Global gene expression profiling of PAX-FKHR fusion-positive alveolar and PAX-FKHR fusion-negative embryonal rhabdomyosarcomas.

Paediatric rhabdomyosarcomas (RMS) are classified into two major subtypes based on histological appearance, embryonal (ERMS) and alveolar (ARMS), but this clinically critical distinction is often difficult on morphological grounds alone. ARMS, the more aggressive subtype, is associated in most cases with unique recurrent translocations fusing the PAX3 or PAX7 transcription factor genes to FKHR. In contrast, ERMS lacks unique genetic alterations. To identify novel diagnostic markers and potential therapeutic targets, we analysed the global gene expression profiles of these two RMS subtypes in 23 ARMS (16 PAX3-FKHR, 7 PAX7-FKHR) and 15 ERMS (all PAX-FKHR-negative) using Affymetrix HG-U133A oligonucleotide arrays. A statistically stringent supervised comparison of the ARMS and ERMS expression profiles revealed 121 genes that were significantly differentially expressed, of which 112 were higher in ARMS, including genes of interest as potential diagnostic markers or therapeutic targets, such as CNR1, PIPOX (sarcosine oxidase), and TFAPbeta. Interestingly, many known or putative downstream targets of PAX3-FKHR were highly overexpressed in ARMS relative to ERMS, including CNR1, DCX, ABAT, ASS, JAKMIP2, DKFZp762M127, and NRCAM. We validated the highly differential expression of five genes, including CNR1, DKFZp762M127, DCX, PIPOX, and FOXF1 in ARMS relative to ERMS by quantitative RT-PCR on an independent set of samples. Finally, we developed a ten-gene microarray-based predictor that distinguished ARMS from ERMS with approximately 95% accuracy both in our data by cross-validation and in an independent validation using a published dataset of 26 samples. The gene expression signature of ARMS provides a source of potential diagnostic markers, therapeutic targets, and PAX-FKHR downstream genes, and can be used to reliably distinguish these sarcomas from ERMS.

Chromosomal translocations in sarcomas: prospects for therapy.

In the past two decades, cytogenetic and molecular genetic investigations have revealed that sarcomas are frequently characterized by specific chromosomal translocations, often involving genes encoding transcription factors. These translocations result in the expression of chimeric oncoproteins that contain functional domains contributed by each of the parental genes. Functioning as deregulated transcription and signaling factors, these novel proteins contribute to the malignant phenotype of the tumor cell by disrupting the tightly regulated process of target gene expression. Several therapeutic strategies that exploit the tumor-specific nature of these oncogenes are currently being investigated. These targeted approaches seek to manipulate the specific biology of these gene fusions, and along with more traditional therapeutic modalities, could augment current approaches to cancer management.

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.

Proliferative and apoptotic differences between alveolar rhabdomyosarcoma subtypes: a comparative study of tumors containing PAX3-FKHR or PAX7-FKHR gene fusions.

BACKGROUND: Most alveolar rhabdomyosarcomas (ARMS) have chromosome translocations and resultant gene fusion products. The more common translocation fuses the PAX3 and FKHR genes; patients who have PAX3-FKHR-positive ARMS have reduced event-free survival compared to patients with ARMS containing the less common translocation that fuses the PAX7 and FKHR genes. PROCEDURE: We examined histology, immunohistochemical markers of differentiation, and cell cycle characteristics of a panel of ARMS containing either PAX3-FKHR or PAX7-FKHR transcript to determine if these features differ between the ARMS subsets. RESULTS: Cell cycle parameters varied significantly: the number of nuclei that stained with either an immunohistochemical marker of proliferation (MIB1), or a TUNEL-based assay for apoptosis was significantly greater in tumors that expressed PAX3-FKHR compared to tumors that expressed PAX7-FKHR transcript. CONCLUSIONS: We conclude that compared to PAX7-FKHR-containing tumors, ARMS that contain PAX3-FKHR transcript have (1) increased cell proliferation, consistent with greater loss of cell cycle regulation, and (2) apoptosis that is increased but insufficient to prevent tumor formation. More marked cell cycle dysregulation may contribute to poorer prognosis for patients with ARMS that have PAX3-FKHR fusion. Med Pediatr Oncol 2001;37:83-89.

Forkhead homologue in rhabdomyosarcoma functions as a bifunctional nuclear receptor-interacting protein with both coactivator and corepressor functions.

