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Clinical Cancer Research Vol. 9, 5295-5298, November 1, 2003
© 2003 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Methylation-Mediated Silencing of SOCS-1 Gene in Hepatocellular Carcinoma Derived from Cirrhosis

Osamu Okochi, Kenji Hibi1, Mitsuru Sakai, Soichiro Inoue, Shin Takeda, Tetsuya Kaneko and Akimasa Nakao

Second Department of Surgery, Nagoya University School of Medicine, Nagoya, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Suppressor of cytokine signaling-1 (SOCS-1) is a negative regulator of Janus kinase and signal transducer and activation of transcription pathway. Recently, it was demonstrated that SOCS-1 gene was silenced frequently by methylation of CpG island in human hepatocellular carcinoma (HCC). We examined the methylation-mediated silencing of SOCS-1 in tumors of HCC patients.

Experimental Design: Fifty patients with HCC were investigated in this study. We examined the methylation status of the SOCS-1 promoter region by methylation-specific PCR and then confirmed the methylation-mediated silencing of SOCS-1 by Northern blot analysis. Furthermore, this methylation status was compared with clinicopathological findings.

Results: Aberrant methylation of the SOCS-1 gene was detected in 30 of 50 (60%) HCC specimens. No corresponding nontumorous liver tissues showed SOCS-1 methylation. Subsequent Northern analysis proved that methylation of the SOCS-1 promoter inactivated translation and diminished expression of SOCS-1 mRNA. We then analyzed the correlation between the clinicopathological data and SOCS-1 aberrant methylation and found that HCC derived from liver cirrhosis had a significant relationship with SOCS-1 methylation (P = 0.0207).

Conclusions: SOCS-1 may be a novel tumor suppressor, and its aberrant methylation may be a key event for HCC transformation of cirrhotic nodules.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HCC2 is a common malignant disease, and its incidence has recently increased around the world (1 , 2) . The main risk factors associated with HCC are infection with hepatitis C virus, infection with hepatitis B virus, and liver cirrhosis followed by these viral infections. According to etiological study, liver cirrhosis is considered to be a premalignant region because HCC occurs frequently in the background of liver cirrhosis.

Recent advances in genetics have proved that accumulated genetic alterations through the repeating destruction and regeneration of hepatocytes are supposed to result in carcinogenesis. A variety of oncogenes such as c-myc, cyclin D1, and ß-catenin and tumor suppressor genes such as p16, p53, and Rb genes were reported to be associated with hepatocarcinogenesis (3, 4, 5, 6) . However, the specific genes that play a leading role have not been identified.

Recently, Yoshikawa et al. (7) demonstrated that the SOCS-1 gene is frequently silenced by methylation of the CpG island in human HCC. SOCS-1 is an intracellular protein that negatively regulates the JAK/STAT signaling pathway, which is a principal cytokine signaling transduction pathway. The JAK/STAT pathway has also been known to play an important role in the regeneration of hepatocytes (8 , 9) , and furthermore, additional studies have indicated that both JAKs and STATs are involved in the oncogenesis of several tumors (10, 11, 12) .

These findings suggested a potential role of SOCS protein, a growth suppressor of HCC, through negative regulation of the JAK/STAT pathway.

To define the role of SOCS-1 in the tumorigenic pathway of the liver, we examined the methylation-mediated silencing of SOCS-1 in tumors of 50 HCC patients. The results obtained were then compared with clinicopathological aspects.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Sample Collection and DNA Preparation.
Fifty primary tumors and corresponding nontumorous liver tissues were collected at Nagoya University Hospital from HCC patients during liver resection surgery. All tissues were confirmed histologically. Collected samples were stored immediately at -80°C until analysis. DNA was prepared as described previously (13) . Clinicopathological profiles of the patients enrolled in the study are shown in Table 1Citation . All patients with liver cirrhosis belonged to class A according to Child-Pugh classification.


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  		Table 1 Clinicopathological findings and methylation status of the SOCS-1 promoter in HCC patients

 
Sodium Bisulfite Modification.
One µg of genomic DNA extracted from tumors and nontumorous tissues was subjected to bisulfite treatment as described previously (14) . Briefly, alkali-denatured DNA was modified by 2.1 M sodium bisulfite/0.5 mM hydroquinone at pH 5.0. The bisulfite-reacted DNA was then treated with NaOH, purified using Wizard DNA Clean-Up System (Promega, Madison, WI), precipitated with ethanol, and resuspended in distilled water.

