Analysis of the molecular and cellular pathways involved in the genesis of rare hepatic tumours

Abstract

According to the European Parliament and the Council of the European Union (Regulation EC n.141/2000), rare diseases are defined solely on the basis of low prevalence and affect not more than five individuals per 10.000 in the European population. RD are a large and diverse group of disorders; they include more than 6.000 conditions and involve all organs and tissues, often with several clinical subtypes within the same disease. Almost all RD may cause early mortality and/or long-term disability and accurate and timely diagnosis is often of great importance for prevention and treatment. Very often information on many RD are insufficient, concerning either diagnosis and/or prognosis. An integrated approach was set on a very rare and severe liver malignancy of childhood, hepatoblastoma. Although its annual incidence in western countries is 1.5 cases per million of individuals younger than 15 years, HB represents the most common liver cancer of childhood (Reynolds et al., 2004; McLaughlin et al., 2006; Roebuck and Perilongo, 2006). Its prognosis depends on numerous clinical and histological factors. Children with a poor prognosis are usually characterized by abnormal α-fetoprotein levels (<100 or >1,000,000 ng/ml) and distant metastases (i.e. lung and lymph nodes), whereas patients with good prognosis appear to have, among others, a decline in circulating AFP levels during chemotherapy (Roebuck and Perilongo, 2006; De Ioris et al., 2007). Scientific evidence points out HB as a multi-factorial condition associated with: genetic conditions (i.e. Beckwith-Wiedemann Syndrome and Familial Adenomatous Polyposis) (Roebuck and Perilongo, 2006), non genetic and environmental factors such as the endocrine-metabolic status of the mother (young age, higher body mass index, use of infertility treatments, smoking) and (pre)eclampsia (McLaughlin et al., 2006). An association with occupational paternal exposure to metals, petroleum products and paints/pigments as well the possible involvement of ubiquitous environmental contaminants, such as phthalates, have been suggested (Buckley et al., 1989; Reynolds et al., 2004; McLaughlin et al., 2006). Moreover, epidemiological data indicate low birth weight and increased survival of LBW newborns as consistently associated with increased risk of HB (Reynolds et al., 2004). In the first part of the project, in vitro studies on 4 liver cancer cell lines and 9 matched biopsis collected from HB patients were performed in order to individuate new molecular markers of childhood liver cancers. Special attention was dedicated to the canonical and non canonical WNT pathways and to the IGF-II signalling, since they are involved in the pathogenesis of FAP and BWS, the genetic disorders that predispose to HB onset. FAP patients carry a germline mutation in APC gene, which codifies for a protein that is part, together with Axin and GSK-3β, of the complex that controls the cytoplasmic levels of β-catenin (Orford et al., 1998), the central effector molecule of the canonical WNT pathway (Rubinfeld et al., 1996). BWS patients show a loss of imprinting LOI in the region 11p15, where IGF-II gene is localized. The LOI causes the biallelic expression and therefore, an increase of IGF-II levels, that could lead, as suggested by Veronese and coworkers, to enhanced cellular proliferation, differentiation failure and tumour development (Veronese et al., 2010). An mRNA- and protein-array, performed on the liver cancer cell lines; showed different signatures in the expression levels of genes and proteins, compared to normal human hepatocytes. The mRNA array analyzed the expression of several genes (96) involved in canonical and non canonical WNT signallings and revealed, in liver cancer cell lines compared to normal human hepatocytes, the contemporary upregulation of antagonists genes of the canonical WNT pathway, such as NLK and SOX17, and the downregulation of its agonists genes, such as TCF7L2, TLE1, SLC9A3R1 and WNT10A. These evidences suggest an inactivation of the canonical WNT signalling; at the same time the overexpression of RHOU transcript indicate the activation of the non canonical WNT signalling. The very innovative protein array technology evaluated the epression levels of 224 proteins involved in biological pathways such as apoptosis, cell cycle, and signal transduction, and revealed that Grb-2 is over-expressed in the cell lines investigated, compared to normal human primary hepatocytes. Grb-2 is an ubiquitously expressed adapter induced by IGF (I and II) signallings, in normal conditions; through the interaction with Raf/MAPK, Grb-2 activation leads to the induction of cellular proliferation (Foulstone et al., 2005). In a second step, we approached the study of 3 snap-frozen and 6 paraffin-embedded matched tissues obtained from HB patients. In these rare samples we could evaluate the markers previously evidenced and validated in liver cancer cell lines (NLK, RHOU and WNT10A transcripts; Grb-2 protein). This study showed similar results in HB matched tissues when compared to liver cancer cell lines, suggesting that these markers are reliable also in HB tissue samples. MicroRNAs are small RNA sequences, 20-22 nucleotides long, that can bind to the 3’UTR of specific mRNAs, and regulate their expression by leading to translational repression, mRNA cleavage, and mRNA decay. Since microRNA expression profiles are altered in several tumours compared to their normal counterparts, these molecules are largely used also in the field of cancer classification (Calin and Croce, 2006). A microRNA-array, performed in the three snap-frozen HB tissues, revealed that 51 microRNAs could distinguish tumour from non-tumour samples. By comparing our results with previously published data (Varnholt et al., 2008), we selected 9 microRNAs to be analysed in our 9 matched samples. The comparison between these microRNAs expression in tumour and in non-tumour counterparts, highlighted four up-regulated microRNAs (miR-125a, -150, -199a and -214), and a down-regulated one (miR-148a). It has been demonstrated that miR-214 binds to the PTEN mRNA and downregulates its protein levels (Yang et al., 2008). PTEN is a negative regulator of the PI3K/Akt pathway which is activated by IGF signalling, in normal conditions (Foulstone et al., 2005). In our