The Beta Subunit of Hemoglobin (HBB2/HBB) Suppresses

Published OnlineFirst October 28, 2016; DOI: 10.1158/0008-5472.CAN-15-2929 Cancer Microenvironment and Immunology Research

The Beta Subunit of Hemoglobin (HBB2/HBB) Suppresses Neuroblastoma Growth and Metastasis Shelly 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 of similarly and its administration in human tumor xenograft mod- inhibiting the viability of cancer cells that metastasize to the lung, els limited the development of adrenal neuroblastoma tumors but the molecular identity was obscure. Here we report the as well as spontaneous lung and bone marrow metastases. Expres- isolation and characterization of the beta subunit of hemoglobin sion studies in mice indicated that HBB2 is produced by alveolar as a lung-derived antimetastatic factor. Peptide mapping in the epithelial and endothelial cells and is upregulated in mice bearing beta subunit of human hemoglobin (HBB) deﬁned a short C- undetectable metastasis. Our work suggested a novel function terminal region (termed Metox) as responsible for activity. In for HBB as a theranostic molecule: an innate antimetastasis factor tissue culture, both HBB and murine HBB2 mediated growth with potential utility as an anticancer drug and a biomarker arrest and apoptosis of lung-metastasizing neuroblastoma cells, signaling the presence of clinically undetectable metastasis. along with a variety of other human cancer cell lines. Metox acted Cancer Res; 77(1); 1–13. 2016 AACR.

Introduction In a previous study (15), we demonstrated that the microenvironment of the normal lung possesses the capacity to restrain Metastasis is the major killer of patients with cancer. Bidi- lung-metastasizing neuroblastoma cells and block their metastat- rectional interaction between cancer cells and their microenvi- ic potential. Factors derived from normal mouse lungs signifi- ronment is a critical determinant of tumor progression and cantly inhibited the viability of neuroblastoma lung-metastasiz- metastasis (1–5). Numerous reports in the last decade deal with ing cells by inducing cell-cycle arrest and apoptosis. Micrometa- mechanisms by which the microenvironment promotes tumor static neuroblastoma cells (MicroNB), generated as described progression (6–9). In contrast, relatively little is known regard- previously (16), were significantly more susceptible to this ing inhibitory microenvironmental cells and molecules (10) growth-restraining function than cells derived from frank neuro- with a few notable exceptions such as immunocytes and their blastoma metastasis (MacroNB). The difference in susceptibility products (11) or granulocytes (12). between micro- and macrometastatic cells raised the hypothesis Neuroblastoma is the most common extracranial solid tumor that factors in the lung microenvironment exert antimetastatic in children. Lung metastasis is a rare event (3%–4%) but its functions including the maintenance of micrometastatic tumor presence is clinically important because it signals a poor prognosis cells in a state of growth arrest thereby blocking progression to (13). Sixty percent to 70% of children with high-risk disease will overt lung metastasis. In the current study, we set out to isolate and ultimately experience relapse due to the presence of micrometas- characterize the lung-derived metastasis-inhibitory factor and tasis (14). As cure after relapse is extremely rare, novel modalities probe its metastasis-restraining activity. for the inhibition and elimination of neuroblastoma metastases are needed. Materials and Methods Cell culture 1Department of Cell Research and Immunology, The George S. Wise Faculty of The human neuroblastoma lung micrometastatic (MicroNB) Life Sciences, Tel Aviv University, Tel Aviv, Israel. 2Institute of Human Virology, and macrometastatic (MacroNB) variants were generated using a University of Maryland School of Medicine, Baltimore, Maryland. 3Department of mouse model for human neuroblastoma metastasis (17) from Clinical Microbiology and Immunology, Faculty of Medicine, Tel Aviv University, 4 the parental cell lines MHH-NB11 (18) and SH-SY5Y (19) as Tel Aviv, Israel. Department of Pharmaceutics, School of Pharmacy, Fudan detailed here (16), and were maintained in culture as described University, Shanghai, P.R. China. previously (17). Primary human pulmonary fibroblasts (HPF) Note: Supplementary data for this article are available at Cancer Research were purchased from Promo-cell. Primary foreskin fibroblasts Online (http://cancerres.aacrjournals.org/). were generated from discarded foreskin tissue. Human pulmo- Corresponding Authors: Isaac P. Witz, Tel Aviv University, Haim Levanon st., nary endothelial cells (HPEC; ref. 20) were kindly provided by Tel Aviv 69978, Israel. Phone: 972-3640-6979; Fax: 972-3640-6974; E-mail: Dr. V. Krump-Konvalinkova (Institute of Pathology, Johannes- [email protected]; and Shelly Maman, [email protected] Gutenberg University, Mainz, Germany). All other mentioned cell doi: 10.1158/0008-5472.CAN-15-2929 lines were purchased from the ATCC. Cells were authenticated 2016 American Association for Cancer Research. every three months according to the ATCC guidelines, as detailed

www.aacrjournals.org OF1

Maman et al.

