7-Dehydrocholesterol is an endogenous suppressor of ferroptosis.

Ferroptosis is a form of cell death that has received considerable attention not only as a means to eradicate defined tumour entities but also because it provides unforeseen insights into the metabolic adaptation that tumours exploit to counteract phospholipid oxidation1,2. Here, we identify proferroptotic activity of 7-dehydrocholesterol reductase (DHCR7) and an unexpected prosurvival function of its substrate, 7-dehydrocholesterol (7-DHC). Although previous studies suggested that high concentrations of 7-DHC are cytotoxic to developing neurons by favouring lipid peroxidation3, we now show that 7-DHC accumulation confers a robust prosurvival function in cancer cells. Because of its far superior reactivity towards peroxyl radicals, 7-DHC effectively shields (phospho)lipids from autoxidation and subsequent fragmentation. We provide validation in neuroblastoma and Burkitt's lymphoma xenografts where we demonstrate that the accumulation of 7-DHC is capable of inducing a shift towards a ferroptosis-resistant state in these tumours ultimately resulting in a more aggressive phenotype. Conclusively, our findings provide compelling evidence of a yet-unrecognized antiferroptotic activity of 7-DHC as a cell-intrinsic mechanism that could be exploited by cancer cells to escape ferroptosis.

Development of a highly cytotoxic, clinical-grade virus-specific T cell product for adoptive T cell therapy.

At present, recipients of allogeneic hematopoietic stem-cells are still suffering from recurrent infections after transplantation. Infusion of virus-specific T cells (VST) post-transplant reportedly fights several viruses without increasing the risk of de novo graft-versus-host disease. This study targeted cytomegalovirus (CMV) for the development of an innovative approach for generating a very specific VST product following Good Manufacturing Practices (GMP) guidelines. We used a sterile disposable compartment named the Leukoreduction System Chamber (LRS-chamber) from the apheresis platelet donation kit as the starting material, which has demonstrated high levels of T cells. Using a combination of IL-2 and IL-7 we could improve expansion of CMV-specific T cells. Moreover, by developing and establishing a new product protocol, we were able to stimulate VST proliferation and favors T cell effector memory profile. The expanded VST were enriched in a closed automated system, creating a highly pure anti-CMV product, which was pre-clinically tested for specificity in vitro and for persistence, biodistribution, and toxicity in vivo using NOD scid mice. Our results demonstrated very specific VST, able to secrete high amounts of interferon only in the presence of cells infected by the human CMV strain (AD169), and innocuous to cells partially HLA compatible without viral infection.

Integrated Proteomics Reveals Apoptosis-related Mechanisms Associated with Placental Malaria.

Malaria in pregnancy is a public health concern in malaria-endemic areas. Accumulation of maternal immune cells in the placenta and increased levels of inflammatory cytokines caused by sequestration of Plasmodium falciparum-infected erythrocytes have been associated to poor neonatal outcomes, including low birth weight because of fetal growth restriction. Little is known about the molecular changes occurring in a P. falciparum-infected placenta that has developed placental malaria during pregnancy but had the parasites cleared by pharmacological treatment (past infection). We conducted an integrated proteome, phosphoproteome and glycoproteome analysis in past P. falciparum-infected placentas aiming to find molecular changes associated with placental malaria. A total of 2946 proteins, 1733 N-linked glycosites and 4100 phosphosites were identified and quantified in this study, disclosing overrepresented processes related to oxidative stress, protein folding and regulation of apoptosis in past-infected placentas Moreover, AKT and ERK signaling pathways activation, together with clinical data, were further correlated to an increased apoptosis in past-infected placentas. This study showed apoptosis-related mechanisms associated with placental malaria that can be further explored as therapeutic target against adverse pregnancy outcomes.

Thiol starvation triggers melanoma state switching in an ATF4 and NRF2-dependent manner.

The cystine/glutamate antiporter xCT is an important source of cysteine for cancer cells. Once taken up, cystine is reduced to cysteine and serves as a building block for the synthesis of glutathione, which efficiently protects cells from oxidative damage and prevents ferroptosis. As melanomas are particularly exposed to several sources of oxidative stress, we investigated the biological role of cysteine and glutathione supply by xCT in melanoma. xCT activity was abolished by genetic depletion in the Tyr::CreER; BrafCA; Ptenlox/+ melanoma model and by acute cystine withdrawal in melanoma cell lines. Both interventions profoundly impacted melanoma glutathione levels, but they were surprisingly well tolerated by murine melanomas in vivo and by most human melanoma cell lines in vitro. RNA sequencing of human melanoma cells revealed a strong adaptive upregulation of NRF2 and ATF4 pathways, which orchestrated the compensatory upregulation of genes involved in antioxidant defence and de novo cysteine biosynthesis. In addition, the joint activation of ATF4 and NRF2 triggered a phenotypic switch characterized by a reduction of differentiation genes and induction of pro-invasive features, which was also observed after erastin treatment or the inhibition of glutathione synthesis. NRF2 alone was capable of inducing the phenotypic switch in a transient manner. Together, our data show that cystine or glutathione levels regulate the phenotypic plasticity of melanoma cells by elevating ATF4 and NRF2.

Ferroptosis: mechanisms and implications for cancer development and therapy response.

