identification and characterization of rab25 as a novel...

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Identification and Characterization of Rab25 as a Novel Tumor Suppressor Gene with

Anti-Angiogenic and Anti-Invasive Activities in Esophageal Squamous Cell Carcinoma

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

Departments of 1Pathology and 2Clinical Oncology, 3Genome Research Centre, Li Ka Shing Faculty of

Medicine, The University of Hong Kong, Hong Kong; 4Department of Clinical Oncology, First Affiliated

Hospital, Zhengzhou University, Zhengzhou, China

Corresponding author: Stephanie Ma, Department of Pathology, Faculty of Medicine, The University of

Hong Kong, Room 53, 10/F, Laboratory Block, Faculty of Medicine Building, 21 Sassoon Road, Pok Fu

Lam, Hong Kong. Phone: +852-2819-9785; Fax: +852-2218-5244; Email: [email protected]

Running title: Rab25 in esophageal cancer.

Conflict of interest statement: None declared.

Authors’ contributions:

Conception and design: M Tong, S Ma, XY Guan

Acquisition of data: M Tong, KY Wong, JN Chen, KH Tang, S Ma

Analysis and interpretation of data: M Tong, S Ma, JY Bao, KW Chan

Writing, review and/or revision of the manuscript: M Tong, S Ma

Administrative, technical, or material support: L Fu, YR Qin, S Lok, PS Kwan

Study supervision: S Ma, XY Guan

Accession code: Transcriptome sequencing data have been submitted to Gene Expression Omnibus (GEO,

http://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE32424.

Keywords: Rab25, esophageal carcinoma, hypermethylation, angiogenesis, RNA-Seq, transcriptome

sequencing

on May 18, 2018. © 2012 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 18, 2012; DOI: 10.1158/0008-5472.CAN-12-1269

http://cancerres.aacrjournals.org/

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Abstract

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

devastating disease characterized by distinctly high incidences and mortality rates. Better understanding

of the molecular events leading to development and progression of the disease is of paramount importance

in identifying putative biomarkers for diagnosis, prognosis and personalized treatment. By high-

throughout transcriptome sequencing (RNA-Seq) profiling of twelve non-tumor and ESCC clinical

samples, we identified a subset of significantly differentially expressed genes involved in integrin

signaling. Of these, Rab25 was found to be the only gene significantly down-regulated and was

subsequently characterized as a novel tumor suppressor critical in ESCC development. In addition to the

samples used for the original RNA-Seq screening, frequent down-regulation of Rab25 expression was

also confirmed in a larger cohort of ESCC tumor specimens using qPCR and immunohistochemistry

analyses. Reduced expression of Rab25 was also found to be correlated with decreased overall survival.

Absent or weaker expression of Rab25 was detected in a panel of ESCC cell lines as compared to a

pooled normal tissue control. Demethylation treatment and bisulfite genomic sequencing analyses found

down-regulation of Rab25 in both ESCC cell lines and clinical samples to be associated with promoter

hypermethylation. Functional studies using lentiviral-based overexpression and suppression systems lent

direct support of Rab25 to function as an important tumor suppressor with both anti-invasive and anti-

angiogenic abilities, through a deregulated FAK-Raf-MEK1/2-ERK signaling pathway. Further

characterization of Rab25 may provide a prognostic biomarker for ESCC outcome prediction and a novel

therapeutic target in ESCC treatment.




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Introduction

Esophageal cancer ranks as the sixth leading cause of cancer-related deaths worldwide, with distinctly

high incidences 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. The disease is

characterized by regional variation in incidences. More than 50% of all ESCC cases in the world occur in

China. In Linzhou and nearby cities in Henan Province of Northern China, ESCC constitutes over 90% of

all esophageal cancer cases in the area, and has the highest incidences and mortality rates of esophageal

cancer reported in the world (1-2). Despite advances in diagnostic techniques and therapeutic modalities,

ESCC remains a devastating malignancy due to late diagnoses and the aggressive nature of the disease. A

better understanding of the recurrent genetic alterations and underlying molecular mechanisms involved

in ESCC development and progression will facilitate the identification of novel targets, allowing for more

sensitive methods of detection, facilitating earlier diagnosis and prolonging patient survival.

