Revealing the Roles of Keratin 8/18-Associated Signaling Proteins Involved in the Development of Hepatocellular Carcinoma
Abstract
:1. Introduction
2. Etiology
2.1. Liver Diseases
2.2. The Mutations and the Molecular Aspects of HCC Driver Genes
3. HCC Management
4. Keratin 8/18 and Liver Diseases
4.1. K8/K18 Mutations Associated with Liver Diseases
4.2. K8/K18-Related Inclusion Body and K18 Apoptotic Fragment as Liver Disease Biomarkers
5. Keratin 8/18-Related Signaling Pathways
6. Conclusions
Author Contributions
Funding
Conflicts of Interest
Abbreviations
APAP | acetaminophen |
APC | adenomatous polyposis coli |
BCLC | Barcelona clinical liver cancer |
CDK | cyclin-dependent kinase |
EMT | epithelial-mesenchymal transition |
G | globular |
HBV | hepatitis B virus |
HCC | hepatocellular carcinoma |
HCV | hepatitis C virus |
IHB | intracellular hyaline body |
K | keratin |
MDB | Mallory-Denk body |
PKB | protein kinase B |
PKC | protein kinase C |
PLC | primary liver cancer |
Rb | retinoblastoma |
SAPK | stress-activated protein kinase |
TACE | trans-arterial chemoembolization |
TERT | telomerase reverse transcriptase |
TERC | telomerase RNA component |
References
- IARC. Cancer Today. Available online: Gco.iarc.fr/today/home (accessed on 22 April 2021).
- Llovet, J.M.; Kelley, R.K.; Villanueva, A.; Singal, A.G.; Pikarsky, E.; Roayaie, S.; Lencioni, R.; Koike, K.; Zucman-Rossi, J.; Finn, R.S. Hepatocellular carcinoma. Nat. Rev. Dis. Prim. 2021, 7, 6–28. [Google Scholar] [CrossRef] [PubMed]
- Huang, J.; Lok, V.; Ngai, C.H.; Chu, C.; Patel, H.K.; Chandraseka, V.T.; Zhang, L.; Chen, P.; Wang, S.; Lao, X.-Q.; et al. Disease burden, risk factors, and recent trends of liver cancer: A global country-level Analysis. Liver Cancer 2021, 1–16. [Google Scholar] [CrossRef]
- Rowe, J.H.; Ghouri, Y.A.; Mian, I. Review of hepatocellular carcinoma: Epidemiology, etiology, and carcinogenesis. J. Carcinog. 2017, 16, 1. [Google Scholar] [CrossRef] [PubMed]
- Akinyemiju, T.; Abera, S.; Ahmed, M.; Alam, N.; Alemayohu, M.A.; Allen, C.; Al-Raddadi, R.; Alvis-Guzman, N.; Amoako, Y.; Artaman, A.; et al. The burden of primary liver cancer and underlying etiologies from 1990 to 2015 at the global, regional, and national level: Results from the global burden of disease study. JAMA Oncol. 2017, 3, 1683–1691. [Google Scholar] [CrossRef]
- Toivola, D.M.; Boor, P.; Alam, C.; Strnad, P. Keratins in health and disease. Curr. Opin. Cell Biol. 2015, 32, 73–81. [Google Scholar] [CrossRef]
- Jacob, J.T.; Coulombe, P.A.; Kwan, R.; Omary, B. Types I and II Keratin Intermediate Filaments. Cold Spring Harb. Perspect. Biol. 2018, 10, a018275. [Google Scholar] [CrossRef] [Green Version]
- Pan, X.; Hobbs, R.P.; A Coulombe, P. The expanding significance of keratin intermediate filaments in normal and diseased epithelia. Curr. Opin. Cell Biol. 2013, 25, 47–56. [Google Scholar] [CrossRef] [Green Version]
- Strnad, P.; Paschke, S.; Jang, K.-H.; Ku, N.-O. Keratins: Markers and modulators of liver disease. Curr. Opin. Gastroenterol. 2012, 28, 209–216. [Google Scholar] [CrossRef]
- Ku, N.-O.; Strnad, P.; Zhong, B.-H.; Tao, G.-Z.; Omary, M.B. Keratins let liver live: Mutations predispose to liver disease and crosslinking generates Mallory-Denk bodies. Hepatology. 2007, 46, 1639–1649. [Google Scholar] [CrossRef]
- Weng, Y.-R.; Cui, Y.; Fang, J.-Y. Biological functions of cytokeratin 18 in cancer. Mol. Cancer Res. 2012, 10, 485–493. [Google Scholar] [CrossRef] [Green Version]
- Loschke, F.; Seltmann, K.; Bouameur, J.-E.; Magin, T.M. Regulation of keratin network organization. Curr. Opin. Cell Biol. 2015, 32, 56–64. [Google Scholar] [CrossRef]
- Marrero, J.A.; Kulik, L.M.; Sirlin, C.B.; Zhu, A.X.; Finn, R.S.; Abecassis, M.M.; Roberts, L.R.; Heimbach, J.K. Diagnosis, staging, and management of hepatocellular carcinoma: 2018 practice guidance by the american association for the study of liver diseases. Hepatology 2018, 68, 723–750. [Google Scholar] [CrossRef] [Green Version]
- Neuveut, C.; Wei, Y.; Buendia, M.A. Mechanisms of HBV-related hepatocarcinogenesis. J. Hepatol. 2010, 52, 594–604. [Google Scholar] [CrossRef] [Green Version]
- Bartosch, B.; Thimme, R.; Blum, H.E.; Zoulim, F. Hepatitis C virus-induced hepatocarcinogenesis. J. Hepatol. 2009, 51, 810–820. [Google Scholar] [CrossRef] [Green Version]
- EASL. EASL clinical practice guidelines: Management of hepatocellular carcinoma. J. Hepatol. 2018, 69, 182–236. [Google Scholar] [CrossRef] [Green Version]
- Ashtari, S.; Pourhoseingholi, M.A.; Sharifian, A.; Zali, M.R. Hepatocellular carcinoma in Asia: Prevention strategy and plan-ning. World J. Hepatol. 2015, 7, 1708. [Google Scholar] [CrossRef]
