The Beta Subunit of Hemoglobin (HBB2/HBB) Suppresses ... · Microenvironment and Immunology The Beta Subunit of Hemoglobin (HBB2/HBB) Suppresses Neuroblastoma Growth and Metastasis

Microenvironment and Immunology

The Beta Subunit of Hemoglobin (HBB2/HBB)Suppresses Neuroblastoma Growth andMetastasisShelly Maman1,2, Orit Sagi-Assif1,Weirong Yuan2, Ravit Ginat1,Tsipi Meshel1, Inna Zubrilov1,Yona Keisari3,Weiyue Lu4,Wuyuan Lu2, and Isaac P.Witz1,2

Abstract

Soluble pulmonary factors have been reported to be capable ofinhibiting the viability of cancer cells that metastasize to the lung,but the molecular identity was obscure. Here we report theisolation and characterization of the beta subunit of hemoglobinas a lung-derived antimetastatic factor. Peptide mapping in thebeta subunit of human hemoglobin (HBB) defined a short C-terminal region (termed Metox) as responsible for activity. Intissue culture, both HBB and murine HBB2 mediated growtharrest and apoptosis of lung-metastasizing neuroblastoma cells,along with a variety of other human cancer cell lines. Metox acted

similarly and its administration in human tumor xenograft mod-els limited the development of adrenal neuroblastoma tumorsaswell as spontaneous lung and bonemarrowmetastases. Expres-sion studies in mice indicated that HBB2 is produced by alveolarepithelial and endothelial cells and is upregulated inmice bearingundetectable metastasis. Our work suggested a novel functionfor HBB as a theranostic molecule: an innate antimetastasis factorwith potential utility as an anticancer drug and a biomarkersignaling the presence of clinically undetectable metastasis.Cancer Res; 77(1); 14–26. �2016 AACR.

IntroductionMetastasis is the major killer of patients with cancer. Bidi-

rectional interaction between cancer cells and their microenvi-ronment is a critical determinant of tumor progression andmetastasis (1–5). Numerous reports in the last decade deal withmechanisms by which the microenvironment promotes tumorprogression (6–9). In contrast, relatively little is known regard-ing inhibitory microenvironmental cells and molecules (10)with a few notable exceptions such as immunocytes and theirproducts (11) or granulocytes (12).

Neuroblastoma is the most common extracranial solid tumorin children. Lung metastasis is a rare event (3%–4%) but itspresence is clinically important because it signals a poor prognosis(13). Sixty percent to 70% of children with high-risk disease willultimately experience relapse due to the presence of micrometas-tasis (14). As cure after relapse is extremely rare, novel modalitiesfor the inhibition and elimination of neuroblastoma metastasesare needed.

In a previous study (15), we demonstrated that the microen-vironment of the normal lung possesses the capacity to restrainlung-metastasizing neuroblastoma cells and block their metastat-ic potential. Factors derived from normal mouse lungs signifi-cantly inhibited the viability of neuroblastoma lung-metastasiz-ing cells by inducing cell-cycle arrest and apoptosis. Micrometa-static neuroblastoma cells (MicroNB), generated as describedpreviously (16), were significantly more susceptible to thisgrowth-restraining function than cells derived from frank neuro-blastoma metastasis (MacroNB). The difference in susceptibilitybetween micro- and macrometastatic cells raised the hypothesisthat factors in the lung microenvironment exert antimetastaticfunctions including the maintenance of micrometastatic tumorcells in a state of growth arrest thereby blocking progression toovert lungmetastasis. In the current study,we set out to isolate andcharacterize the lung-derived metastasis-inhibitory factor andprobe its metastasis-restraining activity.

Materials and MethodsCell culture

The human neuroblastoma lung micrometastatic (MicroNB)and macrometastatic (MacroNB) variants were generated using amouse model for human neuroblastoma metastasis (17) fromthe parental cell lines MHH-NB11 (18) and SH-SY5Y (19) asdetailed here (16), and were maintained in culture as describedpreviously (17). Primary human pulmonary fibroblasts (HPF)were purchased from Promo-cell. Primary foreskin fibroblastswere generated from discarded foreskin tissue. Human pulmo-nary endothelial cells (HPEC; ref. 20) were kindly provided byDr. V. Krump-Konvalinkova (Institute of Pathology, Johannes-GutenbergUniversity,Mainz, Germany). All othermentioned celllines were purchased from the ATCC. Cells were authenticatedevery three months according to the ATCC guidelines, as detailed

1Department of Cell Research and Immunology, The George S. Wise Faculty ofLife Sciences, Tel Aviv University, Tel Aviv, Israel. 2Institute of Human Virology,University of Maryland School of Medicine, Baltimore, Maryland. 3Department ofClinical Microbiology and Immunology, Faculty of Medicine, Tel Aviv University,Tel Aviv, Israel. 4Department of Pharmaceutics, School of Pharmacy, FudanUniversity, Shanghai, P.R. China.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: Isaac P. Witz, Tel Aviv University, Haim Levanon st.,Tel Aviv 69978, Israel. Phone: 972-3640-6979; Fax: 972-3640-6974; E-mail:[email protected]; and Shelly Maman, [email protected]

doi: 10.1158/0008-5472.CAN-15-2929

�2016 American Association for Cancer Research.

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here (15). All cultures were periodically examined for mycoplas-ma contamination.

