Wilms' tumor gene WT1-shRNA as a potent apoptosis-inducing agent for solid tumors

Wilms' tumor gene WT1 is overexpressed in leukemia and various types of solid tumors and plays an important role in leukemogenesis and tumorigenesis. We tested apoptosis-inducing ability of short hairpin RNAs targeting exon 5 (shWTE5), exon10 (shWTE10) and 3'UTR (shWT3U) of the WT1 gene. Among the three WT1-shRNAs, since shWTE5 most effectively induced apoptosis, its ability as an apoptosis-inducing agent was intensively examined. shWTE5 induced mitochondrial damage and resultant apoptosis in five WT1-expressing solid cancer cells originated from gastric (AZ-521), lung (LU99B), ovarian (TYKnuCPr) cancers, fibrosarcoma (HT-1080) and glioblastoma (A172). Moreover, shWTE5 significantly enhanced apoptosis induced by chemotherapeutic agents, doxorubicin (DOX) and etoposide (ETP), or by death ligand TRAIL in all of the four solid tumor cells examined (HT-1080, LU99B, TYK and A172). Transduction of one each of WT1 isoforms with exon 5 [17AA(+)KTS(+) and 17AA(+)KTS(-)] prevented mitochondrial damage induced by ETP or TRAIL and inhibited apoptosis. These results showed that shWTE5 induced apoptosis through the suppression of the WT1 isoform with exon 5. Furthermore, shWTE5 increased expression of proapoptotic Bak and Bax proteins and decreased antiapoptotic Bcl-xL and Bcl-2 proteins in WT1-expressing HT-1080 cells, indicating that WT1 isoforms with exon 5 might play an antiapoptotic role through regulation of Bcl-2 family genes in solid tumor cells. The results presented here demonstrated that WT1-shRNA targeting exon 5 should serve as a potent anti-cancer agent for various types of solid tumors.     

Expression of the Wilms' tumor gene (WT1) in human leukemias.

Leukemic cells from seventy patients with various types of human leukemias were examined for expression of the WT1 gene, the Wilms' tumor gene located at chromosome 11p13. WT1 was expressed in 7 of 16 cases of acute lymphoblastic leukemia, 15 of 22 with acute myelogenous leukemia and 8 of 10 in blast crisis of chronic myelogenous leukemia. No detectable WT1 RNA was found in chronic leukemias, including chronic lymphocytic leukemia, plasma cell leukemia, hairy cell leukemia and chronic myelogenous leukemia in chronic phase. The expression pattern of WT1 in these human leukemia samples indicates the involvement of this gene in the early stage of hematological cell differentiation.     

Intragenic homozygous deletion of the WT1 gene in Wilms' tumor.

One example of intragenic homozygous deletion of the WT1 gene on chromosome 11p13 was found after screening 42 samples of Wilms' tumor DNA from Japanese patients. After construction of a restriction map for the genomic sequence covering the 3' half of the gene, the deletion was analysed at the nucleotide sequence level. The deletion occurred in the patient's germline on his paternal chromosome, and most of the short arm of his maternal chromosome 11 was subsequently lost in the tumor. The size of the deletion was about 8 kb, removing exons 6 and 7 and resulting in premature termination. The deletion seemed to be created by recombination between short homologous sequences found in an Alu repeat, with a 16-bp duplication left at the junction. This case conforms to a two-hit model for the genesis of a certain group of tumors, and supports the hypothesis that WT1 is one of the recessive oncogenes responsible for Wilms' tumor.     

Expression and mutation analysis of the Wilms' tumor 1 gene in human neural tumors.

