?(Fig.2g),2g), on the one hand by Rad51i and on the other hand by the Rad51 deregulation mediated by Elk1. These data suggest that Elk1 is an important transcription factor regulated by MAPK signaling pathway which regulates downstream HRR gene expression like mRNA and protein expression is upregulated in metastatic melanoma cell lines and patient samples and this high expression correlates with a reduced overall survival of melanoma patients. melanoma patients. In addition, Rad51 expression in melanoma cells was regulated on a transcriptional level by the MAPK signaling pathway with Elk1 as the main downstream transcriptional effector. Most strikingly, melanoma cells which developed resistance towards MAPK inhibitors could be efficiently targeted by Rad51 inhibitors similar to their sensitive counterparts, leading to DNA damage, G2/M arrest and apoptosis. Furthermore, the treatment of MAPK inhibitor resistant cells with Rad51 inhibitors enhances the susceptibility of these cells for MAPK inhibitor treatment in vitro and in vivo. These data indicate that Rad51 plays a critical role in the survival of metastatic melanoma cells and is a promising target for the therapy of melanoma irrespective of its MAPK inhibitor resistance status. and other HRR-associated genes in tumor cells is supposed to enhance DNA repair and increase resistance to DNA damaging substances17C19. Several mechanisms for the regulation of RAD51 level are already described. Among them, MAPK signaling pathway is often shown to mediate the transcription of mRNA20C26. MAPK inhibition in melanoma cells ORM-10103 was recently shown to induce a HR deficient phenotype22. Targeted therapy of patients with BRAF-mutated melanoma with either BRAF inhibitors or a combination of BRAF and MEK inhibitors has demonstrated a great success for the treatment of melanoma patients. However, the development of resistance remains the limiting factor for the long-term success of targeted therapy27. Therefore, it is essential to find new critical therapeutic targets in melanoma treatment to enable improved combination therapies. Within this work, we investigated the potential of Rad51 as therapeutic target in metastatic melanomas with or without acquired resistance to inhibitors of the MAPK pathway (MAPKi) as single agents or in combined treatments. We show that Rad51 may be a promising new target for the treatment of melanoma. Materials and methods Cell culture The metastatic melanoma cell lines A375, SK-MEL19 and SK-MEL28 were purchased from ATCC. SbCl2 cell line was a gift of Dr. B. Giovanelli (Stehlin Foundation for Cancer Research, St. Joseph Hospital, Houston, TX). The other metastatic melanoma cell lines used ORM-10103 here and the vemurafenib resistant patient derived xenograft (PDX) cells, WM4205-3, were kindly gifted by M. Herlyn and C. Krepler from the Wistar Institute (Philadelphia, USA). These cells were tested every 6 month to exclude mycoplasm contaminations. The cell lines SbCl2 and SK-MEL2 carry an mutation, whereas all other cell lines used here are and and showed no clear differences (Fig. ?(Fig.1a1a). Open in a separate window Fig. 1 Melanoma cells exhibit a high expression of Rad51 resulting in increased DNA damage repair.a The basal mRNA level of different homologous recombination repair (HRR) and nucleotide excision repair (NER) genes in melanoma cell lines, normalized to respective actin expression is shown relative to expression in melanocytes (RT-qPCR, expression (high expression: blue line, gene has a negative influence on the survival of melanoma patients (Fig. ?(Fig.1b).1b). In contrast, we found no significant differences in patient survival in the two groups expressing ORM-10103 higher or lower levels of the other HRR-associated genes (Supplementary Fig. 1A). Therefore, we focused on Rad51 expression and confirmed that also Rad51 protein was strongly expressed in most human melanoma cell lines, in contrast to primary human melanocytes (FM) and primary human fibroblasts (FF), which have either no or very low levels of Rad51 (Fig. ?(Fig.1c).1c). Similar to the observations in melanoma cell lines, we show high Rad51 expression in metastatic melanoma tumor samples from 17 out of 25 patients by immunohistochemical staining (Fig. ?(Fig.1d,1d, Supplementary Fig. 1B). Since increased HRR genes expression and in particular the expression of mediate an upregulation of HRR capacity in cancer cells, we have analyzed whether Rad51 protects melanoma cells from DNA damage by increasing HRR capacity. Therefore, we have analyzed the formation of nuclear Rad51 foci and pH2AX foci after genotoxic stress via cisplatin treatment in melanoma cells with or without prior Rad51 inhibition. Indeed, Rad51 foci induction by cisplatin treatment was blocked through ORM-10103 previous treatment with Rad51 inhibitor (Rad51i) B02 (Fig. ?(Fig.1e).1e). We also show that treatment with cisplatin leads ORM-10103 to an increased pH2AX foci number, indicating the accumulation of DNA double-strand breaks, which was further enhanced by previous treatment with Rad51i (Fig. ?(Fig.1e1e). These data suggest a critical role of Rad51 overexpression for effective DNA damage repair and thus for the survival of metastatic melanoma cells. gene PR55-BETA expression is regulated by the MAPK signaling pathway in melanoma cells via Elk1 Next, we asked whether the high expression of HRR genes is influenced.