| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Molecular Biology, Pathobiology, and Genetics |
1 at 3p22 in Esophageal Squamous Cell CarcinomaDepartments of 1 Clinical Oncology, 2 Pathology, and 3 Anatomy, The University of Hong Kong, Pokfulam, Hong Kong, China; 4 Department of Clinical Oncology, First Affiliated Hospital, Zhengzhou University, Zhengzhou, China; and 5 State Key Laboratory of Oncology in Southern China, Cancer Center, Sun Yat-Sen University, Guangzhou, China
Requests for reprints: Xin-Yuan Guan, Department of Clinical Oncology, The University of Hong Kong, Room L10-56, Laboratory Block, 21 Sassoon Road, Pokfulam, Hong Kong. Fax: 852-28169126; E-mail: xyguan{at}hkucc.hku.hk.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1 (PLC1), was identified within the 3p22 region. In this study, absent expression of PLC1 was detected in 26 of 50 (52%) primary ESCCs and 4 of 9 (44.4%) ESCC cell lines, which was significantly associated with DNA copy number loss and promoter hypermethylation (P < 0.05). Functional studies showed that PLC1 was able to suppress both in vitro and in vivo tumorigenic ability of ESCC cells, including foci formation, colony formation in soft agar, and tumor formation in nude mice. The tumor-suppressive mechanism of PLC1 was associated with its role in the cell cycle arrest at the G1-S checkpoint by up-regulation of p21 and down-regulation of phosphorylated Akt (Ser473). In addition, down-regulation of PLC1 protein was significantly correlated with ESCC metastasis (P = 0.014), which was associated with its function in increasing cell adhesion and inhibiting cell mobility. Taken together, our results suggest that PLC1 plays an important suppressive role in the development and progression of ESCC. [Cancer Res 2007;67(22):10720–5] |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Deletion of 3p is one of the most common genetic alterations in ESCC detected by comparative genomic hybridization (3–5) and loss of heterozygosity (LOH; refs. 6). In our recent study, single nucleotide polymorphism (SNP) mass spectrophotometric genotyping with 350 SNP markers was used to detect minimal deleted region at 3p in 100 ESCC cases and four minimal deleted regions, including 3p22-21.3, were detected.6 One candidate TSG, phospholipase C (PLC)-1 (PLC1), was identified within the 3p22 region. Loss of one copy of PLC1 gene was detected in 39 of 97 (40.2%) ESCCs by quantitative real-time PCR.6 PLC1 gene is composed of 16 exons and encodes a protein of 756 amino acids. PLC1 belongs to a mammalian phosphoinositide-specific PLC superfamily. To date, 11 PLC isozymes have been cloned from mammalian cells, which can be divided into four groups (ß, 
, , and 
) based on their sequence homology, structure, and activation mechanisms (7, 8). The main function of PLC enzymes is to hydrolyze the minor lipid component, phosphatidylinositol 4,5-bisphosphate (PIP2), and release the second messenger, inositol 1,4,5-trisphosphate, which can release calcium from intracellular stores, and diacylglycerol, which mediates the activation of protein kinase C (PKC; refs. 9, 10). PLCß1 and PLC1 isoforms have been linked with PKC activation (11, 12); however, the direct link between PLC1 and PKC is still unclear.
Although the expression levels, regulation mechanisms, and biochemical features of PLC1 have been widely studied, its role in cancer development is still unclear. In the present study, PLC1 expression pattern in primary ESCCs and ESCC cell lines was investigated. Tumor-suppressive function of PLC1 was shown by both in vitro and in vivo assays. In addition, down-regulation of PLC1 protein was associated with ESCC metastasis. The tumor-suppressive mechanism of PLC1 was also addressed.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Semiquantitative reverse transcription-PCR. Total RNA was extracted from cell lines and frozen ESCC tissues by the TRIzol reagent (Invitrogen). Reverse transcription of total RNA (2 µg) was done using an Advantage RT for PCR kit (Clontech), and cDNA was subjected to PCR for 28 cycles of amplification with the following pair of primers: PLCDFw, 5'-GGCGTCCCCCTACCCTGTCATCC and PLCDRv, 5'-TGCTGCACACGGCTCCTCACTG. GAPDH gene was used as a control.
