The expression of HMGB1 protein and its receptor RAGE

The expression of HMGB1protein and its receptor RAGE in human malignant tumors

Nora Kostova ?Stanislava Zlateva ?

Iva Ugrinova ?Evdokia Pasheva

Received:12June 2009/Accepted:18October 2009/Published online:30October 2009óSpringer Science+Business Media,LLC.2009

Abstract High Mobility Group Box 1(HMGB1)is a nuclear non-histone protein discovered to be released in the extracellular medium as a response to various stimuli and implicated in cancerogenesis.High HMGB1levels are reported in a variety of tumor types,but there are few data relating HMGB1to the histological grade or to a particular cell type and cellular localization.We studied the expres-sion of HMGB1protein in malignant human tumors of different differentiation level and in tumor metastasis.In all tumor tissues,the protein level is elevated.In moder-ately differentiated carcinomas,the localization of the protein is perinuclear,while in the low differentiated;there is a tendency for non-speci?c nuclear localization.HMGB1protein and its receptor RAGE are identi?ed as a ligand–receptor pair that plays an important role in regulating the invasiveness of tumor cells.RAGE is not produced in all of the tested tumor specimens.We found high level of expression in hepatocellular,colorectal,and breast cribri-form carcinomas,but not in malignant testicular speci-mens.Probably,the RAGE synthesis is related to distinctive tumor types.In metastatic cells,RAGE exhibits higher level of expression losing its speci?c granular cytosolic pattern characteristic for the primary tumors.Keywords HMGB1protein á

Receptor for advanced glycation end products áCarcinoma áMetastasis

Introduction

High mobility group box (HMGB)proteins are a set of nuclear non-histone proteins found in a variety of eukary-otic species [1].Initially regarded as structural components of chromatin,studies over the past decade drastically changed the view of their cellular role.They have been implicated in transcriptional regulation,DNA repair,recombination,differentiation,and extracellular signaling [2,3].HMGB proteins are composed of two homologous DNA binding domains (boxes A and B)that form L-shaped structure with three a -helices and a C-terminal domain,containing continuous stretch of glutamate or aspartate residues [2,4].They bind with high af?nity to speci?c structural distortions in the double helix such as synthetic four way junctions [5]and adducts that are formed in DNA modi?ed by the anti-tumor drug cisplatinum [6]and UV light [7].Moreover,HMGB1protein (also known as am-photerin)has the ability to induce bends in DNA,promoting the formation of nucleoprotein complexes,thus facilitating the interaction of DNA binding proteins with their cognate sites in chromatin [8].For example,HMGB1and 2have been reported to enhance binding of several transcription factors to their DNA-responsive elements,such as Hox and Pou proteins [9],p53[10],and the steroid hormone recep-tors [11].Apart from its intranuclear role,HMGB1protein is also shown to be localized in the extracellular medium of certain cell types as matrix-bound and in soluble molecules [12]and to play important role in in?ammation and tumor metastasis [13,14].Monocytes/macrophages can secrete HMGB1actively in response to various stimuli like tumor necrosis factor (TNF a )or interleukin-1(IL-1b )[13].Once secreted,HMGB1is able to activate other cells involved in immune response or in?ammatory reactions and can act as a cytokine itself [15,16].The protein can also be passively

N.Kostova áS.Zlateva áI.Ugrinova áE.Pasheva (&)