With the advent of next-generation sequencing technologies in recent years, a new sequencing platform,

called transcriptome sequencing (RNA-Seq), has been applied to delineate changes at the transcriptomic

level. The development of ESCC, like many other cancers, is believed to be driven by the accumulation of

genetic alterations, causing the transformation of normal cells to malignant cells. Thus, studying recurrent

changes at the levels of functional transcripts in malignant cells compared to non-tumor cells may aid in

the identification of deregulated molecular events and pathways involved in driving ESCC. In the present

study, we performed RNA-Seq analysis on twelve patient-derived non-tumor and ESCC clinical samples

and identified a number of commonly and significantly differentially expressed genes. Pathway

enrichment analysis (DAVID databases) found the deregulated genes to be commonly associated with a

number of cancer-related pathways. Of these, two of the most significantly enriched pathways are related

to integrin signaling, which is commonly known to influence important cellular processes critical to tumor

development and progression, including cell proliferation, cell survival, angiogenesis and cell motility and

invasiveness (3-6). Among the differentially expressed genes involved in integrin signaling, Ras-related

protein Rab25 was found to be the only significantly down-regulated gene in ESCC compared to non-

tumor tissue; and was thus chosen for further studies. Rab25 belongs to the Rab family of small GTPases

and plays a critical role in the maintenance of normal epithelial lining (7-9). Past studies have shown

Rab25 to play very contrasting roles in cancer, depending on the tissue in which it is expressed. It has

previously been implicated in the progression of ovarian and breast cancer (10-12), while in contrast,

more recent studies have identified a tumor suppressive role of Rab25 in colon cancer and triple-negative




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breast cancer (13-14). To date, the role of Rab25 in ESCC has not been explored. In the present study,

frequent Rab25 down-regulation was identified in ESCC clinical specimens compared to its non-tumor

counterparts, while reduced Rab25 expression significantly correlated with worst overall survival. Absent

or significantly weaker expression of Rab25 was also likewise detected in a panel of ESCC cell lines as

compared to a pooled normal tissue control. Down-regulation of Rab25 expression in both ESCC cell

lines and clinical samples was found to be significantly associated with promoter hypermethylation, as

evidenced by our results obtained from 5-aza-2’-deoxycitidine (5-aza-dC) demethylation treatment and

bisulfite genomic sequencing (BGS). Finally, functional studies found Rab25 to possess both anti-

invasive and anti-angiogenic abilities through a dysregulated MAPK/ERK signaling pathway. Taken

together, our results suggest Rab25 to function as a novel tumor suppressor in ESCC by repressing

invasion, angiogenesis and tumorigenicity.




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Materials and methods

Collection of esophageal tissue samples

All clinical specimens used for RNA-Seq and Rab25 expression studies by qPCR, Western blot and

immunohistochemistry were collected from ESCC patients who underwent surgical resection of tumor

tissues at Linzhou Cancer Hospital (Henan, China). When available, paired adjacent non-tumor tissues

from the proximal resection margins (over 5 cm away from the ESCC sample) were also collected. The

patients had received no previous local or systemic treatment prior to operation. All clinical samples used

in this study were approved by the committee for ethical review of research involving human subjects at

Zhengzhou University and The University of Hong Kong. For RNA-Seq, twelve fresh frozen clinical

specimens (including 3 paired tumor and non-tumor, 4 unpaired tumor and 2 unpaired non-tumor samples)

were randomly selected for analysis. Supplemental Table 1 provides a summary of the clinico-

pathological parameters (i.e. patient age, gender, TNM grade) of each of the patients collected for this

purpose. Immunohistochemistry for Rab25 was performed on a tissue microarray (TMA) consisting of

270 pairs of formalin-fixed, paraffin-embedded ESCC tumor and non-tumor specimens (15).

RNA-Seq and differential expression analysis

RNA-Seq was performed 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 non-tumor samples (4N, 5N, 6N, 8N, 9N) were sequenced with 38-base single

reads. Sequencing reads were filtered for polymers, primer adaptors, and ribosomal RNAs and then

mapped against the human genome assembly (NCBI Build 37.1) using CLC Genomic Workbench. The

expression abundance for each gene was measured by RPKM (number of exon reads mapped per kilobase

per million mapped reads) (16). The differential expression between tumor and non-tumor samples was

evaluated using the t-test and Baggerley’s test (17) by treating the same type of individuals as one group.

The genes with a consistent type of regulation for both the t-test and Baggerley’s test were kept, and the

genes with a Bonferroni corrected p-value less than 0.001 were considered to be significant.

