rab25 is a tumor suppressor gene with antiangiogenic and ... · recurrent changes at the levels of...

Tumor and Stem Cell Biology

Rab25 Is a Tumor Suppressor Gene with Antiangiogenic andAnti-Invasive Activities in Esophageal Squamous CellCarcinoma

Man Tong1, Kwok Wah Chan1, Jessie Y.J. Bao3, Kai Yau Wong1, Jin-Na Chen2, Pak Shing Kwan1,Kwan Ho Tang1, Li Fu2, Yan-Ru Qin4, Si Lok3, Xin-Yuan Guan2, and Stephanie Ma1

AbstractEsophageal squamous cell carcinoma (ESCC), the major histologic subtype of esophageal cancer, is a

devastating disease characterized by distinctly high incidences and mortality rates. However, there remainslimited understanding of molecular events leading to development and progression of the disease, which areof paramount importance to defining biomarkers for diagnosis, prognosis, and personalized treatment. Byhigh-throughout transcriptome sequence profiling of nontumor and ESCC clinical samples, we identified asubset of significantly differentially expressed genes involved in integrin signaling. The Rab25 gene implicatedin endocytic recycling of integrins was the only gene in this group significantly downregulated, and itsdownregulation was confirmed as a frequent event in a second larger cohort of ESCC tumor specimens byquantitative real-time PCR and immunohistochemical analyses. Reduced expression of Rab25 correlated withdecreased overall survival and was also documented in ESCC cell lines compared with pooled normal tissues.Demethylation treatment and bisulfite genomic sequencing analyses revealed that downregulation of Rab25expression in both ESCC cell lines and clinical samples was associated with promoter hypermethylation.Functional studies using lentiviral-based overexpression and suppression systems lent direct support ofRab25 to function as an important tumor suppressor with both anti-invasive and -angiogenic abilities,through a deregulated FAK–Raf–MEK1/2–ERK signaling pathway. Further characterization of Rab25 mayprovide a prognostic biomarker for ESCC outcome prediction and a novel therapeutic target in ESCCtreatment. Cancer Res; 72(22); 6024–35. �2012 AACR.

IntroductionEsophageal cancer ranks as the sixth leading cause of

cancer-related deaths worldwide, with distinctly high inci-dences and mortality rates particularly in East Asia, Africa,and North America (1). Esophageal squamous cell carcinoma(ESCC) is the most common form of esophageal cancer. Thedisease is characterized by regional variation in incidences.More than 50% of all ESCC cases in theworld occur in China. In

Linzhou and nearby cities in Henan Province of NorthernChina, ESCC constitutes more than 90% of all esophagealcancer cases in the area and has the highest incidences andmortality rates of esophageal cancer reported in the world (1,2). Despite advances in diagnostic techniques and therapeuticmodalities, ESCC remains a devastatingmalignancy due to latediagnoses and the aggressive nature of the disease. A betterunderstanding of the recurrent genetic alterations and under-lying molecular mechanisms involved in ESCC developmentand progression will facilitate the identification of novel tar-gets, allowing for more sensitive methods of detection, facil-itating earlier diagnosis, and prolonging patient survival.

With the advent of next-generation sequencing technologiesin recent years, a new sequencing platform, called transcrip-tome sequencing (RNA-Seq), has been applied to delineatechanges at the transcriptomic level. The development ofESCCs, like many other cancers, is believed to be driven bythe accumulation of genetic alterations, causing the transfor-mation of normal cells to malignant cells. Thus, studyingrecurrent changes at the levels of functional transcripts inmalignant cells compared with nontumor cells may aid in theidentification of deregulated molecular events and pathwaysinvolved in driving ESCCs. In the present study, we conductedRNA-Seq analysis on 12 patient-derived nontumor and ESCCclinical samples and identified a number of commonly and

Authors' Affiliations: Departments of 1Pathology and 2Clinical Oncology,3GenomeResearchCentre, Li Ka Shing Faculty ofMedicine, TheUniversityof Hong Kong, Hong Kong; and 4Department of Clinical Oncology, FirstAffiliated Hospital, Zhengzhou University, Zhengzhou, China

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

Accession code: Transcriptome sequencing data have been submitted toGeneExpressionOmnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) underthe accession number GSE32424.

Corresponding Author: Stephanie Ma, Department of Pathology, Fac-ulty of Medicine, The University of Hong Kong, Room 53, 10/F, Labo-ratory Block, Faculty of Medicine Building, 21 Sassoon Road, Pok FuLam, Hong Kong. Phone: 852-2819-9785; Fax: 852-2218-5244; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-12-1269

�2012 American Association for Cancer Research.

