The BRAF kinase is mutated, typically V600E, to induce an active oncogenic state in a large fraction of melanoma, thyroid, hairy cell leukemia, and to a lesser extent, a wide spectrum of other cancers1,2. BRAFV600E phosphorylates and activates the kinases MEK1 and MEK2, which in turn phosphorylate and activate the kinases ERK1 and ERK2, stimulating the MAPK pathway to promote cancer3. Targeting MEK1/2 is proving to be an important therapeutic strategy, as a MEK1/2 inhibitor provides a survival advantage in metastatic melanoma4, which is increased when co-administered with a BRAFV600E inhibitor5. In this regard, we previously found that copper (Cu) influx enhances MEK1 phosphorylation of ERK1/2 through a Cu-MEK1 interaction6. We now show that genetic loss of the high affinity Cu transporter Ctr1 or mutations in MEK1 that disrupt Cu binding reduced BRAFV600E-driven signaling and tumorigenesis. Conversely, a MEK1-MEK5 chimera that phosphorylates ERK1/2 independent of Cu or an active ERK2 restored tumor growth to cells lacking Ctr1. Importantly, Cu chelators used in the treatment of Wilson disease7 reduced tumor growth of both BRAFV600E-transformed cells and cells resistant to BRAF inhibition. Taken together, these results suggest that Cu-chelation therapy could be repurposed to treat BRAFV600E mutation-positive cancers.

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c Copper (Cu) is essential for development and proliferation, yet the cellular requirements for Cu in these processes are not well defined. We report that Cu plays an unanticipated role in the mitogen-activated protein (MAP) kinase pathway. Ablation of the Ctr1 high-affinity Cu transporter in flies and mouse cells, mutation of Ctr1, and Cu chelators all reduce the ability of the MAP kinase kinase Mek1 to phosphorylate the MAP kinase Erk. Moreover, mice bearing a cardiac-tissue-specific knockout of Ctr1 are deficient in Erk phosphorylation in cardiac tissue. In vitro investigations reveal that recombinant Mek1 binds two Cu atoms with high affinity and that Cu enhances Mek1 phosphorylation of Erk in a dose-dependent fashion. Coimmunoprecipitation experiments suggest that Cu is important for promoting the Mek1-Erk physical interaction that precedes the phosphorylation of Erk by Mek1. These results demonstrate a role for Ctr1 and Cu in activating a pathway well known to play a key role in normal physiology and in cancer. Copper (Cu) is a metal ion that functions as a redox-active cofactor for a broad range of biochemical reactions, including mitochondrial oxidative phosphorylation, protection from reactive oxygen species, connective tissue maturation, iron absorption, neuropeptide biogenesis, and other processes (28, 43). Numerous studies point to the essentiality of Cu for normal growth and development, while aberrant Cu accumulation in tissues, as manifested in Wilson's disease patients, results in significant pathologies (33,35,42,47,60,61). However, the precise roles Cu plays and the mechanistic processes by which Cu drives cellular proliferation and growth are not well understood.The Ras/mitogen-activated protein kinase (MAPK) signaling pathway is an evolutionarily conserved pathway involved in the control of many fundamental biological processes, including cell proliferation, apoptosis, survival, differentiation, motility, and metabolism (26,30). Aberrant Ras/MAPK signaling has significant consequences; loss of function of several components of the Ras/MAPK signaling cascade results in lethality, whereas gain-offunction mutations in many of the Ras/MAPK signaling components underlie cancer (2,12,26,55).Here we identify the Ctr1 high-affinity Cu ϩ transporter, conserved from yeast to humans, as being important for stimulation of the MAPK Erk in response to extracellular growth factor-mediated activation of the Ras signaling pathway. Moreover, genetic, physiological, and biochemical experiments point to a direct role for Cu in the ability of the MAPK kinase Mek1 to phosphorylate Erk in fruit flies, cultured cells, and mice. These studies suggest that the MAPK signaling pathway is a key cellular proliferation pathway that is stimulated by Cu and may be a direct target of potent cancer chemotherapeutics that function via Cu chelation. MATERIALS AND METHODS Drosophila melanogaster stocks and crosses. Phantom Gal4, UAS mCD8::GFP/TM6, Tb flies were from Michael O'Connor, University of Minnesota (44). The UAS-Ctr1ARNA...

