The Beta Subunit of Hemoglobin (HBB2/HBB) Suppresses Neuroblastoma Growth And

Home , Globin, Hemoglobin subunit alpha, Hemoglobin subunit beta

Author Manuscript Published OnlineFirst on October 28, 2016; DOI: 10.1158/0008-5472.CAN-15-2929 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

The beta subunit of hemoglobin (HBB2/HBB) suppresses neuroblastoma growth and

metastasis

1,2Shelly Maman, 1Orit Sagi-Assif, 2Weirong Yuan, 1Ravit Ginat, 1Tsipi Meshel, 1Inna Zubrilov.

3Yona Keisari, 4Weiyue Lu, 2Wuyuan Lu and 1,2Isaac P. Witz

1Department of Cell Research and Immunology, The George S. Wise Faculty of Life Sciences,

Tel Aviv University, Tel Aviv, Israel; 2Institute of Human Virology, University of Maryland School of

Medicine, Baltimore, Maryland; 3Department of Clinical Microbiology and Immunology, Faculty of

Medicine, Tel Aviv University, Tel Aviv, Israel; 4Department of Pharmaceutics, School of

Pharmacy, Fudan University, Shanghai, 201203, PR China

Running title

HBB2/HBB inhibits neuroblastoma formation and metastasis

Keywords

Hemoglobin beta, beta globin, HBB2, HBB, metastasis, micrometastasis, microenvironment

Financial support

Research was supported by the National Institutes of Health grant AI087423 (to W. Lu), by the

National Basic Research Program of China (973 Program) 2013CB932500 (to W-Y. Lu), by the

German Research Foundation (Deutche Forschungsgemeinschaft DFG) grant BA4027/6-1 (to I.P.

Witz), by the James & Rita Leibman Endowment Fund for Cancer Research (to I.P. Witz), by the

Fred August and Adele Wolpers Charitable Fund (Clifton, NJ, USA) (to I.P. Witz) and by the Sara and Natan Blutinger Foundation (West Orange, NJ, USA) (to I.P. Witz).

Corresponding author

Isaac P. Witz, Dept. of Cell Research and Immunology, George S. Wise Faculty of Life Sciences,

Tel- Aviv University, Tel-Aviv 69978, Israel. Tel: +972-3-640-6979, Fax: +972-3-640-6974, Email: [email protected]

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 28, 2016; DOI: 10.1158/0008-5472.CAN-15-2929 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Conflict of interest

The authors declare no conflict of interests.

Word count

5622 (ALL TEXT excluding references)

Total number of figures and tables

References:

Abbreviations

BSA: Bovine serum albumin

FCS: Fetal calf serum

FITC: Fluorescein isothiocyanate

MacroNB: Macrometastatic neuroblastoma

MicroNB: Micrometastatic neuroblastoma

HBA: Hemoglobin subunit alpha

HBB: Human hemoglobin subunit beta

HBB2: Murine hemoglobin subunit beta

HPLC: High-performance liquid chromatography

LC-MS: Liquid chromatography–mass spectrometry

ESI-MS: Electrospray ionization mass spectrometry qRT-PCR: Quantitative Real-Time PCR

ERK: Extracellular signal-regulated kinase

TAK1: TGF-beta Activated Kinase 1

MTS: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium

XTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

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Abstract

Soluble pulmonary factors have been reported to be capable of inhibiting the viability of cancer cells that metastasize to the lung, but the molecular identity was obscure. Here we report the isolation and characterization of the beta subunit of hemoglobin as a lung-derived anti-metastatic factor. Peptide mapping in the beta subunit of human hemoglobin (HBB) defined a short C-terminal region (termed Metox) as responsible for activity. In tissue culture, both HBB and murine HBB2 mediated growth arrest 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 models limited development of adrenal neuroblastoma tumors as well as spontaneous lung and bone marrow metastases.

Expression studies in mice indicated that HBB2 is produced by alveolar epithelial and endothelial cells and is up-regulated in mice bearing undetectable metastasis. Our work suggested a novel function for HBB as a theranostic molecule: An innate anti-metastasis factor with potential utility as an anti-cancer drug and a biomarker signaling the presence of clinically undetectable metastasis.

Introduction

Metastasis is the major killer of cancer patients. Bidirectional interaction between cancer cells and their microenvironment is a critical determinant of tumor progression and metastasis (1-5).

Numerous reports in the last decade deal with mechanisms by which the microenvironment promotes tumor progression (6-9). In contrast, relatively little is known regarding inhibitory microenvironmental cells and molecules (10) with a few notable exceptions such as immunocytes and their products (11) or granulocytes (12).

Neuroblastoma is the most common extracranial solid tumor in children. Lung metastasis is a rare event (3-4%) but its presence is clinically important because it signals a poor prognosis (13).

60-70% of children with high-risk disease will ultimately experience relapse due to the presence of micrometastasis (14). Since cure after relapse is extremely rare, novel modalities for the inhibition and elimination of neuroblastoma metastases are needed.

