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.
AimsEstrogen 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.MethodshPYGO2 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.ResultshPYGO2 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.ConclusionsOur 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.
Quality control (QC) for immunohistochemistry (IHC) analysis routinely incorporates archived specimens for on-slide control material. We have assessed the utility of cell-line-derived xenograft (CDX) tumours for QC in breast estrogen receptor (ER) and progesterone receptor (PR) biomarker testing. Immunoblot and IHC analyses were used to select cell lines with different steady-state levels of ER and PR expression. CDX tumours all demonstrated consistent and comparable expression of ER and PR with corresponding cell lines from which they were derived. Three pathologists experienced in breast biomarker reporting scored tumours from different locations on mammary fat pads to determine reproducibility. Tumours from different locations were consistently scored as identical, and the CDX tumours representing different levels of biomarker expression were similar to patient-derived controls. Pathologists could not consistently distinguish CDX tumours from patient-derived controls, suggesting that within the appropriate quality management setting, CDX tumours may serve as control material for reporting purposes.
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