Copper transporters are responsible for copper isotopic fractionation in eukaryotic cells.

Copper isotopic composition is altered in cancerous compared to healthy tissues. However, the rationale for this difference is yet unknown. As a model of Cu isotopic fractionation, we monitored Cu uptake in Saccharomyces cerevisiae, whose Cu import is similar to human. Wild type cells are enriched in 63Cu relative to 65Cu. Likewise, 63Cu isotope enrichment in cells without high-affinity Cu transporters is of slightly lower magnitude. In cells with compromised Cu reductase activity, however, no isotope fractionation is observed and when Cu is provided solely in reduced form for this strain, copper is enriched in 63Cu like in the case of the wild type. Our results demonstrate that Cu isotope fractionation is generated by membrane importers and that its amplitude is modulated by Cu reduction. Based on ab initio calculations, we propose that the fractionation may be due to Cu binding with sulfur-rich amino acids: methionine and cysteine. In hepatocellular carcinoma (HCC), lower expression of the STEAP3 copper reductase and heavy Cu isotope enrichment have been reported for the tumor mass, relative to the surrounding tissue. Our study suggests that copper isotope fractionation observed in HCC could be due to lower reductase activity in the tumor.

Hypoxia induces copper stable isotope fractionation in hepatocellular carcinoma, in a HIF-independent manner.

Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer, with increasing incidence worldwide. The unrestrained proliferation of tumour cells leads to tumour hypoxia which in turn promotes cancer aggressiveness. While changes in the concentration of copper (Cu) have long been observed upon cancerization, we have recently reported that the isotopic composition of copper is also altered in several types of cancer. In particular, we showed that in hepatocellular carcinoma, tumour tissue contains heavier copper compared to the surrounding parenchyma. However, the reasons behind such isotopic signature remained elusive. Here we show that hypoxia causes heavy copper enrichment in several human cell lines. We also demonstrate that this effect of hypoxia is pH, HIF-1 and -2 independent. Our data identify a previously unrecognized cellular process associated with hypoxia, and suggests that in vivo tumour hypoxia determines copper isotope fractionation in HCC and other solid cancers.