Review Article 247

Hemoglobins, , and 11p15.5 Chromosomal Region in Cancer Biology and İmmunity with Special Emphasis for Brain Tumors

Meric Adil Altinoz1 Ilhan Elmaci2 Bahri Ince3 Aysel Ozpinar4 Aydin Murat Sav5

1 Department of Immunology, Istanbul University, DETAE - Address for correspondence Meric Adil Altinoz, MD, Department of Experimental Medicine Research Institute, Istanbul, Turkey Immunology, Istanbul University, DETAE - Experimental Medicine 2 Department of Neurosurgery, Memorial Hospital, Istanbul, Turkey Research Institute, Istanbul, Turkey 34300 3 Department of Psychiatry, Bakirkoy Mental Diseases Education and (e-mail: [email protected]). Training Hospital, Istanbul, Turkey 4 Department of Biochemistry, Acibadem University, Istanbul, Turkey 5 Department of Pathology, Acibadem University, Istanbul, Turkey

J Neurol Surg A 2016;77:247–257.

Abstract In systemic cancers, increased hemolysis leads to extracellular hemoglobin (HB), and experimental studies have shown its provoking role on tumor growth and metastasis. However, investigations have shown that HB chains presented by tumor vascular pericytes or serum protein complexes of HB could also induce antitumor immunity, which may be harnessed to treat refractory cancers and brain tumors. Mounting recent evidence shows that expression of HBs is not restricted to erythrocytes and that HBs exist in the cells of lung and kidney, in macrophages, and in neurons and glia of the central nervous system (CNS). HBs mediate coping with hypoxia and free radical stress in normal and tumor cells, and they are increased in certain tumors including breast, lung, colon, and squamous cell cancers. Recent studies showed HBs in meningioma, in the cyst fluid of craniopharyngioma, in the cerebrospinal fluid (CSF) of pediatric patients with posterior fossa tumors, and in glioblastoma cell lines. Hemorphins, abundant brain formed via HB-chain cleavage, exert activity and antiproliferative and immunomodifier effects. Hence mutations in HBs may modify brain tumorigenesis via influencing hemorphins and perturbing regulations of immune surveillance and cell growth in the neuroectodermal tissues. The β-globin gene cluster resides in the chromosome region 11p15.5, harboring important immunity genes and IGF2, H19, PHLDA2/TSSC3, TRIM3, and SLC22A18 genes associated with cancers and gliomas. 11p15.5 is a prominent region subject to epigenetic regulation. Thus the β-globin loci may exert haplotypal interactions with these. Some clues support this theory. It is well established that iron load induces liver cancer in thalassemia major; however iron Keywords load–independent associations also exist. Enhanced rates of hematologic malignancies ► hemoglobins are associated with HB Lepore, association of hemoglobin E with cholangiocarcinoma, ► brain tumors and enhanced gastric cancer rates in the thalassemia trait. In the African Herero ► 11p15.5 gene cluster population, a mutant form of δ-globin is very prevalent, and this population has higher

received © 2016 Georg Thieme Verlag KG DOI http://dx.doi.org/ April 30, 2015 Stuttgart · New York 10.1055/s-0035-1566120. accepted after revision ISSN 2193-6315. August 11, 2015 published online March 2, 2016 248 Novel Pathways of Glial Tumor Formation Altinoz et al.

rates of pediatric brain tumors. Globins are also expressed in healthy endothelia and in tumoral vessels, indicating potential involvement in angiogenesis. Studies on HBs and their cleavage peptides in cancers and brain tumors may lead to innovative treatment strategies.

Introduction Gower-2 (α2ε2), Portland-1 (ζ2γ2) HB, and HB-F (fetal hemo- globin) (α2γ2). In adults, three HBs exist: the major adult HB- Primary central nervous system (CNS) tumors have gained A(α2β2, 97%) and the minor HBs, HB-A2 (α2δ2, < 3%)11 the attention of researchers for many reasons. In high-grade and HB-F (<0.6%).12 HB-A consists of two α- and two β-globin glial tumors, maximum surgical resection, radiotherapy, and globular chain proteins assembling into a α2β2 heterote- adjuvant temozolomide is the new standard of care. Yet the tramer, where each globin molecule binds a heme prosthetic 13 median survival of patients with glioblastoma multiforme group. In adult HB, the carriage of O2 is mediated via (GBM) is 15 months.1 This outcome is alarmingly poor; binding of the ligand to heme iron. Carbon dioxide is trans- hence deciphering novel pathways of glial tumor formation is ported in the blood in solution and by interactions with the essential to develop better treatments. In other brain tumors aminoterminal residues of HB and not via binding to the that are not as invasive as GBM, patients’ morbidity and heme iron.3 HB is also involved in the generation, transport, mortality after surgical resection are still high due to the and scavenging of NO and its metabolic derivatives.3,14,15 The obvious essentiality of all neural tissues in the CNS. In antioxidant functions of intracellular HB and superoxide and systemic cancers, survival ratios are still far from satisfactory hydrogen peroxide scavenger properties were discovered,9,16 for metastatic disease. and its detoxification of oxidizing radicals yields nontoxic In this review, we discuss hemoglobin (HB) chains, hem- ferric states.17 orphins formed via cleavage of globin chains,2 and the 11p15.5 gene locus pertinent to malignancies with special Tumor-Provoking Roles of Extracellular HBs emphasis on brain tumors. The 11p15.5 is a chromosomal 3 site, where β-globin gene cluster and genes in association To expand in size, tumors must recruit new vessels for O2 and with gliomagenesis reside including IGF-2, H19, TSSC3, TRIM3, nutrient supply.18 Elevation of vascular endothelial growth and SLC22A18. Important genes of immunity also reside factor A (VEGF-A)19 and mechanical stress generated by here,4 and loss of heterozygosity (LOH) events at this site expanding tumor cells result in tortuous vessels18 leading are frequent in high-grade glial tumors.5 Because 11p15.5 is a to an uneven blood flow and lower perfusion within prominent chromosomal site subject to epigenetic regulation tumors.20 However, tumors manage to expand despite insuf-

