Expression profiling identifies new function of collapsin response mediator protein 4 as a metastasi

ORIGINAL ARTICLE

Expression pro?ling identi?es new function of collapsin response mediator protein 4as a metastasis-suppressor in prostate cancer

X Gao 1,5,J Pang 1,5,L-Y Li 1,5,W-P Liu 1,J-M Di 1,Q-P Sun 1,Y-Q Fang 1,X-P Liu 1,X-Y Pu 1,D He 2,M-T Li 3,Z-L Su 2and B-Y Li 4

1

Department of Urology,The Third Af?liated Hospital,Sun Yat-sen University,Guangzhou,China;2Department of Pathology,The Third Af?liated Hospital,Sun Yat-sen University,Guangzhou,China;3Proteomics Center,Zhongshan School of Medcine,Sun Yat-sen University,Guangzhou,China and 4Department of Urology,University of Kansas Medical Center,Kansas City,KS,USA

Metastasis is the chief cause of mortality from cancer,but the mechanisms leading to metastasis are poorly under-stood.We used a proteomics approach to screen for metastasis-associated proteins and found that collapsin response mediator protein-4(CRMP4)expression was inversely associated with the lymph node metastasis of prostate cancer (PCa).Subsequent in vitro and in vivo studies revealed that overexpression of CRMP4not only suppressed the invasion ability of PCa cells,but also strongly inhibited tumor metastasis in an animal model.Furthermore,methylation of a CpG island within the promoter region of the CRMP4gene is responsible for downregulation of CRMP4expression.Thus,in this study,we show new function of CRMP4as a metastasis-suppressor in PCa.The ?ndings provide new mechanistic insights into metastasis and therapeutic potential for this most common male cancer.

Oncogene (2010)29,4555–4566;doi:10.1038/onc.2010.213;published online 14June 2010

Keywords:prostate cancer;proteomics;collapsin re-sponse mediator protein-4;metastasis-suppressor gene;methylation

Introduction

Prostate cancer (PCa)is the most common solid organ malignancy affecting men and the second leading cause of cancer death in the United States (Jemal et al.,2008).Metastatic cancers,once established,are the main cause of mortality associated with cancer.Metastasis is a complex and multistage process involving local inva-sion,intravasation,successful transit through the blood-stream,extravasation,and eventual proliferation and survival within a favorable target organ (Loberg et al.,2005).Recently,technological advances permit the genomic characterization of tumors,enhancing our

understanding of cancer initiation and metastasis.Cancers are a heterogeneous mass of neoplastic cells with various properties,including different metastatic activity (Lapointe et al.,2004;Shaffer and Pandol?,2006;Tomlins et al.,2007).During carcinogenesis,some tumor cells probably acquire new phenotypes by increased expression of metastasis-promoting genes or decreased expression of metastasis-suppressor genes (Klein,2008).However,the identi?cation of individuals at increased risk of metastasis,who may then be selected for aggressive systemic therapy,remains a considerable challenge.

PCa often migrates through the lymphatic route,depositing tumor cells into pelvic lymph nodes (Miyake et al.,2007).Among the adverse pathological features,the presence of pelvic lymph node metastasis is the strongest predictor of poor outcome (Burton et al.,2008).Proteomics-based expression pro?ling has been commonly used to compare and contrast heterogeneous groups of human tumors (Hanash,2003).In this study,we used two-dimensional (2D)?uorescence difference gel electrophoresis (DIGE)technique coupled with matrix-assisted laser desorption/ionization time-of-?ight/time-of-?ight mass spectrometry analysis to identify differen-tially expressed proteins in pelvic lymph node metastatic PCa (LNM PCa)compared with localized PCa.We selected the differentially expressed protein,collapsin response mediator protein-4(CRMP4),validated its preferentially suppressed expression in metastatic PCa,and showed its contribution to the metastasis-suppres-sing process in a series of functional studies.

Results

Identi?cation of differentially expressed CRMP4in metastatic PCa

By comparing protein proles from fresh-frozen LNM PCa and localized PCa tissues using the 2D DIGE technique with matrix-assisted laser desorption/ioniza-tion time-of-?ight/time-of-?ight mass spectrometry analysis,CRMP4was identi?ed for the ?rst time to be downregulated by 1.86-fold in the LNM PCa.The expression of CRMP4was signi?cantly decreased in LNM PCa compared with localized PCa (P o 0.001;

Received 7December 2009;revised 20April 2010;accepted 4May 2010;published online 14June 2010

Correspondence:Professor X Gao,Department of Urology,The Third Af?liated Hospital,Sun Yat-sen University,Tianhe Road 600,Guangzhou,Guangdong 510630,China.

E-mail:Xin.Gao.zsu@https://www.360docs.net/doc/5b4934106.html, or gaoxin44@https://www.360docs.net/doc/5b4934106.html, 5

These authors contributed equally to this work.

