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BMC Cancer
Published in: BMC Cancer 1/2018

Open Access 01-12-2018 | Research article

Enrichment of cancer stem cells via β-catenin contributing to the tumorigenesis of hepatocellular carcinoma

Authors: Harshul Pandit, Yan Li, Xuanyi Li, Weizhong Zhang, Suping Li, Robert C. G. Martin

Published in: BMC Cancer | Issue 1/2018

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Abstract

Background

Hepatocellular carcinoma (HCC) is among the deadliest cancers due to its heterogeneity, contributing to chemoresistance and recurrence. Cancer stem-like cells (CSCs) are suggested to play an important role in HCC tumorigenesis. This study investigates the role of Wnt/β-catenin pathway in CSC enrichment and the capabilities of these CSCs in tumor initiation in orthotopic immunocompetent mouse model.

Methods

HCC-CSCs were enriched using established serum-free culture method. Wnt/β-catenin pathway activation and its components were analyzed by western blot and qRT-PCR. The role of β-catenin in enrichment of CSC spheroids was confirmed using siRNA interference. Tumorigenic capabilities were confirmed using orthotopic immunocompetent mouse model by injecting 2 × 106 Hepa1–6 CSC spheroids or control cells in upper left liver lobe.

Results

The serum-free cultured Hepa1–6 cells demonstrated self-renewal, spheroid formation, higher EpCAM expression, increased Hoechst-33342 efflux, and upregulated Wnt/β-catenin signaling. Wnt/β-catenin pathway upregulation was implicated with the downstream targets, i.e., c-MYC, Cyclin-D1, and LEF1. Also, we found that GSK-3β serine-9 phosphorylation increased in Hepa1–6 spheroids. Silencing β-catenin by siRNA reversed spheroid formation phenotype. Mice injected with Hepa1–6 CSC spheroids showed aggressive tumor initiation and growth compared with mice injected with control cells.