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In a previous study (15) we demonstrated that the microenvironment of the normal lung possesses the capacity to restrain lung-metastasizing neuroblastoma cells and block their metastatic potential. Factors derived from normal mouse lungs significantly inhibited the viability of neuroblastoma lung-metastasizing cells by inducing cell cycle arrest and apoptosis.

Micrometastatic neuroblastoma cells (MicroNB), generated as described (16), were significantly more susceptible to this growth-restraining function than cells derived from frank neuroblastoma metastasis (MacroNB). The difference in susceptibility between micro and macro metastatic cells raised the hypothesis that factors in the lung microenvironment exert anti-metastatic functions including the maintenance of micrometastatic tumor cells in a state of growth arrest thereby blocking progression to overt lung metastasis. In the present study we set out to isolate and characterize the lung-derived metastasis-inhibitory factor and probe its metastasis-restraining activity.

Methods (See also Supplementary data)

Cell culture

The human neuroblastoma lung micrometastatic (MicroNB) and macrometastatic (MacroNB) variants were generated using a mouse model for human neuroblastoma metastasis (17) from the parental cell lines MHH-NB11 (18) and SH-SY5Y (19) as detailed here (16), and were maintained in culture as previously described (17). Primary human pulmonary fibroblasts (HPF) were purchased from Promo-cell (Heidelberg, Germany). Primary foreskin fibroblasts were generated from discarded foreskin tissue. Human pulmonary endothelial cells (HPEC) (20) were kindly provided by Dr. V.

Krump-Konvalinkova (Institute of pathology, Johannes-Gutenberg University, Mainz, Germany). All other mentioned cell lines were purchased from the American Type Culture Collection (ATCC)

(Manassas, VA). Cells were authenticated every three months according to the ATCC guidelines, as detailed here (15). All cultures were periodically examined for mycoplasma contamination.

HPLC separation of lung-derived factors

Lungs of one hundred BALB/c athymic nude mice were used to prepare the lung-derived factors as

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previously described (15). Lyophilized lung-derived factors were reconstituted in Milli-Q purified water (EMD Millipore) to a concentration of 1mg/ml, filtered (0.45µm) and subjected to separation by

Alliance reversed-phase high-performance liquid chromatograph (RP-HPLC) (Waters, Milford, MA) system using Waters XBridge C18 column (30X150mm, 5µm) running a gradient of 35% to 50% acetonitrile (Fisher Scientific, Pittsburgh, PA, USA) in water containing 0.1% trifluoroacetic acid

(Halocarbon, Inc., River Edge, NJ, USA) at a flow rate of 15 ml/min. The HPLC-separated fraction found to inhibit the viability of neuroblastoma cells was HPLC-purified running the same conditions.

LC-MS/MS identification of the lung-derived inhibitory factor

The purified inhibitory factor was digested in Coomassie stained polyacrylamide gel. Protein spots were excised from the gel and digested with trypsin according to published procedures (21). The digested inhibitory factor was injected to a Thermo Electron Orbitrap Velos ETD mass spectrometer using a 8cmx75µm Phenomenex Jupiter 10µm C18 capillary column, and the peptides eluted from the column by an acetonitrile-0.1M acetic acid gradient at a flow rate of 0.5µl/min over 30 min. The digest was analyzed using the double play function acquiring full mass spectra followed by ion spectra to determine molecular mass and amino acid sequence in sequential scans. The data were analyzed using the Sequest search algorithm against the Mouse International Protein Index (IPI).

HPLC separation of native human hemoglobin

Native human hemoglobin was dissolved in Milli-Q purified water (EMD Millipore) to a concentration of 1mg/ml and filtered (0.45 µm). Human hemoglobin was then chromatographed by Alliance RP-

HPLC system (Waters) using Waters XBridge C18 column (50X250mm, 10µm) running a gradient of

35% to 50% acetonitrile (Fisher Scientific) in water containing 0.1% trifluoroacetic acid (Halocarbon) at a flow rate of 40ml/min. The separated alpha and beta subunits of hemoglobin (HBA and HBB, respectively) were collected and purified by reversed phase HPLC and their molecular masses were verified by ESI-MS.

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Solid-phase synthesis of Metox and scrambled-Metox peptides

The inhibitory human HBB peptide (ENFRLLGNVLVCVLA) designated Metox, and a control peptide of a scrambled amino acid sequence (ANVLNECVFVGRLLL) designated scrambled-Metox, were chemically synthesized. The synthesis was performed on appropriate PAM resins (Applied

Biosystems) on an 433A peptide synthesizer (Applied Biosystems) using an optimized HBTU

(Oakwood Chemical, West Columbia, SC, USA) activation/DIEA (Sigma-Aldrich) in-situ neutralization protocol for Boc solid-phase peptide synthesis (22). After chain assembly, the peptides were cleaved by anhydrous hydrogen fluoride (Airgas, PA, USA) in the presence of 5% p- cresol (Sigma-Aldrich) at 0°c for 1hr, followed by precipitation with cold ether. The Metox and scrambled-Metox peptides were purified by RP-HPLC, and their molecular masses were ascertained by ESI-MS.