Oncogene (2010)29,4555–4566

&2010Macmillan Publishers Limited All rights reserved 0950-9232/10

https://www.360docs.net/doc/5b4934106.html,/onc

Figure 1).We further determined whether its expression was associated with the metastasis of PCa.Western blot and real-time reverse transcriptase PCR (RT–PCR)were used to verify that the proteomics data were an accurate representation of the CRMP4gene expression patterns in PCa.As predicted,the expression of CRMP4protein was signi?cantly decreased in LNM PCa tissues compared with localized PCa tissues (P ?0.007;Figure 2a).CRMP4gene expression was also signi?-cantly decreased in LNM PCa tissues by real-time PCR (P o 0.001;Figure 2b).CRMP4protein expression was also assessed in archival formalin ?xed PCa and benign prostate hyperplasia (BPH)specimens by immuno-histochemistry (IHC).Notably,CRMP4expression was detected mainly in cytoplasm of epithelial cells and its expression was intensive in all BPH specimens.In constrast,CRMP4expression was signi?cantly down-regulated in LNM PCa tissues compared with localized PCa tissues (P o 0.001;Figure 2c and Supplemental Figure 5A).

To examine the speci?city of CRMP4expression,we analyzed the expression patterns of all ?ve members of the CRMP gene family (CRMP1,CRMP2,CRMP3,CRMP4and CRMP5)in BPH and PCa tissues by real-time RT–PCR.Of all CRMP family members,the CRMP4gene was determined to be the only one differentially expressed in PCa tissue (all P -values >0.05;Figure 2e).We also examined CRMP4expres-sion in other endoblast-derived cancers including lung,gastric and intestine cancers by western blot and con?rmed that the decreased expression pattern of CRMP4is speci?c in LNM PCa (Figure 2d).

Overexpression of CRMP4and inhibition of metastasis in vitro and in vivo

To examine the role of expression of CRMP4in metastasis,a CRMP4expression vector was stably introduced into two PCa cell lines (PC3and LNCaP)and clones that stably expressed CRMP4were isolated.Interestingly,transfected PC3and LNCaP cells with high CRMP4expression became rounded and seldom have ?lopodia protruding from the cell edge following actin ?laments staining with FITC-conjugated phalloi-din,but mock-transfected cells maintained a ?at,spindle-and ?broblast-like morphology and showed numerous ?lopodia (Figure 3a,Supplemental Figure 1).PCa cells transiently transfected with the plasmid pEGFP-CRMP4,carrying the fusion gene for enhanced green ?uorescent protein (EGFP)-tagged CRMP4,were examined directly and found that CRMP4diffusely distributed in the cytoplasm (Figure 3a).Transfected clones expressed higher levels of CRMP4than the mock-transfected clones (Figure 3b).The same ?uores-cence intensity in the two stable cells (PC3-pEGFP-C1and PC3-pEFGP-CRMP4)was observed (Supplemental Figure 6)and a modi?ed

3-(4,5)-dimethyl-thiahiazo

Figure 1The 2D DIGE analysis and matrix-assisted laser desorption/ionization time-of-?ight/time-of-?ight mass spectrometry (MALDI-TOF/TOF MS)identi?cation of CRMP4.The expression of CRMP4was signi?cantly downregulated in LNM PCa compared with localized PCa (P o 0.001,Student’s t-test)by 2D DIGE analysis and MALDI-TOF/TOF MS identi?cation.The image was generated by the DeCyder 2D Differential Analysis V6.0Software.The con?dence interval (CI)was 100%,score 149and 11peptide matched.The sequence of precursor at m/z 1031.54was analyzed by MS/MS to be SAADLISQAR.(a )The spot circled with red line represented localized 2D DIGE map of protein CRMP4.(b )Spot map of CRMP4as shown in three-dimensional view.(c )Graph view of CRMP4.(d )The MS map and MS/MS map of CRMP identi?ed by ABI 4800MALDI TOF/TOF.The coverage of identi?ed peptides from CRMP4was indicated below.In all,9peptides whose sequences were determined by MALDI-TOF/TOF MS were highlighted with red color.

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(-z-y1)-3,5-diphenytetrazoliumromide assay showed that in vitro proliferation was essentially unchanged with altered CRMP4expression.We then used an in vitro -reconstituted basement membrane invasion assay to investigate whether CRMP4expression affected the invasive ability of PC3and LNCaP cells.After a 48-h incubation,we noted a statistically signi?cant reduction in the invasive activity of CRMP4-expressing clones compared with control clones (P o 0.001,P o 0.001,respectively;Figures 3c and d).

We then addressed the potential functional role of CRMP4as a metastasis-suppressor gene by metastatic assays in vivo by assessing the effect of restoring CRMP4expression to PC3cells.PC3cells expressing either empty vector (pEGFP-c1)or CRMP4expression vector (pEGFP-CRMP4)with GFP reporter were injected into mouse prostate (Figure 3e).At speci?ed time,the

organs,including lung,liver,kidney,pancreas and prostate,were dissected from each mouse and GFP activities,representative of metastasis,were recorded.Although the primary tumor weights as well as associated-GFP activities in prostate were comparable both in control and in CRMP4-expressing tumor cells,there were signi?cant decreases in liver,lung and kidney metastases in CRMP4-expressing PC3cells (Figures 3f and g).These results indicate that expression of CRMP4in PCa cells signi?cantly suppresses the in vivo incidence of metastases.

Identi?cation of 50CpG island methylation of the CRMP4gene promoter

To investigate the mechanism for the downregulation of CRMP4expression in metastatic PCa,we further analyzed the structure of the CRMP4promoter

region.

