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Patched Knockout Mouse Models of Basal Cell Carcinoma
Get PDFBasal cell carcinoma (BCC) is the most common human tumor. Mutations in the hedgehog (HH) receptor Patched (PTCH) are the main cause of BCC. Due to their high and increasing incidence, BCC are becoming all the more important for the health care system. Adequate animal models are required for the improvement of current treatment strategies. A good model should reflect the situation in humans (i.e., BCC initiation due to Ptch mutations on an immunocompetent background) and should allow for (i) BCC induction at a defined time point, (ii) analysis of defined BCC stages, and (iii) induction of BCC in 100% of animals. In addition, it should be easy to handle. Here, we compare several currently existing conventional and conditional Ptch knockout mouse models for BCC and their potential use in preclinical research. In addition, we provide new data using conditional Ptchflox/flox mice and the K5-Cre-ERT+/− driver
Patched associated tumors: Modifier genes and pathogenesis
No full textMutationen im Patched-Gen (Ptch), welches
für eine Komponente der Hedgehog (Hh)/Ptch Signalkaskade kodiert,
werden häufig bei Basalzellkarzinomen (BCC), Medulloblastomen (MB)
und Rhabdomyosarkomen (RMS) gefunden. Wie Daten aus dem Ptchneo67/+
Tiermodell zeigen, ist die Tumorinzidenz vom genetischen
Hintergrund abhängig. Der Hintergrund C57BL/6N (B6) prädisponiert
die Tiere zu MB, wohingegen der BALB/c (BALB) Hintergrund zu RMS
prädisponiert. Dies weist auf die Existenz von Modifikatorgenen
hin. Ein Ziel dieser Arbeit war die Kartierung und Identifikation
dieser Gene.
Im Rahmen dieses Projektes wurde an der Charakterisierung des
RMS-Suszeptibilitäts-Lokus Parms1 (Patched-Associated RMS 1)
mitgewirkt. Dieser Lokus liegt auf Chromosom 2 im Intervall
zwischen den Mikrosatellitenmarkern D2Mit37 und D2Mit102. In dieser
Region begünstigen dominante BALB-Allele die Entstehung von RMS.
Diese Daten wurden in der Zeitschrift Genomics publiziert (H Hahn
et al., 2004). In den Mauslinien B6 und BALB wurden für elf
Kandidatengene SNP- oder Genexpressions-Analysen durchgeführt.
Aufgrund der Überexpression von Catenin src im Skelettmuskel von
B6-Mäusen und seiner tumorsuppressorischen Funktion könnte dieses
Gen in die Modulation der RMS-Suszeptibilität involviert sein.
Zusätzlich wurde für Feinkartierungsuntersuchungen des Lokus Parms1
in 218 B6x(BALBxB6)Ptchneo67/+ N2-Mäusen eine genomweite
Kopplungsanalyse mit 97 polymorphen Mikrosatellitenmarkern
durchgeführt. Weiterhin wurden Nachkommen aus Kreuzungen von
B6Ptchneo67/+-Mäusen mit den Inzuchtstämmen 129Sv, DBA2J und FVB/N
generiert und auf die Entwicklung von Tumoren untersucht. In den
F1-Generationen sollte dann auf der Basis der Korrelation von
Segmentidentität und Phänotyp eine weitere Kartierung der
RMS-Suszeptibilität durchgeführt werden. Im Laufe der
Untersuchungen wurde eine genetische Kontamination bei B6-Mäusen,
die im Zeitraum von Herbst 2003 bis Frühjahr 2005 von der Firma
Charles River Laboratories zur Etablierung der entsprechenden
Kreuzungen zugekauft worden waren, festgestellt. Die SNP-Analyse
ließ auf eine Kontamination mit einer DBA-Inzuchtlinie schließen (F
Nitzki et al., 2007). Aufgrund dieser Tatsache wurde das
Kartierungsprojekt abgebrochen.
