Kenneth R. Kao

Antisense Suppression of Pygopus2 Results in Growth Arrest of Epithelial Ovarian Cancer

Purpose: The Pygopus proteins are critical elements of the canonical Wnt/β-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.

The legacy of lithium effects on development.

The simple element lithium causes specific effect on early embryonic development. In amphibians, treatment during early cleavage enhances anterior and dorsal development at the expense of posterior and ventral development. Conversely, treatment at late cleavage reduces head development. While an effect of lithium on the phosphoinositide cycle was suspected, the enhancement of dorsoanterior development is now thought to result primarily from stimulation of the Wnt/beta-catenin pathway. The multiple uses of this pathway in development may account for lithiums various specific effects.

Overexpression of a Novel Xenopus Rel mRNA Gene Induces Tumors in Early Embryos

The Rel family of transcriptional activators form a large diverse group of proteins that are involved in the activation of genes involved in immunity, development, apoptosis and cancer. So far, none of the rel genes cloned in mammals appear to be required for embryonic development. We have cloned and characterized a cDNA from an embryonic cDNA library that encodes a novelXenopus Rel protein, called Xrel3. Xrel3 is a member of the cRel subfamily and is most closely related to but distinct from otherXenopus Rel members. The expression of Xrel3 mRNA was investigated using Northern analysis, RNase protection assay, reverse transcriptase-linked polymerase chain reaction and in situhybridization. Messages are present maternally and are slightly enriched in the equatorial region of the blastula stage embryo. At gastrulation, the accumulation of Xrel3 messages declines to undetectable levels but then increases after neurulation. In situ RNA hybridization was used to determine the spatial location of Xrel3 messenger RNA in embryos. Messages are localized to the developing forebrain, dorsal mid-hindbrain region, the inner ear primordium, or otocyst, and in the notochord. Overexpression by microinjection of Xrel3 RNA induced tumors in the developing embryo that appeared after gastrulation. The location of the tumors depended on the location of the injection site. These results suggest that Xrel3 might have a generalized role in regulation of cell differentiation in the embryo.

Pygopus is required for embryonic brain patterning in Xenopus

We have identified two Xenopus mRNAs that encode proteins homologous to a component of the Wnt/beta-catenin transcriptional machinery known as Pygopus. The predicted proteins encoded by both mRNAs share the same structural properties with human Pygo-2, but with Xpygo-2alpha having an additional 21 N-terminal residues. Xpygo-2alpha messages accumulate in the prospective anterior neural plate after gastrulation and then are localized to the nervous system, rostral to and including the hindbrain. Xpygo-2beta mRNA is expressed in oocytes and early embryos but declines in level before and during gastrulation. In late neurula, Xpygo-2beta mRNA is restricted to the retinal field, including eye primordia and prospective forebrain. A C-terminal truncated mutant of Xpygo-2 containing the N-terminal Homology Domain (NHD) caused both axis duplication when injected at the 2-cell stage and inhibition of anterior neural development when injected in the prospective head, mimicking the previously described effects of Wnt-signaling activators. Inhibition of Xpygo-2alpha and Xpygo-2beta by injection of gene-specific antisense morpholino oligonucleotides into prospective anterior neurectoderm caused brain defects that were prevented by coinjection of Xpygo-2 mRNA. Both Xpygo-2alpha and Xpygo-2beta morpholinos reduced the eye and forebrain markers Xrx-1, Xpax-6, and XBF-1, while the Xpygo-2alpha morpholino also eliminated expression of the mid-hindbrain marker En-2. The differential expression and regulatory activities of Xpygo-2alpha/beta in rostral neural tissue indicate that they represent essential components of a novel mechanism for Wnt signaling in regionalization of the brain.

The transcriptional activity of Pygopus is enhanced by its interaction with cAMP-response-element-binding protein (CREB)-binding protein.

Pygopus is a core component of the beta-catenin/TCF (T-cell factor) transcriptional activation complex required for the expression of canonical Wnt target genes. Recent evidence suggests that Pygopus could interpret histone methylation associated with target genes and it was shown to be required for histone acetylation. The involvement of a specific acetyltransferase, however, was not determined. In this report, we demonstrate that Pygopus can interact with the HAT (histone acetyltransferase) CBP [CREB (cAMP-responsive-element-binding protein)-binding protein]. The interaction is via the NHD (N-terminal homology domain) of Pygopus, which binds to two regions in the vicinity of the HAT domain of CBP. Transfected and endogenous hPygo2 (human Pygopus2) and CBP proteins co-immunoprecipitate in HEK-293 (human embryonic kidney 293) cells and both proteins co-localize in SW480 colorectal cancer cells. The interaction with CBP also enhances both DNA-tethered and TCF/LEF1 (lymphoid enhancing factor 1)-dependent transcriptional activity of Pygopus. Furthermore, immunoprecipitated Pygopus protein complexes displayed CBP-dependent histone acetyltransferase activity. Our data support a model in which the NHD region of Pygopus is required to augment TCF/beta-catenin-mediated transcriptional activation by a mechanism that includes both transcriptional activation and histone acetylation resulting from the recruitment of the CBP histone acetyltransferase.

