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Liver and Metabolic Syndrome
Dig Dis 2010;28:236–246 DOI: 10.1159/000282095
PTEN in Non-Alcoholic Fatty Liver Disease/Non-Alcoholic Steatohepatitis and Cancer
Marion Peyrou Lucie Bourgoin Michelangelo Foti
Department of Cellular Physiology and Metabolism, Geneva Medical Faculty, Geneva , Switzerland
Introduction
Non-alcoholic fatty liver disease (NAFLD) is com-monly associated with obesity and diabetes, and repre-sents a well-established component of the metabolic syn-drome [1] . NAFLD encompasses histological features in-cluding simple hepatic steatosis, steatohepatitis, fibrosis and cryptogenic cirrhosis [2] . Hepatocellular carcinoma (HCC) might occur as a likely end stage of NAFLD [3] , thus NAFLD can be regarded as a preneoplastic state of the liver.
The current concept in the development of NAFLD involves a ‘two hits’ model, in which the first hit leads to hepatic steatosis and the second to non-alcoholic steato-hepatitis (NASH). Insulin resistance may be responsible for both hits, whereas oxidative stress, mitochondrial dysfunctions and deregulated cytokine signaling are likely critical events in the progression towards NASH [4] . Obesity is considered a key etiological condition that predisposes to the development of the metabolic syn-drome and NAFLD [5] . Indeed, elevated plasma levels of free fatty acids, released by adipocytes or coming from high-fat feeding, can deeply affect insulin signaling/sen-sitivity in the liver and the development of NAFLD through mechanisms including: (i) intrahepatocellular accumulation of triglycerides and diacylglycerol, (ii) pro-duction of pro-inflammatory cytokines, (iii) activation of
Key Words PTEN � Obesity � Liver � Insulin resistance � Steatosis � Fibrosis � Hepatocellular carcinoma
Abstract The tumor suppressor PTEN is a protein/phosphoinositide phosphatase regulating the PI3K/Akt signaling pathway and is mutated or deleted in a variety of human cancers, includ-ing hepatocellular carcinoma (HCC). Accumulating evidence indicates that alterations of PTEN expression and activity in hepatocytes are common and recurrent molecular events associated with liver disorders of various etiologies includ-ing obesity, the metabolic syndrome, hepatitis B virus/hepa-titis C virus infection and abusive alcohol consumption. Ge-netic and molecular studies, particularly in the context of non-alcoholic fatty liver disease (NAFLD), support a critical role for PTEN in hepatic insulin sensitivity and the develop-ment of steatosis, steatohepatitis and fibrosis. PTEN muta-tions/deletion or low PTEN expression are also associated with diverse liver malignancies, suggesting a critical role for PTEN in hepatic cancers. This review provides an overview of the current knowledge on pathological dysregulations of PTEN expression/activity in the liver with obesity and the metabolic syndrome, and the role of this enzyme in the de-velopment of non-alcoholic fatty liver disease and hepato-cellular carcinoma. Copyright © 2010 S. Karger AG, Basel
Michelangelo Foti Department of Cellular Physiology and MetabolismCentre Médical Universitaire (CMU) , 1, rue Michel-Servet CH–1211 Geneva 4 (Switzerland) Tel. +41 22 379 5204, Fax +41 22 379 5260, E-Mail michelangelo.foti @ unige.ch
© 2010 S. Karger AG, Basel0257–2753/10/0281–0236$26.00/0
Accessible online at:www.karger.com/ddi
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PTEN in NAFLD/NASH and Cancer Dig Dis 2010;28:236–246 237
Ser/Thr kinases, (iv) impairment of IRS/PI3K/Akt signal-ing, (v) alteration of mitochondrial functions and (vi) modulation of gene expression via activation of the tran-scription factors such as NF � B (nuclear factor kappa B) or PPAR (peroxisome proliferator-activated receptor) [6, 7] . These dysregulated processes trigger alterations in in-sulin signaling, inflammation and oxidative stress in he-patocytes [1] , which are the principal factors contributing to the development or worsening of hepatic insulin resis-tance and NAFLD. Further aberrant activation of hepat-ic stellate and Kupffer cells then trigger the development of liver fibrosis and inflam mation [8] . NASH can then evolve to cirrhosis and the subsequent development of ad-enomas and carcinomas.