HPLC separation of lung-derived factorsLungs of 100 BALB/c athymic nude mice were used to prepare

the lung-derived factors as described previously (15). Lyophilizedlung-derived factors were reconstituted in Milli-Q purified water(EMD Millipore) to a concentration of 1 mg/mL, filtered (0.45mm), and subjected to separation by Alliance reversed-phase high-performance liquid chromatograph (HPLC;Waters) system usingWaters XBridge C18 column (30 � 150 mm, 5 mm) running agradient of 35% to 50% acetonitrile (Thermo Fisher Scientific) inwater containing 0.1% trifluoroacetic acid (Halocarbon, Inc.) at aflowrate of 15mLperminute. TheHPLC-separated fraction foundto inhibit the viability of neuroblastoma cells was HPLC-purifiedrunning the same conditions.

LC/MS-MS identification of the lung-derived inhibitory factorThe purified inhibitory factor was digested in Coomassie-

stained polyacrylamide gel. Protein spots were excised from thegel and digested with trypsin according to the published proce-dures (21). The digested inhibitory factor was injected to aThermo Electron Orbitrap Velos ETD mass spectrometer usinga8 cm�75mmPhenomenex Jupiter 10mmC18capillary column,and the peptides eluted from the column by an acetonitrile-0.1mol/L acetic acid gradient at a flow rate of 0.5 mL per minute over30 minutes. The digest was analyzed using the double playfunction acquiring full mass spectra followed by ion spectra todeterminemolecularmass and amino acid sequence in sequentialscans. The data were analyzed using the Sequest search algorithmagainst the Mouse International Protein Index (IPI).

HPLC separation of native human hemoglobinNative human hemoglobin was dissolved in Milli-Q purified

water (EMDMillipore) to a concentration of 1mg/mL andfiltered(0.45 mm). Human hemoglobin was then chromatographed byAlliance RP-HPLC system (Waters) using Waters XBridge C18column (50�250mm,10mm) running a gradient of 35% to50%acetonitrile (Thermo Fisher Scientific) in water containing 0.1%trifluoroacetic acid (Halocarbon) at a flow rate of 40 mL perminute. The separated alpha and beta subunits of hemoglobin(HBA and HBB, respectively) were collected and purified byreverse-phase HPLC and their molecular masses were verified byelectrospray ionization mass spectrometry (ESI-MS).

Solid-phase synthesis of Metox and scrambled-Metox peptidesThe inhibitory human HBB peptide (ENFRLLGNVLVCVLA)

designated Metox, and a control peptide of a scrambled aminoacid sequence (ANVLNECVFVGRLLL) designated scrambled-Metox, were chemically synthesized. The synthesis was performedon appropriate PAM resins (Applied Biosystems) on an 433Apeptide synthesizer (Applied Biosystems) using an optimizedHBTU (Oakwood Chemical) activation/DIEA (Sigma-Aldrich) insitu neutralization protocol for Boc solid-phase peptide synthesis(22). After chain assembly, the peptides were cleaved by anhy-drous hydrogen fluoride (Airgas) in the presence of 5% p-cresol(Sigma-Aldrich) at 0�C for 1 hour, followed by precipitation withcold ether. The Metox and scrambled-Metox peptides were puri-fied by reverse-phase HPLC, and their molecular masses wereascertained by ESI-MS.

Treating mice with MetoxMice were orthotopically inoculated with MicroNB cells to

the adrenal gland to generate local adrenal tumors and lungand bone marrow micrometastasis as described previously(16). Fourteen days after tumor cell inoculation, mice weretreated intranasally with 15 mg/kg of the human HBB pep-tide, Metox, once a week for 8 weeks. The lyophilized peptidewas dissolved prior to each administration in dimethyl sulf-oxide (Sigma-Aldrich), diluted in sterile PBS, and filtered(0.2 mm). Mice were forced to inhale 20 mL of Metox (0.3mg/mouse) or of the control scrambled peptide, scrambled-Metox (control group).

Statistical analysisPaired or unpaired Student t test was used to compare in vitro

and in vivo results.Formore details onMaterials andMethods, see Supplementary

Data

ResultsIsolation and identification of a mouse-inhibitory lung factor

Soluble lung-derived factors induce growth arrest and apo-ptosis of lung-metastasizing human neuroblastoma cells (15).Here we isolated the inhibitory factor from mouse lungs.Soluble factors derived from the lungs of 100 athymic nudemice were generated as described previously (15). Dialysis ofthe lung-derived factors suggested that the molecular weight ofthe inhibitory factor(s) is higher than 3,500 Da (Supplemen-tary Fig. S1A). The biologically active dialyzed lung-derivedfactors were separated by reverse-phase HPLC to numerousfractions (Fig. 1A). These fractions were incubated with micro-metastatic (MicroNB) and macrometastatic (MacroNB) neuro-blastoma cells for 72 hours (Supplementary Fig. S1B). An MTS-based viability assay indicated that one of the distinct separatedpeaks inhibited the viability of the cells by 25%–50% (P <0.05; Fig. 1B) to the same extent as unseparated lung-derivedfactors (15).

The active inhibitory fraction was subjected to high-resolu-tion purification using reverse-phase HPLC. This resulted in asingle, narrow, and symmetric peak (Fig. 1C) representing,most probably, a single factor. ESI-MS analysis confirmed thatthe purified fraction was indeed a single factor of a molecularmass of 15,824.5 Da (Fig. 1C). This fraction reduced theviability of MicroNB and MacroNB cells by 65% (P < 0.01)and 35% (P < 0.05), respectively (Fig. 1D). Sequence analysisby LC/MS-MS coupled with tryptic digestion followed by adatabase search in the International Protein Index (IPI), iden-tified the fraction as mouse hemoglobin subunit beta-2(HBB2), a protein with 147 amino acid residues (Fig. 1E).N-terminal Edman degradation (23) of the isolated factorfurther verified its sequence identity.

