The major finding of this study is that RIP1, an essential component of inflammation and NF-κB signaling, plays an important role in regulating p53. Our findings indicate that RIP1 activates NF-κB, resulting in upregulation of mdm2 and a complete shutdown of the p53 tumor suppressor signaling network. We show that RIP1 is overexpressed in human glioblastoma (GBM), the most common adult malignant brain tumor, but not in lower grade glioma, and confers a worse prognosis in this disease. Furthermore, our data suggest that the RIP1 may regulate mdm2, lowering p53 function in GBM.
Stressful stimuli, such as DNA damage, results in activation of both p53 and NF-κB pathways. Cross-talk between the NF-κB and p53 signaling pathways is well documented and may play important roles in the pathogenesis of stress/inflammation induced cancer and in resistance to treatment. However, specific mechanisms by which NF-κB and p53 cross-talk are still under intense study. Altered regulation of both the NF-κB and p53 pathways is established in GBM. p53 function is frequently altered in glioma either by direct mutation or changes in regulatory signals, due to mdm2 gene amplification or loss of p14ARF
. Our data suggest that increased expression of RIP1 may be an important additional mechanism of regulating p53 in GBM. It is important to note that mdm2 is known to downregulate both wild type as well as mutant p53 which may result in complex biological outcomes (46
Previous studies have demonstrated that IKK2 or Bcl-3, a protein related to the IκB family of NF-κB inhibitors, regulate p53 via augmentation of mdm2 levels (21
). In the case of IKK2, upregulation of mdm2 is mediated by activation of NF-κB. Our data show that inhibition of NF-κB activation using a dominant-negative IκBαM results in a block of RIP1 mediated mdm2 upregulation and rescues RIP1-mediated p53 inhibition. Also, a RIP1 mutant lacking the intermediate domain known to be deficient in NF-κB activation failed to inhibit p53 induction. Thus, the negative regulation of p53 by NF-κB occurs at multiple nodes and may be important in the pathogenesis of cancer. An increase in IKK2 levels has not been reported in GBM, and our data suggest that RIP1 may be the key player conducting the NF-κB-p53 cross-talk in GBM.
A major finding of this study is that RIP1 is overexpressed in GBM but not in lower grade glioma and confers a worse prognosis in GBM. Increased expression of RIP1 is common in GBM, with about 30% of tumors showing increases in RIP1, and frequently the increase is substantial. Furthermore, increased expression of RIP1 is uncommon in grade II-III gliomas, demonstrating a correlation of RIP1 with increased malignancy. Importantly, in matched pairs of primary low grade glioma that progressed to secondary GBM, RIP1 is usually low in the primary low grade glioma and increased in the secondary GBM. In a study of 70 GBMs we find that increased RIP1 is an independent negative prognostic indicator in GBM. There were no differences in the age, Karnofsky performance status, or treatment in the low versus high RIP1 groups. These findings imply that increased expression of RIP1 promotes a more malignant clinical phenotype in GBM. It should be noted that since most patients in our study had a complete resection, our study does not address whether low RIP1 level would confer a survival advantage in those patients with partial resection or biopsy alone.
RIP1 may contribute to the pathogenesis of GBM by multiple mechanisms. Firstly, increased expression of RIP1 is sufficient to activate NF-κB as shown in and reported previously (40
). Thus, increased RIP1 level in cancer is likely to lead to constitutive and deregulated NF-κB activation. In addition, RIP1 has also been reported to have a role in PI3K-Akt activation (39
). Thus, an augmented cellular RIP1 level in glioma cells appears sufficient to induce sustained activation of at least two pro-survival signaling pathways of central importance in cancer. In this study, we show that RIP1-mediated NF-κB activation leads to upregulation of mdm2 and inhibition of p53 pathways. Thus, cells with increased RIP with activated NF-κB and Akt, and downregulated p53, would favor oncogenic signaling, resistance to DNA damage, and chemotherapy-induced apoptosis, all favoring a more malignant phenotype.
Paradoxically, RIP1 is also involved in cell death when ectopically expressed, in response to inflammatory cytokines, or other forms of cellular stress (24
). However, apoptosis and proliferation are closely linked, and a number of key oncogenic proteins such as Ras, c-Myc and E2F1 can also induce apoptosis or growth arrest (49
). The RIP1 knockout phenotype includes failure to thrive and an early death with substantial apoptosis in lymphoid and adipose tissues (27
), suggesting an important role for RIP1 in survival signaling. RIP1 knockout MEFs are more sensitive to TNF induced cell death and RIP1 protects thymocytes from TNFR-2 induced cell death (50
). The data in this study also support an oncogenic and prosurvival role for RIP1 in GBM, but the effect of RIP1 signaling could be quite complex in the heterogenous tumor populations.
Our data suggest that increased expression of RIP1 identifies a group of glioblastoma patients who have a significantly worse prognosis. The clinical utility of this study may lie in the identification of a subgroup of patients with GBM that have high RIP1 levels, worse prognosis, and are resistant to standard chemotherapy. We propose that these patients may respond better to drugs targeting the NF-κB signaling network.