Estrogen receptor is a key regulator of proliferation and differentiation in mammary epithelia and represents a crucial prognostic indicator and therapeutic target in breast cancer. Mechanistically, estrogen receptor induces changes in gene expression through direct gene activation and also through the biological functions of target loci. Here, we identify the product of human MTA3 as an estrogen-dependent component of the Mi-2/NuRD transcriptional corepressor in breast epithelial cells and demonstrate that MTA3 constitutes a key component of an estrogen-dependent pathway regulating growth and differentiation. The absence of estrogen receptor or of MTA3 leads to aberrant expression of the transcriptional repressor Snail, a master regulator of epithelial to mesenchymal transitions. Aberrant Snail expression results in loss of expression of the cell adhesion molecule E-cadherin, an event associated with changes in epithelial architecture and invasive growth. These results establish a mechanistic link between estrogen receptor status and invasive growth of breast cancers.
Binding of different regulatory subunits and methylation of the catalytic (C) subunit carboxyterminal leucine 309 are two important mechanisms by which protein phosphatase 2A (PP2A) can be regulated. In this study, both genetic and biochemical approaches were used to investigate regulation of regulatory subunit binding by C subunit methylation. Monoclonal antibodies selectively recognizing unmethylated C subunit were used to quantitate the methylation status of wild-type and mutant C subunits. Analysis of 13 C subunit mutants showed that both carboxyterminal and active site residues are important for maintaining methylation in vivo. Severe impairment of methylation invariably led to a dramatic decrease in B␣ subunit binding but not of striatin, SG2NA, or polyomavirus middle tumor antigen (MT) binding. In fact, most unmethylated C subunit mutants showed enhanced binding to striatin and SG2NA. Certain carboxy-terminal mutations decreased B␣ subunit binding without greatly affecting methylation, indicating that B␣ subunit binding is not required for a high steady-state level of C subunit methylation. Demethylation of PP2A in cell lysates with recombinant PP2A methylesterase greatly decreased the amount of C subunit that could be coimmunoprecipitated via the B␣ subunit but not the amount that could be coimmunoprecipitated with A␣ subunit or MT. When C subunit methylation levels were greatly reduced in vivo, B␣ subunits were found complexed exclusively to methylated C subunits, whereas striatin and SG2NA in the same cells bound both methylated and unmethylated C subunits. Thus, C subunit methylation is critical for assembly of PP2A heterotrimers containing B␣ subunit but not for formation of heterotrimers containing MT, striatin, or SG2NA. These findings suggest that methylation may be able to selectively regulate the association of certain regulatory subunits with the A/C heterodimer.
The Cancer Genome Atlas (TCGA) project has generated gene expression data that divides glioblastoma (GBM) into four transcriptional classes: proneural, neural, classical, and mesenchymal. Because transcriptional class is only partially explained by underlying genomic alterations, we hypothesize that the tumor microenvironment may also have an impact. In this study, we focused on necrosis and angiogenesis because their presence is both prognostically and biologically significant. These features were quantified in digitized histological images of TCGA GBM frozen section slides that were immediately adjacent to samples used for molecular analysis. Correlating these features with transcriptional data, we found that the mesenchymal transcriptional class was significantly enriched with GBM samples that contained a high degree of necrosis. Furthermore, among 2422 genes that correlated with the degree of necrosis in GBMs, transcription factors known to drive the mesenchymal expression class were most closely related, including C/EBP-β, C/EBP-δ, STAT3, FOSL2, bHLHE40, and RUNX1. Non-mesenchymal GBMs in the TCGA data set were found to become more transcriptionally similar to the mesenchymal class with increasing levels of necrosis. In addition, high expression levels of the master mesenchymal factors C/EBP-β, C/EBP-δ, and STAT3 were associated with a poor prognosis. Strong, specific expression of C/EBP-β and C/EBP-δ by hypoxic, perinecrotic cells in GBM likely account for their tight association with necrosis and may be related to their poor prognosis.
As genomics advances reveal the cancer gene landscape, a daunting task is to understand how these genes contribute to dysregulated oncogenic pathways. Integration of cancer genes into networks offers opportunities to reveal protein–protein interactions (PPIs) with functional and therapeutic significance. Here, we report the generation of a cancer-focused PPI network, termed OncoPPi, and identification of >260 cancer-associated PPIs not in other large-scale interactomes. PPI hubs reveal new regulatory mechanisms for cancer genes like MYC, STK11, RASSF1 and CDK4. As example, the NSD3 (WHSC1L1)–MYC interaction suggests a new mechanism for NSD3/BRD4 chromatin complex regulation of MYC-driven tumours. Association of undruggable tumour suppressors with drug targets informs therapeutic options. Based on OncoPPi-derived STK11-CDK4 connectivity, we observe enhanced sensitivity of STK11-silenced lung cancer cells to the FDA-approved CDK4 inhibitor palbociclib. OncoPPi is a focused PPI resource that links cancer genes into a signalling network for discovery of PPI targets and network-implicated tumour vulnerabilities for therapeutic interrogation.
Protein phosphatase 2A (PP2A) is a multifunctional serine/threonine phosphatase that is critical to many cellular processes including development, neuronal signaling, cell cycle regulation, and viral transformation. PP2A has been implicated in Ca 2؉ -dependent signaling pathways, but how PP2A is targeted to these pathways is not understood. We have identified two calmodulin (CaM)-binding proteins that form stable complexes with the PP2A A/C heterodimer and may represent a novel family of PP2A B-type subunits. These two proteins, striatin and S/G 2 nuclear autoantigen (SG2NA), are highly related WD40 repeat proteins of previously unknown function and distinct subcellular localizations. Striatin has been reported to associate with the postsynaptic densities of neurons, whereas SG2NA has been reported to be a nuclear protein expressed primarily during the S and G 2 phases of the cell cycle. We show that SG2NA, like striatin, binds to CaM in a Ca 2؉ -dependent manner. In addition to CaM and PP2A, several unidentified proteins stably associate with the striatin-PP2A and SG2NA-PP2A complexes. Thus, one mechanism of targeting and organizing PP2A with components of Ca 2؉-dependent signaling pathways may be through the molecular scaffolding proteins striatin and SG2NA. PP2A,1 an essential serine/threonine protein phosphatase found in all eukaryotic cells, regulates a wide variety of important cellular events, including DNA replication, transcription, translation, development, neuronal signaling and progression of the cell cycle (for reviews see Refs. 1-3). The PP2A heterotrimer consists of a catalytic (C) subunit, a structural (A) subunit, and a regulatory (B-type) subunit (4). Although relatively few C-and A-type subunits have been identified, multiple B-type subunits exist, including B (or B55), BЈ (or B56), and BЈЈ (or PR72/130) classes (5-9). To enable utilization of this phosphatase for numerous substrates in different pathways, PP2A is regulated at multiple levels, including covalent modifications, interaction with inhibitory proteins and lipids, and association with the various B-type subunits. For example, BЈ subunits were recently shown to target PP2A to the adenomatous polyposis coli tumor suppressor scaffolding protein, physically associating PP2A with specific substrates and thus regulating Wnt--catenin signaling (10).PP2A has also been shown to form complexes with CaM-dependent kinase IV (CaMKIV) (11), suggesting a role for PP2A in Ca 2ϩ -dependent signaling. This possibility is further supported by patch clamp experiments with both neuronal (12) and smooth muscle cells (13) that have used both okadaic acid and recombinant PP2A C subunit to implicate PP2A in the regulation of calcium-activated potassium channels and L-type Ca 2ϩ channels (14).To better understand how PP2A is targeted to various microenvironments and signal transduction pathways within the cell, we have looked for additional PP2A targeting subunits. Here we report the identification of two PP2A-associated proteins that may represent a novel ...
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