Transcription Factors GATA4 and GATA6 in Pediatric Liver Disease

GATA transcription factors are required for the normal differentiation of various tissues, including liver, but their expression is low in normal postnatal hepatocytes. Aberrant expression of GATA factors has been connected to several human diseases. Hepatoblastoma (HB) is a rare liver malignancy of small children, with a largely unknown molecular pathology, histologically resembling fetal liver tissue. Biliary atresia (BA) is a neonatal cholestatic condition caused by fibroinflammatory obstruction of the extrahepatic bile ducts, with subsequent histological changes in liver. This thesis work demonstrates that GATA4 is frequently overexpressed in childhood HBs. The effects of GATA4 in HB cell malignancy were studied in vitro utilizing a human HB cell line. The changes in cell function and gene expression caused by modification of GATA4 levels were analyzed. We found that GATA4 1) protects HB cells from the commonly used cytostatic drug doxorubicin by regulating the intrinsic apoptotic pathway, and 2) shifts the transcriptomic profile of HB cells to more mesenchymal-type and enhances their migration. A second focus of this thesis is the role of GATA6 transcription factor in BA pathogenesis. We found that GATA6, normally restricted to biliary epithelium in postnatal liver, is highly expressed in the hepatocytes undergoing ductal metaplasia in livers of BA patients. GATA6 expression correlates to known prognostic markers, including bile ductule expansion and age at portoenterostomy. The expression of GATA6 decreases significantly after resolution of cholestasis by successful portoenterostomy. Analogously to its increased expression in BA, GATA6 is also upregulated in the hepatocytes of two mouse models with biliary obstruction. Furthermore, forced expression of GATA6 in two human hepatocyte cell models drives their gene expression towards cholangiocyte lineage. Taken together, transcription factors GATA4 and GATA6 are essential for the initial differentiation of hepatoblasts and for liver organogenesis. Compared to normal postnatal liver, their expression is upregulated in these two pediatric diseases of the liver. GATA4 and GATA6 are here demonstrated as putative drivers of hepatocyte neoplasia and metaplasia.GATA-geeninsäätelytekijät ovat tärkeitä eri elinten normaalille kehitykselle ja solujen erilaistumiselle. Myös maksan varhainen kehitys on riippuvaista GATA4:stä ja GATA6:sta, mutta syntymän jälkeen niiden ilmentyminen maksasoluissa vähenee selvästi. GATA-proteiinien poikkeava ilmentyminen syntymän jälkeen on yhdistetty moniin eri sairauksiin ja kehityshäiriöihin. Tämä väitöskirja keskittyy GATA-tekijöiden rooliin kahdessa lasten maksasairaudessa. Hepatoblastooma (HB) on pienten lasten harvinainen maksan pahalaatuinen kasvain, joka mikroskooppisesti muistuttaa sikiöaikaista maksakudosta. Sappitieatresia (SA) on synnynnäinen maksanulkoisten sappiteiden arpeutuminen ja tukkeutuminen, jonka syntymekanismi on pitkälti tuntematon. Tässä työssä selvitimme, että HB-kasvaimissa GATA4:n ilmentyminen on selvästi normaalia maksakudosta runsaampaa. Käytimme HB-solulinjaa tutkiaksemme, miten GATA4:n määrän manipuloiminen vaikuttaa solujen toimintaan ja geeninsäätelyyn. GATA4 geenin ilmentymisen estäminen lisää HB solujen herkkyyttä yleisesti käytetylle solunsalpaajalääkkeelle, doksorubisiinille, lisääntyneen ohjelmoidun solukuoleman kautta. GATA4:n estäminen myös vähentää HB-solujen kykyä liikkua ja näin ollen niiden potentiaalia muodostaa etäpesäkkeitä. Tutkimuksemme osoitti, että GATA6:a ilmentyy syntymän jälkeen normaalisti sappitie-epiteelissä, mutta ei maksasoluissa. SA-potilaiden maksasolut pyrkivät uudelleen erilaistumaan sappitie-epiteelisolun kaltaisiksi kompensoidakseen maksaan kertyvän sapen aiheuttamaa stressitilaa. Nämä muuntuneet maksasolut alkavat sikiöaikaisen maksakudoksen tavoin ilmentää GATA6:a. Kun sapen kulku vapautetaan uudelleen maksansisäiset sappitiet ohutsuoleen yhdistävässä leikkauksessa, myös GATA6 häviää maksasoluista. Työssä on käytetty myös kahta SA:ta jäljentelevää hiirimallia, joiden maksasoluissa GATA6 käyttäytyy vastaavasti. Lisäämällä keinotekoisesti GATA6:n määrää ihmisen maksasoluviljelmässä, saadaan solujen geeninsäätely muuttumaan sappitie-epiteelin suuntaan. Yhteenvetona, geeninsäätelytekijä GATA4 vaikuttaa lasten maksasyövässä pahanlaatuisuuteen liittyviin tekijöihin ja GATA6 on mahdollinen maksasolujen uudelleen erilaistumisen säätelijä synnynnäisessä sappitieatresiassa

