WT1 Isoforms Research Articles

Overexpression of WT1 in all molecular subtypes of breast cancer and its impact on survival: exploring oncogenic and tumor suppressor roles of distinct WT1 isoforms.

Breast cancer is a highly heterogeneous solid tumor, posing challenges in developing targeted therapies effective for all mammary carcinoma subtypes. WT1 emerges as a promising target for breast cancer therapy due to its potential oncogenic role in various cancer types. Previous works have yielded inconsistent results. Therefore, further studies are needed to clarify the behavior of this complex gene in breast cancer. In this study, we examined WT1 expression in both Formalin Fixed Paraffin Embedded breast tumors (n = 41) and healthy adjacent tissues (n = 41) samples from newly diagnosed cases of ductal invasive breast cancer. The fold change in gene expression between the tumor and healthy tissue was determined by calculating 2-∆∆Ct. Disease-free survival analysis was computed using the Kaplan-Meier method. To identify the expression levels of different WT1 isoforms, we explored the ISOexpresso database. Relative quantification of the WT1 gene revealed an overexpression of WT1 in most cases. The percentage of patients surviving free of disease at 8years of follow-up was lower in the group overexpressing WT1 compared to the group with down-regulated WT1. Interestingly, this overexpression was observed in all molecular subtypes of invasive breast cancer, underscoring the significance of WT1 as a potential target in all these subtypes. The observed WT1 down-expression in a few cases of invasive breast cancer, associated with better survival outcomes, may correspond to the down-regulation of a particular WT1-KTS (-) isoform: the WT1 A isoform (EX5-/KTS-). The co-expression of this WT1 oncogenic isoform with a regulated WT1- tumor suppressor isoform, such as the major WT1 F isoform (EX5-/KTS +), could also explain such survival outcomes. Due to its capacity to adopt dual roles, it becomes imperative to conduct individual molecular expression profiling of the WT1 gene. Such an approach holds great promise in the development of personalized treatment strategies for breast cancer.

Understanding WT1 Alterations and Expression Profiles in Hematological Malignancies

WT1 is a true chameleon, both acting as an oncogene and tumor suppressor. As its exact role in leukemogenesis is still ambiguous, research with model systems representing natural conditions surrounding the genetic alterations in WT1 is necessary. In a cohort of 59 leukemia/lymphoma cell lines, we showed aberrant expression for WT1 mRNA, which does not always translate into protein levels. We also analyzed the expression pattern of the four major WT1 protein isoforms in the cell lines and primary AML blasts with/without WT1 mutations and demonstrated that the presence of mutations does not influence these patterns. By introduction of key intronic and exonic sequences of WT1 into a lentiviral expression vector, we developed a unique tool that can stably overexpress the four WT1 isoforms at their naturally occurring tissue-dependent ratio. To develop better cellular model systems for WT1, we sequenced large parts of its gene locus and also other important myeloid risk factor genes and revealed previously unknown alterations. Functionally, inhibition of the nonsense-mediated mRNA decay machinery revealed that under natural conditions, the mutated WT1 alleles go through a robust degradation. These results offer new insights and model systems regarding the characteristics of WT1 in leukemia and lymphoma.

Truncated WT1 Protein Isoform Expression Is Increased in MCF-7 Cells with Long-Term Estrogen Depletion.

