Chiara Raggi

Expert consensus document: Cholangiocarcinoma: current knowledge and future perspectives consensus statement from the European Network for the Study of Cholangiocarcinoma (ENS-CCA)

Cholangiocarcinoma (CCA) is a heterogeneous group of malignancies with features of biliary tract differentiation. CCA is the second most common primary liver tumour and the incidence is increasing worldwide. CCA has high mortality owing to its aggressiveness, late diagnosis and refractory nature. In May 2015, the “European Network for the Study of Cholangiocarcinoma” (ENS-CCA: www.enscca.org or www.cholangiocarcinoma.eu) was created to promote and boost international research collaboration on the study of CCA at basic, translational and clinical level. In this Consensus Statement, we aim to provide valuable information on classifications, pathological features, risk factors, cells of origin, genetic and epigenetic modifications and current therapies available for this cancer. Moreover, future directions on basic and clinical investigations and plans for the ENS-CCA are highlighted.

HEPATOCYTE GROWTH FACTOR/C-MET SIGNALING IS REQUIRED FOR STEM-CELL-MEDIATED LIVER REGENERATION IN MICE

Hepatocyte growth factor (HGF)/c‐Met supports a pleiotrophic signal transduction pathway that controls stem cell homeostasis. Here, we directly addressed the role of c‐Met in stem‐cell–mediated liver regeneration by utilizing mice harboring c‐met floxed alleles and Alb‐Cre or Mx1‐Cre transgenes. To activate oval cells, the hepatic stem cell (HSC) progeny, we used a model of liver injury induced by diet containing the porphyrinogenic agent, 3,5‐diethocarbonyl‐1,4‐dihydrocollidine (DDC). Deletion of c‐met in oval cells was confirmed in both models by polymerase chain reaction analysis of fluorescence‐activated cell‐sorted epithelial cell adhesion molecule (EpCam)‐positive cells. Loss of c‐Met receptor decreased the sphere‐forming capacity of oval cells in vitro as well as reduced oval cell pool, impaired migration, and decreased hepatocytic differentiation in vivo, as demonstrated by double immunofluorescence using oval‐ (A6 and EpCam) and hepatocyte‐specific (i.e. hepatocyte nuclear factor 4‐alpha) antibodies. Furthermore, lack of c‐Met had a profound effect on tissue remodeling and overall composition of HSC niche, which was associated with greatly reduced matrix metalloproteinase (MMP)9 activity and decreased expression of stromal‐cell–derived factor 1. Using a combination of double immunofluorescence of cell‐type–specific markers with MMP9 and gelatin zymography on the isolated cell populations, we identified macrophages as a major source of MMP9 in DDC‐treated livers. The Mx1‐Cre‐driven c‐met deletion caused the greatest phenotypic impact on HSCs response, as compared to the selective inactivation in the epithelial cell lineages achieved in c‐Metfl/fl; Alb‐Cre+/− mice. However, in both models, genetic loss of c‐met triggered a similar cascade of events, leading to the failure of HSC mobilization and death of the mice. Conclusion: These results establish a direct contribution of c‐Met in the regulation of HSC response and support a unique role for HGF/c‐Met as an essential growth‐factor–signaling pathway for regeneration of diseased liver. (HEPATOLOGY 2012)

An Integrated Genomic and Epigenomic Approach Predicts Therapeutic Response to Zebularine in Human Liver Cancer

