CANCER BIOLOGY: Expression of transforming growth factor alpha/epidermal growth factor receptor, hepatocyte growth factor/c-met and acidic fibroblast growth factor/fibroblast growth factor receptors during hepatocarcinogenesis

It is widely believed that abnormal production of poly-peptidegrowth factors, together with other molecular alterations, playan important role in neoplastic development Transforming growthfactor alpha (TGF     

Forced expression of hepatocyte-specific fibroblast growth factor 21 delays initiation of chemically induced hepatocarcinogenesis

Inappropriate fibroblast growth factor (FGF) signaling is involved in most tissue-specific pathologies including cancer. Previously we showed that inappropriate expression and chronic activity of FGF receptor (FGFR) 1 in hepatocytes accelerated diethylnitrosamine (DEN)-initiated hepatocarcinogenesis. Here we showed that although widely expressed FGF1 and FGF2 are frequently upregulated in hepatocellular carcinoma (HCC), germline deletion of both FGF1 and FGF2 had no effect on DEN-initiated hepatocarcinogenesis. Thus overexpression of FGF1 or FGF2 may be a consequence rather than contributor to hepatoma progression. FGF21 is the first of 22 homologues whose expression has been reported to be preferentially in the liver. We showed that similar to FGF1 and FGF2, FGF21 mRNA was upregulated in neoplastic and regenerating liver after partial hepatectomy (PH) and CCl4 administration. In situ hybridization analysis confirmed that in contrast to FGF1 and FGF2, expression of FGF21 mRNA was limited to hepatocytes. Forced overexpression of FGF21 in hepatocytes by gene targeting had no apparent impact on normal liver development and compensatory response to injury. Surprisingly, overexpression of FGF21 delayed the appearance of DEN-induced liver tumors. At 8 and 10 mo, only 10% and 30% of transgenic mice, respectively, developed adenomas compared to 50% (all adenomas) and 80% (60% adenoma/20% HCC) in the wild-type (WT) mice. However, the incidence and burden of HCC at 10 mo and later was equal in the FGF21 transgenic and WT mice. We propose that FGF21 may delay development of adenomas through activation of resident hepatocyte FGFR4 at early times, but counteracts the delay by acceleration of progression to HCC through interaction with ectopic FGFR1 once it appears in hepatoma cells. This indicates a dual function of FGF21 that may reflect changes in FGFR isotype during progression of differentiated hepatoma cells.     

Expression of tumor-associated aldehyde dehydrogenase during rat hepatocarcinogenesis using the resistant hepatocyte model

The resistant hepatocyte model was used to study expressionof tumor-associated aldehyde dehydrogenase (ALDH) activity duringthe course of rat hepatocarcinogenesis. The hepatic ALDH phenotypewas determined at intervals over 280 days by histochemical analysis,total ALDH activity assays and gel electrophoresis, using propionaldehydeand NAD (P/NAD) to characterize normal liver ALDH activity orbenzaldehyde and NADP (B/NADP) to determine tumor-associatedALDH activity. By total activity assays and gel electrophoresis,no significant changes in ALDH activity occurred until day 70.However, histochemical analysis clearly demonstrated changesin ALDH activity early in neoplastic development. Intense focalhepatocyte staining with P/NAD and/or B/NADP was first detectableat day 28. The number of P/NAD-positive foci increased untilday 35 then declined until day 70. The number of B/NADP-positivefoci also increased until day 35, but then remained relativelyconstant for the remainder of the experiment. GGT activity ofserial sections indicated that early ALDH-positive lesions representa small subpopulation (9%) of all GGT-positive foci. However,by day 168 a significant portion (80%) of persistent GGT-positiveneoplastic nodules were also B%NADP-positive histochemically.In addition, virtually all hepatocellular carcinomas (96%) generatedby this protocol possessed significantly elevated levels oftumor-associated ALDH by histochemical analysis, total ALDHactivity and gel electrophoresis. These results indicate thatearly appearing ALDH-positive lesions may define one early subpopulationof all initiated cells that have a high probability of progressingto the ultimate neoplasm.     