samples as well, the comparison of tumour and normal counterparts indicated a correlation between high miR-214 levels and low PTEN protein levels in the tumour samples. The study of liver cancer cell lines and HB biopsis revealed the inactivation of the canonical WNT pathway and the contemporary induction of the non canonical one. Furthermore the activation of IGF-signalling is also speculated on the basis of the deregulation of Grb-2 and PTEN protein levels. In the second part of the project the association between HB onset and the exposure to a particular phthalate, DEHP, was investigated. DEHP is the most abundant phthalate in the environment and liver represents one of its main targets. Studies performed on mice and humans demonstrated that DEHP alters the glucose metabolism (Latini et al., 2008). This process involve a family of proteins called PPARs that mediates the IGF-promoted signalling (Feige et al., 2007; Latini et al., 2008), responsible of glycogen storage (Lopez et al., 1999). In utero exposure to phthalates has been shown to be significantly associated with prematurity (Latini et al., 2008), that in turn has been strongly associated with HB (McLaughlin et al., 2006). HB can derive either from developmental disturbances during critical phases of organogenesis or from the interplay of risk factors (e.g. DEHP) during prenatal or early neonatal life (Salvatore et al., 2008). In order to investigate the post-natal effects of DEHP prenatal exposure on liver development, pregnant CD-1 mice were exposed to DEHP (25 and 100 mg/kg BW pro die) during the critical period of liver organogenesis and histogenesis (starting from the 11th day of gestation and till the 19th) (Duncan, 2003). Male and female F1 mice were sacrificed at post natal day 21 and 35, respesenting weaning and puberty, respectively. Histopathological, histochemical and gene expression studies performed on livers of PND35 treated mice did not show significant differences compared to controls, while at PND21 treated mice showed alterations in glucose and lipid metabolism that were absent in controls. The inactivation of GSK-3β was speculated since these mice were characterized by lack of glycogen storage and presence of cytoplasmic β-catenin. Glycogen storage is triggered by IGF-II in fetal life and ensures stable levels of glycaemia at birth when the newborn makes the adjustment to extra-uterine life (Lopez et al., 1999; Hui et al., 2006). The presence of cytoplasmic β-catenin indicates that the canonical WNT pathway is active, and that the transcription of genes involved in cell proliferation is promoted (Rubinfeld et al., 1996). PND21 mice exposed to DEHP showed hepatocytes vacuolization (hepatosteatosis), a feature of an ecess of fatty acid synthesis, as a consequence of glycogen synthesis inhibition (Shimano et al., 2007). AFP levels in mice progressively decrease after birth and are almost completely switched off in the third week of life, when liver accumulates energy as glycogen instead of AFP (Rusyn et al., 2006; Heudorf et al., 2007; Latini et al., 2009) On the basis of the association between AFP expression and embryonal status, this gene is also used as a marker of hepatocytes maturation (Qin and Tang, 2004). Our results showed high levels of AFP gene expression in treated mice compared to controls, suggestable of a role for DEHP exposure in determining both the alteration of post-natal AFP to glycogen switch off, and a delay of hepatocytes maturation. Furthermore the possible role of microRNAs in the DEHP-induced alterations was speculated. IGF-II gene, fundamental in the promotion of glycogen storage during fetal life (Lopez et al., 1999; Hui et al., 2006), harbours a microRNA, miR-483, within its second intron (Fu et al., 2005; www.ensembl.org). The analysis of miR-483 expression showed that this miR can be considered a fetal marker, since its levels progressively decrease after birth. High levels of miR-483 were detected in PND21 mice treated with DEHP 100 mg/kg bw pro die, in confirmation with a role of DEHP exposure in delaying the hepatocytes maturation. We also demonstrated that this miR is able to target and downregulate β-catenin, thus representing a limit in the proliferation stimulus induced by high levels of this protein within the cytoplasm (Rubinfeld et al., 1996). The multidisciplinary approach, performed by using in vivo and in vitro studies, enabled us to identify early and tardive markers of hepatoblastoma. Our studies indicated that the prenatal exposure to DEHP delays liver maturation by affecting carbohydrate and lipid pathways involved in fetal nutrition. The incapability to accumulate glycogen, due to the inactivation of GSK-3β function, is counterbalanced by an enhanced synthesis of lipid, as demonstrated by the presence of hepatocyte vacuolization. Furthermore the improper increased levels of AFP gene expression, suggested that DEHP alters the post-natal AFP-to-glycogen switch and may delay the post natal maturation of hepatocytes, as suggested also by high levels of miR-483, another fetal marker. A biological role for miR-483 in limiting the proliferation activation induced by β-catenin has been also proposed. The characterization of in vitro models (liver cancer cell lines and HB matched tissue samples) has identified new molecular tardive markers of chilhood liver cancers, at mRNA- (WNT10A, NLK and RHOU trascripts), protein- (Grb-2 and PTEN), and at microRNA- (miR-214) level. Gene expression analysis showed an inactivation of the canonical WNT signalling; and the contemporary activation of the non canonical one. At protein level, the upregulation of Grb-2 in tumour compared to non tumour samples, lets speculate the activation of the Raf/MAPK signalling pathway. The upregulation of miR-214 in tumour compared to non tumour samples seemed to affect PTEN protein levels, which resulted to be downregulated in the tumour counterpart. This evidence suggests that the activation of PI3K/Akt pathway could lead to the misregulation of the cellular functions that it controls, such as glucose metabolism, cell proliferation and survival (Foulstone et al., 2005). Even though in in vivo and in vitro models the patterns of deregulation of WNT and IGF-signalling pathways involve different mechanisms, the importance of these pathways and their crosstalks can be considered pivotal in the onset and in the characterization of hepatoblastoma.