Bisulfite treatment and promoter methylation analysis. Genomic DNA was extracted from tumors, normal tissues, and cell lines by phenol-chloroform method followed by bisulfite modification. Methylation specific-PCR (MSP) and bisulfite genomic sequencing (BGS) were done as previously described (14). The bisulfite-treated DNA was amplified with methylation-specific primer set, PLCDm3: 5'-GGTTGGAAGTTTGGACGTC, PLCDm4: 5'-TAACCCGAACCAACGAACG, or the unmethylation-specific primer set, PLCDu3: 5'-GTGGTTGGAAGTTTGGATGTT, PLCDu4: 5'-CCTAACCCAAACCAACAAACA. A 350-bp DNA fragment (–496 to –147) containing 36 CpG sites was amplified by BGS primers, BGS1: 5'-TTTAGGTATTGTTT TTTGAAGATT and BGS2: 5'-AAAAACTAACCAACCCTAACC.
Fluorescent in situ hybridization. A bacterial artificial chromosome (BAC) clone containing PLC1 gene (RP11-213K17) was used to study DNA copy number change in KYSE30 cells. BAC probe and chromosome 3 centromere probe were labeled with Spectrum Orange-dUTP and Spectrum Green-dUTP (Vysis), respectively. Fluorescence in situ hybridization (FISH) reaction was done as described previously (15).
5-Aza-2'-deoxycytidine treatment. To study whether demethylation could restore PLC1 expression in KYSE30 cells, 2 x 105 cells were treated with 50 µmol/L 5-aza-2'-deoxycytidine (5-aza-dC; Sigma-Aldrich Corporation) for 3 days, with changing of 5-aza-dC and medium every 24 h. Total RNA was then extracted and PLC1 expression was detected by reverse transcription-PCR (RT-PCR).
Tissue microarrays and immunohistochemistry. A total of 221 formalin-fixed and paraffin-embedded ESCC tumor specimens used in this study were obtained from the archives of Cancer Center, Sun Yat-Sen University (Guangzhou, China). Two tissue microarrays contained 442 primary ESCC tumor samples (duplicate 0.6-mm tissue cores for each ESCC) and 52 paired nontumorous esophageal tissues were constructed according to a method described previously (16). Multiple sections (5 µm thick) were cut from the tissue microarray blocks and mounted on microscope slides.
Immunohistochemical staining was done using standard streptavidin-biotin-peroxidase complex method (16). In brief, tissue microarray section was deparaffinized and nonspecific bindings were blocked with 10% normal rabbit serum for 10 min. The tissue microarray section was then incubated with anti-PLC1 polyclonal antibody (Santa Cruz Biotechnology, Inc.; 1:50 dilution) at 37°C for 60 min, and subsequently incubated with biotinylated goat anti-rabbit immunoglobulin at a concentration of 1:100 for 30 min at 37°C.
Tumor-suppressive function of PLC1. To test tumor-suppressive function of PLC1, PLC1 was PCR amplified, cloned into pcDNA3.1/V5-His TOPO TA vector (Invitrogen), and transfected into ESCC cell line KYSE30 cells. Stable PLC1-expressing clones (PLC1-c6 and PLC1-c7) were selected for further study. Blank vector–transfected KYSE30 cells (Vec-30) were used as control. For foci formation assay, 1 x 103 PLC1-expressing cells (PLC1-c6 or PLC1-c7) or Vec-30 cells were plated in wells of a six-well plate. After 7 days culture, surviving colonies (>50 cells per colony) were counted with Giemsa staining. Triplicate independent experiments were done.
Colony formation in soft agar was carried out by growing 5 x 103 cells in 0.4% bactoagar on a bottom layer of solidified 0.6% bactoagar in six-well plates. After 3 weeks, colonies consisted of >80 cells were counted and expressed as the means ± SD of triplicate within the same experiment.
Cell growth rate of PLC1-expressing clones (PLC1-c6 and PLC1-c7) and control Vec-30 cells were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded in 96-well plate at a density of 1 x 103 per well. The cell growth rate was detected using cell proliferation MTT kit (Sigma) according to the manufacturer's instruction. Triplicate independent experiments were done.
Tumor formation in nude mice. In vivo tumor-suppressive ability of PLC1 was investigated by tumor xenograft experiment. About 2 x 106 PLC1-expressing KYSE30 cells or control Vec-30 cells were injected s.c. into the right and left hind legs of 4-week-old nude mice (10 mice for PLC1-c6 cells and 10 mice for PLC1-c7 cells), respectively. Tumor formation in nude mice was monitored over a 4-week period. The tumor volume was calculated by the formula V = 0.5 x L x W2 (17).