Resident hepatocyte fibroblast growth factor receptor 4 limits hepatocarcinogenesis

Fibroblast growth factor (FGF) family signaling mediates cell‐to‐cell communication in development and organ homeostasis in adults. Of the FGF receptor (FGFR) isotypes, FGFR4 is the sole resident isotype present in mature parenchymal hepatocytes. FGFR1 that is normally associated with activated nonparenchymal cells appears ectopically in hepatoma cells. Ectopic expression and chronic activity of FGFR1 in hepatocytes accelerates diethylnitrosamine (DEN)‐initiated hepatocarcinogenesis by driving unrestrained cell proliferation and tumor angiogenesis. Hepatocyte FGFR4 mediates liver's role in systemic cholesterol/bile acid and lipid metabolism and affects proper hepatolobular restoration after damage without effect on cell proliferation. Here we ask whether FGFR4 plays a role in progression of hepatocellular carcinoma (HCC). We report that although spontaneous HCC was not detected in livers of FGFR4‐deficient mice, the ablation of FGFR4 accelerated DEN‐induced hepatocarcinogenesis. In contrast to FGFR1 that induced a strong mitogenic response and depressed rate of cell death in hepatoma cells, FGFR4 failed to induce a mitogenic response and increased the rate of cell death. FGFR1 but not FGFR4 induced cyclin D1 and repressed p27 expression. Analysis of activation of Erk, JNK, and PI3K‐related AKT signaling pathways indicated that in contrast to FGFR1, FGFR4 failed to sustain Erk activation and did not activate AKT. These differences may underlie the opposing effects of FGFR1 and FGFR4. These results suggest that in contrast to ectopic FGFR1 that is a strong promoter of hepatoma, resident FGFR4 that mediates differentiated hepatocyte metabolic functions also serves to suppress hepatoma progression. © 2008 Wiley‐Liss, Inc.     

Loss of hepatocyte growth factor/c-Met signaling pathway accelerates early stages of N-nitrosodiethylamine induced hepatocarcinogenesis

Hepatocyte growth factor (HGF) has been reported to have both positive and negative effects on carcinogenesis. Here, we show that the loss of c-Met signaling in hepatocytes enhanced rather than suppressed the early stages of chemical hepatocarcinogenesis. c-Met conditional knockout mice (c-metfl/fl, AlbCre+/-; MetLivKO) treated with N-nitrosodiethylamine developed significantly more and bigger tumors and with a shorter latency compared with control (w/w, AlbCre+/-; Cre-Ctrl) mice. Accelerated tumor development was associated with increased rate of cell proliferation and prolonged activation of epidermal growth factor receptor (EGFR) signaling. MetLivKO livers treated with N-nitrosodiethylamine also displayed elevated lipid peroxidation, decreased ratio of reduced glutathione to oxidized glutathione, and up-regulation of superoxide dismutase 1 and heat shock protein 70, all consistent with increased oxidative stress. Likewise, gene expression profiling done at 3 and 5 months after N-nitrosodiethylamine treatment revealed up-regulation of genes associated with cell proliferation and stress responses in c-Met mutant livers. The negative effects of c-Met deficiency were reversed by chronic p.o. administration of antioxidant N-acetyl-L-cysteine. N-acetyl-L-cysteine blocked the EGFR activation and reduced the N-nitrosodiethylamine-initiated hepatocarcinogenesis to the levels of Cre-Ctrl mice. These results argue that intact HGF/c-Met signaling is essential for maintaining normal redox homeostasis in the liver and has tumor suppressor effect(s) during the early stages of N-nitrosodiethylamine-induced hepatocarcinogenesis.     

Expression of a hepatocyte membrane antigen during hepatocarcinogenesis and in the developing liver of the rat

Changes in the expression of a cell membrane antigen during hepatocarcinogenesis and in the developing liver were analyzed by HAM.4, a monoclonal antibody (MAb) against a membrane glycoprotein of normal rat hepatocyte. Of the precancerous lesions observed during hepatocarcinogenesis induced by diethylnitrosamine, 2-acetylaminofluorene and partial hepatectomy, early neoplastic foci were uniformly stained by HAM.4. In contrast, some cells in the neoplastic nodules at the late stage did not express HAM.4 antigen on the cell surface. Of the cancer tissues, well-differentiated hepatocellular carcinomas were stained by HAM.4 whereas poorly differentiated carcinomas did not bind HAM.4 In developing rat liver, HAM.4 antigen was first expressed on fetal hepatocytes at the 18th day of gestation. It gradually increased until 4 weeks after birth when the intensity of the stain was almost the same as in adult rat liver. These results suggest that the expression of a membrane antigen defined by HAM.4 is closely associated with the differentiation of bile canalicular face and that HAM.4 might be useful in characterizing differentiation of cells during malignant transformation of hepatocytes.     

