Xinping Tan

Platelet-derived growth factor receptor-α: a novel therapeutic target in human hepatocellular cancer

Hepatocellular cancer (HCC) is a disease of poor prognosis. Identifying novel molecular aberrations might present opportunities to identify new therapeutic targets. Due to the similarities between the processes of development and cancer, we used early developing livers to identify genes that might play a primary role in HCC. Platelet-derived growth factor receptor-α (PDGFRα) was identified from microarray using early developing mouse livers. Expression of PDGFRα and its upstream effectors, PDGF-AA and PDGF-CC, were examined in HCC tissues (n = 43) by Western blot, real-time PCR, and immunohistochemistry. Finally, effect of anti-PDGFRα antibody (mAb 3G3, ImClone Systems, Inc.) was examined on human hepatoma cells. A high expression of PDGFRα was observed during early liver development. HCCs (17 of 21) revealed cytoplasmic PDGFRα and activated PDGFRα (phospho-Tyr754) by immunohistochemistry. Additional HCCs (14 of 22) showed elevated PDGFRα levels when compared with the adjacent normal livers by Western blots. Of these 14 patients, 3 showed increased PDGFRα gene expression, 3 showed elevated PDGF-AA, and 4 had higher PDGF-CC levels in the tumors compared with adjacent livers. Multiple hepatoma cell lines, when treated with mAb 3G3, showed significant decreases in cell proliferation and survival (P < 0.05). In conclusion, ∼70% of HCC tissues had elevated PDGFRα levels due to diverse mechanisms. PDGFRα inhibition in hepatoma cells led to diminution of tumor cell survival and proliferation and thus might be of therapeutic significance. [Mol Cancer Ther 2007;6(7):1932–41]

Wnt'er in liver: Expression of Wnt and frizzled genes in mouse

The Wnt signaling pathway is essential for a wide array of developmental and physiological processes. Wnts are extracellular ligands that bind to frizzled (Fz) receptors at the membrane, canonically inducing β‐catenin nuclear translocation and activation. Although β‐catenin has been shown to be critical in liver biology, the expression of the 19 Wnt and 10 Fz genes in liver remains undetermined. We report comprehensive analysis of Wnt and Fz expression in whole liver as well as individual cell types: freshly isolated and plated hepatocytes, biliary epithelial cells, normal and activated stellate and Kupffer cells, and sinusoidal endothelial cells (SECs). Oligonucleotides for the 19 Wnt, 10 frizzled receptors genes, and secreted Frizzled‐related protein‐1 (sFRP or Fzb) were synthesized based on the available sequences. A total of 11 Wnts and 8 Fz genes and Fzb were expressed in normal liver. Although only 6 Wnt and 5 Fz genes were expressed in freshly isolated hepatocytes, 8 Wnt genes, 7 Fz genes, and Fzb were expressed in plated hepatocytes. Although 12 Wnt and 7 Fz genes were expressed in biliary tree, additional Fz9 and Fzb were only expressed in cultured biliary epithelial cells. The same 14 Wnt and 7 Fz genes were expressed in both activated and normal stellate and Kupffer cells; only Fzb was expressed in their activated state. Also, 11 Wnt, seven Fz, and Fzb genes were expressed in SECs. Conclusion: These data indicate that most Wnt and frizzled genes are expressed in the liver and might be playing important roles in liver pathobiology via canonical and noncanonical pathways. HEPATOLOGY 2007;45:195–204.)

Activation of Wnt/β-catenin pathway during hepatocyte growth factor–induced hepatomegaly in mice†

