Summary Reactive oxygen species (ROS) activate NF-E2-related transcription factor 2 (Nrf2), a key transcriptional regulator driving antioxidant gene expression and protection from oxidant injury. Here we report that in response to elevation of intracellular ROS above a critical threshold, Nrf2 stimulates expression of transcription Kruppel-like factor 9 (Klf9), resulting in further Klf9-dependent increases in ROS and subsequent cell death. We demonstrated that Klf9 independently causes increased ROS levels in various types of cultured cells and in mouse tissues and is required for pathogenesis of bleomycin-induced pulmonary fibrosis in mice. Mechanistically, Klf9 binds to the promoters and alters the expression of several genes involved in the metabolism of ROS, including suppression of thioredoxin reductase 2, an enzyme participating in ROS clearance. Our data reveal an Nrf2-dependent feed-forward regulation of ROS and identify Klf9 as a novel ubiquitous regulator of oxidative stress and lung injury.

Malignant melanoma possesses one of the highest metastatic potentials among human cancers. Acquisition of invasive phenotypes is a prerequisite for melanoma metastases. Elucidation of the molecular mechanisms underlying melanoma invasion will greatly enhance the design of novel agents for melanoma therapeutic intervention. Here, we report that guanosine monophosphate synthase (GMPS), an enzyme required for the de novo biosynthesis of GMP, has a major role in invasion and tumorigenicity of cells derived from either BRAF V600E or NRAS Q61R human metastatic melanomas. Moreover, GMPS levels are increased in metastatic human melanoma specimens compared with primary melanomas arguing that GMPS is an attractive candidate for anti-melanoma therapy. Accordingly, for the first time we demonstrate that angustmycin A, a nucleoside-analog inhibitor of GMPS produced by Streptomyces hygroscopius efficiently suppresses melanoma cell invasion in vitro and tumorigenicity in immunocompromised mice. Our data identify GMPS as a powerful driver of melanoma cell invasion and warrant further investigation of angustmycin A as a novel anti-melanoma agent.

GTP is a major regulator of multiple cellular processes, but tools for quantitative evaluation of GTP levels in live cells have not been available. Here we report characterization of genetically encoded GTP sensors, constructed by inserting cpYFP into a region of the bacterial FeoB G-protein that undergoes a GTP-driven conformational change. GTP binding to these sensors results in a ratiometric change in their fluorescence, thereby providing an internally normalized response to changes in GTP levels while minimally perturbing those levels. Mutations introduced into FeoB to alter its affinity for GTP allowed generation of sensors with a wide dynamic range. Critically, in mammalian cells the sensors show consistent changes in fluorescence intensity ratios upon depletion or restoration of GTP pools. These sensors are suitable for detecting spatio-temporal changes in GTP levels in living cells, and for the development of high throughput screenings of molecules modulating intracellular GTP levels.

Melanoma progression is associated with increased invasion and, often, decreased levels of microphthalmia-associated transcription factor (MITF). Accordingly, downregulation of MITF induces invasion in melanoma cells, however little is known about the underlying mechanisms. Here, we report for the first time that depletion of MITF results in elevation of intracellular GTP levels and increased amounts of active (GTP-bound) RAC1, RHO-A and RHO-C. Concomitantly, MITF-depleted cells display larger number of invadopodia and increased invasion. We further demonstrate that the gene for guanosine monophosphate reductase (GMPR) is a direct MITF target, and that the partial repression of GMPR accounts mostly for the above phenotypes in MITF-depleted cells. Reciprocally, transactivation of GMPR is required for MITF-dependent suppression of melanoma cell invasion, tumorigenicity, and lung colonization. Moreover, loss of GMPR accompanies downregulation of MITF in vemurafenib-resistant BRAFV600E-melanoma cells and underlies the increased invasion in these cells. Our data uncover novel mechanisms linking MITF-dependent inhibition of invasion to suppression of guanylate metabolism.

No abstract