C. Bart Rountree

Epigenetic Regulation of Cancer Stem Cell Marker CD133 by Transforming Growth Factor-β

Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide. CD133, a transmembrane glycoprotein, is an important cell surface marker for both stem cells and cancer stem cells in various tissues including liver. CD133 expression has been recently linked to poor prognosis in HCC patients. CD133+ liver cancer cells are characterized by resistance to chemotherapy, self‐renewal, multilineage potential, increased colony formation, and in vivo cancer initiation at limited dilution. Recent studies demonstrate that CD133 expression is regulated by DNA methylation. In this study, we explored the role of transforming growth factor β (TGFβ), a multifunctional cytokine that plays a critical role in chronic liver injury, in the regulation of CD133 expression. TGFβ1 is capable of up‐regulating CD133 expression specifically within the Huh7 HCC cell line in a time‐ and dose‐dependent manner. Most important, TGFβ1‐induced CD133+ Huh7 cells demonstrate increased tumor initiation in vivo. Forced expression of inhibitory Smads, including Smad6 and Smad7, attenuated TGFβ1‐induced CD133 expression. Within CD133− Huh7 cells, TGFβ1 stimulation inhibited the expression of DNA methyltransferases (DNMT) 1 and DNMT3β, which are critical in the maintenance of regional DNA methylation, and global DNMT activity in CD133− Huh7 cells was inhibited by TGFβ1. DNMT3β inhibition by TGFβ1 was partially rescued with overexpression of inhibitory Smads. Lastly, TGFβ1 treatment led to significant demethylation in CD133 promoter‐1 in CD133− Huh7 cells. Conclusion: TGFβ1 is able to regulate CD133 expression through inhibition of DNMT1 and DNMT3β expression and subsequent demethylation of promoter‐1. TGFβ1‐induced CD133+ Huh7 cells are tumorigenic. The mechanism by which TGFβ induces CD133 expression is partially dependent on the Smads pathway. HEPATOLOGY 2010

c‐Met represents a potential therapeutic target for personalized treatment in hepatocellular carcinoma

c‐Met, a high‐affinity receptor for hepatocyte growth factor (HGF), plays a critical role in cancer growth, invasion, and metastasis. Hepatocellular carcinoma (HCC) patients with an active HGF/c‐Met signaling pathway have a significantly worse prognosis. Although targeting the HGF/c‐Met pathway has been proposed for the treatment of multiple cancers, the effect of c‐Met inhibition in HCC remains unclear. The human HCC cell lines Huh7, Hep3B, MHCC97‐L, and MHCC97‐H were used in this study to investigate the effect of c‐Met inhibition using the small molecule selective c‐Met tyrosine kinase inhibitor PHA665752. MHCC97‐L and MHCC97‐H cells demonstrate a mesenchymal phenotype with decreased expression of E‐cadherin and increased expression of c‐Met, fibronectin, and Zeb2 compared with Huh7 and Hep3B cells, which have an epithelial phenotype. PHA665752 treatment blocked phosphorylation of c‐Met and downstream phosphoinositide 3‐kinase/Akt and mitogen‐activated protein kinase/Erk pathways, inhibited cell proliferation, and induced apoptosis in c‐Met–positive MHCC97‐L and MHCC97‐H cells. In xenograft models, administration of PHA665752 significantly inhibited c‐Met–positive MHCC97‐L and MHCC97‐H tumor growth, and PHA665752‐treated tumors demonstrated marked reduction of both c‐Met phosphorylation and cell proliferation. c‐Met–negative Huh7 and Hep3B cells were not affected by c‐Met inhibitor treatment in vitro or in vivo. In addition, c‐Met–positive MHCC97‐L and MHCC97‐H cells demonstrated cancer stem cell–like characteristics, such as resistance to chemotherapy, tumor sphere formation, and increased expression of CD44 and ABCG2, and PHA665752 treatment suppressed tumor sphere formation and inhibited CD44 expression. Conclusion: c‐Met represents a potential target of personalized treatment for HCC with an active HGF/c‐Met pathway. (HEPATOLOGY 2011;)

