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Brief report

Aberrant expression of Wnt antagonist SFRP1 in pancreatic cancer

BU, Xian-min; ZHAO, Cheng-hai; DAI, Xian-wei

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Pancreatic cancer is one of the malignant tumor with a very poor prognosis. Both genetic and epigenetic alterations are involved in the pathogenetic mechanisms of pancreatic cancer. Hypermethylation and subsequent loss of expression of some tumor suppressor genes and tumor-related genes occur frequently in pancreatic cancer, such as loss of expression of p16,1 RASSF1A,2 SOCS-1,3 and hMLH14 genes were repoted.

SFRP1 was found in 1987 by several different groups,5–7 containing a cysteine-rich domain (CRD) which shares 30%-50% sequence similarity with that of Wnt receptor Frizzled protein. Through the CRD, SFRP1 can antagonize Wnt signaling by interacting with Wnt ligand. As Wnt signaling pathway plays important roles not only in embryo development, but also in proliferation, differentiation, and apoptosis in adult tissues. Thus aberrant activation of Wnt pathway may induce tumorigenesis. As a Wnt inhibitor, SFRP1 downregulation caused by hypermethylation has been found in several cancers.8–12

In order to investigate the function of the Wnt antagonists SFRP1 expression, the methylation status and aberrant expression of SFRP1 in pancreatic cancer were investigated in this study, the correlation between the expression of SFRP1 and clinical pathologic characteristics of primary pancreatic cancers were also analyzed.

METHODS

Cell lines, normal pancreatic tissues, tumor and matched adjacent tissue samples preparation

The human pancreatic cancerous cell lines CFPAC-1, PC-3 and PANC-1 were obtained from KUNKEN Bio-reagent Corp. (Shanghai, China). These cell lines were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, penicillin (100 IU/ml), and streptomycin (100 μg/ml). Normal pancreatic tissue samples were obtained from China Medical University Biopsy Center. Primary pancreatic cancer and matched adjacent tissue samples were obtained from patients who underwent operation at the Second Affiliated Hospital, China Medical University. The samples were frozen in liquid nitrogen immediately after surgery. Haematoxylinand eosin-staining were used to assure that cancer samples were mostly consist of tumor cells and no tumor cell was in tumor adjacent tissue samples. The patients characteristics were shown in Table 1.

Table 1
Table 1:
Clinico-pathological characteristics of primary pancreatic cancers (n=36)

Immunohistochemical assay

SABC staining method was used to detect the SFRP1 expression in 6 normal pancreatic samples and 10 samples of primary pancreatic cancers. The procedures followed protocols of the kit. The primary antibody SFRP1 was purchased from Santa Cruz (CA, USA), and was used at a dilution of 1:2000. The second antibody was biotinylated mouse antigoat antibody.

DNA and RNA extraction

DNA was extracted by a standard phenol/chloroform extraction and ethanol precipitation procedure. RNA was isolated by Trizol (Takara Co., Japan) according to the protocols supplied by the manufacturers.

Reverse transcription-polymerase chain reaction (PCR)

Semiquantitative RT-PCR was performed by Takara RNA PCR 3.0 Kit. cDNA was synthesized from 1 μg SFRP1 RNA using random 9 primer and AMV reverse transcriptase. Cycle condition was 1 cycle of 30°C for 10 minutes, 42°C for 25 minutes, 99°C for 5 minutes and 5°C for 5 minutes. For PCR, the SFRP1 primer sequences were (F) 5′-AGATGCTTAAGTGTGACAAGTTCC-3′ and (R) 5′-TCAGATTTCAACTCGTTGTCACAG-3′. Cycle condition was: 1 cycle of 94°C for 2 minutes, 30 cycles of 94°C for 30 seconds, 60°C for 30 seconds and 72°C for 2 minutes.

Methylation-specific PCR (MSP)

The methylation status of SFRP1 was detected by Genmed MSP Kit (Genmed, Shanghai, China). The procedure was performed according to its protocol. The primers for methylated sequence of SFRP1 were (F) 5′-TGTAGTTTTCGGAGTTAGTGTCGCGC-3′ and (R) 5′-CCTACGATCGAAAACGACGCGAACG-3′, and those for unmethylated sequences were (F) 5′-GTTTTGTAGTTTTTGGAGTTAGTGTTGTGT-3′ and (R) 5′-CTCAACCTACAATCAAAAACAACACAAACA-3′. Cycle condition was 1 cycle of 95°C for 5 minutes, 35 cycles of 95°C for 30 seconds, 60°C for 30 seconds and 72°C for 30 seconds.

Statistical analysis

All data were analyzed using SPSS 11.0 software. Methylation and expression status of SFRP1 between primary pancreatic cancer and cancer adjacent tissues were analyzed by Fisher's exact test. Correlation of clinico-pathological data and aberrant expression of SFRP1 were also analyzed by Fisher's exact test. Statistical significance was taken when P value was less than 0.05.

RESULTS

SFRP1 expression in pancreatic cancers and pancreatic tissues

SFRP1 positive expression was found in all 6 normal pancreatic tissue samples and in 3 out of 10 primary pancreatic cancers. Negtive expression was found in other 7 primary pancreatic cancers (Figures 1 and 2).

