Translocations, cancer and the puzzle of specificity.

The finding of acquired chromosomal translocations that are consistently associated with specific tumour types supports the premise of lineage-specific mechanisms of tumorigenesis. We review the evidence indicating that the specificity of these translocations and the corresponding gene fusions is related to biological constraints at the level of recombination, expression, and protein function. A dynamic relationship between the gene fusion and the cellular environment is proposed in which the environment influences the selection of oncogenic fusions and the oncogenic fusion in turn influences the cellular environment.

Rearrangement of the PAX3 paired box gene in the paediatric solid tumour alveolar rhabdomyosarcoma.

We have determined that PAX3 (found previously to be mutated in Waardenburg syndrome) is the chromosome 2 locus rearranged by the t(2;13)(q35;q14) translocation of the paediatric solid tumour alveolar rhabdomyosarcoma. The rearrangement breakpoints occur within an intron downstream of the paired box and homeodomain-encoding regions. Upstream PAX3 sequences hybridize to a novel transcript in t(2;13)-containing lines. Cloning and characterization of this novel transcript indicate that the translocation juxtaposes the PAX3 DNA binding elements with chromosome 13 sequences, suggesting formation of a hybrid transcription factor. Therefore, PAX3 gene alterations are associated with two completely unrelated human diseases.

Fusion of a fork head domain gene to PAX3 in the solid tumour alveolar rhabdomyosarcoma.

We have examined the structure and expression of the products associated with the t(2;13)(q35;q14) translocation associated with alveolar rhabdomyosarcoma. The chromosome 13 gene (FKHR) is identified as a member of the fork head domain family of transcription factors characterized by a conserved DNA binding motif. Polymerase chain reaction analysis demonstrates that a 5'PAX3-3' FKHR chimaeric transcript is expressed in all eight alveolar rhabdomyosarcomas investigated. Immunoprecipitation experiments detect the predicted fusion protein. These findings indicate that the t(2;13) generates a potentially tumorigenic fusion transcription factor consisting of intact PAX3 DNA binding domains, a truncated fork head DNA binding domain and C-terminal FKHR regions.

Genomic hypomethylation and far-5' sequence alterations are associated with carcinogen-induced activation of the hamster thymidine kinase gene.

We have investigated the mechanism of activation of an inactive but functionally intact hamster thymidine kinase (TK) gene by the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine. Following carcinogen treatment of TK- RJK92 Chinese hamster cells, aminopterin-resistant (HATr) colonies appeared at a frequency 50-fold higher than in untreated controls. More than 80% of these HATr variants expressed TK enzymatic activity and were divided into high- and low-activity classes. In all TK+ variants, TK expression was correlated with demethylation in the 5' region of the TK gene and the appearance a 1,400-nucleotide TK mRNA. Using high-performance liquid chromatography to measure the level of genomic methylation, we found that four of five high-activity lines demonstrated extensive genomic hypomethylation (approximately 25% of normal level) that was associated with demethylation of all TK gene copies. Restriction endonuclease analysis of 15 low-activity lines revealed four instances of sequence alterations in the far-5' region of the TK gene and one instance of a tandem low-copy amplification. In these lines, the structurally altered gene copy was demethylated. Thus, we propose that a chemical carcinogen can activate TK expression by several different mechanisms. Focal demethylation with or without gene rearrangement was associated with low TK activity, whereas demethylation throughout the genome was associated with high TK activity.

The PAX3-FKHR fusion protein created by the t(2;13) translocation in alveolar rhabdomyosarcomas is a more potent transcriptional activator than PAX3.

