Japanese Journal of Clinical Oncology 34:191-194 (2004)
© 2004 Foundation for Promotion of Cancer Research
Hypermethylation-associated Inactivation of the SOCS-1 Gene, a JAK/STAT Inhibitor, in Human Pancreatic Cancers
1 Department of Molecular Biology and 2 Department of Pathology, Institute of Gerontology, Nippon Medical School, Kawasaki, Kanagawa, 3 Center for Digestive Diseases, Nippon Medical School, Kawasaki, Kanagawa, 4 Department of Pathology, Nara Medical University, Kashihara, Nara and 5 Department of Medicine III, Osaka University Medical School, Osaka, Japan
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
|---|
Background: SOCS-1, a JAK-binding protein (SSI-1/SOCS-1/JAB), regulates the JAK/STAT signal transduction pathway that relays signals from various cytokines in the extracellular matrix into the cell. Inactivation of the SOCS-1 gene by methylation has been previously described in hepatocellular carcinomas and multiple myeloma. The purpose of the present work was to analyze the expression of the SOCS-1 gene and identify inactivation of this gene by methylation in pancreatic cancers.
Methods: 20 samples were analyzed. We identified the expression of SOCS-1 gene using RT-PCR and the mechanism of inactivation in this gene by methylation assay.
Results: We documented marked suppression of SOCS-1 mRNA and reduction of SOCS-1 protein in 7 of 14 primary pancreatic cancers examined; moreover, CpG-rich regions upstream of the SOCS-1 gene were hypermethylated in 8 of the 14 tumors.
Conclusions: The results suggested that this gene is silenced in a substantial portion of pancreatic cancers through mechanisms that cause methylation in the promoter region.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
|---|
Pancreatic cancer is one of the most malignant diseases of the human digestive system, and prognosis is generally poor. However, the molecular mechanisms underlying transformation of pancreatic cells remain obscure. Recent molecular analyses have revealed inactivation of certain tumor suppressor genes in pancreatic cancers, including p16, SMAD4 and p53, as well as abnormal activation of the K-ras oncogene (1). Microsatellite analysis, comparative genomic hybridization (CGH) analysis and karyotyping have provided evidence that loss of heterozygosity (LOH) is often an important genetic mechanism that causes inactivation of tumor suppressor genes in this type of cancer, as it frequently occurs in chromosomal regions 17p, 9p, 18q, 12q or 6q (2–7). These observations imply that losses of other tumor-suppressor genes, as yet undefined, contribute to the development of pancreatic tumors.
Genetic alterations that have been observed so far in human pancreatic cancers can be classified into four categories: 1) altered expression of genes encoding growth factors/receptors, which contributes to the malignant process by conferring selective advantages to cells; 2) activation of oncogenes, with or without sequence aberrations, which augments expression; 3) loss of function of tumor suppressor genes, signaled by somatic mutations or LOH in specific chromosomal regions; and 4) changes in DNA-methylation status, either hypo- or hyper-methylation, a common feature in cancer cells.
The SOCS-1 gene (suppressor of cytokine-signaling 1, also termed JAB1 or SSI-1), which encodes a JAK-binding protein that regulates the signal transduction pathway of JAK/STAT (Janus kinase signal transducers and activators of transcription) has been cloned by several researchers, including the present authors. SOCS-1 protein relays signals from various cytokines in the extracellular matrix into cells (8–10). Inactivation of the SOCS-1 gene by methylation has been described in hepatocellular carcinomas (HCCs) (11–13). Moreover, Galm has shown that SOCS-1 is frequently silenced by methylation in multiple myeloma (14). Here, we report altered expression of SOCS-1 in human pancreatic cancers and a possible association between this event and hypermethylation in the gene’s promoter region.
|
SUBJECTS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
|---|
Tumor Specimens and DNA Extraction
Tumor samples and corresponding non-cancerous tissues were obtained from 20 patients with primary pancreatic cancer who underwent surgery at Affiliated Hospitals of Nippon Medical School and Nara Medical University (Table 1). The study was approved by the Institutional Review Board, and informed consent was obtained from all participants prior to surgery. Portions of these tissues were frozen immediately after surgery and stored at –80°C. DNA extraction was carried out according to standard procedures.
