21 research outputs found
CD271 determines migratory properties of melanoma cells
Melanoma cell expression of the nerve growth factor receptor CD271 is
associated with stem-like properties. However, the contributing role of the
receptor in melanoma cell migration is elusive. Here, we explored extracranial
(skin, soft tissue, lymph node and liver, n = 13) and matched brain metastases
(BM, n = 12) and observed a heterogeneous distribution of phenotypically
distinct subsets of CD271+ cells. In addition, we observed that CD271
expression gradually rises along with melanoma progression and metastasis by
exploration of publicly available expression data of nevi, primary melanoma (n
= 31) and melanoma metastases (n = 54). Furthermore, we observed highest
levels of CD271 in BM. Sub-clustering identified 99 genes differentially
expressed among CD271high and CD271low (p < 0.05) BM-subgroups. Comparative
analysis of subsets revealed increased ( ≥ 1.5fold, log2) expression of
migration-associated genes and enrichment of CD271-responsible genes involved
in DNA-repair and stemness. Live cell-imaging based scratch-wound assays of
melanoma cells with stable knock-down of CD271 revealed a significantly
reduced cell migration (3.9fold, p = 1.2E-04) and a reduced expression of
FGF13, CSPG4, HMGA2 and AKT3 major candidate regulatory genes of melanoma cell
migration. In summary, we provide new insights in melanoma cell migration and
suggest that CD271 serves as a candidate regulator, sufficient to determine
cellular properties of melanoma brain metastatic cells
Effects of RAL signal transduction in KRAS- and BRAF-mutated cells and prognostic potential of the RAL signature in colorectal cancer
Our understanding of oncogenic signaling pathways has strongly fostered current concepts for targeted therapies in metastatic colorectal cancer. The RALA pathway is novel candidate due to its independent role in controlling expression of genes downstream of RAS. We compared RALA GTPase activities in three colorectal cancer cell lines by GTPase pull-down assay and analyzed the transcriptional and phenotypic effects of transient RALA silencing. Knocking-down RALA expression strongly diminished the active GTP-bound form of the protein. Proliferation of KRAS mutated cell lines was significantly reduced, while BRAF mutated cells were mostly unaffected. By microarray analysis we identified common genes showing altered expression upon RALA silencing in all cell lines. None of these genes were affected when the RAF/MAPK or PI3K pathways were blocked. To investigate the potential clinical relevance of the RALA pathway and its associated transcriptome, we performed a meta-analysis interrogating progression-free survival of colorectal cancer patients of five independent data sets using Cox regression. In each dataset, the RALA-responsive signature correlated with worse outcome. In summary, we uncovered the impact of the RAL signal transduction on genetic program and growth control in KRAS- and BRAF-mutated colorectal cells and demonstrated prognostic potential of the pathway-responsive gene signature in cancer patients
Deciphering mechanisms of brain metastasis in melanoma - the gist of the matter
Abstract Metastasis to distant organs and particularly the brain still represents the most serious obstacle in melanoma therapies. Melanoma cells acquire a phenotype to metastasize to the brain and successfully grow there through complex mechanisms determined by microenvironmental than rather genetic cues. There do appear to be some prerequisites, including the presence of oncogenic BRAF or NRAS mutations and a loss of PTEN. Further mediators of the brain metastatic phenotype appear to be the high activation of the PI3K/AKT or STAT3 pathway or high levels of PLEKHA5 and MMP2 in metastatic cells. A yet undefined subset of brain metastases exhibit a high level of expression of CD271 that is associated with stemness, migration and survival. Hence, CD271 expression may determine specific properties of brain metastatic melanoma cells. Environmental cues – in particular those provided by brain parenchymal cells such as astrocytes - seem to help specifically