Aberrant protein post-translational modification is a hallmark of malignant tumors. Lysine succinylation (Ksucc) plays a vital role in cell energy metabolism in various cancers. However, whether succinylation can be catalyzed by acetyltransferase p300 remains unclear. In this study, we unveiled that p300 is a “writer” for succinylation, and p300-mediated Ksucc promotes cell glycometabolism in lung adenocarcinoma (LUAD). Specifically, our succinylome data revealed that EP300 deficiency leads to the systemic reduction of Ksucc, and 79.55% of the p300-succinylated proteins were found in the cytoplasm, which were primarily enriched in the carbohydrate metabolism process. Interestingly, deleting EP300 led to a notable decrease in Ksucc levels on several glycolytic enzymes, especially Phosphoglycerate Kinase 1 (PGK1). Mutation of the succinylated site of PGK1 notably hindered cell glycolysis and lactic acid excretion. Metabolomics in vivo indicated that p300-caused metabolic reprogramming was mainly attributed to the altered carbohydrate metabolism. In addition, 89.35% of LUAD patients exhibited cytoplasmic localization of p300, with higher levels in tumor tissues than adjacent normal tissues. High levels of p300 correlated with advanced tumor stages and poor prognosis of LUAD patients. Briefly, we disclose the activity of p300 to catalyze succinylation, which contributes to cell glucose metabolic reprogramming and malignant progression of lung cancer.

Patient behavior and physiology can directly affect cancer metabolism. Smoking is the leading risk factor for non-small cell lung cancer (NSCLC). Here, we identified that smoking modulates lung cancer cell metabolism through altered protein post-translational modification. Proteomic analyses identified elevated K251 succinylation (K251-Su) of GAPDH, a key enzyme in glycolysis, in NSCLC samples, and GAPDH K251-Su was significantly higher in patients who smoke compared to non-smokers. Exposure of lung cancer cells to cigarette smoke extract led to increased uptake of glutamine and enhanced GAPDH K251-Su. Glutamine uptake by cancer cells in hypoxic and nutrient-deficient microenvironments provided succinyl-CoA donors for GAPDH succinylation at K251, which was catalyzed by acyltransferase p300. K251-Su increased GAPDH stability by suppressing TRIM4-mediated K254 ubiquitination. GAPDH K251-Su enhanced glycolysis and glutamine reductive carboxylation to meet the demands for cell growth and to support survival in hypoxic and nutrient-depleted conditions, promoting tumor growth and metastasis. These findings indicate that tobacco smoking mediates metabolic reprogramming of cancer cells through succinylation of GAPDH to drive NSCLC progression.

The positive rate of GAPDH K251-Su in the tumor and adjacent normal tissue of NSCLC

The 3D reconstruction video of lung micro-CT scanning in a KrasG12D-driven NSCLC GAPDH WT mouse. The red nodular lesions in the 3D reconstructed video indicate tumor lesions.

Supplementary Figure 6 (Related to Fig.3).

The effects of cigarette smoke extract and various amino acids on GAPDH K251-Su, GAPDH and glutamine uptake in A549 cells under different culture conditions.