Treating mice with Metox

Mice were orthotopically inoculated with MicroNB cells to the adrenal gland to generate local adrenal tumors and lung and bone marrow micrometastasis as previously described (16). Fourteen days post tumor cell inoculation mice were treated intranasally with 15mg/kg of the human HBB peptide,

Metox, once a week for 8 weeks. The lyophilized peptide was dissolved prior to each administration in Dimethyl Sulfoxide (DMSO) (Sigma-Aldrich), diluted in sterile PBS and filtered (0.2µm). Mice were forced to inhale 20µl of Metox (0.3mg/mouse) or of the control scrambled peptide, scrambled-Metox

(control group).

Statistical analysis

Paired or unpaired Student t-test was used to compare in-vitro and in-vivo results.

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Results

Isolation and identification of a mouse inhibitory lung factor

Soluble lung-derived factors induce growth arrest and apoptosis 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 nude mice were generated as previously described (15).

Dialysis of the lung-derived factors suggested that the molecular weight of the inhibitory factor(s) is higher than 3500Da (Supplementary Fig. S1a). The biologically active dialyzed lung-derived factors were separated by reversed-phase HPLC to numerous fractions (Fig. 1a). These fractions were incubated with micrometastatic (MicroNB) and macrometastatic (MacroNB) neuroblastoma cells for 72 hr (Supplementary Fig. S1b). An MTS-based viability assay indicated that one of the distinct separated peaks inhibited the viability of the cells by 25-50% (p<0.05) (Fig. 1b) to the same extent as un-separated lung-derived factors (15).

The active inhibitory fraction was subjected to high-resolution purification using reversed-phase

HPLC. This resulted in a single, narrow and symmetric peak (Fig. 1c) representing, most probably, a single factor. Electrospray ionization mass spectrometry (ESI-MS) analysis confirmed that the purified fraction was indeed a single factor of a molecular mass of 15824.5 Da (Fig. 1c).

This fraction reduced the viability of MicroNB and MacroNB cells by 65% (p<0.01) and 35%

(p<0.05), respectively (Fig. 1d). Sequence analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with tryptic digestion followed by a database search in the

International Protein Index (IPI), identified 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 factor further verified its sequence identity.

The inhibitory activity of the isolated factors against MicroNB and MacroNB cells could be effectively neutralized by anti-mouse HBB2 antibodies, validating HBB2 as the inhibitory factor in mouse lungs (Fig. 1f). The inhibitory activity was not affected by an isotype control (Fig. 1f).

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Elevated levels of HBB2 alert to the presence of micrometastases

MicroNB cells were orthotopically inoculated to the adrenal gland of athymic nude mice generating local tumors. The control group was injected with PBS. Quantitative Real-Time PCR

(qRT-PCR) analyses performed 8 weeks after the intra-adrenal inoculation of the tumor cells, revealed the presence of micrometastatic human neuroblastoma cells in lungs, bone marrow and liver of the inoculated mice (Supplementary Fig. S2). At this point there was no evidence of overt metastasis. qRT-PCR analyses indicated that the level of mouse HBB2 mRNA (Supplementary Table S1) was 25 times higher in lungs of 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 sections stained with anti-mouse HBB2 antibody revealed a higher expression of HBB2 in lungs harboring neuroblastoma micrometastases than in normal lungs (Fig. 2b). In these lungs, an intracellular expression of HBB2 in cells lining the alveoli was observed (Fig. 2b). The higher expression of

HBB2 in lungs harboring micrometastases was verified by western blot analysis of lung tissue lysates (p<0.005) (Fig. 2c). HBB2 expression was also significantly higher in liver (p<0.01) and bone marrow (p<0.05) of micrometastasis-bearing mice than in liver and bone marrow of control normal mice (Fig. 2c). HBB2 concentration in the serum of micrometastasis-bearing mice was 7 times higher (p<0.005) than its concentration in the serum of normal mice (Fig. 2d). The serum concentration of the alpha subunit, HBA, was very low and was similar in normal control mice and in micrometastasis-bearing mice (Fig. 2d). Similarly there was no significant difference between the concentration of the whole, intact hemoglobin protein in the serum of normal and micrometastasis-bearing mice (Fig. 2d). This result excludes the possibility that the high concentrations of HBB2 in serum of tumor bearers were due to hemolysis. It is not unlikely that the lungs are an important contributor to the elevated HBB2 serum levels, since the most significant elevation in the expression of HBB2 in micrometastasis-bearing organs was in the lungs (Fig. 2c, Supplementary Fig. S2). However, other micrometastasis-bearing organs such

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as bone marrow and liver (Fig. 2c, Supplementary Fig. S2) do contribute as well, to the increased HBB2 protein levels in the serum.

