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

Abstract

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.

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

“…This observation is especially relevant as Rb loss is associated with metastatic and castrate resistant PrCa 24 25. The pygo2 gene is responsive to growth stimuli such as oestrogen receptor signalling in breast cancer26 and is required like MYC, for ribosome biogenesis in cancer 27. These observations supported the hypothesis that activation of PYGO2 gene expression is associated with tumour progression.…”

“…Tissue microarrays (TMAs) were constructed26 from archival formalin-fixed, paraffin-embedded (FFPE) donor blocks from all radical prostatectomy specimens collected from Eastern Health at St John’s, NL (human investigation number, Memorial University, HREB 2012.133) from 2010 to 2014. All specimens were reported to have been fixed in 10% neutral buffered formalin for 24–72 hours at room temperature.…”

Augmentation of Myc-Dependent Mitotic Gene Expression by the Pygopus2 Chromatin Effector

“…Results represent averages ± SEM from at least two independent experiments. ChIP-Seq and Bioinformatics ChIPs were performed as described earlier using Pygo2 S3I4 antisera (Tzenov et al, 2016) and 10 8 S3 cells. 20 ng of DNA was sequenced on an Illumina HiSeq 2500 sequencer (McGill University Genome Quebec Innovation Center).…”

New Pharmacological Opportunities for Betulinic Acid

“…Another way that betulinic acid restricts estrogen signaling is through its effect on the Pygopus protein, the expression of which is strongly indicative of breast cancer, both at the cultured cell level and in samples of invasive ER− and ER+ tumors. Betulinic acid affects the coactivation of the estrogen-responsive gene promoter by transcription factor SP-1 (specificity protein-1), a step that entails a synergistic effect with antagonists or selective estrogen modulators [48].…”