Influence of ONCONASE in the therapeutic potential of PARP and BRAF inhibitors in human A375 melanoma cells.

Melanoma is one of the most aggressive form of skin cancer, characterized by high mortality rate due to the metastatic potential of its cells. Several therapies have been approved during last few years. Recently, the melanoma molecular characterization led to development of drugs acting against specific targets. For instance, BRAF inhibitors (BRAFi) have been tested against BRAF-mutated melanoma. Unfortunately, all chemotherapy strategies failed for the resistance acquired by tumor cells. Poly (ADP-ribose) polymerase (PARP) enzymes are crucial in the DNA damage response and the PARP inhibition causes the accumulation of unrepaired DNA, inducing cell death. PARP inhibitors (PARPi) are commonly used in chemotherapy. The most used PARPi in cancer therapy is olaparib, although it can be quickly throwing outside the cells by the P-glycoprotein drug efflux transporter. For this reason, a new PARPi generation has been designed, such as AZD2461 that demonstrated a lower binding affinity to the P-glycoprotein than olaparib. In vitro and in vivo studies showed that the protein onconase (ONC) exerted an antitumor effect in different cancer types, due to its ribonuclease activity. Therefore, in this work, the ONC effects on A375 human melanoma cell line that harbors a mutation in the BRAF kinase gene, has been evaluated. A reduction in A375 cell viability has been observed with ONC treatment, while no cytotoxicity was registered in normal human melanocytes. In A375 cells a reduction in BrdU incorporation and a decrease in Ki67 protein expression occurred, suggesting that ONC elicits a cytostatic effect. In addition, an increase of both Annexin V-FITC fluorescence and cleaved PARP1 expression level were observed, suggesting that ONC induces apoptotic cell death. In order to investigate the ONC intracellular targets, changes in the proteome profile have been investigated using mass spectrometry. A decreased expression level of proteins involved in telomere elongation, in the mesenchymal cell development and in the ribosomal subunit biogenesis has been obtained. A decrease in protein synthesis in ONC-treated cells was also demonstrated. Besides ONC, AZD2461 PARPi can reduce A375 cell viability, conversely, no strong benefits were obtained when ONC was administered in combination with AZD2461, in comparison with each single drug treatment. ONC and AZD2461 have displayed an inhibitory effect on both TNF-α gene transcription and NF-κB DNA binding activity, but no additive effect was observed when they were used in combination. In order to obtain A375 cells resistant to AZD2461, cells were treated for two months with AZD2461, but this long treatment did not induce resistance. Nevertheless, the AZD2461 long-time treated cells resulted more responsive to the ONC pro-apoptotic action, if compared to the parental ones. Subsequently, resistance against the BRAFi dabrafenib has been induced in A375 since long time treatment with increasing drug concentrations of dabrafenib induced a sorting of an A375 resistant cell (A375DR) subpopulation. A375DR cells displayed activation of the cancer stem cells markers CD133 and NANOG and increase expression of epithelial-mesenchymal transition-related proteins N-cadherin and nuclear β-catenin, in comparison with A375 parental (A375P) cells. In addition, A375DR showed an increase in the ERK1/2 phosphorylation level, suggesting a reactivation of the MAPK pathway. By comparing A375P and A375DR, ONC treatment can inhibit the total viability and the proliferation rate in both cell subpopulations and induce apoptotic cell death. Moreover, among its pleiotropic effects, ONC reduced nuclear p65 NF-κB amount and IκB kinase phosphorylation level, as well as metalloproteinase-2 activity in both cell subpopulations. Finally, ONC decreased cell colony formation, migration and invasion capability more extensively in A375DR than in A375P cells. In conclusion, ONC successfully counteracts malignant phenotype of A375 cells especially in BRAFi resistant cells and could become a helpful tool for therapy of melanoma recurrence.