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The redox properties of Copper (Cu) make it both an ideal cofactor for many enzymes, and, in its free form, a highly toxic molecule capable of stimulating production of reactive oxygen species or binding to protein thiol groups. Therefore, living organisms have evolved homeostatic systems to “handle” Cu avoiding dangerous and wasteful aspecific interactions. These systems comprise uptake, carrier, storage and excretion proteins. The importance of Cu-homeostatic systems was initially discovered in humans where alterations of Cu-excretory proteins were shown to be responsible for two lethal genetic disorders; the Wilson and Menkes diseases. The levels of bioavailable Cu in the aquatic environment is important because concentrations in oceanic waters tend to be minute, whilst in some fresh and coastal waters, particularly around areas of mineral extraction, viniculture and farming operations, concentrations can be excessive. In contrast to terrestrial vertebrates, fish are not only exposed to dietary sources of copper but are also exposed to dissolved ionic copper that may enter via the skin and gills. Indeed, the latter route is important in fish and it has been demonstrated in physiological studies that under conditions of dietary deficiency, fish can satisfy their own body requirements by uptake from water. Therefore, fish must have systems relating to both gill and gut to enable maintenance of body homeostasis of this essential, yet toxic, metal. In an attempt to understand the mechanisms of Cu homeostasis in fish, whether under conditions of deficiency, adequacy or excess, it is essential to consider the expression of known Cu-homeostasis proteins. Thus, cDNAs for sea bream (Sparus aurata) homologues of copper transporter 1 (Ctr1), antioxidant protein 1 (Atox1), Menkes protein (ATP7A), Wilson protein (ATP7B), and metallothionein (MT), which are responsible for the uptake, delivery to the secretory pathway and scavenging of intracellular Cu, were cloned and their mRNA tissue expression levels measured. To investigate the molecular basis of the different homeostatic and toxic responses to waterborne or dietary Cu, sea bream were exposed to sub-toxic levels of Cu in the diet (130 mg/Kg of dry diet) or water (0.3 mg/L) and tissue mRNA and Cu levels were measured. Moreover, to discriminate between the effect of different metals on the transcriptional regulation of Cu homeostasis genes in fish, Sparus aurata fibroblast (SAF1) cells were exposed to sub-toxic levels of Cu (25 μM), Zn (100 μM) and Cd (10 μM). In addition, a microarray was used to gain a broader overview of the transcriptional response of SAF1 cells to Cu (25 μM). Waterborne or dietary Cu resulted in distinct expression profiles of Cu-homeostasis genes and markers of oxidative stress. After dietary exposure, Cu increased in intestine and liver, whilst after waterborne exposure Cu increased in gill and liver. Exposure to dietary Cu resulted in decreases in Ctr1 and ATP7A mRNA in both liver and intestine. Renal Ctr1 levels remained unchanged, whilst ATP7A mRNA decreased. In contrast, waterborne Cu exposure increased intestinal Ctr1 and ATP7A mRNA, and increased renal Ctr1 and decreased renal ATP7A mRNA. Both dietary and waterborne Cu increased ATP7B mRNA in liver. Metallothionein (MT) mRNA increased in liver and gill after waterborne Cu. Glutathione reductase (GR), a marker of oxidative stress, increased expression in liver and gill after waterborne Cu exposure, but decreased in intestine. Thus, exposure to Cu via water or diet has different, often opposite effects on Cu-homeostasis genes. The decrease in expression of both Cu-transport genes in intestine after dietary exposure may indicate a defensive mechanism to limit uptake of Cu. The opposite effects in intestine after waterborne exposure are more difficult to explain, but again may reflect a defence mechanism against excess bloodborne Cu coming from the gill. Since both dietary and waterborne Cu increased Cu levels in liver and increased hepatic ATP7B it is likely that well-characterised mammalian route of Cu excretion to bile is active in sea bream. However, only hepatic Cu derived from gill increased the expression of the stress markers MT and GR. This suggests that Cu is delivered to liver in a different form from gill as that from intestine, the intestinally derived pool being less toxic. Thus the increase in copper transport gene expression in intestine after gill exposure might be a mechanism to enable incorporation of excess bloodborne Cu into the intestinal pathway of Cu delivery to liver, thus minimizing toxicity. The in vitro exposure of SAF1 cells to Cu showed a similar response to liver of fish exposed to waterborne Cu indicating similar Cu availability and complexation. ATP7A mRNA levels were induced by Cu but not by Zn or Cd suggesting Cu-specific regulation. Conversely, MT and GR were induced by all metals tested. The transcriptomic analysis highlighted that the biological processes most significantly affected by Cu were secretion, protein trafficking and stress. Overall, these results show that in fish copper has distinct effects on tissue Cu transporter genes and oxidative stress depending on whether it is taken up via the gill or gut and that intestinal absorption may be required for normal uptake and metabolism of Cu, regardless of the route of uptake. Moreover, changes in mRNA levels indicate that Cu homeostasis genes, at least in fish, may be regulated at the transcriptional level. Although more work needs to be done to identify genes that are robust predictors of Cu toxicity, the microarray results presented here show a clear transcriptional fingerprint which may characterize Cu toxicity in fish.

