Molecular insights into mechanisms of hepatoblastoma pathogenesis

Hepatoblastoma (HB) is the most common pediatric liver malignancy with an annual incidence of 1.8 cases per million children. Most cases are sporadic, although certain genetic disorders such as Beckwith-Wiedemann syndrome and familial adenomatous polyposis are associated with increased risk of HB. Developmental pathways, such as WNT/β-catenin and Hedgehog signaling, are often aberrantly activated in HB cells. The overall mutation burden in HB is generally low, but the CTNNB1 gene encoding β-catenin is altered in over 60% of cases. Surgical resection of tumor is a mainstay of HB treatment, generally supported by pre- and post-operative chemotherapy. The survival rate of HB is over 80% if tumor is confined to liver but decreases dramatically when there is extrahepatic involvement, so new treatment options are needed. In this thesis we examined two key factors, GATA4 and neuropilin-2, in the pathobiology of HB. Additionally, we conducted in vitro experiments investigating the potential of chloroquine as a novel HB therapy. Transcription factor GATA4 is crucial for early liver development. We observed that most HB patient samples and cell lines express high levels of GATA4. Moreover, GATA4 expression was associated with mesenchymal-like and motile phenotype in HB cells. Neuropilins (NRP) are multifunctional receptors involved both in physiological and pathological processes. We noted high NRP1 and NRP2 expression in HB patient samples and six HB cell models. Utilizing siRNA transfection, we observed decreased viability and motility in NRP2 knockdown cells compared to cells with intact gene expression suggesting that NRP2 promotes aggressive behavior in HB cells. Chloroquine is a traditional anti-malarial which has demonstrated potential in cancer management. We observed a drastic decrease in HB spheroid viability after chloroquine treatment. Chloroquine treatment also modified the metabolic profile of HB cells with a remarkable decrease in NAD+ and aspartate concentrations. Additionally, we noticed significant decrease in PARP1/2 expression suggesting that chloroquine may have effect on DNA repair system in HB cells. In summary, this thesis shed new light on the molecular mechanisms of HB pathogenesis. In the future, these findings may be utilized in development of novel treatment approaches and diagnostics to improve survival and life quality of HB patients.Hepatoblastooma (HB) on varhaisen lapsuusiän yleisin pahanlaatuinen maksakasvain. Kudostasolla nämä kasvaimet muistuttavat rakenteeltaan sikiöaikaista maksaa. Kehityksenaikaisten signaalireittien aktivoituminen on tyypillistä HB:lle, mutta suurelta osin molekyylibiologinen tausta on edelleen tuntematon. HB-riski on kohonnut Beckwith-Wiedemannin syndroomaa ja familiaalista adenomaattista polypoosia sairastavilla potilailla, mutta useimmissa tapauksista HB esiintyy ilman tunnistettuja periytyviä tekijöitä. HB:n hoito perustuu kasvaimen kirurgiseen poistoon yhdistettynä leikkausta edeltävään ja sen jälkeiseen solunsalpaajalääkitykseen. Tässä väitöskirjatyössä tutkittiin GATA4-geeninsäätelytekijän ja neuropiliini-2 -reseptorin merkitystä HB:n synnyssä. Lisäksi väitöskirjassa selvitettiin malarialääke klorokiinin syöpäsolujen kasvua rajoittavia toimintamekanismeja ja sen käytettävyyttä HB:n hoidossa. Tutkimus toteutettiin hyödyntäen arkistoituja HB-potilaiden kudosnäytteitä sekä useita HB-solumalleja. GATA4-geeninsäätelytekijällä on keskeinen rooli maksan sikiöaikaisen kehityksen ohjaamisessa ja sen poikkeava ilmentyminen on yhdistetty aiemmissa tutkimuksissa maksasyövän kehittymiseen. Tässä väitöskirjassa toistettiin aiemmat havainnot GATA4:n korkeasta ilmentymisestä HB-kudoksissa. Lisäksi yhdistimme GATA4:n ilmentymisen HB-solujen epiteliaalis-mesenkymaaliseen muutokseen, joka on keskeinen prosessi solujen muutoksessa normaalisti toimivista soluista syöpäsoluiksi. Neuropiliini-2 -reseptori välittää useita signaalireittejä pitkin syöpäsolujen kasvua ja levittäytymistä tehostavia viestejä. Väitöskirjatyössä osoitettiin huomattavan korkea neuropiliini-2 ilmentyminen HB-kudoksissa verrattuna normaaliin maksakudokseen. Solutasolla neuropiliini-2:n ilmentyminen liitettiin lisääntyneeseen HB-solujen elinvoimaisuuteen sekä migraatiokykyyn. Klorokiinilla on osoitettu olevan potentiaalia syöpälääkkeenä useissa pre-kliinisissä ja kliinisissä tutkimuksissa. Havaitsimme klorokiinin heikentävän merkittävästi HB-solujen elinvoimaisuutta verrattain alhaisilla konsentraatioilla. Metaboliatutkimukset osoittivat, että klorokiini muuttaa HB-solujen metaboliaa, mm. vähentäen aspartaatin saatavuutta soluissa. Lisäksi tutkimuksemme osoitti klorokiinin vähentävän DNA:n korjauksessa keskeisten PARP-entsyymien ilmentymistä HB-soluissa. Tiivistäen, tässä väitöskirjatyössä tunnistettiin uusia molekyylibiologisia mekanismeja, jotka osallistuvat HB:n syntyyn ja etenemiseen, sekä esitettiin uusi potentiaalinen lääkevaihtoehto aggressiivisen HB:n hoitoon