The inhibitory activity of the isolated factors against MicroNBandMacroNB cells could be effectively neutralized by anti-mouseHBB2 antibodies, validating HBB2 as the inhibitory factor inmouse lungs (Fig. 1F). The inhibitory activity was not affectedby an isotype control (Fig. 1F).

Elevated levels ofHBB2alert to thepresenceofmicrometastasesMicroNB cells were orthotopically inoculated to the adrenal

gland of athymic nude mice generating local tumors. The

HBB2/HBB Inhibits Neuroblastoma Formation and Metastasis

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Figure 1.

Isolation and identification of an inhibitory lung factor. A, Dialyzed lung-derived factors were subjected to separation by analytical C18 reverse-phase HPLC.B, An MTS-based viability assay revealed that one HPLC-separated fraction significantly inhibited cell viability. C, Purification of the inhibitory fraction andanalysis by electrospray ionization mass spectrometry yielded a molecular mass of 15,824.5 Da. D, An MTS-based viability assay verified the inhibitory activityof the HPLC-purified fraction. (Continued on the following page.)

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Cancer Res; 77(1) January 1, 2017 Cancer Research16




control group was injected with PBS. Quantitative real-timePCR (qRT-PCR) analyses performed 8 weeks after the intra-adrenal inoculation of the tumor cells revealed the presenceof micrometastatic human neuroblastoma cells in lungs,bone marrow, and liver of the inoculated mice (SupplementaryFig. S2). At this point, there was no evidence of overt metastasis.

qRT-PCR analyses indicated that the level of mouse HBB2mRNA (Supplementary Table S1) was 25 times higher in lungsof micrometastasis-bearing mice than in lungs of normal mice(P < 0.001; Fig. 2A). Levels of the alpha subunit of hemoglobin(HBA) mRNA were low and similar in the two groups of mice(Fig. 2A). Immunofluorescence analyses of frozen lung sectionsstained with anti-mouse HBB2 antibody revealed a higherexpression of HBB2 in lungs harboring neuroblastoma micro-metastases than in normal lungs (Fig. 2B). In these lungs, anintracellular expression of HBB2 in cells lining the alveoli wasobserved (Fig. 2B). The higher expression of HBB2 in lungsharboring micrometastases was verified by Western blot anal-ysis of lung tissue lysates (P < 0.005; Fig. 2C). HBB2 expressionwas also significantly higher in liver (P < 0.01) and bonemarrow (P < 0.05) of micrometastasis-bearing mice than inliver and bone marrow of control normal mice (Fig. 2C). HBB2concentration in the serum of micrometastasis-bearing micewas 7 times higher (P < 0.005) than its concentration in theserum of normal mice (Fig. 2D). The serum concentration ofthe alpha subunit, HBA, was very low and was similar innormal control mice and in micrometastasis-bearing mice (Fig.2D). Similarly there was no significant difference between theconcentration of the whole, intact hemoglobin protein in theserum of normal and micrometastasis-bearing mice (Fig. 2D).This result excludes the possibility that the high concentrationsof HBB2 in serum of tumor bearers were due to hemolysis. It isnot unlikely that the lungs are an important contributor to theelevated HBB2 serum levels, as the most significant elevation inthe expression of HBB2 in micrometastasis-bearing organs wasin the lungs (Fig. 2C; Supplementary Fig. S2). However, othermicrometastasis-bearing organs such as bone marrow and liver(Fig. 2C; Supplementary Fig. S2) do contribute as well, to theincreased HBB2 protein levels in the serum.

Alveolar epithelial cells are the main source of HBB2We next set out to identify the HBB2-producing lung cells and

in particular those cells in whom transcription is upregulatedin micrometastasis-bearing mice (Fig. 2A). It was previouslyreported that HBB2 is synthesized by pulmonary epithelial cells(24). Confirming these results, we found that such cells do indeedexpress HBB2. A higher expression of HBB2 was seen in epithelialcells lining the alveoli in lung sections ofmicrometastasis-bearingmice than in alveolar epithelial cells from normal mice (Fig. 3A;Supplementary Fig. S3). A higher expression of HBB2 was alsoseen in endothelial cells lining blood vessels of micrometastasis-bearing mice than in endothelial cells of control mice (Fig. 3A;Supplementary Figs. S3 and S4).

To verify that epithelial and endothelial cells from microme-tastasis-bearing mice indeed produce higher levels of HBB2 than

similar cells from control, normal mice, single-cell suspensionswere prepared from lung tissues of control and micrometastasis-bearing mice using the GentleMACS dissociator (25). Lung cellswere separated using magnetic-activated cell sorting (MACS) toisolate immune, epithelial, and endothelial cells using corre-spondingly the lineage cell markers CD45, CD326, and CD31(26). Flow cytometry analysis was performed to verify the efficacyof the separation procedure (Fig. 3B). Confocal microscopyconfirmed the expression of HBB2 protein in the sorted epithelialand endothelial cells (Fig. 3C). qRT-PCR analyses of the separatedcell populations indicated that indeed HBB2 mRNA is producedby pulmonary epithelial and endothelial cells and not by pul-monary immunocytes (Fig. 3D). HBB2 mRNA expression was 30times higher in pulmonary epithelial cells of micrometastasis-bearing mice than in control mice (P < 0.001). HBB2 mRNAexpression was 5 times higher in pulmonary endothelial cells ofmicrometastasis-bearing mice than in pulmonary endothelialcells of normal mice (P < 0.05; Fig. 3D). An additional HBB2mRNA–expressing pulmonary cell population (CD45/CD31/CD326-negative cells, possibly hematopoietic stem cells) wasidentified to express HBB2 mRNA. However, HBB2 expressionby these cells was only 2 times higher in cells derived frommicrometastasis-bearing mice compared with control mice (datanot shown).

