Abstract
Aim: To determine whether JUN (the oncogene encoding c-Jun protein) is amplified and overexpressed in hepatocellular carcinoma (HCC). Materials and Methods: DNA copy number aberrations were investigated using a high-density oligonucleotide microarray. DNA copy numbers were determined by fluorescence in situ hybridization. Genomic DNA and mRNA were quantified using real-time quantitative PCR. Results: A novel amplification was found at the chromosomal region 1p32-31 in a JHH-2 HCC cell line within which JUN is amplified and overexpressed. However, no copy number gain of JUN (>2-fold) was observed in 34 primary HCC tumors. Rather, a loss of JUN (<0.5-fold) was seen in 13 (38%) out of the 34 tumors and expression of JUN was significantly lower in 26 (70%) out of the 37 HCC tumors compared with their nontumorous counterparts. Conclusion: Although JUN was amplified and overexpressed in JHH-2 HCC cells, amplification and overexpression of JUN may be rare in primary HCCs.
Hepatocellular carcinoma (HCC) is the fifth most common malignancy in men and the eighth most common in women worldwide and is estimated to cause approximately half a million deaths annually (1). Although the risk factors for HCC are well characterized, the molecular pathogenesis of this widespread type of cancer remains poorly understood (2). Amplification of DNA in specific chromosomal regions plays a crucial role in the development and progression of human malignancies, specifically when proto-oncogenic target genes within those amplicons are overexpressed. To identify genes potentially involved in HCC, we investigated DNA copy number aberrations in human HCC cell lines using high resolution single nucleotide polymorphism (SNP) arrays (3-5). We found that a novel amplification at the chromosomal region 1p32-31 occurs in HCC cell line and that the human oncogene JUN, which lies within the 1p32-31 amplicon, is amplified and overexpressed.
The oncogene JUN encodes the protein c-Jun, a component of the AP-1 transcriptional complex which regulates a wide range of cellular processes, including cell proliferation, death, survival and differentiation (6-8). JUN is the cellular homologue of v-Jun, the transforming oncogene of the avian sarcoma virus 17 (9, 10). Many experimental approaches have indicated that JUN plays an important role in carcinogenesis. Thus, JUN can transform mammalian cells, when coexpressed with an activated oncogene such as Ras or Src (11). Furthermore, transgenic mice expressing JUN develop osteosarcoma in cooperation with c-Fos (12). Recently, several lines of evidence have suggested that JUN is implicated in human cancer, including highly aggressive sarcomas (13), Hodgkin lymphomas (14) and acute myeloid leukemia (15). Amplification of JUN has also recently been described in highly aggressive sarcomas (13) and malignant pleural mesotheliomas (16). Most important in terms of this study, JUN also appears to be crucial for initiation of HCC development in a mouse model (17).
Therefore, overexpression of JUN following amplification may contribute to the initiation or progression of cancer, including HCC. In this study, based on the findings of the SNP-array analysis, we determined if JUN is indeed amplified and overexpressed in primary HCC tumors.
Materials and Methods
Cell lines and tumor samples. A total of 21 liver cancer cell lines (HCC-derived HLE, HLF, PLC/PRF/5, Li7, Huh7, Hep3B, SNU354, SNU368, SNU387, SNU398, SNU423, SNU449, SNU475, JHH-1, JHH-2, JHH-4, JHH-5, JHH-6, JHH-7, Huh1, and the hepatoblastoma line HepG2) were obtained from the Health Science Research Resources Bank (Osaka, Japan) and the American Type Culture Collection (Manassas, VA, USA) and were examined as described previously (3). All cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Paired tumor and nontumor tissues were obtained from 37 HCC patients who underwent surgery at the Hospital of Tokyo Medical and Dental University. All specimens were frozen immediately in liquid nitrogen and stored at −80°C until required. Genomic DNA was isolated using the Puregene DNA isolation kit (Gentra, Minneapolis, MN, USA) and total RNA was obtained using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Thirty-four tumor samples were available for DNA analyses, and 37 paired tumor and nontumor samples were available for mRNA analyses. Prior to the study, informed consent was obtained and the study was approved by Ethics Committees.
SNP array. DNA copy number changes were analyzed by the GeneChip Mapping 100K array (Affymetrix, Santa Clara, CA, USA), as described previously (3). Copy number changes were calculated using the Copy Number Analyzer for Affymetrix GeneChip Mapping Arrays (CNAG; http://www.genome.umin.jp; (18).
Fluorescence in situ hybridization (FISH). FISH was performed using three bacterial artificial chromosomes (BACs), RP11-1040N24, RP11-63G10 and RP11-960H15 as probes (Invitrogen), as described previously (3). The BACs were selected based on their homology to locations in the human genome according to the database provided by the UCSC (http://genome.ucsc.edu/).
