Agnes Holczbauer

Human hepatic cancer stem cells are characterized by common stemness traits and diverse oncogenic pathways.

Epigenetic mechanisms play critical roles in stem cell biology by maintaining pluripotency of stem cells and promoting differentiation of more mature derivatives. If similar mechanisms are relevant for the cancer stem cell (CSC) model, then epigenetic modulation might enrich the CSC population, thereby facilitating CSC isolation and rigorous evaluation. To test this hypothesis, primary human cancer cells and liver cancer cell lines were treated with zebularine (ZEB), a potent DNA methyltransferase‐1 inhibitor, and putative CSCs were isolated using the side population (SP) approach. The CSC properties of ZEB‐treated and untreated subpopulations were tested using standard in vitro and in vivo assays. Whole transcriptome profiling of isolated CSCs was performed to generate CSC signatures. Clinical relevance of the CSC signatures was evaluated in diverse primary human cancers. Epigenetic modulation increased frequency of cells with CSC properties in the SP fraction isolated from human cancer cells as judged by self‐renewal, superior tumor‐initiating capacity in serial transplantations, and direct cell tracking experiments. Integrative transcriptome analysis revealed common traits enriched for stemness‐associated genes, although each individual CSC gene expression signature exhibited activation of different oncogenic pathways (e.g., EGFR, SRC, and MYC). The common CSC signature was associated with malignant progression, which is enriched in poorly differentiated tumors, and was highly predictive of prognosis in liver and other cancers. Conclusion: Epigenetic modulation may provide a tool for prospective isolation and in‐depth analysis of CSC. The liver CSC gene signatures are defined by a pernicious interaction of unique oncogene‐specific and common stemness traits. These data should facilitate the identifications of therapeutic tools targeting both unique and common features of CSCs. (HEPATOLOGY 2011;)

Specific fate decisions in adult hepatic progenitor cells driven by MET and EGFR signaling

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.

Contribution of Hepatic Lineage Stage-Specific Donor Memory to the Differential Potential of Induced Mouse Pluripotent Stem Cells

Recent studies suggested that induced pluripotent stem cells (iPSCs) retain a residual donor cell gene expression, which may impact their capacity to differentiate into cell of origin. Here, we addressed a contribution of a lineage stage‐specific donor cell memory in modulating the functional properties of iPSCs. iPSCs were generated from hepatic lineage cells at an early (hepatoblast‐derived, HB‐iPSCs) and end stage (adult hepatocyte, AH‐iPSCs) of hepatocyte differentiation as well as from mouse embryonic fibroblasts (MEFs‐iPSCs) using a lentiviral vector encoding four pluripotency‐inducing factors Oct4, Sox2, Klf4, and c‐Myc. All resulting iPSC lines acquired iPSCs phenotype as judged by the accepted criteria including morphology, expression of pluripotency markers, silencing of transducing factors, capacity of multilineage differentiation in teratoma assay, and normal diploid karyotype. However, HB‐iPSCs were more efficient in directed differentiation toward hepatocytic lineage as compared to AH‐iPSCs, MEF‐iPSCs, or mouse embryonic stem cells (mESCs). Extensive comparative transcriptome analyses of the early passage iPSCs, donor cells, and mESCs revealed that despite global similarities in gene expression patterns between generated iPSCs and mESCs, HB‐iPSCs retained a transcriptional memory (seven upregulated and 17 downregulated genes) typical of the original cells. Continuous passaging of HB‐iPSCs erased most of these differences including a superior capacity for hepatic redifferentiation. These results suggest that retention of lineage stage‐specific donor memory in iPSCs may facilitate differentiation into donor cell type. The identified gene set may help to improve hepatic differentiation for therapeutic applications and contribute to the better understanding of liver development. STEM CELLS 2012;30:997–1007

Epigenetic reprogramming modulates malignant properties of human liver cancer

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. 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. (Hepatology 2014;59:2251–2262)