Hepatocyte growth factor (HGF) and β‐catenin both play a crucial role in stimulating hepatocyte proliferation, but whether these 2 pathways cooperate in inducing hepatocyte proliferation is unclear. We have previously reported that β‐catenin forms a complex with c‐Met (HGF receptor) that undergoes dissociation because of β‐catenin tyrosine phosphorylation on stimulation by HGF. It is also known that delivery of the human HGF gene cloned in a plasmid under a CMV promoter results in hepatomegaly in mice. In addition, recently characterized β‐catenin transgenic mice also showed hepatomegaly. The present study was based on the hypothesis that HGF‐induced hepatomegaly is mediated, at least in part, by activation of the Wnt/β‐catenin pathway. Here we report that delivery of the human HGF gene delivery in mice led to hepatomegaly via β‐catenin activation in the liver in 1‐ and 4‐week studies. The mechanisms of β‐catenin activation in the 1‐week study included loss of c‐Met–β‐catenin association as well as canonical β‐catenin activation, leading to its nuclear translocation. In the 4‐week study, β‐catenin activation was observed via canonical mechanisms, whereas the c‐Met–β‐catenin complex remained unchanged. In both studies there was an associated increase in the E‐cadherin–β‐catenin association at the membrane. In addition, we generated liver‐specific β‐catenin knockout mice, which demonstrated significantly smaller livers. HGF gene delivery failed to induce hepatomegaly in these β‐catenin conditionally null mice. In conclusion, β‐catenin‐ and HGF‐mediated signaling pathways cooperate in hepatocyte proliferation, which may be crucial in liver development, regeneration following partial hepatectomy, and pathogenesis of hepatocellular carcinoma. (HEPATOLOGY 2006;44:992–1002.)

Conditional β-catenin loss in mice promotes chemical hepatocarcinogenesis: Role of oxidative stress and platelet-derived growth factor receptor α/phosphoinositide 3-kinase signaling†

Activation of β‐catenin, the central effector of the canonical Wnt pathway and a recognized oncogene, has been implicated in hepatocellular carcinoma. We examined N‐nitrosodiethylamine (DEN)‐induced tumorigenesis in hepatic β‐catenin conditional knockout mice (β‐cat KO). Male β‐cat KO and age‐ and sex‐matched littermate controls were given a single intraperitoneal DEN injection and followed for 6‐12 months for hepatic tumors. Hepatic tumors were characterized for histology, proliferation, apoptosis, oxidative stress, and specific proteins by way of western blot, immunohistochemistry, and coprecipitation studies. For in vivo tumor intervention studies, specific inhibitors were administered intraperitoneally or through drinking water. Intriguingly, β‐cat KO mice showed a paradoxical increase in susceptibility to DEN‐induced tumorigenesis. This accelerated tumorigenesis is due to increased injury and inflammation, unrestricted oxidative stress, fibrosis, and compensatory increase in hepatocyte proliferation secondary to platelet‐derived growth factor receptor α (PDGFRα)/phosphoinositide 3‐kinase (PIK3CA)/Akt activation and c‐Myc overexpression. In vitro suppression of β‐catenin expression in hepatoma cells led to enhanced PDGFRα expression, which was abrogated in the presence of nuclear factor κB (NF‐κB) inhibitor. Daily treatment of 6‐month‐old DEN‐exposed β‐cat KO with PDGFRα inhibitor dramatically reduced tumor numbers and size. Inclusion of N‐acetyl‐L‐cysteine, a known antioxidant and NF‐κB inhibitor, in the drinking water led to complete abolition of tumorigenesis in DEN‐exposed β‐cat KO. Conclusion: Loss of β‐catenin impairs the livers ability to counteract DEN‐induced oxidative stress and enhances tumorigenesis through PDGFRα/PIK3CA/Akt signaling. Blockade of PDGFRα or oxidative stress dramatically affects β‐catenin–deficient tumorigenesis. Also, hepatoma cells use PDGFRα/PIK3CA signaling as an escape mechanism following β‐catenin suppression, and their sequential suppression profoundly impedes tumor proliferation. HEPATOLOGY 2010

Fibroblast growth factor enriches the embryonic liver cultures for hepatic progenitors.

Fibroblast growth factors (FGFs) play an important role in hepatic induction during development. The aim of our study was to investigate the effect of exogenous FGFs on ex vivo liver development. We begin our analysis by examining FGF signaling during early mouse liver development. Phospho-FGF receptor (Tyr653/654) was detected in embryonic day 10 (E10) to E12 livers only. Next, E10 livers were cultured in the presence of FGF1, FGF4, or FGF8 for 72 hours and examined for histology, proliferation, apoptosis, and differentiation. FGFs especially FGF8 promoted sheet-like architecture, cell proliferation, and survival as compared to the control. All FGFs induced a striking increase in the number of c-kit and alpha-fetoprotein-positive progenitors, without altering albumin staining. However these progenitors were CK-19-positive (biliary and bipotential progenitor marker) only in the presence of FGF1 or FGF4 and not FGF8. FGFs also induced beta-catenin, a stem cell renewal factor in these cultures. In conclusion, the presence of activated FGFR indicates a physiological role of FGF during early liver development. FGF1 and FGF4 enrich the embryonic liver cultures for bipotential hepatic progenitors. FGF8 promotes such enrichment and induces a one-step differentiation toward a unipotential hepatocyte progenitor. Thus, FGFs might be useful for enrichment and propagation of developmental hepatic progenitors.

