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The protein tyrosine phosphatase ζ/receptor-type protein tyrosine phosphatase β (PTPζ/RPTPβ) and its ligand pleiotrophin (PTN) are overexpressed in human glioblastomas. Both molecules are involved in neuronal cell migration during CNS development. In addition, PTN can induce glioma cell migration which is at least in part mediated through binding to PTPζ/RPTPβ. To study the relevance of this ligand–receptor pair for glioma growth in vitro and in vivo, we transfected the human glioblastoma cell line U251-MG with small interfering RNA (siRNA) directed against PTPζ/RPTPβ. Stable siRNA transfection resulted in strong down-regulation of PTPζ/RPTPβ expression. When injected subcutaneously into nude mice, clones that expressed normal levels of PTPζ/RPTPβ (PTPζ + clones) formed exponentially growing tumours, whereas tumour growth was almost completely abrogated for clones that expressed reduced PTPζ/RPTPβ levels (PTPζ – clones). Similar results were obtained using an orthotopic intracerebral model. Proliferation of PTPζ – cells in vitro was significantly reduced compared with that of control clones. Matrix-immobilized PTN stimulated the proliferation of PTPζ + cells but not of PTPζ – cells. Haptotactic migration induced by PTN was reduced for PTPζ – clones compared with control clones. Our findings suggest that antagonization of PTPζ/RPTPβ expression can inhibit glioma growth in vivo and may thus represent a potentially promising treatment strategy.
The protein tyrosine phosphatase ζ/receptor-type protein tyrosine phosphatase β (PTPζ/RPTPβ) and its ligand pleiotrophin (PTN) are overexpressed in human glioblastomas (Muller et al. 2003; Ulbricht et al. 2003). PTPζ/RPTPβ is a member of a family of receptor-type transmembrane protein tyrosine phosphatases (Krueger and Saito 1992; Levy et al. 1993). Several different splice variants of human PTPζ/RPTPβ have been described, including a transmembrane long form, a truncated transmembrane short form, and a secreted form termed phosphacan. PTPζ/RPTPβ is predominantly expressed in the CNS. Interestingly, highest levels in both the developing and adult mouse brain occur in regions that have the greatest mitotic potential, such as the embryonic ventricular and subventricular zones, the dentate gyrus, and the subependymal layer of the anterior horn of the lateral ventricle in adult brain (Levy et al. 1993). The transmembrane forms of PTPζ/RPTPβ are mainly localized on migrating neurones during brain development (Maeda and Noda 1998). In addition, radial glial cells express high levels of PTPζ/RPTPβ (Canoll et al. 1993).
Several different ligands can bind to the extracellular domain of PTPζ/RPTPβ, including the growth factors PTN and the closely related midkine (MK), the extracellular matrix molecules tenascin-C and -R, the neuronal cell adhesion molecules contactin, Nr-CAM, neural cell adhesion molecule, L1/Ng-CAM, and TAG-1/axonin-1, as well as amphoterin and fibroblast growth factor-2 (FGF-2) (Peles et al. 1998). Interactions of PTPζ/RPTPβ with neuronal adhesion molecules have been implicated in glial–neuronal interaction and neuronal migration during development. Studies on knockout mice have shown that PTPζ/RPTPβ is also important for myelinization (Harroch et al. 2000, 2002). Interactions of PTPζ/RPTPβ with PTN may be relevant for tumour progression, as PTN can stimulate tumour growth and angiogenesis (Fang et al. 1992; Chauhan et al. 1993). In addition, ribozyme targeting of PTN has been shown to inhibit the growth of gliomas in nude mice (Grzelinski et al. 2005).
Previous findings suggest that PTN can stimulate haptotactic glioma cell migration in vitro, and that up-regulated expression of PTPζ/RPTPβ and PTN in human astrocytic tumour cells may create an autocrine loop that is important for glioma cell migration (Ulbricht et al. 2003; Muller et al. 2004). In the present study we used small interfering RNA (siRNA) transfection to analyse the relevance of PTPζ/RPTPβ to glioma growth in vivo. We performed additional in vitro studies to determine the effects of down-regulated PTPζ/RPTPβ expression on cell proliferation and migration.