ESCC cell lines and culture conditions

ESCC cell lines EC18 and EC109 were kindly provided by Professor George Tsao (Department of

Anatomy, The University of Hong Kong). ESCC cell lines HKESC1 and KYSE520 were provided by

Professor Gopesh Srivastava (Department of Pathology, The University of Hong Kong). The other five

ESCC cell lines, KYSE30, KYSE140, KYSE180, KYSE410 and KYSE510 were obtained from DSMZ




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(Braunschweig, Germany), the German Resource Centre for Biological Material (18). KYSE30 and

KYSE180 cell lines were cultured in DMEM. All other ESCC cell lines were maintained in RPMI. Both

media were supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. All cell lines

used in this study were regularly authenticated by morphological observation and tested for absence of

Mycoplasma contamination (MycoAlert, Lonza Rockland, Rockland, ME).

RNA extraction, cDNA synthesis and quantitative real-time PCR (qPCR)

Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA). First-strand complementary

DNA (cDNA) was synthesized from 1 μg of total RNA using the Advantage RT-for-PCR kit (Clontech

Laboratories, Mountain View, CA) and was then used for qPCR analysis. qPCR was performed using

SYBR Green PCR master mix (Applied Biosystems, Carlsbad, CA) on an ABI Prism 7900HT System

(Applied Biosystems). �-actin was amplified as an internal control. Supplemental Table 2 provides a list

of the primer sequence used to amplify Rab25 in the qPCR assay.

DNA extraction, bisulfite modification and promoter methylation analysis

Genomic DNA was extracted from normal and tumor esophageal tissues and cell lines by phenol-

chloroform method followed by bisulfite modification using the EpiTECT Bisulfite Kit (Qiagen, Valencia,

CA). Methylation-specific PCR (MSP) and bisulfite genomic sequencing (BGS) were performed as

previously described (19) using primers listed in Supplemental Table 2.

Immunohistochemistry (IHC)

Paraffin sections were deparaffinized in xylene and rehydrated in graded alcohols and distilled water.

Slides were heated for antigen retrieval in 10mM citrate (pH 6.0). Sections were incubated with

polyclonal rabbit anti-human Rab25 (1:400, gift from Dr. Kwai Wa Cheng, University of Texas MD

Anderson Cancer Center), rat anti-human CD34 (Biogenex, San Ramon, CA), mouse anti-human PCNA

(Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human cytokeratin 5/6 (Millipore, Billerica,

MA) or rabbit anti-human phospho-ERK1/2 (Cell Signaling, Danvers, MA) antibodies overnight at 4oC.

EnVision Plus System-HRP (DAB) (Dako, Denmark) was used according to manufacturer’s instruction.

Staining was revealed by counter-staining with hematoxylin. Stained slides were scanned by Aperio

Scanscope® CS system (Vista, CA). Evaluation of immunohistochemical staining for Rab25 was

performed by pathologist Chan KW (Department of Pathology, The University of Hong Kong) who had

no prior knowledge of patient data. Staining intensity was divided into three scores: no (0% staining), low




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(less than 50% staining) or high (greater than 50% staining) Rab25 expression. Both relative intensity and

number of cells stained was taken into consideration during the scoring process. The specificity of

polyclonal rabbit anti-human Rab25 antibody has been verified and reported (10). Staining intensities of

PCNA and phospho-ERK1/2 were quantified using Aperio Spectrum® positive pixel count algorithm.

Microvessel density was assessed as previously described (20).




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Results

Differential gene expression profiling by RNA-Seq

We sequenced twelve RNA libraries from 7 ESCC tumor and 5 non-tumor samples, generating a total of

91 million 38-bp single reads and an average of 7.6 million reads per sample (Supplemental Table 3). A

total of 72% of the reads aligned with the human genome, of which 57% aligned with unique locations in

the genome. Across the twelve samples, a total of 14,351 distant genes (42% of the total number of genes

in the reference database) were expressed with at least 10 mappable exon reads. For each gene, the

expression value was measured based on RPKM (16). The Euclidean distance between samples was

calculated, and a hierarchical clustering tree based on this distance showed a two-branch (tumor vs. non-

tumor) partition (Fig. 1A). The 5 non-tumor (N) samples and 7 tumor (T) samples clustered into two

distinct groups, indicating that the gene expression between tumor and non-tumor samples differs

significantly. Among the 7 tumor samples, samples 1T, 6T, 7T, 8T and 9T were more closely clustered

than when compared with 2T and 3T (Fig. 1A). This observation further suggests that RNA-Seq is a

powerful technique in delineating changes at the transcriptomic level. To identify differentially expressed

genes of functional importance, the t-test and Baggerley’s test were performed to address the significance

of the deregulated genes in tumor samples. Of the 14,351 represented genes, 1,598 genes were up-

regulated and 132 were down-regulated with a Bonferroni-corrected p-value of less than 0.001 (red dots

in Fig. 1B). The global profile of the differential expression profiling for the 1,730 deregulated genes is

demonstrated as a heatmap (Fig. 1C), where the genes are listed in descending order according to the

Baggerley’s test analysis. Subsequent pathway enrichment analysis by DAVID found the 1,730

differentially expressed genes to be commonly associated with a number of cancer-related pathways. Of

these, two of the most significantly enriched pathways are related to integrin signaling (Fig. 1D).