|
Expression of the SOCS-1 Gene
Reverse transcription-PCR (RT-PCR) experiments were performed using genomic DNA from resected pancreatic cancer specimens and primers 5'-GGAAGAGCTCCGCGGAGTACAGAGCCCATT-3' and 5'-AACCCAGGCCGGGGAGGGTACCCACAGGT-3'. For semi-quantitative analysis of SOCS-1 expression (8), we used G3PD (primers G3PD-F, 5'-AAGAGGAAGAGAGAGACCCTCACT-3' and G3PD-R, 5'-CATAGGCCCCTCCCCTCTTCAA-3') as a quantity control. Cycling conditions were 94°C for 4 min, then 30 cycles of 94°C for 40 s, 65°C for 30 s and 72°C for 1 min, with final extension for 5 min at 72°C. The PCR products were electrophoresed in a 2% agarose gel.
Methylation Assay
To investigate the mechanism(s) responsible for inactivation of SOCS-1 in pancreatic tumors, we performed methylation assays of the 5' CpG islands around NotI landmark sites, after digesting the genomic region with HpaII, a restriction enzyme that recognizes the CCGG sequence and is sensitive to cytosine methylation.
For this procedure, we digested 1 mg of each DNA in a total volume of 20 ml of reaction solution that contained 10 units of HpaII (Takara, Tokyo, Japan), 10 mM Tris-Cl (pH 7.5), 10 mM MgCl2 and 1 mM Dithiothreitol at 37°C for 3 h. A 1-ul aliquot of reaction solution was used directly for PCR amplification with primers 3' SOCS1-F (5'-GCGCATCACGCGCGCCAGCGCGCTC-3') and 3' SOCS1-R (5'-CTCGTGGGTCCCAGGCCATCTTCACGCTAA-3') to examine methylation status in exonic regions, and 5' SOCS1-F (5'-GGAAGAGCTCCGCGGAGTAC-3') and 5' SOCS1-R (5'-CGGCCGGACAACTCCGGAGG-3') for the promoter region. To detect hypermethylation, a PCR-amplified HpaII digest of genomic DNA from human gastric cancer cell line HuGC-OOHIRA, which yielded no amplification products corresponding to methylation, was used as a negative control.
Analysis of the Genomic Region of SOCS-1 in Tumors
Genomic DNA from ten of the pancreatic cancers were screened for mutations using a single pair of primers for PCR (5'-GGAAGAGCTCCGCGGAGTACAGAGCCCATT-3' and 5'-AACCCAGGCCGGGGAGGGTACCCACAGGT-3'), which were designed to amplify the entire coding region. The products were directly sequenced using primers designed to sequence overlapping segments. Nucleotides were determined by the BigDye terminator cycle-sequencing method (Applied Biosystems, Foster City, CA, USA) using an autosequencer (ABI PRISM 377; Applied Biosystems).
To examine allelic losses at 16p13.13, the chromosomal location of the SOCS-1 gene, we used primers GT356F (5'-TAGGTACAGTGACCTAAAGC-3') and GT356R (5'-CTGCTGAATCATGAAGCTGA-3') to amplify the dinucleotide repeat sequence present on a genomic cosmid clone, super-cos 356d7 (GDB AC002286), which contains both the CA-repeat and the entire SOCS-1 gene (8).
Immunohistochemistry of SOCS-1 Protein
Immunohistochemical staining was performed with goat anti-SOCS-1 polyclonal antibody (Dako, Tokyo, Japan) on formalin-fixed, paraffin-embedded tumor tissues using the SABC (streptoavidin biotin peroxidase complex) method. After heat treatment, peroxidase activity was suppressed with 5% goat serum for 30 min and 1% hydrogen peroxide was added. Primary antibody was applied to the slides and incubated overnight at 4°C. SOCS-1 protein was detected by incubation with biotinylated goat anti-rabbit immunoglobulins (Dako code #E0432) for 1 h. After three washes with PBS, the samples were incubated with 1.25 mg/ml streptoavidin (Dako code #K0377A) and biotinylated horseradish peroxidase (Dako code #K0377B) for 30 min, followed by three more washes in PBS. The slides were covered with 0.05% 3–3' diaminobenzidine (DAB) containing 0.01% hydrogen peroxide for 7 min, rinsed with distilled water, dehydrated in ethanol and overlaid with coverglasses using mounting medium. Expression of SOCS-1 protein was analyzed on the basis of the number of positive cells in a given area.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
|---|
RT-PCR experiments revealed markedly reduced expression of SOCS-1 in 7 of the 14 resected pancreatic cancer specimens in comparison with corresponding normal pancreatic tissues. Figure 1 displays representative results of the RT-PCR assays.