Alveolar epithelial cells are the main source of HBB2

We next set out to identify the HBB2-producing lung cells and in particular those cells in whom transcription is up-regulated in micrometastasis-bearing mice (Fig. 2a). It was previously reported that HBB2 is synthesized by pulmonary epithelial cells (24). Confirming these results we found that such cells do indeed express HBB2. A higher expression of HBB2 was seen in epithelial cells lining the alveoli in lung sections of micrometastasis-bearing mice than in alveolar epithelial cells from normal mice (Fig. 3a, Supplementary Fig. S3). A higher expression of HBB2 was also seen in endothelial cells lining blood vessels of micrometastasis-bearing mice than in endothelial cells of control mice (Fig. 3a, Supplementary Fig. S3, S4).

In order to verify that epithelial and endothelial cells from micrometastasis-bearing mice indeed produce higher levels of HBB2 than similar cells from control, normal mice, single cell suspensions were prepared from lung tissues of control and micrometastasis-bearing mice using the GentleMACS dissociator (25). Lung cells were separated using magnetic-activated cell sorting (MACS) to isolate immune, epithelial and endothelial cells using correspondingly the lineage cell markers CD45, CD326 and CD31 and (26). Flow cytometry analysis was performed to verify the efficacy of the separation procedure (Fig. 3b). Confocal microscopy confirmed the expression of HBB2 protein in the sorted epithelial and endothelial cells (Fig. 3c). qRT-PCR analyses of the separated cell populations indicated that indeed HBB2 mRNA is produced by pulmonary epithelial and endothelial cells and not by pulmonary immunocytes (Fig. 3d). HBB2 mRNA expression was 30 times higher in pulmonary epithelial cells of micrometastasis-bearing mice than in control mice (p<0.001). HBB2 mRNA expression was 5 times higher in pulmonary endothelial cells of micrometastasis-bearing mice than in pulmonary endothelial cells of normal mice (p<0.05) (Fig. 3d). An additional HBB2 mRNA-expressing pulmonary cell population

(CD45/CD31/CD326-negative cells – possibly hematopoietic stem cells) was identified to express

HBB2 mRNA. However HBB2 expression by these cells was only 2 times higher in cells derived

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from micrometastasis-bearing mice compared to control mice (data not shown).

Expression of the alpha subunit of hemoglobin, HBA, was also seen in pulmonary epithelial and endothelial cells, however there was no significant difference in HBA expression in the pulmonary cell populations derived from control and micrometastasis-bearing mice (Fig. 3d).

We next asked if the elevated expression of HBB2 in pulmonary cells of micrometastasis-bearing mice is mediated by a direct interaction between these cells or their soluble products and micrometastatic neuroblastoma cells residing in the lungs. To answer this question we co- cultured MicroNB cells with human pulmonary endothelial cells or with human pulmonary fibroblasts in a Transwell system. Following the co-incubation, total RNA was isolated from the endothelial cells and fibroblasts and a qRT-PCR was performed to examine human HBB expression in these cells. Expression levels of HBB mRNA were 8 times higher (p<0.005) in the endothelial cells co-cultured with soluble factors from MicroNB cells than in control cells (Fig. 3e).

The expression of HBB mRNA was not altered in human pulmonary fibroblasts co-cultured with

MicroNB cells (Fig. 3e). Expression levels of the mRNA of human hemoglobin alpha chain, HBA, remained unchanged in endothelial cells co-cultured with MicroNB cells (Fig. 3e).

The data summarized in this section clearly demonstrate that pulmonary epithelial cells and to a lesser degree pulmonary endothelial cells are an important source for the levels of HBB2 in the lungs of nude mice bearing human neuroblastoma micrometastases. Moreover the results of the in-vitro experiments demonstrate that neuroblastoma–derived soluble factors are capable to stimulate pulmonary endothelial cells to selectively upregulate transcription of the hemoglobin beta chain.

Human HBB inhibits the viability of neuroblastoma cells

The 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 by reversed-phase HPLC, during which the alpha and beta subunits of hemoglobin were fully separated (Supplementary Fig. S5b) and

purified. The separation was verified by mass spectrometry analysis (Fig. 4a and

Supplementary Fig. S5c).

Whereas the alpha subunit (HBA) did not influence neuroblastoma cell viability, incubation with

100µg/ml human HBB decreased the viability of MicroNB cells by 62% (p<0.005) and of

MacroNB cells by 39% (p<0.01) (Fig. 4b). These results were strikingly similar to the viability- inhibitory functions mediated by mouse lung-derived factors (15) and by lung-derived mouse

HBB2 (Fig. 1b, d). The HBB-mediated inhibitory activity was dose dependent (Supplementary

Fig. S5d). Intact human hemoglobin inhibited the viability of neuroblastoma cells, but not to the same extent as its beta subunit (Fig. 4b).

