispos 2000; 28: 131–138. 41. Higami Y, Shimokawa I, Ando K, et al. Dietary restriction reduces hepatocyte proliferation and enhances p53 expression but does not increase apoptosis in normal rats during development. Cell Tissue Res 2000; 299: 363–369. doi:10.1007/s004419900126. 42. Bellamy COC, Clarke AR, Wyllie AH, et al. p53 deficiency in liver reduces local control of survival and proliferation, but does not affect apoptosis after DNA damage. FASEB J 1997; 11: 591–599. 43. McClendon AK, Dean JL, Ertel A, et al. RB and p53 cooperate to prevent liver tumorigenesis in response to tissue damage. Gastroenterology 2011; 141: 1439–1450. doi:10.1053/j.gastro.2011.06.046. 44. Cadoret A, Ovejero C, Saadi-Kheddouci S, et al. Hepatomegaly in transgenic mice expressing an oncogenic form of β-catenin. Cancer Res 2001; 61: 3245–3249. 45. Harada N, Miyoshi H, Murai N, et al. Lack of tumorigenesis in the mouse liver after adenovirus-mediated expression of a dominant stable mutant of β-catenin. Cancer Res 2002; 62: 1971–1977. 46. Harada N, Oshima H, Katoh M, et al. Hepatocarcinogenesis in mice with β-catenin and ha-ras gene mutations. Cancer Res 2004; 64: 48–54. doi:10.1158/0008-5472. 47. Song S, Christova T, Perusini S, et al. Wnt inhibitor screen reveals iron dependence of β-catenin signaling in cancers. Cancer Res 2011; 71: 7628–7639. doi:10.1158/0008-5472. 48. Troadec MB, Courselaud B, Détivaud L, et al. Iron overload promotes cyclin D1 expression and alters cell cycle in mouse hepatocytes. J Hepatol 2006; 44: 391–399. doi:10.1016/j.jhep.2005.07.033. 49. Apte U, Zeng G, Muller P, et al. Activation of Wnt/β-catenin pathway during hepatocyte growth factor-induced hepatomegaly in mice. Hepatology 2006; 44: 992–1002. doi:10.1002/hep.21317. 50. Betts DH, Bain NT, Madan P. The p66(Shc) adaptor protein controls oxidative stress response in early bovine embryos. PLoS One 2014 Jan 24; 9(1): e86978. doi:10.1371/journal.pone.0086978. 51. Favetta LA, Robert C, King WA, et al. Expression profiles of p53 and p66shc during stress-induced senescence in fetal bovine fibroblasts. Exp Cell Res 2004; 299: 36–48. 52. Yuji J, Masaki T, Yoshida S, et al. Identification of p46 shc expressed in the nuclei of hepatocytes with high proliferating activity: study of regenerating rat liver. Int J Mol Med 2004; 13: 721–728. doi:10.3892/ijmm.13.5.721. Cell Biochem Funct 2015; 33: 241–248.
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SUPPORTING INFORMATION Additional supporting information may be found in the online version of this article at the publisher’s web site.
Cell Biochem Funct 2015; 33: 241–248.
