Abstract 1877: A new H-ras specific feedback loop from the TOR-pathway impacts on tumorigenicity

Introduction: The lack of direct Ras inhibitors, has led to the development of inhibitors of downstream components of the Ras pathway albeit with mixed success. Unexpected mechanistic complexity, feedback loops and tumor specific synthetically lethal requirements continue to be major obstacles. In the past we investigated the mechanisms of the Ras membrane signaling system on the nanoscale (nanoclustering). Ras nanoclustering on the plasma membrane is critical for its signaling activity and we and others recently showed how it is exploited in a number of physiological and pathophysiological conditions, including cancer. Importantly, Ras isoform specificity seems to emerge at the level of laterally segregated nanocluster. This is supported by isoform specific nanocluster scaffolds, such as galectin-1 (Gal-1), which specifically augments GTP-H-ras nanocluster. Methods: The effect of compounds and siRNAs targeting the TOR pathway and associated proteins on H- vs. K-ras4B (hereafter K-ras)-nanoclustering was evaluated using FRET-measurements in BHK and HEK cells, or point pattern analysis of electron micrographs from BHK cell plasma membrane sheets. Ras specific effects of treatments were furthermore assessed in PC12 differentiation assays, driven by oncogenic H- or K-ras. Changes in downstream signaling were evaluated using Western Blotting of treated BHK and HEK cells. The effect of treatments (compounds or siRNAs) on mammosphere formation was assessed in MDA-MB-231 cells. Results: Here we describe a new feedback mechanism from the TOR-pathway specifically to H-ras, but not K-ras. Inhibition of FKBP12 inhibitors cycloheximide (CHX), Rapamycin (Rapa) and FK506, as well as knockdown of FKBP12 specifically increased H-ras nanoclustering. While all compounds could also specifically increase differentiation of H- but not K-rasG12V transfected PC12 cells, their effect on cellular signaling pathways differed in BHK cells. Both, CHX and Rapa increased Erk phosphorylation, but only Rapa blocked S6K and S6 phosphorylation, while CHX did the opposite. Surprisingly, siRNA-mediated ablation of FKBP12 upregulated Gal-1 protein levels, suggesting that the observed increase in H-ras nanoclustering is due to this response. This was however not observed for FKBP12 inhibitors. On the other hand overexpression of Gal-1 also increased FKBP12 levels. Intriguingly, only FKBP12 ablation (with concomitant Gal-1 upregulation) was sufficient to increase the mammosphere forming ability, but not Gal-1 overexpression alone. This effect could be mimicked by FKBP12 inhibition with CHX or Rapa, but not FK506. Conclusions: Our data indicate a H-ras specific feedback from inhibition of the TOR pathway, which can lead to increased mammosphere formation. These data suggest a tumor promoting effect by TOR pathway inhibitors via H-ras probably in a specific cellular and genetic context. Citation Format: Itziar M. D. Posada, Benoit Lectez, Christina Oetken-Lindholm, Daniel Abankwa. A new H-ras specific feedback loop from the TOR-pathway impacts on tumorigenicity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1877.