Members of the SSX gene family were initially identified as fusion partners to the SYT gene in synovial sarcomas harboring the t(X;18) translocation. There are currently 6 known SSX genes, 5 of which are fully characterized; all 6 genes map to chromosome X.1, 2, 3, 4, 5SSX gene products have been categorized as CT (cancer-testis) antigens since they are expressed in testis and, in the case of SSX1, 2, 4 and 5, in a wide variety of tumors.4, 6 The identification of SSX, together with other CT antigens, has vastly expanded the possible antigenic targets that can be useful in cancer immunotherapy. The known SSX family members share 70–90% homology at the protein level, and naturally occurring serologic responses mounted by cancer patients against 1 member have been shown to cross-react with other members of the family.4 Data from this laboratory and from others suggested that the SSX gene family comprised more than the 5 genes that had been previously characterized.4, 7 An incomplete sequence of SSX6 was recently defined through database searches.5 In our study, we have extended the search for additional SSX genes by isolating SSX-containing genomic clones from a genomic library and by a search of databases. This resulted in the completion of the SSX6 sequence and the identification of 3 novel SSX genes (SSX7, SSX8 and SSX9) and 10 pseudogenes. All genes and pseudogenes (except ΨSSX10) map to chromosome X.
Human SSX genes comprise a gene family with 6 known members. SSX1, 2 and 4 have been found to be involved in the t(X;18) translocation characteristically found in all synovial sarcomas. Four (SSX1, 2, 4 and 5) are known to be expressed in a subset of tumors and testis, and anti-SSX antibodies have been found in sera from cancer patients. SSX antigens are thus typical cancer-testis (CT) antigens. To identify additional SSX family members, we isolated and characterized human genomic clones homologous to a prototype SSX cDNA. We also searched public databases for sequences similar to SSX. This identified 3 additional SSX genes, SSX7, 8, 9, and also completed the sequence of the formerly partially defined SSX6 gene. In addition to these novel SSX genes, several SSX pseudogenes were identified. With the exception of 1 pseudogene, all SSX genomic SSX sequences map to chromosome X. Among normal tissues, SSX7 mRNA was present only in testis, whereas SSX6, 8 and 9 were not detected in any normal tissue. SSX6 and 7 were expressed in 1 of 9 melanoma cell lines tested, whereas SSX8 and 9 expression was not detected in any tumor tissue or cell lines tested. SSX1, 2, 4 and 5 mRNA expression can be induced in cell lines by 5-aza-2-deoxycytidine or Trichostatin A. These agents also induce SSX6, but not SSX3, 7, 8 or 9 in the tumor cell lines tested, indicating that mechanisms other than methylation or histone acetylation may be responsible for the repressed state of some SSX genes. © 2002 Wiley-Liss, Inc.
MATERIAL AND METHODS
Library screening and evaluation of the clones
An amplified human placenta genomic library (Clontech, Palo Alto, CA) was screened with an SSX probe prepared by RT-PCR amplification from the melanoma cell line SK-MEL-37 using primers MEL40-A and MEL40-B.4 λ-DNA was prepared from the purified positive clones using the WIZARD lambda preparation kit (Promega, Madison, WI). For the initial evaluation of SSX-containing clones, 2 μl of 1:100 diluted phage DNA was amplified in the presence of MEL40-A and MEL40-B2 (5′-CAAATCATTCCCCTGGAAGTC), the PCR products were cloned within the pGEM-T Easy vector (Promega) and sequenced by the DNA sequencing facility of Cornell University (Ithaca, NY). Exons 5, 6, 7 and 9 were individually amplified from each novel clone using primers corresponding to intronic sequences flanking the exons. The primers were designed to anneal to intronic sequences of all known SSX genes contained in the GenBank databases. The amplification products were cloned and sequenced.
Cell lines and tissues
Cell lines were obtained from the repository maintained at the Ludwig Institute for Cancer Research (LICR), New York Branch at the Memorial Sloan-Kettering Cancer Center. Normal and tumor tissues were obtained from the departments of pathology, New York Presbyterian Hospital (NYPH) and Memorial Sloan-Kettering Cancer Center (MSKCC). The 9 melanoma cell lines used were SK-MEL-19, -23, -29, -30, -31, -33, -37, -179 and MZ2-Mel 3.1.
