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Clinical Cancer Research Vol. 8, 3178-3186, October 2002
© 2002 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Investigation in Liver Tissues and Cell Lines of the Transcription of 13 Genes Mapping to the 16q24 Region That Are Frequently Deleted in Hepatocellular Carcinoma

Philippe Riou, Raphaël Saffroy, Jérome Comoy, Marine Gross-Goupil, Jean-Paul Thiéry, Jean-François Emile, Daniel Azoulay, Dominique Piatier-Tonneau, Antoinette Lemoine and Brigitte Debuire1

Service de Biochimie et Biologie moléculaire [P. R., R. S., J. C., M. G-G., J-P. T., A. L., B. D.], Service d’Anatomie pathologique [J-F.E.], and Centre Hépatobiliaire Hôpital Universitaire Paul Brousse [D. A.], UPRES 1596-Faculté de Médecine Paris-Sud, 94804 Villejuif Cedex, France, and Génétique Moléculaire et Biologie du Développement, FRE 2376, Centre National de la Recherche Scientifique, 94801 Villejuif Cedex, France [D. P-T.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Many studies have associated chromosomal deletions in the 16q24 region with human cancers, including hepatocellular carcinoma. A more limited region around the microsatellite D16S402 has been shown implicated in the metastatic spread of hepatocellular carcinoma, prostate cancer, and Wilms’ tumors. It is likely that one or more tumor suppressor genes are located in this 16q24 area.

We used SYBR Green reagents to quantify, by real-time quantitative RT-PCR, the production of mRNA for 13 genes mapping to 16q24. The locations of these genes were determined from published human genome sequencing data. We studied mRNA levels in 10 liver tumor tissues, 10 nontumor liver tissues, five hepatoma cell lines, and in isolated hepatocytes. Results were compared with those for loss of heterozygosity observed in the D16S402 region and recurrence.

No down-regulation was observed in tumor tissues. Two genes were consistently overexpressed: OKL38 and CDH13. CDH13, which functions in cell-cell adhesion, seems to be involved in liver carcinogenesis. However, no relationship was observed between the expression of this gene and changes in the D16S402 microsatellite or tumor recurrence. None of the other genes tested seemed to be a good candidate for a major tumor suppressor gene in liver carcinogenesis.

Our results suggest that additional unknown genes involved in carcinogenesis are located in the 16q24 area.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HCC2 is one of the most frequent human cancers worldwide (1) . However, the molecular mechanisms underlying HCC tumorigenesis and tumor metastasis are still poorly understood. HCC, like other solid tumors, seems to develop following multiple genetic events (2) , including the functional inactivation of TSGs and the activation of oncogenes. Previous studies of LOH have suggested that several genomic regions may be involved in liver carcinogenesis. These regions are mostly located on chromosome arms 1p, 4q, 5q, 6q, 8p, 9p, 11p, 13q, 16p, 16q, and 17p, indicating that dysfunctions of diverse tumor or metastasis suppressor genes located on these chromosomes are involved in the development of HCC (3, 4, 5) .

LOH on the long arm of chromosome 16 has been reported to be frequent in several human cancers, including HCC. Analyses of LOH frequencies (6, 7, 8, 9, 10, 11, 12) , comparative genomic hybridization (13, 14, 15) , and aberrant DNA methylation (16 , 17) have suggested that one or more TSGs involved in the development of HCC map to chromosome 16q. LOH on chromosome 16q has been correlated with clinicopathological characteristics, such as the degree of differentiation, size, and the occurrence of metastases, indicating that LOH on chromosome 16 may be involved in the progression of HCC (6 , 12) .