CancerResearch

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on September 24, 2020. © 2012 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst September 18, 2012; DOI: 10.1158/0008-5472.CAN-12-1269

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significantly differentially expressed genes. Pathway enrich-ment analysis (DAVID databases) found the deregulated genesto be commonly associated with a number of cancer-relatedpathways. Of these, 2 of the most significantly enriched path-ways are related to integrin signaling, which is commonlyknown to influence important cellular processes critical totumor development and progression, including cell prolifera-tion, cell survival, angiogenesis, cell motility, and invasiveness(3–6). Among the differentially expressed genes involved inintegrin signaling, Ras-related protein Rab25 was found to bethe only significantly downregulated gene in ESCC comparedwith nontumor tissue and was thus chosen for further studies.Rab25 belongs to the Rab family of small GTPases and plays acritical role in the maintenance of normal epithelial lining (7–9). Past studies have shownRab25 to play very contrasting rolesin cancer, depending on the tissue in which it is expressed. Ithas previously been implicated in the progression of ovarianand breast cancer (10–12), whereas in contrast, more recentstudies have identified a tumor-suppressive role of Rab25 incolon cancer and triple-negative breast cancer (13, 14). To date,the role of Rab25 in ESCCs has not been explored. In thepresent study, frequent Rab25 downregulation was identifiedin ESCC clinical specimens compared with its nontumorcounterparts, whereas reduced Rab25 expression significantlycorrelated with worst overall survival. Absent or significantlyweaker expression of Rab25 was also likewise detected in apanel of ESCC cell lines as compared with a pooled normaltissue control. Downregulation of Rab25 expression in bothESCC cell lines and clinical samples was found to be signifi-cantly associated with promoter hypermethylation, as evi-denced by our results obtained from 5-aza-20-deoxycitidine(5-aza-dC) demethylation treatment and bisulfite genomicsequencing (BGS). Finally, functional studies found Rab25 topossess both anti-invasive and antiangiogenic abilities througha dysregulated mitogen-activated protein kinase (MAPK)/extracellular signal–regulated kinase (ERK) signaling pathway.Taken together, our results suggest Rab25 to function as anovel tumor suppressor in ESCCs by repressing invasion,angiogenesis, and tumorigenicity.

Materials and MethodsCollection of esophageal tissue samplesAll clinical specimens used for RNA-Seq and Rab25 expres-

sion studies by quantitative real-time PCR (qPCR), Westernblotting, and immunohistochemistry were collected frompatients with ESCCs who underwent surgical resection oftumor tissues at Linzhou Cancer Hospital (Henan, China).When available, paired adjacent nontumor tissues from theproximal resection margins (>5 cm away from the ESCCsample) were also collected. The patients had received noprevious local or systemic treatment before operation. Allclinical samples used in this study were approved by thecommittee for ethical review of research involving humansubjects at Zhengzhou University (Zhengzhou, China) and TheUniversity of Hong Kong (Pok Fu Lam, Hong Kong). For RNA-Seq, 12 fresh-frozen clinical specimens (including 3 pairedtumor and nontumor, 4 unpaired tumor, and 2 unpairednontumor samples) were randomly selected for analysis. Sup-

plementary Table S1 provides a summary of the clinicopath-ologic parameters [i.e., patient age, gender, tumor–node–metastasis (TNM) grade] of each of the patients collected forthis purpose. Immunohistochemistry for Rab25was conductedon a tissue microarray (TMA) consisting of 270 pairs offormalin-fixed, paraffin-embedded ESCC tumor and nontumorspecimens (15).

RNA-Seq and differential expression analysisRNA-Seq was conducted on the Illumina Cluster Station and

GAIIx using the Standard Cluster Generation Kit v4 and the 36-Cycle Sequencing Kit v3. cDNA libraries from 7 ESCC samples(1T, 2T, 3T, 6T, 7T, 8T, 9T) and 5 nontumor samples (4N, 5N, 6N,8N, 9N) were sequenced with 38-base single reads. Sequencingreads were filtered for polymers, primer adaptors, and ribo-somal RNAs and then mapped against the human genomeassembly (NCBI Build 37.1) using CLC Genomic Workbench.The expression abundance for each gene was measured byRPKM (number of exon reads mapped per kilobase per millionmapped reads; ref. 16). The differential expression betweentumor and nontumor samples was evaluated using the t testand Baggerley test (17) by treating the same type of individualsas one group. The genes with a consistent type of regulation forboth the t test and Baggerley test were kept, and the genes witha Bonferroni corrected P less than 0.001 were considered to besignificant.

ESCC cell lines and culture conditionsESCC cell lines EC18 and EC109 were kindly provided by

Professor George Tsao (Department of Anatomy, The Univer-sity of Hong Kong). ESCC cell lines HKESC1 and KYSE520 wereprovided by Professor Gopesh Srivastava (Department ofPathology, The University of Hong Kong). The other 5 ESCCcell lines, KYSE30, KYSE140, KYSE180, KYSE410, and KYSE510,were obtained from DSMZ, the German Resource Centre forBiological Material (18). KYSE30 and KYSE180 cell lines werecultured in Dulbeccos' Modified Eagles' Media. All other ESCCcell lines were maintained in RPMI. Both media were supple-mented with 10% FBS and 1% penicillin/streptomycin. All celllines used in this study were regularly authenticated by mor-phologic observation and tested for absence of Mycoplasmacontamination (MycoAlert, Lonza Rockland).

RNA extraction, cDNA synthesis, and qPCRTotal RNA was isolated using TRIzol reagent (Invitrogen).

