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52 Oncogenic role and specificity of frizzled receptor expression in animal models of hepatocellular carcinoma
180A
AASLD ABSTRACTS
52
ONCOGENIC ROLE AND SPECIFICITY OF FRIZZLED RECEPTOR EXPRESSION IN ANIMAL MODELS OF HEPATOCELLULAR CARCINOMA. Marc Herrmann, Philippe
Merle, Suzanne de la Monte, Liver Research Center, RI Hospital and Brown Medical School, Providence, RI; L Lefrancois, INS~ RM U271, Hotel Dieu, Lyon, France; Sophia Califano, Liver Research Center, RI Hospital and Brown Medical School, Providence, RI; Chrish'an Tr~po, INS~ RM U271, Hotel Dieu, Lyon, France; Shinji Tanaka, Kyushu University, Fukuoka, Japan; Jack Wands, Liver Research Center, RI Hospital and Brown Medical School, Providence, RI BACKGROUND: We are investigating the sequential changes in gene regulation that may be necessary and sufficient to transform hepatocytes in vivo. In this regard, one of the earliest genetic changes is activation of the Wnt/Frizzled signal transduction pathway. This cascade is involved in cell fate determination, cell proliferation and migration. The purpose of this study was to create an experiment paradigm using a series of single, double, and triple transgenic mice strains and evaluate expression of Frizzled genes. These transgenic lines over-express an oncogene (myc) a transforming protein (SV40-T-large-antigen), a viral protein (HBx) associated with transcriptional transactivation of cellular growth related genes, and a constitutive hepatic proliferative stimulus provided by activation of the insulin/IGF-1 signal transduction pathway (IRS-1). METHODS: A quantitative, real-time RT-PCR assay was developed to investigate expression of all the different members of the Frizzled family (FZD1 - 9) during the different steps of hepatocarcinogenesis in single, double, and triple transgenic mice. Frizzled gene expression was measured in the livers of non-transgenic littermates, in tumor-free early dysplastic lesions, in late dysplastic peri-tumoral liver, and in the corresponding HCC tumor as observed over a 40 week period. The /3-catenin gene was also evaluated for mutations by PCR amplification and sequencing. RESULTS: All murine HCC tumors had a homologous wild-type/3-catenin exon-2 gene as assessed by genomic DNA sequencing. There was no expression of FZD-2, 3, 5 , o r 9 . In contrast, a specific RT-PCR signal was detected for FZD1, 4, 6, 7, and 8. In addition, FZD-1 and 6 expression remained stable, whereas FZD-4 and 8 were downregulated in tumor (T) vs. normal liver (NL) (p < 0.5). The most striking observation was the specific and highly upregulated FZD7 gene in 8/13 (67%) of livers with early dysplasia, 7/16 (44%) of tumor-free late dysplastic livers, 12/18 (67%) in surrounding peritumorous liver, and 13/18 (72%) of hepatocellular carcinomas. In addition FZD7 reached the highest levels of over-expression in early and rapidly growing tumors generated from SV40-T-large-antigen transgenic mice, and HBx/IRS-1/myc triple transgenic mice as compared to the single (myc) or double (HBx/myc) transgenic animals with more slowly developing tumors. CONCLUSIONS: There is early, common and specific upregulation of the FZD7 receptor gene as compared to the other eight family members. During hepatocarcinogenesis, higher levels of expression are achieved in the most rapid growing tumors. Therefore, in these transgenic animal models of HCC, FZD7 over-expression is a fundamental, early genetic change that is required during the multi-step process of hepatocyte transformation in vivo. Disclosures: Sophia Califano - No relationships to disclose Suzanne de la Monte - No relationships to disclose Marc Herrmann - No relationships to disclose L Lefrancois - No relationships to disclose Philippe Merle - No relationships to disclose Shinji Tanaka - No relationships to disclose Christian Tr6po - No relationships to disclose Jack Wands - No relationships to disclose
HEPATOLOGY, October 2003
53 THE BLOCKADE OF EPIDERMAL GROWTH FACTOR RECEPTOR TYROSINE KINASE ACTIVITY REDUCES THE EMERGENCE OF HEPATOCELLULAR CARCINOMA WITHIN CIRRHOTIC RAT LIVER. Olivier Rosmorduc, INS~ RM U402,
