Reduced abundance of the E3 ubiquitin ligase E6AP contributes to decreased expression of the INK4/ARF locus in non-small cell lung cancer.

The tumor suppressor p16INK4a, one protein encoded by the INK4/ARF locus, is frequently absent in multiple cancers, including non-small cell lung cancer (NSCLC). Whereas increased methylation of the encoding gene (CDKN2A) accounts for its loss in a third of patients, no molecular explanation exists for the remainder. We unraveled an alternative mechanism for the silencing of the INK4/ARF locus involving the E3 ubiquitin ligase and transcriptional cofactor E6AP (also known as UBE3A). We found that the expression of three tumor suppressor genes encoded in the INK4/ARF locus (p15INK4b, p16INK4a, and p19ARF) was decreased in E6AP-/- mouse embryo fibroblasts. E6AP induced the expression of the INK4/ARF locus at the transcriptional level by inhibiting CDC6 transcription, a gene encoding a key repressor of the locus. Luciferase assays revealed that E6AP inhibited CDC6 expression by reducing its E2F1-dependent transcription. Chromatin immunoprecipitation analysis indicated that E6AP reduced the amount of E2F1 at the CDC6 promoter. In a subset of NSCLC samples, an E6AP-low/CDC6-high/p16INK4a-low protein abundance profile correlated with low methylation of the gene encoding p16INK4a (CDKN2A) and poor patient prognosis. These findings define a previously unrecognized tumor-suppressive role for E6AP in NSCLC, reveal an alternative silencing mechanism of the INK4/ARF locus, and reveal E6AP as a potential prognostic marker in NSCLC.

The p53-Mdm2 loop: a critical juncture of stress response.

The presence of a functional p53 protein is a key factor for the proper suppression of cancer development. A loss of p53 activity, by mutations or inhibition, is often associated with human malignancies. The p53 protein integrates various stress signals into a growth restrictive cellular response. In this way, p53 eliminates cells with a potential to become cancerous. Being a powerful decision maker, it is imperative that p53 will be activated properly, efficiently and temporarily in response to stress. Equally important is that p53 activation will be extinguished upon recovery from stress, and that improper activation of p53 will be avoided. Failure to achieve these aims is likely to have catastrophic consequences for the organism. The machinery that governs this tight regulation is largely based on the major inhibitor of p53, Mdm2, which both blocks p53 activities and promotes its destabilization. The interplay between p53 and Mdm2 involves a complex network of positive and negative feedback loops. Relief from Mdm2 suppression is required for p53 to be stabilized and activated in response to stress. Protection from Mdm2 entails a concerted action of modifying enzymes and partner proteins. The association of p53 with the PML-nuclear bodies may provide an infrastructure in which this complex regulatory network can be orchestrated. In this chapter we use examples to illustrate the regulatory machinery that drives this network.

E6AP ubiquitin ligase regulates PML-induced senescence in Myc-driven lymphomagenesis.

Neoplastic transformation requires the elimination of key tumor suppressors, which may result from E3 ligase-mediated proteasomal degradation. We previously demonstrated a key role for the E3 ubiquitin ligase E6AP in the regulation of promyelocytic leukemia protein (PML) stability and formation of PML nuclear bodies. Here, we report the involvement of the E6AP-PML axis in B-cell lymphoma development. A partial loss of E6AP attenuated Myc-induced B-cell lymphomagenesis. This tumor suppressive action was achieved by the induction of cellular senescence. B-cell lymphomas deficient for E6AP expressed elevated levels of PML and PML-nuclear bodies with a concomitant increase in markers of cellular senescence, including p21, H3K9me3, and p16. Consistently, PML deficiency accelerated the rate of Myc-induced B-cell lymphomagenesis. Importantly, E6AP expression was elevated in ∼ 60% of human Burkitt lymphomas, and down-regulation of E6AP in B-lymphoma cells restored PML expression with a concurrent induction of cellular senescence in these cells. Our findings demonstrate that E6AP-mediated down-regulation of PML-induced senescence is essential for B-cell lymphoma progression. This provides a molecular explanation for the down-regulation of PML observed in non-Hodgkin lymphomas, thereby suggesting a novel therapeutic approach for restoration of tumor suppression in B-cell lymphoma.