Figure 2Validation of CRMP4gene as signi?cantly downregulated in metastatic PCa and its speci?c expression in prostate.(a )Western blot analyses of CRMP4expression in BPH and PCa.There was a signicant difference in CRMP4expression level among groups (P ?0.007,One-way ANOVA).CRMP4expression was signi?cantly downregulated in LNM PCa tissues (P ?0.031,compared with the BPH group;P ?0.007,compared with the localized PCa group,Newman–Keuls test).GAPDH was used as the internal loading control.(b )Real-time RT–PCR analysis of CRMP4expression.There was a signicant difference in CRMP4expression level among groups (P o 0.001,One-way ANOVA).The expression of CRMP4gene was signi?cantly decreased in LNM PCa tissues (P o 0.001,compared with the BPH group;P o 0.001,compared with the localized PCa group,Newman–Keuls test).(c )Immunohistochemical analysis of CRMP4in BPH tissue,localized and LNM PCa.The arrows indicate the positive staining in prostate glandular epithelium.The LNM PCa group had signi?cantly lower expression of CRMP4than that in the localized PCa group.(d )Western blot analyses of speci?c decreased expression of CRMP4in metastatic PCa.There was no decreased expression of CRMP4in all other endoblasts-derived metastatic tumor tissues including lung,gastric,intestine cancer.GAPDH was used as the internal loading control.(e )The expression of all other CRMP family members (CRMP1,CRMP2,CRMP3and CRMP5)in BPH,localized PCa and LNM PCa tissues by real-time RT–PCR.There were no signi?cant differences in all other CRMP family members (C RMP1,CRMP2,CRMP3and CRMP5)among these three groups (all P-values >0.05,one-way ANOVA).

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First,the sequences of CRMP4promoter region were ampli?ed by PCR and sequenced in two metastatic PCa tissues and three PCa cell lines (PC3,PC3M and LNCaP).No mutations were found in the sequences of CRMP4promoter region from à940to the tran-scriptional start site (Supplemental Figure 2B).

However,we identi?ed two CpG islands in the 50

-?anking sequences with the program MethPrimer

(https://www.360docs.net/doc/5b4934106.html,/methprimer/)(Supplemental Figure 2A).We used bisul?te-sequencing PCR to ?nd all methylation alleles in metastatic PCa tissues and PCa cell lines compared with localized PCa and BPH tissues (Figure 4A and Supplemental Figures 3and 4).The results revealed the methylation status of 43CpG dinucleotides in the CpG island 1(Figure 4A)and 27CpG dinucleotides in the CpG island

2

Figure 3CRMP4is a functional metastasis-suppressor gene.(a )Cell Morphology of CRMP4-transfected and mock-transfectd PC3and LNCaP cells.Transfected PC3and LNCap cells with high CRMP4expression became rounded.Enhanced green ?uorescent protein-tagged CRMP4showed the intracellular location of CRMP4under ?uorescence microscopy.(b )PCa cell lines (PC3,LNCaP,DU145)transfected with pcDNA3.1-CRMP4expressed higher levels of CRMP4than the control mock-transfected cells.(c )Expression of CRMP4suppressed the in vitro invasion activity of PC3and LNCaP cells.(d )CRMP4-transfected PC3and LNCaP cells had statistically signi?cantly lower in vitro invasive activity than mock-transfected cells (P o 0.001,P o 0.001,respectively;Student’s t-test).Results were the means of three separate measurements obtained from three separate experiments.(e )PC3cells expressing GFP reporter with either empty vector (pEGFP-c1)or CRMP4expression vector (pEGFP-CRMP4)were injected into mouse prostate,and GFP activity was recorded for each mouse (n ?9mice per group).(f )GFP activity in lung,liver,kidney and pancreas,representative of metastasis,was recorded for each https://www.360docs.net/doc/5b4934106.html,an images from different mice were shown.The color bar represents GFP intensity.(g )GFP activities in the organs from either each control mouse or each CRMP4-expressing mouse were quanti?ed,and the ratios of GFP activities in the metastatic organ relative to primary prostate tumor were shown.There were signi?cant decreases in liver,lung and kidney metastases in the CRMP4-expressing PC3cells compared with mock-transfected PC3cells.

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(Supplemental Figure3).Our data showed that the

CpG sites inà848,à841,à690,à680,à678,à674,à671,à665,à660andà658of CpG island1were highly or moderately methylated in cell lines(PC3,PC3M and

DU145)and metastatic PCa(primary lesion and meta-static lymph node).In contrast,these CpG sites in localized PCa,BPH and normal prostate were mostly unmethylated except a few sporadic5-methylcytosine in some of the clones.Thus,we used methylation speci?c PCR(MSP)to detect the presence of speci?c methylation alleles of CpG island1in?ve of seven metastatic PCa and three PCa cell lines(Figure4B).The treatment with demethylating agent5-aza-2-deoxycytidine in PC3cells was effective for reexpression of CRMP4(Figure4C). Furthermore,western blot was used to validate the correlation of the promoter methylation with decreased CRMP4expression in metastatic PCa(Figure4D)and in PCa cell lines(Figure3b).

