- Email: [email protected]
41 The tissue microenviroment modulates HCC cells invasive phenotype
Parallel Session 4: Hepatocellular Carcinoma ~THE TISSUE MICROENVIROMENT MODULATES HCC CELLS INVASIVE PHENOTYPE
G. Giannelli, C. Bergamini, E. Fransvea, C. Sgarra, S. Antonaci.
Department of Internal Medicine, Immunology and Infectious Diseases, Section of Internal Medicine, University of Bari, Policlinico, Bari, Italy Hepatecellular carcinoma (HCC) is the fifth most frequent malignancy in the world, and the third cause of tumor-related death. Prognosis and survival are still poor. The different behavior of HCC is likely attributable to metastasis occurrence, in particular to blood vessels. Previously we reported that an extracellular matrix protein (ECM) named Laminin-5 (Ln5), absent in normal liver, is expressed in a high percentage of HCC tissues. In particular, the ¥-2 chain, is strongly (96%) related to metastasis occurrence and to shorter survival. Goal of this study was to investigate how HCC cells acquire the ability to spread to surrounding tissue. HCC invasive cells produce detectable levels of the Ln-5 "(-2 chain, whereas non invasive cells do not. However, after TGF-[31 stimulation, non invasive cells also produce the Ln-5 "~-2 chain. In addition, HCC fiwasive and non invasive cells express similar levels and localization of[3-catenin, whereas low and high levels of E-cadherin are expressed in invasive and non invasive cells, respectively. In the presence of Ln-5, the invasive cells strongly scatter after few hours while the non fiwasive do not. In invasive cells E-cadherin is delocalized and [3-catenin translocated in tile nucleus. Non invasive cells, if previously treated with TGF-[31, show"a similar molecular and functional behaviour in the presence of Ln-5. After incubation with Ln-5, both invasive and non invasive HCC cells show an up-regulation of SNAIL and/or SLUG transcription factor followed by a down-regulation of E-cadherin. Therefore, it is likely that microenvironmental factors such as Ln-5 and TGF-['51 induce an epithelialmesenchymal transition (EMT), leading to a more invasive and aggressive HCC phenotype. Finally, different Ln-5 biological events such as scattering and EMT are mediated by 0~3[~1 and a6[M integrins, respectively. In conclusion, we propose a scenario whereby HCC cells undergo EMT under stimulation with ECM proteins such as Ln-5 and with TGF-[31. To our knowledge, this is the first time that Ln-5 has been reported to induce EMT of cancer cells. This opens new strategies for targeting biological drugs and emphasizes tile role of the tissue microenvironment in modulating tile behavior of HCC cancer cells.
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RECURRENT REARRENGEMENT REGION OF CHROMOSOME 19 TARGETED BY HEPATITIS B VIRUS DNA INTEGRATION IN HEPATOCELLULAR CARCINOMA
K. Saigo 1,3, K. Yoshida2, R. lkeda 2, T. Urashima3, T. Ochiai 3, T. Kenmochi I , M. Maruyama 1, C. Iwashita1, K. Otuki 1, T. Asano 4, Y. Murakami 5, I. Inoue 2. 1Department of Surgery, Chiba-East National
Hospital, Chiba, Japan," 2Derision of Genetic Diagnosis, Institute of Medical Science, Unioersity of Tokyo, Tokyo, Japan," SSecond Department of Surgery. Chiba UnioersitySchool of Medicine, Chiba, Japan," 4Digestive Surgery, Chiba Cancer Cente~ Chiba, Japan," 5Laboratory of Human Tumor Virus,Institute For Virus Research, Kyoto University, Kyoto, Japan Hepatitis B virus (HBV) infection is an important risk factor for the development of hepatocellular carcinoma (HCC) and also HBV-DNA integration into tile cellular DNA is frequently detected in HCC. According to a recent reports, about 30% of HBV-related HCCs, HBV-DNA integrated into host genome resulting in the generation of novel fusion transcripts or tile genome instability, which lead to cell proliferation and viability. In woodchuck animal model, Woodchuck hepatitis virus (WHV) frequently integrated into myc family gene and induced liver tumor. However the preferential sites of HBV integration and the mechanism of HBV tumorigenesis remain obscure. We have therefore applied a PCR based method to investigate integration sites of HBV-DNA.