Regulation of expression of the hepatocyte growth factor scatter factor receptor, c-met, by cytokines

The c-met proto-oncogene product is a 190 kDa heterodimeric receptor tyrosine kinase activated by the binding of its li,gand, hepatocyte growth factor/scatter factor (HGF/SF), a cytokine known to stimulate cell growth, motility and morphogenesis. Altered expression of c-met receptor levels in tumour cells may therefore play an important role in regulating the metastatic progression of cancers. We have determined the effects of a number of cytokines on c-met expression in the colon cancer cell line HT29. We report that c-met message and protein levels are up-regulated by the cytokines IL-5, IL-10, TGF-beta, PDGF and basic FCF while down-regulation occurred after treatment with IFN-gamma. We conclude that up-regulation of the HGF/SF receptor in vivo by the above cytokines may enhance tumour cell sensitivity to HGF/SF and therefore be an important step in the progression of metastatic spread.     

肝细胞生长因子及其受体c-met在卵巢上皮性肿瘤组织中的表达及意义

目的:研究肝细胞生长因子(hepatocytegrowthfactor,HGF)和cmet蛋白在卵巢上皮性肿瘤组织中的表达以及与临床病理特征的关系。方法:应用免疫组织化学SP法对45例卵巢上皮性恶性肿瘤及10例卵巢上皮性良性肿瘤组织中HGF和cmet蛋白的表达进行定位分析;应用蛋白印迹法检测HGF、cmet蛋白在卵巢上皮性肿瘤中的表达。结果:在卵巢癌组织中,HGF在上皮细胞的表达强于间质细胞,cmet在上皮细胞强表达,间质细胞无表达。HGF和cmet蛋白在卵巢癌组织中阳性率分别为60.0%(27/45)和73.0%(33/45),较良性肿瘤组织的阳性率20.0%(2/10)和50.0%(5/10)显著升高,P值分别为0.000和0.000。手术病理分期Ⅲ～Ⅳ期者HGF和cmet蛋白表达的阳性率为70.4%(19/27)和88.9%(24/27),较Ⅰ～Ⅱ期者的38.9%(7/18)和50.0%(9/18)显著升高,P值分别为0.048和0.000。淋巴结转移者阳性率分别为78.6%(11/14)和92.8%(13/14),较无淋巴结转移者阳性率51.6%(16/31)和64.5%(21/31)升高显著,P值分别为0.025和0.000。HGF和cmet蛋白表达阳性率与年龄和病理分级无关,P值分别为0.436、0.549、0.465和0.124。HGF和cmet蛋白相对表达强度在卵巢癌组织中为1.29±1.25和1.57±1.25,在良性肿瘤组织中为0.17±0.34和0.44±0.46,两组比较差异有统计学意义,P值分别为0.000和0.007。在卵巢癌组织中HGF和cmet蛋白的表达呈正相关性,P=0.009。结论:卵巢上皮性肿瘤组织中存在HGF和cmet蛋白的表达,HGF和cmet与卵巢癌的发生、侵袭和转移密切相关。     

Expression of the proto-oncogenes c-met and c-kit and their ligands, hepatocyte growth factor/scatter factor and stem cell factor, in SCLC cell lines and xenografts.

We examined a panel of 25 small cell lung cancer (SCLC) cell lines and nude mouse xenografts for expression of the proto-oncogenes c-met and c-kit, and for expression of the corresponding ligands, hepatocyte growth factor (HGF) (also known as scatter factor (SF)), and stem cell factor (SCF), respectively. Expression of mRNA was detected by Northern blotting, and c-met and c-kit protein expression was detected by Western blotting and immunocytochemistry. c-met and c-kit mRNA was expressed in 22 of the examined cell lines or xenografts, and coexpression of the two proto-oncogenes was observed in 20 tumours. Expression of c-met and c-kit protein paralleled in the mRNA expression. HGF/SF mRNA was expressed in two of the examined tumours, and only one of these also expressed the c-met proto-oncogene. SCF mRNA was expressed in 19 of the examined tumours, and in 18 of these coexpression of c-kit and SCF was present. The high percentage of SCLC tumours expressing c-met and c-kit indicates that these proto-oncogenes may have an important function in this disease. The rare coexpression of c-met and HGF/SF is evidence that an autocrine regulatory pathway is not present for this receptor/ligand system in SCLC, while the frequent coexpression of c-kit and SCF indicates that this receptor/ligand system may have an autocrine function in SCLC.     