Real-time quantitative PCR. Genomic DNA and mRNA were quantified using a real-time fluorescence detection method, as described previously (3). The primers used were as follows: JUN DNA forward: 5′-CAGGTGGCACAGCTTAAACA-3′, reverse: 5′-TTTTTCTCTCCGTCGCAACT-3′; JUN mRNA forward: 5′-CCCCAAGATCCTGAAACAGA-3′, reverse: 5′-CCGTTGCTGGA CTGGATTAT-3′. These primers were designed using Primer3Plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) on the basis of sequence data obtained from the NCBI database (http://www.ncbi.nlm.nih.gov/). Endogenous controls for mRNA and genomic DNA levels were GAPDH and long interspersed nuclear element 1 (LINE-1), respectively.
Statistical analysis. All statistical analyses were performed using SPSS 15.0 software (SPSS Inc., Chicago, IL, USA). The Wilcoxon signed-rank test was used to compare JUN mRNA levels between tumorous and non-tumorous tissues. P-values of <0.05 were considered significant.
Results
Detection of 1p32-31 amplification in a JHH-2 HCC cell line by array analyses. Twenty HCC cell lines were screened for DNA copy number aberrations using Affymetrix GeneChip Mapping 100K array analysis. One of the 20 cell lines, JHH-2, exhibited a high-level copy-number gain that is indicative of gene amplification within the chromosomal region 1p32-31 (Figure 1A). The estimated extent of the region of amplification was 3.9 Mb. This chromosomal region lies between the Affymetrix markers SNP_A-1693528 and SNP_A-1722104 and contains 27 known or predicted protein-coding genes including JUN (Figure 1B). To confirm amplification of JUN, we performed FISH analyses on JHH-2 cells using the BACs RP11-1040N24, RP11-63G10 and RP11-960H15 as probes. The BAC RP11-63G10, which contains JUN, generated an amplified FISH signal (Figure 1D). In contrast, neither of the BACs RP11-1040N24 or RP11-960H15, which correspond to chromosomal regions outside of the amplicon, showed an amplified signal (Figure 1C and E).
DNA copy number and expression of JUN in liver cancer cell lines. Amplification of JUN was further determined by assay of the DNA copy number of JUN in 21 liver cancer cell lines (20 HCC cell lines and the hepatoblastoma line HepG2) by real-time quantitative PCR. For this analysis, the copy number values were normalized by assigning the copy number of genomic DNA derived from normal lymphocytes a value of 1. Copy number changes were counted as gains or losses if the copy number value for a given tumor cell was >2.0 or <0.5, respectively. A copy number gain-of-JUN was observed in 3 out of the 21 cell lines: JHH-2, JHH-7 and PLC/PRF/5 (Figure 2A). Loss of JUN was observed in 1 out of the 21 cell lines: Huh7 (Figure 2A).
A common criterion for the designation of a gene as a putative target of amplification is that its gene amplification leads to its overexpression (19). To determine whether gene amplification leads to overexpression of JUN, we assayed the mRNA level of JUN in the same 21 liver cancer cell lines by real-time quantitative PCR. JUN mRNA was overexpressed in 2 out of the 3 cell lines (JHH-2 and JHH-7 but not PLC/PRF/5) that had copy number gains of the JUN gene (Figure 2B). These findings suggested that JUN could be the target of the 1p32-31 amplicon.
DNA copy number and expression of JUN in primary HCC tumors. To determine if the amplification and overexpression of JUN that was observed in HCC cell lines was relevant to primary human carcinomas, we first determined the copy number of JUN in 34 primary HCCs using a similar method to that used for the HCC cell lines. No copy number gain of JUN (>2-fold) was observed in any of the tumors. Instead, a loss of JUN (<0.5-fold) was seen in 13 (38%) out of the 34 tumors (Figure 3A).
We next further assayed the mRNA level of JUN in paired tumor and nontumor tissues from 37 HCC patients (Figure 3B). The expression of JUN was significantly lower in 26 (70%) of the tumors compared with their nontumorous counterparts (Wilcoxon signed-rank test; p=0.002).
Map of the amplicon at 1p32-31 in the JHH-2 cell line. A, Copy number profiles for chromosome 1 in JHH-2 cells. Copy number values were determined by SNP array analyses. B, The position of the Affymetrix SNP probes, the JUN gene and the three BACs chosen as probes for the FISH experiment based on the UCSC genome database (http://genome.ucsc.edu/) is shown. C-E, Representative images of FISH on metaphase chromosomes from JHH-2 cells using the following BAC probes: RP11-1040N24 (C), RP11-63G10 (D) and RP11-960H15 (E). RP11-63G10 shows an amplified signal (arrow; D), while RP11-1040N24 and RP11-960H15 show one copy and two copies per cell, respectively (arrowheads; C and E).