Downregulation of transcription factor GATA4 sensitizes human hepatoblastoma cells to doxorubicin-induced apoptosis

Hepatoblastoma, the most common type of pediatric liver cancer, is treated with a combination of surgery and chemotherapy. An essential drug in the treatment of hepatoblastoma is doxorubicin, which in high doses is cardiotoxic. This adverse effect is due to downregulation of cardiac expression of transcription factor GATA4, leading in turn to diminished levels of anti-apoptotic BCL2 (B-cell lymphoma 2) protein family members. GATA4 is also expressed in early fetal liver, but absent from normal postnatal hepatocytes. However, GATA4 is highly expressed in hepatoblastoma tissue. In this study, we assessed the role of GATA4 in doxorubicin-induced apoptosis of hepatoblastoma cells. Herein, we demonstrate that doxorubicin decreases GATA4 expression and alters the expression pattern of BCL2 family members, most profoundly that of BCL2 and BAK, in the HUH6 hepatoblastoma cell line. Silencing of GATA4 by siRNA prior to doxorubicin treatment sensitizes HUH6 cells to the apoptotic effect of this drug by further shifting the balance of BCL2 family members to the pro-apoptotic direction. Specifically, expression levels of anti-apoptotic BCL2 were decreased and pro-apoptotic BID were increased after GATA4 silencing. On the whole, our results indicate that since high endogenous levels of transcription factor GATA4 likely protect hepatoblastoma cells from doxorubicin-induced apoptosis, these cells can be rendered more sensitive to the drug by downregulation of GATA4.Peer reviewe

Type 1 tyrosinemia in Finland: a nationwide study

Background Introduction of nitisinone and newborn screening (NBS) have transformed the treatment of type 1 tyrosinemia, but the effects of these changes on the long-term outcomes remain obscure. Also, the predictors for later complications, the significance of drug levels and the normalization of laboratory and imaging findings are poorly known. We investigated these issues in a nationwide study. Results Type 1 tyrosinemia was diagnosed in 22 children in 1978-2019 in Finland. Incidence was 1/90,102, with a significant enrichment in South Ostrobothnia (1/9990). Median age at diagnosis was 5 (range 0.5-36) months, 55% were girls and 13 had homozygotic Trp262X mutation. Four patients were detected through screening and 18 clinically, their main findings being liver failure (50% vs. 100%, respectively, p = 0.026), ascites (0% vs. 53%, p = 0.104), renal tubulopathy (0% vs. 65%, p = 0.035), rickets (25% vs. 65%, p = 0.272), growth failure (0% vs. 66%, p = 0.029), thrombocytopenia (25% vs. 88%, p = 0.028) and anaemia (0% vs. 47%, p = 0.131). One patient was treated with diet, seven with transplantation and 14 with nitisinone. Three late-diagnosed (6-33 months) nitisinone treated patients needed transplantation later. Kidney dysfunction (86% vs. 7%, p = 0.001), hypertension (57% vs. 7%, p = 0.025) and osteopenia/osteoporosis (71% vs. 14%, p = 0.017) were more frequent in transplanted than nitisinone-treated patients. Blood/serum alpha-fetoprotein decreased rapidly on nitisinone in all but one patient, who later developed intrahepatic hepatocellular carcinoma. Liver values normalized in 31 months and other laboratory values except thrombocytopenia within 18 months. Imaging findings normalized in 3-56 months excluding five patients with liver or splenic abnormalities. Low mean nitisinone concentration was associated with higher risk of severe complications (r = 0.758, p = 0.003) despite undetectable urine succinylacetone. Conclusions Prognosis of type 1 tyrosinemia has improved in the era of nitisinone, and NBS seems to provide further benefits. Nevertheless, the long-term risk for complications remains, particularly in the case of late diagnosis and/or insufficient nitisinone levels.Peer reviewe

