Although chemoresistance remains a primary challenge in the treatment of pancreatic ductal adenocarcinoma (PDAC), exploiting oxidative stress might offer novel therapeutic clues. Here we explored the potential of targeting cystine/glutamate exchanger (SLC7A11/xCT), which contributes to the maintenance of intracellular glutathione (GSH). Genomic disruption of xCT via CRISPR-Cas9 was achieved in two PDAC cell lines, MiaPaCa-2 and Capan-2, and xCT-KO clones were cultivated in the presence of N-acetylcysteine. Although several cystine/ cysteine transporters have been identified, our findings demonstrate that, in vitro, xCT plays the major role in intracellular cysteine balance and GSH biosynthesis. As a consequence, both xCT-KO cell lines exhibited amino acid stress with activation of GCN2 and subsequent induction of ATF4, inhibition of mTORC1, proliferation arrest, and cell death. Tumor xenograft growth was delayed but not suppressed in xCT-KO cells, which indicated both the key role of xCT and also the presence of additional mechanisms for cysteine homeostasis in vivo. Moreover, rapid depletion of intracellular GSH in xCT-KO cells led to accumulation of lipid peroxides and cell swelling. These two hallmarks of ferroptotic cell death were prevented by vitamin E or iron chelation. Finally, in vitro pharmacologic inhibition of xCT by low concentrations of erastin phenocopied xCT-KO and potentiated the cytotoxic effects of both gemcitabine and cisplatin in PDAC cell lines. In conclusion, our findings strongly support that inhibition of xCT, by its dual induction of nutritional and oxidative cellular stresses, has great potential as an anticancer strategy. Significance: The cystine/glutamate exchanger xCT is essential for amino acid and redox homeostasis and its inhibition has potential for anticancer therapy by inducing ferroptosis.

Cancer cells are characterized as highly proliferative at the expense of enhancement of metabolic rate. Consequently, cancer cells rely on antioxidant defenses to overcome the associated increased production of reactive oxygen species (ROS). The reliance of tumor metabolism on amino acids, especially amino acid transport systems, has been extensively studied over the past decade. Although cysteine is the least abundant amino acid in the cell, evidences described it as one of the most important amino acid for cell survival and growth. Regarding its multi-functionality as a nutrient, protein folding, and major component for redox balance due to its involvement in glutathione synthesis, disruption of cysteine homeostasis appears to be promising strategy for induction of cancer cell death. Ten years ago, ferroptosis, a new form of non-apoptotic cell death, has been described as a result of cysteine insufficiency leading to a collapse of intracellular glutathione level. In the present review, we summarized the metabolic networks involving the amino acid cysteine in cancer and ferroptosis and we focused on describing the recently discovered glutathione-independent pathway, a potential player in cancer ferroptosis resistance. Then, we discuss the implication of cysteine as key player in ferroptosis as a precursor for glutathione first, but also as metabolic precursor in glutathione-independent ferroptosis axis.

The evolution of life from extreme hypoxic environments to an oxygen-rich atmosphere has progressively selected for successful metabolic, enzymatic and bioenergetic networks through which a myriad of organisms survive the most extreme environmental conditions. From the two lethal environments anoxia/high O, cells have developed survival strategies through expression of the transcriptional factors ATF4, HIF1 and NRF2. Cancer cells largely exploit these factors to thrive and resist therapies. In this review, we report and discuss the potential therapeutic benefit of disrupting the major Myc/Hypoxia-induced metabolic pathway, also known as fermentative glycolysis or "Warburg effect", in aggressive cancer cell lines. With three examples of genetic disruption of this pathway: glucose-6-phosphate isomerase (GPI), lactate dehydrogenases (LDHA and B) and lactic acid transporters (MCT1, MCT4), we illuminate how cancer cells exploit metabolic plasticity to survive the metabolic and energetic blockade or arrest their growth. In this context of NRF2 contribution to OXPHOS re-activation we will show and discuss how, by disruption of the cystine transporter xCT (SLC7A11), we can exploit the acute lethal phospholipid peroxidation pathway to induce cancer cell death by 'ferroptosis'.

Lactate is a central metabolite in brain physiology but also contributes to tumor development. Glioblastoma (GB) is the most common and malignant primary brain tumor in adults, recognized by angiogenic and invasive growth, in addition to its altered metabolism. We show herein that lactate fuels GB anaplerosis by replenishing the tricarboxylic acid (TCA) cycle in absence of glucose. Lactate dehydrogenases (LDHA and LDHB), which we found spatially expressed in GB tissues, catalyze the interconversion of pyruvate and lactate. However, ablation of both LDH isoforms, but not only one, led to a reduction in tumor growth and an increase in mouse survival. Comparative transcriptomics and metabolomics revealed metabolic rewiring involving high oxidative phosphorylation (OXPHOS) in the LDHA/B KO group which sensitized tumors to cranial irradiation, thus improving mouse survival. When mice were treated with the antiepileptic drug stiripentol, which targets LDH activity, tumor growth decreased. Our findings unveil the complex metabolic network in which both LDHA and LDHB are integrated and show that the combined inhibition of LDHA and LDHB strongly sensitizes GB to therapy.