In einem zweiten Projekt wurde die in unserem Labor generierte
Mauslinie Ptchflox/flox charakterisiert. Mittels einer
Tamoxifen-induzierbaren Cre-Rekombinase wurde in diesem
konditionellen Ptch-knockout-Mausmodell eine Ptch-Mutation
induziert. Neben hyperproliferativen Veränderungen der Magenwand
und des Mesenteriums sowie einem Entwicklungsdefekt der allgemeinen
lymphoiden Vorläuferzellen im Knochenmark konnten in diesen Mäusen
BCC hervorgerufen werden (A Uhmann et al., 2007). Die BCC wurden
durch eine biallelische Deletion von Ptch initiiert und entstehen
wahrscheinlich unabhängig von der Haarzyklusphase aus sowohl
Basalzellen der Epidermis als auch der äußeren Wurzelscheide des
Haarfollikels. Interessanterweise wurden die BCC nach 200 Tagen
regredient, zeigten einen differenzierteren Phänotyp und eine
verminderte Proliferation. Da in jedem BCC Stadium die
Hh/Ptch-Signalkaskade aktiv war, kann die Regression nicht durch
eine Inaktivierung des Signalweges bedingt sein. Von regredienten
Tumorstadien wurden die Differenzierungsmarker Keratin 1 (K1) und
Keratin 10 (K10) exprimiert, wohingegen K1 und K10 in den frühen
Tumorstadien nicht detektiert wurden. Daher steht die Expression
von K1 und K10 vermutlich im direkten Zusammenhang mit der
Regression der BCC. Diese Daten leisten einen wichtigen Beitrag zum
besseren Verständnis der Pathogenese dieser Tumorentität und
könnten in der Zukunft eventuell zur Etablierung neuer
Therapieansätze beisteuern.
Schließlich wurde die Zelllinie B9 aus Ptchflox/floxERT2+/--Mäusen
generiert. In diesen Zellen lässt sich eine Ptch-Mutation durch
Behandlung mit Tamoxifen induzieren. Die resultierende
Ptch-Deletion bewirkt eine Aktivierung der Hh/Ptch Signalkaskade,
was auch durch Inkubation der Zellen mit Shh hervorgerufen werden
kann. Zusätzlich wurden die Ptch-defiziente Zelllinie Ptch-/-, die
eine pathologisch aktivierte Hh/Ptch Signalkaskade aufweist, und
die Wildtyp-Ptch Zelllinie Ptchflox/flox etabliert. Ptch-/- Zellen
zeigen im Vergleich zur Zelllinie Ptchflox/flox eine stark erhöhte
Aktivität der Hh/Ptch Signalkaskade sowie eine erhöhte
Proliferationsrate. Sowohl in B9- als auch Ptch-/--Zellen lässt
sich die physiologisch bzw. pathologisch aktivierte Hh/Ptch
Signalkaskade durch die Zugabe von Cyclopamin inhibieren. Diese
Zelllinien können dazu beitragen, die Funktion von Ptch besser zu
verstehen.Patched (Ptch), a component of the
Hedgehog (Hh)/Ptch signalling cascade, is frequently mutated in
basal cell carcinoma (BCC), medulloblastoma (MB) and
rhabdomyosarcoma (RMS). Ptchneo67/+ mice, in which exons 6 and 7
are deleted, show genetic background-dependent susceptibility to
the development of these tumors. The C57BL/6N (B6) strain is
genetically susceptible to MB, whereas the BALB/c (BALB) background
predisposes to RMS. The first aim of this work was to map and to
identify the respective modifier genes.
Within the scope of this work the RMS susceptibility modifier locus
Parms1 (Patched-Associated RMS 1) was identified on chromosome 2
between D2Mit37 and D2Mit102. Within this region dominant BALB
alleles confer RMS susceptibility. These data were published in
Genomics (H Hahn et al., 2004). SNP or gene expression analyses
were performed for eleven candidate modifier genes. Due to the
enhanced expression of Catenin src in skeletal muscle of B6 mice
and due to its tumor suppressing function this gene might be
involved in the modulation of RMS susceptibility. In order to
refine the location of Parms1, a second genome wide scan was
performed on 218 B6x(BALBxB6)Ptchneo67/+ N2 mice using 97
polymorphic microsatellite markers. In addition, the progeny of a
cross between B6Ptchneo67/+ mice and either 129Sv, DBA2J or FVB/N
inbred mice were screened for tumor formation. DNA of these mice
should have been used to further narrow down the RMS susceptibility
region on the basis of correlation between segment identity and
tumor phenotype. During the study heterozygosity at some loci of
the C57BL/6N mouse strain derived from Charles River Laboratories
were noticed. A panel of 100 SNP markers was used to confirm and
specify the genetic contamination. Retrospective analyses
demonstrated that the contamination took place as early as autumn
2003 and has persisted ever since at a fairly constant level.
