Copper content is significantly elevated in gastric cancer (GC), especially in malignant tumors.

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Abstract​

Cuproptosis, caused by excessively high copper concentrations, is urgently exploited as a potential cancer therapeutic. However, the mechanisms underlying the initiation, propagation, and ultimate execution of cuproptosis in tumors remain unknown. Here, we show that copper content is significantly elevated in gastric cancer (GC), especially in malignant tumors. Screening reveals that METTL16, an atypical methyltransferase, is a critical mediator of cuproptosis through the m6A modification on FDX1 mRNA. Furthermore, copper stress promotes METTL16 lactylation at site K229 followed by cuproptosis. The process of METTL16 lactylation is inhibited by SIRT2. Elevated METTL16 lactylation significantly improves the therapeutic efficacy of the copper ionophore– elesclomol. Combining elesclomol with AGK2, a SIRT2-specific inhibitor, induce cuproptosis in gastric tumors in vitro and in vivo. These results reveal the significance of non-histone protein METTL16 lactylation on cuproptosis in tumors. Given the high copper and lactate concentrations in GC, cuproptosis induction becomes a promising therapeutic strategy for GC.

 

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Introduction​

Copper, an essential trace element in organisms, is involved in cell growth and metabolism1,2,3,4,5,6. Copper homeostasis disorder is relevant to various tumors, such as gynecological tumors7,8 and colorectal cancer9. Indeed, increased serum copper levels in cancer patients are correlated with cancer grade and chemotherapy resistance10. Copper becomes toxic if its concentration exceeds a certain threshold, inducing a newly discovered form of cell death, named cuproptosis11. Cuproptosis occurs when copper binds directly to lipoylated components of the tricarboxylic acid cycle, leading to subsequent loss of iron-sulfur cluster-containing proteins, proteotoxic stress and ultimately cell death. A recent study using genome-wide CRISPR-Cas9 loss-of-function screening identified ten critical factors of cuproptosis11. Among them, ferredoxin 1 (FDX1) encodes a reductase known to reduce Cu2+ to its more toxic form Cu1+, lipoylates dihydrolipoamide S-acetyltransferase (DLAT) that is essential for cuproptosis12, and is the direct target of elesclomol that is an important copper ionophore13. However, the mechanisms underlying the initiation, propagation, and execution of cuproptosis in tumors remain unknown.

 

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RNA modification, as an epigenetic regulator, has gained increasing attention in recent years14. N6-methyladenosine (m6A), the most abundant RNA modification in eukaryotic cells, plays a crucial role in tumorigenesis15,16. The expression of regulatory proteins associated with m6A is abnormal in most tumors, leading to drug resistance and tumor development17,18. METTL16, followed by classic METTL3/METTL14 methyltransferases19,20, was recently identified as a second m6A writer21. Previous studies have demonstrated that METTL16 is responsible for m6A deposition in many transcripts, including the MAT2A transcript that encodes SAM synthetase22 and U6 snRNA23. METTL16 shows tumorigenesis and tumor-promoting capabilities in an m6A-dependent manner in multiple tumors24,25,26,27,28,29, while the regulatory mechanisms controlling METTL16 activity remain unclear. A comprehensive study about the regulation and function of METTL16 may therefore provide better insights into the prevention of tumor therapy and metastasis.

Recently, lactate-derived lactylation has been identified as a newly discovered post-translational modification (PTM)30. Lactate is taken up by tumor cells and transported to mitochondria for oxidation to provide energy31, meanwhile derives lactylation of histone lysine (K) residue to stimulate gene transcription30. Histone lactylated modification has been proven to involve multiple pathological processes, such as macrophage polarization30,32 and tumorigenesis33. Delactylases and lactyltransferases are also gradually being discovered. Delactylases mainly include SIRT1-3 and HDAC1-334,35,36 while lactyltransferases mainly include P300, MOF, ME-PCT, and RE-PCT30,36,37 The abundance and specificity of non-histone proteins in cells yet were higher than that of histones. Whether there are numerous lactylated modifications on non-histone proteins and how these lactylated non-histone proteins operate and are regulated in tumor progression are urgently needed to be explored38.

In this study, we found that copper content is significantly elevated in gastric cancer (GC), especially in malignant tumor types. Screening revealed that METTL16 is a critical mediator of cuproptosis in GC through the m6A modification on FDX1 mRNA. Interestingly, we found that high copper content promotes non-histone protein METTL16-K229 lactylation through increasing the interaction of potential lactyltransferases AARS1 / AARS2 to METTL16, and ultimately leads to cuproptosis. In addition, delactylases SIRT2 inhibits the process of METTL16 lactylation. Given that gastric tumors (especially malignant tumor types) have higher copper and lactate concentrations than normal tissues, combined treatment with copper ionophore– elesclomol and SIRT2-specific inhibitors to trigger cuproptosis obviously improves the efficacy of gastric cancer treatment. The study findings provide insights into the mechanisms underlying the initiation and execution of cuproptosis and suggest a promising therapeutic strategy for GC.

 

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Results​

Copper content is high in gastric cancer and related to tumor progression​

Cuproptosis is caused by excessively high copper concentration, but the regulatory mechanisms of cuproptosis in tumors still need to be explored11. Given copper is mainly absorbed through the stomach and upper small intestine, tumors of the gastrointestinal system are suitable for studying the initiation and propagation of cuproptosis. We analyzed copper concentrations in 48 pairs of GC tissues and adjacent tissues, finding higher copper concentrations in GC tissues than in normal gastric tissues (Fig. 1a, P < 0.01). Further analysis revealed that the relative copper content in patients with stage III GC was higher than that in patients with stage I and II GC (Fig. 1b, P < 0.05), indicating that copper content is correlated with tumor progression. Additionally, the copper content was negatively correlated with the overall survival (OS) and disease-free survival (DFS) in patients with GC (Fig. 1c, d; log-rank P < 0.05). Meanwhile, the copper content was positively correlated with Ki-67 expression, an indicator of cell proliferation (Fig. 1e). Similarly, the copper content was positively correlated with platelet/lymphocyte and neutrophil/lymphocyte ratios in serum specimens of patients, both of which are inflammation-related prognostic biomarkers for invasive malignancy for GC (Fig. 1f, g). These results indicated that the high content of copper is involved in the progression and development of GC.

 

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Additionally, we examined the differences of copper concentrations in different GC types, especially in malignant tumor types. Intriguingly, we discovered higher copper concentrations in mucinous adenocarcinomas than in overall gastric tumors (Fig. 1h). The copper content was positively correlated with invasive indicators such as lymph node metastasis (Fig. 1i) and vascular invasion (Fig. 1j) in mucinous adenocarcinomas. However, the copper content was not significantly associated with these invasive indicators or tumor differentiation in non-mucinous adenocarcinoma (Supplementary Fig. 1a–h). Mucinous adenocarcinoma, a rare histological subtype of GC, is associated with a more advanced stage and worse 5-year OS and DFS than non-mucinous adenocarcinoma39. As a refractory GC, mucinous adenocarcinoma is characterized by chemoradiotherapy resistance and more effective therapeutic approaches are urgently needed.

Recently, researchers have found that high intracellular copper concentration triggers a reductase FDX1 to reduce Cu2+ to a more toxic form Cu1+, leading to cuproptosis11. We found that FDX1 protein level was higher in GC compared to normal gastric tissues (Fig. 1k). Given both FDX1 protein level and copper content are high in GC, cuproptosis may be more easily triggered. It provides a potential therapeutic strategy for GC, especially for malignant tumors—mucinous adenocarcinomas.