Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jan 23;6(1):e291.
doi: 10.1038/oncsis.2016.88.

The role of the cancer stem cell marker CD271 in DNA damage response and drug resistance of melanoma cells

T Redmer  1   2 , I Walz  2 , B Klinger  2   3 , S Khouja  2 , Y Welte  2 , R Schäfer  1   2 , C Regenbrecht  2   4
Affiliations

The role of the cancer stem cell marker CD271 in DNA damage response and drug resistance of melanoma cells

T Redmer et al. Oncogenesis. .

Abstract

Several lines of evidence have suggested that stemness and acquired resistance to targeted inhibitors or chemotherapeutics are mechanistically linked. Here we observed high cell surface and total levels of nerve growth factor receptor/CD271, a marker of melanoma-initiating cells, in sub-populations of chemoresistant cell lines. CD271 expression was increased in drug-sensitive cells but not resistant cells in response to DNA-damaging chemotherapeutics etoposide, fotemustine and cisplatin. Comparative analysis of melanoma cells engineered to stably express CD271 or a targeting short hairpin RNA by expression profiling provided numerous genes regulated in a CD271-dependent manner. In-depth analysis of CD271-responsive genes uncovered the association of CD271 with regulation of DNA repair components. In addition, gene set enrichment analysis revealed enrichment of CD271-responsive genes in drug-resistant cells, among them DNA repair components. Moreover, our comparative screen identified the fibroblast growth factor 13 (FGF13) as a target of CD271, highly expressed in chemoresistant cells. Further we show that levels of CD271 determine drug response. Knock-down of CD271 in fotemustine-resistant cells decreased expression of FGF13 and at least partly restored sensitivity to fotemustine. Together, we demonstrate that expression of CD271 is responsible for genes associated with DNA repair and drug response. Further, we identified 110 CD271-responsive genes predominantly expressed in melanoma metastases, among them were NEK2, TOP2A and RAD51AP1 as potential drivers of melanoma metastasis. In addition, we provide mechanistic insight in the regulation of CD271 in response to drugs. We found that CD271 is potentially regulated by p53 and in turn is needed for a proper p53-dependent response to DNA-damaging drugs. In summary, we provide for the first time insight in a CD271-associated signaling network connecting CD271 with DNA repair, drug response and metastasis.

