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Phospho-Smad3 signaling is predictive biomarker for hepatocellular carcinoma risk assessment in primary biliary cholangitis patients

Naohiro Nakamura1,Katsunori Yoshida1,*,Rinako Tsuda1,Miki Murata1,Takashi Yamaguchi1,Kanehiko Suwa1,Mayuko Ichimura2,Koichi Tsuneyama2,Koichi Matsuzaki1,Toshiaki Nakano1,Junko Hirohara1,Toshihito Seki1,Kazuichi Okazaki1, M. Eric Gershwin3,Makoto Naganuma1
1
Department of Gastroenterology and Hepatology, Kansai Medical University, 573-1010 Osaka, Japan
2
Department of Pathology & Laboratory Medicine, Institute of Biomedical Sciences, Tokushima University Graduate School, 770-8503 Tokushima, Japan
3
Division of Rheumatology, Allergy and Clinical Immunology, University of California at Davis School of Medicine, Davis, CA 95616, USA
DOI: 10.52586/5042 Volume 26 Issue 12, pp.1480-1492
Submited: 18 September 2021 Revised: 08 November 2021
Accepted: 16 November 2021 Published: 30 December 2021
*Corresponding Author(s):  
Katsunori Yoshida
E-mail:  
yoshidka@hirakata.kmu.ac.jp
Copyright: © 2021 The author(s). Published by BRI. This is an open access article under the CC BY 4.0 license (https://creativecommons.org/licenses/by/4.0/).
Abstract

Introduction: Patients with primary biliary cholangitis (PBC) are at increased risk for development of hepatocellular carcinoma (HCC), particularly in the presence of comorbidities such as excessive alcohol consumption. Although liver fibrosis is an important risk factor for HCC development, earlier predictors of future HCC development in livers with little fibrosis are needed but not well defined. The transforming growth factor (TGF)-ββ/Smad signaling pathway participates importantly in hepatic carcinogenesis. Phosphorylated forms (phospho-isoforms) in Smad-related pathways can transmit opposing signals: cytostatic C-terminally-phosphorylated Smad3 (pSmad3C) and carcinogenic linker-phosphorylated Smad3 (pSmad3L) signals. Methods and results: To assess the balance between Smad signals as a biomarker of risk, we immunohistochemically compared Smad domain-specific Smad3 phosphorylation patterns among 52 PBC patients with various stages of fibrosis and 25 non-PBC patients with chronic hepatitis C virus infection. HCC developed in 7 of 11 PBC patients showing high pSmad3L immunoreactivity, but in only 2 of 41 PBC patients with low pSmad3L. In contrast, 9 of 20 PBC patients with minimal Smad3C phosphorylation developed HCC, while HCC did not occur during follow-up in 32 patients who retained hepatic tumor-suppressive pSmad3C. Further, PBC patients whose liver specimens showed high pSmad3L positivity were relatively likely to develop HCC even when little fibrosis was evident. Conclusion: In this study, Smad phospho-isoform status showed promise as a biomarker predicting likelihood of HCC occurrence in PBC. Eventually, therapies to shift favorably Smad phospho-isoforms might decrease likelihood of PBC-related HCC.

Key words

TGF-β; PBC; Smad; HCC

References

[1] Parkin DM. Global cancer statistics in the year 2000. The Lancet Oncology. 2001; 2: 533–543.

[2] Augustine MM, Fong Y. Epidemiology and risk factors of biliary tract and primary liver tumors. Surgical Oncology Clinics of North America. 2014; 23: 171–188.

[3] Enomoto H, Ueno Y, Hiasa Y, Nishikawa H, Hige S, Takikawa Y, et al. Transition in the etiology of liver cirrhosis in Japan: a nationwide survey. Journal of Gastroenterology. 2020; 55: 353–362.

[4] Rong G, Wang H, Bowlus CL, Wang C, Lu Y, Zeng Z, et al. Incidence and Risk Factors for Hepatocellular Carcinoma in Primary Biliary Cirrhosis. Clinical Reviews in Allergy & Immunology. 2015; 48: 132–141.

[5] Marschall H, Henriksson I, Lindberg S, Söderdahl F, Thuresson M, Wahlin S, et al. Incidence, prevalence, and outcome of primary biliary cholangitis in a nationwide Swedish populationbased cohort. Scientific Reports. 2019; 9: 11525.

[6] Shibuya A, Tanaka K, Miyakawa H, Shibata M, Takatori M, Sekiyama K, et al. Hepatocellular carcinoma and survival in patients with primary biliary cirrhosis. Hepatology. 2002; 35: 1172–1178.

[7] Silveira MG, Suzuki A, Lindor KD. Surveillance for hepatocellular carcinoma in patients with primary biliary cirrhosis. Hepatology. 2008; 48: 1149–1156.

