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

T Redmer,B Klinger,Y Welte,R Schäfer,I Walz,S Khouja,C Regenbrecht

doi:10.1038/oncsis.2016.88

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.

Highlights

Despite recent progress in treatment options, malignant melanoma metastasized to liver, lung or brain remains to be a non-curable disease
235 genes were sensitive melanoma cells upregulated in the two cell lines by CD271 (Figure 2a, We performed microarray-based genome-wide expression profiling of the drug-sensitive melanoma cell line MeWo (MeWoPar) and of derivatives selected for resistance to etoposide (MeWoEto), fotemustine (MeWoFote), vindesine (MeWoVind) or cisplatin (MeWoCis).[22]
We observed a strong activation of mitogen-activated protein kinase signaling (p-ERK1/2) and the p53-signaling pathway following treatment with cisplatin (Cis), etoposide (Eto) or temozolomide, as well as a moderate activation of p38 kinase and checkpoint-kinase 1 (CHK1) (Figure 6a), we did not detect activation of canonical nuclear factor κB (NFκB) signaling (Figure 6a and Supplementary Figure S8A)

Summary

INTRODUCTION

Despite recent progress in treatment options, malignant melanoma metastasized to liver, lung or brain remains to be a non-curable disease. It is well known that intrinsic resistance to chemotherapeutics involves the expression of active membrane transporters,[12,13,14] anti-apoptotic genes,[15,16] genes associated with stemness[17,18] and epigenetic modulators.[17] The increased DNA repair capacity of melanoma cells[18] provides an additional mechanism conferring intrinsic resistance to targeted drugs and chemotherapeutics. It has been shown that acquired resistance of melanoma and glioblastoma to fotemustine is directly linked to high expression of the de-alkylating enzymes.[20] acquired increase of the DNA repair capacity is likely to be a major mechanism of melanoma cells in evading chemotherapeutic interventions (reviewed in Soengas and Lowe[21]). Received 19 July 2016; revised 31 October 2016; accepted 25 November 2016 we analyzed the consequence of drug-induced changes in gene transgene showed increased endogenous and ectopic expression in non-resistant cells

RESULTS

DISCUSSION

Findings

MATERIALS AND METHODS