Association of CRMP4expression with postoperative biochemical relapse,clinical metastasis and VEGF expression in PCa tissue

We used IHC to examine CRMP4expression in archival formalin?xed tissue from65cases of PCa(localized PCa,n?38;LNM PCa,n?27)and20cases of BPH. Staining intensity was quanti?ed using Image-Pro Plus software.As mentioned above,the expression level of CRMP4in LNM PCa specimens(7.21±1.26integrated optical density units)was statistically signi?cantly lower than in localized PCa tissue(17.12±1.37integrated optical density units,P o0.001)and in BPH tissue (27.46±2.84integrated optical density units,P o0.001; Supplemental Figure5A).Four cases with localized PCa (Patients1–4in Supplemental Figure5B),who had relatively lower CRMP4expression level at diagnosis, had a rapid biochemical relapse and clinical metastasis. Furthermore,there was a trend of decreased expression level of CRMP4in these cases when they progressed from localized lesions to metastatic ones(Supplemental Figures5B and C).In contrast,two cases(Patient M3 and M7in Figure5b)in LNM PCa groups had relatively higher expression level of CRMP4and their CpG sites were mostly unmethylated,validated by MSP (Figure4B).The case(Patient M3)had no biochemical relapse(undetectable serum prostate speci?c antigen) and clinical metastatic?ndings until2-year follow-up. We also used IHC to examine simultaneously CRMP4and vascular endothelial growth factor (VEGF)expression in archival formalin?xed tissue from32cases of prostate tissues(BPH,n?10;localized PCa,n?10;LNM PCa,n?12).These results showed that the CRMP4expression was inversely associated with VEGF expression in PCa tissues(Figures5a and b; r?à0.612,P o0.001).

Discussion

To become metastatic,tumor cells must coordinate the increased expression of metastasis-promoting genes and/or the decreased expression of metastasis-suppres-sing genes(Steeg,2003).The advent of DIGE-based proteomics technology,in which the expression of thousands of proteins can be assessed simultaneously,

has greatly facilitated the dissection of this process (Hanash,2003;Go rg et al.,2004).Lymphatic invasion is

one of the major routes for cancer cell dissemination

and lymph node status provides important information

for both diagnosis and treatment of human cancer (Kishimoto et al.,2006;de Boer et al.,2009).Here we describe the use of the2D DIGE proteomics analysis to identify a set of proteins whose expression is associated

with pelvic lymph node metastasis in PCa.We experimentally demonstrate in vivo,for the?rst time,

the functional metastasis-suppressing role of the gene, CRMP4.

CRMP4is a member of the CRMP family of cytosolic phosphoproteins,which may mediate semaphorin/col-lapsin-induced growth cone collapse and are involved in axonal guidance and neuronal differentiation(Goshima

et al.,1995;Gaetano et al.,1997;Fukada et al.,2000). During the last few years,?ve members of the CRMP

gene family(CRMP1,CRMP2,CRMP3,CRMP4and CRMP5),encoding closely related60–66kDa proteins,

have been cloned and they have50–70%sequence homology with one another(Wang and Strittmatter, 1997;Arimura et al.,2000).A previous study has reported that CRMP1is an invasion suppressor and correlates with clinical outcomes in non-small-cell lung cancer.They further characterized that CRMP1was a

novel invasion-suppression gene(Shih et al.,2001, 2003).Here we found that expression of both CRMP4 mRNA and protein was inversely associated with the lymph node metastasis of PCa and that overexpression

of CRMP4in PCa cell lines reduced its in vitro invasive activity.Furthermore,we demonstrated experimentally

that forced expression of CRMP4in PCa cell lines induces a decreased metastatic state in a mouse model of

PCa metastasis.The expression of CRMP1,CRMP2, CRMP3and CRMP5mRNAs in the PCa specimens

was investigated using real-time RT–PCR analyses,but

no association between these CRMP family members

and metastasis in PCa could be established.These

?ndings support the notion that CRMP4has new function as a metastasis suppressor in PCa.

Although efforts over the past two decades have

identi?ed metastasis suppressors ranging from kinases,

such as MAP kinase kinase4,to GTP-binding proteins,

such as Rho-GDI2,only a limited number of metastasis-suppressor genes have been identi?ed to date,and mechanistic details for most of these genes are largely unknown(Rinker-Schaeffer et al.,2006).We showed

that the level of CRMP4protein was statistically

signi?cantly higher in localized PCa compared with metastatic PCa,suggesting that the level of CRMP4 protein decreases during clinical disease when cells acquire the ability to grow at a metastatic site.CRMP4,

on reexpression in metastatic PCa cell lines,reduced metastasis without a signi?cant effect on tumorigenicity.

As such,it will be important to integrate CRMP4 function into the context of other known metastasis suppressors,including both novel(NM23,BRMS1,

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KiSS1and KAI1)and known (MKK4and RKIP)metastasis suppressors (Steeg,2006).

