Different mechanisms could explain the origin and heterogeneity of CSC such as (i) differentiation arrest (stem cells), (ii) dedifferentiation (mature cells) and (iii) transdifferentiation (bone marrow stem cells). It is conceivable that all 3 mechanisms may be corrupted by oncogenic events, resulting in an assortment of CSC and explaining their heterogeneity. Defining and characterizing this heterogeneity is of vital importance for understanding CSC biology, and for effective therapeutic translation. Our most recent results in this project include: (1) Reversal of DNA hypermethylation and associated gene silencing is an emerging cancer therapy approach. Here we addressed the impact of epigenetic alterations and cellular context on functional and transcriptional reprogramming of hepatocellular carcinoma (HCC) cells. Our strategy employed a 3-day treatment of established and primary human HCC-derived cell lines grown as a monolayer at various cell densities with the DNMT1 inhibitor zebularine (ZEB) followed by a 3D culture to identify cells endowed with self-renewal potential. Differences in self-renewal, gene expression, tumorigenicity, and metastatic potential of spheres at generations G1-G5 were examined. Transient ZEB exposure produced differential cell density-dependent responses. In cells grown at low density, ZEB caused a remarkable increase in self-renewal and tumorigenicity associated with long-lasting gene expression changes characterized by a stable overexpression of cancer stem cell-related and key epithelial-mesenchymal transition genes. These effects persisted after restoration of DNMT1 expression. In contrast, at high cell density, ZEB caused a gradual decrease in self-renewal and tumorigenicty, and up-regulation of apoptosis- and differentiation-related genes. A permanent reduction of DNMT1 protein using short hairpin RNA (shRNA)-mediated DNMT1 silencing rendered HCC cells insensitive both to cell density and ZEB effects. Similarly, WRL68 and HepG2 hepatoblastoma cells expressing low DNMT1 basal levels also possessed a high self-renewal, irrespective of cell density or ZEB exposure. Spheres formed by low-density cells treated with ZEB or shDNMT1 displayed a high molecular similarity which was sustained through consecutive generations, confirming the essential role of DNMT1 depletion in the enhancement of cancer stem cell properties. In conclusion, these results identify DNA methylation as a key epigenetic regulatory mechanism determining the pool of cancer stem cells in liver cancer and possibly other solid tumors;(2) Human primary liver cancer is classified into biologically distinct subgroups based on cellular origin. Liver cancer stem cells (CSCs) have been recently described. We investigated the ability of distinct lineages of hepatic cells to become liver CSCs and the phenotypic and genetic heterogeneity of primary liver cancer. We transduced mouse primary hepatic progenitor cells, lineage-committed hepatoblasts, and differentiated adult hepatocytes with transgenes encoding oncogenic H-Ras and SV40LT. The CSC properties of transduced cells and their ability to form tumors were tested by standard in vitro and in vivo assays and transcriptome profiling. Irrespective of origin, all transduced cells acquired markers of CSC/progenitor cells, side populations, and self-renewal capacity in vitro. They also formed a broad spectrum of liver tumors, ranging from cholangiocarcinoma to hepatocellular carcinoma, which resembled human liver tumors, based on genomic and histologic analyses. The tumor cells coexpressed hepatocyte (hepatocyte nuclear factor 4 alpha), progenitor/biliary (keratin 19, epithelial cell adhesion molecule, A6), and mesenchymal (vimentin) markers and showed dysregulation of genes that control the epithelial-mesenchymal transition. Gene expression analyses could distinguish tumors of different cellular origin, indicating the contribution of lineage stage-dependent genetic changes to malignant transformation. Activation of c-Myc and its target genes was required to reprogram adult hepatocytes into CSCs and for tumors to develop. Stable knockdown of c-Myc in transformed adult hepatocytes reduced their CSC properties in vitro and suppressed growth of tumors in immunodeficient mice. From these data we conclude that any cell type in the mouse hepatic lineage can undergo oncogenic reprogramming into a CSC by activating different cell type-specific pathways. Identification of common and cell of origin-specific phenotypic and genetic changes could provide new therapeutic targets for liver cancer;(3) The relative contribution of hepatocyte growth factor (HGF)/MET and epidermal growth factor (EGF)/EGF receptor (EGFR), two key signal transduction systems in the normal and diseased liver, to fate decisions of adult hepatic progenitor cells (HPCs) has not been resolved. Here, we developed a robust culture