Molecular and structural investigation of the p53 regulatory pathway mediated by the numb isoforms
Numb functions as an oncosuppressor in breast cancer. Numb protein is lost or reduced in 20-30% of breast cancers, an event that correlates with adverse prognosis. Loss of Numb causes increased Notch signaling and attenuation of the p53 tumor suppressor pathway, with ensuing increased resistance to genotoxic drugs and expansion of the cancer stem cells.
At mechanistic level, Numb is able to bind Mdm2, thereby inhibiting its ubiquitin-ligase activity on p53. The rescue of Numb levels in Numb-defective tumors, or the pharmacological inhibition of Mdm2, reverts p53 dysfunction and sensitizes cells to chemotherapeutic treatments, arguing that restoration of the Numb:p53 axis might represent an anti-cancer stem cell therapy in these tumors.
We have recently characterized the structural basis of the Numb:Mdm2 interaction. We mapped the binding domains between Numb and Mdm2, identifying the Numb PTB long domain (containing an 11-residue Exon3 loop) and the acidic domain of Mdm2.
In mammals four major isoforms of Numb have been identified. The presence of the Ex3-loop confers a specific ability to Numb isoforms 1 and 2 to regulate Mdm2 and consequently the level and activity of p53. In some breast tumors, the low levels of these two isoforms correlate with adverse prognosis, indicating that the deregulation of the Numb isoforms expression might represent a further mechanism of loss of Numb function in breast tumors.
To better understand the role of the different Numb isoforms, we have identified, by mass-spectrometry analysis, specific and novel interactors of these isoforms, focusing on the isoforms 1 and 2, due to their specificity on p53 regulation.
In this regard, an endocitic protein belonging to the sorting nexins family, seems to be important for the regulation of Numb:p53 axis, also contributing, at molecular level, to the formation of the complex between Numb and p53.
The overall goal of this project is to perform an in depth characterization of this new molecular pathway to better elucidate the mechanisms of regulation of Numb isoforms on p53, that might regard also some p53 cytoplasmic activities, at the moment well described to play an important role in the p53 tumor suppressor activity but poorly investigated.
Finally, considering the relevance of the restoration of the Numb:p53 axis in the breast tumors in which Numb function is alterated, we also aim to gain insights on Numb:p53 interaction at structural level, taking advantage of the sorting nexin protein contribution to this binding.