EWI-2 regulates melanoma growth and lung metastasis in a TGF-β1-dependent manner
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE47978 | GEO | 2013/06/15
SECONDARY ACCESSION(S): PRJNA208517
REPOSITORIES: GEO
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Project description:EWI-2 (IgSF8) plays a novel, bifunctional role in melanoma cells. EWI-2 inhibits migration, metastasis, EMT-like changes, and CD271-dependent invasion in multiple melanoma cell lines. On the other hand, EWI-2 supports melanoma cell proliferation, survival, and xenograft growth. Consistent with these results, EWI-2 levels were elevated in human malignant melanoma, but not in metastatic melanoma samples. Altered melanoma cell functions, caused by EWI-2 ablation, are almost entirely dependent on enhanced TRF-M-NM-21 signaling, and also require contributions from tetraspanin proteins CD9 and CD81. In melanoma cells lacking EWI-2, tetraspanins CD9 and CD81 enhance TRF-M-NM-21 signaling by facilitating TM-NM-2R-2M-bM-^HM-^RTM-NM-2R-1 receptor complex formation. When EWI-2 is present, CD9 and CD81 are diverted into EWI-2 complexes, and thus TRF-M-NM-21 signaling is inhibited. 4 samples = 2 Control + 2 EWI-2 KD.
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Project description:Background: 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. Results: Here we identified the nerve growth factor receptor (CD271) as a crucial determinant of melanoma cell tumorigenicity, stem-like properties, heterogeneity and plasticity. 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 found CD271 linked to the neural crest specifier SOX10 and observed a shared set of 271 differentially regulated genes. To dissect the connection of CD271 and CD133 we analyzed 10 patient-derived melanoma-cell lines for cell-surface expression of both markers compared to established cell lines MeWo and A375. We found CD271+ cells in the majority of cell lines analyzed as well as in a set of 16 different patient-derived melanoma metastases. Strikingly, only 2/12 cell lines 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 in vitro deduced from CD271+ but not CD271 knock-down cells. Conclusions: 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 CD271 is a crucial determinant of stem-like properties of melanoma cells like colony-formation and tumorigenicity. For knock-down of CD271 (NGFR), melanoma cells were transfected with 2 M-BM-5g of each shRNA plasmid; #2: 5M-bM-^@M-^Y-ACAACCTCATCCCTGTCTATT-3M-bM-^@M-^Y; #3: 5M-bM-^@M-^Y-CCCGAGCACATAGA CTCCTTT-3M-bM-^@M-^Y and #4: 5M-bM-^@M-^Y-CCGAGCACATAGACTCCTTTA-3M-bM-^@M-^Y or control shRNA (shCtl 5M-bM-^@M-^Y-GGAATCTCATTCGATGCATAC-3M-bM-^@M-^Y; all from Quiagen) using Lipofectamine2000 (Invitrogen). Cells were selected with puromycin (10M-BM-5g/ ml) over a period of two weeks. Whole genome expression profiling of CD271k.d. cells (shRNA#4) and shCtl. cells was performed with three biological replicates. Illumina raw data of BeadChip HumanHT-12V4 platform were summarized via the BeadStudio without normalization and background correction. Follow-up processing was done via the R/Bioconductor environment employing packages lumi, limma and q-value. Data were normalized with quantile normalization. Genes were termed significantly differentially expressed when the average detection p-value of at least one case was < 0.05 the ratio was outside the interval [0.75,1.33], one of the p-values from limma test, Student's t-test, Welch test and Wilcoxon test was < 0.05. At least one of the q-values corresponding to one of these tests < 0.05.
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