Differential Regulation of Hypoxia-Induced CXCR4 Triggering during B-Cell Development and...

Differential Regulation of Hypoxia-Induced CXCR4 Triggering

during B-Cell Development and Lymphomagenesis

Erich Piovan,1,3Valeria Tosello,

1Stefano Indraccolo,

2Massimo Masiero,

1Luca Persano,

1

Giovanni Esposito,2Rita Zamarchi,

2Maurilio Ponzoni,

4Luigi Chieco-Bianchi,

1

Riccardo Dalla-Favera,1,3and Alberto Amadori

1,2

1Oncology Section, Department of Oncology and Surgical Sciences, University of Padova; 2Istituto di Ricovero e Cura a Carattere Scientifico,Istituto Oncologico Veneto, Padua, Italy; 3Institute for Cancer Genetics and the Departments of Pathology and Genetics and Development,Columbia University, New York, New York; and 4Pathology Unit, San Raffaele Hospital Scientific Institute, Milan, Italy

Abstract

The chemokine receptor CXCR4 plays a central role in organ-specific homing and tumor spreading and is induced byhypoxia. B lymphocytes are exposed to low oxygen tensionsduring their development, but the influence of hypoxia ontheir physiology is poorly understood. Here, we show thathypoxia is associated with up-regulation of CXCR4 expressionin human normal and malignant B cells, through bothtranscriptional and posttranslational mechanisms. However,a dichotomic functional response to CXCR4 triggering wasobserved: both peripheral B cells and lymphomas arisingfrom mature B cells displayed increased responses to CXCR4triggering under hypoxia, whereas germinal center (GC) Bcells as well as GC-derived lymphomas showed CXCR4 receptordesensitization. This phenomenon was associated with differ-ential modulation of key signal-transducing molecules,including mitogen-activated protein kinase phosphatase-1and regulator of G protein signaling molecule-1. The unre-sponsiveness of GC-derived lymphomatous B cells to CXCR4triggering under hypoxia may have implications for thedevelopment and pathogenesis of GC-derived lymphoidtumors. [Cancer Res 2007;67(18):8605–14]

Introduction

Lymphocytes are exposed to low oxygen tensions as theydevelop and acquire effector functions (1). The degree of hypoxiahas been positively correlated with hypoxia-inducible factor 1(HIF-1; ref. 2). HIF-1 is composed of the oxygen- and growthfactor–regulated subunit HIF-1a, and the constitutively expressedHIF-1h subunit (3, 4). HIF-1a has been reported to be expressed ingerminal centers (GC) and in the majority of non–Hodgkin’slymphomas (5). Through the use of chimeric mice, it has beenreported that HIF-1a deficiency results in lineage-specific defectsof B lymphocytes, including defects in development of Blymphocytes and autoimmunity (6). The mechanisms by whichnormal and malignant B lymphocytes adapt and respond tohypoxia are poorly understood.

The presence of hypoxic areas is a common feature to malignanttumors, including lymphomas (7). Clinical studies have shown that

the low oxygen tension within a neoplastic lesion is anindependent indicator of poor outcome and correlates withincreased risk of metastasis (8–10). More than 60 target genesthat are activated by HIF-1 have been identified; these include thechemokine receptor CXCR4 (11) and its ligand stromal cell-derivedfactor-1 (SDF-1/CXCL12; refs. 12, 13). Because B-cell lymphomasoften express CXCR4, which plays a crucial role in numerousprocesses, among which tumor metastasis (14), and arise inlymphoid organs which constitutively produce CXCL12 (15, 16),an understanding of the regulatory role of hypoxia on CXCR4expression and function can provide insight into its role in normalB-cell development as well as into lymphomagenesis.

Here, we report that the function of CXCR4 is differentiallyregulated under hypoxic conditions in lymphomas of differentorigin. We observed CXCR4 desensitization in GC B cells and GC-derived lymphomas and showed that this phenomenon is due toHIF-1a–dependent up-regulation of signal transduction moleculessuch as mitogen-activated protein kinase (MAPK) phosphatase 1(MKP-1) and regulator of G protein signaling protein 1 (RGS1).

Materials and Methods

Cells and culture conditions. Hu/severe combined immunodeficient

(SCID) lymphomas were generated as reported (17, 18). The following

lymphoma cell lines were used: Burkitt’s lymphoma cell lines (BRG and

Ramos; from Dr. M. Ferrarini, National Cancer Research Institute, Genoa,Italy) and diffuse large B-cell lymphoma (DLBCL) cell lines OCI-Ly1, OCI-

Ly7, OCI-Ly8, OCI-Ly10, VAL, and SUDLH4. Hypoxic treatment of cells was

achieved by incubating cells in a PROOX model 110 chamber (Biospherix).

During incubation, 0.1% to 0.5% O2 was maintained. Cobalt chloride (CoCl2;Sigma) was used at a concentration of 100 Amol/L.

Details concerning cytofluorimetric analysis, RNA extraction, and reverse

transcription-PCR (RT-PCR), Northern and Western blot, confocal micros-copy, real-time PCR, CXCR4 compartmentalization, chemotaxis, gene

expression data, ELISA for extracellular signal-regulated kinase 1/2

(ERK1/2), statistical analysis, and B lymphocyte purification are provided

as Supplementary Materials and Methods.Lentiviral small interfering RNA cell lines. The lentiviral plasmids

containing HIF-1a small interfering RNA (siRNA) and LUC siRNA target

sequences (19) were a kind gift of Dr. O.V. Razorenova (Lerner Research

Institute, Cleveland, OH). The lentiviral vector was produced as described(20). Cells expressing the siRNA were selected in puromycin [siRNA (P)] or

sorted for enhanced green fluorescent protein [siRNA (E)].

