Increased Non-Homologous End Joining Makes DNA-PK a Promising Target for Therapeutic Intervention in Uveal Melanoma.

Doherty Doherty,Sisley Sisley,Rennie Rennie,Bryant Bryant,Valluru Valluru

doi:10.3390/cancers11091278

Doherty Doherty, Sisley Sisley + Show 3 more

Open Access

https://doi.org/10.3390/cancers11091278

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Journal: Cancers	Publication Date: Aug 30, 2019
Citations: 15	License type: CC BY 4.0

Affiliation: Royal Hallamshire Hospital

Abstract

Uveal melanoma (UM) is the most common primary intraocular tumour in adults, with a mean survival of six months following metastasis. The survival rates have not improved in over 30 years. This study has shown that sister chromatid exchange (SCE) is low in UM which is likely due to a reduced expression of FANCD2. As FANCD2 can function to suppress non-homologous end joining (NHEJ), this study therefore investigated NHEJ in UM. The activation of the catalytic subunit of the NHEJ pathway protein DNA-dependent protein kinase (DNA-PK) was measured by analysing the foci formation and the ligation efficiency by NHEJ determined using a plasmid-based end-joining assay. Using small-interfering RNA (siRNA) knock-down, and chemical inhibitors of DNA-PK, the survival of primary UM cultures and two cell lines were determined. To assess the homologous recombination capacity in response to the inhibition of DNA-PK, a SCE analysis was performed. In addition, to support the findings, the messenger RNA (mRNA) expression of genes associated with NHEJ was analysed using the Cancer Genome Atlas (TCGA)-UM RNAseq data (n = 79). The NHEJ activity and DNA-PKcs activation was upregulated in UM and the inhibition of DNA-PK selectively induced apoptosis and sensitized to ionising radiation and inter-strand cross-linking agents. The inhibition of the NHEJ protein DNA-PK is lethal to UM, indicating a potentially effective therapeutic option, either alone or as a sensitizer for other treatments.

Highlights

Uveal melanoma (UM) is a rare but an aggressive tumour of the eye that annually affects 5–7 adults per million in Caucasian populations, with mortality rates of approximately 40–50% within ten years of diagnosis [1,2,3]
non-homologous end joining (NHEJ) in UM was first investigated by analysing the activation of DNA-PK measuring the foci formation of phosphorylated DNA-PKcs at the Serine 2056 (Ser2056) residue, an event that initiates
The spontaneous foci formation of Ser2056 DNA-PKcs was significantly higher in both UM cell lines and UM short-term cultures (STCs) when compared to the control cell lines, WM793 and MRC5VA (P < 0.01) (Figure 1)

Summary

Introduction

Uveal melanoma (UM) is a rare but an aggressive tumour of the eye that annually affects 5–7 adults per million in Caucasian populations, with mortality rates of approximately 40–50% within ten years of diagnosis [1,2,3]. The incidence of metastases at presentation is low, they subsequently develop with a peak incidence approximately three to five years after treatment of the primary tumour, underlining the importance of developing new and effective treatments to target this disseminated disease [5,6,7]. Mutations in guanine nucleotide binding proteins GNAQ and GNA11, not prognostic, occur in approximately 80–90% of UM and are considered an early, if not initiating event in UM [13,14,15,16] These mutations produce constitutive activation of the ERK 1/2 / MEK and Yes Associated Protein (YAP) [17]. In cutaneous melanoma (CM), Cancers 2019, 11, 1278; doi:10.3390/cancers11091278 www.mdpi.com/journal/cancers

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