such as imprinting, the β-globin gene cluster and glioma- ficient vascular supply, suggesting other mechanisms of O2 susceptibility genes may exert haplotypal associations. In the delivery. Tumor cells are able to form primitive microvessels – last decade, the presence of HB chains in nonerythroid cells via vasculogenic mimicry (VM),21 23 and highly invasive and their upregulation in malignancies were discovered. tumors line fluid-conducting channels.21 However, an effi- Expressions of HB chains in macrophages,6 lung cells,7,8 renal cient RBC circulation through these channels is problematic mesangium,9 and retinal ganglion cells10 were found, and because these channels exert varying diameters22 and are 21 their roles in oxygen (O2) sensing and protection against free often too small (< 5 µm in diameter). Retraction of endo- radicals were discovered. thelial cell (EC)-lined blood vessels leave empty sleeves composed only of basement membrane24 resembling inver- 23 tebrate vessels. In invertebrates, extraerythrocytic HB-O2 A Short Description of HB Genetics and 25 26 Biochemistry exists and transports O2 through the organism. Although fluid-conducting primitive channels and tumoral vasculo- 27 HBs are heme-containing proteins binding O2,carbonmon- genic mimicry may hamper efficient RBC circulation, they oxide, and nitric oxide (NO). In red blood cells (RBCs), HB may facilitate diffusion of free HB. accounts for 98% of proteins, and during differentiation Hence a four-step process is suggested, where extracellular 28 from reticulocytes, RBCs finish the synthesis of HB before HB contributes to tumoral O2 delivery. First, hemolysis in 2 28 ribosomal degradation. The human α-globin gene cluster tumor vessels generates extraerythrocytic HB-O2. Second,

locates on chromosomal region 16p13.3 and includes seven extraerythrocytic HB-O2 is transported within the small loci2:5′- zeta(ζ) – pseudozeta (Ψζ) – mu(µ) – pseudoalpha1 intercellular channels in plasma. Third, within the hypoxic

(Ψα1) – α2–α1–theta(θ) – 3′.Thehumanβ-globin gene and acidic tumor environment, extraerythrocytic HB-O2

cluster locates on chromosomal region 11p15.5 and includes delivers O2 to adjacent tumor cells. And finally, the deoxy- five loci2:5′–epsilon(ε) – gammaG (Gγ) – gammaA(Aγ) – genated HB close to the EC-lined tumor vessels deprives

delta(δ) – β(β) – 3′. During ontogeny, developing erythro- intraerythrocytic HB-O2 of O2, thus starting a new cycle of 28 blasts sequentially express embryonic Gower-1 (ζ2ε2), O2 transport. It was also demonstrated with experimental

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29 3þ studies that ECs could uptake free HB, yet it remains to be is in the ferric (Fe ) state, it cannot bind O2. In human blood, shown whether this is relevant for tumor vessels. metHB occurs at trace amounts spontaneously (1 to 2% of total Tumor cells enhance procoagulant activity (PCA) and HB) or at higher levels during oxidizing conditions. NO reacts platelet adhesion on their surface shield cancer antigens to with oxyHB to produce metHB, and most of the metHB in facilitate escape from immune surveillance and reduce their RBCs is derived from this oxidation.3 The metHB is also shear stress–associated death in circulation. Tissue factor (TF) present in solid tumors detectable by MR imaging37 and is a transmembrane glycoprotein that complexes with factor associates with high free radical production in cancer cells. 7 to initiate coagulation and is involved in tumor adhesion, Because hemin and HB components are not linked metastasis, and angiogenesis.30 Free HB binds to TF on the covalently, metHB may release hemin, and both were found to cancer cell surface and enhances its PCA and also stimulates propagate glial tumor growth.37 Free HB also increases its expression of TF.30 TF apoprotein purified from human human glioma cell migration along nanofibers.38 Hence melanoma cells exists in monomeric (53 kDa) and hetero- free HBs may be important therapeutic targets. If the same dimeric (58 kDa) forms.30 TF purified from the human brain HB domains are responsible to induce TF, VEGF, and MMP-9, also exists as monomers and heterodimers, and its 58 kDa blocking antibodies against these may exert antitumor effi- form is a heterodimer with the HB α-chain.30 HB also cacy. Because free HB iron accelerates tumor growth, tumors increases matrix metalloproteinase-9 (MMP-9) and VEGF with higher HB levels on MR images may be more sensitive to synthesis in cancer cells, which play crucial roles in tumor iron chelators such as deferoxamine, which showed promise angiogenesis and invasion.31,32 These findings may have as an anticancer drug.39 clinical implications. In renal cancer patients treated with VEGF antagonist bevacizumab, HB levels increase that posi- Expression of HBs in Systemic Cancers tively correlate with survival.33 As healthy ECs synthesize HB, HBs may be released from systemic capillaries to compensate Studies that demonstrated expression of HB chains in sys- the EC toxicity of bevacizumab, and hence HB increases may temic cancers are also very recent. These are discussed briefly reflect the potency of bevacizumab. Indeed, stringent VEGF here. inhibition in primates increases HB, RBC precursors, and erythropoietin (EPO).33 Breast Cancer However, antiangiogenic treatment reduction of the HBs The HB β-chain is significantly higher in human breast directly in tumor tissue may reflect decreased EC density in cancers positive for estrogen and progesterone receptor in tumors. In human breast tumor mouse xenografts treated comparison with those positive only for estrogen receptor.40 with a combination of bevacizumab and doxorubicin (DOX), Progesterone induces breast cancer differentiation, and the tumoral expression of HB chains decreased.34 Basal-like HB β-chain may be involved in this process.41 Interestingly, breast tumors showed improved responses to bevacizumab lesser expression of HB chains (both α and β) were observed and DOX compared with single DOX, and luminal-like breast in benign and malignant canine breast tumors.42 However, tumors responded equally well to DOX with or without decrease of HBs was more in benign breast tumors. As the bevacizumab.34 Themostprominenttumorgrowthdelay tumor stage increased, this decline ceased, and HB levels were was associated with the largest changes in gene expression.34 even higher at stage V disease.42 The initial HB decline may be When the combination treatment was compared with single associated with anaerobic cancer metabolism, yet when the DOX in basal-like breast tumors, six genes were specifically tumor load increases, cancer cells may require additional downregulated with the combined treatment, and two of metabolic pathways. these were genes encoding HB θ-chain and δ-chain.34 In the luminal-like tumor that showed equal responses to DOX Colon Cancer either alone or in combination, the most significant decreases Diffuse reflectance spectroscopy of colon in normal, polyp, occurred only for three, and two of these were genes encoding andcancerpatientsrevealedincreasedHBconcentration HB α1- and α2-chains.34 These findings indicate roles of HBs and decreased oxygenation during progressing stages of in response to chemotherapy and antiangiogenic drugs. colon carcinogenesis.43 Intratumoral RBC lysis may have