Studies in lymphoma-bearing mice. SCID mice were purchased fromCharles River. Procedures involving animals and their care conformed with

institutional guidelines that comply with national and international laws

and policies (EEC Council Directive 86/609, OJ L 358, December 12, 1987).

Groups of 7- to 9-week-old SCID mice were injected i.p. with (a) 70 � 106 to100 � 106 peripheral blood mononuclear cells (PBMC) to generate hu/SCID

lymphomas or (b) 10 � 106 BRG lymphoma cells. Tumors and other tissues

of interest were processed as previously described (18). To identify hypoxic

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

Requests for reprints: Alberto Amadori, Department of Oncology and SurgicalSciences, Oncology Section, University of Padova, via Gattamelata 64, Padua I-35128,Italy. Phone: 39-049-8215891; Fax: 39-049-8072854; E-mail: [email protected].

I2007 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-06-4722

www.aacrjournals.org 8605 Cancer Res 2007; 67: (18). September 15, 2007

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tumor cells, we used pimonidazole hydrochloride (Hypoxyprobe-1, Chem-icon International; see Supplementary Materials and Methods).

Immunohistochemistry. Immunohistochemical analysis was done on

frozen or formalin-fixed, paraffin-embedded sections, with the following

primary antibodies: mouse anti–HIF-1a (ESEE 122, Novus Biologicals),rabbit anti–HIF-1a, rabbit anti–MKP-1, rabbit anti-RGS1 (Santa Cruz

Biotechnology), mouse anti-CD34 (Novocastra), mouse anti-CD3, mouse

anti-CD10, and mouse anti-IgD (Dakocytomation). Controls included

isotype-matched murine or rabbit antibodies of irrelevant specificity.

Results

Hypoxia-induced HIF-1A stabilization is associated withhigh CXCR4 expression in lymphoma cells in vivo . Little isknown on the expression of hypoxia in hematologic malignancies(5). To better understand the role of hypoxia in a B-cell lymphomasetting, B lymphomas of different origin, hu/SCID lymphomacells (originating from circulating B lymphocytes; ref. 21), and cellsof Burkitt’s-like lymphoma (BRG cell line; originated from GC Blymphocytes) were injected i.p. into SCID mice, and the tumormasses were studied for the expression of HIF-1a by immunohis-tochemistry. Hu/SCID lymphomas showed diffuse areas of necrosisin five of seven tumors analyzed, with viable perinecrotic cellsstaining for HIF-1a (Fig. 1A, left), indicating the presence of intensehypoxia. Occasionally, HIF-1a staining was diffusely present withinthe tumor, possibly indicating increased HIF-1a levels driven byoxygen-independent mechanisms (Fig. 1A, middle). On the otherhand, BRG-derived lymphomas showed areas of necrosis in onlyone of six tumors (not shown), with the bulk of the tumor massshowing scattered cells staining for HIF-1a, except for lymphomacells infiltrating adjacent tissues, where intense HIF-1a stainingwas found (Fig. 1A, right).

To better define the extent of hypoxia in our lymphoma models,we determined the presence of pimonidazole-labeled cells by flowcytometry as a surrogate marker of hypoxia. In 10 tumors examined,pimonidazole labeling was present in over 60% of hu/SCID lymphomacells (Fig. 1B), whereas BRG lymphomas showed lower expression ofpimonidazole adducts (<30% of the cells).

We recently showed that hu/SCID lymphomas are composed oftwo phenotypically distinct B-cell subsets (22): A CD23Plow

population that expresses high surface CXCR4 (CXCR4Pint) and aCD23Pint subset that barely expresses CXCR4 (CXCR4Plow; ref. 23).Double staining of tumor cells with anti-CXCR4 and antipimonida-zole antibodies revealed that the two hu/SCID lymphoma cellsubpopulations showed a substantially different staining forpimonidazole (Fig. 1C), with the CXCR4Pint subpopulation stainingmuch more intensely than cells not expressing CXCR4 (left). Similarresults were obtained in BRG lymphomas, where cells expressinghigher levels of surface CXCR4 stained more intensely forpimonidazole (Fig. 1C, right).

Hypoxia increases surface CXCR4 expression on B lympho-ma cells through transcriptional and posttranscriptionalmechanisms. The above data indicated that hypoxia correlateswith preferential expression of CXCR4 in our tumors. To gainfurther insight on the mechanisms underlying this association, hu/SCID lymphoma cells and cells of Burkitt’s-like lymphoma (BRGand Ramos cell lines) were exposed in vitro to severe hypoxia(<0.5%) or CoCl2, which mimics the effects of hypoxia by inducingHIF-1a stabilization (24), for 16 to 18 h. As shown in Fig. 2A ,both hypoxia and CoCl2 significantly increased surface expressionof CXCR4 in all the cell lines tested. Conversely, there was nosignificant increase in surface expression of other chemokine

receptors, such as CCR6, CCR7, CXCR3, and CXCR5 (not shown).This phenomenon was reversible upon reoxygenation, supportingthe causal role of hypoxia in regulating CXCR4 expression (Fig. 2B).