Alveolar rhabdomyosarcomas are pediatric solid tumors with a hallmark cytogenetic abnormality: translocation of chromosomes 2 and 13 [t(2;13) (q35;q14)]. The genes on each chromosome involved in this translocation have been identified as the transcription factor-encoding genes PAX3 and FKHR. The NH2-terminal paired box and homeodomain DNA-binding domains of PAX3 are fused in frame to COOH-terminal regions of the chromosome 13-derived FKHR gene, a novel member of the forkhead DNA-binding domain family. To determine the role of the fusion protein in transcriptional regulation and oncogenesis, we identified the PAX3-FKHR fusion protein and characterized its function(s) as a transcription factor relative to wild-type PAX3. Antisera specific to PAX3 and FKHR were developed and used to examine PAX3 and PAX3-FKHR expression in tumor cell lines. Sequential immunoprecipitations with anti-PAX3 and anti-FKHR sera demonstrated expression of a 97-kDa PAX3-FKHR fusion protein in the t(2;13)-positive rhabdomyosarcoma Rh30 cell line and verified that a single polypeptide contains epitopes derived from each protein. The PAX3-FKHR protein was localized to the nucleus in Rh30 cells, as was wild-type PAX3, in t(2;13)-negative A673 cells. In gel shift assays using a canonical PAX binding site (e5 sequence), we found that DNA binding of PAX3-FKHR was significantly impaired relative to that of PAX3 despite the two proteins having identical PAX DNA-binding domains. However, the PAX3-FKHR fusion protein was a much more potent transcriptional activator than PAX3 as determined by transient cotransfection assays using e5-CAT reporter plasmids. The PAX3-FKHR protein may function as an oncogenic transcription factor by enhanced activation of normal PAX3 target genes.

The role of chimeric paired box transcription factors in the pathogenesis of pediatric rhabdomysarcoma.

Alveolar rhabdomyosarcoma (ARMS) is an aggressive pediatric soft tissue tumor with striated muscle differentiation. Chromosomal studies of these tumors identified 2;13 and 1;13 translocations. Using physical mapping and cloning strategies, we determined that t(2;13) and t(1;13) rearrange PAX3 and PAX7, which encode members of the paired box transcription factor family, and juxtapose these genes with FKHR, which encodes a novel member of the fork head transcription factor family. These translocations result in chimeric transcripts consisting of 5' PAX3 or PAX7 exons fused to 3' FKHR exons, which encode fusion proteins containing the PAX3 or PAX7 DNA-binding domain and the COOH-terminal FKHR transcriptional activation domain. In transfection studies, the PAX3-FKHR fusion activates transcription of reporter genes containing PAX DNA-binding sites, and is 10-100-fold more potent as a transcriptional activator than is wild-type PAX3. This increased function results from the insensitivity of the COOH-terminal FKHR activation domain to the inhibitory effects of NH2-terminal PAX3 domains. In addition to functional alterations, our studies demonstrated PAX3-FKHR and PAX7-FKHR overexpression resulting from two distinct mechanisms, increased transcription of PAX3-FKHR by a copy number-independent mechanism, and gene amplification of PAX7-FKHR. These findings indicate that the genetic changes in these tumors result in high levels of chimeric transcription factors that are hypothesized to inappropriately activate transcription of genes with PAX DNA-binding sites and thereby induce tumorigenic behavior. The differences in overexpression strategies suggest important differences between the mechanisms for regulating PAX3 and PAX7 expression. These differences extend to the phenotypic level, at which clinical differences have been found between the two ARMS subtypes: PAX7-FKHR tumors more often occur as localized lesions in the extremities of younger patients and are associated with longer event-free survival as compared to PAX3-FKHR tumors. Therefore, the clinical heterogeneity within the ARMS category is associated with genetic heterogeneity. Further analysis of the transcriptional function, regulation of expression, and phenotypic effects will help to elucidate the action of these fusion products and the biological basis of the clinical heterogeneity.

Evidence for a 17p tumor related locus distinct from p53 in pediatric primitive neuroectodermal tumors.

Primitive neuroectodermal tumors of the central nervous system are the most common malignant brain tumors in children. Cytogenetic analysis of these tumors has demonstrated alterations of chromosome 17, in particular isochromosome 17q, as the most frequent chromosomal abnormality detected. Since the consistent loss of a specific chromosomal region in a given tumor type most likely indicates the presence of a tumor related gene in that region, we undertook a combined molecular and cytogenetic approach to examine alterations of chromosome 17 in primitive neuroectodermal tumors. Seven of 14 tumors analyzed demonstrated loss of alleles for loci on 17p. In three of the seven tumors tested, a loss in copy number was observed for only the most telomeric locus on 17p13.3, D17S34. Limited sequence analysis of the same seven tumors did not reveal mutations in four highly conserved coding regions of the p53 gene. These data suggest a new tumor associated locus on 17p distinct from and distal to TP53, which is involved in the initiation or progression of at least a subset of primitive neuroectodermal tumors.