Subsequent molecular analyses in HCC (5 , 10, 11, 12 , 15 , 18, 19, 20) , prostate (21 , 22) , ovarian (23, 24, 25) , breast (26 , 27) , bladder (28) , and Wilms’ (29, 30, 31, 32) tumors have identified the 16q24 region as a major region of LOH, associated with metastatic and aggressive behavior of the cancer (21 , 22 , 29 , 32, 33, 34) . Mashimo et al. (33) recently used microcell-mediated chromosome transfer into a highly metastatic rat prostatic cancer cell line to show that microsatellite marker D16S402 was retained in microcell hybrids displaying significant reduction in lung metastasis. We have shown that changes in the D16S402 microsatellite are frequent in European HCC patients and are correlated with a higher risk of recurrence (35) . LOH studies in several other types of tumor have also suggested that there is at least one TSG near the D16S402 locus (19 , 23 , 26 , 31 , 36) .

Data from the human genome sequencing program were recently published (37) , and information concerning the fine mapping of genes is now available on via the internet.3 We identified 13 genes mapping to the 16q23.3–24.1 chromosomal region between the D16S422 and D16S3037 loci, encompassing a region of approximately 2 cM, including D16S402 (Fig. 1)Citation . We selected only known genes and full-length mRNAs corresponding to unknown genes for study. The expression of these genes has not previously been studied in patients with HCC.



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  		Fig. 1. Genetic map and known function of the 13 genes mapping to the 16q23.3–24.1 chromosomal region.

 
We quantified the expression of these genes in tumoral and nontumoral tissues from the liver and in five hepatoma cell lines. We compared the results obtained for gene expression with those concerning LOH specific to D16S402 and surrounding microsatellite markers and metastatic spread.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue and Cell Specimens.
The Institutional Review Board of the Hôpital Universitaire Paul Brousse and the Committee for Research on Human Subjects at the Faculté de Médecine Paris-Sud approved this research. Liver tumor tissue and noncancerous liver tissues were excised during surgery from 10 patients with HCC. The tissues underwent histological examination by the pathologist and were then frozen and stored at -80°C for RNA and DNA extraction. Particular attention was paid to obtain the "core" part of the tumor to avoid the adjacent noncancerous tissue, as proved by histopathological examination. The clinicopathological features of the HCCs are summarized in Table 1Citation . For each patient, we collected paired peripheral blood samples at a time when the patient was not undergoing surgery, to prevent contamination of the blood with hepatocytes, which may occur during surgery (38) , or specimens of nonhepatic tissue—such as gallbladder, if possible—for use as a normal control for microsatellite analysis. Ten NL tissue samples were obtained from biopsies of donated livers during graft harvesting. These samples were treated in the same way as HCC samples and were used for histological analysis to determine the normal expression profile of the genes studied. Human liver tumor cell lines HepG2, PLC/PRF/C, TONG, HA22TNGH and MAHLAVU were obtained from the American Type Culture Collection. Cells were grown in Dulbecco F12–RPMI 1640 (Life Technologies, Inc., Cergy Pontoise, France), containing 10% FCS and gentamycine, and harvested at 70% confluence. Human hepatocytes were isolated by collagenase digestion from NLs from three cadaveric multiple organ donors, as reported previously (39) .


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  		Table 1 Characteristics of the 10 patients

 
DNA Extraction.
DNA was isolated by a standard proteinase K digestion and phenol/chloroform extraction procedure.

RNA Extraction.
RNA was extracted from 20 mg of tissue or 106 cells with a QIAamp Tissue kit (Qiagen, Courtaboeuf, France) according to the manufacturer’s instructions. A final elution volume of 50 µl was used.

Analysis of Microsatellite Alterations.
Seven microsatellite markers (D16S422, D16S3091, D16S402, WFDC1-GT12, WFDC1-GT20, D16S3061, and D16S3037) spanning the chromosomal region 16q23.3–24.1 and their corresponding PCR primers were chosen from the human genome database (Table 2)Citation . PCR was performed as described previously (35 , 40) . The PCR products were separated by capillary electrophoresis, using an ABI PRISM 310 machine (Applied Biosystems, Foster City, CA).


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  		Table 2 Oligonucleotide primer sequences used

 
Real-time Quantitative PCR.
First-strand cDNA was synthesized in a volume of 80 µl, with TaqMan reverse transcription reagents (Applied Biosystems). We mixed 10x reverse transcriptase buffer (8 µl), 5 mM MgCl2, 500 µM of each dNTP, 2.5 µM random hexamers, 32 units of RNase inhibitor, and 100 units of Multiscribe reverse transcriptase with 4 µg of total RNA. The cycling conditions were 10 min at 25°C, 30 min at 48°C, and 5 min at 95°C. Reactions in which the enzyme was omitted were used as negative controls.