Abstract 2653: Direct oncogenic reprogramming of adult mouse hepatocytes into cancer stem cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Objective: Primary human liver cancer (PLC), the third most lethal cancer worldwide, is classified into biologically distinct subgroups, which suggests origin from different hepatic lineage cells. The existence of cancer stem cells (CSCs) was reported in PLC, but the cellular origin of liver CSCs has not been elucidated. Our aim was to investigate the contribution of different hepatic lineage cells to the evolution of CSCs and the phenotypic and genetic heterogeneity of PLC. Methods: Three cell types at different levels of differentiation, including primary mouse hepatic progenitor cells (HPCs), lineage committed hepatoblasts (HBs) and terminally differentiated hepatocytes (AHs) were co-transduced with lentiviral vectors carrying oncogenic H-Ras-Luciferase/EGFP and SV40 large T (LT)-mCherry. CSC properties of FACS sorted H-Ras-EGFP+/SV40LT-mCherry+ cells were tested by standard in vitro and in vivo assays. Individual liver tumors derived from intrasplenic injection of transduced HPCs, HBs and AHs were subjected to immunohistochemistry and whole transcriptome profiling. Results: HPCs, HBs and AHs were susceptible to transformation albeit with a different efficiency as shown by the frequency of tumor initiating cells (1/7, 1/26 and 1/42, respectively). All transduced cells acquired similar attributes of liver CSCs in vitro as judged by self-renewal ability, expression of CSC marker CD133, CD24, CD44 and CD90 and high percentage of side population cells. HPC-, HB- and AH-initiated liver tumors commonly showed a multi-lineage differentiation expressing hepatocyte (HNFα), hepatic progenitor cell (EpCAM, cytokeratin 19, A6) and mesenchymal (vimentin) markers and resembled human PLC. Nevertheless, tumors displayed distinct morphophenotypes according to their cell-of origin: AH tumors showed predominantly hepatocellular carcinoma, HB tumors cholangicarcinoma and HPC tumors epithelial-mesenchymal transition (EMT)-like features. Gene expression analyses revealed the activation of EMT- and embryonic cell-related transcriptional programs in all tumors with the highest number of significant genetic changes in AH (2826) versus HB (574) and HPC tumors (906). Hierarchical clustering distinguished tumors of different cellular origin underscoring the contribution of lineage-stage-dependent genetic changes in malignant transformation. Notably, AH-derived tumors showed specific enrichment of c-Myc target genes. Stable knockdown of c-Myc in transformed AHs reduced their CSC properties and delayed tumor growth. Conclusions: Our results indicate that liver tumors with dominant CSC features can originate from any cell in the hepatocytic lineage. Identification of common and cell-of-origin specific phenotypic and genetic changes may provide novel therapeutic targets for treatment of PLC. Citation Format: Agnes Holczbauer, Valentina M. Factor, Jesper B. Andersen, David E. Kleiner, Jens U. Marquardt, Chiara Raggi, Mitsuteru Kitade, Daekwan Seo, Akita Hirofumi, Marian E. Durkin, Snorri S. Thorgeirsson. Direct oncogenic reprogramming of adult mouse hepatocytes into cancer stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2653. doi:10.1158/1538-7445.AM2013-2653

