Oncogenic activation of the human Pygopus2 promoter by E74-like factor-1.

Pygopus is a component of the T-cell factor/beta-catenin transcriptional complex essential for activation of Wnt target genes and is also required for cell regulation in the absence of Wnt signaling. Human Pygopus2 (hPygo2) is overexpressed in a high proportion of breast and epithelial ovarian malignant tumors and is required for the growth of several cell lines derived from these carcinomas. The mechanisms regulating hPygo2 gene activation, however, are unknown. Here, we have determined cis- and trans-interacting factors responsible for hPygo2 expression in cancer. The minimal region required for a maximal 109-fold activation of the hPygo2 promoter in MCF-7 breast cancer cells is 48 bp upstream of the start of transcription. Within 25 bp of the transcriptional start, there are two overlapping tandem Ets transcription factor-binding sites, which are critical for hPygo2 promoter activity. In vitro DNA pull-down assays and proteomic analyses identified the Ets family members Elk-1 and E74-like factor-1 (Elf-1) as potential hPygo2 promoter binding factors, whereas in vivo chromatin immunoprecipitation assays verified that only Elf-1 specifically bound to the hPygo2 promoter in MCF-7 cells. Modulation of elf-1 in MCF-7 cells by silencing via RNA interference or overexpression caused a corresponding decrease or increase, respectively, in hPygo2 promoter activity. Overexpression of Elf-1 in HeLa cells, in which Elf-1 is expressed at a lower level than in MCF-7 cells, caused a 4-fold increase in endogenous hPygo2 mRNA levels. These results provide new evidence that Elf-1 is involved in transcriptional activation of hPygo2. Like hPygo2, previous studies implicated Elf-1 in breast and ovarian cancer and our present findings suggest that the oncogenic requirement of hPygo2 is fulfilled, in part, by Elf-1.

Antisense suppression of pygopus2 results in growth arrest of epithelial ovarian cancer.

PURPOSE: The Pygopus proteins are critical elements of the canonical Wnt/beta-catenin transcriptional complex. In epithelial ovarian cancer, constitutively active Wnt signaling is restricted to one (endometrioid) tumor subtype. The purpose of this study was to determine the level of expression and growth requirements of human Pygopus2 (hPygo2) protein in epithelial ovarian cancer. EXPERIMENTAL DESIGN: Expression and subcellular localization of hPygo2 was determined in epithelial ovarian cancer cell lines and tumors using Northern blot, immunoblot, and immunofluorescence. Immunohistochemistry was done on 125 archived patient epithelial ovarian cancer tumors representing all epithelial ovarian cancer subtypes. T-cell factor-dependent transcription levels were determined in epithelial ovarian cancer cells using TOPflash/FOPflash in vivo assays. Phosphorothioated antisense oligonucleotides were transfected into cell lines and growth assayed by cell counting, anchorage-independent colony formation on soft agar, and xenografting into severe combined immunodeficient mice. RESULTS: All six epithelial ovarian cancer cell lines and 82% of the patient samples overexpressed nuclear hPygo2 compared with control cells and benign disease. Depletion of hPygo2 by antisense oligonucleotides in both Wnt-active (TOV-112D) and Wnt-inactive serous (OVCAR-3, SKOV-3) and clear cell (TOV-21G) carcinoma cell lines halted growth, assessed using tissue culture, anchorage-independent, and xenograft assays. CONCLUSIONS: hPygo2 is unexpectedly widely expressed in, and required in the absence of, Wnt signaling for malignant growth of epithelial ovarian cancer, the deadliest gynecologic malignancy. These findings strongly suggest that inhibition of hPygo2 may be of therapeutic benefit for treating this disease.