MSP.
The bisulfite-treated DNA was amplified by MSP. The primers for MSP were described previously (7) . The PCR was hot-started at 95°C for 5 min, and the protocol consisted of 35 cycles of 95°C for 30 s, 60°C for 1 min, and 72°C for 1 min and a final extension of 72°C for 5 min. DNA from Hep3B and HuH-7 (HCC cell lines) was used as a positive control for methylated and unmethylated alleles, respectively. Control reaction without DNA was performed for each set of PCR. Ten µl of each PCR product were loaded directly onto nondenaturing 8% polyacrylamide gels, stained with ethidium bromide, and visualized under UV illumination.

Northern Analysis.
Resected specimens were lysed in guanidine thiocyanate, and RNA was extracted using cesium chloride density gradient centrifugation. Northern blot hybridization was performed essentially as described previously (15) . The SOCS-1 cDNA probe for hybridization was generated using the reverse transcription-PCR method. This probe was designed as nucleotide position 679-1030 of the SOCS-1 cDNA sequence. A human ß-actin probe was used as an internal control.

Statistical Analysis.
The association between SOCS-1 methylation status and clinicopathological findings was examined by the {chi}2 test. Statistical significance was considered as P < 0.05.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We first examined the methylation status of the SOCS-1 promoter region in 50 HCC and 50 corresponding nontumorous liver tissue specimens using MSP (Fig. 1)Citation . Aberrant methylation of the SOCS-1 gene was detected in 30 of 50 (60%) HCC specimens. Most of the tumors also exhibited unmethylation, which might account for the contamination of nontumorous cells in tumor specimens. No corresponding nontumorous liver tissues showed SOCS-1 methylation.



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  		Fig. 1. Representative MSP analysis of SOCS-1 in tumors (T) and corresponding nontumorous liver tissues (N) of HCC patients. Unmethylated PCR products are shown as a 175-bp band in Lane U, whereas methylated PCR products are as a 160-bp band in Lane M. DNA from Hep3B and HuH-7 cell lines was used as a positive control for methylated and unmethylated alleles, respectively.

 
To confirm inactivation of the SOCS-1 gene by methylation, we then examined SOCS-1 mRNA expression in primary tumors and corresponding liver tissues by Northern analysis (Fig. 2)Citation . Thirty-one paired RNA samples obtained from the 50 patients described above were available for this examination. Of 18 tumors that showed SOCS-1 methylation, 15 had lower expression of SOCS-1 mRNA than the corresponding nontumorous liver tissues. On the other hand, in the 13 tumors that showed no SOCS-1 methylation, we observed the same or higher level of SOCS-1 expression compared with the corresponding nontumorous tissues. These observations indicated that methylation of the SOCS-1 promoter inactivated the translation to a greater or lesser extent.



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  		Fig. 2. Northern analysis of SOCS-1 gene in tumors (T) and corresponding nontumorous liver tissues (N) of HCC patients. Lower expression of SOCS-1 was observed in tumors with SOCS-1 methylation than in the corresponding nontumorous tissues. The same or higher level of SOCS-1 expression was observed in tumors without SOCS-1 methylation. A human ß-actin probe was used as an internal control.

