Heme oxygenase-1 mitigates liver injury and fibrosis via modulation of LNX1/Notch1 pathway in myeloid cells.

Activation of resident macrophages (Mϕ) and hepatic stellate cells is a key event in chronic liver injury. Mice with heme oxygenase-1 (HO-1; Hmox1)-deficient Mϕ (LysM-Cre:Hmox1 flfl ) exhibit increased inflammation, periportal ductular reaction, and liver fibrosis following bile duct ligation (BDL)-induced liver injury and increased pericellular fibrosis in NASH model. RiboTag-based RNA-sequencing profiling of hepatic HO-1-deficient Mϕ revealed dysregulation of multiple genes involved in lipid and amino acid metabolism, regulation of oxidative stress, and extracellular matrix turnover. Among these genes, ligand of numb-protein X1 (LNX1) expression is strongly suppressed in HO-1-deficient Mϕ. Importantly, HO-1 and LNX1 were expressed by hepatic Mϕ in human biliary and nonbiliary end-stage cirrhosis. We found that Notch1 expression, a downstream target of LNX1, was increased in LysM-Cre:Hmox1 flfl mice. In HO-1-deficient Mϕ treated with heme, transient overexpression of LNX1 drives M2-like Mϕ polarization. In summary, we identified LNX1/Notch1 pathway as a downstream target of HO-1 in liver fibrosis.

Labile Heme and Heme Oxygenase-1 Maintain Tumor-Permissive Niche for Endometriosis-Associated Ovarian Cancer.

Endometriosis, a painful gynecological condition accompanied by inflammation in women of reproductive age, is associated with an increased risk of ovarian cancer. We evaluated the role of peritoneal heme accumulated during menstrual cycling, as well as peritoneal and lesional macrophage phenotype, in promoting an oncogenic microenvironment. We quantified the heme-degrading enzyme, heme oxygenase-1 (HO-1, encoded by Hmox1) in normal peritoneum, endometriotic lesions and endometriosis-associated ovarian cancer (EAOC) of clear cell type (OCCC). HO-1 was expressed primarily in macrophages and increased in endometrioma and OCCC tissues relative to endometriosis and controls. Further, we compared cytokine expression profiles in peritoneal macrophages (PM) and peripheral blood mononuclear cells (PBMC) in women with endometriosis versus controls as a measure of a tumor-promoting environment in the peritoneum. We found elevated levels of HO-1 along with IL-10 and the pro-inflammatory cytokines (IL-1ß, IL-16, IFNγ) in PM but not in PBMC from endometriosis patients. Using LysM-Cre:Hmox1flfl conditional knockout mice, we show that a deficiency of HO-1 in macrophages led to the suppression of growth of ID8 ovarian tumors implanted into the peritoneum. The restriction of ID8 ovarian tumor growth was associated with an increased number of Mac3+ macrophage and B cells in LysM-Cre:Hmox1flfl mice compared to controls. Functional experiments in ovarian cancer cell lines show that HO-1 is induced by heme. Low levels of exogenous heme promoted ovarian cancer colony growth in soft agar. Higher doses of heme led to slower cancer cell colony growth in soft agar and the induction of HO-1. These data suggest that perturbation of heme metabolism within the endometriotic niche and in cancer cells themselves may be an important factor that influences tumor initiation and growth.

HO-1 and Heme: G-Quadruplex Interaction Choreograph DNA Damage Responses and Cancer Growth.

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 DNA G-quadruplexes (G4) leads to altered gene expression in cancer cells that regulate transcription, recombination and replication. Here, we provide new data supporting the nuclear role of HO-1 and heme in modulating DNA damage response, G4 stability and cancer growth. Finally, we discuss an alternative role of labile heme as a nuclear danger signal (NDS) that regulates gene expression and nuclear HO-1 regulated DNA damage responses stimulated by its interaction with G4.

Measurement of labile and protein-bound heme in fixed prostate cancer cells and in cellular fractions.

Labile heme is present in the cells at very low concentrations, either unbound or loosely bound to molecules, and accessible for signaling as alarmin. Our recent work suggests that extracellular heme can be taken up and detected in the nuclei of cancer cells. Here, we describe the detailed protocol for detection of labile and total heme in prostate cancer cells and its measurement in subcellular compartments in vitro. The protocol can be adapted to be used for other cell types. For complete details on the use and execution of this protocol, please refer to Canesin et al. (2020).

