Long-term combination therapy with metformin and oxymetholone in a Fanconi anemia mouse model.

Fanconi anemia (FA) is a disease caused by defective deoxyribonucleic acid (DNA) repair that manifests as bone marrow failure, cancer predisposition, and developmental defects. We previously reported that monotherapy with either metformin (MET) or oxymetholone (OXM) improved peripheral blood (PB) counts and the number and functionality of bone marrow hematopoietic stem progenitor cells (HSPCs) number in Fancd2-/- mice. To evaluate whether the combination treatment of these drugs has a synergistic effect to prevent bone marrow failure in FA, we treated cohorts of Fancd2-/- mice and wildtype controls with either MET alone, OXM alone, MET+OXM, or placebo diet from age 3 weeks to 18 months. The OXM treated animals showed modest improvements in blood parameters including platelet count (p = .01) and hemoglobin levels (p < .05). In addition, the percentage of quiescent hematopoietic stem cell (HSC) (LSK [Lin-Sca+c-Kit+]) was significantly increased (p = .001) by long-term treatment with MET alone. The combination of metformin and oxymetholone did not result in a significant synergistic effect in any hematopoietic parameter. Gene expression analysis of liver tissue from these animals showed that some of the expression changes caused by Fancd2 deletion were partially normalized by metformin treatment. Importantly, no adverse effects of the individual or combination therapies were observed, despite the long-term administration. We conclude that androgen therapy is not a contraindication to concurrent metformin administration in clinical trials. HIGHLIGHTS: Long-term coadministration of metformin in combination with oxymetholone is well tolerated by Fancd2-/- mice. Hematopoietic stem cell quiescence in mutant mice was enhanced by treatment with metformin alone. Metformin treatment caused a partial normalization of gene expression in the livers of mutant mice.

Long-term combination therapy with Metformin and Oxymetholone in a Fanconi Anemia mouse model.

Fanconi Anemia (FA) is a disease caused by defective DNA repair which manifests as bone marrow failure, cancer predisposition, and developmental defects. Mice containing inactivating mutations in one or more genes in the FA pathway partially mimic the human disease. We previously reported that monotherapy with either metformin (MET) or oxymetholone (OXM) improved peripheral blood (PB) counts and the number and functionality of bone marrow (BM) hematopoietic stem progenitor cells (HSPCs) number in Fancd2-/- mice. To evaluate whether the combination treatment of these drugs has a synergistic effect to prevent bone marrow failure in FA, we treated cohorts of Fancd2-/- mice and wild-type controls with either MET alone, OXM alone, MET+OXM or placebo diet. Both male and female mice were treated from age 3 weeks to 18 months. The OXM treated animals showed modest improvements in blood parameters including platelet count (p=0.01) and hemoglobin levels (p<0.05). In addition, the percentage of quiescent HSC (LSK) was significantly increased (p=0.001) by long-term treatment with MET alone. However, the absolute number of progenitors, measured by LSK frequency or CFU-S, was not significantly altered by MET therapy. The combination of metformin and oxymetholone did not result in a significant synergistic effect on any parameter. Male animals on MET+OXM or MET alone were significantly leaner than controls at 18 months, regardless of genotype. Gene expression analysis of liver tissue from these animals showed that some of the expression changes caused by Fancd2 deletion were partially normalized by metformin treatment. Importantly, no adverse effects of the individual or combination therapies were observed, despite the long-term administration.

Pharmacologic Targeting of TFIIH Suppresses KRAS-Mutant Pancreatic Ductal Adenocarcinoma and Synergizes with TRAIL.