Institute of Molecular Biology,Bulgarian Academy of Sciences,1113So?a,Bulgaria

e-mail:eva@obzor.bio21.bas.bg

123

Mol Cell Biochem (2010)337:251–258DOI 10.1007/s11010-009-0305-0

released by damaged or necrotic cells,leading to in?am-mation.In contrast,apoptotic cells retain HMGB1?rmly bound to their chromatin even when they lose the integrity of their membranes(late apoptosis or secondary necrosis) and do not release the protein[3].Furthermore,HMGB1 has been observed to stimulate migration of smooth muscles cells and?broblasts[17].Anti-HMGB1antibodies inhibit the migration of neuroblastoma and glioma cells[18].The protein has been shown to be involved in cell invasion, tumor growth,and metastasis.The administration of anti-HMGB1antibodies suppresses the metastasis formation by Lewis lung tumor cells implanted under the skin of recipient mice[14].HMGB1protein has been found in a variety of tumor 9d56af3e87c24028915fc3ddpared to the normal tissue,it is overex-pressed in gastric and colorectal adenocarcinomas[19].The protein is upregulated in melanomas[20]and high HMGB1 protein levels have been reported in leukemia cells[21].A marked intertumoral variation of HMGB1expression is registered in different breast cancers[22].Although in most tumors,HMGB1is overexpressed,tumors devoid of HMGB proteins have been also found.For example,adrenal gland carcinoma shows no HMGB1expression[23]. HMGB1protein is supposed to affect cell invasion,tumor growth,and metastasis by high af?nity binding to the Receptor for Advanced Glycation End products(RAGE) [14,17].RAGE is the only cellular receptor identi?ed for extracellular HMGB1until now[24]and is associated with the invasive and metastatic ability of gastric,colon,and prostate cancer[25–27].Both HMGB1and RAGE are expressed in colon cancer cell lines[28]and their overex-pression is associated with prostate cancer development [29].The inhibition of HMGB1–RAGE interaction sup-presses tumor growth and metastasis[14].

These data concerning the expression of HMGB1pro-tein and its receptor RAGE in human tumors are mainly quantitative and do not refer their presence to the histo-logical type and cellular localization or to the particular cell type in the tumor population.That is why we were motivated to investigate the expression of HMGB1protein and its receptor RAGE in human malignant tumors of different histological grade.

Materials and methods

Tissue specimens

Tissue surgical pathology specimens routinely?xed in formalin and embedded in paraf?n were obtained from the Medical Faculty,Tracian University,Stara Zagora.The specimens included72cases of ductal breast carcinoma(38 cases of cribriform type),83cases of colorectal carcinoma, 68cases of hepatocellular carcinoma,and45cases of testicular carcinoma.The sections containing metastasis in the liver(24samples)originated from colorectal carcino-mas.The histological grading was made according to Elston and Ellis[30].Pathological examination was performed using hematoxylin–eosin stained sections of each specimen.

Antibodies and immunoreagents

Polyclonal rabbit anti-HMGB1antibody was obtained from Upstate Biotechnology(Lake Placid,NY,USA).Polyclonal rabbit anti-Ki67and monoclonal mouse anti-RAGE anti-bodies were purchased from Santa Cruz Biotechnology,Inc. (Santa Cruz,CA,USA).Mouse and rabbit EnVision?Kits (HRP)were obtained from DakoCytomation(Glostrup, Denmark)and used to visualize antibody binding.Chro-mogens used were3,30-diaminobenzidine(DAB;Glostrup, Denmark).

Immunochistochemistry

Five micrometers thick sections were cut from paraf?n-embedded tissue blocks and mounted on silanized slides. After de-waxing and rehydration,the sections were antigen-retrieved as follows:in0.01M Citrate buffer(pH2.5)and 30min microwave heating at low power(300W)for HMGB1immune reaction.For RAGE antibody antigen-retrieval reaction,best results were obtained with5%Urea, 0.1M Tris–HCl(pH6)and15min pressure cooking.For Ki 67immune reaction,15min pressure cooking was applied in 0.01M Citrate buffer(pH6).Thereafter,the specimens were subsequently treated with3%H2O2and1%bovine serum albumin(BSA)in PBS buffer(pH7.4).Incubation with the primary antibodies diluted with1%BSA in the same buffer as indicated(anti-HMGB11:2,000;anti-RAGE 1:1,000,and anti-Ki671:500)was carried out for24h at4°C. Rabbit and mouse EnVision?HRP-labeled polymers were used as a detection system yielding a brown DAB precipitate. The slides were counterstained with Mayer’s hematoxylin and mounted in Entellan.The described protocols gave us intensive immunohistochemical reactions for all primary antibodies and good cell morphology.The specimens were photographed with a Nikon Eclipse E600W microscope equipped with a DXM1200F digital camera.Negative con-trols were obtained by omitting the primary antibodies. Results