High expression of heparin-binding EGF-like growth factor in rat hepatocarcinogenesis

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a member of the EGF family and is highly expressed in hepatoma tissues but not in normal liver. However, it is unknown when HB-EGF is induced during hepatocarcinogenesis and what are the mechanisms underlying its high expression in hepatoma. To address this issue, the expression of HB-EGF was investigated during hepatocarcinogenesis in LEC (Long-Evans with a cinnamon-like coat color) rats, which spontaneously develop hepatitis and hepatoma. LEA (Long-Evans with an agouti coat color) rats were used as controls. Furthermore, the induction of HB-EGF mRNA by various agents was investigated in a rat hepatoma cell line and hepatocytes in primary culture. Expression of HB-EGF mRNA in the liver was very low at the stage of acute and chronic hepatitis and markedly increased at the stage of hepatoma in LEC rats. Non-involved tissues adjacent to hepatoma showed low expression of HB-EGF mRNA. Immunochemical studies revealed positive staining in hepatoma tissues. Induction of HB-EGF mRNA by several growth factors was observed in a hepatoma cell line but not in normal hepatocytes. Our results suggest that HB-EGF is associated with the early progression steps of hepatoma. © 1996 Wiley-Liss, Inc.     

The scatter factor/hepatocyte growth factor: c-met pathway in human embryonal central nervous system tumor malignancy

Embryonal central nervous system (CNS) tumors, which comprise medulloblastoma, are the most common malignant brain tumors in children. The role of the growth factor scatter factor/hepatocyte growth factor (SF/HGF) and its tyrosine kinase receptor c-Met in these tumors has been until now completely unknown. In the present study, we show that human embryonal CNS tumor cell lines and surgical tumor specimens express SF/HGF and c-Met. Furthermore, c-Met mRNA expression levels statistically significantly correlate with poor clinical outcome. Treatment of medulloblastoma cells with SF/HGF activates c-Met and downstream signal transduction as evidenced by c-Met, mitogen-activated protein kinase, and Akt phosphorylation. SF/HGF induces tumor cell proliferation, anchorage-independent growth, and cell cycle progression beyond the G1-S checkpoint. Using dominant-negative Cdk2 and a degradation stable p27 mutant, we show that cell cycle progression induced by SF/HGF requires Cdk2 function and p27 inhibition. SF/HGF also protects medulloblastoma cells against apoptosis induced by chemotherapy. This cytoprotective effect is associated with reduction of proapoptotic cleaved poly(ADP-ribose) polymerase and cleaved caspase-3 proteins and requires phosphoinositide 3-kinase activity. SF/HGF gene transfer to medulloblastoma cells strongly enhances the in vivo growth of s.c. and intracranial tumor xenografts. SF/HGF-overexpressing medulloblastoma xenografts exhibit increased invasion and morphologic changes that resemble human large cell anaplastic medulloblastoma. This first characterization establishes SF/HGF:c-Met as a new pathway of malignancy with multifunctional effects in human embryonal CNS tumors.     

Modifications of the hepatocyte growth factor/c-met pathway by constitutive expression of transforming growth factor-α in rat liver epithelial cells