Conclusions

Successfully induced Hepa1–6 spheroids were identified with CSC-like properties. Aberrant β-catenin upregulation mediated by GSK-3β was observed in the Hepa1–6 spheroids. The β-catenin mediated CSC enrichment in the induced spheroids possesses the capability of tumor initiation in immunocompetent mice. Our study suggests plausible cell dedifferentiation mediated by β-catenin contributes to CSC-initiated HCC tumor growth in vivo.
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Literature
1.
2.
3.
Bosch FX, Ribes J, Diaz M, Cleries R. Primary liver cancer: worldwide incidence and trends. Gastroenterology. 2004;127:S5–s16.CrossRefPubMed
4.
Altekruse SF, McGlynn KA, Reichman ME. Hepatocellular carcinoma incidence, mortality, and survival trends in the United States from 1975 to 2005. J Clin Oncol. 2009;27:1485–91.CrossRefPubMedPubMedCentral
5.
Chiba T, Kamiya A, Yokosuka O, Iwama A. Cancer stem cells in hepatocellular carcinoma: recent progress and perspective. Cancer Lett. 2009;286:145–53.CrossRefPubMed
6.
Lobo NA, Shimono Y, Qian D, Clarke MF. The biology of cancer stem cells. Annu Rev Cell Dev Biol. 2007;23:675–99.CrossRefPubMed
7.
Dalerba P, Cho RW, Clarke MF. Cancer stem cells: models and concepts. Annu Rev Med. 2007;58:267–84.CrossRefPubMed
8.
Ma S, Chan KW, Hu L, Lee TK, Wo JY, Ng IO, Zheng BJ, Guan XY. Identification and characterization of tumorigenic liver cancer stem/progenitor cells. Gastroenterology. 2007;132:2542–56.CrossRefPubMed
9.
10.
Ma S. Biology and clinical implications of CD133(+) liver cancer stem cells. Exp Cell Res. 2013;319:126–32.CrossRefPubMed
11.
Zhao X, Parpart S, Takai A, Roessler S, Budhu A, Yu Z, Blank M, Zhang YE, Jia HL, Ye QH, et al. Integrative genomics identifies YY1AP1 as an oncogenic driver in EpCAM(+) AFP(+) hepatocellular carcinoma. Oncogene. 2015;34:5095–104.
12.
Yang ZF, Ho DW, Ng MN, Lau CK, Yu WC, Ngai P, Chu PW, Lam CT, Poon RT, Fan ST. Significance of CD90+ cancer stem cells in human liver cancer. Cancer Cell. 2008;13:153–66.CrossRefPubMed
13.
Liu LL, Fu D, Ma Y, Shen XZ. The power and the promise of liver cancer stem cell markers. Stem Cells Dev. 2011;20:2023–30.CrossRefPubMed
14.
Terris B, Cavard C, Perret C. EpCAM, a new marker for cancer stem cells in hepatocellular carcinoma. J Hepatol. 2010;52:280–1.CrossRefPubMed
15.
Yamashita T, Forgues M, Wang W, Kim JW, Ye Q, Jia H, Budhu A, Zanetti KA, Chen Y, Qin LX, et al. EpCAM and alpha-fetoprotein expression defines novel prognostic subtypes of hepatocellular carcinoma. Cancer Res. 2008;68:1451–61.CrossRefPubMed
16.
Yamashita T, Honda M, Nakamoto Y, Baba M, Nio K, Hara Y, Zeng SS, Hayashi T, Kondo M, Takatori H, et al. Discrete nature of EpCAM+ and CD90+ cancer stem cells in human hepatocellular carcinoma. Hepatology. 2013;57:1484–97.CrossRefPubMed
17.
Yamashita T, Ji J, Budhu A, Forgues M, Yang W, Wang HY, Jia H, Ye Q, Qin LX, Wauthier E, et al. EpCAM-positive hepatocellular carcinoma cells are tumor-initiating cells with stem/progenitor cell features. Gastroenterology. 2009;136:1012–24.CrossRefPubMed
18.
19.
Nejak-Bowen KN, Monga SP. Beta-catenin signaling, liver regeneration and hepatocellular cancer: sorting the good from the bad. Semin Cancer Biol. 2011;21:44–58.CrossRefPubMed
20.
Moon RT, Kohn AD, De Ferrari GV, Kaykas A. WNT and beta-catenin signalling: diseases and therapies. Nat Rev Genet. 2004;5:691–701.CrossRefPubMed
21.
Wielenga VJ, Smits R, Korinek V, Smit L, Kielman M, Fodde R, Clevers H, Pals ST. Expression of CD44 in Apc and Tcf mutant mice implies regulation by the WNT pathway. Am J Pathol. 1999;154:515–23.CrossRefPubMedPubMedCentral
22.
Yamashita T, Budhu A, Forgues M, Wang XW. Activation of hepatic stem cell marker EpCAM by Wnt-beta-catenin signaling in hepatocellular carcinoma. Cancer Res. 2007;67:10831–9.CrossRefPubMed
23.
Gangavarapu KJ, Azabdaftari G, Morrison CD, Miller A, Foster BA, Huss WJ. Aldehyde dehydrogenase and ATP binding cassette transporter G2 (ABCG2) functional assays isolate different populations of prostate stem cells where ABCG2 function selects for cells with increased stem cell activity. Stem Cell Res Ther. 2013;4:132.CrossRefPubMedPubMedCentral
24.
25.
Kroger A, Ortmann D, Krohne TU, Mohr L, Blum HE, Hauser H, Geissler M. Growth suppression of the hepatocellular carcinoma cell line Hepa1-6 by an activatable interferon regulatory factor-1 in mice. Cancer Res. 2001;61:2609–17.PubMed
26.
Dan Q, Sanchez R, Delgado C, Wepsic HT, Morgan K, Chen Y, Jeffes EW, Lowell CA, Morgan TR, Jadus MR. Non-immunogenic murine hepatocellular carcinoma Hepa1-6 cells expressing the membrane form of macrophage colony stimulating factor are rejected in vivo and lead to CD8+ T-cell immunity against the parental tumor. Mol Ther. 2001;4:427–37.CrossRefPubMed
