Birt-Hogg-Dubé (BHD) syndrome is a rare disorder caused by mutations in FLCN and associated with increased risk of kidney cancer. It has been shown that FLCN-interacting protein 1 and 2 (FNIP1 and FNIP2) double knockout mice, like the FLCN knockout mice, develop renal carcinoma (Hasumi et al., 2015). However, the molecular mechanisms linking FNIP and FLCN remain unknown. In their new study, Nagashima et al. (2016) show that FNIP2 undergoes proteasome-dependent degradation via β-TRCP and Casein Kinase 1 (CK1)-directed ubiquitination in a nutrition-dependent manner. Degradation of FNIP2 leads to lysosomal dissociation of FLCN and association of mTOR, which promotes the proliferation of renal cancer cells.
The authors started by showing that FNIP protein abundance decreases after nutrient stimulation in starved HeLa cells. Refeeding increased mTOR and S6K phosphorylation, suggesting FNIP downregulation in mTORC1 activation. The presence of a proteasome inhibitor (MG132) blocked nutrient-stimulated FNIP downregulation. These results suggest that FNIP stability is negatively regulated by nutrient stimulation in a proteasome-dependent manner.
The knockdown of β-TRCP, a well-characterized F-box protein known to play a role in regulating various signalling pathways, led to an increase in FNIP protein levels without affecting mRNA expression. β-TRCP specifically bound to exogenous and endogenous FNIP2 suggesting that SCFβ-TRCP ubiquitin ligase complex interacts with FNIP to control their stability.
A potential β-TRCP phosphorylation binding motif in FNIP1 and FNIP2 was identified and the authors generated a mutant without this motif (FNIP2-3A) for subsequent analyses. The FNIP2-3A mutant was unable to interact with β-TRCP, indicating that β-TRCP recognizes FNIP2 through this specific motif. To identify a protein kinase responsible for phosphorylation in the β-TRCP binding motif, FNIP2 protein levels following co-expression with various kinases was evaluated. Expression of CK1 led to a decline in FNIP2 and co-expression of CK1 and β-TRCP enhanced FNIP2 poly-ubiquitination indicating that CK1-mediated phosphorylation promoted SCFβ-TRCP-dependent ubiquitination and subsequent degradation of FNIP2.
β-TRCP was depleted in HeLa cells and the FNIP protein levels examined under starved and refed conditions. Following starvation, FNIP protein accumulated in both control and β-TRCP depleted cells. However, upon refeed, FNIP levels decreased in control cells, while β-TRCP knock-down cells maintained high protein levels of FNIP, despite the decrease in mRNA expression, suggesting that β-TRCP regulates the degradation of FNIP in a nutrient-dependent manner.
Starvation promotes FLCN recruitment to lysosomes and FNIP co-expression facilitatesFLCN accumulation at the lysosome via the formation of a complex (Starling et al., 2016). mTORC1 is recruited and activated in the lysosome in a nutrient-dependent manner. The authors assessed how β-TRCP depletion influenced FLCN distribution in a nutrient-dependent manner. β-TRCP depletion led to a partial enrichment of FLCN to the lysosomal membrane under growth conditions, and enhanced enrichment of FLCN to lysosomes was retained even after nutrition stimulation, whereas mTOR continued to be diffusely localized in the cytoplasm. These results suggest a causal relationship of lysosomal localization between FLCN and mTOR complexes.
To exclude possible pleiotropic effects of β-TRCP knockdown, the authors ectopically expressed Flag-FNIP2 WT and FNIP2-3A mutant in HeLa cells to confirm the results that show that FNIP abundance is critical in lysosomal localization of both FLCN and mTOR, suggesting that β-TRCP-mediated FNIP degradation activates mTORC1 signalling in a nutrient-dependent manner.
FNIP levels and mTORC1 activity were also assessed in the FLCN-null BHD cell line UOK-257 starved and refed. Consistent with a previous report (Baba et al., 2006), loss of FLCN led to activated mTORC1 signalling. Loss of FLCN displayed mTOR enrichment to the lysosomal membrane in a nutrient-independent manner and FLCN restoration in UOK-257-2 cells and its association with lysosomes caused the dissociation of mTOR from lysosomes in a nutrient-dependent manner. To assess the role of β-TRCP-mediated FNIP degradation in renal cancer cell progression, FNIP was depleted from UOK-257-2 cells and FNIP2-WT or non-degradable FNIP2- 3A reintroduced. FNIP knockdown in UOK-257-2 cells led to the activation of mTORC1 signalling. Conversely, reintroducing FNIP2-WT diminished the enhanced mTORC1 activity, while introducing the FNIP2-3A mutant further enhanced mTORC1 activity. FLCN expression suppressed colony formation but these effects were reversed by FNIP depletion in UOK-257-2 cells. Reintroducing FNIP2-WT conversely blocked colony formation whereas reintroducing the FNIP2-3A mutant further suppressed cell growth, suggesting a tumour suppressive role of FNIP2 and a contribution of β-TRCP in promoting renal cancer cell proliferation. To validate the in vitro studies, the authors performed mouse xenograft experiments using UOK-257 and UOK-257-2 cell lines. Tumour growth of UOK-257-2 cells was significantly suppressed when compared to that of UOK-257 cells. FNIP depletion enhanced tumorigenesis. Reintroducing FNIP2-WT in FNIP depleted cells resulted in decreased tumour size and FNIP2-3A mutant presented further suppressive effects. In addition, mTORC1 activity was inversely correlated with FNIP2 levels in xenografts. These data indicate a tumour suppressive role for the FLCN complex in BHD-associated renal cancer tumorigenesis.
In summary, the results indicate that SCFβ-TRCP negatively regulates the FLCN complex by promoting nutrient-dependent FNIP degradation and provide molecular insight into the pathogenesis of BHD-associated renal cancer with β-TRCP and CK1 signalling being potential therapeutic targets.
- Nagashima K, Fukushima H, Shimizu K, Yamada A, Hidaka M, Hasumi H, Ikebe T, Fukumoto S, Okabe K, & Inuzuka H (2016). Nutrient-induced FNIP degradation by SCFβ-TRCP regulates FLCN complex localization and promotes renal cancer progression. Oncotarget PMID: 28039480