Expression of ECRG4 is associated with lower proliferative potential of esophageal cancer cells

Authors


  • Conflict of interest: The authors declare no conflict of interest.

Correspondence: Toshihiko Torigoe, MD, PhD, Department of Pathology, Sapporo Medical University School of Medicine, South-1 West-17 Chuo-ku, Sapporo 060-8556, Japan. Email: torigoe@sapmed.ac.jp

Abstract

We have shown that ECRG4 suppressed Fas-induced apoptosis in Jurkat cells. ECRG4 mRNA expression was ubiquitously detected in normal adult human tissues, suggesting that ECRG4 plays a major role in human tissues. ECRG4 mRNA expression was down-regulated in tumor cells. Expression of ECRG4 suppressed cell growth. We established an anti-ECRG4 monoclonal antibody. Our immunohistochemical analysis demonstrated that ECRG4-positive cells tended to be distributed in the region that was negative for Ki-67 in esophageal squamous cell carcinoma tissues. There was a significant inverse correlation between ECRG4 expression and Ki-67 labeling index in esophageal squamous cell carcinoma. This study provides the first functional evidence for an association of endogenous expression of ECRG4 with cell proliferation. ECRG4 is a candidate tumor suppressor gene that might be involved in the proliferation of esophageal squamous cell carcinoma.

In our recent study, we found that esophageal cancer-related gene 4 (ECRG4) was expressed in resting T-cells but was down-regulated in activated T-cells. Activated T-cells had increased sensitivity to Fas-induced apoptosis and increased proliferative capacity. The results suggested that ECRG4 was involved in reducing Fas-induced apoptosis and suppression of cell growth. Our study clarified that ECRG4 is a novel antiapoptotic gene that might be involved in negative regulation of caspase 8-mediated apoptosis in T-cells.[1] ECRG4 has also been identified as one of the down-regulated genes in esophageal cancer tissues.[2] ECRG4 is down-regulated via promoter hypermethylation in different types of human cancer cells.[3-6] Overexpression of ECRG4 results in suppression of cancer cell growth and inhibition of cancer cell migration and invasion.[7, 8] High expression level of ECRG4 in patients with esophageal squamous cell carcinoma (ESCC) was associated with longer survival than that in patients with low ECRG4 expression level. However, the relationship between ECRG4 expression and cell proliferation in ESCC remains unclear.

In this study, we investigated the relationship between ECRG4 expression and proliferative capacity in ESCC using immunohistochemical staining with clinical samples.

Materials and Methods

Cell lines and culture conditions

Human embryonic kidney cell line 293T, human gastric cancer cell line SNU1, human cervical adenocarcinoma cell line HeLa, and human T cell leukemia cell line Jurkat were obtained from American Type Culture Collection (Manassas, VA). Human oral cancer squamous cell carcinoma cell line HSC2 was obtained from the Human Science Research Resources Bank (HSRRB, Osaka, Japan). Human esophageal squamous cancer cell line TE8 was obtained from RIKEN Bioresource Center (Ibaraki, Japan). Human lung adenocarcinoma cell line 1–87 was obtained from the Cell Resource Center for Biomedical Research, Tohoku University (Sendai, Japan). Human colon adenocarcinoma cell lines Colo320 and SW480 were kind gifts from Dr Kohzoh Imai (Sapporo Medical University, Sapporo, Japan). Human esophageal squamous cancer cell line KE4 was kindly provided by Dr Kyogo Itoh (Kurume University Research Center for Innovation Cancer Therapy, Fukuoka, Japan). Human renal clear cell cancer cell line SMKT R-2 was a kind gift from Dr Noriomi Miyao (Sapporo Medical University, Sapporo, Japan). Human oral squamous cancer cell lines OSC19 and OSC20 and human lung adenocarcinoma cell line LHK2 were established in our laboratory. These cells, except for Jurkat cells, were cultured in DMEM (Sigma-Aldrich, St. Louis MO) with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin G and 100 μg/mL streptomycin. Human T cell leukemia cell line Jurkat cells were cultured in RPMI1640 (Sigma-Aldrich) with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin G and 100 μg/mL streptomycin. The Fas-resistant Jurkat variant clone (Jurkat-FasR) was established and characterized previously.[9]

