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In dieser Arbeit wurde der Einfluss des Transkriptionsfaktors Carbohydrate responsive element binding protein (ChREBP) auf die experimentelle Hepatokarzinogenese untersucht, der eine wichtige Rolle in der protoonkogenen AKT/mTOR-vermittelten Karzinogenese der Leber spielen soll.
Dazu diente das Pankreasinseltransplantationsmodell bei diabetischen C57Bl/6J- Wildtyp- (WT; n = 180) und ChREBP-Knockout-Mäusen (KO; n = 143). Es erfolgten histologische, proliferationskinetische, immunhistochemische und Western-Blot- Analysen.
Das Hauptergebnis dieser Arbeit stellt die Hyperproliferation im Lebergewebe der diabetischen, transplantierten Mäuse dar, aus der sich klarzellige Leberherde (CCF) und im weiteren Verlauf auch hepatozelluläre Adenome (HCA) und Karzinome (HCC) entwickelten. Die Proliferationsaktivität lag in den Herden der Knockout- Mäuse bei 21,77 ± 3,38 % (Mittelwert ± S.E.M.), in denen der Wildtyp-Mäuse bei 29,04 ± 11,97 %. Die Leberherde unterschieden sich in ihrer Morphologie: Die Herde der KO-Mäuse waren kleiner und sie zeigten nur eine starke Glykogenspeicherung, während die Herde der Wildtyp-Mäuse neben dem Glykogen auch viel Fett speicherten.
Die Entstehung manifester Tumoren setzte im Knockout-Stamm später ein und die Tumoren wiesen auch ein langsameres Wachstum auf. Tumoren entstanden nicht nur in diabetischen, transplantierten Mäusen (WT-HCC n = 3; KO-HCC n = 1; KO-HCA n = 2), sondern auch in diabetischen Wildtyp-Kontrollmäusen (n = 4), aber nicht bei diabetischen KO-Tieren. Immunhistochemisch konnte in den Tumoren des KO- Stamms eine verminderte Glykolyse und de novo Lipogenese sowie ein herunterregulierter AKT/mTOR-Signalweg nachgewiesen werden.
Diese Ergebnisse weisen auf einen protoonkogenen Charakter von ChREBP zum einen in der hormonell-induzierten Hepatokarzinogenese, aber auch in einem metabolischen Karzinogeneseprozess im Rahmen eines Insulinmangeldiabetes hin. Der Einfluss von ChREBP auf den Energiestoffwechsel der Zelle und auf den metabolischen Switch vom glykogenotischen zum lipogenen Phänotyp in der experimentellen Hepatokarzinogenese-Sequenz scheint essentiell zu sein. Der Knockout von ChREBP reduziert dementsprechend die Proliferation im Lebergewebe und scheint die Hepatokarzinogenese in diesem Modell zu verzögern.
GPR68 (OGR1) belongs to the proton-sensing G protein-coupled receptors that are involved
in cellular adaptations to pH changes during tumour development. Although expression of GPR68
has been described in many tumour cell lines, little is known about its presence in human tumour
entities. We characterised the novel rabbit monoclonal anti-human GPR68 antibody 16H23L16
using various cell lines and tissue specimens. The antibody was then applied to a large series of
formalin-fixed, paraffin-embedded normal and neoplastic human tissue samples. Antibody specificity
was demonstrated in a Western blot analysis of GPR68-expressing cells using specific siRNAs.
Immunocytochemical experiments revealed pH-dependent changes in subcellular localisation of the
receptor and internalisation after stimulation with lorazepam. In normal tissue, GPR68 was present in
glucagon-producing islet cells, neuroendocrine cells of the intestinal tract, gastric glands, granulocytes,
macrophages, muscle layers of arteries and arterioles, and capillaries. GPR68 was also expressed
in neuroendocrine tumours, where it may be a positive prognostic factor, in pheochromocytomas,
cervical adenocarcinomas, and endometrial cancer, as well as in paragangliomas, medullary thyroid
carcinomas, gastrointestinal stromal tumours, and pancreatic adenocarcinomas. Often, tumour
capillaries were also strongly GPR68-positive. The novel antibody 16H23L16 will be a valuable tool for
basic research and for identifying GPR68-expressing tumours during histopathological examinations.
Hepatocellular carcinoma (HCC) is a deadly form of liver malignancy with limited treatment
options. Amplification and/or overexpression of c-MYC is one of the most frequent genetic events
in human HCC. The mammalian target of Rapamycin Complex 1 (mTORC1) is a major functional
axis regulating various aspects of cellular growth and metabolism. Recently, we demonstrated that
mTORC1 is necessary for c-Myc driven hepatocarcinogenesis as well as for HCC cell growth in vitro.