Contextualisation of the new type of cell death called “ferroptosis” opened a completely new avenue for the development of anti-cancer therapies. Cumulative fundamental research dating back to the mid-20th century, crowned by the extraordinary work of the group led by Dr. Stockwell from Columbia University in 2012, finally got its candidature to be applied in the clinical settings. Although the potential for clinical importance is undoubtedly growing every day, as showed by the increasing number of papers dealing with ferroptosis and its applications, long experience of cancer research and treatment taught us that caution is still necessary. The plasticity of the tumour cells, particularly acute, along with its involvement in the resistance mechanisms, that have been seen, to greater or lesser extent, for almost all currently used therapies, represents the biggest fascinations in biomedical research field and also the biggest challenge to achieving cures in cancer patients. Accordingly, the main features of fundamental research have to be vigilance and anticipation. In this review, we tried to summarize the literature data, accumulated in the past couple of years, which point out the pitfalls in which “ferroptosis inducers” can fall if used prematurely in the clinical settings, but at the same time can provide a great advantage in the exhausting battle with cancer resistance. This is the first comprehensive review focusing on the effects of the cell-to-cell contact/interplay in the development of resistance to ferroptosis, while the contribution of cell-born factors has been summarized previously so here we just listed them.

In our previous study, we showed that a cystine transporter (xCT) plays a pivotal role in ferroptosis of pancreatic ductal adenocarcinoma (PDAC) cells in vitro. However, in vivo xCTKO cells grew normally indicating that a mechanism exists to drastically suppress the ferroptotic phenotype. We hypothesized that plasma and neighboring cells within the tumor mass provide a source of cysteine to confer full ferroptosis resistance to xCTKO PDAC cells. To evaluate this hypothesis, we (co-) cultured xCTKO PDAC cells with different xCT-proficient cells or with their conditioned media. Our data unequivocally showed that the presence of a cysteine/cystine shuttle between neighboring cells is the mechanism that provides redox and nutrient balance, and thus ferroptotic resistance in xCTKO cells. Interestingly, although a glutathione shuttle between cells represents a good alternative hypothesis as a “rescue-mechanism”, our data clearly demonstrated that the xCTKO phenotype is suppressed even with conditioned media from cells lacking the glutathione biosynthesis enzyme. Furthermore, we demonstrated that prevention of lipid hydroperoxide accumulation in vivo is mediated by import of cysteine into xCTKO cells via several genetically and pharmacologically identified transporters (ASCT1, ASCT2, LAT1, SNATs). Collectively, these data highlight the importance of the tumor environment in the ferroptosis sensitivity of cancer cells.

Lactate is a central metabolite in brain physiology, involved in the astrocyte-neuron lactate shuttle, but also contributes to tumor development. Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults, recognized by angiogenic and invasive growth, in addition to its altered metabolism. By adapting their glycolytic or oxidative metabolism, GBM stem-like cells are able to resist chemo- and radiotherapy. We show herein that lactate fuels GBM anaplerosis by replenishing the TCA cycle in absence of glucose. Lactate dehydrogenases (LDH) catalyze the interconversion of pyruvate and lactate. Deletion of either LDHA or LDHB did not alter significantly GBM growth and invasion. However, ablation of both LDH isoforms led to a reduction of tumor growth, and, consequently, to an increase in mouse survival. Comparative transcriptomics and metabolomics revealed metabolic rewiring involving high oxidative phosphorylation (OxPhos) in the double LDHA/B KO group which sensitized tumors to cranial irradiation, massively improving mouse survival. Survival was also increased when control mice were treated with the antiangiogenic treatment, bevacizumab, and the antiepileptic drug, stiripentol which targets LDH activity. Taken together, this highlights the complex metabolic network in which both LDH A and B are integrated and underscores that combined inhibition of LDHA and B is necessary to impact tumor development. Targeting of these enzymes in combination with anti-angiogenic and repurposed drugs may be of therapeutic benefit, especially when associated with radiotherapy.

Myeloid cells are a key determinant of tumor progression and patient outcomes in a range of cancers and are therefore being actively pursued as targets of new immunotherapies. The recent use of high-dimensional single-cell approaches, e.g., mass cytometry and single-cell RNA-sequencing (scRNA-seq) has reinforced the predominance of myeloid cells in the tumor microenvironment and uncovered their phenotypic diversity in different cancers. The cancerous metabolic environment has emerged as a critical modulator of myeloid cell functions in anti-tumor immunity versus immune suppression and immune evasion. Here, we discuss mechanisms of immune-metabolic crosstalk in tumorigenesis, with a particular focus on the tumor-associated myeloid cell’s metabolic programs. We highlight the impact of several metabolic pathways on the pro-tumoral functions of tumor-associated macrophages and myeloid-derived suppressor cells and discuss the potential myeloid cell metabolic checkpoints for cancer immunotherapy, either as monotherapies or in combination with other immunotherapies.