Reverse transcription-PCR

The cDNA mixture was synthesized by reverse transcription using SuperScript III and oligo(dT) primer (Invitrogen) according to the manufacturer's protocol. For detection of mRNA expression, we performed RT-PCR as follows. Briefly, PCR amplification was done in 25 μL PCR mixture containing 1 μL of the cDNA mixture, KOD DASH DNA polymerase (Toyobo, Osaka, Japan), and 200 pmol of a primer. The PCR mixture was initially incubated at 94°C for 5 min followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 2 s and extension at 74°C for 30 s. For specific detection of ECRG4, the primer pair used for RT-PCR analysis was 5'-AAACGAGAAGCACCTGTTCC-3’ and 5'-GTAGTTGACGCTGGCTCCAT-3’ as forward and reverse primers, respectively. As an internal control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was detected by using forward primer 5'-CGAGATCCCTCCAAAATCAA-3’ and reverse primer 5'-GTCTTCTGGGTGGCAGTGAT-3’.

Cell proliferation assay

Briefly, cells were suspended at a concentration of 0.5–2 × 104 cells in 100 μL of culture medium per well on 96-well flat-bottom plates (Corning, NY) for 24–96 h. Then a 1/10 volume of Premix WST-1 (Takara) was added to each well and the plates were incubated at 37°C for 2 to 4 h. The optical density of each well was quantified by using a microplate reader (Model 680 microplate reader, Bio-Rad). The test wavelength was 450 nm and the reference one was 655 nm. For the MTT assay, cells were incubated with MTT reagent (Sigma) for 4 h, and formazan dye was solubilized by the addition of acidic isopropanol. The test wavelength was 570 nm and the reference one was 630 nm.

Plasmids and transfection

To construct expression plasmids, total RNA was extracted from the Fas-resistant Jurkat clone, and cDNA was synthesized from the RNA by reverse transcription. Full-length ECRG4 (GenBank: AF325503.1) with an NH2-terminal myc epitope tag was amplified by using specific forward and reverse primers including NheI and XhoI restriction sites. The PCR product was purified and cloned into mammalian expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA). Full-length ECRG4 with a COOH-terminal flag epitope tag was amplified by using specific forward and reverse primers including BamHI and XhoI restriction sites and cloned into mammalian expression vector pcDNA3.1. Similarly, the PCR product of full-length ECRG4 with an NH2-terminal flag epitope tag was produced and cloned into NheI and XhoI restriction sites of the pcDNA3.1 vector. 293T cells were transfected by using Lipofectamine 2000 reagent (Invitrogen). For the construct of protein expression, an N-terminal deletion mutant of ECRG4 cDNA was inserted into the pQE30 (QIAGEN) vector, which allows the expression of recombinant proteins with an NH2-terminal His6 tag, at the BamHI and SalI sites. The primer pair was 5'-CGCGGATCCCGAGAAGCACCTGTTCCAACT-3’ as a forward primer and 5'-ACGCGTCGACTTAGTAGTCATCGTAGTTGAC-3’ as a reverse primer (underlines indicating BamHI and SalI recognition sites, respectively). The cDNA sequences were confirmed by direct sequencing.

Tissue samples

Tissue specimens were described previously.[1] They were obtained from an esophageal cancer patient who underwent surgery at Kushiro City General Hospital. Tumor tissues and corresponding normal tissues were frozen at −80°C. The patient and family gave informed consent for the use of tissue specimens in research.

Western blotting

Cell lysate with radioimmunoprecipitation assay buffer (150 mmol/L NaCl, 1.0% NP40, 0.5% deoxycholic acid, 0.1% SDS, 50 mmol/L Tris-HCl (pH 8.0), protease inhibitor cocktail (Complete, Roche Diagnostics, Basel, Switzerland)) were boiled with SDS sample buffer and then separated by 12.5% or 15% SDS-PAGE. The proteins were transferred electrophoretically to a polyvinylidene fluoride membrane (Immunobilon-P, Millipore, Billerica, MA) and probed with mouse anti-FLAG m2 (Sigma-Aldrich), mouse anti-myc 9E10, or mouse anti-β-actin mAb AC-15 (Sigma-Aldrich). After three washes with wash buffer (0.1% Tween 20, PBS), the membrane was reacted with a peroxidase-labeled secondary antibody (peroxidase-labeled goat anti-mouse IgG; KPL, Gaithersburg, MD). Finally, the signal was visualized by using an enhanced chemiluminescence detection system (Amersham Life Science, Arlington Heights, IL) according to the manufacturer's protocol.

