Rac family small GTPases regulate actin organization, cell cycle progression, vesicular trafficking and gene expression. Growing evidence indicates that cancer cells exploit Rac activity to drive tumor growth, invasion and metastasis. We have devised a novel selection scheme in C. elegans to identify critical components of Rac effector activity for possible pharmacologic targeting in cancer. This system is based on our finding that constitutively activated CED-10/Rac1 (Q61L missense mutation) in hypodermal cells drives lethal disruption of morphogenesis. We reasoned that second-site mutations that reduce CED-10 function also would decrease CED-10-dependent lethality, creating an unambiguous selection condition for genes required by CED-10/Rac1. Furthermore, titration of CED-10(Q61L) levels to a minimal activity threshold for 100% lethality would sensitize the system to modest drops in CED-10(Q61L) output. To meet these criteria, we developed a conditional expression system for modulation of CED-10(Q61L) levels. We drove hypodermal-specific cDNA expression with a portion of the
lin-26 promoter, and for conditional expression we used an aberrantly long 3''UTR sensitive to Smg system nonsense-mediated mRNA decay. Animals expressing CED-10(Q61L)::UTRNMD in the temperature-sensitive
smg-1(
cc546ts) background were mostly viable at 15 deg C, but were inviable when grown at restrictive temperature (24 deg C). Incremental increases in temperature resulted in progressive severity of morphogenetic defects and lethality. Control animals exhibited a ten-fold increase in GFP expression when grown at 23 deg C relative to 15 deg C. We validated the selection system by introducing mutations in known CED-10/Rac1 effectors.
p21-activated kinases (Paks) are the best-characterized Rac effectors in human cells, and we found that CED-10 signals primarily through MAX-2/Pak during distal tip cell migration. At screening temperature (23 deg C), loss of MAX-2 function suppressed CED-10(Q61L)-dependent lethality to 90%. Partial suppression by MAX-2 indicates that CED-10 uses additional effectors during hypodermal morphogenesis. Loss of the Rac effector PES-7/IQGAP restored low-level viability (0.6%), while no suppression was seen with loss of the Rac effector F46F6.2/PKN or the CED-10 effector UNC-115/AbLIM. A pilot F1 clonal screen of 200 genomes yielded two suppressor alleles that confer 15-20% survival at 23 deg C. We will study novel CED-10/Rac1 signaling components identified with our screen directly in human cancer cell lines to analyze potential involvement in Rac-dependent signaling and oncogenic growth transformation.