Die Untersuchung der Funktion von PTK7 in der Zellmigration

Zellmigration ist ein Prozess der sowohl während der Embryonalentwicklung als auch im adulten Organismus eine wichtige Rolle spielt. Während der Embryonalentwicklung findet Zellmigration bei der Gastrulation, der konvergenten Extension sowie der Neuralleistenzellmigration statt. PTK7 (Proteinthyrosinkinase 7) reguliert embryonale Zellmigration. Die Expression von PTK7 in verschiedenen Tumorzelllinien korreliert mit dem metastatischen Potential dieser Zellen und gibt dadurch Hinweise, dass PTK7 bei der Tumorzellmigration eine Rolle spielen könnte. In Vertebraten ist PTK7 für Zellbewegungsprozesse wie den Schluss des Neuralrohrs oder der Neuralleistenzellwanderung verantwortlich. Da das humane Ortholog von PTK7, Colon Carcinoma Kinase 4 (CCK4), in Darmkrebszellen identifiziert wurde, stellt sich die Frage ob PTK7 die Migration und Invasion von Tumorzellen beeinflussen kann. Um die Funktion von PTK7 im Zusammenhang mit Tumorzellmigration und Metastasierung zu untersuchen, haben wir PTK7 in der gering invasiven Brustkrebszelllinie MCF7 induzierbar exprimiert. Die Analyse dieser Zellen hat ergeben, dass die Überexpression von PTK7 zu einer Zunahme von Filopodia-artigen Zellausläufern führt, sowie zu einer gesteigerten Invasivität von MCF7 Zellen. Da auch für die Überexpression von Ror2 ein sehr ähnlicher Filopodien-Phenotyp beschrieben wurde, wurde die Interaktion von PTK7 und Ror2 in vitro und in vivo in Xenopus laevis Neuralleistenzellen untersucht. In Immunpräzipitationsanalysen konnten wir eine Interaktion von PTK7 und Ror2 zeigen, die auch in der Xenopus laevis Neuralleistenzellmigration konserviert ist. Weiterhin konnten wir, wie bereits für Xenopus PTK7 und Drosophila otk gezeigt wurde, in humanen Zelllinien bestätigen, dass PTK7 an Wnt-Proteine bindet und hier als Wnt Co-Rezeptor fungiert um Wnt3a zu binden und den kanonischen Wnt Signalweg zu inhibieren

A PTK7/Ror2 Co-Receptor Complex Affects Xenopus Neural Crest Migration.

Neural crest cells are a highly migratory pluripotent cell population that generates a wide array of different cell types and failure in their migration can result in severe birth defects and malformation syndromes. Neural crest migration is controlled by various means including chemotaxis, repellent guidance cues and cell-cell interaction. Non-canonical Wnt PCP (planar cell polarity) signaling has previously been shown to control cell-contact mediated neural crest cell guidance. PTK7 (protein tyrosine kinase 7) is a transmembrane pseudokinase and a known regulator of Wnt/PCP signaling, which is expressed in Xenopus neural crest cells and required for their migration. PTK7 functions as a Wnt co-receptor; however, it remains unclear by which means PTK7 affects neural crest migration. Expressing fluorescently labeled proteins in Xenopus neural crest cells we find that PTK7 co-localizes with the Ror2 Wnt-receptor. Further, co-immunoprecipitation experiments demonstrate that PTK7 interacts with Ror2. The PTK7/Ror2 interaction is likely relevant for neural crest migration, because Ror2 expression can rescue the PTK7 loss of function migration defect. Live cell imaging of explanted neural crest cells shows that PTK7 loss of function affects the formation of cell protrusions as well as cell motility. Co-expression of Ror2 can rescue these defects. In vivo analysis demonstrates that a kinase dead Ror2 mutant cannot rescue PTK7 loss of function. Thus, our data suggest that Ror2 can substitute for PTK7 and that the signaling function of its kinase domain is required for this effect

PTK7 and Ror2 co-localize in NC cells and co-precipitate independent of the kinase-homology domain of PTK7.