A CD133‐Expressing Murine Liver Oval Cell Population with Bilineage Potential

Although oval cells are postulated to be adult liver stem cells, a well‐defined phenotype of a bipotent liver stem cell remains elusive. The heterogeneity of cells within the oval cell fraction has hindered lineage potential studies. Our goal was to identify an enriched population of bipotent oval cells using a combination of flow cytometry and single cell gene expression in conjunction with lineage‐specific liver injury models. Expression of cell surface markers on nonparenchymal, nonhematopoietic (CD45−) cells were characterized. Cell populations were isolated by flow cytometry for gene expression studies. 3,5‐Diethoxycarbonyl‐1,4‐dihydrocollidine toxic injury induced cell cycling and expansion specifically in the subpopulation of oval cells in the periportal zone that express CD133. CD133+CD45− cells expressed hepatoblast and stem cell‐associated genes, and single cells coexpressed both hepatocyte and cholangiocyte‐associated genes, indicating bilineage potential. CD133+CD45− cells proliferated in response to liver injury. Following toxic hepatocyte damage, CD133+CD45− cells demonstrated upregulated expression of the hepatocyte gene Albumin. In contrast, toxic cholangiocyte injury resulted in upregulation of the cholangiocyte gene Ck19. After 21–28 days in culture, CD133+CD45− cells continued to generate cells of both hepatocyte and cholangiocyte lineages. Thus, CD133 expression identifies a population of oval cells in adult murine liver with the gene expression profile and function of primitive, bipotent liver stem cells. In response to lineage‐specific injury, these cells demonstrate a lineage‐appropriate genetic response. Disclosure of potential conflicts of interest is found at the end of this article.

Epithelial-to-mesenchymal transition of murine liver tumor cells promotes invasion†‡

Epithelial‐to‐mesenchymal transition (EMT) is predicted to play a critical role in metastatic disease in hepatocellular carcinoma. In this study, we used a novel murine model of EMT to elucidate a mechanism of tumor progression and metastasis. A total of 2 × 106 liver cells isolated from Ptenloxp/loxp/Alb‐Cre+ mice, expanded from a single CD133+CD45− cell clone, passage 0 (P0), were sequentially transplanted to obtain two passages of tumor cells, P1 and P2. Cells were analyzed for gene expression using microarray and real‐time polymerase chain reaction. Functional analysis included cell proliferation, migration, and invasion in vitro and orthotopic tumor metastasis assays in vivo. Although P0, P1, and P2 each formed tumors consistent with mixed liver epithelium, within the P2 cells, two distinct cell types were clearly visible: cells with epithelial morphology similar to P0 cells and cells with fibroblastoid morphology. These P2 mesenchymal cells demonstrated increased locomotion on wound healing; increased cell invasion on Matrigel basement membrane; increased EMT‐associated gene expression of Snail1, Zeb1, and Zeb2; and down‐regulated E‐cadherin. P2 mesenchymal cells demonstrated significantly faster tumor growth in vivo compared with P2 epithelial counterparts, with invasion of intestine, pancreas, spleen, and lymph nodes. Furthermore, P2 mesenchymal cells secreted high levels of hepatocyte growth factor (HGF), which we propose acts in a paracrine fashion to drive epithelial cells to undergo EMT. In addition, a second murine liver cancer stem cell line with methionine adenosyltransferase 1a deficiency acquired EMT after sequential transplantations, indicating that EMT was not restricted to Pten‐deleted tumors. Conclusion: EMT is associated with a high rate of liver tumor proliferation, invasion, and metastasis in vivo, which is driven by HGF secreted from mesenchymal tumor cells in a feed‐forward mechanism. (HEPATOLOGY 2010)

Stem cells in liver diseases and cancer: Recent advances on the path to new therapies

Stem cells have potential for therapy of liver diseases, but may also be involved in the formation of liver cancer. Recently, the American Association for the Study of Liver Diseases Henry M. and Lillian Stratton Basic Research Single Topic Conference “Stem Cells in Liver Diseases and Cancer: Discovery and Promise” brought together a diverse group of investigators to define the status of research on stem cells and cancer stem cells in the liver and identify problems and solutions on the path to clinical translation. This report summarizes the outcomes of the conference and provides an update on recent research advances. Progress in liver stem cell research includes isolation of primary liver progenitor cells (LPCs), directed hepatocyte differentiation of primary LPCs and pluripotent stem cells, findings of transdifferentiation, disease‐specific considerations for establishing a therapeutically effective cell mass, and disease modeling in cell culture. Tumor‐initiating stem‐like cells (TISCs) that emerge during chronic liver injury share the expression of signaling pathways, including those organized around transforming growth factor beta and β‐catenin, and surface markers with normal LPCs. Recent investigations of the role of TISCs in hepatocellular carcinoma have provided insight into the transcriptional and post‐transcriptional regulation of hepatocarcinogenesis. Targeted chemotherapies for TISC are in development as a means to overcome cellular resistance and mechanisms driving disease progression in liver cancer. (HEPATOLOGY 2012;55:298–306)