We also studied the effects of hypoxia on CXCR4 surfaceexpression in a panel of DLBCL cell lines (also originating fromGC B cells) representative of the three different gene expressionsubgroups recently described (25): GC B cell–like (GCB), activatedB cell–like (ABC), and unclassified or type III. Most DLBCL celllines showed a significant increase in surface expression of CXCR4under hypoxic conditions or after CoCl2 treatment, with GCBDLBCL cell lines showing a higher modulation compared with celllines of the other two subgroups (Supplementary Table S1).

It has been shown that in some cell types, CXCR4 expression canbe regulated by hypoxia through HIF-1 activation (11, 26). Thus,we investigated whether a transcriptional mechanism was alsoresponsible for hypoxia-induced CXCR4 up-regulation in ourlymphoma cells. As shown in Fig. 2C , CXCR4 mRNA, but not themRNA of other receptors evaluated, was significantly up-regulatedin response to hypoxia or CoCl2 in hu/SCID lymphomas and BRGlymphoma cells. Northern blot analysis showed a strong increase inCXCR4 mRNA as early as 4 h, and persisting after 18 h, afterincubation with CoCl2 (Fig. 2C, right) or hypoxia (not shown) inBRG lymphoma cells. These results, however, do not exclude thatincreased CXCR4 transcript stability during hypoxia may alsocontribute to higher CXCR4 gene expression.

In view of the possibility that surface CXCR4 expression couldalso be regulated by posttranscriptional mechanisms (16), such asreceptor redistribution, we compared the expression of CXCR4 inthe extracellular and intracellular compartments by flow cytometry(27). In normoxic conditions, CXCR4 was predominantly distrib-uted in the intracellular pool in both hu/SCID and BRG lymphomacells (Fig. 2D); conversely, exposure to hypoxia or CoCl2 determineda significant shift of the distribution of CXCR4 from the intra-cellular to the extracellular compartment. These results were alsoconfirmed by confocal microscopy (not shown).

Differential response to CXCR4 triggering under hypoxia inB-cell lymphomas of different cellular origin may reflect adevelopmental regulation. Hu/SCID lymphomas resemble immu-noblastic lymphomas arising from the expansion of the very fewEBV-infected B lymphocytes present in the injected PBMC (18, 28),representing post-GC B cells. To assess whether the hypoxia-associated changes observed in CXCR4 expression were alsoaccompanied by an effect on CXCR4 function and signaling, hu/SCID lymphoma cells were incubated in hypoxic conditions, andthe number of lymphoma cells migrating in response to CXCL12was recorded. As shown in Fig. 3A , the hypoxia-dependent increasein CXCR4 expression was accompanied by a significant rise in thenumber of hu/SCID lymphoma cells migrating in the presence ofa CXCL12 gradient. We next evaluated the effects of hypoxia onCXCR4-mediated chemotaxis in BRG and Ramos Burkitt’s lympho-ma cells, which are derived from GC centroblasts. Notably, despitetheir higher CXCR4 surface expression under hypoxic conditions,both cell lines displayed a significant inhibition of the migratoryresponse to CXCL12 (Fig. 3A).

A similar evaluation of the effects of hypoxia on CXCR4 functionin DLBCL cell lines disclosed a similar dichotomic functionalresponse (Fig. 3B), with GCB DLBCL cell lines (Ly7; left) displayinga significant inhibition of their chemotactic response to CXCL12under hypoxic conditions, while the non-GCB cell line Ly8showing an increased response (Fig. 3B, right) under the sameconditions.

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To determine whether this different response could be develop-mentally regulated, we investigated the functional response toCXCR4 triggering under hypoxic conditions in the normal counter-part of the lymphomatous cells; that is, GC B cells for Burkitt’slymphomas and DLBCL and peripheral blood B cells for hu/SCIDtumors. As shown in Fig. 3C (left), in analogy to hu/SCID lymphomacells, hypoxia significantly increased the number of peripheral Bcells migrating in the presence of a CXCL12 gradient. GC B cells(CD38+CD44�) instead were unresponsive to CXCL12 independent ofhypoxia, confirming previous results (Fig. 3C, right ; refs. 15, 29).

Using hu/SCID and BRG Burkitt’s lymphoma cells as a model, weevaluated whether distinct alterations of the CXCR4 signaling

pathway could be involved in determining the differentialfunctional responses of naıve/memory versus GC B cells. Thus,we examined the phosphorylation status of p42/p44 MAPK/ERK 1and 2, which are implicated in signal transduction (30, 31). Asshown in Fig. 3D , under normoxic conditions, phosphorylationof ERK1/2 slightly increased in hu/SCID lymphoma cells afterCXCL12 stimulation, whereas it underwent a dramatic increaseunder hypoxic conditions (Fig. 3D). Conversely, BRG cells displayeda marked increase of phosphorylated ERK1/2 after CXCL12stimulation under normoxic conditions, whereas no significantincrease was detectable under hypoxic conditions (Fig. 3D, right).These results indicate that the functional chemotactic response to

Figure 1. High CXCR4 expression in vivo correlates with hypoxia in B-cell lymphomas. A, tumor sections derived from experimental hu/SCID and BRG lymphomaswere analyzed by immunohistochemistry after staining for H&E and with anti–HIF-1a antibodies. N, necrosis; P, infiltrated pancreas; L, lymphoma; C, colon.Original magnifications are shown in each panel. B, tumor-bearing mice were exposed for 3 to 5 h to pimonidazole before sacrifice and lymphoma cells weresubsequently stained with antipimonidazole antibodies, and finally analyzed by flow cytometry. Results of five consecutive experiments. **, P < 0.001, significantreduction compared with hu/SCID lymphomas.MFI, mean fluorescence intensity. C, tumor-bearing mice were treated as above and lymphoma cells were subsequentlystained with antipimonidazole and anti-CXCR4 antibodies. Representative cytofluorimetric profiles of each lymphoma type. PE, phycoerythrin. The experimentwas repeated at least twice with similar results.