The growth-restraining activity spectrum of Human HBB

Human HBB was found to be inhibitory against several additional cancer cell lines (Table 1). The lung carcinoma cell line A549 and the melanoma cell line RALL were inhibited by all 3 doses of

HBB used (1, 10 and 100µg). The highest amount of HBB (100µg), reduced the viability of the breast cancer cell lines T47D and MCF-7, the prostate cancer cell line 22RVi, the cervical cancer cell line Hela, and the melanoma cell line RKTJ. These results demonstrate that the HBB- mediated inhibition of viability is not restricted to neuroblastoma cells.

Human HBB did not influence the viability of the normal (transformed) cell lines HEK293T and human pulmonary endothelial cells, nor did it influence the viability of the normal (non- transformed) 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 in neuroblastoma cells

Flow cytometry analysis of annexin-V and PI apoptotic test indicated that in HBB-treated MicroNB and MacroNB cells the percentage of cells in early apoptosis and late apoptosis was increased by 17% (p<0.01) and 13% (p<0.05), respectively, and that the percentage of necrotic cells was very low and did not change after treatment with HBB (Fig. 4c). However, the viability of HBB- treated MicroNB and MacroNB cells decreased by 62% and 39%, respectively (Fig. 4b).

Apoptosis and/or necrosis are thus not the only mechanisms responsible for the decline in cell viability.

Flow cytometry analysis for cell cycle progression revealed that HBB increased the fraction of

MicroNB and MacroNB cells in the G0-G1 phase by 59% (p<0.01) and 23% (p<0.05), respectively (Fig. 4d). The increased percentage of cells in the G0-G1 phase was accompanied by a decrease in cyclin D1 protein level (p<0.005) (Fig. 4e).

In addition to apoptosis and growth arrest, HBB decreased ERK phosphorylation (p<0.005) and increased p38 phosphorylation (p<0.005), creating a low ERK/p38 signaling ratio in the tumor cells (Fig. 4f, g). The phosphorylation of TAK1 in HBB-treated cells was also increased (p<0.01)

(Fig. 4h).

Taken together these results indicate that human HBB induces apoptosis and cell cycle arrest in neuroblastoma cells, leading to growth arrest of the tumor.

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

In order to identify the functional region of human HBB responsible for the viability inhibitory activity on tumor cells we cleaved the protein to N- and C-terminal fragments by cyanogen bromide (Fig. 5a). MTS-based viability assays showed that the C-terminal fragment is responsible for the growth arrest activity (Fig. 5b). The N-terminal fragment also exhibited an inhibitory effect however to a lower extent (Fig. 5b).

HBB was then synthesized in 14 peptide segments of 15 amino acids each (Supplementary table S2). Each segment was composed of 5 amino acids overlapping those of the preceding segment and 5 amino acids overlapping those of the following segment (Fig. 5c). Each of these segments was assayed for its ability to block the viability of MicroNB cells. While peptides 2, 3 and 8 stimulated tumor viability, peptide 11 with the amino acid sequence ENFRLLGNVLVCVLA

(designated hereafter “Metox”), significantly inhibited the viability of the cells by 23-70% depending on its concentration (Fig. 5d). It is not unlikely that the viability inhibitory function of

intact HBB is a net balance between the growth promoting functions mediated by peptides 2, 3 and 8 and the growth inhibitory functions of peptide 11.

Confocal microscopy suggested that FITC-labeled Metox is internalized into MicroNB and

MacroNB cells (Fig. 5e). Flow cytometry indeed indicated membrane binding and cellular uptake of FITC-labeled Metox after 30 minutes of incubation (Fig. 5f). The uptake was inhibited in 4°c, implying that the process depends on endocytosis (Fig. 5f).

Metox inhibits local tumors and metastasis

Athymic nude mice were orthotopically inoculated with MicroNB cells to the adrenal gland.

Fourteen days following inoculation, mice were treated by the intranasal route (27) once a week for 8 weeks with 15mg/kg Metox or with the same amounts of a control peptide having the identical amino acid composition as Metox but in a scrambled sequence (Fig. 6a).

Twenty days after tumor cell inoculation a difference (p<0.05) was apparent in local tumor volume between mice treated with Metox and mice treated with scrambled-Metox (Fig. 6b). This difference became more significant with time (Fig. 6b). Seventy days post tumor cell inoculation, the weight of local tumors resected from Metox-treated mice was 12 times lower (p<0.001) than that of local adrenal tumors resected from mice treated similarly but with scrambled Metox. (Fig.

6c, d). qRT- PCR analyses indicated that the metastatic load of MicroNB cells was significantly lower

(p<0.001) in lungs and bone marrow of mice treated with Metox compared to that found in organs derived from mice treated with scrambled-Metox (Fig. 6e, f).

