Laboratory Investigation (2004) 84, 513–522 & 2004 USCAP, Inc All rights reserved 0023-6837/04 $25.00 www.laboratoryinvestigation.org Overexpression of autocrine motility factor in metastatic tumor cells: possible association with augmented expression of KIF3A and GDI-b Takashi Yanagawa1, Hideomi Watanabe1, Toshiyuki Takeuchi2, Shuhei Fujimoto3, Hideyuki Kurihara2,4 and Kenji Takagishi1 1Department of Orthopaedic Surgery; 2Department of Molecular Medicine; 3Department of Microbiology and 4Department of Neurosurgery Gunma University Faculty of Medicine, Institute for Molecular and Cellular Regulation, Gunma University, Showa, Maebashi, Gunma 371-8511, Japan Autocrine motility factor (AMF), which is identical to phosphohexose isomerase (PHI)/glucose-6-phosphate isomerase (GPI) , a ubiquitous enzyme essential for glycolysis, neuroleukin (NLK), a neurotrophic growth factor, and maturation factor (MF) mediating the differentiation of human myeloid cells, enhances the motility and metastatic ability of tumor cells. AMF/PHI activity is elevated in the serum or urine in patients with malignant tumors. Here, we constructed an amf/phi/nlk/mf gene using adenovirus vector and transfected into two tumor cell lines. Overexpression of AMF/PHI/NLK/MF enhanced AMF secretion into the culture media in both tumor cell lines. However, upregulation of motility and metastatic ability was found only in metastatic fibrosarcoma cells expressing an AMF receptor, gp78, and was not found in gp78-undetectable osteosarcoma cells. Thus, not only serum AMF activity but also gp78-expression in tumor cells may be required for metastasis-related motility induction. With the use of microarray analyses, we detected two augmented genes, rho GDP dissociation inhibitor beta and kinesin motor 3A, as well as AMF itself. The RNA message and protein expression of these two molecules was confirmed to be upregulated, suggesting a possible association with AMF-induced signaling for cell motility and metastasis. Laboratory Investigation (2004) 84, 513–522, advance online publication, 16 February 2004; doi:10.1038/labinvest.3700057 Keywords: AMF/PHI/GPI/NLK; KIF3A; GDI-b; motility; metastasis One of the major features of malignancy is the identical to phosphohexose isomerase (PHI), also invasive potential of the tumor cells. Liotta1 pro- designated as glucose-6-phosphate isomerase (GPI), posed a three-step hypothesis for the mechanism of which is a ubiquitous enzyme catalyzing the con- tumor cell invasion. The first is tumor cell attach- version of glucose-6-phosphate to fructose-6-phos- ment via cell surface receptors to the matrix, the phate.5 AMF/PHI has also been found to be identical second is local degradation of the matrix, and the to neuroleukin (NLK),6,7 a neurotrophic growth third is tumor cell locomotion into the region of the factor that supports the survival of spinal and matrix. A group of secreted cytokines inducing cell sensory neurons,8 and maturation factor (MF) motile activity in vitro may initiate cell locomotion mediating the differentiation of human myeloid in vitro.2 Among them, autocrine motility factor leukemic HL-60 cells to terminal monocytic cells,9 (AMF) stimulates cell motility in various types of suggesting that this cytokine is multifunctional, tumor cells in an autocrine manner.3,4 We purified probably dependent on the target cells. AMF/PHI AMF from conditioned medium of murine meta- activities have shown to be elevated in the serum or static fibrosarcoma, and demonstrated that AMF is urine of patients with disseminated malignant tumors such as gastrointestinal, kidney, breast, colorectal, and lung carcinomas,10,11, thereby being Correspondence: Dr H Watanabe, MD, PhD (DMSc), Department useful as a tumor-dissemination marker. An immu- of Orthopaedic Surgery, Gunma University Faculty of Medicine, nohistochemical study has demonstrated that the 3-39-22 Showa, Maebashi, Gunma 371-8511, Japan. E-mail: [email protected] prognosis of AMF/PHI-positive patients with pul- Received 14 April 2003; revised 18 June 2003; accepted 8 monary adenocarcinoma is poorer than that of September 2003; published online 16 February 2004 negative patients.12 Furthermore, AMF/PHI was Overexpression of AMF/PHI/NLK in metastatic cells T Yanagawa et al 514 found in rheumatoid joints,13,14 and was implicated fibrosarcoma cell line, Gunma clone-4 protein-free as a synovial self-pathogenic antigen responsible for (Gc-4 PF),5 in which the motility and metastatic the onset of rheumatoid arthritis.15,16 capacity show high responses to AMF by increased AMF binds to a unique 78 kDa glycoprotein expression of the receptor.18 Another was Dunn receptor, and initiates gp78 phosphorylation.4,17 osteosarcoma protein free (DPF), a mouse osteosar- Anti-gp78 monoclonal antibody (3F3A) stimulates coma cell line.33 Gc-4 PF cells were cultured in motility of metastatic tumor cell,18 and reveals the RPMI 1640 medium without serum. DPF cells were localization of gp78 on the leading lamella as well as cultured in RPMI 