Deregulations of numerous signaling pathways lead-ing to insulin resistance, steatosis, NASH, fibrosis, aber-rant cell proliferation and resistance to cell death have been reported. Among these, abnormal regulation of the
PI3K/Akt/PTEN/mTOR pathway critically contributes to the development of NAFLD and HCC [9–12] . Of particu-lar interest is PTEN, a protein/phosphoinositide phos-phatase, which terminates PI3K signaling and acts as a potent tumor suppressor. In the context of liver metabol-ic diseases and cancer, increasing evidence now supports a crucial role of PTEN in the development of these dis-eases.
PTEN
PTEN (phosphatase and tensin homolog) is a protein of 403 amino acid residues with a molecular weight of ap-proximately 47 kDa. The enzyme has a N-terminal phos-phatase domain (residues 7–185) with a typical catalytic core (residues 124–130) and a central C2 domain (resi-dues 186–351), both of which are required for the enzy-
Phosphatasedomain
Phosphorylation
Catalytic core PDZ domain
C2 domain
PDB
7 124 130 185 351 403
S362 to S385S229 T232C71 C124
K125 K128 K289
N C
T319 T321
ROS
Acetylation UbiquitinationOxidation
PCAF
ROCK
S229 T232 T319 T321 S362 T366 S370 S380 T382 T383 S385
GSK3� CK2
GLTSCR2
PEST
PEST
C-terminaldomain
Interaction with other proteinsNHERF1/2, MAST kinases, MAGI2, p53, others ...
NEDD4-1
Fig. 1. PTEN protein structure, functional domains and post-translational modifications. The different PTEN domains, their functions and known proteins interacting with PTEN are indicated. The residues undergoing post-translational modifications such as oxidation, acetylation, ubiquitination and phosphorylation are also highlighted, as well as the cellular enzymes (black shapes) potentially modifying these residues. ROS = Reactive oxygen species; PEST = proline-glutamate-serine-threonine-rich.
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matic activity. The C2 domain is also involved in the binding to the enzyme substrates and in the interactions with membranes. The C-terminal tail (residues 352–403) contains PEST (proline-glutamate-serine-threo-nine-rich) sequences contributing to protein stability, and a PDZ domain allowing interactions with scaffolding proteins [13–15] ( fig. 1 ).
PTEN was first identified as a potent tumor suppres-sor frequently mutated or deleted in a variety of hu-man cancers. It represents the second most mutated/de-leted tumor suppressor after p53 in human cancers [16] . PTEN has a dual function, capable of dephosphorylat-ing both proteins and lipids. However, the best charac-terized function is as a phosphoinositide phosphatase. PTEN removes the 3 � -phosphate of PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 to generate PtdIns(4)P and PtdIns(4,5)P 2 , respectively, and thus terminates signaling downstream of PI3K ( fig. 2 ) [17] . In this regard, numerous studies in-dicate that PTEN is a potent negative regulator of growth factor signaling, insulin/IGF-1 signaling in particular, in peripheral tissues [11] . Its tumor suppression activity is most likely due to its antagonistic effects on the anti-apoptotic, proliferative and hypertrophic activity of PI3K [18] . However, recent studies have demonstrated that PTEN can also shuttle from the cytoplasm to the nucleus, where it is essential for maintaining chromosomal stabil-ity and DNA repair [19] . Interestingly, PTEN was also shown to (i) interact with and dephosphorylate the focal
adhesion kinase FAK, thus negatively regulating cell in-teractions with the extracellular matrix [20] , and (ii) in-teract with and stabilize E-cadherin/ � -catenin adherens junctional complexes, thus preventing cancer cell inva-siveness [21, 22] .
Regulation of PTEN Expression and Activity
PTEN expression and activity are regulated by numer-ous and complex mechanisms, in particular in patholog-ical conditions. These mechanisms include epigeneticsilencing of the gene, transcriptional and post-tran-scriptional modulation of the mRNA expression, and post-translational modifications of the protein, which af-fect PTEN stability, localization, activity and interactions with other cellular partners ( fig. 3 ).