Among the pivotal downstream effectors of mTORC1, upregulation of Fatty Acid Synthase (FASN) and
its mediated de novo lipogenesis is a hallmark of human HCC. Here, we investigated the importance
of FASN on c-Myc-dependent hepatocarcinogenesis using in vitro and in vivo approaches. In mouse
and human HCC cells, we found that FASN suppression by either gene silencing or soluble inhibitors
more effectively suppressed proliferation and induced apoptosis in the presence of high c-MYC
expression. In c-Myc/Myeloid cell leukemia 1 (MCL1) mouse liver tumor lesions, FASN expression
was markedly upregulated. Most importantly, genetic ablation of Fasn profoundly delayed (without
abolishing) c-Myc/MCL1 induced HCC formation. Liver tumors developing in c-Myc/MCL1 mice
depleted of Fasn showed a reduction in proliferation and an increase in apoptosis when compared
with corresponding lesions from c-Myc/MCL1 mice with an intact Fasn gene. In human HCC samples,
a significant correlation between the levels of c-MYC transcriptional activity and the expression
of FASN mRNA was detected. Altogether, our study indicates that FASN is an important effector
downstream of mTORC1 in c-MYC induced HCC. Targeting FASN may be helpful for the treatment
of human HCC, at least in the tumor subset displaying c-MYC amplification or activation.
EGFR is overexpressed in the majority of clear cell renal cell carcinomas (CCRCCs). Although EGFR deregulation was found to be of great significance in CCRCC biology, the EGFR
overexpression is not associated with EGFR-targeted therapy responsiveness. Moreover, the prognostic role of EGFR expression remains controversial. In the present study, we evaluated the role
played by EGFR overexpression in CCRCC and its prognostic significance associated with different
immunohistochemical localization patterns. In our study, the Total Score (TS) related to membranouscytoplasmic EGFR expression showed a significant correlation with grade, pathologic stage (pT),
and Stage, Size, Grade, and Necrosis (SSIGN) score, and a negative correlation with nuclear EGFR
expression. No significant correlations were shown between nuclear EGFR and clinic-pathological
features. Additionally, a correlation between SGLT1 expression levels and pT was described. Multivariate analysis identifies pT and SSIGN score as independent prognostic factors for CCRCC. A
significantly increased survival rate was found in the case of positive expression of nuclear EGFR
and SGLT1. Based on our findings, SGLT1 and nuclear EGFR overexpression defines a subgroup of
CCRCC patients with good prognosis. Membranous-cytoplasmic EGFR expression was shown to be
a poor prognostic factor and could define a CCRCC subgroup with poor prognosis that should be
responsive to anti-EGFR therapies
FAM159B is a so-called adaptor protein. These proteins are essential components in numerous cell signalling pathways. However, little is known regarding FAM159B expression in normal and neoplastic human tissues. The commercially available rabbit polyclonal anti-human FAM159B antibody HPA011778 was initially characterised for its specificity using Western blot analyses and immunocytochemistry and then applied to a large series of formalin-fixed, paraffin-embedded normal and neoplastic human tissue samples. Confirmation of FAM159B’s predicted size and antibody specificity was achieved in BON-1 cells, a neuroendocrine tumour cell line endogenously expressing FAM159B, using targeted siRNA. Immunocytochemical experiments additionally revealed cytoplasmic expression of the adaptor protein. Immunohistochemical staining detected FAM159B expression in neuronal and neuroendocrine tissues such as the cortex, the trigeminal ganglia, dorsal root and intestinal ganglia, the pancreatic islets and the neuroendocrine cells of the bronchopulmonary and gastrointestinal tract, but also in the syncytiotrophoblasts of the placenta. FAM159B was also expressed in many of the 28 tumour entities investigated, with high levels in medullary and anaplastic thyroid carcinomas, parathyroid adenomas, lung and ovarian carcinomas, lymphomas and neuroendocrine tumours of different origins. The antibody HPA011778 can act as a useful tool for basic research and identifying FAM159B expression in tissue samples.