Supplemental Table 4 provides a full list of the significantly differentially expressed genes involved in

integrin signaling pathway (p < 0.001). Among these, Ras-related protein Rab25 was found to be the only

significantly down-regulated gene (p < 1E-20) in ESCC compared to non-tumor tissue (Fig. 1E and

Supplemental Fig. 1); and was thus chosen for further studies. RPKM values obtained from RNA-Seq

were reproducible upon qPCR analysis, showing higher Rab25 expression in normal samples versus

tumor samples (Fig. 1F). The result indicates a high level of concordance of the differential expression

measurements between both platforms.

Rab25 as a novel tumor suppressor gene in human ESCC




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To determine whether down-regulation of Rab25 was a common event in ESCC, we extended our qPCR

analysis to an additional 43 paired non-tumor/primary ESCC samples. Rab25 was significantly down-

regulated in tumor tissues when compared with adjacent non-tumor tissues (paired t-test, p < 0.0001, Fig.

2A). We also examined the expression of Rab25 at the protein level by Western blot and IHC analyses. Of

the 6 esophageal tissue samples examined by Western blot analysis, Rab25 expression was consistently

lower in the 3 ESCC samples as compared with the 3 non-tumor esophageal samples (Fig. 2B). To

investigate the clinical significance of Rab25 expression in ESCC, a TMA comprising 270 paired non-

tumor/ESCC samples, with more detailed patient clinical data, was used for an immunohistochemical

study. A strong Rab25 staining was observed in the non-tumor samples (Fig. 2C). More than half of all

informative non-tumor samples displayed a high Rab25 expression level (89/171, 52.0%), whereas only a

quarter of the informative tumor samples showed high Rab25 expression (42/164, 25.6%) (Fig. 2D, chi

square test p < 0.001). In a Kaplan-Meier survival analysis comparing patients with different Rab25

expression levels, higher Rab25 expression was significantly associated with a longer survival time (log-

rank test, p = 0.009). Among three groups of patients with different Rab25 expression levels, increased

survival was observed with enhanced Rab25 expression (Fig. 2E). Patients with negative Rab25

expression had the worst prognosis, with a mean overall survival of 27.7 months, while patients with low

Rab25 expression displayed a relatively improved survival of 34.3 months. Patients with high Rab25

expression had the longest mean survival time of 45.2 months.

The Rab25 promoter region is frequently hypermethylated in ESCC

In addition to expression analysis in clinical samples, Rab25 expression was also examined at both

genomic and proteomic levels in 8 esophageal cell lines, by qPCR and Western blot analyses. Absent or

significantly down-regulated Rab25 expression was observed in a panel of ESCC cell lines when

compared with a pooled normal tissue control (5 cases of non-tumor esophageal clinical tissue samples

pooled together). In particular, Rab25 was found to be completely absent in the ESCC cell lines EC109

and KYSE520 (Fig. 3A and B). Down-regulation of tumor suppressor genes in cancers is often associated

with hypermethylation and histone deacetylation. To determine whether Rab25 down-regulation was

associated with epigenetic regulation in ESCC, EC109 and KYSE520 cells that lacked Rab25 expression

were treated with varying concentrations of the DNA methyltransferase inhibitor (5-aza-dC) and/or the

histone acetylation agents (TSA or VPA) to investigate the effects of DNA demethylation and histone

acetylation on Rab25 expression. As shown in Fig. 3C, qPCR analysis showed a dose-dependent

restoration of Rab25 expression after demethylation treatment with 5-aza-dC. Treatment with the histone




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acetylation agent TSA or VPA did not significantly alter Rab25 expression, and combined treatment with

10 �M 5-aza-dC and 0.2 �M TSA did not further enhance Rab25 expression. These studies support the

idea that DNA methylation, but not histone modification, is involved in Rab25 inactivation in ESCC.

To substantiate the role of aberrant promoter hypermethylation in Rab25 silencing, we performed MSP or

BGS to investigate the methylation status of the Rab25 promoter region. The 10 kb sequence directly

upstream of the Rab25 gene was analyzed by two publicly available databases for potential CpG islands:

CpG Island Searcher (http://www.cpgislands.com) and EMBOSS-CpGPlot

(http://www.ebi.ac.uk/Tools/emboss/cpgplot). Three common CpG islands were predicted by both

programs, including the regions at -8217 to -6690 (CpG I1), -6689 to -5717 (CpG I2) and -1287 to -453

(CpG I3) 5’ upstream of Rab25 (Fig. 4A). In addition, because a recent study also found the sequence

from -173 to +17 (CpG I4) to contain the core promoter region of the Rab25 gene (21), we also

investigated the methylation status in this region (Fig. 3D). We performed MSP at CpG I1 and BGS at

CpG I2 and CpG I3, however, no difference in methylation status between Rab25 expressing or absent

ESCC cell lines could be detected in these predicted CG dinucleotide-rich regions (data not shown). But

BGS at CpG I4, which contains the promoter region of Rab25 (21), showed a high density of methylation

in Rab25 absent EC109 and KYSE520 cell lines. In contrast, methylation was rarely detected in the same

CpG sites in the Rab25 expressing KYSE30 cell line (Fig. 3D and E). As compared with untreated EC109,

methylation was significantly reduced in EC109 cells treated with 5-aza-dC (Fig. 3E). In addition, we also

investigated the methylation status in a pair of matched ESCC and non-tumor clinical sample. Rab25

expression was found to be significantly down-regulated in ESCC as compared to non-tumor in this case

as confirmed by qPCR analysis. ESCC sample was found heavily methylated (75-100% methylation) in

18 of 21 CG dinucleotides examined, while the non-tumor samples showed significantly lower levels of

methylation (0-25% methylation) in 17 of 21 CG dinucleotides examined (Fig. 3E, representative shown).

Taken together, these observations provide strong evidence in support of the notion that DNA promoter

hypermethylation is implicated in Rab25 inactivation in human ESCC.

Rab25 suppresses in vitro migration, invasion and angiogenesis in ESCC

Frequent epigenetic silencing of Rab25 in ESCC cell lines and human ESCC samples prompted us to

further investigate the function of Rab25 in ESCC. To assess whether Rab25 might possess a tumor-

suppressive function, EC109 cells without Rab25 expression, were stably transduced with lentivirus

packaged with either a Rab25 expressing vector or an empty vector (EV) control to generate Rab25




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overexpressing (EC109 Rab25) or control cells (EC109 EV). Similarly, KYSE30 cells, exhibiting high

levels of Rab25 expression, was lentivirally transduced with either Rab25 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 was confirmed at

both mRNA and protein levels by qPCR and Western blot, respectively (Fig. 4A and B). Stable Rab25

overexpression significantly reduced the ability of the cells to migrate and invade through transwell

chambers. Conversely, suppressing Rab25 expression resulted in an opposing effect (Fig. 4C). Next, we

also investigated whether overexpression of Rab25 has a suppressive effect on angiogenesis. HUVECs

were treated with conditioned media collected from Rab25 overexpressed or suppressed cells and their

respective controls. Morphological changes and differential tube-forming abilities of HUVECs following

treatment with conditioned media is shown in Fig. 4D. HUVECs displayed elongated tube-like structures

following treatment with conditioned media from Rab25 suppressed clones 849 and 852, as compared

with control cell medium treatment. Conversely, treatment with conditioned media from Rab25

overexpressed cells displayed a contrasting effect. These observations suggest a role for Rab25 in the

regulation of invasion, migration and angiogenesis in ESCC.

Rab25 suppresses in vivo tumor formation and angiogenesis in ESCC

An in vivo mouse model was further utilized to support our findings in vitro. Tumor formation ability of

Rab25 overexpressed clone was significantly subdued as compared with EV control cells (Fig. 5A, left

panel). On the contrary, mice injected with Rab25 repressed clone formed larger and more tumors than

compared with NTC cells (Fig. 5A, right panel). Serial sections from the xenograft tumors were then

subjected to H&E staining as well as IHC analysis. Histological analysis and CK5/6 IHC staining found

tumors formed with EC109 Rab25, EC109 EV and KYSE30 shRNA 852 cells resembled an ESCC

phenotype, while xenografts formed from KYSE30 NTC cells were composed mostly of necrotic cells

and fibroblasts. Xenograft tissue sections were also examined for PCNA and CD34 expression by IHC.

As compared with EC109 Rab25 overexpressing xenografts, xenografts generated with EV cells

displayed an enhanced PCNA and CD34 expression, indicative of increased cell proliferation and

microvessel density, respectively. Conversely, reduced PCNA and CD34 expression were observed in

KYSE30 NTC xenografts compared to Rab25 repressed tumors (Fig. 5B).

Rab25 drives ESCC through a deregulated MAPK/ERK signaling pathway




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Rab25 has previously been reported to control recycling of �1 integrin (11-13). FAK, which localizes

with � subunit of integrins, is the major and most extensively studied downstream player activated by

integrins. Activation and auto-phosphorylation of FAK has previously been shown to be critical in driving

a number of cancer processes including promoting cell survival, cell proliferation and cell motility;

through regulating downstream pathways including the MAPK/ERK pathway (4, 22-24). In view of the

importance of MAPK/ERK pathway in regulating cell invasiveness and angiogenesis, we therefore

examined if Rab25 could regulate the expression of activated kinases in this particular signaling cascade.

As compared with EV control, Rab25 overexpression resulted in a reduction of phosphorylated FAK and

c-Raf with a concomitant decrease in the downstream 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 compared with its respective NTC (Fig. 6A). Interestingly, immunohistochemical

staining in the resected xenografts likewise showed a reduced expression of phospho-ERK in Rab25

overexpressing xenografts. Conversely, tumors formed with Rab25 repressed cells displayed an enhanced

expression of phosphor-ERK (Fig. 6B). Taken together, our results suggested that Rab25 exerts its tumor

suppressive function in ESCC through regulating the MAPK/ERK signaling pathway (Fig. 6C).




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Discussion

Past studies on the genetic alterations in ESCC by our group and other research laboratories have mainly

been focused on investigating differential gene expression, chromosomal aberrations and loss of

heterozygosity using microarrays, comparative genomic hybridization and microsatellite DNA marker

analysis techniques (25-28). In recent years, the advent of next-generation sequencing technologies has

provided a new platform for generating vast amounts of data at genomic, epigenomic, transcriptomic and

proteomic levels by means of a variety of high-throughput technologies (29). In particular, the recent

development of RNA sequencing has provided a new approach for mapping and quantifying

transcriptomes (30). Direct tabulation of the transcriptome by RNA-Seq has advantages over existing

technologies in several ways. First, unlike hybridization-based approaches, RNA-Seq is not limited to

detecting known genes but can also include the detection of novel transcripts and alternative splice forms

(provided sequencing is performed to a sufficient depth). A second advantage of RNA-Seq relative to

DNA microarrays is that RNA-Seq has high sensitivity for detecting transcripts of low expressed genes

and that many sequencing reads can be unambiguously assigned to the genome. Finally, it is also shown

to have high levels of reproducibility. RNA-Seq has now been successfully applied in the study of various

disease models including Alzheimer’s disease (31), leukemia (32), prostate (33-34) and breast (35)

cancers, but yet ESCC. In this present study, RNA-Seq was employed to investigate differential gene

expression in twelve non-tumor and ESCC clinical samples. Using a stringent statistical cut-off of p <

0.001, 1,730 commonly differentially expressed genes were identified. Pathway and Gene Ontogeny

analysis by DAVID in the 1,730 genes found 2 of the top 7 most enriched pathways to be associated with

integrin signaling (i.e. integrin cell surface interactions and integrin signaling pathway), suggesting that

this pathway should play a critical role in driving ESCC tumorigenesis. Integrin signaling has been

extensively shown in the past to control initiation and progression of various cancer types. And this is also

true in the context of ESCC, as a number of the identified differentially expressed genes involved in

integrin signaling in this study (listed in Supplemental Table 4) have already been previously implicated

in ESCC pathogenesis. These include CAV1, ROCK1, ITGB1, ICAM1, VCL, PXN and SHC1 (36-44).

Here, we report the identification and characterization of a tumor-suppressor gene Rab25 in ESCC, which

was found to be significantly down-regulated in the integrin signaling associated pathway.

Rab25, with an epithelial distribution, was first discovered with its expression enriched in the normal

gastrointestinal mucosa, kidney and lung, but absent in the brain, liver and skeletal muscle (7). It is a

complex molecule that has been described as an intracellular transport protein (45-46). More recent




