Revealing the Ferroptotic Phenotype of Medulloblastoma.

The interaction of iron and oxygen is an integral part of the development of life on Earth. Nonetheless, this unique chemistry continues to fascinate and puzzle, leading to new biological ventures. In 2012, a Columbia University group recognized this interaction as a central event leading to a new type of regulated cell death named "ferroptosis." The major feature of ferroptosis is the accumulation of lipid hydroperoxides due to (1) dysfunctional antioxidant defense and/or (2) overwhelming oxidative stress, which most frequently coincides with increased content of free labile iron in the cell. This is normally prevented by the canonical anti-ferroptotic axis comprising the cystine transporter xCT, glutathione (GSH), and GSH peroxidase 4 (GPx4). Since ferroptosis is not a programmed type of cell death, it does not involve signaling pathways characteristic of apoptosis. The most common way to prove this type of cell death is by using lipophilic antioxidants (vitamin E, ferrostatin-1, etc.) to prevent it. These molecules can approach and detoxify oxidative damage in the plasma membrane. Another important aspect in revealing the ferroptotic phenotype is detecting the preceding accumulation of lipid hydroperoxides, for which the specific dye BODIPY C11 is used. The present manuscript will show how ferroptosis can be induced in wild-type medulloblastoma cells by using different inducers: erastin, RSL3, and iron-donor. Similarly, the xCT-KO cells that grow in the presence of NAC, and which undergo ferroptosis once NAC is removed, will be used. The characteristic "bubbling" phenotype is visible under the light microscope within 12-16 h from the moment of ferroptosis triggering. Furthermore, BODIPY C11 staining followed by FACS analysis to show the accumulation of lipid hydroperoxides and consequent cell death using the PI staining method will be used. To prove the ferroptotic nature of cell death, ferrostatin-1 will be used as a specific ferroptosis-preventing agent.

Integrin-αvß3 is a Therapeutically Targetable Fundamental Factor in Medulloblastoma Tumorigenicity and Radioresistance.

Medulloblastoma is one of the most prevalent solid tumors found in children, occurring in the brain's posterior fossa. The standard treatment protocol involves maximal resection surgery followed by craniospinal irradiation and chemotherapy. Despite a long-term survival rate of 70%, wide disparities among patients have been observed. The identification of pertinent targets for both initial and recurrent medulloblastoma cases is imperative. Both primary and recurrent medulloblastoma are marked by their aggressive infiltration into surrounding brain tissue, robust angiogenesis, and resistance to radiotherapy. While the significant role of integrin-αvß3 in driving these characteristics has been extensively documented in glioblastoma, its impact in the context of medulloblastoma remains largely unexplored. Integrin-αvß3 was found to be expressed in a subset of patients with medulloblastoma. We investigated the role of integrin-αvß3 using medulloblastoma-derived cell lines with ß3-subunit depletion or overexpression both in vitro and in vivo settings. By generating radioresistant medulloblastoma cell lines, we uncovered an increased integrin-αvß3 expression, which correlated with increased susceptibility to pharmacologic integrin-αvß3 inhibition with cilengitide, a competitive ligand mimetic. Finally, we conducted single-photon emission computed tomography (SPECT)/MRI studies on orthotopic models using a radiolabeled integrin-αvß3 ligand (99mTc-RAFT-RGD). This innovative approach presents the potential for a novel predictive imaging technique in the realm of medulloblastoma. Altogether, our findings lay the foundation for employing SPECT/MRI to identify a specific subset of patients with medulloblastoma eligible for integrin-αvß3-directed therapies. This breakthrough offers a pathway toward more targeted and effective interventions in the treatment of medulloblastoma. SIGNIFICANCE: This study demonstrates integrin-αvß3's fundamental role in medulloblastoma tumorigenicity and radioresistance and the effect of its expression on cilengitide functional activity.

Metabolic Rewiring toward Oxidative Phosphorylation Disrupts Intrinsic Resistance to Ferroptosis of the Colon Adenocarcinoma Cells.