Genetic/Epigenetic Mechanisms Epigenetic PTEN silencing by hypermethylation of its
promoter has been suggested as a potential mechanism contributing to PTEN downregulation in several cancers, including HCC [14, 23] . However, data supporting this concept should be interpreted with caution since meth-ylation of the PTEN promoter does not always strictly correlate with the loss of PTEN protein expression and the promoter of a PTEN pseudogene can also be methyl-ated [24] . Interestingly, histone deacetylase inhibitors
InsR/IGF-1R
IRSsOther stimuli
Plasma membrane
SKIPPTEN
PTEN
?
? ?
? ?
MAPKGrp1 PDK1
Akt
SHIP2
FAK Others
PI3-K
PI(3,4,5)P3 PI(3,4)P2PI(4,5)P2 PI(4)P
PPP P
P
PPP
Fig. 2. Simplified scheme depicting the phosphoinositide phosphatase activity and other potential functions of PTEN in insulin/IGF-1 signaling. Dotted lines and question marks indicate potential PTEN direct or indirect inhibitory functions (line heads) or protein phosphatase activ-ity (arrowheads). PtdIns(3,4,5)P 3 dephos-phorylation on the 5 � position by twoother phosphoinositide phosphatases, i.e. SHIP2 and SKIP, which are involved in insulin/IGF-1 signal transduction, is also highlighted.
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PTEN in NAFLD/NASH and Cancer Dig Dis 2010;28:236–246 239
were also shown to upregulate PTEN transcription in fi-broblastic cells via Egr1 activation, suggesting that his-tone acetylation likely represents a mechanism regulating PTEN expression as well [25] .
At the transcriptional level, several transcription fac-tors have been shown to directly bind the PTEN promot-er and upregulate PTEN transcription. These include Egr1, which is induced by UV radiation and IGF2 [26, 27] ; p53, which undergoes reciprocal regulation by PTEN [28] ; PPAR- � , following activation by the agonist rosigli-tazone [29] ; Spry2, through a poorly characterized mech-anism [30] ; Atf2, in response to resistin stimulation [31] ; and Myc and Cbf-1 in response to Notch1 activation [32] . On the contrary, other transcription factors, such as NF � B [33, 34] , p300/CBP [33] , Hes-1 [32] , Cbf-1 [35, 36] and c-Jun [37] , have been shown to negatively regulate PTEN transcription, in particular following MAPK acti-vation. These transcriptional downregulations of PTEN expression likely play an important role not only in the development of various cancers, but also in liver meta-bolic diseases.
Post-Transcriptional Mechanisms: miRNAs Adding to the complexity of the regulation of PTEN
expression, recent evidence indicated that PTEN might also undergo post-transcriptional repression by specific micro RNAs (miRNAs). miRNAs are a class of genes en-coding single-stranded RNA molecules partially com-
plementary to one or more mRNA molecules. Theirprimary function is to downregulate gene expression through translational repression or degradation of spe-cific mRNAs. Several miRNAs, including miR-21, miR-19a, miR-17–92, miR-214, miR-216a and miR-217, have been shown to specifically modulate PTEN mRNA ex-pression [38–43] . Of particular interest is miR-21, whose expression is regulated by metabolic factors (fatty acids) and which strongly affects PTEN expression/activity in the early stages of NAFLD in hepatocytes, causing steato-sis and inflammation [34, 38, 40] . Elevated levels of miR-21 and PTEN downregulation have also been function-ally correlated with aberrant cell growth, migration and invasiveness of cancer cells in the context of HCC [38, 44] . Finally, miR-21 is upregulated in liver tumor tissues, sug-gesting it may play a fundamental role in liver cancer de-velopment.