Simple Summary
Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly cancers worldwide. The occurrence of oncogenic KRAS mutations is considered a signature event in PDAC, leading to genomic instability. The aim of our study was to evaluate the impact of the oncogenic KRAS G12D mutation on the activity of the error-prone alt-EJ repair mechanism, and to investigate the potential role of Polθ in the development of pancreatic cancer. We found that oncogenic KRAS increases the expression of key alt-EJ proteins in a mouse and human PDAC model. Using TLR assay, we also found increased alt-EJ activity in mouse and human cell lines upon the expression of KRAS D12D. The inactivation/impairment of alt-EJ by polymerase theta (Polθ) depletion delays the development of pancreatic cancer and prolongs the survival of experimental mice, though it does not prevent the PDAC development, which leads to full-blown PDAC with disseminated metastasis. Our studies provide a high-value target as a novel therapeutic candidate for the treatment of pancreatic and other cancers.
Abstract
Pancreatic ductal adenocarcinoma (PDAC), due to its genomic heterogeneity and lack of effective treatment, despite decades of intensive research, will become the second leading cause of cancer-related deaths by 2030. Step-wise acquisition of mutations, due to genomic instability, is considered to drive the development of PDAC; the KRAS mutation occurs in 95 to 100% of human PDAC, and is already detectable in early premalignant lesions designated as pancreatic intraepithelial neoplasia (PanIN). This mutation is possibly the key event leading to genomic instability and PDAC development. Our study aimed to investigate the role of the error-prone DNA double-strand breaks (DSBs) repair pathway, alt-EJ, in the presence of the KRAS G12D mutation in pancreatic cancer development. Our findings show that oncogenic KRAS contributes to increasing the expression of Polθ, Lig3, and Mre11, key components of alt-EJ in both mouse and human PDAC models. We further confirm increased catalytic activity of alt-EJ in a mouse and human model of PDAC bearing the KRAS G12D mutation. Subsequently, we focused on estimating the impact of alt-EJ inactivation by polymerase theta (Polθ) deletion on pancreatic cancer development, and survival in genetically engineered mouse models (GEMMs) and cancer patients. Here, we show that even though Polθ deficiency does not fully prevent the development of pancreatic cancer, it significantly delays the onset of PanIN formation, prolongs the overall survival of experimental mice, and correlates with the overall survival of pancreatic cancer patients in the TCGA database. Our study clearly demonstrates the role of alt-EJ in the development of PDAC, and alt-EJ may be an attractive therapeutic target for pancreatic cancer patients.
In addition to the classical oestrogen receptors, ERα and ERβ, a G protein-coupled oestrogen receptor (GPER) has been identified that primarily mediates the rapid, non-genomic signalling of oestrogens. Data on GPER expression at the protein level are contradictory; therefore, the present study was conducted to re-evaluate GPER expression by immunohistochemistry to obtain broad GPER expression profiles in human non-neoplastic and neoplastic tissues, especially those not investigated in this respect so far. We developed and thoroughly characterised a novel rabbit monoclonal anti-human GPER antibody, 20H15L21, using Western blot analyses and immunocytochemistry. The antibody was then applied to a large series of formalin-fixed, paraffin-embedded human tissue samples. In normal tissue, GPER was identified in distinct cell populations of the cortex and the anterior pituitary; islets and pancreatic ducts; fundic glands of the stomach; the epithelium of the duodenum and gallbladder; hepatocytes; proximal tubules of the kidney; the adrenal medulla; and syncytiotrophoblasts and decidua cells of the placenta. GPER was also expressed in hepatocellular, pancreatic, renal, and endometrial cancers, pancreatic neuroendocrine tumours, and pheochromocytomas. The novel antibody 20H15L21 will serve as a valuable tool for basic research and the identification of GPER-expressing tumours during histopathological examinations.