The CRMP4protein encodes a 62-kDa protein with no signal sequence,transmembrane domain or classical protein interaction domains.However,CRMP4contains a number of consensus phosphorylation sites that may serve as substrates for signaling molecules,such as Cdk,protein kinase C and proline-directed kinases (Gaetano et al.,1997).CRMP4are intracellular phosphoproteins involved in rib-like actin bundles

in

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lamellipodia and functionally regulates the actin cyto-skeleton in motile cells (Rosslenbroich et al.,2005),which may be relevant for metastasis suppressing.However,in the case of a newly characterized metastasis suppressor such as CRMP4,determining its biochemical mechanism of action still remains daunting.

Cancer has long been thought to progress through accumulation of genetic mutations.However,it has become increasingly clear that epigenetic mechanisms have a central role in tumorigenesis (Feinberg et al.,2006;Ting et al.,2006).In eukaryotes,DNA methyla-tion of gene promoters is associated with transcriptional repression (Zilberman,2007).The CRMP4gene,which locates on human chromosome 5q32,contains 15exons,including an exon de?ned by an anaplastic oligoden-droglioma-expressed sequence tag,and spans at least 61.7kb (Matsuo et al.,2000).We found no mutations in the sequences of CRMP4promoter core region in either metastatic PCa or PCa cell lines.Indeed,the promoters of several tumor-suppressor genes,such as p16,p15and E-cadherin ,are highly methylated with a rare gene mutation in human oral squamous-cell carcinoma (Uzawa et al.,2002).Here we detected methylation of the CRMP4gene promoter region in metastatic PCa and in PCa cell lines and validated that the promoter methylation was correlated with the level of CRMP4expression in metastatic PCa and in PCa cell lines.Therefore,these observations suggest that CRMP4promoter methylation,but not mutational inactivation is likely the main mechanism of CRMP4downregulation.

Abnormal methylation in the neighborhood of the start codon has been shown to suppress gene expression in tumor cells by either interfering with RNA polymer-ase II initiation or transcription factor binding (Tate and Bird,1993).Several regions of the promoter of the CRMP4gene have been shown to be potentially relevant for the regulation of its expression (Matsuo et al.,2000).The sequence contained between à439and à163bp from the start codon of the CRMP4gene is rich in potential binding sites for several transcription factors,such as Oct-1,ets-1,c-Myc,MyoD/myogenin,Sp1and Ap-1(Matsuo et al.,2000).Upregulation of these transcription factors in cancer cells would poten-tially correlate with an increase in the levels of CRMP4and an enhanced cancer metastasis-suppressive action.However,abnormal methylation of this promoter region prevents CRMP4expression with concomitant loss of its metastasis-repressor function (Figure 5c).

VEGFs,among major pro-angiogenic and pro-meta-static molecules,are indirect promoters of tumor growth in vivo by inducing angiogenesis and lymphangiogenesis,which is crucial for supporting expansion of tumor mass and metastases (Chi et al.,2009).Some of the above-mentioned transcription factors (Sp1,Ap-1,ets-1)are known to be upregulated in tumor cells and can switch on the expression of VEGF (Schewe et al.,2005;Seth and Watson,2005;Ozanne et al.,2007).Sema3A,also known as collapsin-1,can inhibit the migration or motility of endothelial cells by depolymerization of F-actin and retraction of lamellipodia (Miao et al.,1999).Neuropilin-1and plexin A1are required for Sema3A to initiate the signal transduction cascade.VEGF and Sema3A are competitive inhibitors that have over-lapping neuropilin-1binding sites (Miao et al.,1999).A recent study has reported that VEGF and Sema3A,as antagonistic autocrine neuropilin-1ligands,have a key role in PCa progression (Yacoub et al.,2009).CRMPs can mediate the signal of Sema3A-neuropilins and the expression of CRMPs are positively associated with that of Sema3A (Shih et al.,2001;Deo et al.,2004).Thus,we presume that abnormal methylation of the promoter region in CRMP4prevents CRMP4expression,which might indirectly increase the VEGF expression,thus contributes to PCa metastasis.However,further studies are needed to con?rm the underlying mechanisms.

In conclusion,we have identi?ed,to our knowledge,for the ?rst time,that CRMP4has new function as a metastasis suppressor in PCa.Our studies directly shown that overexpression of CRMP4not only sup-pressed the ability of PCa cells to invade matrigel in vitro ,but also strongly inhibited tumor metastasis in an animal model.Methylation of a CpG island within the promoter region of the CRMP4is responsible for downregulation of CRMP4expression in metastatic PCa.Realization of the mechanism regulating the expression of CRMP4will lead to the development of new strategies for the treatment of PCa,possibly by increasing the amount of CRMP4to halt cancer

metastasis.