Background The wt1 gene codes for a transcription factor that presents several protein isoforms with diverse biological properties, capable of positively and negatively regulating genes involved in proliferation, differentiation, and apoptosis. WT1 protein is overexpressed in more than 90% of breast cancer; however, its role during tumor progression is still unknown. Methodology. In this work, we analyzed the expression of WT1 isoforms in several breast cancer cells with different tumor marker statuses and an in vitro assay using MCF-7 cells cultured with long-term estrogen depletion (MCF-7 LTED cells) with the finality to mimic the process of switching from hormone-dependent to hormone-independent. Moreover, growth kinetics, sensitivity to tamoxifen, and relative expression analysis of ER and Her2/neu were performed. Results Initially, the expression of 52-54 kDa protein isoform of WT1 in the breast cancer cell line ER (+) was detected by western blot and was absent in ER (-), and the 36-38 kDa protein isoform of WT1 was detected in all cell lines analyzed. The analysis of alternative splicing by RT-PCR shows that the 17AA (+)/KTS (-) isoform of WT1 was the most frequent in the four cell lines analyzed. In vitro, the MCF-7 cells in the estrogen depletion assay show an increase in the expression of the 52-54 kDa isoform of WT1 in the first 48 hours, and this was maintained until week 13, and later, this expression was decreased, and the 36-38 kDa isoform of WT1 did not show change during the first 48 hours but from week 1 showed an increase of expression, and this remained until week 27. Growth kinetic analysis showed that MCF-7 LTED cells presented a 1.4-fold decrease in cellular proliferation compared to MCF-7 cells cultured under normal conditions. In addition, MCF-7 LTED cells showed a decrease in sensitivity to the antiproliferative effect of tamoxifen (p ≤ 0.05). Samples collected until week 57 analyzed by qRT-PCR showed an increase in the relative expression of the Her2/neu and ER. Conclusions Modulation of protein isoforms showed differential expression of WT1 isoforms dependent on estrogen receptor. The absence of 52-54 kDa and the presence of the 36-38 kDa protein isoform of WT1 were detected in ER-negative breast cancer cell lines classified as advanced stage cells. Long-term estrogen depletion assay in MCF-7 cells increased the expression of the 36-38 kDa isoform and reduced the 52-54 kDa isoform, and these cells show an increase in the expression of tumor markers of ER and Her2/neu. MCF-7 LTED cells showed low proliferation and insensitivity to tamoxifen compared to MCF-7 cells in normal conditions. These results support the theory about the relationship of the 36-38 kDa isoform of WT1 and the absence of ER function in advanced breast cancer.

Implications of the Wilms' Tumor Suppressor Wt1 in Cardiomyocyte Differentiation.

The Wilms’ tumor suppressor Wt1 is involved in multiple developmental processes and adult tissue homeostasis. The first phenotypes recognized in Wt1 knockout mice were developmental cardiac and kidney defects. Wt1 expression in the heart has been described in epicardial, endothelial, smooth muscle cells, and fibroblasts. Expression of Wt1 in cardiomyocytes has been suggested but remained a controversial issue, as well as the role of Wt1 in cardiomyocyte development and regeneration after injury. We determined cardiac Wt1 expression during embryonic development, in the adult, and after cardiac injury by quantitative RT-PCR and immunohistochemistry. As in vitro model, phenotypic cardiomyocyte differentiation, i.e., the appearance of rhythmically beating clones from mouse embryonic stem cells (mESCs) and associated changes in gene expression were analyzed. We detected Wt1 in cardiomyocytes from embryonic day (E10.5), the first time point investigated, until adult age. Cardiac Wt1 mRNA levels decreased during embryonic development. In the adult, Wt1 was reactivated in cardiomyocytes 48 h and 3 weeks following myocardial infarction. Wt1 mRNA levels were increased in differentiating mESCs. Overexpression of Wt1(-KTS) and Wt1(+KTS) isoforms in ES cells reduced the fraction of phenotypically cardiomyocyte differentiated clones, which was preceded by a temporary increase in c-kit expression in Wt1(-KTS) transfected ES cell clones and induction of some cardiomyocyte markers. Taken together, Wt1 shows a dynamic expression pattern during cardiomyocyte differentiation and overexpression in ES cells reduces their phenotypical cardiomyocyte differentiation.

Immunohistochemical Expression of Wilms’ Tumor 1 Protein in Human Tissues: From Ontogenesis to Neoplastic Tissues

The human Wilms’ tumor gene (WT1) was originally isolated in a Wilms’ tumor of the kidney as a tumor suppressor gene. Numerous isoforms of WT1, by combination of alternative translational start sites, alternative RNA splicing and RNA editing, have been well documented. During human ontogenesis, according to the antibodies used, anti-C or N-terminus WT1 protein, nuclear expression can be frequently obtained in numerous tissues, including metanephric and mesonephric glomeruli, and mesothelial and sub-mesothelial cells, while cytoplasmic staining is usually found in developing smooth and skeletal cells, myocardium, glial cells, neuroblasts, adrenal cortical cells and the endothelial cells of blood vessels. WT1 has been originally described as a tumor suppressor gene in renal Wilms’ tumor, but more recent studies emphasized its potential oncogenic role in several neoplasia with a variable immunostaining pattern that can be exclusively nuclear, cytoplasmic or both, according to the antibodies used (anti-C or N-terminus WT1 protein). With the present review we focus on the immunohistochemical expression of WT1 in some tumors, emphasizing its potential diagnostic role and usefulness in differential diagnosis. In addition, we analyze the WT1 protein expression profile in human embryonal/fetal tissues in order to suggest a possible role in the development of organs and tissues and to establish whether expression in some tumors replicates that observed during the development of tissues from which these tumors arise.