In bone marrow, a factor adheres to human prostate cancer hindered sufficient O2 uptake despite higher levels of HB. In cells much higher than colon cancer cells, and it was revealed colon adenoma versus colon carcinoma patients, proteo- as an HB α-chain, attributed to frequent bone metastases of mics identified 11 upregulated proteins in cancer including prostate cancers.35 Free HB also accelerates the progression of the HB β-chain.44 carcinomatous peritonitis.36 In rats, intraperitoneal injec- tions of antioxidant enzymes inhibited peritoneal carcino- Other Cancers matosis while injection of free HB profoundly stimulated In mice transgenic for the c-raf, a serine-threonine kinase of peritoneal tumor growth. This is likely due to free HB’siron the Raf family, development of lung cancers coincided with an stimulation of the Fenton reaction, where peroxide is con- increase of HB α-chain.45 verted to hydroxyl radicals leading to tissue injury and Proteomic analysis revealed nine differentially expressed inflammation.36 Extracellular methemoglobin (metHB) and proteins between the sera of ovarian cancer patients and hemin released from metHB are also involved in tumor healthy controls, and the most distinctive peptides in cancer biology.37 Because metHB is a HB, in which the heme iron were HB α- and β-chain.46 Furthermore, combined use of

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CA-125 and HB detected ovarian cancer progression with T cells in regressor mice revealed strong reactivity against 99% improved sensitivity.46 MHC-presented tumor peptides.52 Some of these epitopes HB α-andβ-chain expressions are significantly higher in derived from tumor stroma but not from tumor cells. Proteo- cervical carcinoma than in the normal cervix, which did not mics defined two major peptides that derive from murine HB involve common hematopoietic transcription factors.47 In β1- and β2-chains within the most stimulatory fractions,

cervical cancer cells, expression of HBA1 was 10-fold higher which both contained an LVVYPWTQRY (HBB33–42)se- than that of HBB and inducible with oxidant stress. Ectopic quence.52 Although cultured CMS4 tumor cells did not ex-

