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 .

Background NME6 is a member of the nucleoside diphosphate kinase (NDPK/NME/Nm23) family which has key roles in nucleotide homeostasis, signal transduction, membrane remodeling and metastasis suppression. The well-studied NME1-NME4 proteins are hexameric and catalyze, via a phospho-histidine intermediate, the transfer of the terminal phosphate from (d)NTPs to (d)NDPs (NDP kinase) or proteins (protein histidine kinase). For the NME6, a gene/protein that emerged early in eukaryotic evolution, only scarce and partially inconsistent data are available. Here we aim to clarify and extend our knowledge on the human NME6. Results We show that NME6 is mostly expressed as a 186 amino acid protein, but that a second albeit much less abundant isoform exists. The recombinant NME6 remains monomeric, and does not assemble into homo-oligomers or hetero-oligomers with NME1-NME4. Consequently, NME6 is unable to catalyze phosphotransfer: it does not generate the phospho-histidine intermediate, and no NDPK activity can be detected. In cells, we could resolve and extend existing contradictory reports by localizing NME6 within mitochondria, largely associated with the mitochondrial inner membrane and matrix space. Overexpressing NME6 reduces ADP-stimulated mitochondrial respiration and complex III abundance, thus linking NME6 to dysfunctional oxidative phosphorylation. However, it did not alter mitochondrial membrane potential, mass, or network characteristics. Our screen for NME6 protein partners revealed its association with NME4 and OPA1, but a direct interaction was observed only with RCC1L, a protein involved in mitochondrial ribosome assembly and mitochondrial translation, and identified as essential for oxidative phosphorylation. Conclusions NME6, RCC1L and mitoribosomes localize together at the inner membrane/matrix space where NME6, in concert with RCC1L, may be involved in regulation of the mitochondrial translation of essential oxidative phosphorylation subunits. Our findings suggest new functions for NME6, independent of the classical phosphotransfer activity associated with NME proteins.

Cutaneous melanoma is the most aggressive form of skin cancer. Despite the significant advances in the management of melanoma in recent decades, it still represents a challenge for clinicians. The TP53 gene, the guardian of the genome, which is altered in more than 50% of human cancers, is rarely mutated in melanoma. More recently, researchers started to appreciate the importance of shorter p53 isoforms as potential modifiers of the p53-dependent responses. We analyzed the expression of p53 and p73 isoforms both at the RNA and protein level in a panel of melanoma-derived cell lines with different TP53 and BRAF status, in normal conditions or upon treatment with common anti-cancer DNA damaging agents or targeted therapy. Using lentiviral vectors, we also generated stable clones of H1299 p53 null cells over-expressing the less characterized isoforms Δ160p53α, Δ160p53β, and Δ160p53γ. Further, we obtained two melanoma-derived cell lines resistant to BRAF inhibitor vemurafenib. We observed that melanoma cell lines expressed a wide array of p53 and p73 isoforms, with Δ160p53α as the most variable one. We demonstrated for the first time that Δ160p53α, and to a lesser extent Δ160p53β, can be recruited on chromatin, and that Δ160p53γ can localize in perinuclear foci; moreover, all Δ160p53 isoforms can stimulate proliferation and in vitro migration. Lastly, vemurafenib-resistant melanoma cells showed an altered expression of p53 and p73 isoforms, namely an increased expression of potentially pro-oncogenic Δ40p53β and a decrease in tumor-suppressive TAp73β. We therefore propose that p53 family isoforms can play a role in melanoma cells’ aggressiveness.