Scavenging of Labile Heme by Hemopexin Is a Key Checkpoint in Cancer Growth and Metastases.

Hemopexin (Hx) is a scavenger of labile heme. Herein, we present data defining the role of tumor stroma-expressed Hx in suppressing cancer progression. Labile heme and Hx levels are inversely correlated in the plasma of patients with prostate cancer (PCa). Further, low expression of Hx in PCa biopsies characterizes poorly differentiated tumors and correlates with earlier time to relapse. Significantly, heme promotes tumor growth and metastases in an orthotopic murine model of PCa, with the most aggressive phenotype detected in mice lacking Hx. Mechanistically, labile heme accumulates in the nucleus and modulates specific gene expression via interacting with guanine quadruplex (G4) DNA structures to promote PCa growth. We identify c-MYC as a heme:G4-regulated gene and a major player in heme-driven cancer progression. Collectively, these results reveal that sequestration of labile heme by Hx may block heme-driven tumor growth and metastases, suggesting a potential strategy to prevent and/or arrest cancer dissemination.

STAT3 inhibition with galiellalactone effectively targets the prostate cancer stem-like cell population.

Cancer stem cells (CSCs) are a small subpopulation of quiescent cells with the potential to differentiate into tumor cells. CSCs are involved in tumor initiation and progression and contribute to treatment failure through their intrinsic resistance to chemo- or radiotherapy, thus representing a substantial concern for cancer treatment. Prostate CSCs' activity has been shown to be regulated by the transcription factor Signal Transducer and Activator of Transcription 3 (STAT3). Here we investigated the effect of galiellalactone (GL), a direct STAT3 inhibitor, on CSCs derived from prostate cancer patients, on docetaxel-resistant spheres with stem cell characteristics, on CSCs obtained from the DU145 cell line in vitro and on DU145 tumors in vivo. We found that GL significantly reduced the viability of docetaxel-resistant and patient-derived spheres. Moreover, CSCs isolated from DU145 cells were sensitive to low concentrations of GL, and the treatment with GL suppressed their viability and their ability to form colonies and spheres. STAT3 inhibition down regulated transcriptional targets of STAT3 in these cells, indicating STAT3 activity in CSCs. Our results indicate that GL can target the prostate stem cell niche in patient-derived cells, in docetaxel-resistant spheres and in an in vitro model. We conclude that GL represents a promising therapeutic approach for prostate cancer patients, as it reduces the viability of prostate cancer-therapy-resistant cells in both CSCs and non-CSC populations.

A Role of the Heme Degradation Pathway in Shaping Prostate Inflammatory Responses and Lipid Metabolism.

The molecular mechanisms of prostate inflammation are unclear. We hypothesized that heme oxygenase 1 (HMOX1; HO-1), an enzyme responsible for degradation of heme to carbon monoxide, bilirubin, and iron, is an important regulator of inflammation and epithelial responses in the prostate. Injection of non-uropathogenic Escherichia coli (MG1655 strain) or phosphate-buffered saline into the urethra of mice led to increased numbers of CD45+ leukocytes and mitotic markers (phosphorylated histone H3 and phosphorylated ERK1/2) in the prostate glands. Leukocyte infiltration was elevated in the prostates harvested from mice lacking HO-1 in myeloid compartment. Conversely, exogenous carbon monoxide (250 ppm) increased IL-1ß levels and suppressed cell proliferation in the prostates. Carbon monoxide did not affect the number of infiltrating CD45+ cells in the prostates of E. coli- or phosphate-buffered saline-treated mice. Interestingly, immunomodulatory effects of HO-1 and/or carbon monoxide correlated with early induction of the long-chain acyl-CoA synthetase 1 (ACSL1). ACSL1 levels were elevated in response to E. coli treatment, and macrophage-expressed ACSL1 was in part required for controlling of IL-1ß expression and prostate cancer cell colony growth in soft agar. These results suggest that HO-1 and/or carbon monoxide might play a distinctive role in modulating prostate inflammation, cell proliferation, and IL-1ß levels in part via an ACSL1-mediated pathway.

Heme-Derived Metabolic Signals Dictate Immune Responses.

