Although antioxidants promote melanoma metastasis, the role of reactive oxygen species (ROS) in other stages of melanoma progression is controversial. Moreover, genes regulating ROS have not been functionally characterized throughout the entire tumor progression in mouse models of cancer. To address this question, we crossed mice-bearing knock-out of Klf9, an ubiquitous transcriptional regulator of oxidative stress, with two conditional melanocytic mouse models: BrafCA mice, where BrafV600E causes premalignant melanocytic hyperplasia, and BrafCA/Pten−/− mice, where BrafV600E and loss of Pten induce primary melanomas and metastases. Klf9 deficiency inhibited premalignant melanocytic hyperplasia in BrafCA mice but did not affect formation and growth of BrafCA/Pten−/− primary melanomas. It also, as expected, promoted BrafCA/Pten−/− metastasis. Treatment with antioxidant N-acetyl cysteine phenocopied loss of Klf9 including suppression of melanocytic hyperplasia. We were interested in a different role of Klf9 in regulation of cell proliferation in BrafCA and BrafCA/Pten−/− melanocytic cells. Mechanistically, we demonstrated that BRAFV600E signaling transcriptionally upregulated KLF9 and that KLF9-dependent ROS were required for full-scale activation of ERK1/2 and induction of cell proliferation by BRAFV600E. PTEN depletion in BRAFV600E-melanocytes did not further activate ERK1/2 and cell proliferation, but rendered these phenotypes insensitive to KLF9 and ROS. Our data identified an essential role of KLF9-dependent ROS in BRAFV600E signaling in premalignant melanocytes, offered an explanation to variable role of ROS in premalignant and transformed melanocytic cells and suggested a novel mechanism for suppression of premalignant growth by topical antioxidants.
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.
Polyamine inhibition for cancer therapy is, conceptually, an attractive approach but has yet to meet success in the clinical setting. The aryl hydrocarbon receptor (AHR) is the central transcriptional regulator of the xenobiotic response. Our study revealed that AHR also positively regulates intracellular polyamine production via direct transcriptional activation of 2 genes, ODC1 and AZIN1, which are involved in polyamine biosynthesis and control, respectively. In patients with multiple myeloma (MM), AHR levels were inversely correlated with survival, suggesting that AHR inhibition may be beneficial for the treatment of this disease. We identified clofazimine (CLF), an FDA-approved anti-leprosy drug, as a potent AHR antagonist and a suppressor of polyamine biosynthesis. Experiments in a transgenic model of MM (Vk*Myc mice) and in immunocompromised mice bearing MM cell xenografts revealed high efficacy of CLF comparable to that of bortezomib, a first-in-class proteasome inhibitor used for the treatment of MM. This study identifies a previously unrecognized regulatory axis between AHR and polyamine metabolism and reveals CLF as an inhibitor of AHR and a potentially clinically relevant anti-MM agent.
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.
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