19
Methods: We have examined 10 HCC specimens, which are all anti-
HBc positive, with using a PCR based methods to amplify viral-cellular DNA junction. Subsequently amplified DNA was sequenced to examine the integration sites of vital DNA. Results: Using an adaptor-ligation suppression PCR with primers specific for adaptor and HBx sequences, we could isolate 9 viral cellular junctions from 7 samples. Two integration sites were into human telomerase reverse transcriptase (hTERT) gene, four integration sites were into a particular site of chromosome 19q13.1, and the remaining three were into a hypothetical protein and ESTs. The integration sites of the gene on 19q13.1 were within 240bp of flanked with Alu sequences and the gone could be implicated in tumorigenesis. In addition, we detected reciprocal chromosomal translocation between this region of 19q13.1 and specific region of chromosome 17p 11.2 in 22 cases from 26 HCC samples. Conclusions; Our results support the hypothesis that 1) the HBV-DNA integrations may occur randomly throughout the genome but the tumorigenic HBV-DNA integ-ration sites might not be random. 2) Translocation breakpoint is one o f the preferential target for HBV integrations in HCC. 3) HBV integration frequently occurs into or adjacent to genes that most likely have important roles in tumorigenesis.
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P53-SELECTIVE ONCOLYTIC ADENOVIRUS ADP53DPR DOES NOT REPLICATE IN PRIMARY HUMAN HEPATOCYTES BUT EFFICIENTLY LYSES P53-ALTERED TUMORS IN VITRO AND IN VIVO
E Kuehnel, L. Zender, T.C. Wirth, B. Schulte, M.E Manns, S. Kubicka.
Gastroenterology, Hepatology and Endocrinology, Medical School Hannover, Hannover, Germany A major prerequisite of oncolytic virotherapy is tile tight restriction of viral replication to malignant cells. This study presents tile bicistronic adenoviral vector Adp53dpR for virotherapy of tumors with altered p53status. The conditionally replicating Adp53dpR was constructed by inclusion of two interacting cistrons. The first cistron contains E1A under control of an artificial GAL4-affine CMV-promoter which can be efficiently inhibited by GAL4-KtL'kB. The second cistron contained GAL4KRAB controlled by an artificial p53 sensitive promoter (prMinRGC). prMinRGC showed strong upregulation by active p53 but was almost inactive in p53 altered cells. A comparable control vires AdR, lacking the p53 dependent promoter, was also consmtcted. In Doxombicin treated HepG2 cells infected by Adp53dpR viral replication and cell lysis was inhibited whereas the control vires showed normal replication. In Huh7 cells both vector types showed an equivalent replication rate. Doxorubicin pretreatment significantly enhanced tile selectivity of the oncolytic vector by timely upregulation of p53-dependent repressor expression thereby preventing E1 A-mediated inhibilion of p53-dependent transcription. These results were confirmed in a subset of p53 wt and p53 altered cell lines. Most important, p53 selectivity was additionally confirmed in Doxorubicin pretreated primary human hepatecytes where Adp53dpR was unable to replicate, p53 dependent relation between GAL4-KRAB and E1A expression and dose correlation to doxorubicin pretreatment was shown by western blot analysis in Adp53dpR infected p53-wt cells, which could not be observed when the control virus was used. In contrast, in p53 altered cell lines infected with either Adp53dpR or control, no GAL4-KRAB could be detected and E1A was fully expressed. In cell lysis assays Adp53dpR showed slightly reduced oncolytic properties compared to Ad-wt but was more efficient and p53-selective compared to ONYX-015. The vector was also more efficient in the treatment of s.c. grown Hep3B xenografis and resulted in partial remissions. Together these data suggest that Adp53dpR represents an effective tool for specific virotherapy in p53-altered cancer cells and might help to reduce side effects on healthy tissue. Regarding p53 selectivity and oncolytic properties Adp53dpR displays an improved profile compared to ONYX-015.