Establishment of anti-ECRG4 monoclonal antibody

To characterize the expression of endogenous ECRG4, we established mouse antiserum against ECRG4 as described previously.[10] N-terminal deletion mutant of ECRG4 (aa41-148) recombinant protein (100 μg) was used for immunization of BALB/c mice by intraperitoneal (i.p.) injection four times at 2-week intervals. One week after the last injection, spleen cells were collected and fused with the NS-1 mouse myeloma cell line (ATCC) at a 4:1 ratio. All mouse procedures were carried out in accordance with institutional protocol guidelines at Sapporo Medical University School of Medicine. Screening was performed with ELISA using recombinant N-terminal deletion mutant of ECRG4 protein and Western blotting.

Immunohistochemical staining

Immunohistochemical staining was done on formalin-fixed, paraffin-embedded sections as described previously.[11] Four- to 5-μm-thick sections were cut, deparaffinized in xylene, and rehydrated in graded alcohol. Antigen retrieval was done by boiling for 20 min in a microwave oven in a preheated 0.01 mol/L concentration of sodium citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked by 3% hydrogen peroxide in ethanol for 5 min. Slides were incubated for 1 h with the mAb: anti-ECRG4 and anti-Ki67 (clone BGX-Ki-67, Biogenex, Fremont, CA). After incubation, the slides were washed thrice with PBS and incubated for 30 min with Simple Stain MAX-PO (Multi) secondary antibody mixture (Nichirei, Tokyo, Japan). After washing thrice with PBS, staining was done by incubation for 1 to 2 min with 3,3'-diaminobenzidine used as the chromogen, and counterstaining was done with Mayer's hematoxylin.

Statistical analysis

Statistical differences between groups were evaluated using a two-tailed unpaired-t test or a two-tailed Mann-Whitney U-test or the chi square test and anova, with P-values <0.05 considered significant.

Results

Overexpression of ECRG4 suppresses cell growth

First, cell growth rates were assessed and the growth rates of Jurkat-FasR and parental Jurkat cells were compared. Jurkat-FasR cells showed decreased proliferative capacity (Fig. 1a). Since ECRG4 is one of the genes overexpressed in Jurkat-FasR cells, we established stable ECRG4-expressing cells by gene transfection. Jurkat-mock cells were established by transfecting the plasmid vector pIRESpuro3 into Jurkat cells as described previously.[1] Jurkat-ECRG4 cells had a significantly slower growth rate than that of the control cells ( Fig. 1b). Next, we examined proliferative capacity of 293T cells. ECRG4-negative 293T cells were transfected with a plasmid encoding myc-tagged ECRG4, and the resulting 293T-ECRG4 cells transiently expressed the mRNA and protein (Fig. 1c,d). Cell growth rates were assessed and compared among parental 293T cells (293T-WT), 293T-mock transfectant cells and 293T-ECRG4 cells. 293T-ECRG4 cells had a significantly slower growth rate than that of the control cells (Fig. 1e). These findings suggested that ECRG4 might be involved in cell growth as well as Fas-induced apoptotic signals.

Figure 1.

(a) ECRG4 expression suppresses cell growth. Parental Jurkat cells and Jurkat-FasR cells (2 × 103) were plated on 96-well tissue culture dishes. The relative cell numbers were assessed at 24, 48, 72 h after plating by WST-1 assay. (cell number at 24 h = 1.0). The data represent averages of triplicate samples and standard deviation. **, P < 0.01, unpaired t-test. (b) Stable ECRG4-expressing Jurkat (Jurkat-ECRG4) cells were established by transfection with the plasmid pIRESpuro3-ECRG4-FLAG and puromycin selection. Jurkat-mock cells were established by transfection with the mock plasmid vector pIRESpuro3. Parental Jurkat cells and Jurkat transfectants (2 × 103) were plated on 96-well tissue culture dishes. The relative cell numbers were assessed at 24, 48, 72 h after plating by WST-1 assay. (cell number at 24 h = 1.0). The data represent averages of triplicate samples and standard deviation. **, P < 0.01, unpaired t-test. (c,d) 293T cells were transfected with the pcDNA3.1-myc-ECRG4 expression vector or pcDNA3.1-myc expression vector. (e) Parental 293T cells (293T-WT) and the transfectants (2 × 103) were plated on 96-well tissue culture dishes. The relative cell numbers were assessed at 24, 48, 72 h after transfection by MTT assay. (cell number at 24 h = 1.0). The data represent averages of triplicate samples and standard deviation. **, P < 0.01, anova. image, Jurkat; image, Jurkat-FasR; image, ECRG4; image, Mock; image, ECRG4; image, Mock; image, WT.

