Research ArticleExperimental Studies

Clinical Significance of FANCD2 Gene Expression and its Association with Tumor Progression in Hepatocellular Carcinoma

HISATERU KOMATSU, TAKAAKI MASUDA, TOMOHIRO IGUCHI, SHO NAMBARA, KUNIAKI SATO, QUINGJANG HU, HIDENARI HIRATA, SHUHEI ITO, HIDETOSHI EGUCHI, KEISHI SUGIMACHI, HIDETOSHI EGUCHI, YUICHIRO DOKI, MASAKI MORI and KOSHI MIMORI
Anticancer Research March 2017, 37 (3) 1083-1090;

Abstract

Background/Aim: Fanconi anemia complementation group D2 (FANCD2) gene is vitally involved in DNA damage responses. We investigated the clinical significance of FANCD2 expression in hepatocellular carcinoma (HCC). Patients and Methods: FANCD2 mRNA expression of resected HCC tissues was assessed in two HCC cohorts; Our cases (n=111), and The Cancer Genome Atlas (TCGA; n=371). Gene set enrichment analysis (GSEA) was conducted using the TCGA dataset. Proliferation and invasion assays were performed using siRNAs, and the effect of inhibition of the mechanistic target of rapamycin (mTOR) pathway was evaluated. Results: FANCD2 expression was up-regulated in tumor tissues. Cases with high FANCD2 expression had poorer prognoses in both cohorts, and were associated with larger tumor size and invasive phenotypes. FANCD2 knockdown attenuated proliferation and invasion of HCC cells. FANCD2 expression was suppressed by mTOR inhibition. GSEA supported these findings. Conclusion: Elevated FANCD2 expression in HCC could be a novel biomarker for poor prognosis with potential therapeutic relevance.

Hepatocellular carcinoma (HCC) is the major histological subtype of primary liver cancer, accounting for approximately 70-90% of all cases (1). HCC has been recognized as one of the most threatening malignancies because of the limited availability of radical therapeutic options (2, 3). Thus, identification of novel biomarkers that can predict clinical outcomes in HCC and investigation of molecules which are involved in the tumor progression are both very important for patient treatment.

Fanconi anemia (FA) complementation group D2 (FANCD2) gene encodes the FANCD2 protein, which localizes to DNA repair foci and plays crucial roles as a component in the FA pathway (4). The FANC genes are critically involved in the FA pathway and regulate DNA damage responses and maintain genomic integrity (5, 6). It is well established that dysfunction of the FANC genes confers a high risk of occurrence of certain types of hematological and solid tumor, including liver cancer, due to increased genomic instability (7, 8). Therefore, in general, the biological roles of FANC genes, including FANCD2, have mainly been recognized as being tumor-suppressive (9). However, there is increasing evidence that suggests that aberrant FANCD2 expression is associated with tumor aggressiveness in several types of cancers (10-13). Interestingly, there are seemingly paradoxical findings on FANCD2 expression even in the same cancer types. In breast cancer, for instance, accelerated epithelial tumor formation was observed in a FANCD2-deficient mouse model (14), although there was a strong association between FANCD2 expression and the proliferative state of cancer cells (15).

Taken together, the clinical significance and involvement in cancer progression of FANCD2 have not been fully elucidated in solid types of cancer, including HCC. For this reason, we aimed to determine the significance of FANCD2 expression in HCC.

Materials and Methods

Patients and sample collection. Between 2000 and 2004, 111 patients with HCC who underwent liver resection at the Kyushu University Beppu Hospital and affiliated hospitals were enrolled in this study. Resected HCC tissues were immediately frozen in liquid nitrogen and kept at −80°C until RNA extraction. Corresponding noncancerous liver tissues were also stored (available in 65 out of 111 cases). Registration of clinicopathological characteristics and a prognostic follow-up were conducted after surgery. Written informed consent was obtained from each patient. All protocols in this study met the guidelines of relevant governmental agencies and were approved by the Ethics Review Board of Kyushu University after informed consent was obtained from patients.