Tumour promotion versus tumour suppression in chronic hepatic iron overload Steven A. Bloomer1 and Kyle E. Brown2,3,4* 1
Division of Science and Engineering, Penn State Abington College, Abington, PA, USA Iowa City Veterans Administration Medical Center, Iowa City, IA, USA 3 Division of Gastroenterology-Hepatology, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA, USA 4 Program in Free Radical and Radiation Biology, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA, USA 2
Although iron-catalysed oxidative damage is presumed to be a major mechanism of injury leading to cirrhosis and hepatocellular carcinoma in hemochromatosis, these events have been difficult to recapitulate in an animal model. In this study, we evaluated regulators of hepatocarcinogenesis in a rodent model of chronic iron overload. Sprague–Dawley rats were iron loaded with iron dextran over 6 months. Livers were harvested and analysed for markers of oxidative stress, as well as the following proteins: p53, murine double minute 2, the Shc proteins p66, p52, p46; β-catenin, CHOP, C/EBPα and Yes-associated protein. In this model, iron loading is associated with hepatocyte proliferation, and indices of oxidative damage are mildly increased in tandem with augmented antioxidant defenses. Alterations potentially favouring carcinogenesis included a modest but significant decrease in p53 levels and increases in p52, p46 and β-catenin levels compared with control livers. Countering these factors, the iron-loaded livers demonstrated a significant decrease in CHOP, which has recently been implicated in the development of hepatocellular carcinoma, as well as a reciprocal increase in C/EBPα and decrease in Yes-associated protein. Our results suggest that chronic iron overload elicits both tumour suppressive as well as tumour-promoting mechanisms in rodent liver. Copyright © 2015 John Wiley & Sons, Ltd. key words—β-catenin; C/EBPα; CHOP; hemochromatosis; liver; MDM2; oxidative stress; Yes-associated protein
INTRODUCTION Iron-catalysed oxidative damage to cellular components is presumed to be a major mechanism of tissue damage caused by iron overload disorders such as hemochromatosis. As a primary site of iron storage under physiologic conditions, the liver is a target of iron-related damage, which can progress to cirrhosis if untreated. Once cirrhosis is present, there is a substantial risk of hepatocellular carcinoma (HCC) developing within the iron-loaded liver.1 Although less common, HCC can also occur in noncirrhotic livers of hemochromatosis patients,2 and multiple studies have linked excess iron with liver cancer risk in various forms of chronic liver disease.3–7 Taken together, these data support the concept that oxidative damage driven by excess iron may be directly involved in hepatocarcinogenesis. These clinical observations notwithstanding, it has proven difficult to confirm a causative role for iron-catalysed oxidative damage in the genesis of HCC in an animal model. HCC has not been reported in rodents iron-loaded with either dietary carbonyl iron or parenteral iron dextran, which
*Correspondence to: Kyle E. Brown, Division of GastroenterologyHepatology, 4553 JCP, 200 Hawkins Drive, Iowa City, IA 52242, USA. E-mail: [email protected]
Copyright © 2015 John Wiley & Sons, Ltd.
are two of the most commonly used models. Furthermore, the combination of either of these two methods of iron administration with other hepatocarcinogenesis induction protocols has yielded variable results, with iron enhancing the number of preneoplastic foci and/or HCCs in some models, while having no effect or even decreasing numbers of preneoplastic foci in others.8–10 The failure of exogenous iron to promote hepatocarcinogenesis in animal models is particularly surprising in view of the fact that hepatic iron overload stimulates hepatocyte proliferation and commonly results in liver enlargement.11 Hepatomegaly is a frequent physical finding in hemochromatosis and is a consistent consequence of iron loading in rodent models, irrespective of the chemical form of iron used or the route of its administration. Although the mechanisms responsible for liver enlargement have not been characterized in all models of iron overload, we previously reported that iron dextran appears to act as a direct mitogen.11 In that work, we observed upregulation of cyclin D1 protein and messenger RNA in the livers of rats exposed to iron dextran over a period of 6 months. Like other key regulators of hepatocyte proliferation such as transforming growth factor-α and epidermal growth factor, chronic overexpression of cyclin D1 in transgenic mice is sufficient to drive hepatocarcinogenesis.12–14 Thus, the lack of HCC in Received 1 December 2014 Revised 25 February 2015 Accepted 26 March 2015
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rodent models despite chronic proliferative stimulation as well as the potential for iron-induced oxidative damage is intriguing and suggested to us that iron may induce tumour suppressive mechanisms.
expression of which is sometimes affected by experimental variables (Figure S1). After normalization to Ponceau, the mean protein expression within the control group was determined. Samples in the iron-loaded group were then normalized to the control group, which was given a value of 1. All immunoblots were performed in duplicate or triplicate.