expression of wild-type HBA1 and HBB, but not mutant forms press HB β-chain, vaccination of mice with HBB33–42 peptide incapable to bind heme group, suppressed oxidative stress promoted CD8þ T-cell responses.52 Furthermore, these CD8þ and apoptosis of cervical cancer cells.47 T cells protected mice against a subsequent challenge with Because the development of head and neck squamous cell CMS4 tumor and, unexpectedly, with unrelated HB β-chain– carcinoma is a multistep process of field cancerization, negative breast and colon tumors. These vaccines destabilized healthy, tumor-distant, and tumor-adjacent squamous mu- tumor vessels by triggering immunity against HB β-chainþ/ cosae were analyzed via proteomics to reveal tumorigene- SMAþ/NG2þ vascular pericytes.52 Moreover, there were no sis.48 HB chains were among the most differently expressed untoward effects on brain or eye vessels.52 Thus it would be proteins in tumors versus normal tissue; whereas the total HB logical to question why these β-chain peptides on tumor β-chain had reduced in-tumor tissues, a HB β-chain fragment endothelia do not induce spontaneous tumor regression. This 113–146 was found to be profoundly higher.48 may be explained by the delicate balance between immune Proteins of oral squamous (tongue) cancer biopsies and responses. Low doses of HB-chain fragments may induce noncancerous mucosa were defined by proteomics. Six pro- metastatic process as indicated for extracellular HBs, but teins discriminated best the cancerous and healthy tissues, exogenous vaccination with higher peptide doses may pro- and remarkably, one of these proteins was the HB β-chain.49 voke antitumor immunity. Because β-chain fragment vac- HB β-chain was overexpressed in 6 tumors but in none of the cines work in different cancer types, this approach would be 12 normal mucosa.49 promising for refractory cancers and glial tumors with high These studies indicate that during carcinogenesis, dedif- angiogenesis. ferentiating cells activate synthesis and metabolism of HB chains. The critical finding is that HB elevation in cancer cells fi 47 Antitumor Immunity Induction of does not involve erythroid-speci c transcription factors. If Extracellular HB and Serum Protein this is also relevant with other malignancies, selective gene Complexes therapies could be developed that do not perturb healthy erythropoiesis. In mice transplanted with Ehrlich tumor, HB forms a complex with serum proteins but not in tumor-free mice. The HB- associated protein (HAP) complex includes haptoglobin (Htb), HB Expression in Normal and Tumoral α α 53,54 Endothelial Cells and in Cancer Cells serum albumin, an -fetoprotein homolog, and HB -chain. Juxtaposing Tumor Capillaries Injecting this HAP complex led to regression of intraperitoneally and intramuscularly transplanted tumors with apoptosis in HB α-chain is expressed in arterial ECs and enriched at the 34% of tumor cells.54 More strikingly, a 1-year follow-up of the myoendothelial junction. It regulates the effects of NO on HAP-injected animals did not reveal any tumor regrowth.55 The vascular reactivity.50 Cell–cell interactions of human ECs and in vitro addition of the HAP complex into Ehrlich tumor culture smooth muscle cells are necessary for HB α-chain expression, medium did not lead to tumor cell death and even slightly which also indicates that HB studies in pure cancer cell increased cell proliferation,54 which is similar to previously cultures may overlook their function because they could be described phenomena. Extracellular HBs may provide advan- induced via interaction of cancer cells with surrounding ECs tages to cancer cells to survive and metastasize. However, when and smooth muscle cells. Punctuate HB α-chain and little HB HB chains are complexed to peptides in the serum of the cancer- β-chain staining occur at paracellular junctions of thoraco- bearing host, their conformation would change, which may dorsal arteries.50 HB is prominently expressed in human uncover some hidden peptide pockets leading to antitumor breast tumor cells closest to capillaries, which suggest its immunity.

roles in O2 diffusion; breast tumor vessel endothelia are ubiquitously labeled with HB, whereas normal breast tissue 51 Expression of HB Chains in Healthy Brain and is negative. HB chains in tumor ECs may provide metabolic in Neurodegenerative Disease support and also may arise due to tumoral VM. HB chains are expressed in cortical56,57 and A9 dopaminergic Antitumor Immunity Induction of HBs in neurons of the substantia nigra, in a subpopulation of cortical Tumor Endothelia and hippocampal astrocytes, and in virtually all mature oligo- dendrocytes.58 Mesencephalic A9 dopaminergic neurons pres- Tumor vessel HBs are also involved in tumor immunology.52 enthighmetabolicratesandoxidativestressandexerthigher Marrow dendritic cells engineered to secrete high IL-12p70 expression of genes involved in energy metabolism and mito- dramatically inhibit CMS4 sarcoma in mice. Analysis of CD8þ chondrial function.58 When HB α-andβ-chain expressions were