The utilization of fluorescein-guided biopsies has recently been discussed to improve and expedite operative techniques in the detection of tumor-positive tissue, as well as to avoid making sampling errors. In this study, we aimed to report our experience with fluorescein-guided biopsies and elucidate distribution patterns in different histopathological diagnoses in order to develop strategies to increase the efficiency and accuracy of this technique. We report on 45 fluorescence-guided stereotactic biopsies in 44 patients (15 female, 29 male) at our institution from March 2016 to March 2021, including 25 frame-based stereotactic biopsies and 20 frameless image-guided biopsies using VarioGuide®. A total number of 347 biopsy samples with a median of 8 samples (range: 4–18) per patient were evaluated for intraoperative fluorescein uptake and correlated to definitive histopathology. The median age at surgery was 63 years (range: 18–87). Of the acquired specimens, 63% were fluorescein positive. Final histopathology included glioblastoma (n = 16), B-cell non-Hodgkin lymphoma (n = 10), astrocytoma, IDH-mutant WHO grade III (n = 6), astrocytoma, IDH-mutant WHO grade II (n = 1), oligodendroglioma, IDH-mutant and 1p/19q-codeleted WHO grade II (n = 2), reactive CNS tissue/inflammation (n = 4), post-transplantation lymphoproliferative disorder (PTLD; n = 2), ependymoma (n = 1), infection (toxoplasmosis; n = 1), multiple sclerosis (n = 1), and metastasis (n = 1). The sensitivity for high-grade gliomas was 85%, and the specificity was 70%. For contrast-enhancing lesions, the specificity of fluorescein was 84%. The number needed to sample for contrast-enhancing lesions was three, and the overall number needed to sample for final histopathological diagnosis was five. Interestingly, in the astrocytoma, IDH-mutant WHO grade III group, 22/46 (48%) demonstrated fluorescein uptake despite no evidence for gadolinium uptake, and 73% of these were tumor-positive. In our patient series, fluorescein-guided stereotactic biopsy increases the likelihood of definitive neuropathological diagnosis, and the number needed to sample can be reduced by 50% in contrast-enhancing lesions.
Background
The focal form of CHI is caused by an autosomal recessive pathogenic variant affecting the paternal homologue of genes ABCC8 or KCNJ11 and a second somatic event specifically occurring in the affected islet of Langerhans. The approach of this study was to integrate the genetic changes occurring in pancreatic focal lesions of CHI at the genomic and transcriptional level.
Research Design and Methods
Patients receiving therapeutic surgery and with proven ABCC8 or KCNJ11 pathogenic variants were selected and analyzed for loss of heterozygosity (LOH), changes in copy number and uniparental disomy (UPD) on the short am of chromosome 11 by molecular microarray analysis and methylation-specific MLPA. Gene expression was analyzed by RT-PCR and Massive Analysis of cDNA Ends (MACE).
Results
Both genes, ABCC8 and KCNJ11, are located in proximity to the Beckwith-Wiedemann (BWS) imprinting control region on chromosome 11p15. Somatic paternal uniparental isodisomy (UPD) at chromosome 11p was identified as second genetic event in focal lesions resulting in LOH and monoallelic expression of the mutated ABCC8/KCNJ11 alleles. Of five patients with samples available for microarray analysis, the breakpoints of UPD on chromosome 11p were different. Samples of two patients were analyzed further for changes in gene expression. Profound downregulation of growth suppressing genes CDKN1 and H19 was detected in focal lesions whereas growth promoting gene ASCL2 and pancreatic transcription factors of the endocrine cell lineage were upregulated.
Conclusions
Paternal UPD on the short arm of chromosome 11 appears to be the major second genetic event specifically within focal lesions of CHI but no common breakpoint for UDP can be delineated. We show for the first time upregulation of growth promoting ASCL2 (achaete-scute homolog 2) suggestive of a driving factor in postnatal focal expansion in addition to downregulation of growth suppressing genes CDKN1C and H19.
ObjectiveLarge-scale genome sequencing efforts of human tumours identified epigenetic modifiers as one of the most frequently mutated gene class in human cancer. However, how these mutations drive tumour development and tumour progression are largely unknown. Here, we investigated the function of the histone demethylase KDM6A in gastrointestinal cancers, such as liver cancer and pancreatic cancer.DesignGenetic alterations as well as expression analyses of KDM6A were performed in patients with liver cancer. Genetic mouse models of liver and pancreatic cancer coupled with Kdm6a-deficiency were investigated, transcriptomic and epigenetic profiling was performed, and in vivo and in vitro drug treatments were conducted.ResultsKDM6A expression was lost in 30% of patients with liver cancer. Kdm6a deletion significantly accelerated tumour development in murine liver and pancreatic cancer models. Kdm6a-deficient tumours showed hyperactivation of mTORC1 signalling, whereas endogenous Kdm6a re-expression by inducible RNA-interference in established Kdm6a-deficient tumours diminished mTORC1 activity resulting in attenuated tumour progression. Genome-wide transcriptional and epigenetic profiling revealed direct binding of Kdm6a to crucial negative regulators of mTORC1, such as Deptor, and subsequent transcriptional activation by epigenetic remodelling. Moreover, in vitro and in vivo genetic epistasis experiments illustrated a crucial function of Deptor and mTORC1 in Kdm6a-dependent tumour suppression. Importantly, KDM6A expression in human tumours correlates with mTORC1 activity and KDM6A-deficient tumours exhibit increased sensitivity to mTORC1 inhibition.ConclusionKDM6A is an important tumour suppressor in gastrointestinal cancers and acts as an epigenetic toggle for mTORC1 signalling. Patients with KDM6A-deficient tumours could benefit of targeted therapy focusing on mTORC1 inhibition.