Pancreatic ductal adenocarcinoma (PDAC) typically presents as metastatic disease at diagnosis and remains refractory to treatment. Next-generation sequencing efforts have described the genomic landscape, classified molecular subtypes, and confirmed frequent alterations in major driver genes, with coexistent alterations in KRAS and TP53 correlating with the highest metastatic burden and poorest outcomes. However, translating this information to guide therapy remains a challenge. By integrating genomic analysis with an arrayed RNAi druggable genome screen and drug profiling of a KRAS/TP53 mutant PDAC cell line derived from a patient-derived xenograft (PDCL), we identified numerous targetable vulnerabilities that reveal both known and novel functional aspects of pancreatic cancer biology. A dependence on the general transcription and DNA repair factor TFIIH complex, particularly the XPB subunit and the CAK complex (CDK7/CyclinH/MAT1), was identified and further validated utilizing a panel of genomically subtyped KRAS mutant PDCLs. TFIIH function was inhibited with a covalent inhibitor of CDK7/12/13 (THZ1), a CDK7/CDK9 kinase inhibitor (SNS-032), and a covalent inhibitor of XPB (triptolide), which led to disruption of the protein stability of the RNA polymerase II subunit RPB1. Loss of RPB1 following TFIIH inhibition led to downregulation of key transcriptional effectors of KRAS-mutant signaling and negative regulators of apoptosis, including MCL1, XIAP, and CFLAR, initiating caspase-8 dependent apoptosis. All three drugs exhibited synergy in combination with a multivalent TRAIL, effectively reinforcing mitochondrial-mediated apoptosis. These findings present a novel combination therapy, with direct translational implications for current clinical trials on metastatic pancreatic cancer patients. Significance: This study utilizes functional genetic and pharmacological profiling of KRAS-mutant pancreatic adenocarcinoma to identify therapeutic strategies and finds that TFIIH inhibition synergizes with TRAIL to induce apoptosis in KRAS-driven pancreatic cancer.

Microfluidics Formulated Liposomes of Hypoxia Activated Prodrug for Treatment of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) presents as an unmet clinical challenge for drug delivery due to its unique hypoxic biology. Vinblastine-N-Oxide (CPD100) is a hypoxia-activated prodrug (HAP) that selectively converts to its parent compound, vinblastine, a potent cytotoxic agent, under oxygen gradient. The study evaluates the efficacy of microfluidics formulated liposomal CPD100 (CPD100Li) in PDAC. CPD100Li were formulated with a size of 95 nm and a polydispersity index of 0.2. CPD100Li was stable for a period of 18 months when freeze-dried at a concentration of 3.55 mg/mL. CPD100 and CPD100Li confirmed selective activation at low oxygen levels in pancreatic cancer cell lines. Moreover, in 3D spheroids, CPD100Li displayed higher penetration and disruption compared to CPD100. In patient-derived 3D organoids, CPD100Li exhibited higher cell inhibition in the organoids that displayed higher expression of hypoxia-inducible factor 1 alpha (HIF1A) compared to CPD100. In the orthotopic model, the combination of CPD100Li with gemcitabine (GEM) (standard of care for PDAC) showed higher efficacy than CPD100Li alone for a period of 90 days. In summary, the evaluation of CPD100Li in multiple cellular models provides a strong foundation for its clinical application in PDAC.

Proteomic analysis distinguishes extracellular vesicles produced by cancerous versus healthy pancreatic organoids.

Extracellular vesicles (EVs) are produced and released by both healthy and malignant cells and bear markers indicative of ongoing biological processes. In the present study we utilized high resolution flow cytometry to detect EVs in the plasma of patients with pancreatic ductal adenocarcinoma (PDAC) and in the supernatants of PDAC and healthy control (HC) pancreatic organoid cultures. Using ultrafiltration and size exclusion chromatography, PDAC and HC pancreatic organoid EVs were isolated for mass spectrometry analysis. Proteomic and functional protein network analysis showed a striking distinction in that EV proteins profiled in pancreatic cancer organoids were involved in vesicular transport and tumorigenesis while EV proteins in healthy organoids were involved in cellular homeostasis. Thus, the most abundant proteins identified in either case represented non-overlapping cellular programs. Tumor-promoting candidates LAMA5, SDCBP and TENA were consistently upregulated in PDAC EVs. Validation of specific markers for PDAC EVs versus healthy pancreatic EVs will provide the biomarkers and enhanced sensitivity necessary to monitor early disease or disease progression, with or without treatment. Moreover, disease-associated changes in EV protein profiles provide an opportunity to investigate alterations in cellular programming with disease progression.

Development of a Beta Cell-Specific Expression Control Element for Recombinant Adeno-Associated Virus.