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overexpressed in these cells, genes involving in O2 homeostasis VV-5/ suppressed proliferation of tumor cells by and oxidative phosphorylation were particularly affected be- 25 to 95% with maximal activity detected in fibrosarcoma and sides changes in genes involved in free radical defense and iron MCF-7 human breast carcinoma cells; human leukemia cells and NO metabolism.58 Similar HB staining was found in rodent were less sensitive.69 In normal cells, antiproliferative activity and human CNS,58,59 indicating conserved important roles in of valorphin was several-fold lower.69 In mice inoculated with neural physiology. Systemic administration of a mitochondrial syngeneic breast carcinoma, VV-5 and chemotherapeutic toxin markedly decreases mRNAs of HB α-andβ-chains in the epirubicin combination was tested. Simultaneous adminis- nigral dopaminergic neurons.59 Analyses of mitochondrial pro- tration with 1 mg/kg VV-5 and 25 mg/m2 epirubicin achieved teome in the cortical pyramidal neurons in multiple sclerosis a further 22% suppression when compared with epirubicin revealed four proteins that distinguished patients from controls; alone and a 42% tumor suppression when compared with one of these was the increased HB β-chain.60 HB expressions control.70 Prominent decrease of -7 peptides were were analyzed on postmortem human brains obtained from found in the serum of breast cancer patients with respect to patients with Alzheimer disease (AD), Parkinson disease (PD), control (p ¼ 0.002),71 and it was proposed that hemorphin-7 and dementia with Lewy bodies (DLB).61 HB α-andβ-chains may both function as a biomarker and a promising drug for were reduced in practically all neurons with tau deposits or breast cancer.2 The decline of hemorphin-7 in breast cancer is tangles in AD, in 80% of neurons with α-synuclein deposits, attributed to a serum proteasome elevated in cancer andinneuronswithLewybodiesinPDandDLB.61 Studies are patients.2 However, simplistic views on the role of hemor- necessary to illuminate whether HB-chain reductions are the phins on cell growth may not represent the big picture.72 LVV- cause or the results of these particular neuropathologies. HB 7 binds to the -4 (AT-4) receptor with high exists as a canonical α2β2 heterotetramer in neuronal cells in affinity. The receptor of AT-4 is distributed in the CNS vitro and in vivo,62 which strongly suggest a similarity of markedly differently from that of AT1 and AT2, and it exerts function of neuronal HBs to RBC-HBs in binding gaseous ligands. unique functions.72 Although both LVV-7 and AT-4 bind to Hence it is more likely that they exert prosurvival effects, which AT4 receptor, only LVV-7 stimulates 3H-thymidine uptake also indicate their possible misuse by malignant cell clones. (suggesting cell growth) in the neuroblastoma/glioma hybrid NG108–15 cell line.72 LVV-7 also induces 3H-thymidine uptake in the human neuroblastoma cell line SK-N-MC.73 Hemorphins as Peptide Products of HB – Cleavage: Presence in the CNS, Antitumor The shared feature of NG108 15 and SK-N-MC cells is their and Immunomodifier Efficacies neuronal component; therefore LVV-7 may not stimulate tumors of glial origin. Moreover, both in NG-108–15 and Hemorphins are peptides endowing opioid activity and pro- SK-N-MC cells, growth-stimulating effects of LLV-7 occurred duced via HB-chain cleavage. Based on their N-terminal until a level of 0.1 µM and vanished at 1 µM.72,73 Thus sequence, LVV-, VV-, and V-hemorphins are distinguished.63 considering the abundance of LVV-7 in the brain,72 prolifera- LVV and VV-7 are peptides corresponding to β-chain fragments tion induction may not be one of the main actions of LVV-7. 32–41 and 33–41, respectively. These peptides derive from HB Hemorphins are also important regulators of immunity. LVV- fragments excreted by the RBCs, further shortened by tissue- 4 matching a conserved sequence of HB β-chain was first isolated specific enzymes to generate tissue pools. However, comparison from the spine and thus is also called .74 Despite of the hemorphin content in brain and blood revealed that the acting as a calcium-mobilizing agonist at the N-formylpeptide brain peptidome originates minimally from the blood.64 It is receptor, spinorphin blocks neutrophil chemotaxis74 and neu- likely that brain hemorphin peptidome originates from neuro- tralizes the inhibitory effect of cyclosporin-A and trifluoperazine glial HBs. Hemorphins LVV-7 and VV-7 are particularly charac- on interleukin (IL)-2 synthesis by activated blood lymphocytes.75 teristic of the CNS.65,66 Hemorphins also exert several additional LVV-4 suppresses carrageenan-induced accumulation of neu- biological roles: inhibiting angiotensin-converting enzyme, trophils in mouse air pouches and may be an endogenous factor modifying β-endorphin release, and a low affinity toward against inflammation and associating pain.76 Neutrophils of orphan receptor subtype-3.66,67 VV-7, LVV-6, and innate immunity act inefficiently to control tumor growth or VV-6 were identified in pheochromocytoma with lower values they even propagate cancers via the release of angiogenic than the normal adrenal tissue.68 Hemorphins suppress fibro- factors,77 whereas lymphocytes act more specifically in killing sarcoma cell growth both via cytotoxicity and inhibition of cell tumor cells. Hence anti-inflammatory effects and hindering the proliferation with maximal effects at concentrations 105 to suppression of IL-2 release from lymphocytes indicate that 106 M (tested concentrations were between 105 and 1015 hemorphins may provoke more efficient antitumor responses. M).63 VV hemorphins, except VV-4, exhibited cytotoxicity sig- fi ni cantly higher than the respective LVV hemorphins. In the HB Mutations May Result in Aberrant group of VV hemorphins, the activity of VV-5 (valorphin) was fi 63 Hemorphin Metabolism or Exert Haplotypal signi cantly higher than of VV-7, VV-6, and VV-4. The amino Interactions with Tumor Susceptibility Genes acid sequence VVYPWTQ corresponding to valorphin was iden- at 11p15.5: Clues from Systemic Cancer tified as important for both of the cytotoxic and antiproliferative effects.63 VV-5/valorphin resides in position 33–39 of all the HB mutations or polymorphisms may result in aberrant hem- β-, γ-, δ-, or ε-chain of human HB, and thus it may also be orphin metabolism and thereby perturb regulatory mechanisms produced by proteolytic fragmentation of other HB chains. of cell growth and immune surveillance. There exist 1,720

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different HB variants in various globin chains,78 yet only a minor were compared via proteomics.90 Considering the high CSF percentage of these (5.2% of the world population, 37 million) turnover, samples were obtained before and after tumor cause overt clinical signs.79 Yet when clinically silent hemoglo- removal, which allowed the investigation of peptides associ- binopathies are also included, 70 million people in the world ated with tumor load. It was revealed that HB α-chain are carriers of hemoglobinopathies.80 Thalassemias are caused increased in the CSF of three of six cases with pilocytic by mutations modifying HB-chain synthesis rather than HB astrocytoma.90 Even higher increases of HB α-chain were function (yet a minor percentage of mutations could affect determined in the CSF of three of four cases with medullo- both). β-thalassemia is the most common single-gene inherited blastoma. Slightly elevated levels of the HB α-chain were seen condition in the world. Almost 70,000 infants are born with in the CSF in two of three cases with ependymoma. Levels of β-thalassemia major worldwide each year.80 When it comes to HB α-chain were very low in the control hydrocephalus β-thalassemia carriers, many cases are overlooked or misdiag- cases.90 HB β-chain was elevated in three of six cases with nosed as iron deficiency because both conditions are the most pilocytic astrocytoma, with a very similar pattern as seen for frequent causes of microcytic anemia.81,82 It is established that the α-chain.90 HB β-chain was elevated in all patients with iron overload in thalassemia major induces liver cancer, yet very medulloblastoma, and the highest increases occurred in limited studies exist that have analyzed the association of patients who had higher increases in the α-chain. Mildly hemoglobinopathies and thalassemia trait with malignancy elevations of β-chain occurred in two of three cases with risk where iron load is not a causal factor. At present, few studies ependymoma, with a similar pattern as seen for β-chain.90 have indicated certain important associations: enhanced rates of Interestingly, slight elevations of HB β-chain also occurred in hematologic malignancies associated with HB Lepore,83 control hydrocephalus patients, although at much lesser enhanced gastric cancer rates in thalassemia trait,84 significantly degrees than observed in patients with pilocytic astrocytoma higher rates of thalassemia trait in patients with various types of and medulloblastoma.90 cancer,85 and high rates of cholangiocarcinoma in association Despite their prominent cell growth rate, medulloblastomas with HB E.86 Nonetheless, no studies have previously analyzed do not represent signs of hypoxic coagulation necrosis, which rates of β-globin mutations in chemorefractory cancers and is frequently seen in high-grade glial tumors. It would be brain tumors. Such investigations may most likely provide novel worthwhile to assess whether HBs are rich in the cytoplasm of