Diese Arbeit befasst sich mit dem Einfluss des Transkriptionsfaktors ChREBP und eines
Diabetes mellitus auf die hepatische Tumorgenese sowie das Wachstum und die Glykogenspeicherung
der Hepatozyten. Im speziellen sollte untersucht werden, inwieweit sich
die in den Kurzzeitversuchen beobachteten Glykogenspeicherherde nach drei Monaten
Versuchszeitraum weiterentwickeln.
ChREBP beeinflusst als Transkriptionsfaktor maßgeblich den Zucker- und Fettstoffwechsel,
indem es unter anderem die Lipogenese, Glykolyse und den Pentosephosphatweg
aktiviert und die Glukoneogenese hemmt. Zudem ist ChREBP an der Tumorentstehung
in verschiedenen Geweben z.B. der Leber, des Kolons und der Brust beteiligt, indem es
unter anderem den zellulären Metabolismus in Richtung einer starken Proliferation und
einer erhöhten Glykolyse, Laktatbildung und Aktivierung des Pentosephosphatwegs verändert.
Mäusen mit einem durch Streptozotocin induzierten Diabetes wurden je 200 isolierte,
isologe Pankreasinseln intraportal transplantiert. Somit entstand in den Empfängertieren
lokal in der Leber sowohl eine Hyperglykämie als auch eine Hyperinsulinämie im Sinne
eines Typ 2 Diabetes. Der Einfluss von ChREBP auf die Leber wurde durch den Einsatz eines
Knockout-Stammes untersucht. Nach drei Monaten wurden die Tiere nach einer Perfusion
getötet und Paraffinschnittpräparate der Lebern angefertigt und hinsichtlich ihrer
Proliferationsaktivität, Glykogenspeicherung und Herd- bzw. Tumorentstehung histologisch
ausgewertet.
Die Ergebnisse dieser Arbeit zeigen ein deutlich geringeres Auftreten von Glykogenspeicherherden
nach drei Monaten Versuchsdauer im Vergleich zu den Kurzzeitversuchen
nach einer und vierWochen. Auch sind nach drei Monaten noch keine manifesten Leberzelltumoren
entstanden. Es gab keinen Unterschied in der Herdfrequenz zwischen den
beiden Genotypen.
Weiterhin wurde der Einfluss von ChREBP auf die Proliferationsaktivität der Hepatozyten
untersucht. In der nicht diabetischen, transplantierten Gruppe zeigten die ChREBPKnockout-
Tiere eine stärkere und bei den diabetischen nicht transplantierten Mäusen
eine geringere Proliferationsaktivität als die Wildtyp-Tiere.
Die Hepatozyten der diabetischen Knockout-Tiere speicherten weniger und die der nicht
diabetischen mehr Glykogen als ihre Vertreter aus der Wildtyp-Gruppe.
In dieser Arbeit konnte gezeigt werden, dass ChREBP zu einer veränderten Glykogenspeicherung
der Hepatozyten führt. Sein Einfluss zeigt sich bei der diabetischen Stoffwechsellage
durch eine Steigerung und bei der nicht diabetischen durch eine Verminderung
dieser. Auch die Proliferationsaktivität der Hepatozyten wird von ChREBP beeinflusst,
indem es sie beim Vorliegen eines Diabetes mellitus steigert. Da sich auch aus
den vorhandenen klarzelligen Herden keine Tumoren entwickelt haben scheint die Tumorentstehung
bei der Maus bei beiden Genotypen länger als drei Monate zu dauern.
Der Transkriptionsfaktor ChREBP ist ein zentraler Regulator des Fett- und Glukosestoffwechsels
und wird insbesondere in der Leber intensiv erforscht. Allerdings ist seine Rolle in
der Niere, die maßgeblich an der Glukose-Homöstase beteiligt ist und einen wichtigen Ort
der Glukoneogenese darstellt, noch weitgehend unerforscht.
Zentrales Ziel dieser Arbeit ist es, im Rahmen des hepatischen Pankreasinseltransplantationsmodells
die Auswirkungen eines Diabetes mellitus und eines Fehlens des Transkriptionsfaktors
CHREBP auf das renale Tubulusepithel anhand von Wildtyp- und ChREBPKnockout-
Mäusen über einen Versuchszeitraum von drei bzw. sechs Monaten zu evaluieren.
