Update on clinical trials and treatments for RCC

Renal cell carcinoma (RCC) is the most common type of kidney cancer and although the majority of cases are sporadic approximately 3% of cases are caused by genetic conditions such as BHDVHLHLRCC and TSC (Randall et al., 2014). These inherited forms of RCC have provided great insights into sporadic cancer genetics. BHD patients can develop multiple kidney tumours.  In most cases these tumours are small local RCCs that can be surgically removed.  However, these treatments are not without risk, and sometimes complete nephrectomies are carried out which leave patients with severely reduced kidney function and at risk of recurrence. The development of selective drug treatments that target only cancer cells can therefore improve disease outcome and increase patient quality of life. Even though significant advances have been made in the treatment of kidney cancer, there is a need for effective and more tolerable treatments, both for single agent and combination use. This blog summarises recent results from clinical trials assessing new treatments.

  • The U.S. Food and Drug Administration (FDA) very recently approved Lenvima (lenvatinib), a multiple receptor tyrosine kinase inhibitor (TKI), in combination with Everolimus for the treatment of patients with advanced renal cell carcinoma (aRCC) who were previously treated with an anti-angiogenic therapy. This approval was based on the impressive results of aphase II study with 153 patients (Motzer et al., 2015), in which the combination of Lenvima and Everolimus significantly prolonged progression-free survival and median overall survival when compared with Everolimus alone, the standard of care for patients with aRCC following prior anti-angiogenic therapy.
  • AVEO Oncology have announced that the first patient has been dose in the TIVO-3 trial, a randomized study created to compare the vascular endothelial growth factor (VEGF) TKI tivozanib hydrochloride to sorafenib in patients with refractory advanced RCC. The Phase III trial is expected to enrol approximately 322 patients with recurrent or metastatic RCC who have failed at least two prior treatments, including other VEGFR-TKI therapies. The primary endpoint of the study is progression free survival. Secondary endpoints include overall survival, overall response rate, safety and tolerability. Top line readout of the study is currently projected for the first quarter of 2018 and depending on the results the study completion date is currently projected for June 2019.
  • A small phase II study (Muselaers et al., 2016) with only 14 patients highlights modest efficacy and a concerning safety profile associated with the 177Lu-labelled girentuximab compound in patients with metastatic RCC. Carbonic anhydrase IX (CAIX) expression has been investigated as a prognostic biomarker in RCC and as a consequence CAIX-directed treatments have emerged, including 177Lu-labelled girentuximab. However, the questionable efficacy and concerning safety profile of the study might limit research to develop this agent further.
  • Very recently, Pfizer announced positive results from the phase III S-TRAC trial of SUTENT (sunitinib), an oral TKI, as adjuvant therapy in patients with RCC at high risk for recurrence after surgery. The study that includes more than 670 patients met its primary endpoint of improving disease-free survival (DFS) becoming the first RCC trial of a TKI to prolong DFS in the adjuvant setting. The concept of adjuvant therapy is to help lower the risk of cancer recurrence in patients with early-stage cancer. The adverse events observed for the compound in the trial were consistent with its known safety profile. Full efficacy and safety data will be submitted for presentation at the ESMO 2016 Congress in Copenhagen, October 2016 and study completion date is currently projected for August 2017.

The development of new and more efficient drug treatments for RCC is a very active field. Ongoing basic research into the biology of kidney tumorigenesis and the identification of biomarkers, a topic discussed in a past blog post, will enable a more efficient selection of patient cohorts more likely to respond to treatment and the design of more specific and targeted single or combination drugs.

Randall JM, Millard F, & Kurzrock R (2014). Molecular aberrations, targeted therapy, and renal cell carcinoma: current state-of-the-art. Cancer metastasis reviews, 33 (4), 1109-24 PMID: 25365943

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Folliculin is required for embryonic brain development in zebrafish

Birt-Hogg-Dubé syndrome (BHD) is caused by mutations in the gene encoding folliculin (FLCN). How this leads to the BHD clinical manifestations is not yet clear. Since homozygous mutations of FLCN are lethal in mice, rats and dogs at early embryonic stage (Hasumi et al., 2009), zebrafish is a valuable alternative model to study the developmental functions of FLCN. Newly published research from Kenyon et al. (2016) examines the role of FLCN in zebrafish development using morpholino oligonucleotides to generate a zebrafish BHD model and reconcile the expression of FLCN transcripts in the developing embryo with the phenotype associated with the morpholino knock-down of FLCN.