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This study has shown that RNA interference targeting PTPζ/RPTPβ can inhibit glioblastoma growth in vivo. Mechanisms of this antitumour effect appear to include inhibition of tumour cell proliferation as well as migration.
Previous in vitro studies suggested that PTPζ/RPTPβ is mainly involved in glioma cell migration. Although PTN is a secreted growth factor it has little chemotactic activity for glioma cells but stimulates haptotactic migration up to 200-fold (Ulbricht et al. 2003). PTN-induced haptotactic migration of C6 rat glioma cells could specifically be inhibited by an PTPζ/RPTPβ antibody which, however, only recognized rat PTPζ/RPTPβ and not the human form (Ulbricht et al. 2003). The present study showed that the PTN-induced migration of human glioma cells was reduced by 51.7–76.3% when PTPζ/RPTPβ expression was down-regulated. Importantly, no such inhibition of migration was observed when collagen or fibronectin was used as haptoattractant, suggesting that stimulation of migration is specifically mediated by the interaction between PTN and PTPζ/RPTPβ.
Proliferation was also inhibited in cells that expressed reduced levels of PTPζ/RPTPβ. In vivo, the fraction of proliferating cells was less than 3% in all tumours derived from s.c. or i.c. PTPζ – clones, whereas it was greater than 40% in PTPζ + tumours. In vitro, proliferation of PTPζ – cells was reduced by 55.7–89.6% after 9 days of growth. The effect of PTPζ/RPTPβ down-regulation on cell proliferation was to some extent unexpected as we and others were previously unable to detect effects of PTN on glioma cell proliferation in vitro (Mentlein and Held-Feindt 2002; Muller et al. 2003). Interestingly, Lu et al. recently reported that PTN exists in an 18-kDa form and a 15-kDa form, the latter of which is most probably generated by post-translational cleavage of the longer form (Lu et al. 2005). They further showed that the 18-kDa form stimulates haptotactic glioma cell migration by binding PTPζ/RPTPβ, whereas the 15-kDa form stimulates proliferation by binding anaplastic lymphoma kinase (ALK), another PTN receptor. However, previous studies by other groups demonstrated that also the long form of PTN can the have mitogenic and transformic activity (Zhang et al. 1999; Bernard-Pierrot et al. 2001). Differences in protein folding or modifications resulting from diverse strategies of PTN purification or recombinant production are thought to account for the different functional effects.
In the present study we analysed the effect of matrix-immobilized PTN on glioma cell proliferation. Interestingly, when PTPζ + cells were grown on a PTN-coated surface they proliferated at a significantly faster rate than on plastic. This effect is most likely mediated through PTPζ/RPTPβ because (i) the PTPζ + cells but not the PTPζ – cells proliferated at an increased rate on PTN, and (ii) U251-MG cells do not express ALK mRNA (as analysed by RT–PCR, unpublished observation, Ulbricht, U.). It is possible that matrix-immobilized PTN assumes a solid-phase conformation that is capable of triggering mitogenic signals via PTPζ/RPTPβ, whereas PTN in solution lacks this property. Alternatively, binding of U251-MG cells to PTN-coated surfaces may not directly induce mitogenic signalling, but attachment and spreading on PTN may interfere with other signalling pathways, rendering the cells less responsive to other mitogenic signals, and/or causing alterations in cytoskeletal organization that affect cell cycling.