Expression Analysis of novel SSX genes
For RT-PCR analysis of the novel genes, the following primers were used. SSX6-A: 5′-CTAAAGCATCAGAGAAGAGAAGC; SSX6-B: 5′-TTTTGGGTCCAGATCTCTCGTG; SSX7-A: 5′-TTTGCAAGGAGACCTAGGGC; SSX7-B: 5′-GGGGAGTTACTCGTCGTCTTCT; SSX8-A: 5′-AAAGAGACCCAGGGATGATGA; SSX8-B: 5′-CTCTTCATAAATCACCAGCTGG; SSX9-A: 5′-AGACAACGACTGTGCAAAGAGAG; SSX9-B: 5′-TGTGAATCTTCTCAGAAGTATTTGCTC. Thirty-five cycles of PCR amplifications were performed and the products were analyzed by agarose gel electrophoresis. The PCR conditions for SSX1–5 were as previously described.4 Annealing temperature was 60°C for SSX6, 7 and 9, and 65°C for SSX8.
5′-Aza-2′-deoxycytidine (5-DC) and Trichostatin A (TSA) Treatment of Cell Lines
Colon cancer cell lines were plated in 6-well plates in MEM supplemented with FCS, glutamine and with penicillin/streptomycin. 5-DC (Sigma, St. Louis, MO) 1 μM final concentration was added 24 hr after plating, while TSA (USB) (0.3–1 μM final concentration), if included, was added 48 hr after plating. Total RNA was harvested after 96 hr with the Trizol reagent (Sigma) and used for RT-PCR analysis.
Identification of novel SSX genes by plaque hybridization
To identify novel SSX genes, a placenta genomic library was screened using an SSX probe previously described,4 and 26 clones were isolated. A 1.5 kb fragment that included the first to the third protein-coding exons in SSX was amplified from each clone and sequenced. Two of the 26 clones could not be amplified and were not evaluated further. Among the 24 sequenced clones, SSX1, SSX2, SSX4 and SSX5 were identified, represented by 1, 5, 1 and 11 clones, respectively. No genomic counterpart of SSX3 or SSX6 was identified among these clones. In addition to the 4 known genes, 2 novel SSX genes were isolated, represented by 5 clones and 1 clone, respectively. The remaining exon sequences within these 2 novel SSX clones were characterized by PCR amplification using primers derived from intronic sequences flanking each protein-coding exon. The complete coding sequence of 1 of the 2 novel genes was obtained by this approach, and this gene was designated SSX7. The other gene, however, was found to be missing the last protein-coding exon. It was presumed to be a pseudogene, and is designated ΨSSX1. This finding was subsequently confirmed by comparison to the genomic sequence found in the GenBank (see below).
Identification of additional SSX genes by database search
We previously defined 6 protein-coding exons in SSX genes.4 An analysis of all SSX cDNA and expressed sequence tags (ESTs) in GenBank revealed additional exons in the 5′ and 3′ untranslated regions of the RNA transcripts. In total, SSX genes were found to contain 10 exons, with several splice variants being detected (described below). Using an artificially constructed SSX cDNA that contained all possible exons, nucleotide databases available in NCBI (nr, htgs, dbest) were searched for novel SSX genes using BLAST program with the default settings. This search revealed 8 genomic clones, each containing 1 to 8 SSX gene-related sequences. Detailed analysis of these sequences identified and confirmed the sequences of SSX1, 2, 3, 4, 5, 7 and completed the sequence of SSX6. In addition, 2 novel SSX genes (designated SSX8 and SSX9) were found, as well as 10 SSX pseudogenes, including ΨSSX1. Except for a single pseudogene within a chromosome 6 contig (ΨSSX10, Z99496), all other clones were derived from chromosome X. Figure 1 shows the distribution and orientation of SSX genes and pseudogenes in genomic clones from chromosome X.
