Immortalisierung embryonaler Fibroblasten aus Wildtyp und Hupki (Human p53 knock-in) Mäusen durch Veränderungen des p19Arf/p53 Kontrollweges und Erst-Charakterisierung des neuen Prolin 72-kodierenden Hupki-Mausstamms

Aufgrund zunehmenden Wissens um die Regulation des Wachstums maligner Zellen können neue Therapieansätze gefunden werden. Dafür ist es notwendig, mehr über die komplexen Mechanismen der Tumorentstehung und der unbegrenzten Proliferation, der Immortalisierung, von Zellen zu wissen. Immortale Zellen besitzen im Unterschied zu normalen Zellen kein begrenztes Teilungsvermögen, welches abhängig von der Anzahl der Zellteilungen und der Länge der Telomere (Harley et al., 1990) ist. Genauso unterscheiden sich Zelllinien von primären embryonalen (normalen) Zellen. Diese teilen sich bereits nach wenigen Passagen nicht mehr und treten in die Seneszenz ein, die mit charakteristischen morphologischen, zellphysiologischen, molekularen und biochemischen Veränderungen verbunden ist (Kanungo, 1994). Murine und humane Fibroblasten unterscheiden sich in den Mechanismen der Seneszenz: Der Hauptmechanismus der Seneszenz. Die 20%ige Sauerstoff Atmosphäre der Zellkulturbedingungen übt in den primären, embryonalen murinen Fibroblasten (MEFs) einen oxidativen Stress auf die Zellproliferation aus. P19/ARF-p53 scheint der wichtigste Regulationsmechanismus für die Seneszenz in diesen Zellen zu sein. Mit der Inaktivierung von Tumorsuppressorgenen wie z.B. p53 oder pRb durch Punktmutationen oder Deletionen kann die zelluläre Seneszenz unterbrochen und die Zelle zur Immortalisierung gebracht werden. Als Transkriptionsfaktor und Tumorsuppressor weist das p53- Gen Mutationen in 50% aller menschlichen Tumoren auf. Bekannt ist der p53- Polymorphismus im Codon 72 im Exon 4 in der Polyprolindomäne, das entweder für die Aminosäure Arginin (CGC) oder Prolin (CCC) kodiert. Beide Varianten zeigen biochemische Unterschiede im Zellzyklus, der Apoptose und der Tumorgenese-Regulation Gegenstand der vorliegenden Arbeit war die Untersuchung der spontanen Immortalisierungsrate der MEFs und der Hupki Maus Fibroblasten, die einen Teil des humanen Arginin p53- Gens vom Exon 4 zum Exon 9, HUFs R/R genannt, tragen (die HUFs wurden von der Humanen P53 knock in Maus (Hupki Maus) (Luo et al., 2001a) etabliert). Die MEFs weisen eine deutlich höhere Empfindlichkeit gegenüber der 20%igen Sauerstoffatmosphäre der Zellkulturbedingungen als die HUFs R/R auf. Nur ca. 10% (11 von 121) der gesamten primären Zellpopulationen überlebte die Seneszenz und traten in den Immortalisierungsprozess ein. Mehr als 60% der etablierten Zelllinien (7 von 11) weisen entweder p53 missense- Mutationen (5 von 7) vom Typ G>C oder C>G Transversion, mit einer Überexpression an p19 Protein, oder den p19/ARF- DNA Verlust (2 von7) auf. Dagegen verhielten sich die HUFs R/R, die in 90% der primären Zellklone zu immortalisierten Linien auswuchsen (109/120), deutlich resistenter gegenüber den angewandten reaktiven Sauerstoffspezies (reactive oxygene species (ROS)) als die MEFs. Nur 20% der HUFs Zellklone (4 von 20) weisen eine Punkmutation oder eine Deletion in der p53-DNA-Bindungsdomäne auf. 80% der restlichen Zelllinien sind nicht dadurch betroffen Die durchflusszytometrische Analyse der Apoptose mittels Annexin/PI- Kit zeigt, dass die stressigen in vitro Zellkulturbedingungen mehr primäre MEFs in die Apoptose treiben als HUFs R/R. Nachdem die erste Hupki Maus mit dem p53 Arginin Protein ein recht gutes Model für in vivo und in vitro Untersuchungen des humanen p53 Gens darstellt, wurde eine zweite Hupki Maus mit der Prolin- Variante hergestellt, um die multiplen Fragestellungen nach dem p53 Polymorphismus zu beantworten. Die ersten im Rahmen der vorliegenden Arbeit durchgeführten in vitro Studien zeigen in den Hupki Prolin (HUFs PP) Fibroblasten eine hohe Menge an p21 WAF in den klassischen in vitro Zellkulturbedingungen auf, die in den primären, embryonalen Hupki Arginin Fibroblasten. Erst nach Induktion mit gamma-Strahlen erreicht wurde. Dagegen weisen die HUF Zellen mit der p53 Arginin Variante eine Induktion der Perp (P53 apoptosis effactor related to PMP22) nach einer Gamma-Strahlen Exposition auf. Studien zeigten einen Zusammenhang zwischen dem heterozygoten Arg/Pro Genotyp und dem Lungenkarzinom (Fan et al., 2000). Auch das Prolin- Codon Pro/ Pro Genotyp weist eine Assoziierung mit Lungentumoren (Kawajiri et al., 1993) auf. Von 47, mit dem Prokarzinogenen Benzo(a)pyren B(a)P behandelten, primären, embryonalen heterozygoten Hupki Arginin/Prolin (HUFs RP) Zellpopulationen ließen sich 20 (42%) als Zelllinien etablieren: Davon weisen 25% der Zellklone eine p53 G>C, C>G oder C>T Punktmutation auf dem Prolin- Allel auf, gefolgt vom einem Verlust der Heterozygosität (LOH) des Arginin-Allels. Dieses Ergebnis scheint sich von der Immortalisierung der mit B(a)P behandelten Hupki hetereozygoten Arginin Fibroblasten (Liu et al., 2005) deutlich zu unterscheiden