Post-Translational Mechanisms Additional processes in which PTEN protein level
and activity are modulated have been suggested to occur in post-translational steps. These include modifications of the protein, such as phosphorylation, acetylation, ubiquitination and the REDOX state, which have been shown to affect PTEN stability and degradation as well as its enzymatic activity ( fig. 1 ) [14, 45, 46] . Several ki-nases, i.e. CK2 [47, 48] , GSK3 [48, 49] and ROCK [50] , phosphorylate PTEN at its C-terminus and thus regulate
DNA mRNA Translation Protein Transcription
StabilityDegradationLocalization
Activity
PhosphorylationUbiquitination
AcetylationREDOX state
Function
NF-�BPPAR-�CBF-1MYCIGF-2SPRY2c-JunEgr-1p53
ATF-2
miR-21miR-19a
miR-17–92miR-214
miR-216amiR-217
Protein/proteininteractions
Methylation,histone
acetylation Fig. 3. Diagram illustrating the epige-netic, transcriptional, translational and post-translational mechanisms regulating PTEN expression and activity.
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the stability, activity and membrane association of PTEN. The histone acetylase PCAF acetylates PTEN on two lysine residues, which modulates PTEN enzymatic activity [51] . Reactive oxygen species have also been shown to regulate PTEN activity by inducing the forma-tion of intramolecular disulfide bridges between two ac-tive sites of the enzyme [52, 53] . Finally, PTEN ubiqui-tination, in particular by the NEDD4-1 E3 ubiquitin li-gase, has been proposed to either trigger proteasomal degradation of PTEN (when the enzyme is polyubiqui-tinated) [54] or translocation into the nucleus (following monoubiquitination) [55] . Similarly, PTEN sequestra-tion in specific subcellular compartments or membranes through an interaction with distinct proteins, i.e. FAK [20] , MAGI proteins [56] , MAST proteins [57] , p53 [58] , NHERF1/2 [59] , and PICT1 [60] , likely represents addi-tional mechanisms controlling PTEN stability and/or activity ( fig. 1 ).
PTEN Alterations in NAFLD
Hepatic PTEN expression/activity is altered in liver diseases associated with obesity, the metabolic syndrome, viral infection and alcohol consumption. Therefore, it ap-pears that dysregulations of PTEN expression/activity in hepatocytes represents an important and recurrent mo-lecular mechanism contributing to the development of liver disorders with distinct etiological factors ( fig. 4 , 5 ). In this regard, we showed that PTEN expression in hepa-tocytes is downregulated in obese animals and humans displaying steatosis [34] . Others have also reported that similar alterations of PTEN expression are observed in liver tissues of rodents with hepatic fibrosis induced by either biliary stenosis or a choline-deficient diet [61, 62] . We extensively investigated the mechanisms altering PTEN expression in the liver with the metabolic syn-drome and obesity. We could demonstrate that high levels
Hepatocyte
Plasma membrane
UFAs
UFAs
CD36/others?
mTOR +
miR-21
miR-21upregulation
PTENmRNA
degradationPTEN
downregulation
Steatosis
Inflammation
Proliferation
Migrationinvasiveness
Insulin resistance
Nucleus
mTOR mTORNF-�B
NF-�B
NF-�B
NF-�B
P P
P
P
Fig. 4. Downregulation of PTEN expression in hepatocytes by UFAs and effects on hepatocyte functions. UFAs activate a signaling complex formed by mTOR and NF�B. Activated NF�B then translocates into the nucleus and stimulates the synthesis of miR-21, an miRNA, which binds to PTEN mRNA 3 � -UTR and induces its deg-radation. PTEN downregulation, in turn, promotes hepatocyte insulin resistance, steatosis, inflammation, pro-liferation and migration/invasiveness.