G. Giannelli, C. Bergamini, E. Fransvea, C. Sgarra, S. Antonaci.
Department of Internal Medicine, Immunology and Infectious Diseases, Section of Internal Medicine, University of Bari, Policlinico, Bari, Italy Hepatecellular carcinoma (HCC) is the fifth most frequent malignancy in the world, and the third cause of tumor-related death. Prognosis and survival are still poor. The different behavior of HCC is likely attributable to metastasis occurrence, in particular to blood vessels. Previously we reported that an extracellular matrix protein (ECM) named Laminin-5 (Ln5), absent in normal liver, is expressed in a high percentage of HCC tissues. In particular, the ¥-2 chain, is strongly (96%) related to metastasis occurrence and to shorter survival. Goal of this study was to investigate how HCC cells acquire the ability to spread to surrounding tissue. HCC invasive cells produce detectable levels of the Ln-5 "(-2 chain, whereas non invasive cells do not. However, after TGF-[31 stimulation, non invasive cells also produce the Ln-5 "~-2 chain. In addition, HCC fiwasive and non invasive cells express similar levels and localization of[3-catenin, whereas low and high levels of E-cadherin are expressed in invasive and non invasive cells, respectively. In the presence of Ln-5, the invasive cells strongly scatter after few hours while the non fiwasive do not. In invasive cells E-cadherin is delocalized and [3-catenin translocated in tile nucleus. Non invasive cells, if previously treated with TGF-[31, show"a similar molecular and functional behaviour in the presence of Ln-5. After incubation with Ln-5, both invasive and non invasive HCC cells show an up-regulation of SNAIL and/or SLUG transcription factor followed by a down-regulation of E-cadherin. Therefore, it is likely that microenvironmental factors such as Ln-5 and TGF-['51 induce an epithelialmesenchymal transition (EMT), leading to a more invasive and aggressive HCC phenotype. Finally, different Ln-5 biological events such as scattering and EMT are mediated by 0~3[~1 and a6[M integrins, respectively. In conclusion, we propose a scenario whereby HCC cells undergo EMT under stimulation with ECM proteins such as Ln-5 and with TGF-[31. To our knowledge, this is the first time that Ln-5 has been reported to induce EMT of cancer cells. This opens new strategies for targeting biological drugs and emphasizes tile role of the tissue microenvironment in modulating tile behavior of HCC cancer cells.
•
]
RECURRENT REARRENGEMENT REGION OF CHROMOSOME 19 TARGETED BY HEPATITIS B VIRUS DNA INTEGRATION IN HEPATOCELLULAR CARCINOMA
K. Saigo 1,3, K. Yoshida2, R. lkeda 2, T. Urashima3, T. Ochiai 3, T. Kenmochi I , M. Maruyama 1, C. Iwashita1, K. Otuki 1, T. Asano 4, Y. Murakami 5, I. Inoue 2. 1Department of Surgery, Chiba-East National
Hospital, Chiba, Japan," 2Derision of Genetic Diagnosis, Institute of Medical Science, Unioersity of Tokyo, Tokyo, Japan," SSecond Department of Surgery. Chiba UnioersitySchool of Medicine, Chiba, Japan," 4Digestive Surgery, Chiba Cancer Cente~ Chiba, Japan," 5Laboratory of Human Tumor Virus,Institute For Virus Research, Kyoto University, Kyoto, Japan Hepatitis B virus (HBV) infection is an important risk factor for the development of hepatocellular carcinoma (HCC) and also HBV-DNA integration into tile cellular DNA is frequently detected in HCC. According to a recent reports, about 30% of HBV-related HCCs, HBV-DNA integrated into host genome resulting in the generation of novel fusion transcripts or tile genome instability, which lead to cell proliferation and viability. In woodchuck animal model, Woodchuck hepatitis virus (WHV) frequently integrated into myc family gene and induced liver tumor. However the preferential sites of HBV integration and the mechanism of HBV tumorigenesis remain obscure. We have therefore applied a PCR based method to investigate integration sites of HBV-DNA.