Expression of ECRG4 mRNA in tumor cell lines

ECRG4 mRNA expression was investigated in human tumor cell lines (3 oral cancers, 3 esophageal cancers, 1 gastric cancer, 2 colon cancers, 2 lung cancers, 1 cervical cancer, 1 renal cell cancer). ECRG4 mRNA expression was down-regulated in all human tumor cell lines (Fig. 2).

Figure 2.

ECRG4 mRNA expression in various cancer cells. cDNA samples from various cancer cell lines were analyzed for the expression of ECRG4 and GAPDH by RT-PCR with specific primers.

Establishment of anti-ECRG4 monoclonal antibody

Next, we examined whether a decrease in endogenous ECRG4 level alters cell growth. In order to evaluate the endogenous ECRG4 protein expression level, we established an anti-ECRG4 monoclonal antibody (Fig. 3a). We examined ECRG4 protein expression level in an esophageal cancer patient. ECRG4 was down-regulated in esophageal cancer tissues compared with the levels in normal tissues (Fig. 3b). Next, we examined ECRG4 protein levels in formalin-fixed, paraffin-embedded esophageal squamous cell carcinoma specimens by immunohistochemical staining. ECRG4 immunoreactivity was evaluated qualitatively. Since ECRG4 was cloned from the normal esophageal epithelium, cases in which ECRG4 immunoreactivity in the tumor tissues was the same as that in the normal esophageal epithelium were defined as positive (Fig. 3c). ECRG4 was distributed in the cytoplasm. Cases in which ECRG4 immunoreactivity in the tumor tissues was less than that in the normal esophageal epithelium were defined as negative (Fig. 3d).

Figure 3.

Establishment of anti-ECRG4 monoclonal antibody and immunohistochemical staining with the specific antibody. (a) 293T cells were transfected with the pcDNA3.1-ECRG4-FLAG expression vector. The transfected cells were characterized by Western blotting with anti-ECRG4 monoclonal antibody, anti-FLAG tag antibody and anti-β-actin antibody. (b) Expression of ECRG4 was determined by RT-PCR and Western blotting in tissue pairs of noncancerous tissue (Normal) and carcinoma tissue (Tumor) in an esophageal cancer patient. Immunohistochemical staining (x100) showed (c) ECRG4-positive staining and (d) ECRG4-negative staining.

Histopathological analysis of ECRG4 expression and cell growth

Since ECRG4 suppressed growth of cultured cells, we examined whether the ECRG4 protein level affects proliferative capacity of esophageal squamous cell carcinoma cells. We examined the relationship in esophageal squamous cell carcinoma tissues between ECRG4 expression and Ki-67 labeling index. Clinicopathological data for 10 patients diagnosed with ESCC are summarized in Table 1. There were no significant differences between the ECRG4 negative group and ECRG4 positive group. In esophageal squamous cell carcinoma tissues, ECRG4-positive cells tended to be distributed in the region that was negative for Ki-67 in many, but not all, cases examined (Fig. 4a–d). Our immunohistochemical analysis demonstrated a significant inverse correlation between ECRG4 expression and Ki-67 labeling index in esophageal squamous cell carcinoma (Fig. 4e). Results of immunohistochemical staining for the 10 patients diagnosed with ESCC are summarized in Table 2. Furthermore, we confirmed that there was no correlation between ECRG4 positivity and Ki-67 positivity using high magnification data with serial sections (Fig. S1).

Figure 4.

Inverse relationship of the immunoreactivity between ECRG4 and Ki67. Immunohistochemical staining of anti-ECRG4 and anti-Ki67 (x100). (a,b) ECRG4-positive staining case. (c,d) ECRG4-negative staining case. (e) Ki-67 labeling indexes of ECRG4-positive and -negative cases. Each value is the median and standard error of the mean. *, P < 0.05, Mann-Whitney U-test.

Table 1. Association between esophageal cancer-related gene 4 (ECRG4) expression and clinicopathological characteristics of patients with esophageal squamous cell carcinoma (ESCC)
CharacteristicsECRG4ECRG4P-value
Positive n = 5Negative n = 5
  1. Chi square test.
Age, y  0.20
≤6031 
>6024 
Sex  0.30
Male45 
Female10 
Tumor status  0.72
T111 
T221 
T322 
T401 
Lymph node metastasis  0.57
N010 
N133 
N211 
N301 
Stage  0.50
I10 
II21 
III22 
Iva01 
IVb01 
Tumor cell differentiation  0.28
Good20 
Moderate23 
Poor12 
Table 2. Correlation between esophageal cancer-related gene 4 (ECRG4) expression and esophageal squamous cell carcinoma (ESCC) clinicopathological features
CaseAgeGenderDifferentiationpTINFpNLyvStageECRG4Ki-67
 170malegoodT3INFbN2ly1V0III++++
 250malegoodT3INFbN1ly2v1III+
 351malemoderateT2INFbN1ly2v2II+
 458malepoorT2INFbN1ly0v0II+
 569femalemoderateT1bINFaN0ly0v0I+
 659malepoorT2INFcN1ly3v0IVb+++
 762malemoderateT4INFbN1ly1v1IVa+
 873malemoderateT3INFbN1ly2v0III++
 970malemoderateT3INFbN3ly0v1III+++
1067malepoorT1bINFaN2ly1v0II+++