Paris, France;~ duardo Schiller, H6pital Cantonal de Gen~ve, Gen~ve, Switzerland; V&onique Barbu, INS~ RM U402, Paris, France; Dominique Wendum, Anatomie Pathologi~ue, H6pital Saint-Antoine, Paris, France; Colette Rey, H~l~neRobin, 1N~ RM U402, Paris, France; Francois Clergue, H6pital Cantonal de Gen~ve, Gen2ove,Switzerland; Raoul Poupon, Chantal Housset, 1N~ RM U402, Paris, France Different lines of evidence suggest that TGF-a and its receptor EGFR, may contribute to hepatocellular carcinogenesis. The aims of this study were 1) to examine the expression of TGF-a and of EGFR in a model of hepatocellular carcinoma (HCC) within cirrhotic liver, and 2) to test the anti-tumoral effect of an EGFR tyrosine kinase inhibitor (ZD1839, Iressa (r), Astra Zeneca) in this model. Methods: Male Wistar rats received intraperitoneal injections of diethylnitrosamine (DEN, 50 mg/kg weekly) for 12 weeks (n - 3) or 16 weeks (n - 16), and were killed 2 weeks after the end of DEN administration. Normal rats (n - 3) served as healthy controls. Rats submitted to 16-week DEN administration were treated between weeks 12 and 18 with daily intraperitoneal injections of 2 mg/kg ZD 1839 (n - 8) or of vehicle (n - 8). Malignant nodules > - 3 m m in diameter, were counted on the entire liver surface, by two i n d e p e n d e n t observers. One liver section per animal and at least one nodule, if present, were submitted to histological analysis. TGF-a and EGFR transcripts were quantified by means of real-time PCR. ERK and phosphoERK were detected by Western blot. Proliferation and cell death were assessed by Ki67 immunolabelling and by TUNEL assay, respectively. Results: The protocol of DEN administration resulted in the emergence of cirrhosis without HCC after 14 weeks and of HCC within cirrhotic liver after 18 weeks. In the absence of ZD 1839 treatment, the expression of TGF-a (expressed as a number of transcripts x 103/mg RNA) became significantly higher in each one of the following conditions: normal liver (7.5 -+ 1.4), cirrhotic liver without HCC (16.1 -+ 2.8), cirrhotic liver with HCC, in the non-tumoral tissue (43.9 -+ 10.1) and in tumors (96.3 _+ 22.5). Meanwhile, the expression of EGFR, after an initial decrease during the progression from normal (33.0 _+ 5.2) to the cirrhotic liver without HCC (14.9 -+ 4.2), returned to initial levels in the non-tumoral tissue (28.7 -+ 6.9) and tumors (24.4 _+ 5.5) from cirrhotic liver with HCC. In the group of rats treated with ZD 1839, the number of hepatocellular carcinoma nodules at 18 weeks, was significantly lower (3.7 -+ 0.45) than in untreated animals (18.2 _+ 1.25, p < 0.05). In the tumors from ZD 1839-treated rats as compared with those from untreated rats, a significant decrease in the amounts of TGF-a transcripts (32.1 _+ 5.2 vs 96.3 _+ 22.5, p < 0.05) and of EGFR transcripts (11.9 _+ 2.3 vs 24.4 _+ 5,5, p < 0.05) was found. In addition, ERK 1/2 was phosphorylated to a lesser extent in the liver tissue from ZD 1839-treated rats, as compared with untreated rats. Yet no significant difference in the rate of Ki67- or TUNELlabelled ceils within tumors and non-tumoral tissue was found between ZD 1839-treated and untreated animals. In conclusion, these results indicate that liver fibrogenesis and carcinogenesis occurs in parallel with a progressive increase in TGF-a autocrine action. The EGFR tyrosine kinase inhibitor ZD 1839 reduces the emergence of hepatocellular carcinoma in the cirrhotic liver. This effect coincides with a decrease in the local expression of TGF-a and of EGFR, and with a decrease in ERK 1/2 activation. This study suggests that inhibition of the TGF-a/EGFR loop by ZD 1839 could prevent or delay the emergence of hepatocellular carcinoma in cirrhotic patients. Disclosures: V6ronique Barbu - No relationships to disclose Francois Clergue - No relationships to disclose Chantal Housset - No relationships to disclose Raoul Poupon - No relationships to disclose Colette Rey - No relationships to disclose H616ne Robin - No relationships to disclose Olivier Rosmorduc - No relationships to disclose Eduardo Schiffer - No relationships to disclose Dominique W e n d u m - No relationships to disclose
