May Play a Role in the Development of Brain Tumors Cells : Evidence That the Novel PDGF-C and PDGF-D Ligands Regulates Survival and Mitogenic Pathways in Glioblastoma Platelet-derived Growth Factor (PDGF) Autocrine Signaling

Glioblastoma multiforme, the most common form of malignant brain tumor, is resistant to all forms of therapy and causes death within 9–12 months of diagnosis. Glioblastomas are known to contain numerous genetic and physiological alterations affecting cell survival and proliferation; one of the most common alterations being platelet-derived growth factor (PDGF) autocrine signaling characterized by coexpression of PDGF and its receptor. The PDGF family consists of four members, PDGF-A, -B, -C, and -D, that signal through the and PDGF receptor (PDGFR) tyrosine kinases. Numerous studies have demonstrated expression of PDGF-A, PDGF-B, and the PDGFRs in gliomablastomas, but such studies have not been conducted for the newly identified PDGF-C and -D. Therefore, we examined the expression of all PDGF ligands and receptors in 11 glioma cell lines and 5 primary glioblastoma tumor tissues by quantitative reverse transcription-PCR. Expression of PDGF/PDGFR pairs that are known to functionally interact were identified in all of the samples. Interestingly, PDGF-C expression was ubiquitous in brain tumor cells and tissues but was very low or absent in normal adult and fetal brain. PDGF-D was expressed in 10 of 11 brain tumor cell lines and 3 of 5 primary brain tumor samples. As a strategy for blocking PDGFR signaling, CT52923, a potent selective small molecule piperazinyl quinazoline kinase inhibitor of the PDGFR, was identified. In model systems using NIH/3T3 cells, CT52923 blocked PDGF autocrine-mediated phosphorylation of PDGFR, Akt, and mitogen-activated protein kinase (MAPK), while having no effect on v-fms or V12-ras-mediated Akt or extracellular signal-regulated protein kinase (Erk) phosphorylation. More importantly, p.o. administration of CT52923 to nude mice caused a significant 61% reduction (P < 0.006) in tumor growth of NIH/3T3 cells transformed by PDGF, whereas tumor formation by cells expressing v-fms was unaffected. We next characterized PDGF autocrine signaling in five glioblastoma cell lines. In all of the cases, PDGF autocrine signaling was evident because treatment with 1–10 M CT52923 inhibited PDGFR autophosphorylation when present at a detectable level and blocked downstream Akt and/or Erk phosphorylation. The functional significance of PDGF autocrine signaling in these cells was demonstrated by the fact that the CT52923 inhibited soft agar colony formation, and, when given p.o. to nude mice, it effectively reduced tumor formation by 44% (P < 0.0019) after s.c. injection of C6 glioblastoma cells. This study of glioblastoma cells and primary tissues is the first to implicate PDGF-C and -D in brain tumor formation and confirms the existence of autocrine signaling by PDGF-A and -B. More importantly, treatment with the PDGFR antagonist CT52923 inhibited survival and/or mitogenic pathways in all of the glioblastoma cell lines tested and prevented glioma formation in a nude mouse xenograft model. Together these findings demonstrate the potential therapeutic utility of this class of compounds for the treatment of glioblastoma. INTRODUCTION Approximately 17,500 primary central nervous system tumors occur annually in the United States. The majority of these are malignant gliomas that are among the most aggressive and highly invasive of human cancers (1). After diagnosis of glioblastoma multiforme, the median survival time of 9–12 months has remained unchanged despite multiple clinical trials designed to optimize radiation and/or chemotherapy (2). These cytotoxic treatment strategies kill cells by damaging DNA or by disrupting pathways required for cell division but do not target the underlying oncogenic defects in signal transduction that are now being more fully defined. These genetic alterations affecting genomic stability, cell cycle progression, and cell survival pathways begin to be observed in low-grade astrocytomas and become more frequent in the more advanced anaplastic astrocytomas and glioblastoma multiforme (3). One of the most common defects observed in brain tumors at all stages is the establishment of a putative PDGF-autocrine loop attributable to the coexpression of PDGF and its receptor (4–6). The importance of PDGF signaling in brain tumors is underscored by the fact that retrovirus-mediated expression of PDGF-B in the brain of neonatal mice results in the formation of astrocytomas (7). The PDGF family consists of four members, PDGF-A, -B, -C, and -D, which signal through the and PDGF-receptor (PDGFR) tyrosine kinases. Biosynthesis and processing of the PDGFs results in the formation of full-length disulfide-linked homodimers PDGF-AA, BB, CC, and DD and the heterodimer PDGF-AB (reviewed in Ref. 8). Although PDGF-AA, BB, and AB undergo additional processing it is not required for their biological activity. In contrast, PDGF-CC and -DD require proteolytic cleavage for activity (9–12). The PDGF-A and -C chains selectively bind PDGFR, whereas PDGF-D preferentially binds PDGFR, and PDGF-B displays similar affinity for both receptors (8–12). Numerous studies have demonstrated expression of PDGF-A, PDGF-B, and the PDGFRs in glioblastomas, but such studies have not been conducted for the newly identified PDGF-C and -D. Here, we demonstrate that these novel PDGFs are routinely expressed in glioma cell lines and in primary glioblastoma tissues along with their cognate PDGFR, which indicates a potential role in the development of brain tumors. Because of the numerous genetic and physiological alterations observed in human glioma, the relative importance of PDGF signaling is not fully understood. To demonstrate a causative role, reversion of the transformed phenotype of glioblastoma cells or tumor growth has been achieved with antibodies that neutralize PDGF, with dominant negative mutants of PDGF or PDGFR or with the small molecule PDGFR kinase inhibitor STI571 (13–17). Recently, we identified CT52923, a potent selective small molecule antagonist of the PDGFR that effectively inhibits PDGF-induced cell proliferation and migration in cultured cells and the in vivo PDGF-mediated response to Received 1/17/02; accepted 5/2/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at Millennium Pharmaceuticals, Inc., 256 East Grand Avenue, South San Francisco, CA 94080. Phone: (650) 244-6832; Fax: (650) 244-9208; E-mail: [email protected]. 2 Present address: Dartmouth-Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756. 3 The abbreviations used are: PDGF, platelet-derived growth factor; MAPK, mitogenactivated protein kinase; ERK, extracellular signal-regulated protein kinase; PI3k, phosphatidylinositol 3 -kinase; Cyc, cyclophilin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ECL, enhanced chemiluminescence; RT-PCR, reverse transcription-PCR.