Deletion of 3p is one of the most frequent chromosomal alterations in many solid tumors, including esophageal squamous cell carcinoma (ESCC), suggesting the existence of one or more tumor-suppressor genes at 3p. Recently, our loss of heterozygosity study revealed that 3p22 was frequently deleted in ESCC and a candidate tumor-suppressor gene (TSG), phospholipase C-δ1 (PLCδ1), was identified within the 3p22 region. In this study, absent expression of PLCδ1 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 PLCδ1 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 PLCδ1 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 PLCδ1 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 PLCδ1 plays an important suppressive role in the development and progression of ESCC. [Cancer Res 2007;67(22):10720–5]
- esophageal squamous cell carcinoma
- tumor suppressor gene
- loss of heterozygosity
Esophageal squamous cell carcinoma (ESCC), the major histologic subtype of esophageal cancer, is one of the most common malignancies and ranked as the sixth leading cause of cancer death worldwide ( 1). ESCC is characterized by its remarkable geographic distribution, and >50% of ESCC cases in the world occur in China. Linzhou (formerly Linxian) and the nearby counties in Henan province of Northern China have the highest incidence of ESCC in the world ( 1, 2). As other solid tumors, the pathogenesis of ESCC is also a long process with multiple genetic alterations, including inactivation of tumor-suppressor genes and activation of oncogenes. Therefore, identification of the recurrent genetic changes of ESCC is critical for the isolation of ESCC-related genes.
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)-δ1 (PLCδ1), was identified within the 3p22 region. Loss of one copy of PLCδ1 gene was detected in 39 of 97 (40.2%) ESCCs by quantitative real-time PCR. 6 PLCδ1 gene is composed of 16 exons and encodes a protein of 756 amino acids. PLCδ1 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 (β, γ, δ, and ε) 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). PLCβ1 and PLCγ1 isoforms have been linked with PKC activation ( 11, 12); however, the direct link between PLCδ1 and PKC is still unclear.
Although the expression levels, regulation mechanisms, and biochemical features of PLCδ1 have been widely studied, its role in cancer development is still unclear. In the present study, PLCδ1 expression pattern in primary ESCCs and ESCC cell lines was investigated. Tumor-suppressive function of PLCδ1 was shown by both in vitro and in vivo assays. In addition, down-regulation of PLCδ1 protein was associated with ESCC metastasis. The tumor-suppressive mechanism of PLCδ1 was also addressed.
Materials and Methods
Cell lines and primary tumor specimens. Three Chinese ESCC cell lines (HKESC1, EC18, and EC109) and six Japanese ESCC cell lines (KYSE30, KYSE140, KYSE180, KYSE410, KYSE510, and KYSE520; ref. 13) were kindly provided by Professor Srivastava (Department of Pathology, The University of Hong Kong). Two immortalized esophageal cell lines (NE1 and NE3) were kindly provided by Professor Tsao (Department of Anatomy, The University of Hong Kong). The primary ESCC tumor tissues and their paired nontumorous tissues were collected immediately after surgical resection at Linzhou Cancer Hospital (Henan, China). Samples used in this study were approved by the Committees for Ethical Review of Research involving Human Subjects at Zhengzhou University and The University of Hong Kong.
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 PLCδ1 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 PLCδ1 expression in KYSE30 cells, 2 × 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 PLCδ1 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-PLCδ1 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 PLCδ1. To test tumor-suppressive function of PLCδ1, PLCδ1 was PCR amplified, cloned into pcDNA3.1/V5-His TOPO TA vector (Invitrogen), and transfected into ESCC cell line KYSE30 cells. Stable PLCδ1-expressing clones (PLCδ1-c6 and PLCδ1-c7) were selected for further study. Blank vector–transfected KYSE30 cells (Vec-30) were used as control. For foci formation assay, 1 × 103 PLCδ1-expressing cells (PLCδ1-c6 or PLCδ1-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 × 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 PLCδ1-expressing clones (PLCδ1-c6 and PLCδ1-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 × 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 PLCδ1 was investigated by tumor xenograft experiment. About 2 × 106 PLCδ1-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 PLCδ1-c6 cells and 10 mice for PLCδ1-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 × L × 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 1× 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. PLCδ1-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. PLCδ1-c6 or Vec-30 cells (1 × 106 to 2 × 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 PLCδ1, cyclin D1, and β-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 PLCδ1 expression and promoter methylation, LOH, as well as clinicopathologic characteristics were assessed by χ2 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.