Contaminating alleles most probably originated from a DBA strain (F
Nitzki et al., 2007). Due to the contamination the mapping project
was cancelled.
The second project dealt with the characterization of the
conditional Ptchflox/flox mouse line. Upon ubiquitous deletion of
Ptch that was achieved by a tamoxifen inducible Cre recombinase,
the mice developed hyperproliferative changes of the stomach and
the mesenterium, a developmental defect of the common lymphoid
progenitors in the bone marrow and BCC (A Uhmann et al., 2007).
Subsequent analysis of BCC revealed that these tumors were
initiated by biallelic deletion of Ptch and arose independent of
the hair cycle from both the basal cells of the epidermis and the
outer root sheath of the hair follicle. Surprisingly, BCC regressed
after 200 days and showed a more differentiated phenotype
accompanied by reduced proliferation. As the Hh/Ptch signalling
cascade was active at all BCC stages, regression was not caused by
downregulation of the pathway’s activity. Since regressing tumors
expressed the differentiation markers Keratin 1 (K1) and Keratin 10
(K10) that were never detected at earlier tumor stages, K1 and K10
are most likely associated with regression of BCC. These data
contribute to the better understanding of the pathogenesis of these
tumors and might help to establish new therapeutic approaches in
the future.
Finally, the wildtype cell line Ptchflox/flox and the Ptch
deficient cell line Ptch-/- or the cell line B9 were generated from
skin of Ptchflox/flox and Ptchflox/floxERT2+/- mice, respectively.
In B9 cells, deletion of Ptch can be induced by tamoxifen. Similar
to stimulation by Shh, tamoxifen-induced Ptch deletion in these
cells leads to the activation of the Hh/Ptch pathway. Compared to
Ptchflox/flox cells, Ptch-/- cells display constitutive pathway
activity as well as an elevated proliferation rate. In both, B9 and
Ptch-/- cells the activated signalling cascade can be inhibited by
cyclopamine. These cell lines will be employed to improve the
current knowledge about the function of Ptch
Overexpression of mutant Ptch in rhabdomyosarcomas is associated with promoter hypomethylation and increased Gli1 and H3K4me3 occupancy.
No full textMice with heterozygous loss of the tumor suppressor Patched1 (Ptch) develop rhabdomyosarcoma (RMS)-like tumors. However, Ptch transcripts are consistently overexpressed in these tumors. We have recently shown that the upregulated transcripts are derived from the mutated Ptch allele thus leading to the hypothesis that the wild-type allele is repressed during RMS development. Here we describe epigenetic changes taking place at the Ptch locus during RMS development. We showed a lower degree of DNA-methylation in methylation-sensitive CpG regions of the Ptch promoter in RMS compared to normal muscle from heterozygous Ptch animals. In agreement with these results, treatment of heterozygous Ptch mice with the DNA demethylating agent 5-aza-2-deoxycytidine (5-aza-dC) between embryonic days E9.5-E11.5 significantly accelerated RMS formation. Since Ptch promoter methylation occurs after/around E13.5, the window for RMS initiation during embryogenesis, these results provide additional evidence that Ptch promoter hypomethylation may contribute to RMS formation. We have also demonstrated increased trimethylation of histone H3 lysine 4 (H3K4me3) and preferential binding of Gli1, a known Ptch activator, to the mutant locus in RMS. Together, these findings support an alternative model for RMS formation in heterozygous Ptch mice including loss of methylation and concomitant occupancy by activating histone marks of mutant Ptch
Context-dependent modulation of aggressiveness of pediatric tumors by individual oncogenic RAS isoforms