In a search for novel transcriptional intermediary factors for the estrogen receptor (ER), we used the ligand-binding domain and hinge region of ER as bait in a yeast two-hybrid screen of a cDNA library derived from tamoxifen-resistant MCF-7 human breast tumors from an in vivo athymic nude mouse model. Here we report the isolation and characterization of the forkhead homologue in rhabdomyosarcoma (FKHR), a recently described member of the hepatocyte nuclear factor 3/forkhead homeotic gene family, as a nuclear hormone receptor (NR) intermediary protein. FKHR interacts with both steroid and nonsteroid NRs, although the effect of ligand on this interaction varies by receptor type. The interaction of FKHR with ER is enhanced by estrogen, whereas its interaction with thyroid hormone receptor and retinoic acid receptor is ligand-independent. In addition, FKHR differentially regulates the transactivation mediated by different NRs. Transient transfection of FKHR into mammalian cells dramatically represses transcription mediated by the ER, glucocorticoid receptor, and progesterone receptor. In contrast, FKHR stimulates rather than represses retinoic acid receptor- and thyroid hormone receptor-mediated transactivation. Most intriguingly, overexpression of FKHR dramatically inhibits the proliferation of ER-dependent MCF-7 breast cancer cells. Therefore, FKHR represents a bifunctional NR intermediary protein that can act as either a coactivator or corepressor, depending on the receptor type.

Rhabdomyosarcoma and undifferentiated sarcoma in the first two decades of life: a selective review of intergroup rhabdomyosarcoma study group experience and rationale for Intergroup Rhabdomyosarcoma Study V.

PURPOSE: To review the importance of prognostic factors in developing new protocols for children with rhabdomyosarcoma (RMS). PATIENTS AND METHODS: Four studies conducted by the Intergroup Rhabdomyosarcoma Study (IRS) Group from 1972 through 1991. RESULTS: Favorable prognostic factors are: (1) undetectable distant metastases at diagnosis; (2) primary sites in the orbit and nonparameningeal head/neck and genitourinary nonbladder/prostate regions; (3) grossly complete surgical removal of localized tumor at the time of diagnosis; (4) embryonal/botryoid histology; (5) tumor size < or = 5 cm; and (6) age younger than 10 years at diagnosis. The IRS-V protocols are risk-based and refine therapy by reducing exposure to cyclophosphamide and radiation therapy (XRT) in patients at low risk while adding new, active agents such as topotecan or irinotecan to the standard therapy of vincristine, actinomycin D, and cyclophosphamide (VAC) plus XRT for patients with unfavorable histology or advanced disease. Collection of biologic specimens from patients with newly diagnosed disease continues to identify other factors that may distinguish patients with favorable features from those who need more intensive therapy. A new protocol that takes into account their previous treatment is needed for patients with recurrent disease. This program (being planned) does not include bone marrow/stem cell reconstitution because this strategy has thus far failed to improve survival rates of patients with metastases at diagnosis. CONCLUSION: Better understanding of biologic differences and new, active agents are needed to improve outcome of patients with unfavorable features at presentation.

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).

Association of EWS-FLI1 type 1 fusion with lower proliferative rate in Ewing's sarcoma.

The Ewing's sarcoma (ES) family of tumors, including peripheral neuroectodermal tumor (PNET), is defined genetically by specific chromosomal translocations resulting in fusion of the EWS gene with a member of the ETS family of transcription factors, either FLI1 (90-95%) or ERG (5-10%). A second level of molecular genetic heterogeneity stems from the variation in the location of the translocation breakpoints, resulting in the inclusion of different combinations of exons from EWS and FLI1 (or ERG) in the fusion products. The most common type of EWS-FLI1 fusion transcript, type 1, is associated with a favorable prognosis and appears to encode a functionally weaker transactivator, compared to other fusion types. We sought to determine whether the observed covariation of structure, function, and clinical course correlates with tumor cell kinetic parameters such as proliferative rate and apoptosis, and with expression of the receptor for insulin-like growth factor I (IGF-1R). In a group of 86 ES/PNET with defined EWS-ETS fusions (45 EWS-FLI1 type 1, 27 EWS-FLI1 non-type 1, 14 EWS-ERG), we assessed proliferation rate by immunostaining for Ki-67 using MIB1 antibody (n = 85), apoptosis by TUNEL assay (n = 66), and IGF-1R expression by immunostaining with antibody 1H7 (n = 78). Ki-67 proliferative index was lower in tumors with EWS-FLI1 type 1 than those with non-type 1 EWS-FLI1, whether analyzed as a continuous (P = 0.049) or categorical (P = 0.047) variable. Logistic regression analysis suggests that this association was secondary to the association of type 1 EWS-FLI1 and lower IGF-1R expression (P = 0.04). Comparing EWS-FLI1 to EWS-ERG cases, Ki-67 proliferative index was higher in the latter (P = 0.01, Mann-Whitney test; P = 0.02, Fisher's exact test), but there was no significant difference in IGF-1R. TUNEL results showed no significant differences between groups. Our results suggest that clinical and functional differences between alternative forms of EWS-FLI1 are paralleled by differences in proliferative rate, possibly mediated by differential regulation of the IGF-1R pathway.