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PTEN in NAFLD/NASH and Cancer Dig Dis 2010;28:236–246 241
of unsaturated fatty acids (UFAs), but not glucose or in-sulin, significantly decreased PTEN mRNA expression in hepatic cells. UFAs trigger PTEN downregulation through a mechanism involving the sequential activation of mTOR and NF�B, which were found to form a complex in cul-tured cells ( fig. 4 ) [34, 40] . These studies revealed a novel interaction between mTOR and NF � B, and established a molecular link between NF � B-dependent transcriptional regulation of PTEN and the mTOR nutrient-sensing path-way activated by UFAs [34] . We further demonstrated that UFA-mediated mTOR/NF�B activation does not trigger PTEN mRNA downregulation through mechanisms re-lated to methylation of the PTEN promoter, histone deacetylases activities or repression of the PTEN promot-er activity. In contrast, UFA-mediated mTOR/NF�B acti-vation triggers the upregulation of the expression of miR-21, which binds to PTEN mRNA 3 � -UTR and induces its degradation ( fig. 4 ). Supporting these data, the promoter activity of miR-21 was increased by mTOR/NF � B activa-tion and miR-21 expression was increased in the livers of rats on a high-fat diet and in human liver biopsies of obese patients with diminished PTEN expression and steatosis [40] . This aberrant upregulation of miR-21 expression by excessive circulating levels of UFAs and NF � B activation
exemplify a novel regulatory mechanism by which UFAs affect PTEN expression in hepatocytes.
With the metabolic syndrome, in addition to an excess of circulating free fatty acids, deregulated production of inflammatory cytokines by immune cells or adipokines by the adipose tissue have been clearly associated with NAFLD [63] . Inflammatory cytokines also significantly alter PTEN expression as shown in non-liver cells. For example, transforming growth factor- � was demonstrat-ed to decrease PTEN expression [43, 64–66] through post-transcriptional mechanisms possibly involving miRNA-dependent downregulation of PTEN [43, 66] . In cancer cell lines, tumor necrosis factor- � was shown to affect PTEN expression via NF�B-dependent mecha-nisms [33, 67] . In myeloma cells, interleukin-6 (IL-6) was shown to upregulate miR-21, a miRNA inhibiting PTEN expression in hepatocytes [38] , via Stat3-dependent mechanisms [68] , whereas IL-1 suppresses PTEN expres-sion in endothelial cells [69] . Similarly, various adipo-kines including leptin, resistin and adiponectin have been reported to either up- or downregulate PTEN ex-pression or activity in nonhepatic cells via CK2-depen-dent phosphorylation, p38/ATF-2 activation and REDOX regulation, respectively [31, 70, 71] . Of note, PPAR- � , a
PTENUpregulation
Downregulation
Mutations/deletions
Normal
Ethanol
Steatosis
Steatohepatitis
Fibrosis
HCA/HCC/CC
Insulin sensitivityGlucose tolerance
Insulin sensitivityGlucose tolerance
FFACytokines
miRNAsHBV, HCV
PTEN
PTEN
Fig. 5. Spectrum of liver pathologies as-sociated with modulation of PTEN ex-pression levels in hepatocytes by various etiological factors. FFA = Free fatty acids; HBV = hepatitis B virus; HCV = hepatitis C virus; HCA = hepatocellular adenomas; CC = cholangiocellular carcinomas.
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transcription factor modulated by fatty acids and cyto-kines, upregulates PTEN expression in cancer and hepa-toma cells [29, 72, 73] , thus suggesting a potential mecha-nism by which PPAR- � agonists may alleviate cellular dysfunctions associated with the metabolic syndrome. Although most of these cytokines/adipokines are clearly involved in liver insulin sensitivity and steatosis/fibrosis/inflammation [63, 74] , it remains to be firmly established whether PTEN expression/activity is also modulated by these factors in the liver and whether there is a causal re-lationship between potential PTEN alterations induced by these cytokines/adipokines and their beneficial/detri-mental effects on the liver physiology.
PTEN Loss of Function in NAFLD and Cancer Development
The first evidence supporting a critical role for PTEN in the liver came from genetic studies in mice where het-erozygous deletion of PTEN was shown to induce atypi-cal adenomatous liver hyperplasia [75] . More recently, ge-netic inhibition of PTEN expression, specifically in the liver of rodents, was shown to trigger liver steatosis and steatohepatitis at early stages of development, as well as hepatomegaly and HCC later in life [76, 77] .
