Pleiotrophin Signaling through Anaplastic Lymphoma Kinase Is Rate-limiting for Glioblastoma Growth

Abstract

Glioblastoma multiforme is the most common highly aggressive human brain cancer, and receptor tyrosine kinases have been implicated in the progression of this malignancy. We have recently identified anaplastic lymphoma kinase (ALK) as a tyrosine kinase receptor for pleiotrophin, a secreted growth factor that is highly expressed during embryonic brain development and in tumors of the central nervous system. Here we report on the contribution of pleiotrophin-ALK signaling to glioblastoma growth. We found ALK overexpressed in human glioblastoma relative to normal brain and detected ALK mRNA in glioblastoma cell lines. We reduced the endogenous ALK in glioblastoma cells by ribozyme targeting and demonstrated that this prevents pleiotrophin-stimulated phosphorylation of the anti-apoptotic protein Akt. Furthermore, this depletion of ALK reduced tumor growth of xenografts in athymic nude mice and prolonged survival of the animals because of increased apoptosis in the tumors. These findings directly implicate ALK signaling as a rate-limiting factor in the growth of glioblastoma multiforme and suggest potential utility of therapeutic targeting of ALK. Glioblastoma multiforme is the most common highly aggressive human brain cancer, and receptor tyrosine kinases have been implicated in the progression of this malignancy. We have recently identified anaplastic lymphoma kinase (ALK) as a tyrosine kinase receptor for pleiotrophin, a secreted growth factor that is highly expressed during embryonic brain development and in tumors of the central nervous system. Here we report on the contribution of pleiotrophin-ALK signaling to glioblastoma growth. We found ALK overexpressed in human glioblastoma relative to normal brain and detected ALK mRNA in glioblastoma cell lines. We reduced the endogenous ALK in glioblastoma cells by ribozyme targeting and demonstrated that this prevents pleiotrophin-stimulated phosphorylation of the anti-apoptotic protein Akt. Furthermore, this depletion of ALK reduced tumor growth of xenografts in athymic nude mice and prolonged survival of the animals because of increased apoptosis in the tumors. These findings directly implicate ALK signaling as a rate-limiting factor in the growth of glioblastoma multiforme and suggest potential utility of therapeutic targeting of ALK. Tumors of glial origin including astrocytomas, oligodendrogliomas, and ependymomas account for almost 80% of all primary brain malignancies. Glioblastoma multiforme is both the single most common glial tumor and the most lethal with a mean survival of only 1 year despite aggressive treatment (1.Holland E.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6242-6244Crossref PubMed Scopus (545) Google Scholar). Although these tumors exhibit multiple genetic alterations, including loss or mutation of the tumor suppressors PTEN (2.Wang S.I. Puc J. Li J. Bruce J.N. Cairns P. Sidransky D. Parsons R. Cancer Res. 1997; 57: 4183-4186PubMed Google Scholar), p53 (3.Albertoni M. Daub D.M. Arden K.C. Viars C.S. Powell C. Van Meir E.G. Oncogene. 1998; 16: 321-326Crossref PubMed Scopus (39) Google Scholar), and INK4a-ARF (4.James C.D. Olson J.J. Curr. Opin. Oncol. 1996; 8: 188-195Crossref PubMed Scopus (29) Google Scholar), receptor tyrosine kinase (RTK) 1The abbreviations used are: RTKreceptor tyrosine kinaseALKanaplastic lymphoma kinaseECDextracellular domainPDGFplatelet-derived growth factorntnucleotidesPBSphosphate-buffered salinePI3-kinasephosphatidylinositol 3-kinaseMAPKmitogen-activated protein kinaseTUNELterminal deoxynucleotidyltransferase-mediated dUTP nick end-labelingCMVcytomegalovirusPTNpleiotrophin1The abbreviations used are: RTKreceptor tyrosine kinaseALKanaplastic lymphoma kinaseECDextracellular domainPDGFplatelet-derived growth factorntnucleotidesPBSphosphate-buffered salinePI3-kinasephosphatidylinositol 3-kinaseMAPKmitogen-activated protein kinaseTUNELterminal deoxynucleotidyltransferase-mediated dUTP nick end-labelingCMVcytomegalovirusPTNpleiotrophinsignaling seems to play a particularly important role in tumor development and growth. Glioblastomas and glioblastoma cell lines have been shown to overexpress the tyrosine kinase receptors for epidermal growth factor (5.Nishikawa R. Ji X.D. Harmon R.C. Lazar C.S. Gill G.N. Cavenee W.K. Huang H.