Glutathione peroxidase 4 (GPX4) has been reported as one of the major targets for ferroptosis induction, due to its pivotal role in lipid hydroperoxide removal. However, recent studies pointed toward alternative antioxidant systems in this context, such as the Coenzyme Q-FSP1 pathway. To investigate how effective these alternative pathways are in different cellular contexts, we used human colon adenocarcinoma (CRC) cells, highly resistant to GPX4 inhibition. Data obtained in the study showed that simultaneous pharmacological inhibition of GPX4 and FSP1 strongly compromised the survival of the CRC cells, which was prevented by the ferroptosis inhibitor, ferrostatin-1. Nonetheless, this could not be phenocopied by genetic deletion of FSP1, suggesting the development of resistance to ferroptosis in FSP1-KO CRC cells. Considering that CRC cells are highly glycolytic, we used CRC Warburg-incompetent cells, to investigate the role metabolism plays in this phenomenon. Indeed, the sensitivity to inhibition of both anti-ferroptotic axes (GPx4 and FSP1) was fully revealed in these cells, showing typical features of ferroptosis. Collectively, data indicate that two independent anti-ferroptotic pathways (GPX4-GSH and CoQ10-FSP1) operate within the overall physiological context of cancer cells and in some instances, their inhibition should be coupled with other metabolic modulators, such as inhibitors of glycolysis/Warburg effect.

Genetic Disruption of the γ-Glutamylcysteine Ligase in PDAC Cells Induces Ferroptosis-Independent Cell Death In Vitro without Affecting In Vivo Tumor Growth.

The conceptualization of a novel type of cell death, called ferroptosis, opens new avenues for the development of more efficient anti-cancer therapeutics. In this context, a full understanding of the ferroptotic pathways, the players involved, their precise role, and dispensability is prerequisite. Here, we focused on the importance of glutathione (GSH) for ferroptosis prevention in pancreatic ductal adenocarcinoma (PDAC) cells. We genetically deleted a unique, rate-limiting enzyme for GSH biosynthesis, γ-glutamylcysteine ligase (GCL), which plays a key role in tumor cell proliferation and survival. Surprisingly, although glutathione peroxidase 4 (GPx4) has been described as a guardian of ferroptosis, depletion of its substrate (GSH) led preferentially to apoptotic cell death, while classical ferroptotic markers (lipid hydroperoxides) have not been observed. Furthermore, the sensitivity of PDAC cells to the pharmacological/genetic inhibition of GPx4 revealed GSH dispensability in this context. To the best of our knowledge, this is the first time that the complete dissection of the xCT-GSH-GPx4 axis in PDAC cells has been investigated in great detail. Collectively, our results revealed the necessary role of GSH in the overall redox homeostasis of PDAC cells, as well as the dispensability of this redox-active molecule for a specific, antioxidant branch dedicated to ferroptosis prevention.

NUPR1 inhibitor ZZW-115 induces ferroptosis in a mitochondria-dependent manner.

Ferroptosis is an iron-dependent cell death characterized by the accumulation of hydroperoxided phospholipids. Here, we report that the NUPR1 inhibitor ZZW-115 induces ROS accumulation followed by a ferroptotic cell death, which could be prevented by ferrostatin-1 (Fer-1) and ROS-scavenging agents. The ferroptotic activity can be improved by inhibiting antioxidant factors in pancreatic ductal adenocarcinoma (PDAC)- and hepatocellular carcinoma (HCC)-derived cells. In addition, ZZW-115-treatment increases the accumulation of hydroperoxided lipids in these cells. We also found that a loss of activity and strong deregulation of key enzymes involved in the GSH- and GPX-dependent antioxidant systems upon ZZW-115 treatment. These results have been validated in xenografts induced with PDAC- and HCC-derived cells in nude mice during the treatment with ZZW-115. More importantly, we demonstrate that ZZW-115-induced mitochondrial morphological changes, compatible with the ferroptotic process, as well as mitochondrial network disorganization and strong mitochondrial metabolic dysfunction, which are rescued by both Fer-1 and N-acetylcysteine (NAC). Of note, the expression of TFAM, a key regulator of mitochondrial biogenesis, is downregulated by ZZW-115. Forced expression of TFAM is able to rescue morphological and functional mitochondrial alterations, ROS production, and cell death induced by ZZW-115 or genetic inhibition of NUPR1. Altogether, these results demonstrate that the mitochondrial cell death mediated by NUPR1 inhibitor ZZW-115 is fully rescued by Fer-1 but also via TFAM complementation. In conclusion, TFAM could be considered as an antagonist of the ferroptotic cell death.