No full textAbstract A prototypic pediatric cancer that frequently shows activation of RAS signaling is embryonal rhabdomyosarcoma (ERMS). ERMS also show aberrant Hedgehog (HH)/GLI signaling activity and can be driven by germline mutations in this pathway. We show, that in ERMS cell lines derived from sporadic tumors i.e. from tumors not caused by an inherited genetic variant, HH/GLI signaling plays a subordinate role, because oncogenic mutations in HRAS , KRAS , or NRAS (collectively named oncRAS) inhibit the main HH target GLI1 via the MEK/ERK-axis, but simultaneously increase proliferation and tumorigenicity. oncRAS also modulate expression of stem cell markers in an isoform- and context-dependent manner. In Hh -driven murine ERMS that are caused by a Patched mutation, oncHRAS and mainly oncKRAS accelerate tumor development, whereas oncNRAS induces a more differentiated phenotype. These features occur when the oncRAS mutations are induced at the ERMS precursor stage, but not when induced in already established tumors. Moreover, in contrast to what is seen in human cell lines, oncRAS mutations do not alter Hh signaling activity and marginally affect expression of stem cell markers. Together, all three oncRAS mutations seem to be advantageous for ERMS cell lines despite inhibition of HH signaling and isoform-specific modulation of stem cell markers. In contrast, oncRAS mutations do not inhibit Hh-signaling in Hh-driven ERMS. In this model, oncRAS mutations seem to be advantageous for specific ERMS populations that occur within a specific time window during ERMS development. In addition, this window may be different for individual oncRAS isoforms, at least in the mouse.Abstract A prototypic pediatric cancer that frequently shows activation of RAS signaling is embryonal rhabdomyosarcoma (ERMS). ERMS also show aberrant Hedgehog (HH)/GLI signaling activity and can be driven by germline mutations in this pathway. We show, that in ERMS cell lines derived from sporadic tumors i.e. from tumors not caused by an inherited genetic variant, HH/GLI signaling plays a subordinate role, because oncogenic mutations in HRAS , KRAS , or NRAS (collectively named oncRAS) inhibit the main HH target GLI1 via the MEK/ERK-axis, but simultaneously increase proliferation and tumorigenicity. oncRAS also modulate expression of stem cell markers in an isoform- and context-dependent manner. In Hh -driven murine ERMS that are caused by a Patched mutation, oncHRAS and mainly oncKRAS accelerate tumor development, whereas oncNRAS induces a more differentiated phenotype. These features occur when the oncRAS mutations are induced at the ERMS precursor stage, but not when induced in already established tumors. Moreover, in contrast to what is seen in human cell lines, oncRAS mutations do not alter Hh signaling activity and marginally affect expression of stem cell markers. Together, all three oncRAS mutations seem to be advantageous for ERMS cell lines despite inhibition of HH signaling and isoform-specific modulation of stem cell markers. In contrast, oncRAS mutations do not inhibit Hh-signaling in Hh-driven ERMS. In this model, oncRAS mutations seem to be advantageous for specific ERMS populations that occur within a specific time window during ERMS development. In addition, this window may be different for individual oncRAS isoforms, at least in the mouse
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
The role of Hedgehog signaling in Ependymoma
No full textIm Rahmen dieser Arbeit wurde die Aktivität der Hedgehog (HH) Signalkaskade in Ependymomen untersucht. Dabei erfolgte zunächst der Nachweis von Sonic Hedgehog (SHH) an Gewebeschnitten mittels immunhistochemischer Färbungen (IHC). Dafür wurden 86 Paraffinschnitte analysiert. Die meisten Ependymome wiesen eine Expression von SHH auf. Anhand dieser Untersuchungen zeigte sich die höchste SHH Expression in Grad II und III Ependymomen. Tumore der hinteren Schädelgrube wiesen ein etwas höheres SHH Expressionsniveau auf als supratentorielle und spinale Ependymome. Interessanterweise zeigte sich in Abhängigkeit der prognostisch ungünstigen Fusion des RELA-Protoonkogenes, NF-kB Untereinheit (RELA) kein Unterschied in fusionspositiven und -negativen Ependymomen. Neben Ependymomen wurde auch eine SHH Expression in Ependymzellen nachgewiesen, die als Ursprungszellen für Ependymome gelten. Darüber hinaus wurde die Aktivität des Signalweges anhand von quantitativer real time-PCR (qRT-PCR) von Zielgenen an RNA untersucht, die aus 25 tiefgefrorenen Gewebeproben von Ependymomen isoliert wurde. So konnten eine erhöhte Expression des glioma-associated oncogene (GLI) - GLI1 und eine verringerte Expression von HHIP im Vergleich zu normalem Kontrollgewebe gezeigt werden. Zusammengefasst weisen diese Ergebnisse auf eine aktive HH Signalkaskade in Ependymomen hin. Schließlich konnte die Expression von GLI1 Transkripten auch exemplarisch an Gewebeschnitten von Ependymomen mittels RNAscope nachgewiesen werden. Des Weiteren wurde in dieser Arbeit mittels IHC an Paraffinschnitten