here (15). All cultures were periodically examined for mycoplas- Treating mice with Metox ma contamination. Mice were orthotopically inoculated with MicroNB cells to the adrenal gland to generate local adrenal tumors and lung HPLC separation of lung-derived factors and bone marrow micrometastasis as described previously Lungs of 100 BALB/c athymic nude mice were used to prepare (16). Fourteen days after tumor cell inoculation, mice were the lung-derived factors as described previously (15). Lyophilized treated intranasally with 15 mg/kg of the human HBB pep- lung-derived factors were reconstituted in Milli-Q purified water tide, Metox, once a week for 8 weeks. The lyophilized peptide (EMD Millipore) to a concentration of 1 mg/mL, filtered (0.45 was dissolved prior to each administration in dimethyl sulf- fi mm), and subjected to separation by Alliance reversed-phase high- oxide (Sigma-Aldrich), diluted in sterile PBS, and ltered m m performance liquid chromatograph (HPLC; Waters) system using (0.2 m).Micewereforcedtoinhale20 LofMetox(0.3 Waters XBridge C18 column (30 150 mm, 5 mm) running a mg/mouse) or of the control scrambled peptide, scrambled- gradient of 35% to 50% acetonitrile (Thermo Fisher Scientific) in Metox (control group). water containing 0.1% trifluoroacetic acid (Halocarbon, Inc.) at a flow rate of 15 mL per minute. The HPLC-separated fraction found Statistical analysis to inhibit the viability of neuroblastoma cells was HPLC-purified Paired or unpaired Student t test was used to compare in vitro running the same conditions. and in vivo results. For more details on Materials and Methods, see Supplementary LC/MS-MS identification of the lung-derived inhibitory factor Data The purified inhibitory factor was digested in Coomassie- stained polyacrylamide gel. Protein spots were excised from the Results gel and digested with trypsin according to the published proce- Isolation and identification of a mouse-inhibitory lung factor dures (21). The digested inhibitory factor was injected to a Soluble lung-derived factors induce growth arrest and apo- Thermo Electron Orbitrap Velos ETD mass spectrometer using ptosis of lung-metastasizing human neuroblastoma cells (15). m m a8cm 75 m Phenomenex Jupiter 10 m C18 capillary column, Here we isolated the inhibitory factor from mouse lungs. and the peptides eluted from the column by an acetonitrile-0.1 Soluble factors derived from the lungs of 100 athymic nude fl m mol/L acetic acid gradient at a ow rate of 0.5 L per minute over mice were generated as described previously (15). Dialysis of 30 minutes. The digest was analyzed using the double play the lung-derived factors suggested that the molecular weight of function acquiring full mass spectra followed by ion spectra to the inhibitory factor(s) is higher than 3,500 Da (Supplemen- determine molecular mass and amino acid sequence in sequential tary Fig. S1A). The biologically active dialyzed lung-derived scans. The data were analyzed using the Sequest search algorithm factors were separated by reverse-phase HPLC to numerous against the Mouse International Protein Index (IPI). fractions (Fig. 1A). These fractions were incubated with micrometastatic (MicroNB) and macrometastatic (MacroNB) neuro- HPLC separation of native human hemoglobin blastoma cells for 72 hours (Supplementary Fig. S1B). An MTS- Native human hemoglobin was dissolved in Milli-Q purified based viability assay indicated that one of the distinct separated water (EMD Millipore) to a concentration of 1 mg/mL and filtered peaks inhibited the viability of the cells by 25%–50% (P < (0.45 mm). Human hemoglobin was then chromatographed by 0.05; Fig. 1B) to the same extent as unseparated lung-derived Alliance RP-HPLC system (Waters) using Waters XBridge C18 factors (15). column (50 250 mm, 10 mm) running a gradient of 35% to 50% The active inhibitory fraction was subjected to high-resolu- acetonitrile (Thermo Fisher Scientific) in water containing 0.1% tion purification using reverse-phase HPLC. This resulted in a trifluoroacetic acid (Halocarbon) at a flow rate of 40 mL per single, narrow, and symmetric peak (Fig. 1C) representing, minute. The separated alpha and beta subunits of hemoglobin most probably, a single factor. ESI-MS analysis confirmed that (HBA and HBB, respectively) were collected and purified by the purified fraction was indeed a single factor of a molecular reverse-phase HPLC and their molecular masses were verified by mass of 15,824.5 Da (Fig. 1C). This fraction reduced the electrospray ionization mass spectrometry (ESI-MS). viability of MicroNB and MacroNB cells by 65% (P < 0.01) and 35% (P < 0.05), respectively (Fig. 1D). Sequence analysis Solid-phase synthesis of Metox and scrambled-Metox peptides by LC/MS-MS coupled with tryptic digestion followed by a The inhibitory human HBB peptide (ENFRLLGNVLVCVLA) database search in the International Protein Index (IPI), iden- fi designated Metox, and a control peptide of a scrambled amino ti ed the fraction as mouse hemoglobin subunit beta-2 acid sequence (ANVLNECVFVGRLLL) designated scrambled- (HBB2), a protein with 147 amino acid residues (Fig. 1E). Metox, were chemically synthesized. The synthesis was performed N-terminal Edman degradation (23) of the isolated factor fi on appropriate PAM resins (Applied Biosystems) on an 433A further veri ed its sequence identity. peptide synthesizer (Applied Biosystems) using an optimized The inhibitory activity of the isolated factors against MicroNB HBTU (Oakwood Chemical) activation/DIEA (Sigma-Aldrich) in and MacroNB cells could be effectively neutralized by anti-mouse situ neutralization protocol for Boc solid-phase peptide synthesis HBB2 antibodies, validating HBB2 as the inhibitory factor in (22). After chain assembly, the peptides were cleaved by anhy- mouse lungs (Fig. 1F). The inhibitory activity was not affected drous hydrogen fluoride (Airgas) in the presence of 5% p-cresol by an isotype control (Fig. 1F). (Sigma-Aldrich) at 0C for 1 hour, followed by precipitation with cold ether. The Metox and scrambled-Metox peptides were puri- Elevated levels of HBB2 alert to the presence of micrometastases fied by reverse-phase HPLC, and their molecular masses were MicroNB cells were orthotopically inoculated to the adrenal ascertained by ESI-MS. gland of athymic nude mice generating local tumors. The

OF2 Cancer Res; 77(1) January 1, 2017 Cancer Research

HBB2/HBB Inhibits Neuroblastoma Formation and Metastasis

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 and analysis by electrospray ionization mass spectrometry yielded a molecular mass of 15,824.5 Da. D, An MTS-based viability assay verified the inhibitory activity of the HPLC-purified fraction. (Continued on the following page.) www.aacrjournals.org Cancer Res; 77(1) January 1, 2017 OF3