- Chang, M.H.; You, S.-L.; Chen, C.-J.; Liu, C.-J.; Lai, M.-W.; Wu, T.-C.; Wu, S.-F.; Lee, C.-M.; Yang, S.-S.; Chu, H.-C.; et al. Long-term effects of hepatitis B immunization of infants in preventing liver cancer. Gastroenterology 2016, 151, 472–480. [Google Scholar] [CrossRef] [Green Version]
- Paterlini-Bréchot, P.; Saigo, K.; Murakami, Y.; Chami, M.; Gozuacik, D.; Mugnier, C.; Lagorce, D.; Bréchot, C. Hepatitis B virus-related insertional mutagenesis occurs frequently in human liver cancers and recurrently targets human telomerase gene. Oncogene 2003, 22, 3911–3916. [Google Scholar] [CrossRef]
- Lucey, M.R.; Mathurin, P.; Morgan, T.R. Alcoholic hepatitis. N. Engl. J. Med. 2009, 360, 2758–2769. [Google Scholar] [CrossRef]
- Mancebo, A.; González–Diéguez, M.L.; Cadahía, V.; Varela, M.; Pérez, R.; Navascués, C.A.; Sotorríos, N.G.; Martínez, M.; Rodrigo, L.; Rodríguez, M. Annual incidence of hepatocellular carcinoma among patients with alcoholic cirrhosis and identification of risk groups. Clin. Gastroenterol. Hepatol. 2013, 11, 95–101. [Google Scholar] [CrossRef]
- Lin, C.-W.; Mo, L.-R.; Chang, C.-Y.; Perng, D.-S.; Hsu, C.-C.; Lo, G.-H.; Chen, Y.-S.; Yen, Y.-C.; Hu, J.-T.; Yu, M.-L.; et al. Heavy alcohol consumption increases the incidence of hepatocellular carcinoma in hepatitis B virus-related cirrhosis. J. Hepatol. 2013, 58, 730–735. [Google Scholar] [CrossRef] [PubMed]
- Schulze, K.; Imbeaud, S.; Letouzé, E.; Alexandrov, L.B.; Calderaro, J.; Rebouissou, S.; Couchy, G.; Meiller, C.; Shinde, J.; Soysouvanh, F.; et al. Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets. Nat. Genet. 2015, 47, 505–511. [Google Scholar] [CrossRef] [PubMed]
- Ally, A.; Balasundaram, M.; Carlsen, R.; Chuah, E.; Clarke, A.; Dhalla, N.; Holt, R.A.; Jones, S.J.; Lee, D.; Ma, Y. Comprehensive and integrative genomic characterization of hepatocellular carcinoma. Cell 2017, 169, 1327–1341. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Daddadifagagna, F.; Reaper, P.M.; Clay-Farrace, L.; Fiegler, H.; Carr, P.; von Zglinicki, T.; Saretzki, G.; Carter, N.P.; Jackson, S.P. A DNA damage checkpoint response in telomere-initiated senescence. Nat. Cell Biol. 2003, 426, 194–198. [Google Scholar] [CrossRef]
- Günes, C.; Rudolph, K.L. The role of telomeres in stem cells and cancer. Cell 2013, 152, 390–393. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Calado, R.T.; Young, N.S. Telomere Diseases. N. Engl. J. Med. 2009, 361, 2353–2365. [Google Scholar] [CrossRef] [PubMed]
- Levine, A.J. Reviewing the future of the P53 field. Cell Death Differ. 2018, 25, 1–2. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Delbridge, A.R.D.; Strasser, A. The BCL-2 protein family, BH3-mimetics and cancer therapy. Cell Death Differ. 2015, 22, 1071–1080. [Google Scholar] [CrossRef] [Green Version]
- Vogelstein, B.; Lane, D.; Levine, A.J. Surfing the p53 network. Nat. Cell Biol. 2000, 408, 307–310. [Google Scholar] [CrossRef]
- Engeland, K. Cell cycle arrest through indirect transcriptional repression by p53: I have a DREAM. Cell Death Differ. 2018, 25, 114–132. [Google Scholar] [CrossRef] [Green Version]
- Janic, A.; Valente, L.J.; Wakefield, M.J.; Di Stefano, L.; Milla, L.; Wilcox, S.; Yang, H.; Tai, L.; Vandenberg, C.J.; Kueh, A.J.; et al. DNA repair processes are critical mediators of p53-dependent tumor suppression. Nat. Med. 2018, 24, 947–953. [Google Scholar] [CrossRef] [PubMed]
- Fujimoto, A.; Furuta, M.; Totoki, Y.; Tsunoda, T.; Kato, M.; Shiraishi, Y.; Tanaka, H.; Taniguchi, H.; Kawakami, Y.; Ueno, M.; et al. Whole-genome mutational landscape and characterization of noncoding and structural mutations in liver cancer. Nat. Genet. 2016, 48, 500–509. [Google Scholar] [CrossRef] [PubMed]
- Kent, L.N.; Leone, G. The broken cycle: E2F dysfunction in cancer. Nat. Rev. Cancer 2019, 19, 326–338. [Google Scholar] [CrossRef] [PubMed]
- Knudsen, E.S.; Wang, J.Y.J. Targeting the RB-pathway in Cancer Therapy. Clin. Cancer Res. 2010, 16, 1094–1099. [Google Scholar] [CrossRef] [Green Version]
- Marshall, A.E.; Roes, M.V.; Passos, D.T.; Deweerd, M.C.; Chaikovsky, A.C.; Sage, J.; Howlett, C.J.; Dick, F.A. RB1 Deletion in Retinoblastoma Protein Pathway-Disrupted Cells Results in DNA Damage and Cancer Progression. Mol. Cell. Biol. 2019, 39, 39. [Google Scholar] [CrossRef] [Green Version]
- Hauer, M.; Gasser, S.M. Chromatin and nucleosome dynamics in DNA damage and repair. Genes Dev. 2017, 31, 2204–2221. [Google Scholar] [CrossRef] [Green Version]
- Hoeijmakers, J. DNA Damage, Aging, and Cancer. N. Engl. J. Med. 2009, 361, 1475–1485. [Google Scholar] [CrossRef]