 
Subsequently, we analyzed the correlation between the clinicopathological data and aberrant SOCS-1 methylation (Table 1)Citation . As a result, we found that HCC derived from liver cirrhosis had a significant relationship with SOCS-1 methylation (P = 0.0207). No other findings such as gender, hepatitis virus infection, tumor size, distribution of tumors, differentiation of tumor, and vascular invasion were correlated with SOCS-1 methylation status.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Cytokines are crucial secreted proteins that regulate cellular proliferation and differentiation. The stimuli of these mediators are mainly led to the transcriptional activation of cytokine-induced genes through the JAK/STAT signaling pathway (16) . Recently, the potential role of the JAK/STAT pathway in oncogenesis has been proposed in many kinds of tumors (10, 11, 12 , 17 , 18) . On the other hand, the SOCS family has been identified as a negative feedback protein of cytokine-induced signaling pathway (19, 20, 21) . These proteins are activated by STATs and negatively regulate the JAK/STAT pathway by inhibiting the JAKs directly or blocking the access of the STATs. Although the mechanism by which SOCS proteins regulate cytokine signaling has been studied to an extent, their biological roles continue to be examined. In recent studies, it was reported that SOCS-1 expression was suppressed through aberrant methylation of the CpG island in several HCC cell lines and that restored SOCS-1 expression suppressed both the growth rate and anchorage-independent growth of cells (7 , 22) . Another study (23) has shown that GFI-1B, a proto-oncogenic protein derived from GFI-1 in T-cell lymphoma, repressed reporter activity of SOCS-1/SOCS-3 promoters. In the current study, we frequently observed aberrant methylation of the SOCS-1 promoter in the tumors of HCC patient, whereas the same methylation was not detected in the corresponding nontumorous liver tissues. Furthermore, we showed methylation-mediated silencing of SOCS-1 expression in the tumors using Northern analysis. These observations suggested that SOCS-1 might act as a tumor suppressor, at least in certain HCCs.

Subsequently, we compared the methylation status of SOCS-1 in the tumors of HCC patients with their clinicopathological features, and we demonstrated that SOCS-1 methylation was observed more frequently in HCCs derived from cirrhosis than in those that were not derived from cirrhosis. With regard to this result, it was supposed that inactivation of SOCS-1 might be an important factor for hepatocarcinogenesis, especially in patients with cirrhosis. Growth factors and cytokines are critical for maintaining liver volume and physiology (24) , and the JAK/STAT pathway activated in response to these agents is associated with the proliferation of hepatocytes. In particular, TNF-{alpha} and IL-6 are important components of the signaling pathway that lead to liver regeneration (9) . Plasma TNF-{alpha} and IL-6 levels were significantly higher than in patients with liver cirrhosis than in those without it, and the severity of liver cirrhosis was an important factor for the occurrence of increased IL-6 level (25 , 26) . This phenomenon was supposed to be caused by decreased cytokine clearance of the liver as well as enhanced endogeneous lipopolysaccharide levels. High levels of plasma TNF-{alpha} and IL-6 after liver cirrhosis may induce the activation of the JAK/STAT pathway. Both activated cytokine pathway and inactivated negative regulators such as SOCS-1 may result in unrestricted proliferation of hepatocytes.

Cirrhotic nodules have long been considered to be premalignant lesions followed by HCC (27) . It was suggested that accumulated genetic alterations induced by continuous regeneration of hepatocytes might lead to HCC. Recently, it was reported that several cirrhotic nodules already had some chromosomal aberrations, and more allelic imbalances appeared in the progression to HCC (28) . In this study, methylation of the SOCS-1 promoter was closely related to the pathogenesis of HCC patients with liver cirrhosis. Besides, SOCS-1 methylation was detected in early stages of HCC, whereas it was undetectable in the corresponding nontumorous liver tissues. These findings indicated that SOCS-1 might be a novel tumor suppressor and that its aberrant methylation might be a key event for HCC transformation of cirrhotic nodules.

This study provided solid evidence for additional studies on the molecular mechanism of SOCS-1 in HCC and also confirmed that SOCS-1 might play an important role in the carcinogenic pathway in the liver of patients with cirrhosis. These observations offered the possibility that tumor formation in liver cirrhosis might be controlled by inducing the expression of silenced SOCS-1 using demethylating reagents.


   ACKNOWLEDGMENTS
 
We thank Y. Nishikawa and M. Taguchi for technical assistance.


   FOOTNOTES
 
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.