HPV16 E6 augments Wnt signaling in an E6AP-dependent manner.

In this study we investigated the effect of HPV16 E6 on the Wnt/beta-catenin oncogenic signaling pathway. Luciferase reporter assays indicated that ectopically expressed E6 significantly augmented the Wnt/beta-catenin/TCF-dependent signaling response in a dose-dependent manner. This activity was independent of the ability of E6 to target p53 for degradation or bind to the PDZ-containing E6 targets. Epistasis experiments suggested that the stimulatory effect is independent of GSK3beta or APC. Coexpression, half-life determination, cell fractionation and immunofluorescence analyses indicated that E6 did not alter the expression levels, stability or cellular distribution of beta-catenin. Further experiments using E6 mutants defective for E6AP binding and E6AP knockdown cells indicated the absolute requirement of the ubiquitin ligase E6AP for enhancement of the Wnt signal by E6. Thus, this study suggests a role for the E6/E6AP complex in augmentation of the Wnt signaling pathway which may contribute to HPV induced carcinogenesis.

Mdm2 in growth signaling and cancer.

Genetic and biochemical evidence have demonstrated a direct link between Mdm2 and cancer development. Elevated expression of Mdm2 is observed in a significant proportion of different types of cancer. The major contribution of Mdm2 to the development of cancer is through a tight inhibition of the activities and stability of the tumor suppressor p53. However, extensive studies over the past few years have identified p53-independent functions of Mdm2, in the regulation of several important cellular processes and multiple signaling pathways. The promotion of cell cycle progression by Mdm2 is mediated via p53 inhibition, and by regulating the pRb/E2F complex. Mdm2 is an important mediator of growth and survival signaling in the PI3K/Akt pathway, an activator of certain steroid hormone receptors, and an inhibitor of the TGF-beta growth restrictive pathway. Thus, the impact on these pathways by deregulated Mdm2, as often observed in cancer, can be oncogenic in a permissible environment. This renders Mdm2 as an important target for the development of anti-cancer drugs.

C-Abl as a modulator of p53.

P53 is renowned as a cellular tumor suppressor poised to instigate remedial responses to various stress insults that threaten DNA integrity. P53 levels and activities are kept under tight regulation involving a complex network of activators and inhibitors, which determine the type and extent of p53 growth inhibitory signaling. Within this complexity, the p53-Mdm2 negative auto-regulatory loop serves as a major route through which intra- and extra-cellular stress signals are channeled to appropriate p53 responses. Mdm2 inhibits p53 transcriptional activities and through its E3 ligase activity promotes p53 proteasomal degradation either within the nucleus or following nuclear export. Upon exposure to stress signals these actions of Mdm2 have to be moderated, or even interrupted, in order to allow sufficient p53 to accumulate in an active form. Multiple mechanisms involving a variety of factors have been demonstrated to mediate this interruption. C-Abl is a critical factor that under physiological conditions is required for the maximal and efficient accumulation of active p53 in response to DNA damage. C-Abl protects p53 by antagonizing the inhibitory effect of Mdm2, an action that requires a direct interplay between c-Abl and Mdm2. In addition, c-Abl protects p53 from other inhibitors of p53, such as the HPV-E6/E6AP complex, that inhibits and degrades p53 in HPV-infected cells. Surprisingly, the oncogenic form of c-Abl, the Bcr-Abl fusion protein in CML cells, also promotes the accumulation of wt p53. However, in contrast to the activation of p53 by c-Abl, its oncogenic form, Bcr-Abl, counteracts the growth inhibitory activities of p53 by modulating the p53-Mdm2 loop. Thus, it appears that by modulating the p53-Mdm2 loop, c-Abl and its oncogenic forms critically determine the type and extent of the cellular response to DNA damage.