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

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.


Patients
All patients were treated at the Sestre milosrdnice University Hospital Center and clinical data were available. The study was approved by the Ethics Review Committee of Sestre milosrdnice UHC and the

RNA extraction, RT and qPCR analysis
Total RNA was extracted from 50 mg of frozen tissues using the TRIzol Reagent and purified on PureLink (Hs01119974_m1) and GLI3 (Hs00609233_m1) genes, and ß-glucuronidase (GUSB; Hs00939627_m1) and TATA-binding protein (TBP; Hs00427620_m1) as reference genes. The qPCR analysis of TP73 isoforms (TAp73 and ΔNN'p73) was performed in duplicate using TaqMan Gene Expression custom primers and TAMRA-labeled probes (Metabion, Germany) with TBP as reference gene (Supplementary   Table S5), in 25 μL reaction mixture containing 125 ng of cDNA, 1 × TaqMan Gene Expression Master Mix (Thermo Fisher Scientific), forward and reverse primers (900 nM each) and labeled probe (250 nM).
Gene expression analyses were performed according to protocol suggested by the manufacturer on the 7300 Real-Time PCR System (Thermo Fisher Scientific). Threshold cycle values were normalized to reference gene(s) (ΔCt) and relative expression was calculated using the 2 −ΔCt method.
To distinguish the three different N-terminal TP53 isoforms, a nested qPCR approach was used. For longer N-terminal isoforms (full-length and Δ40), a new DNA template was initially made in pre-amplification PCR using long F and R primers with 25 ng of cDNA. For shorter Δ133 isoforms, a template was initially made in pre-amplification step using short F and same R primer with 50 ng of cDNA. PCR was performed on 2720 Thermal Cycler (Thermo Fisher Scientific) Table S6). For full-length and Δ40 isoforms, p53_F and d40p53_F were used, respectively, both in combination with each of the three reverse primers (p53a_R, p53b_R and p53g_R) on "long" cDNA templates. For Δ133 isoforms, d133p53_F was used in combination with each of the three reverse primers on "short" templates. On both types of pre-amplified templates, a qPCR reaction for all TP53 mRNA isoforms was also performed using all_p53_F and all_p53_R primers. qPCR was performed on the CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) in 10 μL reaction mix containing 1 × Takyon Low Rox SYBR MasterMix dTTP Blue (Eurogentec, Belgium), forward and reverse primers (400 nM each), 1 μL of diluted pre-amplified cDNA template and Milli-Q water. qPCR conditions were the following: [95°C for 3 min (1 cycle)], [95°C for 15 s, 63°C for 20 s, 72°C for 10 s (40 cycles)], and finally melting curve analysis from 72°C to 95°C with ramp of 0.5°C in 5 s.
Threshold cycle values for nine TP53 mRNA isoforms were normalized to appropriate all TP53 mRNA variants' value (ΔCt) and relative expression was calculated using the 2 −ΔCt method.