Arayo Haga

Autocrine motility factor signaling induces tumor apoptotic resistance by regulations Apaf-1 and Caspase-9 apoptosome expression

Autocrine motility factor (AMF) is a cytokine that regulates locomotion and metastasis of tumor cells. It is well known that expression levels of AMF secretion and its receptor (AMF R) are closely related to tumor malignancy and rheumatoid arthritis. We have established that AMF signaling induced anti‐apoptotic activity and that human fibrosarcoma HT‐1080 line that secreted high levels of AMF were resistant to drug‐induced apoptosis. These cells did not express the apoptotic protease activating factor‐1 (Apaf‐1) and Caspase‐9 genes that encode for the proteins that form the “apoptosome” complex. The disappearance of the Apaf‐1 and Caspase‐9 gene was recovered by a cellular signaling inhibitor of protein kinase C, phosphatidylinositol 3‐phosphate kinase and mitogen‐activated protein kinase of the in vitro cultured human fibrosarcoma HT‐1080 line. Treatment with these inhibitors favored apoptotic cell death induced by anti‐cancer drugs of the murine ascites Ehrlich line. Apoptotic resistance of tumor cells allows them to escape death from cancer chemotherapy, so an understanding of malignant anti‐apoptotic activities is important. Antibodies against AMF induced Ehrlich ascites apoptosis in vitro, and effectively aided in vivo apoptosis induced by anti‐cancer drugs. The results might indicate a novel route by which tumor cells protect themselves with products, such as AMF, and proliferate despite various stresses and chemical insults; AMF regulates expression of Apaf‐1 and caspase‐9 genes via a complex signaling pathway and indirectly regulates formation of the apoptosome.

Tumor Autocrine Motility Factor Is an Angiogenic Factor That Stimulates Endothelial Cell Motility

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.

Silencing of Autocrine Motility Factor Induces Mesenchymal-to-Epithelial Transition and Suppression of Osteosarcoma Pulmonary Metastasis

Phosphoglucose isomerase (PGI) is a multifunctional enzyme that functions in glucose metabolism as a glycolytic enzyme catalyzing an interconversion between glucose and fructose inside the cell, while it acts as cytokine outside the cell, with properties that include autocrine motility factor (AMF)-regulating tumor cell motility. Overexpression of AMF/PGI induces epithelial-to-mesenchymal transition with enhanced malignancy. Recent studies have revealed that silencing of AMF/PGI resulted in mesenchymal-to-epithelial transition (MET) of human lung fibrosarcoma cells and breast cancer cells with reduced malignancy. Here, we constructed a hammerhead ribozyme specific against GUC triplet at the position G390 in the human, mouse, and rat AMF/PGI mRNA sequence. Mesenchymal human osteosarcoma MG-63, HS-Os-1, and murine LM8 cells were stably transfected with the ribozyme specific for AMF/PGI. The stable transfectant cells showed effective downregulation of AMF/PGI expression and subsequent abrogation of AMF/PGI secretion, which resulted in morphologic change with reduced growth, motility, and invasion. Silencing of AMF/PGI induced MET, in which upregulation of E-cadherin and cytokeratins, as well as downregulation of vimentin, were noted. The MET guided by AMF/PGI gene silencing induced osteosarcoma MG-63 to terminally differentiate into mature osteoblasts. Furthermore, MET completely suppressed the tumor growth and pulmonary metastasis of LM8 cells in nude mice. Thus, acquisition of malignancy might be completed in part by upregulation of AMF/PGI, and waiver of malignancy might also be controlled by downregulation of AMF/PGI.

Autocrine motility factor secreted by tumor cells upregulates vascular endothelial growth factor receptor (Flt-1) expression in endothelial cells

The autocrine motility factor (AMF) is known as a cytokine regulating tumor cells motility via AMF receptor (AMFR) and promotes their metastasis. Recently, AMFRs have been found on the surface of host cells and it was showed that AMF possibly affects them. The signaling of AMF‐AMFR in the host endothelial cells induces expression of a vascular endothelial growth factor receptor (VEGFR) Flt‐1 and AMFR feedback that is regulated at the transcriptional level. AMF‐exposure stimulated the Flt‐1 expression on human umbilical vein endothelial cells (HUVECs) surface and this AMF‐treated cells exhibited high‐responsibility against VEGF. The protein kinase C (PKC) and phosphatidylinositol 3 kinase (PI3K) play an important role in this signal transduction. The findings of our study suggest the possibility of “tumor AMF→host AMFR→PKC, PI3K→→VEGFR or AMFR→angiogenesis, metastasis” as a new signal cross talk between the tumor and the host.

Inhibition mechanism of cytokine activity of human autocrine motility factor examined by crystal structure analyses and site-directed mutagenesis studies.