Expression of HMGB1protein in primary human malignant tumors

We applied the immunochistochemical approach for the determination of the production of HMGB1protein in

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different types of human malignant tumors.In all tested specimens,the protein exhibited nuclear localization.We examined ductal breast carcinomas,moderately differenti-ated and cribriform type invasive carcinomas.In all cases,the HMGB1immunoreactivity in the malignant tissue sections was stronger in comparison with the normal tissue (Fig.1,compare panels b and d with panel a).An inter-esting ?nding was that in the case of moderately differ-entiated ductal breast carcinomas in most of the cells that ?lled the enlarged mammary ducts the localization of the protein was perinuclear (Fig.1,panel c);while in the case of the invasive cribriform carcinomas,the speci?c peri-nuclear localization was lost and a strong immune reaction was registered in the whole nucleus of the cells which form a duct like pattern consisting of cellular bridges (Fig.1,panel e).

An intense immunostaining with an anti-HMGB1anti-body was observed in the colorectal carcinoma specimens compared to the normal ones (Fig.2,compare panels b and c with panel a).The positive signal was detected in the lumen localized cells as well as in the epithelium cells.The speci?c perinuclear localization was registered in most of the cases of moderately differentiated carcinomas (Fig.2,panel b),while in the low differentiated tumors with bad

prognosis again the HMGB1protein level was highly elevated,but the signal was spread all over the nucleus (Fig.2,panel c).In the primary hepatocellular carcinomas,the speci?c localization of the HMGB1protein persisted (Fig.3,panel b).In contrast,for the testicular carcinomas,the immunohistochemical reaction resulted in heteroge-neous cell staining and in all specimens,the positive cells exhibited uniform nuclear distribution of HMGB1protein (Fig.4,panel a).As mentioned above,in all tested normal tissue samples,the immune staining was faint and uni-formly distributed in the nucleus.No speci?c protein localization was ever detected (see panels a in Figs.1,2,and 3).

Expression of RAGE in primary human malignant tumors

We used the immunochistochemical technique to estimate the expression level of the cellular receptor for HMGB1protein (RAGE)in different tumor tissue specimens as well as its speci?c localization in the cell.In all primary malignant tumors,the RAGE immunoreactivity was always cytoplasmic.The most intensive signal was detec-ted in the hepatocellular carcinomas.No RAGE

expression

Fig.1Immunochistochemical analysis of the expression level of HMGB1protein and RAGE in breast carcinomas.The

surgical specimens after antigen retrieval were incubated either with anti-HMGB1antibody (1:2,000)or with anti-RAGE antibody (1:1,000)as indicated.Panels a,f :normal mammary gland,b,c,g,and h :ductal breast carcinoma,d,e,i,and j :ductal (cribriform)breast

carcinoma.Magni?cation 9200for a,b,d,f,g,and i ,and 9500for c,e,h,and j .The selected objects of interest are marked by arrows

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was observed in the normal hepatocytes (Fig.3,compare panels c with d).The immune reaction resulted in a cyto-plasmic dot-like granular pattern of RAGE localization (Fig.3,panels d and e).High level of RAGE production with the same granular pro?le was observed in the colo-rectal carcinomas,but only in the cells that had penetrated the lumen (Fig.2,panels d and e).In contrast to the HMGB1positive epithelium cells,no RAGE expression was registered in this cellular type.In breast cancer sam-ples,the RAGE level revealed some variation with respect to the tumor type.In all cribriform carcinoma specimens,a positive cytoplasmic diffuse signal was seen in the duct like structure (Fig.1,panels i and j).In contrast,the immunoreactivity in the moderately differentiated ductal carcinoma samples was weak and only a few cells with granular positive immune reaction could be seen (Fig.1,panels g and h).Interestingly,no RAGE expression was ever detected in all testicular carcinomas (Fig.4,panel b).It should be stressed that in contrast to the HMGB1background immune staining in the normal tissue speci-mens,we did not register any RAGE synthesis in the control samples (see Fig.1,panel f,Fig.3panel c).