METHODS Chronic iron administration Male Sprague–Dawley rats weighing 200–250 g (Harlan Laboratories, Indianapolis, IN) were individually housed in polyethylene cages with stainless steel tops and were fed a standard rat diet (Dyets, Inc., Bethlehem, PA) and allowed water ad libitum. Iron dextran (50 mg iron per rat for the first four injections, 100 mg per rat thereafter) was administered by intraperitoneal injection every 2 weeks for 6 months as previously described.15 Iron-loaded animals received a total of 900 mg of iron during the course of this experiment. Control animals received intraperitoneal injections of an equivalent quantity of dextran on an identical schedule. The animals were cared for in accordance with criteria from the National Research Council, and the protocol was approved by the Animal Research Committee of the John Cochran Veterans Administration Medical Center. Rats (n = 5 per group) were euthanized by exsanguination while anesthetized with pentobarbital sodium (65 mg kg 1). At the time of death, the livers were quickly excised, weighed and divided for analysis as described in the succeeding text. Biochemical assays Frozen liver tissue was assayed for nonheme iron using the spectrophotometric method of Torrance and Bothwell.16 Thiobarbituric acid reactive species (TBARS) were measured in liver samples as described previously.15 Protein carbonyls were determined by the dinitrophenyl hydrazine method as described previously.15 Immunoblotting Liver tissue was homogenized in radioimmunoprecipitation assay buffer (50 mM Tris, 150 mM NaCl, 0.25% sodium deoxycholate, 1% triton-X, 1 mM EDTA and 1 mM Na3VO4), protein concentrations were determined by the Bradford protein assay (BioRad, Hercules, CA) and immunoblotting was performed as previously described.15 Antibody information and conditions are given in Table S1. Images were taken with the ChemiDoc XRS+ system (BioRad, Hercules, CA), and brightness of bands was quantified with Image Lab software (BioRad). To confirm equal loading and transfer, membranes were subsequently stained with Ponceau S staining solution, and all protein bands within each sample lane were quantified using Image Lab software. Band densities of the protein of interest were normalized to the density of a consistent band on the Ponceau-stained membrane for each sample (shown in Figure S1). Ponceau staining has been shown by us and others17 to quantify protein loading more accurately than routinely used loading controls such as β-actin, the Copyright © 2015 John Wiley & Sons, Ltd.
Statistics Biochemical measures and protein values were compared between groups by t-test for independent samples, controlling for variance as appropriate. Results were considered significant at a p-value of less than 0.05. RESULTS Iron loading resulted in a highly significant increase in hepatic nonheme iron concentration (10 706 vs. 189 μg g 1, p < 0.001). As previously reported, liver sections from rats iron-loaded in this manner show heavy iron deposition in both hepatocytes and sinusoidal lining cells with no conspicuous inflammatory reaction and no histologically evident fibrosis.15,18 No tumours were observed in control or iron-loaded livers. To assess for evidence of iron-induced oxidative damage, we measured TBARS and protein carbonyls. Iron loading was associated with significant (p < 0.05), but rather modest increases in both TBARS (60% increase, Figure 1A) and protein carbonyls (33% increase; Figure 1B). In contrast, levels of the redox-sensitive protein, thioredoxin-1 (Trx-1) were elevated nearly fourfold with iron loading compared with controls (Figure 1C). To further examine cellular pathways controlling hepatocyte proliferation in this model, we first determined the effects of iron loading on the tumour suppressor protein, p53. In the iron-loaded rat livers, there was a subtle but significant decrease in p53 compared with control animals (Figure 2). Because murine double minute 2 (MDM2) is a negative regulator of p53 that has been reported to be responsive to iron,19 we evaluated its expression in this model. Protein levels of MDM2 were increased twofold by iron loading (Figure 2), consistent with the role of MDM2 in stimulating degradation of p53. The oncogenic protein β-catenin is a mediator of the Wnt pathway, and iron has been implicated in Wnt signalling;20 therefore, we evaluated the expression of β-catenin in this model. Iron overload was associated with a twofold increase in β-catenin protein abundance (Figure 3). However, immunohistochemical staining showed no nuclear localization of β-catenin in either the iron-loaded livers or the controls; only membranous staining was observed with no appreciable differences between the groups (not shown). Consistent with this result, levels of c-myc, another β-catenin target, were similar in iron-loaded and control livers (Figure 3). The Src-homology and collagen homolog (Shc) family of adaptor proteins contains three related redox-sensitive proteins (p66, p52 and p46) transcribed from the same gene, which contain overlapping amino acid sequences. Initially, these proteins were shown to transform NIH 3T3 cells and Cell Biochem Funct 2015; 33: 241–248.