The results reported above showed that endogenous mouse HBB2 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 mice treated with scrambled-Metox (a treatment that does not reduce tumor and metastasis load) were significantly higher (p<0.01) than its expression levels in organs of mice treated with Metox (which reduces tumor and metastasis load) (Fig. 6g, h).

Similar results were obtained in an additional in-vivo experiment in which Metox was administered either by the intranasal route or intravenously to MicroNB cell inoculated mice

(Supplementary Fig. S7). Both forms of Metox administration inhibited the growth of local adrenal tumors and of lung and bone marrow metastasis, however intranasal administration of

Metox was more effective.

Discussion

The present study is the first to report that the β subunit of hemoglobin belongs to the group of moonlighting proteins which are capable of performing multiple physiological functions (24). In addition to its oxygen transport functions HBB also exhibits an anti-tumor reactivity and as such joins the arsenal of innate resistance factors such as defensins (28,29) that regulate cancer progression. Interestingly, a novel function was also recently reported for the α subunit of hemoglobin in regulation of the effects of nitric oxide in non-erythroid cells (30).

In neuroblastoma patients, lung metastasis is a relatively late event (31). This delay may be caused by the inhibitory function of the lung-derived HBB restraining the further progression of lung-residing micrometastases. Overt neuroblastoma lung metastasis may develop if a subset of such lung-residing cells develops resistance to HBB or if the expression of HBB is down- regulated. The latter possibility is supported by an Oncomine meta-analysis (32) of gene- expression profiling accumulated by several research groups (33-37), demonstrating that a 17-40 fold decrease in HBB expression occurred in overt lung metastatic lesions as compared to its expression in normal lung tissues.

In addition to neuroblastoma cells, other tumor cells are sensitive to the proliferation-restraining function of HBB. Breast cancer, lung cancer and melanoma are among the sensitive cancer types. However different tumors belonging to a certain cancer type display a heterogeneous response to the growth-retaining function of HBB; Only 2 of the 5 breast cancer cell lines tested were sensitive. Ongoing experiments are aimed to identity the common factor that confers HBB- sensitivity to the growth-restraining function of this protein upon different types of tumor cells.

The bearing of a local neuroblastoma tumor and of micrometastasis triggered an adaptive

enhanced synthesis of HBB2 by pulmonary epithelial cells and to a lesser degree by pulmonary endothelial cells. An up-regulated synthesis of HHB2 was also detected in bone marrow and liver cells. Assuming that endothelial cells in these organs are another source for circulating HBB2 and the fact that endothelial cells constitute a very large overall mass in the body, these cells could play a significant role in resisting the propagation of neuroblastoma (and other tumors). Whereas the synthesis of hemoglobin or its subunits by non-erythroid cells such as pulmonary epithelial cells, mesangial cells in the kidney and neurons in the brain was reported (24,38-42), we are not aware of studies reporting the synthesis of the beta subunit by pulmonary endothelial cells.

Interestingly, cathepsin proteases capable of proteolytic degradation of both α- and β-globin are also expressed by pulmonary epithelial cells, where these proteases are involved in post- translational processing of surfactant proteins (43-45).

The upregulated synthesis of HBB2 is apparently mediated by a direct contact between these host cells and soluble factors derived from the tumor cells; In-vitro experiments demonstrated that co-culturing pulmonary endothelial cells with culture supernatants of tumor cells stimulated HBB synthesis by the former cells but not by pulmonary fibroblasts.

What drives the up-regulation of HBB2 in micrometastasis bearing mice? First we demonstrated that the up-regulation of HBB2 is transcriptional and occurs at the mRNA level as well as at the protein level. We then experimentally excluded the possibility that hemolysis occurs in nude mice bearing human neuroblastoma xenografts. Free hemoglobin is therefore not the source for the upregulated expression of HBB2 in these mice. By demonstrating that the concentrations of nitric oxide metabolites, nitrite and nitrate were similar in the lungs and serum of normal and micrometastasis-bearing mice (Supplementary Fig. S8), we also excluded the possibility that free hemoglobin sequestered nitric oxide, depleting its amounts and causing endothelial dysfunction

(41). On the other hand and as indicated above we provided evidence that the up-regulation of

HBB2 is triggered in pulmonary cells by tumor-derived factors.

The adaptive up-regulated expression of HBB2 in tumor-bearing mice suggests that this protein may alert for danger signals delivered by invading foreign cells such as microorganisms or cancer

cells sharing patterns which are recognized by HBB2 (46). The in-vivo experiments performed in this study indicate that the up-regulated levels of HBB2 in tumor and metastasis bearers are insufficient to eradicate micrometastatic tumor cells and that an exogenous administration of

HBB2 or of its derivative Metox is needed in order to efficiently halt metastasis formation.