Frequent down-regulation of PLCδ1 in ESCCs. The gene expression of PLCδ1 in nine ESCC cell lines and 50 primary ESCC tumors and their paired nontumorous tissues were studied by semiquantitative RT-PCR. Expression of PLCδ1 was observed in all 50 tested nontumorous tissues. However, absent expression of PLCδ1 was detected in 26 of 50 (52%) primary ESCCs ( Fig. 1A ). Similarly, absent expression of PLCδ1 was detected in four of nine (EC109, KYSE30, 180, and 510) ESCC cell lines ( Fig. 1B). PLCδ1 expression in protein level was also studied using ESCC tissue microarray containing 221 ESCC cases. Expression of PLCδ1 was detected in all 26 informative normal esophageal epithelia. However, absent expression of PLCδ1 protein was detected in 82 of 197 (41.6%) informative ESCC cases ( Fig. 1C).
Furthermore, clinical association study showed that the frequency of absent PLCδ1 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 PLCδ1 and other clinicopathologic variables ( Table 1 ).
Frequent methylation of PLCδ1 promoter region in ESCC. To determine whether down-regulation of PLCδ1 in ESCC is caused by hypermethylation in its promoter region, the methylation status of PLCδ1 was analyzed. MSP using methylation- or unmethylation-specific primers was done to investigate the methylation status of PLCδ1. In four ESCC cell lines (EC109, KYSE30, KYSE180, and KYSE510) with absent expression of PLCδ1, only methylated allele of PLCδ1 was detected ( Fig. 2A ). In two cell lines (KYSE140 and KYSE410) with normal or weak expression of PLCδ1, 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 PLCδ1 expression ( Fig. 2A). To further explore the methylation details of PLCδ1 in ESCC, three ESCC cell lines with different degrees of PLCδ1 methylation were characterized by BGS. BGS results revealed a high density of methylated CpG sites in KYSE30 cell line with absent expression of PLCδ1, whereas most of the CpG sites were unmethylated in the immortalized esophageal epithelial cells (NE1) and HKESC1 cell line with PLCδ1 expression ( Fig. 2B). These results showed that methylation of the PLCδ1 promoter was associated with its transcriptional repression.
We next investigated the methylation frequency of PLCδ1 promoter in 50 primary ESCC tumors and their paired nontumorous tissues by MSP. Methylation of PLCδ1 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 PLCδ1 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 PLCδ1 was caused by the inactivation of both alleles. The DNA copy number of PLCδ1 gene was studied by FISH with a BAC clone containing PLCδ1 gene. The result showed that loss of two copies of PLCδ1 gene was observed in KYSE30 cells ( Fig. 3A ). The remaining two alleles of PLCδ1 in KYSE30 cells were completely methylated ( Fig. 3B). To further show that the remaining two alleles of PLCδ1 were inactivated by methylation, KYSE30 cells were treated with the demethylating agent 5-aza-dC. After treatment, expression of PLCδ1 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 PLCδ1 gene.
We then investigated the correlation of down-regulation of PLCδ1 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 PLCδ1, inactivation of PLCδ1 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 PLCδ1 was significantly associated with LOH and methylation of PLCδ1 (P < 0.05, Fisher's exact test).
Tumor-suppressive ability of PLCδ1. To determine if PLCδ1 has tumor-suppressive ability, two stably PLCδ1-expressing clones (PLCδ1-c6 and PLCδ1-c7) were selected from PLCδ1-transfected KYSE30 cells. PLCδ1 gene and protein expression in PLCδ1-c6 and PLCδ1-c7 were confirmed by RT-PCR and Western blot analysis ( Fig. 4A ). Tumor-suppressive function of PLCδ1 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 PLCδ1-c6 and PLCδ1-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 PLCδ1-c6 and PLCδ1-c7 cells compared with Vec-30 cells (P < 0.001; Fig. 4C). Cell growth assay also revealed that the cell growth rates in PLCδ1-c6 and PLCδ1-c7 cells were significantly inhibited by PLCδ1 (P < 0.001) compared with Vec-30 cells ( Fig. 4D, left).
To further explore the in vivo tumor-suppressive ability of PLCδ1, tumor formation in nude mouse was tested by injection of PLCδ1-c6 cells (n = 10) or PLCδ1-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 PLCδ1-c6 and PLCδ1-c7 cells, respectively. In addition, the size of tumors caused by PLCδ1-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 PLCδ1 has a strong tumor-suppressive ability both in vitro and in vivo.