Maman et al.

control group was injected with PBS. Quantitative real-time similar cells from control, normal mice, single-cell suspensions PCR (qRT-PCR) analyses performed 8 weeks after the intra- were prepared from lung tissues of control and micrometastasis- adrenal inoculation of the tumor cells revealed the presence bearing mice using the GentleMACS dissociator (25). Lung cells of micrometastatic human neuroblastoma cells in lungs, were separated using magnetic-activated cell sorting (MACS) to bone marrow, and liver of the inoculated mice (Supplementary isolate immune, epithelial, and endothelial cells using corre- Fig. S2). At this point, there was no evidence of overt metastasis. spondingly the lineage cell markers CD45, CD326, and CD31 qRT-PCR analyses indicated that the level of mouse HBB2 (26). Flow cytometry analysis was performed to verify the efficacy mRNA (Supplementary Table S1) was 25 times higher in lungs of the separation procedure (Fig. 3B). Confocal microscopy of micrometastasis-bearing mice than in lungs of normal mice confirmed the expression of HBB2 protein in the sorted epithelial (P < 0.001; Fig. 2A). Levels of the alpha subunit of hemoglobin and endothelial cells (Fig. 3C). qRT-PCR analyses of the separated (HBA) mRNA were low and similar in the two groups of mice cell populations indicated that indeed HBB2 mRNA is produced (Fig.2A).Immunofluorescence analyses of frozen lung sections by pulmonary epithelial and endothelial cells and not by pul- stained with anti-mouse HBB2 antibody revealed a higher monary immunocytes (Fig. 3D). HBB2 mRNA expression was 30 expression of HBB2 in lungs harboring neuroblastoma micro- times higher in pulmonary epithelial cells of micrometastasis- metastases than in normal lungs (Fig. 2B). In these lungs, an bearing mice than in control mice (P < 0.001). HBB2 mRNA intracellular expression of HBB2 in cells lining the alveoli was expression was 5 times higher in pulmonary endothelial cells of observed (Fig. 2B). The higher expression of HBB2 in lungs micrometastasis-bearing mice than in pulmonary endothelial harboring micrometastases was verified by Western blot anal- cells of normal mice (P < 0.05; Fig. 3D). An additional HBB2 ysis of lung tissue lysates (P < 0.005; Fig. 2C). HBB2 expression mRNA–expressing pulmonary cell population (CD45/CD31/ was also significantly higher in liver (P < 0.01) and bone CD326-negative cells, possibly hematopoietic stem cells) was marrow (P < 0.05) of micrometastasis-bearing mice than in identified to express HBB2 mRNA. However, HBB2 expression liver and bone marrow of control normal mice (Fig. 2C). HBB2 by these cells was only 2 times higher in cells derived from concentration in the serum of micrometastasis-bearing mice micrometastasis-bearing mice compared with control mice (data was7timeshigher(P < 0.005) than its concentration in the not shown). serum of normal mice (Fig. 2D). The serum concentration of Expression of the alpha subunit of hemoglobin, HBA, was also the alpha subunit, HBA, was very low and was similar in seen in pulmonary epithelial and endothelial cells; however, there normal control mice and in micrometastasis-bearing mice (Fig. was no significant difference in HBA expression in the pulmonary 2D). Similarly there was no significant difference between the cell populations derived from control and micrometastasis-bear- concentration of the whole, intact hemoglobin protein in the ing mice (Fig. 3D). serum of normal and micrometastasis-bearing mice (Fig. 2D). We next asked whether the elevated expression of HBB2 in This result excludes the possibility that the high concentrations pulmonary cells of micrometastasis-bearing mice is mediated by a of HBB2 in serum of tumor bearers were due to hemolysis. It is direct interaction between these cells or their soluble products and not unlikely that the lungs are an important contributor to the micrometastatic neuroblastoma cells residing in the lungs. To elevated HBB2 serum levels, as the most significant elevation in answer this question, we cocultured MicroNB cells with human the expression of HBB2 in micrometastasis-bearing organs was pulmonary endothelial cells or with human pulmonary fibro- in the lungs (Fig. 2C; Supplementary Fig. S2). However, other blasts in a Transwell system. Following the coincubation, total micrometastasis-bearing organs such as bone marrow and liver RNA was isolated from the endothelial cells and fibroblasts and a (Fig. 2C; Supplementary Fig. S2) do contribute as well, to the qRT-PCR was performed to examine human HBB expression in increased HBB2 protein levels in the serum. these cells. Expression levels of HBB mRNA were 8 times higher (P < 0.005) in the endothelial cells cocultured with soluble factors Alveolar epithelial cells are the main source of HBB2 from MicroNB cells than in control cells (Fig. 3E). The expression We next set out to identify the HBB2-producing lung cells and of HBB mRNA was not altered in human pulmonary fibroblasts in particular those cells in whom transcription is upregulated cocultured with MicroNB cells (Fig. 3E). Expression levels of the in micrometastasis-bearing mice (Fig. 2A). It was previously mRNA of human hemoglobin alpha chain, HBA, remained reported that HBB2 is synthesized by pulmonary epithelial cells unchanged in endothelial cells cocultured with MicroNB cells (24). Confirming these results, we found that such cells do indeed (Fig. 3E). express HBB2. A higher expression of HBB2 was seen in epithelial The data summarized in this section clearly demonstrate that cells lining the alveoli in lung sections of micrometastasis-bearing pulmonary epithelial cells and to a lesser degree pulmonary mice than in alveolar epithelial cells from normal mice (Fig. 3A; endothelial cells are an important source for the levels of HBB2 Supplementary Fig. S3). A higher expression of HBB2 was also in the lungs of nude mice bearing human neuroblastoma seen in endothelial cells lining blood vessels of micrometastasis- micrometastases. Moreover, the results of the in vitro experi- bearing mice than in endothelial cells of control mice (Fig. 3A; ments demonstrate that neuroblastoma-derived soluble factors Supplementary Figs. S3 and S4). are capable to stimulate pulmonary endothelial cells to selec- To verify that epithelial and endothelial cells from microme- tively upregulate the transcription of the hemoglobin beta tastasis-bearing mice indeed produce higher levels of HBB2 than chain.

