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

Fanconi anemia (FA) is a rare recessive disorder, characterized by congenital skeletal abnormalities, progressive bone marrow failure and an increased cancer susceptibility [1]. The disease is caused by bi-allelic mutations in one of 13 FA genes, all of which have now been identified [2]. The FA genes appear to act in a common pathway of DNA damage signalling and DNA remodelling, distal parts of which interact with regulators of cell cycle control and DNA repair, especially the repair of DNA interstrand-crosslinks.

There are three consecutive compartments of the FA pathway [3]. The proximal compartment consists of eight FA proteins (A, B, C, E, F, G, L, and M), which form a nuclear FA core complex upon activation. This complex functions as an E3 ligase and mediates the monoubiquitination of FANCD2 [4], which represents the central FA pathway protein. FANCI also becomes monoubiquitinated during this process [5, 6] and cooperates with FANCD2 in the ID (FANCI/FANCD 2) complex [7, 8]. The activated proteins of the ID complex subsequently co-localize with proteins of the distal FA pathway compartment (FANCD1/BRCA2, FANCN, and FANCJ) and with other DNA-repair proteins such as RAD51 at sites of DNA-damage. Cells having a defect in one of the proximal FA core complex genes are deficient in FANCD2/FANCI monoubiquitination and FANCD2/FANCI nuclear focus formation. Similarly, cells having a defect in one of the distal FA pathway genes FANCD1 or FANCN are deficient in RAD51 focus formation [8‐12]. Cells with a defect in one of the ID complex proteins lack the respective protein and are defective in monoubiquitination of the other. Thus, inactivation of the FA pathway can comprehensively be identified at the cellular level by assays detecting FANCD2 monoubiquitination and FANCD2/RAD51 focus formation.

FA pathway inactivation occurs sporadically in a variety of tumor types of non-FA patients, suggesting a role of the FA genes in tumor suppression or maintenance of genomic stability among the general population. Distal FA pathway inactivation via mutations in FANCD1 occurs in familial cases of breast (2-25%) [13] and ovarian cancer (2-6%) [14], in familial cases of pancreatic cancer (17%) [15] and in sporadic cancers of various tumor entities [16‐18]. In comparison, genetic inactivation of the proximal FA pathway appears to occur infrequently in tumors among the general population and has, in terms of GI cancers, yet only been reported in pancreatic cancer, where it was associated with rare mutations in FANCC or FANCG [19, 20]. In addition, germline mutations of FANCC might contribute to the tumorigenesis or tumor progression of pancreatic cancer [20‐22]. Finally, epigenetic inactivation of the proximal FA pathway via hypermethylation of FANCF has been reported in a variety of tumor entities [23‐27], but its significance is not yet well understood [28, 29].

Unlike the setting in FA patients, FA pathway-deficient tumors arising in patients of the general population harbor the FA gene defect exclusively in the tumor cells, whereas stroma and all other non-malignant cells lack the defect, thus representing a tumor-specific, absolute biochemical difference [30]. As FA pathway-deficient cells are hypersensitive to ICL-agents and PARP inhibitors, FA pathway inactivation in tumors represents a promising target for rational, genotype-based anticancer therapy [31‐38].

The prevalence of FA pathway defects has not yet been systematically investigated in GI cancer. We assessed proximal and distal FA pathway function in 48 cell lines derived from gastric, pancreatic, colorectal, hepatocellular and cholangiocellular carcinomas applying assays for FANCD2 monoubiquitination and FANCD2/RAD51 focus formation [39]. We newly identified a single cell line, HuH-7, derived from a hepatocellular carcinoma (HCC), which exhibited a proximal FA pathway defect, ascribable to genetic FANCC inactivation. When compared to four other HCC cell lines, HuH-7 cells exhibited an increased sensitivity towards ICL-agents, which was reversible in these cells by genetic correction through FANCC overexpression. Our data represent the first evidence for genetic inactivation of the proximal FA pathway in HCC and further support the assumption that genetic inactivation of the proximal FA pathway is a rare event in solid tumors among the general population.

We report here the identification and functional characterization of an inactivating nonsense FANCC mutation in the HCC cell line HuH-7. This cell line was established from a well-differentiated HCC of a 57 year-old Japanese male patient [42], displays an aneuploid phenotype with an average number of 60 chromosomes, and is negative for HBV and HCV [43, 44]. To our knowledge, this is the first evidence of genetic inactivation of the proximal FA pathway in a GI tumor entity other than pancreatic cancer.