As cancer cells develop resistance to apoptosis, non-apoptotic cell death modalities, such as ferroptosis, have emerged as promising strategies to combat therapy-resistant cancers. Cells that develop resistance to conventional therapies or metastatic cancer cells have been shown to have increased sensitivity to ferroptosis. Therefore, targeting the regulatory elements of ferroptosis in cancer could offer novel therapeutic opportunities. In this review, we first provide an overview of the known ferroptosis regulatory networks and discuss recent findings on how they contribute to cancer plasticity. We then expand into the critical role of selenium metabolism in regulating ferroptosis. Finally, we highlight specific cases where induction of ferroptosis could be used to sensitize cancer cells to this form of cell death.

LRP8-mediated selenocysteine uptake is a targetable vulnerability in MYCN-amplified neuroblastoma.

Ferroptosis has emerged as an attractive strategy in cancer therapy. Understanding the operational networks regulating ferroptosis may unravel vulnerabilities that could be harnessed for therapeutic benefit. Using CRISPR-activation screens in ferroptosis hypersensitive cells, we identify the selenoprotein P (SELENOP) receptor, LRP8, as a key determinant protecting MYCN-amplified neuroblastoma cells from ferroptosis. Genetic deletion of LRP8 leads to ferroptosis as a result of an insufficient supply of selenocysteine, which is required for the translation of the antiferroptotic selenoprotein GPX4. This dependency is caused by low expression of alternative selenium uptake pathways such as system Xc- . The identification of LRP8 as a specific vulnerability of MYCN-amplified neuroblastoma cells was confirmed in constitutive and inducible LRP8 knockout orthotopic xenografts. These findings disclose a yet-unaccounted mechanism of selective ferroptosis induction that might be explored as a therapeutic strategy for high-risk neuroblastoma and potentially other MYCN-amplified entities.

Glutaminolysis dynamics during astrocytoma progression correlates with tumor aggressiveness.

BACKGROUND: Glioblastoma is the most frequent and high-grade adult malignant central nervous system tumor. The prognosis is still poor despite the use of combined therapy involving maximal surgical resection, radiotherapy, and chemotherapy. Metabolic reprogramming currently is recognized as one of the hallmarks of cancer. Glutamine metabolism through glutaminolysis has been associated with tumor cell maintenance and survival, and with antioxidative stress through glutathione (GSH) synthesis. METHODS: In the present study, we analyzed the glutaminolysis-related gene expression levels in our cohort of 153 astrocytomas of different malignant grades and 22 non-neoplastic brain samples through qRT-PCR. Additionally, we investigated the protein expression profile of the key regulator of glutaminolysis (GLS), glutamate dehydrogenase (GLUD1), and glutamate pyruvate transaminase (GPT2) in these samples. We also investigated the glutathione synthase (GS) protein profile and the GSH levels in different grades of astrocytomas. The differential gene expressions were validated in silico on the TCGA database. RESULTS: We found an increase of glutaminase isoform 2 gene (GLSiso2) expression in all grades of astrocytoma compared to non-neoplastic brain tissue, with a gradual expression increment in parallel to malignancy. Genes coding for GLUD1 and GPT2 expression levels varied according to the grade of malignancy, being downregulated in glioblastoma, and upregulated in lower grades of astrocytoma (AGII-AGIII). Significant low GLUD1 and GPT2 protein levels were observed in the mesenchymal subtype of GBM. CONCLUSIONS: In glioblastoma, particularly in the mesenchymal subtype, the downregulation of both genes and proteins (GLUD1 and GPT2) increases the source of glutamate for GSH synthesis and enhances tumor cell fitness due to increased antioxidative capacity. In contrast, in lower-grade astrocytoma, mainly in those harboring the IDH1 mutation, the gene expression profile indicates that tumor cells might be sensitized to oxidative stress due to reduced GSH synthesis. The measurement of GLUD1 and GPT2 metabolic substrates, ammonia, and alanine, by noninvasive MR spectroscopy, may potentially allow the identification of IDH1mut AGII and AGIII progression towards secondary GBM.

Glypican-3 induces a mesenchymal to epithelial transition in human breast cancer cells.

Breast cancer is the disease with the highest impact on global health, being metastasis the main cause of death. To metastasize, carcinoma cells must reactivate a latent program called epithelial-mesenchymal transition (EMT), through which epithelial cancer cells acquire mesenchymal-like traits.Glypican-3 (GPC3), a proteoglycan involved in the regulation of proliferation and survival, has been associated with cancer. In this study we observed that the expression of GPC3 is opposite to the invasive/metastatic ability of Hs578T, MDA-MB231, ZR-75-1 and MCF-7 human breast cancer cell lines. GPC3 silencing activated growth, cell death resistance, migration, and invasive/metastatic capacity of MCF-7 cancer cells, while GPC3 overexpression inhibited these properties in MDA-MB231 tumor cell line. Moreover, silencing of GPC3 deepened the MCF-7 breast cancer cells mesenchymal characteristics, decreasing the expression of the epithelial marker E-Cadherin. On the other side, GPC3 overexpression induced the mesenchymal-epithelial transition (MET) of MDA-MB231 breast cancer cells, which re-expressed E-Cadherin and reduced the expression of vimentin and N-Cadherin. While GPC3 inhibited the canonical Wnt/ß-Catenin pathway in the breast cancer cells, this inhibition did not have effect on E-Cadherin expression. We demonstrated that the transcriptional repressor of E-Cadherin - ZEB1 - is upregulated in GPC3 silenced MCF-7 cells, while it is downregulated when GPC3 was overexpressed in MDA-MB231 cells. We presented experimental evidences showing that GPC3 induces the E-Cadherin re-expression in MDA-MB231 cells through the downregulation of ZEB1.Our data indicate that GPC3 is an important regulator of EMT in breast cancer, and a potential target for procedures against breast cancer metastasis.