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7727-7731Crossref PubMed Scopus (806) Google Scholar), platelet-derived growth factor (PDGF) (6.Nister M. Claesson-Welsh L. Eriksson A. Heldin C.H. Westermark B. J. Biol. Chem. 1991; 266: 16755-16763Abstract Full Text PDF PubMed Google Scholar), hepatocyte growth factor (7.Bowers D.C. Fan S. Walter K.A. Abounader R. Williams J.A. Rosen E.M. Laterra J. Cancer Res. 2000; 60: 4277-4283PubMed Google Scholar), nerve growth factor (8.Singer H.S. Hansen B. Martinie D. Karp C.L. J. Neurooncol. 1999; 45: 1-8Crossref PubMed Scopus (48) Google Scholar), and vascular endothelial growth factor (9.Plate K.H. Breier G. Weich H.A. Risau W. Nature. 1992; 359: 845-848Crossref PubMed Scopus (2107) Google Scholar). In addition, these tumors frequently overexpress the ligands for these RTKs, suggesting a potential role for autocrine RTK signaling in glioblastoma growth. The importance of RTK signaling is supported by the finding that the combined activation of two downstream targets of RTK signaling (Ras and Akt) in neural progenitor cells induces glioblastoma-like tumors in mice (10.Holland E.C. Celestino J. Dai C. Schaefer L. Sawaya R.E. Fuller G.N. Nat. Genet. 2000; 25: 55-57Crossref PubMed Scopus (739) Google Scholar).Pleiotrophin is a secreted heparin-binding growth factor highly expressed in the developing nervous system and down-regulated in the adult. Relative to normal brain, pleiotrophin expression is increased following acute ischemic injury (11.Yeh H.J. He Y.Y. Xu J. Hsu C.Y. Deuel T.F. J. Neurosci. 1998; 18: 3699-3707Crossref PubMed Google Scholar) and in tumors (12.Schulte A.M. Bicknell R. Lewis C.E. Ferrara N. Tumor Angiogenesis. 1. Oxford University Press, Oxford, United Kingdom1997: 273-289Google Scholar). This suggests a potential role of pleiotrophin as a tumor growth factor insofar as the reactivation of a developmentally regulated signaling pathway may provide a tumor with a powerful growth signal. In fact, pleiotrophin expression induces tumor growth and metastasis of NIH3T3 cells (13.Chauhan A.K. Li Y.S. Deuel T.F. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 679-682Crossref PubMed Scopus (139) Google Scholar) and has a rate-limiting role both as an angiogenic factor (14.Choudhuri R. Zhang H.T. Donnini S. Ziche M. Bicknell R. Cancer Res. 1997; 57: 1814-1819PubMed Google Scholar) and a tumor growth factor for different tumors including melanoma and choriocarcinoma (15.Czubayko F. Riegel A.T. Wellstein A. J. Biol. Chem. 1994; 269: 21358-21363Abstract Full Text PDF PubMed Google Scholar, 16.Czubayko F. Schulte A.M. Berchem G.J. Wellstein A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14753-14758Crossref PubMed Scopus (148) Google Scholar, 17.Schulte A.M. Lai S. Kurtz A. Czubayko F. Riegel A.T. Wellstein A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14759-14764Crossref PubMed Scopus (177) Google Scholar) (reviewed in Ref. 12.Schulte A.M. Bicknell R. Lewis C.E. Ferrara N. Tumor Angiogenesis. 1. Oxford University Press, Oxford, United Kingdom1997: 273-289Google Scholar). In addition, pleiotrophin activates both the Ras-MAPK and the PI3K-Akt signaling axes (18.Souttou B. Ahmad S. Riegel A.T. Wellstein A. J. Biol. Chem. 1997; 272: 19588-19593Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), and both pathways are implicated in glial tumorigenesis (10.Holland E.C. Celestino J. Dai C. Schaefer L. Sawaya R.E. Fuller G.N. Nat. Genet. 