The ARF tumor suppressor is a nucleolar protein that activates p53-dependent checkpoints by binding Mdm2, a p53 antagonist. Despite persuasive evidence that ARF can bind and inactivate Mdm2 in the nucleoplasm, the prevailing view is that ARF exerts its growth-inhibitory activities from within the nucleolus. We suggest ARF primarily functions outside the nucleolus and provide evidence that it is sequestered and held inactive in that compartment by a nucleolar phosphoprotein, nucleophosmin (NPM). Most cellular ARF is bound to NPM regardless of whether cells are proliferating or growth arrested, indicating that ARF-NPM association does not correlate with growth suppression. Notably, ARF binds NPM through the same domains that mediate nucleolar localization and Mdm2 binding, suggesting that NPM could control ARF localization and compete with Mdm2 for ARF association. Indeed, NPM knockdown markedly enhanced ARF-Mdm2 association and diminished ARF nucleolar localization. Those events correlated with greater ARF-mediated growth suppression and p53 activation. Conversely, NPM overexpression antagonized ARF function while increasing its nucleolar localization. These data suggest that NPM inhibits ARF's p53-dependent activity by targeting it to nucleoli and impairing ARF-Mdm2 association.

The ARF tumor suppressor protects us against cancer through protein-protein interactions in partially defined p53-dependent and p53-independent pathways. We performed a two-hybrid screen using ARF as bait and present the identification of several new ARF partners that may regulate its growth inhibitory signaling. The potential physiological roles of these novel ARF binding proteins in regulating ARF signaling are discussed.

Mechanisms of neuroendocrine tumor (NET) proliferation are poorly understood and therapies that effectively control NET progression and metastatic disease are limited. We found amplification of a putative oncogene, RABL6A, in primary human pancreatic NETs(PNETs) that correlated with high level RABL6A protein expression. Consistent with those results, stable silencing of RABL6A in cultured BON-1 PNET cells revealed that it is essential for their proliferation and survival. Cells lacking RABL6A predominantly arrested in G1 phase with a moderate mitotic block. Pathway analysis of microarray data suggested activation of the p53 and retinoblastoma (Rb1) tumor suppressor pathways in the arrested cells. Loss of p53 had no effect on the RABL6A knockdown phenotype, indicating RABL6A functions independent of p53 in this setting. By comparison, Rb1 inactivation partially restored G1 to S phase progression in RABL6A knockdown cells although it was insufficient to override the mitotic arrest and cell death caused by RABL6A loss. Thus, RABL6A promotes G1 progression in PNET cells by inactivating Rb1, an established suppressor of PNET proliferation and development. This work identifies RABL6A as a novel negative regulator of Rb1 that is essential for PNET proliferation and survival. We suggest RABL6A is a new potential biomarker and target for anticancer therapy in PNET patients.

Conflict of interest: The Icahn School of Medicine has filed patents covering composition of matter on the small molecules disclosed herein for the treatment of human cancer and other diseases and for methods of use for using these small-molecule PP2A activators (patent no. 9,937,186 B2). RAPPTA Therapeutics LLC has optioned this intellectual property for the clinical and commercial development of this series of small-molecule PP2A activators. GN has an ownership interest in RAPPTA Therapeutics LLC.

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