Dazu wurden die tubuläre Glykogenspeicherung in Form von Glykogenspeicherkernen und
Armanni-Ebstein-Läsionen, die Proliferationsaktivität des renalen Tubulusepithels sowie das
Auftreten von renalen Tumoren untersucht.
Das Vorliegen eines Diabetes mellitus führt in der Niere der Wildtyp-Mäuse zu einer tubulären
Glykogenakkumulation, die durch einen ChREBP-Knockout deutlich verstärkt wurde.
Diese Glykogenspeicherung fehlte bei nicht diabetischen Tieren völlig. Gleichzeitig ließ sich
bei diabetischen Wildtyp-Tieren eine zum Teil erhöhte Proliferationsaktivität sowie das Auftreten
von Nierentumoren beobachten. Das Ausschalten des Transkriptionsfaktors ChREBP
führte nicht nur zu einem vermehrten Auftreten von Glykogenspeicherkernen und Armanni-
Ebstein-Läsionen, sondern resultierte auch in einem deutlich gesteigertem Vorkommen von
renalen Tumoren. Diese Tumoren traten hier auch ohne das gleichzeitige Vorliegen eines
Diabetes mellitus auf. Auch die Proliferationsaktivität der Tubulusepithelien war bei den
ChREBP-Knockout-Mäusen gesteigert.
Somit führt ein Diabetes mellitus, insbesondere bei einem gleichzeitigem Knockout von
ChREBP, zu einer Akkumulation von Glykogen innerhalb der renalen Tubulusepithelien
und fördert die Entstehung von Tumoren in der Niere bei Mäusen. Auch ein Knockout von
ChREBP scheint- unabhängig vom Vorliegen eines Diabetes- einen fördernden Einfluss auf
die renale Tumorgenese zu haben. Allerdings sind die daran beteiligten Mechanismen noch
weitgehend unbekannt. Daher sollte die Rolle von ChREBP und die Bedeutung der diabetisch
bedingten tubulären Veränderungen in Form der Glykogenspeicherkerne und Armanni-
Ebstein-Läsionen
Simple Summary
The tumor suppressor protein P53 is a major player in preventing liver cancer development and progression. In this study we could show that P53 negatively regulates the expression of Helicase, lymphoid specific (HELLS), previously described as an important pro-tumorigenic epigenetic regulator in hepatocarcinogenesis. The regulatory mechanism included induction of the P53 target gene P21 (CDKN1A) resulting in repression of HELLS via downregulation of the transcription factor Forkhead Box Protein M1 (FOXM1). Our in vitro and in vivo findings indicate an important additional aspect of the tumor suppressive function of P53 in liver cancer linked to epigenetic regulation.
Abstract
The major tumor suppressor P53 (TP53) acts primarily as a transcription factor by activating or repressing subsets of its numerous target genes, resulting in different cellular outcomes (e.g., cell cycle arrest, apoptosis and senescence). P53-dependent gene regulation is linked to several aspects of chromatin remodeling; however, regulation of chromatin-modifying enzymes by P53 is poorly understood in hepatocarcinogenesis. Herein, we identified Helicase, lymphoid specific (HELLS), a major epigenetic regulator in liver cancer, as a strong and selective P53 repression target within the SNF2-like helicase family. The underlying regulatory mechanism involved P53-dependent induction of P21 (CDKN1A), leading to repression of Forkhead Box Protein M1 (FOXM1) that in turn resulted in downregulation of HELLS expression. Supporting our in vitro data, we found higher expression of HELLS in murine HCCs arising in a Trp53−/− background compared to Trp53+/+ HCCs as well as a strong and highly significant correlation between HELLS and FOXM1 expression in different HCC patient cohorts. Our data suggest that functional or mutational inactivation of P53 substantially contributes to overexpression of HELLS in HCC patients and indicates a previously unstudied aspect of P53′s ability to suppress liver cancer formation.
Simple Summary
Hepatocellular carcinoma (HCC) ranks second among the leading causes of cancer-related death. Since current therapeutic options are very limited, a deeper understanding of the molecular mechanisms underlying the tumor onset and progression of HCC holds great potential for improved therapeutic options. Although it has been shown that deleted in liver cancer 1 (DLC1) acts as a tumor suppressor whose allele is lost in 50% of liver cancers, alterations in gene expression initiated by DLC1 loss have not yet been the primary focus of liver cancer research. To identify novel gene targets that allow for a personalized medicine approach for HCC therapy, we performed gene expression profiling for HepG2 cells stably expressing DLC1shRNA. We provide evidence that TSPAN5 is required for HCC growth, migration and invasion, and dissected the underlying molecular mechanisms involving myocardin-related transcription factors. Thus, TSPAN5 represents a novel therapeutic target for the treatment of HCC characterized by DLC1 loss.