Up-regulation of MET but not neural cell adhesion molecule expression by the PAX3-FKHR fusion protein in alveolar rhabdomyosarcoma.

The 2;13 chromosomal translocation in alveolar rhabdomyosarcoma generates the chimeric protein PAX3-FKHR, which is a powerful transcriptional activator. We hypothesize that PAX3-FKHR regulates downstream effector genes involved in rhabdomyosarcoma tumorigenesis. We evaluated alterations in expression of MET and neural cell adhesion molecule that were proposed previously as downstream targets of wild-type PAX3. We used a myogenic tumor cell culture system and rhabdomyosarcoma tumor specimens to assess candidate gene expression in relationship to various PAX3-FKHR expression levels. We demonstrate that the expression of MET, but not neural cell adhesion molecule, correlates significantly with PAX3-FKHR expression. These findings indicate that MET, which encodes a receptor involved in growth and motility signaling, is a downstream target of PAX3-FKHR in alveolar rhabdomyosarcoma.

Fusion of PAX7 to FKHR by the variant t(1;13)(p36;q14) translocation in alveolar rhabdomyosarcoma.

Although the t(2;13)(q35;q14) translocation has been found in most cases of the pediatric cancer alveolar rhabdomyosarcoma, several cases have been reported with a variant t(1;13)(p36;q14) translocation. Our findings indicate that this t(1;13) rearranges PAX7 on chromosome 1 and fuses it to FKHR on chromosome 13. This fusion results in a chimeric transcript consisting of 5' PAX7 and 3' FKHR regions, which is similar to the 5' PAX3-3' FKHR transcript formed by the t(2;13). The 5' PAX3 and PAX7 regions encode related DNA binding domains, and therefore we postulate that these translocations create similar chimeric transcription factors that alter expression of a common group of target genes.

Predominant expression of alternative PAX3 and PAX7 forms in myogenic and neural tumor cell lines.

PAX3 and PAX7 are closely related paired box family members expressed during early neural and myogenic development. Assay of PAX3 and PAX7 mRNA expression in embryonal rhabdomyosarcoma, neuroblastoma, Ewing's sarcoma, and melanoma cell lines revealed tumor-specific expression patterns similar to the corresponding embryonic lineages. Although the mammalian PAX3 and PAX7 genes were reported to contain eight exons, we found that the predominant PAX3 and PAX7 transcripts in these tumor lines contain previously uncharacterized ninth exons. These splicing events alter the COOH-terminal coding regions of the encoded products but do not alter the transcriptional activity as assayed using a reporter gene with a model PAX3/PAX7 binding site. However, the findings of nearly identical COOH-terminal regions within the corresponding genes of the avian and fish genomes suggest conserved functional roles for these regions that require further investigation.

Gene fusions involving PAX and FOX family members in alveolar rhabdomyosarcoma.

