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

Abstract

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 WT1expressing 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.

WT1 gene silencing by aerosol delivery of PEI–RNAi complexes inhibits B16-F10 lung metastases growth

“…These results show the activation of the intrinsic apoptotic pathway by WT1 gene silencing and corroborate, in part, the report that shRNA silencing of WT1 induces apoptosis of fibrosarcoma cells. 56 WT1-1 RNAi treatment significantly increased (P ¼ 0.003) the mean survival time of tumor-bearing mice in comparison with the mice treated with WT1-2 RNAi or the untreated mice. Studies are ongoing to assess the potential of using 5% CO 2 during nebulization to increase the efficacy of aerosol delivery because it has been reported that this addition enhances transgene expression in the lung for PEI-DNA vectors.…”

Mapping of novel peptides of WT-1 and presenting HLA alleles that induce epitope-specific HLA-restricted T cells with cytotoxic activity against WT-1+ leukemias

“…27 Inhibition of WT-1 expression by ShRNA reverses these effects, thereby eliminating leukemic cells with clonogenic potential. 28 Several studies have demonstrated that T cells specific for certain peptides of WT-1 that are expected to be immunogenic based on their predicted binding to specific HLA alleles can be generated from healthy donors and some tumor-bearing patients. [29][30][31][32] Furthermore, these T cells can lyse WT-1 ϩ tumor cells in vitro 29,30,32 and inhibit their growth in vivo in immunodeficient mice bearing human WT-1 ϩ tumor xenografts.…”

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

“…15,34 Functional studies suggest that many of WT1 effects are related to particular WT1 isoforms. 8,20,[35][36][37][38][39][40] The available data on WT1 isoform expression patterns in normal and malignant hematopoiesis are scarce and the methods so far used for the detection of WT1 major isoforms (reverse transcriptase PCR, GeneScan, quantitative PCR of EX5[ þ ] and KTS[ þ ] variants) have only yielded an approximate estimation of WT1 isoform levels.…”