Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq and ChIP-seq identification of unique and overlapping targets of GLI transcription factors in melanoma cell lines

ABSTRACT: Despite significant progress in therapy, melanoma is still the most lethal form of skin cancer, with a rising incidence worldwide. Little is known about the impact of deregulated Hedgehog-GLI (HH-GLI) signalling pathway in the progression of this disease. Based on previous research, we hypothesized that in melanoma activation of HH-GLI signaling pathway is non- canonical due to its crosstalk with MAPK signaling pathway, which is the most deregulated pathway in melanoma. In order to investigate the link between the two pathways and to find novel GLI transcriptional targets that could be considered for potential combination therapy, we performed RNA sequencing on three melanoma cell lines with overexpressed GLI1, GLI2 and GLI3 and combined them with results of ChIP sequencing on endogenous GLI1, GLI2 and GLI3 proteins on the same cell lines. RNA-seq revealed a total of 808 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. After combining these results, 21 targets were selected for validation by qPCR. Fifteen of these targets were validated in the tested cell lines, 6 of which were detected by both RNA-seq and ChIP-seq.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Homo sapiens

SUBMITTER: Nenad Bartonicek

PROVIDER: E-MTAB-11936 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Aberrant activation of Hedgehog (HH) signaling has been identified as a key etiologic factor of many human malignancies. Signal strength, target gene specificity, and oncogenic activity of HH signaling profoundly depend on interactions with other pathways such as epidermal growth factor receptor-mediated signaling which has been shown to cooperate with HH/GLI in basal cell carcinoma and pancreatic cancer. We demonstrate that the human medulloblastoma cell line Daoy possesses a fully inducible endogenous HH pathway. Treatment of Daoy cells with Sonic Hedgehog or Smoothened agonist induced expression of GLI1 protein and prevented processing of GLI3 to its repressor form. To study interactions between HH- and EGF-induced signaling in greater detail, time-resolved measurements were carried out and analyzed on the transcriptomic as well as proteomic level. Daoy cells responded to the co-treatment by downregulating GLI1, PTCH, and HHIP on the transcript level which was also seen when Amphiregulin (AREG) was used instead of EGF. The finding that EGFR signaling silences proteins acting as negative regulators of HH signaling is firstly described here as a novel crosstalk mechanism. Furthermore, combined EGFR/HH signaling maintains high GLI1 protein levels contrasting its downregulation on the transcript level. On the other hand, high level synergism was observed with respect to a strong and significant upregulation of numerous canonical EGF-targets with putative tumor-promoting properties such as MMP7, VEGFA, and IL-8. In conclusion, synergistic effects between EGFR and HH signaling can selectively induce a switch from a canonical HH/GLI profile to a modulated specific target gene profile pointing to more wide-spread, yet context-dependent, interactions between HH/GLI and growth factor receptor signaling in human malignancies. To study interactions between HH- and EGF-induced signaling, time-resolved measurements were carried out over a period of 24 h at 14 different time points after stimulation by EGF with and without additional stimulation by SHH. Furthermore, as a control, cells without any stimulation by EGF and SHH (control) and cells in the presents of SHH were analyzed. Overall, three biological replicates of 60 different treatment/timepoints were analyzed yielding 180 different samples.

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RNA-seq and ChIP-seq identification of unique and overlapping targets of GLI transcription factors in melanoma cell lines

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