The chromosomal translocations t(2;13)(q35;q14) and t(1;13)(p36;q14) are characteristic of alveolar rhabdomyosarcoma, a pediatric soft tissue cancer related to the striated muscle lineage. These translocations rearrange PAX3 and PAX7, members of the paired box transcription factor family, and juxtapose these genes with FKHR, a member of the fork head transcription factor family. This juxtaposition generates PAX3-FKHR and PAX7-FKHR chimeric genes that are expressed as chimeric transcripts that encode chimeric proteins. The fusion proteins, which contain the PAX3/PAX7 DNA binding domain and the FKHR transcriptional activation domain, activate transcription from PAX-binding sites with higher potency than the corresponding wild-type PAX proteins. This increased function results from the insensitivity of the FKHR activation domain to inhibitory effects of N-terminal PAX3/PAX7 domains. In addition to altered function, the fusion products are expressed in ARMS tumors at higher levels than the corresponding wild-type PAX products due to two distinct mechanisms. The PAX7-FKHR fusion is overexpressed as a result of in vivo amplification while the PAX3-FKHR fusion is overexpressed due to a copy number-independent increase in transcriptional rate. Finally, though FKHR subcellular localization is regulated by an AKT-dependent pathway, the fusion proteins are resistant to these signals and show exclusively nuclear localization. Therefore, these translocations alter biological activity at the levels of protein function, gene expression, and subcellular localization with the cumulative outcome postulated to be aberrant regulation of PAX3/PAX7 target genes. This aberrant gene expression program is then hypothesized to contribute to tumorigenic behavior by impacting on the control of growth, apoptosis, differentiation and motility.

Translin recognition site sequences flank chromosome translocation breakpoints in alveolar rhabdomyosarcoma cell lines.

Alveolar rhabdomyosarcoma is characterized by a t(2;13)(q35;q14) chromosome translocation, which leads to the fusion of the PAX3 and the FKHR genes. The resulting fusion gene encodes a chimeric protein which has aberrant transcriptional activity. We report the molecular definition of the genomic breakpoints on both derivative chromosomes in one case and the derivative chromosome 13 breakpoints in two other cases. The DNA sequences adjacent to the breakpoints on the derivative chromosome 13 are remarkable for their resemblance to recognition sequences for the protein translin. Gel shift analyses confirm that these sequences bind translin. These findings suggest that translin may not only be important in the genesis of chromosomal translocations in lymphoid malignancy, but also in translocations found in solid tumours.

Transcriptional modulation of the anti-apoptotic protein BCL-XL by the paired box transcription factors PAX3 and PAX3/FKHR.

The aberrant expression of the transcription factors PAX3 and PAX3/FKHR associated with rhabdomyosarcoma (RMS), solid tumors displaying muscle cell features, suggests that these proteins play an important role in the pathogenesis of RMS. We could previously demonstrate that one of the oncogenic functions of PAX3 and PAX3/FKHR in RMS is protection from apoptosis. BCL-XL is a prominent anti-apoptotic protein present in normal skeletal muscle and RMS cells. In the present study, we establish that BCL-XL is transcriptionally modulated by PAX3 and PAX3/FKHR, since enhanced expression of both PAX proteins stimulates transcription of endogenous BCL-XL mRNA in a cell type specific manner. Further, we present evidence that both PAX3 and PAX3/FKHR can transcriptionally activate the Bcl-x gene promoter in cotransfection assays. Using electrophoretic mobility shift assays, an ATTA binding site for PAX3 and PAX3/FKHR could be localized in the upstream promoter region (position -42 to -39). Finally, ectopic overexpression of either PAX3, PAX3/FKHR or BCL-XL can rescue tumor cells from apoptosis induced by antisense treatment. These results suggest that at least part of the anti-apoptotic effect of PAX3 and PAX3/FKHR is mediated through direct transcriptional modulation of the prominent anti-apoptotic protein BCL-XL. Oncogene (2000).

PAX3 and PAX7 exhibit conserved cis-acting transcription repression domains and utilize a common gain of function mechanism in alveolar rhabdomyosarcoma.

The t(2;13) and t(1;13) translocations of alveolar rhabdomyosarcoma (ARMS) result in chimeric PAX3-FKHR or PAX7-FKHR transcription factors, respectively. In each chimera, a PAX DNA-binding domain is fused to the C-terminal FKHR transactivation domain. Previously we demonstrated that PAX3-FKHR is more potent than PAX3 because the FKHR transactivation domain is resistant to repression mediated by the PAX3 N-terminus. Here we test the hypothesis that the cis-acting repression domain is a conserved feature of PAX3 and PAX7 and that PAX7-FKHR gains function similarly. Using PAX-specific DNA-binding sites, we found that PAX7 was virtually inactive, while PAX7-FKHR exhibited activity 600-fold above background and was comparable to PAX3-FKHR. Deletion analysis showed that the transactivation domains of PAX7 and PAX7-FKHR are each more potent than either full-length protein, and resistance to cis-repression is responsible for the PAX7-FKHR gain of function. Further deletion mapping and domain swapping experiments with PAX3 and PAX7 showed that their transactivation domains exhibit subtle dose-dependent differences in potency, likely due to regions of structural divergence; while their repression domains are structurally and functionally conserved. Thus, the data support the hypothesis and demonstrate that PAX3 and PAX7 utilize a common gain of function mechanism in ARMS.