Diabetes mellitus, caused by loss or dysfunction of the insulin-producing beta cells of the pancreas, is a promising target for recombinant adeno-associated virus (rAAV)-mediated gene therapy. To target potential therapeutic payloads specifically to beta cells, a cell type-specific expression control element is needed. In this study, we tested a series of rAAV vectors designed to express transgenes specifically in human beta cells using the islet-tropic rAAV-KP1 capsid. A small promoter, consisting of only 84 bp of the insulin core promoter was not beta cell-specific in AAV, but highly active in multiple cell types, including tissues outside the pancreas. A larger 363 bp fragment of the insulin promoter (INS) also lacked beta cell specificity. However, beta cell-specific expression was achieved by combining two regulatory elements, a promoter consisting of two copies of INS (INS × 2) and microRNA (miRNA) recognition elements (MREs). The INS × 2 promoter alone showed some beta cell preference, but not tight specificity. To reduce unspecific transgene expression in alpha cells, negative regulation by miRNAs was applied. MREs that are recognized by miRNAs abundant in alpha cells effectively downregulated the transgene expression in these cells. The INS2 × -MRE expression vector was highly specific to human beta cells and stem cell-derived beta cells.

Generation of functional ciliated cholangiocytes from human pluripotent stem cells.

The derivation of mature functional cholangiocytes from human pluripotent stem cells (hPSCs) provides a model for studying the pathogenesis of cholangiopathies and for developing therapies to treat them. Current differentiation protocols are not efficient and give rise to cholangiocytes that are not fully mature, limiting their therapeutic applications. Here, we generate functional hPSC-derived cholangiocytes that display many characteristics of mature bile duct cells including high levels of cystic fibrosis transmembrane conductance regulator (CFTR) and the presence of primary cilia capable of sensing flow. With this level of maturation, these cholangiocytes are amenable for testing the efficacy of cystic fibrosis drugs and for studying the role of cilia in cholangiocyte development and function. Transplantation studies show that the mature cholangiocytes generate ductal structures in the liver of immunocompromised mice indicating that it may be possible to develop cell-based therapies to restore bile duct function in patients with biliary disease.

A Stromal Lysolipid-Autotaxin Signaling Axis Promotes Pancreatic Tumor Progression.

Pancreatic ductal adenocarcinoma (PDAC) develops a pronounced stromal response reflecting an aberrant wound-healing process. This stromal reaction features transdifferentiation of tissue-resident pancreatic stellate cells (PSC) into activated cancer-associated fibroblasts, a process induced by PDAC cells but of unclear significance for PDAC progression. Here, we show that PSCs undergo a dramatic lipid metabolic shift during differentiation in the context of pancreatic tumorigenesis, including remodeling of the intracellular lipidome and secretion of abundant lipids in the activated, fibroblastic state. Specifically, stroma-derived lysophosphatidylcholines support PDAC cell synthesis of phosphatidylcholines, key components of cell membranes, and also facilitate production of the potent wound-healing mediator lysophosphatidic acid (LPA) by the extracellular enzyme autotaxin, which is overexpressed in PDAC. The autotaxin-LPA axis promotes PDAC cell proliferation, migration, and AKT activation, and genetic or pharmacologic autotaxin inhibition suppresses PDAC growth in vivo. Our work demonstrates how PDAC cells exploit the local production of wound-healing mediators to stimulate their own growth and migration. SIGNIFICANCE: Our work highlights an unanticipated role for PSCs in producing the oncogenic LPA signaling lipid and demonstrates how PDAC tumor cells co-opt the release of wound-healing mediators by neighboring PSCs to promote their own proliferation and migration.See related commentary by Biffi and Tuveson, p. 578.This article is highlighted in the In This Issue feature, p. 565.

Diabetes relief in mice by glucose-sensing insulin-secreting human α-cells.

