IL1RAP Pulls a Double Shift in the Cysteine Factory

The metastatic spread of cancer cells to distal sites within the body is responsible for the majority of cancer-related deaths (1). However, cancer cells encounter many barriers to metastatic spread, including a form of cell death induced by extracellular matrix (ECM) detachment termed anoikis (“state of being without a home”; ref. 2). Interestingly, transformation of cells with oncogenes including v-Ha-ras and v-src confers anoikis resistance (2). ECM detachment results in loss of growth factor signaling, and the activation of receptor tyrosine kinase pathways, small GTPases, or epithelial-to-mesenchymal transition of cancer cells has been shown to promote their survival following ECM detachment (1). ECM detachment also profoundly alters cellular metabolism. Detachment of breast epithelial cells from the ECM resulted in a loss of glucose transport and NADPH generation via the pentose phosphate pathway, leading to reactive oxygen species (ROS) generation (3). Importantly, antioxidant treatment could rescue detachment-induced ROS and viability (3). Similarly, metastasizing melanoma cells rely on NADPH-generating enzymes in the folate pathway to overcome oxidative stress during metastasis (4), which is promoted by antioxidant treatment (4, 5). Thus, multiple pathways can influence anoikis sensitivity, but whether they function through discrete or common effectors is less clear.

The detoxification of ROS relies on the tripeptide antioxidant glutathione (GSH), which is recycled by NADPH-dependent enzymes (6). GSH is synthesized from the amino acids glutamate, glycine, and cysteine in a highly regulated manner, with cysteine being limiting for GSH synthesis under most conditions (6, 7). At the organismal level, most cysteine is obtained from the diet, but it is also synthesized de novo from methionine and serine via the transsulfuration pathway (TSS) in the liver (6). In the first step of the TSS, cystathionine β-synthase catalyzes the irreversible production of cystathionine from homocysteine and serine (7). In the next step, cystathionine γ-lyase (CTH) cleaves cystathionine to produce cysteine (7). Most nonhepatic tissues rely on circulating sources of cysteine, which is predominantly found in its oxidized form, cystine. Cystine is transported into cells via the xCT/CD98 transporter (also known as system X_c⁻) in exchange for glutamate (7). Cancer cells upregulate the expression and/or activity of xCT and have been shown to be particularly reliant on xCT and extracellular cystine to maintain GSH pools and redox homeostasis (7). However, a recent study demonstrated that cysteine synthesis via the TSS contributes significantly to GSH synthesis following cystine depletion (8). Thus, both pathways have the potential to mitigate oxidative stress following ECM detachment in cancer cells.

Although suppression of anoikis plays a critical role in metastasis, much is still unknown about anoikis regulation in tumors of mesenchymal origin. In this issue of Cancer Discovery, Zhang and colleagues (9) demonstrate that the cell surface protein IL1 receptor accessory protein (IL1RAP) maintains redox homeostasis and anoikis resistance via the regulation of cyst(e)ine and GSH pools (Fig. 1) in the highly metastatic childhood cancer Ewing sarcoma. To investigate novel anoikis suppressors, the authors generated a three-dimensional (3-D) culture system to model the anoikis phenotype in the mesenchymal NIH3T3 fibroblast model system. Transformation with KRAS^G12D or ETV6–NTRK3 could suppress anoikis, consistent with prior observations that oncogenic transformation promotes anoikis resistance (2). To identify anoikis resistance mechanisms, the authors performed both global proteome and acute translatome analyses and identified eight anoikis suppressor candidates, including IL1RAP. IL1RAP was selected for follow-up due to its extremely high mRNA and protein expression in Ewing sarcoma and its cell surface expression, which allows for targeting for immunotherapy. High IL1RAP expression correlated with poor survival in Ewing sarcoma, and IL1RAP depletion reduced the growth of Ewing sarcoma in 3-D and soft agar cultures, reduced the growth of xenograft tumors in vivo, and dramatically reduced metastasis. Furthermore, IL1RAP depletion induced oxidative stress in cell culture and in vivo and reduced the levels of GSH. Thus, these findings demonstrate IL1RAP is a novel mediator of redox homeostasis and anoikis resistance in Ewing sarcoma.