The Wilms' tumor 1 gene, WT1, encodes a zinc-finger protein that is implicated in the development of Wilms' tumor. Mutant or aberrantly expressed WT1 isoforms have also been described in desmoplastic small round cell tumor, acute leukemias, mesothelioma, breast tumors and melanoma. During early development, WT1 is expressed in the brain and spinal cord, however its role in the malignancies that affect these tissues has not been previously investigated. In our study we have examined neural tumors including brain tumors and neuroblastomas for WT1 expression and for mutations affecting the zinc-fingers. Although WT1 expression was detected in gliomas, medulloblastomas and neuroblastomas, neither zinc-finger region mutations nor splicing anomalies affecting the KTS site were detected. We therefore conclude that WT1 does not play a significant role in the etiology of human neural tumors.     

Overexpression of the Wilms' tumor gene WT1 in pancreatic ductal adenocarcinoma

The expression of the Wilms' tumor gene WT1 was examined by immunohistochemistry in 40 cases of pancreatic ductal adenocarcinoma. WT1 protein was expressed in 30 (75%) of the 40 pancreatic ductal adenocarcinomas, but not in the remaining 10 (25%). In normal pancreatic ductal cells, WT1 protein was undetectable. No correlations between WT1 expression and clinicopathological parameters such as age, sex, T or N stage, tumor location, and tumor differentiation were observed. Treatment with WT1 antisense oligomers significantly inhibited the growth of five human pancreatic cancer cell lines, PSN1, MiaPaCa2, ASPC1, BxPC3, and PCI6, expressing the WT1 gene. These results indicate an important role of the WT1 gene in the tumorigenesis of pancreatic ductal adenocarcinoma expressing WT1 and provide a rationale for new treatment strategies to treat pancreatic ductal adenocarcinoma by targeting the WT1 gene and its product.     

RNA expression of the WT1 gene in Wilms' tumors in relation to histology.

BACKGROUND: On the basis of accumulating data, the recently isolated WT1 gene is a Wilms' tumor gene and a putative tumor suppressor gene. These findings include expression in developing fetal kidney, intragenic deletions in tumors, and germline mutations in predisposed individuals. Wilms' tumors, which exhibit a broad range of differentiation, are composed of three cell types: blastema, epithelium, and stroma. PURPOSE: The purpose of this study was to investigate the relationship between WT1 gene expression and histologic composition in Wilms' tumors in an effort to elucidate how the WT1 gene functions in proliferation of these histologic components. METHODS: We used Northern blot hybridization to study WT1 gene expression by messenger RNA (mRNA) accumulation in 20 tumors of varying histology and in adjacent uninvolved kidney tissue. In two patients, tumors were also compared before and after therapy. RESULTS: Tumors that were predominantly blastemal expressed high amounts of WT1 mRNA, whereas predominantly stromal tumors expressed either low or undetectable amounts. Blastemal tumors that were predominantly poorly differentiated expressed WT1 mRNA at higher levels than those that were more well differentiated. Although we expected that a putative tumor suppressor gene like WT1 would generally be expressed at lower levels in tumor than in normal kidney, this was true only in predominantly stromal cells. One of the two patients studied before and after therapy had a dramatic response to therapy accompanied by a decline in WT1 gene expression and disappearance of blastemal and epithelial elements. CONCLUSIONS: A correlation was observed between WT1 gene expression and histology of the tumors. Level of expression was inversely related to the degree of differentiation in blastemal tumors and in the patient with a dramatic response to therapy. These results, in conjunction with the observation that WT1 mRNA is abundant in normal fetal kidney, suggest that WT1 gene expression is related to kidney development, especially in differentiation of blastemal components. IMPLICATIONS: Further studies to search for alterations of the WT1 gene in tumors and to identify regulatory factors in gene expression will increase understanding of the role of this gene in normal development and tumorigenesis.     

