Notch1-Induced Survival Signaling And Its Implications In Cancer Therapeutics

Abstract

Notch receptors and ligands are type I transmembrane proteins that regulate development and differentiation during cell-cell contact. There are four Notch receptor homologues and five notch ligands, identified in humans till date. Upon ligand activation, Notch1 intracellular domain (NIC-1) is released into the cytoplasm, which binds to several proteins as well as translocates into the nucleus to effect the Notch signaling. In the absence of the activated Notch signaling, the Notch target genes are kept repressed by the transcriptional repressor C protein binding factor 1 (CBF1) also known as RBPjk or CSL for CBF1/Su(H)/Lag1. RBPjk binds to the sequence “CGTGGGAA” and acts as a constitutive repressor. Upon ligand dependent activation, NIC-1 enters into the nucler and converts RBPjk from transcriptional repressor to an activator. Notch binding to CSL replaces the SMRT corepressor complex with a coactivator complex including SKIP, Mastermind like 1 (MAML1) (Mastermind in Drosophila), and histone acetyl transferases PCAF, GCN5 and p300 activating the transcription of target genes. Mastermind-like (MAML), a family of transcriptional activator proteins comprising of 3 members 1 to 3, has been shown to be required for Notch signaling. MAML forms a ternary complex with RBPjk-NIC by directly interacting with NIC. In turn, MAML recruits the histone acetyl transferase p300/CBP, which acetylates the histones, thereby altering the structure of chromatin amenable for transcription. Activation of Notch pathway induces oncogenesis, which can be divided into two categories including 1) Inhibition of Apoptosis and 2) Induction of proliferation. In T cells, activation of Notch1 protects cells from T cell receptor, dexamethasone and etoposide-mediated apoptosis, Fas receptor-mediated signaling by up regulating IAP (Inhibitor of Apoptosis) and Bcl-2 families, as well as FLIP (FLICE-like inhibitor protein). Notch signaling also promotes the survival of T cells through maintenance of cell size as well as through the promotion of glucose uptake and metabolism. Notch-1 has been shown to protect against anoikis (apoptosis induced by matrix withdrawal) or p53-mediated apoptosis in immortalized epithelial cells, T cell receptor-induced apoptosis in mature cells and dexamethasone-mediated apoptosis in thymocytes. This study was carried out to functionally characterize NIC-1 (human Notch1-intracellular domain) as an inhibitor of apoptosis and to evaluate the therapeutic potential of reversal of this apoptosis inhibition. The main objectives of this study are 1. Construction of recombinant adenovirus expressing human Notch1-intracellular domain (Ad-NIC-1) and characterization of NIC-1 as an inhibitor of chemotherapy and p53-induced cytotoxicity and apoptosis. 2. Role of PI3 kinase -Akt/PKB -mTOR pathway in NIC-1-mediated inhibition of p53-induced apoptosis. 3. Essential role of association between mTOR and NIC-1 and the dependent NIC-1 phosphorylation in Notch1-mediated transcription and survival signaling. 4. Identification of NIC-1 as an inhibitor of E1A-induced apoptosis and the role of mTOR in NIC-1-mediated inhibition of E1A-induced apoptosis. Activated Notch1 was first linked to tumorigenesis through identification of a recurrent t(7;9)(q34;q34.3) chromosomal translocation involving the human Notch1 gene that is found in a subset of human pre-T-cell acute lymphoblastic leukemia’s (T-ALL). Deregulated Notch signaling is oncogenic, inhibits apoptosis and promotes survival. In order to understand survival signaling induced by Notch1 and its possible role in chemoresistance, we have generated a replication deficient recombinant adenovirus expressing human Notch1-intracellular domain (Ad-NIC-1) and shown that it produces functional NIC-1 protein. Using this overexpression system, we characterized that activated Notch1-inhibits chemotherapy and in particular p53 induced apoptosis. Notch1-mediated inhibition of p53-induced apoptosis does not include coactivator squelching. p53 was inefficient in binding to its DNA in NIC-1 overexpressing cells. The levels of phosphorylation at Ser15, Ser20, and Ser392 of p53 expressed from Ad-p53 significantly reduced in NIC-1 preinfected cells. These results suggest that NIC-1-mediated inhibition of p53-mediated apoptosis involves reduced DNA binding, reduced nuclear localization and reduced post translational modifications and thus reduced transactivation of its target genes. Notch1-mediated inhibition of p53 was found to occur mainly through mammalian target of rapamycin (mTOR) using PI3 kinase-Akt/PKB pathway, as the mTOR inhibitor; rapamycin treatment was able to reverse Notch-1 mediated inhibition of p53 and chemoresistance. Consistent with this, rapamycin failed to reverse NIC-1 induced chemoresistance in cells expressing rapamycin resistant mTOR. Our results also suggest that the N-terminal HEAT repeat and the kinase function of mTOR are essential for Notch mediated inhibition of p53. Further, ectopic expression of eIF4E, a translational regulator that acts downstream