2000; 25: 55-57Crossref PubMed Scopus (739) Google Scholar). Recently we identified ALK, an orphan RTK, as the receptor for pleiotrophin (19.Stoica G.E. Kuo A. Aigner A. Sunitha I. Souttou B. Malerczyk C. Caughey D. Wen D. Karavanov A. Riegel A.T. Wellstein A. J. Biol. Chem. 2001; 276: 16772-16779Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). The ALK tyrosine kinase was originally discovered as a fusion protein with nucleophosmin due to a t(2;5) translocation (20.Morris S.W. Kirstein M.N. Valentine M.B. Dittmer K.G. Shapiro D.N. Saltman D.L. Look A.T. Science. 1994; 263: 1281-1284Crossref PubMed Scopus (1936) Google Scholar). This fusion resulted in constitutive activation of the intracellular ALK kinase and was shown to induce anaplastic lymphoma (20.Morris S.W. Kirstein M.N. Valentine M.B. Dittmer K.G. Shapiro D.N. Saltman D.L. Look A.T. Science. 1994; 263: 1281-1284Crossref PubMed Scopus (1936) Google Scholar). More recently, the full-length ALK receptor has been shown to be highly expressed in the developing nervous system and down-regulated postnatally (21.Iwahara T. Fujimoto J. Wen D. Cupples R. Bucay N. Arakawa T. Mori S. Ratzkin B. Yamamoto T. Oncogene. 1997; 14: 439-449Crossref PubMed Scopus (546) Google Scholar), very similarly to the expression profile of its ligand, pleiotrophin (19.Stoica G.E. Kuo A. Aigner A. Sunitha I. Souttou B. Malerczyk C. Caughey D. Wen D. Karavanov A. Riegel A.T. Wellstein A. J. Biol. Chem. 2001; 276: 16772-16779Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). Here we report that this tyrosine kinase receptor is overexpressed in human glioblastoma and is rate-limiting for the growth of a xenograft model of glioblastoma.DISCUSSIONWe recently identified ALK, an orphan RTK, as the receptor for the growth factor pleiotrophin (19.Stoica G.E. Kuo A. Aigner A. Sunitha I. Souttou B. Malerczyk C. Caughey D. Wen D. Karavanov A. Riegel A.T. Wellstein A. J. Biol. Chem. 2001; 276: 16772-16779Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). Previous studies have shown that a t(2;5) translocation can generate a fusion protein of the intracellular ALK kinase domain with nucleophosmin and that this constitutively active kinase can induce anaplastic lymphoma (20.Morris S.W. Kirstein M.N. Valentine M.B. Dittmer K.G. Shapiro D.N. Saltman D.L. Look A.T. Science. 1994; 263: 1281-1284Crossref PubMed Scopus (1936) Google Scholar). In contrast with this limited role of the nucleophosmin/ALK fusion protein for lymphoid neoplasms, the full-length ALK receptor has been shown to be expressed in the developing nervous system and by virtue of its expression pattern is implicated with nervous system physiology (21.Iwahara T. Fujimoto J. Wen D. Cupples R. Bucay N. Arakawa T. Mori S. Ratzkin B. Yamamoto T. Oncogene. 1997; 14: 439-449Crossref PubMed Scopus (546) Google Scholar). We now report that one of the highly malignant tumors of the brain, glioblastoma, overexpresses ALK, and we demonstrate that the pleiotrophin-ALK pathway is essential for tumor growth of one of the commonly used model systems, U87MG cells.Furthermore, our initial studies on the expression of ALK in cancers of epithelial origin showed ALK expression in one-third of breast cancer cell lines (7 of 18) and in a little over one-half of breast cancer tissues from patients (8 of 13). Still, the expression levels of ALK in breast cancers relative to normal tissues were not as prominent as in glioblastoma, and we thus focused the studies reported here on this latter tumor type. Interestingly, depletion of ALK from Hs578T breast cancer cells that express high levels of the ALK receptor as well as pleiotrophin significantly inhibited tumor growth in animals 2C. Powers, A. Aigner, G. E. Stoica, K. McDonnell, and A. Wellstein, unpublished data. very similar to the data reported here for the U87MG cells. This suggests a potentially rate-limiting role of ALK for tumor types other than glioblastoma that utilize this growth factor receptor pathway.As outlined above, the major pathway affected by the pleiotrophin-ALK interaction is apoptosis, and this begs the question as to whether