890 Manipulation of epithelial-to-mesenchymal transition reveals metastatic breast cancers support immune suppression via heme metabolism

BackgroundOncogenic epithelial-to-mesenchymal transition (EMT) enhances triple negative breast cancer (TNBC) aggressiveness and immune suppression. To identify alternative immunotherapy targets for metastatic TNBC, we reversed EMT in mesenchymal-like human TNBC and mouse mammary carcinoma models by restoring the micro-RNA-200c. This approach identified several tumor cell metabolizing enzymes with potential immune modulatory functions such as heme oxygenase-1 (HO-1). HO-1 converts heme to bilirubin, an established immune suppressor in other diseases that has never been tested as such in cancer. We hypothesize that tumor cell-HO-1 activity and subsequent bilirubin secretion enhance TNBC metastasis by supporting a pro-tumor immune microenvironment (figure 1).MethodsWe tested the impact of tumor cell-HO-1 and bilirubin on macrophage immune suppression and efferocytic capacity (engulfment of dead tumor cells) using qRT-PCR, flow cytometry and live cell imaging. Human and mouse macrophages were analyzed after treatment with exogenous bilirubin or bilirubin-depleted conditioned medium collected from tumor cells treated with tin mesoporphyrin (SnMP), an enzymatic HO-1 inhibitor. Primary tumor growth and lung metastatic burden were observed in syngeneic mice harboring HO-1 depleted 66Cl-4 mammary tumors (shRNA). Breast cancer specimens were analyzed via CIBERSORT to predict immune cell abundance in patients with high versus low levels of heme metabolism genes.ResultsMacrophages cultured with conditioned medium from tumor cells treated with the HO-1 inhibitor SnMP demonstrated a 35–65% decrease in immune suppressive genes (Arg1, Cd274, Tgfb1) compared to those treated with control conditioned medium. This effect was rescued by exogenous treatment with 2.5 µM bilirubin. Direct bilirubin treatment enhanced macrophage PD-L1 mRNA and protein expression by at least 6-fold. In contrast, bilirubin decreased expression of macrophage efferocytosis genes (Mertk, Tyro3) by at least 50%, resulting in decreased efferocytic capacity. To test whether bilirubin supports tumor progression via modulation of macrophages, we evaluated tumor growth and metastasis after tumor cell-HO-1 depletion. While mice with shHO1 tumors had enhanced primary tumor growth compared to those with shCnt tumors, HO-1 depletion decreased lung metastatic capacity. Although immune cell infiltration and activation is currently underway in this mouse model, CIBERSORT analysis revealed that breast cancer specimens with high levels of heme metabolism genes have a predicted increase in M2 macrophage presence.Abstract 890 Figure 1Project modelHeme oxygease-1 (HO-1) breaks down heme into immune modulatory products such as bilirubin (BR). We demonstrated that tumor cell-secreted BR may enhance TNBC lung metastasis by supporting macrophage immune suppression and dysfunction. This can be blocked by genetic (shRNA) or pharmacologic (SnMP) inhibition of HO-1 in TNBC cells. Figure made with biorender.com.ConclusionsTumor cell-HO-1 may support immune cell suppression and dysfunction during breast cancer metastasis via bilirubin. Since HO-1 inhibitors including SnMP are FDA approved for treatment of other diseases, these findings could rapidly be translated to provide an additional immunotherapy for metastatic TNBC.

New Insights into the Pivotal Role of Iron/Heme Metabolism in TLR4/NF-κB Signaling-Mediated Inflammatory Responses in Human Monocytes

Iron metabolism and heme biosynthesis are essential processes in cells during the energy cycle. Alteration in these processes could create an inflammatory condition, which results in tumorigenesis. Studies are conducted on the exact role of iron/heme metabolism in induced inflammatory conditions. This study used lipopolysaccharide (LPS)- or high-glucose-induced inflammation conditions in THP-1 cells to study how iron/heme metabolism participates in inflammatory responses. Here, we used iron and heme assays for measuring total iron and heme. We also used flow cytometry and Western blotting to analyze molecular responses. Our results demonstrated that adding LPS or high-glucose induced iron formation and heme synthesis and elevated the expression levels of proteins responsible for iron metabolism and heme synthesis. We then found that further addition of heme or 5-aminolevulinic acid (ALA) increased heme biosynthesis and promoted inflammatory responses by upregulating TLR4/NF-κB and inflammatory cytokine expressions. We also demonstrated the inhibition of heme synthesis using succinylacetone (SA). Moreover, N-MMP inhibited LPS- or high-glucose-induced inflammatory responses by inhibiting TLR4/NF-κB signaling. Hence, iron/heme metabolism checkpoints could be considered a target for treating inflammatory conditions.

