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Despite being the leading cause of cancer deaths, metastasis remains a poorly understood process. To identify novel regulators of metastasis in melanoma, we performed a large-scale RNA sequencing screen of 48 samples from patient-derived xenograft (PDX) subcutaneous melanomas and their associated metastases. In comparison with primary tumors, expression of glycolytic genes was frequently decreased in metastases, whereas expression of some tricarboxylic acid (TCA) cycle genes was increased in metastases. Consistent with these transcriptional changes, melanoma metastases underwent a metabolic switch characterized by decreased levels of glycolytic metabolites and increased abundance of TCA cycle metabolites. A short isoform of glyceraldehyde-3-phosphate dehydrogenase, spermatogenic (GAPDHS) lacking the N-terminal domain suppressed metastasis and regulated this metabolic switch. GAPDHS was downregulated in metastatic nodules from PDX models as well as in human patients. Overexpression of GAPDHS was sufficient to block melanoma metastasis, whereas its inhibition promoted metastasis, decreased glycolysis, and increased levels of certain TCA cycle metabolites and their derivatives including citrate, fumarate, malate, and aspartate. Isotope tracing studies indicated that GAPDHS mediates this shift through changes in pyruvate carboxylase activity and aspartate synthesis, both metabolic pathways critical for cancer survival and metastasis. Together, these data identify a short isoform of GAPDHS that limits melanoma metastasis and regulates central carbon metabolism.Significance:This study characterizes metabolic changes during cancer metastasis and identifies GAPDHS as a novel regulator of these processes in melanoma cells.

Supplementary Table from A Short Isoform of Spermatogenic Enzyme GAPDHS Functions as a Metabolic Switch and Limits Metastasis in Melanoma

Supplementary Figure from A Short Isoform of Spermatogenic Enzyme GAPDHS Functions as a Metabolic Switch and Limits Metastasis in Melanoma

Despite being the leading cause of cancer deaths, metastasis remains a poorly understood process. To identify novel regulators of metastasis in melanoma, we performed a large-scale RNA sequencing screen of 48 samples from patient-derived xenograft (PDX) subcutaneous melanomas and their associated metastases. In comparison with primary tumors, expression of glycolytic genes was frequently decreased in metastases, whereas expression of some tricarboxylic acid (TCA) cycle genes was increased in metastases. Consistent with these transcriptional changes, melanoma metastases underwent a metabolic switch characterized by decreased levels of glycolytic metabolites and increased abundance of TCA cycle metabolites. A short isoform of glyceraldehyde-3-phosphate dehydrogenase, spermatogenic (GAPDHS) lacking the N-terminal domain suppressed metastasis and regulated this metabolic switch. GAPDHS was downregulated in metastatic nodules from PDX models as well as in human patients. Overexpression of GAPDHS was sufficient to block melanoma metastasis, whereas its inhibition promoted metastasis, decreased glycolysis, and increased levels of certain TCA cycle metabolites and their derivatives including citrate, fumarate, malate, and aspartate. Isotope tracing studies indicated that GAPDHS mediates this shift through changes in pyruvate carboxylase activity and aspartate synthesis, both metabolic pathways critical for cancer survival and metastasis. Together, these data identify a short isoform of GAPDHS that limits melanoma metastasis and regulates central carbon metabolism.Significance:This study characterizes metabolic changes during cancer metastasis and identifies GAPDHS as a novel regulator of these processes in melanoma cells.

Metastasis is a very inefficient process in which few disseminated cancer cells survive. We developed a patient-derived xenograft assay in which melanomas engraft and spontaneously metastasize (Quintana Nature 456:593-598). Using this assay, we discovered that melanoma cells experience a spike in reactive oxygen species during metastasis and that distant metastasis is limited by oxidative stress (Piskounova Nature 527:186-191). Successfully metastasizing cells undergo reversible metabolic changes during metastasis that increase oxidative stress resistance. For example, melanoma cells in lymph experience less oxidative stress and form more metastases than melanoma cells in the blood (Ubellacker Nature 585:113-120). This is true in both patient-derived melanomas growing in immunocompromised mice and mouse melanomas growing in syngeneic immunocompetent mice. Cells metastasizing through blood, but not lymph, appear to undergo ferroptosis, a form of cell death marked by lipid oxidation. Multiple differences between lymph fluid and blood plasma may contribute to this difference in oxidative stress, including higher levels of oleic acid in lymph. Oleic acid is a monounsaturated fatty acid that protects melanoma cells from ferroptosis, apparently by reducing the abundance of oxidizable polyunsaturated fatty acids (PUFAs) in phospholipids. In the current study we found that some PUFAs sensitize melanoma cells to lipid peroxidation and cell death more than others. Moreover, we analyzed the lipid profiles of common sites of distant metastasis – liver, brain, kidney, pancreas, bone marrow, lung, and liver – and found that the PUFA compositions of these tissues varies significantly. This raises the possibility that metastasizing melanoma cells may incorporate more PUFAs into their phospholipid membranes and exhibit increased ferroptosis sensitivity in some metastatic sites as compared to others. We found that melanoma cells isolated from metastatic sites by flow cytometry had higher levels of PUFAs in their phospholipid membranes compared to those isolated from subcutaneous tissues. These cells isolated from distal sites also exhibited higher levels of lipid ROS and contained higher levels of oxidized lipid species compared to subcutaneous tumors. These data suggest that the lipid metabolism of metastatic tumors reflects both lipid availability in the metastatic site as well as cell-intrinsic differences in lipid metabolism, both of which contribute to lipid ROS. Both cell-intrinsic and cell-extrinsic lipid metabolism likely influence the ability of melanoma cells to survive in distinct metastatic sites during metastasis. Citation Format: Thomas P. Mathews, Sarah Muh, Arin B. Aurora, Sean J. Morrison. Differences in melanoma lipid metabolism among distinct metastatic sites [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr PR017.