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\. Abkürzungsverzeichnis 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 Überexpression 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 für die Erhaltung der Pluripotenz 51 6.2 Der Verlust von E-Cadherin führt 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 führt 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 führt 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 für die Bildung der kompakten Morula, ein frühes 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 für die Morphologie von mES-Zellen essentiell ist. Eine starke Reduktion des E-Cadherin Levels in mES-Zellen führte 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 führte. 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, führte die Überexpression 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 Schlüsselfunktion.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 25 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

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, &beta;-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 &lt;10 and &gt;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&ndash;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

The Nerve Growth Factor Receptor CD271 Is Crucial to Maintain Tumorigenicity and Stem-Like Properties of Melanoma Cells

<div><p>Background</p><p>Large-scale genomic analyses of patient cohorts have revealed extensive heterogeneity between individual tumors, contributing to treatment failure and drug resistance. In malignant melanoma, heterogeneity is thought to arise as a consequence of the differentiation of melanoma-initiating cells that are defined by cell-surface markers like CD271 or CD133.</p><p>Results</p><p>Here we confirmed that the nerve growth factor receptor (CD271) is a crucial determinant of tumorigenicity, stem-like properties, heterogeneity and plasticity in melanoma cells. Stable shRNA mediated knock-down of CD271 in patient-derived melanoma cells abrogated their tumor-initiating and colony-forming capacity. A genome-wide expression profiling and gene-set enrichment analysis revealed novel connections of CD271 with melanoma-associated genes like CD133 and points to a neural crest stem cell (NCSC) signature lost upon CD271 knock-down. In a meta-analysis we have determined a shared set of 271 differentially regulated genes, linking CD271 to SOX10, a marker that specifies the neural crest. To dissect the connection of CD271 and CD133 we have analyzed 10 patient-derived melanoma-cell strains for cell-surface expression of both markers compared to established cell lines MeWo and A375. We found CD271⁺ cells in the majority of cell strains analyzed as well as in a set of 16 different patient-derived melanoma metastases. Strikingly, only 2/12 cell strains harbored a CD133⁺ sub-set that in addition comprised a fraction of cells of a CD271⁺/CD133⁺ phenotype. Those cells were found in the label-retaining fraction and <i>in vitro</i> deduced from CD271⁺ but not CD271 knock-down cells.</p><p>Conclusions</p><p>Our present study provides a deeper insight into the regulation of melanoma cell properties and points CD271 out as a regulator of several melanoma-associated genes. Further, our data strongly suggest that CD271 is a crucial determinant of stem-like properties of melanoma cells like colony-formation and tumorigenicity.</p></div

Expression of CD271 in melanoma cells is a crucial determinant of proliferation and tumorigenicity.

<p>(<b>A</b>) Absence of subcutaneous tumors 45 days after injection of 1×10⁶ CD271 knock-down (CD271^k.d.) cells into NSG mice. Cells were stably transfected with shRNA#4. (<b>B–C</b>) Simultaneous injection of cells stably transfected with a shRNA control plasmid (shCtl.) led to proper formation of tumors (arrows in B). Tumor growth is shown as mean values ± SD of biological triplicates. (<b>D</b>) Tumors of (B) showed expression of CD271, MITF and TYR. H&E indicates tumor histology. (<b>E</b>) Heat map of 10 significantly regulated melanoma-associated genes selected from a genome-wide expression profiling. Different splice forms of ras homolog family member J (<i>RHOJ</i>), v-Erb-B2 erythroblastic leukemia viral oncogene homolog 3 (<i>ERBB3</i>), SRY-box 2 (<i>SOX2</i>) and follistatin (<i>FST</i>) are included. Independent biological triplicates of either CD271^k.d. cells or shCtl. cells were analyzed. Up-regulated or down-regulated genes are indicated in red or blue, respectively. (<b>F</b>) Validation of genome-wide expression profiling by qPCR for the regulation of CD271, CD133, SOX10, SOX2, ERBB3, insulin-like growth factor binding protein 2 (<i>IGFBP-2</i>), GLI-family zinc finger 2 (<i>GLI-2</i>), RHOJ and forkhead-box D3 (<i>FOXD3</i>) as an additional gene in patient-derived melanoma cells stably transfected with shRNA plasmids #2, #3 or #4 (CD271^k.d.). Expression levels of shRNA control (shCtl.) cells and CD271^k.d. cells are shown as ΔΔCT values normalized to β-actin and related to shCtl. cells as mean values ± SD of biological triplicates. *p≤0.05; ***p≤0.001 (t-test). The scale is logarithmic (log). (<b>G</b>) Comparison of data sets of shCtl. and CD271^k.d. cells with data sets of human embryonic stem cell derived neural crest stem cells (NCSC) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092596#pone.0092596-Lee1" target="_blank">[50]</a> by gene-set enrichment analysis (GSEA). GSEA revealed the presence of a NCSC-specific gene signature in shCtl. cells that is lost upon CD271 silencing (non-NCSC signature). Genes found in the signature among others are CD271, <i>ERBB3</i>, <i>SOX10</i>, microphthalmia-associated transcription factor (<i>MITF</i>), snail homolog 2 (<i>SNAI2</i>) and semaphorin 3C (<i>SEMA3C</i>).</p