Drug-induced transcription and epigenetic changes can predict whether liver cancer can be successfully treated with an epigenome-targeting drug. A Crystal Ball for Cancer Therapy If you ask most first-year medical students, they are not likely to list fortune-telling as a required skill for a successful clinician. Yet, predicting how different patients will respond to therapy could save time, money, and lives. Andersen et al. peer into their crystal ball and provide an approach for determining whether patients with liver cancer will respond to treatment or not. Liver cancer causes more than half a million deaths annually. Although early cases may respond to treatment, it is often diagnosed at later stages when symptoms become more severe. Only a subset of patients with advanced-stage liver cancer is eligible for the most successful current therapy, sorafenib, leaving a large number of patients with few options. Epigenetic changes, or changes in gene expression not due to differences in DNA sequence, contribute to the initiation and progression of liver cancer. These changes are reversible, and epigenetic modifier drugs such as zebularine can reactivate tumor suppressor genes and decrease tumor cell growth. Andersen et al. characterized the epigenetic changes induced by zebularine in liver cancer cell lines. They found a distinct signature that identified two groups of cells—those sensitive and those resistant to zebularine. In an animal xenograft model, zebularine treatment of liver tumors with the “sensitive” profile resulted in increased survival and decreased metastasis to the lungs. Moreover, the zebularine-sensitive signature predicted prognosis of patients with liver cancer with an accuracy ranging from 84 to 96%. Therefore, this study not only provides a proof of principle that epigenetic modification may be a viable therapy for liver cancer but also gives us a glimpse into the future, allowing clinicians to predict clinical outcome for liver cancer patients, as well as the success or failure of this emerging treatment regimen. Epigenomic changes such as aberrant hypermethylation and subsequent atypical gene silencing are characteristic features of human cancer. Here, we report a comprehensive characterization of epigenomic modulation caused by zebularine, an effective DNA methylation inhibitor, in human liver cancer. Using transcriptomic and epigenomic profiling, we identified a zebularine response signature that classified liver cancer cell lines into two major subtypes with different drug responses. In drug-sensitive cell lines, zebularine caused inhibition of proliferation coupled with increased apoptosis, whereas drug-resistant cell lines showed up-regulation of oncogenic networks (for example, E2F1, MYC, and TNF) that drive liver cancer growth in vitro and in preclinical mouse models. Assessment of zebularine-based therapy in xenograft mouse models demonstrated potent therapeutic effects against tumors established from zebularine-sensitive but not zebularine-resistant liver cancer cells, leading to increased survival and decreased pulmonary metastasis. Integration of the zebularine gene expression and demethylation response signatures allowed differentiation of patients with hepatocellular carcinoma according to their survival and disease recurrence. This integrated signature identified a subclass of patients within the poor-survivor group that is likely to benefit from therapeutic agents that target the cancer epigenome.

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;)

Definition of Ubiquitination Modulator COP1 as a Novel Therapeutic Target in Human Hepatocellular Carcinoma

The development of targeted therapeutics for hepatocellular carcinoma (HCC) remains a major challenge. The ubiquitination modulator COP1 regulates p53 activity by ubiquitination and it is frequently overexpressed in human HCC. In this study, we tested the hypothesis that COP1 blockade by short interfering RNA (siRNA)-mediated inhibition could affect the course of HCC progression. The COP1 isoform COP1-1 was selected as the most effective target for siRNAs in terms of growth inhibition and apoptotic induction in several HCC cell lines. Growth inhibition occurred in HCC cells that retained wild-type p53 or expressed mutant p53 (Y220C or R249S), whereas p53-null Hep3B cells were resistant. Microarray expression analysis revealed that the antiproliferative effects of COP1 blockade were driven by a common subset of molecular alterations including a p53-associated functional network. In an orthotopic mouse xenograft model of HCC, systemic delivery of a modified COP1 siRNA by stable nucleic acid-lipid particles suppressed neoplastic growth in liver without unwanted immune responses. Our findings offer a first proof of principle that COP1 can be a promising target for systemic therapy of HCC.

Antitumor effects in hepatocarcinoma of isoform-selective inhibition of HDAC2

Histone deacetylase 2 (HDAC2) is a chromatin modifier involved in epigenetic regulation of cell cycle, apoptosis, and differentiation that is upregulated commonly in human hepatocellular carcinoma (HCC). In this study, we show that specific targeting of this HDAC isoform is sufficient to inhibit HCC progression. siRNA-mediated silencing of HDAC inhibited HCC cell growth by blocking cell-cycle progression and inducing apoptosis. These effects were associated with deregulation of HDAC-regulated genes that control cell cycle, apoptosis, and lipid metabolism, specifically, by upregulation of p27 and acetylated p53 and by downregulation of CDK6 and BCL2. We found that HDAC2 silencing in HCC cells also strongly inhibited PPARγ signaling and other regulators of glycolysis (ChREBPα and GLUT4) and lipogenesis (SREBP1C and FAS), eliciting a marked decrease in fat accumulation. Notably, systemic delivery of HDAC2 siRNA encapsulated in lipid nanoparticles was sufficient to blunt the growth of human HCC in a murine xenograft model. Our findings offer preclinical proof-of-concept for HDAC2 blockade as a systemic therapy for liver cancer.

