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Autocrine motility factor (AMF) stimulates the motility of cancer cells via an autocrine route and has been implicated in tumor progression and metastasis. Overexpression of AMF is correlated with the aggressive nature of breast cancer and is negatively associated with clinical outcomes. In contrast, AMF also has the ability to suppress cancer cells. In this study, AMFs from different cancer cells were demonstrated to have suppressive activity against MCF-7 and MDA-MB-231 breast cancer cells. In a growth and colony formation assay, AMF from AsPC-1 pancreatic cancer cells (ASPC-1:AMF) was determined to be more suppressive compared to other AMFs. It was also demonstrated that AsPC-1:AMF could arrest breast cancer cells at the G0/G1 cell cycle phase. Quantified by Western blot analysis, AsPC-1:AMF lowered levels of the AMF receptor (AMFR) and G-protein-coupled estrogen receptor (GPER), concomitantly regulating the activation of the AKT and ERK signaling pathways. JAK/STAT activation was also decreased. These results were found in estrogen receptor (ER)-positive MCF-7 cells but not in triple-negative MDA-MB-231 cells, suggesting that AsPC-1:AMF could work through multiple pathways led to apoptosis. More importantly, AsPC-1:AMF and methyl jasmonate (MJ) cooperatively and synergistically acted against breast cancer cells. Thus, AMF alone or along with MJ may be a promising breast cancer treatment option.

The E3 ubiquitin (Ub) ligase gp78 plays an important role in endoplasmic reticulum (ER)-associated degradation (ERAD) and regulation of lipid biogenesis. Although a variety of substrates of gp78 have been described, the regulation of the degradation of gp78 itself remains poorly understood. To address this problem, we used co-immunoprecipitation-coupled liquid chromatography-tandem mass spectrometry (Co-IP/LC-MS/MS) to identify novel proteins interacting with gp78. One of the proteins identified in this study is the deubiquitylating (DUB) enzyme USP34 (Ub-specific protease 34). We demonstrate that knockdown of USP34 facilitates proteasomal degradation of gp78 and consequently impairs the function of gp78 in regulating lipid droplet formation. This study unveils a previously unknown function of USP34 in regulating the metabolic stability of gp78 and adds to our understanding of the relevance of partnering of DUBs and E3s in regulation of protein ubiquitylation.

Polyubiquitin chain linkage specificity or topology is essential for its role in diverse cellular processes. Previous studies pay more attentions to the linkage specificity of the first ubiquitin moieties, whereas, little is known about the editing mechanism of linkage specificity in longer polyubiquitin chains. gp78 and its cognate E2-Ube2g2 catalyze lysine48 (K48)-linked polyubiquitin chains to promote the degradation of targeted proteins. Here, we show that the linkage specificity of the entire polyubiquitin chain is determined by the conjugation manner of the first ubiquitin molecule but not the following ones. Further study discovered that the gp78 CUE domain works as a proofreading machine during the growth of K48-linked polyubiquitin chains to ensure the linkage specificity. Together, our studies uncover a novel mechanism underlying the linkage specificity determination of longer polyubiquitin chains.

Endoplasmic reticulum (ER) stress occurs during early embryonic development. The aim of this study is to determine whether ER stress occurs during human embryonic stem cell differentiation induced by retinoic acid (RA). H9 human embryonic stem cells were subjected to RA treatment for up to 29days to induce differentiation. HEK293 cells were treated with RA as a control. The results demonstrate that several ER stress-responsive genes are differentially regulated in H9 and HEK293 cells in response to 5days of RA treatment. GRP78/Bip was upregulated in H9 cells but downregulated in HEK293 cells. eIF2α was downregulated in H9 cells but not in HEK293 cells. Phosphorylation of eIF2α was downregulated in H9 cells but upregulated in HEK293 cells. XBP-1 was downregulated immediately after RA treatment in H9 cells, but its downregulation was much slower in HEK293 cells. Additionally, two ER-resident E3 ubiquitin ligases, gp78 and Hrd1, were both upregulated in H9 cells following 5 days of exposure to RA. Moreover, the protein Bcl2 was undetectable in H9 cells and H9-derived cells but was expressed in HEK293 cells, and it expression in the two types of cells was unaltered by RA treatment. In H9 cells treated with RA for 29 days, GRP78/Bip, XBP-1 and Bcl2 were all upregulated. These results suggest that ER stress is involved in H9 cell differentiation induced by RA.