The identified FANCC nonsense mutation c.553C > T, p.R185X in HuH-7 represents a known FA mutation, first described by Gibson et al. [45]. Interestingly, non-splice site nonsense mutations can cause exon-skipping through aberrant splicing [46], and accordingly, the c.553C > T mutation has been reported to cause partial transcriptional skipping of exon 6 of FANCC in an FA patient [45], a mechanism confirmed for HuH-7 in our study.

Unfortunately, no matched non-malignant tissue is available for HuH-7, precluding definitive genomic copy number analyses in regard to whether the identified FANCC mutation represents a homo- or hemizygous mutation in this cell line. However, according to copy number analyses by the Sanger Institute (Cambridge, UK) using high-density single nucleotide polymorphism (SNP) arrays (SNP 6.0) [47], HuH-7 harbors three nearly identical copies of the chromosomal arm 9q, where FANCC is located at 9q22.3, as evidenced by virtually exclusive homozygosity of all SNPs assessed on 9q. According to proposed evaluation models for the identification of LOH events where no matching normal tissue is available, these data are strongly suggestive (although not definitely evidentiary) of allelic loss of one copy of chromosome 9q including the non-mutated FANCC allele in the original tumor (or its precursor cells), followed by repeated duplication of the remaining chromosome 9q, including the mutated FANCC allele, later on [48‐50]. Typical recurrent numerical chromosomal aberrations in HCC include losses on 1p, 4q, 8p, 13q, 16q, and 17p and gains on 1q, 8q, 16p and 20q [51]. Although chromosome 9 is rarely clonally altered on the cytogenetic level in HCC, LOH has been reported for several regions on chromosome 9 including the loci of the FANCC (9q22.3) and the FANCG (9p13) genes [52].

FA pathway defects in tumors require bi-allelic inactivation of one of at least 13 FA genes. On the one hand, these bi-allelic mutations could both be inherited, as applies to tumors occurring in FA-patients. On the other hand, mono-allelic germline mutations of distal FA pathway genes, such as FANCD1, FANCN or FANCJ, confer low to medium penetrance susceptibility for breast/ovarian cancer [12, 13, 53] and, as applies to FANCD1 and FANCN, also for pancreatic cancer [15, 54‐57]. In addition, inherited mono-allelic mutations of proximal FA pathway genes have been associated with the predisposition for or the accelerated development of certain tumors [21, 54, 55, 58]. In particular, germline mutations of FANCC have been described in pancreatic cancer, associated with LOH in the tumor [21, 22]. However, germline and somatic changes in FANCC and FANCG may have comparatively low penetrance for pancreatic cancer [55], which is supported by studies investigating germline mutations of upstream FA pathway genes in sporadic, yet FA-typical tumors among the general population [59]. Nevertheless, as the FANCC mutation in HuH-7 reported in our study represents an established FA mutation and was therefore most likely present in the germline of the patient in mono-allelic form, our data might indicate an increased risk for the development of HCC in individuals of the general population harboring this or other FANCC mutations.

The occurrence of an FA-associated FANCC mutation in HCC could also denote a tissue-specific susceptibility for the development of HCC in FA patients; The majority of solid malignancies seen in FA patients consists of head and neck or gynaecologic carcinomas (5.3%), as reported in a large meta-analysis of 1300 cases [60], but also 2.8% of all FA patients developed liver tumors. These tumors manifested at a significantly younger age than other solid malignancies (median age: 13 years for liver tumors as compared to 26 years for other solid malignancies). In fact, the cumulative probability of liver tumors in FA patients has been estimated to be 46% by age 50 [60]. However, the significance of these data regarding a potential liver-specific cancer susceptibility of FA patients is complicated by the observation that many liver tumors do not proceed to malignancy during the life span of FA patients [60, 61]. In addition, there appears to be a strong association between androgen therapy, which is frequently used for the treatment of bone marrow failure in FA, and the occurrence of liver tumors [60‐63]. Nevertheless, HCC represented the majority (58%) of tumors of 36 FA patients with androgen-related liver tumors in one study [61]. Thus, the association of FA with HCC could be attributable both to the primary tumorigenic effects of FA pathway inactivation in hepatocytes, as well as to potential secondary, amplifying or accelerating effects of androgen therapy in FA patients.