B-Cell Lymphoma Hypoxia-Induced CXCR4 Desensitization




CXCR4 triggering under hypoxic conditions in B cells correlateswith distinct alterations of the CXCR4 signaling pathway and thatthis response may be developmentally regulated, with peripheralB cells exhibiting increased CXCR4 function and GC B cellsundergoing CXCR4 desensitization.

Hypoxia-induced CXCR4 uncoupling in GC-derived lympho-ma cells is associated with up-regulation of MKP-1 and RGSproteins. RGS proteins increase the intrinsic GTPase activity of Gasubunits (32) and can thus modulate chemokine responses. Inaddition, MKP-1 attenuates p44/p42 MAPK activity, which isrequired for chemokine-mediated activation and migration (33).Both mechanisms could be at play in hypoxia-mediated CXCR4desensitization in GC-derived lymphoma cells. Of the RGS proteins

evaluated by RT-PCR, after incubation under hypoxic conditions, aconsistent increase was observed only for RGS1 transcript in bothBRG and Ramos cell lines (Fig. 4A), whereas no significant up-modulation of any RGS transcript was found in hu/SCIDlymphomas (not shown). Increased expression of RGS1 wasconfirmed by Western blotting in both cell lines (Fig. 4B), whereasno increased protein levels were recorded for hu/SCID lymphomas(not shown). Similar results were obtained for MKP-1, where asubstantial increase was found by both quantitativeRT-PCR and Western blotting in the Burkitt’s cell lines BRG andRamos (Fig. 4C and D , respectively). A similar evaluation of theeffects of hypoxia or CoCl2 on RGS1 and MKP-1 expression inDLBCL cell lines disclosed how only GCB DLBCL cell lines (Ly1 and

Figure 2. Hypoxia up-regulates CXCR4expression in human B-cell lymphomasby transcriptional and posttranscriptionalmechanisms. A, hu/SCID, BRG, andRamos lymphoma cells were cultured for18 h in normoxia (Norm ) in the absenceor presence of CoCl2 or hypoxic (Hyp )conditions and analyzed for CXCR4surface expression by flow cytometry.Results of four consecutive experimentsare summarized after conversion ofCXCR4 mean fluorescence intensity interms of molecules of equivalent solublefluorochrome (MESF ). *, P < 0.001,significant increase compared withnormoxia. B, Burkitt’s lymphoma cells,BRG, were cultured for 18 h in hypoxicconditions and subsequently reexposedto normoxia for the indicated times.CXCR4 expression was determined byflow cytometry. C, hu/SCID and BRGlymphoma cells were cultured in the aboveconditions, and changes in chemokinereceptor gene expression were determinedby quantitative PCR. Fold changerepresents increases in chemokinereceptor expression under hypoxia orCoCl2 conditions compared with normoxiacontrols. Columns, mean of threeconsecutive experiments; bars, SD.*, P < 0.05, significant increase comparedwith normoxia. Extreme right panel, BRGlymphoma cells were cultured for differenttimes in normoxia in the absence orpresence of CoCl2, as indicated. TotalRNA was analyzed by Northern blot forCXCR4 mRNA expression. D, thedistribution of CXCR4 in intracellular versuscell surface compartments was assessedby flow cytometric quantification in cellscultured under the indicated conditions.Columns, mean of three consecutiveexperiments; bars, SD. *, P < 0.05,significant increase compared withnormoxia.

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Ly7) but not ABC (Ly10) or type III (unclassified; Ly8) DLBCL celllines displayed a significant increase in their expression levels(Supplementary Fig. S2).

Numerous other mechanisms of receptor desensitization havebeen described (34). We found that hypoxia or CoCl2 treatment didnot modulate the expression of CD26 and CD45 (not shown),transforming growth factor-h1 and its receptor, or members of theh-arrestin family (ARRB1, ARRB2, or ARRB3) shown to be involvedin receptor desensitization in other models (Supplementary Fig. S1).Also, phosphorylation of G protein–coupled receptors is thought tobe essential for their ligand-induced endocytosis. We found thatCXCR4 exhibited a low level of tyrosine phosphorylation in Burkitt’slymphoma cells (BRG) cultured under normoxic conditions, whichdid not appreciably change after hypoxic exposure (not shown). Thus,MKP-1 and RGS1 proteins are the main determinants of CXCR4desensitization in GC-derived lymphoma cells, although we cannotexclude that additional mechanisms may be responsible for theobserved receptor desensitization in GC-derived lymphoma cells.