- Ribeiro-Silva, C.; Vermeulen, W.; Lans, H. SWI/SNF: Complex complexes in genome stability and cancer. DNA Repair 2019, 77, 87–95. [Google Scholar] [CrossRef]
- Vasileiou, G.; Ekici, A.B.; Uebe, S.; Zweier, C.; Hoyer, J.; Engels, H.; Behrens, J.; Reis, A.; Hadjihannas, M.V. Chroma-tin-remodeling-factor ARID1B represses Wnt/β-catenin signaling. Am. J. Hum. Genet. 2015, 97, 445–456. [Google Scholar] [CrossRef] [Green Version]
- Jiang, H.; Cao, H.-J.; Ma, N.; Bao, W.-D.; Wang, J.-J.; Chen, T.-W.; Zhang, E.-B.; Yuan, Y.-M.; Ni, Q.-Z.; Zhang, F.-K.; et al. Chromatin remodeling factor ARID2 suppresses hepatocellular carcinoma metastasis via DNMT1-Snail axis. Proc. Natl. Acad. Sci. USA 2020, 117, 4770–4780. [Google Scholar] [CrossRef]
- Perugorria, M.J.; Olaizola, P.; Labiano, I.; Esparza-Baquer, A.; Marzioni, M.; Marin, J.J.G.; Bujanda, L.; Banales, J.M. Wnt–β-catenin signalling in liver development, health and disease. Nat. Rev. Gastroenterol. Hepatol. 2019, 16, 121–136. [Google Scholar] [CrossRef]
- Stamos, J.L.; Weis, W.I. The β-catenin destruction complex. Cold Spring Harb. Perspect. Biol. 2013, 5, a007898. [Google Scholar] [CrossRef]
- Kim, E.; Lisby, A.; Ma, C.; Lo, N.; Ehmer, U.; Hayer, K.E.; Furth, E.E.; Viatour, P. Promotion of growth factor signaling as a critical function of β-catenin during HCC progression. Nat. Commun. 2019, 10, 1–17. [Google Scholar] [CrossRef]
- Wang, W.; Smits, R.; Hao, H.; He, C. Wnt/β-Catenin Signaling in Liver Cancers. Cancers 2019, 11, 926. [Google Scholar] [CrossRef] [Green Version]
- Monga, S.P. β-catenin signaling and roles in liver homeostasis, injury, and tumorigenesis. Gastroenterology 2015, 148, 1294–1310. [Google Scholar] [CrossRef] [Green Version]
- Waisberg, J.; Saba, G.T. Wnt-/-β-catenin pathway signaling in human hepatocellular carcinoma. World J. Hepatol. 2015, 7, 2631. [Google Scholar] [CrossRef] [Green Version]
- Llovet, J.M.; Montal, R.; Sia, D.; Finn, R.S. Molecular therapies and precision medicine for hepatocellular carcinoma. Nat. Rev. Clin. Oncol. 2018, 15, 599–616. [Google Scholar] [CrossRef]
- Smith, S.F.; Collins, S.E.; Charest, P.G. Ras, PI3K and mTORC2–three’s a crowd? J. Cell Sci. 2020, 133. [Google Scholar] [CrossRef]
- Muñoz-Maldonado, C.; Zimmer, Y.; Medová, M. A Comparative Analysis of Individual RAS Mutations in Cancer Biology. Front. Oncol. 2019, 9, 1088. [Google Scholar] [CrossRef] [Green Version]
- Guijarro, L.; Sanmartin-Salinas, P.; Pérez-Cuevas, E.; Toledo-Lobo, M.; Monserrat, J.; Zoullas, S.; Sáez, M.; Álvarez-Mon, M.; Bujan, J.; Noguerales-Fraguas, F.; et al. Possible Role of IRS-4 in the Origin of Multifocal Hepatocellular Carcinoma. Cancers 2021, 13, 2560. [Google Scholar] [CrossRef]
- Bruix, J.; Reig, M.; Sherman, M. Evidence-Based Diagnosis, Staging, and Treatment of Patients With Hepatocellular Carcinoma. Gastroenterology 2016, 150, 835–853. [Google Scholar] [CrossRef] [Green Version]
- Forner, A.; Gilabert, M.; Bruix, J.; Raoul, J.-L. Treatment of intermediate-stage hepatocellular carcinoma. Nat. Rev. Clin. Oncol. 2014, 11, 525–535. [Google Scholar] [CrossRef]
- Bruix, J.; Han, K.-H.; Gores, G.; Llovet, J.M.; Mazzaferro, V.M. Liver cancer: Approaching a personalized care. J. Hepatol. 2015, 62, S144–S156. [Google Scholar] [CrossRef] [Green Version]
- Foerster, F.; Galle, P.R. Comparison of the current international guidelines on the management of HCC. JHEP Rep. 2019, 1, 114–119. [Google Scholar] [CrossRef] [Green Version]
- Yang, J.D.; Hainaut, P.; Gores, G.J.; Amadou, A.; Plymoth, A.; Roberts, L.R. A global view of hepatocellular carcinoma: Trends, risk, prevention and management. Nat. Rev. Gastroenterol. Hepatol. 2019, 16, 589–604. [Google Scholar] [CrossRef]
- Matsui, J.; Funahashi, Y.; Uenaka, T.; Watanabe, T.; Tsuruoka, A.; Asada, M. Multi-kinase inhibitor E7080 suppresses lymph node and lung metastases of human mammary breast tumor MDA-MB-231 via inhibition of vascular endothelial growth factor-receptor (VEGF-R) 2 and VEGF-R3 kinase. Clin. Cancer Res. 2008, 14, 5459–5465. [Google Scholar] [CrossRef] [Green Version]
- Wilhelm, S.M.; Dumas, J.; Adnane, L.; Lynch, M.; Carter, C.A.; Schütz, G.; Thierauch, K.-H.; Zopf, D. Regorafenib (BAY 73-4506): A new oral multikinase inhibitor of angiogenic, stromal and oncogenic receptor tyrosine kinases with potent preclinical antitumor activity. Int. J. Cancer 2011, 129, 245–255. [Google Scholar] [CrossRef] [PubMed]
- Ribas, A.; Wolchok, J.D. Cancer immunotherapy using checkpoint blockade. Science 2018, 359, 1350–1355. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Ku, N.-O.; Omary, M.B. A disease-and phosphorylation-related nonmechanical function for keratin. J. Cell Biol. 2006, 174, 115–125. [Google Scholar] [CrossRef] [Green Version]
