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Background The finding of hemizygous or homozygous deletions at band 14 on chromosome 13 in a variety of neoplasms suggests the presence of a tumor-suppressor locus telomeric to the RB1 gene.
Methods We studied samples from 216 patients with various types of sporadic tumors or idiopathic pancytopenia, peripheral-blood samples from 109 patients with familial cancer or multiple cancers, and control blood samples from 475 healthy people or patients with diseases other than cancer. We performed functional studies of cell lines lacking ARLTS1 expression with the use of both the full-length ARLTS1 gene and a truncated variant.
Results We found a gene at 13q14, ARLTS1, a member of the ADP-ribosylation factor family, with properties of a tumor-suppressor gene. We analyzed 800 DNA samples from tumors and blood cells from patients with sporadic or familial cancer and controls and found that the frequency of a nonsense polymorphism, G446A (Trp149Stop), was similar in controls and patients with sporadic tumors but was significantly more common among patients with familial cancer than among those in the other two groups (P=0.02; odds ratio, 5.7; 95 percent confidence interval, 1.3 to 24.8). ARLTS1 was down-regulated by promoter methylation in 25 percent of the primary tumors we analyzed. Transfection of wild-type ARLTS1 into A549 lung-cancer cells suppressed tumor formation in immunodeficient mice and induced apoptosis, whereas transfection of truncated ARLTS1 had a limited effect on apoptosis and tumor suppression. Microarray analysis revealed that the wild-type and Trp149Stop-transfected clones had different expression profiles.
Conclusions A genetic variant of ARLTS1 predisposes patients to familial cancer.
To identify putative tumor-suppressor genes at 13q14.3, we sequenced and characterized a 790-kb segment spanning the minimal region of loss1 and performed a detailed mutational study of most of the known genes in this region (DLEU1, DLEU2, miR-15a, miR-16-1, RFP2, KCNRG, DLEU6, DLEU7, and DLEU8).1,11,12 Using computational and experimental approaches, we identified a gene encoding a member of the Ras superfamily, ARLTS1 (also referred to as ARL11 by the Human Genome Organisation Gene Nomenclature Committee). Here, we describe the results of studies to determine whether ARLTS1 is a tumor-suppressor gene.
Methods
Study Samples
We studied 215 samples of sporadic tumors or blood (from 65 patients with a thyroid tumor, 58 with colorectal adenocarcinoma, 48 with breast carcinoma, 39 with CLL, and 5 with lung carcinoma) and 1 sample from a patient with idiopathic pancytopenia. The specimens of colorectal cancer and idiopathic pancytopenia were from patients in Bucharest, Romania; the breast-cancer specimens were from patients in Ferrara, Italy (38 specimens), and Aarhus, Denmark (10 specimens); the CLL samples were from patients overseen at the CLL Consortium in the United States; the lung-cancer specimens were also from patients in Ferrara; and the thyroid-tumor specimens were from patients in Naples, Italy.
We obtained 109 peripheral-blood samples from patients with familial or multiple cancers: they included 69 women with BRCA1- and BRCA2-negative familial breast cancer, 17 men with both prostate cancer and malignant melanoma (negative for mutations in the p16 gene), 17 patients with familial CLL (at least two first-degree relatives affected), and 6 patients with pancreatic cancer or melanoma (with no mutations in the p16 or p14 gene and a family history of at least one case of pancreatic cancer or melanoma). A sample from only one affected member per family was analyzed. The familial CLL specimens were from patients in Paris (11 specimens) and the CLL consortium in the United States (6 specimens). All other specimens from patients with familial or multiple cancer were from Philadelphia.
We also obtained 475 control blood samples from healthy people or patients with diseases other than cancer. The control samples were from 156 renal-transplantation donors in Bucharest, 203 blood donors in Ferrara, and 116 blood donors in Philadelphia.
Written informed consent was obtained for the use of all specimens in accordance with the guidelines for the protection of human subjects at each participating institution. All subjects were white, as indicated by medical records in the case of the patients and information obtained during interviews with the controls. Of the patients with cancer, 58 percent were European and 42 percent were American. Of the controls, 76 percent were European and 24 percent were American.
Molecular Studies
Molecular studies, examination for the loss of heterozygosity, cloning of ARLTS1, and methylation studies were performed as described previously.13,14,15,16 We searched a computer database using Exofish (www.genoscope.cns.fr) and found a 182-bp, evolutionarily conserved region and obtained the full-length complementary DNA (cDNA) with the use of expressed sequence tags and rapid amplification of cDNA ends.
Detection of ARLTS1 Mutations
We directly sequenced DNA on both strands from 597 samples using a DNA-sequencing system (model 377, Applied Biosystems). DNA from the 203 Italian control subjects was analyzed by denaturing high-performance liquid chromatography (Transgenomics), and all the samples with abnormal patterns were directly sequenced.