the activity of apoptosis-inducing cytotoxic drugs might cooperate with this pathway. To test this hypothesis we studied the efficacy of cisplatinum, an alkylating agent used to treat glioblastoma, in the U87MG cell panel. Indeed, cells with reduced residual levels of endogenous ALK become more sensitive to cisplatinum-induced apoptosis.2 This suggests the potential for a positive interaction between ALK tyrosine kinase-targeted and conventional cytotoxic therapies.In the current paper, we have utilized ribozyme targeting of ALK to generate a panel of cells with a range of residual endogenous expression of this receptor tyrosine kinase. The residual receptor levels correlated with 1) pleiotrophin induction of Akt phosphorylation, 2) tumor growth, 3) median survival, and 4) apoptosis in a gene dose-dependent manner. Finally, the tumor cells became more sensitive to a cytotoxic agent, cisplatinum, upon the reduction of ALK.2 This suggests to us that drugs that specifically inhibit the pleiotrophin-stimulated ALK kinase activity hold the promise to be efficacious against tumors that utilize this growth stimulatory axis. Tumors of glial origin including astrocytomas, oligodendrogliomas, and ependymomas account for almost 80% of all primary brain malignancies. Glioblastoma multiforme is both the single most common glial tumor and the most lethal with a mean survival of only 1 year despite aggressive treatment (1.Holland E.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6242-6244Crossref PubMed Scopus (545) Google Scholar). Although these tumors exhibit multiple genetic alterations, including loss or mutation of the tumor suppressors PTEN (2.Wang S.I. Puc J. Li J. Bruce J.N. Cairns P. Sidransky D. Parsons R. Cancer Res. 1997; 57: 4183-4186PubMed Google Scholar), p53 (3.Albertoni M. Daub D.M. Arden K.C. Viars C.S. Powell C. Van Meir E.G. Oncogene. 1998; 16: 321-326Crossref PubMed Scopus (39) Google Scholar), and INK4a-ARF (4.James C.D. Olson J.J. Curr. Opin. Oncol. 1996; 8: 188-195Crossref PubMed Scopus (29) Google Scholar), receptor tyrosine kinase (RTK) 1The abbreviations used are: RTKreceptor tyrosine kinaseALKanaplastic lymphoma kinaseECDextracellular domainPDGFplatelet-derived growth factorntnucleotidesPBSphosphate-buffered salinePI3-kinasephosphatidylinositol 3-kinaseMAPKmitogen-activated protein kinaseTUNELterminal deoxynucleotidyltransferase-mediated dUTP nick end-labelingCMVcytomegalovirusPTNpleiotrophin1The abbreviations used are: RTKreceptor tyrosine kinaseALKanaplastic lymphoma kinaseECDextracellular domainPDGFplatelet-derived growth factorntnucleotidesPBSphosphate-buffered salinePI3-kinasephosphatidylinositol 3-kinaseMAPKmitogen-activated protein kinaseTUNELterminal deoxynucleotidyltransferase-mediated dUTP nick end-labelingCMVcytomegalovirusPTNpleiotrophinsignaling seems to play a particularly important role in tumor development and growth. Glioblastomas and glioblastoma cell lines have been shown to overexpress the tyrosine kinase receptors for epidermal growth factor (5.Nishikawa R. Ji X.D. Harmon R.C. Lazar C.S. Gill G.N. Cavenee W.K. Huang H.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7727-7731Crossref PubMed Scopus (806) Google Scholar), platelet-derived growth factor (PDGF) (6.Nister M. Claesson-Welsh L. Eriksson A. Heldin C.H. Westermark B. J. Biol. Chem. 1991; 266: 16755-16763Abstract Full Text PDF PubMed Google Scholar), hepatocyte growth factor (7.Bowers D.C. Fan S. Walter K.A. Abounader R. Williams J.A. Rosen E.M. Laterra J. Cancer Res. 2000; 60: 4277-4283PubMed Google Scholar), nerve growth factor (8.Singer H.S. Hansen B. Martinie D. Karp C.L. J. Neurooncol. 1999; 45: 1-8Crossref PubMed Scopus (48) Google Scholar), and vascular endothelial growth factor (9.Plate K.H. Breier G. Weich H.A. Risau W. Nature. 