[8] Murillo Perez CF, Hirschfield GM, Corpechot C, Floreani A, Mayo MJ, van der Meer A, et al. Fibrosis stage is an independent predictor of outcome in primary biliary cholangitis despite biochemical treatment response. Alimentary Pharmacology & Therapeutics. 2019; 50: 1127–1136.

[9] Inagaki Y, Okazaki I. Emerging insights into Transforming growth factor beta Smad signal in hepatic fibrogenesis. Gut. 2007; 56: 284–292.

[10] Dooley S, ten Dijke P. TGF-β in progression of liver disease. Cell and Tissue Research. 2012; 347: 245–256.

[11] Moses HL, Serra R. Regulation of differentiation by TGF-beta. Current Opinion in Genetics & Development. 1996; 6: 581–586.

[12] Bellam N, Pasche B. Tgf-beta signaling alterations and coloncancer. Cancer Treatment and Research. 2010; 155: 85–103.

[13] Heldin CH, Miyazono K, ten Dijke P. TGF-beta signalling from cell membrane to nucleus through SMAD proteins. Nature. 1997; 390: 465–471.

[14] Gordeeva O. TGFβ Family Signaling Pathways in Pluripotent and Teratocarcinoma Stem Cells’ Fate Decisions: Balancing Between Self-Renewal, Differentiation, and Cancer. Cells. 2019; 8: 1500.

[15] Matsuzaki K. Smad phosphoisoform signals in acute and chronic liver injury: similarities and differences between epithelial and mesenchymal cells. Cell and Tissue Research. 2012; 347: 225–243.

[16] Wrighton KH, Lin X, Feng X. Phospho-control of TGF-beta superfamily signaling. Cell Research. 2009; 19: 8–20.

[17] Hannon GJ, Beach D. P15INK4B is a potential effector of TGFbeta-induced cell cycle arrest. Nature. 1994; 371: 257–261.

[18] Staller P, Peukert K, Kiermaier A, Seoane J, Lukas J, Karsunky H, et al. Repression of p15INK4b expression by Myc through association with Miz-1. Nature Cell Biology. 2001; 3: 392–399.

[19] Lasorella A, Noseda M, Beyna M, Yokota Y, Iavarone A. Id2 is a retinoblastoma protein target and mediates signalling by Myc oncoproteins. Nature. 2000; 407: 592–598.

[20] Feng XH, Lin X, Derynck R. Smad2, Smad3 and Smad4 cooperate with Sp1 to induce p15(Ink4B) transcription in response to TGF-beta. The European Molecular Biology Organization Journal. 2000; 19: 5178–5193.

[21] Pardali K, Kurisaki A, Morén A, ten Dijke P, Kardassis D, Moustakas A. Role of Smad Proteins and Transcription Factor Sp1 in p21Waf1/Cip1 Regulation by Transforming Growth Factor-β. Journal of Biological Chemistry. 2000; 275: 29244–29256.

[22] Frederick JP, Liberati NT, Waddell DS, Shi Y, Wang X. Transforming growth factor beta-mediated transcriptional repression of c-myc is dependent on direct binding of Smad3 to a novel repressive Smad binding element. Molecular and Cellular Biology. 2004; 24: 2546–2559.

[23] Hui L, Zatloukal K, Scheuch H, Stepniak E, Wagner EF. Proliferation of human HCC cells and chemically induced mouse liver cancers requires JNK1-dependent p21 downregulation. The Journal of Clinical Investigation. 2008; 118: 3943–3953.

[24] Nagata H, Hatano E, Tada M, Murata M, Kitamura K, Asechi H, et al. Inhibition of c-Jun NH2-terminal kinase switches Smad3 signaling from oncogenesis to tumor- suppression in rat hepatocellular carcinoma. Hepatology. 2009; 49: 1944–1953.

[25] Matsuzaki K, Murata M, Yoshida K, Sekimoto G, Uemura Y, Sakaida N, et al. Chronic inflammation associated with hepatitis C virus infection perturbs hepatic transforming growth factor beta signaling, promoting cirrhosis and hepatocellular carcinoma. Hepatology. 2007; 46: 48–57.

[26] Murata M, Matsuzaki K, Yoshida K, Sekimoto G, Tahashi Y, Mori S, et al. Hepatitis B virus X protein shifts human hepatic transforming growth factor (TGF)-beta signaling from tumor suppression to oncogenesis in early chronic hepatitis B. Hepatology. 2009; 49: 1203–1217.

[27] Deng Y, Yoshida K, Jin QL, Murata M, Yamaguchi T, Tsuneyama K, et al. Reversible phospho-Smad3 signalling between tumour suppression and fibrocarcinogenesis in chronic hepatitis B infection. Clinical and Experimental Immunology. 2014; 176: 102–111.

