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Wilms' tumor gene 1 in different types of cancer
Umeå University, Faculty of Medicine, Department of Medical Biosciences.
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The Wilms’ tumor gene 1 (WT1) was first reported as a tumor suppressor gene in Wilms’ tumor. However, later studies have shown the oncogenic properties of WT1 in a variety of tumors. It was recently proposed that WT1 was a chameleon gene, due to its dual functions in tumorigenesis. We aimed to investigate the clinical significance of WT1 as biomarker in acute myeloid leukemia (AML) and clear cell renal cell carcinoma (ccRCC) and to elucidate the function of WT1 as an oncogene in squamous cell carcinoma of head and neck (SCCHN).

In AML, it was suggested that WT1 expression was an applicable marker of minimal residual disease (MRD). In adult patients with AML, we found a good correlation between WT1 expression levels normalized to two control genes, β-actin and ABL. Outcome could be predicted by a reduction in WT1 expression in bone marrow (≥ 1-log) detected less than 1 month after diagnosis, when β-actin was used as control. Also, irrespective of the control gene used, outcome could be predicted by a reduction in WT1 expression in peripheral blood (≥ 2-log) detected between 1 and 6 months after treatment initiation.

Previous studies in RCC demonstrated that WT1 acted as a tumor suppressor. Thus, we tested whether single nucleotide polymorphisms (SNPs) or mutations in WT1 might be associated with WT1 expression and clinical outcome in patients with ccRCC. We performed sequencing analysis on 10 exons of the WT1 gene in a total of 182 patient samples, and we identified six different SNPs in the WT1 gene. We found that at least one or two copies of the minor allele were present in 61% of ccRCC tumor samples. However, no correlation was observed between WT1 SNP genotypes and RNA expression levels. Moreover, none of the previously reported WT1 mutations were found in ccRCC. Nevertheless, we found that a favorable outcome was associated the homozygous minor allele for WT1 SNP. We then further investigated whether WT1 methylation was related to WT1 expression and its clinical significance. Methylation array and pyrosequencing analyses showed that the WT1 promoter region CpG site, cg22975913, was the most frequently hypermethylated CpG site. We found a trend that showed nearly significant correlation between WT1 mRNA levels and hypermethylation in the 5’-untranslated region. Hypermethylation in the WT1 CpG site, cg22975913, was found to be associated with patient age and a worse prognosis.

One previous study reported that WT1 was overexpressed in SCCHN. That finding suggested that WT1 might play a role in oncogenesis. We found that both WT1 and p63 could promote cell proliferation. A positive correlation between WT1 and p63 expression was observed, and we identified p63 as a WT1 target gene. Furthermore, several known WT1 and p63 target genes were affected by knocking down WT1. Also, co-immunoprecipitation analyses demonstrated a protein interaction between WT1 and p53.

In summary, WT1 gene expression can provide useful information for MRD detection during treatment of patients with AML. In RCC, our results suggested that the prognostic impact of WT1 SNPs was limited to the subgroup of patients that were homozygous for the minor allele, and that WT1 promoter hypermethylation could be used as a prognostic biomarker. In SCCHN, WT1 and p63 acted as oncogenes by affecting multiple genes involved in cancer cell growth.

Place, publisher, year, edition, pages
Umeå: Umeå University , 2015. , 59 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1717
Keyword [en]
WT1, AML, MRD, ccRCC, SNPs, DNA methylation, SCCHN, p63
National Category
Cancer and Oncology
Research subject
Clinical Chemistry
Identifiers
URN: urn:nbn:se:umu:diva-103389ISBN: 978-91-7601-263-5 (print)OAI: oai:DiVA.org:umu-103389DiVA: diva2:813042
Public defence
2015-06-12, Hörsal Betula, 6M, Norrlands universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2015-05-22 Created: 2015-05-21 Last updated: 2015-09-02Bibliographically approved
List of papers
1. Reduction in WT1 Gene Expression During Early Treatment Predicts the Outcome in Patients With Acute Myeloid Leukemia
Open this publication in new window or tab >>Reduction in WT1 Gene Expression During Early Treatment Predicts the Outcome in Patients With Acute Myeloid Leukemia
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2012 (English)In: Diagnostic molecular pathology (Print), ISSN 1052-9551, E-ISSN 1533-4066, Vol. 21, no 4, 225-233 p.Article in journal (Refereed) Published
Abstract [en]

Wilms tumor gene 1 (WT1) expression has been suggested as an applicable minimal residual disease marker in acute myeloid leukemia (AML). We evaluated the use of this marker in 43 adult AML patients. Quantitative assessment of WT1 gene transcripts was performed using real-time quantitative-polymerase chain reaction assay. Samples from both the peripheral blood and the bone marrow were analyzed at diagnosis and during follow-up. A strong correlation was observed between WT1 normalized with 2 different control genes (beta-actin and ABL1, P < 0.001). WT1 mRNA level at diagnosis was of no prognostic relevance (P > 0.05). A >= 1-log reduction in WT1 expression in bone marrow samples taken < 1 month after diagnosis significantly correlated with an improved overall survival (P = 0.004) and freedom from relapse (P = 0.010) when beta-actin was used as control gene. Furthermore, a reduction in WT1 expression by >= 2 logs in peripheral blood samples taken at a later time point significantly correlated with a better outcome for overall survival (P = 0.004) and freedom from relapse (P = 0.012). This result was achieved when normalizing against both b-actin and ABL1. These results therefore suggest that WT1 gene expression can provide useful information for minimal residual disease detection in adult AML patients and that combined use of control genes can give more informative results.