guide melanoma cells that express CCR4 or CD271, potential “homing receptors”. Upon entering the brain, these cells interact with brain parenchyma cells and are thereby reprogrammed to adopt a neurological phenotype. Several lines of evidence suggest that current therapies may have a negative effect by activating a program that drives tumor cells toward stemness and metastasis. Yet significant improvements have expanded the therapeutic options for treating brain metastases from melanoma, by combining potent BRAF inhibitors such as dabrafenib with checkpoint inhibitors or stereotactic surgery. Further progress toward developing new therapeutic strategies will require a more profound understanding of the mechanisms that underlie brain metastasis in melanoma
Deciphering the role of E-cadherin in pluripotency and reprogramming
1\. Inhalt 2\. AbkĂĽrzungsverzeichnis 6 3\. Zusammenfassung 7 4\. Einleitung 10
4.1 Zelladhäsion und Zelladhäsionsmoleküle 10 4.2 Die Cadherin-Superfamilie 11
4.3 E-Cadherin, der erste Vertreter der Cadherine 15 4.3.1 Molekulare
Grundlagen der E-Cadherin-vermittelten Zelladhäsion 17 4.3.2 Transkriptionelle
und Posttranslationale Regulationen der E-Cadherin Expression 21 4.3.3
E-Cadherin während der frühen Embryogenese und Organogenese 25 4.4 Embryonale
Stammzellen (ES-Zellen) 27 4.4.1 Isolierung Embryonaler Stammzellen 28 4.5 Das
transkriptionelle Netzwerk in murinen embryonalen Stammzellen 29 4.5.1 ES-
Zellen und Epigenetik 33 4.6 Induzierte Pluripotente Stammzellen 35 4.6.1 iPS-
Induktion nach Takahashi und Yamanaka 35 4.7 Aufklärung von Signalwegen und
Mechanismen während der iPS-Induktion 36 5\. Materialien und Methoden 40 5.1
Zellkultur 40 5.1.1 Kultivierung von mES- und iPS-Zellen 40 5.1.2 Isolierung
und Kultivierung von murinen embryonalen Fibroblasten (MEF) 40 5.1.3
Kultivierung von Platinum-E (Plat-E) Zellen, Virusproduktion und Transduktion
von Zellen 41 5.1.4 Gewinnung von iPS Zellen 42 5.2 RNA-Isolierung und cDNA
Synthese aus Zellen und Tumorgewebe 42 5.2.1 cDNA Synthese 43 5.2.2
Quantitative real-time RT-PCR (qPCR) und RT-PCR 44 5.3
Proteinexpressionsanalysen 46 5.3.1 Immunfluoreszenz und Immunhistochemie 46
5.3.2 Fluoreszenz-aktivierte Zell-Sortierung (FACS) und Durchflusszytometrie
47 5.3.3 Western-Blot Analyse 48 5.4 In vitro Differenzierung von iPS Zellen
(Embryoid Body Formation) 48 5.5 In vivo Differenzierung 49 5.5.1
Teratokarzinom-Bildung 49 5.5.2 Blastozysten-Injektionen 49 5.6
Expressionsverlust und Ăśberexpression von E-Cadherin 50 5.6.1 E-Cadherin shRNA
und Expressionsplasmid 50 5.6.2 HTNcre/loxP vermittelte Reduktion des
E-Cadherin und b-Catenin Levels 50 5.7 Alkalische Phosphatase Aktivität 50 6\.
Ergebnisse 51 6.1 Die Expression von E-Cadherin in mES-Zellen ist essentiell
fĂĽr die Erhaltung der Pluripotenz 51 6.2 Der Verlust von E-Cadherin fĂĽhrt zur
Reduktion des Proteinlevels von b-Catenin und p120 Catenin aber nicht zu einer
Kernlokalisierung 53 6.3 Die Expression von E-Cadherin und SSEA-1
klassifiziert induzierte pluripotente Zellen 54 6.4 Hepatozyten-iPS (Hep-iPS)
können direkt, ohne Selektion generiert werden 58 6.5 Die Menge an E-Cadherin/
SSEA-1 positiven Zellen in OSKM Kolonien ist gering 60 6.6 Der
E-Cadherin/b-Catenin Komplex reguliert die Differenzierung 61 6.7 Ecadhigh iPS
Zellen differenzieren in vivo unter Bildung von Teratokarzinomen 63 6.8
Ecadhigh Zellen integrieren in die Blastozyste und partizipieren an der
Embryogenese 65 6.9 Die Abwesenheit von E-Cadherin und b-Catenin fĂĽhrt zu
einer ineffizienten Reprogrammierung von Fibroblasten 66 6.10 Die
Überexpression von E-Cadherin als zusätzlicher Faktor führt nicht zu einer
Erhöhung der Effizienz der Reprogrammierung 69 6.11 E-Cadherin Überexpression
fĂĽhrt zur Bildung von stabilen iPS Zellen bei Abwesenheit von exogenem Oct4 70
6.12 ESKM Zellen besitzen alle Eigenschaften vollständig reprogrammierter iPS
Zellen 72 6.13 Die in vivo Differenzierung von ESKM Zellen resultiert in der
Bildung differenzierter Tumore 74 6.14 H3K4me3 und H3K27me3 Methylierungen
sind präsent im Chromatin vollständig reprogrammierter iPS Zellen aber
abwesend in partiell reprogrammierten Zellen 76 7\. Diskussion 78 8\.
