Abstract 4293: URI and PP1γ constitute a negative feedback loop regulating MAPK signaling in BRAF-V600E mutated cancer cells.
Authors: Britschgi Christian , Lipinski Kamil A. , Theurillat Jean-Philippe , Metzler Stefan C. , Flueckiger Stefanie , Frischknecht Lukas , Schmidt Juliane , Krek Wlhelm 
Published: August 2014

Malignant transformation of cells involves acquisition of so-called hallmarks of cancer. Such hallmark capabilities are brought about by functional activation of oncogenes and inactivation of tumor suppressor genes. Much has been learned about the actions of individual such cancer genes, however, how an entire set of acquired mutations shapes cancer phenotypes in a context-dependent manner remains elusive in many cases. It has for example increasingly become clear that feedback mechanisms control activated signaling pathways and might thus mitigate oncogenic signaling. Therefore, functional inactivation of such feedback loops might represent an additional event in carcinogenesis. Along this line we have recently shown that URI (unconventional prefoldin RPB5-interactor) can act as a negative regulatory subunit for the protein phosphatase 1γ (PP1γ) and form a negative feedback loop regulating mTOR-S6K1-BAD apoptotic signaling at mitochondria.

Since the URI-PP1γ interaction in this context is regulated by URI-phosphorylation at S371, we performed a mass spectrometric search for other phospho-sites regulating URI-PP1γ-complex formation. We identified a potential MAPK-target site in URI's PP1γ-interaction domain, which is phosphorylated by extracellular signal-related kinase (ERK) in vitro. In in vitro kinase / in vitro binding assays, we demonstrate that this phosphorylation event provokes URI-PP1γ complex disruption. Moreover, PP1γ can dephosphorylate MEK in vitro. These observations lead to the intriguing conclusion that URI and PP1γ form a negative feedback on MEK-ERK-signaling. Indeed, in co-immunoprecipitation assays, introducing the BRAF-V600E-mutation (but not KRAS-G13D) into HEK293T cells promotes complex disruption, which can be rescued by co-treatment with the BRAF-V600E-specific inhibitor PLX4720. To address the question of whether this feedback loop is important in vivo, we analyzed a set of colorectal cancers by TMA (n=357). We identified low PP1γ expression in 28% of all CRC, however, in the BRAF-V600E-mutated subgroup, PP1γ expression was significantly more frequently low (50%) and it correlated with worse overall survival in this subgroup only. This observation is also mirrored by cell culture experiments in the CRC cell line RKO: Downregulation of PP1γ by RNAi increases colony formation in long term proliferation assays, particularly under low serum conditions, and this is rescued by re-introducing rat PP1γ in cells.

In summary, we have identified a novel negative feedback loop tuning signaling output of the BRAF-V600E-MEK-ERK pathway. Deregulating this feedback by reducing PP1γ levels might provide a subgroup of BRAF-V600E-mutated cancer cells with a further proliferative advantage by de-repressing MEK-ERK-signaling.

Citation Format: Christian Britschgi, Kamil A. Lipinski, Jean-Philippe Theurillat, Stefan C. Metzler, Stefanie Flueckiger, Lukas Frischknecht, Juliane Schmidt, Wlhelm Krek. URI and PP1γ constitute a negative feedback loop regulating MAPK signaling in BRAF-V600E mutated cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4293. doi:10.1158/1538-7445.AM2013-4293