Stable Transfection of A549 Cells
A549 is a highly tumorigenic, non–small-cell lung-carcinoma cell line that has wild-type TP53 and RB1 genes but does not express the p16INK4a gene. We constructed ARLTS1 expression vectors, one containing the full-length gene (pMV7–ARLTS1-sense) and the other containing a truncated gene encoding a protein product lacking a C-terminal and identical to the polymorphic variant implicated by the genetic data (pMV7–ARLTS1-
C-terminal) by ligating the relevant open reading frame in a sense orientation into a mammalian expression vector (pMV7). All sequenced constructs were transfected with the use of FuGENE6, according to the manufacturer's instructions (Boehringer Mannheim). Stably transfected cells were selected with the use of G418 and examined for the transformed phenotype by establishing in vivo tumorigenicity in Nu/Nu nude mice and selected for apoptosis with the use of the Active Caspase-3 phycoerythrin monoclonal antibody apoptosis kit (Pharmingen, BD Biosciences). Cell-cycle profiles were identified with the use of flow cytometry of cells stained with propidium iodide, and gene-expression profiles were determined with the use of a Kimmel Cancer Center/Thomas Jefferson University human 18.5K expression bioarray (Compugen Human Oligo Set 1.0), as described previously.17
Statistical Analysis
Statistical analysis of categorical results was performed with the use of Fisher's exact test. A logistic-regression model was used to determine the odds ratio for cancer in association with specific mutations. Tumor weights in immunodeficient mice were examined in an analysis-of-variance model, which included the treatment group and the time at which the animal was killed; two-sided P values for specific comparisons between groups were calculated. P values of less than 0.05 were considered to indicate statistical significance.
Results
Deletion of ARLTS1 in Various Types of Cancer
Using Exofish18 on 1.4 Mb of the assembled genomic sequence at chromosome 13q141,11,19,20 and rapid amplification of cDNA ends, we cloned cDNA from bone marrow and spleen that encodes a conceptual protein of 196 amino acids with a predicted molecular mass of 21 kD. Analysis of protein databases with the basic local alignment search tool (BLAST) revealed significant homology with the ADP-ribosylation factor (ARF) and ARF-like (ARL) protein family of the Ras superfamily21,22; we therefore named the gene ARLTS1 (for ADP-ribosylation factor–like tumor-suppressor gene 1; GenBank accession number, AF441378 [GenBank] ; European Molecular Biology Laboratory–European Bioinformatics Institute Minimum Information about a Microarray Experiment accession number, E-MEXP-274). The genomic structure of ARLTS1 is very similar to that of class III ARF (ARF6): it has two exons, and the second contains the entire open reading frame. We found highly conserved protein homologues in mouse and rat and similar proteins in zebrafish, Drosophila melanogaster, and Arabidopsis thaliana (data not shown), indicating that ARLTS1 has been evolutionarily conserved over time.
Previously, we reported that the genomic region at 13q14.3 is hemizygously deleted in approximately 20 percent of the CLL samples we analyzed.14 We confirmed that ARLTS1 is within the region targeted by deletions by using loss-of-heterozygosity analysis on DNA samples from 20 colorectal carcinomas; 10 percent of the specimens (2 of 20) had the hemizygous deletion (data not shown). We therefore hypothesized that monoallelic loss of ARLTS1 occurs in a fraction of both hematopoietic and solid tumors. To test this idea, we sought to identify secondary events, such as a mutation or an epigenetic alteration, that could inactivate the remaining allele.
Association of a Germ-Line Truncating Polymorphism in ARLTS1 With Familial Cancer
We identified a germ-line polymorphism — the substitution of adenine for guanine at position 446 (G446A), resulting in a stop codon at position 149 (Trp149Stop) (Figure 1A) — in samples from both patients with cancer and controls. The position of the stop codon predicts premature termination of translation, leading to the synthesis of a 148-amino-acid protein. Trp149 is a conserved amino acid in 12 other ARF or ARF-related proteins, including all six ARF members, whereas the stretch of 25 amino acids in the C-terminal (which is lost in the truncated form) is conserved in both mouse- and rat-homologue genes.