Expression of the alpha subunit of hemoglobin, HBA, was alsoseen inpulmonary epithelial and endothelial cells; however, therewas no significant difference in HBA expression in the pulmonarycell populations derived from control and micrometastasis-bear-ing mice (Fig. 3D).

We next asked whether the elevated expression of HBB2 inpulmonary cells ofmicrometastasis-bearingmice ismediated by adirect interaction between these cells or their soluble products andmicrometastatic neuroblastoma cells residing in the lungs. Toanswer this question, we cocultured MicroNB cells with humanpulmonary endothelial cells or with human pulmonary fibro-blasts in a Transwell system. Following the coincubation, totalRNA was isolated from the endothelial cells and fibroblasts and aqRT-PCR was performed to examine human HBB expression inthese cells. Expression levels of HBBmRNAwere 8 times higher (P< 0.005) in the endothelial cells cocultured with soluble factorsfromMicroNB cells than in control cells (Fig. 3E). The expressionof HBB mRNA was not altered in human pulmonary fibroblastscocultured with MicroNB cells (Fig. 3E). Expression levels of themRNA of human hemoglobin alpha chain, HBA, remainedunchanged in endothelial cells cocultured with MicroNB cells(Fig. 3E).

The data summarized in this section clearly demonstrate thatpulmonary epithelial cells and to a lesser degree pulmonaryendothelial cells are an important source for the levels of HBB2in the lungs of nude mice bearing human neuroblastomamicrometastases. Moreover, the results of the in vitro experi-ments demonstrate that neuroblastoma-derived soluble factorsare capable to stimulate pulmonary endothelial cells to selec-tively upregulate the transcription of the hemoglobin betachain.

(Continued.) E, Sequence analysis of the inhibitory fraction by LC/MS-MS coupled with tryptic digestion and database search positively identified theinhibitory protein as mouse hemoglobin subunit beta 2 (HBB2) of 147 amino acid residues. F, HBB2 was verified as the inhibitory lung factor when the addition ofa specific anti-mouse HBB2 antibody, and not IgG control, blocked the inhibitory activity of lung-derived factors incubated with MicroNB and MacroNB cells,as indicated in anMTS-based viability assay. Control bars indicate incubationwith growthmedia, treatment bars indicate incubationwith lung factors (LF) solubilizedin growth media. Data are means of three independent experiments þ SD. Significance was evaluated using Student t test. � , P < 0.05; �� , P < 0.01; ��, P < 0.005.






Human HBB inhibits the viability of neuroblastoma cellsThe next set of experiments was aimed to find out if similarly to

mouse HBB2, the beta subunit of human hemoglobin (HBB)would also inhibit the viability of human neuroblastoma cells.Native human hemoglobin was subjected to separation byreverse-phase HPLC, during which the alpha and beta subunitsof hemoglobinwere fully separated (Supplementary Fig. S5B) andpurified. The separation was verified by mass spectrometry anal-ysis (Fig. 4A; Supplementary Fig. S5C).

Whereas the alpha subunit (HBA) did not influence neuroblas-toma cell viability, incubation with 100 mg/mL human HBBdecreased the viability of MicroNB cells by 62% (P < 0.005) andof MacroNB cells by 39% (P < 0.01; Fig. 4B). These results were

strikingly similar to the viability-inhibitory functionsmediated bymouse lung-derived factors (15) and by lung-derived mouseHBB2 (Fig. 1B and D). The HBB-mediated inhibitory activity wasdose dependent (Supplementary Fig. S5D). Intact human hemo-globin inhibited the viability of neuroblastoma cells, but not tothe same extent as its beta subunit (Fig. 4B).

The growth-restraining activity spectrum of human HBBHuman HBB was found to be inhibitory against several addi-

tional cancer cell lines (Table 1). The lung carcinoma cell lineA549 and themelanoma cell line RALLwere inhibited by all threedoses of HBB used (1, 10, and 100 mg). The highest amount ofHBB (100 mg), reduced the viability of the breast cancer cell lines

Figure 2.

The expression of the inhibitory factor HBB2 is elevated in micrometastasis-bearing organs. Organs of mice that were orthotopically inoculated to the adrenal glandwith either PBS (normal mice) or MicroNB cells (micrometastasis-bearing mice) were harvested and examined for mHBB2 expression by qRT-PCR, immunostainingof frozen lung sections, and Western blot analysis. A, qRT-PCR quantification of HBA and HBB2 mRNA in lungs of normal and micrometastasis-bearingmice.B, Frozen sections of normal andmicrometastasis-bearing lungs immunostainedwith anti-mouseHBB2 (green) andDAPI (blue). Top, scale bar, 50mm;bottom,scale bar, 7.5 mm. Negative control was stained with secondary antibody and DAPI. C,Western blot analysis for the expression of HBA and HBB2 in normal (Norm)lungs, liver, and bone marrow and in the corresponding micrometastasis-bearing (Mic) organs. Whole cell lysates of mouse fibroblasts served as negativecontrol; Mouse heart extract served as positive control. D, Serum separated from blood of normal and micrometastasis-bearing mice was examined forhemoglobin (Hb), HBA, and HBB2 expression by ELISA. Data are means of mice in each group (n¼ 18, 9 mice in each group)þSD. Significance was evaluated usingStudent t test. � , P < 0.05; �� , P < 0.01; �� , P < 0.005; �� , P < 0.001.