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Figure 2. Effect of iron loading on the murine double minute 2 (MDM2)– p53 axis. Top panel: representative immunoblots for MDM2 (115 kDa) and p53 (53 kDa). Bottom panel: quantitation of MDM2 and p53 expression. Band densities were normalized to the Ponceau stain, and results are pre* sented as this ratio. Significant difference between control and iron-loaded animals, n = 5 animals per group
Figure 1. Assessment of the redox environment with iron loading. Assays for thiobarbituric acid reactive species (TBARS; Panel A), and protein carbonyls (Panel B) were performed as described in the Methods section. Panel C: immunoblot analysis of thioredoxin-1 (Trx-1; 12 kDa) in control and iron-loaded livers. Top panel: representative immunoblot. Bottom panel: quantitation of Trx-1 expression. Band densities of Trx-1 were normalized * to the Ponceau stain, and results are presented as this ratio. Significant difference between control and iron-loaded animals, n = 5 animals per group
induce tumourigenesis in mice.21 More recently, p66 was shown to have a stimulatory role on p53 expression22 and to enhance mitochondrial H2O2 generation.23 Additionally, in human and animal models of HCC, p46 and p52 are overexpressed.24,25 In agreement with reports in various types of cultured cells, the abundance of p66 was low compared with that of p46 and p52 in both control and ironloaded livers.26 We found no significant difference in p66 expression between the control and iron groups; however, Copyright © 2015 John Wiley & Sons, Ltd.
iron loading was associated with robust increases in both p46 and p52 proteins (Figure 4). The absence of HCC despite the decrease in the tumour suppressor p53 and the increase of several proteins associated with oncogenesis prompted us to investigate mechanisms that may oppose cancer development. In this regard, CCAAT/enhancer binding protein α (C/EBPα) is of particular interest because it is an inhibitor of cellular proliferation27 and it is induced by iron overload.28 In addition, C/EBPα is a negative regulator of YAP. Overexpression of YAP induces hepatomegaly and strongly stimulates development of HCC in murine liver.29,30 YAP expression has also been described in human HCC, in which it is suggested to be a prognostic marker.31,32 We first confirmed the responsiveness of C/EBPα to iron in our model, showing an approximate 50% increase in C/EBPα protein levels in the iron-loaded livers (Figure 5). Consistent with the increase in C/EBPα, YAP expression was decreased nearly 50% in the iron-loaded livers compared with controls (Figure 5). To examine an upstream mechanism for the iron-induced increase in C/EBPα, we analysed C/EBP homologous protein (CHOP), which is a negative regulator of C/EBPα.29 CHOP has recently been implicated in liver injury and in Cell Biochem Funct 2015; 33: 241–248.
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Figure 3. Iron loading increases hepatic β-catenin levels. Top panels: representative immunoblots for β-catenin (82 kDa) and c-myc (50 kDa). Bottom panel: quantitation of β-catenin and c-myc levels, normalized to the * Ponceau stain. Significant difference between control and iron-loaded animals, n = 5 animals per group
the development of HCC.33,34 Interestingly, iron loading was associated with a ~30% decrease in CHOP protein expression (Figure 5).
DISCUSSION Clinical observations suggest a role for iron in the genesis of HCC in patients with cirrhosis due to hemochromatosis and other etiologies.3,4,35,5–7 Iron excess may promote hepatocarcinogenesis by several routes, including (1) enhancement of oxidative damage to DNA, leading to mutagenesis; (2) acceleration of the development of cirrhosis, which is the primary risk factor for HCC in humans; and (3) stimulation of proliferation. Despite these potentially pro-carcinogenic actions of iron, rodent livers have proven to be quite resistant to the development of HCC in the context of exogenous iron overload. One major difference between rodent models and human liver disease is that iron overload models in rodents fail to induce cirrhosis or even severe fibrosis, thus eliminating this important risk factor for HCC. Although oxidative damage and growth promotion may be sufficient to induce the development of HCC in the absence of cirrhosis, this appears to be a rare phenomenon, based on the observation that the occurrence of Copyright © 2015 John Wiley & Sons, Ltd.
Figure 4. Abundance of p66, p52 and p46 Shc was detected via immunoblot. Top panels: representative immunoblots for p66, p52 and p46 (66, 52 and 46 kDa, respectively). The p66 and p52/p46 bands are shown separately because the conditions required to detect p66 caused the p52 and p46 bands to be indistinguishable. Because p52 was undetectable in control livers, * band densities for this protein were not quantified. Significant difference between control and iron-loaded animals, n = 5 animals per group
HCC in noncirrhotic patients with hemochromatosis remains the subject of case reports. Thus, the objective of this work was to examine specific mechanisms that may account for resistance of rodent liver iron-induced hepatocarcinogenesis. The present results demonstrate that although potentially pro-carcinogenic pathways are activated by iron excess, there is also upregulation of mechanisms that suppress tumour development. Given the nearly 60-fold increase in iron concentration achieved in our model, the observation that Cell Biochem Funct 2015; 33: 241–248.