erstmalig gezeigt, dass SHH in Astroblastomen, Pineozytomen und Pineoblastomen exprimiert wird. Ebenso ließ sich SHH in Glioblastomen, Astrozytomen und Neurozytomen nachweisen. Zusammengefasst stellen diese Daten eine wichtige Grundlage für weiterführende Analysen im Hinblick auf mögliche neue Therapieoptionen von Ependymomen dar. Eine Vielzahl von verschiedenen Inhibitoren der HH Signalkaskade, die an unterschiedlichen Zielmolekülen ihre Wirkung entfalten, wird bereits in der Therapie anderer Tumore verwendet. Somit könnten auf der Grundlage dieser Arbeit in der Zukunft alternative Behandlungsoptionen überprüft und umgesetzt werden.In this thesis the activity of Hedgehog (HH) signaling in ependymoma was analyzed. To this end, immunohistochemical stainings were performed on 86 paraffin sections to examine the expression of Sonic Hedgehog (SHH). The results demonstrated that the majority of ependymomas exhibited positive staining for SHH. The highest levels of expression were observed in grade II and III ependymoma. A slightly higher expression level was observed in tumors located in the posterior fossa compared to those in supratentorial or spinal regions. Notably, tumors exhibiting the unfavorable fusion of the protooncogene RELA, which is a subunit of NF-kB, demonstrated no significant variation in SHH expression when compared to fusion-negative ependymoma.
Ependymal cells, regarded as the cells of origin for ependymoma, also exhibited positive staining for SHH.
Furthermore, the activity of HH signaling was analyzed by means of quantitative real-time PCR (qRT-PCR) using RNA isolated from 25 frozen tissue samples of ependymoma. These analyses revealed an increased expression of the glioma-associated oncogene (GLI) and reduced expression of hedgehog interacting protein (HHIP) compared to control tissue without tumor.
The collective findings of these analyses suggest the presence of active HH signaling in ependymoma.
Finally, GLI1 transcripts were detected in a few representative paraffin sections by RNAscope.
Furthermore, this study provides the initial documentation of positivity for SHH by immunohistochemistry in astroblastoma, pineocytoma, and pineoblastoma. Glioblastoma, astrocytoma, and neurocytoma also demonstrate SHH expression.
In summary, these findings provide a significant foundation for further analyses, particularly regarding the exploration of novel therapeutic approaches for ependymoma. Several different inhibitors of HH signaling that interact at various levels of the signaling cascade are already used for treatment of other tumor entities. Consequently, this study provides a foundation for the future investigation and potential implementation of alternative therapeutic options that target HH signaling in ependymoma.2025-04-1
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
DMBA/TPA Treatment Is Necessary for BCC Formation from Patched Deficient Epidermal Cells in Ptchflox/floxCD4Cre+/− Mice
No full textThe development of basal cell carcinoma (BCC), the most frequently diagnosed tumor among persons with European ancestry, is closely linked to mutations in the Hedgehog (Hh) receptor and tumor suppressor Patched1 (Ptch). Using Ptchflox/floxCD4Cre+/− mice, in which Ptch was ablated in CD4Cre-expressing cells, we demonstrate that the targeted cells can give rise to BCC after treatment with DMBA (7,12-dimethylbenz(a)anthracene)/TPA (12-O-tetradecanoylphorbol-13-acetate), but not after wounding of the skin. In addition, in this model, BCC are not caused by malfunctioning of Ptch-deficient T cells, as BCC did not develop when bone marrow (BM) of Ptchflox/floxCD4Cre+/− mice was transplanted into Ptch wild-type mice. Instead, lineage-tracing experiments and flow cytometric analyses suggest that the tumors are initiated from rare Ptch-deficient stem cell-like cells of the epidermis that express CD4. As DMBA/TPA is a prerequisite for BCC development in this model, the initiated cells need a second stimulus for expansion and tumor formation. However, in contrast to papilloma, this stimulus seems to be unrelated to alterations in the Ras signaling cascade. Together, these data suggest that biallelic loss of Ptch in CD4+ cells does not suffice for BCC formation and that BCC formation requires a second so far unknown event, at least in the Ptchflox/floxCD4Cre+/− BCC mouse model
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