Optimization of Extracellular signal-regulated kinase 5 (ERK5) targeting in melanoma.

Melanoma is the deadliest skin cancer with a very poor prognosis in advanced stages. Although targeted and immune therapies have improved survival, not all patients benefit from these treatments. It has been reported that the mitogen-activated protein kinase ERK5 is consistently express in human melanoma and supports melanoma cell proliferation in vitro and in vivo. However, ERK5 inhibition results in cell-cycle arrest rather than appreciable apoptosis. To clarify the role of ERK5 in melanoma growth, we performed transcriptomic analyses following ERK5 knockdown in melanoma cells expressing BRAFV600E and found that cellular senescence was among the most affected processes. In melanoma cells expressing either wild-type or mutant (V600E) BRAF, both genetic and pharmacologic inhibition of ERK5 elicited cellular senescence, as observed by a marked increase in senescence-associated b-galactosidase activity and p21 expression. In addition, depletion of ERK5 from melanoma cells resulted in increased levels of CXCL1, CXCL8, and CCL20, proteins typically involved in the senescence-associated secretory phenotype. Knockdown of p21 suppressed the induction of cellular senescence by ERK5 blockade, pointing to p21 as a key mediator of this process. In vivo, ERK5 knockdown or inhibition with XMD8-92 in melanoma xenografts promoted cellular senescence. Based on these results, small-molecule compounds targeting ERK5 constitute a rational series of prosenescence drugs that may be exploited for melanoma treatment. ERK5 pro-proliferative activities are linked to its presence in the nucleus, but the mechanisms involved in ERK5 nuclear translocation are poorly characterized. We focused on the elucidation of this process to found novel targets and compounds able to prevent ERK5 nuclear shuttling, in order to design new strategies for cancer treatment. To achieve single ERK5 tracking in living cells, we used a Super-Resolution microscope. HeLa cells have been transfected with an expression vector for ERK5, linked to HaloTag, alone or with a vector for a constitutively active form of the ERK5 activator MEK5 (MEK5DD). The cell-permeable photoactivatable chromophore JaneliaFluor646, able to recognise Halo Tag, has been used as detection technique for super resolution imaging. As a complementary approach, HeLa cells, transfected with ERK5 and MEK5DD, have been treated with the alpha/beta importin-mediated transport inhibitor Ivermectin (IVM). MTT and 2D-colony forming assays were performed in A375 cells treated with IVM in combination with the ERK5-i AX-15836. The HaloTag technology provides the JaneliaFluor646 selective binding to ERK5 and Highly Inclined and Laminated Optical sheet (HILO) microscopy allow us to collect the signal of individual chromophores. Data showed that in ERK5-transfected cells, the protein is mainly localized in the cytoplasm, whereas it moves in the nucleus with the activator MEK5DD and this effect is partially reverted in cells treated with IVM. Moreover, ERK5 amount in the nuclear fraction of lysates from IVM treated-cells is lower compared to control, suggesting a role of alpha/beta importins in ERK5 nuclear transport. Finally, we found that ERK5i AX-15836, which has been reported to induce ERK5 nuclear translocation in a paradoxical way, reduced melanoma cell proliferation only in combination with IVM. The HaloTag-JaneliaFluor646 method has proven effective for the localization and tracking of single ERK5 molecules with nanometre accuracy, thus providing a novel approach to evaluate how ERK5 moves to the nucleus. The described technique will also help future studies to investigate the mechanism of action of ERK5 in the nucleus. The actors involved in these processes could be identify as novel targets for ERK5 inhibition, and therefore for a possible anticancer therapy.