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Clinical Trial
. 2014 May 5;9(5):e92596.
doi: 10.1371/journal.pone.0092596. eCollection 2014.

The nerve growth factor receptor CD271 is crucial to maintain tumorigenicity and stem-like properties of melanoma cells

Torben Redmer  1 , Yvonne Welte  2 , Diana Behrens  3 , Iduna Fichtner  3 , Dorothea Przybilla  4 , Wasco Wruck  5 , Marie-Laure Yaspo  6 , Hans Lehrach  6 , Reinhold Schäfer  4 , Christian R A Regenbrecht  7
Affiliations
Clinical Trial

The nerve growth factor receptor CD271 is crucial to maintain tumorigenicity and stem-like properties of melanoma cells

Torben Redmer et al. PLoS One. .

Erratum in

  • PLoS One. 2014;9(8):e105274

Abstract

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 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 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 that CD271 is a crucial determinant of stem-like properties of melanoma cells like colony-formation and tumorigenicity.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Expression of CD271 in melanoma cells is a crucial determinant of proliferation and tumorigenicity.
(A) Absence of subcutaneous tumors 45 days after injection of 1×106 CD271 knock-down (CD271k.d.) cells into NSG mice. Cells were stably transfected with shRNA#4. (B–C) 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. (D) Tumors of (B) showed expression of CD271, MITF and TYR. H&E indicates tumor histology. (E) 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 (RHOJ), v-Erb-B2 erythroblastic leukemia viral oncogene homolog 3 (ERBB3), SRY-box 2 (SOX2) and follistatin (FST) are included. Independent biological triplicates of either CD271k.d. cells or shCtl. cells were analyzed. Up-regulated or down-regulated genes are indicated in red or blue, respectively. (F) Validation of genome-wide expression profiling by qPCR for the regulation of CD271, CD133, SOX10, SOX2, ERBB3, insulin-like growth factor binding protein 2 (IGFBP-2), GLI-family zinc finger 2 (GLI-2), RHOJ and forkhead-box D3 (FOXD3) as an additional gene in patient-derived melanoma cells stably transfected with shRNA plasmids #2, #3 or #4 (CD271k.d.). Expression levels of shRNA control (shCtl.) cells and CD271k.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). (G) Comparison of data sets of shCtl. and CD271k.d. cells with data sets of human embryonic stem cell derived neural crest stem cells (NCSC) 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, ERBB3, SOX10, microphthalmia-associated transcription factor (MITF), snail homolog 2 (SNAI2) and semaphorin 3C (SEMA3C).
Figure 2
Figure 2. CD271+ and CD133+ melanoma cells hold comparable tumor-initiating capacities.
(A) Flow cytometry of enriched CD271+ cells show a high content of CD271 expressing cells before transplantation. (B) Immunofluorescence analysis and immunohistochemistry on paraffin sections (2 µm) of tumors derived from 1×105 of either CD271+, CD133−, CD133+ or unsorted cells for CD133 (upper row) and HMB45 (center row), respectively. A representative tumor out of n = 3 is shown. H&E shows histological morphology of tumors (lower row). Haematoxylin served as counter stain for HMB45. Bar size is 50 µm. (C) Immunofluorescence analysis for CD271 and CD133 on sections of tumors described in (A) showing co-localization and discrete expression of both markers. Scale bars indicate 50 µm. Nuclei were stained with DAPI (blue). (D) Growth of tumors following injection of 1×105 of either CD271+, CD133−, CD133+ or unsorted cells was monitored for 57 days. Tumor volumes are shown as mean values ± SD of biological triplicates. Growth of 106 of CD271+ cells is not shown. (E) Comparison of mRNA expression levels of either CD271+ cells with respective xenograft tumors derived from injection of CD271+ cells at different cell numbers (105, 106; left chart) or comparison of CD133+ cells with tumors as pointed (right chart). Expression in CD133+ cells was compared to xenograft tumors shown in (B) derived from injection of 105 of CD133−, CD133+ or unsorted cells. mRNA expression levels of CD271, CD133, SOX10, MITF, MART-1 and TYR were determined by qPCR as indicated. Expression levels reveal the independence of the in vivo differentiation capacity of melanoma cells from the initial cell number or the cells phenotype. Shown are ΔCT values normalized to β-actin as mean value ± SD of biological triplicates. *p≤0.05; **p≤0.01 (t-test).
