Human Papilloma Virus (HPV) E7-Mediated Attenuation of Retinoblastoma (Rb) Induces hPygopus2 Expression via Elf-1 in Cervical Cancer

Abstract

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.

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

“…Immunoblotting, RNA collection and cDNA generation were performed as described 25. cDNAs were subjected to quantitative real-time PCR (qRT-PCR) using RT2 SYBR green master mix (SA Biosciences) and analysed by the relative quantitative comparative threshold cycle (ΔΔC t ) method 25.…”

“…cDNAs were subjected to quantitative real-time PCR (qRT-PCR) using RT2 SYBR green master mix (SA Biosciences) and analysed by the relative quantitative comparative threshold cycle (ΔΔC t ) method 25. Expression was normalised to β-actin.…”

“…siRNAs against SP1 (siSP1),40 E74-like factor 1 (ELF1)34 and a non-targeting negative control (siNTC),25 were purchased from Dharmacon (see online supplementary table S2). Cells were seeded and forward transfected with siRNA oligos at final concentrations of 10 nM using Lipofectamine RNAiMAX TM (Invitrogen) as per the manufacturer's instructions.…”

“…Chromatin immunoprecipitation (ChIP) was performed essentially as described25 using anti-ERα, and anti-SP1 antibodies, normal rabbit and mouse IgGs as negative controls. hPYGO2 , cathepsin D ( CTSD ) and baculoviral Inihibtor of Apoptosis Protein (IAP) repeat containing 5 ( BIRC5 ) promoters were amplified by qRT-PCR (sequences given in online supplementary table S2).…”

Akt Phosphorylates Wnt Coactivator and Chromatin Effector Pygo2 at Serine 48 to Antagonize Its Ubiquitin/Proteasome-mediated Degradation

“…Additionally, Pygo2 facilitates ␤-catenin-induced hair follicle stem cell activation and is required for skin overgrowth in K14-⌬N-␤-catenin transgenic mice (21). Overexpression of PYGO2 has been reported for human breast, lung, colon, brain, cervical, and ovarian cancer cells (22)(23)(24)(25)(26)(27)(28), and PYGO2 resides in a chromosomal region that is frequently amplified in breast cancer (29,30). These findings implicate the importance of controlling Pygo2/PYGO2 levels for proper Wnt signaling function in development, regeneration, and tumorigenesis.…”