RNA-seq and ChIP-seq Identification of Unique and Overlapping Targets of GLI Transcription Factors in Melanoma Cell Lines

Abstract

Background: Despite significant progress in therapy, melanoma still has a rising incidence worldwide, and novel treatment strategies are needed. Recently, researchers have recognized the involvement of the Hedgehog-GLI (HH-GLI) signaling pathway in melanoma and its consistent crosstalk with the MAPK pathway. In order to further investigate the link between the two pathways and to find new target genes that could be considered for combination therapy, we set out to find transcriptional targets of all three GLI proteins in melanoma. Methods: We performed RNA sequencing on three melanoma cell lines (CHL-1, A375, and MEL224) with overexpressed GLI1, GLI2, and GLI3 and combined them with the results of ChIP-sequencing on endogenous GLI1, GLI2, and GLI3 proteins. After combining these results, 21 targets were selected for validation by qPCR. Results: RNA-seq revealed a total of 808 differentially expressed genes (DEGs) for GLI1, 941 DEGs for GLI2, and 58 DEGs for GLI3. ChIP-seq identified 527 genes that contained GLI1 binding sites in their promoters, 1103 for GLI2 and 553 for GLI3. A total of 15 of these targets were validated in the tested cell lines, 6 of which were detected by both RNA-seq and ChIP-seq. Conclusions: Our study provides insight into the unique and overlapping transcriptional output of the GLI proteins in melanoma. We suggest that our findings could provide new potential targets to consider while designing melanoma-targeted therapy.

GLI Transcriptional Targets S100A7 and KRT16 Show Upregulated Expression Patterns in Epidermis Overlying the Tumor Mass in Melanoma Samples

“…In our previous study, we performed RNA-sequencing and ChIP-sequencing on melanoma cell lines in order to identify new HH-GLI-regulated targets [ 12 ]. qPCR validation of these results allowed us to narrow down the gene list to a few genes that show consistent and stable expression in the majority of the tested melanoma cell lines.…”

“…Human melanoma cell lines A375 (RRID: CVCL_0132), CHL-1 (RRID: CVCL_1122), and MEL224 (RRID: CVCL_U915) were kindly provided by Andreja Ambriović Ristov, PhD, and Neda Slade, PhD [ 12 , 62 ]. Cell lines A375 and CHL-1 were maintained in the recommended medium: Dulbecco’s Modified Eagle Medium (Merck KGaA, Darmstadt, Germany), while MEL224 was maintained in RPMI 1640 medium (Merck KGaA, Darmstadt, Germany), both supplemented with 10% FBS (Merck KGaA, Darmstadt, Germany), 1 mM sodium pyruvate, 1% streptomycin/penicillin, and 4 mM L-glutamine (Gibco Thermo Fisher Scientific, Waltham, MA, USA).…”

“…Fold change was calculated relative to the RPLP0 housekeeping gene using the 2 −ΔΔCt method. Primer sequences for GLI1 , GLI2 , GLI3 , PTCH1 , S100A7 , and KRT16 used for qPCR were published before [ 12 ].…”

“…This is also true for the role of HH-GLI signaling in melanoma [ 10 , 11 ]. In our previous research [ 12 ], we have identified novel targets of GLI proteins in melanoma using RNA sequencing. Among the 15 targets that were successfully validated on a panel of melanoma cell lines, two of them, namely, S100A7 and KRT16, have emerged as particularly interesting.…”

Editorial for Special Issue “Molecular Mechanisms and Signaling Pathways in Melanoma”

“…By using an RNA-sequencing strategy in conjunction with ChIP-seq techniques on melanoma cell lines with a high expression of GLI1-3 proteins in the hedgehog signaling pathway, Kurtovic et al [7] were able to validate 21 targets via subsequent quantitative PCR. These proteins may represent potential targets for therapy, although they require further validation.…”