First-strand cDNA was synthesized from 1 mg of total RNAusing the Advantage RT-for-PCR kit (Clontech Laboratories)and was then used for qPCR analysis. qPCR was carried outusing SYBR Green PCRmaster mix (Applied Biosystems) on anABI Prism 7900HT System (Applied Biosystems). b-Actin wasamplified as an internal control. Supplementary Table S2provides a list of the primer sequence used to amplify Rab25in the qPCR assay.

DNA extraction, bisulfite modification, and promotermethylation analysis

Genomic DNA was extracted from normal and tumoresophageal tissues and cell lines by phenol–chloroform

Rab25 in Esophageal Cancer

www.aacrjournals.org Cancer Res; 72(22) November 15, 2012 6025




method followed by bisulfite modification using the EpiTECTBisulfite Kit (Qiagen). Methylation-specific PCR (MSP) andBGS were conducted as previously described (19) using pri-mers listed in Supplementary Table S2.

ImmunohistochemistryParaffin sections were deparaffinized in xylene and rehy-

drated in graded alcohols and distilled water. Slides wereheated for antigen retrieval in 10 mmol/L citrate (pH 6.0).Sections were incubated with polyclonal rabbit anti-humanRab25 (1:400, gift from Dr. Kwai Wa Cheng, University of TexasMD Anderson Cancer Center, Houston, TX), rat anti-humanCD34 (Biogenex), mouse anti-human PCNA (Santa Cruz Bio-technology), mouse anti-human cytokeratin 5/6 (Millipore), orrabbit anti-human phospho-ERK1/2 (Cell Signaling) antibo-dies overnight at 4�C. EnVision Plus System-HRP (DAB; Dako)was used according to manufacturer's instruction. Stainingwas revealed by counterstaining with hematoxylin. Stainedslides were scanned by Aperio Scanscope CS system (Vista).Evaluation of immunohistochemical staining for Rab25 wasconducted by pathologist K.W. Chan (Department of Pathol-ogy, TheUniversity ofHongKong)who hadnoprior knowledgeof patient data. Staining intensity was divided into 3 scores: no(0% staining), low (<50% staining), or high (>50% staining)Rab25 expression. Both relative intensity and number of cellsstained were taken into consideration during the scoringprocess. The specificity of polyclonal rabbit anti-human Rab25antibody has been verified and reported (10). Staining inten-sities of proliferating cell nuclear antigen (PCNA) and phos-pho-ERK1/2 were quantified using Aperio Spectrum positivepixel count algorithm. Microvessel density was assessed aspreviously described (20).

ResultsDifferential gene expression profiling by RNA-Seq

We sequenced 12 RNA libraries from 7 ESCC tumor and 5nontumor samples, generating a total of 91million 38-bp singlereads and an average of 7.6 million reads per sample (Supple-mentary Table S3). A total of 72% of the reads aligned with thehuman genome, of which 57% aligned with unique locations inthe genome. Across the 12 samples, a total of 14,351 distantgenes (42% of the total number of genes in the referencedatabase) were expressed with at least 10 mappable exonreads. For each gene, the expression value was measured onthe basis of RPKM (16). The Euclidean distance betweensamples was calculated, and a hierarchical clustering treebased on this distance showed a 2-branch (tumor vs. non-tumor) partition (Fig. 1A). The 5 nontumor (N) samples and 7tumor (T) samples clustered into 2 distinct groups, indicatingthat the gene expression between tumor and nontumor sam-ples differs significantly. Among the 7 tumor samples, samples1T, 6T, 7T, 8T, and 9T were more closely clustered than whencompared with 2T and 3T (Fig. 1A). This observation furthersuggests that RNA-Seq is a powerful technique in delineatingchanges at the transcriptomic level. To identify differentiallyexpressed genes of functional importance, the t test andBaggerley test were conducted to address the significance ofthe deregulated genes in tumor samples. Of the 14,351 repre-

sented genes, 1,598 genes were upregulated and 132 weredownregulated with a Bonferroni-corrected P less than 0.001(red dots in Fig. 1B). The global profile of the differentialexpression profiling for the 1,730 deregulated genes is shownas a heatmap (Fig. 1C), where the genes are listed in descendingorder according to the Baggerley test analysis. Subsequentpathway enrichment analysis by DAVID found the 1,730 dif-ferentially expressed genes to be commonly associated with anumber of cancer-related pathways. Of these, 2 of the mostsignificantly enriched pathways are related to integrin signal-ing (Fig. 1D). Supplementary Table S4 provides a full list of thesignificantly differentially expressed genes involved in integrinsignaling pathway (P < 0.001). Among these, Ras-related pro-tein Rab25 was found to be the only significantly downregu-lated gene (P< 1E-20) in ESCC comparedwith nontumor tissue(Fig. 1E and Supplementary Fig. S1) and was thus chosen forfurther studies. RPKM values obtained from RNA-Seq werereproducible upon qPCR analysis, showing higher Rab25expression in normal samples versus tumor samples (Fig.1F). The result indicates a high level of concordance of thedifferential expressionmeasurements between both platforms.

