All models used here rely on WT

All models used here rely on WT FGFR3, which is activated by exogenous FGF ligand to alter chondrocyte proliferation and differentiation. This approach toward modeling the aberrant FGFR signaling in cartilage differs from the actual situation in ACH or TD, where the chondrocytes are exposed to long-term FGFR signal mediated by constitutively active FGFR3. If statins affect, from an unknown reason, a biogenesis and/or signaling of mutated, but not the WT, FGFR3 as suggested earlier, they could not show any activity in the models used here. Moreover, statin treatment may change the cholesterol level in the plasma membrane to interfere with signaling and internalization of mutant but not WT FGFR3. Reduced receptor activity and Fatostatin A receptor attributed to disrupted cholesterol level in plasma membrane upon statin treatment has been previously described for LOX-1, a primary receptor for oxidized low-density lipoprotein. Since the effect of statins may vary according to which statin is used and also among different cell lines46, 47, the models used here do not have to necessarily respond to the statins used. Alternatively, statins may only produce a subtle inhibitory effect on FGFR3 signaling which is sufficient to rescue the FGFR phenotypes in vivo, but not in the models used here where a robust FGFR3 activation is achieved by addition of FGF ligand. With the partial exception of cultured embryonal tibias, the models used here by no means reproduce the complex spatiotemporal interactions among several signaling systems involved in precise coordination of chondrocyte proliferation and differentiation which underlies the proper skeletal growth. It is therefore possible that, in vivo, statins rescue the FGFR3 inhibitory effect on skeletal growth indirectly, via modulation of other signaling pathway(s) essential in growth plate regulation. The fact that statins are known to affect several signaling pathways essential for skeletal growth, such as AKT, BMP and Hedgehog signaling lends support to the above-mentioned hypothesis48, 49, 50, 51, 52. In summary, the evidence collected here argues against a direct statin effect on inhibition of FGFR3 signaling in chondrocytes. Further investigations should be carried-out to unravel the exact mechanism of statin-mediated rescue of the FGFR3-related skeletal dysplasia.
Author contributions All authors contributed substantially to this work including drafting the experiments, critically commenting the intellectual content and approving the manuscript for publication. Specifically BF and PK designed the experiments and wrote the manuscript; BF, NR, LB, MH, MB, MKB, IG, IV and MV collected the data and prepared figures; PK obtained funding. Dr. Pavel Krejci (krejcip@med.muni.cz) takes whole responsibility for integrity of this work.
Competing interest statement
Acknowledgements This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (KONTAKT LH12004, MUNI/A/0810/2016) and the Czech Science Foundation (14-31540S).
Introduction Hepatocellular carcinoma (HCC) is one of the most common types (70%–90%) of liver cancer and a leading cause of cancer-related death worldwide [1]. Because HCC is a highly vascularized tumor type [2], targeting pro-angiogenic signaling such as vascular endothelial growth factor (VEGF) is a reasonable treatment option for patients with HCC. The multi-targeted tyrosine kinase inhibitor sorafenib, which has inhibitory activity against VEGFR1–3, RAF kinases, PDGFRα and β, FLT3, RET, and KIT, has been used as a first-line systemic therapy for patients with advanced unresectable HCC (uHCC) for 10 years, but tumor responses have been limited, and further improvements are required [3]. The FGF family is composed of 18 ligands and 4 receptors. FGF signaling pathways regulate fundamental development processes and many physiological roles in adult organisms [4], and are thought to underlie the mechanism of escape from anti-VEGF therapies [5]. Deregulated FGF signaling pathways including receptor mutations and amplifications have been implicated in several tumor types, and recent genomic studies of HCC tumors have revealed potential alterations of FGF signaling pathways [6,7]. In particular, FGF19 expression has been shown to correlate with poor prognosis of HCC patients, and overexpression of Fgf19 induces hepatocellular carcinogenesis in mouse models [8,9]. FGFR4-specific inhibitors recently demonstrated tumor responses in HCC patients with FGF19-expression in clinical trials [10].