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Figure 5. Reciprocal regulation of C/EBP homologous protein (CHOP), CCAAT/enhancer binding protein α (C/EBPα) and Yes-associated protein (YAP) with iron administration. Top panel: representative immunoblots for CHOP (37 kDa), C/EBPα (45 kDa) and YAP (75 kDa). Bottom panel: quanti* tation of protein expression, normalized to the Ponceau stain. Significant difference between control and iron-loaded animals, n = 5 animals per group
levels of the tumour suppressor protein p53 were decreased may seem surprising. p53 is stabilized by DNA damage and thus would be expected to increase in the context of widespread oxidative damage. However, in keeping with previous findings in this model,15 indices of oxidative damage were only modestly increased in the iron-loaded livers. We also found that iron loading was associated with an increase in Trx-1; Trx-1 has been shown to have a protective role in other models of liver damage.36,37 We and others have previously shown induction of additional protective responses by iron loading including increased glutathione and cysteine concentrations and enhanced activity of the glutathione Copyright © 2015 John Wiley & Sons, Ltd.
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synthetic machinery, as well as increased expression of metallothionein and heme oxygenase-1.15,38,18 Iron loading of rats with carbonyl iron and ferrocene for as long as 32 months has proven to be the exception to the rule that iron overload fails to induce liver cancer. In those studies, the development of preneoplastic nodules and a single HCC was accompanied by robust increases in multiple indices of oxidative damage, including oxidatively modified DNA.39 Taken together, we postulate that an adaptive response leading to enhanced cellular defenses in our model mitigates oxidative damage, which likely plays an important role in inhibiting carcinogenesis. It is worth pointing out that the decrease in p53 levels in the iron-loaded livers in the present study, while statistically significant, is both of modest magnitude and associated with increased proliferation as previously reported.11 Diverse models, which result in induction of benign hepatocyte proliferation [partial hepatectomy (PH), acute treatment with carbon tetrachloride or the nongenotoxic direct mitogen 1,4 bis [2-(3,5-dichloropyridyloxy)] benzene], have consistently shown an inverse relationship between proliferation and p53 expression.40–42 Furthermore, in support of the general finding that decreases in p53 do not invariably cause carcinogenesis, conditional knockdown of p53 in the liver has also been shown to increase proliferation without tumourigenesis. Other studies have demonstrated that ablation of p53 alone is insufficient to cause hepatocellular carcinoma; however, cancers developed when p53 ablation was combined with knockout of retinoblastoma protein and exposure to the carcinogen diethylnitrosamine.43 Thus, the observation that small decreases in p53 levels are not associated with tumourigenesis in the absence of a strong carcinogenic stimulus is consistent with prior data. At a mechanistic level, the decrease in p53 protein in our model appears to be because, at least in part, of ironmediated upregulation of MDM2. It is interesting to note that our data regarding the effects of iron on MDM2 and p53 conflict with those reported by Dongiovanni et al.19 Those authors found that treatment with the iron chelator deferoxamine increased MDM2 and diminished p53 levels, both in immortalized foetal mouse hepatocytes and in rat liver. Conversely, iron in the form of ferric ammonium citrate suppressed MDM2 and elevated p53 levels in immortalized hepatocytes—the inverse of our findings. There are important experimental differences that may explain the discrepant results. First, Dongiovanni et al. did not examine the effects on MDM2 and p53 of chronic iron overload in a whole animal model, limiting their whole animal study to a relatively short-term (2 weeks) chelation experiment. The current paper is the first to examine the effects of chronic iron excess on MDM2 and p53 in a whole animal. Second, in the study of Dongiovanni and colleagues, the treatment of cultured hepatocytes with ferric ammonium citrate was of short duration (24 h). The acute nature of the exposure is important in view of their observation that iron-related changes in p53 expression were blunted by co-treatment with antioxidants.19 Given prior and current data showing that long-term iron overload upregulates multiple Cell Biochem Funct 2015; 33: 241–248.