PCR was performed with an ABI PRISM 7700 Sequence Detector and SYBR Green reagents (Applied Biosystems). Specific primers for human ß-actin, LOC083693, AK022605, CDH13, HSBP1, HUMCLPB, KIAA0190, KIAA0703, KIAA1609, MLYCD, OKL38, S1P, TAF1C, and WFDC1 were designed to work in the same cycling conditions (50°C for 2 min to permit uracil N-glycosylase cleavage, 95°C for 10 min, followed by 50 cycles of 95°C for 15 s, and 60°C for 1 min). The specificity of the nucleotide sequences chosen was confirmed by conducting basic local alignment search tool searches. To prevent the amplification of contaminating genomic DNA, the two primers bound to different exons. TaqMan RNase control reagents (Applied Biosystems), designed for ribosomal S18 RNA amplification, were used as a reference to normalize the results. We used 2.5 µl of the reverse transcriptase product for PCR in a final volume of 25 µl. For each PCR, a standard curve was produced, using four 1:10 dilutions of a positive sample. All samples were run in triplicate. The relative amounts of mRNA for each tested gene in the samples were calculated by comparison with standard curves. For each sample, results were normalized, using the S18 RNA value of the calibrator to obtain a final R-gene value. All samples were resolved in a 1.8% agarose gel to confirm the specificity of the PCR.

CDH13 Sequencing Analysis.
PCR was performed in coding exons (exons 1–14) and their surrounding regions using oligonucleotides as previously described (41) . PCR reactions were carried out in a 50 µl volume containing 0.5 µM of each primer, 50 µM of each dNTP, 2.5 µl of formamide, and 2 units of Taq (Q.biogene, Illkirch, France) using a gene Amp PCR system 2400 (Applied Biosystems) and using the following conditions : (a) 94°C (4 min); (b) 40 cycles of 92°C (1 min), 58°C (1 min), 72°C (1 min); and a final extension step at 72°C (7 min). Sequencing was performed on both strands using the ABI Prism dichloro-rhodamine terminator cycle sequencing ready reaction kit (Applied Biosystems) after purification of the PCR products using the Qiaquick PCR purification kit (Qiagen). The sequences were analyzed on an ABI 310 automated sequencer unit (Applied Biosystems).

Statistical Analysis.
Statistical analysis was performed using the SAS software system (SAS Institute Inc., Cary, NC), and the Student’s t test was used to determine the statistical significance of differences of gene expression data according to the samples analyzed.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Analysis of Gene Expression in Patients’ Samples and Cell Lines.
We assessed the expression of genes by evaluating RNA levels in 10 healthy liver tissues to calculate R-gene values for each gene tested, as described in "Materials and Methods." The cutoff point for changes in gene expression, as determined from RNA levels, was set at 3 SDs above or below the mean value for the genes in NL tissues. A 10-fold difference in R-gene values between samples was the maximum observed (for HSBP1 and CDH13 genes). Mean R-gene values in NL, were then compared with the R-gene values of 10 matched NTLs and TLs (Table 3)Citation . Three genes (CDH13, OKL38, and KIAA0190) displayed significantly higher levels of expression in liver samples from patients with HCC than in NL samples. Significantly higher levels of CDH13 (P < 0.05) and OKL38 (P < 0.01) expression were observed in TL than in NL samples. For OKL38, expression levels were also higher in NTL adjacent to TL (P < 0.02). For KIAA0190, a significant difference was observed only between TL and NTL (P < 0.05). No significant up- or down-regulation was observed for the other genes. No significant difference was observed for ß-actin, used as a control.