RNA preparation, reverse transcription (RT), and quantitative polymerase chain reaction (qPCR). Total RNA from frozen tissue specimens and HCC cell lines was extracted using ISOGEN (Nippon Gene, Tokyo, Japan). The quality assessment of extracted RNA was performed by measuring absorbance, and we confirmed that all samples were of satisfactory quality. cDNA was synthesized by RT from 8 μg total RNA with M-MLV reverse transcriptase (Invitrogen, CA, USA). qPCR was performed using a LightCycler 480 Probe Master kit (Roche Applied Science, Penzberg, Germany). mRNA expression of each gene was quantified using the following specific oligonucleotide primers: FANCD2: 5’-AACTTGGAGGAGATTGATGGTC -3’ (sense) and 5’-CGCTCTTTAGCAGACATGGA-3’ (antisense); glyceraldehyde-3-phosphate dehydrogenase (GAPDH): 5’-AGCCACATCGCTCAGACAC-3’ (sense) and 5’-GCCCAATACGACCAAATCC-3’ (antisense). mRNA amplification conditions consisted of initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 62°C for 10 s, and elongation at 67°C for 10 s.

Acquisition of profiles of gene expression, copy number, and clinical information from The Cancer Genome Atlas (TCGA) dataset. We obtained data on RNA sequencing, single nucleotide polymorphism (SNP) arrays, and corresponding clinical information on HCC cases from The Cancer Genome Atlas (TCGA), via the Broad Institute's Firehose (http://gdac.broadinstitute.org/runs/stddata__2015_11_01/data/LIHC/20151101/). Of the 371 cases with mRNA expression profiles, clinical information was available in 370 cases and information on SNPs was available in 364 cases. Expression profiles of 50 paired noncancerous liver samples were also acquired. Gene copy number alteration profiles calculated from the results of SNP arrays were analyzed to assess the relationships between the copy number alteration and expression of FANCD2. Copy number gain was defined as log-ratios of 0.10 or more.

Gene set enrichment analysis (GSEA). The correlations between FANCD2 mRNA expression and predefined gene signatures in public datasets listed above by GSEA (16) were investigated using the TCGA dataset. Gene sets extracted from the Broad Institute database were as follows: LEE_LIVER_CANCER_SURVIVAL_DN (http://www.broadinstitute.org/gsea/msigdb/cards/LEE_LIVER_CANCER_SURVIVAL_DN), CHIANG_LIVER_CANCER_SUBCLASS_PROLIFERATION_UP (http://www.broadinstitute.org/gsea/msigdb/cards/CHIANG_LIVER_CANCER_SUBCLASS_PROLIFERATION_UP), REACTOME_CELL_CYCLE, (http://www.broadinstitute.org/gsea/msigdb/cards/REACTOME_CELL_CYCLE), and HALLMARK_ PI3K_AKT_MTOR_SIGNALING (http://www.broadinstitute.org/gsea/msigdb/cards/HALLMARK_PI3K_AKT_MTOR_SIGNALING).

Cell lines. Human Hepatoma cell lines HepG2 and PLC/PRF/5 were obtained from the cell bank of RIKEN BioResource Center (Tsukuba, Japan) and the Cell Resource Center for Biomedical Research, Institute of Development, Aging, and Cancer, Tohoku University (Sendai, Japan), respectively. Cells were maintained in Dulbecco's modified Eagle's medium (Gibco, CA, USA) supplemented with 10% fetal bovine serum and (FBS) 1% streptomycin sulfate. All cells were cultured at 37°C in a humidified atmosphere containing 5% CO2.

Transfection with small interfering RNA (siRNA). A FANCD2-specific siRNA, a specific siRNA against mechanistic target of rapamycin (mTOR), and a negative control siRNA were obtained from Thermo Fisher Scientific (Waltham, MA, USA). siRNA oligonucleotides were transfected into the cells using Lipofectamine RNAiMAX (Thermo Fisher Scientific) following the manufacturer's instructions.