DNA and RNA binding by the Wilms' tumour gene 1 (WT1) protein +KTS and -KTS isoforms-From initial observations to recent global genomic analyses.

The Wilms' tumour gene 1 protein (WT1) is a zinc finger transcription factor found indispensable for foetal development. WT1 has also been implicated in the development of tumours in several organ systems, including acute myeloid leukaemia (AML). Four main WT1 isoforms come from 2 alternative splice events. One alternative splice results in the inclusion or exclusion of 3 amino acids, KTS, between zinc fingers 3 and 4 in the WT1 protein. The KTS insert has been extensively investigated due to the functional implications for DNA and RNA binding. In this review, we provide an overview of the research into the isoforms containing or lacking the KTS insert in leukaemic cells, as well as the research into the binding patterns of the WT1 -KTS and +KTS isoforms to DNA and RNA. Finally, we connect the results of the DNA binding research to the ChIP-CHIP and ChIP-Seq investigations into the global genomic binding of the WT1 protein that have recently been performed.

The Wilms tumor protein WT1 stimulates transcription of the gene encoding insulin-like growth factor binding protein 5 (IGFBP5)

Insulin-like growth factor (IGF) binding proteins (IGFBPs) constitute a family of six secreted proteins that regulate the signaling of insulin-like growth factors (IGFs). IGFBP5 is the most conserved family member in vertebrates and the major IGF binding protein in bone. IGFBP5 is required for normal development of the musculoskeletal system, and various types of cancer frequently express high levels of IGFP5. Here we identify the gene encoding IGFBP5 as a novel downstream target of the Wilms tumor protein WT1. IGFBP5 and WT1 are expressed in an overlapping pattern in the condensing metanephric mesenchyme of embryonic murine kidneys. Down-regulation of WT1 by transfection with antisense vivo-morpholino significantly decreased Igfbp5 transcripts in murine embryonic kidney explants. Likewise, silencing of Wt1 in a mouse mesonephros-derived cell line reduced Igfbp5 mRNA levels by approximately 80%. Conversely, induction of the WT1(−KTS) isoform, whose role as transcriptional regulator has been firmly established, significantly increased IGFBP5 mRNA and protein levels in osteosarcoma cells. IGFBP5 expression was not significantly changed by WT1(+KTS) protein, which exhibits lower DNA binding affinity than the WT1(−KTS) isoform and has a presumed role in post-transcriptional gene regulation. Luciferase reporter constructs harboring 0.8 and 1.6 kilobases of the murine Igfbp5 promoter, respectively, were stimulated approximately 5-fold by co-transfection of WT1(−KTS). The WT1(+KTS) variant had no significant effect on IGFBP5 promoter activity. Binding of WT1(−KTS), but not of WT1(+KTS) protein, to the IGFBP5 promoter in human osteosarcoma cells was proven by chromatin immunoprecipitation (ChIP) and confirmed by electrophoretic mobility shift assay. These findings demonstrate that WT1 activates transcription of the IGFBP5 gene with possible implications for kidney development and bone (patho)physiology.

Dysregulation of WTI (\u2212KTS) is Associated with the Kidney-Specific Effects of the LMX1B R246Q Mutation

Mutations in the LIM homeobox transcription factor 1-beta (LMX1B) are a cause of nail patellar syndrome, a condition characterized by skeletal changes, glaucoma and focal segmental glomerulosclerosis. Recently, a missense mutation (R246Q) in LMX1B was reported as a cause of glomerular pathologies without extra-renal manifestations, otherwise known as nail patella-like renal disease (NPLRD). We have identified two additional NPLRD families with the R246Q mutation, though the mechanisms by which LMX1BR246Q causes a renal-specific phenotype is unknown. In this study, using human podocyte cell lines overexpressing either myc-LMX1BWT or myc-LMX1BR246Q, we observed dominant negative and haploinsufficiency effects of the mutation on the expression of podocyte genes such as NPHS1, GLEPP1, and WT1. Specifically, we observed a novel LMX1BR246Q-mediated downregulation of WT1(−KTS) isoforms in podocytes. In conclusion, we have shown that the renal-specific phenotype associated with the LMX1BR246Q mutation may be due to a dominant negative effect on WT1(−KTS) isoforms that may cause a disruption of the WT1 (−KTS):(+KTS) isoform ratio and a decrease in the expression of podocyte genes. Full delineation of the LMX1B gene regulon is needed to define its role in maintenance of glomerular filtration barrier integrity.