|
Figure 2 shows representative results of methylation assays, showing hypermethylation of the 5' promoter region in tumor DNAs. Hypermethylation was observed upstream of SOCS-1 in 8 of the 14 pancreatic cancers examined (cases 1, 4, 7, 9 and 14). Of the ten pancreatic cancer tissues examined for LOH, none had lost alleles of a microsatellite marker (GTcos356) within the SOCS-1 locus. Direct sequencing of the entire coding region revealed no somatic mutations of SOCS-1 in any of those ten tumors.
|
Figure 3 displays immunohistochemical staining of the representative pancreatic cancer sections with anti-SOCS-1 antibody. All seven specimens that showed suppression of the gene on histological examination, showed marked reductions in SOCS-1 protein.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
|---|
The present study documents a reduction in SOCS-1 expression in human primary pancreatic cancers, often accompanied by hypermethylation of the gene’s promoter region, which silenced the gene in 7 of the 14 tumors examined. Unchanged expression in the other seven tumors might indicate that at least one allele escaped inactivation. Hypermethylation of SOCS-1 appears to have a predominant role in blocking the JAK/STAT signaling pathway during development of this type of tumor (15).
The SOCS-1 gene product is structurally related to a cytokine-inducible SH2 protein that reduces JAK1/JAK2/JAK3 tyrosine kinase activity and suppresses the phosphorylation of tyrosine and activation of STATs. Thus, SOCS-1 functions as a negative regulator of the JAK/STAT signaling pathway (10). Members of the STAT family bind tyrosine-phosphorylated cytokine receptors through their SH2 domains. Once bound to the receptor, STATs are phosphorylated by JAKs, causing them to dissociate from the receptor and form homo-hetero-dimers. STAT dimers then translocate to the nucleus where they interact with specific DNA elements in the promoter regions of the target genes and thereby regulate transcription (11). Emerging evidence strongly implicates abnormal activation of STAT signaling in oncogenic transformation. Constitutive activation of STAT3 proteins occurs frequently in tumor cells (12), suggesting that aberrant STAT3 signaling plays a role in malignant progression. Recent studies have demonstrated an absolute requirement of STAT3 signaling in transformation by the Src oncoprotein (13).
The results of the present study suggest that dysregulation of the JAK/STAT signal transduction pathway plays a role in the development of pancreatic tumors. The mechanisms that subvert normal, highly regulated STAT signaling by means of Src and other oncoproteins have not yet been defined completely. Understanding how oncoproteins alter STAT signaling should provide further insight into the role of abnormal STAT activation in oncogenesis, and may suggest a mechanistic basis for circumventing the oncogenic process. The uniqueness of STAT3 as the sole STAT family member that is constitutively active in Src-transformed fibroblasts makes this system an excellent model for investigating such mechanisms.
In a previous study, we had demonstrated inactivation of the SOCS-1 gene in HCCs. In that study, expression level of SOCS-1 mRNA was markedly suppressed in 50% of HCCs (4/8). In an additional series of methylation analysis using 30 HCCs, 16 (53%) showed hypermethylation of the gene. As a further step, it would be useful to identify the mechanism that regulates the methylation status of the SOCS-1 gene in development and progression of human pancreatic cancer. The results of this study showed that reduction of SOCS-1 expression correlated with its methylation in promoter region of SOCS-1 gene in five (case 1, 4, 7, 14 and 16) of seven cases (71%).
|
Acknowledgments |
|---|
|
TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
|---|
The authors thank Aya Yabe, Sizuyo Miyamoto, Junko Sato, Mayumi Tanaka and Naoko Tsuruta for their technical assistance. This work was supported by special grants for Strategic Advanced Research on Cancer from the Ministry of Education, Science, Sports and Culture of Japan; by a Research Grant from the Ministry of Health and Welfare of Japan; and by a Research for the Future Program Grant of The Japan Society for the Promotion of Science.
|
FOOTNOTES |
|---|
+ For reprints and all correspondence: Mitsuru Emi, Department of Molecular Biology, Institute of Gerontology, Nippon Medical School, 1–396, Kosugi-cho, Nakahara-ku, Kawasaki 211–8533, Japan. E-mail: memi{at}nms.ac.jp
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
|---|
1 Hruban RH, Lacobuzio-Donahue C, Wilentz RE. Molecular pathology of pancreatic cancer. Cancer J 2001;7:251–8.[Web of Science][Medline]
2 Hahn SA, Seymour AB, Hoque AT, Schutte M, da Costa LT, Redston MS, et al. Allelotype of pancreatic adenocarcinoma using xenograft enrichment. Cancer Res 1995;55:4670–5.