of mTOR, inhibited p53-induced apoptosis and conferred protection against p53-mediated cytotoxicity to similar extent as that of NIC-1 overexpression, but was not reversed by rapamycin, which indicates that eIF4E is the major target of mTOR in Notch1-mediated survival signaling. Notch1-intracellular domain (NIC-1), following proteolytic cleavage, binds to RBPjk and regulates transcription. Active NIC-1 located in the nucleus is phosphorylated, which makes it more stable and bind better to RBPjk. NIC-1 was also shown to bind to Deltex1 in the cytoplasm. Next, we studied the requirement of components of Notch1 signaling pathway for this function. By using variety of approaches, we found that both RBPjk and Maml1 and hence transcription activation is required for NIC-1-mediated survival signaling and inhibition of p53 functions. Interestingly, while we found the other Notch1 effector, Deltex1 is also required for above functions, Notch1 failed to activate PI3 kinase -Akt/PKB -mTOR pathway in Deltex1, but not in RBPjk silenced cells. Our results suggest that Notch-Deltex1 pathway activates PI3 kinase. Previous studies show that NIC-1 interacts with Deltex1 and Grb2 interacts with PI3 kinase. Our data shows that Deltex1 interacts with SH3 domain of Grb2. Since Notch1-Deltex1 and PI3 kinase-Grb2 interactions are known, we conclude that Notch1 activation of PI3 kinase involves Deltex1 and Grb2. We found activated mTOR was able to binds to NIC-1 and regulates its phosphorylation. Inhibition of mTOR either by PI3 kinase inhibitors or mTOR inhibitor treatment or silencing of Akt/PKB or mTOR reduced the phosphorylation of NIC-1 with the concomitant reduction in NIC-1-mediated transcription. Further, endogenous Notch1 receptor activated by the DSL ligand failed to activate transcription efficiently in rapamycin treated cells, implying a positive role for mTOR in mammalian Notch signaling. These studies reveal that Notch1 activates PI3 kinase -Akt/PKB -mTOR signaling through Deltex1 and subsequently activated mTOR modulates Notch1 signaling by direct binding and possibly thorough phosphorylation of the intracellular domain of Notch. Adenoviral E1A, in the absence of cooperating oncogene, suppresses primary tumor growth and reverses the transformed phenotype of human tumor cells by inducing apoptosis. E1A requires p53 for efficient induction of apoptosis and was shown to induce apoptosis by down regulating Akt and the activation of pro apoptotic factor p38 MAP kinase. Since our results suggest Notch1 inhibits chemotherapy and p53-induced apoptosis, we analyzed the ability of Notch1 to protect cells from E1A-induced apoptosis. Here we show that NIC-1 suppresses the ability of E1A to induce apoptosis. NIC-1 requires mTOR-dependent signal to inhibit E1A-mediated apoptosis, as the rapamycin, an mTOR inhibitor was able to completely reverse the ability of Notch1 to protect cells against E1A-induced apoptosis. The role of mTOR in NIC-1-mediated survival signaling was further confirmed by using the cells stably expressing rapamycin resistant mTOR. Rapamycin was able to reverse Notch1-mediated protection in cells expressing wild type mTOR but not in rapamycin resistant mTOR expressing cells. We also found that E1A was able to induce apoptosis in cells silenced for the pro apoptotic factor p38 and NIC-1 continued to inhibit E1A-induced apoptosis in these cells. These results confirm that Notch1 requires the activation of mTOR signaling but not p38 MAP kinase for inhibition of E1A-induced apoptosis. These results also suggest that the combination therapy utilizing E1A-mediated gene delivery in combination with inhibition of mTOR pathway may prove successful in treating Notch overexpressing cancers. Chemotherapy remains a major treatment modality for human cancers. Chemoresistance is a clinical problem that severely limits treatment success. It can be divided into two forms: intrinsic and acquired. Intrinsic resistance is the essence of oncogenic transformation, resulting from activation of oncogenes and the loss of tumor suppressors, and manifests itself as alterations in cell cycle checkpoints and apoptotic pathways. It is now widely accepted that the apoptotic capacity of the cancer cell is crucial in determining the response to chemotherapeutic agents. Indeed, several gene products that regulate apoptosis, i.e., p53, Akt and PI3K are frequently altered in cancer cells. In this study, we identified that cells with aberrant Notch1 signaling are chemoresistant. Activated Notch1 overexpression makes cells resistant to chemotherapy in a wild type p53 dependent manner. Notch protected p53 wild type cells but not p53 mutated or p53 deleted cells against chemotherapy induced cytotoxicity. Further, inactivation of p53 by specific silencing abrogated the ability of NIC-1 to protect H460 cells against adriamycin induced cytotoxicity. Most importantly, NIC-1 mediated chemoresistance can be reversed by blocking PI3 kinase -Akt/PKB -mTOR pathway. Collectively, these results suggest that cancers with activated Notch1 signaling are chemoresistant and provide basis for the reversal of chemoresistance.