Olivier Govaere1, Jasper Wouters2, Michaela Petz3, Yves-Paul Vandewynckel4, Kathleen Van den Eynde2, Anke Van den Broeck5, Stefaan Verhulst6, Laurent Dollé6, Lies Gremeaux7, An Ceulemans2, Frederik Nevens8, Leo A van Grunsven6, Baki Topal5, Hugo Vankelecom7, Gianluigi Giannelli9, Hans Van Vlierberghe4, Wolfgang Mikulits3, Mina Komuta2, Tania Roskams2. 1. Department of Imaging and Pathology, KU Leuven and University Hospitals Leuven, Leuven, Belgium. Electronic address: olivier.govaere@med.kuleuven.be. 2. Department of Imaging and Pathology, KU Leuven and University Hospitals Leuven, Leuven, Belgium. 3. Department of Medicine I, Institute of Cancer Research, Medical University of Vienna, Vienna, Austria. 4. Department of Hepatology & Gastroenterology, Ghent University, Ghent, Belgium. 5. Department of Abdominal Surgery, KU Leuven and University Hospitals Leuven, Leuven, Belgium. 6. Department of Biomedical Sciences, Liver Cell Biology Laboratory, Vrije Universiteit Brussel, Brussels, Belgium. 7. Department of Development and Regeneration, KU Leuven, Leuven, Belgium. 8. Department of Hepatology, KU Leuven and University Hospitals Leuven, Leuven, Belgium. 9. Department of Medical Biosciences and Human Oncology, Padiglione Semeiotica Medica, Bari, Italy.
Abstract
BACKGROUND & AIMS: Cancer stem cells (CSCs) are thought to be persistent in tumours due to their chemoresistance and to cause relapse and metastasis. Hepatic carcinomas displaying hepatic progenitor cell (HPC) features have been associated with a poor prognosis, though it remains unclear how CSCs relate to these different histological subtypes. METHODS: Candidate CSCs were isolated using the side population (SP) technique from primary tissue samples diagnosed as keratin(K)19-negative or -positive hepatocellular carcinoma (HCC) or as combined hepatocellular/cholangiocarcinoma and analysed for gene and protein expression. The effect of laminin-332 was analysed in vitro by using HCC cell lines and in vivo using a xenograft mouse model. RESULTS: The size of the SP correlated with the degree of HPC features found in human hepatic cancer, and also showed an elevated mRNA expression of biliary/HPC markers and the extracellular matrix marker LAMC2, the gene encoding the laminin γ2-chain. Immunopositivity for the γ2-chain of laminin-332 was seen in the extracellular matrix surrounding small HPC-like tumour cells with a low proliferation rate. In vitro, laminin-332 increased K19 expression, phosphorylated mTOR and decreased phospho-histone H3 expression, indicating reduced cell mitosis. The effect of laminin-332 was enhanced upon mTORC1 inhibition and diminished when inhibiting mTORC1+C2. Resistance to doxorubicin and sorafenib treatment, and the SP fraction increased in the coated condition. In vivo, laminin-332 reduced tumour growth and sustained K19 expression. CONCLUSIONS: In this study we identified a prominent role for laminin-332 as part of the specialised CSC niche in maintaining and supporting cell 'stemness', which leads to chemoresistance and quiescence.
BACKGROUND & AIMS: Cancer stem cells (CSCs) are thought to be persistent in tumours due to their chemoresistance and to cause relapse and metastasis. Hepatic carcinomas displaying hepatic progenitor cell (HPC) features have been associated with a poor prognosis, though it remains unclear how CSCs relate to these different histological subtypes. METHODS: Candidate CSCs were isolated using the side population (SP) technique from primary tissue samples diagnosed as keratin(K)19-negative or -positive hepatocellular carcinoma (HCC) or as combined hepatocellular/cholangiocarcinoma and analysed for gene and protein expression. The effect of laminin-332 was analysed in vitro by using HCC cell lines and in vivo using a xenograft mouse model. RESULTS: The size of the SP correlated with the degree of HPC features found in humanhepatic cancer, and also showed an elevated mRNA expression of biliary/HPC markers and the extracellular matrix marker LAMC2, the gene encoding the laminin γ2-chain. Immunopositivity for the γ2-chain of laminin-332 was seen in the extracellular matrix surrounding small HPC-like tumour cells with a low proliferation rate. In vitro, laminin-332 increased K19 expression, phosphorylated mTOR and decreased phospho-histone H3 expression, indicating reduced cell mitosis. The effect of laminin-332 was enhanced upon mTORC1 inhibition and diminished when inhibiting mTORC1+C2. Resistance to doxorubicin and sorafenib treatment, and the SP fraction increased in the coated condition. In vivo, laminin-332 reduced tumour growth and sustained K19 expression. CONCLUSIONS: In this study we identified a prominent role for laminin-332 as part of the specialised CSC niche in maintaining and supporting cell 'stemness', which leads to chemoresistance and quiescence.
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