Abstract
Human hepatocellular carcinoma (HCC) is among the most lethal and common cancers in the human population, and new molecular targets for therapeutic intervention are urgently needed. Deleted in liver cancer 1 (DLC1) was originally identified as a tumor suppressor gene in human HCC. DLC1 is a Rho-GTPase-activating protein (RhoGAP) which accelerates the return of RhoGTPases to an inactive state. We recently described that the restoration of DLC1 expression induces cellular senescence. However, this principle is not amenable to direct therapeutic targeting. We therefore performed gene expression profiling for HepG2 cells depleted of DLC1 to identify druggable gene targets mediating the effects of DLC1 on senescence induction. This approach revealed that versican (VCAN), tetraspanin 5 (TSPAN5) and N-cadherin (CDH2) were strongly upregulated upon DLC1 depletion in HCC cells, but only TSPAN5 affected the proliferation of HCC cells and human HCC. The depletion of TSPAN5 induced oncogene-induced senescence (OIS), mediated by the p16INK4a/pRb pathways. Mechanistically, silencing TSPAN5 reduced actin polymerization and thereby myocardin-related transcription factor A- filamin A (MRTF-A-FLNA) complex formation, resulting in decreased expression of MRTF/SRF-dependent target genes and senescence induction in vitro and in vivo. Our results identify TSPAN5 as a novel druggable target for HCC.
Die vorliegende Arbeit liefert ein Modell zur Erzeugung PI3K-mutierter Tumoren in der
Mausleber, je nach Mutationsart mit unterschiedlichem onkogenen Potenzial. Bekannte und
bisher unbekannte nachgeschaltete Ziele der PI3K konnten identifiziert werden. Diese
können im Rahmen von künftigen Target Therapien von Bedeutung sein.
Die in diesem Versuch verwendete Methode des hydrodynamischen Gentransfers bietet eine
zuverlässige Möglichkeit, ohne aufwendige Zucht von transgenen Mausstämmen,
verschiedene (Onko)Gene in der Mausleber langfristig zu exprimieren und ihre
Auswirkungen in vivo zu untersuchen.
Congenital hyperinsulinism (CHI) causes hypoglycemia due to irregular insulin secretion. In infants, a rapid diagnosis and appropriate management to avoid severe hypoglycemia is mandatory. CHI is a heterogeneous condition at the clinical and genetic level, and disease-causing genes have been identified in about half of the patients. The majority of mutations have been identified in the ABCC8 and KCNJ11 genes encoding subunits of the K<sub>ATP</sub> channel responsible for two distinct histological forms. The diffuse form is caused by autosomal recessive or dominant inherited mutations, whereas the focal form is caused by a paternally transmitted recessive mutation and a second somatic event. We report on an unselected cohort of 136 unrelated patients from the German CHI registry. Mutations in either the ABCC8 or KCNJ11 gene were identified in 61 of these patients (45%). In total, 64 different mutations including 38 novel ones were detected in this cohort. We observed biparental (recessive) inheritance in 34% of mutation-positive patients, dominant inheritance in 11% and paternal transmission of a mutation associated with a focal CHI type in 38%. In addition, we observed inheritance patterns that do not exactly follow the classical recessive or dominant mode, further adding to the genetic complexity of this disease.