PubMed Disclaimer

Figures

Figure 1
Figure 1
CD271 expression discriminates chemoresistant and sensitive cells. (a) Venn diagram depicts unique and commonly top upregulated genes of resistant MeWo cells (fotemustine, MeWoFote, Fote), (vindesine, MeWoVind, Vind) or (etoposide, MeWoEto, Eto) as compared with parental (MeWoPar, Par). (b) Left panel: verification of CD271 expression in resistant cells by qPCR, shown are relative expression levels±s.d. of independent triplicates. Right panels: immunoblot analysis of cells described in a, cisplatin resistant (MeWoCis, Cis), as well as parental cells for levels of CD271. (c) Comparative flow cytometry analysis of resistant and parental MeWo cells for levels of CD271. (d, e) Immunoblot analysis of MeWoPar, MeWoEto and MeWoCis cells following a dose (etoposide: 0.1–10 μM and cisplatin: 0.3–30 μM both 24 h) and time dependent (2, 4, 6 and 24 h, *etoposide or cisplatin was withdrawn after 6 h, cells were maintained for additional 18 h) treatment for levels of CD271. (f) Left panels: analysis of MeWoPar cells following treatment with either etoposide (10 μM), cisplatin (10 μM), fotemustine (30 μg/ml) or vindesine 1 ng/ml for 24 h for cell surface expression of CD271. Right panel: quantification of Ki67-positive MeWoPar cells following drug treatment for 24 h. Shown are numbers of Ki67-positive cells in %±s.d. related to 4′,6-diamidin-2-phenylindol (DAPI). In (b, d, e), whole-cell lysates were analyzed, β-tubulin served as loading control; in (c, f) 10 000 cells were recorded following incubation with either CD271-PE (top panels) or mouse IgG1 isotype control (mIgG1, lower panels). Shown are mean values±s.d. as indicated.
Figure 2
Figure 2
Integrative analysis of melanoma cells with overexpression and/or knock-down of CD271 links CD271 with DNA repair. (a) Venn diagram depicting commonly upregulated genes induced in A375 and T20/02 cells engineered to stably express CD271 (A375NGFR/CD271, T20/02NGFR/CD271) or stably transfected with a CD271-targeting shRNA (shCD271, T20/02k.d.). Numbers of identified genes found commonly upregulated in A375NGFR/CD271 and T20/02NGFR/CD271 (235 genes) or differentially regulated in T20/02NGFR/CD271 and T20/02k.d. cells (577 genes, comprising up and downregulated genes) are shown. Right panel: DNA repair cluster from DAVID analysis of a set of 340 CD271-responsive genes upregulated in T20/02 cells, red or blue indicates positive or negative GO-contribution, respectively. (b) GSEA of profiling data of T20/02NGFR/CD271 and T20/02k.d. cells using a DNA repair signature (230 genes) as provided by MSigDB. (c) Verification of the CD271-dependent regulation of DNA repair genes RAD21, RAD51, RAD51AP1, DNA damage-binding protein 2 (DDB2) and xeroderma pigmentosum genes (XPC) in T20/02NGFR/CD271 (left panel) or cells stably transfected with CD271 specific shRNAs (#2, #3, #4) by qPCR (right panel). Expression levels are shown in logarithmic scale, indicating relative expression levels±s.d. of independent triplicates, compared with cells either expressing GFP or knock-down control (shCtl.). *P⩽0.05; **P⩽0.01; ***P⩽0.001. (d) CD271-induced expression of DNA repair genes in A375NGFR/CD271 cells as determined by qPCR. (e) Expression levels of CD271, RAD51AP1, RAD51 and MSH6 in cells of patient-tumor derived cell strains (Mel91, T20/02 and Mel41) sorted for presence or absence of CD271 by fluorescence-assisted cell sorting (FACS). Shown are three independent experiments.
Figure 3
Figure 3
