metabolites H OH OH Article Iron-Bound Lipocalin-2 Protects Renal Cell Carcinoma from Ferroptosis Julia K. Meier 1 , Matthias Schnetz 1 , Susanne Beck 2 , Tobias Schmid 1 , Monica Dominguez 1 , Sanela Kalinovic 3 , Andreas Daiber 3 , Bernhard Brüne 1,4,5,6 and Michaela Jung 1, *   Citation: Meier, J.K.; Schnetz, M.; Beck, S.; Schmid, T.; Dominguez, M.; Kalinovic, S.; Daiber, A.; Brüne, B.; Jung, M. Iron-Bound Lipocalin-2 Protects Renal Cell Carcinoma from Ferroptosis. Metabolites 2021, 11, 329. https://doi.org/10.3390/metabo 11050329 Academic Editor: James McCullagh Received: 7 April 2021 Accepted: 17 May 2021 Published: 19 May 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Faculty of Medicine, Institute of Biochemistry I, Goethe-University Frankfurt, 60590 Frankfurt am Main, Germany; meier@biochem.uni-frankfurt.de (J.K.M.); matthias.schnetz@t-online.de (M.S.); t.schmid@biochem.uni-frankfurt.de (T.S.); dominguez@biochem.uni-frankfurt.de (M.D.); b.bruene@biochem.uni-frankfurt.de (B.B.) 2 Institute of Pathology, University Hospital Heidelberg, 69120 Heidelberg, Germany; Susanne.Beck@med.uni-heidelberg.de 3 Department of Cardiology 1, Molecular Cardiology, Medical Center of the Johannes Gutenberg University, 55131 Mainz, Germany; sanelakalinovic@gmail.com (S.K.); Daiber@uni-mainz.de (A.D.) 4 Fraunhofer Institute for Translational Medicine and Pharmacology, 60596 Frankfurt am Main, Germany 5 German Cancer Consortium (DKTK), Partner Site Frankfurt, 60590 Frankfurt am Main, Germany 6 Frankfurt Cancer Institute, Goethe-University Frankfurt, 60596 Frankfurt am Main, Germany * Correspondence: m.jung@biochem.uni-frankfurt.de 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 pro-survival pathways initiated by iron-loaded Lcn-2. Keywords: lipocalin-2; iron; ROS; SLC7A11; Nrf2; ISR; p-eIF2α; erastin; ferroptosis 1. Introduction One of the most recurrent urologic tumors is renal cell carcinoma (RCC). Due to its clinically silent development and progression, RCC represents a major, yet underestimated health problem, since the detection of the majority of RCC cases is by incidental radiologic discovery [1]. Resistance of RCC against chemo- and radiation-therapy further restricts therapeutic medical options [2]. Currently, no reliable diagnostic approach for early RCC detection and no effective method for recurrence surveillance or therapy response are available. It appears pivotal to identify suitable diagnostic and prognostic biomarkers, in order to enable detection of premetastatic tumors and to install efficient therapy approaches such as partial rather than radical nephrectomy. An unmet need also represents a more Metabolites 2021, 11, 329. https://doi.org/10.3390/metabo11050329 https://www.mdpi.com/journal/metabolites