The Hedgehog-GLI Pathway Regulates MEK5-ERK5 Expression and Activation in Melanoma Cells

Malignant melanoma is the deadliest skin cancer, with a poor prognosis in advanced stages. We recently showed that the extracellular signal-regulated kinase 5 (ERK5), encoded by the MAPK7 gene, plays a pivotal role in melanoma by regulating cell functions necessary for tumour development, such as proliferation. Hedgehog-GLI signalling is constitutively active in melanoma and is required for proliferation. However, no data are available in literature about a possible interplay between Hedgehog-GLI and ERK5 pathways. Here, we show that hyperactivation of the Hedgehog-GLI pathway by genetic inhibition of the negative regulator Patched 1 increases the amount of ERK5 mRNA and protein. Chromatin immunoprecipitation showed that GLI1, the major downstream effector of Hedgehog-GLI signalling, binds to a functional non-canonical GLI consensus sequence at the MAPK7 promoter. Furthermore, we found that ERK5 is required for Hedgehog-GLI-dependent melanoma cell proliferation, and that the combination of GLI and ERK5 inhibitors is more effective than single treatments in reducing cell viability and colony formation ability in melanoma cells. Together, these findings led to the identification of a novel Hedgehog-GLI-ERK5 axis that regulates melanoma cell growth, and shed light on new functions of ERK5, paving the way for new therapeutic options in melanoma and other neoplasms with active Hedgehog-GLI and ERK5 pathways

PO-099 Targeting the mitogen activated protein kinase ERK5 in human melanoma

Introduction Melanoma is the most aggressive skin cancer with a poor prognosis in advanced stages. Available treatments for melanoma are unsatisfactory, because rapidly lead to an acquired resistance in the majority of cases. Therefore, there is urgent need to identify novel possible targets involved in melanoma growth. ERK5/BMK1 is a member of the Mitogen-Activated Protein Kinases (MAPK) family and regulates cell functions critical for tumour development. Indeed, several studies reported a direct involvement of ERK5 in several types of cancer including prostate and breast cancer and hepatocellular carcinoma. However, no data have been reported about a possible role of ERK5 in melanoma. Material and methods Cell lines and patient-derived primary melanoma cells (wild type B-RAF: SSM2c and M26c; BRAFV600E: A375, SK-Mel-5, SK-Mel-28, 501-Mel, expressing; NRASQ61R: SK-Mel-2; MeWo) have been used for in vitro and in vivo experiments. HEK293T cells were used for protein overexpression. ERK5 inhibition was achieved using ERK5 and MEK5 inhibitors or lentiviral vectors encoding shRNA specific for ERK5. BRAF inhibition was achieved using Vemurafenib, a BRAFV600E inhibitor. Results and discussions In silico data analysis indicated that components of the ERK5 pathway are upregulated in up to 47% melanoma patients. Accordingly, we found that ERK5 is consistently expressed and active in commercial and patients derived melanoma cell lines. On that basis, we investigated the role of ERK5 in melanoma cell growth. In vitro , pharmacological or genetic inhibition of ERK5 decreased the number of viable cells in several melanoma cell lines. Moreover, xenografts performed using LV-shERK5-transduced A375 or SSM2c cells showed a reduced tumour growth when compared to those transduced with control LV-shC. We also found that oncogenic BRAF positively regulates expression, phosphorylation and nuclear localization of exogenous and endogenous ERK5. Accordingly, combined pharmacological inhibition of BRAFV600E and MEK5 is required to decrease nuclear ERK5, that is critical for the regulation of cell proliferation. Furthermore, the combination of MEK5 or ERK5 inhibitors with vemurafenib is more effective than single treatments in reducing 2D colony formation and growth of BRAFV600E melanoma cells and xenografts. Conclusion Our results identify ERK5 as a critical regulator of melanoma growth in vitro and in vivo , and point toward the possibility of targeting ERK5, alone or in combination with BRAF-MEK1/2 inhibitors, for the treatment of melanoma