Structural analysis of PAX7 rearrangements in alveolar rhabdomyosarcoma.

In the pediatric cancer alveolar rhabdomyosarcoma, the common 2;13 and less frequent 1;13 translocations fuse PAX3 and PAX7, respectively, with FKHR to produce chimeric genes. To compare structural features of these rearrangements, we cloned and mapped a 64-kb genomic region containing PAX7 exons 5 through 8. With the use of Southern blot methodology, rearrangements of the 30-kb PAX7 intron 7 were detected in 9 of 9 PAX7-FKHR-positive cases. Similar to our t(2;13) studies, the t(1;13) breakpoints were randomly distributed within the seventh intron. In contrast with the > 90% frequency of reciprocal rearrangements in the t(2;13), reciprocal rearrangements involving the 3' PAX7 region were detected in only 4 of 9 cases. Furthermore, we detected PAX7-FKHR genomic amplification in 10 of 11 cases, in contrast with the < 5% frequency of PAX3-FKHR amplification. The differences in occurrence, reciprocity, and amplification between the PAX3-FKHR and PAX7-FKHR fusions indicate important differences in the mechanism of the two associated chromosomal translocation events.

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.

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.

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.

Cytokeratin-negative desmoplastic small round cell tumor: a report of two cases emphasizing the utility of reverse transcriptase-polymerase chain reaction.

Desmoplastic small round cell tumor (DSRCT) is a unique, highly aggressive neoplasm that chiefly affects male adolescents and young adults. This tumor is characterized by nests of small undifferentiated cells that show immunohistochemical evidence of epithelial, mesenchymal, and neural differentiation. We report two cases of DSRCT that lacked immunohistochemical evidence of epithelial differentiation, but were found to have the fusion transcripts characteristic of this tumor. Both patients (a 41-year-old male and a 31-year-old female) presented with large intra-abdominal masses. After diagnostic biopsy, both were treated with multi-agent chemotherapy. One patient expired 18 days after diagnosis, and the other is currently alive 28 months later. Histologically, both tumors had the characteristic features of DSRCT and were composed of small round cells with hyperchromatic nuclei and scanty cytoplasm. In one of the cases, perinuclear intracytoplasmic hyaline inclusions were seen. Immunohistochemically, neither case expressed any of the epithelial markers tested, including AE1/AE3, CAM 5.2 and EMA. Both tumors were diffusely immunoreactive for desmin with a prominent globoid "dot-like" pattern of staining in one case. Both tumors stained for vimentin, neuron specific enolase, and synaptophysin, but were negative for CD99, muscle-specific actin, and myogenin. Reverse transcriptase-polymerase chain reaction revealed EWS-WT1 fusion transcripts characteristic of this neoplasm. In conclusion, we describe two cases of DSRCT that lacked immunohistochemical evidence of epithelial differentiation but had histologic and other immunohistochemical features which suggested this diagnosis. The ability to confirm the diagnosis of this rare tumor using molecular genetic techniques is particularly useful in those cases with unusual histologic or immunophenotypic features.

Conservation of an insulin response unit between mouse and human glucose-6-phosphatase catalytic subunit gene promoters: transcription factor FKHR binds the insulin response sequence.

Because overexpression of the glucose-6-phosphatase catalytic subunit (G-6-Pase) in both type 1 and type 2 diabetes may contribute to the characteristic increased rate of hepatic glucose production, we have investigated whether the insulin response unit (IRU) identified in the mouse G-6-Pase promoter is conserved in the human promoter. A series of human G-6-Pase-chloramphenicol acetyltransferase (CAT) fusion genes was transiently transfected into human HepG2 hepatoma cells, and the effect of insulin on basal CAT expression was analyzed. The results suggest that the IRU identified in the mouse promoter is conserved in the human promoter, but that an upstream multimerized insulin response sequence (IRS) motif that is only found in the human promoter appears to be functionally inactive. The G-6-Pase IRU comprises two distinct promoter regions, designated A and B. Region B contains an IRS, whereas region A acts as an accessory element to enhance the effect of insulin, mediated through region B, on basal G-6-Pase gene transcription. We have previously shown that the accessory factor binding region A is hepatocyte nuclear factor-1, and we show here that the forkhead protein FKHR is a candidate for the insulin-responsive transcription factor binding region B.

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.

Absence of SYT-SSX fusion products in soft tissue tumors other than synovial sarcoma.

