ELUCIDATING THE MECHANISMS OF STRESS-MEDIATED REGULATION OF P73 STABILITY

Abstract

p73, the structural and functional homologue of p53, exists as two major forms: the pro-apoptotic TAp73 or the anti-apoptotic DNp73. Unlike p53, p73 is rarely mutated in cancers. However, both TAp73 and DNp73 are often up-regulated in many tumors. Upon genotoxic stresses such as chemotherapeutic drugs, TAp73 is stabilized mainly through post-translational modifications, leading to its pro-apoptotic activities and concomitantly, DNp73 is degraded through poorly understood mechanisms. Presence of dominant-negative oncogenic DNp73 is often associated with chemoresistance in many cancers. Expectedly, a balance in the expression of both these major forms has to be coordinated precisely to achieve the desired cellular outcome. In the first part of this study, we have therefore investigated the possible mechanisms of stress-induced DNp73 degradation and show here that c-Jun, the AP-1 family member activated by stress signals and involved in stabilizing TAp73, promotes DNp73 degradation. Genotoxic stress-mediated DNp73 degradation was found to occur in a c-Jun-dependent manner through an ubiquitin-independent but proteasome-dependent mechanism. Absence or down-regulation of c-Jun expression abrogated reduction of DNp73 levels upon stress insults whereas over-expression of c-Jun led to its degradation. c-Jun controlled DNp73 degradation through the non-classical, polyamine-induced antizyme (Az) pathway, by regulating the latter¿s processing during stress response. Consistently, expression of c-Jun or Az1, or addition of polyamines promoted DNp73 degradation, whereas silencing Az1 expression or inhibiting Az1 activity in cells exposed to stress reduced c-Jun-dependent DNp73 degradation. Moreover Az1 was able to bind to DNp73. These data together demonstrate the existence of a novel c-Jun-dependent mechanism regulating the abundance of the anti-apoptotic DNp73 in response to genotoxic stress. Non-genotoxic stresses such as hypoxia play an important role in supporting tumorigenesis. Several studies have recently shown that TAp73 and DNp73 have also the ability to promote cell growth and survival. Here we show that hypoxia induces the stabilization of both TAp73 and DNp73. This process did not require VHL E3 ubiquitin ligase, a key regulator known to modulate hypoxia-regulated proteins but involved a prolyl-4-hydroxylase enzyme. Additionally, we found that AMPK signaling pathway was required for p73 stabilization under low oxygen tension and this was abrogated in the absence of the two catalytic subunits, AMPKa1 and AMPKa2. Lastly, autophagy, a downstream effect of hypoxia, regulated partly by AMPK, was reduced in the absence of p73. Overall, we have demonstrated the mechanism via which oncogenic DNp73 is degraded upon genotoxic stresses. We have also found a possible novel function of p73 in mediating autophagy in response to non-genotoxic stress, hypoxia.