COVID-19:Attacks the 1-Beta Chain of Hemoglobin and Captures the Porphyrin to Inhibit Human Heme Metabolism

The novel coronavirus pneumonia is a contagious acute respiratory disease caused by the SARS-COV-2 coronavirus. The pathogenic mechanism of the novel coronavirus is unknown, which presents a significant impediment to the patient rescue. A conserved domain search strategy was utilized in this work to determine that a large number of viral proteins could bind to hemoglobin. S could bind to extracellular hemoglobin. SARS-COV-2 virus proteins interacted with porphyrins. SARS-COV-2 viruses could synthesize heme from porphobilinogen and encode all the similar enzymes required for the process. Both E and ORF3a contained heme-binding sites. ORF3a's Arg134 and E's Cys44 were the heme-iron binding sites, respectively. ORF3a also contained homologous domains to human cytochrome C reductase and bacterial EFeB protein. The molecular docking analysis revealed that ORF3a and ORF8 proteins were shown to be capable of attacking hemoglobin's 1-beta chain, whereas ORF3a was found to be effective in capturing heme for dissociation to iron and porphyrin. Deoxyhemoglobin was more susceptible to viral attack than oxidized hemoglobin. In summary, the combination of viral proteins to porphyrins and their metal compounds would improve the ability to permeate cell membranes and generate oxygen free radicals (ROS). It may be associated with viral infections and epidemic transmission. Viral proteins regulated the production and function of NO, CO and CO2 by inhibiting the activity of hemoglobin, thereby affecting immune cell function. Viral proteins' attack on hemoglobin could result in symptoms such as respiratory distress and blood clotting, damage to numerous organs and tissues, and disruption of normal human heme metabolism.

CYTOCHROMES OF MITOCHONDRIES AND ACTIVITY OF HEME METABOLISM ENZYMES IN THE LIVER UNDER DIFFERENT NUTRIENT REGIMES

The content of mitochondrial cytochromes and the activity of key enzymes of heme metabolism in the liver of rats under conditions of different dietary supply of protein and sucrose were investigated. The quantitative determination of mitochondrial cytochrome was performed by differential spectrophotometry, δ-aminolevulinate synthase activity was determined spectrophotometrically taking into account the molar extinction coefficient of 0.023x10(3) M(-1)sm(-1). Hemoxygenase activity was determined using the amount of formed bilirubin. It was found that under conditions of consumption of high-sucrose diet a significant decrease in the content of all mitochondrial cytochromes is noted: the content of cytochromes aa3, b and c1 decreases within 1.2-1.7 times, and content of cytochrome c decreases in two times. In the case of excessive consumption of sucrose on the background of alimentary protein deprivation the content of cytochromes b and c1 in the liver of rats does not differ statistically from similar indicators of the group of animals kept on a high-sucrose diet. At the same time, the content of cytochromes aa3 and c is significantly reduced. According to the activity of δ-aminolevulinate synthase under conditions of consumption of a high-sucrose diet, the studied enzymatic activity decreases by about 1.5 times with a simultaneous increase in the activity of heme oxygenase. Thus, there is a marked decrease in heme synthesis against the background of increased catabolism, which explains the decrease in the content of cytochromes in the mitochondria of the liver of rats under conditions of excess sucrose in the diet. The maximum increase in the activity of heme oxygenase (almost threefold) is observed in animals that were kept on a high-sugar diet deficient in protein content. Thus, dietary protein deficiency is a critical factor affecting the heme metabolism in the mitochondria of liver cells. The established changes in the content of mitochondrial cytochromes and the activities of key enzymes of heme metabolism in the liver could be considered as prerequisites for deepening its energy imbalance in conditions of different supply of sucrose and protein in diet.