Rab25 as a novel tumor suppressor gene in humanESCCsTo determine whether downregulation of Rab25 was a

common event in ESCCs, we extended our qPCR analysis toan additional 43 paired nontumor/primary ESCC samples.Rab25 was significantly downregulated in tumor tissues whencompared with adjacent nontumor tissues (paired t test, P <0.0001; Fig. 2A). We also examined the expression of Rab25 atthe protein level by Western blot and immunohistochemicalanalyses. Of the 6 esophageal tissue samples examined byWestern blot analysis, Rab25 expressionwas consistently lowerin the 3 ESCC samples than in the 3 nontumor esophagealsamples (Fig. 2B). To investigate the clinical significance ofRab25 expression in ESCCs, a TMA comprising 270 pairednontumor/ESCC samples, with more detailed patient clinicaldata, was used for an IHC study. A strong Rab25 staining wasobserved in the nontumor samples (Fig. 2C). More than half ofall informative nontumor samples displayed a high Rab25expression level (89 of 171, 52.0%), whereas only a quarter ofthe informative tumor samples showed high Rab25 expression(42 of 164, 25.6%; Fig. 2D; c2 test, P < 0.001). In a Kaplan–Meiersurvival analysis comparing patients with different Rab25expression levels, higher Rab25 expression was significantlyassociatedwith a longer survival time (log-rank test, P¼ 0.009).Among 3 groups of patients with different Rab25 expressionlevels, increased survival was observed with enhanced Rab25expression (Fig. 2E). Patients with negative Rab25 expressionhad the worst prognosis, with a mean overall survival of 27.7months, whereas patients with lowRab25 expression displayeda relatively improved survival of 34.3 months. Patients withhigh Rab25 expression had the longest mean survival time of45.2 months.

The Rab25 promoter region is frequentlyhypermethylated in ESCCs

In addition to expression analysis in clinical samples, Rab25expression was also examined at both genomic and proteomic

Tong et al.

Cancer Res; 72(22) November 15, 2012 Cancer Research6026




levels in 8 esophageal cell lines, by qPCR and Western blotanalyses. Absent or significantly downregulated Rab25 expres-sion was observed in a panel of ESCC cell lines whencompared with a pooled normal tissue control (5 cases ofnontumor esophageal clinical tissue samples pooled togeth-er). In particular, Rab25 was found to be completely absentin the ESCC cell lines EC109 and KYSE520 (Fig. 3A and B).Downregulation of tumor suppressor genes in cancers isoften associated with hypermethylation and histone deace-tylation. To determine whether Rab25 downregulation wasassociated with epigenetic regulation in ESCCs, EC109 andKYSE520 cells that lacked Rab25 expression were treatedwith varying concentrations of the DNA methyltransferaseinhibitor (5-aza-dC) and/or the histone acetylation agents[trichostatin A (TSA) or valproic acid (VPA)] to investigate

the effects of DNA demethylation and histone acetylation onRab25 expression. As shown in Fig. 3C, qPCR analysisshowed a dose-dependent restoration of Rab25 expressionafter demethylation treatment with 5-aza-dC. Treatmentwith the histone acetylation agent TSA or VPA did notsignificantly alter Rab25 expression, and combined treat-ment with 10 mmol/L 5-aza-dC and 0.2 mmol/L TSA did notfurther enhance Rab25 expression. These studies support theidea that DNA methylation, but not histone modification, isinvolved in Rab25 inactivation in ESCC.

To substantiate the role of aberrant promoter hypermethy-lation in Rab25 silencing, we conducted MSP or BGS toinvestigate the methylation status of the Rab25 promoterregion. The 10-kb sequence directly upstream of the Rab25gene was analyzed by 2 publicly available databases for

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Figure 1. RNA-Seq expression profiling data. A, cladogram clustering diagram based on the gene expression values for 12 ESCC and nontumor samples.B, volcano plot of the P value as a function of weighted fold-change for the 5 nontumor versus 7 tumor samples. Blue dots represent genes not significantlydifferentially expressed (Bonferroni-corrected P > 0.001) and red dots represent genes significantly differentially expressed (Bonferroni-correctedP� 0.001). C, heatmap of expression profiles for the 1,730 genes that showed significant expression changes (1,598 upregulated and 132 downregulated).D, pathway enrichment analysis of significantly differentially expressed genes. E, expression tiling of Rab25 in 3 pairs of matched ESCCs andcorresponding nontumor clinical samples. F, qPCR analysis of the relative expression of Rab25 in the original 12 ESCC and nontumor clinical samplesused for RNA-Seq. Table shows the RPKMexpression values of the 12 samples analyzed by RNA-Seq. Validation study to determine the correlation betweenqPCR and RNA-Seq data.