Heme is one of the most abundant molecules in the body acting as the functional core of hemoglobin/myoglobin involved in the O2/CO2 carrying in the blood and tissues, redox enzymes and cytochromes in mitochondria. However, free heme is toxic and therefore its removal is a significant priority for the host. Heme is a well-established danger-associated molecular pattern (DAMP), which binds to toll-like receptor 4 (TLR4) to induce immune responses. Heme-derived metabolites including the bile pigments, biliverdin (BV) and bilirubin (BR), were first identified as toxic drivers of neonatal jaundice in 1800 but have only recently been appreciated as endogenous drivers of multiple signaling pathways involved in protection from oxidative stress and regulators of immune responses. The tissue concentration of heme, BV and BR is tightly controlled. Heme oxygenase-1 (HO-1, encoded by HMOX1) produces BV by heme degradation, while biliverdin reductase-A (BLVR-A) generates BR by the subsequent conversion of BV. BLVR-A is a fascinating protein that possesses a classical protein kinase domain, which is activated in response to BV binding to its enzymatic site and initiates the downstream mitogen-activated protein kinases (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways. This links BLVR-A activity to cell growth and survival pathways. BLVR-A also contains a bZip DNA binding domain and a nuclear export sequence (NES) and acts as a transcription factor to regulate the expression of immune modulatory genes. Here we will discuss the role of heme-related immune response and the potential for targeting the heme system for therapies directed toward hepatitis and cancer.

Deletion of Biliverdin Reductase A in Myeloid Cells Promotes Chemokine Expression and Chemotaxis in Part via a Complement C5a--C5aR1 Pathway.

Biliverdin reductase (BVR)-A is a pleotropic enzyme converting biliverdin to bilirubin and a signaling molecule that has cytoprotective and immunomodulatory effects. We recently showed that biliverdin inhibits the expression of complement activation fragment 5a receptor one (C5aR1) in RAW 264.7 macrophages. In this study, we investigated the role of BVR-A in determining macrophage inflammatory phenotype and function via regulation of C5aR1. We assessed expression of C5aR1, M1-like macrophage markers, including chemokines (RANTES, IP-10), as well as chemotaxis in response to LPS and C5a in bone marrow-derived macrophages from BVR fl/fl and LysM-Cre:BVR fl / fl mice (conditional deletion of BVR-A in myeloid cells). In response to LPS, macrophages isolated from LysM-Cre:BVR fl/fl showed significantly elevated levels of C5aR1 as well as chemokines (RANTES, IP10) but not proinflammatory markers, such as iNOS and TNF. An increase in C5aR1 expression was also observed in peritoneal macrophages and several tissues from LysM-Cre:BVR fl/fl mice in a model of endotoxemia. In addition, knockdown of BVR-A resulted in enhanced macrophage chemotaxis toward C5a. Part of the effects of BVR-A deletion on chemotaxis and RANTES expression were blocked in the presence of a C5aR1 neutralizing Ab, confirming the role of C5a-C5aR1 signaling in mediating the effects of BVR. In summary, BVR-A plays an important role in regulating macrophage chemotaxis in response to C5a via modulation of C5aR1 expression. In addition, macrophages lacking BVR-A are characterized by the expression of M1 polarization-associated chemokines, the levels of which depend in part on C5aR1 signaling.

Heme detoxification by heme oxygenase-1 reinstates proliferative and immune balances upon genotoxic tissue injury.

Phenotypic changes of myeloid cells are critical to the regulation of premature aging, development of cancer, and responses to infection. Heme metabolism has a fundamental role in the regulation of myeloid cell function and activity. Here, we show that deletion of heme oxygenase-1 (HO-1), an enzyme that removes heme, results in an impaired DNA damage response (DDR), reduced cell proliferation, and increased cellular senescence. We detected increased levels of p16INK4a, H2AXγ, and senescence-associated-ß-galactosidase (SA-ß-Gal) in cells and tissues isolated from HO-1-deficient mice. Importantly, deficiency of HO-1 in residential macrophages in chimeric mice results in elevated DNA damage and senescence upon radiation-induced injury. Mechanistically, we found that mammalian target of rapamycin (mTOR)/S6 protein signaling is critical for heme and HO-1-regulated phenotype of macrophages. Collectively, our data indicate that HO-1, by detoxifying heme, blocks p16INK4a expression in macrophages, preventing DNA damage and cellular senescence.