Elevated JNK activation contributes to the pathogenesis of human brain tumors Marc A Antonyak 1 , Lawrence C Kenyon 2 , Andrew K Godwin 3 , David C James 4 , David R Emlet 1 , Isamu Okamoto 1 , Mehdi Tnani 1 , Marina Holgado-Madruga 1 , David K Moscatello 5 and Albert J Wong* ,1 1 The Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania, PA 19107, USA; 2 Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, PA 19107, USA; 3 Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania, PA 19111, USA; 4 Department of Laboratory Medicine and Pathology, Mayo Clinic and Research Foundation, Rochester, Minnesota, MN 55905, USA; 5 Differentiated Cell Laboratory, Coriell Institute for Medical Research, Camden, New Jersey, NJ 08103, USA The ERK pathway is typically associated with activation of the EGF receptor and has been shown to play a major role in promoting several tumor phenotypes. An analogous signaling module, the JNK pathway, has not been shown to be consistently activated by the EGF receptor but is instead more uniformly stimulated by cellular stresses and cytokines. The function of the JNK pathway in primary tumors is unclear as it has been implicated in both promoting apoptosis and cell growth in vitro, which may be a reflection of the cell lines chosen. Primary human brain tumors frequently show overexpression of the EGF receptor. To clarify the role of JNK in tumorigenesis, we have investigated the role of JNK in a large panel of primary human brain tumors and tumor derived cell lines. Here we present evidence that JNK has a major role in promoting tumorigenesis both in vivo and in vitro. Western blot analysis demonstrated that 86% (18 of 21) of primary brain tumors showed evidence of JNK activation but only 38% (8 of 21) showed evidence of ERK activation. Kinase assays revealed that 77% of brain tumor cell lines activated JNK in response to EGF (7 of 13) or had high levels of basal activity (3 of 13), whereas none of six normal cell lines analysed, including astrocytes, had these properties. Of several growth factors examined, EGF produced the highest level of JNK induction in tumor cell lines and the duration of activation was greater than that seen for ERK. Expression of a dominant-negative (dn) form of JNK potently inhibited EGF mediated anchorage independent growth and protection from cell death in two glial tumor cell lines. These findings demonstrate that enhanced JNK activa- tion is frequently found in primary brain tumors and that this activation contributes to phenotypes related to transformation. Oncogene (2002) 21, 5038 – 5046. doi:10.1038/sj.onc. 1205593 Keywords: apoptosis; brain tumor; glioblastoma multi- forme; EGF receptor; JNK; MAP kinase Introduction The MAP kinase signaling modules, which include the ERK, JNK and p38 cascades, have an essential role in linking cell surface receptors to a variety of cellular processes. In mammalian cells, the ERK pathway is typically activated by growth factors and has a major role in cell proliferation and tumorigenesis since expression of constitutively active forms of components of this pathway can transform cells (Kyriakis, 1994; Mansour, 1994; Kyriakis, 1992). Indeed, elevated ERK activation has been associated with several human cancers (Mandell, 1998; Sivaraman et al., 1997; Xing and Imagawa, 1999). On the other hand, it is possible that some tumors may not rely on the ERK pathway for tumorigenesis. Constitutive ERK activation appears to be more common in certain types of cancer such as those of the pancreas, colon, lung, ovary, and kidney, while low frequencies were noted in tumors from the brain, stomach, and liver (Hoshino, 1999). Furthermore, expression of potent oncogenes including Tpr-Met, the EGF variant type III (EGFRvIII), and BCR-ABL in cells have been shown to induce transformation but do not elevate ERK activity (Moscatello, 1996; Raitano et al., 1995; Rodrigues et al., 1997). The JNK pathway is not consistently activated by growth factors but is more uniformly stimulated by cellular stresses and cytokines (Whitmarsh and Davis, 1996). The evidence that the JNK pathway has a role in tumorigenesis is conflicting. A major physiological role for JNK is to induce apoptosis in various cell types (Abreu-Martin et al., 1999; Chen et al., 1996). It has been shown that the loss of ERK activation coupled with JNK activation promotes cell death (Xia et al., 1995) and MKK4, which phosphorylates JNK, has been implicated as a tumor suppressor gene (Teng, 1997). On the other hand, the prevention of apoptosis and the promotion of cell growth has been shown to be Received 7 August 2001; revised 5 April 2002; accepted 15 April 2002 *Correspondence: AJ Wong, 233 S. 10th Street, BLSB 1002, Philadelphia, PA 19107, USA; E-mail: albert.wong@mail.tju.edu Oncogene (2002) 21, 5038 – 5046 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc