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Carcinogenesis Advance Access originally published online on February 17, 2008
Carcinogenesis 2008 29(3):629-637; doi:10.1093/carcin/bgm291
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Epigenetic inactivation of protein kinase D1 in gastric cancer and its role in gastric cancer cell migration and invasion

Mirang Kim1,8, Hay-Ran Jang1, Jeong-Hwan Kim1, Seung-Moo Noh2, Kyu-Sang Song3, June-Sik Cho4, Hyun-Yong Jeong5, Jim C. Norman6, Patrick T. Caswell6, Gyeong Hoon Kang7, Seon-Young Kim1, Hyang-Sook Yoo1 and Yong Sung Kim1,8,*

1 Medical Genomics Research Center, KRIBB, Daejeon 305-806, Korea
2 Department of General Surgery
3 Department of Pathology
4 Department of Diagnostic Radiology and
5 Department of Internal Medicine, College of Medicine, Chungnam National University, Daejeon 301-747, Korea
6 Integrin Cell Biology Laboratory, Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD, UK
7 Department of Pathology, Seoul National University College of Medicine, Seoul 110-744, Korea
8 Department of Functional Genomics, University of Science and Technology, Daejeon 305-806, Korea

* To whom correspondence should be addressed. Tel: +82 42 879 8110; Fax: +82 42 879 8119; Email: yongsung{at}kribb.re.kr


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
Protein kinase D (PKD) 1 influences cell migration by mediating both trans-Golgi vesicle fission and integrin recycling to the cell surface. Using restriction landmark genomic scanning methods, we found that the promoter region of PKD1 was aberrantly methylated in gastric cancer cell lines. Silencing of PKD1 expression was detected in 72.7% of gastric cancer cell lines examined, and the silencing was associated with CpG hypermethylation in the promoter region of PKD1. Treatment with 5-aza-2'-deoxycytidine and trichostatin A partially reversed PKD1 methylation and restored gene expression in PKD1-silenced cell lines. Real-time reverse transcription–polymerase chain reaction analysis of 96 paired clinical primary gastric cancer samples revealed that 59% of the analyzed tumors had a >2-fold decrease in PKD1 expression compared with each normal-appearing tissue and that this downregulation of PKD1 expression was significantly correlated with increased methylation. We also observed a gradual increase in the level of promoter methylation of PKD1 in aging, normal-appearing mucosal tissues, suggesting that PKD1 methylation may be one of the earliest events that predispose an individual to gastric cancer. PKD1 expression was required for directional migration of gastric cancer cells. Furthermore, knock down of PKD1 by RNA interference promoted the invasiveness of cell lines that expressed PKD1 at relatively high levels. Based on these results, we propose that PKD1 is frequently silenced by epigenetic regulation, which plays a role in cell migration and metastasis in gastric cancer.

Abbreviations: 5-aza-dC, 5-aza-2'-deoxycytidine; ChIP, chromatin immunoprecipitation; LOE, loss of expression; PCR, polymerase chain reaction; PKD, protein kinase D; RLGS, restriction landmark genomic scanning; RT, reverse transcription; siRNA, small interfering RNA; TSA, trichostatin A


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
The protein kinase D (PKD) family of serine/threonine protein kinases, now classified as a subfamily of the calcium/calmodulin-dependent protein kinase superfamily (1), comprises PKD1 (also known as PKCµ), PKD2 and PKD3 in human and PKD1 in mouse (2–5). PKD1 is the most extensively characterized member of the family and is a target of diacylglycerol and a direct substrate of protein kinase C. PKD1 is present in the cytoplasm of resting cells and is recruited to multiple cellular compartments such as the plasma membrane, the nucleus or the Golgi in response to certain stimuli. This intracellular mobility of PKD1 enables it to function as a communicator between different subcellular compartments. PKD1 has been implicated in multiple biological processes including membrane trafficking, signal transduction, cell adhesion, migration, survival, proliferation, differentiation and apoptosis (6–8).

PKD1 plays a major role in cell motility, migration and invasion. PKD1 regulates the fission of transport vesicles budding from the trans-Golgi network to the cell surface (9,10). This PKD1-mediated polarized membrane transport is required for fibroblast motility (11). PKD1 also influences cell migration by promoting the recycling of {alpha}vβ3 integrin to form a polarized distribution of focal adhesions at the leading edge of migrating cells (12). PKD1 is downregulated in prostate cancer and is associated with altered cellular aggregation and motility in prostate cancer cells (13,14). Additionally, PKD1 forms a complex with the actin-binding protein, cortactin, and the focal adhesion protein, paxillin, at invadopodia (sites of extracellular matrix degradation) in breast cancer cells (15), suggesting a possible role for this kinase in controlling the ability of cancer cells to invade the extracellular matrix.

The altered pattern of gene expression within cancer cells is derived from genetic and epigenetic changes of genomic DNA. Methylation of genomic DNA is one of the most studied epigenetic gene regulation mechanisms in carcinogenesis. Aberrant DNA methylation of specific genes is one of the earliest and most frequent alterations in cancer cells, such that methylation can be used for early detection of cancers and to monitor cancer progression (16–19). Discovery of new methylated genes in cancer can lead to the identification of new factors that are important for tumor initiation and progression (16). We have studied global DNA methylation patterns in gastric cancer cell lines and gastric cancer tissues using restriction landmark genomic scanning (RLGS). RLGS is a powerful tool for detecting >2000 restriction landmarks distributed on an entire genome in one procedure that employs direct end labeling of the genomic DNA digested with a methylation-sensitive restriction enzyme followed by high-resolution two-dimensional electrophoresis (20,21). In this study, we found using RLGS (22) that the PKD1 promoter is aberrantly methylated in gastric cancer. We observed a high frequency of PKD1 methylation in primary gastric tumors and gastric cancer cell lines and found a significant association between this methylation and the inactivation of PKD1 expression. Furthermore, we demonstrated that downregulation of PKD1 by RNA interference increased the invasiveness of gastric cancer cells through a three-dimensional matrix.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 Funding
 References
 
Cell lines and tissue samples
Gastric cancer cell lines (SNU-001, SNU-005, SNU-016, SNU-216, SNU-484, SNU-520, SNU-601, SNU-620, SNU-638, SNU-668 and SNU-719) were obtained from the Korean Cell Line Bank (http://cellbank.snu.ac.kr/index.htm) and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% antibiotic–antimycotic solution (Invitrogen, Carlsbad, CA). Ninety-six frozen tumors paired with adjacent normal tissues were collected from Chungnam National University Hospital. The samples included 35 stage I, 15 stage II, 33 stage III and 13 stage IV tumors from 30 females and 66 males, 23–82 years of age (average of 58 years). All samples were obtained with informed consent, and their use was approved by the Internal Review Board at Chungnam National University Hospital.

Formalin-fixed paraffin samples
Five archival samples of endoscopically resected gastric adenomas and 10 archival samples of endoscopically obtained non-neoplastic gastric mucosa (five intestinal metaplasia and five chronic gastritis) were collected from Seoul National University Hospital. After identifying adenoma, intestinal metaplasia or chronic gastritis on hematoxylin and eosin-stained slides, a region corresponding to the identified lesion was scraped from 20 mm thick paraffin sections. The materials collected were dewaxed by washing in xylene and then by rinsing in ethanol. The dried tissues were digested with proteinase K and subjected to the standard method of DNA extraction using phenol–chloroform–isoamyl alcohol and ethanol precipitation.

RLGS analysis
Genomic DNA was extracted using the phenol–chloroform method, and RLGS was performed as described (23). Briefly, 5 µg of genomic DNA was incubated in the presence of DNA polymerase I, ddTTP, ddATP, dGTP({alpha}S) and dCTP({alpha}S) to fill in randomly broken ends. The treated DNA was then digested with NotI (a methylation-sensitive enzyme), end labeled with ({alpha}-32P)dGTP and ({alpha}-32P)dCTP using Sequenase and subsequently digested with EcoRV. The labeled DNA (1.5 µg) was separated by size on a 0.8% agarose gel for 12 h. Thereafter, DNA was digested in the gel with HinfI to further fragment the DNA. The gel was fused with the polyacrylamide gel by adding melted agarose to fill up the gap. Second-dimension electrophoresis was carried out for 7 h. The gel was dried and exposed to X-ray film. RLGS gels were run for paired samples of primary gastric cancer and adjacent normal tissue. For cell line DNA, RLGS gels were also run in pairs consisting of cell line DNA alone and DNA of the cell line mixed with DNA from normal tissue. Differences between cell line DNA were detected as described (24). Spot intensities were compared with the Master RLGS profile (25), or with our RLGS profile (22), to identify its sequence.

Real-time reverse transcription–polymerase chain reaction
Total RNA was isolated from gastric cancer cell lines or paired normal and tumor tissues using the RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions and treated with DNase I (Promega, Madison, WI). DNase-treated RNA (5 µg) was reverse transcribed with Superscript II reverse transcriptase (Invitrogen) according to the manufacturer’s guidelines. Real-time reverse transcription (RT)–polymerase chain reaction (PCR) was performed using the Exicycler Quantitative Thermal Block (Bioneer, Daejeon, Korea). The RT reaction product (100 ng) was amplified in a 15 µl reaction with 2x SYBR Premix EX Taq (Takara, Shiga, Japan) using the primers 5'-CAAGGGCTACAATCGCTCTC-3' (located in exon 15, Figure 1B) and 5'-TGTGGCTAGCACTTGGATTG-3' (located in exon 16, Figure 1B). Samples were heated to 95°C for 1 min and then amplified for 45 cycles consisting of 95°C for 30 s, 60°C for 30 s and 72°C for 30 s. β-Actin was amplified as a control. Relative quantification of PKD1 was performed by comparative threshold cycle (CT) methods (26).


Figure 1
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  		Fig. 1. Silencing of PKD1 expression and CpG hypermethylation in gastric cancer cell lines. (A) Comparison of the intensity of spot 7C10 in NotI–EcoRV–HinfI RLGS profiles. Arrowheads indicate spots corresponding to 7C10 in the previous master RLGS profile (22) for DNA samples from four gastric cancer cell lines. Spot 7C10 is missing in cell lines SNU-601 and -638 but present in SNU-484 and -668. (B) Schematic diagram of PKD1 . The map of PKD1 is taken from the UCSC Genome Browser (http://genome.ucsc.edu) and shows the position of the PKD1 coding regions and a CpG island. Sites analyzed by bisulfite sequencing (CpG number 1–72) and pyrosequencing (CpG number 13–19) are indicated. The location of the DNA fragment amplified by the ChIP assay is indicated. The cloned EcoRV–NotI fragment (1727 bp) of RLGS spot 7C10 is located at the upstream region of the gene. Vertical bars indicate 122 CpG sites. E, EcoRV site; H, HinfI site; N, NotI site; TSS, transcription start site and ATG, start codon. (C) RT–PCR analysis of PKD1 in 11 gastric cancer cell lines and normal gastric mucosa. β-Actin expression was used as a control. PKD1 methylation status was analyzed by bisulfite sequencing (D) and pyrosequencing (E) in gastric cancer cell lines. (D) For the bisulfite sequencing results, numerical values in parentheses indicate the percentage of methylation. Each row represents one cloned and sequenced PCR product, and the columns contain data for each of the CpG sites analyzed (filled square, methylated sites; open square, unmethylated sites). The region that was also analyzed by pyrosequencing is indicated. (E) Pyrogram of six gastric cancer cell lines. The seven CpG sites analyzed are shaded. The expected sequence in this region is GGAAGGGTYGYGTTTGTYGAGGYGGTTTYGAGTTTYGTTATTY (Y = T or C). The percentage of methylation at each individual site is indicated, and the last column shows the average percentage of methylation at the seven CpG sites. (F) Comparison of PKD1 expression and methylation in gastric cancer cell lines. PKD1 expression (at left) was analyzed by real-time RT–PCR and was normalized to β-actin expression in each sample. The percentage of CpG methylation at right was analyzed by pyrosequencing.

 
Bisulfite sequencing
Genomic DNA (1 µg) was modified by sodium bisulfite using the Ez DNA Methylation kit (ZYMO Research, Orange, CA) according to the manufacturer’s instructions. Bisulfite-modified DNA was amplified in a 20 µl reaction with the primers 5'-ATTTGGAAGAGGTGGTAGGG-3' and 5'-CTAAACCAAAAACTTCTTTCTCC-3', resulting in a 659 bp product. All samples were heated to 95°C for 12 min and then amplified for 35 cycles of 95°C for 45 s, 55°C for 35 s and 72°C for 60 s, followed by a final extension at 72°C for 10 min. PCR products were electrophoresed through a 1% agarose gel and visualized by ethidium bromide staining. Bands were purified from the gel using the Qiagen Gel Extraction kit and cloned using the pGEM-T Easy Vector (Promega). Ten clones were randomly chosen for sequencing. Complete bisulfite conversion was verified by the fact that <0.01% of the cytosines in non-CG dinucleotides were unconverted in the final sequence.

5-aza-2'-deoxycytidine and trichostatin A treatment
Gastric cancer cells (SNU-601 and SNU-638) were seeded in 10 cm dishes at a density of 1 x 106 cells 1 day before the drug treatment. The cells were treated with 1 µM 5-aza-2'-deoxycytidine (5-aza-dC; Sigma, St Louis, MO) every 24 h for 3 days and then harvested. Another culture of cells was treated with 250 nM trichostatin A (TSA; Sigma) for 1 day. To test the combined effect of 5-aza-dC and TSA, cells were treated with 1 µM 5-aza-dC for 3 days followed by one treatment with 250 nM TSA for 1 day. DNA was prepared and tested for reversion of PKD1 methylation by pyrosequencing. Total RNA and protein were prepared and tested for restoration of PKD1 expression by real-time RT–PCR and western blotting. The value of each point of pyrosequencing and RT–PCR was calculated as the average ± standard deviation from three independent experiments and a total of six independent PCR analyses.

Chromatin immunoprecipitation assay
Chromatin immunoprecipitation (ChIP) assay was performed with a ChIP assay kit (Upstate Biotechnology, Lake Placid, NY) according to the manufacturer’s protocol with some modifications. Briefly, proteins were cross-linked to DNA by addition of formaldehyde directly to the culture medium to a final concentration of 1% for 10 min at 37°C. The cross-linking reaction was quenched by adding glycine to a final concentration of 0.125 M for 5 min at room temperature. The collected cells were washed twice with ice-cold phosphate-buffered saline with proteinase inhibitors and resuspended in 200 µl sodium dodecyl sulfate lysis buffer (Upstate Biotechnology) per 1 x 106 cells. Lysates were sonicated 21 times for 5 s with 30-s intervals on ice, at power setting 3 (Fisher Sonicator Dismembrator 100). These shearing conditions yielded DNA fragments ranging from 200 to 500 bp. The sheared samples were centrifuged at 13 000 r.p.m. for 10 min at 4°C, and the supernatants were diluted in nine volumes of ChIP dilution buffer (Upstate Biotechnology). The cell supernatants were precleaned with salmon sperm DNA/protein A agarose beads (Upstate Biotechnology). The supernatants were immunoprecipitated with 5 µg of anti-acetyl-histone H3 (Upstate Biotechnology, catalog no. 06-599), 5 µl of anti-acetyl-histone H4 (Upstate Biotechnology, catalog no. 06-866), 5 µg of anti-trimethyl-histone H3 (Lys9) (Upstate Biotechnology, catalog no. 07-442) or no antibody. Immunoprecipitated DNA was recovered using the QIAquick PCR Purification kit (Qiagen) and analyzed by real-time PCR. The DNA was amplified in a 15 µl reaction with 2x SYBR Premix EX Taq (Takara) using the primers 5'-CAGGAAGCGGGTCTGAGG-3' and 5'-CCGGGATAGGACCGAGTG-3' (located in PKD1 CpG island, Figure 1B). Samples were heated to 95°C for 1 min and then amplified for 45 cycles consisting of 95°C for 30 s, 60°C for 30 s and 72°C for 30 s. The amount of immunoprecipitated DNA was normalized to the input DNA. The value of each point was calculated as the average ± standard deviation from two independent ChIP experiments and a total of four independent PCR analyses.

Pyrosequencing
The promoter region of PKD1 (108 bp) was amplified using forward primer (5'-AGAGAGGAAGGGAATTTTTGTA-3') and biotinylated reverse primer (biotin-5'-TTCCCCAAACCTAAACCTC-3') designed by PSQ Assay Design (Biotage AB, Kungsgatan, Sweden). Bisulfite-modified DNA (100 ng) was amplified in a 20 µl reaction with HotStart PCR Premix (Bioneer) using the primers described above. All samples were heated to 95°C for 5 min and then amplified for 50 cycles of 95°C for 30 s, 58°C for 40 s and 72°C for 30 s, followed by a final extension at 72°C for 5 min. Pyrosequencing reactions were performed according to the manufacturer’s specifications with a sequencing primer (5'-GGGAATTTTTGTAGGGAG-3') using the PSQ HS 96A System (Biotage AB).

Wound-healing assay
SNU-484 and SNU-601 cells were grown to confluency, and a wound was established by scratching one time with a 1 mm thick pipette tip. Wounded monolayers were placed on the stage of a Zeiss Axiovert S100 inverted microscope in an atmosphere of 5% CO2 at 37°C. Cells were observed using a x20 phase-contrast objective, and images were collected every 20 min using a Hamamatsu C4742-95 digital camera. Cell tracks were analyzed using Andor Bioimaging (London, UK) software. The persistence and speed of migration were extracted from the track plots. Persistence is defined as the ratio of the vectorial distance traveled to the total path length followed by the cell.

Small interfering RNA transfections
Double-stranded small interfering RNA (siRNA) oligonucleotides targeting PKD1 were synthesized by Dharmacon (Lafayette, CO). The sequences used were 5'-GAAGAGAUGUAGCUAUUAAUU-3' for the sense sequence and 5'-UUAAUAGCUACAUCUCUUCUU-3' for the antisense sequence. Each siRNA oligonucleotide (1 µM, in solution T) was transfected into SNU-484 or SNU-668 cells using a Nucleofector instrument according to the manufacturer’s protocol (Amaxa Biosystems, Cologne, Germany). Knock down of PKD1 protein was examined by western blotting using an antibody against PKD1 (PKCµ, D-20) (Santa Cruz Biotechnology, Santa Cruz, CA). {alpha}-Tubulin was measured as a control in the same samples.

Inverted invasion assay
An inverted invasion assay was performed as described (27) with minor modifications. Briefly, Matrigel (BD Biosciences, Erembodegem, Belgium) was polymerized with 25 µg/ml fibronectin (Sigma) in 8 µm pore size, 6.5 mm polycarbonate transwell inserts (Corning) for 1 h at 37°C. Inserts were then inverted, and 4 x 104 cells in 100 µl RPMI containing 10% fetal bovine serum were seeded directly onto the opposite face of the filter. Inserts were covered with the base of a 24-well plate and incubated for 5 h to allow the cells to attach and were then turned right-side-up and dipped into serum-free RPMI to wash. Finally, inserts were placed in serum-free RPMI, and RPMI supplemented with 10% fetal bovine serum and 30 ng/ml of epidermal growth factor was applied to the top of the Matrigel to provide a chemotactic gradient. The plate was incubated at 37°C for 5 days. Living cells were stained with 4 µM calcein–acetoxymethyl ester (Invitrogen C1430) in serum-free RPMI and visualized by confocal microscopy using a x10 objective. Optical sections (Z-sections) were scanned at 10 µm intervals moving up from the underside of the filter into the Matrigel to produce serial images. Invading cells in each section were quantified using ImageJ software from the Research Service Branch Web site of the National Institutes of Health (http://rsb.info.nih.gov/).

Statistical methods for analysis
The Student’s unpaired t-test was used to detect differences in PKD1 expression or promoter methylation between gastric tumors and adjacent normal tissues. Results with P values <0.05 were considered significant. Correlation between the level of PKD1 expression and PKD1 CpG methylation or between methylation and aging was determined using the Pearson’s correlation coefficient (r), and its significance was inferred from a t-test using a t value calculated from the following formula t = r((n – 2)/(1 – r2))1/2 with n – 2 degrees of freedom. Results with P values <0.05 were considered significant. The clinicopathologic factors in various groups of patients either positive or negative for PKD1 were compared using the {chi}2 test.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 Funding
 References
 
Identification of PKD1 as a novel epigenetic target in gastric cancer using RLGS analysis
RLGS assays were performed on gastric cancer cell lines to look for aberrant methylation of genomic DNA as compared with normal gastric mucosal tissue. We identified a DNA spot that was absent in 8 of 11 (72.7%) gastric cancer cell lines. Figure 1A shows representative changes in the spot intensity in two gastric cancer cell lines (SNU-601 and SNU-638) but no changes in SNU-484 and SNU-668. This DNA spot corresponded to spot number 7C10 (GenBank accession number CG465101), which we had previously identified by mixing RLGS gels with NotI-linked clones (22). By searching the UCSC Genome Bioinformatics database (http://genome.ucsc.edu/), we found that the NotI-end sequence of the clone 7C10 matched the NotI-end sequence of a NotI/EcoRV fragment in the promoter region of PKD1 on human chromosome 14q12 and overlapped a CpG island covering the 5' upstream region and the first exon (Figure 1B).

PKD1 silencing and CpG hypermethylation in gastric cancer cell lines
We assessed the level of PKD1 mRNA expression by RT–PCR using RNA purified from gastric cancer cell lines. PKD1 expression was silenced in 8 of 11 cell lines tested, SNU-001, -005, -016, -520, -601, -620, -638 and -719 (Figure 1C). Bisulfite sequencing analysis identified extensive hypermethylation (83.5–96.4%) at 72 CpG sites in PKD1-silenced cell lines (SNU-001, -601 and -638). Conversely, very low levels of methylation (3.1–10.5%) were observed in PKD1-expressing cell lines (SNU-216, -484 and -668) (Figure 1D). Pyrosequencing analysis was also performed to quantitatively examine the methylation status of seven PKD1 CpG sites (Figure 1E). We next measured PKD1 expression by real-time RT–PCR and compared it with the methylation status measured by pyrosequencing. Figure 1F clearly shows that silencing of PKD1 correlates with CpG hypermethylation.

Restoration of PKD1 expression by treatment with 5-aza-dC and TSA
To examine whether PKD1 silencing in gastric cancer cells could be restored, we treated SNU-601 and -638 cell lines with the DNA methylation inhibitor, 5-aza-dC (28), and/or the histone deacetylase inhibitor, TSA (29). After treatment, cells were harvested and analyzed for changes in PKD1 methylation and expression compared with untreated cells. Pyrosequencing revealed that the CpG sites of PKD1 were partially demethylated by 5-aza-dC alone or 5-aza-dC and TSA in both cell lines (Figure 2A). In addition, real-time RT–PCR and western blotting revealed an increase in PKD1 expression (Figure 2B and C). TSA was more effective than 5-aza-dC at restoring PKD1 expression in SNU-638. Combining 5-aza-dC with TSA had a synergistic effect in both cell lines. These results suggested that PKD1 expression in these gastric cancer cells was regulated by an epigenetic mechanism that includes both DNA methylation and histone acetylation. To decipher whether DNA methylation and gene silencing in the PKD1 promoter are related, we examined local histone acetylation and methylation in the chromatin associated with the PKD1 promoter region using the ChIP assay. The histone-associated DNAs, immunoprecipitated with antibodies against acetyl-H3 (K9 and K14), acetyl-H4 (K5, K8, K12 and K16) or H3K9me3, were amplified with primer sets corresponding to the PKD1 CpG island. Acetylation levels of histones H3 and H4 at the PKD1 CpG island were elevated in SNU-484 cells, in which the PKD1 CpG island is unmethylated and transcriptionally active, compared with SNU-601 or -638 cells, in which the PKD1 CpG island is hypermethylated and transcriptionally silent. In contrast, H3K9me3 was enriched in SNU-601 or -638 cells (Figure 2D). These results clearly indicate that histone modification is also a feasible mechanism for explaining transcriptional silencing of PKD1 in gastric cancer.


Figure 2
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  		Fig. 2. Effect of 5-aza-dC and TSA on PKD1 expression in gastric cancer cell lines. SNU-601 and SNU-638 cells were treated with 5-aza-dC and TSA as described in the Materials and Methods. (A) PKD1 methylation status was analyzed by pyrosequencing at the end of treatment. (B) PKD1 expression was analyzed by real-time RT–PCR and was normalized to β-actin expression in each sample. (C) PKD1 protein expression was analyzed by western blotting with {alpha}-tubulin as a control. (D) ChIP assays of the PKD1 CpG island. Chromatin DNA was immunoprecipitated with antibodies specific for acetyl-H3 (AcH3), acetyl-H4 (AcH4) or trimethyl-H3-K9 (H3K9me3). DNA fragments corresponding the PKD1 CpG island (see Figure 1B) were amplified by PCR. The amount of immunoprecipitated DNA was normalized to the input DNA.

 
Hypermethylation of PKD1 in primary gastric cancers
We also observed PKD1 hypermethylation in three pairs of randomly selected primary gastric tumor and normal tissue. Bisulfite sequencing showed that 37.5–76.7% of 72 CpG sites were methylated in tumors, whereas only 0.7–15.6% of CpG sites were methylated in adjacent normal tissues (Figure 3A). Pyrosequencing analysis revealed hypermethylation (41.7–71.1%) of seven PKD1 CpG sites in tumors and hypomethylation (4.4–5.9%) in normal adjacent tissues (Figure 3B).


Figure 3
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  		Fig. 3. Expression and methylation status of PKD1 in primary gastric cancers. PKD1 methylation status was analyzed by bisulfite sequencing (A) and pyrosequencing (B) in gastric tumor tissues (T) and normal adjacent tissues (N). The diagrams are as described in the legend to Figure 1. (C) The expression level of PKD1 was measured by real-time RT–PCR in 96 paired normal and tumor tissues. Expression levels were normalized to the β-actin level in each sample. (D) Pyrosequencing was performed at seven CpG sites as shown in (B) for 83 paired normal and tumor tissues to evaluate the level of methylation at each site. The box plot analysis shows the median, 25th and 75th percentiles and outliers. (E) The relationship between methylation change and relative PKD1 expression level. Expression values are expressed as the log2 ratio of tumor samples over normal samples. Methylation change is expressed as the difference in methylation between paired tumor and normal tissues (tumor minus normal). Methylation of PKD1 with respect to age in normal tissues (F) and in tumor tissues (G) in the 83 paired normal and tumor tissues analyzed by pyrosequencing. The extent to which PKD1 was methylated was plotted against the age in years. (H) Methylation of PKD1 in chronic gastritis, intestinal metaplasia and adenoma. Pyrosequencing was performed at PKD1 seven CpG sites for 15 paraffin sections. The methylation levels are shown by dots with their means by bars.

 
To evaluate PKD1 expression during gastric carcinogenesis, we quantified the levels of PKD1 mRNA using real-time RT–PCR in paired normal and tumor tissues obtained from 96 individuals with gastric cancer. After normalization by comparison with β-actin expression, we determined that the expression of PKD1 in tumors was significantly lower than that in normal tissues (P < 0.0001) (Figure 3C). We then arbitrarily defined as loss of expression (LOE) those tumors in which expression was less than half that in normal tissue. PKD1 LOE was found in 59% (57 of 96) of primary tumors. When PKD1 LOE was compared within each clinicopathologic category, such as tumor depth (early and advanced gastric cancer), TNM staging (stage I, II, III and IV), or Lauren’s classification (intestinal and diffuse type), we found no significant difference within each clinical parameter (data not shown).

We next measured the extent of methylation of the seven CpG sites evaluated by pyrosequencing using 83 paired normal and tumor DNAs that were used in the real-time RT–PCR described above. DNA from the 83 normal samples showed methylation of 9.8, 8.6, 11.8, 8.6, 12.3, 11.1 and 14.3% (mean values) at each of the seven CpG sites with an average of 10.9%, whereas DNA from the 83 tumor samples showed methylation of 18.6, 16.3, 20.5, 14.9, 21.7, 20.6 and 23.8% with an average of 19.5%. The overall methylation status in tumors was significantly higher than that in normal tissues (P < 0.0001) (Figure 3D). To assess whether methylation of the PKD1 CpG sites was associated with LOE in the clinical samples, we calculated a Pearson’s correlation coefficient between relative expression and methylation and inferred its significance by t-test. For each paired tumor and normal sample, we used the log2-transformed ratio between tumor and normal samples as the relative expression and defined methylation change as the difference in methylation between tumor and normal samples. Figure 3E shows a significant negative correlation between relative expression and methylation change (r = –0.1629, P = 0.0283), indicating that a decrease in PKD1 expression was associated with an increase in CpG hypermethylation in the clinical samples.

Interestingly, we observed a gradual increase in methylation in aging, normal-appearing mucosal tissue. Methylation was significantly increased in an age-dependent manner in normal tissues (r = 0.3748, P = 0.0005) (Figure 3F), suggesting that PKD1 methylation is age related. This age-related methylation was not observed in tumor tissues (r = 0.0034, P = 0.9750) (Figure 3G). The normal gastric mucosa used in this study actually may be normal-appearing gastric mucosa harboring precancerous cells, and the tumor also may include normal mucosa or precancerous cells in part because samples for analysis taken from the gross clinical specimens were not isolated using a technique such as a laser-captured microdissection. To elucidate the methylation status of PKD1 in multistep gastric carcinogenesis, we performed pyrosequencing analysis on the promoter region of PKD1 in five sets of gastric adenomas, intestinal metaplasia and chronic gastritis. Pyrosequencing results showed that PKD1 was methylated at all stages of progression but showed a marked increase in methylation frequency from chronic gastritis (mean 17.0%, range 4.5–28.5%) or intestinal metaplasia (mean 19.9%, range 11.2–31.5%) to adenomas (39.9%, range 10.4–72.3%) (Figure 3H). This result demonstrates a marked methylation event in non-neoplastic gastric mucosa as well as in premalignant lesions.

PKD1 expression is required for directional migration of gastric cancer cells
PKD1 has been implicated in regulation of cell adhesion and migration (11,12,30,31). PKD1 controls {alpha}vβ3 integrin recycling to influence directional migration (12,32). We therefore investigated the effect of PKD1 downregulation on gastric cancer cell migration by comparing the migratory properties of SNU-484 cells (in which the PKD1 promoter was hypomethylated and PKD1 expression was high) and SNU-601 cells (in which the PKD1 promoter was hypermethylated and PKD1 expression was low). SNU-484 cells migrated directionally in a rapid and persistent manner in wound-healing assays (Figure 4A, left and supplementary Video 1, available at Carcinogenesis Online). However, SNU-601 cells were unable to close the wound and migrated randomly, although these cells moved rapidly (Figure 4A, right and supplementary Video 2, available at Carcinogenesis online). These findings demonstrated that PKD1 expression was required for directional migration of gastric cancer cells on a two-dimensional substrate, whereas the migration of cells expressing only low levels of PKD1 was rapid but random (Figure 4B and C).


Figure 4
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  		Fig. 4. Reduced PKD1 levels are associated with increased random migration and invasiveness of gastric cancer cells. (A) Migration tracks of SNU-484 and SNU-601 cells. The position of the cell nucleus was monitored every 20 min over a period of 5 h. Ten cell tracks from fields representative of those obtained from three independent experiments are shown; the starting position of each cell is denoted by a red dot. (B) Analysis of migration speed of SNU-484 and SNU-601 cells in wound-healing assays. Mean ± SD of ~100 cells analyzed in three independent assays is shown. (C) Analysis of persistence (the tendency to continue traveling in the same direction without turning) of migrating SNU-484 and SNU-601 defined as the ratio of the vectorial distance traveled to the total path length followed by the cell. Videos of the migration tracks are available as supplementary data, available at Carcinogenesis Online.

 
siRNA-mediated depletion of PKD1 promotes gastric cancer cell invasion
Metastasizing tumor cells often move randomly and rapidly undergo amoeboid shape changes (33). It is interesting to note that the SNU-484 cell line was derived from a primary tumor, whereas the SNU-601 cell line was derived from malignant ascites (34). Indeed, studies indicate that cell behavior, including migration, differs considerably when cells encounter a three-dimensional microenvironment (35). We therefore investigated the effect of PKD1 on gastric cancer cell invasion using gastric cancer cell lines that express PKD1. We depleted PKD1 in SNU-484 and SNU-668 cells by siRNA transfection and investigated invasion through Matrigel supplemented with fibronectin. We first confirmed that PKD1-specific siRNA effectively knocked down PKD1 protein expression in these cells (Figure 5A). We next performed an inverted invasion assay, in which cells were seeded onto the bottom of the filter and migrated upward through the Matrigel (27). PKD1-depleted SNU-484 and SNU-668 cells were more invasive (10.6 ± 3.1% for SNU-484 and 9.2 ± 2.3% for SNU-668) than cells transfected with control siRNA (1.6 ± 0.9% for SNU-484 and 4.3 ± 1.7% for SNU-668) (Figure 5B and C). These data suggested that PKD1 silencing could promote tumor aggressiveness of gastric cancer by increasing the invasiveness of gastric cancer cells.


Figure 5
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  		Fig. 5. siRNA-mediated depletion of PKD1 promotes gastric cancer cell invasion. (A) Knock down of PKD1 by siRNA. SNU-668 or SNU-484 cells were transfected with PKD1-specific or control siRNA, and PKD1 knockdown was confirmed by western blotting with anti-PKD1. {alpha}-Tubulin was evaluated as a control. (B) Inverted Matrigel plug invasion assay. PKD1-specific or control siRNA was transfected into SNU-484 and SNU-668 cells, and the cells were allowed to invade through a Matrigel plug for 5 days. Cells were stained with calcein–acetoxymethyl ester and visualized by confocal microscopy using a x10 objective. Fluorescence intensity values relating to the number of cells at each 10 µm layer within the Matrigel were quantified using ImageJ software. Invasion assays were performed in triplicate. (C) Data bars represent the percentage of cells that invaded the Matrigel plug beyond 40–90 µm. Data represent mean ± SD.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 Funding
 References
 
We suggest that PKD1 is a novel epigenetic target in gastric cancer and that inactivation of PKD1 increases tumor cell invasiveness. PKD1, which was recently reclassified as a member of the PKD family (1), is emerging as an important modulator of several kinase signal transduction pathways (36,37). It has been suggested that PKD1 may play an important role in tumor progression, although little genetic and epigenetic information concerning the control of PKD1 expression has been described in any type of cancer until now.

Loss of heterozygosity at chromosome 14q11–12 in the vicinity of PKD1 has been frequently detected in several tumors such as ovarian carcinoma (38), neuroblastoma (39) and stromal tumors of the gastrointestinal tract (40). To our knowledge, however, no tumor suppressor gene has been identified in this genomic region. Recent studies demonstrated that downregulation of PKD1 may impact the progression of prostate cancer to androgen independence through cell-signaling cascades (13) and that phosphorylation of E-cadherin by PKD1 increases cellular aggregation and decreases cellular motility in prostate cancer (14). Comprehensive mutational analysis of the consensus coding sequences in human cancers revealed that PKD1 is mutated in colorectal cancers (41). Together, these data suggest that PKD1 may be associated with carcinogenesis in a number of cancers.

We identified PKD1 as a novel hypermethylated gene in gastric cancer by RLGS analysis. PKD1 expression was downregulated in 73% of gastric cancer cell lines analyzed, and dysfunction was associated with CpG hypermethylation of PKD1. We also analyzed PKD1 expression in clinical tumor samples and found that 59% of gastric tumors had a 2-fold reduction in PKD1 expression compared with their normal tissue counterparts and that PKD1 downregulation also correlated with CpG hypermethylation in these samples. Furthermore, PKD1 expression was restored after treatment with a DNA methyltransferase inhibitor and/or a histone deacetylase inhibitor. These results demonstrate that PKD1 is frequently downregulated due to an epigenetic mechanism in gastric cancer, suggesting that this gene may play an important role in gastric carcinogenesis.

Although recent research has provided insights into the molecular pathology of sporadic gastric cancers, the mechanism by which carcinogenesis is initiated in human gastric mucosal tissues remains largely unknown (42). It is widely accepted that epigenetic alterations are a prerequisite for tumor formation and that these alterations facilitate the accumulation of further genetic abnormalities that result in cancer progression through clonal expansion of cells with a proliferative advantage (43). Our data show that the CpG island in the PKD1 promoter of normal-appearing gastric tissues became gradually methylated as cells aged. This observation is consistent with our previous data and the data of other groups. For example, the LIMS2 promoter is hypermethylated in gastric mucosa in an age-dependent manner (24). Likewise, the incidence of sporadic gastric tumors correlates strongly with age in neoplasia (44), and the ER-{alpha} promoter is hypermethylated in colon carcinomas in an age-dependent manner (45). Our observations suggest that methylation in normal-appearing gastric mucosa can be due to a ‘field cancerization effect’ (43) in which hypermethylation of the PKD1 promoter may represent one of the earliest events that predispose an individual to gastric cancer. Additional observations for a marked methylation event in non-neoplastic gastric mucosa as well as in premalignant lesions indicate that CpG island hypermethylation of PKD1 occurs early in multistep gastric carcinogenesis and tends to accumulate along the pathway to carcinoma.

Our present results show that inactivation of PKD1 promotes cancer cell aggressiveness and metastasis. PKD1-specific siRNA significantly promoted invasiveness of SNU-484 and SNU-668 cells, which express PKD1 at relatively high levels. Furthermore, downregulation of PKD1 in fibroblasts causes a loss of directional migration on two-dimensional substrates in much the same way as we report here for gastric cancer cells (12). We recently found that this reduced migrational persistence is due to a reciprocal relationship between the trafficking of {alpha}vβ3 and {alpha}5β1 integrins, such that decreased recycling of {alpha}vβ3 increases the trafficking of {alpha}5β1 and its ability to communicate with the Rho–ROCK–phosphocofilin axis (32), a pathway intrinsically linked to tumor cell invasion (46,47). Downregulation of PKD1 in gastric cancers may therefore relieve the negative regulatory influence of {alpha}vβ3 on {alpha}5β1 trafficking and Rho–ROCK signaling, thus promoting tumor cell invasion. In breast cancer cells, PKD1 forms a complex with the actin-binding protein cortactin and the focal adhesion protein paxillin, at sites of extracellular matrix degradation (15). Interestingly, Rho is thought to promote a protease-independent, amoeboid mode of tumor invasion (46). Therefore, PKD1 and {alpha}vβ3 could act in concert to suppress invasion, but when PKD1 is downregulated, protease-independent migration may be activated. Further molecular studies and more detailed descriptions of the vesicular trafficking and signaling downstream of {alpha}vβ3 and {alpha}5β1 integrins are needed to elucidate the precise contribution of PKD1 to the invasiveness of tumor cells.

We report that PKD1 is a novel epigenetic target frequently silenced by promoter hypermethylation in gastric cancer, perhaps as an early event during gastric carcinogenesis. We suggest that epigenetic silencing of PKD1 increases tumor cell invasiveness in gastric cancer, thus highlighting the need to study signaling mechanisms involved in PKD1 silencing-induced cancer cell aggressiveness. Finally, PKD1 may be a useful molecular biomarker for evaluating the risk of developing gastric cancer, and it may serve as a therapeutic target of agents that could increase its expression and activity in gastric cancer cells.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
Funding
 References
 
Supplementary Videos 1 and 2 are found at http://carcin.oxfordjournals.org/


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
References
 
21C Frontier Functional Human Genome Project from the Ministry of Science and Technology of Korea (FG06-11-01).


   Acknowledgments
 
Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 

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Received September 3, 2007; revised November 27, 2007; accepted November 27, 2007.


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M. Kim, J.-H. Kim, H.-R. Jang, H.-M. Kim, C.-W. Lee, S.-M. Noh, K.-S. Song, J.-S. Cho, H.-Y. Jeong, Y. Hahn, et al.
LRRC3B, Encoding a Leucine-Rich Repeat-Containing Protein, Is a Putative Tumor Suppressor Gene in Gastric Cancer
Cancer Res., September 1, 2008; 68(17): 7147 - 7155.
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