Discussion
In the present study, we show that JUN is amplified and overexpressed in the JHH-2 HCC cell line. This cell line is derived from a Japanese HCC patient who was seronegative for hepatitis B virus (HBV) surface antigen (20). Furthermore, a copy number gain of JUN (>2-fold) was observed in 3 out of the 21 liver cancer cell lines (including JHH-2) that were tested and 2 of these 3 cell lines (JHH-2 and JHH-7) also showed enhanced mRNA expression of JUN. However, no copy number gain of JUN was observed in 34 primary HCC tumors that were examined. In fact, loss of JUN (<0.5-fold) was seen in 13 (38%) out of the 34 tumors. This loss of JUN in primary HCCs is consistent with previous studies that identified a frequent loss of DNA in HCCs at the chromosomal location where JUN resides (1p32-31) in comparative genomic hybridization and DNA microarray studies (21, 22). These findings suggest that amplification of JUN is rare in primary HCCs. However, the number of primary tumor samples examined in this study was relatively small. Further analysis of a greater number of primary samples is required to confirm the status of JUN amplification in primary HCCs.
The overall levels of the JUN protein product c-Jun are regulated by transcriptional and translational mechanisms and fine-tuning is achieved by post-translational modification, primarily by phosphorylation (23). Our analysis focused on JUN mRNA levels and further study is required to determine the mechanism and functional significance of the regulation of c-Jun protein levels in HCC. Real-time quantitative PCR analyses of primary HCC samples showed that expression of JUN mRNA is lower in tumors than in their nontumorous counterparts. These data are consistent with the fact that JUN is usually not overexpressed in human tumors (7). However, the low level of JUN in tumors does not necessarily imply that JUN does not have oncogenic potential in HCC. Several lines of evidence demonstrate a liver-specific function of JUN for cell survival and cell-cycle progression. JUN is essential for normal hepatogenesis during embryonic development (24, 25). Differentiated hepatocytes also require JUN for cell-cycle progression since conditional knockout of JUN in adult livers reduced the proliferation capacity of hepatocytes after partial hepatectomy, a strong inducer of cell-cycle reentry (26).
DNA copy number and expression of JUN in liver cancer cell lines. A, DNA copy number of JUN in 21 liver cancer cell lines (20 HCC cells and one hepatoblastoma line HepG2) and four different peripheral blood lymphocyte samples (normal cell controls) was evaluated by real-time quantitative PCR with reference to LINE-1 controls. Values are normalized such that the average copy number in genomic DNA derived from normal lymphocytes has a value of 1 (solid horizontal line). A value greater than the cut-off value of 2, which represents a twofold increase in copy number over that of normal lymphocytes, was used to determine copy number gain and is shown as a broken line. A value less than the cut-off value of 0.5, which represents one half of the copy number of normal lymphocytes, was used to determine copy number loss and is shown as a dotted line. B, Relative expression levels of JUN in 21 liver cancer cell lines, as evaluated by real-time quantitative PCR, are shown. The results are presented as a ratio between the expression level of JUN and a reference gene (GAPDH) to correct for variation in the amount of RNA.
DNA copy number and expression level of JUN in primary HCC tumors. A, The copy number of JUN in 34 primary HCC tumors and four normal peripheral blood lymphocyte samples was determined as described in the Materials and Methods Section. B, The relative expression of JUN in paired tumor (T) and nontumor tissues (NT) from 37 patients with primary HCC was determined as described in the Materials and Methods Section.
However, the exact mechanism by which JUN contributes to tumorigenicity remains to be elucidated. JUN is located at the end of cellular signaling cascades that include oncogenes that are important for human tumorigenesis (7). The terminal position of JUN in cellular signaling pathways make JUN a participant in numerous and diverse mechanisms of oncogenesis. In order to fully understand the role of JUN in HCC it is therefore important to clarify the network of downstream target genes of JUN in HCC. Although our examination did not demonstrate the amplification of JUN in primary HCCs, the data presented in this work clearly show JUN amplification and overexpression in JHH-2 HCC cells. The JHH-2 cell line therefore provides an efficient tool for analyses of the relationship between JUN and oncogenesis.
Acknowledgements
This work was supported by Grants-in-Aid for Scientific Research (20590408) from the Japan Society for the Program of Science (to K.Y.).
- Received July 16, 2009.
- Revision received November 3, 2009.
- Accepted November 5, 2009.
- Copyright© 2009 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