19
Methods: We have examined 10 HCC specimens, which are all anti-
HBc positive, with using a PCR based methods to amplify viral-cellular DNA junction. Subsequently amplified DNA was sequenced to examine the integration sites of vital DNA. Results: Using an adaptor-ligation suppression PCR with primers specific for adaptor and HBx sequences, we could isolate 9 viral cellular junctions from 7 samples. Two integration sites were into human telomerase reverse transcriptase (hTERT) gene, four integration sites were into a particular site of chromosome 19q13.1, and the remaining three were into a hypothetical protein and ESTs. The integration sites of the gene on 19q13.1 were within 240bp of flanked with Alu sequences and the gone could be implicated in tumorigenesis. In addition, we detected reciprocal chromosomal translocation between this region of 19q13.1 and specific region of chromosome 17p 11.2 in 22 cases from 26 HCC samples. Conclusions; Our results support the hypothesis that 1) the HBV-DNA integrations may occur randomly throughout the genome but the tumorigenic HBV-DNA integ-ration sites might not be random. 2) Translocation breakpoint is one o f the preferential target for HBV integrations in HCC. 3) HBV integration frequently occurs into or adjacent to genes that most likely have important roles in tumorigenesis.
[
•
T
H
E
P53-SELECTIVE ONCOLYTIC ADENOVIRUS ADP53DPR DOES NOT REPLICATE IN PRIMARY HUMAN HEPATOCYTES BUT EFFICIENTLY LYSES P53-ALTERED TUMORS IN VITRO AND IN VIVO
E Kuehnel, L. Zender, T.C. Wirth, B. Schulte, M.E Manns, S. Kubicka.
Gastroenterology, Hepatology and Endocrinology, Medical School Hannover, Hannover, Germany A major prerequisite of oncolytic virotherapy is tile tight restriction of viral replication to malignant cells. This study presents tile bicistronic adenoviral vector Adp53dpR for virotherapy of tumors with altered p53status. The conditionally replicating Adp53dpR was constructed by inclusion of two interacting cistrons. The first cistron contains E1A under control of an artificial GAL4-affine CMV-promoter which can be efficiently inhibited by GAL4-KtL'kB. The second cistron contained GAL4KRAB controlled by an artificial p53 sensitive promoter (prMinRGC). prMinRGC showed strong upregulation by active p53 but was almost inactive in p53 altered cells. A comparable control vires AdR, lacking the p53 dependent promoter, was also consmtcted. In Doxombicin treated HepG2 cells infected by Adp53dpR viral replication and cell lysis was inhibited whereas the control vires showed normal replication. In Huh7 cells both vector types showed an equivalent replication rate. Doxorubicin pretreatment significantly enhanced tile selectivity of the oncolytic vector by timely upregulation of p53-dependent repressor expression thereby preventing E1 A-mediated inhibilion of p53-dependent transcription. These results were confirmed in a subset of p53 wt and p53 altered cell lines. Most important, p53 selectivity was additionally confirmed in Doxorubicin pretreated primary human hepatecytes where Adp53dpR was unable to replicate, p53 dependent relation between GAL4-KRAB and E1A expression and dose correlation to doxorubicin pretreatment was shown by western blot analysis in Adp53dpR infected p53-wt cells, which could not be observed when the control virus was used. In contrast, in p53 altered cell lines infected with either Adp53dpR or control, no GAL4-KRAB could be detected and E1A was fully expressed. In cell lysis assays Adp53dpR showed slightly reduced oncolytic properties compared to Ad-wt but was more efficient and p53-selective compared to ONYX-015. The vector was also more efficient in the treatment of s.c. grown Hep3B xenografis and resulted in partial remissions. Together these data suggest that Adp53dpR represents an effective tool for specific virotherapy in p53-altered cancer cells and might help to reduce side effects on healthy tissue. Regarding p53 selectivity and oncolytic properties Adp53dpR displays an improved profile compared to ONYX-015.