Interestingly, liver-specific PTEN knockout mice have also improved systemic insulin sensitivity and glucose tol-erance [76, 77] . However, whether this is related to in-creased insulin sensitivity specifically in the liver or to a complex in vivo systemic crosstalk between a PTEN-deficient liver and other peripheral tissues is, to date, still unclear. In support of this latter hypothesis, PTEN dele-tion in the liver is accompanied by decreased circulating levels of leptin and body fat content [77] . In addition, we observed that although constitutive Akt activity is in-creased in hepatocytes with downregulated PTEN, insu-lin signaling upstream of Akt, i.e. insulin receptor/IRS1 expression and phosphorylation, is impaired as it has been described in cancer cells [34, 78] . Consistent with these findings, we observed a lack of insulin responsiveness in PTEN-deficient hepatocytes regarding the insulin-in-duced upregulation of genes such as FAS and sterol regu-latory binding protein (SREBP)-1c [34] . These data raise the hypothesis that PTEN downregulation in hepatocytes may paradoxically cause insulin resistance despite an in-creased activation of specific insulin effectors such as Akt. Further studies are needed to clarify whether PTEN downregulation in hepatocytes, e.g. with the metabolic syndrome, is also a causal factor for insulin resistance.
One indubitable effect of PTEN deletion or downregu-lation in the liver is the development of steatosis, an evi-dent phenotype in liver-specific PTEN knockout mice [76, 77] . We also showed that PTEN downregulation is associated with steatosis in obese and insulin resistant rat animal models and humans [34] . Furthermore, in vitro studies clearly demonstrated that PTEN downregulation, either induced by UFAs or through the use of specific siRNAs targeting PTEN, triggers lipid accumulation in hepatoma cells ( fig. 4 ) [34] . The mechanisms by which PTEN loss of function induces steatosis are, however, not completely clear. In one study, analyses of liver-specific PTEN knockout mice suggested that steatosis was in-duced by a strong upregulation of PPAR- � and a moder-ate increase in expression of SREBP-1c and its target genes [76] , whereas another report indicated that de novo fatty acid synthesis was enhanced but fatty acid uptake was unchanged [77] . On the contrary, our in vitro studies showed that basal FAS and SREBP-1c expressions were not affected by PTEN downregulation. In contrast, we observed a strong upregulation of the fatty acid trans-porter FAT/CD36 and of other enzymes involved in fatty acid esterification (PEPCK and GAPT1). Functional as-says also indicated that PTEN downregulation strongly promotes fatty acid uptake, while it decreases the release of fatty acid metabolites in cultured hepatic cells [34] . These discrepancies between in vivo and in vitro data may originate from the different methodologies used to assess fatty acid uptake and release by hepatocytes or from the different extent of PTEN repression (i.e. com-plete deletion in knockout mice versus 40–80% down-regulation induced by UFAs or siRNA in cultured cells). Partial PTEN downregulation or total deletion can po-tentially mediate very different effects as has been ele-gantly demonstrated with studies examining the role of PTEN in prostate tumor progression [46] .
Knockout mice for PTEN in the liver have provided convincing evidence that PTEN loss of function leads, with time and in a stepwise manner, also to the develop-ment of steatohepatitis, fibrosis and (with ageing) the oc-currence of hepatocellular adenoma and carcinoma [75–77] . These phenotypes are reminiscent of the develop-ment of NAFLD and liver cancer, which may occur in humans with the metabolic syndrome as an etiological factor. Other studies also support an important role for PTEN in preventing tissue inflammation and fibrosis. Indeed, decreased levels of PTEN expression in mice models developing steatohepatitis and fibrosis following a choline-deficient diet or biliary stenosis were reported [61, 62] . Alterations of PTEN expression have also been
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functionally involved in fibrosis pathologies of other tis-sues, e.g. idiopathic pulmonary fibrosis [79–81] and the development of heart interstitial fibrosis [82] . Finally, PTEN was shown to control epithelial-to-mesenchymal transition in mesoderm cells in the chick embryo [83] . In support of these data, we reported that PTEN depletion in hepatocytes, either through genetic tools (siRNAs) or by exposure to UFAs, induces the expression of genes promoting inflammation (tumor necrosis factor- � and IL-8), epithelial-to-mesenchymal transition and fibrosis ( � -SMA, Twist, Snail and transforming growth factor- � ) [44] . These studies suggest that pathological dysregula-tions of PTEN expression/activity causing steatosis may also promote progression of the disease towards different clinical stages of increasing severity and potentially to the development of cancer. Additional studies are now re-quired to better delineate the PTEN-dependent molecu-lar mechanisms, cell processes and cellular interactions governing inflammatory and fibrotic processes in theliver.
