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Melanoma is skin cancer developed from melanocytes and is considered to be one of the most aggressive tumor types due to high metastatic potential. One of the reasons why melanoma is prone to metastasis is a process known as a phenotype switching which is similar to epithelial–mesenchymal transition (EMT) since it includes EMT-related genes and signaling pathways. In addition, this process is shown to be implicated in resistance to targeted therapy. Depending on the BRAF mutation status, first line treatment options for melanoma can include targeted therapy or immunotherapy for BRAF-mutated melanoma, or immunotherapy for wild- type BRAF melanoma. Around 60% of melanoma carry BRAF mutation, of which almost 90% harbor BRAF V600E mutation that causes continuous activation of the MAPK signaling pathway. BRAF V600E mutated kinase can be targeted by BRAF inhibitor (BRAFi), vemurafenib, which is a first targeted therapy licensed for treatment of advanced melanoma. Although initial response in the clinic was very powerful, the disease relapses within several months of therapy introduction due to occurrence of acquired resistance to BRAFi therapy. Reactivation of the MAPK pathway occurs in the majority of BRAFi-resistant tumors, while the second most frequently activated signaling pathway is PI3K/AKT. So to investigate the underlying molecular mechanisms of BRAFi targeted therapy resistance in more detail, we generated and characterized two melanoma cell lines with acquired resistance to vemurafenib. The resistant cell lines exhibited specific features of slow cycling cells possibly as a consequence of vemurafenib-driven phenotype switching. These characteristics included morphological and molecular features of EMT-like cells, enhanced resistance to chemotherapy, changed levels of cell-cycle regulators and thus reduced proliferation. Additional features included decreased migration ability but tendency toward collective migration, and reactivation of MAPK or activation of PI3K/AKT signaling pathways, depending on the cell line. Reduced levels of NME1 and NME2 metastasis suppressor proteins were observed in primary vemurafenib-resistant cell line, which could originate from vemurafenib- acquired resistance and is one of the reasons for increased PI3K/AKT signaling. Further studies are required to reveal the vemurafenib-dependent suppressors of NME proteins, their association with PI3K/AKT signaling, and their impact on vemurafenib-resistant melanoma cell characteristics.

In metastatic melanoma tumor suppressor protein p53 is not functional, although it is rarely mutated (less than 10%). Despite the low rate of mutations, p53 fails to function as tumor suppressor and tumor cells continue to proliferate and spread. Therefore, the question arises what in the metastatic melanoma prevents the wild type p53 protein to function. Since p53 interactions play a significant role in tumorigenesis, we have investigated whether the p53 protein function can be altered by interactions with p53 low molecular weight isoforms, p73 isoforms, and NME and GLI families of proteins. Therefore, we studied the protein expression profile of p53 and its potential interaction partners (p73/NME/GLI) in metastatic melanoma and in adjacent healthy skin tissue. In the study on 38 patients with metastatic melanoma, the expression of p53, p73, NME and GLI protein families was determined by western blot analysis. Protein expression analysis showed elevated expression of Δ133p53α, Δ160p53α, ΔNp73α, NME1, NME2, GLI1_160 kDa, GLI1_130 kDa and GLI2_133 kDa in tumor samples compared with healthy tissue samples. Strong correlations were detected only between TAp73β and p53α, and GLI3R and p53α. The remaining correlations were mostly moderate (p53α with Δ160p53α, TAp73α, ΔNp73α, GLI1_118 and GLI1_160). Interestingly, Δ133p53β was negatively correlated with p53α. We have shown that p53 forms complexes with p53 and p73 isoforms hence inhibiting its transcriptional and apoptotic activity. Although NME and GLI did not form complexes with p53, they were able to modify its activity. Defining the interactions between protein p53 and other proteins could contribute to better understanding of the molecular basis of melanoma and finally lead to the development of new approaches in melanoma treatment.