We demonstrated that HCC cells having an inactivated FA pathway display the classic cellular FA phenotype, including a specific hypersensitivity towards ICL-agents, illustrated in HuH-7 by a pronounced cell cycle arrest in G2 upon treatment with MMC at low concentrations and a strongly decreased proliferation rate as compared to a panel of non-isogenic HCC lines. Importantly, this ICL-hypersensitivity phenotype was reversed using an isogenic HuH-7 model of exogenous FANCC expression, confirming that ICL-hypersensitivity in these cells was attributable specifically to inactivation of FANCC. Of note however, IC50-ratios between FANCC-deficient and FANCC-proficient cells were partly smaller in the isogenic model than could have been expected from our results using the non-isogenic model. This observation could be attributable to FA pathway-independent ICL-sensitivity differences among the non-isogenic HCC cell lines, but could also provide further support for our previous hypothesis that constitutive exogenous FANCC over-expression does not completely substitute for physiologically regulated, endogenous FANCC expression [37, 38].

It is well established that systemic chemotherapy lacks effectiveness in unselected HCC patients [64, 65] and HCC are therefore considered largely chemoresistant, at least partially explaining the poor prognosis of this tumor entity [66]. Accordingly, guidelines are currently lacking also regarding the choice of chemotherapeutic agent to use in transarterial chemoembolization (TACE), one of the major treatment modalities for non-surgical patients at advanced HCC stages [67]. Our data indicate that non-FA patients having a FA-deficient HCC might predictably benefit from treatment using ICL-agents. Consequently, assessment of FA pathway function in HCC could facilitate individualized therapeutic approaches, using genotype-based patient stratification in regard to both systemic chemotherapy and TACE.

To get an estimate of the prevalence of FANCC inactivation in HCC, we sequenced cDNA derived from 18 surgical HCC tissue specimens to screen for genetic FANCC inactivation. We further screened these samples for hypermethylation of the FANCC promoter region and for lack of FANCC mRNA expression, as epigenetic FANCC inactivation has previously been reported in acute leukaemia and breast cancer [68, 69]. Additionally, we included FANCG and FANCF in these analyses, as FANCG represents another proximal FA gene that has been described to be mutated in GI cancer, specifically in pancreatic cancer [20, 22], while FANCF has been reported to be epigenetically inactivated in various tumor types [23‐25, 27]. On the genetic level, we found no further inactivating alterations, especially no evidence for complete homozygous gene deletions, inactivating point mutations, small deletions or insertions, in FANCC, FANCG or FANCF, respectively. The detected FANCG variant c.20C > T, p.S7F has been reported in an FA patient of the complementation group G in addition to pathogenic FANCG mutations [70]. There is no information available on the nature of the c.643C > A, p.Q215K variant in FANCG. However, LOH or a second sequence variant was not detected in that tumor either. Additionally, since only two to three overlapping PCR reactions were used to amplify the complete coding sequences of FANCC, FANCG and FANCF, respectively, most potential large intragenic deletions would have been detected. However, this mechanism of proximal FA gene inactivation occurs almost exclusively in FANCA, whereas it appears to be extremely rare in FANCC and has not at all been described in FANCG [20, 71‐73]. On the epigenetic level, we found no evidence for hypermethylation of FANCC, FANCG or FANCF in any of the 18 samples. Consistently, FANCC, FANCG and FANCF were expressed in all samples on the mRNA level.

Our negative screening results for proximal FA pathway inactivation in HCC were not unexpected, as a hypothetical high prevalence should have become evident earlier during clinical trials - manifesting as a selective chemosensitivity of the majority of HCC towards ICL-agents. Nevertheless, the lack of FA mutations in 18 HCC does not rule out rare occurrences of proximal FA pathway inactivation in HCC and is consistent with previous reports on the low prevalence of proximal FA pathway inactivation in various tumor entities among the general population [20‐22, 26, 29, 68]. Future studies applying a higher sample number are required to definitely determine the prevalence of FA pathway inactivation in HCC.

In summary, we identified a HCC cell line harboring an inactivating mutation of the FANCC gene, specifically causing proximal FA pathway inactivation and the classic cellular ICL-hypersensitivity phenotype. Assessment of FA pathway function in HCC could thus represent a novel approach to identify small subgroups of HCC patients expected to predictably benefit from individualized treatment protocols using ICL-agents. However, as proximal FA pathway inactivation occurs at low frequency in HCC and other tumors among the general population, the development of rapid, economic and clinically applicable screening tests for proximal FA pathway inactivation, particularly in primary tumor tissues, remains an indispensable requirement to facilitate future clinical studies [38].