Abstract
Simple SummaryThe xCT transporter of oxidized form of cysteine has been recognized as fundamental for cellular amino acid and redox homeostasis. Increasing number of data suggests that xCT inhibition-induced ferroptosis has great potential for development of novel anti-cancer therapeutics for pancreatic cancer patients. The aim of this study was to investigate potential resistance mechanisms that cancer cells with genetically disrupted xCT (xCTKO) may exploit in order to develop resistance to ferroptosis. Our data clearly showed that shuttle of reduced cysteine between cancer xCTKO and neighboring cells provide protection of the former. Importantly, this shuttle seems to be fueled by the import and reduction of oxidized cysteine by xCT-proficient feeder layer. In summary, two important findings are: (1) supply of the reduced cysteine has to be taken in consideration when xCT-based ferroptosis inducers are used, and (2) systemic inhibition of xCT could be potential approach in overcoming this resistant mechanism.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.
Highlights
Despite great efforts in the development of new diagnostics and treatments, cancer is still a major threat for human health with rising incidence and mortality rates [1,2]
In our previous study [10], we showed that genetic disruption of the xCT transporter in pancreatic ductal adenocarcinoma (PDAC), one of the most aggressive types of cancer with a dismal 5-year survival rate of 7% [11], promotes intracellular cysteine depletion, inhibition of GSH synthesis followed by accumulation of membrane lipid hydroperoxides and ferroptotic cell death in vitro
Considering our previous observations [10] and in order to understand the resistance to ferroptotic cell death of xCTKO cells in vivo, this study aimed to investigate the role of adjacent fibroblasts in the resistance of xCTKO PDAC cells, while identifying the importance of the amino acid transporters involved in the interplay between xCT-expressing cells and xCT-disrupted cells
Summary
Despite great efforts in the development of new diagnostics and treatments, cancer is still a major threat for human health with rising incidence and mortality rates [1,2]. Ferroptosis is a regulated, iron-dependent type of cell death characterized by the accumulation of lipid hydroperoxides (LOOH) in response to oxidative stress and oxidation of membrane lipids, mostly polyunsaturated fatty acids (PUFAs), which compromises the permeability and fluidity of the membrane lipid bilayer, eventually leading to cell death [6]. Intracellular level of GSH depends on the import of cysteine in its oxidized (dominant) form ( known as cystine, CySSCy), through the system xc-. This transport system, consisting of the substrate-specific subunit named xCT (SLC7A11) and the chaperone CD98 (SLC3A2), promotes the import of CySSCy at the expense of glutamate (Glu) efflux in a Na+-independent way [6,9,10]
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