potential CpG islands: CpG Island Searcher (http://www.cpgis-lands.com) and EMBOSS-CpGPlot (http://www.ebi.ac.uk/Tools/emboss/cpgplot). Three common CpG islands werepredicted by both programs, including the regions at �8,217to �6,690 (CpG I1), �6,689 to �5,717 (CpG I2), and �1,287 to�453 (CpG I3) 50 upstream of Rab25 (Fig. 4A). In addition,because a recent study also found the sequence from �173 toþ17 (CpG I4) to contain the core promoter region of theRab25 gene (21), we also investigated the methylation statusin this region (Fig. 3D). We conducted MSP at CpG I1 andBGS at CpG I2 and CpG I3; however, no difference inmethylation status between Rab25 expressing or absent

ESCC cell lines could be detected in these predicted CGdinucleotide–rich regions (data not shown). But BGS at CpGI4, which contains the promoter region of Rab25 (21),showed a high density of methylation in Rab25-absent EC109and KYSE520 cell lines. In contrast, methylation was rarelydetected in the same CpG sites in the Rab25-expressingKYSE30 cell line (Fig. 3D and E). As compared with untreatedEC109, methylation was significantly reduced in EC109 cellstreated with 5-aza-dC (Fig. 3E). In addition, we also inves-tigated the methylation status in a pair of matched ESCCsand nontumor clinical sample. Rab25 expression was foundto be significantly downregulated in ESCC as compared with

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Tong et al.





nontumor in this case as confirmed by qPCR analysis. ESCCsample was found heavily methylated (75%–100% methyla-tion) in 18 of 21 CG dinucleotides examined, whereas thenontumor sample showed significantly lower levels of meth-ylation (0%–25% methylation) in 17 of 21 CG dinucleotidesexamined (Fig. 3E, representative shown). Taken together,these observations provide strong evidence in support of the

notion that DNA promoter hypermethylation is implicatedin Rab25 inactivation in human ESCCs.

Rab25 suppresses in vitro migration, invasion, andangiogenesis in ESCCs

Frequent epigenetic silencing of Rab25 in ESCC cell lines andhuman ESCC samples prompted us to further investigate the

Figure 3. Rab25 promoter region isfrequently hypermethylated inESCCs. A and B, measurement ofgenomic and proteomic Rab25expression levels in a panel of ESCCcell lines compared with a poolednormal tissue control by qPCR andWestern blotting. C, qPCR analysisof Rab25 expression afterdemethylation and/or histoneacetylation treatment with 5-aza-dC(blue bars), TSA (pink bars), and/orVPA (green bars) in ESCC cell lineswithout Rab25 expression. Double(gray bars), combined 10 mmol/L 5-aza-dC and 200 nmol/L TSAtreatment. D, a schematic diagramshowing the distribution of 4predicted CpG islands 50-upstreamof the Rab25 gene. The CpG islandscover the regions �8,217 to �6,690(CpG I1),�6,689 to�5,717 (CpG I2),�1,287 to �453 (CpG I3), and �173to þ17 (CpG I4). MSP wasconducted at CpG I1 and BGS wasconducted at CpG I2, CpG I3, andCpG I4. Specifically, 23 CpGdinucleotides are present at CpG I4.BGS primers (4BGS-F and 4BGS-R)were designed to amplify thesequence in this region.Transcription factor–binding sites forSp1 and CRE are predicted withinthis core promoter region. E,mapping of themethylation status ofCpGdinucleotides within CpG I4 in acase of matched ESCC andnontumor samples, Rab25-expressing KYSE30 cells, Rab25-negative KYSE520 and EC109 cells,and EC109 cells treated with 5-aza-dC by BGS. The percentage ofmethylation at each CpGdinucleotide is displayed in the piecharts.

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function of Rab25 in ESCCs. To assess whether Rab25 mightpossess a tumor-suppressive function, EC109 cells withoutRab25 expression were stably transduced with lentiviruspackaged with either a Rab25-expressing vector or an emptyvector (EV) control to generate Rab25-overexpressing(EC109 Rab25) or control cells (EC109 EV). Similarly, KYSE30cells, exhibiting high levels of Rab25 expression, were lenti-virally transduced with either Rab25 short hairpin RNA(shRNA; clones 849 and 852) or a non-target control (NTC)sequence to generate cells with Rab25 stably repressed(KYSE30 shRNA 849 or 852) or control cells (KYSE30 NTC).Stable Rab25 overexpression and knockdown were con-firmed at both mRNA and protein levels by qPCR andWestern blotting, respectively (Fig. 4A and B). Stable Rab25overexpression significantly reduced the ability of the cellsto migrate and invade through Transwell chambers. Con-

versely, suppressing Rab25 expression resulted in an oppos-ing effect (Fig. 4C). Next, we also investigated whetheroverexpression of Rab25 has a suppressive effect on angio-genesis. Human umbilical vein endothelial cells (HUVEC)were treated with conditioned media collected from Rab25overexpressed or suppressed cells and their respective con-trols. Morphologic changes and differential tube-formingabilities of HUVECs following treatment with conditionedmedia are shown in Fig. 4D. HUVECs displayed elongatedtube-like structures following treatment with conditionedmedia from Rab25 suppressed clones 849 and 852, as com-pared with control cell medium treatment. Conversely,treatment with conditioned media from Rab25-overex-pressed cells displayed a contrasting effect. These observa-tions suggest a role for Rab25 in the regulation of invasion,migration, and angiogenesis in ESCCs.

