1. Functional role of Pak1/Erk signaling in Rac-related diseases Daniela Araiza-Olivera, Jennifer Rhodes, and Jonathan Chernoff
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INTRODUCTION
The RAS/MAPK signaling pathway regulates key cellular processes, including proliferation, differentiation, and survival. Somatic mutations of the RAS pathway are found in 30% of human cancers, including pancreatic, colon, thyroid, lung and melanoma. Furthermore, germline mutations in this pathway, collectively termed Rasopathy syndromes, cause abnormal development.
As p2-1activated kinases (Paks) have been shown to be required for the activation of ERK by Ras, we evaluated Pak1 and its activator Rac1 as potential therapeutic targets in both Rasopathies and in melanoma.
Figure 3. Rac1 expression and its treatment can modify the developmental phenotypes.
A. Treatment with 1 mM Rac1 (pink) and BRAF (yellow) inhibitors affect the development of Rac1P29S and BRAFV600E mutants. B. Zebrafish Western blotting reveals the activity of p-ERK and p-PAK in presence or absence of the inhibitors. C. Microinjection of Rac1 promotes changes in the heart development at 72 hpf and can be rescued by PAK or ERK inhibitors, but not a BRAF inhibitor. A B C
Figure 1. Overview of Ras/MAPK pathway
We then studied the effect of these small molecule inhibitors in human malignant melanoma cell lines bearing activating mutations in BRAF, PREX (an activator of Rac1), and/or RAC1. Treatment with PAK or RAC inhibitors decreased the migration and proliferation of cells with mutant PREX or RAC1 mutations, but not those with BRAF mutations; the reverse was true when these cells were treated with a BRAF inhibitor. These data suggest that Pak1 might serve as a useful therapeutic target in PREX or RAC1driven cancers such as malignant melanoma. A B
Rasopathies are developmental abnormalities in newborns caused by germline gain-of-function mutations in the RAS pathway. An underlying theme in Rasopathy syndromes is the sustained activation of ERK during embryonic development, which often manifests as craniofacial aberrancies, skin lesions, cardiac defects, and cognitive defects (Zenker M. Current opinion in pediatrics, 2011).
In zebrafish, expression of gain-of-function mutants of RAS pathways factors N-RAS, H-RAS, SOS, SHP2, BRAF, and MEK1 induce aberrant jaw cartilage development, as well as cardiac defects. These abnormalities are consistent with the aberrancies found in the human syndromes and are associated with defective migration of neural crest cells (Kelly ML, et al. Dev Cell,. 2014).
Despite current knowledge, it remains poorly understood whether PAK1 has a role in the etiology of Rasopathy syndromes.
PAK1 is a serine/threonine kinase that acts downstream of the small GTPases CDC42 and RAC1 to regulate a multitude of cellular processes. It phosphorylates c-RAF and MEK1, and we and others have shown that loss of PAK1 activity leads to loss of c-RAF, MEK1, and subsequent ERK activation in many cell types. Importantly, we have recently shown that zPAK1 is essential for ERK activation and neural crest migration in zebrafish. Given the essential role of PAK1 in ERK activation in vertebrates, we speculated that this kinase might play a role in the pathogenesis of Rasopathies and might serve as a useful therapeutic target.
Using a zebrafish model, we first asked if the phenotypic consequences of Ras pathway overactivation during development could be blocked by Pak small molecule inhibitors. Introducing gain-of-function genes such as BRAFWT, BRAFV600E, KRAS4AG12V and Rac1P29S into one-cell zebrafish embryos produced a Rasopathy phenotype, characterized by abnormal head and heart development. These phenotypes were suppressed by a Pak-specific small molecule inhibitor. pERK and pPAK levels were increased in embryos expressing BRAFV600E, KRAS4AG12V and Rac1P29S, and were restored to baseline upon addition of Pak1 or Rac1 small molecule inhibitors. C
Figure 4. Consequences of small molecule inhibitors in melanoma cell signaling and proliferation.
Melanoma cell lines with mutations on BRAF, NRAS, PREX and RAC1 treated with 1 mM of BRAF (blue), MEK (red) and PAK1 (green) inhibitors. A. Cell migration is completely affected by MEK inhibitor, however PAK1 inhibitor only affects the PREX and RAC1 cell lines. B. Western blot analysis with p-AKT, p-ERK, p-PAK1 and RAC1 demonstrate that the specific drugs reduce the cell signaling. C. Cell proliferation response is decreased by MEK inhibitor, while PAK1 inhibitor decrease its specific targets.
Figure 2. Small molecule inhibitors prevents Rasopathy phenotype.
A. Developomental defects produced by the microinjection of BRAFV600E, KRAS-4AG12D, and RAC1P29S mRNA were reverted by the addition of 1 mM PD (red), Frax 1036, and PF (green). B. Western blotting of the zebrafish lysates demonstrates the levels of p-ERK and p-PAK 1/2, and the effect of the inhibitors. b-actin was used as loading control. A B
CONCLUSION
Overactivation of the RAS/MAPK pathway in zebrafish by BRAFV600E, KRAS-4AG12V, or RAC1P29S causes a phenotype consistent with Rasopathies in humans, which includes defects in cardiac development.
Small molecule inhibitors of the PAK family might be of therapeutic benefit in treatment of melanoma caused by mutations of RAC1P29S .
Our data suggests that there may be multiple access points that could have therapeutic utility in the treatment of Rasopathies or melanoma associated with elevated ERK activity.