The elucidation of the mechanism underlying the growth-restraining activity of HBB and its derived Metox peptide is outside the scope of the present study. We do speculate however, that the presence of HBB2 at the apical surface of endothelial and epithelial cells may indicate that

HBB2 is secreted from these cells. The fact that proliferation inhibitory and pro apoptotic signaling were activated in tumor cells by HBB2 also supports the suggestion that this HBB2/HBB- mediated signaling is activated by secreted forms of these proteins. The findings that HBB induces both apoptosis and cell cycle arrest of tumor cells, that the HBB-derived Metox binds the outer membrane and is internalized into the tumor cells and that HBB activated TAK1 and P38, down regulated ERK phosphorylation and Cyclin D1 stability serve as basis for a working hypothesis as to its mode of action. We hypothesize that these growth arrest-inducing activities are mediated by binding of soluble HBB2/HBB to a yet un-identified HBB receptor. Such a receptor could facilitate the internalization of HBB and Metox into the target cells.

A low ERK/P38 phosphorylation ratio may lead to tumor dormancy (47). Although other signaling mechanisms that do not induce dormancy may also act in conjunction with a low ERK/p38 signaling ratio (48,49), we hypothesize that in addition to apoptosis, HBB induces tumor dormancy. Future work will confirm or negate this hypothesis.

The present study provides proof of concept that microenvironmental control, in the form of a naturally-occurring protein, HBB, exerts proliferation-restraining functions (apoptosis and cell cycle arrest) on tumor cells; neuroblastoma being the case in point. Similar microenvironmental control mechanisms that block the proliferation of incipient cancer cells are postulated to operate in healthy people (10,50).

The bioactive tumor-restraining region of HBB (ENFRLLGNVLVCVLA) was found to exert significant anti-tumor and anti-metastasis activities both in-vivo as well as in-vitro. This peptide offers promising opportunities for the development of novel therapies for the treatment of both

primary as well as residual disease. In addition, the inducible expression of HBB in the serum and organs of individuals harboring clinically undetectable metastasis could be exploited for the early detection of minimal residual disease preceding relapse.

Acknowledgments

The authors thank Dr. Mickey Harlev and Dr. Maya Levin Arama (Animal Care Facilities, Sackler

Faculty of Medicine, Tel-Aviv University), Dr. Joe Bryant and Dr. Eugene Ateh (Animal Core

Facility, Institute of Human Virology, University of Maryland School of Medicine, Baltimore,

Maryland) for the help with animal experiments. We also thank the W.M. Keck Biomedical Mass

Spectrometry Laboratory at the University of Virginia Biomedical Research Facility for the MS and

MS/MS analyses. The authors disclose no potential conflicts of interest.

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Tables

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

Tumor type Cell line % difference % difference % difference in cell viability in cell viability in cell viability (10µg HBB) (1µg HBB) (100µg HBB) B reast Breast MDA-231 No change +5% No change MDA-MB-468 No change No change No change T47D 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 change Lung Lung A549 -17% -18% -29% Prostate 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 -23% -42% -62% (MicroNB)

Normal HEK293T No change No change No change (transformed) HPEC No change No change No change Foreskin No change No change No change Normal fibroblasts (non-transformed) HPF No change No change No change *HPEC – Human pulmonary endothelial cells; HPF – Human pulmonary fibroblasts

HBB isolated from native human hemoglobin was incubated with numerous human cancer cell lines and cell viability was assessed using MTS-based viability assays. 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's t-test, p<0.05)

Figure legends

Figure 1. Isolation and identification of an inhibitory lung factor. a. Dialyzed lung-derived factors were subjected to separation by analytical C18 reversed-phase HPLC. b. An MTS-based viability assay revealed that one HPLC-separated fraction significantly inhibited cell viability. c.

Purification of the inhibitory fraction and analysis by electrospray ionization mass spectrometry

(ESI-MS) yielded a molecular mass of 15824.5 Da. d. An MTS-based viability assay verified the inhibitory activity of the HPLC-purified fraction. e. Sequence analysis of the inhibitory fraction by liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with tryptic digestion and database search positively identified the inhibitory 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 of a 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 an

MTS-based viability assay. Control bars indicate incubation with growth media, treatment bars indicate incubation with lung factors (LF) solubilized in growth media. Data are means of three independent experiments + SD. Signiﬁcance was evaluated using Student’s t-test. *p<0.05,

**p<0.01, ***p<0.005.

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 gland with either PBS (normal mice) or MicroNB cells (micrometastasis-bearing mice) were harvested and examined for mHBB2 expression by qRT-PCR, immunostaining of frozen lung sections and western blot analysis. a. qRT- PCR quantification of HBA and HBB2 mRNA in lungs of normal and micrometastasis-bearing mice. b. Frozen sections of normal and micrometastasis-bearing lungs immunostained with anti-mouse HBB2 (green) and DAPI (blue). Upper panel, scale bar =

50µm; Lower panel, scale bar = 7.5µm. 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 negative control; Mouse heart extract served as

positive control. d. Serum separated from blood of normal and micrometastasis-bearing mice was examined for hemoglobin (Hb), HBA and HBB2 expression by enzyme-linked immunosorbent assay (ELISA). Data are means of mice in each group (n=18, 9 mice in each group) +SD.

