Generation and Analysis of Genetically Defined Liver Carcinomas Derived from Bipotential Liver Progenitors

Zender, Lars; Xue, Wei; Cordon‐Cardo, Carlos; Hannon, Gregory J.; Lucito, Robert; Powers, Scott; Flemming, Peer; Spector, Mona S.; Lowe, Scott W.

doi:10.1101/sqb.2005.70.059

“…Generation and analysis of liver tumours. Isolation, culture and retroviral infection of murine hepatoblasts were as described 4,5 . Doxycycline treatment (BD Biosciences) and bioluminescence imaging (Xenogen) was performed according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas

Xue

¹

,

Zender

²

,

Miething

³

et al. 2007

Nature

View full text Add to dashboard Cite

Although cancer arises from a combination of mutations in oncogenes and tumour suppressor genes, the extent to which tumour suppressor gene loss is required for maintaining established tumours is poorly understood. p53 is an important tumour suppressor that acts to restrict proliferation in response to DNA damage or deregulation of mitogenic oncogenes, by leading to the induction of various cell cycle checkpoints, apoptosis or cellular senescence 1,2 . Consequently, p53 mutations increase cell proliferation and survival, and in some settings promote genomic instability and resistance to certain chemotherapies 3 . To determine the consequences of reactivating the p53 pathway in tumours, we used RNA interference (RNAi) to conditionally regulate endogenous p53 expression in a mosaic mouse model of liver carcinoma 4,5 . We show that even brief reactivation of endogenous p53 in p53-deficient tumours can produce complete tumour regressions. The primary response to p53 was not apoptosis, but instead involved the induction of a cellular senescence program that was associated with differentiation and the upregulation of inflammatory cytokines. This program, although producing only cell cycle arrest in vitro, also triggered an innate immune response that targeted the tumour cells in vivo, thereby contributing to tumour clearance. Our study indicates that p53 loss can be required for the maintenance of aggressive carcinomas, and illustrates how the cellular senescence program can act together with the innate immune system to potently limit tumour growth.p53 mutations are common in human liver cancer 6 , which is typically highly aggressive and resistant to non-surgical therapies. To determine the requirement for p53 loss in the maintenance of such carcinomas, we used reversible RNAi 7 to control p53 in a chimaeric liver cancer mouse model (Fig. 1a) 4,5 . Purified embryonic liver progenitor cells (hepatoblasts) were transduced with retroviruses expressing oncogenic ras (HrasV12), the tetracycline transactivator protein tTA ('tet-off') and a tet-responsive p53 miR30 design short hairpin RNA (shRNA; Supplementary Fig. 1a) 7,8 , and seeded into the livers of athymic nude mice following intrasplenic injection 4,5 . To facilitate in vivo imaging, the oncogenic ras retrovirus co-expressed green fluorescent protein (GFP) and, in some experiments, hepatoblasts were also co-transduced with a luciferase reporter.p53 expression was efficiently suppressed in the absence of doxycycline (Dox) and rapidly restored following Dox addition ( Supplementary Fig. 1b, c). On transplantation into the livers of recipient mice, hepatoblast populations co-expressing Ras and the conditional p53 shRNA rapidly produced invasive hepatocarcinomas in the absence of Dox (Fig. 1b), whereas cells expressing each vector alone did not (data not shown). These tumours were GFP-positive and, if expressing luciferase, could be visualized externally by bioluminescence imaging (Fig. 1b).

show abstract

“…Moreover, because the conditional alleles cause the alteration to be present throughout the entire organ, the majority of GEMMs do not recapitulate the human microenvironment where cancer cells and normal cells are both present and interact. Faster and more flexible in vivo models would greatly facilitate the functional characterization of candidate driver alterations and therapeutic targets, as well as produce tractable preclinical models for testing novel therapies.Our laboratory has previously established an orthotopic allograft mouse model for hepatocellular carcinoma that is based on the genetic manipulation of liver progenitor cells followed by the orthotopic transplantation into recipient mice (15,16). This model enabled us to rapidly study genetic interactions during tumorigenesis as well as to identify and functionally validate tumor suppressor genes and oncogenes.…”

mentioning

confidence: 99%

Mouse model of intrahepatic cholangiocarcinoma validates FIG–ROS as a potent fusion oncogene and therapeutic target