Background: Among the five somatostatin receptors (sst<sub>1</sub>-sst<sub>5</sub>), the sst<sub>3</sub> receptor displays a distinct pharmacological profile. Like sst<sub>2</sub>, the sst<sub>3</sub> receptor efficiently internalizes radiolabeled somatostatin analogs. Unlike sst<sub>2</sub>, however, internalized sst<sub>3</sub> receptors are rapidly transferred to lysosomes for degradation. Apart from this, very little is known about the clinical relevance of the sst<sub>3</sub> receptor, which may in part be due to the lack of specific monoclonal sst<sub>3</sub> antibodies. Methods: Here, we have extensively characterized the novel rabbit monoclonal anti-human sst<sub>3</sub> antibody UMB-5 using transfected cells and receptor-expressing tissues. UMB-5 was then subjected to immunohistochemical staining of a series of 190 formalin-fixed, paraffin-embedded normal and neoplastic human tissues. Results: Specificity of UMB-5 was demonstrated by detection of a broad band migrating at a molecular weight of 70,000–85,000 in immunoblots from human pituitary. After enzymatic deglycosylation, the size of this band decreased to a molecular weight of 45,000. Tissue immunostaining was completely abolished by pre-adsorption of UMB-5 with its immunizing peptide. In addition, UMB-5 detected distinct cell populations in human tissues like pancreatic islands, anterior pituitary, adrenal cortex, adrenal medulla, and enteric ganglia, similar to that seen with a rabbit polyclonal antibody generated against a different carboxyl-terminal epitope of the sst<sub>3</sub> receptor. In a comparative immunohistochemical study, UMB-5 yielded predominant plasma membrane staining in the majority of pituitary adenomas, pheochromocytomas, and a subset of neuroendocrine tumors. The sst<sub>3</sub> receptor was also present in many glioblastomas, pancreatic, breast, cervix, and ovarian carcinomas. Conclusion: The rabbit monoclonal antibody UMB-5 may prove of great value in the identification of sst<sub>3</sub>-expressing tumors during routine histopathological examinations. Given its unique trafficking properties, these tumors may be potential candidates for sst<sub>3</sub>-directed receptor radiotherapy.
In dieser Arbeit wurde erstmalig das zuvor an Ratten etablierte insulin-vermittelte Hepatokarzinogenesemodel mit der intraportalen Pankreasinseltransplantation auf die Maus übertragen und sowohl an einem Wildtyp -als auch einem Carbohydrate responsive-element bindung protein (ChREBP)-knockout-Stamm durchgeführt. CHREBP ist ein wichtiger Transkriptionsfaktor des Glukose- und Lipidmetabolismus, welcher auch bei der Entstehung des Diabetes mellitus Typ 2 bzw. des metabolischen Syndroms involviert ist. Bei Streptozotozin-induzierter diabetischer Stoffwechsellage erfolgte nach 5 Tagen die intraportale Transplantation von 70, zuvor isolierten, isologen Pankreasinseln. Zur Darstellung der Hepatozytenproliferation erhielten die Tiere das Basenanalogon Bromodesoxyuridin (BrdU), welches zum Teil über eine subkutan- implantierte osmotische Minipumpe bzw. über dreimalige Injektionen appliziert wurde. Mit dem BrdU-Labeling-Index konnte die Proliferationsaktivität der im Abstromgebiet der Pankreasinseln entstandenen hepatozytären Wildtyp- und ChREBP-Knockout-Herde ermittelt und mit dem jeweiligen unveränderten Lebergewebe verglichen werden. Nach einer Versuchsdauer von 1 Woche bzw. 4 Wochen erfolgte die Tötung im Rahmen der retrograden Organperfusion und gleichzeitiger Organfixation in Narkose. In direkter Transplantatumgebung entstanden in beiden diabetischen Genotypen klarzellige Hepatozytenherde, welche sich jedoch in Morphologie, im Glykogen- und Lipidgehalt, in der Proliferationsaktivität und der Frequenz und der Verteilung im Leberparenchym unterscheiden. So entsprachen die klarzelligen Herde der diabetischen Wildtyp- Maus denen des Rattenmodels und den humanen Herden, welche durch einen gesteigerten Glykogen- und Lipidgehalt, vergrößerten Zellkernen sowie einer nach 4 Wochen vierfach gesteigerten Proliferationsaktivität (Herd vs. unverändertes Lebergewebe: 11,34 ± 2,24 MW ± S.E.M vs. 2,4 ± 0,71 MW ± S.E.M.; p = 0,006) charakterisiert sind und somit auch bei der Maus als präneoplastische Läsionen einzustufen sind. Die hier erstmals beschriebenen Herde der diabetischen ChREBP-KO-Maus zeichnen sich hingegen durch große, balloniert erscheinende Hepatozyten mit einem exzessiven Glykogen- und nur minimalen Lipidgehalt, einem kleineren Zellkern, einem konfluierenden Verteilungsmuster und einer geringeren Proliferationsaktivität (Herd vs. unverändertes Lebergewebe: 6,08 ± 0,7 MW ±S.E.M. vs. 1,91 ± 0,02 MW ± S.E.M.; p = 0,0002), als die der Wildtyp-Herde, aus. Diese Resultate unterstützen die Annahme, dass ChREBP einen wesentlichen Einfluss auf den hepatischen Glukose-und Lipidmetabolismus hat und weiterhin einen protoonkogenen Faktor in der insulin-vermittelten Hepatokarzinogenese darstellt.