Type 1 tyrosinemia in Finland: a nationwide study

Background Introduction of nitisinone and newborn screening (NBS) have transformed the treatment of type 1 tyrosinemia, but the effects of these changes on the long-term outcomes remain obscure. Also, the predictors for later complications, the significance of drug levels and the normalization of laboratory and imaging findings are poorly known. We investigated these issues in a nationwide study. Results Type 1 tyrosinemia was diagnosed in 22 children in 1978-2019 in Finland. Incidence was 1/90,102, with a significant enrichment in South Ostrobothnia (1/9990). Median age at diagnosis was 5 (range 0.5-36) months, 55% were girls and 13 had homozygotic Trp262X mutation. Four patients were detected through screening and 18 clinically, their main findings being liver failure (50% vs. 100%, respectively, p = 0.026), ascites (0% vs. 53%, p = 0.104), renal tubulopathy (0% vs. 65%, p = 0.035), rickets (25% vs. 65%, p = 0.272), growth failure (0% vs. 66%, p = 0.029), thrombocytopenia (25% vs. 88%, p = 0.028) and anaemia (0% vs. 47%, p = 0.131). One patient was treated with diet, seven with transplantation and 14 with nitisinone. Three late-diagnosed (6-33 months) nitisinone treated patients needed transplantation later. Kidney dysfunction (86% vs. 7%, p = 0.001), hypertension (57% vs. 7%, p = 0.025) and osteopenia/osteoporosis (71% vs. 14%, p = 0.017) were more frequent in transplanted than nitisinone-treated patients. Blood/serum alpha-fetoprotein decreased rapidly on nitisinone in all but one patient, who later developed intrahepatic hepatocellular carcinoma. Liver values normalized in 31 months and other laboratory values except thrombocytopenia within 18 months. Imaging findings normalized in 3-56 months excluding five patients with liver or splenic abnormalities. Low mean nitisinone concentration was associated with higher risk of severe complications (r = 0.758, p = 0.003) despite undetectable urine succinylacetone. Conclusions Prognosis of type 1 tyrosinemia has improved in the era of nitisinone, and NBS seems to provide further benefits. Nevertheless, the long-term risk for complications remains, particularly in the case of late diagnosis and/or insufficient nitisinone levels.Peer reviewe

Incidence and long-term outcomes of surgically treated childhood hepatic malignancies in Finland

Aim To analyse incidence, treatment and outcomes of paediatric liver malignancies in Finland during 1987-2017. Methods Medical records and national cancer registry data of 47 children with liver malignancies were reviewed. Survival was calculated with the Kaplan-Meier method. Results During follow-up, liver malignancy incidence remained stable at 1.1:10(6). Altogether, 42 patients with hepatoblastoma (n = 24), hepatocellular carcinoma (n = 11) and undifferentiated embryonal sarcoma (n = 7) underwent surgery at median age 4.6 (interquartile range, 2.0-9.6) years and were followed up for 13 (7.0-19) years. Cumulative 5-year survival was 86% for hepatoblastoma, 41% for hepatocellular carcinoma and 67% for undifferentiated embryonal sarcoma. Five-year survival was decreased among hepatoblastoma patients aged >= 2.4 years (73% versus 100%, P = .040), with PRETreatment EXTent of disease IV (PRETEXT, 60% vs 100%, P = .004), and with recurrent disease (67% vs 88%, P = .029). Recurrent/residual disease associated with decreased 5-year survival in hepatocellular carcinoma (0% vs 83%, P = .028). Survival was similar among 19 transplanted and 23 resected patients. In total, 14 deaths occurred either for the underlying malignancy (n = 8), adverse effects of chemotherapy (n = 5) or unrelated reasons (n = 1). Conclusion Outcomes for PRETEXT I-III hepatoblastoma and un-metastasized hepatocellular carcinoma were encouraging. Adverse effects of chemotherapy significantly contributed to mortality.Peer reviewe

Germline Sequencing Identifies Rare Variants in Finnish Subjects with Familial Germ Cell Tumors