Signiﬁcance was evaluated using Student’s t-test. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001.

Figure 3. Mouse HBB2 is synthesized in alveolar epithelial and endothelial cells. a. Frozen sections of lungs from normal and micrometastasis-bearing mice were immunostained for mouse

HBB2 (green), the epithelial cell marker CD326 (red) or the endothelial cell marker CD31 (red) and DAPI (blue). Upper panel, scale bar = 25µm (epithelial cells) or 50µm (endothelial cells);

Lower panel, magnification of upper panel, scale bar = 5µm (epithelial cells) or 10µm (endothelial cells). b. Flow cytometry analysis for the expression of CD31 or CD326 before and after isolation of epithelial and endothelial cells from pulmonary single cell suspensions of normal and micrometastasis-bearing mice using magnetic-activated cell sorting (MACS). An appropriate isotype control was analyzed for each cell marker. c. Pulmonary endothelial and epithelial cells were immunostained for HBB2 (green), CD31 (red) or CD326 (red) and DAPI (blue) after MACS separation from pulmonary single cell suspensions of normal and micrometastasis-bearing mice.

Scale bar = 7.5µm. d. qRT- PCR quantification of mouse HBA and HBB2 mRNA in immunocytes, endothelial and epithelial cells separated from lungs of normal and micrometastasis-bearing mice using MACS. e. qRT- PCR quantification of human HBA and HBB mRNA in primary human pulmonary fibroblasts (HPF) and human pulmonary endothelial cells (HPEC) after incubation with

MicroNB cells in a Transwell system that enables the passage of soluble factors between the co- cultured cells. Data are means of mice in each group (n=12, 6 mice in each group) or of 3 independent in-vitro experiments +SD. Signiﬁcance was evaluated using Student’s t-test.

*p<0.05, **p<0.01, ***p<0.005, ****p<0.001.

Figure 4. Human HBB inhibits neuroblastoma cell viability by inducing apoptosis and growth arrest. a. RP-HPLC separation of native human hemoglobin resulted in the isolation of the beta subunit of a molecular mass of 15867 Da. b. An MTS-based viability assay indicated that

human HBB inhibits the viability of MicroNB and MacroNB cells. The alpha subunit of hemoglobin

(HBA) did not influence cell viability. The whole human hemoglobin protein (Hb) also inhibited cell 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 with human 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 of MicroNB and MacroNB cells incubated with human HBB were subjected to western blot analysis and immunostaining. Cyclin D1 expression was calculated in reference to β-tubulin. f. Whole cell lysates of MicroNB and MacroNB cells incubated with human HBB were subjected to western 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 are means of three independent experiments +SD. Signiﬁcance was evaluated using Student’s t-test. *p<0.05, **p<0.01,

***p<0.005, ****p<0.001.

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 acid methionine 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 is in 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 high performance liquid chromatography. Each segment was designed to overlap in 5 amino acids with its preceding and following segment. d. An MTS-based viability assay indicated that peptide 11 of human HBB (designated hereafter Metox) significantly inhibited the viability of

MicroNB cells. e. MicroNB and MacroNB cells incubated with FITC-conjugated Metox at a concentration of 10µg/ml for 0, 5 and 30 min 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 = 10µm. f.

MicroNB cells incubated with FITC-conjugated Metox at a concentration of 10µg/ml for 30 minutes were either washed with PBS to remove unbound Metox or with trypsin to remove

unbound and membrane bound Metox. The percentage of positive FITC-Metox cells is presented for surface bound and internalized Metox (washed with PBS) and for internalized Metox (washed with trypsin). Data are means of three independent experiments ± SD. Signiﬁcance was evaluated using Student’s t-test. *p < 0.05, **p <0.01, ***p < 0.005

Figure 6. A short fragment of human HBB (Metox) inhibits neuroblastoma local tumor growth and metastasis. a. Mice were orthotopically inoculated to the adrenal gland with neuroblastoma micrometastases. Fourteen days post inoculation, mice were intranasally treated with Metox or with a scrambled Metox peptide (control group), 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 were harvested and examined for the presence of human neuroblastoma cells and for mHBB2 expression using qRT-PCR. b. Volume measurements of local tumors of mice treated with Metox or scrambled-Metox. c. Mice treated with Metox or scrambled-Metox were photographed right before the extraction of 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 of the 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 means of mice in each group (n=24, 12 mice in each group) +SD. Signiﬁcance was evaluated using Student’s t-test. *p < 0.05, **p <0.01, ***p < 0.005, ****p<0.001.

The beta subunit of hemoglobin (HBB2/HBB) suppresses neuroblastoma growth and metastasis

Shelly Maman, Orit Sagi-Assif, Weirong Yuan, et al.

Cancer Res Published OnlineFirst October 28, 2016.

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