Phillip G. Andrews

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 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.

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.

Selective estrogen receptor modulators and betulinic acid act synergistically to target ERα and SP1 transcription factor dependent Pygopus expression in breast cancer

Aims Estrogen and progesterone hormone receptor (ER and PR) expression in invasive breast cancer predicts response to hormone disruptive therapy. Pygopus2 (hPYGO2) encodes a chromatin remodelling protein important for breast cancer growth and cell cycle progression. The aims of this study were to determine the mechanism of expression of hPYGO2 in breast cancer and to examine how this expression is affected therapeutically. Methods hPYGO2 and ER protein expression was examined in a breast tumour microarray by immunohistochemistry. hPYGO2 RNA and protein expression was examined in ER+ and ER− breast cancer cell lines in the presence of selective estrogen hormone receptor modulator drugs and the specificity protein-1 (SP1) inhibitor, betulinic acid (BA). The effects of these drugs on the ability for ER and SP1 to bind the hPYGO2 promoter and affect cell cycle progression were studied using chromatin immunoprecipitation assays. Results hPYGO2 was expressed in seven of eight lines and in nuclei of 98% of 65 breast tumours, including 3 Ductal carcinoma in situ and 62 invasive specimens representing ER-negative (22%) and ER-positive (78%) cases. Treatment with either 4-Hydroxytamoxifen (OHT) or fulvestrant reduced hPYGO2 mRNA 10-fold and protein 5–10-fold within 4 h. Promoter analysis indicated an ER/SP1 binding site at nt −225 to −531 of hPYGO2. SP1 RNA interference and BA reduced hPYGO2 protein and RNA expression by fivefold in both ER- and ER+ cells. Further attenuation was achieved by combining BA and 4-OHT resulting in eightfold reduction in cell growth. Conclusions Our findings reveal a mechanistic link between hormone signalling and the growth transcriptional programme. The activation of its expression by ERα and/or SP1 suggests hPYGO2 as a theranostic target for hormone therapy responsive and refractory breast cancer.

PYGOPUS2 expression in prostatic adenocarcinoma is a potential risk stratification marker for PSA progression following radical prostatectomy

Aims Prostate cancer (PrCa) is the most frequently diagnosed non-cutaneous cancer in men. Without clear pathological indicators of disease trajectory at diagnosis, management of PrCa is challenging, given its wide-ranging manifestation from indolent to highly aggressive disease. This study examines the role in PrCa of the Pygopus (PYGO)2 chromatin effector protein as a risk stratification marker in PrCa. Methods RNA expression was performed in PrCa cell lines using Northern and RT-PCR analyses. Protein levels were assessed using immunoblot and immunofluorescence. Immunohistochemistry was performed on tissue microarrays constructed from radical prostatectomies with 5-year patient follow-up data including Gleason score tumour staging, margin and lymph node involvement and prostate serum antigen (PSA) levels. Biochemical recurrence (BR) was defined as a postoperative PSA level of >0.2 nL. Univariate and multivariate analyses were performed using SAS and Kaplan-Meier curves using graphPad (Prism). Results In vitro depletion of PYGO2 by RNAi in both androgen receptor positive and negative PrCa cell lines attenuated growth and reduced Ki67 and 47S rRNA expression, while PYGO2 protein was localised to the nuclei of tumours as determined by immunohistochemistry. High expression levels of PYGO2 in tumours (n=156) were correlated with BR identified as PSA progression, after 7-year follow-up independent of other traditional risk factors. Most importantly, high PYGO2 levels in intermediate grade tumours suggested increased risk of recurrence over those with negative or weak expression. Conclusion Our data suggest that elevated PYGO2 expression in primary prostate adenocarcinoma is a potential risk factor for BR.

Abstract 3108: A novel role for hPygo2 in ribosomal RNA transcription.

Background: Several ribosomes are required for each translated messenger RNA. During cell growth and division, a significant proportion of cellular resources are devoted to the production of ribosomes, but the epigenetic mechanisms by which cells adjust to this requirement are not fully understood. We have previously demonstrated that human Pygopus 2 (hPygo2) is highly expressed in, and required for the growth of a number of different cancers. hPygo2 functions as a chromatin remodeling protein through direct association with trimethylated Histone H3 at lysine4 (H3K4me3). It is here that hPygo2 likely serves as an adapter to which histone acetyltransferases can bind and promote further chromatin remodeling events required for transcription. We now present evidence that hPygo2 is involved in chromatin remodeling at the ribosomal (r)RNA promoter during proliferative growth of cancer cells. Methods: Immunoprecipitation (IP) and immunofluorescence (IF) was performed in breast (MCF7), ovarian (SKOV3) and cervical (HeLa) cancer cells. siRNAs were designed to specifically deplete hPygo2. For hPygo2 binding to the ribosomal gene promoter, chromatin IPs were used and analyzed by conventional or quantitative (q) PCR. 47S rRNA expression was measured by qPCR or by pulse-chase with either Br-UTP or 3 H-uridine. Cell cycle analysis was performed by flow cytometry. Results: We found that hPygo2 interacted with UBF-1 and Treacle, two nucleolar proteins involved in 47S rRNA transcription. hPygo2 co-localized with UBF-1 and Treacle along with newly synthesized de novo rRNA in the nucleoli of cancer cells. hPygo2 was further detected at the ribosomal gene promoter along with core components of the rDNA transcription complex, including RNA polymerase I. Furthermore, hPygo2 was required for Histone H4 acetylation at the rRNA promoter and for transcription of the 47S pre-rRNA. Depletion of hPygo2 was accompanied by activation of the ribosomal stress response, detected by the binding of ribosomal protein L11 to hdm2, thereby inhibiting its E3 ubiquitin ligase activity resulting in p53 dependent growth arrest. Conclusion: Our results suggest that chromatin remodeling function of hPygo2 may serve to augment or maintain 47S pre-rRNA transcription required for ribosome biogenesis in cancer. Citation Format: Phillip G. P. Andrews, Kenneth R. Kao. A novel role for hPygo2 in ribosomal RNA transcription. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3108. doi:10.1158/1538-7445.AM2013-3108