Scientific Report 2007

Molecular and Experimental Medicine

Division of Molecular Oncology

Pleiotrophin: A Cytokine With Critical Roles in Growth and in Development and Progression of Human Neoplasms

T.F. Deuel, Y. Chang, P. Perez-Pinera, W. Zhang

We previously identified and cloned pleiotrophin, an 18-kD cytokine with diverse roles in normal growth and in the development and progression of malignant tumors. Pleiotrophin signals by inactivating the receptor protein tyrosine phosphatase (RPTP) β/ζ. Inactivation of RPTPβ/ζ leads to increased levels of tyrosine phosphorylation of the substrates of RPTPβ/ζ by unknown tyrosine kinases that phosphorylate the same sites that normally are dephosphorylated by RPTPβ/ζ in cells not stimulated with pleiotrophin. Known substrates of RPTPβ/ζ include β-catenin, β-adducin, Fyn, histone deacetylase 2, anaplastic lymphoma kinase (ALK), and TrkA, the receptor of nerve growth factor. Through this unique signaling mechanism, pleiotrophin regulates levels of tyrosine phosphorylation of important proteins in different cellular systems.

An Alternative Mechanism of Receptor Tyrosine Kinase Activation

Receptor protein tyrosine kinases such as the platelet-derived growth factor (PDGF) receptor undergo ligand-enforced dimerization and induce conformational changes in the active-site domains of the receptor that initiate autophosphorylation and autoactivation. In the past year, we found that pleiotrophin stimulates tyrosine phosphorylation of ALK through a mechanism entirely different from that of PDGF with the PDGF receptor; the pleiotrophin-stimulated tyrosine phosphorylation and activation of ALK are the consequence of the pleiotrophin-enforced dimerization of its cognate receptor, RPTPβ/ζ, and occurs without a direct interaction of pleiotrophin with the extracellular domain of ALK. We found that chemically enforced dimerization of RPTPβ/ζ alone is sufficient to stimulate tyrosine phosphorylation of ALK, that ALK is a substrate of RPTPβ/ζ, and that ALK phosphorylated through enforced dimerization of RPTPβ/ζ is dephosphorylated by RPTPβ/ζ at the same site in ALK that is phosphorylated when RPTPβ/ζ is inactivated through enforced dimerization of RPTPβ/ζ. We also showed that pleiotrophin activates the tyrosine kinase activity of ALK in pleiotrophin-stimulated cells and that the activated ALK is the kinase that phosphorylates β-catenin in pleiotrophin-stimulated cells. The site phosphorylated in β-catenin by ALK furthermore is recognized and dephosphorylated by RPTPβ/ζ.

These studies suggest a model in which in unstimulated cells, the tyrosine phosphatase activity of RPTPβ/ζ regulates the steady-state levels of tyrosine phosphorylation of ALK. The levels of expression of pleiotrophin thus critically regulate and determine the relative catalytic activity of RPTPβ/ζ through the degree to which pleiotrophin enforces dimerization and thus inactivation of RPTPβ/ζ. In pleiotrophin-stimulated cells, inactivation of RPTPβ/ζ permits increased tyrosine phosphorylation of ALK that is not restrained by the tyrosine phosphatase activity of RPTPβ/ζ. These findings thus suggest that the levels of expression of pleiotrophin regulate the steady-state levels of the tyrosine phosphatase activity of RPTPβ/ζ, which, in turn, regulates its steady-state levels of tyrosine phosphorylation of ALK and, presumably, regulates the steady-state catalytic activity. We have also identified an alternative mechanism of activation of receptor protein tyrosine kinases.

Our results also indicate that β-catenin is a downstream target of ALK. This tyrosine phosphorylation site in β-catenin is potentially important, because when it is phosphorylated in pleiotrophin-stimulated cells, it disrupts the association of β-catenin with N-cadherin needed for cells to adhere to each other. Because disruption of homophilic cell-cell adhesion is characteristic of highly malignant cells that express Ptn, the gene for pleiotrophin, our data suggest that one mechanism through which pleiotrophin stimulates a more aggressive phenotype in malignant cells is disruption of normal cytoskeletal architecture.