Inhibition of ERK5 elicits cellular senescence in melanoma via the cyclin-dependent kinase inhibitor p21

2021 The Authors.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. The mitogen-activated protein kinase ERK5 supports the growth of melanoma cells 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 β-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.The work in E. Rovida’s lab was supported by grants from Associazione Italiana per la Ricerca sul Cancro (AIRC, IG-15282 and IG-21349), by Ente Fondazione Cassa di Risparmio di Firenze (ECRF), and Universita degli Studi di Firenze (Fondo di Ateneo ex-60%). A. Tubita was supported by a “Carlo Zanotti” Fondazione Italiana per la Ricerca sul Cancro (FIRC)-AIRC fellowship (ID-23847)

ERK5 is activated by oncogenic BRAF and promotes melanoma growth

Malignant melanoma is among the most aggressive cancers and its incidence is increasing worldwide. Targeted therapies and immunotherapy have improved the survival of patients with metastatic melanoma in the last few years; however, available treatments are still unsatisfactory. While the role of the BRAF-MEK1/2-ERK1/2 pathway in melanoma is well established, the involvement of mitogen-activated protein kinases MEK5-ERK5 remains poorly explored. Here we investigated the function of ERK5 signaling in melanoma. We show that ERK5 is consistently expressed in human melanoma tissues and is active in melanoma cells. Genetic silencing and pharmacological inhibition of ERK5 pathway drastically reduce the growth of melanoma cells and xenografts harboring wild-type (wt) or mutated BRAF (V600E). We also found that oncogenic BRAF positively regulates expression, phosphorylation, and nuclear localization of ERK5. Importantly, ERK5 kinase and transcriptional transactivator activities are enhanced by BRAF. Nevertheless, combined pharmacological inhibition of BRAFV600E and MEK5 is required to decrease nuclear ERK5, that is critical for the regulation of cell proliferation. Accordingly, combination of MEK5 or ERK5 inhibitors with BRAFV600E inhibitor vemurafenib is more effective than single treatments in reducing colony formation and growth of BRAFV600E melanoma cells and xenografts. Overall, these data support a key role of the ERK5 pathway for melanoma growth in vitro and in vivo and suggest that targeting ERK5, alone or in combination with BRAF-MEK1/2 inhibitors, might represent a novel approach for melanoma treatment

ERK5-dependent mechanisms regulate melanoma cell proliferation

Melanoma is the deadliest skin cancer, with a very poor prognosis in advanced stages. Available treatments for melanoma, especially in its intermediate or advanced stages, are unsatisfactory. ERK5 is a member of the Mitogen-Activated Protein kinase family and regulates cell functions critical for tumor development, such as proliferation, invasion and angiogenesis. In silico data analysis indicated that ERK5 pathway components are upregulated in 47% of human melanomas. On this basis, we hypothesized that ERK5 could play a relevant role in melanoma. To study the possible role of ERK5 in the biology of melanoma, we employed a number of human melanoma cell lines. During my PhD, we found that ERK5 is required for the growth of melanoma cells and xenografts harboring wild type (wt) or mutated BRAF (V600E). We later identified a new interplay between ERK5 and the mutated form of B-RAF (V600E) frequently present in melanoma patients. Indeed, using melanoma cells harboring wt BRAF (M26c) and HEK293T cells we found that B-RAFV600E positively regulates ERK5 expression and phosphorylation and increases ERK5 nuclear localization, including that in the chromatin-bound fraction. Accordingly, combined pharmacological inhibition of BRAFV600E and MEK5 is required to decrease nuclear ERK5, that is critical for the regulation of cell proliferation. Furthermore, the combination of MEK5 or ERK5 inhibitors with Vemurafenib is more effective than single treatments in reducing 2D colony formation and growth of BRAFV600E melanoma cells and xenografts. To deepen the knowledge of the molecular mechanisms by which ERK5 controls the growth of melanoma cells, we performed microarray analysis to quantify mRNAs following genetic silencing of ERK5, using two different ERK5-targeting shRNAs (shERK5-275 and shERK5-262), in two different BRAF-mutated melanoma cell lines (A375 and SKMel-5). In particular, by meta-analysis of microarray data, we identified 34 differentially expressed genes (DEGs) (logFC > 1.5) shared by A375 and SKMel-5 cells. Among the above-mentioned genes we further identified 16 upregulated and 18 downregulated DEGs. The idea is to use the upregulated genes in order to identify possible compensative pathways upon ERK5 genetic silencing and to use the downregulated genes to identify new functions of ERK5 in melanoma. The results of the present study shed light on the fact that ERK5 is involved in several mechanisms including, surely, mechanisms involved in melanoma proliferation

