- Email: [email protected]
112 INACTIVATION OF CYCLIN E1 INHIBITS CHEMICAL HEPATOCARCINOGENESIS IN MICE
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Saturday, April 25
111 TRANSIENT TELOMERE DYSFUNCTION PROMOTES HEPATOCARCINOGENESIS Y. Begus-Nahrmann, A. Lechel, D. Hartmann, K.L. Rudolph. Molecular Medicine, Ulm University, Ulm, Germany E-mail: [email protected] Background: Chromosomal instability, short telomeres, and telomerase activation are hallmarks of human hepatocellular carcinoma. Studies in mouse models have shown that telomere dysfunction induces chromosomal instability and tumor initiation. However, telomere stabilization is required for tumor progression since too high levels of instability impair tumor cell survival. These results suggested that a sequence of telomere dysfunction followed by telomerase re-activation may enhance tumor initiation and progression during human carcinogenesis. Aims: The aim of this study was to test the hypothesis that telomere dysfunction followed by telomerase activation promote tumorigenesis. Methods: We produced transgenic mice allowing doxycycline inducible and transient expression of TRF2DBDM (inducing a transient peak of telomere dysfunction) in the liver. These mice were injected at postnatal day 15 with di-ethyl-nitrosamine. The same mice were treated with intrasplenic injection of doxycycline to induce transient expression of TRF2DBDM and telomere dysfunction. Control mice lacked the TRF2DBDM transgene but were treated the same way. The animals were analysed at an age of 13 months. Results: Doxycycline induced expression of TRF2DBDM in the liver of double-transgenic mice correlated with transient induction of telomere dysfunction in 2−4 month old mice as measured by induction of anaphase bridges (morphological correlate of chromosomal fusions). After 13 months both the LAPrtTA-TRF2DBDM (n = 23) and the control group (n = 29) developed HCC. However, transient telomere dysfunction in LAPrtTA-TRF2DBDM double transgenic mice strongly increased tumor formation. The tumor size was significantly increased by more than two fold in male LAPrtTA-TRF2DBDM mice compared to controls (p < 0.0001). In addition, the number of tumors was significantly increased in female LAPrtTA-TRF2DBDM mice compared to controls (p = 0.019). Chromosomal instability measured by array CGH revealed an increase of aberrations in double-transgenic mice. Conclusions: The results provide first experimental evidence that transient induction of telomere dysfunction during early stages of hepatocarcinogenesis leads to a significant acceleration of tumor formation. These results appear to be important for human hepatocarcinogenesis, which is characterized by telomere shortening and chromosomal instability. 112 INACTIVATION OF CYCLIN E1 INHIBITS CHEMICAL HEPATOCARCINOGENESIS IN MICE N. Moro1 , N. Gassler2 , Y.A. Nevzorova1 , P. Sicinski3 , C. Trautwein1 , C. Liedtke1 . 1 Department of Medicine III, 2 Institute of Pathology, University Hospital Aachen, Aachen, Germany; 3 Dana-Farber Cancer Institute, Boston, MA, USA E-mail: [email protected] Background and Aims: E-type cyclins E1 and E2 are important for the transition of quiescent cells into the cell cycle. The aim of our study was to investigate the consequences of cyclin E1 depletion in a chemical model of hepatocarcinogenesis. Methods: Constitutive cyclin E1 knockout (E1−/−) mice and wild-type controls (WT) were injected once with 25 mg/kg (i.p.) of the carcinogen diethyl-nitrosamine (DEN) at the age of 14 days to induce hepatocellular carcinoma (HCC). Alternatively, 6−8 weeks old WT and E1−/− mice were injected with a single dose of 200 mg/kg DEN i.p. and analyzed 0–168 hours after injection for early tumorigenic effects. Results: 22 weeks after DEN administration 40% of WT mice (n = 15) had small, macroscopic visible tumor nodules 1 mm in diameter. In contrast, none of the E1−/− mice (n = 15) displayed any tumor 22 weeks after DEN treatment. 38 weeks after DEN treatment 100% of WT controls showed overall strong tumorigenesis with an average of 23 tumors/per liver