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Figure 3.

Mouse HBB2 is synthesized in alveolar epithelial and endothelial cells. A, Frozen sections of lungs from normal and micrometastasis-bearing mice wereimmunostained for mouse HBB2 (green), the epithelial cell marker CD326 (red), or the endothelial cell marker CD31 (red) and DAPI (blue). Top, scale bar, 25 mm(epithelial cells) or 50 mm (endothelial cells); bottom, magnification of top panel, scale bar, 5 mm (epithelial cells) or 10 mm (endothelial cells). B, Flowcytometry analysis for the expression of CD31 or CD326 before and after isolation of epithelial and endothelial cells from pulmonary single-cell suspensions of normalandmicrometastasis-bearingmice usingmagnetic-activated cell sorting. An appropriate isotype control was analyzed for each cell marker.C,Pulmonary endothelialand epithelial cells were immunostained for HBB2 (green), CD31 (red), or CD326 (red) and DAPI (blue) after magnetic-activated cell sorting separation frompulmonary single-cell suspensions of normal and micrometastasis-bearing mice. Scale bar, 7.5 mm. D, qRT- PCR quantification of mouse HBA and HBB2mRNA in immunocytes, endothelial, and epithelial cells separated from lungs of normal and micrometastasis-bearing mice using magnetic-activated cell sorting.E, qRT- PCR quantification of human HBA and HBB mRNA in primary human pulmonary fibroblasts (HPF) and human pulmonary endothelial cells (HPEC) afterincubation with MicroNB cells in a Transwell system that enables the passage of soluble factors between the cocultured cells. Data are means of mice in eachgroup (n ¼ 12, 6 mice in each group) or of three independent in vitro experiments þSD. Significance was evaluated using Student t test. � , P < 0.05; �� , P < 0.01;�� , P < 0.005; �� , P < 0.001.






T47D and MCF-7, the prostate cancer cell line 22RVi, the cervicalcancer cell line Hela, and the melanoma cell line RKTJ. Theseresults demonstrate that theHBB-mediated inhibition of viabilityis not restricted to neuroblastoma cells.

Human HBB did not influence the viability of the normal(transformed) cell lines HEK293T and human pulmonary endo-thelial cells, nor did it influence the viability of the normal(nontransformed) human foreskin and pulmonary fibroblasts

(Table 1). HBB did not cause hemolysis of human erythrocytes(Supplementary Fig. S6).

HBB mediates apoptosis of and cell-cycle arrest inneuroblastoma cells

Flow cytometry analysis of Annexin-V and PI apoptotic testindicated that in HBB-treated MicroNB and MacroNB cells, thepercentage of cells in early apoptosis and late apoptosis was

Figure 4.

Human HBB inhibits neuroblastoma cell viability by inducing apoptosis and growth arrest. A, Reverse-phase HPLC separation of native human hemoglobinresulted in the isolation of the beta subunit of a molecular mass of 15,867 Da. B, An MTS-based viability assay indicated that human HBB inhibits the viability ofMicroNB and MacroNB cells. The alpha subunit of hemoglobin (HBA) did not influence cell viability. The whole human hemoglobin protein (Hb) also inhibitedcell viability but not to the same extent as HBB. C, Flow cytometry analysis of Annexin-V and PI apoptosis assay for MicroNB and MacroNB cells incubated withhuman HBB. D, Cell-cycle analysis was performed using flow cytometry to determine the percentage of cells in sub-G0 and G0–G1 phases. E, Whole cell lysates ofMicroNB and MacroNB cells incubated with human HBB were subjected to Western blot analysis and immunostaining. Cyclin D1 expression was calculated inreference to b-tubulin. F,Whole cell lysates of MicroNB andMacroNB cells incubatedwith humanHBBwere subjected toWestern blot analysis and immunostaining.ERK1/2 (F), p38 (G), and TAK1 (H) phosphorylation was calculated in reference to total ERK2, p38, and TAK1, respectively, as measured by densitometry. Data aremeans of three independent experiments þSD. Significance was evaluated using Student t test. � , P < 0.05; �� , P < 0.01; �� , P < 0.005; �� , P < 0.001.

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increased by 17% (P < 0.01) and 13% (P < 0.05), respectively, andthat the percentage of necrotic cells was very low and did notchange after treatment with HBB (Fig. 4C). However, the viabilityof HBB-treated MicroNB and MacroNB cells decreased by 62%and 39%, respectively (Fig. 4B). Apoptosis and/or necrosis arethus not the only mechanisms responsible for the decline in cellviability.

Flow cytometry analysis for cell-cycle progression revealed thatHBB increased the fraction of MicroNB and MacroNB cells in theG0–G1 phase by 59% (P < 0.01) and 23% (P < 0.05), respectively(Fig. 4D). The increased percentage of cells in the G0–G1 phasewas accompanied by a decrease in cyclin D1 protein level (P <0.005; Fig. 4E).

In addition to apoptosis and growth arrest, HBB decreased ERKphosphorylation (P < 0.005) and increased p38 phosphorylation(P < 0.005), creating a low ERK/p38 signaling ratio in the tumorcells (Fig. 4F andG). The phosphorylation of TAK1 inHBB-treatedcells was also increased (P < 0.01; Fig. 4H).

Taken together, these results indicate that human HBB inducesapoptosis and cell-cycle arrest in neuroblastoma cells, leading togrowth arrest of the tumor.