Wilms Tumor Suppressor, WT1, Cooperates with MicroRNA-26a and MicroRNA-101 to Suppress Translation of the Polycomb Protein, EZH2, in Mesenchymal Stem Cells

Hereditary forms of Wilms arise from developmentally arrested clones of renal progenitor cells with biallelic mutations of WT1; recently, it has been found that Wilms tumors may also be associated with biallelic mutations in DICER1 or DROSHA, crucial for miRNA biogenesis. We have previously shown that a critical role for WT1 during normal nephrogenesis is to suppress transcription of the Polycomb group protein, EZH2, thereby de-repressing genes in the differentiation cascade. Here we show that WT1 also suppresses translation of EZH2. All major WT1 isoforms induce an array of miRNAs, which target the 3' UTR of EZH2 and other Polycomb-associated transcripts. We show that the WT1(+KTS) isoform binds to the 5' UTR of EZH2 and interacts directly with the miRNA-containing RISC to enhance post-transcriptional inhibition. These observations suggest a novel mechanism through which WT1 regulates the transition from resting stem cell to activated progenitor cell during nephrogenesis. Our findings also offer a plausible explanation for the fact that Wilms tumors can arise either from loss of WT1 or loss of miRNA processing enzymes.

Correlation between expression profile of Wilms tumor 1 gene isoforms and neuroblastoma grade malignancy

Wilms tumor 1 gene (WT1) is expressed in neuroblastoma (NB) which represents the most aggressive extracranial pediatric tumor. This latter may transform into a more benign form such as ganglioneuroblastoma and ganglioneuroma or progress into a highly aggressive metastatic cancer with a poor survival rate. WT1 acts as tumor suppressor gene in NB by inducing the maturation in a less invasive mass. To date, it has been identified 13 mRNA WT1 variants encoding 13 proteins, however, most of the studies have focused their attention exclusively on isoform of ~49 kDa molecular weight (1, 2). In the present study, we have analyzed, the expression profile of WT1 isoforms, in undifferentiated and all-trans retinoic acid (RA) differentiated NB cells in order to evaluate their involvement in tumor malignancy. Results have shown that different isoforms are expressed both in untreated and RA treated NB cells. Their expression is significantly increased in RA treated cells, suggesting that WT1 isoforms are inversely related to NB malignancy grade. In accord to this hypothesis, WT1 isoforms and nestin expression are inversely related in undifferentiated and RA treated cells. Furthermore, the inhibition of the two signalling pathways specifically involved in differentiation of NB, PI3K/Akt and MAPK/ERK respectively, trigger an overexpression of all WT1 isoforms. In conclusion, these data suggest that overexpression of WT1 isoforms might promote trans-differentiation of NB into a more benign tumor such as ganglioneuroblastoma or ganglioneuroma.

Identification of a Novel C-Terminal Truncated WT1 Isoform with Antagonistic Effects against Major WT1 Isoforms.

The Wilms’ tumor gene WT1 consists of 10 exons and encodes a zinc finger transcription factor. There are four major WT1 isoforms resulting from alternative splicing at two sites, exon 5 (17AA) and exon 9 (KTS). All major WT1 isoforms are overexpressed in leukemia and solid tumors and play oncogenic roles such as inhibition of apoptosis, and promotion of cell proliferation, migration and invasion. In the present study, a novel alternatively spliced WT1 isoform that had an extended exon 4 (designated as exon 4a) with an additional 153 bp (designated as 4a sequence) at the 3’ end was identified and designated as an Ex4a(+)WT1 isoform. The insertion of exon 4a resulted in the introduction of premature translational stop codons in the reading frame in exon 4a and production of C-terminal truncated WT1 proteins lacking zinc finger DNA-binding domain. Overexpression of the truncated Ex4a(+)WT1 isoform inhibited the major WT1-mediated transcriptional activation of anti-apoptotic Bcl-xL gene promoter and induced mitochondrial damage and apoptosis. Conversely, suppression of the Ex4a(+)WT1 isoform by Ex4a-specific siRNA attenuated apoptosis. These results indicated that the Ex4a(+)WT1 isoform exerted dominant negative effects on anti-apoptotic function of major WT1 isoforms. Ex4a(+)WT1 isoform was endogenously expressed as a minor isoform in myeloid leukemia and solid tumor cells and increased regardless of decrease in major WT1 isoforms during apoptosis, suggesting the dominant negative effects on anti-apoptotic function of major WT1 isoforms. These results indicated that Ex4a(+)WT1 isoform had an important physiological function that regulated oncogenic function of major WT1 isoforms.

