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CD271 is a molecular switch with divergent roles in melanoma and melanocyte development

ABSTRACT: Dysregulation of signaling networks controlling self-renewal and migration of developmental cell lineages is closely linked to the proliferative and invasive properties of tumors. Identification of such signaling pathways and their critical regulators is vital for successful design of effective targeted therapies against neoplastic tissue growth. The neurotrophin receptor (CD271/NGFR/p75/NTR) is a key regulator of the melanocytic cell lineage through its ability to mediate cell growth, survival, and differentiation. Using clinical melanoma samples, normal melanocytes and global gene expression profiling we have investigated the role of CD271 in rewiring signal transduction networks of melanoma cells during neoplastic transformation. Our analysis demonstrates that depending on the cell fate, tumor initiation vs normal development, elevated levels of CD271 can serve as a switch between proliferation/survival and differentiation/cell death. Two divergent arms of neurotrophin signaling hold the balance between positive regulators of proliferation, selfrenewal, and survival controlled by E2F, MYC, SREBP1 and AKT3 pathways on the one hand, and differentiation, senescence, and apoptosis controlled by TRAF6/IRAK-dependent activation of AP1 and TP53 mediated processes on the other hand. A molecular network map revealed in this study uncovers CD271 as a context-specific molecular switch between normal development and malignant transformation. https://doi.org/10.1038/s41598-019-42773-y http://orcid.org/0000-0001-9889-5727

ORGANISM(S): Homo sapiens

PROVIDER: GSE130244 | GEO | 2019/06/10

REPOSITORIES: GEO

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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.

Project description:Background: Cutaneous squamous cell carcinoma (cSCC) is the second most prevalent form of skin cancer, showing a rapid increasing incidence worldwide. Although most cSCC can be cured by surgery, a sizeable number of cases are diagnosed at advanced stages, with local invasion and distant metastatic lesions. In the skin, neurotrophins (NTs) and their receptors (CD271 and Trk) form a complex network regulating epidermal homeostasis. Recently, several works suggested a significant implication of NT receptors in cancer. However, CD271 functions in epithelial tumors are controversial and its precise role in cSCC is still to be defined. Methods: Spheroids from cSCC patients with low-risk (In situ or Well-Differentiated cSCC) or high-risk tumors (Moderately/Poorly Differentiated cSCC), were established to explore histological features, proliferation, invasion abilities, and molecular pathways modulated in response to CD271 overexpression or activation in vitro. The effect of CD271 activities on the response to therapeutics was also investigated. The impact on the metastatic process and inflammation was explored in vivo and in vitro, by using zebrafish xenograft and 2D/3D models. Results: Our data proved that CD271 is upregulated in Well-Differentiated tumors as compared to the more aggressive Moderately/Poorly Differentiated cSCC, both in vivo and in vitro. We demonstrated that CD271 activities reduce proliferation and malignancy marker expression in patient-derived cSCC spheroids at each tumor grade, by increasing neoplastic cell differentiation. CD271 overexpression significantly increases cSCC spheroid mass density, while it reduces their weight and diameter, and promotes a major fold-enrichment in differentiation and keratinization genes. Moreover, both CD271 overexpression and activation decrease cSCC cell invasiveness in vitro. A significant inhibition of the metastatic process by CD271 was observed in a newly established zebrafish cSCC model. We found that the recruitment of leucocytes by CD271-overexpressing cells directly correlates with tumor killing and this finding was further highlighted by monocyte infiltration in a THP-1-SCC13 3D model. Finally, CD271 activity synergizes with Trk receptor inhibition, by reducing spheroid viability, and significantly improves the outcome of photodynamic therapy (PTD) or chemotherapy in spheroids and zebrafish. Conclusion: Our study provides evidence that CD271 could prevent the switch between low to high-risk cSCC tumors. Because CD271 contributes to maintaining active differentiative paths and favors the response to therapies, it might be a promising target for future pharmaceutical development.

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CD271 is a molecular switch with divergent roles in melanoma and melanocyte development

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