and an average tumor diameter of 6 mm (±3 mm), whereas only 80% of E1−/− mice developed liver tumors with a significantly lower average frequency (3 tumors/liver) and size (median diameter of 2±1 mm).To explore the molecular mechanism leading to tumor inhibition in E1−/−, we investigated the immediate response to DEN. Maximal liver damage in WT and E1−/− animals was evident 48 h after DEN as shown by serum transaminase levels. E1−/− mice performed twofold higher liver apoptosis compared to controls as monitored by TUNEL assays and caspase-3 activity, which was correlated with a modified expression profile of the cell cycle inhibitor p21. Surprisingly, we also detected a strongly reduced expression of the cytokine interleukin-6. The proliferative response following acute DEN-mediated liver injury was delayed in E1−/− mice by two days further indicating a prolonged cell cycle arrest in these animals. Conclusion: Depletion of cyclin E1 protects from HCC formation through mechanisms involving delayed cell cycle progression and increased apoptosis. We conclude that cyclin E1 is an essential factor for the earliest steps in tumor formation which can not be compensated by cyclin E2. 113 MODULATION OF DEMETHYLATION AND ANTITUMOR POTENTIAL OF ZEBULARINE IN LIVER CANCER J.B. Andersen, C. Raggi, J.U. Marquardt, E.A. Conner, Y.H. Lee, V.M. Factor, S.S. Thorgeirsson. Laboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA E-mail: [email protected] Background and Aims: Aberrant promoter hypermethylation at 5 -methylcytosine is associated with atypical gene silencing, affecting genes involved in tumorigenesis. Zebularine (ZEB) is a potent inhibitor of DNA methyltransferase and is an effective demethylating agent in cancer cells. Here, we examined the transcriptomic and epigenomic changes following ZEB-treatment of 10 liver cancer cell lines using microarray analysis, characterizing the methylation and re-expression of silenced genes, in order to evaluate ZEB as a potential therapy for liver cancer. Methods: Each cell line (7 HCC, 3 CCC and 2 normal) was treated with 100–200mM ZEB for 7 days and gene lists were computed (p0.001) using corresponding controls as reference. The therapeutic efficacy of ZEB in vivo was evaluated in a model of liver cancer metastases. The tumor cells expressing luciferase were transplanted intrasplenic into SCID/Beige mice. Results: Gene signatures included 631 genes changed by ZEB in HCC and 451 in CCC cell lines. Comparison of these gene lists to the corresponding normal cell lines identified unique genes to either HCC (238 genes) or CCC (85 genes). Hierarchical clustering of the HCC “ZEB”-signature grouped the cell lines in two clusters. Apoptosis, cell cycle analysis and tumor growth potential of ZEB-pretreated cell lines transplanted subcutaneous into athymic mice differentiated the clusters into responder and non-responders. Methylation profiling of 807 cancerspecific genes following ZEB-treatment confirmed the differential ZEBsensitivity. Responders showed a preferred re-expression of genes involved in apoptosis and tumor-suppression. Integration of the “ZEB”-signature with 53 HCCs differentiated the patients according to survival (p = 0.016) and multivariate analysis of clinicopathological variables revealed significant association with metastasis. Moreover, HUH7 cells expressing luciferase transplanted intrasplenic into SCID/Beige mice showed a 2-fold decrease in bioluminescent signal 14 days following ZEB-therapy. The addition of a short-term pretreatment with raloxifene, a known inhibitor of ZEB metabolism, further decreased bioluminescence up to 7-fold with decreased lung metastasis and overall increase in survival (p = 0.0004). The ZEB-therapy was confirmed in vivo in HUH1 and KMCH (responders) and WRL68 (non-responder). Conclusion: The results suggest that transcriptome analysis can identify a subset of HCC patients who may benefit from treatment with ZEB/raloxifene and/or other demethylating agents.
