Due to its high efficacy, low cost, and safety, the biguanide metformin is the most widely used oral anti-diabetic drug. In addition, potential clinical applications other than diabetes have been proposed, such as cancer prevention and treatment, anti-aging ,anti-haze activity, and other applications. Among them, validation of metformin's anti-tumor clinical efficacy and exploration of the underlying mechanism have become a hot spot in the field of cancer research. Over time, more and more novel anti-tumorigenic mechanisms of metformin are gradually being revealed.
Metformin might influence tumorigenesis both indirectly, through systemic reduction of insulin levels, and directly, via induction of energetic stress. On the one hand, metformin has been shown to suppress tumorigenesis by altering systemic endocrine and metabolic states and thereby reducing serum glucose and insulin levels. On the other hand, accumulating evidence has shown that metformin acts directly on tumor cells and exerts its antitumor biological effects through AMPK-dependent and/or non-AMPK-dependent signaling pathways. Specifically, metformin can activate AMP-activated protein kinase (AMPK) to inactivate mammalian target of rapamycin (mTOR), change intracellular reactive oxygen species (ROS) levels, and inhibit mitochondrial functions. Metformin has also been shown to regulate iron homeostasis in cells, which may be linked to cell metabolism. With the advancement of research, the mechanism by which metformin regulates apoptosis, autophagy, and other cell death pathways has gradually been revealed, but the dominant cell death mechanism by which metformin mediates tumor suppression is still not clear. One of the key challenges in cancer research is how to effectively kill cancer cells while leaving healthy cells intact. Dissecting the principal molecular mechanisms of metformin's antitumor activities and the major forms of death mediated by metformin can help us to develop novel treatments.
To proliferate and progress, cancer cells show a higher iron requirement than healthy cells. The dependence of cancer cells on iron has implications in a number of cell death pathways, including ferroptosis, an iron-dependent form of cell death. Uniquely, both iron excess and iron depletion can be utilized in anticancer therapies. The absorption, transportation, storage and utilization of iron are mediated by the membrane protein including transferrin receptor 1 (TFR1), the ferrireductase activity of STEAP3, divalent metal transporter 1 (DMT1, also termed SLC11A2), and ferritin (an iron storage protein complex), all affect the sensitivity of ferroptosis. When iron is depleted, ferritinophagy can maintain iron homeostasis through autophagic degradation of the iron-storage protein ferritin. Ferritin is recruited by the specific cargo receptor NCOA4 to autophagosomes and undergoes lysosomal degradation to release free iron. In addition, iron metabolism can be regulated by mitochondria,which are the sole sites of heme synthesis and the major sites for iron–sulfur cluster (ISC) biogenesis. Iron is an essential trace element for proper cell functioning.Exploring its physiological functions and mechanism of action may provide potential therapeutic directions against cancer and other diseases.
Their dependence on iron makes cancer cells more susceptible to iron-catalyzed necrosis, known as ferroptosis. Ferroptosis was first proposed by Stockwell as a novel regulated cell death in 2012. Unlike autophagy and apoptosis, ferroptosis is defined as an iron-dependent and reactive oxygen species (ROS)-reliant cell death with characteristic cytological changes, including decreased or vanished mitochondria cristae, ruptured outer mitochondrial membranes, and condensed mitochondrial membranes. Many diseases, including tumors, have been reported to be associated with ferroptosis, and targeting ferroptosis to eliminate tumors is increasingly becoming a promising new approach for tumor treatment.The biological significance of ferroptosis is expanding rapidly by virtue of the discovery that GPX4 and system xc− are crucial regulators of ferroptosis and by the use of ferrostatins to inhibit ferroptosis in diverse contexts. One of the key bottlenecks for protection from ferroptosis is the availability of GSH, which serves as a redox equivalent for GPXs, including GPX4. GSH synthesis depends on the availability of intracellular cysteine, which can be generated from cystine imported from the extracellular space via the sodium-independent cystine/glutamate antiporter system xc−.
System xc− is a heterodimer consisting of a heavy chain (4F2, gene name SLC3A2) and a light chain (xCT, gene name SLC7A11). xCT is often aberrantly overexpressed in many cancers, making it a weak spot for overcoming cancer by induction of ferroptosis. Interestingly, several FDA-approved drugs have been identified to function via ferroptosis induction in different cancer entities. Additionally, ferroptosis-inducing compounds and mechanisms have been identified and are broadly categorized as system xc− inhibitors (Erastin, Sulfasalazine, Sorafenib), glutathione depleters (FIN56), direct GPX4 inhibitors (RSL3), or as an iron scavenger (Deferoxamine). Among these, sulfasalazine (SAS) is commonly used for treatment of rheumatoid arthritis and has been previously identified as an inhibitor of SLC7A11 transporter activity. In addition, Slc7a11 KO mice are viable with no obvious phenotype. Therefore, SLC7A11 likely represents a better therapeutic target for cancer treatment than GPX4 because inhibiting SLC7A11 would presumably cause less toxicity in patients than inhibiting GPX4. Considering this, we have primarily focused on Sulfasalazine (which inhibits SLC7A11) in our studies.
UFM1 represents a small subclass of ubiquitin proteins discovered in 2004. It is composed of 85 amino acids and has a molecular weight of 9.1 kD. UFM1 has a tertiary structure similar to the ubiquitin molecule, but only 16% of its amino acid sequence is similar, and it is located in the nucleus and cytoplasm. Recent studies have demonstrated that as a novel posttranslational modification, UFMylation is involved in the occurrence and development of various diseases, including breast cancer. Here, we found that UFM1 can act as a target protein for metformin, and that UFM1 can modify SLC7A11, which can thus be regulated by metformin.
In this study, for the first time, we describe the involvement of ferroptosis in metformin-induced cell death and tumor inhibition, as well as the mechanism of metformin in regulating ferroptosis, and evaluate the strategies of metformin combined with ferroptosis inducers (Sulfasalazine) in the treatment of cancer. In short, our study demonstrates the function and mechanism of metformin in regulating ferroptosis in cancer cells through SLC7A11 UFMylation.