"Beige" Cross Talk Between the Immune System and Metabolism

With thymic senescence the epithelial network shrinks to be replaced by adipose tissue. Transcription factor TBX-1 controls thymus organogenesis, however, the same TBX-1 has also been reported to orchestrate beige adipose tissue development. Given these different roles of TBX-1, we have assessed if thymic TBX-1 expression persists and demonstrates this dualism during adulthood. We have also checked whether thymic adipose involution could yield beige adipose tissue. We have used adult mouse and human thymus tissue from various ages to evaluate the kinetics of TBX-1 expression, as well as mouse (TEP1) and human (1889c) thymic epithelial cells (TECs) for our studies. Electron micrographs show multi-locular lipid deposits typical of beige adipose cells. Histology staining shows the accumulation of neutral lipid deposits. qPCR measurements show persistent and/or elevating levels of beige-specific and beige-indicative markers (TBX-1, EAR-2, UCP-1, PPAR-gamma). We have performed miRNome profiling using qPCR-based QuantStudio platform and amplification-free NanoString platform. We have observed characteristic alterations, including increased miR21 level (promoting adipose tissue development) and decreased miR34a level (bias toward beige adipose tissue differentiation). Finally, using the Seahorse metabolic platform we have recorded a metabolic profile (OCR/ECAR ratio) indicative of beige adipose tissue. In summary, our results support that thymic adipose tissue emerging with senescence is bona fide beige adipose tissue. Our data show how the borders blur between a key immune tissue (the thymus) and a key metabolic tissue (beige adipose tissue) with senescence. Our work contributes to the understanding of cross talk between the immune system and metabolism

