Identification of Pygopus 2 as a component of the ribosomal RNA transcription complex in cancer

Abstract

Background: Pygopus was initially validated as a co-activator for the canonical Wnt signaling pathway, a critical intracellular cascade implicated both in development and cancer. Further studies indicated that Pygopus appears to have both Wnt-dependent and Wnt-independent functions. Our lab has demonstrated that human Pygopus 2 (hPygo2) was overexpressed and required for Epithelial Ovarian Cancer cell growth in the absence of Wnt signaling. Purpose: The purpose of this thesis was to investigate the mechanism of Wnt-independent requirement of hPygo2 for Epithelial Ovarian Cancer cell growth. -- Hypothesis and Objectives: I hypothesized that hPygo2 achieves Wnt-independent functions in Epithelial Ovarian Cancer via interacting with protein factors not related to Wnt signaling. Therefore, three objectives were proposed in this thesis: 1) to determine minimal domain(s) of hPygo2 required for Wnt-independent Epithelial Ovarian Cancer cell growth; 2) to identify potential protein factors that interact with this critical domain; and 3) to characterize the identified interactions. -- Results: Objective 1). The transfection of hPygo2-specific antisense oligonucleotides in Wnt-inactive Epithelial Ovarian Cancer cell line, SKOV-3 led to cell growth arrest, as shown previously. To assess the sequence requirements of hPygo2 for SKOV-3 cell growth, plasmids encoding various mutant hPygo2 with site-specific alterations within the transcriptional activation domain (N-terminal Homology Domain, NHD) or Wnt-mediating domain (Plant Homeodomain, PHD) were transfected in the hPygo-2-specific antisense treated SKOV-3 cells. Like wild-type hPygo2, mutant hPygo2 proteins with alterations in the PHD that prevented their Wnt-mediating ability restored the growth levels of antisense-treated SKOV-3 cells. On the other hand, hPygo2 protein with mutations in the transcriptional activation NHD domain, failed to restore cell growth. Objective 2). Using a proteomics approach, I identified several possible proteins including p68, RNA helicases II/Gu, Nop56, and Treacle protein from SKOV-3 nuclear extracts that bound to the NHD domain. Objective 3). The treacle protein was identified to have the highest probability of interaction with hPygo2. This interaction was confirmed in vitro and in vivo, in cancer cells. Immunofluorescence assays indicated that Treacle colocalized with hPygo2 in the nucleoli of SKOV-3 and HeLa cancer cells. Actinomycin D treatment in HeLa cells suggested that hPygo2 is a fibrillar component of the nucleolus and may be involved in transcription or early modification of premature ribosomal RNAs. -- Conclusions: The NHD, but not the PHD domain, of hPygo2 protein is required for Wnt-independent SKOV-3 cell proliferation. The hPygo2 interacts with Treacle through its NHD domain and co-localizes with Treacle to the fibrillar compartment of nucleolus in cancer cells. Thus, these results suggest one of the Wnt-independent functions of hPygo2 is involved in ribosomal biogenesis.