We have previously shown that rat liver epithelial cells (RLEC) transfected with and constitutively expressing transforming growth factor-α (TGF-α) have an enhanced mitogenic response to hepatocyte growth factor (HGF). In the study reported here, we examined tumor clones derived from the TGF-α transfectants with respect to mitogenic response to HGF. Tumor cell lines that expressed TGF-α responded to HGF with a greater increase in DNA synthesis than did the nontransfected parental RLEC (pRLEC). The tumor clones had also acquired a lower threshold for HGF response, which enabled them to undergo significant DNA synthesis at a low concentration of HGF that did not evoke a response in the pRLEC or TGF-α transfectants. We investigated the mechanisms by which TGF-α expression may influence the HGF/c-met pathway. We showed that most TGF-α transfectants and tumor cells displayed increases in c-met mRNA and protein, indicating that the enhanced HGF response may be due in part to an increase in the amount of receptor present. However, in all transfectants and tumor clones that constitutively expressed TGF-α, c-met was tyrosine phosphorylated in the absence of ligand (HGF) or another exogenous growth factors. These data suggest that induction of c-met mRNA and transactivation of c-met may be a sequela of the constitutive expression of TGF-α and that constitutive activation of the epidermal growth factor receptor pathway leads to phosphorylation and activation of c-met. These studies provide evidence for a novel mechanism of communication between epidermal growth factor receptor and c-met pathways that may partially explain the synergistic effects reported between TGF-α and HGF. Mol. Carcinog. 18:244–255, 1997. © 1997 Wiley-Liss, Inc.     

Expression and localization of scatter factor/hepatocyte growth factor in human astrocytomas

Scatter factor/hepatocyte growth factor (SF/HGF) is a pleiotropic cytokine that has been implicated in glioma invasion and angiogenesis. The SF/HGF receptor, MET, has been found to be expressed in neoplastic astrocytes as well as in endothelial cells of the tumor vasculature. Both SF/HGF and MET expression have also been described to correlate with the malignancy grade of human gliomas. However, most glioblastoma cell lines lack SF/HGF expression, raising the question of the cellular origin of SF/HGF in vivo. Using in situ hybridization, we analyzed glioblastomas, anaplastic astrocytomas, diffuse astrocytomas, pilocytic astrocytomas, and normal brain for the expression of SF/HGF mRNA. We detected strong SF/HGF expression by the majority of the tumor cells and by vascular endothelial cells in all glioblastoma specimens analyzed. Combined use of in situ hybridization with fluorescence immunohistochemistry confirmed the astrocytic origin of the SF/HGF-expressiong cells. In contrast, CD68-immunoreactive microglia/macrophages, as well as vascular smooth muscle cells reactive to alpha-smooth muscle actin, lacked SF/HGF expression. In anaplastic, diffuse, and pilocytic astrocytomas, SF/HGF expression was confined to a subset of tumor cells, and signals were less intense than in glioblastomas. In addition, we detected SF/HGF mRNA in cortical neurons. SF/HGF expression was not up regulated around necroses or at tumor margins. MET immunoreactivity was observed in GFAP-expressing astrocytic tumor cells and endothelial cells as well as in a subset of microglia/macrophages. We conclude that in vivo, both autocrine and paracrine stimulation of tumor cells and endothelium through the SF/HGF-MET system are likely to contribute to tumor invasion and angiogenesis. Lack of SF/HGF expression by most cultured glioblastoma cells is not representative of the in vivo situation and most likely represents a culture artifact.     

肝癌干细胞标志CD90在化学诱癌过程中的动态变化

目的检测CD90在化学诱导C57BL/6J小鼠肝癌过程中各时期的动态变化。方法实验组通过化学法诱导50只C57BL/6J雄性小鼠肝癌,对照组为50只正常C57BL/6J雄性小鼠。观察小鼠成瘤情况和生长状态。对每4周定期处死的小鼠组织标本进行病理学观察,应用RT-PCR和荧光实时定量PCR(FQ-RT-PCR)、Western印迹法技术检测100只小鼠肝组织及相应阶段正常肝组织CD90 mRNA及其蛋白表达情况。结果在化学诱癌第16周时,小鼠肝脏表面开始出现结节样变化,直径约2mm,呈灰白色,多发性。病理改变主要是肝细胞排列轻度紊乱,细胞轻度异型增生。第20周时肝癌结节直径在5～25mm之间,镜下为中、高分化的肝癌细胞;RT-PCR检测结果显示,实验组在16周以前CD90蛋白不表达。FQ-RT-PCR、Western blot检测结果显示,CD90 mRNA在化学诱癌16周以前,实验组与对照组比较差异无统计学意义(P>0.05),但在诱癌16周以后,CD90表达呈显著性增加,实验组与对照组比较,差异具有统计学意义(P<0.05)。结论肝癌干细胞标志物CD90在肝癌的发生、发展过程中,其蛋白表达随着诱癌时间的延长而逐渐增加,认为CD90对肝癌的发生、发展有一定的调控作用。     