1992; 359: 845-848Crossref PubMed Scopus (2107) Google Scholar). In addition, these tumors frequently overexpress the ligands for these RTKs, suggesting a potential role for autocrine RTK signaling in glioblastoma growth. The importance of RTK signaling is supported by the finding that the combined activation of two downstream targets of RTK signaling (Ras and Akt) in neural progenitor cells induces glioblastoma-like tumors in mice (10.Holland E.C. Celestino J. Dai C. Schaefer L. Sawaya R.E. Fuller G.N. Nat. Genet. 2000; 25: 55-57Crossref PubMed Scopus (739) Google Scholar). receptor tyrosine kinase anaplastic lymphoma kinase extracellular domain platelet-derived growth factor nucleotides phosphate-buffered saline phosphatidylinositol 3-kinase mitogen-activated protein kinase terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling cytomegalovirus pleiotrophin receptor tyrosine kinase anaplastic lymphoma kinase extracellular domain platelet-derived growth factor nucleotides phosphate-buffered saline phosphatidylinositol 3-kinase mitogen-activated protein kinase terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling cytomegalovirus pleiotrophin Pleiotrophin is a secreted heparin-binding growth factor highly expressed in the developing nervous system and down-regulated in the adult. Relative to normal brain, pleiotrophin expression is increased following acute ischemic injury (11.Yeh H.J. He Y.Y. Xu J. Hsu C.Y. Deuel T.F. J. Neurosci. 1998; 18: 3699-3707Crossref PubMed Google Scholar) and in tumors (12.Schulte A.M. Bicknell R. Lewis C.E. Ferrara N. Tumor Angiogenesis. 1. Oxford University Press, Oxford, United Kingdom1997: 273-289Google Scholar). This suggests a potential role of pleiotrophin as a tumor growth factor insofar as the reactivation of a developmentally regulated signaling pathway may provide a tumor with a powerful growth signal. In fact, pleiotrophin expression induces tumor growth and metastasis of NIH3T3 cells (13.Chauhan A.K. Li Y.S. Deuel T.F. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 679-682Crossref PubMed Scopus (139) Google Scholar) and has a rate-limiting role both as an angiogenic factor (14.Choudhuri R. Zhang H.T. Donnini S. Ziche M. Bicknell R. Cancer Res. 1997; 57: 1814-1819PubMed Google Scholar) and a tumor growth factor for different tumors including melanoma and choriocarcinoma (15.Czubayko F. Riegel A.T. Wellstein A. J. Biol. Chem. 1994; 269: 21358-21363Abstract Full Text PDF PubMed Google Scholar, 16.Czubayko F. Schulte A.M. Berchem G.J. Wellstein A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14753-14758Crossref PubMed Scopus (148) Google Scholar, 17.Schulte A.M. Lai S. Kurtz A. Czubayko F. Riegel A.T. Wellstein A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14759-14764Crossref PubMed Scopus (177) Google Scholar) (reviewed in Ref. 12.Schulte A.M. Bicknell R. Lewis C.E. Ferrara N. Tumor Angiogenesis. 1. Oxford University Press, Oxford, United Kingdom1997: 273-289Google Scholar). In addition, pleiotrophin activates both the Ras-MAPK and the PI3K-Akt signaling axes (18.Souttou B. Ahmad S. Riegel A.T. Wellstein A. J. Biol. Chem. 1997; 272: 19588-19593Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), and both pathways are implicated in glial tumorigenesis (10.Holland E.C. Celestino J. Dai C. Schaefer L. Sawaya R.E. Fuller G.N. Nat. Genet. 2000; 25: 55-57Crossref PubMed Scopus (739) Google Scholar). Recently we identified ALK, an orphan RTK, as the receptor for pleiotrophin (19.Stoica G.E. Kuo A. Aigner A. Sunitha I. Souttou B. Malerczyk C. Caughey D. Wen D. Karavanov A. Riegel A.T. Wellstein A. J. Biol. Chem. 2001; 276: 16772-16779Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). The ALK tyrosine kinase was originally discovered as a fusion protein with nucleophosmin due to a t(2;5) translocation (20.Morris S.W. Kirstein M.N. Valentine M.B. Dittmer K.G. Shapiro D.N. Saltman D.L. Look A.T. Science. 1994; 263: 1281-1284Crossref PubMed Scopus (1936) Google Scholar). This fusion resulted in constitutive activation of the intracellular ALK kinase and was shown to induce anaplastic lymphoma (20.Morris S.W. Kirstein M.N. Valentine M.B. Dittmer K.G. Shapiro D.N. Saltman D.L. Look A.T. Science. 