Our long term goals are to understand the molecular mechanisms underlying glioblastoma multiforme, the most common and deadly among human brain tumors. The EGF receptor has been strongly implicated and much research on its downstream signaling has focused on the ERK signaling module. Unexpectedly, our work has instead led us to study a pathway not commonly considered downstream of the EGF receptor or in tumorigenesis, the JNK pathway. We have found that 86 percent of primary glioblastoma tumors show activation of a 54 kDa JNK isoform. EGF induces strong JNK activation in 69 percent of cell lines derived from glioblastoma tumors but only weak activation was observed in 6 normal cell lines. Further work in two tumor cell lines has indicated that JNK is important for anchorage independent growth and the prevention of apoptosis. We hypothesize that an important difference between glioblastoma tumors and normal tissues is that EGF receptor signals are also directed towards the JNK pathway. The goals of this application are to further study the mechanisms by which JNK becomes activated and how it contributes to multiple phenotypes.
In Specific Aim #1, we will examine the mechanisms underlying the enhanced EGF induced activation of JNK seen in glioblastoma cell lines. Several points at which signals can be directed towards the JNK signaling module will be examined: 1) the small GTPases, Rac and Cdc42; 2) PI 3-kinase; and 3) Gab1. These molecules will be tested for increased activity/binding following EGF addition in glioblastoma cell lines as compared to normal astrocyte cell lines. Dominant negative versions of these molecules will be used to confirm their roles in JNK activation.
In Specific Aim #2, we will determine which JNK isoform the 54 kDa form corresponds to using RNase protection. Next, we will identify the mechanisms by which it became preferentially phosphorylated. Most importantly, we will determine what properties this isoform has that might contribute to tumorigenesis. The localization of the 54 kDa isoform will be studied in tumor sections and the transcription factors that bind to this isoform in tumors will also be studied.
In Specific Aim #3, we will evaluate the relative contribution of JNK and ERK to glial tumorigenesis. The notion that JNK contributes to tumorigenesis is relatively new and not well studied, especially in animal models, but there is a much more extensive literature on the contribution of ERK to tumorigenesis. Thus, we will attempt to clarify the relative contribution of these two signaling modules to these critical phenotypes in vivo: 1) tumor formation in athymic mice, 2) angiogenesis, 3) cell proliferation, and 4) prevention of apoptosis. This work will further enhance our knowledge of this novel signaling pathway in this human cancer and provide new avenues for diagnostics and therapeutics.
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