Figure 3
Figure 3. The label-retaining melanoma cell-fraction comprises CD271/CD133 double positive cells.
(A) Co-expression of CD271 and CD133 in melanoma cell cultures shown by flow cytometry. The double positive fractions are highlighted and indicated as % ± SD of biological triplicates. (B) Immunofluorescence microscopy for co-expression of CD271 and CD133 in cell cultures shows a discrete or simultaneous expression of these markers. Scale bars indicate 50 µm. Nuclei were stained with DAPI (blue). Representatives of n = 5 are shown. (C–D) Isolation of highly fluorescent cells that retained the lipophilic dye PKH26 of two melanoma cell strains (Mel9-1 and Mel4-7, left panels) by FACS, 7 days after labeling. PKH26high fractions are indicated as % ± SD of n = 3 independent FACS experiments. Confirmation of isolated, dye retaining fractions for presence of CD271+ and CD133+ cells by co-labeling and flow cytometry (right panels). Analysis shows the presence of cells with discrete and co-expression of markers. Representative plots indicate fractions as % ± SD of n = 3 independent experiments. (E) Analysis of isolated dye-retaining cells 7 days after sorting supports the co-localization of CD271 (green) and PKH26 (red, white arrows, first panel) or co-expression of CD271 (red) and CD133 (green) (panels 2–4). (F) Co-localization of CD271 (green) and PKH26 (red, white arrows, first panel) and low expression of CD133 in Mel4-7 cells. Scale bars indicate 50 µm. Nuclei were stained with DAPI (blue). Representatives of n = 3 are shown.
Figure 4
Figure 4. Asymmetrically dividing cells are CD133+ or double positive.
(A) Flow cytometry for CD271 and CD133 in CD133+ enriched cells. Results depict the presence of single and double positive cells. The experiment was carried out in independent biological triplicates, a representative example is shown. (B) Distribution of CD271 and CD133 in symmetrically and asymmetrically dividing CD133+ cells detected by immunofluorescence microscopy for both markers. (C) Asymmetric cell division in unsorted cells is illustrated by the asymmetrical retention of PKH26. (D) Symmetric cell division is demonstrated by the symmetric orientation of microtubules indicated by α-Tubulin staining (red) and symmetrical distribution of CD133 (green). Scale bars indicate 50 µm. Nuclei were stained with DAPI (blue).
Figure 5
Figure 5. Melanoma cells can adopt a CD133+ phenotype by changes of extracellular cues.
(A) Proposed model of phenotypical transitions of melanoma cells in dependence of FGF2. In this model, a CD271+/CD133− cell could become CD271+/CD133+. In a second step, this cell may convert into a CD271−/CD133+ cell. (B–C) Unsorted melanoma cells were grown in standard (Q263) or human embryonic stem cell (hESC)-medium in absence or presence of FGF2 for 5 days. The amount of CD271 and CD133 positive cells was determined by flow cytometry. Bar graphs indicate the amount of CD133+ or CD271+ fractions in % ± SD of biological triplicates. (D) Immunofluorescence microscopy of untransfected (Mock) cells cultured either in Q263 (upper row) or hESC-medium in absence of FGF2 (lower row) for CD271 (red) and CD133 (green). Double positive colonies are shown in the bordered area and in the magnification. Scale bars indicate 50 µm. (E) Growth of melanoma cells is shown as cell counts ± SD of n = 3 experiments. 1×105 cells were plated initially and cultured for 5 days under different conditions as indicated. *p≤0.05; **p≤0.01 (t-test). (F) Immunofluorescence microscopy of shCtl. and CD271k.d. cells grown under colony-forming conditions indicating the formation of CD271+/CD133+ colonies by shCtl. cells but not CD271k.d. cells. Phase contrast (PH) depicts cellular morphology. Scale bars indicate 50 µm. Nuclei were stained with DAPI (blue). Representatives of biological triplicates are shown.