Silencing of HIF-1A reverses hypoxia-induced CXCR4 up-regulation and prevents receptor uncoupling in Burkitt’s-likelymphoma cells. Having established that hypoxia is associatedwith CXCR4 desensitization through complex alterations of thesignaling pathways downstream of the CXCR4 receptor, weaddressed whether HIF-1 plays a major role in hypoxia-associatedmodulation of CXCR4 expression and function by investigating theeffects of its functional knockdown. To this end, we used lentiviralvectors expressing a siRNA specific for HIF-1a (>70% knockdownas determined by quantitative RT-PCR and Western blot; Fig. 5A),and investigated the effects of HIF-1a siRNA on CXCR4 expressionand function under hypoxic conditions in BRG lymphoma cells. Asshown in Fig. 5B , BRG cells stably expressing HIF-1a siRNA showedan important inhibition of CXCR4 up-regulation under hypoxicconditions as evaluated by quantitative flow cytometry comparedwith control cells. Similarly, by quantitative RT-PCR, there was asignificant inhibition of CXCR4 transcript induction by hypoxia(not shown) or CoCl2 in HIF-1a knockout BRG cells (Fig. 5B, right).

Figure 3. Hypoxia induces a dichotomic functional response to CXCR4 triggering in normal and malignant B cells. A, lymphoma cells were cultured for 18 h in normoxicor hypoxic conditions. Migration of cells was then assayed by chemotaxis microchamber technique. Columns, mean of at least three experiments; bars, SD. *, P < 0.05,significant increase compared with cells cultured in normoxic conditions; **, P < 0.05, significant decrease compared with cells cultured in normoxic conditions.B, DLBCL lymphoma cells were cultured for 18 h in normoxic conditions or exposed to CoCl2 or hypoxic conditions. Migration of cells was then assayed by chemotaxismicrochamber technique. Columns, mean of at least three experiments; bars, SD. *, P < 0.05, significant increase compared with cells cultured in normoxicconditions; **, P < 0.05, significant decrease compared with cells cultured in normoxic conditions. C, peripheral blood B cells or human GC B cells were separatedand evaluated for CXCL12-induced migratory responsiveness under normoxic and hypoxic conditions. Columns, mean of at least three experiments; bars, SD.*, P < 0.05, significant increase compared with cells cultured in normoxic conditions; **, P < 0.05, significant decrease compared with cells cultured in normoxicconditions. D, serum-starved lymphoma cells were incubated for 18 h under normoxic or hypoxic conditions and then stimulated with CXCL12 (100 ng/mL) for differenttime intervals before evaluation of the activation status of MAPK by ELISA. One representative experiment of three for each lymphoma type.





This inhibition of CXCR4 modulation under hypoxic conditionswas associated with a substantial protection from hypoxia-inducedCXCR4 uncoupling (Fig. 5C), with HIF-1a knockout BRG cellsmaintaining their responsiveness to CXCL12 under hypoxicconditions. To evaluate whether the induction of RGS1 and MKP-1 could play a role in hypoxia-induced CXCR4 desensitization, wealso determined by quantitative RT-PCR the effect of HIF-1aknockdown on RGS1 and MKP-1 expression under hypoxicconditions. BRG cells stably expressing HIF-1a siRNA, in contrastto control cells, showed no significant increase in RGS1 and MKP-1expression after CoCl2 treatment (Fig. 5D) or hypoxic exposure (notshown). These findings were also confirmed at protein level byconfocal microscopy of MKP-1 expression (Fig. 5D) and RGS1 (notshown). These data suggest that RGS1 and MKP-1 are downstreamtargets of HIF-1a and that this latter is required for hypoxia-induced CXCR4 up-regulation and receptor uncoupling in Burkitt’s-like lymphoma cells.

Expression of HIF-1A and downstream molecules in thenormal counterpart of Burkitt’s-like lymphomas and primaryGC-derived B-cell lymphomas. As previously mentioned, humanGC B cells have been shown to have high CXCR4 expression and yetrespond poorly to chemokines (15, 29); because Burkitt’s lympho-mas and DLBCL are presumed to arise from GC B cells and wehave shown that under hypoxic conditions these cells undergoCXCR4 desensitization, we were interested in studying the

expression of HIF-1a and the downstream molecules MKP-1 andRGS1 in these samples. As shown in Fig. 6A , HIF-1a seemed to bepredominantly expressed in tonsil GCs, given the good overlap ofHIF-1a staining with the GC B-cell marker CD10. This result wasconfirmed at higher magnification (Fig. 6B, inset) where thestaining for HIF-1a was found to be predominantly within GCs likeCD10 staining and unlike IgD staining, which outlines GCs (notshown). The largest portion of HIF-1a staining was found in thecytoplasmic compartment of GC B cells, with occasional nuclearsignal being detected. Interestingly, GCs were also found to beavascular, as shown by the lack of CD34 reactivity (Fig. 6A),whereas a rich vascular network was present outside of GCs,further suggesting that hypoxia is probably present within GCs. Onthe other hand, CXCR4 expression was found to be diffuselyexpressed both in GC and parafollicular areas. We next evaluatedthe expression of MKP-1 and RGS1 proteins in tonsil sections byimmunohistochemistry and immunofluorescence. As shown inFig. 6C , RGS1 and MKP-1 proteins were expressed predominantlywithin GCs, much like HIF-1a, but unlike IgD that stained mantlezone B cells. Confocal microscopic analysis of tonsil sectionsconfirmed these data, as the fluorescence distribution of RGS1 andMKP-1 (Fig. 6C, right) was found to be predominantly within GCs.Thus, the expression of MKP-1 and RGS1 in GCs, possibly inducedby HIF-1a, may indeed contribute to GC B-cell desensitization ofCXCR4.