There are an increasing number of ubiquitin ligases (E3s) implicated in endoplasmic reticulum (ER)-associated degradation (ERAD) in mammals. The two for which the greatest amount of information exists are the RING finger proteins gp78 and Hrd1, which are the structural orthologs of the yeast ERAD E3 Hrd1p. We now report that Hrd1, also known as synoviolin, targets gp78 for proteasomal degradation independent of the ubiquitin ligase activity of gp78, without evidence of a reciprocal effect. This degradation is observed in mouse embryonic fibroblasts lacking Hrd1, as well as with acute manipulation of Hrd1. The significance of this is underscored by the diminished level of a gp78-specific substrate, Insig-1, when Hrd1 expression is decreased and gp78 levels are consequently increased. These finding demonstrate a previously unappreciated level of complexity of the ubiquitin system in ERAD and have potentially important ramifications for processes where gp78 is implicated including regulation of lipid metabolism, metastasis, cystic fibrosis and neurodegenerative disorders.

Accumulation of ascites fluid often observed in some solid tumors is one of the most devastating conditions of a patient's difficulty in responding to treatment, and to a decrease in the quality of life. Various factors are thought to be associated with the formation of cancer-induced fluid accumulation and hyperpermeability of a blood vessel is thought to go with this process. Here, we report a new factor that is involved in this process, e.g., autocrine motility factor (AMF). AMF is a tumor-related cytokine which stimulates the tumor cell locomotion and migration and promotes tumor cell invasion during metastasis. AMF secretion and its receptor (AMFR) expression in tumor cells are closely correlated with disease aggravation of convalescence. The response of endothelial or mesothelial cellular morphological alternation to AMF leads to motile enhancement and vascular permeability. Tumor AMF induces gaps in an endothelial or mesothelial monolayer by stimulating a cellular movement, and accelerates the ascites accumulation. And treatment experiment with anti-AMF antibody succeeded in the reduction of the ascites accumulation, which renders AMF to the target molecule. It is suggested that AMF is one of the significant factors which relates to various pathological malignancies induced by tumor mass, and understanding of its function could benefit prognosis and treatment.

Autocrine motility factor interacts with its cell surface receptor (AMF-R) to stimulate tumor cell motility. To study AMF-R expression following transformation of polarized epithelial MDCK cells, we have used the invasive Moloney sarcoma virus transformed MDCK (MSV-MDCK) cell population. Decreased E-cadherin expression of the transformed MSV-MDCK clones is associated with both increased cellular motility and increased AMF-R expression. Increased AMF-R expression is due to MSV transformation as differentially motile MSV-MDCK clones, which either retain low E-cadherin, exhibit equivalent high levels of AMF-R. Loss of the polarized epithelial phenotype and increased cellular motility following transformation of MDCK cells is thus associated with a shift from a high E-cadherin/low AMF-R to a low E-cadherin/low AMF-R phenotype.

Deficiency of circulating α-1-antitrypsin (AAT) is the most widely recognized abnormality of a proteinase inhibitor that causes lung disease. AAT-deficiency is caused by mutations of the AAT gene that lead to AAT protein retention in the endoplasmic reticulum (ER). Moreover, the mutant AAT accumulated in the ER predisposes the homozygote to severe liver injuries, such as neonatal hepatitis, juvenile cirrhosis, and hepatocellular carcinoma. Despite the fact that mutant AAT protein is subject to ER-associated degradation (ERAD), yeast genetic studies have determined that the ubiquitination machinery, Hrd1/Der3p-cue1p-Ubc7/6p, which plays a prominent role in ERAD, is not involved in degradation of mutant AAT. Here we report that gp78, a ubiquitin ligase (E3) pairing with mammalian Ubc7 for ERAD, ubiquitinates and facilitates degradation of ATZ, the classic deficiency variant of AAT having a Z mutation (Glu 342 Lys). Unexpectedly, gp78 over-expression also significantly increases ATZ solubility. p97/VCP, an AAA ATPase essential for retrotranslocation of misfolded proteins from the ER during ERAD, is involved in gp78-mediated degradation of ATZ. Surprisingly, unlike other ERAD substrates that cause ER stress leading to apoptosis when accumulated in the ER, ATZ, in fact, increases cell proliferation when over-expressed in cells. This effect can be partially inhibited by gp78 over-expression. These data indicate that gp78 assumes multiple unique quality control roles over ATZ, including the facilitation of degradation and inhibition of aggregation of ATZ.