Saborowski

¹

,

Saborowski

²

,

Davare

³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Cholangiocarcinoma is the second most common primary liver cancer and responds poorly to existing therapies. Intrahepatic cholangiocarcinoma (ICC) likely originates from the biliary tree and develops within the hepatic parenchyma. We have generated a flexible orthotopic allograft mouse model of ICC that incorporates common genetic alterations identified in human ICC and histologically resembles the human disease. We examined the utility of this model to validate driver alterations in ICC and tested their suitability as therapeutic targets. Specifically, we showed that the fused-in-glioblastoma-c-ros-oncogene1 (FIG-ROS1(S); FIG-ROS) fusion gene dramatically accelerates ICC development and that its inactivation in established tumors has a potent antitumor effect. Our studies establish a versatile model of ICC that will be a useful preclinical tool and validate ROS1 fusions as potent oncoproteins and therapeutic targets in ICC and potentially other tumor types.C holangiocarcinoma, the second most common primary liver cancer, has a dismal prognosis due to its poor responsiveness to existing therapies. It represents 10-25% of primary liver cancers worldwide (1), but is more frequent in Asia, due to a higher prevalence of risk factors such as parasitic infections, biliary-duct cysts, hepatolithiasis, and primary sclerosing cholangitis (2). Intrahepatic cholangiocarcinoma (ICC) develops within the liver parenchyma, shows markers of cholangiocyte differentiation, including positivity for cytokeratin 19, and leads to abundant stromal desmoplasia. Whereas surgical resection can be curative, most patients are not eligible for this procedure due to advanced disease, and no effective systemic therapy has been developed to date (3, 4).Sequencing efforts are beginning to generate in-depth information about the somatic alterations that occur in cholangiocarcinoma. Among the most frequently mutated genes are the tumor suppressor tumor protein p53 (TP53) (37-44%) (5, 6) and the kirsten rat sarcoma viral oncogene homolog (KRAS) (17-54%) (6-8), although mutations in MLL3 and SMAD4 are also common (15% and 17% of cases, respectively) (6). Despite their high mutational frequency, none of these signature genes is amenable to targeted therapy.Recently, the mutational spectrum of cholangiocarcinoma has been extended by the identification of ROS1 fusions in nearly 9% of cholangiocarcinoma patients (9). Oncogenic fusion kinases involving the orphan receptor tyrosine kinase ROS1 have been previously reported at low frequency in lung adenocarcinoma and glioblastoma and result from chromosomal rearrangements that lead to constitutive activation of the ROS1 kinase activity. Consequently, ROS1 presents a potential drug target for a subset of patients with ICC.Mouse models have proven a powerful tool for understanding the relationship between cancer genetics and tumor behavior, but only a few genetically engineered mouse models (GEMMs) of ICC exist and many display a mixed ICC/hepatocellular carcinoma (HCC) histology (10-13). A histopathologica...

show abstract

“…To determine the requirement for p53 loss in the maintenance of such carcinomas, we used reversible RNAi 7 to control p53 in a chimaeric liver cancer mouse model (Fig. 1a) 4,5 . Purified embryonic liver progenitor cells (hepatoblasts) were transduced with retroviruses expressing oncogenic ras (HrasV12), the tetracycline transactivator protein tTA ('tet-off') and a tet-responsive p53 miR30 design short hairpin RNA (shRNA; Supplementary Fig.…”

mentioning

confidence: 99%

“…Consequently, p53 mutations increase cell proliferation and survival, and in some settings promote genomic instability and resistance to certain chemotherapies 3 . To determine the consequences of reactivating the p53 pathway in tumours, we used RNA interference (RNAi) to conditionally regulate endogenous p53 expression in a mosaic mouse model of liver carcinoma 4,5 . We show that even brief reactivation of endogenous p53 in p53-deficient tumours can produce complete tumour regressions.…”