Figure 4. Effect of hypoxia on MKP-1 and RGS1 proteinsin lymphoma cells. A, Burkitt’s-like lymphoma cells BRGand Ramos or hu/SCID lymphoma cells were cultured for18 h in normoxic or hypoxic conditions. Total RNA wasextracted and analyzed by semiquantitative RT-PCR forRGS1 mRNA expression. As a control for RNA loading,primers specific for h2-m were used. *, number of PCRcycles are different between samples (see SupplementaryMaterials and Methods). B, BRG and Ramos cells werecultured in normoxia or hypoxia, or incubated with CoCl2 for18 h. Cell lysates were then analyzed by immunoblottingfor RGS-1 expression. Anti–a-tubulin antibodies wereused as a control of protein loading. C, total RNA wasextracted from cells cultured under appropriate conditionsand analyzed by semiquantitative and quantitativeRT-PCR for MKP-1 mRNA expression. Columns, meanof three consecutive experiments; bars, SD. Left *, numberof PCR cycles are different between samples (seeSupplementary Materials and Methods). * (in graph ),P < 0.05, significant increase compared with normoxia.D, BRG, Ramos, and hu/SCID lymphoma cells werecultured in normoxia, hypoxia, or incubated with CoCl2 for18 h. Cell lysates were then analyzed by immunoblottingfor MKP-1 expression. Anti–a-tubulin antibodies wereused as a control of protein loading.

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Similar evaluation of the expression of CXCR4 and HIF-1a inhuman B-cell lymphomas of GC origin (DLBCL and Burkitt’slymphomas) and non-GC origin (marginal zone and mantle celllymphomas) disclosed how these two proteins were more highlyexpressed in GC-derived B-cell lymphomas (Fig. 6D ; SupplementaryFig. S3), suggesting that in these tumors HIF-1a expression maymodulate CXCR4 expression and function in vivo . In addition, theevaluation of the expression of the downstream molecules MKP-1and RGS1 in primary Burkitt’s lymphoma disclosed that the

expression of these molecules is rather diffuse. As shown in Fig. 6D(left), in most cases (9 of 10) HIF-1a staining was predominantlycytoplasmic with few cells showing nuclear staining, similarly toGC B cells; only rarely (1 of 10) was nuclear HIF-1a staining diffusethroughout the tumor mass, in the absence of evident necrosis(Fig. 6D, bottom left). The expression of MKP-1 was also ratherdiffuse (10 of 10; Fig. 6D, top middle), with a predominance in thecytoplasmic compartment, much like has been recently reported inother tumor types (35). On the other hand, RGS1 expression was

Figure 5. Silencing of HIF-1a reverses hypoxia-induced up-regulation of CXCR4, MKP-1, and RGS1 and prevents receptor uncoupling in Burkitt’s-like lymphomacells. A, BRG cells were transduced with HIF-1a siRNA vectors [HIF-1a siRNA (P) or HIF-1a siRNA (E)] or control vectors [LUC siRNA (P) or LUC siRNA (E)] andsubsequently evaluated for HIF-1a gene knockdown at the mRNA level by quantitative RT-PCR or at the protein level by Western blot after hypoxic exposure.Antinucleoporin p62 antibody was used as a control of protein loading. **, P < 0.05, significant decrease in HIF-1a gene expression in HIF-1a siRNA–transduced cellscompared with mock-transduced cells cultured in normoxic conditions. B, flow cytometric evaluation and quantitative RT-PCR evaluation of lentiviral HIF-1a siRNA– orcontrol LUC siRNA–transduced BRG cells for CXCR4 expression after exposure to hypoxia or CoCl2. Columns, mean of five experiments; bars, SD. **, P < 0.05,significant decrease compared with control-transduced cells in hypoxic conditions. C, transduced cells were cultured for 18 h in normoxic or hypoxic conditions.Migration of cells was then assayed by chemotaxis microchamber technique. *, P < 0.05, significant decrease compared with mock-transduced cells cultured in hypoxicconditions. D, transduced cells were cultured for 18 h in normoxia or in the presence of CoCl2 and analyzed by quantitative RT-PCR for vascular endothelialgrowth factor (top left , as control), MKP-1 (top middle ), and RGS1 mRNA expression (top right ).





diffuse and found to be prevalently nuclear in all cases analyzed

(10 of 10; Fig. 6D, bottom middle). CXCR4 expression was also found

to be diffuse, with predominant cytoplasmic staining (10 of 10;

Fig. 6D, right).These results show that the expression of the hypoxia-associated

marker HIF-1a and CXCR4 (together with RGS1 and MKP-1) are

diffusely present in GC-derived B-cell lymphomas and suggest that

the conditions for hypoxia-induced CXCR4 desensitization may be

present in vivo .

Discussion

CXCR4 is one of the most common chemokine receptorsexpressed by tumor cells. Previous reports have shown that lowoxygen concentrations can induce high expression of CXCR4 inmonocytes and epithelial-derived tumors (11) through theactivation and stabilization of HIF-1a.