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studies have also shown that it is a tumor modulator as it can regulate tumor growth and angiogenesis. A

hand-full of studies have now been conducted to investigate the deregulated expression of Rab25 in

different cancer types including ovarian (10, 47), breast (14, 48) and colon cancers (13). Based on these

studies, Rab25 seems to display very contradicting roles dependent on cellular context. Concordant with

findings in triple-negative breast cancer and colon cancer (13-14, 48), we also found Rab25 to function as

a tumor suppressor in ESCC. And as Nam et al. reported for colon cancer (13), Rab25 was also found to

have prognostic value in ESCC. Cheng et al. has previously reported that Rab25 can act through multiple

pathways to enhance apoptosis and to suppress angiogenesis and invasion in triple-negative breast cancer

by modulating VEGF-A and VEGFR-1 expression (14). Contrary to their findings, our preliminary

findings by qPCR show 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 Rab25

mediates tumorigenicity, metastasis and angiogenesis in ESCC – through a deregulated FAK-Raf-

MEK1/2-ERK signaling. This study is also the first comprehensive study to identify a role of promoter

hypermethylation in the inactivation of Rab25 expression in ESCC. It is interesting to note that Rab25 has

been suggested, along with a number of other genes (CDCA8, ATAD2, AURKA, etc.) to have a strong

correlation for methylation dependent expression changes in ovarian cancer; although data is yet to be

validated (49). On the contrary, loss of Rab25 expression as a result of chromosome 1q22-23 mutation

has previously been suggested for breast cancer cell lines (48) while up-regulated Rab25 expression has

been suggested to be associated with chromosome amplification in ovarian cancer (10) and PKA-

dependent regulation of the proximal promoter in gastric cancer cell line model (21). Our preliminary

studies of DNA copy number in two ESCC cell lines (KYSE520 and EC109) and two ESCC clinical

samples with no or low Rab25 expression suggested that down-regulation of Rab25 is not associated with

deletion of the Rab25 gene (Supplemental Fig. 2). Although there is no solid explanation to conclusively

explain the different outcomes observed by different groups with regard to Rab25 expression, its

regulation, its functional role and its mediated pathways, several important factors should be considered.

Most of the data that indicates Rab25 as an oncogene is derived from studies in ovarian and breast cancers,

which is in a completely different cellular context from ESCC. The stage of the disease can also

determine whether a certain gene acts as a tumor promoter or suppressor. For instance, TGF-beta has been

shown to act as a tumor suppressor in early stages of pancreatic cancer, but then switches to become an

oncogene in metastatic pancreatic cancer (50). Further, the cellular / mechanistic pathway most impacted

may also be vital in determining the nature of Rab25’s action in different cellular models. For instance,

Cheng et al. found Rab25 to exert tumor suppressive properties in MDA-MB-231 breast cancer cells in




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part through effects on VEGF-A secretion and VEGFR-1 expression (14) while our present study found

Rab25 to exert tumor suppressive effects in ESCC through a deregulated MAPK/ERK pathway. In

ovarian and breast cancers, however, Rab25 has been shown to possess oncogenic functions through

suppressing Bak/Bax pro-apopototic molecules and activation of PI3K/Akt pathway (10). These factors

may potentially contribute to determine whether Rab25 promotes or impedes tumor growth. Regardless,

all these data do suggest a multi-functional role of Rab25 in different cellular contexts and either a gain or

loss of Rab25 expression could potentially lead to tumorigenesis in different organ models.




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Acknowledgements

The authors thank Dr. Kwai Wa Cheng of University of Texas MD Anderson Cancer Centre (Texas, USA)

for his generous gift of the monoclonal rabbit anti-human Rab25 antibody.

Grant Support

This work was supported by a grant from the National Natural Science Foundation of China (30971606)

and the Sun Yat-Sen University “Hundred Talents Program” (85000-3171311).




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Figure Legends

Figure 1. RNA-Seq expression profiling data. A, cladogram clustering diagram based on the gene

expression values for twelve ESCC and non-tumor samples. B, volcano plot of the p-value as a function

of weighted fold-change for the 5 non-tumor vs. 7 tumor samples. Blue dots represent genes not

significantly differentially expressed (Bonferroni-corrected p-value > 0.001), and red dots represent genes

significantly differentially expressed (Bonferroni-corrected p-value � 0.001). C, heatmap of expression

profiles for the 1,730 genes that showed significant expression changes (1,598 up-regulated and 132

down-regulated). D, pathway enrichment analysis of significantly differentially expressed genes. E,

expression tiling of Rab25 in 3 pairs of matched ESCC and corresponding non-tumor clinical samples. F,

qPCR analysis of the relative expression of Rab25 in the original twelve ESCC and non-tumor clinical

samples used for RNA-Seq. Table shows the RPKM expression values of the twelve samples analyzed by

RNA-Seq. Validation study to determine the correlation between real-time qPCR and RNA-Seq data.