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In addition to the G446A (Trp149Stop) variant, we identified four other variations in ARLTS1: C65T (Ser22Leu) and G490A (Glu164Lys), both of which were found in thyroid adenomas, and C392T (Pro131Leu) and T442C (Cys148Arg), which were present in heterozygous form in 6.2 percent and 66.9 percent, respectively, of the controls (Table 1). Glu164 is well conserved in homologues of ARLTS1 protein, suggesting that it is critical to protein function. Interestingly, we found two C65T missense mutations, one G446A nonsense mutation, and one G490A missense mutation among 23 thyroid adenomas of follicular origin, whereas wild-type ARLTS1 was present in all 42 samples of the nonfollicular type. It is unlikely that this allelic distribution is random (P=0.005 by Fisher's exact test). A member of a family with CLL who was homozygous for the G446A polymorphism also had thyroid adenoma (Table 2).
Down-Regulation of ARLTS1 by Promoter Hypermethylation
Analysis of RNA from normal hematopoietic and solid tissues with the use of an ARLTS1 probe revealed ubiquitous expression of a 2.2-kb transcript and additional 1.3- and 5.5-kb transcripts resulting from the use of different polyadenylation sites. Northern blotting, a semiquantitative reverse-transcriptase–polymerase-chain-reaction assay, or both showed a reduction or absence of ARLTS1 expression in 4 of 16 fresh tumor samples (2 of 7 lung carcinomas and 2 of 9 samples of CLL cells) for which cDNA, RNA, or both were available, as compared with the levels of expression in their normal counterparts (Figure 2).
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Induction of Apoptosis in Vivo by Full-Length ARLTS1
ARLTS1 expression was dramatically decreased in the A549 cell line. This line was transfected with the use of the pMV7 vector containing the full-length ARLTS1 coding sequence (ARLTS1-FL), the C-terminal–deleted cDNA (ARLTS1-Stop), or the control (empty) vector. The transfectants were selected according to the level of expression of the transfected ARLTS1 minigene (Figure 3A). We evaluated the ability of these transfected cells to form tumors in Nu/Nu mice, which lack an immune system. During eight weeks of observation, all ARLTS1-FL–transfected cells consistently formed smaller tumors (i.e., tumors that weighed 80 percent less) than did cells transfected with empty vector or wild-type A549 cells (P<0.001). Furthermore, tumor size was intermediate in the group of mice injected with A549 clones expressing the C-terminal protein (i.e., tumors weighed 50 percent less than those of wild-type A549 clones), and we found a significant difference between the size of ARLTS1-FL–induced tumors and ARLTS1-Stop–induced tumors (P=0.04) (Figure 3C and Figure 3D). Thus, ARLTS1 by itself has tumor-suppressor activity in A549 cells, and this activity is partially lost in the presence of the truncated protein.
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C–transfected A549 cells could be induced to undergo apoptosis. Furthermore, several members of the small GTPase family (such as ARF6) were expressed at significantly lower levels in the truncated transfectants (P=0.005). Together these data suggest that the full-length product increases the propensity of the cell to undergo apoptosis. Discussion
We identified ARLTS1, a widely expressed member of the ARF–ARL family that functions as a tumor-suppressor gene in cancers in humans. ARFs are 20-kD guanine nucleotide-binding proteins, members of the Ras GTPase superfamily involved in various cellular functions, including vesicular transport and membrane transport.21 ARLs are structurally very similar to ARFs, and there is a continuum of ARF–ARL functions. Of the 18 known members of this family, only ARL5 has been found to be overexpressed in hepatocellular carcinoma,24 and levels of ARF6 protein correlate with the invasiveness of breast-cancer cells.25 The most common mechanism of ARLTS1 inactivation in cancers in humans seems to be biallelic down-regulation by hypermethylation of the promoter. In data consistent with the properties of a classic tumor-suppressor gene, ARLTS1 alterations were found to consist of combinations of a hypomorphic polymorphism plus loss of heterozygosity in a case of breast cancer and the polymorphism plus hypermethylation in a case of thyroid adenoma.
Since the ARLTS1 G446A mutation was nearly three times as frequent among patients with familial cancers and nearly twice as high among patients with sporadic cancers as among persons in the general population, we propose that ARLTS1 is a low-penetrance tumor-suppressor gene that accounts for a small percentage of familial melanoma or familial CLL. Some kindreds may carry the polymorphism but not have cancer. A similar situation was described for other tumor-suppressor genes, such as the BRCA2 germ-line mutations in pancreatic cancer.26 An apparently neutral polymorphic stop codon has been identified in a BRCA2 gene,27 but wild-type and truncated ARLTS1 proteins were distinguishable because the truncated protein induced lower levels of apoptosis than the full-length protein when expressed in A549 cells. This observation suggests that ARLTS1 is a dose-sensitive gene, a hypotheses in accord with the variations in the levels of expression that we found in tumor samples and cell lines.