Purpose: Pediatric germ cell tumors are rare, representing about 3% of childhood malignancies in children less than 15 years of age, presenting in neonates or adolescents with a greater incidence noted in older adolescents. Aberrations in primordial germ cell proliferation/differentiation can lead to a variety of neoplasms, including teratomas, embryonal carcinoma, choriocarcinoma, and yolk sac tumors. Patients and Methods: Three Finnish families with varying familial germ cell tumors were identified, and whole-genome sequencing was performed using an Illumina sequencing platform. In total, 22 unique subjects across the three families were sequenced. Family 1 proband (female) was affected by malignant ovarian teratoma, Family 2 proband (female) was affected by sacrococcygeal teratoma with yolk sac tumor in the setting of Cornelia de Lange syndrome, and Family 3 proband (male) was affected by malignant testicular teratoma. Rare variants were identified using an autosomal recessive or de novo model of inheritance. Results: For family 1 proband (female), an autosomal recessive or de novo model of inheritance identified variants of interest in the following genes: CD109, IKBKB, and CTNNA3, SUPT6H, MUC5AC, and FRG1. Family 2 proband (female) analysis identified gene variants of interest in the following genes: LONRF2, ANO7, HS6ST1, PRB2, and DNM2. Family 3 proband (male) analysis identified the following potential genes: CRIPAK, KRTAP5-7, and CACNA1B. Conclusion: Leveraging deep pedigrees and next-generation sequencing, rare germline variants were identified that were enriched in three families from Finland with a history of familial germ cell tumors. The data presented support the importance of germline mutations when analyzing complex cancers with a low somatic mutation landscape.Peer reviewe

Chloroquine Triggers Cell Death and Inhibits PARPs in Cell Models of Aggressive Hepatoblastoma

Background:Hepatoblastoma (HB) is the most common pediatric liver malignancy. Despite advances in chemotherapeutic regimens and surgical techniques, the survival of patients with advanced HB remains poor, underscoring the need for new therapeutic approaches. Chloroquine (CQ), a drug used to treat malaria and rheumatologic diseases, has been shown to inhibit the growth and survival of various cancer types. We examined the antineoplastic activity of CQ in cell models of aggressive HB. Methods:Seven human HB cell models, all derived from chemoresistant tumors, were cultured as spheroids in the presence of relevant concentrations of CQ. Morphology, viability, and induction of apoptosis were assessed after 48 and 96 h of CQ treatment. Metabolomic analysis and RT-qPCR based Death Pathway Finder array were used to elucidate the molecular mechanisms underlying the CQ effect in a 2-dimensional cell culture format. Quantitative western blotting was performed to validate findings at the protein level. Results:CQ had a significant dose and time dependent effect on HB cell viability both in spheroids and in 2-dimensional cell cultures. Following CQ treatment HB spheroids exhibited increased caspase 3/7 activity indicating the induction of apoptotic cell death. Metabolomic profiling demonstrated significant decreases in the concentrations of NAD(+)and aspartate in CQ treated cells. In further investigations, oxidation of NAD(+)decreased as consequence of CQ treatment and NAD(+)/NADH balance shifted toward NADH. Aspartate supplementation rescued cells from CQ induced cell death. Additionally, downregulated expression of PARP1 and PARP2 was observed. Conclusions:CQ treatment inhibits cell survival in cell models of aggressive HB, presumably by perturbing NAD(+)levels, impairing aspartate bioavailability, and inhibiting PARP expression. CQ thus holds potential as a new agent in the management of HB.Peer reviewe

SLC-0111, an inhibitor of carbonic anhydrase IX, attenuates hepatoblastoma cell viability and migration

