Regulation of Normal and Neoplastic Steroidogenic Cell Differentiation in the Adrenal Gland and Ovary

The main steroidogenic organs, adrenal cortex and ovary, arise from a common pool of progenitors in the developing embryo. Similar signaling pathways regulate the differentiation, growth, and survival of cells in these tissues. Proper development of the adrenal cortex and ovary requires precise spatiotemporal control of gene expression and apoptosis; disruption of these processes may lead to congenital disorders or malignant transformation. Earlier in vitro studies demonstrated that transcription factor GATA6 regulates the expression of multiple steroidogenic genes in the adrenal cortex. To show that GATA6 is a crucial regulator of adrenocortical development and function in vivo, we generated a mouse model in which Gata6 is conditionally deleted in steroidogenic cells. These mice exhibited a complex adrenal phenotype that includes cortical thinning, blunted aldosterone production, lack of an X-zone, impaired apoptosis, and subcapsular cell hyperplasia. These results offer genetic proof that GATA6 regulates the differentiation of steroidogenic progenitors into adrenocortical cells. Ovarian granulosa cell tumors (GCTs), the most common sex-cord stromal tumors in women, are thought to be caused by aberrant granulosa cell apoptosis during folliculogenesis. A somatic missense mutation in transcription factor FOXL2 (402C→G) is present in vast majority of human GCTs. FOXL2 plays a key role in the development and function of normal granulosa cells. Wild type (wt) FOXL2 induces GCT cell apoptosis, while mutated FOXL2 is less effective. To clarify the molecular pathogenesis of GCTs, we investigated the impact of FOXL2 and two other factors implicated in granulosa cell function, GATA4 and SMAD3, on gene expression and cell viability in GCTs. We found that these factors physically interact and that GATA4 and SMAD3 synergistically induce CCND2 promoter transactivation, which is reduced by both wt and mutated FOXL2. Finally, we demonstrated that GATA4 and SMAD3 protect GCT cells from wt FOXL2 induced apoptosis without affecting the apoptosis induced by mutated FOXL2. These findings suggest that mutated FOXL2 disrupts the balance between growth and apoptosis in granulosa cells, leading to malignant transformation. The treatment of recurrent or metastatic GCTs is challenging, and biologically targeted treatment modalities are needed. Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) activates the extrinsic apoptotic pathway. Interestingly, TRAIL is able to induce apoptosis in malignant cells without affecting normal cells. Vascular endothelial growth factor (VEGF) is the key regulator of both physiological and pathological angiogenesis. Cancer cells often express VEGF receptor, and an autocrine VEGF/VEGFR signaling loop exists in several types of cancer cells. We found that GCT cells express functional TRAIL and VEGF receptors, and that treatment with TRAIL and the VEGF-binding antibody (bevacizumab) induce GCT cell apoptosis. These findings establish a preclinical basis for targeting these two pathways in the GCT treatment.Kaksi tärkeintä steroideja tuottavaa elintä, lisämunuaisen kuorikerros ja munasarja, kehittyvät samasta esiasteesta yksilönkehityksen aikana. Samat signalointireitit säätelevät solujen erilaistumista ja kasvua näissä kudoksissa. Lisämunuaisen kuorikerroksen ja munasarjan normaali kehitys edellyttää tarkkaan kontrolloitua geenien ilmentymisen ja apoptoosin (ohjelmoitu solukuolema) säätelyä. Häiriö näissä säätelyjärjestelmissä voi johtaa synnynnäisiin kehityshäiriöihin tai syövän kehittymiseen. Transkriptiotekijät ovat geenien luentaa sääteleviä proteiineja. Tässä väitöskirjatutkimuksessa osoitettiin muuntogeenistä hiirimallia hyödyntäen, että transkriptiotekijä GATA6 on välttämätön lisämunuaisen kuorikerroksen normaalille kehitykselle. Hiirillä, joiden lisämunuaisen kuorikerroksen soluista GATA6 on poistettu, ilmenee vakavia lisämunuaiskuoren kehityshäiriöitä ja lisäksi niiden aldosteronituotanto on häiriintynyt. Granuloosasolukasvaimet (GSK) ovat yleisimpiä munasarjan sukupienan kasvaimia. GSK:n syntymekanismit ovat vielä suurelta osin tuntemattomia, mutta niiden oletetaan liittyvän granuloosasolujen jakautumisen ja ohjelmoidun kuoleman säätelykoneiston häiriöihin munarakkulan kasvuvaiheen aikana. GSK-soluista on löytynyt somaattinen mutaatio transkriptiotekijä FOXL2:a koodaavasta geenistä. FOXL2 on välttämätön normaalille granuloosasolun kasvulle ja kehitykselle ja sen on osoitettu ajavan GSK-soluja apoptoosiin, kun taas mutatoitunut FOXL2 ei tähän kykene. Selvittääksemme GSK:n syntyyn johtavia mekanismeja tutkimme normaalin ja mutatoidun FOXL2:n, sekä kahden muun GSK-biologiaan liitetyn tekijän, GATA4:n ja SMAD3:n, yhteisvaikutuksia GSK-solujen geenien ilmentymiseen sekä solujen elinkykyyn. Tuloksemme osoittavat, että GATA4 yhdessä SMAD3:n kanssa suojelee GSK-soluja normaalin FOXL2:n aiheuttamalta apoptoosilta, mutta ei vaikuta mutatoidun FOXL2:n toimintaan. Mutaatio FOXL2-geenissä näyttäisi siis häiritsevän granuloosasolujen kasvun ja apoptoosin välistä tasapainoa, mikä johtaa hallitsemattomaan kasvuun ja kasvaimen syntyyn. Levinneen tai uusiutuneen GSK:n hoito on haastavaa ja uusille biologisesti kohdennetuille hoitomuodoille on tarvetta. TRAIL-tekijä on yksi solun ulkoisen apoptoosireitin käynnistävistä molekyyleistä. Kiinnostavan TRAIL:sta tekee se, että se aktivoi apoptoosin vain pahanlaatuisissa soluissa vaikuttamatta terveisiin soluihin. VEGF on verisuonikasvutekijä, joka ohjaa verisuonten kehitystä niin terveissä kuin pahanlaatuisissakin kudoksissa. Tutkimuksessamme osoitamme, että GSK-solut ilmentävät aktiivisia TRAIL- ja VEGF-reseptoreita, ja että soluviljelmissä TRAIL ja VEGF-vasta-aine (bevasitsumabi) ajavat kasvainsolut apoptoosiin. Nämä prekliiniset tulokset luovat pohjaa eläinkokeille ja kliinisen vaiheen jatkotutkimuksille TRAIL:n ja VEGF-inhibition hyödyntämisessä GSK-potilaiden hoidossa