Tumor supresorski gen p53 ima kritičnu ulogu u kontroli staničnog rasta i održavanju genetske stabilnosti. Ove aktivnosti posljedica su činjenice da p53 stvara tetramere koji se vežu za određeni slijed na DNA i aktiviraju transkripciju. Gubitkom tumor supresorske aktivnosti, mutirani proteini p53 dobivaju onkogeni potencijal što rezultira nekontroliranim rastom stanica tumora. Otkrivena su i dva srodnika gena p53- p63 i p73, koji pokazuju veliku strukturnu sličnost s genom p53, ali funkcionalno velike razlike. Sva tri gena imaju dva promotora, što zajedno s alternativnim izrezivanjem i alternativnim početkom translacije daje brojne izoforme proteina s različitim funkcijama. Tako geni p63 i p73 imaju vrlo složenu strukturu i kodiraju dvije skupine izoformi proteina koje imaju suprotno funkcionalno djelovanje- izoforme s transaktivacijskim područjem (TAp63/73) koje imaju tumor supresorsko djelovanje i izoforme bez transaktivacijskog područja (ΔNp63/73) s potencijalnim onkogenim djelovanjem djeluju kao transdominantni inhibitori TAp73 i divljeg tipa p53. Nedavno je utvrđeno da i p53 sadrži još jedan promotor, te da dolazi do sinteze višestrukih izoformi, čija funkcija nije još u potpunosti jasna. U ovom preglednom radu opisane su različite izoforme p63, p73 i p53 te njihova uloga u razvoju i tumorigenezi. Istraživanja članova obitelji p53, posebice njihovih međusobnih interakcija pridonijet će rasvjetljavanju uloge ovih proteina u nastanku i razvoju tumora.

Stanična senescencija je trajni izlazak stanica iz ciklusa do kojeg dolazi zbog skraćivanja telomera, aktivacije onkogena i oštećenja DNA. Protein p73 (član obitelji proteina p53) ima niz uloga u odgovoru na oštećenje DNA, a njegova uloga u tumorigenezi je složena. Postoje mnogobrojne izoforme proteina p73 svrstane u dvije glavne skupine: TAp73 i ΔNp73. Potaknuti potencijalnim onkogenim djelovanjem izoforme ΔNp73α ispitali smo njezin utjecaj na ulazak stanica u senescenciju izazvanu oštećenjem DNA γ-zračenjem u normalnim fibroblastima i staničnoj liniji osteosarkoma čovjeka. Senescentne stanice detektirane su praćenjem endogene β-galaktozidazne aktivnosti povezane sa starenjem, pri čemu je pojačana ekspresija ΔNp73α dovela do umjerenog porasta udjela senescentnih stanica. Metodom Western hibridizacije ispitana je ekspresija proteina uključenih u regulaciju staničnog ciklusa nakon oštećenja DNA, a dobiveni rezultati ukazuju na višu ekspresiju markera stanične proliferacije pod utjecajem ΔNp73α. Dobiveni rezultati predstavljaju vrijednu podlogu za buduća istraživanja uloge izoforme ΔNp73α u tumorigenezi. Cellular senescence is permanent cell cycle exit caused by telomere shortening, oncogene activation and DNA damage. P73 protein (a p53 protein family member) has different functions in DNA damage response, and its role in tumorigenesis is complex. There are multiple p73 protein isoforms divided into two main groups: TAp73 and ΔNp73. Encouraged by potentially oncogenic activity of ΔNp73α isoform, we investigated its impact on senescence onset after DNA damage induced by γ-irradiation in normal human fibroblasts and osteosarcoma cell line. Senescent cells were detected by analysis of endogenous senescence associated β-galactosidase activity, showing moderate increase in the frequency of senescence in cells overexpressing ΔNp73α. Expression of proteins involved in cell cycle regulation after DNA damage was analyzed by Western blot, suggesting higher expression of the markers of cellular proliferation caused by overexpression of ΔNp73α. These results represent a valuable basis for future investigation of the ΔNp73α role in tumorigenesis.