Western blot analysis. Total cellular protein (35 μg) was extracted from cultured cells with RIPA lysis buffer and electrophoresed on 10% Tris-Glycine gels (Thermo Fisher Scientific) and then electroblotted onto Immobilon-P Transfer Membranes (Merck Millipore, Billerica, MA, USA) at 70 V for 4 h at 4°C. Proteins were detected using primary antibodies as follows. Rabbit polyclonal antibodies against FANCD2 (Abcam, Cambridge, UK) and mTOR (Cell Signaling Technology, Danvers, MA, USA), and mouse monoclonal antibodies against β-Actin (Santa Cruz Biotechnology, Dallas, TX, USA) were used at a dilution of 1:1000. We used horseradish peroxidase-linked anti-rabbit or anti-mouse immunoglobulin (GE Healthcare Japan, Tokyo, Japan) diluted 1:5000 as secondary antibodies. Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, Billerica, MA, USA) was used for emission and a FUSION Solo 7S SYSTEM (Vilber Lourmat, Marne-la-Vallée, France) was used for detection of chemiluminescence.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation assays. For cell proliferation assays, cells were seeded at 1.0×104 cells/well with 500 μl culture medium per well in 24-well plates 24 h before analysis. On the next day (day 1), cell lines were transfected with each siRNA. MTT assays were conducted on the days 1, 3, 5, and 7 using a Cell Proliferation Kit I (Roche Applied Science) following the manufacturer's protocols. The contents of each well was moved to 96-well plates and their absorbance was measured using an iMark Microplate Absorbance Reader (Bio-Rad Laboratories, Hercules, CA, USA) at a wavelength of 570 nm corrected to 655 nm.

In vitro invasion assays. In vitro Invasion assays were conducted using BD Biocoat Matrigel Invasion Chambers (pore size: 8 mm, 24-well; BD Biosciences). In assays, cells were transfected with each siRNA 24 h before the assays. The cells (5.0×104/500 μl/well) were then placed in the upper chambers of the wells with serum-free medium. The bottom chamber contained medium with 10% FBS as a chemoattractant. After 72 h, the bottom of the chamber insert was stained with Calcein AM (Thermo Fisher Scientific). The cells that had invaded through the membrane to the lower surface were evaluated using a fluorescence plate reader at excitation/emission wavelengths of 485/530 nm.

Treatment of HCC cells with AZD8055 (mTOR kinase inhibitor). Cells were seeded at 5×105/well with 3 ml of culture medium per well in 3 wells each of a 6-well plates. After 24 h, the cells in a well were removed for protein extraction. Next, an mTOR kinase inhibitor AZD8055 (Selleck Chemicals, Houston, TX, USA) was added to the two wells left (total concentration of 1 μM) and cells were incubated for 48 h, after which cells in one of the wells was removed for protein extraction. AZD8055 was then removed by medium exchange. After 48 h incubation, cells in the remaining well were removed for protein extraction.

Statistical analysis. Data were expressed as means±standard deviation, and statistical analysis was performed using Welch's t-tests for continuous variables. Categorical variables were compared using chi-square tests or Fisher's exact tests. Overall survival (OS) was estimated using the Kaplan–Meier method, and the survival curves were compared using the log-rank tests. p-Values less than 0.05 were defined as statistically significant. Data analysis was performed using JMP 11 software (SAS Institute, Cary, NC, USA) or R version 3.1.1 (The R Foundation for Statistical Computing, Vienna, Austria).

Results

FANCD2 mRNA expression was up-regulated in tumor tissues, and high FANCD2 expression predicted poor prognosis and was associated with malignant phenotypes. Firstly, we analyzed FANCD2 expression in HCC tissues and non-cancerous liver tissues using two independent cohorts of HCC cases. In both datasets, FANCD2 expression in HCC was significantly higher (p<0.001) than that in liver tissue (Figure 1A). Analysis of the TCGA dataset revealed a statistically significant correlation between copy number and expression of FANCD2. Copy number gain of FANCD2 was observed in 21.2 % (77 out of 364 cases) of all cases (Figure 1B). Both cohorts were then divided into two groups according to FANCD2 expression level in HCC tissues using the minimum p-value approach (17). In both cohorts, 5-year OS in the group with high FANCD2 expression was significantly poorer than that of those in the low FANCD2 expression group [our HCC cases, p=0.037 (Figure 1C left); TCGA, p<0.001, (Figure 1C right)]. Finally, statistical associations between FANCD2 expression and clinicopathological factors were evaluated. The group with high FANCD2 expression exhibited significantly larger tumor sizes and higher rate of invasion to the portal vein (Table I).

Correlations between FANCD2 expression and predefined gene signature by GSEA. In order to investigate whether the expression levels of FANCD2 were associated with known gene signatures, we applied GSEA to HCC cases from the TCGA datasets. GSEA revealed that FANCD2 expression levels positively correlated with an unfavorable prognostic gene signature (Figure 2A), a proliferative gene signature (Figure 2B), activity of genes involved in cell cycle (Figure 2C), and activity of target genes of the phosphoinositide 3-kinase (PI3K)/RAC-alpha serine-threonine kinases (AKT)/mTOR pathway signaling (Figure 2D).