- Baribault, H.; Penner, J.; Iozzo, R.; Wilson-Heiner, M. Colorectal hyperplasia and inflammation in keratin 8-deficient FVB/N mice. Genes Dev. 1994, 8, 2964–2973. [Google Scholar] [CrossRef] [Green Version]
- Toivola, D.M.; Nieminen, M.I.; Hesse, M.; He, T.; Magin, T.M.; Omary, M.B.; Eriksson, J.E. Disturbances in hepatic cell-cycle regulation in mice with assembly-deficient keratins 8/18. Hepatology 2001, 34, 1174–1183. [Google Scholar] [CrossRef]
- Loranger, A.; Duclos, S.; Grenier, A.; Price, J.; Wilson-Heiner, M.; Baribault, H.; Marceau, N. Simple epithelium keratins are required for maintenance of hepatocyte integrity. Am. J. Pathol. 1997, 151, 1673–1683. [Google Scholar] [PubMed]
- Gilbert, S.; Loranger, A.; Daigle, N.; Marceau, N. Simple epithelium keratins 8 and 18 provide resistance to Fas-mediated apoptosis. The protection occurs through a receptor-targeting modulation. J. Cell Biol. 2001, 154, 763–774. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Tao, G.-Z.; Toivola, D.M.; Zhong, B.; Michie, S.A.; Resurreccion, E.Z.; Tamai, Y.; Taketo, M.M.; Omary, B. Keratin-8 null mice have different gallbladder and liver susceptibility to lithogenic diet-induced injury. J. Cell Sci. 2003, 116, 4629–4638. [Google Scholar] [CrossRef] [Green Version]
- Magin, T.M.; Schröder, R.; Leitgeb, S.; Wanninger, F.; Zatloukal, K.; Grund, C.; Melton, D.W. Lessons from Keratin 18 Knockout Mice: Formation of Novel Keratin Filaments, Secondary Loss of Keratin 7 and Accumulation of Liver-specific Keratin 8-Positive Aggregates. J. Cell Biol. 1998, 140, 1441–1451. [Google Scholar] [CrossRef]
- Bettermann, K.; Mehta, A.K.; Hofer, E.M.; Wohlrab, C.; Golob-Schwarzl, N.; Svendova, V.; Schimek, M.G.; Stumptner, C.; Thüringer, A.; Speicher, M.R. Keratin 18-deficiency results in steatohepatitis and liver tumors in old mice: A model of steato-hepatitis-associated liver carcinogenesis. Oncotarget 2016, 7, 73309. [Google Scholar] [CrossRef] [Green Version]
- Baribault, H.; Price, J.; Miyai, K.; Oshima, R.G. Mid-gestational lethality in mice lacking keratin 8. Genes Dev. 1993, 7, 1191–1202. [Google Scholar] [CrossRef] [Green Version]
- Zatloukal, K.; French, S.W.; Stumptner, C.; Strnad, P.; Harada, M.; Toivola, D.M.; Cadrin, M.; Omary, M.B. From Mallory to Mallory–Denk bodies: What, how and why? Exp. Cell Res. 2007, 313, 2033–2049. [Google Scholar] [CrossRef]
- Lee, J.; Jang, K.-H.; Kim, H.; Lim, Y.; Kim, S.; Yoon, H.-N.; Chung, I.K.; Roth, J.; Ku, N.-O. Predisposition to apoptosis in keratin 8-null liver is related to inactivation of NF-κB and SAPKs but not decreased c-Flip. Biol. Open 2013, 2, 695–702. [Google Scholar] [CrossRef] [Green Version]
- Omary, B.; Ku, N.-O.; Toivola, D.M. Keratins: Guardians of the liver. Hepatology 2002, 35, 251–257. [Google Scholar] [CrossRef]
- Kucukoglu, O.; Guldiken, N.; Chen, Y.; Usachov, V.; El-Heliebi, A.; Haybaeck, J.; Denk, H.; Trautwein, C.; Strnad, P. High-fat diet triggers Mallory-Denk body formation through misfolding and crosslinking of excess keratin. Hepatology 2014, 60, 169–178. [Google Scholar] [CrossRef] [PubMed]
- Guldiken, N.; Zhou, Q.; Kucukoglu, O.; Rehm, M.; Levada, K.; Gross, A.; Kwan, R.; James, L.P.; Trautwein, C.; Omary, M.B.; et al. Human keratin 8 variants promote mouse acetaminophen hepatotoxicity coupled with c-jun amino-terminal kinase activation and protein adduct formation. Hepatology 2015, 62, 876–886. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Yoon, H.-N.; Yoon, S.-Y.; Hong, J.-H.; Ku, N.-O. A mutation in keratin 18 that causes caspase-digestion resistance protects homozygous transgenic mice from hepatic apoptosis and injury. J. Cell Sci. 2017, 130, 2541–2550. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Ku, N.-O.; Toivola, D.M.; Strnad, P.; Omary, M.B. Cytoskeletal keratin glycosylation protects epithelial tissue from injury. Nat. Cell Biol. 2010, 12, 876–885. [Google Scholar] [CrossRef] [Green Version]
- Ku, N.-O.; Michie, S.; Resurreccion, E.Z.; Broome, R.L.; Omary, B. Keratin binding to 14-3-3 proteins modulates keratin filaments and hepatocyte mitotic progression. Proc. Natl. Acad. Sci. USA 2002, 99, 4373–4378. [Google Scholar] [CrossRef] [Green Version]
- Omary, M.B.; Ku, N.-O.; Tao, G.-Z.; Toivola, D.M.; Liao, J. ‘Heads and tails’ of intermediate filament phosphorylation: Multiple sites and functional insights. Trends Biochem. Sci. 2006, 31, 383–394. [Google Scholar] [CrossRef]
- Ku, N.-O.; Michie, S.; Oshima, R.G.; Omary, M.B. Chronic hepatitis, hepatocyte fragility, and increased soluble phospho-glycokeratins in transgenic mice expressing a keratin 18 conserved arginine mutant. J. Cell Biol. 1995, 131, 1303–1314. [Google Scholar] [CrossRef]