3 Kimura M, Abe T, Sunamura M, Matsuno S, Horii A. Detailed deletion mapping on chromosome arm 12q in human pancreatic adenocarcinoma; identification of a 1-cM region of common allelic loss. Genes Chromosome Cancer 1996;17:88–93.[CrossRef][Web of Science][Medline]
4 Bardi G, Johansson B, Pandis N, Mandahl N, Bak-Jensen E, Andren-Sandberg A, et al. Karyotypic abnormalities in tumours of the pancreas. Br J Cancer 1993;67:1106–12.[Web of Science][Medline]
5 Johansson B, Bardi G, Pandis N, Gorunova L, Backman PL, Mandahl N, et al. Karyotypic pattern of pancreatic adenocarcinomas correlates with survival and tumour grade. Int J Cancer 1994;58:8–13.[Web of Science][Medline]
6 Kimura M, Furukawa T, Abe T, Yatsuoka T, Youssef EM, Yokoyama T, et al. Identification of two common regions of allelic loss in chromosome arm 12q in human pancreatic cancer. Cancer Res 1998;58:2456–60.
7 Abe T, Makino M, Furukawa T, Ouyang H, Kimura M, Yatsuoka T, et al. Identification of three commonly deleted regions on chromosome arm 6q in human pancreatic cancer. Genes Chromosome Cancer 1999;25:60–4.[CrossRef][Web of Science][Medline]
8 Naka T, Narazaki M, Hirata M, Matsumoto T, Minamoto S, Aono A, et al. Structure and function of a new STAT-induced STAT inhibitor. Nature 1997;387:924–7.[CrossRef][Medline]
9 Jones PA, Laird PW. Cancer epigenetics comes of age. Nature Genet 1999;21:163–7.[CrossRef][Web of Science][Medline]
10 Wen Z, Zhong Z, Darnell JE Jr. Maximal activation of transcription by Stat1 and Stat3 require both tyrosine and serine phosphorylation. Cell 1995;82:241–50.[CrossRef][Web of Science][Medline]
11 Krebs DL, Hilton DJ. SOCS: physiological suppressors of cytokine signalling. J Cell Science 2000;113:2813–9.[Abstract]
12 Garcia R, Jove R. Activation of STAT transcription factors in oncogenic tyrosine kinase signaling. J Biomed Sci 1998;5:79–85.[Web of Science][Medline]
13 Yoshikawa H, Matsubara K. SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity. Nature Genet 2001;28(1):29–35.[CrossRef][Web of Science][Medline]
14 Galm O, Yoshikawa H. SOCS-1, a negative regulator of cytokine signaling, is frequently silenced by methylation in multiple myeloma. Blood 2003;101(7):2784–8.
15 Turkson J, Bowman T, Adnane J, Zhang Y, Djeu JY, Sekharam M, et al. Requirement for Ras/Rac1-mediated p38 and c-Jun N-terminal kinase signaling in Stat3 transcriptional activity induced by the Src oncoprotein. Mol Cell Biol 1999;19:7519–28.
Received October 27, 2003; accepted January 27, 2004
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. G. Francipane, V. Eterno, V. Spina, M. Bini, G. Scerrino, G. Buscemi, G. Gulotta, M. Todaro, F. Dieli, R. De Maria, et al. Suppressor of Cytokine Signaling 3 Sensitizes Anaplastic Thyroid Cancer to Standard Chemotherapy Cancer Res., August 1, 2009; 69(15): 6141 - 6148. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. L. Wu, H. Miao, and S. Khan JAK kinases promote invasiveness in VHL-mediated renal cell carcinoma by a suppressor of cytokine signaling-regulated, HIF-independent mechanism Am J Physiol Renal Physiol, December 1, 2007; 293(6): F1836 - F1846. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Komyod, M. Bohm, D. Metze, P. C. Heinrich, and I. Behrmann Constitutive Suppressor of Cytokine Signaling 3 Expression Confers a Growth Advantage to a Human Melanoma Cell Line Mol. Cancer Res., March 1, 2007; 5(3): 271 - 281. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||