Literaturverzeichnis 95 9\. Abbildungsverzeichnis 105 10\. Anhang I 106 11\.
Anhang II 107 12\. Danksagung 108 13\. Lebenslauf 109Die Prozesse der frühen Embryogenese sind abhängig von der streng
kontrollierten Expression von Zelladhäsionsmolekülen. Besonders die Expression
des zur Familie der Cadherine gehörenden Moleküls E-Cadherin ist entscheidend
fĂĽr die Bildung der kompakten Morula, ein frĂĽhes Stadium der
Embryonalentwicklung, aus der sich die Blastozyste bildet. Die Blastozyste
enthält die innere Zellmasse (ICM), eine Ansammlung pluripotenter Zellen, die
nicht nur alle Zellen des späteren Embryos bilden, sondern, aus denen in vitro
auch embryonale Stammzellen (ES-Zellen) abgeleitet werden können. Embryonale
Stammzellen stellen ein in vitro Modellsystem dar, dass Einblicke in diese
frühen Differenzierungsprozesse ermöglicht und, wie die Zellen der ICM, einen
hohen Expressionslevel von E-Cadherin sowie der Pluripotenzmarker Oct4 und
Nanog zeigen. In der vorliegenden Arbeit wurde die Funktion von E-Cadherin in
murinen embryonalen Stammzellen (mES-Zellen) und im Prozess der
Reprogrammierung somatischer Zellen untersucht. Dabei konnte gezeigt werden,
dass E-Cadherin nicht nur fĂĽr die Morphologie von mES-Zellen essentiell ist.
Eine starke Reduktion des E-Cadherin Levels in mES-Zellen fĂĽhrte zu deren
Differenzierung sowie zu einer damit verbundenen Reduktion der Expression der
Pluripotenzgene Oct4 und Nanog. Weiterhin konnte durch die Verwendung des
Cre/loxP Systems gezeigt werden, dass die Störung der E-Cadherin bzw. beta-
Catenin Expression während der OSKM (Oct4, Sox2, Klf4, c-Myc)-induzierten
Reprogrammierung zu einer starken Reduktion der Bildung von iPS-Kolonien
fĂĽhrte. Die Etablierung des E-Cadherin/beta-Catenin Komplexes ist somit ein
entscheidender Schritt während der Konvertierung von Fibroblasten zu iPS-
Zellen. Obwohl die E-Cadherin vermittelte Zelladhäsion nicht unmittelbar mit
der Regulation von Oct4 beteiligt zu sein scheint, fĂĽhrte die Ăśberexpression
von E-Cadherin in Kombination mit SKM, bemerkenswerterweise zur Bildung
stabiler ESKM-iPS Klone. Die aus den Klonen erhaltenen iPS Zellen exprimierten
endogene Level von Oct4 und Nanog, obwohl Oct4 nicht als exogener Faktor
während der Reprogrammierung anwesend war. Weiterhin konnten die ESKM-iPS
Zellen durch die Bildung von Embryoid Bodies und Teratomen differenziert
werden und daher alle Eigenschaften von mES-Zellen. Durch diese
unkonventionell erzeugten iPS-Zellen wird deutlich, dass Pluripotenz neben der
transkriptionellen Regulation auch über die Etablierung von Zelladhäsions-
verbindungen definiert werden kann. Vor allem die E-Cadherin vermittelte
Zelladhäsion übernimmt für die Etablierung und die Erhaltung des pluripotenten
Status eine SchlĂĽsselfunktion.During early embryogenesis the expression of cell adhesion molecules and the
establishment of cell-cell contacts are crucial steps to ensure proper
development of the embryo. Especially expression of the molecule E-Cadherin, a
member of the cadherin superfamily, is crucial for the formation of the
compacted morula, comprising an early stage of embryogenesis that forms the
blastocyst. The latter bears the inner cells mass (ICM), a group of
pluripotent cells, that forms all cells of the adult body. Cells of the ICM
can be used to establish embryonic stem cells (ESCs) as an in vitro system
that allows providing insights in these early differentiation processes.