- Ku, N.; Soetikno, R.M.; Omary, M.B. Keratin mutation in transgenic mice predisposes to Fas but not TNF-induced apoptosis and massive liver injury. Hepatology 2003, 37, 1006–1014. [Google Scholar] [CrossRef]
- Strnad, P.; Tao, G.; Zhou, Q.; Harada, M.; Toivola, D.M.; Brunt, E.M.; Omary, M.B. Keratin Mutation Predisposes to Mouse Liver Fibrosis and Unmasks Differential Effects of the Carbon Tetrachloride and Thioacetamide Models. Gastroenterology 2008, 134, 1169–1179. [Google Scholar] [CrossRef] [Green Version]
- Weerasinghe, S.V.; Ku, N.-O.; Altshuler, P.J.; Kwan, R.; Omary, M.B. Mutation of caspase-digestion sites in keratin 18 interferes with filament reorganization, and predisposes to hepatocyte necrosis and loss of membrane integrity. J. Cell Sci. 2014, 127, 1464–1475. [Google Scholar] [CrossRef] [Green Version]
- Coulombe, P.A.; Hutton, M.; Letal, A.; Hebert, A.; Paller, A.S.; Fuchs, E. Point mutations in human keratin 14 genes of epidermolysis bullosa simplex patients: Genetic and functional analyses. Cell 1991, 66, 1301–1311. [Google Scholar] [CrossRef]
- Strnad, P.; Zhou, Q.; Hanada, S.; Lazzeroni, L.C.; Zhong, B.H.; So, P.; Davern, T.J.; Lee, W.M.; Omary, B. Keratin Variants Predispose to Acute Liver Failure and Adverse Outcome: Race and Ethnic Associations. Gastroenterology 2010, 139, 828–835.e1-3. [Google Scholar] [CrossRef] [Green Version]
- Li, R.; Liao, X.-H.; Ye, J.-Z.; Li, M.-R.; Wu, Y.-Q.; Hu, X.; Zhong, B.-H. Association of keratin 8/18 variants with non-alcoholic fatty liver disease and insulin resistance in Chinese patients: A case-control study. World J. Gastroenterol. 2017, 23, 4047–4053. [Google Scholar] [CrossRef]
- Zhong, B.; Strnad, P.; Selmi, C.; Invernizzi, P.; Tao, G.-Z.; Caleffi, A.; Chen, M.; Bianchi, I.; Podda, M.; Pietrangelo, A.; et al. Keratin variants are overrepresented in primary biliary cirrhosis and associate with disease severity. Hepatology 2009, 50, 546–554. [Google Scholar] [CrossRef] [Green Version]
- Ye, J.; Wu, Y.; Li, M.; Gong, X.; Zhong, B. Keratin 8 mutations were associated with susceptibility to chronic hepatitis B and related progression. J. Infect. Dis. 2019, 221, 464–473. [Google Scholar] [CrossRef]
- Ku, N.-O.; Gish, R.; Wright, T.L.; Omary, M.B. Keratin 8 Mutations in Patients with Cryptogenic Liver Disease. N. Engl. J. Med. 2001, 344, 1580–1587. [Google Scholar] [CrossRef]
- Ku, N.; Lim, J.K.; Krams, S.M.; Esquivel, C.O.; Keeffe, E.B.; Wright, T.L.; Parry, D.A.; Omary, B. Keratins as Susceptibility Genes for End-Stage Liver Disease. Gastroenterology 2005, 129, 885–893. [Google Scholar] [CrossRef]
- Strnad, P.; Lienau, T.C.; Tao, G.-Z.; Lazzeroni, L.C.; Stickel, F.; Schuppan, D.; Omary, M.B. Keratin variants associate with progression of fibrosis during chronic hepatitis C infection. Hepatology 2006, 43, 1354–1363. [Google Scholar] [CrossRef]
- Strnad, P.; Lienau, T.C.; Tao, G.-Z.; Ku, N.-O.; Magin, T.M.; Omary, M.B. Denaturing temperature selection may underestimate keratin mutation detection by DHPLC. Hum. Mutat. 2006, 27, 444–452. [Google Scholar] [CrossRef]
- Omary, M.B. Intermediate filament proteins of digestive organs: Physiology and pathophysiology. Am. J. Physiol. Gastrointest. Liver Physiol. 2017, 312, G628–G634. [Google Scholar] [CrossRef]
- Zatloukal, K.; Stumptner, C.; Fuchsbichler, A.; Janig, E.; Denk, H. Intermediate Filament Protein Inclusions. Methods Cell Biol. 2004, 78, 205–228. [Google Scholar] [CrossRef]
- Cairns, N.J.; Lee, V.M.-Y.; Trojanowski, J.Q. The cytoskeleton in neurodegenerative diseases. J. Pathol. 2004, 204, 438–449. [Google Scholar] [CrossRef] [Green Version]
- Strnad, P.; Zatloukal, K.; Stumptner, C.; Kulaksiz, H.; Denk, H. Mallory–Denk-bodies: Lessons from keratin-containing hepatic inclusion bodies. Biochim. Biophys. Acta (BBA) Mol. Basis Dis. 2008, 1782, 764–774. [Google Scholar] [CrossRef] [Green Version]
- Strnad, P.; Nuraldeen, R.; Guldiken, N.; Hartmann, D.; Mahajan, V.; Denk, H.; Haybaeck, J. Broad Spectrum of Hepatocyte Inclusions in Humans, Animals, and Experimental Models. In Comprehensive Physiology; Wiley: Hoboken, NJ, USA, 2013; Volume 3, pp. 1393–1436. [Google Scholar]
- Aigelsreiter, A.; Neumann, J.; Pichler, M.; Halasz, J.; Zatloukal, K.; Berghold, A.; Douschan, P.; Rainer, F.; Stauber, R.; Haybaeck, J.; et al. Hepatocellular carcinomas with intracellular hyaline bodies have a poor prognosis. Liver Int. 2017, 37, 600–610. [Google Scholar] [CrossRef]
- Stumptner, C.; Omary, B.; Fickert, P.; Denk, H.; Zatloukal, K. Hepatocyte Cytokeratins Are Hyperphosphorylated at Multiple Sites in Human Alcoholic Hepatitis and in a Mallory Body Mouse Model. Am. J. Pathol. 2000, 156, 77–90. [Google Scholar] [CrossRef] [Green Version]
