Tumor Invasion and Metastasis Section Staff

Dr. Elliott Schiffman initially began to study protein factors which regulate tumor cell motility. He found secreted factors in the conditioned media of some human melanoma cells and termed the phenomenon the autocrine motility hypothesis, which suggests that tumor cells may initiate, sustain, and regulate their own locomotion. One such factor, termed autotaxin (ATX), was purified by Dr. Mary Stracke from a melanoma cell line. ATX is a novel 125 kDa glycoprotein and ectoenzyme that has now been shown to be a potent stimulator or tumor cell motility, invasion, and metastasis.

ATX stimulates random and directed motility (ED50 ~ 300-500 pM) in a variety of tumor cell lines, including those from prostate and breast carcinomas, melanomas, and neuroblastomas. Many of these same cell lines as well as teratocarcinoma cells have been shown to synthesize and secrete the ATX protein. ATX was cloned and seuqenced by Dr. Jun Murata. The cDNA sequence analysis revealed significant homology to a family of cell surface type I phosphodiesterases that are now called NPPs (nucleotide phosphodiesterase and pyrophospatases). Members of this family include a marker of B cell activiation (PC-1/NPP-1), a neural differentiation antigen (B-10/NPP-3), and ATX (NPP-2). Tim Clair found that native ATX possesses phosphodiesterase activity at neutral and alkaline pH, binds ATP non-covalently, and has pyrophosphatase, ATPase, ADPase, and AMPase activities. Homogeneously purified recombinant ATX, based on the teratocarcinoma sequence, retains all of these activities, as well as a capacity to stimulate motility.

No physiological function had been linked to the phosphodiesterase catalytic site until Dr. Hoi Young Lee utilized site-directed mutagenesis to demonstrate that the intact active site is necessary for motility stimulation. In fact, a single amino acid, T210, in the catalytic site is necessary for motility stimulation, phosphodiesterase activity, and auto-phosphorylation by ATX. The mutant recombinant proteins, A210-ATX and D210-ATX, lack motility stimulation and both enzymatic activities, while S210-ATX possesses intermediate activities. By demonstrating that the phosphodiesterase active site is linked to motility stimulation, these data opened up the possibility that extracellular enzymatic cascades play a role in regulation of cellular motility.

Northern analysis and reverse-transciptase-PCR amplication of mRNA from the mouse fibroblastic cell line, NIH3T3, indicated that these cells do not synthesize ATX. Dr. Suk Woo Nam transfected ATX cDNA into both parental (clone 7) and ras-tranformed NIH3T3 cells. In Matrigel invasion assays, the subclones that secreted ATX were found to be more invasive, though secretion of gelatinase activity was unchanged. In vivo studies with athymic nude mice demonstrated that atx-transfected NIH3T3 cells were weakly tumorigenic with rare experimental metastases. However, combination of ATX expression with ras transformation resulted in greatly amplified tumorigenesis and metastatic potential compared to ras-tranformed controls. This suggests that ATX augments tumor aggressiveness. In addition, the ATX-secreting tumors were found to be more vascular than appropriate control tumors. We utilized in vivo matrigel plug assays in which protein was mixed with Matrigel prior to injection into the flanks of Balb/c mice to assay for angiogensis. Purified ATX resulted in new blood vessel formation that was comparable to that stimulated by VEGF. ATX was only a weak chemoattractant for HUVECs as well as for human skin or lung microvascular cells, though the protein strongly stimulated motility in coronary artery smooth muscle cells. However, ATX stimulated tubule formation on HUVECs grown on Matrigel and ATX expression in HUVECs was found to be up-regulated by basic FGF. Taken together, these results imply that ATX might contribute to the metastatic cascade through multiple mechanisms, perhaps by supporting an invasive microenvironment for both normal and tumor cells.

Recent Publications

• Nam, S.W., et al. Cancer Res 2001:61:6938-44.

• Nam, S.W., et al. Oncogene 2000:19:241-7.

• Clair, T, et al. J Biol Chem 1997; 272:996-1001.

• Lee, HY, et al. J Biol Chem 1996; 271:24408-12.

• Murata, J, et al. J Biol Chem 1994; 269:30479-84.

• Stracke, ML, et al. J Biol Chem 1992; 267:2524-9.