(Continued.) E, Sequence analysis of the inhibitory fraction by 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 þ SD. Significance was evaluated using Student t test. , P < 0.05; , P < 0.01; , P < 0.005.

OF4 Cancer Res; 77(1) January 1, 2017 Cancer Research

HBB2/HBB Inhibits Neuroblastoma Formation and Metastasis

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). Top, scale bar, 50 mm; 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 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 ELISA. Data are means of mice in each group (n ¼ 18, 9 mice in each group) þSD. Significance was evaluated using Student t test. , P < 0.05; , P < 0.01; , P < 0.005; , P < 0.001.

Human HBB inhibits the viability of neuroblastoma cells strikingly similar to the viability-inhibitory functions mediated by The next set of experiments was aimed to find out if similarly to mouse lung-derived factors (15) and by lung-derived mouse mouse HBB2, the beta subunit of human hemoglobin (HBB) HBB2 (Fig. 1B and D). The HBB-mediated inhibitory activity was would also inhibit the viability of human neuroblastoma cells. dose dependent (Supplementary Fig. S5D). Intact human hemo- Native human hemoglobin was subjected to separation by globin inhibited the viability of neuroblastoma cells, but not to reverse-phase HPLC, during which the alpha and beta subunits the same extent as its beta subunit (Fig. 4B). of hemoglobin were fully separated (Supplementary Fig. S5B) and purified. The separation was verified by mass spectrometry anal- The growth-restraining activity spectrum of human HBB ysis (Fig. 4A; Supplementary Fig. S5C). Human HBB was found to be inhibitory against several addi- Whereas the alpha subunit (HBA) did not influence neuroblas- tional cancer cell lines (Table 1). The lung carcinoma cell line toma cell viability, incubation with 100 mg/mL human HBB A549 and the melanoma cell line RALL were inhibited by all three decreased the viability of MicroNB cells by 62% (P < 0.005) and doses of HBB used (1, 10, and 100 mg). The highest amount of of MacroNB cells by 39% (P < 0.01; Fig. 4B). These results were HBB (100 mg), reduced the viability of the breast cancer cell lines

www.aacrjournals.org Cancer Res; 77(1) January 1, 2017 OF5

Maman et al.

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). 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, 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. 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 magnetic-activated cell sorting separation from pulmonary single-cell suspensions of normal and micrometastasis-bearing mice. Scale bar, 7.5 mm. 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 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) after incubation with MicroNB cells in a Transwell system that enables the passage of soluble factors between the cocultured cells. Data are means of mice in each group (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.

OF6 Cancer Res; 77(1) January 1, 2017 Cancer Research

HBB2/HBB Inhibits Neuroblastoma Formation and Metastasis

Figure 4. Human HBB inhibits neuroblastoma cell viability by inducing apoptosis and growth arrest. A, Reverse-phase HPLC separation of native human hemoglobin resulted 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 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 b-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 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 cervical (Table 1). HBB did not cause hemolysis of human erythrocytes cancer cell line Hela, and the melanoma cell line RKTJ. These (Supplementary Fig. S6). results demonstrate that the HBB-mediated inhibition of viability is not restricted to neuroblastoma cells. HBB mediates apoptosis of and cell-cycle arrest in Human HBB did not influence the viability of the normal neuroblastoma cells (transformed) cell lines HEK293T and human pulmonary endo- Flow cytometry analysis of Annexin-V and PI apoptotic test thelial cells, nor did it influence the viability of the normal indicated that in HBB-treated MicroNB and MacroNB cells, the (nontransformed) human foreskin and pulmonary fibroblasts percentage of cells in early apoptosis and late apoptosis was

www.aacrjournals.org Cancer Res; 77(1) January 1, 2017 OF7

Maman et al.

Table 1. The spectrum of human cancer cells inhibited by human HBB % Difference % Difference % Difference in cell viability in cell viability in cell viability Tumor type Cell line (1 mg HBB) (10 mg HBB) (100 mg HBB) Breast MDA-231 No change þ5% 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 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 change Normal (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 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 t test, P < 0.05). Abbreviations: HPEC, human pulmonary endothelial cells; HPF, human pulmonary fibroblasts.