Long-term outcomes of biliary atresia patients surviving with their native livers

Publisher Copyright: © 2021 The Author(s)Portoenterostomy (PE) has remained as the generally accepted first line surgical treatment for biliary atresia (BA) for over 50 years. Currently, close to half of BA patients survive beyond 10 years with their native livers, and most of them reach adulthood without liver transplantation (LT). Despite normalization of serum bilirubin by PE, ductular reaction and portal fibrosis persist in the native liver. The chronic cholangiopathy progresses to cirrhosis, complications of portal hypertension, recurrent cholangitis or hepatobiliary tumors necessitating LT later in life. Other common related health problems include impaired bone health, neuromotor development and quality of life. Only few high-quality trials are available for evidence-based guidance of post-PE adjuvant medical therapy or management of the disease complications. Better understanding of the pathophysiological mechanisms connecting native liver injury to clinical outcomes is critical for development of accurate follow-up tools and novel therapies designed to improve native liver function and survival.Peer reviewe

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

Circulating levels of TNF-related apoptosis inducing-ligand are decreased in patients with large adult-type granulosa cell tumors-implications for therapeutic potential

Targeted treatments are needed for advanced adult-type granulosa cell tumors (AGCTs). We set out to assess tumor tissue and circulating levels of TNF-related apoptosis-inducing ligand (TRAIL), a promising anti-cancer cytokine, in patients affected by AGCT. We analyzed tissue expression of TRAIL in 127 AGCTs using immunohistochemistry or RT-PCR. Soluble TRAIL was measured by means of ELISA from 141 AGCT patient serum samples, as well as the conditioned media of 15 AGCT patient-derived primary cell cultures, and the KGN cell line. Tissue and serum TRAIL levels were analyzed in relationship with clinical parameters, and serum estradiol, FSH, and LH levels. We found that AGCT samples expressed TRAIL mRNA and protein at levels comparable to normal granulosa cells. AGCT cells did not release soluble TRAIL. TRAIL protein levels were decreased in tumors over 10 cm in diameter (p = 0.04). Consistently, circulating TRAIL levels correlated negatively to tumor dimension (p = 0.01). Circulating TRAIL levels negatively associated with serum estradiol levels. In multiple regression analysis, tumor size was an independent factor contributing to the decreased levels of soluble TRAIL in AGCT patients. AGCTs associate with significantly decreased tumor tissue and serum TRAIL levels in patients with a large tumor mass. These findings encourage further study of agonistic TRAIL treatments in patients with advanced or recurrent AGCT.Peer reviewe