Metastasis-suppressing ability of PLCδ1. Because our tissue microarray result showed that down-regulation of PLCδ1 protein was closely associated with ESCC metastasis, the metastatic role of PLCδ1 was studied by cell adhesion and wound-healing assays. The result revealed that exogenous expression of PLCδ1 could significantly increase cell adhesion ability in PLCδ1-c6 cells (P < 0.05) compared with Vec-30 cells ( Fig. 5A ). The wound-healing assay showed that PLCδ1 was able to inhibit cell mobility ( Fig. 5B).
Cell cycle arrest at G1-S-phase by PLCδ1. To explore the mechanism underlying growth inhibition by PLCδ1, the cell cycle distributions of PLCδ1-c6 and Vec-30 cells were determined by flow cytometry. Before treatment, the percentage of cells in S phase was obviously decreased in PLCδ1-c6 cells compared with Vec-30 cells ( Fig. 5C). After 3 days serum starvation followed by addition of 10% serum for 12 h, the PLCδ1-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 PLCδ1. To investigate the potential molecular mechanism of PLCδ1 in cell cycle arrest, we examined the effects of PLCδ1 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 PLCδ1 can arrest cell cycle at G1-S checkpoint.
Deletion of 3p is one of the most frequent chromosomal alterations in many malignancies, including ESCC. Several candidate TSGs at 3p have been reported, including FHIT at 3p14.2 ( 19), RASSF1A ( 20), CACNA2D2 ( 21), and DLEC1 at 3p21.3 ( 22). Although these TSGs have been widely studied, most of the studies were done in lung and renal carcinomas. As for ESCC, deletion of 3p14.2-p21.2 ( 23, 24) was frequently detected and several candidate TSGs within the region have been studied, including DLEC1, VHL, RAR-β, RASSF1A, FHIT, and ADAMTS9 ( 22, 25, 26). In our recent study, we provided the first evidence that the deletion of 3p22 was one of the most frequently deleted regions in ESCC and PLCδ1 was identified as a candidate TSG within the deleted region. 6
In the present study, we found that mRNA expression of PLCδ1 was down-regulated in 44.4% of ESCC cell lines and 52% of primary ESCC tumors. Tissue microarray study showed that absent expression of PLCδ1 in protein level was detected in 41.6% primary ESCCs. In addition to the frequent allelic loss at the PLCδ1 locus, methylation of CpGs at its promoter region was also frequently observed in primary ESCC tumors with absence of PLCδ1 expression (17 of 26, 65.4%), suggesting that promoter hypermethylation plays an important role in the inactivation of PLCδ1 during the tumorigenesis of esophageal cancer. In ESCC cell line KYSE30, we clearly showed that the inactivation of PLCδ1 gene was caused by the loss of two copies of PLCδ1 gene and promoter methylation of the other two copies of PLCδ1. Collectively, these results suggested that the promoter methylation and DNA copy loss of PLCδ1 gene represent the “first hit” and the “second hit,” respectively, in the inactivation of PLCδ1 during the ESCC development.
The tumor-suppressive function of PLCδ1 has been investigated by both in vitro and in vivo assays in this study. The results showed that PLCδ1 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 PLCδ1 was able to inhibit G1-S phase transition through the activation of p21. A PLCδ1-interacting protein, GTPase-activating protein p122 (p122RhoGAP), was isolated from rat and activates the PIP2-hydrolyzing activity of PLCδ1 ( 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 PLCδ1 might be associated with DLC1 because the later can stimulate the activity of PLCδ1 ( 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 PLCδ1 could be an important member in DLC1 tumor-suppressing pathway.
Another clue that PLCδ1 might collaborate with DLC1 in tumor suppression was that PLCδ1 can increase cell adhesion ability. Our tissue microarray results also suggested that the down-regulation of PLCδ1 was significantly associated with lymph node metastasis in ESCC. Interestingly, activation of PLCδ1 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 PLCδ1. 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 PLCδ1 could down-regulate phosphorylated Akt expression, suggesting that the tumor-suppressive function of PLCδ1 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 PLCδ1 was able to down-regulate Akt and up-regulate p21, which provides new insight into the mechanism of PLCδ1 in cell cycle arrest. In summary, we provided evidences that PLCδ1 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 PLCδ1 in human cancers.
Grant support: Research Grant Council Central Allocation grant HKUST 2/06C and Sun Yat-Sen University “Hundred Talents Program” grant 85000-3171311.
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.
↵6 Y. Qin, et al. High-throughput loss of heterozygosity study of chromosome 3p in esophageal cancer using single nucleotide polymorphism markers, submitted for publication.
- Received June 27, 2007.
- Revision received August 30, 2007.
- Accepted September 24, 2007.
- ©2007 American Association for Cancer Research.