Cell-identity switches, in which terminally differentiated cells are converted into different cell types when stressed, represent a widespread regenerative strategy in animals, yet they are poorly documented in mammals. In mice, some glucagon-producing pancreatic α-cells and somatostatin-producing δ-cells become insulin-expressing cells after the ablation of insulin-secreting ß-cells, thus promoting diabetes recovery. Whether human islets also display this plasticity, especially in diabetic conditions, remains unknown. Here we show that islet non-ß-cells, namely α-cells and pancreatic polypeptide (PPY)-producing γ-cells, obtained from deceased non-diabetic or diabetic human donors, can be lineage-traced and reprogrammed by the transcription factors PDX1 and MAFA to produce and secrete insulin in response to glucose. When transplanted into diabetic mice, converted human α-cells reverse diabetes and continue to produce insulin even after six months. Notably, insulin-producing α-cells maintain expression of α-cell markers, as seen by deep transcriptomic and proteomic characterization. These observations provide conceptual evidence and a molecular framework for a mechanistic understanding of in situ cell plasticity as a treatment for diabetes and other degenerative diseases.

Identification and Isolation of Clonogenic Cholangiocyte in Mouse.

Cholangiocytes are proliferative and are one of the sources for liver progenitor cells. Clonogenic cholangiocytes are defined as cells capable of clonally proliferating and differentiating cholangiocytes both in vitro and in vivo. In this protocol, we describe the method for isolation of primary cholangiocytes from mouse. To study the heterogeneity of cholangiocytes, we used flow cytometry-based cell sorting to isolate different subsets of cholangiocytes. Organoid-forming efficiencies from sorted single cells are compared within different cholangiocyte populations to identify clonogenic cholangiocytes.

Long-Term Correction of Diabetes in Mice by In Vivo Reprogramming of Pancreatic Ducts.

Direct lineage reprogramming can convert readily available cells in the body into desired cell types for cell replacement therapy. This is usually achieved through forced activation or repression of lineage-defining factors or pathways. In particular, reprogramming toward the pancreatic ß cell fate has been of great interest in the search for new diabetes therapies. It has been suggested that cells from various endodermal lineages can be converted to ß-like cells. However, it is unclear how closely induced cells resemble endogenous pancreatic ß cells and whether different cell types have the same reprogramming potential. Here, we report in vivo reprogramming of pancreatic ductal cells through intra-ductal delivery of an adenoviral vector expressing the transcription factors Pdx1, Neurog3, and Mafa. Induced ß-like cells are mono-hormonal, express genes essential for ß cell function, and correct hyperglycemia in both chemically and genetically induced diabetes models. Compared with intrahepatic ducts and hepatocytes treated with the same vector, pancreatic ducts demonstrated more rapid activation of ß cell transcripts and repression of donor cell markers. This approach could be readily adapted to humans through a commonly performed procedure, endoscopic retrograde cholangiopancreatography (ERCP), and provides potential for cell replacement therapy in type 1 diabetes patients.

Genome-wide genetic and epigenetic analyses of pancreatic acinar cell carcinomas reveal aberrations in genome stability.

Pancreatic acinar cell carcinoma (ACC) is an aggressive exocrine tumor with largely unknown biology. Here, to identify potential targets for personalized treatment, we perform integrative genome-wide and epigenome-wide analyses. The results show frequently aberrant DNA methylation, abundant chromosomal amplifications and deletions, and mutational signatures suggesting defective DNA repair. In contrast to pancreatic ductal adenocarcinoma, no recurrent point mutations are detected. The tumor suppressors ID3, ARID1A, APC, and CDKN2A are frequently impaired also on the protein level and thus potentially affect ACC tumorigenesis. Consequently, this work identifies promising therapeutic targets in ACC for drugs recently approved for precision cancer therapy.

Glycoprotein 2 is a specific cell surface marker of human pancreatic progenitors.

PDX1+/NKX6-1+ pancreatic progenitors (PPs) give rise to endocrine cells both in vitro and in vivo. This cell population can be successfully differentiated from human pluripotent stem cells (hPSCs) and hold the potential to generate an unlimited supply of ß cells for diabetes treatment. However, the efficiency of PP generation in vitro is highly variable, negatively impacting reproducibility and validation of in vitro and in vivo studies, and consequently, translation to the clinic. Here, we report the use of a proteomics approach to phenotypically characterize hPSC-derived PPs and distinguish these cells from non-PP populations during differentiation. Our analysis identifies the pancreatic secretory granule membrane major glycoprotein 2 (GP2) as a PP-specific cell surface marker. Remarkably, GP2 is co-expressed with NKX6-1 and PTF1A in human developing pancreata, indicating that it marks the multipotent pancreatic progenitors in vivo. Finally, we show that isolated hPSC-derived GP2+ cells generate ß-like cells (C-PEPTIDE+/NKX6-1+) more efficiently compared to GP2- and unsorted populations, underlining the potential therapeutic applications of GP2.Pancreatic progenitors (PPs) can be derived from human pluripotent stem cells in vitro but efficiency of differentiation varies, making it hard to sort for insulin-producing cells. Here, the authors use a proteomic approach to identify the secretory granule membrane glycoprotein 2 as a marker for PDX1+/NKX6-1+ PPs.