β-Catenin and Met Deregulation in Childhood Hepatoblastomas

ABSTRACTActivation of the Wnt/β-catenin and hepatocyte growth factor/Met signaling has been implicated in various tumors. Owing to the cross-talk between these pathways and aberrant redistribution of β-catenin in hepatoblastomas, we examined their status in this tumor. This study examined changes in β-catenin and Met in paired pretreatment and post-treatment hepatoblastoma tissues in relation to their effects on proliferation and target genes such as c-myc and cyclin-D1. In this study we compared proliferation indices, β-catenin staining and its known molecular targets, c-myc and cyclin-D1, and Met, a tyrosine kinase receptor for hepatocyte growth factor in pretreatment and post-treatment specimens. Pretreatment and post-treatment sections from 13 children, ages 11 weeks to 9 years, were analyzed for these markers by immunohistochemistry. All tumors (13 of 13) displayed increased proliferation and β-catenin (cytoplasmic and nuclear) staining in pretreatment biopsies that remained relatively unaffected after treatment. Aberrant Met staining (cytoplasmic) was observed in all pretreatment samples that decreased considerably after treatment in 11 of 13 patients. A significant subset of these tumors showed increased c-myc and cyclin-D1 staining in pretreatment biopsies that decreased after chemotherapy in most cases. β-Catenin redistribution in tumor cells corresponds to proliferation in hepatoblastomas. However, β-catenin nuclear localization remains unaffected in viable hepatoblastoma tissue after chemotherapy. In contrast, Met undergoes a prominent decrease after treatment and thus might be important in pathogenesis of hepatoblastoma.

β-Catenin Regulates Vitamin C Biosynthesis and Cell Survival in Murine Liver

Because the Wnt/β-catenin pathway plays multiple roles in liver pathobiology, it is critical to identify gene targets that mediate such diverse effects. Here we report a novel role of β-catenin in controlling ascorbic acid biosynthesis in murine liver through regulation of expression of regucalcin or senescence marker protein 30 and l-gulonolactone oxidase. Reverse transcription-PCR, Western blotting, and immunohistochemistry demonstrate decreased regucalcin expression in β-catenin-null livers and greater expression in β-catenin overexpressing transgenic livers, HepG2 hepatoma cells (contain constitutively active β-catenin), regenerating livers, and in hepatocellular cancer tissues that exhibit β-catenin activation. Interestingly, coprecipitation and immunofluorescence studies also demonstrate an association of β-catenin and regucalcin. Luciferase reporter and chromatin immunoprecipitation assays verified a functional TCF-4-binding site located between −163 and −157 (CTTTGCA) on the regucalcin promoter to be critical for regulation by β-catenin. Significantly lower serum ascorbate levels were observed in β-catenin knock-out mice secondary to decreased expression of regucalcin and also of l-gulonolactone oxidase, the penultimate and last (also rate-limiting) steps in the synthesis of ascorbic acid, respectively. These mice also show enhanced basal hepatocyte apoptosis. To test if ascorbate deficiency secondary to β-catenin loss and regucalcin decrease was contributing to apoptosis, β-catenin-null hepatocytes or regucalcin small interfering RNA-transfected HepG2 cells were cultured, which exhibited significant apoptosis that was alleviated by the addition of ascorbic acid. Thus, through regucalcin and l-gulonolactone oxidase expression, β-catenin regulates vitamin C biosynthesis in murine liver, which in turn may be one of the mechanisms contributing to the role of β-catenin in cell survival.