Changes in poly (A) + RNA translational pattern during chemically induced hepatocarcinogenesis

Hepatocarcinoma was induced by administration of diethylnitrosamine to rats. The rats were sacrificed 70 weeks after the administration and the carcinoma nodules were separated from the perinodular parenchymental cells after perfusion of liver with collagenase. The in vitro translational pattern of mRNAs from hepatocellular carcinomas, from perinodular hepatocytes and from regenerating liver after partial hepatectomy were compared by one- and two-dimensional electrophoreses to the pattern obtained with RNA from normal hepatocytes. An increased synthesis of several peptides was observed with RNAs from carcinoma and from regenerating liver and to a lesser extent with RNA from perinodular hepatocytes, which suggests that the increase in synthesis is at least partly related to cell proliferation. A decreased synthesis of several other peptides was observed with RNA from carcinoma nodules and to a lesser extent with RNA from perinodular hepatocytes, but not with RNA from regenerating liver, which suggests that this decrease in synthesis is related to some transformation specific process. These changes are observed as soon as 22 weeks after carcinogen administration. These observations also suggest that at least part of the perinodular hepatocytes have some characteristics of the transformed cells.     

Expression of hepatocyte growth factor and c-MET in skull base chordoma

BACKGROUND: Hepatocyte growth factor (HGF) is a multipotent cytokine that is mediated by its receptor, c-MET. HGF/c-MET contributes to tumor progression in many human malignancies; however, HGF/c-MET is inversely correlated with aggressive biologic behavior in other cancers. Conversely, to the authors' knowledge, little is known regarding the significance of HGF/c-MET expression in skull base chordoma. METHODS: Using immunohistochemical techniques, the authors investigated HGF/c-MET expression in 46 primary and 25 recurrent lesions, and compared it with the expression of proteinases and cell differentiation markers, proliferative ability, and other clinicopathologic parameters. RESULTS: c-MET was found to be expressed in 70.0% of primary and 88.0% of recurrent lesions. HGF expression was scarcely detected. Higher c-MET expression was found to be correlated with younger patient age. Lesions with a higher expression of low molecular weight cytokeratin (CAM5.2) demonstrated significantly higher c-MET scores in both primary and recurrent lesions compared with those with lower CAM5.2 expression. In recurrent lesions, higher c-MET expression was found to be associated with the scores of matrix metalloproteinase (MMP)-1, MMP-2, tissue inhibitor of matrix metalloproteinase-1, and urokinase plasminogen activator (uPA); however, only uPA was found to be correlated with higher c-MET expression in primary lesions. c-MET expression did not appear to be correlated with MIB-1 labeling index. Patients with higher c-MET expression were found to have longer survival. CONCLUSIONS: In the current study, c-MET expression was a common event, and was found to be correlated with CAM5.2 expression, younger patient age, and a favorable prognosis in patients with skull base chordoma. However, HGF/c-MET paracrine signaling also may contribute to its invasive ability, especially in recurrent lesions.     

Deletion type hepatocyte growth factor has different effects on growth and c-met expression in 10 different human hepatocellular carcinoma cell lines

We examined expression of c-met protein, and the mitogenic and morphologic effects of deletion type hepatocyte growth factor (dHGF) by using 10 human hepatocellular carcinoma (HCC) cell lines having different morphologic and biologic features. c-met protein was detected at varying levels in all cells, regardless of the histological grades. Among the 7 lines expressing c-met at high levels, mitogenic effects of dHGF were stimulative for 2 lines; suppressive for 3 lines; and not distinguishable for the other 2 lines. Furthermore, mitogenic effects of dHGF were different in two clonally related cell lines, having different morphologic and biologic features, even though expression of c-met protein was comparable. dHGF induced scattering of cells and morphologic changes in two lines with suppressing and unaffected growth. In the 3 lines expressing c-met at relatively low levels, no remarkable mitogenic or morphogenic effects were detected. These results suggest that the expression levels of c-met protein were not related to the differentiation levels of HCC cells, and dHGF may cause different biological effects on the cells with almost identical c-met protein expression.