Genetic inactivation of the Fanconi anemia gene FANCC identified in the hepatocellular carcinoma cell line HuH-7 confers sensitivity towards DNA-interstrand crosslinking agents

Andreas Palagyi, Kornelia Neveling, Ursula Plinninger, Andreas Ziesch, Bianca Sabrina Targosz, Gerald U. Denk, Stephanie Ochs, Antonia Rizzani, Daniel Meier, Wolfgang E. Thasler, Helmut Hanenberg, Enrico N. De Toni, Florian Bassermann, Claus Schäfer, Burkhard Göke, Detlev Schindler, Eike Gallmeier

Research output: Contribution to journalArticle

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Abstract

Background: Inactivation of the Fanconi anemia (FA) pathway through defects in one of 13 FA genes occurs at low frequency in various solid cancer entities among the general population. As FA pathway inactivation confers a distinct hypersensitivity towards DNA interstrand-crosslinking (ICL)-agents, FA defects represent rational targets for individualized therapeutic strategies. Except for pancreatic cancer, however, the prevalence of FA defects in gastrointestinal (GI) tumors has not yet been systematically explored.Results: A panel of GI cancer cell lines was screened for FA pathway inactivation applying FANCD2 monoubiquitination and FANCD2/RAD51 nuclear focus formation and a newly identified FA pathway-deficient cell line was functionally characterized. The hepatocellular carcinoma (HCC) line HuH-7 was defective in FANCD2 monoubiquitination and FANCD2 nuclear focus formation but proficient in RAD51 focus formation. Gene complementation studies revealed that this proximal FA pathway inactivation was attributable to defective FANCC function in HuH-7 cells. Accordingly, a homozygous inactivating FANCC nonsense mutation (c.553C > T, p.R185X) was identified in HuH-7, resulting in partial transcriptional skipping of exon 6 and leading to the classic cellular FA hypersensitivity phenotype; HuH-7 cells exhibited a strongly reduced proliferation rate and a pronounced G2 cell cycle arrest at distinctly lower concentrations of ICL-agents than a panel of non-isogenic, FA pathway-proficient HCC cell lines. Upon retroviral transduction of HuH-7 cells with FANCC cDNA, FA pathway functions were restored and ICL-hypersensitivity abrogated. Analyses of 18 surgical HCC specimens yielded no further examples for genetic or epigenetic inactivation of FANCC, FANCF, or FANCG in HCC, suggesting a low prevalence of proximal FA pathway inactivation in this tumor type.Conclusions: As the majority of HCC are chemoresistant, assessment of FA pathway function in HCC could identify small subpopulations of patients expected to predictably benefit from individualized treatment protocols using ICL-agents.

Original languageEnglish (US)
Article number127
JournalMolecular Cancer
Volume9
DOIs
StatePublished - May 28 2010
Externally publishedYes

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Fanconi Anemia
Hepatocellular Carcinoma
Cell Line
DNA
Genes
Hypersensitivity
G2 Phase Cell Cycle Checkpoints
Neoplasms
Gastrointestinal Neoplasms
Nonsense Codon
Clinical Protocols
Pancreatic Neoplasms
Epigenomics

ASJC Scopus subject areas

  • Cancer Research
  • Molecular Medicine
  • Oncology

Cite this

Genetic inactivation of the Fanconi anemia gene FANCC identified in the hepatocellular carcinoma cell line HuH-7 confers sensitivity towards DNA-interstrand crosslinking agents. / Palagyi, Andreas; Neveling, Kornelia; Plinninger, Ursula; Ziesch, Andreas; Targosz, Bianca Sabrina; Denk, Gerald U.; Ochs, Stephanie; Rizzani, Antonia; Meier, Daniel; Thasler, Wolfgang E.; Hanenberg, Helmut; De Toni, Enrico N.; Bassermann, Florian; Schäfer, Claus; Göke, Burkhard; Schindler, Detlev; Gallmeier, Eike.

In: Molecular Cancer, Vol. 9, 127, 28.05.2010.