Wild type PAX3 protein and the PAX3-FKHR fusion protein of alveolar rhabdomyosarcoma contain potent, structurally distinct transcriptional activation domains.

Alveolar rhabdomyosarcoma (ARMS) is characterized cytogenetically by a t(2;13)(q35;q14) chromosomal translocation involving two transcription factor genes: PAX3 and FKHR. ARMS cells express a PAX3-FKHR fusion protein containing the complete N-terminal, DNA-binding domain of PAX3 and the C-terminus of FKHR. Recently we demonstrated that PAX3-FKHR is a more potent transcriptional activator than PAX3 despite impaired binding to canonical PAX3 binding sites. Therefore, we propose that the gene fusion results in switching of PAX3 and FKHR transactivation domains with distinct structure, potency or function. To compare the PAX3 and putative PAX3-FKHR transactivation domains, we fused C-terminal test fragments to the heterologous GAL4 DNA-binding domain and tested activation of a reporter gene co-transfected into four cell types. GAL4-PAX3 and GAL4-PAX3-FKHR were found to be potent activators exhibiting different concentration-dependent transactivation profiles and distinct structural motifs. Deletion mapping demonstrated essential acidic and/or serine/threonine-rich domains in the extreme 3' ends of their respective coding regions and positive modifying elements in adjacent 5' sequences. These data demonstrate that PAX3 and PAX3-FKHR contain structurally distinct transcriptional activation domains and suggest that a consequent difference in function is important for oncogenesis.

Regulation of the forkhead transcription factor FKHR, but not the PAX3-FKHR fusion protein, by the serine/threonine kinase Akt.

Akt, a proto-oncogene that encodes a cytosolic serine/threonine kinase, can phosphorylate and modulate the activity of several proteins involved in cellular metabolism and survival. Recently, two mammalian highly related forkhead transcription factors FKHRL1 and AFX and their nematode homologue Daf-16 have been found to be targets of this kinase. Here we show that Akt, but not inactive Akt, represses the transcriptional activity of FKHR, another member of the forkhead family. FKHR mutants with alanine substitutions at three Akt phosphorylation consensus sites (T24, S256 and S319) were inhibited by Akt, but mutation of all three sites rendered FKHR resistant to suppression. By contrast, the transcriptional activity of the oncogenic PAX3-FKHR fusion protein, containing two consensus phosphorylation sites, was not inhibited by Akt. Importantly, Akt inhibited the translocation of FKHR to the nucleus, providing a mechanism by which Akt might regulate the transcriptional activity of FKHR. Consistent with this model, the localization of the PAX3-FKHR fusion protein was nuclear and was not altered by Akt. These results provide evidence that Akt inhibits the transcriptional activity of FKHR by controlling its trafficking into the nucleus and that oncogenic PAX3-FKHR can escape this negative regulation by Akt.

Common and variant gene fusions predict distinct clinical phenotypes in rhabdomyosarcoma.