Canonical WNT/β-Catenin Signaling Plays a Subordinate Role in Rhabdomyosarcomas

The development of skeletal muscle from immature precursors is partially driven by canonical WNT/β-catenin signaling. Rhabdomyosarcomas (RMS) are immature skeletal muscle-like, highly lethal cancers with a variably pronounced blockade of muscle differentiation. To investigate whether canonical β-catenin signaling in RMS is involved in differentiation and aggressiveness of RMS, we analyzed the effects of WNT3A and of a siRNA-mediated or pharmacologically induced β-catenin knock-down on proliferation, apoptosis and differentiation of embryonal and alveolar RMS cell lines. While the canonical WNT pathway was maintained in all cell lines as shown by WNT3A induced AXIN expression, more distal steps including transcriptional activation of its key target genes were consistently impaired. In addition, activation or inhibition of canonical WNT/β-catenin only moderately affected proliferation, apoptosis or myodifferentiation of the RMS tumor cells and a conditional knockout of β-catenin in RMS of Ptchdel/+ mice did not alter RMS incidence or multiplicity. Together our data indicates a subordinary role of the canonical WNT/β-catenin signaling for RMS proliferation, apoptosis or differentiation and thus aggressiveness of this malignant childhood tumor

WNT4 overexpression and secretion in thymic epithelial tumors drive an autocrine loop in tumor cells in vitro

BackgroundWNT4-driven non-canonical signaling is crucial for homeostasis and age-related involution of the thymus. Abnormal WNT signaling is important in many cancers, but the role of WNT signaling in thymic tumors is largely unknown.Materials & MethodsExpression and function of WNT4 and FZD6 were analyzed using qRT–PCR, Western blot, ELISA, in biopsies of non-neoplastic thymi (NT), thymoma and thymic carcinomas. ShRNA techniques and functional assays were used in primary thymic epithelial cells (pTECs) and TC cell line 1889c. Cells were conventionally (2D) grown and in three-dimensional (3D) spheroids.ResultsIn biopsy, WHO classified B3 thymomas and TCs showed increased WNT4 expression compared with NTs. During short-term 2D culture, WNT4 expression and secretion declined in neoplastic pTECs but not in 3D spheroids or medium supplemented with recombinant WNT4 cultures. Under the latter condition, the growth of pTECs was accompanied by increased expression of non-canonical targets RAC1 and JNK. Down-regulation of WNT4 by shRNA induced cell death in pTECs derived from B3 thymomas and led to decreased RAC1, but not JNK protein phosphorylation. Pharmacological inhibition of NF-κB decreased both RAC1 and JNK phosphorylation in neoplastic pTECs.ConclusionsLack of the age-related decline of non-canonical WNT4 expression in TETs and restoration of declining WNT4 expression through exogeneous WNT4 or 3D culture of pTECs hints at an oncogenic role of WNT4 in TETs and is compatible with the WNT4 autocrine loop model. Crosstalk between WNT4 and NF-κB signaling may present a promising target for combined interventions in TETs

Pulmonary cancers across different histotypes share hybrid tuft cell/ionocyte-like molecular features and potentially druggable vulnerabilities

Tuft cells are chemosensory epithelial cells in the respiratory tract and several other organs. Recent studies revealed tuft cell-like gene expression signatures in some pulmonary adenocarcinomas, squamous cell carcinomas (SQCC), small cell carcinomas (SCLC), and large cell neuroendocrine carcinomas (LCNEC). Identification of their similarities could inform shared druggable vulnerabilities. Clinicopathological features of tuft cell-like (tcl) subsets in various lung cancer histotypes were studied in two independent tumor cohorts using immunohistochemistry (n = 674 and 70). Findings were confirmed, and additional characteristics were explored using public datasets (RNA seq and immunohistochemical data) (n = 555). Drug susceptibilities of tuft cell-like SCLC cell lines were also investigated. By immunohistochemistry, 10–20% of SCLC and LCNEC, and approximately 2% of SQCC expressed POU2F3, the master regulator of tuft cells. These tuft cell-like tumors exhibited “lineage ambiguity” as they co-expressed NCAM1, a marker for neuroendocrine differentiation, and KRT5, a marker for squamous differentiation. In addition, tuft cell-like tumors co-expressed BCL2 and KIT, and tuft cell-like SCLC and LCNEC, but not SQCC, also highly expressed MYC. Data from public datasets confirmed these features and revealed that tuft cell-like SCLC and LCNEC co-clustered on hierarchical clustering. Furthermore, only tuft cell-like subsets among pulmonary cancers significantly expressed FOXI1, the master regulator of ionocytes, suggesting their bidirectional but immature differentiation status. Clinically, tuft cell-like SCLC and LCNEC had a similar prognosis. Experimentally, tuft cell-like SCLC cell lines were susceptible to PARP and BCL2 co-inhibition, indicating synergistic effects. Taken together, pulmonary tuft cell-like cancers maintain histotype-related clinicopathologic characteristics despite overlapping unique molecular features. From a therapeutic perspective, identification of tuft cell-like LCNECs might be crucial given their close kinship with tuft cell-like SCLC