Wnt in liver development: Role in biliary cell survival and proliferation

Wnt/13-catenni pathway plays a sigmficant role during development and carcinogenesis. We have earlier detected presence of Wnt-1 as early as in embryonic day 10 livers (El0). We have also reported effect of 13-catenin inhibition on liver growth and lineage specification during liver development. In the present study we investigate direct effect of Wnt on liver development utilizing embryonic liver cultures and Wnt conditioned media. (Wnt-3a). El0 livers were isolated and cultured in an organ culture system. Livers were cultured in conditioned medium from control L929 cells or Win-conditioned media (L cells transfected with Wnt-3a) alone or in presence of Hepatocyte growth factor (HGF). At least 3 livers were cultured for each growth condition. Embryonic liver cultures grown in DMEM supplemented with 10% fetal calf serum (FCS) were utilized as positive controls. After 72 hours all organ cultures were harvested, llxed and processed and finally analyzed for histology, proliferation, apoptosis and lineage. Negative conditioned media grown embryonic liver cultures demonstrated loss of architecture, enhanced apoptosia and deficit proliferation. There were almost no viable cells observed in these cultures. In the presence of Wnt-conditioned media, embryonic liver cultures showed several islands of survived cells and demonstrated a ductular morphology. These cells displayed proliferation as seen by PCNA and Ki-67 posinvity. Also this growth condition displayed an overall decrease in TUNEL positivity especially in the ductular structures. These cells were strongly positive for CK-19 and negative for cr ein and c-kit. Some CK-19 positive cells were also positive for albumin. Embryonic livers cultured in presence of Wnt and HGF show phenotype resembling the positive control (cultures grown in 10% FCS). These cultures exhibited normal cellular architecture with active proliferation and decreased apoptosis and displaying stem cells, hepatocytes (immature & mature) and primitive bile ducts. We conclude that Wnt plays an important role in bihary commitment of the bipotential stem cells by providing them a survival advantage. It also induces transdifferentiation of mature hepatocytes into biliary cells as shown by the presence of some hepato-biliary intermediates. HGF and Wnt seem to be sufficient to maintain a normal embryonic liver culture growth and differentiation supporting stem cells, hepatocytes & bile ducts. Our results suggest an existence of a precise balance of these growth factors for normal liver development

Fibroblast growth factors promote hepatocyte stem cell renewal in embryonic liver cultures

Fibroblast growth factors (FGFs) have been shown to play a definite role in hepatic induction. Stage and tissue specific expression of FGF 1,4 and 8 are necessary for initiation of hepatic bud from the foregut endoderm FGF is also routinely used to maintain and propagate embryonic stem ceils in culture. The aim of this study was to investigate the effect of FGF 1, 4 and 8 on liver development utilizing an in vitro embryonic liver culture system and recotnbinant FGF proteins. Livers were harvested from day 10 embryos (El0) and cultured as an organ in DMEM supplemented with 10% FCS (control) with or without additional FGF 1 or FGF 4 or FGF 8. After 72 hours, the cultured livers were fixed and processed and 4/zm sections were utilized for histologic and immunohistochemical evaluation. Proliferation (PCNA and Ki-67) and apoptosis (TUNEL) assay was also performed. The major difference in the histology of the FGF 1/4/8 treated cultures was in the arrangement of cells in sheet like architecture only as compared to the controls that displayed cells in both ductular and sheet like arrangements. There was no significant change in number of apoptotic cells in presence of FGF 1/4 but there was a decrease in number of apoptotic nuclei in FGF8 treated cultures. Almost all cells in all FGF cultures exhibited PCNA positivity and a sigmficant number of cells were Ki-67 (S-phase) positive. There was a striking difference in the number of c-kn positive stem cells in the presence of any of the three FGFs used as compared to the controls. These stem cells were also positive for a-fetoprotein and albumin and very few were positive for CK-19. There was an overall decrease in number of CK-19 positive cells in presence of the FGFs. To conclude, FGF 1, 4 and 8 are excellent growth factors to maintain and propagate liver stem cells in development. These factors support prohferation and affect apoptosis and thus promote liver stem cell renewal. Also, lineage analysis shows a preponderance of unipotential stem cells (more hepatocytic than biliary) in the FGF treated cultures that suggest existence of selective hepatocyte stem ceils, h appears that FGFs promotes self-renewal of such a unipotential stem cell population in the liver.