Adrenocortical zonation, renewal, and remodeling

The adrenal cortex is divided into concentric zones. In humans the major cortical zones are the zona glomerulosa, zona fasciculata, and zona reticularis. The adrenal cortex is a dynamic organ in which senescent cells are replaced by newly differentiated ones. This constant renewal facilitates organ remodeling in response to physiological demand for steroids. Cortical zones can reversibly expand, contract, or alter their biochemical profiles to accommodate needs. Pools of stem/progenitor cells in the adrenal capsule, subcapsular region, and juxtamedullary region can differentiate to repopulate or expand zones. Some of these pools appear to be activated only during specific developmental windows or in response to extreme physiological demand. Senescent cells can also be replenished through direct lineage conversion; for example, cells in the zona glomerulosa can transform into cells of the zona fasciculata. Adrenocortical cell differentiation, renewal, and function are regulated by a variety of endocrine/paracrine factors including adrenocorticotropin, angiotensin II, insulin-related growth hormones, luteinizing hormone, activin, and inhibin. Additionally, zonation and regeneration of the adrenal cortex are controlled by developmental signaling pathways, such as the sonic hedgehog, delta-like homolog 1, fibroblast growth factor, and WNT/1-catenin pathways. The mechanisms involved in adrenocortical remodeling are complex and redundant so as to fulfill the offsetting goals of organ homeostasis and stress adaptation.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

The origin of plasma neutrophil gelatinase-associated lipocalin in cardiac surgery

Background: Acute kidney injury (AKI) is common after heart surgery. Neutrophil gelatinase-associated lipocalin (NGAL) is produced in injured kidney. NGAL has been used as an early plasma biomarker for AKI in patients undergoing heart surgery. Neutrophils contain all isoforms (25-kDa, 45-kDa and 145-kDa) but the kidney produces almost exclusively the 25-kDa isoform of NGAL. We investigated first, whether there is association between NGAL and neutrophil activation, and second whether activated neutrophils are a significant source of circulating NGAL in plasma in patients undergoing cardiac surgery. Methods: Two separate patient cohorts were studied: 1) the "kinetic cohort" (n = 29) and 2) the "FINNAKI cohort" (n = 306). As NGAL is strictly co-localized with lactoferrin in neutrophils, NGAL and lactoferrin were measured with enzyme-linked immunosorbent assay in all patients. In sixty-one patients of the "FINNAKI cohort" Western blot was used to separate NGAL isoforms according to their molecular size. Mann-Whitney U, Kruskal-Wallis H, Pearson's and Spearman's tests were used as appropriate. Results: There was strong intraoperative association between NGAL and lactoferrin at all four time-points in the "kinetic cohort". In the "FINNAKI cohort", NGAL and lactoferrin concentrations correlated preoperatively (R = 0.59, p <0.001) and at admission to the intensive care unit (R = 0.69, p <0.001). At admission to intensive care unit, concentrations of NGAL and lactoferrin were higher in AKI than in non-AKI patients (NGAL: p <0.001; lactoferrin: p <0.029). In Western blot analyses, neutrophil specific 45-kDa isoform (median 41% [IQR 33.3-53.1]) and mostly neutrophil derived 145-kDa isoform (median 53.5% [IQR 44.0-64.9%]) together represented over 90% of total NGAL in plasma. Potentially kidney derived NGAL isoform (25-kDa) accounted for only 0.9% (IQR 0.3 - 3.0%) of total NGAL in plasma. There were no statistically significant differences in the distribution of NGAL isomers between AKI and non-AKI patients. Conclusions: Plasma NGAL during cardiac surgery is associated with neutrophil activation. Based on molecular size, the majority of circulating NGAL is derived from neutrophils. Neutrophil activation is a confounding factor when interpreting increased plasma NGAL in cardiac surgery.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

Laser capture microdissection-reduced representation bisulfite sequencing (LCM-RRBS) maps changes in DNA methylation associated with gonadectomy-induced adrenocortical neoplasia in the mouse

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