CD133+ liver cancer stem cells from methionine adenosyl transferase 1A–deficient mice demonstrate resistance to transforming growth factor (TGF)‐β–induced apoptosis

Methionine adenosyltransferase (MAT) is an essential enzyme required for S‐adenosylmethionine biosynthesis. Hepatic MAT activity falls during chronic liver injury, and mice lacking Mat1a develop spontaneous hepatocellular carcinoma by 18 months. We have previously demonstrated that CD133+CD45− oval cells isolated from 16‐month‐old Mat1a−/− mice represent a liver cancer stem cell population. The transforming growth factor β (TGF‐β) pathway constitutes a central signaling network in proliferation, apoptosis, and tumorigenesis. In this study, we tested the response of tumorigenic liver stem cells to TGF‐β. CD133+CD45− oval cells were isolated from premalignant 16‐month‐old Mat1a−/− mice by flow cytometry and expanded as five clone lines derived from a single cell. All clone lines demonstrated expression of both hepatocyte and cholangiocyte markers and maintained a small population (0.5% to 2%) of CD133+ cells in vitro, and three of five clone lines produced tumors. Although TGF‐β1 inhibited cell growth equally in CD133− and CD133+ cells from each clone line, the CD133+ population demonstrated significant resistance to TGF‐β–induced apoptosis compared with CD133− cells. Furthermore, CD133+ cells demonstrated a substantial increase in mitogen‐activated protein kinase (MAPK) pathway activation, as demonstrated by phosphorylated extracellular signal‐regulated kinase levels before and after TGF‐β stimulation. MAPK inhibition using mitogen‐activated protein kinase kinase 1 (MEK1) inhibitor PD98059 led to a significant increase in TGF‐β–induced apoptosis in CD133+ cells. Conversely, a constitutively active form of MEK1 blocked the apoptotic effects of TGF‐β in CD133− cells. Conclusion: CD133+ liver cancer stem cells exhibit relative resistance to TGF‐β–induced apoptosis. One mechanism of resistance to TGF‐β–induced apoptosis in CD133+ cancer stem cells is an activated mitogen‐activated protein kinase/extracellular signal‐regulated kinase pathway. (HEPATOLOGY 2009.)

Snail1 induces epithelial-to-mesenchymal transition and tumor initiating stem cell characteristics

BackgroundTumor initiating stem-like cells (TISCs) are a subset of neoplastic cells that possess distinct survival mechanisms and self-renewal characteristics crucial for tumor maintenance and propagation. The induction of epithelial-mesenchymal-transition (EMT) by TGFβ has been recently linked to the acquisition of TISC characteristics in breast cancer. In HCC, a TISC and EMT phenotype correlates with a worse prognosis. In this work, our aim is to elucidate the underlying mechanism by which cells acquire tumor initiating characteristics after EMT.MethodsGene and protein expression assays and Nanog-promoter luciferase reporter were utilized in epithelial and mesenchymal phenotype liver cancer cell lines. EMT was analyzed with migration/invasion assays. TISC characteristics were analyzed with tumor-sphere self-renewal and chemotherapy resistance assays. In vivo tumor assay was performed to investigate the role of Snail1 in tumor initiation.ConclusionTGFβ induced EMT in epithelial cells through the up-regulation of Snail1 in Smad-dependent signaling. Mesenchymal liver cancer post-EMT demonstrates TISC characteristics such as tumor-sphere formation but are not resistant to cytotoxic therapy. The inhibition of Snail1 in mesenchymal cells results in decreased Nanog promoter luciferase activity and loss of self-renewal characteristics in vitro. These changes confirm the direct role of Snail1 in some TISC traits. In vivo, the down-regulation of Snail1 reduced tumor growth but was not sufficient to eliminate tumor initiation. In summary, TGFβ induces EMT and TISC characteristics through Snail1 and Nanog up-regulation. In mesenchymal cells post-EMT, Snail1 directly regulates Nanog expression, and loss of Snail1 regulates tumor growth without affecting tumor initiation.