Embryonic stem cells like cells of the ICM display high expression levels of
E-Cadherin and of the pluripotency markers Oct4 and Nanog. In the present
work, the function of E-Cadherin in murine embryonic stem cells and during the
process of somatic cell reprogramming was analysed. It could been shown in a
shRNA mediated knock-down of E-Cadherin that its expression not only
determines the morphology of ESCs but is also linked to regulation of the
pluripotency genes Oct4 and Nanog. Further, cre/loxP mediated ablation of
E-cadherin and beta-catenin expression followed by the reprogramming with four
viral factors Oct4, Sox2, Klf4 and c-Myc (OSKM) led to a strong reduction in
the number of induced pluripotent stem cell (iPS-cell) colonies. Therefore,
the establishment of the E-Cadherin/beta-Catenin complex is a crucial step in
the process that governs the conversion of fibroblasts to iPS-cells. Although
the link between E-Cadherin mediated cell adhesion and the regulation of Oct4
seems to be indirect, overexpression of E-cadherin in combination with SKM
surprisingly, led to the formation of stable ESKM-iPS clones. Cells of these
clones displayed endogenous expression of Oct4 and Nanog although exogenous
Oct4 was absent during the reprogramming process. Established ESKM-iPS cells
fulfilled all criteria tested so far, to establish their pluripotent state.
ESKM-iPS cells were able to undergo embryoid body mediated differentiation and
formed teratoma. The generation of these unconventional generated iPS cells
points out that pluripotency, beside transcription factors, is also defined by
the adhesive integrity of iPS and ES-cells. Especially E-Cadherin mediated
cell adhesion plays a crucial for the establishment and maintenance of the
pluripotent state
Additional file 1: of Deciphering mechanisms of brain metastasis in melanoma - the gist of the matter
Table S1. Clinical trials. Results of clinical trials as provided by ( https://www.clinicaltrials.gov/ ) of melanoma patients with brain metastases are summarized. OS = overall survival, PFS = progression free survival, BORR = best overall response rate, assessed by IRC (independent reviewer commitee) is defined as percentage of participants who were responders [with best overall response (BOR) documented as confirmed complete response (CR) or partial response (PR)]. OIR = overall intracranial response, defined as the number of participants whose intracranial response was a confirmed complete response (CR) or partial response (PR) assessed by investigators using modified Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1. Metadata 1: provides information about the therapeutic interventions performed within the study as well as the study stage. Metadata 2: provides detailed study data particularly drug applications as well as data additional data of NCT01378975, the time to the development of new brain metastases in responders. (ZIP 25Ă‚Â kb
Tracking of Melanoma Cell Plasticity by Transcriptional Reporters
Clonal evolution and cellular plasticity are the genetic and non-genetic driving forces of tumor heterogeneity, which in turn determine tumor cell responses towards therapeutic drugs. Several lines of evidence suggest that therapeutic interventions foster the selection of drug-resistant neural crest stem-like cells (NCSCs) that establish minimal residual disease (MRD) in melanoma. Here, we establish a dual-reporter system, enabling the tracking of NGFR expression and mRNA stability and providing insights into the maintenance of NCSC states. We observed that a transcriptional reporter that contained a 1-kilobase fragment of the human NGFR promoter was activated only in a minor subset (0.72 ± 0.49%, range 0.3–1.5), and ~2–4% of A375 melanoma cells revealed stable NGFR mRNA. The combination of both reporters provides insights into phenotype switching and reveals that both cellular subsets gave rise to cellular heterogeneity. Moreover, whole transcriptome profiling and gene-set enrichment analysis (GSEA) of the minor cellular subset revealed hypoxia-associated programs that might serve as potential drivers of an in vitro switching of NGFR-associated phenotypes and relapse of post-BRAF inhibitor-treated tumors. Concordantly, we observed that the minor cellular subset increased in response to dabrafenib over time. In summary, our reporter-based approach provides insights into plasticity and identified a cellular subset that might be responsible for the establishment of MRD in melanoma
Decoding the Role of CD271 in Melanoma
The evolution of melanoma, the most aggressive type of skin cancer, is triggered by driver mutations that are acquired in the coding regions of particularly BRAF (rat fibrosarcoma serine/threonine kinase, isoform B) or NRAS (neuroblastoma-type ras sarcoma virus) in melanocytes. Although driver mutations strongly determine tumor progression, additional factors are likely required and prerequisite for melanoma formation. Melanocytes are formed during vertebrate development in a well-controlled differentiation process of multipotent neural crest stem cells (NCSCs). However, mechanisms determining the properties of melanocytes and melanoma cells are still not well understood. The nerve growth factor receptor CD271 is likewise expressed in melanocytes, melanoma cells and NCSCs and programs the maintenance of a stem-like and migratory phenotype via a comprehensive network of associated genes. Moreover, CD271 regulates phenotype switching, a process that enables the rapid and reversible conversion of proliferative into invasive or non-stem-like states into stem-like states by yet largely unknown mechanisms. Here, we summarize current findings about CD271-associated mechanisms in melanoma cells and illustrate the role of CD271 for melanoma cell migration and metastasis, phenotype-switching, resistance to therapeutic interventions, and the maintenance of an NCSC-like state
