In vitro anti-melanoma activity of the amphibian ribonuclease onconase (ONC): comparison between monomeric and dimeric ONC species and correlation between ONC-elicited microRNAs upregulation and lower expression of specific onco-proteins

Melanoma, displaying a high mortality rate and an increasing incidence worldwide is considered the most aggressive forms of skin cancer. Several therapies have been approved during the last few years including target and immunotherapy but unfortunately, all strategies failed for the resistance acquired by tumor cells or for the severe side effects. In this context new therapeutical strategies urge to be discovered and the cytotoxic onconase (ONC), an RNase variant deriving from Rana pipiens’ oocytes, may become a valid choice. This ribonuclease shows remarkable activity against different types of cancer in patients, reaching phase II and phase III clinical trials for non-small cell lung cancer and for malignant mesothelioma, respectively. Unfortunately, nowadays the most important limit for a therapeutic application of ONC is its renal toxicity even if reversible. Oligomeric species of this enzyme can be generated to overcome this problem, since enlarging ONC moiety dimension through dimerization should limit glomerular filtration and increase its circulating half-life. Interestingly, the dimeric form of ONC (ONC-D) displays an enzymatic activity similar to that of ONC. In this thesis, different human cancer cell lines were incubated with increasing concentrations of ONC and ONC-D with the aim of comparing their biological activities. Besides two human cell lines from melanoma (A375 and MeWo), glioblastoma cell line (U251) and hepatocellular carcinoma cell line (HepG2) were also tested. In all cell lines, ONC-D exhibits slightly lower cytotoxic and cytostatic effects compared to the monomer. Therefore, the molecular mechanism of ONC and ONC-D antitumor activity has been deeply investigated in A375 and MeWo melanoma cells. Indeed, both ONC species demonstrated to inhibit the phosphorylation level of the proto-oncogene tyrosine-kinase Src (Src). Importantly, a strong inhibition of Tyr705 phosphorylation of the Signal Transducer and Activator of Transcription 3 (STAT3) was also registered with both ONC and ONC-D. This event could depend on the reduced Src activity because STAT3 is a target substrate of Src kinase. In addition, both Ser727 phosphorylation of STAT3 and the STAT3 total protein expression level were decreased. This suggests a complete downregulation of STAT3 transcriptional activity, which is highly activated in cancer, thus partially explaining the antitumor action of both ONC species. Moreover, the number of apoptotic cells increased after ONC and ONC-D treatments. This result correlates with the lower expression level of the anti-apoptotic B cell lymphoma 2 (Bcl2) protein that is upregulated by STAT3. Even if non-coding RNA species seem to be the preferential targets of ONC ribonucleolytic activity, the target specificity of this enzyme is still unclear. So, in this thesis we investigated the ONC ability to modulate the expression of several microRNAs (miRNAs) in the A375 and FO1 BRAF-mutated melanoma cell lines. RT-PCR, used to measure the expression levels of miRNAs, revealed that the onco-suppressors miR-20a-3p, miR-29a-3p and miR-34a-5p were highly expressed in 48-h ONC-treated A375 cells. Moreover, immunoblots were carried out to study the expression level of some proteins that are codified by target genes of such overexpressed miRNAs. Here it was also confirmed that ONC inhibits A375 cell proliferation, and its cytostatic effect was mechanistically explained by the sharp inhibition of proteins involved in cell cycle progression such as cyclins D1 and A2, as well as retinoblastoma protein and cyclin-dependent kinase 2. In addition, the expression of kinases ERK1/2 and Akt and of the hypoxia inducible factor-1α, proteins controlling pro-survival pathways, was inhibited by ONC. Finally, many crucial proteins involved in cell metabolism, migration, invasion, and metastatic potential were downregulated, as well. Since all mRNAs coding these proteins are predicted or validated targets of miR-20a-3p, miR-29a-3p and/or miR-34a-5p which are upregulated by ONC, we suggest that ONC anti-proliferative and anti-metastatic activities in A375 melanoma cells might depend on the upregulation of these onco-suppressor miRNAs. In conclusion, the results obtained strongly suggest that ONC displays a pleiotropic anti-tumor effect against different human melanoma cell lines. Again, the possible oligomerization of this enzyme could break down the limit for ONC therapeutic application.