A short C-terminal region in human HBB mediates the growtharrest in neuroblastoma cells

To identify the functional region of humanHBB responsible forthe viability-inhibitory activity on tumor cells, we cleaved theprotein to N- and C-terminal fragments by cyanogen bromide(Fig. 5A). MTS-based viability assays showed that the C-terminalfragment is responsible for the growth arrest activity (Fig. 5B). TheN-terminal fragment also exhibited an inhibitory effect, however,to a lower extent (Fig. 5B).

HBB was then synthesized in 14 peptide segments of 15amino acids each (Supplementary table S2). Each segment wascomposed of 5 amino acids overlapping those of the precedingsegment and 5 amino acids overlapping those of the followingsegment (Fig. 5C). Each of these segments was assayed for itsability to block the viability of MicroNB cells. While peptides 2,3, and 8 stimulated tumor viability, peptide 11 with the aminoacid sequence ENFRLLGNVLVCVLA (designated hereafter

"Metox"), significantly inhibited the viability of the cells by23%–70% depending on its concentration (Fig. 5D). It is notunlikely that the viability-inhibitory function of intact HBB is anet balance between the growth-promoting functions mediatedby peptides 2, 3, and 8 and the growth-inhibitory functions ofpeptide 11.

Confocal microscopy suggested that FITC-labeled Metox isinternalized into MicroNB and MacroNB cells (Fig. 5E). Flowcytometry indeed indicated membrane binding and cellularuptake of FITC-labeled Metox after 30 minutes of incubation(Fig. 5F). The uptake was inhibited at 4�C, implying that theprocess depends on endocytosis (Fig. 5F).

Metox inhibits local tumors and metastasisAthymic nude mice were orthotopically inoculated with

MicroNB cells to the adrenal gland. Fourteen days after inocula-tion,micewere treatedby the intranasal route (27)once aweek for8 weeks with 15 mg/kg Metox or with the same amounts of acontrol peptide having the identical amino acid composition asMetox but in a scrambled sequence (Fig. 6A).

Twenty days after tumor cell inoculation, a difference (P< 0.05)was apparent in the local tumor volume between mice treatedwithMetox andmice treatedwith scrambled-Metox (Fig. 6B). Thisdifference became more significant with time (Fig. 6B). Seventydays after tumor cell inoculation, the weight of local tumorsresected from Metox-treated mice was 12 times lower (P <0.001) than that of local adrenal tumors resected from micetreated similarly but with scrambled Metox. (Fig. 6C and D).

qRT-PCR analyses indicated that the metastatic load ofMicroNB cells was significantly lower (P < 0.001) in lungs andbone marrow of mice treated with Metox compared with thatfound in organs derived frommice treated with scrambled-Metox(Fig. 6E and F).

The results reported above showed that endogenous mouseHBB2 expression is upregulated in micrometastasis-bearing mice(Fig. 2C; Supplementary Fig. S2). In accordance with these results,the endogenous HBB2 mRNA expression levels in organs of micetreated with scrambled-Metox (a treatment that does not reducetumor and metastasis load) were significantly higher (P < 0.01)

Table 1. The spectrum of human cancer cells inhibited by human HBB

Tumor type Cell line

% Differencein cell viability(1 mg HBB)



Breast MDA-231 No change þ5% No changeMDA-MB-468 No change No change No changeT47D No change �8% �30%MCF-7 No change No change �26%SKBR3 No change No change No change

Colon SW480 No change No change No changeLung A549 �17% �18% �29%Prostate 22RVi No change �14% �45%Cervix HeLa No change No change �30%Melanoma RKTJ No change No change �65%

RALL �11% �25% �55%Neuroblastoma MHH-NB11 (MicroNB) �23% �42% �62%Normal (transformed) HEK293T No change No change No change

HPEC No change No change No changeNormal (non-transformed) Foreskin fibroblasts No change No change No change

HPF No change No change No change

NOTE: HBB isolated from native human hemoglobin was incubated with numerous human cancer cell lines and cell viability was assessed using MTS-based viabilityassays. Data are means of four independent experiments per cell line. Presented are cell lines in which the difference in cell viability was statistically significant(Student t test, P < 0.05).Abbreviations: HPEC, human pulmonary endothelial cells; HPF, human pulmonary fibroblasts.






Figure 5.

A short C-terminal fragment of human HBB is responsible for the inhibitory effect of the protein. A, Cleavage of human HBB protein in the amino acidmethionine using CNBr resulted in N- and C-terminal fragments. B, An MTS-based viability assay revealed that most of the inhibitory activity of human HBB isin the C-terminal region of the protein. C, Fifteen amino acid segments of human HBB were synthesized using FMOC solid-phase synthesis and purified to>95% by HPLC. Each segment was designed to overlap in 5 amino acids with its preceding and following segment. D, An MTS-based viability assayindicated that peptide 11 of human HBB (designated hereafter Metox) significantly inhibited the viability of MicroNB cells. E, MicroNB and MacroNB cellsincubated with FITC-conjugated Metox at a concentration of 10 mg/mL for 0, 5, and 30 minutes were analyzed by confocal microscopy for Metox cell entry.Incubation with unlabeled Metox served as control. Shown are confocal microscopy images of FITC-conjugated Metox and DAPI staining in MicroNB cells.Scale bar, 10 mm. F, MicroNB cells incubated with FITC-conjugated Metox at a concentration of 10 mg/mL for 30 minutes were either washed withPBS to remove unbound Metox or with trypsin to remove unbound and membrane bound Metox. The percentage of positive FITC-Metox cells is presentedfor surface bound and internalized Metox (washed with PBS) and for internalized Metox (washed with trypsin). Data are means of three independentexperiments þ SD. Significance was evaluated using Student t test. � , P < 0.05; �� , P < 0.01; �� , P < 0.005.