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antioxidants,15,19,18 this suggests that adaption to chronic iron-induced oxidative stress may modulate its effects of excess iron on MDM2 and p53. Previous studies have shown that overexpression of β-catenin is associated with hepatomegaly.44,45 For this reason and given the important role of β-catenin in hepatocellular carcinoma,46,35 as well as the observation that iron might be involved in Wnt signalling,47 we were interested in characterizing β-catenin in our model. While we noted a significant increase in immunoreactive β-catenin with iron loading, we did not observe nuclear localization of β-catenin nor did the iron-loaded livers demonstrate alterations in levels of c-myc, a β-catenin target (Figure 3). Cyclin D1 is also a β-catenin target, and while we and others have previously shown that cyclin D1 is upregulated with iron loading,11,48 our data suggest that this may involve mechanisms other than activation of β-catenin. The increase in β-catenin levels in the iron-loaded livers in the absence of nuclear translocation suggests that the protein may be sequestered at the cell membrane. In murine liver, prolonged proliferative stimulation induced by hepatocyte growth factor results in an increase in membrane-bound β-catenin that has been proposed to serve as a tumour suppressive mechanism by limiting availability of β-catenin.49 Although the mechanism and significance of the increase in β-catenin in this model will require additional study, the current data provide further evidence for the concept that β-catenin is an iron-responsive protein. The Shc family of proteins is involved in diverse functions, including mitogenic signalling,21 cell migration and adhesion, and generation of oxidative stress and apoptosis.23 Members of this family are also implicated in various cancers including HCC.24,25 We observed that iron induced robust increases in p46 and p52 with no consistent change in levels of p66. Prior studies have shown that expression of p66 is induced by oxidative stress.50,51 We speculate that the lack of an increase in p66 levels in response to chronic iron overload may reflect the relatively low level of oxidative stress in this model. Furthermore, although previous studies have indicated that p66 antagonizes the growth stimulatory effects of p52,26 relatively little is known about the biology of these proteins in benign proliferation in the liver. One study in which proliferation was induced by PH reported a marked increase in p46 levels in regenerating rat livers.52 Conversely, p66 appears to act as a ‘brake’ for proliferative processes, and impaired regeneration after PH in aged mice has been linked to enhanced oxidative stress and increased apoptosis mediated by p66.53 Further studies will be required to confirm this relationship, but taken together with our data, these observations suggest that p46 and p52 may be involved in benign proliferation of hepatocytes. Finally, our results suggest that an additional important factor mitigating the risk of HCC in our model is the increase in C/EBPα, which is a powerful tumour suppressor.54,55 The elevation in C/EBPα likely occurs via iron-mediated downregulation of CHOP, which negatively regulates both protein levels of C/EBPα as well as its transcriptional activity.56 Copyright © 2015 John Wiley & Sons, Ltd.
Induction of CHOP expression appears to involve the unfolded protein response and/or the integrated stress response; less is known regarding mechanisms leading to downregulation of CHOP.57 In view of the lack of significant fibrosis in our model, it is interesting to note that CHOP-deficient mice are protected from fibrosis in other models of hepatic injury.33 Also interesting and of potential direct relevance to the inhibition of fibrogenesis, two studies have shown that CHOP knockout mice are protected from tumourigenesis, thus assigning a causative role for CHOP in HCC.33,34 Combined with the observations that C/EBPα causes both apoptosis and downregulation of the pro-oncogenic YAP in cancer cell lines,58 a reciprocal relation between the roles of CHOP and C/EBPα in carcinogenesis has been established. Overexpression of YAP causes massive HCC in animal models, increases the resistance of cancer cell lines to doxorubicin and is a putative prognostic marker of HCC in humans.30,59,32 Thus, the decrease in YAP expression in our model is likely protective against iron-mediated HCC. Research to identify the mechanism by which iron overload suppresses CHOP levels and to test whether forced expression of CHOP enhances fibrogenesis and tumourigenesis in iron overload would be of great interest, as would direct assessment of the protective role of C/EBPα in this model. Future studies should also address whether similar alterations in tumour promoters versus tumour suppressors occur in iron-loaded human livers.
ACKNOWLEDGEMENTS The authors thank Kimberly Broadhurst for expert technical assistance. The c-myc antibody was a kind gift of Dr. Lori Rink at Fox Chase Cancer Center. S. A. B was supported by start-up funds from Penn State Abington. K. E. B. was supported by the Veterans’ Administration.
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Cell Biochem Funct 2015; 33: 241–248.