<p><b>A</b> Co-localization of PTK7 and Ror2. NC cells co-expressing Ror2-EGFP and PTK7-RFP show distinct areas of co-localization (yellow) as well as membrane areas where only PTK7-RFP (red) or Ror2-EGFP (green) is localized. The right panel shows a higher magnification of the single cell in the left panel (indicated by a dashed square), scale bar = 10 μm. <b>B,C</b> Co-immunoprecipitation of PTK7 and Ror2. Full-length myc-tagged PTK7 (PTK7-MT) and a myc-tagged PTK7 deletion construct lacking the kinase homology domain (∆kPTK7-MT) as well as a full-length HA-tagged Ror2 construct were expressed in MCF7 cells. <b>B</b> Constructs and their protein domains are depicted in the top panel. Abbreviations are as follows: IG (immunoglobulin domain), CRD (cysteine-rich domain), K (kringle domain), TM (transmembrane domain), KH (kinase homology domain), TK (tyrosine kinase), S/T (serine/threonine-rich domain), P (proline-rich domain). <b>C</b> Immunoprecipitation experiment; the cell transfection scheme is indicated at the top. Co-immunoprecipitation was carried out using either anti-myc (IP <b>α</b>-MT, upper panel) or anti-HA antibodies (IP <b>α</b>-HA, middle panel). The respective cell lysates are shown in the bottom panel. Antibodies used for Western blotting and molecular weights are indicated at the right.</p

The kinase domain of Ror2 is required to rescue the NC migration defect in PTK7 morphant embryos.

<p><i>Xenopus</i> embryos were injected with different constructs in combination with 100 pg <i>LacZ</i> RNA as a lineage tracer and analyzed by whole-mount <i>in situ</i> hybridization using a <i>twist</i> antisense RNA probe. <b>A</b> Embryo injected with 10 ng control MO and 100 pg <i>GFP</i> RNA shows normal NC migration. <b>B</b> Embryo injected with 10 ng PTK7 MO and 100 pg <i>GFP</i> RNA shows inhibition of NC migration on the injected side, while NC migration is normal on the uninjected side. <b>C</b> Co-injection of 10 ng PTK7 MO together with 100 pg <i>Ror2</i> RNA rescues the NC migration defect. <b>D</b> Embryo injected with 10 ng PTK7 MO and 100 pg of <i>Ror2Δ469</i> RNA. The embryo shows a NC migration defect on the injected side. <b>E</b> Embryo injected with PTK7 MO and a kinase dead mutant of Ror2 (Ror2-3I) showing a NC migration defect on the injected side. <b>F</b> Graph summarizing the percentage of NC migration defects of a minimum of 5 independent experiments for each experimental condition. Asterisks indicates a p-value in a Student’s t-test < 0.001. Scale bar = 500 μm.</p

Loss of function of PTK7 affects NC cell shape and inhibits migration of explanted NC.

<p><b>A</b> Time series showing explanted NC cells injected with 7.5 ng control or PTK7 MO in combination with 50 pg <i>mGFP</i> RNA and 250 pg <i>H2B-mcherry</i>. Cranial NC explants were excised at stage 16–17 and explanted on a fibronectin matrix and incubated until they had stably adhered to the matrix. NC migration was monitored for 5 hours using spinning disk microscopy (10x objective NA 0.45). Images for representative explants injected either with control or PTK7 MO are shown at the start of the experiment (0 h) or after 1, 3 or 5 hours; scale bar = 50 μm. <b>B</b> Cell tracking and Delaunay triangulation for explants injected with 7.5 ng co MO. The upper panel shows a single frame of the spinning disk movie and the lower panel the Delaunay triangulation at the start of the experiment (0 h) or after 5 hours. Cells were tracked over the whole five-hour time interval using the H2B staining of single nuclei. These tracks are shown for single cells as differently colored lines in the images taken after 5 hours, scale bar = 50 μm. <b>C</b> Cell tracking and Delaunay triangulation for explants injected with 7.5 ng PTK7 MO. <b>D</b> Time series showing explants injected with 7.5 ng co MO (upper panel) or 7.5 ng PTK7 MO (lower panel) at a higher magnification. Injected NC cells were explanted at stage 17, cultured for 1.5 hours and imaged with a 63x objective (NA 1.4); scale bar = 20 μm. Images are shown at the start of the experiment (0 min) and after 45 and 90 minutes.</p

Ror2 rescues the PTK7 loss of function phenotype in explanted NC cells.