KRAS est parmi les gènes les plus fréquemment mutés dans les cancers humains, tel que ~ 45% des cancers colorectaux (CCR). Malgré les efforts déployés pour réduire son potentiel oncogénique, KRAS muté est fréquemment associé à la résistance aux médicaments et est extrêmement difficile à cibler sur le plan thérapeutique. Les protéines à la surface cellulaire sont souvent dérégulées dans les cancers et sont des cibles thérapeutiques attrayantes en raison de leur accessibilité aux anticorps. Nous avons séquençé les ARNm de cellules épithéliales intestinales exprimant KRAS muté et observé que ces dernières présentaient des changements importants dans les gènes codant pour des protéines de surface cellulaire. Par conséquent, notre objectif était d'identifier de nouvelles cibles thérapeutiques exprimées à la surface de cellules transformées par l’oncogène KRAS. En utilisant une approche de pointe en protéomique de surface cellulaire, nous avons identifié plusieurs protéines différentiellement exprimées dans les cellules avec KRAS muté par rapport à leurs homologues de type sauvage. Nous avons ensuite effectué un crible CRISPR/Cas9 basé sur les protéines de surface cellulaire, qui a révélé que la perte de la protéine Atp7a affectait de manière différentielle les cellules épithéliales intestinales, en fonction de leur statut KRAS. De façon intéressante, nous avons constaté que ATP7A était régulé à la hausse dans les cellules avec KRAS muté par rapport à leurs homologues de type sauvage. ATP7A a un double rôle dans les cellules; alors qu'il est essentiel pour la maturation des enzymes dépendantes du cuivre (Cu), ATP7A protège les cellules d'une toxicité excessive induite par le Cu (cuproptose). Chez l'homme, les mutations dans ATP7A entraînent des troubles caractérisés par des déficiences systémiques dans le transport et les niveaux de Cu. Chez les animaux et dans les modèles de culture cellulaire, tel que les cellules épithéliales intestinales, les niveaux intracellulaires de Cu sont directement corrélés avec l'abondance post-transcriptionnelle d'ATP7A. Dans le même ordre d'idées, nous avons observé que les cellules de CCR avec KRAS muté avaient relativement plus de Cu intracellulaire, et la surexpression d'ATP7A protégeait les cellules KRAS muté de la cuproptose, par rapport à leurs homologues de type sauvage. Nous avons également observé que la croissance in vivo des xénogreffes KRAS mutées était réduite lorsque les souris étaient nourries avec un régime pauvre en Cu. Le Cu est utilisé par plusieurs enzymes qui régulent des fonctions cellulaires critiques, notamment la respiration mitochondriale, la motilité cellulaire et la prolifération. Nous montrons que les cellules mutantes KRAS étaient plus sensibles au chélateur de Cu, ammonium tetrathiomolybdate (TTM), par rapport aux cellules de type sauvage. De plus, les cellules avec KRAS muté traitées avec le TTM ont présenté des activités réduites de MEK1/2 dépendant du Cu et de l'enzyme de la chaîne de transport d'électrons mitochondriale, cytochrome c oxidase (CCO). Nous avons été surpris de constater que le transporteur de Cu de haute affinité, CTR1, est régulé à la baisse dans les cellules avec KRAS muté, et avons donc émis l'hypothèse que les cellules KRAS mutées doivent absorber le Cu par d'autres moyens. Ainsi, nous avons constaté que la macropinocytose agit comme une voie non canonique d'approvisionnement en Cu dans les cellules avec KRAS muté. Le traitement de cellules in vivo avec l'inhibiteur de la macropinocytose, EIPA, a inhibé l'expression d'ATP7A et diminué le Cu biodisponible dans les xénogreffes KRAS mutées. En conclusion, nos résultats montrent que les cellules avec KRAS muté augmentent les niveaux de Cu et d'ATP7A pour soutenir la tumorigenèse en augmentant l'activité cuproenzymatique et diminuant la cuproptose. Cette étude est pertinente pour le cancer, car les tissus tumoraux contiennent fréquemment des niveaux de Cu plus élevés que les tissus normaux. Des études récentes ont mis en évidence un potentiel de repositionnement du