Using genome level analysis of the zebrafish transcriptome authors analysed FLCN expression at different stages of development. In situ hybridization was used to identify FLCN expression during embryo development. Raised FLCN expression was seen at different developmental stages in fin bud, somites, eye and regions of the brain.  Overlap in the expression of FLCN and the proliferating cell nuclear antigen (PCNA) gene was observed in brain regions suggesting that FLCN may play a role regulating proliferation in the embryo brain. To test this and other FLCN functions the authors used morpholino oligonucleotides to block FLCN expression. Efficiency of morpholino gene knockdown was assessed by PCR, however, the authors were unable to assess protein levels. Knockdown of the FLCN gene by microinjection of FLCN morpholino resulted in clear phenotypic effects at different development stages. Embryos injected with FLCN morpholino showed developmental arrest, increased cell death in the brain, brain oedema, problems with tail circulation and larger and thinner yolk extension when compared with mismatch control morpholino injected embryos. This phenotype was rescued by injection of FLCN RNA in embryos, suggesting that the was caused by reduced expression of FLCN and that FLCN may be required for the development of the zebrafish embryo in the brain.

As BHD patients have an increased risk of developing kidney tumours (Menko et al., 2009) the authors studied the effect of FLCN on kidney development in zebrafish embryos using a pronephros and exocrine pancreas transgenic cell line. Injecting FLCN morpholino into the cell line did not cause defects in pronephric development when compared with controls. BHD was also recently described as a novel ciliopathy (Luijten et al., 2013), however, in this study, there was no difference in cilia expression in the pronephros and central canal cilia in embryos injected with FLCN morpholino. Author discuss that it is possible that although cilia seem to be present in the FLCN knockdown embryos, they may not be functional.

Since there were no obvious defects in motile cilia morphology and in the developing kidney, the authors examined other processes that might be affected by FLCN deficiency. FLCN has been shown to affect cell cycle progression (Nahorski et al., 2012; Laviolette et al., 2013; Kawai et al., 2013; Lu et al., 2014). Authors used an in vivo labelling tool to monitor cell cycle regulation in zebrafish embryos after FLCN knockdown. The zFucci system is composed of two transgenes that fluorescently label G1 and S-M phase nuclei in the living zebrafish embryo. zFucci transgenic embryos were injected with FLCN morpholino or mismatch control and S-M and G1 phases were monitored. Results showed a significant drop in the number of cells in S-M phases and a corresponding increase in G1 cells in FLCN morpholino injected embryos particularly in the retina and brain suggesting a disruption of the cell cycle in the brain as a result of FLCN knockdown.  The authors did not try to rescue the phenotype.  To determine if the change in cell cycle behaviour could be attributed to a specific development stage authors used time-lapse confocal microscopy in FLCN morpholino injected and mismatch control embryos. Results showed that initially embryos showed comparable levels of cells in G1 phase but as time progressed there was an increase in cells in G1 phase in FLCN morpholino injected embryos suggesting that there is no specific stage with a dramatic change in cell cycle behaviour but instead a gradual loss of proliferation of the cells. These results show that FLCN plays a role in cell cycle, however, the exact nature of the role remains to be determined and authors discuss that the cell cycle effects might be secondary to other consequences of FLCN knockdown like the regulation of AMPK signalling.

In summary, the study shows that FLCN is transcribed during embryonic development with elevated transcription levels in proliferating tissues of the zebrafish embryo, that FLCN is probably required for embryonic brain morphogenesis and that it affects the cell cycle particularly in the brain. BHD patients show no defects in the brain, however, all known BHD patients only show a mutation in one copy of the FLCN gene (Wei et al., 2009). This study provides previously undescribed and informative insights into the role of FLCN in vertebrates.