The mitogenic effect of immobilized PTN, however, cannot explain why PTPζ – clones proliferated significantly slower on plastic than PTPζ + clones. Although it is possible that some of the PTN secreted by the U251-MG cells was adsorbed on to the culture plate during the assay and could function in a similar fashion as precoated PTN, such an effect should then also be expected in assays in which recombinant 18-kDa PTN in solution was added to the cells. It is therefore likely that other PTPζ/RPTPβ ligands also contributed to the stimulation of proliferation of U251-MG cells. Several other PTPζ/RPTPβ ligands with mitogenic effects have been described, including MK, which is closely related to PTN, FGF-2, amphoterin and tenascin-C. The MK protein is also expressed by U251-MG cells (unpublished observation, Ulbricht, U.); MK expression is known to be increased in human malignant gliomas (Mishima et al. 1997; Kato et al. 1999), and MK can stimulate the growth of various tumours (Takei et al. 2005). Amphoterin is also expressed by glioma cells (Punnonen et al. 1999) and can stimulate the growth of tumours in mice (Taguchi et al. 2000; Huttunen and Rauvala 2004). FGF-2 is expressed by glioma cells including the U251-MG cell line (unpublished observation, Ulbricht, U.); FGF-2 can stimulate the growth of various tumours, and treatment with a monoclonal FGF-2 antibody was shown to inhibit glioma growth in vivo (Stan et al. 1995). Finally, tenascin-C is strongly expressed in human malignant gliomas and can stimulate glioma cell proliferation and angiogenesis (Herold-Mende et al. 2002; Zagzag et al. 2002; Ruiz et al. 2004). However, little is known about the interactions between PTPζ/RPTPβ and these ligands, and the picture is further complicated by the fact that all four ligands have alternative receptors: MK can also bind to ALK, amphoterin can bind to the receptor for advanced glycation end-products (RAGE), FGF-2 binds to the FGF receptors 1–4, and tenascin-C binds to integrins and annexin II. Further research is required to elucidate the complex interactions between the components of this multiligand–multireceptor conglomerate.
Little is known about the intracellular binding partners of PTPζ/RPTPβ and its signalling pathways. The involvement of a phosphatase in glioma formation and/or progression may seem unexpected. Phosphorylation cascades usually have stimulatory effects on tumour cell proliferation whereas phosphatases are generally assumed to antagonize such pathways, a well known example being the pro-apoptotic phosphatase and tensin homolog (PTEN). However, phosphatases are not always tumour suppressive; for example, the phosphatase PTPα can dephosphorylate and thereby activate kinases of the src family, which leads to cell transformation (Zheng et al. 1992). Thus, depending on the dephosphorylation target and the signalling pathway, phosphatases can also have oncogenic activity. Meng et al. reported that PTPζ/RPTPβ interacts with β-catenin (Meng et al. 2000). PTN dephosphorylates the constitutively active PTPζ/RPTPβ, which upon binding PTN dimerizes and becomes inactivated, resulting in an increase in tyrosine-phosphorylated β-catenin. Tyrosine phosphorylation of β-catenin is known to cause loss of cell–cell adhesion. It further leads to increased levels of cytoplasmic β-catenin which translocates to the nucleus where it binds to the transcription factor TCF/LEF; this causes increased expression of MYC and Cyclin D1, stimulating cell cycling and proliferation (Nelson and Nusse 2004). Activation of this pathway could explain the mitogenic effects of immobilized PTN on PTPζ + glioma cells in our study. In addition, a recent report suggested that the nuclear factor-κB signalling pathway can also be activated through PTPζ/RPTPβ (Lorente et al. 2005).
To conclude, we showed that siRNA transfection targeting PTPζ/RPTPβ can inhibit glioma growth in vivo. Given the strong up-regulation of both PTPζ/RPTPβ in human malignant gliomas, antagonization of PTPζ/RPTPβ expression and/or signalling may be a promising strategy to inhibit the growth of these tumours. This approach appears particularly promising as multiple PTPζ/RPTPβ ligands with mitogenic and motogenic properties, including PTN, MK, FGF-2, amphoterin and tenascin-C, are also simultaneously expressed by glioma cells.