Figure 4Detection of methylation of the CRMP4promoter.(A )Methylation status of bisul?te-modi?ed CRMP4CpG island 1.Each line represented one single clone and each circle represented 1of the 43CpG dinucleotides in this DNA fragment.Methylated CpGs are shown as ?lled circles and unmethylated ones as open circles.The special 10CpGs were marked by rectangles.The percentage of methylated CpGs was 87,79,44,16and 19%in PC3,PC3M,DU145,M6and M6-LN,respectively,which were signi?cantly higher than those in localized PCa,BPH and normal prostate.(a )Prostate cancer cell lines (PC3,PC3M and DU145).(b )Localized PCa (Patient L2and L4).(c )Metastatic PCa (Patient M3and M6).(d )Lymph node of metastatic PCa (Patient M6).(e )BPH.(Patient BPH1).(f )Normal prostate (N1).(B )MSP analysis of CRMP4promoter region in representative cases of localized PCa (Patient L1-5),metastatic PCa (Patient M1-7),cell lines (PC3,PC3M,DU145),BPH (Patient BPH1-2),normal prostate (Patient N1-2),lymph node of local PCa (Patient L3,L4,L5)and lymph node of metastatic PCa (Patient M5,M6,M7).Primer sets used for ampli?cation are designated as methylated (M)and unmethylated (U).All DNA samples were bisul?te-treated.The H 2O control was included in the MSP reaction.Methylation in ?ve of seven metastatic PCa and all of cell lines was observed.(C )Western blot was used to analyze reexpression of CRMP4in PC3cells following treatment with demethylating agent 0.5,5and 12.5m M 5-aza-2-deoxycytidine,respectively.PC3cells showed obvious reexpression of CRMP4.(D )Western blot analyses validated the correlation of the promoter methylation with decreased CRMP4expression in metastatic PCa.CRMP4expression was signi?cantly decreased in metastatic PCa tissues in which the promoter methylation of CRMP4was found (L1-5,localized PCa group;M1-7,lymph node metastatic PCa group;N1-2,normal prostate group).

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Materials and methods

Human PCa specimens and cell line

Primary PCa specimens and pelvic lymph nodes were freshly collected from patients during surgery in the Third Af?liated

Hospital of Sun Yat-sen University.All procedures were approved by the research committee of the hospital and all samples were obtained with the consent of the research subjects.For 2D DIGE proteome analysis,10cases of localized PCa,7cases of LNM PCa and 10cases of

BPH

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were collected.With regard to con?rmatory studies,real-time RT–PCR was performed on 16localized PCa tissues,16LNM PCa tissues and 16BPH tissues for determining the expression of CRMP1,CRMP2,CRMP3,CRMP4and CRMP5.Western blot was performed on nine localized PCa tissues,nine LNM PCa tissues and nine BPH tissues for determining the expression of CRMP4.Paraf?n-embedded specimens from 38cases of localized PCa,27cases of LNM PCa and 20cases of BPH were used to validate the expression of CRMP4using IHC.Human prostate cancer cell lines (PC3,PC3M,DU145and LNCaP)were obtained from the American Type Culture Collection (ATCC,Manassas,VA,USA).

Proteomic analysis

A modi?ed manual microdissection method was used to obtain pure tumor tissue (Paris et al.,2004).Tissue (

B 0.1g)was homogenized in lysis buffer (7M urea,2M thiourea,30m M Tris,4%(w/v)CHAPS,40m M dithiothreitol,0.6m M phenyl-methylsulfonyl ?uoride).The supernatant was collected,puri?ed with a 2D cleanup kit (GE Healthcare BioSciences,Little Chalfont,UK),quanti?ed with the 2D Quant kit (GE Healthcare),and labeled with CyDye DIGE Fluors following the Ettan DIGE user manual as described previously (Fu et al.,2007).A total of 2800–3000protein spots were analyzed across all samples.The ratios of protein abundance that increased or decreased X 1.5-fold (P o 0.01;Jackson et al.,2006)were considered signi?cantly changed.The corresponding differen-tial protein spots with statistical signi?cance and high abundance in both preparative gels were selected for spot picking.The selected protein spots in the preparative gels were automatically picked up and handled in the Ettan Spot Handling Workstation (GE Healthcare).The criteria of successfully identi?ed protein as follows:ion score con?dence interval (CI%)for PMF and MS/MS data X 95%,peptide count (hit)X 4and at least two peptides of distinct sequence were identi?ed in MS/MS analysis.

Real-time PCR

Total RNA was extracted after homogenization of tissue samples using TRIzol (Invitrogen,Carlsbad,CA,USA),followed by DNase digestion (RNase-free DNase set;Qiagen,Valencia,CA,USA)and on-column clean up with the RNeasy MinElute kit (Qiagen).Total RNA (2m g)was reverse transcribed with the Superscript II RNase H-reverse tran-scriptase kit (Invitrogen)for complementary DNA (cDNA)synthesis using random hexamer primers.Expression level and genomic copy number were evaluated by real-time PCR on an ABI PRISM 7000Sequence Detector Thermocycler (Applied Biosystems,Foster City,CA,USA),according to the manufacturer’s protocol.Glyceraldehyde 3-phosphate dehy-drogenase (GAPDH)was applied as the input references.Sequences of PCR primers of CRMP1–5are available on

request.All quantitative PCR reactions were performed in triplicate and repeated at least twice.The D Ct for gene-speci?c mRNA expression was calculated relative to the Ct (threshold cycle)of GAPDH.The DD Ct was calculated by normalizing to the average D Ct of the 16BPH tissue samples.

Western blot

In total,60m g of each protein sample was subjected to western blot analysis using anti-CRMP4(1:7500;Chemicon Inc.,Temecula,CA,USA).GAPDH was used as an internal control in all blotting membranes (anti-GAPDH,1:1000;Cell Signaling Technology,Inc.,Beverly,MA,USA).Immuno-reactive proteins were then visualized using the ECL western blotting system (Pierce Biotechnology,Rockford,IL,USA).Images were scanned by the ImageMaster II scanner (GE Healthcare)and analyzed using ImageQuant TL v2003.03(GE Healthcare).Background binding was subtracted out,and the band signals were expressed as relative protein amounts compared with GAPDH.