Human islets contain four distinct subtypes of ß cells.

Human pancreatic islets of Langerhans contain five distinct endocrine cell types, each producing a characteristic hormone. The dysfunction or loss of the insulin-producing ß cells causes diabetes mellitus, a disease that harms millions. Until now, ß cells were generally regarded as a single, homogenous cell population. Here we identify four antigenically distinct subtypes of human ß cells, which we refer to as ß1-4, and which are distinguished by differential expression of ST8SIA1 and CD9. These subpopulations are always present in normal adult islets and have diverse gene expression profiles and distinct basal and glucose-stimulated insulin secretion. Importantly, the ß cell subtype distribution is profoundly altered in type 2 diabetes. These data suggest that this antigenically defined ß cell heterogeneity is functionally and likely medically relevant.

Identification of tissue-specific cell death using methylation patterns of circulating DNA.

Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed a method of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic ß-cell DNA was identified in the circulation of patients with recently diagnosed type-1 diabetes and islet-graft recipients; oligodendrocyte DNA was identified in patients with relapsing multiple sclerosis; neuronal/glial DNA was identified in patients after traumatic brain injury or cardiac arrest; and exocrine pancreas DNA was identified in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be determined in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally invasive window for diagnosing and monitoring a broad spectrum of human pathologies as well as providing a better understanding of normal tissue dynamics.

Novel surface markers directed against adult human gallbladder.

Novel cell surface-reactive monoclonal antibodies generated against extrahepatic biliary cells were developed for the isolation and characterization of different cell subsets from normal adult human gallbladder. Eleven antigenically distinct gallbladder subpopulations were isolated by fluorescence-activated cell sorting. They were classified into epithelial, mesenchymal, and pancreatobiliary (PDX1(+)SOX9(+)) subsets based on gene expression profiling. These antigenically distinct human gallbladder cell subsets could potentially also reflect different functional properties in regards to bile physiology, cell renewal and plasticity. Three of the novel monoclonal antibodies differentially labeled archival sections of primary carcinoma of human gallbladder relative to normal tissue. The novel monoclonal antibodies described herein enable the identification and characterization of antigenically diverse cell subsets within adult human gallbladder and are putative tumor biomarkers.

The organoid-initiating cells in mouse pancreas and liver are phenotypically and functionally similar.

Pancreatic Lgr5 expression has been associated with organoid-forming epithelial progenitor populations but the identity of the organoid-initiating epithelial cell subpopulation has remained elusive. Injury causes the emergence of an Lgr5(+) organoid-forming epithelial progenitor population in the adult mouse liver and pancreas. Here, we define the origin of organoid-initiating cells from mouse pancreas and liver prior to Lgr5 activation. This clonogenic population was defined as MIC1-1C3(+)/CD133(+)/CD26(-) in both tissues and the frequency of organoid initiation within this population was approximately 5% in each case. The transcriptomes of these populations overlapped extensively and showed enrichment of epithelial progenitor-associated regulatory genes such as Sox9 and FoxJ1. Surprisingly, pancreatic organoid cells also had the capacity to generate hepatocyte-like cells upon transplantation to Fah(-/-) mice, indicating a differentiation capacity similar to hepatic organoids. Although spontaneous endocrine differentiation of pancreatic progenitors was not observed in culture, adenoviral delivery of fate-specifying factors Pdx1, Neurog3 and MafA induced insulin expression without glucagon or somatostatin. Pancreatic organoid cultures therefore preserve many key attributes of progenitor cells while allowing unlimited expansion, facilitating the study of fate determination.

p21 promotes sustained liver regeneration and hepatocarcinogenesis in chronic cholestatic liver injury.