- Fortier, A.-M.; Riopel, K.; Desaulniers, M.; Cadrin, M. Novel insights into changes in biochemical properties of keratins 8 and 18 in griseofulvin-induced toxic liver injury. Exp. Mol. Pathol. 2010, 89, 117–125. [Google Scholar] [CrossRef]
- Guzman, R.E.; Solter, P.F. Characterization of Sublethal Microcystin-LR Exposure in Mice. Veter. Pathol. 2002, 39, 17–26. [Google Scholar] [CrossRef] [PubMed]
- Yuan, Q.; Nagao, Y.; Gaal, K.; Hu, B.; French, S. Mechanisms of Mallory Body Formation Induced by Okadaic Acid in Drug-Primed Mice. Exp. Mol. Pathol. 1998, 65, 87–103. [Google Scholar] [CrossRef] [PubMed]
- Kwan, R.; Hanada, S.; Harada, M.; Strnad, P.; Li, D.H.; Omary, B. Keratin 8 phosphorylation regulates its transamidation and hepatocyte Mallory-Denk body formation. FASEB J. 2012, 26, 2318–2326. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Nan, L.; Dedes, J.; French, B.A.; Bardag-Gorce, F.; Li, J.; Wu, Y.; French, S.W. Mallory body (cytokeratin aggresomes) formation is prevented in vitro by p38 inhibitor. Exp. Mol. Pathol. 2006, 80, 228–240. [Google Scholar] [CrossRef] [PubMed]
- Haybaeck, J.; Stumptner, C.; Thueringer, A.; Kolbe, T.; Magin, T.M.; Hesse, M.; Fickert, P.; Tsybrovskyy, O.; Müller, H.; Trauner, M.; et al. Genetic background effects of keratin 8 and 18 in a DDC-induced hepatotoxicity and Mallory-Denk body formation mouse model. Lab. Investig. 2012, 92, 857–867. [Google Scholar] [CrossRef] [Green Version]
- Nakamichi, I.; Toivola, D.M.; Strnad, P.; Michie, S.A.; Oshima, R.G.; Baribault, H.; Omary, M.B. Keratin 8 overexpression promotes mouse Mallory body formation. J. Cell Biol. 2005, 171, 931–937. [Google Scholar] [CrossRef]
- Mahajan, V.; Klingstedt, T.; Simon, R.; Nilsson, K.P.R.; Thueringer, A.; Kashofer, K.; Haybaeck, J.; Denk, H.; Abuja, P.M.; Zatloukal, K. Cross β-Sheet Conformation of Keratin 8 Is a Specific Feature of Mallory–Denk Bodies Compared with Other Hepatocyte Inclusions. Gastroenterology 2011, 141, 1080–1090.e7. [Google Scholar] [CrossRef]
- Liu, H.; Gong, M.; French, B.A.; Liao, G.; Li, J.; Tillman, B.; French, S.W. Aberrant modulation of the BRCA1 and G1/S cell cycle pathways in alcoholic hepatitis patients with Mallory Denk Bodies revealed by RNA sequencing. Oncotarget 2015, 6, 42491–42503. [Google Scholar] [CrossRef] [Green Version]
- Liu, H.; French, B.A.; Nelson, T.; Li, J.; Tillman, B.; French, S.W. IL-8 signaling is up-regulated in alcoholic hepatitis and DDC fed mice with Mallory Denk Bodies (MDBs) present. Exp. Mol. Pathol. 2015, 99, 320–325. [Google Scholar] [CrossRef] [Green Version]
- Liu, Y.; Trnka, M.J.; Guan, S.; Kwon, D.; Kim, D.-H.; Chen, J.-J.; Greer, P.A.; Burlingame, A.L.; Correia, M.A. A Novel Mechanism for NF-κB-activation via IκB-aggregation: Implications for Hepatic Mallory-Denk-Body Induced Inflammation. Mol. Cell. Proteom. 2020, 19, 1968–1986. [Google Scholar] [CrossRef]
- Komatsu, M.; Kurokawa, H.; Waguri, S.; Taguchi, K.; Kobayashi, A.; Ichimura, Y.; Sou, Y.-S.; Ueno, I.; Sakamoto, A.; Tong, K.I.; et al. The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1. Nat. Cell Biol. 2010, 12, 213–223. [Google Scholar] [CrossRef]
- Schwabe, R.F.; Luedde, T. Apoptosis and necroptosis in the liver: A matter of life and death. Nat. Rev. Gastroenterol. Hepatol. 2018, 15, 738–752. [Google Scholar] [CrossRef]
- Caulín, C.; Salvesen, G.S.; Oshima, R.G. Caspase Cleavage of Keratin 18 and Reorganization of Intermediate Filaments during Epithelial Cell Apoptosis. J. Cell Biol. 1997, 138, 1379–1394. [Google Scholar] [CrossRef]
- Ku, N.-O.; Omary, M.B. Effect of mutation and phosphorylation of type I keratins on their caspase-mediated degradation. J. Biol. Chem. 2001, 276, 26792–26798. [Google Scholar] [CrossRef] [Green Version]
- Church, R.J.; Kullak-Ublick, G.A.; Aubrecht, J.; Bonkovsky, H.L.; Chalasani, N.; Fontana, R.J.; Goepfert, J.C.; Hackman, F.; King, N.M.; Kirby, S. Candidate biomarkers for the diagnosis and prognosis of drug-induced liver injury: An international collabo-rative effort. Hepatology 2019, 69, 760–773. [Google Scholar] [CrossRef]
- Mueller, S.; Nahon, P.; Rausch, V.; Peccerella, T.; Silva, I.; Yagmur, E.; Straub, B.K.; Lackner, C.; Seitz, H.K.; Rufat, P.; et al. Caspase-cleaved keratin-18 fragments increase during alcohol withdrawal and predict liver-related death in patients with alcoholic liver disease. Hepatology 2017, 66, 96–107. [Google Scholar] [CrossRef] [Green Version]
- Feldstein, A.E.; Alkhouri, N.; De Vito, R.; Alisi, A.; Lopez, R.; Nobili, V. Serum cytokeratin-18 fragment levels are useful bi-omarkers for nonalcoholic steatohepatitis in children. Am. J. Gastroenterol. 2013, 108, 1526–1531. [Google Scholar] [CrossRef]