Genetic screens reveal a central role for heme metabolism in artemisinin susceptibility

Artemisinins have revolutionized the treatment of Plasmodium falciparum malaria; however, resistance threatens to undermine global control efforts. To broadly explore artemisinin susceptibility in apicomplexan parasites, we employ genome-scale CRISPR screens recently developed for Toxoplasma gondii to discover sensitizing and desensitizing mutations. Using a sublethal concentration of dihydroartemisinin (DHA), we uncover the putative transporter Tmem14c whose disruption increases DHA susceptibility. Screens performed under high doses of DHA provide evidence that mitochondrial metabolism can modulate resistance. We show that disrupting a top candidate from the screens, the mitochondrial protease DegP2, lowers porphyrin levels and decreases DHA susceptibility, without significantly altering parasite fitness in culture. Deleting the homologous gene in P. falciparum, PfDegP, similarly lowers heme levels and DHA susceptibility. These results expose the vulnerability of heme metabolism to genetic perturbations that can lead to increased survival in the presence of DHA.

Disruption of Plasmodium Falciparum Histidine-Rich Protein II can affect Heme Metabolism in the Blood Stage

Background. Heme is a key metabolic factor in the life of malaria parasite. In the blood stage it acquires host hemoglobin to generate amino acids for its own protein synthesis and by-product heme for metabolic use. The malaria parasite also can de novo synthesize heme by itself. Plasmodium falciparum-specific histidine-rich protein 2 (PfHRP2) is a histidine-rich protein and has a heme-binding site to mediate hemozoin formation, a bio-crystallized form of heme-aggregates. It is interesting to investigate the vibration of hemoglobin-derived heme metabolism and de novo heme-biosynthetic pathway in the Pfhrp2 disrupted parasites during the intraerythrocytic stages. Methods. A CRISPR-Cas9 system was used to disrupt the gene locus of Pfhrp2. DNA was extracted from the transgenic parasites and polymerase chain reaction (PCR), western blotting and southern blotting were used to manifest the successful establishment of transgenic parasites. RNA-seq and comparative transcriptome analysis were performed to identify the difference in gene expression between 3D7 and Pfhrp2- 3D7 parasites. Results. Pfhrp2- transgenic parasites were successfully established by the CRISPR/Cas9 system. The disruption of the exon 2 of Pfhrp2 can down-regulate the gene expression of Pfhrp3 which involved in hemoglobin-derived heme metabolism. It also up-regulates the gene expression level of enzymes of heme biosynthesis.Conclusion: These data support that although Pfhrp2 is a dispensable gene for intraerythrocytic stages parasite but heme metabolism’s stabilizing is very important. The disruption of Pfhrp2 can both affect the pathway of heme metabolism and biosynthesis. A co-operation mechanism may exist between the heme biosynthesis and metabolism pathways for parasite growth in blood stage.

Hereditary Ataxia: A Focus on Heme Metabolism and Fe-S Cluster Biogenesis

Heme and Fe-S clusters regulate a plethora of essential biological processes ranging from cellular respiration and cell metabolism to the maintenance of genome integrity. Mutations in genes involved in heme metabolism and Fe-S cluster biogenesis cause different forms of ataxia, like posterior column ataxia and retinitis pigmentosa (PCARP), Friedreich’s ataxia (FRDA) and X-linked sideroblastic anemia with ataxia (XLSA/A). Despite great efforts in the elucidation of the molecular pathogenesis of these disorders several important questions still remain to be addressed. Starting with an overview of the biology of heme metabolism and Fe-S cluster biogenesis, the review discusses recent progress in the understanding of the molecular pathogenesis of PCARP, FRDA and XLSA/A, and highlights future line of research in the field. A better comprehension of the mechanisms leading to the degeneration of neural circuity responsible for balance and coordinated movement will be crucial for the therapeutic management of these patients.

Nutrients and Porphyria: An Intriguing Crosstalk

Porphyria refers to a group of fascinating diseases from a metabolic and nutritional standpoint as it provides an example of how metabolic manipulation can be used for therapeutic purposes. It is characterized by defects in heme synthesis, particularly in the erythrocytes and liver. Specific enzymes involved in heme biosynthesis directly depend on adequate levels of vitamins and minerals in the tissues. Moreover, micronutrients that are required for producing succinyl CoA and other intermediates in the Krebs (TCA) cycle are indirectly necessary for heme metabolism. This review summarizes articles that describe the nutritional status, supplements intake, and dietary practices of patients affected by porphyria, paying special attention to the therapeutic use of nutrients that may help or hinder this group of diseases.