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.

Wilms' tumor gene WT1 17AA(-)/KTS(-) isoform induces morphological changes and promotes cell migration and invasion in vitro.

The wild-type Wilms' tumor gene WT1 is overexpressed in human primary leukemia and in a wide variety of solid cancers. All of the four WT1 isoforms are expressed in primary cancers and each is considered to have a different function. However, the functions of each of the WT1 isoforms in cancer cells remain unclear. The present study demonstrated that constitutive expression of the WT1 17AA(-)/KTS(-) isoform induces morphological changes characterized by a small-sized cell shape in TYK-nu.CP-r (TYK) ovarian cancer cells. In the WT1 17AA(-)/KTS(-) isoform-transduced TYK cells, cell-substratum adhesion was suppressed, and cell migration and in vitro invasion were enhanced compared to that in mock vector-transduced TYK cells. Constitutive expression of the WT1 17AA(-)/KTS(-) isoform also induced morphological changes in five (one gastric, one esophageal, two breast and one fibrosarcoma) of eight cancer cell lines examined. No WT1 isoforms other than the WT1 17AA(-)/KTS(-) isoform induced the phenotypic changes. A decrease in alpha-actinin 1 and cofilin expression and an increase in gelsolin expression were observed in WT1 17AA(-)/KTS(-) isoform-transduced TYK cells. In contrast, co-expression of alpha-actinin 1 and cofilin or knockdown of gelsolin expression by small interfering RNA restored WT1 17AA(-)/KTS(-) isoform-transduced TYK cells to a phenotype that was comparable to that of the parent TYK cells. These results indicated that the WT1 17AA(-)/KTS(-) isoform exerted its oncogenic functions through modulation of cytoskeletal dynamics. The present results may provide a novel insight into the signaling pathway of the WT1 gene for its oncogenic functions.

Overexpression of the Wilms' tumor gene W T1 in primary astrocytic tumors.

Expression of the Wilms' tumor gene W T1 in primary astrocytic tumors was examined using a quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) or immunohistochemistry. Real-time RT-PCR showed that W T1 mRNA was expressed at various levels in all of the 25 astrocytic tumors examined. Immunohistochemical analysis showed that W T1 protein was expressed in 5 of 6 low-grade astrocytic tumors (grade I-II) and all of 18 high-grade ones (grade III-IV), and that expression levels of W T1 protein in high-grade tumors were significantly higher than those in low-grade ones. W T1 protein was not detected in the normal glial cells contained in the tumor specimens. Furthermore, treatment with W T1 antisense oligomers specifically inhibited growth of glioblastoma cell lines, U87-MG, A172, and T-98G. These results may indicate that the W T1 gene plays an important role in tumorigenesis of primary astrocytic tumors.

Overexpression of the Wilms' tumor gene WT1 in esophageal cancer.

BACKGROUND: The Wilms' tumor gene WT1 is overexpressed in various kinds of solid cancers. However, it remains unclear whether WT1 is expressed in esophageal squamous cell carcinoma. MATERIALS AND METHODS: Expression of the WT1 gene was examined by real-time RT-PCR in 12 esophageal squamous cell carcinoma (ESCC) and by immunohistochemistry in 9 of these 12 and another 29. RESULTS: Real-time RT-PCR showed that the WT1 mRNA was overexpressed in all of the 12 ESCC examined Immunohistochemical analysis showed that the WT1 protein was overexpressed in ESCC cells in 36 (95%) of the 38 examined Furthermore, expression of the WT1 protein was examined in 20 esophageal squamous dysplasia. The WT1 protein was overexpressed in 5 (45%) out of 11 mild dysplasia and in 8 (89%) out of 9 moderate to severe dysplasia. CONCLUSION: These results may indicate an important role of the WT1 gene in the tumorigenesis of ESCC.

Overexpression of the Wilms' tumor gene WT1 in colorectal adenocarcinoma.

Expression of the Wilms' tumor gene WT1 was examined in 59 cases of colorectal adenocarcinoma to examine the involvement of WT1 in tumorigenesis. Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) showed that WT1 mRNA was expressed in the range from 7.2 x 10(-5) to 4.9 x 10(-1) levels (WT1 expression level in K562 leukemic cells was defined as 1.0) in all (100%) of the 28 cases of colorectal adenocarcinoma examined, and that the WT1 mRNA expression levels were higher in 20 (71%) of the 28 cases compared to those of normal-appearing mucosal tissues examined. Immunohistochemical analysis using an anti-WT1 antibody was performed on 46 cases of colorectal adenocarcinoma (15 of the 28 cases with WT1 mRNA expression and 31 newly collected cases), and the expression of WT1 protein was detected in 41 (89%) of the 46 cases. The direct sequencing analysis of the WT1 genomic DNA showed no mutations in any of the 10 exons of the WT1 gene in any of 5 different colorectal adenocarcinomas. These results may indicate an important role of the wild-type WT1 gene in tumorigenesis of colorectal adenocarcinoma.