European Integrated Care Horizon 2020: increase societal participation; reduce care demands and costs in Finnish context

[EN] This project is ptart of larger European level integrated care project led by HU University of Applied Sciences. In Finland, the integration of social and health care services has taken center stage in both the policy and practice arenas. The needs of many client groups, e.g. mental health client or older people, are many and varied. Poor mental health considerably impairs well-being of the population and has considerable economic consequences like absence from work, early retirement and productive losses. In this, professionals with different training backgrounds co-ordinate their expertise in providing care for their shared clients. It provides a safe nexus for the exchange of knowledge and opinions, as well as a framework for reaching a consensus about appropriate health care delivery for a particular client or client cohort. The client should have an immediate access to integrated care, with a focus on rehabilitation in patient’s social roles. The aim of this project is support societal participation, quality of live and reduce care demand and costs in social and health care client. There is a need to better understand different integrated care approaches for social and health care and guide future implementation of new integrated care models.Heikkinen, K.; Lahti, M.; Berg, J.; Kiseleva, A.; Eloranta, S. (2019). European Integrated Care Horizon 2020: increase societal participation; reduce care demands and costs in Finnish context. En Proceedings 5th CARPE Conference: Horizon Europe and beyond. Editorial Universitat Politècnica de València. 228-235. https://doi.org/10.4995/CARPE2019.2019.10208OCS22823

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

Analysis of Non-Relapsed and Relapsed Adult Type Granulosa Cell Tumors Suggests Stable Transcriptomes during Tumor Progression

Adult-type granulosa cell tumor (AGCT) is a rare ovarian malignancy characterized by slow growth and hormonal activity. The prognosis of AGCT is generally favorable, but one-third of patients with low-stage disease experience a late relapse, and over half of them die of AGCT. To identify markers that would distinguish patients at risk for relapse, we performed Lexogen QuantSeq 3′ mRNA sequencing on formalin-fixed paraffin-embedded, archival AGCT tissue samples tested positive for the pathognomonic Forkhead Box L2 (FOXL2) mutation. We compared the transcriptomic profiles of 14 non-relapsed archival primary AGCTs (follow-up time 17–26 years after diagnosis) with 13 relapsed primary AGCTs (follow-up time 1.7–18 years) and eight relapsed tumors (follow-up time 2.8–18.9 years). Non-relapsed and relapsed primary AGCTs had similar transcriptomic profiles. In relapsed tumors three genes were differentially expressed: plasmalemma vesicle associated protein (PLVAP) was upregulated (p = 0.01), whereas argininosuccinate synthase 1 (ASS1) (p = 0.01) and perilipin 4 (PLIN4) (p = 0.02) were downregulated. PLVAP upregulation was validated using tissue microarray RNA in situ hybridization. In our patient cohort with extremely long follow-up, we observed similar gene expression patterns in both primary AGCT groups, suggesting that relapse is not driven by transcriptomic changes. These results reinforce earlier findings that molecular markers do not predict AGCT behavior or risk of relapse

Analysis of non-relapsed and relapsed adult type granulosa cell tumors suggests stable transcriptomes during tumor progression

Adult-type granulosa cell tumor (AGCT) is a rare ovarian malignancy characterized by slow growth and hormonal activity. The prognosis of AGCT is generally favorable, but one-third of patients with low-stage disease experience a late relapse, and over half of them die of AGCT. To identify markers that would distinguish patients at risk for relapse, we performed Lexogen QuantSeq 3\u27 mRNA sequencing on formalin-fixed paraffin-embedded, archival AGCT tissue samples tested positive for the pathognomonic Forkhead Box L2

Analysis of Non-Relapsed and Relapsed Adult Type Granulosa Cell Tumors Suggests Stable Transcriptomes during Tumor Progression

Adult-type granulosa cell tumor (AGCT) is a rare ovarian malignancy characterized by slow growth and hormonal activity. The prognosis of AGCT is generally favorable, but one-third of patients with low-stage disease experience a late relapse, and over half of them die of AGCT. To identify markers that would distinguish patients at risk for relapse, we performed Lexogen QuantSeq 3′ mRNA sequencing on formalin-fixed paraffin-embedded, archival AGCT tissue samples tested positive for the pathognomonic Forkhead Box L2 (FOXL2) mutation. We compared the transcriptomic profiles of 14 non-relapsed archival primary AGCTs (follow-up time 17–26 years after diagnosis) with 13 relapsed primary AGCTs (follow-up time 1.7–18 years) and eight relapsed tumors (follow-up time 2.8–18.9 years). Non-relapsed and relapsed primary AGCTs had similar transcriptomic profiles. In relapsed tumors three genes were differentially expressed: plasmalemma vesicle associated protein (PLVAP) was upregulated (p = 0.01), whereas argininosuccinate synthase 1 (ASS1) (p = 0.01) and perilipin 4 (PLIN4) (p = 0.02) were downregulated. PLVAP upregulation was validated using tissue microarray RNA in situ hybridization. In our patient cohort with extremely long follow-up, we observed similar gene expression patterns in both primary AGCT groups, suggesting that relapse is not driven by transcriptomic changes. These results reinforce earlier findings that molecular markers do not predict AGCT behavior or risk of relapse