Saturday, April 25
111 TRANSIENT TELOMERE DYSFUNCTION PROMOTES HEPATOCARCINOGENESIS Y. Begus-Nahrmann, A. Lechel, D. Hartmann, K.L. Rudolph. Molecular Medicine, Ulm University, Ulm, Germany E-mail: [email protected] Background: Chromosomal instability, short telomeres, and telomerase activation are hallmarks of human hepatocellular carcinoma. Studies in mouse models have shown that telomere dysfunction induces chromosomal instability and tumor initiation. However, telomere stabilization is required for tumor progression since too high levels of instability impair tumor cell survival. These results suggested that a sequence of telomere dysfunction followed by telomerase re-activation may enhance tumor initiation and progression during human carcinogenesis. Aims: The aim of this study was to test the hypothesis that telomere dysfunction followed by telomerase activation promote tumorigenesis. Methods: We produced transgenic mice allowing doxycycline inducible and transient expression of TRF2DBDM (inducing a transient peak of telomere dysfunction) in the liver. These mice were injected at postnatal day 15 with di-ethyl-nitrosamine. The same mice were treated with intrasplenic injection of doxycycline to induce transient expression of TRF2DBDM and telomere dysfunction. Control mice lacked the TRF2DBDM transgene but were treated the same way. The animals were analysed at an age of 13 months. Results: Doxycycline induced expression of TRF2DBDM in the liver of double-transgenic mice correlated with transient induction of telomere dysfunction in 2−4 month old mice as measured by induction of anaphase bridges (morphological correlate of chromosomal fusions). After 13 months both the LAPrtTA-TRF2DBDM (n = 23) and the control group (n = 29) developed HCC. However, transient telomere dysfunction in LAPrtTA-TRF2DBDM double transgenic mice strongly increased tumor formation. The tumor size was significantly increased by more than two fold in male LAPrtTA-TRF2DBDM mice compared to controls (p < 0.0001). In addition, the number of tumors was significantly increased in female LAPrtTA-TRF2DBDM mice compared to controls (p = 0.019). Chromosomal instability measured by array CGH revealed an increase of aberrations in double-transgenic mice. Conclusions: The results provide first experimental evidence that transient induction of telomere dysfunction during early stages of hepatocarcinogenesis leads to a significant acceleration of tumor formation. These results appear to be important for human hepatocarcinogenesis, which is characterized by telomere shortening and chromosomal instability. 112 INACTIVATION OF CYCLIN E1 INHIBITS CHEMICAL HEPATOCARCINOGENESIS IN MICE N. Moro1 , N. Gassler2 , Y.A. Nevzorova1 , P. Sicinski3 , C. Trautwein1 , C. Liedtke1 . 1 Department of Medicine III, 2 Institute of Pathology, University Hospital Aachen, Aachen, Germany; 3 Dana-Farber Cancer Institute, Boston, MA, USA E-mail: [email protected] Background and Aims: E-type cyclins E1 and E2 are important for the transition of quiescent cells into the cell cycle. The aim of our study was to investigate the consequences of cyclin E1 depletion in a chemical model of hepatocarcinogenesis. Methods: Constitutive cyclin E1 knockout (E1−/−) mice and wild-type controls (WT) were injected once with 25 mg/kg (i.p.) of the carcinogen diethyl-nitrosamine (DEN) at the age of 14 days to induce hepatocellular carcinoma (HCC). Alternatively, 6−8 weeks old WT and E1−/− mice were injected with a single dose of 200 mg/kg DEN i.p. and analyzed 0–168 hours after injection for early tumorigenic effects. Results: 22 weeks after DEN administration 40% of WT mice (n = 15) had small, macroscopic visible tumor nodules 1 mm in diameter. In contrast, none of the E1−/− mice (n = 15) displayed any tumor 22 weeks after DEN treatment. 38 weeks after DEN treatment 100% of WT controls showed overall strong tumorigenesis with an average of 23 tumors/per liver
