It is well known that mutations in the folliculin gene (FLCN) cause BHD syndrome; a disorder that can result in fibrofolliculomas, lung and kidney cysts, pneumothorax and renal cancers. The FLCN protein has long been suspected of acting as a tumour suppressor and has been identified as a modulator of the AMPK-mTOR pathway (Baba et al. 2006), a signaling cascade involved in cell metabolic processes and cancer. Unfortunately, the precise mechanism by which FLCN exerts its tumour suppressor role remains unknown. This is addressed by a recent research paper by Laviolette et al. 2017, which finds that FLCN acts as a GTPase activating protein (GAP) for RAB7A and proposes that normal FLCN-RAB7A interactions may suppress tumour growth by modulating the receptor tyrosine kinase (RTK) EGFR (epidermal growth factor receptor).
Elevated EGFR signaling has already been heavily associated with cancer mechanisms (reviewed in Jones et al. 2014) and consistent with this, the paper demonstrates increased levels of EGFR and EFGR signaling in FLCN defective cells (human follicular thyroid carcinoma cell line) relative to FLCN wildtype cells. Upon stimulation with EGF ligand, higher levels of phosphorylated EGFR (and of phosphorylated downstream effector molecules pERK and pS6) were detected in both FLCN deficient cells and those expressing tumour-associated FLCN mutations. In addition to this, high levels of EGFR-activated signaling molecules were detected (via immunohistochemistry) in the renal cysts and tumours from a conditional FLCN knockout mouse and in various types of renal cell carcinomas biopsied from BHD patients. Taken together, this is evidence for the involvement of heightened EGFR signaling in the tumorigenesis associated with FLCN dysfunction. The implications of this finding are that a loss of FLCN function may result in increased mTOR activity (which is downstream of the EGFR pathway) and the paper states that this is contrary to studies claiming FLCN activates mTOR (Petit et al. 2013). However, the authors acknowledge that many of those experiment were performed under different stimulating conditions (amino acid vs EGF excitation) and raises the hypothesis that the interactions and dynamics of FLCN could vary according to the stimulus.
This study identifies a novel interaction between FLCN and GTPase RAB7A by co-immunoprecipitation experiments and mass spectrometry. The FLCN-RAB7A interaction was investigated in more detail using RAB7A proteins locked in several conformations, including a constitutively active GTP-bound form (induced by a Q67L mutation) and a dominant negative form (T22N mutation). Here, preferential precipitation of both the constitutively active and wildtype forms of RAB7A suggested that FLCN could be acting as a GTPase activating protein (GAP) that promotes hydrolysing activity in RAB7A. This hypothesis was confirmed in a functional assay that revealed FLCN enhances the GTPase activity of RAB7A and that GAP activity was lost in FLCN when it housed the tumour-associated mutation K508R.
FLCN is known to regulate the spatial distribution of lysosomes via regulating Rab-RILP interactions in a process that is dependent on nutrient status (Starling et al. 2016). Furthermore, Rab7A is known to regulate the endocytic recycling and lysosomal degradation of EGFRs (Ceresa et al. 2006, Rush et al. 2013). Therefore, the authors of this paper looked to examine how FLCN, as a Rab7A GAP, effects EGFR trafficking and signaling. Intricate imaging experiments showed that FLCN-deficient cells (renal carcinoma cell line) had slower endocytic trafficking of EGFRs compared to normal cells and this manifested as an accumulation of EGFR in early endosomes, alongside a delayed transition into late endosomes. The authors noted that delayed endocytic trafficking could result in more recycled and less degraded EGFR, causing an increase and prolonging of EGFR signaling. Accordingly, this provides a potential explanation for the observed heightened EGFR signaling in FLCN deficient cells and BHD tumours (see above).
Having implicated the involvement of Rab7A and EGFR signaling in FLCN’s tumour suppressor role, the authors examined the mechanism in vivo. Afatinib, an inhibitor of EGFR signaling, was used to treat FLCN-negative induced tumour xenografts in mice. Consistent with the general findings of this paper, that there is heighten EGFR signaling in FLCN defective cells, the study found that Afatinib significantly hindered the growth of these tumours compared to the control-treated mice but, notably, there was no tumour regression. Although the focus of this paper was on EGFR signaling, the authors highlight the importance of investigating the role of other RTK pathways in FLCN-associated renal cancers. Furthermore, it would be interesting to see, with further research and study, whether or not Afatinib holds any promise as a future treatment of BHD.