Cell adhesion assay. Cell adhesion ability was measured by seeding 5,000 cells into 96-well plates precoated with 10 g/mL collagen I (Invitrogen). After incubation for 30 to 90 min, the cells were gently washed with 1x PBS. The number of adherent cells was determined by incubation with 0.1% crystal violet for 30 min, followed by extraction in 10% acetic acid overnight and spectrophotometric analysis at 540 nm. The experiment was done thrice in replicates of six wells.
Wound-healing assay. Cell mobility was studied by a scratch wound-healing assay. PLC1-c6 or Vec-30 cells were cultured in a six-well plate until confluent. The cell layer was wounded using a sterile tip. After incubation for 24 h, the cells were photographed under a phase-contrast microscope. The experiment was done in triplicate.
Cell cycle analysis. PLC1-c6 or Vec-30 cells (1 x 106 to 2 x 106) were cultured in RPMI medium containing 10% fetal bovine serum (FBS). Serum was withdrawn from culture medium when cells were 70% confluent. After 72 h, 10% FBS was added in the medium for an additional 12 h. Cells were fixed in 70% ethanol, stained with propidium iodide, and DNA content was analyzed by Cytomics FC (Beckman Coulter).
Western blot analysis. Western blotting was done according to the standard protocol with antibodies for PLC1, cyclin D1, and ß-actin (Santa Cruz Biotechnology); and p21, total Akt, and Akt (Ser473; Cell Signaling Technology).
Statistical analysis. Statistical analysis was done with the SPSS standard version 13.0. The statistical significance of the correlations between PLC1 expression and promoter methylation, LOH, as well as clinicopathologic characteristics were assessed by 
2 test or Fisher's exact tests. Results expressed as mean ± SD were analyzed using the Student's t test. Differences were considered significant when P value was <0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1 in ESCCs. The gene expression of PLC1 in nine ESCC cell lines and 50 primary ESCC tumors and their paired nontumorous tissues were studied by semiquantitative RT-PCR. Expression of PLC1 was observed in all 50 tested nontumorous tissues. However, absent expression of PLC1 was detected in 26 of 50 (52%) primary ESCCs (Fig. 1A
). Similarly, absent expression of PLC1 was detected in four of nine (EC109, KYSE30, 180, and 510) ESCC cell lines (Fig. 1B). PLC1 expression in protein level was also studied using ESCC tissue microarray containing 221 ESCC cases. Expression of PLC1 was detected in all 26 informative normal esophageal epithelia. However, absent expression of PLC1 protein was detected in 82 of 197 (41.6%) informative ESCC cases (Fig. 1C).
|
1 expression was significantly higher in primary ESCCs with lymph node metastasis (49 of 97 cases, 51%) than that in primary ESCCs without metastasis (33 of 100, 33%, P = 0.014, Fisher's exact test). No significant association was observed between down-regulation of PLC1 and other clinicopathologic variables (Table 1
).
|
1 promoter region in ESCC. To determine whether down-regulation of PLC1 in ESCC is caused by hypermethylation in its promoter region, the methylation status of PLC1 was analyzed. MSP using methylation- or unmethylation-specific primers was done to investigate the methylation status of PLC1. In four ESCC cell lines (EC109, KYSE30, KYSE180, and KYSE510) with absent expression of PLC1, only methylated allele of PLC1 was detected (Fig. 2A
). In two cell lines (KYSE140 and KYSE410) with normal or weak expression of PLC1, both methylated and unmethylated alleles were detected. In contrast, no methylated allele was observed in immortalized esophageal epithelial cells (NE1) and ESCC cell lines (HKESC1, EC18, and KYSE520) with PLC1 expression (Fig. 2A). To further explore the methylation details of PLC1 in ESCC, three ESCC cell lines with different degrees of PLC1 methylation were characterized by BGS. BGS results revealed a high density of methylated CpG sites in KYSE30 cell line with absent expression of PLC1, whereas most of the CpG sites were unmethylated in the immortalized esophageal epithelial cells (NE1) and HKESC1 cell line with PLC1 expression (Fig. 2B). These results showed that methylation of the PLC1 promoter was associated with its transcriptional repression.
|
1 promoter in 50 primary ESCC tumors and their paired nontumorous tissues by MSP. Methylation of PLC1 was detected in 19 of 50 (38%) of the primary ESCCs (Fig. 2C). In contrast, methylation was only found in 3 of 50 (6%) of the paired nontumorous tissues.