IHC

Immunohistochemical staining was respectively performed with anti-CRMP4(1:1500)and anti-VEGF (1:100,Santa Cruz Biotechnology Inc.,Santa Cruz,CA,USA)using the DAKO En-Vision System (Dako Diagnostics,Zug,Switzerland)according to the manufacturer’s instructions.Positive immu-nostaining was visualized and recorded by an experienced pathologist (Z-L S)in a blinded manner.Staining intensity was quanti?ed over three randomly selected,high-powered ?elds per section using Image Pro-Plus software,version 4.0(Media Cybernetics,Bethesda,MD,USA)and is expressed as integrated optical density units of staining intensity per ?eld of vision (Cathcart et al.,2008;Yang et al.,2008).

Molecular cloning and plasmid constructs

cDNA encoding the entire human CRMP4coding region (GenBank accession number NM_001387)was ampli?ed by PCR using above cDNA synthesis from BPH tissues.Primer sequences were as follows:50-GAATTCGGTACCATGTCC TACCAAGGCAAGAAGA-30(sense),50-CTCGAGCCCGGG GTTAACTCAGAGATGTGATATTAG-30(antisense;1737bps).The CRMP4cDNA fragment was cloned into a pMD19-T simple vector and sequenced with an autosequencer (model ABI 377;PE Applied Biosystems,Foster City,CA,USA).Sequence analysis showed a 100%match to the published sequence for CRMP4cDNA.pcDNA3.1-CRMP4was created by inserting CRMP4cDNA between the Kpn I and Xhol I sites of a pcDNA3.1mammalian expression vector (Promega Corp.,Madison,WI,USA).At the same time,the CRMP4cDNA was inserted in frame between the Kpn I and Smal I sites of an EGFP expression vector,pEGFP-C1(Clontech,Palo Alto,CA,USA),yielding pEGFP-CRMP4,

which

Figure 5Correlations of CRMP4expression with VEGF expression in PCa.(a )The representative images of CRMP4and VEGF expression in archival formalin ?xed tissue from 32cases of prostate tissues (BPH,n ?10;Localized PCa,n ?10;LNM PCa,n ?12)by immunohistochemistry detection.(b )The quantitation analysis of CRMP4and VEGF expression in every case of prostate tissue.Staining intensity was quanti?ed using Image-Pro Plus software.The results showed that the CRMP4expression was inversely associated with VEGF expression in PCa tissues (r ?à0.612,P o 0.001).(c )Working model of driving the metastasis for methylation of CRMP4promoter.Abnormal methylation in the neighborhood of the start codon suppresses gene expression in tumor cells by interfering with transcription factor binding.The sequence contained between à439and à163bp from the start codon of the CRMP4gene is rich in potential binding sites for several transcription factors (Ap1,ets 1,Sp1).Abnormal methylation of this promoter region prevents CRMP4expression with concomitant loss of its metastasis repressor function.CRMPs mediate the signal of Sema3A-neuropilins.VEGF and Sema3A are competitive inhibitors that have overlapping neuropilin-1binding sites.Thus,we presume abnormal methylation of CRMP4promoter prevents CRMP4expression and accordingly downregulates Sema3A expression,which may facilitate the binding of neuropilin-1with VEGF,thus promotes cell motility and angiogenesis.

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produced the EGFP-tagged CRMP4protein.These constructs were isolated from plasmid clones and sequenced on both strands with an autosequencer(model ABI377;PE Applied Biosystems).

Transfection and selection

pcDNA3.1-CRMP4and pEGFP-CRMP4plasmids were transfected into70%con?uent PC3cells with DMRIE-C reagent(Invitrogen Inc.)according to the manufacturer’s instructions.Other PC3cells were transfected with the pcDNA3.1(t)plasmids or pEGFP-c1vector containing no insert(mock transfected)as a control.Gentamicin(G418; Life Technologies,Inc.,Rockville,MD,USA)was added to 500ng/ml for the selection of stable transfectants.Selection medium was changed every3days for a3-week period.Clones of resistant cells were isolated and allowed to proliferate for further characterization.For transient transfections,70% con?uent cultures of LNCap and PC3cells were transfected with the pEGFP-CRMP4plasmid as above.Living cells were examined directly and photographed under inverted?uores-cence microscope,after48h.

Matrigel invasion assay

The invasive activities of transfected clones PC3-pcDNA3.1-CRMP4and LNCap-pcDNA3.1-CRMP4were examined by using a membrane invasion culture system,in which a polycarbonate membrane with8-m m pores(Nucleopore Corp., Pleasanton,CA,USA)coated with Matrigel at5mg/ml was placed between the upper and lower well plates of a membrane invasion culture system chamber.The mock transfected cells were used as a control(Cai et al.,2008).Similar data were obtained from at least three independent experiments.