Overexpression of the Wilms' tumor gene WT1 in de novo lung cancers

Expression of the Wilms' tumor gene WT1 in de novo lung cancer was examined using quantitative real-time RT-PCR and immunohistochemistry. Overexpression of the WT1 gene was detected by RT-PCR in 54/56 (96%) de novo non-small cell lung cancers examined and confirmed by detection of WT1 protein with an anti-WT1 antibody. Overexpression of the WT1 gene was also demonstrated in 5/6 (83%) de novo small cell lung cancers by immunohistochemistry. Furthermore, when the WT1 gene was examined for mutations by direct sequencing of genomic DNA in 7 lung cancers, no mutations were found. These results suggest that the nonmutated, wild-type WT1 gene plays an important role in tumorigenesis of de novo lung cancers and may provide us with the rationale for new therapeutic strategies for lung cancer targeting the WT1 gene and its products.     

Absence of mutations in the Wilms' tumor gene WT1 in primary breast cancer

BACKGROUND: It was recently demonstrated that the WT1 gene was overexpressed in primary breast cancer and that the high expression levels of WT1 mRNA significantly correlated with poor prognosis. However, it remained undetermined whether or not the WT1 gene expressed in breast cancer had mutations. METHODS: Breast cancer tissues were obtained from 36 patients with breast cancer. WT1 genomic DNA was PCR-amplified and examined for mutations by direct sequencing. RESULTS: The sequencing analysis showed the absence of mutations through the whole 10 exons of the WT1 gene in the 36 cases of primary breast cancer. Two different single nucleotide polymorphisms (SNP) without an amino acid change (Pro42, C to T in exon 1, and/or Arg300, A to G in exon 7) were detected in the WT1 gene in 31 (86%) of the 36 cases examined. CONCLUSION: The results indicate that the wild-type WT1 gene plays an important role in the tumorigenesis of primary breast cancer.     

The Wilms' tumor gene WT1-GFP knock-in mouse reveals the dynamic regulation of WT1 expression in normal and leukemic hematopoiesis.

The Wilms' tumor gene WT1 is overexpressed in most of human leukemias regardless of disease subtypes. To characterize the expression pattern of WT1 during normal and neoplastic hematopoiesis, we generated a knock-in reporter green fluorescent protein (GFP) mouse (WT1(GFP/+)) and assayed for WT1 expression in normal and leukemic hematopoietic cells. In normal hematopoietic cells, WT1 was expressed in none of the long-term (LT) hematopoietic stem cells (HSC) and very few (<1%) of the multipotent progenitor cells. In contrast, in murine leukemias induced by acute myeloid leukemia 1 (AML1)/ETO+TEL/PDGFbetaR or BCR/ABL, WT1 was expressed in 40.5 or 38.9% of immature c-kit(+)lin(-)Sca-1(+) (KLS) cells, which contained a subset, but not all, of transplantable leukemic stem cells (LSCs). WT1 expression was minimal in normal fetal liver HSCs and mobilized HSCs, both of which are stimulated for proliferation. In addition, overexpression of WT1 in HSCs did not result in proliferation or expansion of HSCs and their progeny in vivo. Thus, the mechanism by which expansion of WT1-expressing cells occurs in leukemia remains unclear. Nevertheless, our results demonstrate that the WT1(GFP/+) mouse is a powerful tool for analyzing WT1-expressing cells, and they highlight the potential of WT1, as a specific therapeutic target that is expressed in LSCs but not in normal HSCs.     

Structural rearrangements of the WT1 gene in Wilms' tumour cells.

We have analysed 55 Wilms' tumour DNAs using the cDNA from the candidate Wilms' predisposition gene, WT1. One tumour, GOS 129, shows a partial homozygous deletion involving only the 3'-most exon of the gene. An adjacent 3' DNA sequence, J7-18, which lies on the same NotI fragment as WT1, is present in GOS 129. Thus, this partial deletion does not extend to the adjacent unmethylated 3' HTF island. These data support the candidature of WT1 as a Wilms' predisposition gene. Tumour GOS 129 has become homozygous as a result of a mitotic recombination event proximal to WT1. Three other tumours showed abnormally sized bands on Southern blot analysis which appear to reflect internal heterozygous rearrangements involving the 5' end of the gene. One of these tumours was from a bilaterally-affected patient and the other 3 were from stage III or IV tumours.