27.
Li Y, Shi X, Zhang J, Zhang X, Martin RC. Hepatic protection and anticancer activity of curcuma: a potential chemopreventive strategy against hepatocellular carcinoma. Int J Oncol. 2014;44:505–13.CrossRefPubMed
28.
Knowles BB, Howe CC, Aden DP. Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science. 1980;209:497–9.CrossRefPubMed
29.
Dontu G, Abdallah WM, Foley JM, Jackson KW, Clarke MF, Kawamura MJ, Wicha MS. In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev. 2003;17:1253–70.CrossRefPubMedPubMedCentral
30.
Ponti D, Costa A, Zaffaroni N, Pratesi G, Petrangolini G, Coradini D, Pilotti S, Pierotti MA, Daidone MG. Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res. 2005;65:5506–11.CrossRefPubMed
31.
Lee J, Kotliarova S, Kotliarov Y, Li A, Su Q, Donin NM, Pastorino S, Purow BW, Christopher N, Zhang W, et al. Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines. Cancer Cell. 2006;9:391–403.CrossRefPubMed
32.
33.
Maric D, Fiorio Pla A, Chang YH, Barker JL. Self-renewing and differentiating properties of cortical neural stem cells are selectively regulated by basic fibroblast growth factor (FGF) signaling via specific FGF receptors. J Neurosci. 2007;27:1836–52.CrossRefPubMedPubMedCentral
34.
Mokkapati S, Niopek K, Huang L, Cunniff KJ, Ruteshouser EC, deCaestecker M, Finegold MJ, Huff V. Beta-catenin activation in a novel liver progenitor cell type is sufficient to cause hepatocellular carcinoma and hepatoblastoma. Cancer Res. 2014;74:4515–25.
35.
Kunjithapatham R, Karthikeyan S, Geschwind JF, Kieserman E, Lin M, Fu DX, Ganapathy-Kanniappan S. Reversal of anchorage-independent multicellular spheroid into a monolayer mimics a metastatic model. Sci Rep. 2014;4:6816.CrossRefPubMedPubMedCentral
36.
Chen Q, Kon J, Ooe H, Sasaki K, Mitaka T. Selective proliferation of rat hepatocyte progenitor cells in serum-free culture. Nat Protoc. 2007;2:1197–205.CrossRefPubMed
37.
Kanwar SS, Yu Y, Nautiyal J, Patel BB, Majumdar AP. The Wnt/beta-catenin pathway regulates growth and maintenance of colonospheres. Mol Cancer. 2010;9:212.CrossRefPubMedPubMedCentral
38.
Calvet CY, Andre FM, Mir LM. The culture of cancer cell lines as tumorspheres does not systematically result in cancer stem cell enrichment. PLoS One. 2014;9:e89644.CrossRefPubMedPubMedCentral
39.
Barker N, van Es JH, Kuipers J, Kujala P, van den Born M, Cozijnsen M, Haegebarth A, Korving J, Begthel H, Peters PJ, Clevers H. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature. 2007;449:1003–7.CrossRefPubMed
40.
41.
Schepers AG, Snippert HJ, Stange DE, van den Born M, van Es JH, van de Wetering M, Clevers H. Lineage tracing reveals Lgr5+ stem cell activity in mouse intestinal adenomas. Science. 2012;337:730–5.CrossRefPubMed
42.
Marjanovic ND, Weinberg RA, Chaffer CL. Cell plasticity and heterogeneity in cancer. Clin Chem. 2013;59:168–79.CrossRefPubMed
43.
44.
Chen Y, Wong PP, Sjeklocha L, Steer CJ, Sahin MB. Mature hepatocytes exhibit unexpected plasticity by direct dedifferentiation into liver progenitor cells in culture. Hepatology. 2012;55:563–74.CrossRefPubMed
45.
Herreros-Villanueva M, Zhang JS, Koenig A, Abel EV, Smyrk TC, Bamlet WR, de Narvajas AA, Gomez TS, Simeone DM, Bujanda L, Billadeau DD. SOX2 promotes dedifferentiation and imparts stem cell-like features to pancreatic cancer cells. Oncogenesis. 2013;2:e61.CrossRefPubMedPubMedCentral
46.
Jabari S, Meissnitzer M, Quint K, Gahr S, Wissniowski T, Hahn EG, Neureiter D, Ocker M. Cellular plasticity of trans- and dedifferentiation markers in human hepatoma cells in vitro and in vivo. Int J Oncol. 2009;35:69–80.PubMed
47.
Jopling C, Boue S, Izpisua Belmonte JC. Dedifferentiation, transdifferentiation and reprogramming: three routes to regeneration. Nat Rev Mol Cell Biol. 2011;12:79–89.CrossRefPubMed
48.
Kumar SM, Liu S, Lu H, Zhang H, Zhang PJ, Gimotty PA, Guerra M, Guo W, Xu X. Acquired cancer stem cell phenotypes through Oct4-mediated dedifferentiation. Oncogene. 2012;31:4898–911.CrossRefPubMedPubMedCentral
49.
Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126:663–76.CrossRefPubMed
50.
Fessler E, Dijkgraaf FE, De Sousa EMF, Medema JP. Cancer stem cell dynamics in tumor progression and metastasis: is the microenvironment to blame? Cancer Lett. 2013;341:97–104.CrossRefPubMed
51.
Metadata
Title
Enrichment of cancer stem cells via β-catenin contributing to the tumorigenesis of hepatocellular carcinoma
Authors
Harshul Pandit
Yan Li
Xuanyi Li
Weizhong Zhang
Suping Li
Robert C. G. Martin
Publication date
01-12-2018
Publisher
BioMed Central
Published in
BMC Cancer / Issue 1/2018
Electronic ISSN: 1471-2407
DOI
https://doi.org/10.1186/s12885-018-4683-0

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