1 To whom requests for reprints should be addressed, at Second Department of Surgery, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. Phone: 81-52-744-2245; Fax: 81-52-744-2255; E-mail: khibi{at}med.nagoya-u.ac.jp Back

2 The abbreviations used are: HCC, hepatocellular carcinoma; JAK, Janus kinase; STAT, signal transducer and activation of transcription; MSP, methylation-specific PCR; TNF-{alpha}, tumor necrosis factor {alpha}; IL-6, interleukin 6. Back

Received 2/10/03; revised 7/ 3/03; accepted 7/ 3/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. El-Serag H. B., Mason A. C. Rising incidence of hepatocellular carcinoma in the United States. N. Engl. J. Med., 340: 745-750, 1999.[Abstract/Free Full Text]
  2. Taylor-Robinson S. D., Foster G. R., Arora S., Hargreaves S., Thomas H. C. Increase in primary liver cancer in the UK, 1979–94. Lancet, 350: 1142-1143, 1997.[Medline]
  3. Tabor E. Tumor suppressor genes, growth factor genes, and oncogenes in hepatitis B virus-associated hepatocellular carcinoma. J. Med. Vir., 42: 357-365, 1994.
  4. Nishida N., Fukuda Y., Komeda T., Kita R., Sando T., Furukawa M., Amenomori M., Shibagaki I., Nakao K., Ikenaga M. Amplification and overexpression of the cyclin D1 gene in aggressive human hepatocellular carcinoma. Cancer Res., 54: 3107-3110, 1994.[Abstract/Free Full Text]
  5. de la Costa A., Romagnolo B., Billuart P., Renard C. A., Buendia M. A., Soubrane O., Fabre M., Chelly J., Beldjord C., Kahn A., Perret C. Somatic mutations of the ß-catenin gene are frequent in mouse and human hepatocellular carcinomas. Proc. Natl. Acad. Sci. USA, 95: 8847-8851, 1998.[Abstract/Free Full Text]
  6. Emi M., Fujiwara Y., Ohata H., Tsuda H., Hirohashi S., Koike M., Miyaki M., Monden M., Nakamura Y. Allelic loss at chromosome band 8p21.3-p22 is associated with progression of hepatocellular carcinoma. Genes Chromosomes Cancer, 7: 152-157, 1993.[Medline]
  7. Yoshikawa H., Matsubara K., Qian G. S., Jackson P., Groopman J. D., Manning J. E., Harris C. C., Herman J. G. SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity. Nat. Genet., 28: 29-35, 2001.[CrossRef][Medline]
  8. Cressman D. E., Diamond R. H., Taub R. Rapid activation of the Stat3 transcription complex in liver regeneration. Hepatology, 21: 1443-1449, 1995.[CrossRef][Medline]
  9. Cressman D. E., Greenbaum L. E., DeAngelis R. A., Ciliberto G., Furth E. E., Poli V., Taub R. Liver failure and defective hepatocyte regeneration in interleukin-6-deficient mice. Science (Wash. DC), 274: 1379-1383, 1996.[Abstract/Free Full Text]
  10. Lacronique V., Boureux A., Valle V. D., Poirel H., Quang C. T., Mauchauffe M., Berthou C., Lessard M., Berger R., Ghysdael J., Bernard O. A. A TEL-JAK2 fusion protein with constitutive kinase activity in human leukemia. Science (Wash. DC), 278: 1309-1312, 1997.[Abstract/Free Full Text]
  11. Bromberg J. F., Wrzeszczynska M. H., Devgan G., Zhao Y., Pestell R. G., Albanese C., Darnell J. E., Jr. Stat3 as an oncogene. Cell, 98: 295-303, 1999.[CrossRef][Medline]
  12. Yoshida T., Hanada T., Tokuhisa T., Kosai K., Sata M., Kohara M., Yoshimura A. Activation of STAT3 by the hepatitis C virus core protein leads to cellular transformation. J. Exp. Med., 196: 641-653, 2002.[Abstract/Free Full Text]
  13. Hibi K., Taguchi M., Nakayama H., Takase T., Kasai Y., Ito K., Akiyama S., Nakao A. Molecular detection of p16 promoter methylation in the serum of patients with esophageal squamous cell carcinoma. Clin. Cancer Res., 7: 3135-3138, 2001.[Abstract/Free Full Text]