Place, publisher, year, edition, pages
Lippincott Williams & Wilkins, 2012
Keyword
WT1 gene expression, acute myeloid leukemia, minimal residual disease, RQ-PCR, control genes
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-62781 (URN)10.1097/PDM.0b013e318257ddb9 (DOI)000311221400005 ()
Available from: 2013-01-02 Created: 2012-12-18 Last updated: 2017-12-06Bibliographically approved
2. Single Nucleotide Polymorphisms in the Wilms' Tumour Gene 1 in Clear Cell Renal Cell Carcinoma
Open this publication in new window or tab >>Single Nucleotide Polymorphisms in the Wilms' Tumour Gene 1 in Clear Cell Renal Cell Carcinoma
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 3, e58396Article in journal (Refereed) Published
Abstract [en]

The Wilms' tumour gene 1 (WT1) single nucleotide polymorphism (SNP) rs16754 has recently been described as an independent prognostic factor in acute myeloid leukaemia (AML) patients. It is of great interest to test whether WT1 SNPs can be used as a molecular marker in other cancer types in order to improve risk and treatment stratification. We performed sequencing analysis on all 10 exons of the WT1 gene in a total of 182 patients with clear cell renal cell carcinoma (ccRCC). Six different SNPs were identified, in descending order for minor allele frequency: rs2234582, rs16754, rs1799925, rs5030315, rs2234583, and rs2234581. At least one minor allele for WT1 SNP was identified in 61% of ccRCC patients. In the entire study population, only 6% carried two copies of the minor allele. The genotypes of WT1 SNPs in 78 tumour-free kidney tissue specimens were found to be in 95% concordance with corresponding tumour samples. No correlation was observed between WT1 SNP genotypes and RNA expression level. WT1 SNP genotypes did not associate with clinical and pathological characteristics. We found favourable outcomes associated with the homozygous minor allele for WT1 SNP. However, SNP genotypes did not show to be of prognostic significance when comparing wild-type versus homozygous or heterozygous for the minor allele in the entire cohort. None of the previously reported WT1 mutations in AML was found in the present study. A novel WT1 missense mutation was identified in only one patient. Our data suggest that common WT1 mutations are not involved in ccRCC. Due to too few cases harbouring the homozygous minor allele, the prognostic impact needs to be verified in larger study populations.

National Category
Cancer and Oncology
Identifiers
urn:nbn:se:umu:diva-70351 (URN)10.1371/journal.pone.0058396 (DOI)000316936100116 ()
Available from: 2013-05-16 Created: 2013-05-14 Last updated: 2017-12-06Bibliographically approved
3. Prognostic Significance of Hypermethylation in the Promoter Region of the Wilms’ Tumour Gene 1 in Clear Cell Renal Cell Carcinoma
Open this publication in new window or tab >>Prognostic Significance of Hypermethylation in the Promoter Region of the Wilms’ Tumour Gene 1 in Clear Cell Renal Cell Carcinoma
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(English)Manuscript (preprint) (Other academic)
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-103388 (URN)
Available from: 2015-05-21 Created: 2015-05-21 Last updated: 2015-05-21
4. Wilms' tumor gene 1 regulates p63 and promotes cell proliferation in squamous cell carcinoma of the head and neck
Open this publication in new window or tab >>Wilms' tumor gene 1 regulates p63 and promotes cell proliferation in squamous cell carcinoma of the head and neck
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2015 (English)In: BMC Cancer, ISSN 1471-2407, E-ISSN 1471-2407, Vol. 15, 342Article in journal (Refereed) Published
Abstract [en]

Background: Wilms' tumor gene 1 (WT1) can act as a suppressor or activator of tumourigenesis in different types of human malignancies. The role of WT1 in squamous cell carcinoma of the head and neck (SCCHN) is not clear. Overexpression of WT1 has been reported in SCCHN, suggesting a possible oncogenic role for WT1. In the present study we aimed at investigating the function of WT1 and its previously identified protein partners p63 and p53 in the SCCHN cell line FaDu. Methods: Silencing RNA (siRNA) technology was applied to knockdown of WT1, p63 and p53 in FaDu cells. Cell proliferation was detected using MTT assay. Chromatin immunoprecipitation (ChIP)/PCR analysis was performed to confirm the effect of WT1 on the p63 promoter. Protein co-immunoprecipitation (co-IP) was used to find protein interaction between WT1 and p53/p63. Microarray analysis was used to identify changes of gene expression in response to knockdown of either WT1 or p63. WT1 RNA level was detected using real-time quantitative PCR (RT-qPCR) in patients with SCCHN. Results: We found that WT1 and p63 promoted cell proliferation, while mutant p53 (R248L) possessed the ability to suppress cell proliferation. We reported a novel positive correlation between WT1 and p63 expression. Subsequently, p63 was identified as a WT1 target gene. Furthermore, expression of 18 genes involved in cell proliferation, cell cycle regulation and DNA replication was significantly altered by downregulation of WT1 and p63 expression. Several known WT1 and p63 target genes were affected by WT1 knockdown. Protein interaction was demonstrated between WT1 and p53 but not between WT1 and p63. Additionally, high WT1 mRNA levels were detected in SCCHN patient samples. Conclusions: Our findings suggest that WT1 and p63 act as oncogenes in SCCHN, affecting multiple genes involved in cancer cell growth.

Keyword
WT1,  p63,  p53,  Cell proliferation,  Squamous cell carcinoma of the head and neck (SCCHN)
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:umu:diva-103386 (URN)10.1186/s12885-015-1356-0 (DOI)000354016900001 ()25929687 (PubMedID)
Available from: 2015-05-21 Created: 2015-05-21 Last updated: 2017-12-04Bibliographically approved

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