1994; 263: 1281-1284Crossref PubMed Scopus (1936) Google Scholar). More recently, the full-length ALK receptor has been shown to be highly expressed in the developing nervous system and down-regulated postnatally (21.Iwahara T. Fujimoto J. Wen D. Cupples R. Bucay N. Arakawa T. Mori S. Ratzkin B. Yamamoto T. Oncogene. 1997; 14: 439-449Crossref PubMed Scopus (546) Google Scholar), very similarly to the expression profile of its ligand, pleiotrophin (19.Stoica G.E. Kuo A. Aigner A. Sunitha I. Souttou B. Malerczyk C. Caughey D. Wen D. Karavanov A. Riegel A.T. Wellstein A. J. Biol. Chem. 2001; 276: 16772-16779Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). Here we report that this tyrosine kinase receptor is overexpressed in human glioblastoma and is rate-limiting for the growth of a xenograft model of glioblastoma. DISCUSSIONWe recently identified ALK, an orphan RTK, as the receptor for the growth factor pleiotrophin (19.Stoica G.E. Kuo A. Aigner A. Sunitha I. Souttou B. Malerczyk C. Caughey D. Wen D. Karavanov A. Riegel A.T. Wellstein A. J. Biol. Chem. 2001; 276: 16772-16779Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). Previous studies have shown that a t(2;5) translocation can generate a fusion protein of the intracellular ALK kinase domain with nucleophosmin and that this constitutively active kinase can induce anaplastic lymphoma (20.Morris S.W. Kirstein M.N. Valentine M.B. Dittmer K.G. Shapiro D.N. Saltman D.L. Look A.T. Science. 1994; 263: 1281-1284Crossref PubMed Scopus (1936) Google Scholar). In contrast with this limited role of the nucleophosmin/ALK fusion protein for lymphoid neoplasms, the full-length ALK receptor has been shown to be expressed in the developing nervous system and by virtue of its expression pattern is implicated with nervous system physiology (21.Iwahara T. Fujimoto J. Wen D. Cupples R. Bucay N. Arakawa T. Mori S. Ratzkin B. Yamamoto T. Oncogene. 1997; 14: 439-449Crossref PubMed Scopus (546) Google Scholar). We now report that one of the highly malignant tumors of the brain, glioblastoma, overexpresses ALK, and we demonstrate that the pleiotrophin-ALK pathway is essential for tumor growth of one of the commonly used model systems, U87MG cells.Furthermore, our initial studies on the expression of ALK in cancers of epithelial origin showed ALK expression in one-third of breast cancer cell lines (7 of 18) and in a little over one-half of breast cancer tissues from patients (8 of 13). Still, the expression levels of ALK in breast cancers relative to normal tissues were not as prominent as in glioblastoma, and we thus focused the studies reported here on this latter tumor type. Interestingly, depletion of ALK from Hs578T breast cancer cells that express high levels of the ALK receptor as well as pleiotrophin significantly inhibited tumor growth in animals 2C. Powers, A. Aigner, G. E. Stoica, K. McDonnell, and A. Wellstein, unpublished data. very similar to the data reported here for the U87MG cells. This suggests a potentially rate-limiting role of ALK for tumor types other than glioblastoma that utilize this growth factor receptor pathway.As outlined above, the major pathway affected by the pleiotrophin-ALK interaction is apoptosis, and this begs the question as to whether the activity of apoptosis-inducing cytotoxic drugs might cooperate with this pathway. To test this hypothesis we studied the efficacy of cisplatinum, an alkylating agent used to treat glioblastoma, in the U87MG cell panel. Indeed, cells with reduced residual levels of endogenous ALK become more sensitive to cisplatinum-induced apoptosis.2 This suggests the potential for a positive interaction between ALK tyrosine kinase-targeted and conventional cytotoxic therapies.In the current paper, we have utilized ribozyme targeting of ALK to generate a panel of cells with a range of residual endogenous expression of this receptor tyrosine kinase. The residual receptor levels correlated with 