increased by 17% (P < 0.01) and 13% (P < 0.05), respectively, and "Metox"), significantly inhibited the viability of the cells by that the percentage of necrotic cells was very low and did not 23%–70% depending on its concentration (Fig. 5D). It is not change after treatment with HBB (Fig. 4C). However, the viability unlikely that the viability-inhibitory function of intact HBB is a of HBB-treated MicroNB and MacroNB cells decreased by 62% net balance between the growth-promoting functions mediated and 39%, respectively (Fig. 4B). Apoptosis and/or necrosis are by peptides 2, 3, and 8 and the growth-inhibitory functions of thus not the only mechanisms responsible for the decline in cell peptide 11. viability. Confocal microscopy suggested that FITC-labeled Metox is Flow cytometry analysis for cell-cycle progression revealed that internalized into MicroNB and MacroNB cells (Fig. 5E). Flow HBB increased the fraction of MicroNB and MacroNB cells in the cytometry indeed indicated membrane binding and cellular G0–G1 phase by 59% (P < 0.01) and 23% (P < 0.05), respectively uptake of FITC-labeled Metox after 30 minutes of incubation (Fig. 4D). The increased percentage of cells in the G0–G1 phase (Fig. 5F). The uptake was inhibited at 4 C, implying that the was accompanied by a decrease in cyclin D1 protein level (P < process depends on endocytosis (Fig. 5F). 0.005; Fig. 4E). In addition to apoptosis and growth arrest, HBB decreased ERK Metox inhibits local tumors and metastasis phosphorylation (P < 0.005) and increased p38 phosphorylation Athymic nude mice were orthotopically inoculated with (P < 0.005), creating a low ERK/p38 signaling ratio in the tumor MicroNB cells to the adrenal gland. Fourteen days after inocula- cells (Fig. 4F and G). The phosphorylation of TAK1 in HBB-treated tion, mice were treated by the intranasal route (27) once a week for cells was also increased (P < 0.01; Fig. 4H). 8 weeks with 15 mg/kg Metox or with the same amounts of a Taken together, these results indicate that human HBB induces control peptide having the identical amino acid composition as apoptosis and cell-cycle arrest in neuroblastoma cells, leading to Metox but in a scrambled sequence (Fig. 6A). growth arrest of the tumor. Twenty days after tumor cell inoculation, a difference (P < 0.05) was apparent in the local tumor volume between mice treated A short C-terminal region in human HBB mediates the growth with Metox and mice treated with scrambled-Metox (Fig. 6B). This arrest in neuroblastoma cells difference became more significant with time (Fig. 6B). Seventy To identify the functional region of human HBB responsible for days after tumor cell inoculation, the weight of local tumors the viability-inhibitory activity on tumor cells, we cleaved the resected from Metox-treated mice was 12 times lower (P < protein to N- and C-terminal fragments by cyanogen bromide 0.001) than that of local adrenal tumors resected from mice (Fig. 5A). MTS-based viability assays showed that the C-terminal treated similarly but with scrambled Metox. (Fig. 6C and D). fragment is responsible for the growth arrest activity (Fig. 5B). The qRT-PCR analyses indicated that the metastatic load of N-terminal fragment also exhibited an inhibitory effect, however, MicroNB cells was significantly lower (P < 0.001) in lungs and to a lower extent (Fig. 5B). bone marrow of mice treated with Metox compared with that HBB was then synthesized in 14 peptide segments of 15 found in organs derived from mice treated with scrambled-Metox amino acids each (Supplementary table S2). Each segment was (Fig. 6E and F). composed of 5 amino acids overlapping those of the preceding The results reported above showed that endogenous mouse segment and 5 amino acids overlapping those of the following HBB2 expression is upregulated in micrometastasis-bearing mice segment (Fig. 5C). Each of these segments was assayed for its (Fig. 2C; Supplementary Fig. S2). In accordance with these results, ability to block the viability of MicroNB cells. While peptides 2, the endogenous HBB2 mRNA expression levels in organs of mice 3, and 8 stimulated tumor viability, peptide 11 with the amino treated with scrambled-Metox (a treatment that does not reduce acid sequence ENFRLLGNVLVCVLA (designated hereafter tumor and metastasis load) were significantly higher (P < 0.01)

OF8 Cancer Res; 77(1) January 1, 2017 Cancer Research

HBB2/HBB Inhibits Neuroblastoma Formation and Metastasis

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 HPLC. 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 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 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 þ SD. Significance was evaluated using Student t test. , P < 0.05; , P < 0.01; , P < 0.005.

www.aacrjournals.org Cancer Res; 77(1) January 1, 2017 OF9

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 gland with neuroblastoma micrometastases. Fourteen days postinoculation, 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 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.