Inactivation of both alleles of PLC1 in ESCC. According to two-hit inactivation theory (18), functional loss of both alleles is necessary for the inactivation of TSG. ESCC cell line KYSE30 (tetraploid in chromosome number) was used to test whether the absent expression of PLC1 was caused by the inactivation of both alleles. The DNA copy number of PLC1 gene was studied by FISH with a BAC clone containing PLC1 gene. The result showed that loss of two copies of PLC1 gene was observed in KYSE30 cells (Fig. 3A
). The remaining two alleles of PLC1 in KYSE30 cells were completely methylated (Fig. 3B). To further show that the remaining two alleles of PLC1 were inactivated by methylation, KYSE30 cells were treated with the demethylating agent 5-aza-dC. After treatment, expression of PLC1 was restored, with concomitant demethylation of its promoter (Fig. 3C). These findings indicated that deletion of one allele and hypermethylation of the other allele may play a synergetic role in the silencing of the PLC1 gene.
|
1 with its allelic loss and hypermethylation in 50 primary ESCCs. LOH and hypermethylation were detected in 21 and 19 cases, respectively. In 26 ESCCs with absent expression of PLC1, inactivation of PLC1 in 22 (84.6%) cases was correlated with either LOH (n = 5) or methylation (n = 7) only, or both LOH and methylation (n = 10, Fig. 3D). Statistical analysis showed that down-regulation of PLC1 was significantly associated with LOH and methylation of PLC1 (P < 0.05, Fisher's exact test).
Tumor-suppressive ability of PLC1. To determine if PLC1 has tumor-suppressive ability, two stably PLC1-expressing clones (PLC1-c6 and PLC1-c7) were selected from PLC1-transfected KYSE30 cells. PLC1 gene and protein expression in PLC1-c6 and PLC1-c7 were confirmed by RT-PCR and Western blot analysis (Fig. 4A
). Tumor-suppressive function of PLC1 was assessed by foci formation assay, soft agar assay, cell growth assay, and tumor xenograft experiment. Foci formation assay showed that the efficiency of foci formation was significantly inhibited (P < 0.001) in PLC1-c6 and PLC1-c7 cells compared with Vec-30 cells (Fig. 4B). A similar result was obtained from soft agar assay, in which the colony formation in soft agar was significantly reduced in PLC1-c6 and PLC1-c7 cells compared with Vec-30 cells (P < 0.001; Fig. 4C). Cell growth assay also revealed that the cell growth rates in PLC1-c6 and PLC1-c7 cells were significantly inhibited by PLC1 (P < 0.001) compared with Vec-30 cells (Fig. 4D, left).
|
1, tumor formation in nude mouse was tested by injection of PLC1-c6 cells (n = 10) or PLC1-c7 cells (n = 10), whereas Vec-30 cells were used as controls. Within 4 weeks, solid tumors were readily visible in left hind legs of all 20 mice (injected with Vec-30 cells), but only observed in 4 of 10 and 5 of 10 mice injected with PLC1-c6 and PLC1-c7 cells, respectively. In addition, the size of tumors caused by PLC1-expressing cells (tumor volume, 50 ± 30 mm3) was significantly smaller than tumors (840 ± 200 mm3) induced by Vec-30 cells (P < 0.001; Fig. 4D, right). These results showed that PLC1 has a strong tumor-suppressive ability both in vitro and in vivo.
Metastasis-suppressing ability of PLC1. Because our tissue microarray result showed that down-regulation of PLC1 protein was closely associated with ESCC metastasis, the metastatic role of PLC1 was studied by cell adhesion and wound-healing assays. The result revealed that exogenous expression of PLC1 could significantly increase cell adhesion ability in PLC1-c6 cells (P < 0.05) compared with Vec-30 cells (Fig. 5A
). The wound-healing assay showed that PLC1 was able to inhibit cell mobility (Fig. 5B).
|
1. To explore the mechanism underlying growth inhibition by PLC1, the cell cycle distributions of PLC1-c6 and Vec-30 cells were determined by flow cytometry. Before treatment, the percentage of cells in S phase was obviously decreased in PLC1-c6 cells compared with Vec-30 cells (Fig. 5C). After 3 days serum starvation followed by addition of 10% serum for 12 h, the PLC1-c6 cells showed even more obvious G1-S–phase arrest compared with Vec-30 cells (Fig. 5C).