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)

Impact of microenvironment and stem-like plasticity in cholangiocarcinoma: molecular networks and biological concepts.

Clinical complexity, anatomic diversity and molecular heterogeneity of cholangiocarcinoma (CCA) represent a major challenge in the assessment of effective targeted therapies. Molecular and cellular mechanisms underlying the diversity of CCA growth patterns remain a key issue of clinical concern. Crucial questions comprise the nature of the CCA-origin, the initial target for cellular transformation as well as the relationship with the cancer stem cells (CSC) concept. Additionally, since CCA often develops in the context of an inflammatory milieu (cirrhosis and cholangitis), the stromal compartment or tumour microenvironment (TME) likely promotes initiation and progression of this malignancy, contributing to its heterogeneity. This review will emphasize the dynamic interplay between stem-like intrinsic and TME-extrinsic pathways, which may represent novel options for multi-targeted therapies in CCA.

Cholangiocarcinoma stem-like subset shapes tumor-initiating niche by educating associated macrophages

BACKGROUND & AIMS A therapeutically challenging subset of cells, termed cancer stem cells (CSCs) are responsible for cholangiocarcinoma (CCA) clinical severity. Presence of tumor-associated macrophages (TAMs) has prognostic significance in CCA and other malignancies. Thus, we hypothesized that CSCs may actively shape their tumor-supportive immune niche. METHODS CCA cells were cultured in 3D conditions to generate spheres. CCA sphere analysis of in vivo tumorigenic-engraftment in immune-deficient mice and molecular characterization was performed. The in vitro and in vivo effect of CCA spheres on macrophage precursors was tested after culturing healthy donor cluster of differentiation (CD)14+ with CCA-sphere conditioned medium. RESULTS CCA spheres engrafted in 100% of transplanted mice and revealed a significant 20.3-fold increase in tumor-initiating fraction (p=0.0011) and a sustained tumorigenic potential through diverse xenograft-generations. Moreover, CCA spheres were highly enriched for CSC, liver cancer and embryonic stem cell markers both at gene and protein levels. Next, fluorescence-activated cell sorting analysis showed that in the presence of CCA sphere conditioned medium, CD14+ macrophages expressed key markers (CD68, CD115, human leukocyte antigen-D related, CD206) indicating that CCA sphere conditioned medium was a strong macrophage-activator. Gene expression profile of CCA sphere activated macrophages revealed unique molecular TAM-like features confirmed by high invasion capacity. Also, freshly isolated macrophages from CCA resections recapitulated a similar molecular phenotype of in vitro-educated macrophages. Consistent with invasive features, the largest CD163+ set was found in the tumor front of human CCA specimens (n=23) and correlated with a high level of serum cancer antigen 19.9 (n=17). Among mediators released by CCA spheres, only interleukin (IL)13, IL34 and osteoactivin were detected and further confirmed in CCA patient sera (n=12). Surprisingly, a significant association of IL13, IL34 and osteoactivin with sphere stem-like genes was provided by a CCA database (n=104). In vitro combination of IL13, IL34, osteoactivin was responsible for macrophage-differentiation and invasion, as well as for in vivo tumor-promoting effect. CONCLUSION CCA-CSCs molded a specific subset of stem-like associated macrophages thus providing a rationale for a synergistic therapeutic strategy for CCA-disease. LAY SUMMARY Immune plasticity represents an important hallmark of tumor outcome. Since cancer stem cells are able to manipulate stromal cells to their needs, a better definition of the key dysregulated immune subtypes responsible for cooperating in supporting tumor initiation may facilitate the development of new therapeutic approaches. Considering that human cholangiocarcinoma represents a clinical emergency, it is essential to move to predictive models in order to understand the adaptive process of macrophage component (imprinting, polarization and maintenance) engaged by tumor stem-like compartment.

Methylation and liver cancer.

Cancer evolution at all stages (including initiation, progression and invasion) is driven by both epigenetic abnormalities and genetic alterations. Epigenetics refer to any structural modification of genomic regions, which lead to modification in gene expression without alterations in DNA sequence. Progressive deregulation of epigenetic process is being increasingly recognized in liver carcinogenesis. This review will provide an overview of DNA methylation, one of the most commonly epigenetic events, which profoundly contributes to liver cancer initiation and progression. Furthermore, the recent advancements in the knowledge of epigenetic reprogramming underlying hepatic cancer stem cells will be highlighted.