MEK inhibitors and DA-Raf, a dominant-negative antagonist of the Ras-ERK pathway, prevent the migration and invasion of KRAS-mutant cancer cells.

The Ras-induced ERK pathway (Raf-MEK-ERK signaling cascade) regulates a variety of cellular responses including cell proliferation, survival, and migration. Activating mutations in RAS genes, particularly in the KRAS gene, constitutively activate the ERK pathway, resulting in tumorigenesis, cancer cell invasion, and metastasis. DA-Raf1 (DA-Raf) is a splicing isoform of A-Raf and contains the Ras-binding domain but lacks the kinase domain. Consequently, DA-Raf antagonizes the Ras-ERK pathway in a dominant-negative manner and can serve as a tumor suppressor that targets mutant Ras protein-induced tumorigenesis. We show here that MEK inhibitors and DA-Raf interfere with the in vitro collective cell migration and invasion of human KRAS-mutant carcinoma cell lines, the lung adenocarcinoma A549, colorectal carcinoma HCT116, and pancreatic carcinoma MIA PaCa-2 cells. DA-Raf expression was silenced in these cancer cell lines. All these cell lines had high collective migration abilities and invasion properties in Matrigel, compared with nontumor cells. Their migration and invasion abilities were impaired by suppressing the ERK pathway with the MEK inhibitors U0126 and trametinib, an approved anticancer drug. Expression of DA-Raf in MIA PaCa-2 cells reduced the ERK activity and hindered the migration and invasion abilities. Therefore, DA-Raf may function as an invasion suppressor protein in the KRAS-mutant cancer cells by blocking the Ras-ERK pathway when DA-Raf expression is induced in invasive cancer cells.

DA-Raf and the MEK inhibitor trametinib reverse skeletal myocyte differentiation inhibition or muscle atrophy caused by myostatin and GDF11 through the non-Smad Ras-ERK pathway.

Myostatin (Mstn) and GDF11 are critical factors that are involved in muscle atrophy in the young and sarcopenia in the elderly, respectively. These TGF-ß superfamily proteins activate not only Smad signalling but also non-Smad signalling including the Ras-mediated ERK pathway (Raf-MEK-ERK phosphorylation cascade). Although Mstn and GDF11 have been shown to induce muscle atrophy or sarcopenia by Smad2/3-mediated Akt inhibition, participation of the non-Smad Ras-ERK pathway in atrophy and sarcopenia has not been well determined. We show here that both Mstn and GDF11 prevented skeletal myocyte differentiation but that the MEK inhibitor U0126 or trametinib restored differentiation in Mstn- or GDF11-treated myocytes. These MEK inhibitors induced the expression of DA-Raf1 (DA-Raf), which is a dominant-negative antagonist of the Ras-ERK pathway. Exogenous expression of DA-Raf in Mstn- or GDF11-treated myocytes restored differentiation. Furthermore, administration of trametinib to aged mice resulted in an increase in myofiber size or recovery from muscle atrophy. The trametinib administration downregulated ERK activity in these muscles. These results imply that the Mstn/GDF11-induced Ras-ERK pathway plays critical roles in the inhibition of myocyte differentiation and muscle regeneration, which leads to muscle atrophy. Trametinib and similar approved drugs might be applicable to the treatment of muscle atrophy in sarcopenia or cachexia.