mentioning

confidence: 99%

“…Purified embryonic liver progenitor cells (hepatoblasts) were transduced with retroviruses expressing oncogenic ras (HrasV12), the tetracycline transactivator protein tTA ('tet-off') and a tet-responsive p53 miR30 design short hairpin RNA (shRNA; Supplementary Fig. 1a) 7,8 , and seeded into the livers of athymic nude mice following intrasplenic injection 4,5 . To facilitate in vivo imaging, the oncogenic ras retrovirus co-expressed green fluorescent protein (GFP) and, in some experiments, hepatoblasts were also co-transduced with a luciferase reporter.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Erratum: Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas

Xue¹,

Zender²,

Miething³

et al. 2011

Nature

View full text Add to dashboard Cite

Although cancer arises from a combination of mutations in oncogenes and tumour suppressor genes, the extent to which tumour suppressor gene loss is required for maintaining established tumours is poorly understood. p53 is an important tumour suppressor that acts to restrict proliferation in response to DNA damage or deregulation of mitogenic oncogenes, by leading to the induction of various cell cycle checkpoints, apoptosis or cellular senescence 1,2 . Consequently, p53 mutations increase cell proliferation and survival, and in some settings promote genomic instability and resistance to certain chemotherapies 3 . To determine the consequences of reactivating the p53 pathway in tumours, we used RNA interference (RNAi) to conditionally regulate endogenous p53 expression in a mosaic mouse model of liver carcinoma 4,5 . We show that even brief reactivation of endogenous p53 in p53-deficient tumours can produce complete tumour regressions. The primary response to p53 was not apoptosis, but instead involved the induction of a cellular senescence program that was associated with differentiation and the upregulation of inflammatory cytokines. This program, although producing only cell cycle arrest in vitro, also triggered an innate immune response that targeted the tumour cells in vivo, thereby contributing to tumour clearance. Our study indicates that p53 loss can be required for the maintenance of aggressive carcinomas, and illustrates how the cellular senescence program can act together with the innate immune system to potently limit tumour growth.p53 mutations are common in human liver cancer 6 , which is typically highly aggressive and resistant to non-surgical therapies. To determine the requirement for p53 loss in the maintenance of such carcinomas, we used reversible RNAi 7 to control p53 in a chimaeric liver cancer mouse model (Fig. 1a) 4,5 . Purified embryonic liver progenitor cells (hepatoblasts) were transduced with retroviruses expressing oncogenic ras (HrasV12), the tetracycline transactivator protein tTA ('tet-off') and a tet-responsive p53 miR30 design short hairpin RNA (shRNA; Supplementary Fig. 1a) 7,8 , and seeded into the livers of athymic nude mice following intrasplenic injection 4,5 . To facilitate in vivo imaging, the oncogenic ras retrovirus co-expressed green fluorescent protein (GFP) and, in some experiments, hepatoblasts were also co-transduced with a luciferase reporter.p53 expression was efficiently suppressed in the absence of doxycycline (Dox) and rapidly restored following Dox addition ( Supplementary Fig. 1b, c). On transplantation into the livers of recipient mice, hepatoblast populations co-expressing Ras and the conditional p53 shRNA rapidly produced invasive hepatocarcinomas in the absence of Dox (Fig. 1b), whereas cells expressing each vector alone did not (data not shown). These tumours were GFP-positive and, if expressing luciferase, could be visualized externally by bioluminescence imaging (Fig. 1b).

show abstract

Generation and Analysis of Genetically Defined Liver Carcinomas Derived from Bipotential Liver Progenitors

Cited by 63 publications

References 49 publications

Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas

Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas

Mouse model of intrahepatic cholangiocarcinoma validates FIG–ROS as a potent fusion oncogene and therapeutic target

Erratum: Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas

Contact Info

Product

Resources

About