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

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.