CD271-responsive genes are predominantly expressed in chemoresistant cells. (a) GSEA with profiling data of MeWoFote, MeWoVind and MeWoEto cells and a consensus signature, comprising 516 CD271-responsive genes. FDR, false discovery rate; NES, nominal enrichment score; NS, no significant enrichment. (b) Venn diagram depicting commonly found CD271-responsive genes in chemoresistant cells. FGF13 represents the most common gene in MeWoFote and MeWoVind cells. (c) Left panel: qPCR of MeWoFote and MeWoPar cells and right panel: MeWoFote cells stably transfected with a validated CD271-targeting shRNA (shCD271) or control (shCtl.) for expression of CD271 and FGF13. (d) Immunoblot analysis of whole-cell lysates of MeWoPar cells either non-transfected (Mock) or engineered to stably express CD271 (NGFR, GFP-tagged) for levels of CD271. (e) Expression level of FGF13 in MeWoPar cells either stably or transiently expressing CD271. (f) Determination of viability of MeWoCis (Cis) and MeWoFote (Fote) cells as compared with stably GFP (Par-GFP) or NGFR/CD271-expressing MeWoPar (Par-NGFR) cells following a dose-dependent treatment with either cisplatin (μM) or fotemustine (μg/ml). Viability is indicated in %±s.d. of n=8 technical replicates. A representative out of n=3 biological replicates is shown.
Figure 4
Figure 4
Levels of CD271 expression modulate drug response. (a) Left panels: IF microscopy of MeWoFote cells stably transfected with a validated CD271-targeting shRNA (shCD271_5-2, clone 5-2) or control (shCtl.) for CD271 (red, left panels), phalloidin (red, center panels), presence of Ki67 (green, right panels) or bright field is shown. Numbers represent % of Ki67-positive cells±s.d. of n=4 counts related to 4′,6-diamidin-2-phenylindol (DAPI) that served as nuclear stain, scale bars indicate 50 μm. Right panel: IF microscopy of clone 5-1 (shCD271_5-1) for phalloidin or bright field is shown. (b) Left and right panels: determination of cell viability and apoptosis of MeWoFote cells, clone 5-2/clone 5-1 modified as described in a following a dose-dependent treatment with fotemustine (μg/ml) for 48 h. Viability is indicated in %±s.d. of n=8 replicates, related to untreated (100% viability) cells, a representative out of three biological replicates is shown, UT=untreated; 0=solvent control. Apoptosis was determined by an ELISA-based measurement of apoptosis induced DNA-fragmentation of clone 5-2 and clone 5-1 after 48 h, scales indicate apoptosis as OD405 values, *P⩽0.05; **P⩽0.01; ***P⩽0.001. (c) Left panels: IF microscopy of MeWoVind cells following stable knock-down of CD271 by a validated shRNA (sh#4) for levels of CD271 (red), Ki67 (green) or bright field. Center and right panel: determination of cell viability CD271 knock-down cells (pool) following a dose-dependent treatment with vindensine and fotemustine for 48 h. Representative experiments out of n=3 are shown.
Figure 5
Figure 5
Expression of CD271-responsive genes facilitates discrimination of primary tumor and melanoma metastases. (a) Left panels: migratory capacity of A375 cells stably transfected with either GFP or CD271 (NGFR/CD271, GFP positive) at time points 0, 7, 15 and 19 h after wounding. Wound closure is indicated by an increasing migration front (blue) and a decreasing wound area (yellow). Right panel: quantification of wound closure. Shown are average wound widths (μm) of n=8 replicates relative to time of imaging (h) of GFP (black) or NGFR (red) transfected cells. Scale bars indicate 200 μm. (b, c) GSEA of profiling data of A375NGFR/CD271 or T20/02NGFR/CD271 or T20/02k.d. cells and signatures indicating melanoma metastasis or melanoma relapse. Top enriched genes are indicated. (d) Expression levels of potential mediators of drug resistance NEK2, HMMR and RAD51AP1 in MeWoFote cells as compared with MeWoPar. Center and right panel: validation of a CD271-dependent expression of NEK2 and HMMR or NEK2 in T20/02NGFR/CD271 or A375NGFR/CD271 cells, respectively. (e) Venn diagram depicting overlapping genes found among data sets of melanoma metastasis (1054 genes) of study GSE8401 as provided by the GEO database and CD271-responsive genes identified in T20/02 cells (340 genes). (f) Supervised clustering of selected CD271-regulated genes depicting distribution in a set of primary tumors and melanoma metastasis. Tumor site is color coded. (g) Expression levels of known ligands potentially binding to CD271, for example, NGF, brain-derived neurotrophic factor (BDNF) and neurotrophin 4 (NTF4) in MeWoPar cells following treated with fotemustine (30, 100 μg/ml) for 24 h (left and center panels) as determined by qPCR. In (d, g), scales indicate relative expression levels±s.d. of independent triplicates, *P⩽0.05; **P⩽0.01; ***P⩽0.001.
