Ubiquitin‐mediated proteolysis has emerged as a key mechanism of regulation in eukaryotic cells. During cell division, a multi‐subunit ubiquitin ligase termed the anaphase promoting complex (APC) targets critical regulatory proteins such as securin and mitotic cyclins, and thereby triggers chromosome separation and exit from mitosis. Previous studies in the yeast Saccharomyces cerevisiae identified the conserved WD40 proteins Cdc20 and Hct1 (Cdh1) as substrate‐specific activators of the APC, but their precise mechanism of action has remained unclear. This study provides evidence that Hct1 functions as a substrate receptor that recognizes target proteins and recruits them to the APC for ubiquitylation and subsequent proteolysis. By co‐immunoprecipitation, we found that Hct1 interacted with the mitotic cyclins Clb2 and Clb3 and the polo‐related kinase Cdc5, whereas Cdc20 interacted with the securin Pds1. Failure to interact with Hct1 resulted in stabilization of Clb2. Analysis of Hct1 derivatives identified the C‐box, a motif required for APC association of Hct1 and conserved among Cdc20‐related proteins. We propose that proteins of the Cdc20 family are substrate recognition subunits of the ubiquitin ligase APC.

The relevance of angiogenesis in tumor biology and as a therapeutic target is well established. MFG-E8 (also termed lactadherin) and developmental endothelial locus 1 (Del1) constitute a two-gene family of A v B 3 /B 5 ligands that regulate angiogenesis. After detecting MFG-E8 mRNA in murine tumor cell lines, we sought to determine if MFG-E8 influenced tumorigenesis in Rip1-Tag2 transgenic mice, a cancer model in which angiogenesis is critical. MFG-E8 mRNA and protein were increased in angiogenic islets and tumors in Rip1-Tag2 mice compared with normal pancreas. Frequencies of angiogenic islets and tumor burdens were decreased in MFG-E8-deficient Rip1-Tag2 mice compared with those in control Rip1-Tag2 mice. Invasive carcinomas were modestly underrepresented in MFG-E8-deficient mice, but tumor frequencies and survivals were comparable in these two strains. Absence of MFG-E8 also led to decreases in tumor vascular permeability without obvious changes in vascular morphology. Decreased proliferation was noted in angiogenic islets and increases in apoptotic cells were detected in islets and tumors. Compensatory increases in mRNA encoding proangiogenic proteins, including FGF2, in angiogenic islets, and Del1, in angiogenic islets and tumors, were also detected in MFG-E8-deficient mice. MFG-E8 and its homologue Del1 may represent relevant targets in cancer and other diseases in which angiogenesis is prominent. [Cancer Res 2007;67(14):6777-85]

To identify novel regulators of endoplasmic reticulum (ER)-linked protein degradation and ER function, we determined the entire inventory of membrane-spanning RING finger E3 ubiquitin ligases localized to the ER. We identified 24 ER membraneanchored ubiquitin ligases and found Nixin/ZNRF4 to be central for the regulation of calnexin turnover. Ectopic expression of wild type Nixin induced a dramatic down-regulation of the ER-localized chaperone calnexin that was prevented by inactivation of the Nixin RING domain. Importantly, Nixin physically interacts with calnexin in a glycosylation-independent manner, induces calnexin ubiquitination, and p97-dependent degradation, indicating an ER-associated degradation-like mechanism of calnexin turnover.The endoplasmic reticulum (ER) 3 is a major cellular site for production, folding, quality control, and distribution of proteins. Many regulatory mechanisms are in place to keep these processes in balance and therefore to ensure cellular fitness and survival, with ubiquitin-dependent protein degradation playing an important part (1). One major challenge the ER faces is an overload with unfolded or folding proteins. An excess of folding proteins in the ER triggers a cellular response called the unfolded protein response (UPR) (2, 3). UPR entails lowering of the protein load by the attenuation of protein translation and the up-regulation of chaperones thereby increasing the protein folding capacity of the cells. If the capacity of UPR is exceeded, the cell utilizes ER-associated degradation (ERAD), a system for the recognition of terminally misfolded proteins and their disposal (4). Misfolded proteins destined for ERAD are ubiquitinated by the RING domain containing ubiquitin ligases (5), Hrd1 (6), and Doa1 (7, 8), retrotranslocated across the ER membrane into the cytosol by the AAA-ATPase p97 (9), and then degraded by the 26 S proteasome.Upon entry into the ER, most nascent polypeptides are recognized by glycosidases and modified on specific asparagine residues (Asn-Xaa-(Thr/Ser)) with the N-glycan GlcNAc 2 Man 9 Gluc 3 (10). Core glycosylation of nascent polypeptides decreases their overall hydrophobicity. Trimming of the terminal two glucose residues by glucosidase I allows for binding of the lectins/chaperones calreticulin and calnexin thereby facilitating the proper folding of the newly synthesized protein (10,11).Although the importance of regulated degradation of ER resident proteins is firmly established, only a small number of RING finger-containing ubiquitin ligases are known to be involved in such processes to date, namely SYVN1/hHrd1 (6, 12), AMFR/gp78 (13), TEB4/MARCH6 (14), RNF5/Rma1 (15), RNF77/TRIM13 (16), and RNF13 (17). Given the importance of protein metabolism and degradation in the ER and the vast number of ubiquitin ligases encoded in the human genome, we asked whether other ubiquitin ligases are involved in the regulation of ER-related degradation processes.Based on the assumption that the ER lumen is devoid of E1 and E2 ubiquitination activity and on the...