Thymoma related myasthenia gravis in humans and potential animal models

Thymoma-associated Myasthenia gravis (TAMG) is one of the anti-acetylcholine receptor MG (AChR-MG) sub-types. The clinico-pathological features of TAMG and its pathogenesis are described here in comparison with pathogenetic models suggested for the more common non-thymoma AChR-MG subtypes, early onset MG and late onset MG. Emphasis is put on the role of abnormal intratumorous T cell selection and activation, lack of intratumorous myoid cells and regulatory T cells as well as deficient expression of the autoimmune regulator (AIRE) by neoplastic thymic epithelial cells. We review spontaneous and genetically engineered thymoma models in a spectrum of animals and the extensive clinical and immunological overlap between canine, feline and human TAMG. Finally, limitations and perspectives of the transplantation of human and murine thymoma tissue into nude mice, as potential models for TAMG, are addressed. (C) 2015 Elsevier Inc. All rights reserved

Insulin‐like growth factor 2 expression in prostate cancer is regulated by promoter‐specific methylation

Deregulation of the insulin‐like growth factor (IGF) axis and dysbalance of components of the IGF system as potential therapeutic targets have been described in different tumor types. IGF2 is a major embryonic growth factor and an important activator of IGF signaling. It is regulated by imprinting in a development‐ and tissue‐dependent manner and has been implicated in a broad range of malignancies including prostate cancer (PCa). Loss of imprinting (LOI) usually results in bi‐allelic gene expression and increased levels of IGF2. However, the regulatory mechanisms and the pathophysiological impact of altered IGF2 expression in PCa remain elusive. Here, we show that in contrast to many other tumors, IGF2 mRNA and protein levels were decreased in 80% of PCa in comparison with non‐neoplastic adjacent prostate and were independent of LOI status. Instead, IGF2 expression in both tumors and adjacent prostate depended on preferential usage of the IGF2 promoters P3 and P4. Decreased IGF2 expression in tumors was strongly related to hypermethylation of these two promoters. Methylation of the A region in promoter P4 correlated specifically with IGF2 expression in the 20% of PCa where IGF2 was higher in tumors than in adjacent prostate. We conclude that IGF2 is downregulated in most PCa and may be particularly relevant during early stages of tumor development or during chemotherapy and androgen deprivation. PCa differs from other tumors in that IGF2 expression is mainly regulated through methylation of promoter‐specific and not by imprinting. Targeting of promoter‐specific regions may have relevance for the adjuvant treatment of PCa

Influence of radiation on cell proliferation.

<p>Comparison between (<b>A</b>) porous group 1 (+por-rad) and group 3 (+por+rad) and (<b>B</b>) non-porous group 2 (-por-rad) and group 4 (-por+rad) respectively with and without radiation. All values are expressed as mean values ± standard deviation.</p

Molecular pathology of thymomas: implications for diagnosis and therapy

Thymomas exhibit a unique genomic landscape, comprising the lowest on average total mutational burden among adult human cancers; a unique point mutation in the GTF2I gene in WHO type A and AB thymomas (and rarely others); almost unique KMT2A-MAML2 translocations in rare WHO type B2 and B3 thymomas; a unique YAP1-MAML2 translocation in almost all metaplastic thymomas; and unique miRNA profiles in relation to GTF2I mutational status and WHO histotypes. While most thymomas can be diagnosed solely on the basis of morphological features, mutational analyses can solve challenging differential diagnostic problems. No molecular biomarkers have been identified that predict the response of unresectable thymomas to chemotherapy or agents with known molecular targets. Despite the common and strong expression of PDL1 in thymomas, immune checkpoint inhibitors are rarely applicable due to the poor predictability of common, life-threatening autoimmune side effects that are related to the unrivaled propensity of thymomas towards autoimmunity