Figure 6
Figure 6
Mechanistic insights in the regulation of CD271. (a) Immunoblot analysis of MeWoPar cells treated with either cisplatin (Cis), etoposide (Eto) or temozolomide (TMZ) (all at a final concentration of 10 μM) for levels of CD271 (s/l=short/long exposure), p-p53S15, p21CIP, γH2AX, p-ERK1/2 and ERK1/2, p-AKTS473 and AKT, p-IκBα, p-CHK1 and p-p38 kinase. (b) Immunoblot analysis of MeWoEto, MeWoCis or MeWoPar cells following dose-dependent treatment with etoposide or cisplatin (3, 10 μM both) for 24 h, respectively. Levels of CD271, γH2AX and phosphorylated (activated) p53 (p-p53S15) are shown indicating the different response of sensitive and resistant cells to drugs. (c) Regulation of p53-targets MDM2, p21CIP (CDKN1A), CD95, NOXA, XIAP, as well as MSH6 and CD271 in MeWoPar and MeWoEto cells treated with etoposide (3, 10 μM) for 24 h. Shown are relative expression levels of representative experiments (I, II). (d) Immunoblot analysis of T20/02 cells stably transfected with shCD271 or control, following dose-dependent treatment with etoposide for levels of CD271, p-p53S15, p21CIP and γH2AX. (e) Analysis of cells described in d but stably transfected with different CD271-targeting shRNAs (sh#2, sh#3, sh#4) or control (shCtl.) for levels of p53, p21CIP, CD95, NOXA and BAX. Scale indicates relative expression levels±s.d. of independent triplicates, *P⩽0.05; **P⩽0.01; ***P⩽0.001. In (a, b, d), whole-cell lysates were analyzed, β-tubulin served as loading control. (f) Panel-based next-generation sequencing (NGS) revealed detection of a TP53 mutation (p.E258K, 5′-GAA->AAA-3' antisense (3′-CTT→TTT-5′)) in MeWoPar cells, not present in wild-type T20/02 cells. Shown are sequences, as well as read depths. (g) Left panel: immunoblot analysis of cells described in d for levels of CD271 and p21CIP following a dose-dependent treatment with MDM2 inhibitor nutlin-3a. Right panel: nutlin-3a treatment of MeWoPar cells.
Figure 7
Figure 7
Proposed model for the CD271 regulation and potential downstream effects mediating drug resistance and metastasis. (a) Alkylating drugs (fotemustine, TMZ), TOP2A inhibitors (etoposide) or inter-cross-linking agents (cisplatin) induce expression of CD271 in MeWoPar cells. (b) Drug-dependent DNA damage results in activation of the ataxia telangiectasia mutated (ATM)/ataxia telangiectasia and RAD3-related (ATR) pathway leading to formation of stabilized p53 (p-p53S15) and γH2AX. Stabilized p53 may regulate expression of CD271. (c) Phenotypic consequences associated with increased CD271 expression levels. Left: drug-sensitive cell population (CD271 low), middle: Short-term drug treatment of sensitive cell population (CD271 intermediate), right: drug-resistant cell population (CD271 high), prone to metastasis. (d) Left: Downstream consequences of enhanced CD271/NGFR expression by drug treatment or overexpression (NGFR-GFP). Right: sustained expression of CD271 regulating drug resistance (FGF13, TOP2A, NEK2) and metastasis-associated (RAD51AP1, TOP2A) genes.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, Larkin J et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med 2011; 364: 2507–2516. - PMC - PubMed
    1. Spagnolo F, Ghiorzo P, Orgiano L, Pastorino L, Picasso V, Tornari E et al. BRAF-mutant melanoma: treatment approaches, resistance mechanisms, and diagnostic strategies. OncoTargets Ther 2015; 8: 157–168. - PMC - PubMed
    1. Nazarian R, Shi H, Wang Q, Kong X, Koya RC, Lee H et al. Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation. Nature 2010; 468: 973–977. - PMC - PubMed
    1. Sun C, Wang L, Huang S, Heynen GJ, Prahallad A, Robert C et al. Reversible and adaptive resistance to BRAF(V600E) inhibition in melanoma. Nature 2014; 508: 118–122. - PubMed
    1. Lehraiki A, Cerezo M, Rouaud F, Abbe P, Allegra M, Kluza J et al. Increased CD271 expression by the NF-kB pathway promotes melanoma cell survival and drives acquired resistance to BRAF inhibitor vemurafenib. Cell Discov 2015; 1: 15030. - PMC - PubMed