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per

field

No. of tu

bes fo

rmed

8.7

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EC109 KYSE30

EC109

EV NTC 849 852Rab25

EV

Invasion

Migration

NTC 849 852Rab25

EV NTC 849 852Rab25

EV NTC 849 852Rab25

EV NTC 849 852Rab25

KYSE30

EC109 KYSE30

EC109 KYSE30

EC109 KYSE30

EV NTC 849 852Rab25

EC109 KYSE30

EV NTC 849 852Rab25

EC109 KYSE30

102

23

41

25

15

10

14

0

109

2833

58

108

187

Figure 4. Rab25 from ESCC cell lines suppressesmigration, invasion, and angiogenesis in vitro. Stable repression or overexpression of Rab25 in KYSE30 andEC109 cells, respectively, by lentiviral transduction was confirmed by qPCR (A) and Western blotting (B). C, representative migration and invasionassays in Rab25-overexpressed or -repressed clones as compared with their controls. Bar chart summary of the number of migrated or invaded cellsper field (�, P < 0.05). D, representative images showing morphologic changes and differential tube-forming abilities of HUVECs following treatmentwith conditioned media from Rab25-overexpressed or -repressed clones as compared with their controls. Bar chart summary showing the number of tubesformed in each assay (��, P � 0.01; ��, P � 0.001).

Tong et al.





Rab25 suppresses in vivo tumor formation andangiogenesis in ESCCsAn in vivo mouse model was further used to support our

findings in vitro. Tumor formation ability of Rab25-overex-pressed clone was significantly subdued as compared with EVcontrol cells (Fig. 5A, left). On the contrary, mice injected withRab25-repressed clone formed larger and more tumors thanNTC cells (Fig. 5A, right). Serial sections from the xenografttumors were then subjected to hematoxylin and eosin (H&E)staining as well as IHC analysis. Histologic analysis and CK5/6IHC staining found that tumors formed with EC109 Rab25,EC109 EV, and KYSE30 shRNA 852 cells resembled an ESCCphenotype, whereas xenografts formed fromKYSE30 NTC cellswere composed mostly of necrotic cells and fibroblasts. Xeno-graft tissue sections were also examined for PCNA and CD34expression by IHC. As compared with EC109 Rab25–overex-pressing xenografts, xenografts generated with EV cells dis-played an enhanced PCNA and CD34 expression, indicative of

increased cell proliferation and microvessel density, respec-tively. Conversely, reduced PCNA and CD34 expression wereobserved in KYSE30 NTC xenografts compared with Rab25-repressed tumors (Fig. 5B).

Rab25 drives ESCCs through a deregulated MAPK/ERKsignaling pathway

Rab25 has previously been reported to control recycling ofb1 integrin (11–13). Focal adhesion kinase (FAK), which loca-lizes with b-subunit of integrins, is the major and mostextensively studied downstream player activated by integrins.Activation and autophosphorylation of FAK has previouslybeen shown to be critical in driving a number of cancerprocesses including promoting cell survival, cell proliferation,and cell motility, through regulating downstream pathwaysincluding the MAPK/ERK pathway (4, 22–24). In view of theimportance of MAPK/ERK pathway in regulating cell invasive-ness and angiogenesis, we therefore examined whether Rab25

A EC109 EV (4/5) KYSE30 shRNA 852 (5/6)

EC109 Rab25 (2/5)

H&E

(×100)

CK5/6

(×200)

*

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PCNA

(×200)

CD34

(×100)

KYSE30 NTC (3/6)

EC109 EV KYSE30 shRNA 852EC109 Rab25 KYSE30 NTCB

300

200

100

0

PC

NA

sta

inin

g index

50

40

30

20

10

0Mic

rove

ssel density

EV NTC 852Rab25

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EV NTC 852Rab25

EC109 KYSE30

Figure 5. Rab25 suppresses in vivo tumor initiation and angiogenesis in ESCCs. A, representative images of the subcutaneous tumors formed in nude micefollowing injection of Rab25-overexpressed or -repressed clones and their respective controls. Tumor incidence denoted in parentheses. B, H&E andIHC staining for CK5/6, PCNA, and CD34 expression in the resected xenografts. Bar chart summary of average staining intensity of PCNA and microvesseldensity in 5 randomly selected "hot spot" regions (�, P < 0.05; ��, P � 0.01).






could regulate the expression of activated kinases in thisparticular signaling cascade. As compared with EV control,Rab25 overexpression resulted in a reduction of phosphory-lated FAK and c-Raf with a concomitant decrease in thedownstream phosphorylation of MEK1/2 and ERK (Fig. 6A).Conversely, increased phosphorylation of FAK, c-Raf, MEK1/2,and ERK was detected in Rab25-suppressed cells, as comparedwith its respective NTC (Fig. 6A). Interestingly, IHC staining inthe resected xenografts likewise showed a reduced expressionof phospho-ERK in Rab25-overexpressing xenografts. Con-versely, tumors formed with Rab25-repressed cells displayedan enhanced expression of phospho-ERK (Fig. 6B). Takentogether, our results suggested that Rab25 exerts its tumor-suppressive function in ESCCs through regulating the MAPK/ERK signaling pathway (Fig. 6C).