Expression level of HMGB1protein and RAGE in metastatic cells

The expression of RAGE and its ligand HMGB1protein was usually associated with invasion and poor prognosis.This motivated us to examine the co-expression of both proteins in liver metastasis generated from primary colorectal carcinomas.In all cases,the immunochisto-chemical reactions resulted in a very intense signal for anti-HMGB1and RAGE antibodies (see Fig.5,panels a and b).HMGB1was overexpressed in the nucleus and in all specimens tested,no speci?c perinuclear localization was detected (Fig.5,panel a).The RAGE positive met-astatic cells exhibited strong immunoreactivity and the typical cytoplasmic granular pattern was no longer seen.The dense brown signal that masked the hematoxylin-stained nuclei might be attributed to predominant mem-brane localization of the receptor (Fig.5,panel b).The specimens containing liver metastasis were treated with anti-Ki-67antibody.Ki-67is a nuclear protein used as a marker for cell proliferation,and in some cases,for dis-tinguishing between malignant and benign tumors.In

all

Fig.2Immunochistochemical analysis of the expression level of HMGB1protein and RAGE in colorectal carcinomas.The surgical specimens after antigen retrieval were incubated either with anti-HMGB1antibody (1:2,000)or with anti-RAGE antibody (1:1,000)as indicated.Panel a :normal tissue,panel b :moderately differentiated

colorectal carcinoma,panels c,d and e :low differentiated colorectal carcinoma.

Magni?cation 9200for (a –d )and 9500for (e ).The selected objects of interest are marked by arrows

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probes,we obtained strong positive Ki-67signal (Fig.5,panel c).

Discussion

High Mobility Group Box 1(HMGB1)is a nuclear protein that is reported to be involved in the cancer process [31].HMGB1is upregulated in several tumors as colon adeno-mas and carcinomas and overexpressed in prostate and malignant melanoma cells [20,29,32,33],although background expression is also detected [23].Breast carci-nomas samples show variation in the expression level of HMGB1[22].We tested surgical pathology specimens derived from different malignant tumors as breast,colo-rectal,hepatocellular,and testicular carcinomas,and in all of them,the amount of HMGB1protein was elevated when compared to the normal tissues.In fact,in all normal samples,the HMGB1protein exhibited background nuclear expression that was expected,as the protein is chromatin associated,and is not tissue speci?c.An inter-esting ?nding was the well-de?ned perinuclear localization in the moderately differentiated breast,hepatocellular,and colorectal carcinomas;whereas in the low differentiated cribriform breast and colorectal carcinomas,the speci?c pattern was lost and the intense immune reaction was

detected all over the nucleus.A preferential association of HMGB1protein with the nuclear matrix was previously reported for colorectal tumors [34].We may speculate that the typical localization pattern of HMGB1might be related to the malignant potential of the tumors tested.

In contrast to HMGB1,its receptor RAGE was entirely absent in the normal tissue samples,and was not produced in all of the tested tumor specimens.We found a high level of expression in the sections derived from hepatocellular carcinomas and in those cells of the colorectal carcinomas that ?ll in the duct,but not in the epithelium ones.In both cases,the localization was cytosolic with well-de?ned granular structure.The dot-like cytoplasmic staining was reported in several cell types [35]when using an antibody against the C-terminally truncated endogenous secretory RAGE (esRAGE)ef?ciently secreted from cultured cells and thought to work as a decoy-type receptor by capturing the ligands.In ductal breast carcinoma samples,the char-acteristic granular pattern was preserved,but the signal was weaker and detected in a restricted number of cells.Higher level of RAGE expression was registered in the low dif-ferentiated cribriform breast carcinomas exhibiting diffuse cytoplasmic 9d56af3e87c24028915fc3dding immunochistochemical analysis,we detected no signal for RAGE in all testicular carcinoma specimens.Therefore,we may speculate that the expression of the receptor does not correlate with

the

Fig.3Immunochistochemical analysis of the expression level of HMGB1protein and RAGE in hepatocellular carcinomas.The surgical specimens after antigen retrieval were incubated either with anti-HMGB1

antibody (1:2,000)or with anti-RAGE antibody (1:1,000)as indicated.Panels a ,c :normal tissue,panels b ,d ,and e :hepatocellular carcinoma.Magni?cation 9200for (a –d )and 9800for (e )