Maman et al.





Figure 6.

A short fragment of human HBB (Metox) inhibits neuroblastoma local tumor growth and metastasis. A, Mice were orthotopically inoculated to the adrenal glandwith neuroblastoma micrometastases. Fourteen days postinoculation, mice were intranasally treated with Metox or with a scrambled Metox peptide (controlgroup) once a week for 8 weeks. Mice were monitored weekly for tumor volume. At the end of the experiment, local tumors were weighed and organs wereharvested and examined for the presence of human neuroblastoma cells and for mHBB2 expression using qRT-PCR. B, Volume measurements of localtumors of mice treated with Metox or scrambled-Metox. C, Mice treated with Metox or scrambled-Metox were photographed right before the extractionof local adrenal tumors. Local adrenal tumors were photographed as well. D, Local adrenal tumors were weighed right after extraction from mice.E, qRT-PCR quantification of MicroNB cells in mouse lungs. F, qRT-PCR quantification of MicroNB cells in mouse bone marrow. G, qRT-PCR quantification ofthe expression of mouse HBB2 in the lungs of mice. H, qRT-PCR quantification of the expression of mouse HBB2 in the bone marrow of mice. Data are meansof mice in each group (n ¼ 24, 12 mice in each group) þSD. Significance was evaluated using Student t test. �, P < 0.05; �� , P < 0.01; ��, P < 0.005; ��P < 0.001.






than its expression levels in organs of mice treated with Metox(which reduces tumor and metastasis load; Fig. 6G and H).

Similar results were obtained in an additional in vivo experi-ment in which Metox was administered either by the intranasalroute or intravenously to MicroNB cell–inoculated mice (Sup-plementary Fig. S7). Both forms of Metox administration inhib-ited the growth of local adrenal tumors and of lung and bonemarrow metastasis; however, intranasal administration of Metoxwas more effective.

DiscussionThe current study is the first to report that the b subunit of

hemoglobin belongs to the group of moonlighting proteins thatare capable of performingmultiple physiologic functions (24). Inaddition to its oxygen transport functions, HBB also exhibits anantitumor reactivity and as such joins the arsenal of innateresistance factors such as defensins (28, 29) that regulate cancerprogression. Interestingly, a novel function was also recentlyreported for the a subunit of hemoglobin in regulation of theeffects of nitric oxide in non-erythroid cells (30).

In patients with neuroblastoma, lung metastasis is a relativelylate event (31). This delay may be caused by the inhibitoryfunction of the lung-derived HBB restraining the further progres-sion of lung-residingmicrometastases. Overt neuroblastoma lungmetastasis may develop if a subset of such lung-residing cellsdevelops resistance to HBB or if the expression of HBB is down-regulated. The latter possibility is supported by an Oncominemeta-analysis (32) of gene expression profiling accumulated byseveral research groups (33–37), demonstrating that a 17- to 40-fold decrease in HBB expression occurred in overt lung metastaticlesions as compared with its expression in normal lung tissues.

In addition to neuroblastoma cells, other tumor cells aresensitive to the proliferation-restraining function of HBB. Breastcancer, lung cancer, andmelanoma are among the sensitive cancertypes. However, different tumors belonging to a certain cancertype display a heterogeneous response to the growth-retainingfunction ofHBB; only 2 of the 5 breast cancer cell lines testedweresensitive. Ongoing experiments are aimed to identity the com-mon factor that confers HBB sensitivity to the growth-restrainingfunction of this protein upon different types of tumor cells.

The bearing of a local neuroblastoma tumor and of microme-tastasis triggered an adaptive enhanced synthesis of HBB2 bypulmonary epithelial cells and to a lesser degree by pulmonaryendothelial cells. An upregulated synthesis of HHB2 was alsodetected in bonemarrow and liver cells. Assuming that endothelialcells in these organs are another source for circulatingHBB2and thefact that endothelial cells constitute a very large overall mass in thebody, these cells could play a significant role in resisting thepropagation of neuroblastoma (and other tumors). Whereas thesynthesis of hemoglobin or its subunits by nonerythroid cells suchas pulmonary epithelial cells, mesangial cells in the kidney andneurons in the brain was reported (24, 38–42), we are not aware ofstudies reporting the synthesis of the beta subunit by pulmonaryendothelial cells. Interestingly, cathepsin proteases capable ofproteolytic degradation of both a- and b-globin are also expressedbypulmonary epithelial cells,where these proteases are involved inpost-translational processing of surfactant proteins (43–45).

The upregulated synthesis of HBB2 is apparentlymediated by adirect contact between these host cells and soluble factors derivedfrom the tumor cells; in vitro experiments demonstrated that

coculturing pulmonary endothelial cells with culture superna-tants of tumor cells stimulated HBB synthesis by the former cellsbut not by pulmonary fibroblasts.

What drives the upregulation of HBB2 in micrometastasisbearing mice? First we demonstrated that the upregulation ofHBB2 is transcriptional and occurs at the mRNA level as well as atthe protein level. We then experimentally excluded the possibilitythat hemolysis occurs in nude mice bearing human neuroblas-toma xenografts. Free hemoglobin is therefore not the source forthe upregulated expression of HBB2 in these mice. By demon-strating that the concentrations of nitric oxidemetabolites, nitrite,and nitrate were similar in the lungs and serum of normal andmicrometastasis-bearing mice (Supplementary Fig. S8), we alsoexcluded the possibility that free hemoglobin sequestered nitricoxide, depleting its amounts and causing endothelial dysfunction(41). On the other hand and as indicated above, we providedevidence that the upregulation of HBB2 is triggered in pulmonarycells by tumor-derived factors.