chélateur de Cu TTM, qui est disponible en clinique et utilisé pour traiter les troubles du Cu. Nos résultats démontrent que la biodisponibilité du Cu pourrait être exploitée pour traiter le CCR avec KRAS muté avec de tels inhibiteurs. Les travaux futurs comprennent l'identification de stratégies combinatoires qui peuvent être améliorer les effets anti-cancéreux de la chélation du Cu. / KRAS is amongst the most frequently mutated genes driving human cancers, including ~ 45% of colorectal cancers (CRC). Despite intense efforts to curb its oncogenic potential, mutant KRAS is frequently associated with drug resistance and is extremely challenging to target therapeutically. Cell-surface proteins are often spatially dysregulated in cancers and are attractive therapeutic targets due to their easy accessibility. We performed RNA sequencing of mutant KRAS-expressing intestinal epithelial cells and observed that cells undergoing transformation exhibited dramatic changes in cell surface-coding genes. Therefore, our goal was to identify novel druggable targets expressed at the cell surface of mutant KRAS-transformed cells. Using a cutting-edge cell surface proteomics approach, we identified several differentially expressed proteins at the surface of KRAS-mutant cells compared to wild-type counterparts. We then performed a cell surface based CRISPR/Cas9 screen, which revealed that loss of the copper exporter Atp7a differentially affected the fitness of intestinal epithelial cells, depending on their KRAS status. Interestingly, we found that ATP7A was upregulated in KRAS-mutant cells compared to wild-type counterparts. ATP7A has a dual role in cells; while it is essential for maturation of copper (Cu)-dependent enzymes, ATP7A protects cells from excess Cu-induced toxicity (cuproptosis). In humans, ATP7A mutations result in disorders characterized by systemic deficiencies in Cu transport and levels. In animals and in tissue culture models, including intestinal epithelial cells, intracellular Cu levels are directly correlated with the post-transcriptional abundance of ATP7A. In line with this, we observed that KRAS-mutant CRC cells and tissues had relatively more intracellular Cu, and ATP7A-overexpression protected KRAS-mutant cells from cuproptosis, compared to wild-type counterparts. We also observed that in vivo growth of KRAS-mutant xenografts was reduced when mice were fed a Cu-deficient diet. Cu is utilized by several enzymes that regulate critical cellular functions including mitochondrial respiration, cell motility and proliferation. We show that KRAS-mutant cells were more sensitive to the Cu chelating drug ammonium tetrathiomolybdate (TTM), compared to wild-type cells. Moreover, TTM-treated KRAS-mutant cells displayed reduced activities of Cu-dependent MEK1/2 and mitochondrial electron transport chain enzyme, cytochrome c oxidase (CCO). We were surprised to find that the high-affinity CTR1 importer is downregulated in KRAS-mutant cells, and so we hypothesized that KRAS cells must uptake Cu through alternate means. In accordance with this, we found that macropinocytosis acts as a non-canonical Cu-supply route in KRAS-mutant cells. In vivo, treating cells with the macropinocytosis inhibitor EIPA, inhibited the expression of ATP7A and decreased bioavailable Cu in KRAS xenografts. In conclusion, our results show that KRAS-mutant cells increase Cu and ATP7A levels, likely to support tumorigenesis by elevating cuproenzymatic activity and parallelly dealing with cuproptosis. This study is relevant to cancer as tumor tissues and patients contain higher Cu levels than normal controls. Recent studies have highlighted a potential for repurposing the clinically available copper chelator TTM, which is used to treat Cu disorders. Our results demonstrate that copper bioavailability could be exploited to treat KRAS-mutated CRC with such inhibitors. Future work includes identification of combinatorial strategies that may be synthetic lethal to copper chelation.

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