PURPOSE: We evaluated the clinical features of the common PAX3-FKHR and variant PAX7-FKHR gene fusions observed in rhabdomyosarcoma. PATIENTS AND METHODS: Reverse-transcriptase polymerase chain reaction (RT-PCR) assays were used to detect the gene fusions in 34 cases of rhabdomyosarcoma. Clinical data were obtained retrospectively and compared with the molecular results. RESULTS: The PAX3-FKHR and PAX7-FKHR gene fusions were present in tumors from 18 and 16 patients, respectively. The group with a PAX7-FKHR fusion was younger (P = .01) and presented more often with an extremity lesion (82% v 22%; P = .001). PAX7-FKHR tumors were more often localized than PAX3-FKHR tumors (P = .03). In patients with metastatic disease at diagnosis, the patterns were different: PAX7-FKHR patients had metastatic disease that involved only bone (n = 2) and distant nodes (n = 2), while the PAX3-FKHR group had multiple sites involved, including bone (n = 7), marrow (n = 7), lungs (n = 3), distant nodes (n = 2), skin (n = 1), and brain (n = 1). No significant difference in relapse rate was observed. A trend toward improved overall survival in the PAX7-FKHR group was noted (P = .09). Event-free survival for this PAX7-FKHR group was significantly longer (P = .04). CONCLUSION: Our results suggest that the common PAX3-FKHR and the variant PAX7-FKHR fusions are associated with distinct clinical phenotypes. Identification of fusion gene status may be a useful diagnostic tool in rhabdomyosarcoma.

EWS-FLI1 and EWS-ERG gene fusions are associated with similar clinical phenotypes in Ewing's sarcoma.

PURPOSE: There are a variety of solid tumors in which alternative chromosomal translocations generate related fusion products. In alveolar rhabdomyosarcoma and synovial sarcoma, these variant fusions have been found to have major clinical significance. We investigated whether the two alternative gene fusion products, EWS-FLI1 and EWS-ERG, define different clinical subsets within the Ewing's sarcoma family of tumors. PATIENTS AND METHODS: We selected 30 cases of Ewing's sarcoma with the EWS-ERG gene fusion and 106 cases with the EWS-FLI1 fusion. Clinical data were obtained for each case and compared with the molecular diagnostic findings. RESULTS: There were no significant clinical differences observed between the two groups in age of diagnosis, sex, metastasis at diagnosis, primary site, event-free survival, or overall survival. CONCLUSION: Differences in the C-terminal partner in the Ewing's sarcoma family gene fusions are not associated with significant phenotypic differences.

EWS-FLI1 fusion transcript structure is an independent determinant of prognosis in Ewing's sarcoma.

PURPOSE: More than 90% of Ewing's sarcomas (ES) contain a fusion of the EWS and FLI1 genes, due to the t(11;22)(q24;q12) translocation. At the molecular level, the EWS-FLI1 rearrangements show great diversity. Specifically, many different combinations of exons from EWS and FLI1 encode in-frame fusion transcripts and result in differences in the length and composition of the chimeric protein, which functions as an oncogenic aberrant transcription factor. In the most common fusion type (type 1), EWS exon 7 is linked in frame with exon 6 of FLI1. As the fundamental pathogenetic lesion in ES, the molecular heterogeneity of these fusion transcripts may have functional and clinical significance. PATIENTS AND METHODS: We performed a clinical and pathologic analysis of 112 patients with ES in which EWS-FLI1 fusion transcripts were identified by reverse-transcriptase polymerase chain reaction (RT-PCR). Adequate treatment and follow-up data were available in 99 patients treated with curative intent. Median follow-up in these 99 patients was 26 months (range, 1 to 140 months). Univariate and multivariate survival analyses were performed that included other prognostic factors, such as age, tumor location, size, and stage. RESULTS: Among the 99 patients suitable for survival analysis, the tumors in 64 patients contained the type 1 fusion and in 35 patients contained less common fusion types. Stage at presentation was localized in 74 patients and metastatic in 25. Metastases (relative risk [RR] = 2.6; P = .008), and type 1 EWS-FLI1 fusion (RR = 0.37; P = .014) were, respectively, independent negative and positive prognostic factors for overall survival by multivariate analysis. Among 74 patients with localized tumors, the type 1 EWS-FLI1 fusion was also a significant positive predictor of overall survival (RR = 0.32; P = .034) by multivariate analysis. CONCLUSION: EWS-FLI1 fusion type appears to be prognostically relevant in ES, independent of tumor site, stage, and size. Further studies are needed to clarify the biologic basis of this phenomenon.