Research output: Contribution to journalArticle

Palagyi, A, Neveling, K, Plinninger, U, Ziesch, A, Targosz, BS, Denk, GU, Ochs, S, Rizzani, A, Meier, D, Thasler, WE, Hanenberg, H, De Toni, EN, Bassermann, F, Schäfer, C, Göke, B, Schindler, D & Gallmeier, E 2010, 'Genetic inactivation of the Fanconi anemia gene FANCC identified in the hepatocellular carcinoma cell line HuH-7 confers sensitivity towards DNA-interstrand crosslinking agents', Molecular Cancer, vol. 9, 127. https://doi.org/10.1186/1476-4598-9-127
Palagyi, Andreas ; Neveling, Kornelia ; Plinninger, Ursula ; Ziesch, Andreas ; Targosz, Bianca Sabrina ; Denk, Gerald U. ; Ochs, Stephanie ; Rizzani, Antonia ; Meier, Daniel ; Thasler, Wolfgang E. ; Hanenberg, Helmut ; De Toni, Enrico N. ; Bassermann, Florian ; Schäfer, Claus ; Göke, Burkhard ; Schindler, Detlev ; Gallmeier, Eike. / Genetic inactivation of the Fanconi anemia gene FANCC identified in the hepatocellular carcinoma cell line HuH-7 confers sensitivity towards DNA-interstrand crosslinking agents. In: Molecular Cancer. 2010 ; Vol. 9.
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abstract = "Background: Inactivation of the Fanconi anemia (FA) pathway through defects in one of 13 FA genes occurs at low frequency in various solid cancer entities among the general population. As FA pathway inactivation confers a distinct hypersensitivity towards DNA interstrand-crosslinking (ICL)-agents, FA defects represent rational targets for individualized therapeutic strategies. Except for pancreatic cancer, however, the prevalence of FA defects in gastrointestinal (GI) tumors has not yet been systematically explored.Results: A panel of GI cancer cell lines was screened for FA pathway inactivation applying FANCD2 monoubiquitination and FANCD2/RAD51 nuclear focus formation and a newly identified FA pathway-deficient cell line was functionally characterized. The hepatocellular carcinoma (HCC) line HuH-7 was defective in FANCD2 monoubiquitination and FANCD2 nuclear focus formation but proficient in RAD51 focus formation. Gene complementation studies revealed that this proximal FA pathway inactivation was attributable to defective FANCC function in HuH-7 cells. Accordingly, a homozygous inactivating FANCC nonsense mutation (c.553C > T, p.R185X) was identified in HuH-7, resulting in partial transcriptional skipping of exon 6 and leading to the classic cellular FA hypersensitivity phenotype; HuH-7 cells exhibited a strongly reduced proliferation rate and a pronounced G2 cell cycle arrest at distinctly lower concentrations of ICL-agents than a panel of non-isogenic, FA pathway-proficient HCC cell lines. Upon retroviral transduction of HuH-7 cells with FANCC cDNA, FA pathway functions were restored and ICL-hypersensitivity abrogated. Analyses of 18 surgical HCC specimens yielded no further examples for genetic or epigenetic inactivation of FANCC, FANCF, or FANCG in HCC, suggesting a low prevalence of proximal FA pathway inactivation in this tumor type.Conclusions: As the majority of HCC are chemoresistant, assessment of FA pathway function in HCC could identify small subpopulations of patients expected to predictably benefit from individualized treatment protocols using ICL-agents.",
author = "Andreas Palagyi and Kornelia Neveling and Ursula Plinninger and Andreas Ziesch and Targosz, {Bianca Sabrina} and Denk, {Gerald U.} and Stephanie Ochs and Antonia Rizzani and Daniel Meier and Thasler, {Wolfgang E.} and Helmut Hanenberg and {De Toni}, {Enrico N.} and Florian Bassermann and Claus Sch{\"a}fer and Burkhard G{\"o}ke and Detlev Schindler and Eike Gallmeier",
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AU - Palagyi, Andreas

AU - Neveling, Kornelia

AU - Plinninger, Ursula

AU - Ziesch, Andreas

AU - Targosz, Bianca Sabrina

AU - Denk, Gerald U.

AU - Ochs, Stephanie

AU - Rizzani, Antonia

AU - Meier, Daniel

AU - Thasler, Wolfgang E.

AU - Hanenberg, Helmut

AU - De Toni, Enrico N.

AU - Bassermann, Florian

AU - Schäfer, Claus

AU - Göke, Burkhard

AU - Schindler, Detlev

AU - Gallmeier, Eike

PY - 2010/5/28

Y1 - 2010/5/28

N2 - Background: Inactivation of the Fanconi anemia (FA) pathway through defects in one of 13 FA genes occurs at low frequency in various solid cancer entities among the general population. As FA pathway inactivation confers a distinct hypersensitivity towards DNA interstrand-crosslinking (ICL)-agents, FA defects represent rational targets for individualized therapeutic strategies. Except for pancreatic cancer, however, the prevalence of FA defects in gastrointestinal (GI) tumors has not yet been systematically explored.Results: A panel of GI cancer cell lines was screened for FA pathway inactivation applying FANCD2 monoubiquitination and FANCD2/RAD51 nuclear focus formation and a newly identified FA pathway-deficient cell line was functionally characterized. The hepatocellular carcinoma (HCC) line HuH-7 was defective in FANCD2 monoubiquitination and FANCD2 nuclear focus formation but proficient in RAD51 focus formation. Gene complementation studies revealed that this proximal FA pathway inactivation was attributable to defective FANCC function in HuH-7 cells. Accordingly, a homozygous inactivating FANCC nonsense mutation (c.553C > T, p.R185X) was identified in HuH-7, resulting in partial transcriptional skipping of exon 6 and leading to the classic cellular FA hypersensitivity phenotype; HuH-7 cells exhibited a strongly reduced proliferation rate and a pronounced G2 cell cycle arrest at distinctly lower concentrations of ICL-agents than a panel of non-isogenic, FA pathway-proficient HCC cell lines. Upon retroviral transduction of HuH-7 cells with FANCC cDNA, FA pathway functions were restored and ICL-hypersensitivity abrogated. Analyses of 18 surgical HCC specimens yielded no further examples for genetic or epigenetic inactivation of FANCC, FANCF, or FANCG in HCC, suggesting a low prevalence of proximal FA pathway inactivation in this tumor type.Conclusions: As the majority of HCC are chemoresistant, assessment of FA pathway function in HCC could identify small subpopulations of patients expected to predictably benefit from individualized treatment protocols using ICL-agents.

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