View this table:
Table I.

Relations between mRNA expression of Fanconi anemia complementation group D2 (FANCD2) and clinicopathological factors for patients with hepatocellular carcinoma (n=111).

FANCD2 knockdown reduced the proliferative and invasive capacity of HCC cells. Next, we performed knockdown experiments in two Hepatoma cell lines, HepG2 and PLC/PRF/5. We confirmed that expression of FANCD2 mRNA and protein were suppressed in the cells transfected with FANCD2-specific siRNA (Figure 3A and B). We then performed cell proliferation and invasion assays and found that FANCD2 knockdown significantly reduced the proliferative capacity of HepG2 and PLC/PRF/5 cells (Figure 3C), and the invasive capacity of PLC/PRF/5 cells (Figure 3D).

Figure 1.
Figure 1.

Clinical significance of Fanconi anemia complementation group D2 (FANCD2) gene expression in hepatocellular carcinoma (HCC) cases. A: FANCD2 mRNA expression levels in our HCC cases and The Cancer Genome Atlas (TCGA) dataset. FANCD2 expression was up-regulated in tumor tissues compared to adjacent liver parenchyma (p<0.001, both cohorts, Welch's t-test). B: FANCD2 expression was positively correlated with its copy number in the TCGA dataset (Spearman's rank correlation coefficient=0.385, p<0.001). Copy number gain of FANCD2 was observed in 21.2% of all cases. C: Five-year overall survival in patients with high FANCD2 expression was significantly poorer than that in patients with low FANCD2 expression (our HCC cases: log-rank p=0.037, TCGA: log-rank p<0.001).

FANCD2 expression was down-regulated by knockdown of mTOR and addition of the mTOR kinase inhibitor AZD8055. FANCD2 has been reported to be targeted by the mTOR pathway in several other cancer types (18-20). We assessed whether such targeting was also found in HCC cells. First, we found that mTOR knockdown led to reduced expression of FANCD2 (Figure 4A). We then assessed the effect of AZD8055, an mTOR pathway inhibitor in clinical application. We found that expression of FANCD2 before AZD8055 treatment (Figure 4B-a) diminished 48 h after treatment (Figure 4B-b). Re-expression of FANCD2 was observed 48 h after removal of AZD8055 (Figure 4B-C).

Figure 2.
Figure 2.

Correlations between Fanconi anemia complementation group D2 (FANCD2) expression and predefined gene signatures by Gene set enrichment analysis in The Cancer Genome Atlas (TCGA) dataset. FANCD2 expression had a significant correlation with an unfavorable prognostic gene signature (A), a proliferative gene signature (B), activity of genes involved in cell-cycle regulation (C), and activity of target genes of the phosphoinositide 3-kinase (PI3K)/RAC-alpha serine-threonine kinases (AKT)/mechanistic target of rapamycin (mTOR) pathway signaling pathway (D).

Discussion

In our clinical study, we found that FANCD2 expression was up-regulated in HCC tissues compared to non-cancerous liver tissues. In survival analysis, higher FANCD2 expression predicted poorer OS in two independent cohorts. GSEA also showed statistically significant relevance between FANCD2 expression and unfavorable prognostic gene signature. Concordantly, analysis of clinicopathological factors showed that higher FANCD2 expression was associated with a larger tumor size and higher rate of invasion to the portal vein, which are recognized as markers of tumor aggressiveness and factors predicting poor therapeutic outcomes in patients with HCC (21, 22). Analysis of the TCGA dataset suggested that FANCD2 expression was at least partially influenced by the extent of its copy number alteration. Copy number gain was observed in approximately 20% of cases and this was considered to be one of the possible causes of elevated expression of FANCD2 in HCC, which is a novel discovery. It is interesting that we found the expression of FANCD2, which is generally assumed to have a tumor-suppressive role, to be up-regulated in tumor tissue compared to non-cancerous tissue. This is an intriguing finding, as this is not a common feature of tumor-suppressor genes (23). These observations suggest that FANCD2 expression is a biomarker for predicting tumor aggressiveness and poor prognosis in HCC. To the best of our knowledge, we have clarified for the first time the clinical significance of FANCD2 expression in HCC.

Figure 3.
Figure 3.