clues in understanding carcinogenesis. medulloblastoma cells and provide enhanced O2 uptake. It was observed mostly that CSF hemorphins were low/absent when 90 HB Expressions in Benign Intracranial brain tumors are not successfully removed. The LVV and VV Tumors hemorphins exhibited the most repeatable trend over other peptides, which led to interesting remarks. In fact, the LVV- and When cerebrospinal fluid (CSF) from patients with meningioma VV-h7 were absent in the postsurgical samples of a medullo- and patients with nontumoral lesions were analyzed via prote- blastoma patient with spinal drop metastasis and in two omics, 10 distinguishing proteins expressed by more than pilocytic astrocytoma patients with residual tumor.90 But twofold in meningioma were identified, and one of these was LVV- and VV-7 were detectable in three of the five control 87 HB Ypsilanti. HB Ypsilanti is a rare high-O2 affinity HB due to CSFs. The absence of these hemorphins in two other controls

β-globin mutation. With slight increases in O2 affinity, HB may associate with the sample heterogeneity because these Ypsilanti carriers are asymptomatic and, at most, mildly poly- CSF samples were obtained from newborn and preterm cythemic.88 Progenitor cells among arachnoid cap cells (where patients in whom cerebral HB metabolisms may differ.90 meningiomas origin) may be programmed to die under certain Lack of hemorphins when there is a tumor load is a striking

O2 pressures, and high-O2 affinity HB variants may block this finding and parallel to the findings that hemorphins exert process. Further analyses pertinent to this possibility may reveal antitumor efficacy. There are no studies on the effects of unique pathways in meningioma formation. Intracystic fluid of hemorphins on glial tumor cells, which may lead to unique pediatric adamantinomatous craniopharyngioma (ACP) was strategies of treatment. analyzed via proteomics. Unique HB α-chain fragments α 2–33, 2–34, and 2–79 were found among 7, 5, and 11 samples 89 HB Expressions in Human GBM Tumor Cell of 13 investigated ACP patients, respectively. Furthermore, a Lines β-chain fragment was found in six patients.89 ACPs are associat- ed with catenin-β1 mutations, which also exist in medulloblas- It was demonstrated initially that globin types neuroglobin tomas;andHBchainsarealsopresentintheCSFofpatientswith and cytoglobins are expressed in human primary GBM medulloblastoma. In the future, investigations on likely associ- tumors and cell lines.91,92 Thereafter, analyses confirmed ations between the catenin-β1 pathway and HB chains would the presence of different HBs (α, β, γ, δ, ζ,andε)inhuman illuminate their biogenesis pathways. GBM cells under normal conditions, indicating reactivation of fetal and embryonic genes and their potential in inherent 93 HB Chains and Hemorphins in Posterior Fossa resistance of GBMs. However, expression of these HBs did Tumors not differ between GBM cells with differing resistances to hypoxia and radiation.93 Nonetheless, these results were CSF samples from 14 children with posterior fossa tumors at obtained from monolayer cultures that do not mimic the diagnosis and from 5 controls with congenital hydrocephalus pathophysiologic features of tumors with differential