Nucleoside diphosphate kinases (NDPK/NME/Nm23) are enzymes composed of subunits NME1/NDPK A and NME2/NDPK B, responsible for the maintenance of the cellular (d)NTP pool and involved in other cellular processes, such as metastasis suppression and DNA damage repair. Although eukaryotic NDPKs are active only as hexamers, it is unclear whether other NME functions require the hexameric form, and how the isoenzyme composition varies in different cellular compartments. To examine the effect of DNA damage on intracellular localization of NME1 and NME2 and the composition of NME oligomers in the nucleus and the cytoplasm, we used live-cell imaging and the FRET/FLIM technique. We showed that exogenous NME1 and NME2 proteins co-localize in the cytoplasm of non-irradiated cells, and move simultaneously to the nucleus after gamma irradiation. The FRET/FLIM experiments imply that, after DNA damage, there is a slight shift in the homomer/heteromer balance between the nucleus and the cytoplasm. Collectively, our results indicate that, after irradiation, NME1 and NME2 engage in mutual functions in the nucleus, possibly performing specific functions in their homomeric states. Finally, we demonstrated that fluorophores fused to the N-termini of NME polypeptides produce the largest FRET effect and thus recommend this orientation for use in similar studies.

Liquid chromatography coupled with electrospray ionization mass spectrometry (ESI-MS) is routinely used in proteomics research. Mass spectrometry-based peptide analysis is performed de facto in positive-ion mode, except for the analysis of some post-translationally modified peptides (e.g., phosphorylation and glycosylation). Collected mass spectrometry data after peptide negative ionization analysis is scarce, because of a lack of negatively charged amino acid side-chain residues that would enable efficient ionization (i.e., on average, every 10th amino acid residue is negatively charged). Also, several phenomena linked to negative ionization, such as corona discharge, arcing, and electrospray destabilization, because of the presence of polar mobile-phase solutions or acidic mobile-phase additives (e.g., formic or trifluoroacetic acid), reduce its use. Named phenomena influence microflow and nanoflow electrospray ionization (ESI) of peptides in a way that prevents the formation of negatively charged peptide ions. In this work, we have investigated the effects of post-column addition of isopropanol solutions of formaldehyde, 2,2-dimethylpropanal, ethyl methanoate, and 2-phenyl-2-oxoethanal as the negative-ion-mode mobile-phase modifiers for the analysis of peptides. According to the obtained data, all four modifiers exhibited significant enhancement of peptide negative ionization, while ethyl methanoate showed the best results. The proposed mechanism of action of the modifiers includes proton transfer reactions through oxonium ion formation. In this way, mobile phase protons are prevented from interfering with the process of negative ionization. To the best of our knowledge, this is the first study that describes the use and reaction mechanism of aforementioned modifiers for enhancement of peptide negative ionization.

Nucleoside diphosphate kinases (NDPKs) catalyze the exchange of the terminal phosphate from trinucleotides to dinucleotides through a highenergy phosphohistidine intermedier. They are encoded by NME genes and have been found, with a few exceptions, in all living beings. Besides their well-known function as key regulators of the cellular nucleotide homeostasis, they have been appointed numerous additional biochemical and biological functions. The discovery of NME1/NDPK A as the first metastasis suppressor opened new avenues in cancer research. Although the precise role of NME genes/proteins in cancer dissemination is not yet revealed, it seems that further intensive research in this field may lead to new advances in cancer diagnosis and prognosis, as well as encourage new therapeutic strategies.