BackgroundIn response to hypoxia, tumor cells undergo transcriptional reprogramming including upregulation of carbonic anhydrase (CA) IX, a metalloenzyme that maintains acid-base balance. CAIX overexpression has been shown to correlate with poor prognosis in various cancers, but the role of this CA isoform in hepatoblastoma (HB) has not been examined. MethodsWe surveyed the expression of CAIX in HB specimens and assessed the impact of SLC-0111, a CAIX inhibitor, on cultured HB cells in normoxic and hypoxic conditions. ResultsCAIX immunoreactivity was detected in 15 out of 21 archival pathology HB specimens. The CAIX-positive cells clustered in the middle of viable tumor tissue or next to necrotic areas. Tissue expression of CAIX mRNA was associated with metastasis and poor clinical outcome of HB. Hypoxia induced a striking upregulation of CAIX mRNA and protein in three HB cell models: the immortalized human HB cell line HUH6 and patient xenograft-derived lines HB-295 and HB-303. Administration of SLC-0111 abrogated the hypoxia-induced upregulation of CAIX and decreased HB cell viability, both in monolayer and spheroid cultures. In addition, SLC-0111 reduced HB cell motility in a wound healing assay. Transcriptomic changes triggered by SLC-0111 administration differed under normoxic vs. hypoxic conditions, although SLC-0111 elicited upregulation of several tumor suppressor genes under both conditions. ConclusionHypoxia induces CAIX expression in HB cells, and the CAIX inhibitor SLC-0111 has in vitro activity against these malignant cells.Peer reviewe

Transcription factor GATA4 associates with mesenchymal-like gene expression in human hepatoblastoma cells

GATA4, a transcription factor crucial for early liver development, has been implicated in the pathophysiology of hepatoblastoma, an embryonal tumor of childhood. However, the molecular and phenotypic consequences of GATA4 expression in hepatoblastoma are not fully understood. We surveyed GATA4 expression in 24 hepatoblastomas using RNA in situ hybridization and immunohistochemistry. RNA interference was used to inhibit GATA4 in human HUH6 hepatoblastoma cells, and changes in cell migration were measured with wound healing and transwell assays. RNA microarray hybridization was performed on control and GATA4 knockdown HUH6 cells, and differentially expressed genes were validated by quantitative polymerase chain reaction or immunostaining. Plasmid transfection was used to overexpress GATA4 in primary human hepatocytes and ensuring changes in gene expression were measured by quantitative polymerase chain reaction. We found that GATA4 expression was high in most hepatoblastomas but weak or negligible in normal hepatocytes. GATA4 gene silencing impaired HUH6 cell migration. We identified 106 differentially expressed genes (72 downregulated, 34 upregulated) in knockdown versus control HUH6 cells. GATA4 silencing altered the expression of genes associated with cytoskeleton organization, cell-to-cell adhesion, and extracellular matrix dynamics (e.g. ADD3, AHNAK, DOCK8, RHOU, MSF, IGFBP1, COL4A2). These changes in gene expression reflected a more epithelial (less malignant) phenotype. Consistent with this notion, there was reduced F-actin stress fiber formation in knockdown HUH6 cells. Forced expression of GATA4 in primary human hepatocytes triggered opposite changes in the expression of genes identified by GATA4 silencing in HUH6 cells. In conclusion, GATA4 is highly expressed in most hepatoblastomas and correlates with a mesenchymal, migratory phenotype of hepatoblastoma cells

Evolving Up-regulation of Biliary Fibrosis-Related Extracellular Matrix Molecules After Successful Portoenterostomy

Successful portoenterostomy (SPE) improves the short-term outcome of patients with biliary atresia (BA) by relieving cholestasis and extending survival with native liver. Despite SPE, hepatic fibrosis progresses in most patients, leading to cirrhosis and a deterioration of liver function. The goal of this study was to characterize the effects of SPE on the BA liver transcriptome. We used messenger RNA sequencing to analyze global gene-expression patterns in liver biopsies obtained at the time of portoenterostomy (n = 13) and 1 year after SPE (n = 8). Biopsies from pediatric (n = 2) and adult (n = 2) organ donors and other neonatal cholestatic conditions (n = 5) served as controls. SPE was accompanied by attenuation of inflammation and concomitant up-regulation of key extracellular matrix (ECM) genes. Highly overexpressed genes promoting biliary fibrosis and bile duct integrity, such as integrin subunit beta 6 and previously unreported laminin subunit alpha 3, emerged as candidates to control liver fibrosis after SPE. At a cellular level, the relative abundance of activated hepatic stellate cells and liver macrophages decreased following SPE, whereas portal fibroblasts (PFs) and cholangiocytes persisted. Conclusion: The attenuation of inflammation following SPE coincides with emergence of an ECM molecular fingerprint, a set of profibrotic molecules mechanistically connected to biliary fibrosis. The persistence of activated PFs and cholangiocytes after SPE suggests a central role for these cell types in the progression of biliary fibrosis.Peer reviewe