Although previous studies have found that IL1RAP functions as a cofactor for IL1–1L1R signaling, Zhang and colleagues (9) found that IL1RAP did not regulate IL1 signaling in Ewing sarcoma. Rather, the interactome analysis identified an association between IL1RAP and CD98, the heavy chain of the system X_c⁻ cystine transporter, suggesting IL1RAP may influence cystine uptake. Indeed, the authors found that CD98 and IL1RAP directly interacted via disulfide bond formation between CD98^C109 and IL1RAP^C362. Furthermore, IL1RAP also interacted with xCT, which was independent of CD98, suggesting the formation of a larger complex with unknown stoichiometry. Surprisingly, IL1RAP did not affect xCT plasma membrane localization or CD98 association but instead influenced the activity of system X_c⁻. They found IL1RAP depletion decreased cystine consumption and glutamate export, both indicators of system X_c⁻ activity. Consistently, intracellular cysteine and GSH levels were reduced, with lower cysteine levels also seen in vivo in Ewing sarcoma xenografts. Finally, cystine deficiency and xCT inhibition induced the iron-dependent form of cell death known as ferroptosis in many cancer cell lines (10), and, similarly, IL1RAP depletion promoted ferroptosis in Ewing sarcoma cells in combination with the xCT inhibitor erastin. In sum, these data demonstrate that IL1RAP is a novel member of the xCT–CD98 complex that promotes cystine–glutamate exchange for GSH synthesis.

Surprisingly, Zhang and colleagues (9) observed that IL1RAP depletion further decreased GSH and increased ROS levels following erastin treatment or xCT knockdown, which is unexpected if IL1RAP influences GSH solely through xCT-mediated cystine uptake. However, de novo cysteine synthesis via the TSS pathway can contribute to GSH levels and cell viability under cystine-deprived conditions in other cancer cell types (8). Importantly, CTH, which mediates the second step of the TSS pathway, was among the top downregulated hits in a proteomics analysis of IL1RAP knockdown cells. Thus, the authors surmised that IL1RAP may also promote cysteine de novo synthesis through the TSS pathway in Ewing sarcoma. They also found that IL1RAP promoted CTH mRNA expression, TSS pathway activity, and the contribution of serine to the cysteine and GSH pools. Importantly, CTH could significantly rescue the viability and lipid ROS of shIL1RAP cells following xCT inhibition. CTH was also important for Ewing sarcoma cell growth in vivo, and CTH knockdown dramatically reduced the invasion and metastasis of tumors. Like IL1RAP, CTH also contributed to cysteine pools in tumors, and CTH knockdown elevated oxidative stress. These data demonstrate that IL1RAP plays a dual role to support intracellular cysteine pools via the regulation of both cystine important and TSS activity.

Prior studies have established a role for cellular transformation and growth factor signaling pathway activation in anoikis resistance (1, 2). The finding that IL1RAP expression was induced by KRAS^G12V and ETV6–NTRK3, together with these prior studies, suggests a more general connection between oncogenic signaling and IL1RAP. To investigate this connection in Ewing sarcoma, Zhang and colleagues (9) examined whether the most common Ewing sarcoma fusion oncoprotein, EWS–FLI1, could induce IL1RAP expression. Indeed, EWS–FLI1 directly controlled IL1RAP expression by recruiting the BAF/p300 complex to the IL1RAP enhancer. In addition, EWS–FLI1 knockdown decreased CTH expression and promoted ROS accumulation and anoikis, as well as ferroptosis, when combined with erastin treatment. Finally, the authors investigated whether IL1RAP could be targeted therapeutically in Ewing sarcoma due to its presence on the plasma membrane. To this end, a human antibody domain (VH) phage–displayed library was screened against a recombinant ectodomain of IL1RAP to identify binders. One was highly specific and could induce antibody-dependent cellular cytotoxicity. These results demonstrate that IL1RAP not only promotes anoikis resistance via cysteine metabolism but is also a potential target for immunotherapy in Ewing sarcoma.

Overall, the work of Zhang and colleagues (9) connects two important observations about the role of growth factor signaling and antioxidants in anoikis resistance and warrants further work into the role of IL1RAP and its regulation by oncogenic signaling in other cancer types. Moreover, the finding that IL1RAP promotes both xCT activity and CTH expression has important implications for our understanding of cysteine metabolism and the interplay between these two pathways. However, the exact mechanism by which IL1RAP regulates CTH transcription remains to be determined and will hopefully provide further insight into the cross-talk between these pathways. Furthermore, as the authors point out, a significant contribution of the TSS to the cysteine pool has been shown only in neuroblastoma and Ewing sarcoma (8, 9), two tumor types of neuroectodermal origin, and raises interesting questions about the role of tissue/lineage of origin and dependence on the TSS versus xCT. Finally, this study demonstrates the promise of targeting IL1RAP for immunotherapy, which has the potential to both disrupt cysteine metabolism and induce antitumor immune responses for therapy.

No disclosures were reported.