MAPK15 protects from oxidative stress-dependent cellular senescence by inducing the mitophagic process

Mitochondria are the major source of reactive oxygen species (ROS), whose aberrant production by dysfunctional mitochondria leads to oxidative stress, thus contributing to aging as well as neurodegenerative disorders and cancer. Cells efficiently eliminate damaged mitochondria through a selective type of autophagy, named mitophagy. Here, we demonstrate the involvement of the atypical MAP kinase family member MAPK15 in cellular senescence, by preserving mitochondrial quality, thanks to its ability to control mitophagy and, therefore, prevent oxidative stress. We indeed demonstrate that reduced MAPK15 expression strongly decreases mitochondrial respiration and ATP production, while increasing mitochondrial ROS levels. We show that MAPK15 controls the mitophagic process by stimulating ULK1-dependent PRKN Ser108 phosphorylation and inducing the recruitment of damaged mitochondria to autophagosomal and lysosomal compartments, thus leading to a reduction of their mass, but also by participating in the reorganization of the mitochondrial network that usually anticipates their disposal. Consequently, MAPK15-dependent mitophagy protects cells from accumulating nuclear DNA damage due to mitochondrial ROS and, consequently, from senescence deriving from this chronic DNA insult. Indeed, we ultimately demonstrate that MAPK15 protects primary human airway epithelial cells from senescence, establishing a new specific role for MAPK15 in controlling mitochondrial fitness by efficient disposal of old and damaged organelles and suggesting this kinase as a new potential therapeutic target in diverse age-associated human diseases

Glutamine Availability Controls BCR/Abl Protein Expression and Functional Phenotype of Chronic Myeloid Leukemia Cells Endowed with Stem/Progenitor Cell Potential

International audienceThis study was directed to characterize the role of glutamine in the modulation of the response of chronic myeloid leukemia (CML) cells to low oxygen, a main condition of hematopoietic stem cell niches of bone marrow. Cells were incubated in atmosphere at 0.2% oxygen in the absence or the presence of glutamine. The absence of glutamine markedly delayed glucose consumption, which had previously been shown to drive the suppression of BCR/Abl oncoprotein (but not of the fusion oncogene BCR/abl) in low oxygen. Glutamine availability thus emerged as a key regulator of the balance between the pools of BCR/Abl protein-expressing and -negative CML cells endowed with stem/progenitor cell potential and capable to stand extremely low oxygen. These findings were confirmed by the effects of the inhibitors of glucose or glutamine metabolism. The BCR/Abl-negative cell phenotype is the best candidate to sustain the treatment-resistant minimal residual disease (MRD) of CML because these cells are devoid of the molecular target of the BCR/Abl-active tyrosine kinase inhibitors (TKi) used for CML therapy. Therefore, the treatments capable of interfering with glutamine action may result in the reduction in the BCR/Abl-negative cell subset sustaining MRD and in the concomitant rescue of the TKi sensitivity of CML stem cell potential. The data obtained with glutaminase inhibitors seem to confirm this perspective