Human papilloma virus (HPV) E7-mediated attenuation of retinoblastoma (Rb) induces hPygopus2 expression via Elf-1 in cervical cancer.

The human papillomavirus (HPV) is the etiologic agent of cervical cancer. In this study, we provide evidence for the human Pygopus (hPygo)2 gene as a cellular biomarker for HPV-related disease. In a tumor microarray of cervical cancer progression, hPygo2 levels were greater in high-grade lesions and squamous cell carcinomas than in normal epithelia. Similarly, hPygo2 mRNA and protein levels were greater in HPV-positive cervical cancer cells relative to uninfected primary cells. RNA interference (RNAi)-mediated depletion of HPV-E7 increased whereas E74-like factor (Elf)-1 RNAi decreased association of Retinoblastoma (Rb) tumor suppressor with the hPygo2 promoter in cervical cancer cell lines. Transfection of dominant-active Rb inhibited Elf-1-dependent activation of hPygo2, whereas Elf-1 itself increased hPygo2 expression. Chromatin immunoprecipitation assays showed that Rb repressed hPygo2 by inhibiting Elf-1 at the Ets-binding site in the hPygo2 promoter. These results suggested that abrogation of Rb by E7 resulted in derepression of Elf-1, which in turn stimulated expression of hPygo2. Thus, initiation of hPygo2 expression by Elf-1 was required for proliferation of cervical cancer cells and its expression therefore may act as a surrogate marker for dysplasia. Mol Cancer Res; 11(1); 19–30. ©2012 AACR.

Negative regulation of dorsal patterning in early embryos by overexpression of XrelA, a Xenopus homologue of NF-κB

Recent results by Richardson et al. (Mech. Dev., 52 (1995) 165-177) suggest that the Xenopus Rel gene XrelA may be involved in the formation of the head and tail of the early embryo. We present evidence to suggest that wild-type XrelA also has a role in dorsoventral development. XrelA overexpression in the dorsal side of embryos reduces dorsal development and attenuates in vitro dorsal morphogenetic movements. XrelA also strongly reduces axis duplication caused by overexpression of a dominant negative mutant of Xenopus glycogen synthase kinase-3 beta. Our results indicate that XrelA may have a role in dorsoventral patterning in early embryos.

Oncogenic Activation of the Human Pygopus2 Promoter by E74-Like Factor-1

Pygopus is a component of the T-cell factor/β-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. (Mol Cancer Res 2008;6(2):259–66)

A co‐dependent requirement of xBcl9 and Pygopus for embryonic body axis development in Xenopus

The Wnt/β‐catenin transcriptional activation complex requires the adapter protein Pygopus (Pygo), which links the basal transcription machinery to β‐catenin, by its association with legless (Lgs)/ B‐cell lymphoma‐9 (Bcl9). Pygo was shown to be required for development in vertebrates, but the role of Lgs/Bcl9 is unknown. We identified an amphibian orthologue of Lgs/Bcl9, XBcl9, which interacted biochemically with Xβ‐catenin and XPygo2. The body axis promoting ability of Xβ‐catenin was diminished when residues required for its interaction with XBcl9 were mutated. In blastula embryos, XBcl9 was transiently preferentially expressed in nuclei of dorsoanterior cells and ectopically expressed XBcl9 required XPygo2 to localize to nuclei. Furthermore, while neither XBcl9 nor XPygo2 alone affected development when ectopically expressed, both were required to induce supernumerary axis and dorsal gene activation. Like XPygo2, depletion of maternal XBcl9 alone caused dorsal defects. These results indicated an essential role of the Pygo‐Bcl9 duet in vertebrate body axis formation. Developmental Dynamics 239:271–283, 2010.

Differential nuclear localization of ER1 protein during embryonic development in Xenopus laevis.

The er1 gene is a novel fibroblast growth factor (FGF)-regulated immediate-early gene, first isolated from Xenopus blastulae, that encodes a nuclear protein with potent transcription transactivational activity (Paterno et al., 1997). We report here the expression pattern of the ER1 protein during Xenopus embryonic development. ER1 protein is present in the early embryo but does not begin to appear in the nucleus until the mid-blastula stage. The first cells to show nuclear localization of ER1 are the presumptive mesodermal cells of the stage 8 blastula. ER1 gradually becomes localized to the nucleus of the remaining cells, first in the presumptive ectoderm and finally, in the presumptive endoderm such that by late blastula, all nuclei in the animal hemisphere are stained. By early gastrula, nuclear staining is ubiquitous. During subsequent development, ER1 protein gradually disappears from the nuclei of various tissues. In tailbud stages, ER1 begins to disappear from the nucleus of ectodermally-derived tissues, such as epidermis and brain, while remaining localized in the nucleus of endodermal cells and of mesodermal tissues, such as somites and notochord. In tadpoles, ER1 is no longer detectable in the nucleus of any cells, except for a few endodermal cells. Cytoplasmic staining, on the other hand, is observed in some mesodermal tissues, including somites and muscle cells. Neural tissue is largely unstained except for weak cytoplasmic staining in the eye.