DiscussionPast studies on the genetic alterations in ESCCs by our group

and other research laboratories have mainly been focused on

investigating differential gene expression, chromosomal aber-rations, and LOH using microarrays, comparative genomichybridization, and microsatellite DNA marker analysis tech-niques (25–28). In recent years, the advent of next-generationsequencing technologies has provided a new platform forgenerating vast amounts of data at genomic, epigenomic,transcriptomic, and proteomic levels by means of a variety ofhigh-throughput technologies (29). In particular, the recentdevelopment of RNA sequencing has provided a new approachfor mapping and quantifying transcriptomes (30). Direct tab-ulation of the transcriptome by RNA-Seq has advantages overexisting technologies in several ways. First, unlike hybridiza-tion-based approaches, RNA-Seq is not limited to detectingknown genes but can also include the detection of noveltranscripts and alternative splice forms (provided sequencingis conducted to a sufficient depth). A second advantage of RNA-Seq relative to DNA microarrays is that RNA-Seq has highsensitivity for detecting transcripts of low expressed genes andthatmany sequencing reads can be unambiguously assigned to

A

B

C

ββ α

Rab25

FAK

Raf

MEK

ERK

Migration

invasionTumorigenesis Angiogenesis

P

P

PP

PP

EC109

EV NTC 849 852Rab25

1.0 1.0 1.42 1.950.54

1.0 1.0 0.93 1.021.09

1.0 1.0 1.49 2.090.65

1.0 1.0 0.93 0.990.98

1.0 1.0 2.14 1.760.66

1.0 1.0 4.45 3.510.64

1.0 1.0 0.93 0.850.99

1.0 1.0 1.04 1.011.05

KYSE30

** **

200

150

100

50

0p-E

RK

sta

inin

g index

p-E

RK

(×2

00)

EV NTC 852Rab25

EC109 KYSE30

EC109 EV

KYSE30 shRNA 852

EC109 Rab25

KYSE30 NTC

p-FAK

Total FAK

p-c-Raf (Ser259)

p-MEK1/2 (Ser217/221)

p-ERK (Thr202/Tyr204)

Total c-Raf

Total ERK

ββ-Actin

Figure6. Rab25drivesESCC tumorigenesis via aderegulatedMAPK/ERKsignalingpathway.A, detectionof expression of phospho-FAK, total FAK, phospho-c-Raf (Ser259), total c-Raf, phospho-MEK1/2, phospho-ERK, and total ERK in Rab25-overexpressed or -repressed clones and their respectivecontrols by Western Blotting. b-Actin was used as a loading control. B, phospho-ERK IHC staining in the resected xenografts generated fromRab25-overexpressed (EC109 Rab25) or -suppressed (KYSE30 shRNA 852) cells and their respective control cells. Bar chart summary of averagestaining intensity of phospho-ERK in 5 randomly selected "hot spot" regions (��,P� 0.01). C, a schematic diagram illustrating the proposed Rab25-regulatedtumor-suppressive mechanism in ESCC tumorigenesis.

Tong et al.





the genome. Finally, it is also shown to have high levels ofreproducibility. RNA-Seq has now been successfully appliedin the study of various disease models including Alzheimerdisease (31), leukemia (32), prostate (33, 34), and breast (35)cancers but not yet ESCCs. In this present study, RNA-Seqwas used to investigate differential gene expression in 12nontumor and ESCC clinical samples. Using a stringentstatistical cutoff of P < 0.001, 1,730 commonly differentiallyexpressed genes were identified. Pathway and Gene Ontog-eny analysis by DAVID in the 1,730 genes found 2 of the top 7most enriched pathways to be associated with integrinsignaling (i.e., integrin cell surface interactions and integrinsignaling pathway), suggesting that this pathway should playa critical role in driving ESCC tumorigenesis. Integrin sig-naling has been extensively shown in the past to controlinitiation and progression of various cancer types. And thisis also true in the context of ESCCs, as a number of theidentified differentially expressed genes involved in integrinsignaling in this study (listed in Supplementary Table S4)have already been previously implicated in ESCC pathogen-esis. These include CAV1, ROCK1, ITGB1, ICAM1, VCL, PXN,and SHC1 (36–44). Here, we report the identification andcharacterization of a tumor suppressor gene Rab25 inESCCs, which was found to be significantly downregulatedin the integrin signaling–associated pathway.Rab25, with an epithelial distribution, was first discovered