Breast Cancer

In previous studies of pleiotrophin in neoplasia, we used a dominant-negative Ptn and found that it reversed the malignant phenotype of human breast cancer cells in vitro and in vivo. We have now found that pleiotrophin signaling cooperates with the signaling pathways stimulated by PyMT, the gene for polyoma virus middle T antigen, driven by the mouse mammary tumor virus (MMTV) promoter in a well-established mammary tumor model. MMTV-Ptn expression in MMTV-PyMT-Ptn transgenic mice induced rapid growth of morphologically identified foci of "scirrhous" carcinoma; tumor angiogenesis; remodeling of the microenvironment; and striking increases in mouse protocollagens Iα2, IVα5, and XIα1 and in tropoelastin in the breast cancers of MMTV-PyMT-Ptn mice compared with breast cancers of MMTV-PyMT mice. The data establish that inappropriate expression of Ptn promotes breast cancer progression to a tumor stage with characteristics of the most advanced and dangerous of human breast cancers. These findings add to the growing recognition of the roles of stromal cells and paracrine signaling in the progression of tumors.

Midkine also signals through RPTPβ/ζ. To establish the relevance of the studies in MMTV-PyMT-Ptn transgenic mice to human breast cancer, we showed that each of the components of the pleiotrophin-midkine/RPTPβ/ζ-ALK signaling pathway, that is, pleiotrophin, midkine, RPTPβ/ζ, and ALK, is expressed in human breast cancers, suggesting this pathway may be relevant in the pathogenesis of human breast cancer. Surprisingly, the immunohistochemical patterns of RPTPβ/ζ and ALK in breast cancers relative to normal breast tissue were different in each of the breast cancers studied, supporting the possibility that this pathway may be important in the pathogenesis of breast cancer. Although the basis of the different patterns of RPTPβ/ζ and ALK are unknown, the data suggest that ALK may be constitutively activated through RPTPβ/ζ and thus may be a major factor in the pathogenesis of human breast cancer.

Publications

Chang, Y., Berenson, J.R., Wang, Z., Deuel, T.F. Dominant negative pleiotrophin induces tetraploidy and aneuploidy in U87MG human glioblastoma cells. Biochem. Biophys. Res. Commun. 351:336, 2006.

Chang, Y., Zuka, M., Perez-Pinera, P., Astudillo, A., Mortimer, J., Berenson, J.R., Deuel, T.F. Secretion of pleiotrophin stimulates breast cancer progression through remodeling of the tumor microenvironment. Proc. Natl. Acad. Sci. U. S. A. 104:10888, 2007.

Chen, H., Gordon, M.S., Campbell, R.A., Li, M., Wang, C.S., Lee, H.J., Sanchez, E., Manyak, S.J., Gui, D., Shalitin, D., Said, J., Chang, Y., Deuel, T.F., Baritaki, S., Bonavida, B., Berenson, J.R. Pleiotrophin is highly expressed by myeloma cells and promotes myeloma tumor growth. Blood 110:287, 2007.

Deuel, T.F., Chang, Y. Growth factors. In: Principles of Tissue Engineering, 3rd ed. Lanza, R., Langer, R., Vacanti, J.P. (Eds.). Elsevier, Philadelphia, 2007, p. 193.

Ezquerra, L., Perez-Garcia, C., Garrido, E., Diez-Fernandez, C., Deuel, T.F., Alguacil, L.F., Herradon, G. Morphine and yohimbine regulate midkine gene expression in the rat hippocampus. Eur. J. Pharmacol. 557:147, 2007.

Perez-Pinera, P., Alcantara, S., Dimitrov, T., Vega, J.A., Deuel, T.F. Pleiotrophin disrupts calcium-dependent homophilic cell-cell adhesion and initiates an epithelial-mesenchymal transition. Proc. Natl. Acad. Sci. U. S. A. 103:17795, 2006.

Perez-Pinera, P., Chang, Y., Astudillo, A., Mortimer, J., Deuel, T.F. Anaplastic lymphoma kinase is expressed in different subtypes of human breast cancer. Biochem. Biophys. Res. Commun. 358:399, 2007.

Perez-Pinera, P., Garcia-Suarez, O., Menendez-Rodriguez, P., Mortimer, J., Chang, Y., Astudillo, A., Deuel, T.F. The receptor protein tyrosine phosphatase (RPTP)β/ζ is expressed in different subtypes of human breast cancer. Biochem. Biophys. Res. Commun. 362:5, 2007.

Perez-Pinera, P., Zhang, W., Chang, Y., Vega, J.A., Deuel, T.F. Anaplastic lymphoma kinase (ALK) is activated through the pleiotrophin (PTN)/receptor protein tyrosine phosphatase (RPTP)β/ζ signaling pathway: an "alternative mechanism of receptor tyrosine kinase (RTK) activation." J. Biol. Chem., in press.