Wilms' tumor gene WT1 promotes homologous recombination-mediated DNA damage repair.

The Wilms' tumor gene WT1 is overexpressed in leukemia and various types of solid tumors and plays an oncogenic role in these malignancies. Alternative splicing at two sites yields four major isoforms, 17AA(+)KTS(+), 17AA(+)KTS(-), 17AA(-)KTS(+), and 17AA(-)KTS(-), and all the isoforms are expressed in the malignancies. However, among the four isoforms, function of WT1[17AA(-)KTS(+)] isoform still remains undetermined. In the present study, we showed that forced expression of WT1[17AA(-)KTS(+)] isoform significantly inhibited apoptosis by DNA-damaging agents such as Doxorubicin, Mitomycin, Camptothesisn, and Bleomycin in immortalized fibroblast MRC5SV and cervical cancer HeLa cells. Knockdown of Rad51, an essential factor for homologous recombination (HR)-mediated DNA repair canceled the resistance to Doxorubicin induced by WT1[17AA(-)KTS(+)] isoform. GFP recombination assay showed that WT1[17AA(-)KTS(+)] isoform alone promoted HR, but that three other WT1 isoforms did not. WT1[17AA(-)KTS(+)] isoform significantly upregulated the expression of HR genes, XRCC2, Rad51D, and Rad54. Knockdown of XRCC2, Rad51D, and Rad54 inhibited the HR activity and canceled resistance to Doxorubicin in MRC5SV cells with forced expression of WT1[17AA(-)KTS(+)] isoform. Furthermore, chromatin immunoprecipitation (ChIP) assay showed the binding of WT1[17AA(-)KTS(+)] isoform protein to promoters of XRCC2 and Rad51D. Immunohistochemical study showed that Rad54 and XRCC2 proteins were highly expressed in the majority of non-small-cell lung cancer (NSCLC) and gastric cancer, and that expression of these two proteins was significantly correlated with that of WT1 protein in NSCLCs. Our results presented here showed that WT1[17AA(-)KTS(+)] isoform had a function to promote HR-mediated DNA repair.

WT1 regulates angiogenesis in Ewing Sarcoma.

Angiogenesis is required for tumor growth. WT1, a protein that affects both mRNA transcription and splicing, has recently been shown to regulate expression of vascular endothelial growth factor (VEGF), one of the major mediators of angiogenesis. In the present study, we tested the hypothesis that WT1 is a key regulator of tumor angiogenesis in Ewing sarcoma. We expressed exogenous WT1 in the WT1-null Ewing sarcoma cell line, SK-ES-1, and we suppressed WT1 expression using shRNA in the WT1-positive Ewing sarcoma cell line, MHH-ES. Suppression of WT1 in MHH-ES cells impairs angiogenesis, while expression of WT1 in SK-ES-1 cells causes increased angiogenesis. Different WT1 isoforms result in vessels with distinct morphologies, and this correlates with preferential upregulation of particular VEGF isoforms. WT1-expressing tumors show increased expression of pro-angiogenic molecules such as VEGF, MMP9, Ang-1, and Tie-2, supporting the hypothesis that WT1 is a global regulator of angiogenesis. We also demonstrate that WT1 regulates the expression of a panel of pro-angiogenic molecules in Ewing sarcoma cell lines. Finally, we found that WT1 expression is correlated with VEGF expression, MMP9 expression, and microvessel density in samples of primary Ewing sarcoma. Thus, our results demonstrate that WT1 expression directly regulates tumor angiogenesis by controlling the expression of a panel of pro-angiogenic genes.

WT1 isoform expression pattern in acute myeloid leukemia

WT1 plays a dual role in leukemia development, probably due to an imbalance in the expression of the 4 main WT1 isoforms. We quantify their expression and evaluate them in a series of AML patients. Our data showed a predominant expression of isoform D in AML, although in a lower quantity than in normal CD34+ cells. We found a positive correlation between the total WT1 expression and A, B and C isoforms. The overexpression of WT1 in AML might be due to a relative increase in A, B and C isoforms, together with a relative decrease in isoform D expression.