OF10 Cancer Res; 77(1) January 1, 2017 Cancer Research

HBB2/HBB Inhibits Neuroblastoma Formation and Metastasis

than its expression levels in organs of mice treated with Metox coculturing pulmonary endothelial cells with culture superna- (which reduces tumor and metastasis load; Fig. 6G and H). tants of tumor cells stimulated HBB synthesis by the former cells Similar results were obtained in an additional in vivo experi- but not by pulmonary fibroblasts. ment in which Metox was administered either by the intranasal What drives the upregulation of HBB2 in micrometastasis route or intravenously to MicroNB cell–inoculated mice (Sup- bearing mice? First we demonstrated that the upregulation of plementary Fig. S7). Both forms of Metox administration inhib- HBB2 is transcriptional and occurs at the mRNA level as well as at ited the growth of local adrenal tumors and of lung and bone the protein level. We then experimentally excluded the possibility marrow metastasis; however, intranasal administration of Metox that hemolysis occurs in nude mice bearing human neuroblas- was more effective. toma 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, Discussion and nitrate were similar in the lungs and serum of normal and The current study is the first to report that the b subunit of micrometastasis-bearing mice (Supplementary Fig. S8), we also hemoglobin belongs to the group of moonlighting proteins that excluded the possibility that free hemoglobin sequestered nitric are capable of performing multiple physiologic functions (24). In oxide, depleting its amounts and causing endothelial dysfunction addition to its oxygen transport functions, HBB also exhibits an (41). On the other hand and as indicated above, we provided antitumor reactivity and as such joins the arsenal of innate evidence that the upregulation of HBB2 is triggered in pulmonary resistance factors such as defensins (28, 29) that regulate cancer cells by tumor-derived factors. progression. Interestingly, a novel function was also recently The adaptive upregulated expression of HBB2 in tumor-bearing reported for the a subunit of hemoglobin in regulation of the mice suggests that this protein may alert for danger signals effects of nitric oxide in non-erythroid cells (30). delivered by invading foreign cells such as microorganisms or In patients with neuroblastoma, lung metastasis is a relatively cancer cells sharing patterns that are recognized by HBB2 (46). late event (31). This delay may be caused by the inhibitory The in vivo experiments performed in this study indicate that the function of the lung-derived HBB restraining the further progres- upregulated levels of HBB2 in tumor and metastasis bearers are sion of lung-residing micrometastases. Overt neuroblastoma lung insufficient to eradicate micrometastatic tumor cells and that an metastasis may develop if a subset of such lung-residing cells exogenous administration of HBB2 or of its derivative Metox is develops resistance to HBB or if the expression of HBB is down- needed to efficiently halt metastasis formation. regulated. The latter possibility is supported by an Oncomine The elucidation of the mechanism underlying the growth- meta-analysis (32) of gene expression profiling accumulated by restraining activity of HBB and its derived Metox peptide is several research groups (33–37), demonstrating that a 17- to 40- outside the scope of the current study. We do speculate, however, fold decrease in HBB expression occurred in overt lung metastatic that the presence of HBB2 at the apical surface of endothelial and lesions as compared with its expression in normal lung tissues. epithelial cells may indicate that HBB2 is secreted from these cells. In addition to neuroblastoma cells, other tumor cells are The fact that proliferation inhibitory and proapoptotic signaling sensitive to the proliferation-restraining function of HBB. Breast were activated in tumor cells by HBB2 also supports the sugges- cancer, lung cancer, and melanoma are among the sensitive cancer tion that this HBB2/HBB-mediated signaling is activated by types. However, different tumors belonging to a certain cancer secreted forms of these proteins. The findings that HBB induces type display a heterogeneous response to the growth-retaining both apoptosis and cell-cycle arrest of tumor cells, that the HBB- function of HBB; only 2 of the 5 breast cancer cell lines tested were derived Metox binds the outer membrane and is internalized into sensitive. Ongoing experiments are aimed to identity the com- the tumor cells, and that HBB-activated TAK1 and P38, down- mon factor that confers HBB sensitivity to the growth-restraining regulated ERK phosphorylation, and Cyclin D1 stability serve as function of this protein upon different types of tumor cells. basis for a working hypothesis as to its mode of action. We The bearing of a local neuroblastoma tumor and of microme- hypothesize that these growth arrest–inducing activities are medi- tastasis triggered an adaptive enhanced synthesis of HBB2 by ated by binding of soluble HBB2/HBB to a yet unidentified HBB pulmonary epithelial cells and to a lesser degree by pulmonary receptor. Such a receptor could facilitate the internalization of endothelial cells. An upregulated synthesis of HHB2 was also HBB and Metox into the target cells. detected in bone marrow and liver cells. Assuming that endothelial A low ERK/P38 phosphorylation ratio may lead to tumor cells in these organs are another source for circulating HBB2 and the dormancy (47). Although other signaling mechanisms that do fact that endothelial cells constitute a very large overall mass in the not induce dormancy may also act in conjunction with a low ERK/ body, these cells could play a significant role in resisting the p38 signaling ratio (48, 49), we hypothesize that in addition to propagation of neuroblastoma (and other tumors). Whereas the apoptosis, HBB induces tumor dormancy. Future work will con- synthesis of hemoglobin or its subunits by nonerythroid cells such firm or negate this hypothesis. as pulmonary epithelial cells, mesangial cells in the kidney and The current study provides proof of concept that microenvi- neurons in the brain was reported (24, 38–42), we are not aware of ronmental control, in the form of a naturally occurring protein, studies reporting the synthesis of the beta subunit by pulmonary HBB, exerts proliferation-restraining functions (apoptosis and endothelial cells. Interestingly, cathepsin proteases capable of cell-cycle arrest) on tumor cells; neuroblastoma being the case proteolytic degradation of both a-andb-globin are also expressed in point. Similar microenvironmental control mechanisms that by pulmonary epithelial cells, where these proteases are involved in block the proliferation of incipient cancer cells are postulated to post-translational processing of surfactant proteins (43–45). operate in healthy people (10, 50). The upregulated synthesis of HBB2 is apparently mediated by a The bioactive tumor-restraining region of HBB (ENFRLLGNV- direct contact between these host cells and soluble factors derived LVCVLA) was found to exert significant antitumor and anti- from the tumor cells; in vitro experiments demonstrated that metastasis activities both in vivo as well as in vitro. This peptide

www.aacrjournals.org Cancer Res; 77(1) January 1, 2017 OF11

Maman et al.

offers promising opportunities for the development of novel Acknowledgments therapies for the treatment of both primary as well as residual The authors thank Dr. Mickey Harlev and Dr. Maya Levin Arama (Animal disease. In addition, the inducible expression of HBB in the Care Facilities, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel), serum and organs of individuals harboring clinically undetect- Dr. Joe Bryant, and Dr. Eugene Ateh (Animal Core Facility, Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD) for the able metastasis could be exploited for the early detection of help with animal experiments. We also thank the W.M. Keck Biomedical Mass minimal residual disease preceding relapse. Spectrometry Laboratory at the University of Virginia Biomedical Research Facility for the MS and MS/MS analyses. Disclosure of Potential Conﬂicts of Interest Y. Keisari is a consultant/advisory board member for SURI Technologies. No potential conﬂicts of interest were disclosed by the other authors. Grant Support This work was supported by the NIH grant AI087423 (W. Lu), by the Authors' Contributions National Basic Research Program of China (973 Program) 2013CB932500 Conception and design: S. Maman, O. Sagi-Assif, Wuyuan Lu (W-Y. Lu), by the German Research Foundation (Deutche Forschungsge- Development of methodology: S. Maman, O. Sagi-Assif meinschaft DFG) grant BA4027/6-1 (I.P. Witz), by the James & Rita Leibman Acquisition of data (provided animals, acquired and managed patients, Endowment Fundfor Cancer Research (I.P. Witz), by the Fred August and Adele provided facilities, etc.): S. Maman, W. Yuan, T. Meshel, Weiyue Lu Wolpers Charitable Fund (I.P. Witz), and by the Sara and Natan Blutinger Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Foundation (I.P. Witz). computational analysis): S. Maman, O. Sagi-Assif, W. Yuan, R. Ginat, T. Meshel, The costs of publication of this article were defrayed in part by the Y. Keisari, W. Lu payment of page charges. This article must therefore be hereby marked advertisement Writing, review, and/or revision of the manuscript: S. Maman, O. Sagi-Assif, in accordance with 18 U.S.C. Section 1734 solely to indicate T. Meshel, Y. Keisari, Weiyue Lu, Wuyuan Lu this fact. Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Maman, W. Yuan, I. Zubrilov Received October 25, 2015; revised October 1, 2016; accepted October 21, Study supervision: O. Sagi-Assif, W. Lu, Wuyuan Lu 2016; published OnlineFirst October 28, 2016.