Nine SSX genes are found in these contigs, SSX1 through SSX9. For 7 of these (SSX2, 3, 4, 6, 7, 8 and 9), all exons are present within these clones. SSX1 and 5, however, are at the 5′ and 3′ ends of AL606490 and AL356464, respectively, and therefore are only partially contained within these genomic clones. Each complete gene occupies approximately 8–10 kb, with the distance between different SSX genes (or pseudogenes) ranging from 10–50 kb (Figure 1). SSX1, 3, 5, 8 and 9 were represented by 1 clone each, whereas SSX2 and SSX4 were found in 2 clones. SSX6, is contained within sequences Z98304, AL356797 and AL356464. Of interest, 2 copies of SSX2 gene were found in AL450023 within a 70 kb stretch. Since the 2 genes are identical throughout the promoter and introns as well as the exons, this might reflect a recent duplication/inversion event. Comparison of AL445236 and AL450023, 2 completely sequenced clones, showed that both contain SSX2, 7 and ΨSSX5. SSX8, Ψ4 and Ψ1, however, were only found in AL450023 but not in the corresponding location of AL445236. One possible explanation would be that clone AL445235 represents a 3′ continuation of AL450023 and that the entire region encompassing SSX2, 7 and Ψ5 are all part of the gene duplication/inversion. A similar gene duplication process has indeed been described, giving rise to 2 exact copies of another CT gene, NY-ESO-1, on chromosome Xq28.8 Except for a single pseudogene on chromosome 6, SSX sequences are clustered within 2 loci, about 3 Mb apart, on chromosome X. A philogenetic analysis of SSX genes demonstrates that SSX3, 9 and 1 are most closely related to SSX2, 7 and 8, respectively (Fig. 2). SSX3, 9 and 1 are located at Xp11.23-p11.3, whereas SSX2, 7 and 8 are at Xp11.4, suggesting that the 2 SSX gene clusters arose from a duplication encompassing an approximately100 Kb region. This concept is further supported by the conserved order and orientation of these homologous genes within the 2 clusters (Fig. 1).
Exon structure of SSX genes and the pseudogenes
Figure 3 shows the exon structure of SSX genes and the pseudogenes. Of the 10 defined exons, only 8 exons (exons 1, 2, 4, 5, 6, 7, 9 and 10) were utilized in all SSX genes. Exon 3 occurred only within 1 sequence tag (BC016640) derived from SSX5 gene, and exon 8 was found only as part of an SSX2 cDNA (XM_033605). The exon-intron junctions, sizes of individual exons and most of the introns are conserved among all SSX genes. The open-reading frame (ORF) is contained between exons 2 and exons 9.
All pseudogenes contained within chromosome X contigs represent incomplete genes. They lack at least 1 exon (ΨSSX3), whereas others are present only as gene segments containing 1 or a few exons (Fig. 3). ΨSSX10, found in a contig derived from chromosome 6 (Z99496), has a cDNA-like structure, including a polyA-tract and no exons. No long ORF can be found in this sequence. It is therefore likely to have been generated as the result of a retrotransposition event. Other SSX pseudogenes are interspersed on X chromosome between the functional genes (Fig. 1). ΨSSX1, ΨSSX4 and ΨSSX5 are from AL450023 and AL445236; ΨSSX2 is within AL356797, ΨSSX3 is within AL356464, and ΨSSX6, 7, 8 and 9 are within AL606490. In contrast to the 9 SSX genes, which all have similar 5′ regions that likely harbor transcriptional control elements, all SSX pseudogenes, except Ψ3, lack these sequences (Fig. 1).
Structural characterization of SSX transcripts and putative proteins
SSX protein homology between members ranges from 73–92%, and homology at the cDNA level is from 87–96% (Table I). The KRAB domain is encoded by 2 exons, as is the case with most other Kruppel-like transcription factors.9 However, the most highly conserved domain among SSX proteins is one (SSXRD) that has recently been implied in the transcription-repression function of SSX.10 This region is also the most conserved region between human and mouse SSX proteins (Chen and Güre, unpublished).
Of the newly defined SSX genes, SSX6, SSX7 and putative SSX9 cDNAs contain an uninterrupted ORF similar to SSX1-5, coding for a protein of 188 amino acids. In comparison, SSX8 has an extra nucleotide in its 7th exon, which results in premature termination and a shorter ORF of 142 amino acids (Fig. 4). Alternatively spliced transcripts have been found for SSX4,4SSX2, SSX5 and SSX7. By RT-PCR and sequencing, splice variants lacking exon 7 were found for SSX4 and 7, and another SSX7 variant missing both exons 4 and 7 was also identified4 (and data not shown). These variant transcripts, if translated, can lead to SSX proteins of various sizes, ranging from 153 amino acids (SSX4 lacking exon 7) to 60 amino acids (SSX7, without exons 4 and 7). Whether these products exist in vivo is unknown.