  14. Frommer M., McDonald L. E., Miller D. S., Collis C. M., Watt F., Grigg G. W., Molloy P. L., Paul C. L. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc. Natl. Acad. Sci. USA, 89: 1827-1831, 1992.[Abstract/Free Full Text]
  15. Hibi K., Nakamura H., Hirai A., Fujikake Y., Kasai Y., Akiyama S., Ito K., Takagi H. Loss of H19 imprinting in esophageal cancer. Cancer Res., 56: 480-482, 1996.[Abstract/Free Full Text]
  16. Darnell J. E., Jr., Kerr I. M., Stark G. R. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science (Wash. DC), 264: 1415-1421, 1994.[Abstract/Free Full Text]
  17. Migone T. S., Lin J. X., Cereseto A., Mulloy J. C., O’Shea J. J., Franchini G., Leonard W. J. Constitutively activated Jak-STAT pathway in T cells transformed with HTLV-1. Science (Wash. DC), 269: 79-81, 1995.[Abstract/Free Full Text]
  18. Danial N. N., Pernis A., Rothman P. B. Jak-STAT signaling induced by the v-abl oncogene. Science (Wash. DC), 269: 1875-1877, 1995.[Abstract/Free Full Text]
  19. Starr R., Willson T. A., Viney E. M., Murray L. J. L., Rayner J. R., Jenkins B. J., Gonda T. J., Alexander W. S., Metcalf D., Nicola N. A., Hilton D. J. A family of cytokine-inducible inhibitors of signaling. Science (Wash. DC), 387: 917-921, 1997.
  20. Endo T. A., Masuhara M., Yokouchi M., Suzuki R., Sakamoto H., Mitsui K., Matsumoto A., Tanimura S., Ohtsubo M., Misawa H., Miyazaki T., Leonor N., Taniguchi T., Fujita T., Kanakura Y., Komiya S., Yoshimura A. A new protein containing an SH2 domain that inhibits JAK kinases. Science (Wash. DC), 387: 921-924, 1997.[CrossRef]
  21. Naka T., Narazaki M., Hirata M., Matsumoto T., Minamoto S., Aono A., Nishimoto N., Kajita T., Taga T., Yoshizaki K., Akira S., Kishimoto T. Structure and function of a new STAT-induced STAT inhibitor. Science (Wash. DC), 387: 924-929, 1997.[CrossRef]
  22. Nagai H., Kim Y. S., Lee K. T., Chu M. Y., Konishi N., Fujimoto J., Baba M., Matsubara K., Emi M. Inactivation of SSI-1, a JAK/STAT inhibitor, in human hepatocellular carcinomas, as revealed by two-dimensional electrophoresis. J. Hepatol., 34: 416-421, 2001.[CrossRef][Medline]
  23. Jegalian A. G., Wu H. Regulation of Socs gene expression by the proto-oncoprotein GFI-1B: two routes for STAT5 target gene induction by erythropoietin. J. Biol. Chem., 277: 2345-2352, 2002.[Abstract/Free Full Text]
  24. Andus T., Bauer J., Gerok W. Effects of cytokines on the liver. Hepatology, 13: 364-375, 1991.[CrossRef][Medline]
  25. Tilg H., Wilmer A., Vogel W., Herold M., Nolchen B., Judmaier G., Huber C. Serum levels of cytokines in cirrhotic liver diseases. Gastroenterology, 103: 264-274, 1992.[Medline]
  26. Lee F. Y., Lu R. H., Tsai Y. T., Lin H. C., Hou M. C., Li C. P., Liao T. M., Lin L. F., Wang S. S., Lee S. D. Plasma interleukin-6 levels in patients with cirrhosis. Relationship to endotoxemia, tumor necrosis factor-{alpha}, and hyperdynamic circulation. Scand. J. Gastroenterol., 31: 500-505, 1996.[Medline]
  27. Tsukuma H., Hiyama T., Tanaka S., Nakao M., Yabuuchi T., Kitamura T., Nakanishi K., Fujimoto I., Inoue A., Yamazaki H., Kawashima T. Risk factors for hepatocellular carcinoma among patients with chronic liver disease. N. Engl. J. Med., 328: 1797-1801, 1993.[Abstract/Free Full Text]
  28. Yeh S. H., Chen P. J., Shau W. Y., Chen Y. W., Lee P. H., Chen J. T., Chen D. S. Chromosomal allelic imbalance evolving from liver cirrhosis to hepatocellular carcinoma. Gastroenterology, 121: 699-709, 2001.[CrossRef][Medline]




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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