Intrauterine growth restriction leads to a dysregulation of Wilms’ tumour supressor gene 1 (WT1) and to early podocyte alterations

Intrauterine growth restriction (IUGR) leads to low nephron number and higher incidence of renal disease. We hypothesized that IUGR induces early podocyte alterations based on a dysregulation of Wilms' tumour suppressor gene 1 (WT1), a key player of nephrogenesis and mediator of podocyte integrity. IUGR was induced in rats by maternal protein restriction during pregnancy. Kidneys were harvested from male offspring at Days 1 and 70 of life. qRT-PCR, immunohistochemistry and electron microscopy were performed in renal tissue. Albuminuria was assessed by enzyme-linked immunosorbent assay. At Day 70 of life, higher albuminuria and overt alterations of podocyte ultrastructure were detected in IUGR animals in spite of normal blood pressure. Moreover, we found increased glomerular immunoreactivity and expression of desmin, while synaptopodin and nephrin were decreased. Glomerular immunoreactivity and expression of WT1 were increased in IUGR animals at this time point with an altered expressional ratio of WT1 +KTS and -KTS isoforms. These changes of WT1 expression were already present at the time of birth. IUGR results in early podocyte damage possibly due to a dysregulation of WT1. We suggest that an imbalance of WT1 isoforms to the disadvantage of -KTS affects nephrogenesis in IUGR rats and that persistent dysregulation of WT1 results in a reduced ability to maintain podocyte integrity, rendering IUGR rats more susceptible for renal disease.

Detection of an Unbalanced Ratio Between WT1 Isoforms in Acute Myeloid Leukemia and Its Correlation with Molecular Abnormalities.

AbstractAbstract 2500Wilms’ tumor gene 1 (WT1) is a tumor suppressor gene coding for a zinc finger transcriptional factor which was found overexpressed in acute myeloid leukemias (AML). In AML WT1 functions as an oncogene rather than a tumor suppressor. This may depend on the unbalanced expression of its different isoforms derived from alternative splicing in exon 5 (EX5+/−) or the presence of KTS in exon 9 ( KTS+/KTS-). In normal hematopoietic cells the physiological ratio between KTS+/KTS- isoforms is 1:1. KTS are inserted between the third and fourth zinc fingers thus influencing the DNA binding and therefore the transcriptional activity.The aim of the study was to investigate the possibility of an unbalanced ratio between the isoforms thus leading to the disruption of WT1 function. In addition we correlated KTS+ isoform with clinical and biological features. Methods: we analyzed KTS+/KTS- isoforms in 120 AML patients ( 102 BM samples, 18 PB), 63 chronic phase CML patients (48 BM and 15 PB). In addition we evaluated 5 samples of selected blast cells from AML patients. In a subset of 38 adult acute myeloid leukemia (AML) patients we measured all WT1 isoforms (A[EX5 -/KTS -], B[+/−], C[-/+] and D[+/+]. Quantitative PCR was used for the WT1 isoforms detection and measurement. Results:KTS+ isoform is significantly overexpressed with a ratio KTS+/KTS− 2:1 in both AML and CML patients and it is confirmed in blast cell samples. In the subgroup of patients analyzed for the expression pattern of all WT1 isoforms we detected a significant overexpression of isoform D (EX5+/KTS+) (median value 821) while the isoform A (EX5−/KTS−) is the less represented ( median value 303). In this subgroup the expression of KTS+ isoforms (B+D) is higher than KTS- ( A+C) with a median value of 642 compared to 421. The ratio of WT1 isoforms was not significantly different among FAB subgroups or according to cytogenetic risk, FLT3 (fms-like tyrosine kinase receptor-3) internal tandem duplication or exon 17 mutation, NPM (nucleophosmin) gene mutations and BAALC (brain and acute leukemia, cytoplasmic) gene expression. Conclusion:Although WT1 is overexpressed in AML and CML patients it lacks its typical tumor suppressor function. In this study we have demonstrated the overexpression of WT1 KTS+ isoforms. In addition we have previously demonstrated that KTS+ isoform does not have transcriptional activity since it is mainly localized within the cytoplasm. In conclusion, WT1 is overexpressed in AML but the unbalanced ratio between the isoforms may probably influence the transcriptional activity. Any possible correlation between the unbalanced ratio of WT1 isoforms and clinical outcome requires further prospective studies to be established. Disclosures:No relevant conflicts of interest to declare.