Expression analysis of novel SSX genes
Testis expresses all previously defined SSX genes (SSX1 through SSX5), and tumors and tumor cell lines often express either 1 or more of the SSX genes SSX1, 2, 4 and 5 and seldomly, SSX3.4, 6, 11 To examine the expression of the novel SSX genes, primers specific for SSX6, SSX7, SSX8, SSX9 and for the pseudogene ΨSSX1 were used for RT-PCR analysis. Total RNA from normal testis, brain, lung, liver, kidney and colon and from melanoma cell lines were tested. SSX6 was found to be expressed in SK-MEL-37 but not in any normal tissues, including testis or in 8 other melanoma cell lines. SSX7 was expressed in testis and at very low levels in SK-MEL-37, but not in any other tissue or cell line. SSX8, SSX9 and ΨSSX1 expression could not be detected in any of the tissue or cell lines used. PCR products from SSX6 and SSX7 amplification, obtained from SK-MEL-37, were sequenced and confirmed to be the correct SSX6 and SSX7 sequences. RT-PCR results of SSX expression in testis are shown in Figure 5.
Induction of SSX expression
Methylation has been implicated as a major factor in regulating CT antigen gene expression, and genome-wide demethylation is thought to be a major cause for CT upregulation in cancer.12, 13 We tested whether 5′-aza-2′ deoxycytidine (5-DC) or the histone acetylase inhibitor Trichostatin A (TSA) could induce SSX gene expression. Seven colon cancer cell lines (SW1116, NCI-H508, LIM1215, SK-CO-1, HT29, CaCo2, and Colo205) that express no or minimal SSX transcripts were treated with 5-DC, TSA, or both. We observed that 5-DC or TSA alone, or in combination, could induce or upregulate SSX1, 2, 4 and 5 in colon cancer cell lines (Fig. 6). Among the novel SSX genes, SSX6 was weakly induced by 5-DC in HT29 but not in the other colon cancer cell lines tested. TSA by itself or in combination with 5-DC could induce SSX6 in SW1116 and CaCo2 (Fig. 6). SSX3, SSX7, SSX8 or SSX9 expression could not be induced by either agent or their combination in these cells (data not shown). In general, 5-DC is more effective in inducing SSX expression. For example TSA, although capable of inducing SSX4, failed to induce SSX1 and SSX5 expression and had minor effects on SSX2 expression. In contrast, 5-DC induced expression of SSX1, 2, 4 and 5 in most cell lines. SW1116, CaCo2 and HT29 seem to be most responsive to SSX induction by 5DC and/or TSA, and SSX1, 2, 4, 5 and 6 were inducible by 5DC and/or TSA in these cell lines. In comparison, induction experiments on SK-CO-1 showed that only SSX4 can be readily induced and other SSX genes are minimally affected. Among the inducible SSX genes, SSX4 is most frequently induced among the cell line panel tested, followed by SSX2, SSX5, SSX1 and SSX6.
Our previous attempt to identify SSX family members resulted in the identification of 5 SSX genes.4 We have now identified additional SSX genes through genomic library screening and database mining. Four complete genes, SSX6, 7, 8 and 9, as well as 10 pseudogenes or gene fragments, were identified. Similar to the MAGE family,14 and most other CT antigen genes,15 all SSX genes, except for a single pseudogene on chromosome 6, are located on chromosome X.