References 1. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 16. Edry Botzer L, Maman S, Sagi-Assif O, Meshel T, Nevo I, Bauerle T, et al. 2011;144:646–74. Lung-residing metastatic and dormant neuroblastoma cells. Am J Pathol 2. Quail DF, Joyce JA. Microenvironmental regulation of tumor progression 2011;179:524–36. and metastasis. Nat Med 2013;19:1423–37. 17. Nevo I, Sagi-Assif O, Edry Botzer L, Amar D, Maman S, Kariv N, et al. 3. Witz IP.Tumor-microenvironment interactions: dangerous liaisons. Adv Generation and characterization of novel local and metastatic human Cancer Res 2008;100:203–29. neuroblastoma variants. Neoplasia 2008;10:816–27. 4.Klein-GoldbergA,MamanS,WitzIP.Theroleplayedbythe 18. Pietsch T, Gottert E, Meese E, Blin N, Feickert HJ, Riehm H, et al. Char- microenvironment in site-specific metastasis. Cancer Lett 2014;352: acterization of a continuous cell line (MHH-NB-11) derived from 54–8. advanced neuroblastoma. Anticancer Res 1988;8:1329–33. 5. Maman S, Witz IP. The metastatic microenvironment. In: Shurin MR, 19. Biedler JL, Roffler-Tarlov S, Schachner M, Freedman LS. Multiple neuro- Umansky V, Malyguine A, editors. The tumor immunoenvironment. New transmitter synthesis by human neuroblastoma cell lines and clones. York, NY: Springer; 2013. p. 745. Cancer Res 1978;38:3751–7. 6. Hanahan D, Coussens LM. Accessories to the crime: functions of cells 20. Krump-Konvalinkova V, Bittinger F, Unger RE, Peters K, Lehr HA, Kirkpa- recruited to the tumor microenvironment. Cancer Cell 2012;21:309–22. trick CJ. Generation of human pulmonary microvascular endothelial cell 7. Whiteside TL.The tumor microenvironment and its role in promoting lines. Lab Invest 2001;81:1717–27. tumor growth. Oncogene 2008;27:5904–12. 21. Rosenfeld J, Capdevielle J, Guillemot JC, Ferrara P. In-gel digestion of 8. Spill F, Reynolds DS, Kamm RD, Zaman MH. Impact of the physical proteins for internal sequence analysis after one- or two-dimensional gel microenvironment on tumor progression and metastasis. Curr Opin Bio- electrophoresis. Anal Biochem 1992;203:173–9. technol 2016;40:41–48. 22. Schnolzer M, Alewood P, Jones A, Alewood D, Kent SB. Insitu neutralization 9. Zigrino P, Loffek S, Mauch C. Tumor-stroma interactions: their role in the in Boc-chemistry solid phase peptide synthesis. Rapid, high yield assembly control of tumor cell invasion. Biochimie 2005;87:321–8. of difficult sequences. Int J Peptide Protein Res 1992;40:180–93. 10. Bissell MJ, Hines WC. Why don't we get more cancer? A proposed role of the 23. Edman P.A method for the determination of amino acid sequence in microenvironment in restraining cancer progression. Nat Med 2011; peptides. Arch Biochem 1949;22:475. 17:320–9. 24. Newton DA, Rao KM, Dluhy RA, Baatz JE. Hemoglobin is expressed by 11. Bindea G, Mlecnik B, Fridman WH, Galon J. The prognostic impact of anti- alveolar epithelial cells. J Biol Chem 2006;281:5668–76. cancer immune response: a novel classification of cancer patients. Semin 25. Jungblut M, Oeltze K, Zehnter I, Hasselmann D, Bosio A. Standardized Immunopathol 2011;33:335–40. preparation of single-cell suspensions from mouse lung tissue using the 12. Granot Z, Henke E, Comen EA, King TA, Norton L, Benezra R. Tumor gentleMACS Dissociator. J Vis Exp 2009;29:1266. entrained neutrophils inhibit seeding in the premetastatic lung. Cancer 26. Bantikassegn A, Song X, Politi K. Isolation of epithelial, endothelial, and Cell 2011;20:300–14. immune cells from lungs of transgenic mice with oncogene-induced lung 13. Kammen BF, Matthay KK, Pacharn P, Gerbing R, Brasch RC, Gooding adenocarcinomas. Am J Respir Cell Mol Biol 2015;52:409–17. CA. Pulmonary metastases at diagnosis of neuroblastoma in pediatric 27. Rashid G, Ophir R, Pecht M, Lourie S, Meshorer A, Ben-Efraim S, et al. patients: CT findings and prognosis. AJR Am J Roentgenol 2001;176: Inhibition of murine Lewis lung carcinoma metastases by combined 755–9. chemotherapy and intranasal THF-gamma 2 immunotherapy. J Immun- 14. Smith MA, Seibel NL, Altekruse SF, Ries LA, Melbert DL, O'Leary M, et al. other Emphasis Tumor Immunol 1996;19:324–33. Outcomes for children and adolescents with cancer: challenges for the 28. Suarez-Carmona M, Hubert P, Delvenne P, Herfs M. Defensins: "Simple" twenty-first century. J Clin Oncol 2010;28:2625–34. antimicrobial peptides or broad-spectrum molecules? Cytokine Growth 15. Maman S, Edry-Botzer L, Sagi-Assif O, Meshel T, Yuan W, Lu W, et al. Factor Rev 2015;26:361–70. The metastatic microenvironment: lung-derived factors control the 29. Conrad DM, Hoskin DW, Liwski R, Naugler C. A re-examination of the role viability of neuroblastoma lung metastasis. Int J Cancer 2013;133: of the acute phase protein response in innate cancer defence. Med Hypoth- 2296–306. eses 2016;93:93–6.