Journal of Neurological Surgery—Part A Vol. 77 No. A3/2016 Novel Pathways of Glial Tumor Formation Altinoz et al. 253 distribution of hypoxic areas and cell-to-cell contacts. stem cell–like tumor subpopulation with increased cell size Changes in HB expressions with experimental hypoxia in having single giant nuclei or multinuclear with differing GBM cell lines were also analyzed.94 Under normoxia, ε globin shapes and chromatin that develop through either endore- was predominant within all cell lines compared with total duplication or cell fusion.100 PGCCs provide vasculogenic globins.94 Exposure to hypoxia time dependently decreased mimicry (VM) and are frequent in high-grade tumors that ε-globin expression in all cell lines accompanied by increases usually correlate with poor prognosis. Aggressive cancers in α- and γ-globin expression. The order of relative increases including inflammatory breast and liver cancers frequently in different globin mRNAs after hypoxia was α > γ > δ > β utilize VM.100 Cobalt chloride treatment (hypoxia mimetic) of > ζ globin.94 Hypoxia also stimulated EPO and the EPO cancer cells induces PGCCs, and unexpectedly, these PGCCs receptor in four of five GBM cell lines. EPO, which does not were supposed to form RBCs both in vitro and in vivo.101 only stimulate erythropoiesis but also HB synthesis, is in- Although this observation seems somewhat eccentric, it may versely correlated with O2 availability and propagates growth, not be impossible. In the fetus, RBCs are first produced in the invasiveness, and chemotherapy and radiation resistance of so-called blood islands of the mesodermal layer by the astrocytic tumors.94 It would be logical to analyze whether hemocytoblasts differentiated from angioblasts. Angioblasts enhanced synthesis of HB chains is involved in EPO propaga- are also progenitors for vessel endothelia.102 Thus undiffer- tion of GBM aggressiveness. entiated cell populations in cancers may regain angioblast cell The prominent increase of α-andγ-chains (which togeth- features. In human glioma, the number of PGCCs increases er form HB-F) following exposure of GBM cells to hypoxia is with tumor grade, and they were proposed to generate consistent with the physiologic functions of HB-F, which is RBCs.100 Very noteworthy, these red bodies were positively the main O2 transport protein in the human fetus during the labeled with a polyclonal antibody binding HB β/δ/γ/ε last 7 months of development. Functionally, HB-F differs from chains.100 Even if these red corpuscles are still not RBCs adult HB-A with its greater affinity to O2 due to its poor contrary to author claims and only represent large HB ag- binding to 2,3-bisphosphoglycerate (2,3-BPG). In adult RBCs, gregates, their staining with globin chains strongly indicate this substance decreases the affinity of HB for O2. HB Gower-2 their production in the endothelia of glial tumor vessels. and HB-A exhibit similar O2-binding characteristics, and HB 95 Gower-1 tightly binds 2,3-BPG, which is synthesized by an β 96 Likely Haplotypal Associations Between - enzyme rich in embryonic and cancerous tissues. Consid- Globin and Glioma-Susceptibility Genes ering the high rates of glycolysis in malignant cells, HB-F with lesser affinity to 2,3-BPG may provide better oxygenation at Genomic imprinting is a gamete-specificmodification lead- low O2 tensions, which explains its highest stimulation with ing to parental specific gene expression in somatic cells of the hypoxia. This switch is similar to the phenomenon when offspring.103 The 11p15.5 is an important gene region har- embryonic HB expression in primitive erythrocytes is fol- boring several cancer-associated genes subject to epigenetic lowed by HB-F dominance.94 It is likely that the fetus, reach- regulation by imprinting. At this region, aberrant imprinting ing a bigger cell mass, exerts a higher demand for O2 than the or LOH of tumor suppressor genes and vice versa aberrant loss embryo and its HB-F dependent mechanism to cope with an of imprinting (LOI) of genes encoding growth factors may

O2 crisis is mimicked by malignant cells. stimulate tumorigenesis. Clustering of high-frequency LOH There is also a report about an unusual globin in human for 11p15.5 occurs in a broad spectrum of adult cancers and glial tumors that is not localized either in the β-orα-globin brain tumors.5 Several distinct areas of LOH were observed in gene cluster. Human androglobin gene ADGB at the 6q24.3 breast and lung tumors, suggesting the presence of multiple locus encodes a protein with a globin domain and a hexa- tumor suppressor genes in this chromosomal region. The coordination of the heme iron.97 ADGB expression is insensi- overall frequency of LOH is between 26% and 35% in malig- tive to experimental hypoxia, and its highest expression nant glial tumors, and these LOH events are more specificfor occurs in the testis associated with postmeiotic stages of higher grade glial tumors.5 Interesting clues exist suggesting spermatogenesis.97 In comparison with testis, ADGB exerted that globin genes and genes of gliomagenesis could exert an 100-fold lower expression in some tissues including haplotypal interactions. HB-A2′, also called HB-B2 or HB- brain.97 However, silencing ADGB reduces growth, colony A2δ′, is the most common δ chain variant of HB-A2, which is formation, and enhances apoptosis in human GBM cell lines clinically and hematologically silent.104 with increased cleaved caspase-3 and decreased bcl-2.98 HB-B2occurswithhighfrequencyamongAfricanswiththe These results provide further impetus to analyze the role of highest prevalence in the Herero population,104 which belongs different globins in glial tumor biogenesis. to the Bantu-speaking blacks from Namibia.105 The β-globin gene cluster haplotype associated with the δ-globin variant HB- HB Expressions in Glial Tumor Vessels B2 was determined in Herero individuals. The high gene fre- quency of HB-B2 resulted from a founder effect.106 Interestingly, In 2008, it was first shown that HB α-andδ-chains are the incidence of pediatric CNS tumors in the Herero population expressed in glial tumor vessels.99 Thereafter, the presence (26 per million) was found to be profoundly greater than the of HB chains in human glial tumor vessels was also demon- generalpopulationinNamibia,107 which is at a low rate (9.3 per strated, but these findings were discussed in regard to million).108 Reproductive histories of Botswana Hereros also polyploid giant cancer cells (PGCCs). PGCCs constitute a provide valuable information. In this population, female infants