Weak expression or mutations/deletion of PTEN, as well as upregulation of miRNAs specifically targeting PTEN for degradation, are frequently observed in human HCC [38, 84, 85] . In mice, loss of PTEN function in the liver leads (with ageing) to the occurrence of HCA (hepa-tocellular adenomas)/HCC in almost 70% of male mice [76] , thus confirming the critical tumor suppressor role of PTEN in the liver. Dysregulated PTEN expression was also previously shown to favor migration and invasive-ness of cancer cells [16, 38, 86–88] . At the molecular level, PTEN loss of function is thought to mediate its tumor suppressor activity by stimulating PI3K signaling [18] , by perturbing chromosomal stability and DNA repair [19] , and by interfering with cell-cell or cell-matrix adhesive interactions [20–22] . Since inflammation, epithelial-to-mesenchymal transition and genomic alterations are typ-ical features of HCC [89, 90] , impaired PTEN expression or activity can thus represent an important step in pro-gression of NAFLD towards HCC. In this regard, our studies demonstrated that UFA-mediated PTEN down-regulation promotes cell proliferation, migration and in-vasiveness, in addition to modulating a set of genes in-volved in cell cycle regulation and HCC [44] . These data were further supported by in vivo experiments using nude mice xenografts to assess the stimulatory effects of UFA-enriched diets for the development and growth of subcutaneously implanted hepatoma-derived tumors [44] . Overall, our findings supported the notion that UFAs, by downregulating hepatic PTEN expression, pro-mote liver tumor initiation and progression ( fig. 4 , 5 ) [44] .
Conclusion
Diabetes and obesity have reached pandemic propor-tions, and NAFLD, which is commonly associated with these diseases, is likely to become the most prevalent liv-er pathology worldwide. The incidence of HCA/HCC with NAFLD etiology is also expected to dramatically in-crease in the future. Understanding the molecular basis of these disorders is, thus, of crucial importance for de-signing and implementing new therapeutic tools to alle-viate and cure these diseases. Although hepatic steatosis is currently regarded as a benign disease, progression to NASH and cirrhosis can lead to liver failure and the de-velopment of HCC. There are multiple molecular factors involved in the progression of hepatic steatosis towards more severe stages of NAFLD and HCC. In this regard, dysregulation of PTEN expression/activity, more than PTEN mutations or deletions, could be a critical step in the occurrence and development of NAFLD, as well as for progression towards HCC. In addition, PTEN alterations induced by high levels of free fatty acids or inflammatory cytokines provide an interesting link between insulin re-sistance and steatosis, which may also explain (at least in part) the high-risk factor for HCA/HCC associated with diabetes and obesity [91, 92] . Given the tumor suppressor activity of PTEN, the role of steatosis and steatohepatitis as preneoplastic states in the hepatocyte malignant trans-formation should also be re-evaluated. Indeed, our stud-ies support the recent concept that downregulation of a tumor suppressor such as PTEN, e.g. by UFAs/cytokines, or PTEN haploinsufficiency is enough to promote carci-nogenesis, thus challenging Knudson’s ‘two hits’ model of a tumor suppressor [93] . Additional studies are needed to evaluate PTEN as a prognostic marker for NAFLD and HCC, and to assess the pertinence of future therapeutic interventions restoring physiological PTEN expression in the liver, e.g. low-fatty acid diets or miRNA inhibitors, to prevent or alleviate metabolic liver disorders and HCC.
Acknowledgements
This work was supported by the Swiss National Science Foun-dation (grant No. 310000-120280/1), the Swiss Research against Cancer Foundation (grant No. KFS-02502-08-2009) and the Ea-gle Foundation (M.F.).
Disclosure Statement
The authors declare that no financial or other conflict of inter-est exists in relation to the content of the article.
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