Characterization of oncogenic human pygopus2 expression and regulation

Tzenov, Youlian Roumenov (2012) Characterization of oncogenic human pygopus2 expression and regulation. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Human Pygopus2 (hPYGO2) is a general chromatin remodeler that is overexpressed in, and required for the growth of, a number of tumours of diverse origin and their cell lines. This thesis explores the expression profile of hPygopus2 throughout the cancer cell cycle and identifies novel mechanisms by which it is activated. Examination of hPygopus2 expression and characterization of factors that enhance its expression could lead to better understanding of the signalling networks at play in cancer. It was hypothesized that hPygopus2 expression is induced in the G1 phase of the cell cycle by factors that promote cell cycle progression. The tumour specific expression of hPYGO2 and its requirement for cell proliferation, specifically at the G1/S transition, suggests that hPYGO2 induction is closely correlated with the cell cycle. -- In a set of normal and cancer cell lines, hPygopus2 mRNA and protein levels were significantly reduced in G₀, highest in G1 and moderate in S and G2/M relative to unsynchronized samples. This cell cycle dependent expression suggested that hPygopus2 may be a marker of the G1 phase in proliferating cells. Furthermore, hPYGO2 protein levels in the G2/M phase relative to the G1 phase were inversely proportional to cell cycle length, highlighting a potential utility of hPYGO2 in determining tumour cell proliferation rate. -- The requirement of hPygopus2 for cancer cell proliferation and its high expression during the G1 phase suggested that it is induced during this phase. Due to its well established role in breast cancer (BrCa), the induction of hPygopus2 by 17β-estradiol (E₂) was studied. High levels of E₂ and its receptor, Estrogen receptor alpha (ERα), are well established risk factors for the development and progression of BrCa. However, our knowledge of ERα transcriptional complex components (and their dynamics) at E₂ target gene promoters, particularly in ERα negative (ERα-) BrCa, is incomplete. E₂ treatment of BrCa cells enhanced hPygopus2 mRNA and protein expression via the binding of ERα and SP1 transcription factor (SP1) complexes at the hPYGO2 promoter. Promoter occupancy of these transcription factors required an intact estrogen response element half-site and GC-box and functional DNA binding domains of both proteins. While ERα binding to the hPYGO2 promoter could be prevented by pretreatment with the ERα antagonist Fulvestrant, SP1 binding was only reduced by RNAi. The ability of these proteins to independently occupy the hPYGO2 promoter suggested that SP1 may still play a role in hPYGO2 activation in ERα- BrCa. In Erα-BrCa cells, SP1 expression was required for the activation of hPYGO2 and several other "ERα-SP1 " target genes and a reduction of SP1 resulted in cell cycle arrest. Together, these findings suggested that in ERα+ BrCa, ERα and SP1 have important roles in the regulation of hPygopus2. The ability of SP1 to modulate hPygopus2 expression in the absence of ERα, suggested that hPYGO2 expression might assist in chemotherapy selection in endocrine disruptor unresponsive BrCa tumours. -- The previously demonstrated induction of hPygopus2 by the E74-like factor 1 (ELF1), a Retinoblastoma (RB) tumour suppressor regulated protein, suggested that deregulation of the RB-ELF1 axis leads to overexpression of hPygopus2. The role of the human papillomavirus (HPV) in the inactivation of RB is well characterized. Evidence was provided for hPYGO2 as a cellular biomarker expressed in response to gene deregulation by the main effector of HPV, the E7 oncoprotein, in cervical cancer (CxCa). hPYGO2 levels were greater in high-grade lesions and squamous cell carcinomas as compared to normal epithelia. Similarly, hPygopus2 mRNA and protein levels were greater in HPV-positive CxCa cells relative to uninfected primary cells. RNAi mediated depletion of HPV-E7 increased, while ELF RNAi decreased, association of RB with the hPYGO2 promoter in CxCa cell lines. Transfection of dominant active RB inhibited ELF1-dependent activation of hPYGO2, while ELF1 itself increased hPygopus2 expression. Chromatin immunoprecipitation assays showed that RB repressed hPygopus2 by inhibiting ELF1 at the E26 transformation specific binding site in the hPYGO2 promoter. These results suggested that abrogation of RB by E7 resulted in derepression of ELF1, which in turn stimulated expression of hPygopus2. Thus, initiation of hPygopus2 expression by ELF1 was required for proliferation of CxCa cells and its expression therefore may act as a surrogate marker for dysplasia.

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