Up-regulation of p21 and down-regulation of phosphorylated Akt by PLC1. To investigate the potential molecular mechanism of PLC1 in cell cycle arrest, we examined the effects of PLC1 on several key cell cycle regulators, including p21, cyclin D1, and Akt from phosphatidylinositol 3-kinase/Akt pathway. Increased expression of p21 and down-regulation of phosphorylated Akt at Ser473 were detected (Fig. 5D). However, no significant difference of cyclin D1 was detected in this study. These results supported that PLC1 can arrest cell cycle at G1-S checkpoint.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1 was identified as a candidate TSG within the deleted region.6
In the present study, we found that mRNA expression of PLC1 was down-regulated in 44.4% of ESCC cell lines and 52% of primary ESCC tumors. Tissue microarray study showed that absent expression of PLC1 in protein level was detected in 41.6% primary ESCCs. In addition to the frequent allelic loss at the PLC1 locus, methylation of CpGs at its promoter region was also frequently observed in primary ESCC tumors with absence of PLC1 expression (17 of 26, 65.4%), suggesting that promoter hypermethylation plays an important role in the inactivation of PLC1 during the tumorigenesis of esophageal cancer. In ESCC cell line KYSE30, we clearly showed that the inactivation of PLC1 gene was caused by the loss of two copies of PLC1 gene and promoter methylation of the other two copies of PLC1. Collectively, these results suggested that the promoter methylation and DNA copy loss of PLC1 gene represent the "first hit" and the "second hit," respectively, in the inactivation of PLC1 during the ESCC development.
The tumor-suppressive function of PLC1 has been investigated by both in vitro and in vivo assays in this study. The results showed that PLC1 could effectively suppress cell growth, decrease foci formation and colony formation in soft agar, and suppress tumor formation in nude mice. Further study revealed that PLC1 was able to inhibit G1-S phase transition through the activation of p21. A PLC1-interacting protein, GTPase-activating protein p122 (p122RhoGAP), was isolated from rat and activates the PIP2-hydrolyzing activity of PLC1 (27). Later, a human homologue of the p122 gene, named deleted in liver cancer 1 (DLC1) gene, was identified as a tumor-suppressor gene (28). The tumor-suppressive role of DLC1 in the development of hepatocellular carcinoma has been widely studied (29–31). The tumor-suppressing function of PLC1 might be associated with DLC1 because the later can stimulate the activity of PLC1 (27). Interestingly, a recent study suggested that DLC1 could also increase p21 expression in breast cancer cells (32). G1-S phase transition is a major checkpoint for cell cycle progression and p21 is one of the critical negative regulators during this transition (33). This result suggested that PLC1 could be an important member in DLC1 tumor-suppressing pathway.
Another clue that PLC1 might collaborate with DLC1 in tumor suppression was that PLC1 can increase cell adhesion ability. Our tissue microarray results also suggested that the down-regulation of PLC1 was significantly associated with lymph node metastasis in ESCC. Interestingly, activation of PLC1 by DLC1 could reduce the amount of PIP2 (27). PIP2 can bind to actin-associated cytoskeletal proteins (e.g., vinculin and talin) and increase the formation of focal adhesion (34). On the basis of these findings, Kawai et al. (35) further showed that p122RhoGAP localized in focal adhesions may inhibit tumor cell migration through the activation of PLC1. In a more recent report, DLC1 was found to be able to inhibit cell migration in human HCC cells (36).
In this study, we also found that PLC1 could down-regulate phosphorylated Akt expression, suggesting that the tumor-suppressive function of PLC1 might be associated with the inhibition of phosphatidylinositol 3-kinase/AKT signal transduction pathway. Previous studies have shown that Akt promotes cell cycle progression directly through inactivation of p21 (37, 38). Our results did show that PLC1 was able to down-regulate Akt and up-regulate p21, which provides new insight into the mechanism of PLC1 in cell cycle arrest. In summary, we provided evidences that PLC1 is a potent tumor-suppressor gene and plays important suppressive role in ESCC development and progression. Further studies will be necessary to elucidate the mechanism of suppressive effect of PLC1 in human cancers.
|
Acknowledgments |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|
Footnotes |
|---|
Received 6/27/07. Revised 8/30/07. Accepted 9/24/07.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
): a novel Ras effector. EMBO J 2001;20:743–54.[CrossRef][Medline] stimulating activities. EMBO J 1995;14:286–91.[Medline]1-binding protein, p122RhoGAP, is localized in focal adhesions. Biochem Soc Trans 2004;32:1107–9.[CrossRef][Medline]This article has been cited by other articles:
|
|
 |
|
  M. Li, H. Edamatsu, R. Kitazawa, S. Kitazawa, and T. Kataoka Phospholipase C{varepsilon} promotes intestinal tumorigenesis of ApcMin/+ mice through augmentation of inflammation and angiogenesis Carcinogenesis, August 1, 2009; 30(8): 1424 - 1432. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Correction: PLC{delta}1 as a Tumor Suppressor in ESCC Cancer Res., May 1, 2008; 68(9): 3549 - 3549. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