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  		Table 3 Gene expression in TL, NTL, and NL samples

 
R-gene values were determined for three independent preparations of isolated normal human hepatocytes and for five HCC cell lines (Table 4)Citation . Except for the HSBP1 gene, expression levels tended to be lower in isolated hepatocytes than in NL samples. We compared mRNA levels in isolated hepatocyte preparations and cell lines and found no significant difference in expression level (<10-fold) between any of the cell lines tested, for ß-actin, HSBP1, MLYCD, OKL38 and TAF1C. In contrast, overexpression (expression level more than 10 times that in isolated hepatocytes) was observed for one or several of the following genes: CDH13, S1P, LOC083693, KIAA0703, KIAA1609, HUMCLPB, AK022605, and KIAA0190. Diverse associations were observed between the overexpressed genes, depending on the cell line, with each cell line displaying a specific gene expression pattern. Strong overexpression was observed for KIAA1609, with mRNA levels >50 and >100 times higher in HA22T and MAHLAVU cell lines, respectively, than normal. The MAHLAVU cell line displayed levels of CDH13 gene expression more than 300 times higher than normal, whereas this gene was not expressed in the other cell lines. Similarly, the WFDC1 gene, which was weakly expressed in isolated hepatocytes and four hepatoma cell lines, was not expressed in HepG2.


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  		Table 4 Gene expression in isolated hepatocytes and cell lines

 
Relationship between LOH at 16q23.3–24.1 and Gene Transcription.
LOH determination for the 16q23.3–24.1 region is summarized in Fig. 2Citation . Four of 10 patients (patients 4, 5, 7, and 8) exhibited LOH in this chomosomal region in TL samples. This LOH was also present in the NTL of patients 4 and 5. No tumor sample displayed down-regulation of expression of the genes investigated. LOH of microsatellite loci within or close to each gene was not related to variations in gene expression. The OKL38 and CDH13 genes were overexpressed in four of six patients without LOH. Three of the four patients with LOH displayed OKL38 overexpression whereas only one displayed CDH13 overexpression. In the nontumor sample from patient 4 that displayed LOH, CDH13 and OKL38 were overexpressed (data not shown).



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  		Fig. 2. Gene expression levels and LOH analysis for the 10 patients studied. +, gene expression >3 SD; n, gene expression <=3 SD; {square}, retention of heterozygosity; , not informative; {blacksquare}, LOH.

 
We also checked for LOH in 16q23.3–24.1 microsatellite markers in the five cell lines (Fig. 3)Citation . The presence of one allele in a cell line may be indicative of this allele being uninformative or of LOH, but the presence of both alleles eliminates the possibility of LOH. PLC/PRF/C, TONG, HA22TNGH, and MAHLAVU cell lines displayed one allele for all of the microsatellite markers tested, which is in favor of the existence of LOH at the 16q23.3–24.1 region, taking into account that the seven microsatellite markers tested have a 50–80% probability of being informative. However, the MAHLAVU cell line showed high levels of CDH13 mRNA. In contrast, although two alleles were observed for the D16S402 and for the WFDC1 microsatellite markers in the HepG2 cell line, this latter did not express CDH13 and WFDC1 at the mRNA level.



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  		Fig. 3. Allele numbers in the cell lines.

 
Correlation between mRNA Levels and Clinical and Pathological Features.
The clinical and pathological features or histological characteristics of tumors, such as the number and diameter of nodules, recurrences, and extrahepatic metastases, were correlated with LOH and mRNA gene expression. Despite the small number of subjects, recurrence seems associated with LOH at the D16S402 locus. Seventy-five percent of patients exhibiting LOH had recurrence, whereas only 33% of patients exempt of LOH recurred. This was in agreement with our previous results on a larger series of subjects (35) , although here no statistical significance can be drawn. Overexpression seems not correlated with recurrence or other histological features for any of the genes tested. In contrast, three of the five patients who did not display CDH13 overexpression, four of whom displayed LOH at this locus, suffered metastatic recurrence within 15 months of surgery. Surprisingly, all of the patients (n = 3) without cirrhosis or underlying liver disease displayed overexpression of at least four genes whereas six of the seven patients with cirrhosis or hepatitis displayed no overexpression or the overexpression of only one or two genes. CDH13 was overexpressed in two of seven patients with cirrhosis or hepatitis and in three of three patients without liver disease. In contrast, overexpression of OKL38 seemed to be independent of underlying liver disease (three of three versus four of seven).

