cells Communication HO-1 and Heme: G-Quadruplex Interaction Choreograph DNA Damage Responses and Cancer Growth Giacomo Canesin 1,† , Anindhita Meena Muralidharan 1,†, Kenneth D. Swanson 2 and Barbara Wegiel 1,*,† 1 Department of Surgery, Division of Surgical Oncology, Cancer Research Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02214, USA; [email protected] (G.C.); [email protected] (A.M.M.) 2 Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02214, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-617-735-2846 † Current address: Vor Biopharma, 100 Cambridgepark Dr, Suite 400, Cambridge, MA 02140, USA. Abstract: Many anti-cancer therapeutics lead to the release of danger associated pattern molecules (DAMPs) as the result of killing large numbers of both normal and transformed cells as well as lysis of red blood cells (RBC) (hemolysis). Labile heme originating from hemolysis acts as a DAMP while its breakdown products exert varying immunomodulatory effects. Labile heme is scavenged by hemopexin (Hx) and processed by heme oxygenase-1 (HO-1, Hmox1), resulting in its removal and the generation of biliverdin/bilirubin, carbon monoxide (CO) and iron. We recently demonstrated that labile heme accumulates in cancer cell nuclei in the tumor parenchyma of Hx knockout mice and contributes to the malignant phenotype of prostate cancer (PCa) cells and increased metastases. Additionally, this work identified Hx as a tumor suppressor gene. Direct interaction of heme with Citation: Canesin, G.; Muralidharan, DNA G-quadruplexes (G4) leads to altered gene expression in cancer cells that regulate transcription, A.M.; Swanson, K.D.; Wegiel, B. HO-1 recombination and replication. Here, we provide new data supporting the nuclear role of HO-1 and and Heme: G-Quadruplex Interaction Choreograph DNA Damage heme in modulating DNA damage response, G4 stability and cancer growth. Finally, we discuss an Responses and Cancer Growth. Cells alternative role of labile heme as a nuclear danger signal (NDS) that regulates gene expression and 2021, 10, 1801. https://doi.org/ nuclear HO-1 regulated DNA damage responses stimulated by its interaction with G4. 10.3390/cells10071801 Keywords: G-quadruplex; heme; nuclear signaling; transcriptional control Academic Editors: Józef Dulak, Agnieszka Ja´zwa-Kusiorand Agnieszka Łoboda 1. Introduction Received: 3 June 2021 The evolution and maintenance of a permissive tumor immune microenvironment Accepted: 13 July 2021 (TIME) is essential for cancer evolution. The dynamics of the interactions among tumor Published: 16 July 2021 and resident immune and non-immune stroma cells is now appreciated to dictate clinical outcomes in response to a variety of treatments. A primary axis that influences the functions Publisher’s Note: MDPI stays neutral within the TIME and as well as response to treatment involves the production and response with regard to jurisdictional claims in to DAMPs. We and others have found that labile heme functions as a DAMP through its published maps and institutional affil- iations. interactions with toll like receptors (TLRs) and as a NDS via binding to regulatory DNA structures that control stress, survival and proliferation related gene sets. In addition, many genes associated with promoting transformed phenotypes are increasingly identified with immune modulatory functions that also affect the TIME and have also been shown to be regulated by heme. Copyright: © 2021 by the authors. Labile heme released from dying RBC or necrotic cells is scavenged by Hx and de- Licensee MDPI, Basel, Switzerland. livered to macrophages (Mφ) for its degradation by heme oxygenase-1 (HO-1, Hmox1) This article is an open access article to immunomodulatory products: carbon monoxide (CO), iron and the bile pigments, distributed under the terms and Hmox1 conditions of the Creative Commons biliverdin (BV)/bilirubin (BR) (Figure1). is a stress-induced gene, but it is also φ Attribution (CC BY) license (https:// expressed basally in resident M [1–4] and regulates inflammatory responses during creativecommons.org/licenses/by/ infection [5], tissue injury [5–7] and carcinogenesis [1,8]. We have recently shown that flfl 4.0/). Mφ either lacking Hmox1 (LysM-Cre:Hmox1 ) or exposed to heme exhibited a marked Cells 2021, 10, 1801. https://doi.org/10.3390/cells10071801 https://www.mdpi.com/journal/cells Cells 2021, 10, x 2 of 15 Cells 2021, 10, 1801 tion [5], tissue injury [5–7] and carcinogenesis [1,8]. We have recently shown that Mφ ei- 2 of 14 ther lacking Hmox1 (LysM-Cre:Hmox1flfl) or exposed to heme exhibited a marked senescent phenotype, increased p16INK4a expression and DNA damage [9,10]. Further, Mφ lacking Hmox1 senescentare deficient