AASLD ABSTRACTS
52
ONCOGENIC ROLE AND SPECIFICITY OF FRIZZLED RECEPTOR EXPRESSION IN ANIMAL MODELS OF HEPATOCELLULAR CARCINOMA. Marc Herrmann, Philippe
Merle, Suzanne de la Monte, Liver Research Center, RI Hospital and Brown Medical School, Providence, RI; L Lefrancois, INS~ RM U271, Hotel Dieu, Lyon, France; Sophia Califano, Liver Research Center, RI Hospital and Brown Medical School, Providence, RI; Chrish'an Tr~po, INS~ RM U271, Hotel Dieu, Lyon, France; Shinji Tanaka, Kyushu University, Fukuoka, Japan; Jack Wands, Liver Research Center, RI Hospital and Brown Medical School, Providence, RI BACKGROUND: We are investigating the sequential changes in gene regulation that may be necessary and sufficient to transform hepatocytes in vivo. In this regard, one of the earliest genetic changes is activation of the Wnt/Frizzled signal transduction pathway. This cascade is involved in cell fate determination, cell proliferation and migration. The purpose of this study was to create an experiment paradigm using a series of single, double, and triple transgenic mice strains and evaluate expression of Frizzled genes. These transgenic lines over-express an oncogene (myc) a transforming protein (SV40-T-large-antigen), a viral protein (HBx) associated with transcriptional transactivation of cellular growth related genes, and a constitutive hepatic proliferative stimulus provided by activation of the insulin/IGF-1 signal transduction pathway (IRS-1). METHODS: A quantitative, real-time RT-PCR assay was developed to investigate expression of all the different members of the Frizzled family (FZD1 - 9) during the different steps of hepatocarcinogenesis in single, double, and triple transgenic mice. Frizzled gene expression was measured in the livers of non-transgenic littermates, in tumor-free early dysplastic lesions, in late dysplastic peri-tumoral liver, and in the corresponding HCC tumor as observed over a 40 week period. The /3-catenin gene was also evaluated for mutations by PCR amplification and sequencing. RESULTS: All murine HCC tumors had a homologous wild-type/3-catenin exon-2 gene as assessed by genomic DNA sequencing. There was no expression of FZD-2, 3, 5 , o r 9 . In contrast, a specific RT-PCR signal was detected for FZD1, 4, 6, 7, and 8. In addition, FZD-1 and 6 expression remained stable, whereas FZD-4 and 8 were downregulated in tumor (T) vs. normal liver (NL) (p < 0.5). The most striking observation was the specific and highly upregulated FZD7 gene in 8/13 (67%) of livers with early dysplasia, 7/16 (44%) of tumor-free late dysplastic livers, 12/18 (67%) in surrounding peritumorous liver, and 13/18 (72%) of hepatocellular carcinomas. In addition FZD7 reached the highest levels of over-expression in early and rapidly growing tumors generated from SV40-T-large-antigen transgenic mice, and HBx/IRS-1/myc triple transgenic mice as compared to the single (myc) or double (HBx/myc) transgenic animals with more slowly developing tumors. CONCLUSIONS: There is early, common and specific upregulation of the FZD7 receptor gene as compared to the other eight family members. During hepatocarcinogenesis, higher levels of expression are achieved in the most rapid growing tumors. Therefore, in these transgenic animal models of HCC, FZD7 over-expression is a fundamental, early genetic change that is required during the multi-step process of hepatocyte transformation in vivo. Disclosures: Sophia Califano - No relationships to disclose Suzanne de la Monte - No relationships to disclose Marc Herrmann - No relationships to disclose L Lefrancois - No relationships to disclose Philippe Merle - No relationships to disclose Shinji Tanaka - No relationships to disclose Christian Tr6po - No relationships to disclose Jack Wands - No relationships to disclose
HEPATOLOGY, October 2003
53 THE BLOCKADE OF EPIDERMAL GROWTH FACTOR RECEPTOR TYROSINE KINASE ACTIVITY REDUCES THE EMERGENCE OF HEPATOCELLULAR CARCINOMA WITHIN CIRRHOTIC RAT LIVER. Olivier Rosmorduc, INS~ RM U402,
