Introduction

Selenoprotein O is one of only 25 selenoproteins that incorporate the 21^st genetically encoded amino acid, selenocysteine. Despite the wide range of physiological importance, the biochemical activity of several selenoproteins remains a mystery. Selenoprotein O (SelO) is an evolutionarily conserved enzyme that catalyzes AMPylation, the covalent addition of AMP from ATP to protein substrates. SelO localizes to the mitochondria to AMPylate multiple key enzymes in cellular energy metabolism and redox biology. Excitingly, AMPylation is a new post translational modification in the mitochondria, which may be analogous to phosphorylation in defining a new branch of mitochondrial signaling. Given the importance of redox homeostasis and metabolic rewiring in cancer metastasis, we examined the role of SelO in melanoma metastasis.

Methods

We used a murine immunocompetent model of melanoma metastasis with luciferase tagged isogenic cell lines, YUMM 3.3,  with defined genetic mutations (Braf^V600E/wt ,Cdkn2^−/−). We generated SelO deficient YUMM3.3 cell lines using CRISPR/Cas9 and verified the deletion using protein immunoblotting and DNA sequencing. To test the importance of SelO in metastasis, we performed subcutaneous or intravenous injection of YUMM3.3 or YUMM 3.3 SelO^−/− in C57BL6 mice and detected micro- and macrometastases by bioluminescence imaging. 

To determine the molecular and cellular role of SelO in the mitochondria, we incubated SelO with highly enriched mitochondria in AMPylation assays using biotin labeled ATP analog. We identified mitochondrial substrates of SelO using avidin pulldown followed by mass spectrometry analysis. Furthermore, we analyzed proliferation, invasion, migration, ROS and oxygen consumption rates of wildtype YUMM3.3 in comparison to YUMM 3.3 SelO^−/−.

Results

Here, we show that SelO deficiency limits metastasis in an immunocompetent model of melanoma. Such impaired metastasis is rescued by antioxidant treatment, suggesting that SelO-deficiency impairs metastasis at least partly by increasing oxidative stress. Mechanistically, SelO AMPylates multiple mitochondrial proteins to alter mitochondrial metabolism and redox adaptations. 

Conclusion

This work provides the first evidence that suggests that melanoma cells become more dependent upon SelO during metastasis. Congruently, high SelO expression is associated with increased risk for death in metastatic melanoma patients. Here, we show that SelO AMPylates mitochondrial proteins to promote melanoma metastasis. Our results highlight the importance of selenoproteins in metastasizing cancer cells and shed light on a novel post-translational modification in cancer biology.