Ubiquitination, the covalent attachment of the small protein modifier ubiquitin to a substrate protein is involved in virtually all cellular processes by mediating the regulated degradation of proteins. Aside from proteasomal degradation, ubiquitination plays important roles in transcriptional regulation, protein trafficking, including endocytosis and lysosomal targeting, and activation of kinases involved in signalling processes. A three-tiered enzymatic cascade consisting of E1 or ubiquitin-activating enzyme, E2 or ubiquitin-conjugating enzyme, and E3, or ubiquitin ligases, is necessary to achieve the many forms of ubiquitination known to date. In this chapter, we summarize the current knowledge on the enzymatic machinery necessary for ubiquitin activation and ligation, as well as its removal, and provide some insight into the complexity of regulatory processes governed by ubiquitination.

An epithelial-mesenchymal transition (EMT) has been implicated in cancer metastasis, drug resistance, and in conferring stem cell-like traits to cancer cells. Most studies investigating EMT in cancer have either utilized immortalized or cancer cell lines that are already primed to undergo an EMT and do not adequately represent a fully differentiated epithelial state in the absence of an EMT induction. Hence, model systems are required which recapitulate all stages of EMT in cancer cells. Here, we report the derivation and characterization of epithelial PyMT-1099 cancer cells from the MMTV-PyMT mouse model of breast cancer. We demonstrate that PyMT-1099 cells undergo an EMT upon TGFβ treatment, while upon TGFβ withdrawal they go through a mesenchymal-epithelial transition (MET), as assessed by changes in cell morphology and marker expression and comparable to normal murine mammary gland NMuMG cells. However, in contrast to NMuMG cells, PyMT-1099 cells show an increase in cell migration and are highly tumorigenic and metastatic when transplanted into immunocompromised mice. Finally, we report cancer cell-specific changes in gene expression during EMT of PyMT-1099 cells not found in non-transformed NMuMG cells. Thus, PyMT-1099 cells are a versatile tool to study breast cancer-associated EMT and MET in vitro and in vivo.

The vast majority of breast cancer-associated deaths are due to metastatic spread of cancer cells, a process aided by epithelial-mesenchymal transition (EMT). Mounting evidence has indicated that long non-coding RNAs (lncRNAs) also contribute to tumor progression. We report the identification of 114 novel lncRNAs that change their expression during TGFβ-induced EMT in murine breast cancer cells (referred to as EMT-associated transcripts; ETs). Of these, the ET-20 gene localizes in antisense orientation within the Tenascin C (Tnc) gene locus. Tnc is an extra-cellular matrix protein which is critical for EMT and metastasis formation. Both ET-20 and Tnc are regulated by the EMT master transcription factor Sox4. Notably, ablation of ET-20 lncRNA effectively blocks Tnc expression and with it EMT. Mechanistically, ET-20 interacts with desmosomal proteins, thereby impairing epithelial desmosomes and promoting EMT. A short transcript variant of ET-20 is found upregulated in invasive human breast cancer cell lines where it also promotes EMT. Targeting ET-20 appears a therapeutically attractive lead to restrain EMT and breast cancer metastasis in addition to its potential utility as a biomarker for invasive breast cancer.

