In this study, we have uncovered a novel STAT3-dependent tumor suppressive mechanism in human glioblastoma cells (see model in ). We found that cytokine-induced endogenous STAT3 signaling was specifically impaired in PTEN-deficient but not PTEN-expressing glioblastoma cells. Inhibition of endogenous STAT3 blocked cytokine-suppression of proliferation in PTEN-expressing glioblastoma cells. Conversely, reactivation of STAT3 in PTEN-deficient glioblastoma cells inhibited their proliferation, invasiveness, and spreading on myelin. We also identified the chemokine IL8 as a direct repressed target gene of STAT3 in glioblastoma cells. IL8 repression mediated the ability of STAT3 to inhibit glioblastoma cell proliferation and invasiveness. Thus, deregulation of the STAT3-IL8 signaling link may confer PTEN loss with the ability to stimulate glioblastoma cell proliferation and invasiveness.
A major conclusion of our study is that STAT3, a protein with oncogenic behavior outside the brain (Bromberg et al., 1999
; Bowman et al., 2000
), exerts tumor suppressor effects in human glioblastoma cells. This finding provides pathophysiological significance to our recent studies indicating that STAT3 suppresses malignant transformation of mouse astrocytes (de la Iglesia et al., 2008
). In some reports, it has been suggested that STATs may promote human glioblastoma tumor cell proliferation and survival (Rahaman et al., 2002
; Konnikova et al., 2003
), and STAT3 activation has been reported in human glial tumors (Weissenberger et al., 2004
). On the other hand, other studies have provided evidence of the absence of STAT3 activation in human gliomas (Wang et al., 2004
). We recently used a genetic approach in mouse astrocytes to directly assess STAT3 function in glial cell transformation. We found that the predominant role of STAT3 is to suppress malignant glial transformation including in the context of PTEN deficiency (de la Iglesia et al., 2008
). In agreement with these mouse genetic studies, our present findings reveal that STAT3 suppresses the malignant properties of PTEN-deficient glioblastoma cells including their proliferation and invasiveness. Interestingly, expression of the glioblastoma-associated protein EGFRvIII in mouse astrocytes triggers an oncogenic switch in STAT3 function (de la Iglesia et al., 2008
). In human glioblastomas, EGFRvIII expression correlates with STAT3 activation in both immunoblotting and immunohistochemical analyses (Mizoguchi et al., 2006
; de la Iglesia et al., 2008
). Therefore, it will be interesting to determine in future studies if and how STAT3 might promote the malignant behavior of EGFRvIII-expressing human glioblastoma cells.
Inhibition of the gliogenesis-promoting STAT3 signaling pathway in glioblastoma cells may serve to couple loss of a general tumor suppressor such as PTEN to the glioblastoma cell-specific behavior of invasiveness and adhesion and spreading on myelin. The PTEN loss-triggered suppression of STAT3 signaling in glioblastoma cells illustrates how interactions between ubiquitously-acting tumor suppressors and developmental signaling pathways implicated in cell differentiation and fate specification may produce tumor-specific pathological behaviors.
The identification of IL8 as a novel STAT3-repressed target gene in glioma cells, whose repression mediates STAT3-inhibition of glioma cell proliferation, invasiveness, and spreading on myelin defines a novel cytokine-chemokine connection (). Although STAT3 is commonly known to induce transcription (Darnell, 1997
; Levy, 2003
), our findings suggest that STAT3 may also act as a transcriptional repressor in glial cells. STAT3 appears to repress the IL8 promoter via inhibition of the NFkB reponse element. Interestingly, STAT3 inhibits expression of the inducible nitric oxide synthase (iNOS) gene in endothelial cells by directly inhibiting NFkB (Yu et al., 2002
; Yu and Kone, 2004
). The DNA binding domain of STAT3 directly interacts with NFkB and thereby inhibits NFkB-dependent iNOS transcription (Yu and Kone, 2004
). These results are consistent with the finding that the DNA binding domain of STAT3 is required for STAT3’s ability to repress IL8 expression in glioblastoma cells (). The candidate glial tumor suppressor ING4 reportedly inhibits glioblastoma-induced angiogenesis via repression of IL8, whereby ING4 inhibits NFkB-dependent IL8 transcription (Garkavtsev et al., 2004
). Collectively, these observations raise the possibility that STAT3 and ING4 may cooperate at the IL8 promoter to inhibit NFkB and thereby repress IL8 gene expression and thus control glioblastoma cell proliferation, invasiveness, and glioblastoma-induced angiogenesis.
The elucidation of STAT3 and IL8 in this study as critical regulators of glioblastoma cell proliferation and invasiveness provides new targets for therapeutic intervention. The chemokine IL8 and its cell surface receptors, CXCR1 and CXCR2, are particularly attractive as potential targets in the treatment of PTEN-deficient glioblastoma. These chemokine receptors have been studied in the context of acute inflammation (Rollins, 1997
; Schraufstatter et al., 2001
), and small molecule inhibitors of CXCR1 and CXCR2 have been characterized (White et al., 1998
; Bertini et al., 2004
). It will be important to determine the potential of CXCR1 and CXCR2 antagonists to inhibit the malignant features of PTEN-deficient glioblastoma cells. These studies may provide the foundation for patient-tailored therapy in the management of brain tumors.