Fanconi anemia complementation group D2 (FANCD2) knockdown reduced proliferative and invasive capacities of hepatocellular carcinoma (HCC) cells. A: FANCD2 mRNA expression in siRNA- and negative control siRNA (si-control)-transfected HCC cells. B: Expression of FANCD2 protein in siRNA- and negative control siRNA-transfected HCC cells. C: FANCD2 knockdown reduced proliferation of HepG2 and PLC/PRF/5 cells. D: FANCD2 knockdown reduced invasion of PLC/PRF/5 cells. *Significantly different from si-control at p<0.05, n.s., not significantly different.

In our experimental study, we showed FANCD2 knockdown significantly reduced the proliferative and invasive capacities of HCC cells in accordance with our clinical findings. Xia et al. recently reported FANCD2 knockdown led to cell-cycle arrest in osteosarcoma cells (24). Consistent with this, GSEA showed a significant correlation between FANCD2 expression and signatures representing the proliferative phenotype or the activity of cell cycle-related genes in our current study. These results may support the reduced proliferation of HCC cells with FANCD2 suppression. Surprisingly, including the influence of FANCD2 on the invasive capacity, these findings suggest that FANCD2 has a tumor-promotory aspect in HCC, and might be a potential therapeutic target in HCC.

We showed a significant correlation between the mTOR signaling pathway activity and FANCD2 expression by GSEA. Moreover, in vitro, we actually found that FANCD2 expression was suppressed by mTOR inhibition in HCC cells. This result agrees with several reports for other malignancies (18-20). These findings indicate that FANCD2 expression is partially regulated by the mTOR signaling pathway as well as the copy number alteration, and also imply that mTOR inhibitor may be a therapy for oncogenic FANCD2.

Figure 4.
Figure 4.

Fanconi anemia complementation group D2 (FANCD2) protein expression was down-regulated by knockdown of the Mechanistic target of rapamycin (mTOR) and addition of an mTOR kinase inhibitor, AZD8055. A: mTOR knockdown led to reduced expression of FANCD2 protein. B: Expression of FANCD2 protein before AZD8055 treatment (a) was diminished after treatment (b). Re-expression of FANCD2 was observed 48 h after removal of AZD8055 (c).

Our findings demonstrate that FANCD2 might play an oncogenic role in HCC progression, and could be a potential therapeutic target, as well as a biomarker for predicting tumor aggressiveness and poor prognosis in HCC. However, the mechanism of how FANCD2 functions as oncogene in HCC remains to be further explored. Consistent with the fact that up-regulation of the FA pathway helps cancer cells maintain resistance to DNA-damaging anticancer agents through increasing the activity of the DNA repair response (25), suppression of FANCD2 expression reduces chemoresistance of HCC cells (26). Further biological investigations are needed to elucidate the detailed molecular mechanisms underlying these unexpected phenomena.

In conclusion, we showed the clinical significance of FANCD2 expression as well as possible involvement of increased FANCD2 expression in tumor progression of HCC. Our findings suggest that FANCD2 expression may serve as a novel biomarker for predicting clinical outcomes and possibly have therapeutic relevance as a promising target molecule in HCC.

Acknowledgements

HCC samples were partly provided by Oita Red Cross Hospital (Oita, Japan), the Hiroshima Red Cross Hospital and Atomic-bomb Survivors Hospital (Hiroshima, Japan), and Iizuka Hospital (Fukuoka, Japan). This research used the super-computing resource provided by the Human Genome Center at the Institute of Medical Science, University of Tokyo (http://sc.hgc.jp/shirokane.html). We thank K. Oda, M. Kasagi, and M Sakuma for their excellent technical assistance. This work was supported by the Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (grant number 15K10168) and OITA Cancer Research Foundation.

Footnotes

  • Disclosure

    All Authors declare that they have no conflicts of interest to disclose.

  • Received December 16, 2016.
  • Revision received February 5, 2017.
  • Accepted February 8, 2017.

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Clinical Significance of FANCD2 Gene Expression and its Association with Tumor Progression in Hepatocellular Carcinoma
HISATERU KOMATSU, TAKAAKI MASUDA, TOMOHIRO IGUCHI, SHO NAMBARA, KUNIAKI SATO, QUINGJANG HU, HIDENARI HIRATA, SHUHEI ITO, HIDETOSHI EGUCHI, KEISHI SUGIMACHI, HIDETOSHI EGUCHI, YUICHIRO DOKI, MASAKI MORI, KOSHI MIMORI
Anticancer Research Mar 2017, 37 (3) 1083-1090;
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