BACKGROUND AND AIMS: The cyclin-dependent kinase inhibitor p21 has been implicated as a tumour suppressor. Moreover, recent genetic studies suggest that p21 might be a potential therapeutic target to improve regeneration in chronic diseases. The aim of this study was to delineate the role of p21 in chronic liver injury and to specify its role in hepatocarcinogenesis in a mouse model of chronic cholestatic liver injury. METHODS: The degree of liver injury, regeneration and tumour formation was assessed in Mdr2(-/-) mice and compared with Mdr2/ p21(-/-) mice. Moreover, the role of p21 was evaluated in hepatoma cells in vitro and in human hepatocellular carcinoma (HCC). RESULTS: Mdr2(-/-) mice developed HCCs as a consequence of chronic inflammatory liver injury. In contrast, tumour development was profoundly delayed in Mdr2/ p21(-/-) mice. Delayed tumour development was accompanied by markedly impaired liver regeneration in Mdr2/ p21(-/-) mice. Moreover, the regenerative capacity of the Mdr2/ p21(-/-) livers in response to partial hepatectomy declined with age in these mice. Hepatocyte transplantation experiments revealed that impaired liver regeneration was due to intrinsic factors within the cells and changes in the Mdr2/ p21(-/-) microenvironment. In human HCCs, a subset of tumours expressed p21, which was associated with a significant shorter patient survival. CONCLUSIONS: We provide experimental evidence that p21 is required for sustained liver regeneration and tumour development in chronic liver injury indicating that p21 needs to be tightly regulated in order to balance liver regeneration and cancer risk. Moreover, we identify p21 as a negative prognostic marker in human HCC.

In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration.

The Wnt target gene Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5) marks actively dividing stem cells in Wnt-driven, self-renewing tissues such as small intestine and colon, stomach and hair follicles. A three-dimensional culture system allows long-term clonal expansion of single Lgr5(+) stem cells into transplantable organoids (budding cysts) that retain many characteristics of the original epithelial architecture. A crucial component of the culture medium is the Wnt agonist RSPO1, the recently discovered ligand of LGR5. Here we show that Lgr5-lacZ is not expressed in healthy adult liver, however, small Lgr5-LacZ(+) cells appear near bile ducts upon damage, coinciding with robust activation of Wnt signalling. As shown by mouse lineage tracing using a new Lgr5-IRES-creERT2 knock-in allele, damage-induced Lgr5(+) cells generate hepatocytes and bile ducts in vivo. Single Lgr5(+) cells from damaged mouse liver can be clonally expanded as organoids in Rspo1-based culture medium over several months. Such clonal organoids can be induced to differentiate in vitro and to generate functional hepatocytes upon transplantation into Fah(-/-) mice. These findings indicate that previous observations concerning Lgr5(+) stem cells in actively self-renewing tissues can also be extended to damage-induced stem cells in a tissue with a low rate of spontaneous proliferation.

Notch signaling inhibits hepatocellular carcinoma following inactivation of the RB pathway.

Hepatocellular carcinoma (HCC) is the third cancer killer worldwide with >600,000 deaths every year. Although the major risk factors are known, therapeutic options in patients remain limited in part because of our incomplete understanding of the cellular and molecular mechanisms influencing HCC development. Evidence indicates that the retinoblastoma (RB) pathway is functionally inactivated in most cases of HCC by genetic, epigenetic, and/or viral mechanisms. To investigate the functional relevance of this observation, we inactivated the RB pathway in the liver of adult mice by deleting the three members of the Rb (Rb1) gene family: Rb, p107, and p130. Rb family triple knockout mice develop liver tumors with histopathological features and gene expression profiles similar to human HCC. In this mouse model, cancer initiation is associated with the specific expansion of populations of liver stem/progenitor cells, indicating that the RB pathway may prevent HCC development by maintaining the quiescence of adult liver progenitor cells. In addition, we show that during tumor progression, activation of the Notch pathway via E2F transcription factors serves as a negative feedback mechanism to slow HCC growth. The level of Notch activity is also able to predict survival of HCC patients, suggesting novel means to diagnose and treat HCC.