Inhibition of the Ras-Net (Elk-3) Pathway by a Novel Pyrazole that Affects Microtubules

Abstract

Net (Elk-3/SAP-2/Erp) is a transcription factor that is phosphorylated and activated by the Ras-extracellular signal-regulated kinase (Erk) signaling pathway and is involved in wound healing, angiogenesis, and tumor growth. In a cellbased screen for small molecule inhibitors of Ras activation of Net transcriptional activity, we identified a novel pyrazole, XRP44X. XRP44X inhibits fibroblast growth factor 2 (FGF-2)-induced Net phosphorylation by the Ras-Erk signaling upstream from Ras. It also binds to the colchicine-binding site of tubulin, depolymerizes microtubules, stimulates cell membrane blebbing, and affects the morphology of the actin skeleton. Interestingly, Combretastin-A4, which produces similar effects on the cytoskeleton, also inhibits FGF-2 RasNet signaling. This differs from other classes of agents that target microtubules, which have either little effect (vincristine) or no effect (docetaxel and nocodazole) on the Ras-Net pathway. XRP44X inhibits various cellular properties, including cell growth, cell cycle progression, and aortal sprouting, similar to other molecules that bind to the tubulin colchicine site. XRP44X has the potentially interesting property of connecting two important pathways involved in cell transformation and may thereby represent an interesting class of molecules that could be developed for cancer treatment.

Involvement of Net and Hif1α in Distinct yet Intricately Linked Hypoxia-induced Signaling Pathways

“…We confirmed that these bands are down-regulated by siRNAs against Net in 444 (data not shown), similar to other cell lines we have studied (8,21). As reported previously, there is an additional non-identified band (n.i.)…”

“…Levels-To study the integration of Net in the hypoxia/PHD/ HIF1␣ signaling pathway, we compared the hypoxic regulation (8,21). We confirmed that these bands are down-regulated by siRNAs against Net in 444 (data not shown), similar to other cell lines we have studied (8,21).…”

ELK3 promotes the migration and invasion of liver cancer stem cells by targeting HIF-1α

“…Previous studies demonstrated that ELK3 participates in the regulation of various genes, including c-fos, egr-1, and PAI-1. In addition, ELK3 is known as a regulator of HIF-1α expression by modulating HIF-1α stability (21)(22)(23)30,31). HIF-1α plays a key role in the induction of cancer invasion, metastasis and angiogenesis, as well as in cancer growth, glucose metabolism, and other metastasis-associated signaling pathways (32)(33)(34)(35)(36).…”

“…ELK3 is activated by mitogen-activated protein kinase (MAPK)-associated pathways, such as the Ras/extracellular signal-regulated kinase (ERK) and p38 pathways, and it plays an important role in various physiological processes, including cell migration, invasion, wound healing, angiogenesis and tumorigenesis, via the regulation of c-fos, early growth response protein 1 (egr-1), and plasminogen activator inhibitor-1 (PAI-1) (19)(20)(21)(22)(23). Mutations in ELK3 disrupt vasculogenesis, angiogenesis and wound healing in mice during development and in adulthood (24,25).…”

“…Previous studies revealed that several genes including Egr-1, PAI-1 and HIF-1α are ELK3 target genes by forming ternary complexes. Among these target genes, HIF-1α is known to stimulate angiogenic and metastatic responses by activating transcription of the genes encoding several growth factors, including VEgF and MMP-2 (19)(20)(21)(22)(23)35,36).…”