IntroductionTP53 is the most frequently mutated gene in human cancer. However, in metastatic melanoma mutations of TP53 occur infrequently and p53 fails to function as a tumour suppressor. The altered expression of p53 family members, including p53/p73 isoforms, as well as of the interactions among them could affect normal function of p53. Furthermore, somatic BRAF mutations have been found in 37%–50% of all melanomas, of which almost 90% harbour the activating V600E mutation. Although initial response to BRAF inhibitors is highly effective, the resistant clones frequently develop, and, in treated patients disease progression is observed within 6 to 8 months. To address this, a better understanding of the genetic basis of melanoma initiation and progression is needed.Material and methodsThe expression profile of p53 and its potential interaction partners - p53 and p73 isoforms was determined in a panel of melanoma cell lines by western blot analysis and quantitative RT-PCR. We have determined the protein levels of p53/p73 isoforms in response to DNA damage treatment (γ-irradiation and etoposide) in cell lines with different TP53 mutational status using western blot analysis. Furthermore, vemurafenib resistant cells are generated and resistance was confirmed by MTT assay. Expression of p53/p73 isoforms was determined in these cells.Results and discussionsRelative expression analysis of metastatic melanoma cell lines revealed that the most expressed p53 isoforms are p53α and Δ133p53α, while Δ40p53ß, Δ40p53γ and Δ133p53γ are least expressed. Also, interestingly, relative expression of full length TAp73 was higher than ΔNp73. Furthermore, the most expressed proteins were p53α, Δ40p53α, Δ133p53γ and Δ160p53γ. Contrary to gene expression, the most expressed p73 isoform is oncogenic ΔNp73β. γ-irradiation induced accumulation of all p53α isoforms in p53 mutant melanoma cell lines, but not in p53 wild type cells. Levels of p53 beta isoforms remained the same, while gamma isoforms were undetectable. Upon γ-irradiation, accumulation of ΔNp73 isoforms was observed in p53 mutant cells. Treatment with etoposide induced expression of p53α isoform, and both TAp73 and ΔNp73 isoforms in p53 wild type cells. Furthermore, in vemurafenib resistant clones the changes in p53/p73 protein expression were observed.ConclusionTaken together, these analyses enabled us to detect p53/p73 isoforms in melanoma cell lines and gave us insight into their abundance in melanoma cell lines for further analyses of p53 interacting partners.

IntroductionMalignant melanoma is the most aggressive form of skin cancer and resistant to available therapies, therefore new molecular approaches for better understanding of disease are needed. Although TP53 is rarely mutated in melanoma, it fails to function as a tumour suppressor. This may result from alterations in p53 family members, including the diverse isoforms of p53 and its homologue p73. Moreover, we assume that p53 functions in melanoma might be altered through interactions with small molecular weight variants of p53 and p73 isoforms, NME and GLI families of proteins. In this study, we conducted a gene/protein expression profiling for p53 and its potential interaction partners (p73/NME/GLI) in metastatic melanoma tissue.Material and methodsMetastatic melanoma and adjacent healthy skin tissues were obtained from 38 patients during surgery in the Sestre milosrdnice University Hospital Centre, Zagreb. Expression of 9 TP53 isoforms, both N- (full-length, Δ40 and Δ133) and C-terminal (α, β and γ), 2 TP73 isoforms (TAp73 and ΔNN’p73), NME1, NME2, GLI1, GLI2, GLI3 and PTCH1, was analysed by RT-qPCR. Expression of p53 (p53α, p53β, Δ40p53α, Δ133p53α, Δ133p53β and Δ160p53α isoforms), p73 (TAp53α, TAp53β, ΔNp73α and ΔNp73β), NME1, NME2, GLI1 (130 and 160 kDa isoforms), GLI2 (133 and 250 kDa) and GLI3 (activator/repressor forms) was analysed by western blot.Results and discussionsRelative expression of ‘long’ TP53 isoforms in tumour tissue was as follows: p53α>p53β > Δ40α>p53γ > Δ40β > Δ40γ. Expression of ‘short’ TP53 isoforms was: Δ133α > Δ133β > Δ133γ. Only Δ40β and Δ40γ were significantly downregulated in tumours. Expression of full length TAp73 was higher than ΔNp73, and both were significantly downregulated in tumours. Significant downregulation in tumours was also observed for PTCH1, GLI1 and GLI2; while NME1 and NME2 were generally the most expressed genes but without significant difference between healthy and tumour tissue. In addition, in metastatic melanomas the most expressed proteins were p53α and NME1, while ΔNp73β, GLI2 (250 kDa) and Δ133p53α showed lowest expression. Eight proteins showed significantly higher expression in tumours compared with healthy skin: 2 GLI1 isoforms (130 and 160 kDa), 133 kDa GLI2 isoform, NME1 and NME2, ΔNp73Δ and 2 p53 isoforms with shortest N- and longest C-terminus.ConclusionWe have shown that TP53/TP73/NME/GLI genes are generally downregulated in metastatic melanoma tissue compared with healthy skin, while, on the contrary, their protein products seem to be upregulated in tumours.