The G446A (Trp149Stop) polymorphism is probably maintained in the general population, because the ARLTS1-C protein retains some functions of the full-length protein and is in the same intracellular location (unpublished data); it retains an antiapoptotic function but at a significantly lower level than does the normal product. We propose that ARLTS1-C predisposes patients to cancer in several ways. First, transfected cells harboring the truncated gene up-regulate fewer proapoptotic genes than cells with the normal gene. Second, because ARLTS1 transfection influences the levels of expression of several other members of the ARF–ARL family, the ARLTS1 product is probably involved in some common functions with other members of this family. The ARLTS-C protein induces these genes at significantly lower levels, suggesting a partial loss of common functions from the ARF–ARL spectrum. Supporting this hypothesis is the fact that the truncated protein lacks the C-terminal motif involved in nucleotide binding and hydrolysis, which are characteristic of Ras-related GTPases.28
The participation of ARLTS1 in an apoptosis pathway is in accord with data showing that the yeast homologue of ARL1 has a role in programmed cell death.29 Furthermore, a substitution in ARL1 near the position corresponding to the stop mutation that we have described inhibits the promotion of programmed cell death induced by Bax in yeast. Therefore, human ARLTS1 and yeast ARL1 may be involved in a conserved apoptosis pathway.
Supported by Program Project grants (P01CA76259, P01CA81534, and P30CA56036) from the National Cancer Institute, a Kimmel Scholar award (to Dr. Bullrich), and grants from the Italian Ministry of Public Health, Italian Ministry of University Research, and Italian Association for Cancer Research (to Drs. Russo and Negrini).
We are indebted to Nathalie Innocent for the confocal-microscopy analysis.
Source Information
From Thomas Jefferson University, Philadelphia (G.A.C., F.T., M.S., C.D.D., S.Y., D.C., S.R., H.A., H.M., T.S., R.B., C.-G.L., F.B., M.N., C.M.C.); Fox Chase Cancer Center, Philadelphia (A.K.G., B.M.); University of Ferrara, Ferrara, Italy (M.F., G. Bernardi, M.N.); the National Cancer Institute, Aviano, Italy (G. Baldassarre); Aarhus University, Aarhus, Denmark (L.L.H., J.O.); Fundeni Hospital, Bucharest, Romania (V.H.); University La Sapienza, Rome (F.R.M.); the Pasteur Institute, Paris (G.D.); the University of California San Diego, La Jolla (L.R., T.K.); the University of Catanzaro, Catanzaro, Italy (A.F.); Kyushu University, Beppu, Japan (M.M.); Istituto di Ricovero e Cura a Carattere Scientifico, Rome (G.R.); and the Dana–Farber Cancer Institute, Boston (D.N.).
Address reprint requests to Dr. Croce at Ohio State University Comprehensive Cancer Center, 385K Wiseman Hall, 400 W. 12th Ave., Columbus, OH 43210, or at carlo.croce{at}osumc.edu.
References
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Related Letters:
Familial Cancer and ARLTS1
De Brakeleer S., Teugels E., De Grève J., Frank B., Klaes R., Burwinkel B., Calin G. A., Croce C. M., Neuberg D.
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N Engl J Med 2005;
353:313-314, Jul 21, 2005.
Correspondence
Familial Cancer Associated with a Polymorphism in ARLTS1
Sellick G. S., Catovsky D., Houlston R. S., Calin G. A., Croce C. M., Neuberg D.
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N Engl J Med 2006;
354:1204-1205, Mar 16, 2006.
Correspondence
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-actin (lanes 9, 10, and 11). Panel B shows that the level of ARLTS1 expression correlates with the level of methylation of this locus analyzed by Southern blotting of digested genomic DNA with BglII alone (B) or in combination with HpaII (BH). The combination of BglII and MspI (BM) was used to determine the fragment length without respect to the degree of methylation. The presence or absence of ARLTS1 expression is shown by the plus and minus signs, respectively; in the restriction-enzyme map, BglII (B) is denoted by thick vertical lines and HpaII (H) by thin vertical lines. The position of the open-reading-frame probe used is indicated by the asterisk. Burkitt's lymphoma (AS283), T-cell acute lymphocytic leukemia (HSB2), and hairy-cell leukemia (MOT) were used for the experiments. Panel C presents the correlation between the level of ARLTS1 expression, analyzed by reverse-trancriptase–polymerase-chain-reaction assays, and the extent of methylation of the cytidine–phosphate–guanosine (CpG) site, analyzed by bisulfite sequencing in paired samples of lung-tumor tissue and normal tissue from fresh tumor, blood samples from patients with CLL (CLL 120, CLL 188, and CLL 222), and other tumor cell lines. White and black squares represent unmethylated and methylated CpG dinucleotides, respectively, and gray squares partially methylated CpG sites. As a control we used Epstein–Barr virus–transformed lymphoblastoid cell lines. N denotes normal, and T tumor.