Abstract 4252: Mechanistic and clinical implications of epigenetic modulation in liver cancer stem cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Epigenetic mechanisms play critical roles in both stem cell and cancer biology. Epigenetic modulation directs stem cells towards pluripotency and promotes differentiation of more mature derivatives. We hypothesized, that if similar mechanisms are relevant to the cancer stem cell (CSC) model, then epigenetic modulation might enrich the CSC population, thereby facilitating CSCs isolation and rigorous evaluation. Five hepatoma cell lines were treated with Zebularine (ZEB), an effective DNMT1-inhibitor. Putative CSC were isolated using the “side population” (SP) approach. FACS-sorted SP and non-SP fractions were examined for tumorigenic capacity and differences in gene expression. SP-derived signatures were integrated with HCC microarray database and tested for a prognostic value. ZEB-treatment significantly reduced the SP size while remarkably increasing the proportion of cells with CSC properties as judged by a superior tumor-initiation capacity relative to the untreated SP cells. This was further confirmed in serial transplantation as well as direct cell tracking experiments using genetically labeled SP and non-SP cells. Furthermore, integrative transcriptome analysis of the ZEB-enriched CSC populations identified common stemness traits although various oncogenic pathways (e.g. EGFR, SRC and MYC) were activated in individual hepatoma cell lines. Moreover, each individual as well as the common CSC signatures were associated with poorly differentiated tumors, high recurrence rate and poor survival in liver as well as lung cancers. In conclusion, we demonstrate that modulation of the liver cancer epigenome is a useful tool to increase the representation of highly tumorigenic cells with CSC properties within the SP fraction. The integrative analysis of the CSC transcriptome revealed a pernicious interaction of common CSC stemness genes with variety of known oncogenic pathways in liver cancer. The significance of CSC- enriched gene signature for the prognosis of liver and other cancers provides a rationale for therapeutic targeting of CSCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4252.

Abstract 4181: Comparison of tumorigenic proficiency at different stages of hepatocyte differentiation

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC BACKGROUND: Current evidence suggests that any cell that can proliferate repeatedly is susceptible to neoplastic transformation. In the liver, these include all cells within the lineage from hepatoblast and adult liver stem cell to hepatocyte and cholangiocyte. Lentiviral vectors with their ability to transduce non-dividing cells are effective tools to introduce oncogenes at different stages of lineage commitment. AIM: To address whether (1) the transforming potential of defined oncogenes is dependent on the stage of hepatocytic differentiation and (2) the genotype and phenotype of developed tumors reflect the stage of differentiation. METHODS: To generate genetically defined liver carcinomas, constitutive lentiviral vectors were developed carrying oncogenic H-Ras and luciferase/EGFP double reporter as well as SV40 large T (LT) antigen and mCherry fluorescent reporter. The E-cadherin-positive hepatoblasts (ED16.5) and newborn (1day) hepatocytes as well as adult (3month) hepatocytes were transduced ex vivo by lentiviral vectors and FACS-sorted before intrasplenic transplantation into NOD/SCID mice. Liver tumors larger than 3mm derived from fetal (n=20), newborn (n=18) and adult hepatocytes (n=10) transfected with H-Ras and SV40 LT were macrodissected and subjected to immunohistochemistry and microarray analyses. RESULTS: Infection with H-Ras alone was not sufficient to transform hepatocytes at any state of differentiation. SV40 LT was capable to transform only fetal hepatocytes but not hepatocytes as judged by robust anchorage-independent growth in vitro and rapid tumor formation in vivo. The combination of activated H-Ras and SV40 LT significantly accelerated the tumor potential of all infected hepatocytes. In all cases, tumors resembled the histopathological subtypes of human hepatocellular carcinoma (HCC) albeit with variable degrees of malignancy. HCCs derived from hepatoblasts were more aggressive with a predominance of pseudoglandular component and more frequent extrahepatic growth. The pseudoglandular structures were strongly positive for cytokeratin 19 (CK19). Unsupervised hierarchical clustering of the expression profiles grouped the tumors according to CK19 associated gene expression signature with highest levels found in fetal- to lowest in adult hepatocyte-derived tumors. HCCs established from hepatoblasts were genetically more heterogeneous and clustered with tumors derived from both newborn and adult hepatocytes. CONCLUSIONS: There is a reciprocal relationship between the transforming potential of defined oncogenes and extent of commitment to hepatocyte lineage. Transformation of hepatocytes at the different stages of differentiation may induce a cancer stem cell-like phenotype that displays the markers characteristic of hepatic stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4181.