OF12 Cancer Res; 77(1) January 1, 2017 Cancer Research

HBB2/HBB Inhibits Neuroblastoma Formation and Metastasis

30. Straub AC, Lohman AW, Billaud M, Johnstone SR, Dwyer ST, Lee MY, et al. 40. Skawran B, Dierich M, Steinemann D, Hohlfeld J, Haverich A, Schlegel- Endothelial cell expression of haemoglobin alpha regulates nitric oxide berger B, et al. Bronchial epithelial cells as a new source for differential signalling. Nature 2012;491:473–7. transcriptome analysis after lung transplantation. Eur J Cardiothorac Surg 31. Cowie F, Corbett R, Pinkerton CR. Lung involvement in neuroblastoma: 2009;36:715–21. incidence and characteristics. Med Pediatr Oncol 1997;28:429–32. 41. Nishi H, Inagi R, Kato H, Tanemoto M, Kojima I, Son D, et al. Hemoglobin 32. Rhodes DR, Yu J, Shanker K, Deshpande N, Varambally R, Ghosh D, et al. is expressed by mesangial cells and reduces oxidant stress. J Am Soc ONCOMINE: a cancer microarray database and integrated data-mining Nephrol 2008;19:1500–8. platform. Neoplasia 2004;6:1–6. 42. Richter F, Meurers BH, Zhu C, Medvedeva VP, Chesselet MF. Neurons 33. Bhattacharjee A, Richards WG, Staunton J, Li C, Monti S, Vasa P, et al. express hemoglobin alpha- and beta-chains in rat and human brains. J Classification of human lung carcinomas by mRNA expression profiling Comp Neurol 2009;515:538–47. reveals distinct adenocarcinoma subclasses. Proc Natl Acad Sci U S A 43. Fruitier I, Garreau I, Lacroix A, Cupo A, Piot JM. Proteolytic degradation of 2001;98:13790–5. hemoglobin by endogenous lysosomal proteases gives rise to bioactive 34. Beer DG, Kardia SL, Huang CC, Giordano TJ, Levin AM, Misek DE, et al. peptides: hemorphins. FEBS Lett 1999;447:81–6. Gene-expression profiles predict survival of patients with lung adenocar- 44. Weaver TE, Lin S, Bogucki B, Dey C. Processing of surfactant protein B cinoma. Nat Med 2002;8:816–24. proprotein by a cathepsin D-like protease. Am J Physiol 1992;263:L95–103. 35. Selamat SA, Chung BS, Girard L, Zhang W, Zhang Y, Campan M, et al. 45. Guttentag S, Robinson L, Zhang P, Brasch F, Buhling F, Beers M. Cysteine Genome-scale analysis of DNA methylation in lung adenocarcinoma protease activity is required for surfactant protein B processing and lamellar and integration with mRNA expression. Genome Res 2012;22: body genesis. Am J Respir Cell Mol Biol 2003;28:69–79. 1197–211. 46. Gambara G, De Cesaris P, De Nunzio C, Ziparo E, Tubaro A, Filippini A, 36. Wachi S, Yoneda K, Wu R. Interactome-transcriptome analysis reveals the et al. Toll-like receptors in prostate infection and cancer between bench and high centrality of genes differentially expressed in lung cancer tissues. bedside. J Cell Mol Med 2013;17:713–22. Bioinformatics 2005;21:4205–8. 47. Aguirre-Ghiso JA, Estrada Y, Liu D, Ossowski L. ERK(MAPK) activity as a 37. Stearman RS, Dwyer-Nield L, Zerbe L, Blaine SA, Chan Z, Bunn PAJr, et al. determinant of tumor growth and dormancy; regulation by p38(SAPK). Analysis of orthologous gene expression between human pulmonary Cancer Res 2003;63:1684–95. adenocarcinoma and a carcinogen-induced murine model. Am J Pathol 48. Hutchison MR.BDNF alters ERK/p38 MAPK activity ratios to promote 2005;167:1763–75. differentiation in growth plate chondrocytes. Mol Endocrinol 2012; 38. Ishikawa N, Ohlmeier S, Salmenkivi K, Myllarniemi M, Rahman I, Mazur 26:1406–16. W, et al. Hemoglobin alpha and beta are ubiquitous in the human lung, 49. Chaudhary SC, Kurundkar D, Elmets CA, Kopelovich L, Athar M. Metfor- decline in idiopathic pulmonary fibrosis but not in COPD. Respir Res min, an antidiabetic agent reduces growth of cutaneous squamous cell 2010;11:123. carcinoma by targeting mTOR signaling pathway. Photochem Photobiol 39. Grek CL, Newton DA, Spyropoulos DD, Baatz JE. Hypoxia up-regulates 2012;88:1149–56. expression of hemoglobin in alveolar epithelial cells. Am J Respir Cell Mol 50. Klein G, Imreh S, Zabarovsky ER. Why do we not all die of cancer at an early Biol 2011;44:439–47. age? Adv Cancer Res 2007;98:1–16.

www.aacrjournals.org Cancer Res; 77(1) January 1, 2017 OF13

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.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-15-2929

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2016/10/28/0008-5472.CAN-15-2929.DC1

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/early/2016/12/15/0008-5472.CAN-15-2929. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.