Figure 1.
Figure 1.:
SFRP1 positive expression in normal pancreatic tissue (SABC, original magnification ×400)
Figure 2.
Figure 2.:
SFRP1 loss of expression in primary pancreatic cancer (SABC, original magnification ×400).

SFRP1 mRNA expression in pancreatic cell lines and pancreatic cancers

Three pancreatic cell lines, 36 primary pancreatic cancers and matched adjacent tissue samples were investigated for SFRP1 mRNA expression by RT-PCR. SFRP1 was positively expressed in PC-3 cell line, but was negatively expressed in CFPAC-1 and PANC-1 cell lines (Figure 3). SFRP1 loss of expression was detected in 20 out of the 36 primary pancreatic cancers and 6 out of 36 matched tumor adjacent tissue samples, respectively (P <0.01).

Figure 3.
Figure 3.:
SFRP1 mRNA expression was positive in PC-3 cell line, negtive in CFPAC and PANC-1 cell lines. Lane 1: CFPAC-1 cell line; Lane 2: PC-3 cell line; Lane 3: PANC-1 cell line.

Hypermethylation and expression of SFRP1 in pancreatic cancers and pancreatic cell lines

SFRP1 methylation status was analyzed in 3 pancreatic cell lines, 36 primary pancreatic cancers and matched adjacent tissue samples by MSP. SFRP1 hypermethylation was detected in CFPAC-1 and PANC-1 cell lines (Figure 4). SFRP1 hypermethylation was detected in 23 out of 36 primary pancreatic cancers and 8 out of 36 matched tumor adjacent tissue samples, respectively (P <0.01, Figure 5).

Figure 4.
Figure 4.:
SFRP1 hypermethylation was detected in CFPAC-1 and PANC-1 cell lines, but not detected in PC-3 cell line. 1: CFPAC-1; 2: PC-3; 3: PANC-1. M: methylated; U: unmethylated.
Figure 5.
Figure 5.:
Some of the methylated was shown in primary pancreatic cancers. M: methylated; U: unmethylated. 4, 11, 19, 23 was from different patient.

Correlation of SFRP1 expression with SFRP1 hypermethylation and clinico-pathological features in primary pancreatic cancers

The correlation between SFRP1 expression and SFRP1 methylation, together with other clinico-pathologic data was analyzed by Fisher's exact test. A significant correlation (P <0.01) between SFRP1 hypermethylation and SFRP1 loss of expression were found. SFRP1 expression was also significantly correlated with tumor stage (P=0.02) and lymph node metastasis status (P=0.04), but not correlated with patient gender, age, differentiation status (Table 2).

Table 2
Table 2:
Correlation of SFRP1 expression with SFRP1 hypermethylation and clinico-pathologic data

DISCUSSION

The role of aberrant activation of Wnt signaling in tumorigenesis has been reported frequently. Overexpression of members of Wnt family, such as Wnt1, Wnt2 and Wnt3a, has been found in several human cancers.13–15 Downregulation of Wnt inhibitor DKK family was also found in human cancers.16,17 Through the CRD that shares homology with Frizzled CRD, SFRPs family can bind directly to Wnt and alter their ablility to bind on Wnt receptors. It was thought to be another kind of Wnt inhibitor after some experiments were done which showed that SFRP1 could inhibit XWnt8 function in Xenopus embryo5,18 and Wnt1-induced accumulation of β-catenin in cultured cells.19 SFRP1 may promote cell apoptosis. One experiment indicated SFRP1 could sensitize MCF-7 breast cancer cells to TNF-induced apoptosis.6 Additionally, the SFRP1 gene was found at 8p11.2, a site of frequent loss of heterozygosity, so it may be a putative tumor suppressor gene.

Recently, SFRP1 downregulation was found in several human cancers. Most of these reports showed that SFRP1 loss of expression was mainly caused by promoter hypermethylation, an important epigenetic gene silencing mechanism. As a very lethal carcinoma, the pathogenesis of pancreatic cancer has been poorly understood. Our early studies have shown that in normal pancreatic tissues, SFRP1 was strongly expressed. In this study, we investigated the function of the expression status of SFRP1 in pancreatic cancer furtherly. The results showed that SFRP1 loss of expression was found at most of the primary pancreatic cancers which had a significantly correlation with SFRP1 hypermethylation. The hypermethylation rate of primary pancreatic cancer was significantly higher than that of tumor adjacent tissues, which suggested SFRP1 hypermethyaltion and subsequent loss of expression occurred in the early stage of carcinogenesis and played an important role in these cancers development. At the same time, the results also showed SFRP1 loss of expression was significantly correlated with tumor stage and lymph node metastasis status, which meant SFRP1 loss of expression might associate with poor prognosis in pancreatic cancers.

As it is known, Wnt signaling is divided into canonical pathway/β-catenin and non-canonical pathway which includes planar cell polarity pathway and Wnt/Ca2+ pathway. As we did not measure the level of β-catenin, we could not determine the pathway through which SFRP1 loss of expression took part in the pancreatic carcinogenesis. Further study is needed. Because the epigenetic alteration usually is reversible, demethylated drug might be a new therapy of pancreatic cancer.

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Keywords:

SFRP1; Wnt; hypermethylation; pancreatic cancer

© 2008 Chinese Medical Association