Expansion of CD133‐Expressing Liver Cancer Stem Cells in Liver‐Specific Phosphatase and Tensin Homolog Deleted on Chromosome 10‐Deleted Mice

PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a lipid phosphatase that regulates mitogenic signaling pathways, and deficiency of PTEN results in cell proliferation, survival, and malignancy. Murine liver‐specific Pten deletion models develop liver malignancy by 12 months of age. Using this model, we describe a population of CD133+ liver cancer stem cells isolated during the chronic injury phase of disease progression and before primary carcinoma formation. We performed immunohistochemistry and flow cytometry isolation using livers from 3‐ and 6‐month‐old PtenloxP/loxP; Alb−Cre+ mice (mutants) and controls. CD133+CD45− nonparenchymal (NP) cells were analyzed for gene expression profile and protein levels. Single CD133+CD45− oval cells were isolated for clonal expansion and tumor analysis. Cultured and freshly isolated liver CD133+CD45− and CD133−CD45− NP cells were injected into immune‐deficient and immune‐competent mice. In mutant mice, the NP fraction increased in CD133+CD45− cells in 3‐ and 6‐month‐old Pten‐deleted animals compared with controls. Clone lines expanded from single CD133+CD45− cells demonstrated consistent liver progenitor cell phenotype, with bilineage gene expression of hepatocyte and cholangiocyte markers. CD133+ cells from expanded clone lines formed robust tumors in immune‐deficient and immune‐competent mice. Furthermore, freshly isolated CD133+CD45− NP liver cells from 6‐month‐old mutants formed tumors in vivo, and CD133−CD45− NP cells did not. Consistent with a cancer stem cell phenotype, CD133+ cells demonstrate resistance to chemotherapy agents compared with CD133− cells. CD133+CD45− nonparenchymal cells from chronic injury PtenloxP/loxP; Alb−Cre+ mice represent a bipotent liver progenitor cell population with cancer stem cell phenotype. STEM CELLS 2009;27:290–299

Expansion of hepatic tumor progenitor cells in Pten-null mice requires liver injury and is reversed by loss of AKT2.

BACKGROUND & AIMS The tumor suppressor PTEN inhibits AKT2 signaling; both are aberrantly expressed in liver tumors. We investigated how PTEN and AKT2 regulate liver carcinogenesis. Loss of PTEN leads to spontaneous development of liver tumors from progenitor cells. We investigated how the loss of PTEN activates liver progenitor cells and induces tumorigenesis. METHODS We studied mice with liver-specific disruptions in Pten and the combination of Pten and Akt2 to investigate mechanisms of liver carcinogenesis. RESULTS PTEN loss leads to hepatic injury and establishes selective pressure for tumor-initiating cells (TICs), which proliferate to form mixed-lineage tumors. The Pten-null mice had increasing levels of hepatic injury before proliferation of hepatic progenitors. Attenuation of hepatic injury by deletion of Akt2 reduced progenitor cell proliferation and delayed tumor development. In Pten/Akt2-null mice given 3,5-diethoxycarbonyl-1,4 dihydrocollidine (DDC), we found that the primary effect of AKT2 loss was attenuation of hepatic injury and not inhibition of progenitor-cell proliferation in response to injury. CONCLUSIONS Liver carcinogenesis in Pten-null mice requires not only the transformation of TICs but selection pressure from hepatic injury and cell death, which activates TICs. Further research is required to elucidate the mechanism for hepatic injury and its relationship with TIC activation.

Clinical application for the preservation of phospho- proteins through in-situ tissue stabilization

BackgroundProtein biomarkers will play a pivotal role in the future of personalized medicine for both diagnosis and treatment decision-making. While the results of several pre-clinical and small-scale clinical studies have demonstrated the value of protein biomarkers, there have been significant challenges to translating these findings into routine clinical care. Challenges to the use of protein biomarkers include inter-sample variability introduced by differences in post-collection handling and ex vivo degradation of proteins and protein modifications.ResultsIn this report, we re-create laboratory and clinical scenarios for sample collection and test the utility of a new tissue stabilization technique in preserving proteins and protein modifications. In the laboratory setting, tissue stabilization with the Denator Stabilizor T1 resulted in a significantly higher yield of phospho-protein when compared to standard snap freeze preservation. Furthermore, in a clinical scenario, tissue stabilization at collection resulted in a higher yield of total phospho-protein, total phospho-tyrosine, pErkT202/Y204 and pAktS473 when compared to standard methods. Tissue stabilization did not have a significant effect on other post-translational modifications such as acetylation and glycosylation, which are more stable ex-vivo. Tissue stabilization did decrease total RNA quantity and quality.ConclusionStabilization at the time of collection offers the potential to better preserve tissue protein and protein modification levels, as well as reduce the variability related to tissue processing delays that are often associated with clinical samples.