Discussion

We have shown that ECRG4-negative ESCC has high proliferative capacity by using clinical samples obtained from surgical resections. We previously reported that ECRG4 was a novel antiapoptotic protein and that it suppressed the activation of caspase 8.[1] The ECRG4 gene was previously cloned from human normal esophageal epithelium and identified as one of the down-regulated genes in esophageal squamous cell carcinoma tissues.[2] The mechanism of inactivation of this gene in tumor tissues involves promoter hypermethylation, which is a frequent molecular event in esophageal squamous cell carcinoma.[3] We previously reported ECRG4 expression in normal adult tissues and tumor tissues. ECRG4 mRNA expression was detected ubiquitously in normal adult tissues, and expression of ECRG4 mRNA was investigated in several pairs of tumor tissues and corresponding normal tissues derived from surgical specimens of the same patient (1 esophageal cancer, 4 gastric cancers, 4 colon cancers, 2 liver cancers, and 2 kidney cancers). ECRG4 mRNA expression was down-regulated in 11 of the 13 stomach, kidney, esophagus and colon tumor tissues.[1] ECRG4 mRNA expression is decreased in esophageal cancer cells, gastric cancer cell lines, colorectal cancer cells and glioma cells.[3, 4, 6] Our data showed that ECRG4 mRNA expression was decreased in many other types of human cancer cell lines including oral cancer, esophageal cancer, gastric cancer, colorectal cancer, lung cancer, cervical cancer and renal cancer cell lines. ECRG4 contains a CDC45 homology domain that overlaps with the APC10 homology domain in the middle region of the protein.[1] Since CDC45 and APC10 homolog domains are cell cycle control domains, ECRG4 might be involved in cell growth. It has been shown that exogenous ECRG4 suppresses cell growth.[4, 6-8] Our data also showed that exogenous ECRG4 suppressed proliferation of Jurkat cells and 293T cells. However, the relationship between endogenous ECRG4 expression and cell proliferation remains unclear. To clarify the relationship between endogenous ECRG4 expression and cell proliferation, we established an anti-ECRG4 monoclonal antibody.

Our immunohistochemical staining results demonstrated that downregulation of endogenous ECRG4 was associated with high proliferative capacity of ESCC cells. ECRG4 protein expression has been shown to be associated with primary tumor size, regional lymph node metastasis, and tumor stage in ESCC in Chinese patients.[8] Similarly, ECRG4 mRNA expression level in patients with locally invasive T2-4 tumors was significantly lower than that in the less invasive T1 tumors, and the levels in stage 4 tumors was significantly lower than the levels in tumors in stages 0–3. However, ECRG4 mRNA expression levels were not significantly different with respect to lymph node status in Japanese patients.[12] Ethnic variations in tumor biology have been reported.[13, 14] Our clinicopathological characteristics were no significant differences in primary tumor size (T1-3 versus T4, P = 0.29), regional lymph node metastasis (N0-1 versus N2-3, P = 0.49), and tumor stage (stages 1–3 versus stage 4, P = 0.11) in ESCC. With respect to tumor size and tumor stage, the small population of our study might be the reason for the observed statistically insignificant. However, with respect to regional lymph node metastasis, no significant differences might be due to ethnicity of the patients. Since a high expression level of ECRG4 in patients with ESCC is associated with longer survival than that in patients with low ECRG4 expression level,[12] ECRG4 expression is a candidate prognostic marker for ESCC. Therefore, our anti-ECRG4 antibody might contribute to prediction of the prognosis for patients with ESCC.

In conclusion, the present clinicopathological study clarified the relationship between ECRG4 expression and cell proliferation capacity in ESCC. Our results indicate that ECRG4 staining might be a good clinicopathological prognostic marker for patients with ESCC.

Acknowledgment

This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Ancillary