The receptor for the autocrine motility factor/phosphoglucose isomerase cytokine (gp78 or AMFR) has been extensively characterized using the 3F3A monoclonal antibody. Cloning of AMFR identified a seven-transmembrane domain G-protein-coupled receptor ubiquitin E3 ligase whose identity as AMFR was based on prior expression cloning with the 3F3A mAb that generated a truncated sequence. We show here that the gp78/AMFR gene product is indeed recognized by the 3F3A mAb. The FLAG-tagged AMFR immunoprecipitated with an anti-FLAG antibody was recognized by the 3F3A mAb in Western blot analysis and cells transfected with AMFR exhibit increased labeling with the 3F3A mAb. The 3F3A mAb does not however recognize higher molecular weight isoforms of AMFR. 3F3A labeling colocalizes with tagged AMFR in a peripheral ER network but does not recognize FLAG- or GFP-tagged AMFR localized to a perinuclear ER domain that likely corresponds to misfolded forms of the protein retained in the ER. These data indicate that 3F3A antibody binding is highly specific for a subpopulation of AMFR localized to an ER subdomain. Coexpression of AMFR-GFP and a lumenal ER-targeted RFP presented extensive colocalization in living cells and AMFR-GFP is concentrated in a basal ER network morphologically similar to that labeled by the 3F3A mAb in fixed cells. The 3F3A anti-AMFR mAb therefore selectively recognizes a subpopulation of expressed AMFR localized to a subdomain of the ER.

Autocrine motility factor (AMF) is a type of tumor-secreted cytokine which primarily stimulates tumor cell motility via receptor-mediated signaling pathways, and is thought to be connected to tumor progression and metastasis. Using in vivo models, we showed that critical neovascularization responded to a biological amount of AMF. This angiogenic activity was fixed by specific inhibitors against AMF. AMF stimulated in vitro motility of human umbilical vein endothelial cells (HUVECs), inducing the expression of cell surface AMF receptor localizing a single predominant perinuclear pattern closely correlated with its motile ability. AMF also elicited the formation of tube-like structures mimicking angiogenesis when HUVECs were grown in three-dimensional type I collagen gels. We further immunohistochemically detected AMF receptors on the surrounding sites of newborn microvessels. These findings suggest that AMF is a possible tumor progressive angiogenic factor which may act in a paracrine manner for the endothelial cells in the clinical neoplasm, and it will be a new target for antiangiogenic treatment.

We have isolated from a human placenta cosmid library a 0.7 kb genomic clone that contains the 5' terminal portion of the autocrine motility factor receptor (hAMFR) coding region. Chloramphenicol Acetyl Transferase (CAT) reporter gene assays have identified this region as the promoter of the hAMFR gene. A single transcription initiation site (+1) has been mapped to 129 bp upstream of the ATG start codon by primer extension. DNA sequence analysis and CAT assay revealed a TATA element at the position -485/-468 which was able to conduct only a marginal transcription (less than 5% of the total activity). The majority of the hAMFR promoter's activity is contributed by a transcription initiator (Inr) element overlapping the initiation site (+1) which independently controls the transcription of the hAMFR gene. Gel mobility shift assays showed that DNA-binding proteins in HeLa cells nuclear extract can bind specifically to both promoter's elements. DNA-binding proteins were found to be differentially expressed by sparse and dense cultured normal fibroblasts. The nuclear-binding protein expressed by sparse NIH-3T3 cells induced a DNA mobility shift similarly to the nuclear protein of HeLa cells, while a different DNA-protein complex size was observed with nuclear proteins extracted from dense cultured NIH-3T3 cells. Also CAT-reporter gene analysis revealed a significant lower activity in dense NIH-3T3 cells as compared with the sparse-cultured counterparts. These results help to explain the previously observed cell-cell contact regulation of AMFR expression in normal cells and its consecutive expression in tumor cells.

Autocrine motility factor (AMF) is a 55 kDa cytokine which is produced and secreted by cancer cells and which regulates cell motility via binding to its receptor, a 78 kDa cell surface glycoprotein (gp78-AMFR), and activating a pertussis toxin (PT)-sensitive G-protein. AMF purified from HT-1080 human fibrosarcoma cells stimulates the growth and motility of 3T3-A31-fibroblasts at a concentration of 0.1 ng/ml or less. The expression of total as well as cell surface gp78-AMFR is down-regulated in contact-inhibited A31-fibroblasts and AMF stimulates the healing of experimentally wounded, density-arrested A31 monolayer cultures. This is the first report of the paracrine and mitogenic actions of AMF and the results presented here show that AMF functions as a growth factor and suggest a possible role for its activity in normal tissue regeneration and tumor cell dissemination.