Expression profiles of p53/p73, NME and GLI families in metastatic melanoma tissue and cell lines

Petar Ozretić,Maja Sabol,Vesna Musani,Ivan Milas,Diana Trnski,Zvonimir Puljiz,Bastien Proust,Sonja Levanat,Martina Radić,Nikolina Hanžić,Neda Slade,Yari Ciribilli,Maja Herak Bosnar

doi:10.1038/s41598-019-48882-y

Petar Ozretić, Maja Sabol + Show 11 more

Open Access

https://doi.org/10.1038/s41598-019-48882-y

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Abstract

Unlike other tumours, TP53 is rarely mutated in melanoma; however, it fails to function as a tumour suppressor. We assume that its functions might be altered through interactions with several families of proteins, including p53/p73, NME and GLI. To elucidate the potential interplay among these families we analysed the expression profiles of aforementioned genes and proteins in a panel of melanoma cell lines, metastatic melanoma specimens and healthy corresponding tissue. Using qPCR a higher level of NME1 gene expression and lower levels of Δ40p53β, ΔNp73, GLI1, GLI2 and PTCH1 were observed in tumour samples compared to healthy tissue. Protein expression of Δ133p53α, Δ160p53α and ΔNp73α isoforms, NME1 and NME2, and N′ΔGLI1, GLI1FL, GLI2ΔN isoforms was elevated in tumour tissue, whereas ∆Np73β was downregulated. The results in melanoma cell lines, in general, support these findings. In addition, we correlated expression profiles with clinical features and outcome. Higher Δ133p53β and p53α mRNA and both GLI1 mRNA and GLI3R protein expression had a negative impact on the overall survival. Shorter overall survival was also connected with lower p53β and NME1 gene expression levels. In conclusion, all examined genes may have implications in melanoma development and functional inactivity of TP53.

Highlights

Malignant melanoma remains the most aggressive and treatment-resistant form of skin cancer with increasing incidence[1]
The TP53 isoforms were pre-amplified in two separate pre-amplification reactions, giving a “long” and “short” template for quantitative real-time PCR amplification and analysis
One out of nine TP53 isoforms was significantly downregulated in tumour tissue (∆40p53β, p = 0.017) (Fig. 2)

Summary

Introduction

Malignant melanoma remains the most aggressive and treatment-resistant form of skin cancer with increasing incidence[1]. In metastatic melanoma TP53 gene is relatively rarely mutated It fails to function as a tumour suppressor and reduced levels of p53 contribute to aggressiveness and resistance to therapy[2]. Transcription from alternative promoters, alternative splicing and diverse translation initiation sites contribute to the family complexity[6,7], and twelve protein isoforms with different N- and C-termini are encoded by the single TP53 gene (Fig. 1)[8]. Twelve protein isoforms with different N- and C-termini are encoded by the human TP53 gene – alternative splicing of human intron 2 gives rise to Δ40p53 (truncated transactivation domain, TAD) and intron 9 to α, β and γ isoforms; the usage of the alternative promoter produces Δ133p53 and Δ160p53 (both lacking the entire TAD), while alternative translation initiation site produces Δ40p53 and Δ160p53

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