phenotype, in their ability increased to undergo p16INK4a maturationexpression and polarization DNA damage [2,11], [9,10 ].to Further, acti- Mφ vate multiplelacking inflammation-relatedHmox1 are deficient in signaling their ability pathways to undergo [12,13] maturation and to clear and polarizationpathogens as [2 ,11], to well as activateto release multiple cytokines inflammation-related [5]. signaling pathways [12,13] and to clear pathogens as well as to release cytokines [5]. Figure 1. A model explaining the role of heme metabolism in the regulation of heme levels and cancer growth. Labile heme released from dying cells and/or erythrocytes (hemolysis) regulates gene Figure 1. A model explaining the role of heme metabolism in the regulation of heme levels and expression via G4 DNA binding, ROS generation and TLR4 binding. BLVR- biliverdin reductase. cancer growth. Labile heme released from dying cells and/or erythrocytes (hemolysis) regulates gene expression via G4 DNA binding, ROS generation and TLR4 binding. BLVR- biliverdin reduc- Labile heme is a constituent of the cell debris or hemolyzed RBC and can drive tase. inflammation as a DAMP via binding to toll-like receptor 4 (TLR4) and promote oxidative Labilestress heme in the is cell a constituent due to presence of the of cell reactive debris ferrous or hemolyzed ions (Figure RBC1)[ and14]. can Low drive levels in- of HO-1, its altered subcellular localization (nuclear isoform), or low levels of Hx are associated flammation as a DAMP via binding to toll-like receptor 4 (TLR4) and promote oxidative with increased labile heme levels within the TIME [15–17]. Repeated heme exposures may stress in the cell due to presence of reactive ferrous ions (Figure 1) [14]. Low levels of HO- occur due to bleeding during cancer progression, trauma, or hemorrhage (i.e., surgical 1, its altered subcellular localization (nuclear isoform), or low levels of Hx are associated removal of the tumor). Recently, Panigrahy et al. has demonstrated that tumor cells killed with increased labile heme levels within the TIME [15–17]. Repeated heme exposures may by chemotherapy or targeted therapy (“tumor cell debris”) stimulate cancer growth in a occur due to bleeding during cancer progression, trauma, or hemorrhage (i.e., surgical model of tumor dormancy [18]. Our published data indicate that labile heme or lack of Hx removal of the tumor). Recently, Panigrahy et al. has demonstrated that tumor cells killed in the tumor stroma clearly promote cancer progression [10]. This might be in part due to by chemotherapy or targeted therapy (“tumor cell debris”) stimulate cancer growth in a immune imbalance, but primarily due to altered gene expression in response to heme:G4 model of tumor dormancy [18]. Our published data indicate that labile heme or lack of Hx interaction within the promoter regions of key oncogenes including c-MYC [19] as well as in the tumor stroma clearly promote cancer progression [10]. This might be in part due to heme-induced HO-1 expression. immune imbalance,Our recent but findings primarily emphasize due to altered the importance gene expression of labile in heme response in the todirect heme:G4 regulation interactionof gene within expression the promoter via modulation regions of ofkey G4 oncogenes stability [ 19including] (Figure c-MYC1). G4 are[19] well-defined as well as sec- heme-inducedondary DNAHO-1 orexpression. RNA structures resulting from Hoogstein hydrogen base pairings between Ourconsecutive recent findings guanine emphasize nucleic acidsthe importance coordinated of labile with aheme metal in cation the direct [20,21 regulation]. G4 structures of geneare expression found throughout via modulation the genome of G4 stability and play [19] regulatory (Figure 1). rolesG4 are during well-defined transcription sec- [22], ondaryrecombination DNA or RNA [ 23structures,24] and replicationresulting fr [25om–27 Hoogstein]. As such, hydrogen G4 structures base act pairings as key regulatorsbe- tween consecutiveof oncogenes guanine and cancer-driving nucleic acids coordina gene promotersted with including a metal cationKRas [20,21]., c-MYC G4, bcl2 struc-, PDGF-A, tures areRb ,foundVEGF-A throughout, hTERT [ 28the,29 genome] and telomeres and play [30 regulatory–35]. G4 structures roles during form transcription transiently during [22], recombinationS phase of the [23,24] cell cycle and whenreplication DNA [25–27]. is temporarily As such, single G4 structures stranded andact as are key subsequently reg- ulators unwoundof oncogenes by endogenous and cancer-d helicases.riving gene G4 regions promoters are known including to drive KRas genomic, c-MYC instability, bcl2, [36] PDGF-Aand, Rb application, VEGF-A, hTERT of G4