  • Kenyon EJ, Luijten MN, Gill H, Li N, Rawlings M, Bull JC, Hadzhiev Y, van Steensel MA, Maher E, & Mueller F (2016). Expression and knockdown of zebrafish folliculin suggests requirement for embryonic brain morphogenesis. BMC developmental biology, 16 (1) PMID: 27391801
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FNIP1 and FNIP2 inhibit Hsp90 chaperone cycle and enhance drug binding

Heat shock protein-90 (Hsp90) is a molecular chaperone required for folding, stability and activity of many proteins, known as clients, including drivers of tumour initiation, progression and metastasis (Rohl et al. 2013). ATPase binding and hydrolysis is essential for the chaperone function of Hsp90. ATPase function is regulated by other proteins known as co-chaperones. In an interesting new study, Woodford et al. (2016) show that the stability of the tumour suppressor folliculin (FLCN), whose mutations cause Birt-Hogg-Dubé syndrome, is dependent on the chaperone function of Hsp90. Authors report that folliculin-interacting protein (FNIP)1 and FNIP2 act as co-chaperones of Hsp90 by regulating its ATPase activity and chaperoning. They also show that the Aha1 co-chaperone competes with FNIPs and can stimulate Hsp90 ATPase activity and that FNIPs enhance the binding of Hsp90 to its inhibitors.

Using human embryonic kidney 293 (HEK293) cells, authors used mass spectrometry to show that that FLCN interacts with the chaperones Hsp70 and Hsp90, their regulators and members of the chaperonin system TRiC. Hsp90 helps to protect its clients from degradation (Rohl et al. 2015), so the authors treated HEK293 cells with the Hsp90 inhibitor ganetespib (GB) and found that FLCN is then ubiquitinated and degraded in the proteasome.

FNIPs were shown to interact with Hsp90 by immunoprecipitation. Treating HEK293 cells with GB did not affect FNIP stability, suggesting that the FNIPs act as co-chaperones of Hsp90 rather than clients. Direct interaction between FNIP1 and Hsp90 was shown and although Hsp90 and FLCN did not directly interact, pre-incubation of Hsp90 with FNIP1 facilitated the Hsp90–FNIP1–FLCN complex formation suggesting that FNIPs are involved in loading of FLCN to Hsp90. Silencing either FNIP1 or FNIP2 with small interfering RNA (siRNA) caused a small decrease in Hsp90 ‘client’ protein levels, while silencing both significantly decreased the stability of ‘clients’ suggesting that the FNIP1 and FNIP2 are functionally redundant here. Overexpression of FNIPs caused an increase in the activity of Hsp90 ‘clients’, including FLCN. Because Hsp90 chaperone function is coupled to its ATPase activity (Panaretou et al. 1998), the impact of FNIPs on Hsp90 ATPase activity was assessed. The presence of FNIPs significantly inhibited the ATPase activity of Hsp90 suggesting that they are potent inhibitors of the chaperone cycle. The authors depleted FNIP protein levels in HEK293 cells to study FNIP effects on Hsp90 interactions with other co-chaperones. Silencing FNIPs increased Hsp90 interaction with the co-chaperones Aha1 and PP5, while overexpressing FNIPs led to significantly reduced Hsp90 interaction with Aha1 and PP5. While FNIPs inhibited the ATPase activity of Hsp90, subsequently adding Aha1 stimulated Hsp90 ATPase activity. These data suggest that Aha1 and the FNIPs compete to bind Hsp90. The impact of FNIPs on Hsp90 binding to ATP or GB was also examined. Overexpression of FNIPs in HEK293 cells led to increase in Hsp90 binding to ATP and to the inhibitor GB. Silencing of FNIPs did not have an impact on Hsp90 binding to ATP but significantly decreased binding to GB.

Tumour cells were already known to be sensitive to Hsp90 inhibitors (Dunn et al. 2015; Woodford et al. 2016), so the authors tested the effect of FNIPs on this sensitivity. FNIPs were highly expressed in a variety of different cancer cell lines and Hsp90 interaction with FNIPs was detected via immunoprecipitation. Two apoptotic markers were abundant in cancer cells treated with GB inhibitor but this effect was not seen in samples lacking FNIPs suggesting that overexpression of FNIPs contributes towards cancer cell sensitivity to Hsp90 inhibitors. Interestingly for BHD, tumours and adjacent normal tissues from patients with renal cell carcinoma (RCC) were analysed and the data showed that FNIPs were overexpressed in RCC tumours compared with adjacent tissues and the Hsp90 from tumours had a higher affinity for binding to GB. As expected, there was a greater association between Hsp90 and FNIPs in tumours compared with normal tissues. However, Hsp90 bound more strongly to Aha1 in normal tissues than in tumours even though both tissues expressed equal levels of Aha1. Addition of FNIP to the protein lysates from normal tissues displaced Aha1 interaction with Hsp90 and increased Hsp90 binding to GB suggesting that FNIPs make renal tumours sensitive to Hsp90 inhibitors.