Real-time PCR quantification of major Wilms’ tumor gene 1 (WT1) isoforms in acute myeloid leukemia, their characteristic expression patterns and possible functional consequences

Wilms' tumor gene 1 (WT1) functions including some contradictory effects may be explained by the presence and interactions of its isoforms, however, their evaluation has been so far complicated by several technical problems. We designed unique quantitative PCR systems for direct quantification of the major WT1 isoforms A[EX5-/KTS-], B[+/-], C[-/+] and D[+/+] and verified their sensitivity, specificity and reproducibility in extensive testing. With this method we evaluated WT1 total and isoform expression in 23 normal bone marrow (BM) samples, 73 childhood acute myeloid leukemia (AML), 20 childhood myelodysplastic syndrome (MDS), 9 childhood severe aplastic anemia (SAA), 30 adult AML and 29 adult MDS patients. WT1 isoform patterns showed differences among these samples and clustered them into groups representing the specific diagnoses (P<0.0001). Isoform profiles were independent of total WT1 expression and possess certain common features-overexpression of isoform D and EX5[+] variants. The KTS[+]/KTS[-] ratio was less variable than the EX5[+]/EX5[-] ratio and differed between children and adults (P<0.001); the EX5[+]/EX5[-] ratio varied between diagnoses (AML vs MDS, P<0.001). These findings bring new insights into WT1 isoform function and suggest that the ratio of WT1 isoforms, particularly EX5 variants, is probably crucial for the process of malignant transformation.

Nuclear Transport of Wilms′ Tumour Protein Wt1 Involves Importins α and β

Background/Aims: Wilms′ tumour protein, Wt1, is a zinc finger molecule, which is required for normal embryonic development. Mutations of the WT1 gene can give rise to childhood cancer of the kidneys. Different Wt1 isoforms exist, which function either as transcription factors or have a presumed role in mRNA processing. Previous studies suggested that Wt1 undergoes nucleocytoplasmic shuttling, and cytoplasmic Wt1 was higher in malignant than in normal cells. The aim of this study was to analyse the molecular pathways along which Wt1 shuttles between the cytoplasm and nucleus. Methods: Interaction of Wt1 protein with various importin α subtypes and importin β was assessed in pull-down assays and co-immunoprecipitation experiments. Nuclear localisation signals (NLS) were identified by combining site-directed mutagenesis with subcellular immunodetection of the transfected Wt1 variants. Results: Wt1(+/-KTS) proteins were found to interact with importin α1 and importin β in vitro and in living cells in vivo. A NLS that was necessary and sufficient for nuclear import could be mapped to the third Wt1 zinc finger. Mutation of this NLS strongly weakened binding of Wt1 to importins. Conclusion: Nuclear translocation of Wilms′ tumour protein involves importins α and β, and a NLS in the third zinc finger.

Expression Pattern of WT1 Isoforms in Patients with Acute Myeloid Leukemia (AML), Myelodysplastic Syndrome (MDS) and Severe Aplastic Anemia (SAA)