Autocrine motility factor (AMF), a tumor-secreted cytokine, stimulates cell migration in vitro and metastasis in vivo. AMF is genetically identical with the extracellular cytokines neuroleukin (NLK) and maturation factor (MF) and, interestingly, the intracellular enzyme phosphohexose isomerase (PHI). The crystal structures of the inhibitor-free open form and the inhibitor (erythrose 4-phosphate, E4P, a strong inhibitor of AMFs cytokine activity)-bound closed form of human AMF have been determined at 1.9 A and 2.4 A resolution, respectively. Upon E4P binding, local conformation changes (open to closed) occur around the inhibitor-binding site. The E4P-bound structure shows that the location of the inhibitor (of cytokine activity) binding site of human AMF is very similar to those of the inhibitor (of enzymatic activity) binding sites of PHIs. The present study shows clearly that there is structural overlap of the regions responsible for the enzymatic and cytokine functions of AMF and PHI and suggests two scenarios for the inhibition mechanism of cytokine activity of AMF by the carbohydrate phosphate group. One likely scenario is that the compound could compete for AMF binding with the carbohydrate moiety of the AMF receptor (AMFR), which is a glycosylated seven-transmembrane helix protein. The other scenario is that the local conformation changes upon inhibitor binding may affect the AMF-AMFR interactions. To examine roles of the residues in the inhibitor-binding site, two mutant AMFs were prepared. Replacements of His389, which is hydrogen-bonded to the hydroxyl group of E4P by Phe, and Thr215, which is hydrogen-bonded to the phosphate group of E4P by Asp, result in mutant AMFs that are impaired in cytokine activity. These results suggest a role for these amino acids in recognition of a carbohydrate moiety of the AMFR. Since the E4P is one of the smallest compounds having AMF inhibitor activity, knowledge of the present crystal structure would provide an insight into the lead compound design of more effective AMF inhibitors.

Tumor autocrine motility factor induces hyperpermeability of endothelial and mesothelial cells leading to accumulation of ascites fluid

Accumulation of ascites fluid often observed in some solid tumors is one of the most devastating conditions of a patients 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.

Enhanced invasiveness of tumour cells after host exposure to heavy metals

The invasiveness of tumour cells to heavy metal-exposed host cells or tissues was investigated. Human fibrosarcoma cell invasion of heavy metal-treated fibroblast or endothelial cell was enhanced in a treatment-time-dependent manner although tumour cell attachment to host cells was not affected. This enhancement was correlated with an increase in metallothioneins in the cytosol of fibroblasts or endothelial cells. Mouse melanoma cell invasion of organ samples obtained from syngeneic mice who had been administered heavy metals was also enhanced. The results suggest that heavy metal-induced metallothioneins serve as a host-derived factor in malignant disease and closely relate to metastasis.

AMF/G6PI induces differentiation of leukemic cells via an unknown receptor that differs from gp78

Autocrine Motility Factor (AMF)/maturation factor (MF)/neuroleukin (NLK) is a multifunctional protein, which acts as a glucose 6-phosphate isomerase (G6PI) intracellularly. Exto-G6PI stimulates invasion and metastasis of tumor cells, neurotropic growth and differentiation of leukemic cells. The cell motility and proliferation receptor is known to be gp78 (78 kilo-Dalton glycoprotein), which has seven transmembrane domains in its N-terminal region, but the maturation factor receptor remains unclear. The human acute monocytic leukemia line does not express gp78 and its motile activity is not enhanced by AMF though it is well differentiated by AMF exposure. The forced expression of gp78 in leukemic cells recovered acceptable motile stimulation, concomitant with reduced differentiation ability. Two unknown proteins were detected by crosslinking between AMF and leukemic cells. The results of this report suggest that the receptor molecule for AMF/NLK/MF in leukemic differentiation is not gp78.

Effect of metallothioneins on transformation of gelatinase A from human fibroblast WI-38 cells

The effect of metallothioneins (MTs) on gelatinase A activity was investigated. The collagenolytic activity of gelatinase A from human fibroblast WI-38 cells was enhanced by the addition of MTs. This enhancement may be caused by the transformation of the inactive 62 kDa intermediate species of gelatinase A to the 59 kDa active enzyme. This enhancement was also observed in the conditioned medium of WI-38 cells exposed to heavy metals, but intracellular 72 kDa pro-gelatinase A did not change. Furthermore, degradation of gelatinase A occurred in the reaction between gelatinase A with substrate and MTs. Our results suggest that MTs may be an endogenous activator of gelatinase A, and may provide a host factor in cancer metastasis.

Crystallization and preliminary X-ray crystallographic study of phosphoglucose isomerase from Plasmodium falciparum.

Phosphoglucose isomerase (PGI) is a key enzyme in glycolysis and glycogenesis that catalyses the interconversion of glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P). For crystallographic studies, PGI from the human malaria parasite Plasmodium falciparum (PfPGI) was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data to 1.5 A resolution were collected from an orthorhombic crystal form belonging to space group P2(1)2(1)2(1) with unit-cell parameters a = 103.3, b = 104.1, c = 114.6 A. Structural analysis by molecular replacement is in progress.