Tracking of Melanoma Cell Plasticity by Transcriptional Reporters
Clonal evolution and cellular plasticity are the genetic and non-genetic driving forces of tumor heterogeneity, which in turn determine tumor cell responses towards therapeutic drugs. Several lines of evidence suggest that therapeutic interventions foster the selection of drug-resistant neural crest stem-like cells (NCSCs) that establish minimal residual disease (MRD) in melanoma. Here, we establish a dual-reporter system, enabling the tracking of NGFR expression and mRNA stability and providing insights into the maintenance of NCSC states. We observed that a transcriptional reporter that contained a 1-kilobase fragment of the human NGFR promoter was activated only in a minor subset (0.72 ± 0.49%, range 0.3-1.5), and ~2-4% of A375 melanoma cells revealed stable NGFR mRNA. The combination of both reporters provides insights into phenotype switching and reveals that both cellular subsets gave rise to cellular heterogeneity. Moreover, whole transcriptome profiling and gene-set enrichment analysis (GSEA) of the minor cellular subset revealed hypoxia-associated programs that might serve as potential drivers of an in vitro switching of NGFR-associated phenotypes and relapse of post-BRAF inhibitor-treated tumors. Concordantly, we observed that the minor cellular subset increased in response to dabrafenib over time. In summary, our reporter-based approach provides insights into plasticity and identified a cellular subset that might be responsible for the establishment of MRD in melanoma
Tracking of Melanoma Cell Plasticity by Transcriptional Reporters
Clonal evolution and cellular plasticity are the genetic and non-genetic driving forces of tumor heterogeneity, which in turn determine tumor cell responses towards therapeutic drugs. Several lines of evidence suggest that therapeutic interventions foster the selection of drug-resistant neural crest stem-like cells (NCSCs) that establish minimal residual disease (MRD) in melanoma. Here, we establish a dual-reporter system, enabling the tracking of NGFR expression and mRNA stability and providing insights into the maintenance of NCSC states. We observed that a transcriptional reporter that contained a 1-kilobase fragment of the human NGFR promoter was activated only in a minor subset (0.72 ± 0.49%, range 0.3-1.5), and ~2-4% of A375 melanoma cells revealed stable NGFR mRNA. The combination of both reporters provides insights into phenotype switching and reveals that both cellular subsets gave rise to cellular heterogeneity. Moreover, whole transcriptome profiling and gene-set enrichment analysis (GSEA) of the minor cellular subset revealed hypoxia-associated programs that might serve as potential drivers of an in vitro switching of NGFR-associated phenotypes and relapse of post-BRAF inhibitor-treated tumors. Concordantly, we observed that the minor cellular subset increased in response to dabrafenib over time. In summary, our reporter-based approach provides insights into plasticity and identified a cellular subset that might be responsible for the establishment of MRD in melanoma
Tumor Cell Plasticity in Equine Papillomavirus-Positive Versus-Negative Squamous Cell Carcinoma of the Head and Neck
Squamous cell carcinoma of the head and neck (HNSCC) is a common malignant tumor in humans and animals. In humans, papillomavirus (PV)-induced HNSCCs have a better prognosis than papillomavirus-unrelated HNSCCs. The ability of tumor cells to switch from epithelial to mesenchymal, endothelial, or therapy-resistant stem-cell-like phenotypes promotes disease progression and metastasis. In equine HNSCC, PV-association and tumor cell phenotype switching are poorly understood. We screened 49 equine HNSCCs for equine PV (EcPV) type 2, 3 and 5 infection. Subsequently, PV-positive versus -negative lesions were analyzed for expression of selected epithelial (keratins, β-catenin), mesenchymal (vimentin), endothelial (COX-2), and stem-cell markers (CD271, CD44) by immunohistochemistry (IHC) and immunofluorescence (IF; keratins/vimentin, CD44/CD271 double-staining) to address tumor cell plasticity in relation to PV infection. Only EcPV2 PCR scored positive for 11/49 equine HNSCCs. IHC and IF from 11 EcPV2-positive and 11 EcPV2-negative tumors revealed epithelial-to-mesenchymal transition events, with vimentin-positive cells ranging between <10 and >50%. CD44- and CD271-staining disclosed the intralesional presence of infiltrative tumor cell fronts and double-positive tumor cell subsets independently of the PV infection status. Our findings are indicative of (partial) epithelial–mesenchymal transition events giving rise to hybrid epithelial/mesenchymal and stem-cell-like tumor cell phenotypes in equine HNSCCs and suggest CD44 and CD271 as potential malignancy markers that merit to be further explored in the horse