Here, we showed that hypoxia mediates selective up-regulationof CXCR4 through stabilization of HIF-1a in normal B cells andmalignant B cells of different non–Hodgkin’s B-cell lymphomas

Figure 6. Expression of HIF-1a, RGS1, MKP-1, and CXCR4 within lymphoid GC and primary Burkitt’s-like lymphomas. A, serial tonsil sections were stained withantibodies against IgD, CD10, HIF-1a, CD34, and CXCR4. B, immunohistochemical staining documenting the predominant expression of HIF-1a within lymphoid GC.Inset, a higher magnification of HIF-1a staining in GC cells. A subset of GC cells show nuclear expression of HIF-1a (arrows in right ). C, immunohistochemicaland immunofluorescence analysis of tonsil sections evaluating the expression and distribution of RGS1 and MKP-1. Tissue sections were stained with antibodiesagainst HIF-1a, IgD, RGS1, or MKP-1 (green signals in confocal images ) and propidium iodide (red signal , only for confocal images). The latter was used to highlightcell nuclei. The fluorescence distribution of each marker (RGS1, MKP-1) is also depicted as a pseudocolored densitometric map with the strongest signal showingas red, a modest signal as yellow green, and its absence as blue. D, immunohistochemical staining documenting the heterogeneous expression patterns of HIF-1a,RGS1, MKP-1, and CXCR4 within primary Burkitt’s lymphomas. Top left, a tumor sample showing predominant cytoplasmic expression of HIF-1a; bottom left,a case with predominant nuclear HIF-1a expression. Insets, a higher magnification of HIF-1a staining in lymphoma cells. Middle top, the predominant cytoplasmicMKP-1 expression within lymphomas; middle bottom, predominant nuclear expression of RGS1 in the same samples. Right, membrane and cytoplasmic staining ofCXCR4 within lymphoma samples. Insets, a higher magnification of each marker studied. Original magnifications, �100 (A and C, left ); �200 (B, C middle panels ,and D); and �1,000 (B and D inset ).

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such as PBMC-derived hu/SCID lymphomas, GC B cell–derivedBurkitt’s, and DLBCL lymphoma cells. This receptor up-regulationpersisted under chronic hypoxia, a situation that may be closer towhat cancer cells are subjected to in the growing tumor mass, andmore importantly it was reversible under normoxic conditions.Further, we expand the knowledge on the mechanisms that may beresponsible for hypoxia-induced CXCR4 up-regulation. Indeed, weshowed that hypoxia is not only able to act at the transcriptionallevel but also at the posttranslational level through a process ofreceptor redistribution.

Responsiveness to CXCL12 correlates with positioning of B cellswithin a peripheral lymphoid organ and is regulated by thedifferentiation state and by B-cell receptor engagement (29). It hasbeen described that two rescue signals, such as antigen and CD40ligand, raise RGS1 and RGS13 expression levels, respectively, in Bcells (36, 37). Hypoxia, through modulation of CXCR4 expressionand of the regulatory molecules RGS1 and MKP-1, may contributeto help GC B cells to negotiate a complex chemokine milieu such asthat present in GC. Indeed, HIF-1a may participate in chemokinereceptor desensitization, thereby allowing the cell to desensitizeto a primary chemokine gradient and respond to a second one. Alikely site of conflicting chemoattractant gradients is the dark andlight zones of GC, where B cells have several alternative fates. Infact, CXCL12 amounts in the dark zone are higher than in theadjacent light zone (16). On the other hand, CXCL13 has adistribution in the GC that is opposite to that of CXCL12, beingmore abundant in the light zone than in the dark zone (16). HIF-1amay assist B cells in their navigation through the GC, modulatingtheir responsiveness to CXCL12.

Concerning the functional importance of hypoxia-induced CXCR4up-regulation in B cells, we found that lymphomas of differentmaturational stage (hu/SCID lymphomas originating from maturecirculating B cells and Burkitt’s/GCB-DLBCL lymphomas originatingfrom GC B cells) showed an opposite response to CXCR4 triggeringunder hypoxic conditions. Indeed, CXCR4 function was enhancedby hypoxia in hu/SCID lymphomas, as shown by increasedchemotactic responsiveness to CXCL12 and increased ERK1/2phosphorylation after CXCR4 triggering, whereas this receptorunderwent desensitization in hypoxic GC-derived lymphoma cells.

Several studies have shown that GC B cells respond poorly tochemokines, despite the expression of CXCR4. CXCR4 expressionand response of B cells within lymphoid organs is heterogeneous.Indeed, within human tonsils, IgD+CD38-negative B cells residingin the mantle zone (considered as naive B cells) and IgD�CD38–negative B cells (considered as memory B cells) express moderatelevels of CXCR4 but undergo efficient migration to CXCL12;meanwhile, GC B cells (CD38+ and IgD+ or IgD�), which expresshigh levels of CXCR4, are essentially refractory to CXCL12-inducedmigration (36). This refractoriness has been attributed, at least inpart, to high-level expression of RGS1 (36) and RGS13 (37). Also, Bcells from RGS1�/� mice migrate better to CXCL12 than do thosefrom wild-type mice (38). On the other hand, a recent study thatused GC B cells from Bcl-2 transgenic mice (which had extendedGC B cell survival) showed robust migratory responses to CXCL12and CXCL13 (16). The authors argued that the lack of responsive-ness of GC B cells to B-cell chemoattractants was caused by theirpoor in vitro viability, impeded by Bcl-2 overexpression. Analternative explanation (39) could be that Bcl-2 overexpressionalters GC B-cell selection, thus allowing B cells to survive in theabsence of normal rescue signals (such as antigen and CD40ligand), which normally lead to increased expression of RGS1 and

RGS13, thus leading to low levels of these RGS proteins andprobably other factors in the Bcl-2 transgenic GC B cells.