In summary, the authors show that FNIPs expression correlates with the cellular response to Hsp90 inhibitors, that they are co-chaperones of Hsp90, and that they inhibit Hsp90 ATPase activity.  The results suggest that FNIPs expression level could potentially serve as a predictive indicator of tumour response to Hsp90 inhibitors used in cancer therapy.

  • Woodford MR, Dunn DM, Blanden AR, Capriotti D, Loiselle D, Prodromou C, Panaretou B, Hughes PF, Smith A, Ackerman W, Haystead TA, Loh SN, Bourboulia D, Schmidt LS, Marston Linehan W, Bratslavsky G, & Mollapour M (2016). The FNIP co-chaperones decelerate the Hsp90 chaperone cycle and enhance drug binding. Nature communications, 7 PMID: 27353360
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Findacure workshop: “How rare disease patient groups can work with researchers”

Although collectively, rare diseases affect an estimated 6 to 8 per cent of the EU population (EURORDIS), the low prevalence of individual rare diseases means they have historically received little attention from government or industry research funders (Forman et al., 2012; Litterman et al., 2014). In the absence of these resources, support from rare disease patient groups is crucial for the initiation and progress of biomedical research. The benefits of rare disease patient groups working with researchers, the different ways patient groups can support research and how to establish partnerships were the themes of last week’s Findacure workshop in London.

  • Flora Raffai from Findacure started the workshop by introducing Findacure and their mission of empowering patient groups and promoting collaboration.
  • Dr Sara Ellis from the AMRC presented the talk ‘Peer review matters: Using peer review to invest in health and medical research with the greatest chance of having an impact’, Dr Ellis started by showing that AMRC member charities, several of them rare disease charities, fund over a third of all publically funded medical research in the UK. To decide what research to fund, AMRC members use peer review to decide if a research project is valuable. In peer review, the research proposal is evaluated by people with similar interests and expertise to those who wrote the project proposal and the reviewers can be not only researchers but also other professionals, patients, patient groups or members of the public. In the involvement of patients and patient groups, it was emphasised that expertise does not necessarily mean academics and that patients/patient groups are often involved in peer review as experts in their own condition. In the second part of her talk Dr Ellis mentioned the importance of charities having a comprehensive research strategy where is clear what are their priorities and the research they wish to fund.
  • Heather Band from the Batten Disease Family Association (BDFA) talked about the different research projects, PhD studentships and research equipment they have been able to fund and the importance of communicating with researchers and of continuity of funding. The BDFA joined with other organisations to create a patient registry, a powerful tool that shows how patients’ groups and researchers can work together. In addition, the BDFA provides mechanisms for individual families to directly fund research. Another good example of bringing patients, patient groups and researchers together mentioned was their Lab Open day where patients and funders meet the researchers and get to see first-hand the progress of a project and interact with researchers.
  • Professor David White from the Cavernoma Alliance UK presented the talk ‘Setting a research agenda with the James Lind Alliance Priority Setting Partnership (PSP)’ explaining how creating a partnership of patients, representatives of patient support organisations, carers and clinicians they have determined, having started with over 2000 uncertainties, the 10 most important questions for research into the rare disease cavernoma. The PSP was published and promoted to researchers and research funders.
  • Dr Jutta Roth from the Oxford Rare Disease Initiative presented the talk ‘New ways for patient organisations to get involved’ explaining how the initiative brings together rare disease stakeholders such as researchers, patient organisations, industry and funders to advance the development of new therapies for patients. Dr Jutta gave examples of patient organisations driven collaborations and of how important is it for patient organisations to participate in meetings with researchers and receive research updates.

There is a global growing interest in strengthening rare disease patient organisations engagement in research (Montserrat et al., 2013; Forsythe et al., 2014). Studies based in the US (Landy et al., 2012), the EU (EURORDIS, 2012) and Australia (Pinto et al., 2016) have characterized patient organisations participation and impact in clinical research. Patient groups are experts in the disease, know the expert clinicians, the researchers and, more importantly, the patients. They can help establish priorities in the field and setting research strategies, design more accessible and suitable clinical trials, mobilise patients and motivate researchers. Collaborations benefit the patient groups as there is a focus on their disease, increasing awareness and knowledge and the researchers by motivating them and reminding them why research matters.