and an average tumor diameter of 6 mm (±3 mm), whereas only 80% of E1−/− mice developed liver tumors with a significantly lower average frequency (3 tumors/liver) and size (median diameter of 2±1 mm).To explore the molecular mechanism leading to tumor inhibition in E1−/−, we investigated the immediate response to DEN. Maximal liver damage in WT and E1−/− animals was evident 48 h after DEN as shown by serum transaminase levels. E1−/− mice performed twofold higher liver apoptosis compared to controls as monitored by TUNEL assays and caspase-3 activity, which was correlated with a modified expression profile of the cell cycle inhibitor p21. Surprisingly, we also detected a strongly reduced expression of the cytokine interleukin-6. The proliferative response following acute DEN-mediated liver injury was delayed in E1−/− mice by two days further indicating a prolonged cell cycle arrest in these animals. Conclusion: Depletion of cyclin E1 protects from HCC formation through mechanisms involving delayed cell cycle progression and increased apoptosis. We conclude that cyclin E1 is an essential factor for the earliest steps in tumor formation which can not be compensated by cyclin E2. 113 MODULATION OF DEMETHYLATION AND ANTITUMOR POTENTIAL OF ZEBULARINE IN LIVER CANCER J.B. Andersen, C. Raggi, J.U. Marquardt, E.A. Conner, Y.H. Lee, V.M. Factor, S.S. Thorgeirsson. Laboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA E-mail: [email protected] Background and Aims: Aberrant promoter hypermethylation at 5 -methylcytosine is associated with atypical gene silencing, affecting genes involved in tumorigenesis. Zebularine (ZEB) is a potent inhibitor of DNA methyltransferase and is an effective demethylating agent in cancer cells. Here, we examined the transcriptomic and epigenomic changes following ZEB-treatment of 10 liver cancer cell lines using microarray analysis, characterizing the methylation and re-expression of silenced genes, in order to evaluate ZEB as a potential therapy for liver cancer. Methods: Each cell line (7 HCC, 3 CCC and 2 normal) was treated with 100–200mM ZEB for 7 days and gene lists were computed (p0.001) using corresponding controls as reference. The therapeutic efficacy of ZEB in vivo was evaluated in a model of liver cancer metastases. The tumor cells expressing luciferase were transplanted intrasplenic into SCID/Beige mice. Results: Gene signatures included 631 genes changed by ZEB in HCC and 451 in CCC cell lines. Comparison of these gene lists to the corresponding normal cell lines identified unique genes to either HCC (238 genes) or CCC (85 genes). Hierarchical clustering of the HCC “ZEB”-signature grouped the cell lines in two clusters. Apoptosis, cell cycle analysis and tumor growth potential of ZEB-pretreated cell lines transplanted subcutaneous into athymic mice differentiated the clusters into responder and non-responders. Methylation profiling of 807 cancerspecific genes following ZEB-treatment confirmed the differential ZEBsensitivity. Responders showed a preferred re-expression of genes involved in apoptosis and tumor-suppression. Integration of the “ZEB”-signature with 53 HCCs differentiated the patients according to survival (p = 0.016) and multivariate analysis of clinicopathological variables revealed significant association with metastasis. Moreover, HUH7 cells expressing luciferase transplanted intrasplenic into SCID/Beige mice showed a 2-fold decrease in bioluminescent signal 14 days following ZEB-therapy. The addition of a short-term pretreatment with raloxifene, a known inhibitor of ZEB metabolism, further decreased bioluminescence up to 7-fold with decreased lung metastasis and overall increase in survival (p = 0.0004). The ZEB-therapy was confirmed in vivo in HUH1 and KMCH (responders) and WRL68 (non-responder). Conclusion: The results suggest that transcriptome analysis can identify a subset of HCC patients who may benefit from treatment with ZEB/raloxifene and/or other demethylating agents.