system that permitted expansion and genetic manipulation of cells capable of multilineage differentiation in vitro and in vivo to examine the individual roles of HGF/MET and EGF/EGFR in HPC self-renewal and binary cell fate decision. By employing loss-of-function and rescue experiments in vitro, we showed that both receptors collaborate to increase the self-renewal of HPCs through activation of the extracellular signal-regulated kinase (ERK) pathway. MET was a strong inducer of hepatocyte differentiation by activating AKT and signal transducer and activator of transcription (STAT3). Conversely, EGFR selectively induced NOTCH1 to promote cholangiocyte specification and branching morphogenesis while concomitantly suppressing hepatocyte commitment. Furthermore, unlike the deleterious effects of MET deletion, the liver-specific conditional loss of Egfr facilitated rather than suppressed progenitor-mediated liver regeneration by switching progenitor cell differentiation toward hepatocyte lineage. These data provide new insight into the mechanisms regulating the stemness properties of adult HPCs and reveal a previously unrecognized link between EGFR and NOTCH1 in directing cholangiocyte differentiation. (4) Activation of c-MYC is an oncogenic hallmark of many cancers including liver cancer,and is associated with a variety of adverse prognostic characteristics. Despite a causative role during malignant transformation and progression in hepatocarcinogenesis, consequences of c- MYC activation for the biology of hepatic cancer stem cells (CSCs) are undefined. Here, distinct levels of c-MYC over-expression were established by using two dose-dependent tetracycline inducible systems in 4 hepatoma cell lines with different p53 mutational status. CSCs were evaluated using side-population approach as well as standard in vitro and in vivo assays. Functional repression of p53 was achieved by lentiviral shRNA transduction. The results show that c-MYC expression levels have a differential impact on liver CSC characteristics. At low levels, c-MYC activation led to increased proliferation and enhanced CSC properties including activation of reprogramming transcription factors and CSC marker expression (e.g. NANOG, OCT4 and EpCAM), expansion of side population and acceleration of tumor growth upon subcutaneous transplantation into immunocompromised mice. However, when exceeding a threshold level, c-MYC induced a pro-apoptotic program and loss of CSC potential both in vitro and in vivo. Mechanistically, c-MYC induced self-renewal capacity of liver cancer cells was exerted in a p53 dependent manner. Low c-MYC activation increased spheroid formation in p53-deficient tumor cells, whereas p53-dependent effects were blunted in the absence of MYC overexpression. Our results confirm the role of c-MYC as a master regulator during hepatocarcinogenesis and establish a new gatekeeper role for p53 in repressing c-MYC induced CSC phenotype in liver cancer cells.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Investigator-Initiated Intramural Research Projects (ZIA)
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Marquardt, Jens U; Thorgeirsson, Snorri S (2014) SnapShot: Hepatocellular carcinoma. Cancer Cell 25:550.e1
Raggi, Chiara; Factor, Valentina M; Seo, Daekwan et al. (2014) Epigenetic reprogramming modulates malignant properties of human liver cancer. Hepatology 59:2251-62
Marquardt, Jens U; Thorgeirsson, Snorri S (2013) Sall4 in ""stemness""-driven hepatocarcinogenesis. N Engl J Med 368:2316-8
Holczbauer, Ágnes; Factor, Valentina M; Andersen, Jesper B et al. (2013) Modeling pathogenesis of primary liver cancer in lineage-specific mouse cell types. Gastroenterology 145:221-231
Kitade, Mitsuteru; Factor, Valentina M; Andersen, Jesper B et al. (2013) Specific fate decisions in adult hepatic progenitor cells driven by MET and EGFR signaling. Genes Dev 27:1706-17
Xin, Hong-Wu; Ambe, Chenwi M; Ray, Satyajit et al. (2013) Wnt and the cancer niche: paracrine interactions with gastrointestinal cancer cells undergoing asymmetric cell division. J Cancer 4:447-57
Xin, Hong-Wu; Ambe, Chenwi M; Hari, Danielle M et al. (2013) Label-retaining liver cancer cells are relatively resistant to sorafenib. Gut :
Xin, Hong-Wu; Hari, Danielle M; Mullinax, John E et al. (2012) Tumor-initiating label-retaining cancer cells in human gastrointestinal cancers undergo asymmetric cell division. Stem Cells 30:591-8
Marquardt, Jens U; Raggi, Chiara; Andersen, Jesper B et al. (2011) Human hepatic cancer stem cells are characterized by common stemness traits and diverse oncogenic pathways. Hepatology 54:1031-42
Woo, Hyun Goo; Wang, Xin Wei; Budhu, Anuradha et al. (2011) Association of TP53 mutations with stem cell-like gene expression and survival of patients with hepatocellular carcinoma. Gastroenterology 140:1063-70

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