The adaptive upregulated expression ofHBB2 in tumor-bearingmice suggests that this protein may alert for danger signalsdelivered by invading foreign cells such as microorganisms orcancer cells sharing patterns that are recognized by HBB2 (46).The in vivo experiments performed in this study indicate that theupregulated levels of HBB2 in tumor and metastasis bearers areinsufficient to eradicate micrometastatic tumor cells and that anexogenous administration of HBB2 or of its derivative Metox isneeded to efficiently halt metastasis formation.

The elucidation of the mechanism underlying the growth-restraining activity of HBB and its derived Metox peptide isoutside the scope of the current study. We do speculate, however,that the presence of HBB2 at the apical surface of endothelial andepithelial cellsmay indicate thatHBB2 is secreted from these cells.The fact that proliferation inhibitory and proapoptotic signalingwere activated in tumor cells by HBB2 also supports the sugges-tion that this HBB2/HBB-mediated signaling is activated bysecreted forms of these proteins. The findings that HBB inducesboth apoptosis and cell-cycle arrest of tumor cells, that the HBB-derivedMetox binds the outer membrane and is internalized intothe tumor cells, and that HBB-activated TAK1 and P38, down-regulated ERK phosphorylation, and Cyclin D1 stability serve asbasis for a working hypothesis as to its mode of action. Wehypothesize that these growth arrest–inducing activities aremedi-ated by binding of soluble HBB2/HBB to a yet unidentified HBBreceptor. Such a receptor could facilitate the internalization ofHBB and Metox into the target cells.

A low ERK/P38 phosphorylation ratio may lead to tumordormancy (47). Although other signaling mechanisms that donot induce dormancymay also act in conjunctionwith a lowERK/p38 signaling ratio (48, 49), we hypothesize that in addition toapoptosis, HBB induces tumor dormancy. Future work will con-firm or negate this hypothesis.

The current study provides proof of concept that microenvi-ronmental control, in the form of a naturally occurring protein,HBB, exerts proliferation-restraining functions (apoptosis andcell-cycle arrest) on tumor cells; neuroblastoma being the casein point. Similar microenvironmental control mechanisms thatblock the proliferation of incipient cancer cells are postulated tooperate in healthy people (10, 50).

The bioactive tumor-restraining region of HBB (ENFRLLGNV-LVCVLA) was found to exert significant antitumor and anti-metastasis activities both in vivo as well as in vitro. This peptide

Maman et al.





offers promising opportunities for the development of noveltherapies for the treatment of both primary as well as residualdisease. In addition, the inducible expression of HBB in theserum and organs of individuals harboring clinically undetect-able metastasis could be exploited for the early detection ofminimal residual disease preceding relapse.

Disclosure of Potential Conflicts of InterestY. Keisari is a consultant/advisory boardmember for SURI Technologies. No

potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: S. Maman, O. Sagi-Assif, Wuyuan LuDevelopment of methodology: S. Maman, O. Sagi-AssifAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Maman, W. Yuan, T. Meshel, Weiyue LuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S.Maman,O. Sagi-Assif,W. Yuan, R.Ginat, T.Meshel,Y. Keisari, W. LuWriting, review, and/or revision of the manuscript: S. Maman, O. Sagi-Assif,T. Meshel, Y. Keisari, Weiyue Lu, Wuyuan LuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Maman, W. Yuan, I. ZubrilovStudy supervision: O. Sagi-Assif, W. Lu, Wuyuan Lu

AcknowledgmentsThe authors thank Dr. Mickey Harlev and Dr. Maya Levin Arama (Animal

Care Facilities, Sackler Faculty ofMedicine, Tel-Aviv University, Tel-Aviv, Israel),Dr. Joe Bryant, and Dr. Eugene Ateh (Animal Core Facility, Institute of HumanVirology, University of Maryland School of Medicine, Baltimore, MD) for thehelp with animal experiments. We also thank the W.M. Keck Biomedical MassSpectrometry Laboratory at the University of Virginia Biomedical ResearchFacility for the MS and MS/MS analyses.

Grant SupportThis work was supported by the NIH grant AI087423 (W. Lu), by the

National Basic Research Program of China (973 Program) 2013CB932500(W-Y. Lu), by the German Research Foundation (Deutche Forschungsge-meinschaft DFG) grant BA4027/6-1 (I.P. Witz), by the James & Rita LeibmanEndowment Fundfor Cancer Research (I.P. Witz), by the Fred August and AdeleWolpers Charitable Fund (I.P. Witz), and by the Sara and Natan BlutingerFoundation (I.P. Witz).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received October 25, 2015; revised October 1, 2016; accepted October 21,2016; published OnlineFirst October 28, 2016.

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2017;77:14-26. Published OnlineFirst October 28, 2016.Cancer Res Shelly Maman, Orit Sagi-Assif, Weirong Yuan, et al. Neuroblastoma Growth and MetastasisThe Beta Subunit of Hemoglobin (HBB2/HBB) Suppresses

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The Beta Subunit of Hemoglobin (HBB2/HBB) Suppresses ... · Microenvironment and Immunology The Beta Subunit of Hemoglobin (HBB2/HBB) Suppresses Neuroblastoma Growth and Metastasis