In vivo metastasis assay

All procedures using animals were approved by and were in accordance with the guidelines of the Animal Care and Use Committee of Third Af?liated Hospital of Sun Yat-sen University.PC3cells expressing either empty vector(pEGFP-C1)or CRMP4expression vector(pEGFP-CRMP4)with GFP reporter were sorted and recollected with?uoresence-activated cell sorting(Becton Dickinson,Franklin Lakes,NJ,USA),and injected into the prostate of male BALB/c nude mice(n?9mice per group,6weeks of age,2?105cells per mouse).At45days after injection,mice were killed and the images of GFP intensity were taken(Singh et al.,2007).The prostate,lung,liver,kidney and pancreas were dissected from each mouse and GFP activities, representative of metastasis,in different organs including lung, liver,kidney and pancreas were recorded with in vivo Imaging System IVIS200(Xenogen,Alameda,CA,USA).

Detection of methylation of the CRMP4promoter Demethylation drug treatment.PC3cells were?rst cultured in 100-mm plates until40%conuent and subsequently treated with0.5,5and12.5m M5-aza-2-deoxycytidine(Sigma, St Louis,MO,USA)for5days,respectively.The cells were then collected for protein extraction by western blot analysis. DNA isolation

DNA was extracted from frozen tissue samples using universal genomic DNA extraction kit(TaKaRa Bio Inc.,Otsu,Shiga, Japan)according to the manufacturer’s instructions. Sequence analysis of promoter region

The promoter region(Matsuo et al.,2000),containingà940to the transcriptional start site,was ampli?ed by PCR.Genomic DNA was extracted from cancer samples,from metastatic PCa patients M3and M6,and from cell lines PC3,PC3M and LNCaP.The retrieved DNA was used directly as templates in PCR reactions consisting of0.02m M each of the forward (50-ATGGGGAGCAATAGCAGTTCTACTC-30)and reverse (50-AGCGGACAGGGAGCGAGCGAGG-30)primers using LA Taq polymerase(TaKaRa).The puri?ed products were then cloned into the TA cloning vector pMD18-T Simple Vector(TaKaRa),and20positive clones were randomly selected for sequence analysis.

Sodium bisul?te modi?cation of DNA

Bisul?te modi?cation of genomic DNA was performed as described previously(Clark et al.,1994;Herman et al.,1996), using MethylDetector Bisul?te Modi?cation Kit(Active Motif)and stored atà801C until use.

Bisul?te-sequencing PCR

In50-?anking sequences of CRMP4,two CpG islands were found using the program MethPrimer.CpG island1contained partial sequences of exon anaplastic oligodendroglioma,and CpG island2contained partial sequences of exon anaplastic oligodendroglioma and exon 1.The nested PCR was conducted to determine the methylation patterns of the CpG islands of CRMP4promoter using the outer and inner primers (Supplemental Table1).The puri?ed PCR products were subcloned into pMD19-T Simple Vector(TaKaRa),and10 positive clones of each product were randomly selected for sequence analysis.

MSP

MSP was used to detect the presence of methylated alleles for speci?c genes.According to the bisulfate-sequencing PCR results,the MSP primer pairs for CRMP4were designed complying with the standard rules(Li and Dahiya,2002; Brandes et al.,2007)to detect the methylation status of CpG island1.The?rst step and second step PCR were ampli?ed,as the same as bisulfate-sequencing PCR.The third step PCR were performed with a primer set speci?c to the methylated or unmethylated sequence(M or U set)described in Supple-mental Table2.PCR products were loaded onto3%agarose gels,stained with ethidium bromide and visualized under ultraviolet light.

Statistical analysis

To compare the relative abundance of individual protein spot features among multiple groups,we performed a one-way ANOVA followed by Newman–Keuls test.For comparisons between two groups,Student’s t test was used.One-way ANOVA followed by Newman–Keuls test was also used to evaluate the differences in western blot,real-time RT–PCR and IHC results.Data were analyzed using SPSS16.0software package(SPSS,Chicago,IL,USA)and a two-tailed P-value of o0.05was considered statistically signi?cant. Abbreviations

2D,two-dimensional;BPH,benign prostate hyperplasia; CRMP,collapsin response mediator protein;DIGE,?uores-cence difference gel electrophoresis;EGFP,enhanced green ?uorescent protein;GAPDH,glyceraldehyde3-phosphate dehydrogenase;IHC,immunohistochemistry;LNM,lymph node metastasis;MSP,methylation speci?c PCR;PCa, prostate cancer;VEGF,vascular endothelial growth factor.

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Con?ict of interest

The authors declare no con?ict of interest. Acknowledgements

We thank Professor Quentin Liu(State Key Laboratory of Oncology in South China,Cancer Center,Sun Yat-Sen University)for providing excellent technical assistances. Special thanks to Professor Zhou Zhu(Johns Hopkins University,School of Medicine,USA)for valuable suggestions and critical reading of this paper.This work was supported by Chinese National Natural Science Foundation30772178, 30973011,30901496,Key Project of Guangdong Provincial Science and Technology Research7117362,Key Project of Chinese Ministry of Health and Chinese National Hi-Tech Research and Development Program2007AA021906.

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Supplementary Information accompanies the paper on the Oncogene website (https://www.360docs.net/doc/5b4934106.html,/onc)

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