- Joka, D.; Wahl, K.; Moeller, S.; Schlue, J.; Vaske, B.; Bahr, M.J.; Manns, M.P.; Schulze-Osthoff, K.; Bantel, H. Prospective biopsy-controlled evaluation of cell death biomarkers for prediction of liver fibrosis and nonalcoholic steatohepatitis. Hepatology 2012, 55, 455–464. [Google Scholar] [CrossRef]
- Ku, N.-O.; Strnad, P.; Bantel, H.; Omary, B. Keratins: Biomarkers and modulators of apoptotic and necrotic cell death in the liver. Hepatology 2016, 64, 966–976. [Google Scholar] [CrossRef] [Green Version]
- Golob-Schwarzl, N.; Bettermann, K.; Mehta, A.K.; Kessler, S.M.; Unterluggauer, J.; Krassnig, S.; Kojima, K.; Chen, X.; Hoshida, Y.; Bardeesy, N.M. High keratin 8/18 ratio predicts aggressive hepatocellular cancer phenotype. Transl. Oncol. 2019, 12, 256–268. [Google Scholar] [CrossRef]
- Kim, S.; Coulombe, P.A. Intermediate filament scaffolds fulfill mechanical, organizational, and signaling functions in the cytoplasm. Genes Dev. 2007, 21, 1581–1597. [Google Scholar] [CrossRef] [Green Version]
- Lee, S.-Y.; Kim, S.; Lim, Y.; Yoon, H.-N.; Ku, N.-O. Keratins regulate Hsp70-mediated nuclear localization of p38 mito-gen-activated protein kinase. J. Cell Sci. 2019, 132. [Google Scholar] [CrossRef] [Green Version]
- Ku, N.-O.; Fu, H.; Omary, B. Raf-1 activation disrupts its binding to keratins during cell stress. J. Cell Biol. 2004, 166, 479–485. [Google Scholar] [CrossRef]
- Bordeleau, F.; Galarneau, L.; Gilbert, S.; Loranger, A.; Marceau, N. Keratin 8/18 modulation of protein kinase C-mediated in-tegrin-dependent adhesion and migration of liver epithelial cells. Mol. Biol. Cell 2010, 21, 1698–1713. [Google Scholar] [CrossRef] [Green Version]
- Ku, N.-O.; Liao, J.; Omary, M. Phosphorylation of human keratin 18 serine 33 regulates binding to 14-3-3 proteins. EMBO J. 1998, 17, 1892–1906. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Hermeking, H.; Benzinger, A. 14-3-3 proteins in cell cycle regulation. Semin. Cancer Biol. 2006, 16, 183–192. [Google Scholar] [CrossRef] [PubMed]
- He, T.; Stepulak, A.; Holmström, T.H.; Omary, M.B.; Eriksson, J.E. The intermediate filament protein keratin 8 is a novel cyto-plasmic substrate for c-Jun N-terminal kinase. J. Biol. Chem. 2002, 277, 10767–10774. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Fogh, B.S.; Multhaupt, H.A.; Couchman, J.R. Protein Kinase C, Focal Adhesions and the Regulation of Cell Migration. J. Histochem. Cytochem. 2014, 62, 172–184. [Google Scholar] [CrossRef] [Green Version]
- Li, J.-J.; Xie, D. RACK1, a versatile hub in cancer. Oncogene 2014, 34, 1890–1898. [Google Scholar] [CrossRef]
- Cooke, M.; Magimaidas, A.; Casado-Medrano, V.; Kazanietz, M.G. Protein kinase C in cancer: The top five unanswered ques-tions. Mol. Carcinog. 2017, 56, 1531–1542. [Google Scholar] [CrossRef]
- Sanghvi-Shah, R.; Weber, G.F. Intermediate Filaments at the Junction of Mechanotransduction, Migration, and Development. Front. Cell Dev. Biol. 2017, 5, 81. [Google Scholar] [CrossRef]
- Osmanagic-Myers, S.; Wiche, G. Plectin-RACK1 (receptor for activated C kinase 1) scaffolding: A novel mechanism to regulate protein kinase C activity. J. Biol. Chem. 2004, 279, 18701–18710. [Google Scholar] [CrossRef] [Green Version]
- Meng, X.; Franklin, D.A.; Dong, J.; Zhang, Y. MDM2–p53 pathway in hepatocellular carcinoma. Cancer Res. 2014, 74, 7161–7167. [Google Scholar] [CrossRef] [Green Version]
- Beg, A.A.; Baltimore, D. An essential role for NF-κB in preventing TNF-α-induced cell death. Science 1996, 274, 782–784. [Google Scholar] [CrossRef]
Pathway | Gene | Alteration | Ref. |
---|---|---|---|
Telomere maintenance | TERT promoter | Gain of function | [23,24] |
Cell cycle | TP53 | Loss of function | |
RB1 | Loss of function | ||
CDKN2A | Loss of function | ||
Chromatin remodeling | ARID2 | Loss of function | |
ARID1A/B | Loss of function | ||
Wnt pathway | CTNNB1 | Gain of function | |
AXIN1 | Loss of function | ||
Ras-PI3K pathway | PIK3CA | Gain of function | |
RPS6KA3 | Unclassified | ||
PTEN | Loss of function | ||
KRAS | Gain of function | ||
NRAS | Gain of function | ||
Oxidative stress | KEAP1 | Gain of function | |
NFE2L2 | Gain of function |
Drug | Targets | HCC Stage | Phase | Comparison | Primary Outcome | NCT Number ‡ |
---|---|---|---|---|---|---|
Atezolizumab + Lenvatinib | PD-L1 + VEGFRs, FGFRs, PDGFRβ, RET, and KIT | Advanced or metastatic | ΙΙΙ | Sorafenib | OS | NCT04770896 |
Toripalimab + Lenvatinib | PD-1 + VEGFRs, FGFRs, PDGFRβ, RET, and KIT | BCLC B or C stage | ΙΙ | – † | ORR | NCT04368078 |
SHR-1210 + Apatinib | PD-1 + VGFR-2 | Advanced | ΙΙΙ | Sorafenib | OS/PFS | NCT03764293 |
