Iron‐bound lipocalin‐2 protects renal cell carcinoma from ferroptosis

Abstract

While the importance of the iron‐load of lipocalin‐2 (Lcn‐2) in promoting tumor progression is widely appreciated, underlying molecular mechanisms largely remain elusive. Considering its role as an iron‐transporter, we aimed at clarifying iron‐loaded, holo‐Lcn‐2 (hLcn‐2)‐dependent signaling pathways in affecting renal cancer cell viability. Applying RNA sequencing analysis in renal CAKI1 tumor cells to explore highly upregulated molecular signatures in response to hLcn‐2, we identified a cluster of genes (SLC7A11, GCLM, GLS), which are implicated in regulating ferroptosis. Indeed, hLcn‐2‐stimulated cells are protected from erastin‐induced ferroptosis. We also noticed a rapid increase in reactive oxygen species (ROS) with subsequent activation of the antioxidant Nrf2 pathway. However, knocking down Nrf2 by siRNA was not sufficient to induce erastin‐dependent ferroptotic cell death in hLcn‐2‐stimulated tumor cells. In contrast, preventing oxidative stress through N‐acetyl‐L‐cysteine (NAC) supplementation was still able to induce erastin‐dependent ferroptotic cell death in hLcn‐2‐stimulated tumor cells. Besides an oxidative stress response, we noticed activation of the integrated stress response (ISR), shown by enhanced phosphorylation of eIF‐2α and induction of ATF4 after hLcn‐2 addition. ATF4 knockdown as well as inhibition of the ISR sensitized hLcn‐2‐treated renal tumor cells to ferroptosis, thus linking the ISR to pro‐tumor characteristics of hLcn‐2. Our study provides mechanistic details to better understand tumor prosurvival pathways initiated by iron‐loaded Lcn‐2.