Figure 2. Down-regulation of Rab25 in ESCC. A, Rab25 expression in matched non-tumor (NT) and

ESCC tumor samples (n = 43) as detected by qPCR. �-actin was used as an internal control. Values

displayed as average delta Ct values. Boxes in the box plot contain the values between the 25th and 75th

percentiles. The lines across the boxes indicate the median. The whiskers extend to the highest values,

excluding outliers and extremes. B, Rab25 protein expression in ESCC tumor (T) and non-tumor (N)

tissues as detected by Western blot. C, representative IHC staining of Rab25 in ESCC tumor and paired

non-tumor squamous epithelium tissue from one patient sample. D, bar chart summary of the distribution

of different Rab25 expression levels in non-tumor versus tumor for all informative cases on the TMA. (E)

Kaplan-Meier survival analysis comparing the overall survival time of ESCC patients with different

Rab25 expression levels.

Figure 3. Rab25 promoter region is frequently hypermethylated in ESCC. A and B, measurement of

genomic and proteomic Rab25 expression levels in a panel of ESCC cell lines compared with a pooled

normal tissue control by qPCR and Western blot. C, real-time qPCR analysis of Rab25 expression after

demethylation and/or histone acetylation treatment with 5-aza-dC (blue bars), TSA (pink bars) and/or

VPA (green bars) in ESCC cell lines without Rab25 expression. Double (grey bars) – combined 10μM 5-

aza-dC and 200nM TSA treatment. D, a schematic diagram showing the distribution of four predicted

CpG islands 5’ upstream of the Rab25 gene. The CpG islands cover the regions -8217 to -6690 (CpG I1),

-6689 to -5717 (CpG I2), -1287 to -453 (CpG I3) and -173 to +17 (CpG I4). MSP was performed at CpG




22

I1 and BGS was performed at CpG I2, CpG I3 and CpG I4. Specifically, 23 CpG dinucleotides are

present at CpG I4. BGS primers (4BGS-F and 4BGS-R) were designed to amplify the sequence in this

region. Transcription factor binding sites for Sp1 and CRE are predicted within this core promoter region.

E, mapping of the methylation status of CpG dinucleotides within CpG I4 in a case of matched ESCC and

non-tumor samples, Rab25 expressing KYSE30 cells, Rab25 negative KYSE520 and EC109 cells and

EC109 cells treated with 5-aza-dC by BGS. The percentage of methylation at each CpG dinucleotide is

displayed in the pie charts.

Figure 4. Rab25 from ESCC cell lines suppresses migration, invasion and angiogenesis in vitro. Stable

repression or overexpression of Rab25 in KYSE30 and EC109 cells, respectively, by lentiviral

transduction was confirmed by A, qPCR and B, Western blot. C, representative migration and invasion

assays in Rab25 overexpressed or repressed clones as compared to their controls. Bar chart summary of

the number of migrated or invaded cells per field (*p < 0.05). D, representative images showing

morphological changes and differential tube-forming abilities of HUVECs following treatment with

conditioned media from Rab25 overexpressed or repressed clones as compared to their controls. Bar chart

summary showing the number of tubes formed in each assay (**p � 0.01 and ***p � 0.001).

Figure 5. Rab25 suppresses in vivo tumor initiation and angiogenesis in ESCC. A, representative images

of the subcutaneous tumors formed in nude mice following injection of Rab25 overexpressed or repressed

clones and their respective controls. Tumor incidence denoted in parentheses. B, H&E and IHC staining

for CK5/6, PCNA and CD34 expression in the resected xenografts. Bar chart summary of average

staining intensity of PCNA and microvessel density in five randomly selected ‘hot spot’ regions (*p <

0.05, **p � 0.01).

Figure 6. Rab25 drives ESCC tumorigenesis via a deregulated MAPK/ERK signaling pathway. A,

detection of 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 respective

controls by Western Blot. �-actin was used as a loading control. B, phospho-ERK IHC staining in the

resected xenografts generated from Rab25 overexpressed (EC109 Rab25) or suppressed (KYSE30

shRNA 852) cells and their respective control cells. Bar chart summary of average staining intensity of

phospho-ERK in five randomly selected ‘hot spot’ regions (**p � 0.01). C, a schematic diagram

illustrating the proposed Rab25 regulated tumor suppressive mechanism in ESCC tumorigenesis.




Published OnlineFirst September 18, 2012.Cancer Res Man Tong, Kwok Wah Chan, Jessie YJ Bao, et al. anti-invasive activities in esophageal squamous cell carcinomaRab25 is a tumor suppressor gene with anti-angiogenic and

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