1) pleiotrophin induction of Akt phosphorylation, 2) tumor growth, 3) median survival, and 4) apoptosis in a gene dose-dependent manner. Finally, the tumor cells became more sensitive to a cytotoxic agent, cisplatinum, upon the reduction of ALK.2 This suggests to us that drugs that specifically inhibit the pleiotrophin-stimulated ALK kinase activity hold the promise to be efficacious against tumors that utilize this growth stimulatory axis. We recently identified ALK, an orphan RTK, as the receptor for the growth factor pleiotrophin (19.Stoica G.E. Kuo A. Aigner A. Sunitha I. Souttou B. Malerczyk C. Caughey D. Wen D. Karavanov A. Riegel A.T. Wellstein A. J. Biol. Chem. 2001; 276: 16772-16779Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). Previous studies have shown that a t(2;5) translocation can generate a fusion protein of the intracellular ALK kinase domain with nucleophosmin and that this constitutively active kinase can induce anaplastic lymphoma (20.Morris S.W. Kirstein M.N. Valentine M.B. Dittmer K.G. Shapiro D.N. Saltman D.L. Look A.T. Science. 1994; 263: 1281-1284Crossref PubMed Scopus (1936) Google Scholar). In contrast with this limited role of the nucleophosmin/ALK fusion protein for lymphoid neoplasms, the full-length ALK receptor has been shown to be expressed in the developing nervous system and by virtue of its expression pattern is implicated with nervous system physiology (21.Iwahara T. Fujimoto J. Wen D. Cupples R. Bucay N. Arakawa T. Mori S. Ratzkin B. Yamamoto T. Oncogene. 1997; 14: 439-449Crossref PubMed Scopus (546) Google Scholar). We now report that one of the highly malignant tumors of the brain, glioblastoma, overexpresses ALK, and we demonstrate that the pleiotrophin-ALK pathway is essential for tumor growth of one of the commonly used model systems, U87MG cells. Furthermore, our initial studies on the expression of ALK in cancers of epithelial origin showed ALK expression in one-third of breast cancer cell lines (7 of 18) and in a little over one-half of breast cancer tissues from patients (8 of 13). Still, the expression levels of ALK in breast cancers relative to normal tissues were not as prominent as in glioblastoma, and we thus focused the studies reported here on this latter tumor type. Interestingly, depletion of ALK from Hs578T breast cancer cells that express high levels of the ALK receptor as well as pleiotrophin significantly inhibited tumor growth in animals 2C. Powers, A. Aigner, G. E. Stoica, K. McDonnell, and A. Wellstein, unpublished data. very similar to the data reported here for the U87MG cells. This suggests a potentially rate-limiting role of ALK for tumor types other than glioblastoma that utilize this growth factor receptor pathway. As outlined above, the major pathway affected by the pleiotrophin-ALK interaction is apoptosis, and this begs the question as to whether the activity of apoptosis-inducing cytotoxic drugs might cooperate with this pathway. To test this hypothesis we studied the efficacy of cisplatinum, an alkylating agent used to treat glioblastoma, in the U87MG cell panel. Indeed, cells with reduced residual levels of endogenous ALK become more sensitive to cisplatinum-induced apoptosis.2 This suggests the potential for a positive interaction between ALK tyrosine kinase-targeted and conventional cytotoxic therapies. In the current paper, we have utilized ribozyme targeting of ALK to generate a panel of cells with a range of residual endogenous expression of this receptor tyrosine kinase. The residual receptor levels correlated with 1) pleiotrophin induction of Akt phosphorylation, 2) tumor growth, 3) median survival, and 4) apoptosis in a gene dose-dependent manner. Finally, the tumor cells became more sensitive to a cytotoxic agent, cisplatinum, upon the reduction of ALK.2 This suggests to us that drugs that specifically inhibit the pleiotrophin-stimulated ALK kinase activity hold the promise to be efficacious against tumors that utilize this growth stimulatory axis.