Figure 6
Figure 6. CD271 is a predominant marker of malignant melanoma.
(A) Immunohistochemistry (IHC) of CD271 and melanoma differentiation markers TYR, HMB45 and MITF of a representative melanoma metastasis (skin). The mutually exclusive expression of CD271 is marked by the red dashed line. Haematoxylin was used as a counter stain in IHC and H&E discriminates differentiation marker positive cells with large nuclei from negative cells with small nuclei. Scale bars indicate 50 µm. (B) Immunofluorescence microscopy of serial sections of a hepatic metastasis (patient T20/15) shows expression of CD271 and absence of CD133. (C) Co-expression and membranous localization of CD133 and the melanoma antigen MART-1 of a representative out of 16 tumors. Scale bars indicate 50 µm. Nuclei were stained with DAPI (blue). (D) Illustration of patient-derived melanoma metastases (PM) derivatives: PMX, PM derived xenografted tumors; PMC, PM derived established cell strains; PMCX, PMC derived xenograft tumors. (E) Roundup of qPCR results of PM, PMX and PMC. Shown are the expression levels of CD271, CD133, ABCB5, SOX10, SOX2, NES, TYR, MART-1 and MITF (MITF-M) (n = 6). The color code indicates high (red) or median (yellow) expression or low/absence (green). A375, MeWo and human melanocytes served as controls (metastases of SKIN  =  cutaneous; PUL  =  pulmonary/lung and LN  =  lymph node).
Figure 7
Figure 7. CD271 and SOX10 are main regulators of a melanoma-associated signaling network.
(A) Venn-diagram depicting the meta-analysis of genes differentially regulated in shCtl. vs. CD271k.d. cells (green) and a data set of shCtl. vs. SOX10k.d. cells (red) . The analysis revealed 271 shared regulated genes among them 65 were found as commonly down-regulated (ratio <0.75) and 145 as commonly up-regulated (ratio >1.33). (B) Model of a yet unknown signaling mechanism that leads to activation of a SOX10-dependent network triggered by CD271. The scheme depicts several genes we found regulated by CD271/SOX10 in (A). Up-regulated genes were representatives of the NFκB family e.g. baculoviral IAP repeat containing 3 (BIRC3), FOS-Like antigen 2 (FOSL2), interleukins 6 and 8 (IL6/8), SRY-box transcription factor 9 (SOX9) and the nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (NFKBIA). Down-regulated genes were the microphthalmia-associated transcription factor (MITF), endothelin receptor b (EDNRB), neural precursor cell expressed, developmentally down-regulated 9 (NEDD9), DEP domain containing 1 (DEPDC1) and cell division cycle associated 3 (CDCA3). (C–D) Comparison of data sets of shCtl. and CD271k.d. cells with data sets of either a whole-in situ hybridization study in Xenopus or a RNAse-protection screen for AP2α binding sites by gene-set enrichment analysis (GSEA). GSEA revealed the presence of TCF4 and TFAP2A (AP2α) regulated genes in shCtl. cells. The TCF4 and TFAP2A signatures are lost upon CD271 silencing. Beside known WNT/TCF4 target genes MITF, DCT and FST (Follistatin), SOX5, SOX6 and NEDD9 were identified (first panels). GSEA for putative AP2α regulated genes revealed SOX10, POU3F2 and SOX5 enriched in shCtl. cells. (E) Analysis of mRNA expression levels of modifiers SOX5 and SOX6 and inducers SOX8, MYB, ETS1, TFAP2A, TFAP2C and NFκB1 of SOX10 expression by qPCR. Expression levels of shRNA control (shCtl.) cells and CD271k.d. cells are shown as ΔΔCT values normalized to β-actin and related to shCtl. cells as mean value ± SD of biological triplicates. **p≤0.01; ***p≤0.001 (t-test). The scale is logarithmic (log). (F) Scheme of putative CD271-dependent signaling pathways. GSEA and qPCR may point out a connection of CD271 and a WNT-dependent regulation of TCF/LEF target genes, activation of AP2-dependent target genes like SOX5 that in turn regulates SOX6 or activation of NFκB signaling.
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