The MAP kinase ERK5 signaling pathway is activated in response to growth factors and different forms of stress. During the last few years, several works have shown that ERK5 plays an important role in regulating cell proliferation and survival in different cancer paradigms. However, these experimental evidences were achieved using ERK5 inhibitors that, subsequently, were shown to be non-specific. Among others, first generation ERK5 inhibitors also impair the activity of the transcriptional regulator BRD4 (a known antitumor target). Consequently, the role of ERK5 as a regulator of tumor proliferation and survival has recently been questioned. Here, we have characterized JWG-071, a new generation competitive and specific ERK5 inhibitor, without BRD4 activity. This small compound exhibits excellent pharmacokinetic parameters, and inhibits ERK5 efficiently in cell lines and in xenograft tumors (systemic administration). To perform the preclinical development of JWG-071, we have used endometrioid and cervical gynecological cancer models. The role of ERK5 in these cancer models has not been previously reported. We show that ERK5 regulates proliferation of gynecological cancer cell lines, through activation of the transcription factor AP-1. Thus, pharmacological (JWG-071) or genetic (CRISPR/Cas9 MEK5-/- cells) inhibition of ERK5 impairs proliferation and growth of endometrioid cancer cells and xenografts (Ishikawa cells). Moreover, in this work we present evidences supporting that ERK5 kinase activity is required for the survival of endometrioid and cervical cancer cell lines. Thus, ERK5 inhibition induces apoptosis in cell lines and in xenografts tumors (Ishikawa cells), by impairing the canonical NF-kB pathway. The NF-kB pathway regulates endometrioid cancer proliferation and survival. Mechanistically, ERK5 inhibition results in a drastic reduction of the upstream regulator NEMO/IKKg, leading to inhibition of p65/RELA expression, nuclear localization and transcriptional activity. Furthermore, we show that inhibition of NF-kB in response to JWG-071 results in activation of the JNK-regulated apoptotic pathway, both in vitro (cell lines) and in vivo (tumor xenografts). In agreement with this, human endometrioid tumors show elevated levels of ERK5 and RELA proteins, compared to peritumoral tissue. Finally, we show that ERK5 inhibition sensitizes endometrioid and cervical cancer cells to standard chemotherapy. Of interest, systemic administration of JWG-071 sensitizes endometrioid xenografts tumors (Ishikawa cells) to paclitaxel treatment. Overall, we propose ERK5 as a new target to tackle endometrioid cancer. We also provide preliminary evidences showing that nuclear ERK5 plays a role in modulating neuroblastoma cancer survival and resistance to chemotherapy. Using a panel of NBL cell lines, we found that chemosensitive (functional p53) NBL cells are sensitive to ERK5 inhibitors, and show a cytosolic ERK5 localization. On the contrary, high-risk chemoresistant (non-functional p53) NBL cells are less sensitive to ERK5 inhibitors and show nuclear ERK5 localization. Interestingly, chemosensitive NBL IMR-32 and SH-SY5Y cells became resistant to cisplatin in response to overexpression of a constitutively nuclear form of ERK5, by a mechanism that impairs p53 expression and transcriptional activity. Finally, we performed a transcriptome (DNA microarrays) analysis to investigate the impact of ERK5 silencing (shRNAi) in high-risk chemoresistant NBL CHLA-90 and SK-N-BE(2) cell lines. Preliminary analysis show that depletion of the ERK5 protein induces changes in the expression of genes related to cell cycle, transcription and apoptosis. These results leads us to propose chemoresistant NBL as a model to validate the antitumor action of ERK5 degrader drugs (PROTACs).
MAP quinasa ERK5: una nueva diana antitumoral en cáncer endometrioide y neuroblastoma
Diéguez Martínez, N. (Author). 30 Jun 2021
Student thesis: Doctoral thesis
Student thesis: Doctoral thesis