A yeast-based small molecule screen identifies a novel activator of human HSF1 and protein chaperone expression and which appears to alleviate the toxicity of protein misfolding diseases.

Copper (Cu) is an essential cofactor for a variety of metabolic functions and the regulation of systemic Cu metabolism is critical to human health. While dietary Cu is absorbed through the intestine, stored in the liver and mobilized into the circulation, systemic Cu homeostasis is poorly understood. We generated mice with a cardiac specific knock out of the Ctr1 Cu transporter, resulting in cardiac Cu deficiency (Ctr1hrt/hrt) and severe cardiomyopathy. Unexpectedly, Ctr1hrt/hrt mice exhibited an increase in serum Cu levels and a concomitant decrease in hepatic Cu stores. Expression of the ATP7A Cu exporter, thought to function predominantly in intestinal Cu acquisition, was strongly increased in liver and intestine of Ctr1hrt/hrt mice. These studies identify ATP7A as a candidate for hepatic Cu mobilization in response to peripheral tissue demand and illuminate systemic regulation that signals the Cu status of the heart to Cu uptake and storage organs.

Copper is an essential trace element required by all aerobic organisms as a cofactor for enzymes involved in normal growth, development, and physiology. Ctr1 proteins are members of a highly conserved family of copper importers responsible for copper uptake across the plasma membrane. Mice lacking Ctr1 die during embryogenesis from widespread developmental defects, demonstrating the need for adequate copper acquisition in the development of metazoan organisms via as yet uncharacterized mechanisms. Whereas the fruit fly, Drosophila melanogaster, expresses three Ctr1 genes, ctr1A, ctr1B, and ctr1C, little is known about their protein isoform-specific roles. Previous studies demonstrated that Ctr1B localizes to the plasma membrane and is not essential for development unless flies are severely copper-deficient or are subjected to copper toxicity. Here we demonstrate that Ctr1A also resides on the plasma membrane and is the primary Drosophila copper transporter. Loss of Ctr1A results in copper-remedial developmental arrest at early larval stages. Ctr1A mutants are deficient in the activity of copper-dependent enzymes, including cytochrome c oxidase and tyrosinase. Amidation of Phe-Met-Arg-Pheamides, a group of cardiomodulatory neuropeptide hormones that are matured via the action of peptidylglycine ␣-hydroxylating monooxygenase, is defective in neuroendocrine cells of Ctr1A mutant larvae. Moreover, both the Phe-Met-Arg-Pheamide maturation and heart beat rate defects observed in Ctr1A mutant larvae can be partially rescued by exogenous copper. These studies establish clear physiological distinctions between two Drosophila plasma membrane copper transport proteins and demonstrate that copper import by Ctr1A is required to drive neuropeptide maturation during normal growth and development.

The diagnosis, classification, and management of cancer are traditionally dictated by the site of tumor origin, for example, breast or lung, and by specific histologic subtypes of site-oforigin cancers (e.g., non-small cell versus small cell lung cancer). However, with the advent of sequencing technologies allowing for rapid, low cost, and accurate sequencing of clinical samples, new observations suggest an expanded or different approach to the diagnosis and treatment of cancer-one driven by the unique molecular features of the tumor. We discuss a genomically driven strategy for cancer treatment using BRAF as an example. Several key points are highlighted: (i) molecular aberrations can be shared across cancers; (ii) approximately 15% of all cancers harbor BRAF mutations; and (iii) BRAF inhibitors, while approved only for melanoma, have reported activity across numerous cancers and related disease types bearing BRAF aberrations. However, BRAF-mutated colorectal cancer has shown poor response rate to BRAF inhibitor monotherapy, striking a cautionary note. Yet, even in this case, emerging data suggest BRAF-mutated colorectal cancers can respond well to BRAF inhibitors, albeit when administered in combination with other agents that impact resistance pathways. Taken together, these data suggest that molecular aberrations may be the basis for a new nosology for cancer. Mol Cancer Ther; 15(4); 533-47. Ó2016 AACR.

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