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cancerogenesis in general.More probably its synthesis is related to distinctive tumor types.Our observations are supported by several reported data.For example,in non-small lung carcinomas,RAGE is reduced and the process is independent of the histological subtype [36],while pan-creatic cell lines with high metastatic ability display strong RAGE expression [37].Inactivation of the receptor in rhabdomyosarcoma cells results in reduced myogenesis and increased proliferation and tumorogenesis [38].

The initial discovery of HMGB1–RAGE interactions in the modulation of neurite outgrowth [39]suggested that HMGB1–RAGE signaling might also be involved in cancer metastasis.Co-expression of RAGE and HMGB1was associated with metastasis to lymph nodes and distal organs and poor prognosis of colorectal cancer [26].HMGB1–RAGE interactions mediated migration,growth,and spread of implanted C6gliomas.The blockade of this interaction by soluble or mutated RAGE resulted in sup-pression of the migration of the glioma,forcing the tumor to undergo prolonged dormancy with decreased prolifera-tion and invasion [14].HMGB1protein was found in the serum and extracellular ?uids [24]when secreted from the cell.It was released passively by necrotic cells or actively

by macrophages/monocytes in response to exogenous and endogenous in?ammatory stimuli in a time-and dose-dependent manner [13,40],and in this way,the interaction with its speci?c receptor (RAGE)was accomplished.The different cellular types present in the investigated tumor tissue specimens did not secrete the protein which explained the fact that in all of them HMGB1was detected in the nucleus.An increased number of necrotic cells were observed in the samples containing metastasis (data not shown)which probably stimulated the ligand/receptor interaction and provoked increased cellular motility

and

Fig.4Immunochistochemical analysis of the expression level of HMGB1protein and RAGE in testicular carcinomas.The surgical specimens after antigen retrieval were incubated either with anti-HMGB1antibody (1:2,000)or with anti-RAGE antibody (1:1,000)as indicated.Magni?cation 9200.The selected objects of interest are marked by

arrows

Fig.5Immunochistochemical analysis of the expression level of HMGB1protein and RAGE in liver metastasis.The surgical specimens after antigen retrieval were incubated either with anti-HMGB1antibody (1:2,000),anti-RAGE antibody (1:1,000),and anti-Ki67(1:500)as indicated.Magni?cation 9200

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invasion.We studied the expression level of HMGB1and its receptor RAGE in metastatic cells in liver specimens originated from primary colorectal carcinomas.Both pro-teins exhibited very intense immune reaction and their speci?c localization pattern was lost.The typical perinu-clear immune reaction characteristic for HMGB1in mod-erately differentiated ductal breast,colorectal,and hepatocellular carcinomas changed to intense signal in the whole nucleus of the metastatic cells.The same holds true for RAGE expression,the dot-like cytoplasmic localization in primary hepatocellular and colorectal carcinomas turned to dense brown staining all over the metastatic cells which might be due to membraneous expression.Data linking the cellular localization of RAGE to prognostic evaluation were reported for colorectal adenomas.The cytosolic pat-tern was associated with mild atypia and small tumor size, while membranous pattern was associated with severe atypia[33].In this respect,the possible correlation between HMGB1and RAGE expression and their speci?c locali-zation,and the state of tumor differentiation could have a prognostic meaning in carcinogenesis.

Acknowledgments We thank Dr.Gulubova and Dr.Vlaykova from the Medical Faculty,Tracian University,and Stara Zagora for the surgical specimens and fruitful advices.This study was supported by grant CRP/BUL06-01from International Centre for Genetic Engi-neering and Biotechnology.

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