CDH13 Mutation Analysis in Cell Lines.
Except for the MAHLAVU cell line, which displays high levels of CDH13 mRNA, a lack of expression was observed in the other cell lines. In the aim to explore whether point mutations in the CDH13 coding sequence might give rise to down-expression of this gene, exons 1–14 and their surrounding regions were sequenced. No base change altering the amino acid sequence was detected in the five cell lines analyzed.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Many studies have shown that chromosomal deletions at the 16q24 region are associated with human cancers, including HCC. A more limited region around the microsatellite D16S402 has been implicated in the metastatic spread of HCC (19 , 35) , prostate cancer (33) , and Wilms’ tumors (31) . There is probably at least one TSG in this 16q24 area. However, no TSG involved in liver carcinogenesis has been identified in this chromosomal region. The inactivation of a TSG requires complete loss or inactivation of both alleles. This occurs generally by mutation of one allele and deletion of the other. This mechanism, initially proposed by Knudson (42) for the retinoblastoma gene, has been confirmed for a large variety of genes and was usually associated with the lack of or down-expression of the gene. The aim of this study was to use quantitative RT-PCR on total RNA isolated from human liver tissues to identify known sequences mapping to this chromosomal region that were down-regulated. To assess the respective contributions of tumor and stromal cells to this phenomenon, gene expression was also studied on total RNA isolated from isolated normal hepatocytes and from hepatocellular carcinoma cell lines.

We studied 13 genes mapping to 16q23.3–24.1. The locations of these genes were determined from published human genome sequencing data (37) . The patterns of expression of several of these genes have never been studied in human tissues.

We quantified mRNA levels by real-time quantitative RT-PCR with SYBR Green reagents. This technique can be used to study a larger number of samples simultaneously than can be studied by Northern blotting. It also provides more accurate and reproducible RNA quantification and requires smaller quantities of tumor tissue. We used two controls to normalize the results: S18 RNase and the ß-actin gene. The ß-actin gene tended to be overexpressed (doubling of expression) in tumor samples. This phenomenon has already been reported in hepatoma and other types of tumor (43, 44, 45) . We, therefore, decided to normalize the final results with S18 RNA. Nevertheless, identical conclusion could be drawn from the results obtained with ß-actin as the reference (data not shown).

Some genes, such as TAF1C, KIAA0703, and HSBP1, were expressed similarly in NL, TL, and adjacent NTL samples, as well as in isolated hepatocytes and hepatoma cell lines. This suggests that these genes are not involved in liver carcinogenesis. Considerable heterogeneous variation was observed in the expression of the other genes, and their role in liver carcinogenesis is unclear. However, the liver carcinogenesis seemed to be most closely associated with the expression of two genes: OKL38 and CDH13.

The expression of OKL38, described as a pregnancy-induced growth inhibitor gene, was similar in cell lines and isolated normal hepatocytes, but was much stronger in tumors and in samples taken from tissue adjacent to tumor than in normal healthy liver tissues. These observations are not consistent with a role for this gene as a TSG in liver carcinogenesis, and the biological significance of the observed changes is unclear.