with its expression enriched in the normal gastrointestinalmucosa, kidney, and lung but absent in the brain, liver, andskeletal muscle (7). It is a complex molecule that has beendescribed as an intracellular transport protein (45, 46). Morerecent studies have also shown that it is a tumor modulator asit can regulate tumor growth and angiogenesis. A handful ofstudies have now been conducted to investigate the deregu-lated expression of Rab25 in different cancer types includingovarian (10, 47), breast (14, 48), and colon cancers (13). On thebasis of these studies, Rab25 seems to display very contra-dicting roles dependent on cellular context. Concordant withfindings in triple-negative breast and colon cancers (13, 14, 48),we also found Rab25 to function as a tumor suppressor inESCCs. And as Nam and colleagues reported for colon cancer(13), Rab25 was also found to have prognostic value in ESCCs.Cheng and colleagues have previously reported that Rab25 canact through multiple pathways to enhance apoptosis and tosuppress angiogenesis and invasion in triple-negative breastcancer by modulating VEGF-A and VEGFR-1 expression (14).Contrary to their findings, our preliminary findings by qPCRshow unaltered VEGF-A and VEGFR-1 expression in Rab25-overexpressed or -repressed ESCC cells (data not shown).However, we did identify another pathway by which Rab25mediates tumorigenicity, metastasis, and angiogenesis inESCC—through a deregulated FAK-Raf-MEK1/2-ERK signal-ing. This study is also the first comprehensive study to identifya role of promoter hypermethylation in the inactivation ofRab25 expression in ESCCs. It is interesting to note that Rab25has been suggested, along with a number of other genes(CDCA8, ATAD2, AURKA) to have a strong correlation formethylation-dependent expression changes in ovarian cancer;although data are yet to be validated (49). On the contrary, loss

of Rab25 expression as a result of chromosome 1q22-23mutation has previously been suggested for breast cancer celllines (48), whereas upregulated Rab25 expression has beensuggested to be associated with chromosome amplification inovarian cancer (10) and PKA-dependent regulation of theproximal promoter in gastric cancer cell line model (21). Ourpreliminary studies of DNA copy number in 2 ESCC cell lines(KYSE520 and EC109) and 2 ESCC clinical samples with no orlow Rab25 expression suggested that downregulation of Rab25is not associated with deletion of the Rab25 gene (Supplemen-tary Fig. S2). Although there is no solid explanation to con-clusively explain the different outcomes observed by differentgroups with regard to Rab25 expression, its regulation, itsfunctional role, and its mediated pathways, several importantfactors should be considered. Most of the data that indicateRab25 as an oncogene is derived from studies in ovarian andbreast cancers, which is in a completely different cellularcontext from ESCCs. The stage of the disease can alsodetermine whether a certain gene acts as a tumor promoteror suppressor. For instance, TGF-b has been shown to act asa tumor suppressor in early stages of pancreatic cancer butthen switches to become an oncogene in metastatic pan-creatic cancer (50). Furthermore, the cellular/mechanisticpathway most impacted may also be vital in determining thenature of the action of Rab25 in different cellular models. Forinstance, Cheng and colleagues found Rab25 to exert tumor-suppressive properties in MDA-MB-231 breast cancer cellsin part through effects on VEGF-A secretion and VEGFR-1expression (14), whereas our present study found Rab25 toexert tumor-suppressive effects in ESCCs through a deregu-lated MAPK/ERK pathway. In ovarian and breast cancers,however, Rab25 has been shown to possess oncogenic func-tions through suppressing Bak/Bax pro-apopototic mole-cules and activation of PI3K/Akt pathway (10). These factorsmay potentially contribute to determine whether Rab25promotes or impedes tumor growth. Regardless, all thesedata do suggest a multifunctional role of Rab25 in differentcellular contexts and either a gain or loss of Rab25 expres-sion could potentially lead to tumorigenesis in differentorgan models.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M. Tong, K.W. Chan, S. Lok, X.-Y. Guan, S. MaDevelopment of methodology: M. Tong, K.Y. Wong, J.-N. ChenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):M. Tong, K.W. Chan, K.Y.Wong, J.-N. Chen, P.S. Kwan,K.H. Tang, Y.-R. Qin, S. Lok, S. MaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Tong, K.W. Chan, J.Y.J. Bao, S. Lok, S. MaWriting, review and/or revision of themanuscript:M. Tong, K.W. Chan, J.Y.J. Bao, S. Lok, X.-Y. Guan, S. MaAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases):K.W. Chan, P.S. Kwan, L. Fu, Y.-R. Qin, SLok, S. MaStudy supervision: K.W. Chan, X.-Y. Guan, S. Ma

AcknowledgmentsThe authors thank Dr. Kwai Wa Cheng of University of Texas MD Anderson

Cancer Center (Houston, TX) for his generous gift of the monoclonal rabbit anti-human Rab25 antibody.






Grant SupportThis work was supported by a grant from the National Natural Science

Foundation of China (30971606) and the Sun Yat-Sen University "HundredTalents Program" (85000–3171311).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received April 3, 2012; revised July 23, 2012; accepted September 5, 2012;published OnlineFirst September 18, 2012.

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2012;72:6024-6035. Published OnlineFirst September 18, 2012.Cancer Res Man Tong, Kwok Wah Chan, Jessie Y.J. Bao, et al. Anti-Invasive Activities in Esophageal Squamous Cell CarcinomaRab25 Is a Tumor Suppressor Gene with Antiangiogenic and

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