[28] Inoue K, Hirohara J, Nakano T, Seki T, Sasaki H, Higuchi K, Ohta Y, et al. Prediction of prognosis of primary biliary cirrhosis in Japan. Liver. 1995; 15: 70–77.

[29] Furukawa F, Matsuzaki K, Mori S, Tahashi Y, Yoshida K, Sugano Y, et al. P38 MAPK mediates fibrogenic signal through Smad3 phosphorylation in rat myofibroblasts. Hepatology. 2003; 38: 879–889.

[30] Scheuer P. Primary biliary cirrhosis. Proceedings of the Royal Society of Medicine. 1967; 60: 1257–1260.

[31] Desmet VJ, Gerber M, Hoofnagle JH, Manns M, Scheuer PJ. Classification of chronic hepatitis: diagnosis, grading and staging. Hepatology. 1994; 19: 1513–1520.

[32] YOUDEN WJ. Index for rating diagnostic tests. Cancer. 1950; 3: 32–35.

[33] Greenland S, Schwartzbaum JA, Finkle WD. Problems due to small samples and sparse data in conditional logistic regression analysis. American Journal of Epidemiology. 2000; 151: 531–539.

[34] Abe M, Onji M. Natural history of primary biliary cirrhosis. Hepatology Research. 2008; 38: 639–645.

[35] Arthur MJ. Fibrogenesis II. Metalloproteinases and their inhibitors in liver fibrosis. American Journal of Physiology. Gastrointestinal and Liver Physiology. 2000; 279: G245–G249.

[36] Ha H, Oh EY, Lee HB. The role of plasminogen activator inhibitor 1 in renal and cardiovascular diseases. Nature Reviews Nephrology. 2009; 5: 203–211.

[37] Wells RG. The role of matrix stiffness in regulating cell behavior. Hepatology. 2008; 47: 1394–1400.

[38] Pinzani M, Macias-Barragan J. Update on the pathophysiology of liver fibrosis. Expert Review of Gastroenterology & Hepatology. 2010; 4: 459–472.

[39] Kawamata S, Matsuzaki K, Murata M, Seki T, Matsuoka K, Iwao Y, et al. Oncogenic Smad3 signaling induced by chronic inflammation is an early event in ulcerative colitis-associated carcinogenesis. Inflammatory Bowel Diseases. 2011; 17: 683–695.

[40] Sekimoto G, Matsuzaki K, Yoshida K, Mori S, Murata M, Seki T, et al. Reversible Smad-dependent signaling between tumor suppression and oncogenesis. Cancer Research. 2007; 67: 5090–5096.

[41] Yamagata H, Matsuzaki K, Mori S, Yoshida K, Tahashi Y, Furukawa F, et al. Acceleration of Smad2 and Smad3 phosphorylation via c-Jun NH (2)-terminal kinase during human colorectal carcinogenesis. Cancer Research. 2005; 65: 157–165.

[42] Yamaguchi T, Matsuzaki K, Inokuchi R, Kawamura R, Yoshida K, Murata M, et al. Phosphorylated Smad2 and Smad3 signaling: Shifting between tumor suppression and fibrocarcinogenesis in chronic hepatitis C. Hepatology Research. 2013; 43: 1327–1342.

[43] Suwa K, Yamaguchi T, Yoshida K, Murata M, Ichimura M, Tsuneyama K, Seki T, et al. Smad Phospho-Isoforms for Hepatocellular Carcinoma Risk Assessment in Patients with Nonalcoholic Steatohepatitis. Cancers. 2020; 12: 286.

[44] Suzuki A, Lymp J, Donlinger J, Mendes F, Angulo P, Lindor K. Clinical predictors for hepatocellular carcinoma in patients with primary biliary cirrhosis. Clinical Gastroenterology and Hepatology. 2007; 5: 259–264.

[45] Zhang X. Primary biliary cirrhosis-associated hepatocellular carcinoma in Chinese patients: Incidence and risk factors. World Journal of Gastroenterology. 2015; 21: 3554.

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Naohiro Nakamura, Katsunori Yoshida, Rinako Tsuda, Miki Murata, Takashi Yamaguchi, Kanehiko Suwa, Mayuko Ichimura, Koichi Tsuneyama, Koichi Matsuzaki, Toshiaki Nakano, Junko Hirohara, Toshihito Seki, Kazuichi Okazaki, M. Eric Gershwin, Makoto Naganuma. Phospho-Smad3 signaling is predictive biomarker for hepatocellular carcinoma risk assessment in primary biliary cholangitis patients. Frontiers in Bioscience-Landmark. 2021. 26(12); 1480-1492.