The chromosomal translocation t(X;18), which generates SYT-SSX1 and SYT-SSX2 fusion products, is a sensitive marker for synovial sarcoma; most synovial sarcomas test positive for this marker. However, few studies have addressed the presence of t(X;18) or its fusion products in spindle cell sarcomas in the differential diagnosis of synovial sarcoma. We studied the presence of the SYT-SSX fusion products with reverse transcriptase polymerase chain reaction on frozen tissue samples of 24 synovial sarcomas and 24 other spindle cell sarcomas, including 12 malignant peripheral nerve sheath tumors. In cases histopathologically diagnosed as synovial sarcoma, SYT-SSX fusion products were detected in 21 of 24 (87%) lesions. No evidence of these fusions was found in 12 malignant peripheral nerve sheath tumors, 2 hemangiopericytomas, 3 leiomyosarcomas, 2 fibrosarcomas, 1 poorly differentiated sarcoma (malignant fibrous histiocytoma), 1 sarcoma with rhabdoid features, and 2 sarcomas not otherwise specified. One lesion with histologic, immunohistologic, and ultrastructural features indeterminate for a diagnosis of synovial sarcoma or malignant peripheral nerve sheath tumor was studied and was positive for SYT-SSX1. The SYT-SSX fusion products appear specific for synovial sarcoma and are not seen in other spindle cell lesions in its differential diagnosis.

Genetics and the biologic basis of sarcomas.

Identification of genetic alterations has contributed greatly to the understanding of sarcoma biology. Additionally, detection of these abnormalities is providing new tools for the diagnosis of sarcomas. In this paper, three important new genetic findings from the past year are reviewed, including the t(12;15) translocation of congenital fibrosarcoma, mutation of the putative tumor suppressor gene hSNF5/INI1 in malignant rhabdoid tumor, and the association of c-kit mutations with gastrointestinal stromal tumor. Highlighted are important studies concerning mechanisms of chromosomal translocation, functions of sarcoma-specific fusion proteins, genetic abnormalities other than translocations, molecular diagnosis, and molecular profiling of gene expression. Particular emphasis is placed on information obtained with comparative genomic hybridization and microarray techniques, because these powerful technologies will facilitate the rapid acquisition of data that provide insight into the molecular genetic and biologic basis of sarcomas.

FKHR binds the insulin response element in the insulin-like growth factor binding protein-1 promoter.

The insulin response element (IRE) in the IGFBP-1 promoter, and in other gene promoters, contains a T(A/G)TTT motif essential for insulin inhibition of transcription. Studies presented here test whether FKHR may be the transcription factor that confers insulin inhibition through this IRE motif. Immunoblots using antiserum to the synthetic peptide FKHR413-430, RNase protection, and Northerns blots show that FKHR is expressed in HEP G2 human hepatoma cells. Southwestern blots, electromobility shift assays, and DNase I protection assays show that Escherichia coli-expressed GST-FKHR binds specifically to IREs from the IGFBP-1, PEPCK and TAT genes; however, unlike HNF3beta, another protein proposed to be the insulin regulated factor, GST-FKHR does not bind the insulin unresponsive G/C-A/C mutation of the IGFBP-1 IRE. When HEP G2 cells were cotransfected with FKHR expression vectors and with IGFBP-1 promoter plasmids containing either native or mutant IREs, FKHR expression induced a 5-fold increase in activity of the native IGFBP-1 promoter but no increase in activity of promoter constructs containing insulin unresponsive IRE mutants. These data suggest that FKHR, and/or a related family member, is the important T(G/A)TTT binding protein that confers the inhibitory effect of insulin on gene transcription.

Negative regulation of the forkhead transcription factor FKHR by Akt.

The FKHR gene was first identified from its disruption by the t(2;13) chromosomal translocation seen in the pediatric tumor alveolar rhabdomyosarcoma. It encodes for a member of the forkhead family of transcription factors. Recently, a homolog of FKHR in the nematode Caenorhabditis elegans was identified called DAF-16, which is a downstream target of two Akt homologs in an insulin-related signaling pathway. We have examined the possible role of Akt in the regulation of FKHR. We find that FKHR can bind in vitro to the insulin-responsive sequence (IRS) in the insulin-like growth factor-binding protein 1 promoter and can activate transcription from a reporter plasmid containing multiple copies of the IRS. Expression of active but not inactive Akt can suppress FKHR-mediated transcriptional activation. Akt can phosphorylate FKHR in vitro on three phosphoacceptor sites, at least a subset of which can also be phosphorylated by Akt in vivo. Importantly, mutation of these three sites to alanine residues enhances the transcriptional activity of FKHR and renders it resistant to inhibition by Akt. Expression of an Akt-resistant mutant of FKHR causes apoptosis in 293T cells in a manner dependent on DNA binding. These results suggest that FKHR may be a direct nuclear regulatory target for Akt in both metabolic and cell survival pathways.