With the possible exception of weak expression in thyroid,16 none of the nontesticular tissues examined exhibited SSX mRNA expression, an expected characteristic of cancer-testis (CT) antigens. Expression of SSX transcripts in testis and in cancer, however, varies for different SSX genes. Normal testis expresses SSX1, 2, 3, 4, 5 and 7 but not 6, 8 and 9. Among tumor tissues, SSX1, 2 and 4 expression is found at substantial frequencies, whereas SSX3, 5 and 6 are rarely expressed and SSX7, 8 and 9 expression have not been detected.6, 11 Previous attempts to identify SSX genes have relied on cDNA library screening2, 3, 4 or analysis of RT-PCR products generated from primers that could amplify any SSX cDNA.4 The absence of SSX8 and SSX9 mRNA in testis and the low expression levels of SSX6 and 7 thus explain why these genes were not isolated previously. Although we could not detect SSX8 and 9 transcripts in any tissue tested, both genes are structurally complete, including an exon-intron organization similar to other expressed SSX genes, homologous putative promoter regions and an ORF identical to other SSX genes. We have thus decided to designate SSX8 and SSX9 as genes, rather than pseudogenes.
Our study has identified 10 possible exons that could be utilized by SSX genes. Evidence for utilization of some of the exons (3 and 8) is provided only by a single EST entry in the NCBI database. The alternatively spliced SSX4 transcript lacking exon 7, on the other hand, is regularly observed in testis and in tumor tissue by RT-PCR.4 Since the splice sites from different SSX genes are identical, it is difficult to explain the apparent bias in exon usage among SSX genes. Comparison of regions up to 3 kb 5′ to transcription start sites shows that all 9 genes have similar 5′ regions that could harbor transcriptional control elements, while most SSX pseudogenes do not. As is the case with MAGE genes,13SSX promoter activity is methylation-sensitive. Because not all SSX genes are inducible by 5-DC or TSA, methylation of crucial CpG residues or histone-mediated gene repression cannot be the only mechanism regulating the expression of these genes. Differences in the expression pattern of SSX genes could be due to subtle differences in the promoter sequences, or in the case of SSX genes that are not expressed, their residence within large silenced chromosomal regions. If the latter is the case, these regions would not encompass more than a few genes at a time, since expressed and nonexpressed SSX genes are interspersed in the genome, often in close proximity. For example, SSX2, SSX7 and SSX8 are within a genomic contig of 90 kb and SSX1 and SSX9 are within a 50 kb region.
The function of SSX gene products is most likely that of a transcriptional repressor.10 This has been attributed to 2 functional domains. One is the KRAB domain close to the N-terminal region. This domain in the SSX proteins, however, is less active in transcriptional repression than in other KRAB-containing proteins.10 The other domain, located at the acidic C-terminus end of SSX proteins and designated SSXRD, was also found to be an active repressor of transcription;10 this SSXRD region is indeed the most conserved domain among SSX proteins. With regard to these domain structures, the KRAB domain is coded by exons 4 and 5, while the SSXRD is encoded by exon 9.
SSX genes were originally discovered by sequencing the breakpoint of the t(X; 18) translocation frequently occurring in synovial sarcomas.1, 2 This event generates a fusion product between SYT and 1 of the SSX genes, the N-terminal of which being almost the entire SYT gene, which reads into the 3′ end of the SSX1, 2 or 4.5, 17, 18 The exons contributed by the SSX genes to this fusion product show some variation. Most frequently, exon 7 and other 3′ exons of SSX1, 2 or 4 are found to fuse with SYT. As a result, the Kruppel-like box of SSX is deleted, while the SSXRD domain remains intact in the fusion product.5 Since the SYT-SSX fusion is widely believed to be a critical element in the etiology of synovial sarcoma, the SSXRD domain would likely be the crucial component contributed by SSX. Challenging this notion, however, is the finding of an SYT-SSX fusion variant with a truncated 3′ end lacking SSXRD.19 The exact role of SSX in the pathogenesis of synovial sarcoma thus remains to be further explored.
As with other CT antigens, the aberrant expression of SSX in malignancy is an immunogenic event, and antibodies against SSX1, 2, 3 and 4 antigens have been demonstrated in sera from cancer patients with a variety of tumors, including melanoma, colon and breast cancer4, 20 (Scanlan et al., unpublished). Due to the strong homology among different SSX proteins, antigenic cross-reactivity among SSX proteins with patient serum would be expected. Recently, CD8+ T-cell immunity against a peptide epitope derived from SSX2 has also been demonstrated,21 indicating that SSX proteins are capable of eliciting both humoral and cellular immune responses spontaneously in cancer patients. SSX proteins are thus potentially useful targets in cancer vaccine-based immunotherapy.