<p><b>A</b> NC explants injected with 7.5 ng MO in combination with 50 pg <i>mGFP</i> RNA, 250 pg <i>H2B-mcherry</i> and 150 pg <i>Ror2</i> RNA. Time-lapse images (upper panel) and Delaunay triangulations (lower panel) at the start of the experiment (0 h) and after 4 or 8 hours are shown for the different conditions. <b>B</b> Graph summarizing percentage of migration defects of 3 independent experiments (total of 39 explants). Standard error of the means are shown. Asterisks indicates a p-value in a Student’s t-test < 0.05. Scale bars = 200 μm.</p

The intracellular domain and the CRD domain of Ror2 are not required for PTK7/Ror2 interaction.

<p>Myc-tagged PTK7 (PTK7-MT) together with FLAG- or HA-tagged Ror2 deletions were expressed in MCF7 cells. <b>A</b> The respective constructs and their protein domains are depicted in the top panel. <b>B</b> Immunoprecipitation using anti-FLAG antibodies; the cell transfection scheme is indicated at the top. Co-precipitated PTK7 was detected using anti-myc antibodies. Immunoprecipitated PTK7 is shown in the top panel, immunoprecipitated Ror2 constructs in the middle panel and cell lysates in the bottom panel. Antibodies used for Western blotting and molecular weights are indicated at the right. <b>C</b> Immunoprecipitation using anti-myc antibodies; the cell transfection scheme is indicated at the top. Co-precipitated full-length Ror2 and sRor2 were detected using anti-HA antibodies (top panel). Immunoprecipitated PTK7 is shown in the middle panel and cell lysates in the bottom panel. Antibodies used for Western blotting and molecular weights are indicated at the right.</p

FLT3 internal tandem duplication in 234 children with acute myeloid leukemia: Prognostic significance and relation to cellular drug resistance

FLT3 is a receptor tyrosine kinase involved in the proliferation and differentiation of hematopoietic stem cells. FLT3 internal tandem duplications (FLT3/ITDs) are reported in acute myeloid leukemia (AML) and predict poor clinical outcome. We found FLT3/ITDs in 11.5% of 234 children with de novo AML. FLT3/ITD-positive patients were significantly older and had higher percentages of normal cytogenetic findings or French-American-British (FAB) classification M1/M2 and lower percentages of 11q23 abnormalities or FAB MS. FLT3/ITD-positive patients had lower remission induction rates (70% vs 88%; P = .01) and lower 5-year probability rates of event-free survival (pEF) (29% vs 46%; P = .0046) and overall survival (32% vs 58%; P = .037). Patients with high ratios (higher than the median) between mutant and wild-type FLT3 had significantly worse 2-year EFS rates than FLT3/ITD-negative patients (pEFS 20% vs 61%, P = .037), whereas patients with ratios lower than the median did not (pEFS 44% vs 61%; P = .26). FLT3/ITD was the strongest independent predictor for pEFS, with an increase in relative risk for an event of 1.92 (P = .01). Using an MTT (methyl-thiazol-tetrazolium)-based assay, we studied cellular drug resistance on 15 FLT3/ITD-positive and 125 FLT3/ITD-negative AML samples, but we found no differences in cellular drug resistance that could explain the, poor outcomes in FLT3/ITD-positive patients, We conclude that FLT3/ITD is less common in pediatric than in adult AML. FLT3/ITD is a strong and independent adverse prognostic factor, and high ratios between mutant and WT-FLT3 further compromise prognosis. However, poor outcomes in FLT3/ITD-positive patients could not be attributed to increased in vitro cellular drug resistance