The Inhibition of Anti-Apoptotic Pathways in Brain Glioma

Brain glioma in its various forms is a fatal disease with very low rate of survival. In about 40% to 50% of high grade gliomas, tumor robustness is correlated with the overexpression of the truncated form of the EGFR (EGFRD), which possesses persistent kinase activity (Ekstrand et al.,1992; Liberman et al., 1985, Malden et aL, 1988, Schlegel et al., 1994). EGFR overexpression correlates with shorter survival and faster recurrence. Indeed human glioma cells,U87MG which express the truncated persistently active receptor (U87MG. EGFRD)induce enhanced tumorigenicity in nude mice (Nishikawa et al., 1994). The enhanced tumorigenicity is correlated with high expression of the antiapoptotic protein BCl-XL. Overexpression of Bcl-XL has been shown to be associated with drug resistance (Ibrado et a., 1996, Minn et al., 1995) which is also a hallmark of glioma cells which express EGFRD (Nagane et al., 1996; Nagane et aL, 1998). Nagane et al reported that EGFRD expression in glioma cells confers resistance to cis-platin(CDDP). This drug resistance is attributed to EGFRA signaling since it does not occur in "isogenic" cells expressing the wild type receptor or a kinase dead EGFRD. It seems therefore that in this malignant form of glioma the truncated receptor not only drives the tumor to grow fast but also confers to the tumor anti-apoptotic signals. Blocking the EGFR kinase by selective inhibitors such as AG 1478 and AG 1517 induces a reduction of Bcl-XL expression and sensitizes the cells to CDDP (cis-platin). It is likely that in other forms of brain tumors anti-apoptotic signals are also enhanced but this feature has so far not been described. Similar synergistic effects of tyrphostins with cytotoxic drugs were obtained in HER-2 overexpressing cells from non-small cell lung carcinoma (NSCL), where a selective inhibitor of HER-2 resensitizes the cells to cytotoxic drugs (Tsai et al., 1996). It has been pointed out that enhanced activity of HER family receptors leads to the persistent activation of Src family kinases, especially pp60c-Src (Levitzki, 1996 for review).

In a recent study we have demonstrated that Src family kinases, especially pp60c-Src is constitutively active in tumor cells which overexpress EGFR and its ligands (Osherov and Levitzki,1994), and the HER-2 kinase (Levitzki 1996).We have recently extended this observation to HPV 16 immortalized keratinocytes which overexpress the EGFR and its ligands (Ben-Bassat et al.,in preparation). Our preliminary results (see below) show that expression of Src antisense RNA or the application of Src selective kinase inhibitors to cells which overexpress EGFR or HER-2 kinase, result in the reduction of Bcl-XL expression, inhibition of cell growth and the inhibition of colony formation in soft agar. Another branch of anti-apoptotic signaling pathway is regulated by PKB which is downstream to PI’-3 kinase (Kennedy et al., 1997; Alessi and Cohen, 1998). For example, a recent study suggests that PKB/c-Akt phosphorylation of BAD, a pro-aopototic family member of the Bcl-2/Bcl-X family of proteins, couples survival signals to the death machinery (Datta et al., 1997). This study provides a link between the PKB/c-Akt pathway and the Bcl/Bcl-X family of proteins. During the grant period we propose to explore the roles of Src kinase and of the PKB pathway in the robustness of glioma and examine their role, if any, in conferring to the ghoma cells their robustness and drug resistance.