Initiation of PINK1-and PRKN-dependent mitophagy is a highly regulated process involving the activity of the AAA-ATPase VCP/p97, a cofactor-guided multifunctional protein central to handling ubiquitinated client proteins. Removal of ubiquitinated substrates such as the mitofusin MFN2 from the outer mitochondrial membrane by VCP is critical for PRKN accumulation on mitochondria, which drives mitophagy. Here we characterize the role of the UBA and UBX-domain containing VCP cofactor UBXN1/SAKS1 during mitophagy. Following mitochondrial depolarization and depending on PRKN, UBXN1 translocated alongside VCP to mitochondria. Prior to mitophagy, loss of UBXN1 led to mitochondrial fragmentation, diminished ATP production, and impaired ER-mitochondrial apposition. When mitophagy was induced in cells lacking UBXN1, mitochondrial translocation of VCP and PRKN was impaired, diminishing mitophagic flux. In addition, UBXN1 physically interacted with PRKN in a UBX-domain depending manner. Interestingly, ectopic expression of the pro-mitophagic VCP cofactor UBXN6/UBXD1 fully reversed impaired PRKN recruitment in UBXN1 -/cells. Mechanistically, UBXN1 acted downstream of PINK1 by facilitating MFN2 removal from mitochondria. In UBXN1 -/cells exposed to mitochondrial stress, MFN2 formed para-mitochondrial blobs likely representing blocked intermediates of the MFN2 removal process partly reversible by expression of UBXN6. Presence of these MFN2 blobs strongly correlated with impaired PRKN translocation to depolarized mitochondria. Our observations connect the VCP cofactor UBXN1 to the initiation and maintenance phase of PRKNdependent mitophagy, and indicate that, upon mitochondrial stress induction, MFN2 removal from mitochondria occurs through a specialized process.

◥Pygopus 2 (Pygo2) is a coactivator of Wnt/b-catenin signaling that can bind bi-or trimethylated lysine 4 of histone-3 (H3K4me 2/3 ) and participate in chromatin reading and writing. It remains unknown whether the Pygo2-H3K4me 2/3 association has a functional relevance in breast cancer progression in vivo. To investigate the functional relevance of histone-binding activity of Pygo2 in malignant progression of breast cancer, we generated a knock-in mouse model where binding of Pygo2 to H3K4me 2/3 was rendered ineffective. Loss of Pygo2-histone interaction resulted in smaller, differentiated, and less metastatic tumors, due, in part, to decreased canonical Wnt/b-catenin signaling. RNA-and ATAC-sequencing analyses of tumor-derived cell lines revealed downregulation of TGFb signaling and upregulation of differentiation pathways such as PDGFR signaling. Increased differentiation correlated with a luminal cell fate that could be reversed by inhibition of PDGFR activity. Mechanistically, the Pygo2-histone interaction potentiated Wnt/b-catenin signaling, in part, by repressing the expression of Wnt signaling antagonists. Furthermore, Pygo2 and b-catenin regulated the expression of miR-29 family members, which, in turn, repressed PDGFR expression to promote dedifferentiation of wild-type Pygo2 mammary epithelial tumor cells. Collectively, these results demonstrate that the histone binding function of Pygo2 is important for driving dedifferentiation and malignancy of breast tumors, and loss of this binding activates various differentiation pathways that attenuate primary tumor growth and metastasis formation. Interfering with the Pygo2-H3K4me 2/3 interaction may therefore serve as an attractive therapeutic target for metastatic breast cancer.Significance: Pygo2 represents a potential therapeutic target in metastatic breast cancer, as its histone-binding capability promotes b-catenin-mediated Wnt signaling and transcriptional control in breast cancer cell dedifferentiation, EMT, and metastasis.