Abstract 2502 Introduction:The Wilms' tumor gene 1 (WT1) is highly expressed in a large proportion of human acute leukemias and other hematological malignancies. It has been demonstrated that WT1 protein is produced in more than 36 different isoforms. These variants have distinct, partially overlapping functions and their ratio is supposed to influence the final effect of WT1. However, only limited information on WT1 isoforms has been published so far and the relevance of their expression pattern remains unclear. Aims:Our main aim was to determine the expression pattern of four WT1 isoforms characterized by the presence or absence of exon 5 and KTS insert (A[−/−], B[+/−], C[−/+], D[+/+]) in patients with AML, MDS and SAA. Materials and Methods:We designed a unique qPCR system for detection and quantification of 4 major WT1 isoforms. Using this method we analyzed the ratio of WT1 isoforms in 8 leukemic cell lines (Kasumi-1, NB-4, K562, MV4;11, REH, NALM6, UOCB6, RS4;11) and diagnostic BM samples of 73 childhood AML, 30 adult AML, 20 childhood MDS, 9 childhood SAA as well as 23 healthy controls. Results:Median expression level of total WT1 in healthy donors was 29 WT1/ABL×104 NCN. Childhood and adult AML expressed WT1 on significantly higher level compared to healthy controls (2058 and 3446 WT1/ABL×104 NCN respectively; p<0.0001). The expression level of total WT1 in patients with MDS was 196 WT1/ABL×104 NCN, whereas children with SAA had total WT1 comparable or lower than control samples (4 WT1/ABL×104 NCN).We found an excellent correlation between the total WT1 expression and the sum of WT1 isoforms (rho=0.916, p<0.0001). However, very low levels of total WT1 precluded detection of WT1 isoforms in majority of healthy donors and SAA samples, since we reached the limit of the sensitivity of qPCR method (as defined by limiting dilution experiments). For the same reason (very low total WT1 levels), 18 patients with AML (mostly AML M5) as well as 8 MDS samples were excluded from further analyses.The analysis of WT1 isoforms ratio showed a diverse expression patterns of WT1 variants in the particular cell lines (p<0.0001). Interestingly, a similar pattern of WT1 isoforms was present in cell lines with MLL/AF4 rearrangement, independent of the lineage (myeloid - MV4;11 and lymphoid - RS4;11).We could successfully determine the expression profile of WT1 isoforms in 1 healthy BM, which showed a substantial overexpression of isoform D with the ratio of 1.2: 1.8: 1.7: 5.3 for A, B, C and D variants, respectively. Rather surprisingly, we found a uniform expression pattern of WT1 isoforms (D>B>C∼A) in patients with childhood AML as well as adult AML (0.9: 2.5: 1.1: 5.5 and 1.3: 3.1: 1.3: 4.5 for isoforms A, B, C and D respectively). In contrast to AML samples, a different and more variable ratio of WT1 variants was found in children with MDS with predominance of variant D and similar levels of isoforms A, B and C (1.6: 1.5: 1.7: 4.6 for A, B, C and D respectively). Moreover, the unsupervised hierarchical cluster analysis according to the expression pattern of WT1 isoforms indicated that children with MDS belonged to a distinct cluster compared to childhood and adult AML. Conclusion:This is the first report of the analysis of WT1 isoforms expression pattern in AML and MDS using a unique qPCR method for the detection of WT1 variants. Our data suggest that WT1 isoforms expression pattern is surprisingly uniform in pediatric and adult AML with predominant expression of Exon5[+] isoforms. To the contrary, the ratio of WT1 isoforms in childhood MDS significantly differ from AML indicating that the expression pattern of WT1 variants is related to the type of malignant cells.Careful pre-analytical testing of the detection system parameters suggests the technical limitations in detection of WT1 isoforms in samples with very low total WT1 levels. Therefore, the previously reported differences in WT1 isoforms ratio between normal bone marrow and AML samples should be interpreted with caution.Supported by MSM0021620813, GAUK 81709, IGA NS10488–3/2009 Disclosures:No relevant conflicts of interest to declare.

Abstract 5007: WT1 promotes tumor growth and vascularization by regulating matrix metalloproteinase 9 in Ewing's sarcoma xenografts

Abstract WT1, a zinc-finger transcription factor, is expressed in tumors of varied origins including sarcomas. High level WT1 expression often correlates with poor patient survival. The role of WT1 in sarcoma biology and the mechanism by which high WT1 expression confers a poor prognosis are both unclear. The aim of this study is to investigate the effect of WT1 expression on sarcoma growth and angiogenesis. We transfected the WT1-null Ewing's sarcoma cell line SK-ES-1 with one of two major WT1 isoforms, and used shRNA to silence WT1 expression in the WT1-positive Ewing's sarcoma cell line MHH-ES. Cells were injected into the flanks of immune deficient mice and tumor growth was monitored. Tumors arising fromWT1-expressing SK-ES-1 cells grew faster than control, while tumors arising from WT1-silenced MHH-ES cells grew more slowly than control. Because our laboratory and others have shown that WT1 regulates VEGF expression, we evaluated tumor vascularity using the endothelial cell marker CD31 and the pericyte marker αSMA. In xenografted SK-ES-1 cells, quantification of CD31 positive area in WT1 expressing tumors showed 60 -70 % more compared to the controls. In contrast, CD31 expression is abolished in tumors arising from WT1-silenced MHH-ES cells. To investigate the mechanism by which WT1 affects tumor growth and vascularity, we performed a gene expression profiling in SK-ES-1 cells expressing different isoforms of WT1. We identified a number of genes up- or downregulated by WT1, including matrix metalloproteinase-9 (MMP9). Luciferase reporter assays showed that WT1 can upregulate MMP9 promoter activity in transiently transfected NIH3T3 cells. Also, immunohistochemical analysis showed increased MMP9 expression in WT1-expressing tumors. Finally, imunohistochemical analysis of primary Ewing's sarcoma samples showed correlated expression of WT1, VEGF, and MMP9. Taken together, our data suggest that WT1 promotes tumor growth and angiogenesis, perhaps via upregulation of MMP9. Increased tumor angiogenesis may lead to more aggressive tumors and a worse outcome for patients, explaining the effect of WT1 on prognosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5007. doi:10.1158/1538-7445.AM2011-5007