CDH13 was the only gene to display significantly higher expression in tumor samples than in NL and NTL samples. In contrast, little or no expression of this gene was detected in four of five cell lines tested in this work. CDH13 encodes a protein belonging to the cadherin family of cell surface glycoproteins responsible for selective cell recognition and adhesion (46) . Down-regulation of the CDH13 gene in HepG2 cells, as observed here, was also reported in a previous study (47) . No mutation of the CDH13 gene was found in the cell lines analyzed. Mechanisms such as changes in the trans-acting elements specific for the CDH13 gene promoter or methylation of the promoter region leading to gene silencing may be involved in the down-regulation of expression observed in cell lines. It is known that the cell-cell adhesion of cadherin is mediated by cytoplasmic proteins such as catenins. Mutation of the ß-catenin gene occurs in 15–20% of HCCs (48, 49, 50) , which can contribute to cancer invasion and metastasis. Some experimental and clinical reports have previously suggested a role for CDH13 in breast and lung carcinogenesis and metastasis progression (41 , 47 , 51, 52, 53) . In particular, decreases in CDH13 production have frequently been observed in breast cancer (47 , 52 , 54) . The transfection of a breast carcinoma cell line displaying low levels of CDH13 gene expression with a cDNA encoding H-cadherin resulted in significant inhibition of tumor growth (47) . In contrast, the study carried out by Mashimo et al. (33) on microcell-mediated chromosome 16 transfer in a highly metastatic rat prostatic cancer cell line suggested that CDH13 is not involved in the metastasis of this tumor type. Wyder et al. (55) recently showed that mouse H-cadherin is overexpressed on the membranes of endothelial cells of tumor-penetrating blood vessels, whereas it is present on only a subset of endothelial cells in healthy organs, suggesting a role for H-cadherin in the mechanism of angiogenesis. This finding is consistent with our results. We found that the level of CDH13 expression was 20 times higher in NL than in isolated hepatocytes, suggesting that cells other than hepatocytes play an important role in CDH13 expression in the liver. This is the first report of CDH13 expression in HCC and hepatoma cell lines. Five of 10 tumor samples and one of five cell lines displayed overexpression of CDH13. A similar discrepancy has already been observed for the maspin gene, originally described as a TSG, which affects cell motility and invasion (56) . Maass et al. (57) detected maspin staining in 23 of 24 pancreatic cancer tissues, which suggests that maspin expression is a common event in pancreatic cancer cells. Although the molecular and biological mechanisms underlying the functions of maspin and CDH13 are unknown, several authors have suggested that these proteins act by blocking tumor cell migration and proliferation, thereby preventing invasion and metastasis (48 , 58 , 59) . Maass’ findings for pancreatic cancer, together with our findings for HCC, provide new information about the factors regulating cell development.

One of the most frequent types of genetic alteration in HCC is somatic LOH; this results in the inactivation of TSGs, which normally regulate cell growth and prevent abnormal cell proliferation. In our study, four of the tumor samples tested displayed 16q23.3–24.1 LOH. All these samples displayed LOH at the D16S402 locus. In three of these cases, recurrence was observed within 15 months of surgery. These results confirm the prognostic value of changes in the D16S402 microsatellite for HCC metastasis that we previously reported (35) . No correlation was observed between the pattern of gene expression and LOH status at loci in the vicinity of the investigated genes for the 10 patients and for the five cell lines studied. No mutation of the CDH13 gene was found in cell lines exhibiting LOH. However, the frequent correlation observed between changes in the D16S402 microsatellite in HCC and the higher risk of recurrence are more likely to be related to a TSG other than CDH13.

In conclusion, we studied the expression patterns, in human liver tissue samples and hepatoma cell lines, of 13 genes mapping to a chromosomal region likely to harbor one or several TSG. Abnormalities of CDH13, a gene involved in cell-cell adhesion, have been described in several human cancers and seem to play a role in HCC. However, no clear relationship has been observed between expression of this gene and changes in the D16S402 microsatellite or tumor recurrence. None of the other genes tested is a good candidate for a major TSG in liver carcinogenesis. Our results suggest that additional unknown genes may be involved in carcinogenesis and located in the 16q23.3–24.1 area.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Service de Biochimie et Biologie moléculaire, Hôpital Universitaire Paul Brousse, UPRES 1596-Faculté de Médecine Paris-Sud, 14 avenue Paul Vaillant Couturier, 94804 Villejuif Cedex, France. Back

2 The abbreviations used are: HCC, hepatocellular carcinoma; LOH, loss of heterozygosity; TSG, tumor suppressor gene; NL, normal liver RNA; NTL, nontumor liver RNA; TL, tumoral liver RNA. Back

3 Internet address: www.genome.cse.ucsc.edu. Back

Received 10/19/01; revised 4/22/02; accepted 6/ 4/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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