Russell O. Pieper

BACKGROUND: Protein tyrosine kinases are important regulators of cellular homeostasis with tightly controlled catalytic activity. Mutations in kinase-encoding genes can relieve the autoinhibitory constraints on kinase activity, can promote malignant transformation, and appear to be a major determinant of response to kinase inhibitor therapy. Missense mutations in the EGFR kinase domain, for example, have recently been identified in patients who showed clinical responses to EGFR kinase inhibitor therapy. METHODS AND FINDINGS: Encouraged by the promising clinical activity of epidermal growth factor receptor (EGFR) kinase inhibitors in treating glioblastoma in humans, we have sequenced the complete EGFR coding sequence in glioma tumor samples and cell lines. We identified novel missense mutations in the extracellular domain of EGFR in 13.6% (18/132) of glioblastomas and 12.5% (1/8) of glioblastoma cell lines. These EGFR mutations were associated with increased EGFR gene dosage and conferred anchorage-independent growth and tumorigenicity to NIH-3T3 cells. Cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR kinase inhibitors. CONCLUSIONS: Our results suggest extracellular missense mutations as a novel mechanism for oncogenic EGFR activation and may help identify patients who can benefit from EGFR kinase inhibitors for treatment of glioblastoma.

Temozolomide (TMZ) is a DNA-methylating agent that has recently been introduced into Phase II and III trials for the treatment of gliomas. TMZ produces O6-methylguanine in DNA, which mispairs with thymine during the next cycle of DNA replication. Subsequent futile cycles of DNA mismatch repair can lead to a p53-associated apoptotic cell death, although this mechanism has been described mostly in hematopoietic neoplasms. We studied the action of TMZ in gliomas and the role p53 might play by using U87 glioma cells that were either p53-wild-type or p53-deficient (by virtue of expression of the viral oncoprotein E6). LN-Z308 cells, in which p53 gene is deleted, were also used. p53-proficient U87 MG cells underwent a prolonged, p53- and p21(Waf1/Cip1)-associated G2-M arrest beginning 2 days after TMZ treatment. Although very few of these cells underwent apoptosis, most underwent senescence over a 10-day period. p53-deficient (E6-transfected U87 and LN-Z308) cells similarly underwent G2-M arrest in response to TMZ, but this arrest was accompanied by only minor changes in p53 or p21(Waf1/Cip1) and was reversed within 7 days of TMZ treatment in association with the appearance of cells with either 8n or subG1 DNA content. These results suggest that glioma cells respond to TMZ by undergoing G2-M arrest. p53 is not necessary for this G2-M arrest to occur but is important in the duration of G2-M arrest and in the ultimate fate of TMZ-treated cells. Therefore, the integrity of the G2-M cell cycle checkpoint may be important in the cytotoxicity of TMZ in glioma cells.

We have previously proposed that intravascular thrombosis and subsequent vasoocclusion contribute to the development of pseudopalisading necrosis, a pathologic hallmark that distinguishes glioblastoma (WHO grade 4) from lower grade astrocytomas. To better understand the potential prothrombotic mechanisms underlying the formation of these structures that drive tumor angiogenesis, we investigated tissue factor (TF), a potent procoagulant protein known to be overexpressed in astrocytomas. We hypothesized that PTEN loss and tumor hypoxia, which characterize glioblastoma but not lower grade astrocytomas, could up-regulate TF expression and cause intravascular thrombotic occlusion. We examined the effect of PTEN restoration and hypoxia on TF expression and plasma coagulation using a human glioma cell line containing an inducible wt-PTEN cDNA. Cell exposure to hypoxia (1% O(2)) markedly increased TF expression, whereas restoration of wt-PTEN caused decreased cellular TF. The latter effect was at least partially dependent on PTEN's protein phosphatase activity. Hypoxic cells accelerated plasma clotting in tilt tube assays and this effect was prevented by both inhibitory antibodies to TF and plasma lacking factor VII, implicating TF-dependent mechanisms. To further examine the genetic events leading to TF up-regulation during progression of astrocytomas, we investigated its expression in a series of human astrocytes sequentially infected with E6/E7/human telomerase, Ras, and Akt. Cells transformed with Akt showed the greatest incremental increase in hypoxia-induced TF expression and secretion. Together, our results show that PTEN loss and hypoxia up-regulate TF expression and promote plasma clotting by glioma cells, suggesting that these mechanisms may underlie intravascular thrombosis and pseudopalisading necrosis in glioblastoma.