1640 medium including trace 19 the trailing edge of motile cells. The expression of elements (5 mM FeCl3,5mM ZnSO4,and10mM gp78 is a predicting factor for prognosis in patients sodium potassium tartrate) without serum. Both with various malignant neoplasms including eso- cells were incubated at 371C in a humidified phageal, colorectal, bladder, gastric, and lung carci- atmosphere of 5% CO2 in air. noma.20–25 The molecular pathway and regulatory mechan- Construction of Recombinant Adenovirus isms by which AMF manifests its effect on cells via its receptor have been elucidated in part. The The cDNA of AMF/PHI was cloned by RT-PCR using signaling pathway involves receptor phosphoryla- mRNA extracted from BALB/cAnNCrj mouse tion,4 and the inhibitory effect of pertussis toxin (PT) liver as a template and the following primers: on the AMF-mediated cell motility suggests a 50-CTTCCGAGCACGTCCTGC-30 and 50-CTAGC regulatory role of G-binding protein.26 It is also TGGGGTGTGAAATACAG-30. The PCR product involved in the activation of phospholipases and the was inserted into TA cloning vector pcR2.1 (Invitro- inositol cycle27 with two downstream components, gen Corp., Carlsbad, CA, USA), cut with NotI and 12-lipoxygenases and protein kinase C.28 Analysis of HindIII, and inserted into pcDNA3 vector (Invitro- the protein kinase cascade(s) in the AMF-stimulated gen Corp., Carlsbad, CA, USA) containing CMV cell motility using specific kinase inhibitors reveals promoter lesion. The whole sequence of the insert the involvement of protein kinase C and tyrosine was analyzed and contained no mutation. An kinase, but not kinase A, reiterating that the signal- expression unit was excised with NruI and SmaI ing is independent of the adenylate cyclase path- and inserted into the SwaI site of the cassette cosmid way.29 Recently, AMF stimulation has been shown to pAdex1cw. The DNA sample was packaged using result in stress-fiber formation, which is associated Gigapack XL (Stratagene, La Jolla, CA, USA). After with activation of two family members of the Rho- plating the transduced Escherichia coli, we could like GTPases, RhoA and Rac1, but not Cdc42 obtain a clone containing the desired insert. Re- activation. This association of the specific small combinant adenovirus was constructed by homo- GTPase Rho activation is accompanied by AMF- logous recombination in human embryonic kidney induced redistribution of the small GTPase from a cell line 293 cells using the DNA samples and the uniform cellular distribution to the lamellipodia and EcoT22I-digested DNA-terminal protein complex filopodia at the leading edges of the cells’ periph- (DNA-TPC). Viruses were propagated in the 293 ery.30 Interestingly, transfection of AMF/PHI-tagged cells and purified by two rounds of CsCl density hemagglutinin into Cos7 cells reveals its intracellu- centrifugation. We constructed two kinds of vectors, lar localization within actin-rich pseudopodial one of which contained lacZ gene (AdlacZ) and domains.31 Torimura et al32 have demonstrated that the other amf/phi gene (Adamf/phi). The cells AMF/PHI enhances Rho activity and phosphoryla- were infected by viral solutions to cell monolayers tion of MEK1 and MEK2 in human hepatoma cell and incubated at 371C for 1 h with brief agita- lines. Rho is one of the most important molecules in tion every 20 min. Culture medium was added, the AMF/PHI signal transduction. and the infected cells were returned to the 371C In this study, to elucidate the metastatic mechan- incubator. isms induced by AMF, we constructed an amf/phi transfection system using adenovirus, and then b-Galactosidase Transduction Assay investigated its secretion, effects on cell motility, and metastasis. Furthermore, the expression of the To assess the efficiency of the adenovirus-mediated molecules related to downstream reaction of AMF/ gene transfer, b-galactosidase activity of Gc-4 PF and PHI stimulation was analyzed with microarray DPF cells infected by adenovirus at 10 multiplicity analysis. of infection (MOI) and 100 MOI was examined 2 and 7 days after infection. At each point, cells were washed two times with PBS, fixed with 0.25% Materials and methods glutaraldehyde, washed four times with PBS, and Cells and Culture Conditions stained in a 1 mg/ml 5-bromo-4-chloro-3-indolyl- b-D-galactopyranoside (X-gal) solution consisting To investigate the function of AMF/PHI as an of 0.2 mM MgCl2, 5 mM K3[Fe(CN)6] and 5 mM autocrine cytokine, we used two cell lines able to K4[Fe(CN)6] in PBS. b-Galactosidase-positive cells grow in a protein-free condition. One was a murine in each well were counted microscopically. Laboratory Investigation (2004) 84, 513–522 Overexpression of AMF/PHI/NLK in metastatic cells T Yanagawa et al 515 Immunoblot Analysis and 100 MOI were injected intravenously at the tail vein into C3H/He mice. Mice were killed 3 weeks Immunoblot analysis was performed to detect AMF/ later, and the lungs were examined for lung- PHI and the receptor, gp78 in Gc-4 PF1 and DPF colonizing nodules.