Protein post-translational modifications (PTMs) are crucial for cancer cells to adapt to hypoxia; however, the functional significance of lysine crotonylation (Kcr) in hypoxia remains unclear. Herein we report a quantitative proteomics analysis of global crotonylome under normoxia and hypoxia, and demonstrate 128 Kcr site alterations across 101 proteins in MDA-MB231 cells. Specifically, we observe a significant decrease in K131cr, K156cr and K220cr of phosphoglycerate kinase 1 (PGK1) upon hypoxia. Enoyl-CoA hydratase 1 (ECHS1) is upregulated and interacts with PGK1, leading to the downregulation of PGK1 Kcr under hypoxia. Abolishment of PGK1 Kcr promotes glycolysis and suppresses mitochondrial pyruvate metabolism by activating pyruvate dehydrogenase kinase 1 (PDHK1). A low PGK1 K131cr level is correlated with malignancy and poor prognosis of breast cancer. Our findings show that PGK1 Kcr is a signal in coordinating glycolysis and the tricarboxylic acid (TCA) cycle and may serve as a diagnostic indicator for breast cancer.

Fatty acid uptake is essential for cell physiological function, but detailed mechanisms remain unclear. Here, we generated an acetyl-CoA carboxylases (ACC1/2) double-knockout cell line, which lacked fatty acid biosynthesis and survived on serum fatty acids and was used to screen for fatty acid uptake inhibitors. We identified a Food and Drug Administration–approved tricyclic antidepressant, nortriptyline, that potently blocked fatty acid uptake both in vitro and in vivo. We also characterized underlying mechanisms whereby nortriptyline provoked lysosomes to release protons and induce cell acidification to suppress macropinocytosis, which accounted for fatty acid endocytosis. Furthermore, nortriptyline alone or in combination with ND-646, a selective ACC1/2 inhibitor, significantly repressed tumor growth, lipogenesis, and hepatic steatosis in mice. Therefore, we show that cells actively take up fatty acids through macropinocytosis, and we provide a potential strategy suppressing tumor growth, lipogenesis, and hepatic steatosis through controlling the cellular level of fatty acids.

Sulforaphane (SFN) was demonstrated to induce apoptosis in a variety of cancers via multiple mechanisms. However, owing to a short half-life in circulation, SFN was not used for clinical treatment yet. Interestingly, SFN analog, sulforaphane-cysteine (SFN-Cys) has a longer half-life in metabolism, and we previously demonstrated that SFN-Cys inhibited invasion in human prostate cancer cells. Here, we would investigate whether SFN-Cys induces apoptosis and find the underlying mechanisms in human non-small cell lung cancer (NSCLC) cells. Western blots were used to test the molecular linkages among extracellular signal-regulated kinases 1/2 (ERK1/2) and downstream signal molecules. Flow cytometry and fluorescence microscopy were used to detect cell death. Cell proliferation assay showed that SFN-Cys inhibited cell viability following a dose-dependent manner. Abnormal cell morphology was viewed after the cells were exposed to SFN-Cys. Flow cytometry showed that SFN-Cys induced cell apoptosis via a dose-dependent manner. Further, SFN-Cys triggered the activation of ERK1/2, which resulted in the upregulation of maspin, Bax, cleaved caspase-3 and downregulation of pro-caspase-3, Bcl-2, α-tubulin. Meanwhile, we demonstrated that recombinant caspase-3 cleaved α-tubulin in the lysate of cells, which were treated by SFN-Cys. These data indicated that SFN-Cys activated the ERK1/2-mediated mitochondria signaling pathway with maspin upregulation and α-tubulin downregulation leading to apoptosis. These findings will help to develop a novel therapeutic to target NSCLC cells.