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were almost three times more likely to survive then males TRIM3: The TRIM family of genes is involved in the devel- (p ¼ 0.000001), and female children were twice as likely to opment of cell differentiation and cancer. Of these, the fly- survive than males (p ¼ 0.01).109 In neonatology it is established ortholog brat is restricted to the embryonic ventral nerve that newborn girls adapt and show greater resistance to cerebral cord and brain and suppresses tumorlike proliferation of hypoxia.110 secondary neuroblasts.119 One of these, TRIM3, sequesters Minor adult HB-A2 carrying the normal δ-chain increases p21WAF1/CIP1 and prevents it from accumulating cyclin D1- in mountain dwellers and during coronary ischemia, after- cdk4 complexes. In humans, LOH of TRIM3 at the 11p15.5 ward returning to basal levels during the healing phase, occurs in 20% of GBMs, and reducing TRIM3 expression suggesting its roles in coping with hypoxia.111 GATA-1, a increases PDGF-induced mouse gliomas.120 Vice versa, its transcription factor involved in δ-chain synthesis, is stimu- reconstitution impairs the growth of glial tumor cells and lated by the female hormone progesterone.111 Hence the HB- attenuates their stemlike qualities.119 A2 variant may have provided survival advantage in female SLC22A18: Solute carrier family 22, member 18 (SLC22A18) children of the Herero population via an unknown and gene at 11p15.5, also known as IMPT1/BWR1A/TSSC5, is a superior protective function of the variant δ-chain. If this member of the polyspecific transporters. SLC22A18 expres- variant δ-chain exerts higher cytoprotection, it is possible sion is significantly decreased in human gliomas compared that the survival chances of neuroectodermal malignant cell with normal brain.121 SLC22A18 expression is significantly clones could also increase, explaining higher pediatric brain lower in gliomas that recur early after surgery.121 SLC22A18 tumors in this population. Alternatively, the δ-chain gene at promoter methylation occurs in 50% of the gliomas, and lack 11p15.5 may exert haplotypal interactions with genes asso- of the SLC22A18 protein predicts a significantly shorter ciated with glial tumors. The chromosomal region 11p15.5, survival in glioma patients receiving temozolomide.122 If where the β-globin gene cluster exists, harbors eight genes haplotypal associations between the β-globin gene cluster with profound roles in immune regulation: STIM1, CD151, and genes of gliomagenesis are proven in the future, patients TC21/RRAS2, SIGIRR/TOLL/IL1R8, pp52/LSP1 (lymphocyte-spe- with hemoglobinopathies could be screened for CNS tumors cific protein), TRIM21, toll-interacting protein (TOLLIP), and and innovative treatment strategies could be developed. SLEN3. These were discussed in a previous publication of our 4 study group. Genes at the same region and associated with Conclusions susceptibility to glial tumors are as follows. IGF-2: Several studies demonstrated the association Focusing on HBs, hemorphins, and the 11p15.5 chromosomal between circulating levels of IGF-1 and -2 and cancer risk, region may lead to several novel strategies in the treatment of and the IGF system has been implicated in the oncogenesis of refractory systemic cancers and CNS tumors. HB expressions essentially all malignancies.112 The IGF-2 gene is normally may be silenced with gene therapy to reduce inherent resistance imprinted in the brain subcortex; studies revealed LOI of IGF- of these malignancies. If peptide domains of free HBs responsible 2 gene in 57% of glial tumors.113 Among high-grade for inducing TF, VEGF, and MMP-9 are defined, monoclonal astrocytomas, 2% of grade III tumors and 13% of grade IV antibodies could be developed to reduce tumor metastasis tumors express IGF-2 mRNA levels that are dramatically and invasion. It will be analyzed whether HBs form serum higher (> 50-fold the sample population median). IGF-2 protein complexes (HAPs) or their tryptic peptides are presented overexpressing tumors frequently display PTEN loss; they by tumoral vascular pericytes in human cancers. If so, injections are highly proliferative and characterized by poor survival.114 of HAPs and tryptic HB-peptides may lead to new immunother- H19: The H19 gene at 11p15.5 encodes a noncoding RNA apy strategies. Studies regarding hemorphin effects on the strongly expressed during embryogenesis.115 H19 and its neigh- growth of refractory cancers and CNS tumors may significantly boring IGF-2 gene are coexpressed in embryonic endoderm and enhance our armamentarium against neoplasms. Lastly, inves- mesoderm, suggesting a common regulation, and also H19 tigations about whether genes at the β-globin gene cluster and influences cell growth by a cis control on IGF-2.115 H19 expres- genes associated with gliomagenesis exert haplotypal interac- sion is especially high in high-grade gliomas, and its depletion tions would provide precious data to predict risk populations inhibits glioma invasion.116 Further, H19 positively correlates among those carrying HB mutations. During the past 60 years, with its derivate miR-675, and its reduction inhibits miR-675 the study of human HB, probably more than any other molecule, expression, which modulates cadherin-13 expression.116 has facilitated the birth of molecular medicine.3 But it is PHLDA2/TSSC3: TSSC3 is implicated in Fas-mediated apo- surprising that the HB expression in nonerythroid cells and in ptosis and imprinted in the placenta and fetal tissues during malignant tissues remained to be discovered in the last decade. human development.117 However, PHLDA2/TSSC3 is not im- There is no doubt that such findings will open an exciting new printed in normal adult brain and blood.118 In contrast, strong era in neurosciences, immunology, and oncology. allelic bias resembling imprinting was detected in most of the glial tumor specimens.118 The expression of Fas and Fas ligand increases from low grade to anaplastic astrocytoma, and despite their coexpression, glial tumor cells do not undergo References TSSC3 apoptosis. Retention of imprinting of in brain tumors is 1 Dubrow R, Darefsky AS, Jacobs DI, et al. Time trends in glioblas- thought to silence the apoptotic pathway through blocking toma multiforme survival: the role of temozolomide. Neuro the Fas pathway.118 Oncol 2013;15(12):1750–1761

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