Durvalumab + Bevacizumab | PD-L1 + VEGF-A | High risk of recurrence | ΙΙΙ | Placebo | RFS | NCT03847428 |
Nivolumab + Ipilimumab | PD-1 + CTLA-4 | Advanced | ΙΙΙ | Sarafenib or Lenvatinib | OS | NCT04039607 |
Pembrolizumab + Bavituximab | PD-1 + PS | Advanced or metastatic | ΙΙ | – † | ORR | NCT03519997 |
Durvalumab + Tremelimumab | PD-1 + CTLA-4 | Advanced | ΙΙΙ | Sorafenib | OS | NCT03298451 |
Gene | Mouse Genotype | Keratin Filament | Liver | Ref. | |||
---|---|---|---|---|---|---|---|
Basal Phenotype | Fragility (Basal Conditions) | Phenotype (Stressed Conditions) | Induced Stresses | ||||
K8 | K8-/- (C57B1/6) | Absent | Embryonic lethality Liver hemorrhage | - | - | - | [68] |
K8-/- (FVB/n) | Absent | Mild hepatitis | ↑ | ↑, Decreased MDBs | Pentobarbital, MLR, high fat diet, Fas | [61,63,65,69,70,71] | |
K8 over-expression | Normal | MDBs | Normal | Increased MDBs | DDC, high fat diet | [69,72] | |
K8 G62C | Normal | Normal | Normal | ↑ | Fas, MLR, APAP | [60,73] | |
K8 S74A | Normal | Normal | Normal | ↑ | Fas | [60] | |
K8 R341H | Normal | Normal | Normal | ↑ | APAP | [73] | |
K18 | K18-/- | Absent | Mild hepatitis MDBs Steatohepatitis (Old mice) | ↑ | ↑ | Fas | [66,67,74] |
K18 over-expression | Normal | Normal | Normal | Decreased MDBs | DDC | [69] | |
K18 S30/31/49A | Normal | Normal | Normal | ↑ | STZ, Fas + PUGNAc | [75] | |
K18 S34A | Normal | Normal | Normal | Mitoticfeatures | PH | [76,77] | |
K18 S53A | Normal | Normal | Normal | ↑ | MLR | [10] | |
K18 R90C | Disrupted | Mild hepatitis | ↑ | ↑ | Fas, CCl4, TAA | [78,79,80] | |
K18 D238/397E (mouse K18expressed FVB/n) | Normal | Normal | Normal | ↑ | Fas | [81] | |
Normal | Normal | Normal | - | Fas | [74] | ||
K18 D238/397E (mouse K18 knocked out FVB/n) | Normal | Normal | Normal | ↓ | Fas, MLR | [74] |
Screened Gene | Ethnicity | No. of Variant Carriers/Total (%) | p Value | Ref. | |
---|---|---|---|---|---|
Liver Disease Cohort | Controls | ||||
AllK8/K18 exons | US | 58/467 (12.4%) | 13/349 (3.7%) | <0.0001 | [87,88] |
Germany | 19/329 (5.8%) | - | 0.001 | [89] | |
US | 45/344 (13.1%) | 9/268 (3.4%) a | 0.01 a | [83] | |
Italy | 17/201 (8.5%) | 4/200 (2%) | p < 0.004 | [85] | |
China | 10/200 (5%) | 1/173 (0.58%) | p = 0.015 | [84] | |
China | 21/540 (3.89%) | 1/173 (0.58%) | p = 0.03 | [86] | |
K8 exon 1 and 6 | Germany | 12/151 (7.9%) | - | - | [90] |
A Role of Signaling Protein | K8/K18-Associated Signaling Protein | K8/K18 Mutation | Effects of K8/K18 Mutation | Ref. |
---|---|---|---|---|
Cell cycle regulator | 14-3-3 | K8-/- K18-/- K18 R90C (disrupted filament) | Arrest in S-G2 phage (in vivo) ǂ | [62] |
K18 S34A (blocked pK18 S34, disturbed 14-3-3 binding) | Accumulation of mitotic figures (mitotic arrest) (in vivo) ǂ | [76] | ||
Transcription factor | p53 | - | ND # | [70] |
NF-kB | K8-/- | High susceptibility to Fas treatment Inhibited NF-kB translocation to the nucleus | [70] | |
Kinase | Stress-activated protein kinases (SAPKs, such as ERK, JNK, and p38) | K8 G62C * (natural mutation, inhibited pK8S74) K8 S74A(blocked pK8 S74) | High susceptibility to Fas treatment Higher activation of SAPK substrates (in vivo) ǂ | [60] |
K8 R148/149E, L159/161A (blocked p38 binding) K18 I150V * (natural mutation) | Dissociation of p38 Translocation of phosphorylated p38 to nucleus (in vitro) ʌ | [120] | ||
Protein kinase B (PKB also known as Akt) | K18 S30/31/49A (K18 glycosylation-deficient mutant) | Akt hyper-glycosylation Inhibition of Akt T308 phosphorylation (in vivo) ǂ | [75] | |
Raf | - | ND # | [121] | |
Protein kinase C (PKC) | K8-/- | Reduced migration of liver epithelial cells (in vitro) ʌ | [122] |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Lim, Y.; Ku, N.-O. Revealing the Roles of Keratin 8/18-Associated Signaling Proteins Involved in the Development of Hepatocellular Carcinoma. Int. J. Mol. Sci. 2021, 22, 6401. https://doi.org/10.3390/ijms22126401
Lim Y, Ku N-O. Revealing the Roles of Keratin 8/18-Associated Signaling Proteins Involved in the Development of Hepatocellular Carcinoma. International Journal of Molecular Sciences. 2021; 22(12):6401. https://doi.org/10.3390/ijms22126401
Chicago/Turabian StyleLim, Younglan, and Nam-On Ku. 2021. "Revealing the Roles of Keratin 8/18-Associated Signaling Proteins Involved in the Development of Hepatocellular Carcinoma" International Journal of Molecular Sciences 22, no. 12: 6401. https://doi.org/10.3390/ijms22126401
APA StyleLim, Y., & Ku, N. -O. (2021). Revealing the Roles of Keratin 8/18-Associated Signaling Proteins Involved in the Development of Hepatocellular Carcinoma. International Journal of Molecular Sciences, 22(12), 6401. https://doi.org/10.3390/ijms22126401