We find here that hypoxia determines induction/up-regulationof RGS1 in GC-derived lymphoma cells. We also showed in situexpression of RGS1 predominantly in GC and that GC are poorlyvascularized given the lack of CD34 staining; thus, it could betempting to speculate that hypoxia may be involved with highexpression of RGS1 in GC B cells, and thus contribute to their poorchemotactic response to CXCL12. The expression of RGS1 in GC Bcells and primary Burkitt’s lymphoma was unexpectedly found tobe predominantly nuclear. It has previously been reported that RGSproteins can localize in the nucleus, in the cytoplasm, or shuttlebetween the nucleus and cytoplasm (40).

Recently, it has been reported that hypoxia is able to inhibitmacrophage migration to numerous chemokines and N-formyl-Met-Leu-Phe, possibly through up-regulation of MKP-1 (41). Inagreement with this report, we found that hypoxia up-regulatedMKP-1 in GC-derived lymphoma cells. As phosphorylation andeventual activation of MAPKs are required for CXCL12-mediatedmigration (33), these results suggest that dephosphorylation ofMAPK by MPK-1 may be required for hypoxia-induced inhibition ofB lymphoma cell migration in response to CXCL12. We showedin situ expression of MKP-1 in GC, with a distribution similar toRGS1 and particularly to HIF-1a, thus providing evidence thathypoxia may contribute to CXCR4 desensitization in GC B cells,through expression of MKP-1.

Using immunochemical hypoxia marker techniques, we wereable to show a rather diffuse presence of hypoxia in experi-mental hu/SCID lymphomas and BRG Burkitt’s lymphomas.Nuclear HIF-1a expression in experimental GC B cell–derivedBRG Burkitt’s lymphomas was found predominantly in lymphomacells infiltrating surrounding tissues, implicating that thephenomenon of CXCR4 desensitization may be localized andrelevant when lymphoma cells acquire a more invasive phenotype.Primary GC-derived B-cell lymphoma samples and GC B cells hada more heterogeneous and variable expression of HIF-1a. In mostcases, the expression was predominantly cytoplasmic. Thefunction of cytoplasmic HIF-1a expression is not known, but asimilar pattern of expression has been described in T lymphocytesinfiltrating the synovial tissue of patients with rheumatoidarthritis (42). The unique aspects of HIF-1a expression in GC Bcells and GC-derived lymphomas (presence of HIF-1a both in thecytoplasm and nucleus) indicate the possible involvement ofoxygen-independent pathways of HIF-1a regulation.

Available gene expression data (Supplementary Fig. S4) fromLymphochip arrays done on lymphomas (25) showed that theexpression levels of HIF-1a, RGS1, and MKP-1 were higher in GC-derived lymphomas (DLBCL and follicular lymphomas) comparedwith chronic lymphocytic leukemia samples, and that theexpression levels of these three transcripts are related.

Lymphomas arise in lymphoid organs that constitutivelyproduce the CXCR4 ligand, CXCL12 (15, 16). In addition,lymphoma cells are able to produce CXCL12 themselves (14).The significance of CXCL12 expression at the primary site is notfully understood. By analogy with its role in the bone marrow,high levels of CXCL12 should retain the tumor cells in situ ratherthan encourage their dissemination. Thus, CXCR4 up-regulationby hypoxia in primary tumors expressing CXCL12 could beexpected to retain tumor cells at this site if functionality ofCXCR4 is maintained, which could encourage their growth andsurvival, but discourage invasion and metastasis. Our results





provide evidence that hypoxia is an important modulator ofCXCR4 responsiveness in normal and malignant B cells and thathypoxia-induced CXCR4 receptor uncoupling may represent anovel mechanism for guiding normal and malignant cellmigration/dissemination in sites of conflicting chemoattractantgradients; this phenomenon may be particularly relevant in earlystages of nodal lymphomas where the integrity of the lymphoidtissue is almost intact.

Acknowledgments

Received 12/21/2006; revised 7/9/2007; accepted 7/13/2007.

Grant support: Ministero dell’Istruzione, dell’Universita e della Ricerca (MIUR)-Fondo per gli Investimenti della Ricerca di Base and MIUR-Programmi di ricerca diRilevante Interesse Nazionale; Istituto Superiore di Sanita (AIDS Project); ItalianAssociation for Research on Cancer (AIRC), and Italian Foundation for Research onCancer; Ministero dell’Universita e Ricerca Scientifica e Tecnologica 60%; PaduaUniversity grants; Ministero della Salute, Ricerca Oncologica Project RF-IOV-2006-408212. Azione Biotec 2005-Regione Veneto. V. Tosello was a recipient of an AIRCfellowship.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Drs. P. Marson and F. Comacchio (Padua, Italy) for precious help inlymphapheresis and tonsil supply, respectively; Dr. O.V. Razorenova (Lerner ResearchInstitute, Cleveland, OH) for providing the pLSLG and pLSLP-HIF-1a/LUC-siRNAlentiviral plasmids; and Prof. N. Fujii (Kyoto University, Kyoto, Japan) for comments onthe manuscript.

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2007;67:8605-8614. Cancer Res Erich Piovan, Valeria Tosello, Stefano Indraccolo, et al. during B-Cell Development and LymphomagenesisDifferential Regulation of Hypoxia-Induced CXCR4 Triggering

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