Paris, France;~ duardo Schiller, H6pital Cantonal de Gen~ve, Gen~ve, Switzerland; V&onique Barbu, INS~ RM U402, Paris, France; Dominique Wendum, Anatomie Pathologi~ue, H6pital Saint-Antoine, Paris, France; Colette Rey, H~l~neRobin, 1N~ RM U402, Paris, France; Francois Clergue, H6pital Cantonal de Gen~ve, Gen2ove,Switzerland; Raoul Poupon, Chantal Housset, 1N~ RM U402, Paris, France Different lines of evidence suggest that TGF-a and its receptor EGFR, may contribute to hepatocellular carcinogenesis. The aims of this study were 1) to examine the expression of TGF-a and of EGFR in a model of hepatocellular carcinoma (HCC) within cirrhotic liver, and 2) to test the anti-tumoral effect of an EGFR tyrosine kinase inhibitor (ZD1839, Iressa (r), Astra Zeneca) in this model. Methods: Male Wistar rats received intraperitoneal injections of diethylnitrosamine (DEN, 50 mg/kg weekly) for 12 weeks (n - 3) or 16 weeks (n - 16), and were killed 2 weeks after the end of DEN administration. Normal rats (n - 3) served as healthy controls. Rats submitted to 16-week DEN administration were treated between weeks 12 and 18 with daily intraperitoneal injections of 2 mg/kg ZD 1839 (n - 8) or of vehicle (n - 8). Malignant nodules > - 3 m m in diameter, were counted on the entire liver surface, by two i n d e p e n d e n t observers. One liver section per animal and at least one nodule, if present, were submitted to histological analysis. TGF-a and EGFR transcripts were quantified by means of real-time PCR. ERK and phosphoERK were detected by Western blot. Proliferation and cell death were assessed by Ki67 immunolabelling and by TUNEL assay, respectively. Results: The protocol of DEN administration resulted in the emergence of cirrhosis without HCC after 14 weeks and of HCC within cirrhotic liver after 18 weeks. In the absence of ZD 1839 treatment, the expression of TGF-a (expressed as a number of transcripts x 103/mg RNA) became significantly higher in each one of the following conditions: normal liver (7.5 -+ 1.4), cirrhotic liver without HCC (16.1 -+ 2.8), cirrhotic liver with HCC, in the non-tumoral tissue (43.9 -+ 10.1) and in tumors (96.3 _+ 22.5). Meanwhile, the expression of EGFR, after an initial decrease during the progression from normal (33.0 _+ 5.2) to the cirrhotic liver without HCC (14.9 -+ 4.2), returned to initial levels in the non-tumoral tissue (28.7 -+ 6.9) and tumors (24.4 _+ 5.5) from cirrhotic liver with HCC. In the group of rats treated with ZD 1839, the number of hepatocellular carcinoma nodules at 18 weeks, was significantly lower (3.7 -+ 0.45) than in untreated animals (18.2 _+ 1.25, p < 0.05). In the tumors from ZD 1839-treated rats as compared with those from untreated rats, a significant decrease in the amounts of TGF-a transcripts (32.1 _+ 5.2 vs 96.3 _+ 22.5, p < 0.05) and of EGFR transcripts (11.9 _+ 2.3 vs 24.4 _+ 5,5, p < 0.05) was found. In addition, ERK 1/2 was phosphorylated to a lesser extent in the liver tissue from ZD 1839-treated rats, as compared with untreated rats. Yet no significant difference in the rate of Ki67- or TUNELlabelled ceils within tumors and non-tumoral tissue was found between ZD 1839-treated and untreated animals. In conclusion, these results indicate that liver fibrogenesis and carcinogenesis occurs in parallel with a progressive increase in TGF-a autocrine action. The EGFR tyrosine kinase inhibitor ZD 1839 reduces the emergence of hepatocellular carcinoma in the cirrhotic liver. This effect coincides with a decrease in the local expression of TGF-a and of EGFR, and with a decrease in ERK 1/2 activation. This study suggests that inhibition of the TGF-a/EGFR loop by ZD 1839 could prevent or delay the emergence of hepatocellular carcinoma in cirrhotic patients. Disclosures: V6ronique Barbu - No relationships to disclose Francois Clergue - No relationships to disclose Chantal Housset - No relationships to disclose Raoul Poupon - No relationships to disclose Colette Rey - No relationships to disclose H616ne Robin - No relationships to disclose Olivier Rosmorduc - No relationships to disclose Eduardo Schiffer - No relationships to disclose Dominique W e n d u m - No relationships to disclose