Forman, J., Taruscio, D., Llera, V. A., Barrera, L. A., Coté, T. R., Edfjäll, C., … Henter, J. I. (2012). The need for worldwide policy and action plans for rare diseases. Acta Paediatrica, International Journal of Paediatrics.

Forsythe, L. P., Szydlowski, V., Murad, M. H., Ip, S., Wang, Z., Elraiyah, T. A., … Hickam, D. H. (2014). A systematic review of approaches for engaging patients for research on rare diseases. Journal of General Internal Medicine.

Landy, D. C., Brinich, M. a., Colten, M. E., Horn, E. J., Terry, S. F., & Sharp, R. R. (2012). How disease advocacy organizations participate in clinical research: a survey of genetic organizations. Genetics in Medicine

Litterman, N. K., Rhee, M., Swinney, D. C., & Ekins, S. (2014). Collaboration for rare disease drug discovery research.

Montserrat Moliner, A., & Waligora, J. (2013). The European union policy in the field of rare diseases. Public Health Genomics, 16(6), 268–277.

Pinto, D., Martin, D., & Chenhall, R. (2016). The involvement of patient organisations in rare disease research: a mixed methods study in Australia. Orphanet Journal of Rare Diseases, 11, 2.

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FNIP1 negatively regulates AMPK activity

Birt-Hogg-Dubé (BHD) syndrome is caused by mutations of the folliculin (FLCN) gene. FLCN is a tumour-suppressor protein which associates with two homologous proteins, folliculin-interacting protein (FNIP)1 and FNIP2. Previous studies have reported that the FLCN/FNIP complex acts as a positive regulator of the AMP-activated protein kinase (AMPK) complex (Hasumi et al., 2015), while other studies suggest the opposite (Reyes et al., 2015). To address this issue, a new report from Siggs et al. (2016) uses a new mouse model of FNIP1 deficiency to find that Fnip1 mutation leads to B-cell deficiency and development of cardiomyopathy with gain-of-function mutations in AMPK supporting the idea that FNIP acts as a negative regulator of AMPK.

Using whole-genome sequencing in mouse mutants with an autosomal recessive B-cell deficiency phenotype the authors identified that this was associated with a splice donor variant mutation in Fnip1. By flow cytometry they showed that B cells were absent from the peritoneum and spleen of Fnip1 homozygous mutants. In the bone marrow (BM) there was an absence of IgM+ or IgD+ cells, although B-cell precursors were still present. Heterozygous mutants revealed a reduced frequency of the marginal zone B-cell population. Because the antiapoptotic protein BCL2 is important for B-cell survival the effects of its overexpression on B-cell development in Fnip1 mutants were measured. Unlike previously reported (Baba et al., 2012), BCL2 overexpression only partially restored B-cell values in the BM, peritoneum and spleen. Examination of the hearts of homozygous Fnip1 mutants revealed that they were enlarged due to hypertrophy and that skeletal muscle was darker than heterozygous or wild-type mice. Histology and biochemical analysis revealed that the mutant myocardium had a marked accumulation of glycogen. Homozygous Fnip1 mutant mice also showed high expression of Nppa, Nppb and Acta1, markers of cardiac stress. Due to the known interaction between FNIP1 and AMPK (Baba et al., 2006) and the cardiac phenotype observed, authors assessed the effect of the Fnip1 mutation on cardiac AMPK activity. A basal activation of the γ2 subunit-containing AMPK complexes but reduced AMP responsiveness, in homozygous Fnip1 mutants, was observed in cardiac tissue but not in the liver. This suggests that Fnip1 and Fnip2 may be functionally redundant in the liver as they are in the kidney (Hasumi et al., 2015). One of the roles of AMPK is to promote autophagy via different pathways including the unc-51-like autophagy activating kinase 1 (ULK1) (Gwinn et al., 2008; Egan et al., 2011) which is activated via phosphorylation at serine 555. In the present study, S555 p-ULK1 is higher in Fnip1 mutant BM cells suggesting that autophagy is enhanced. This was supported by the staining of LC3, an autophagosome marker, in Fnip1 mutant B cells. Overexpression of BLC2 reduced LC3 staining in mutant and wild type cells indicating that BLC2 could oppose the enhanced autophagy in Fnip1 mutant B cells while increasing survival (Cash et al., 2011). AMPK promotes cell-cycle arrest by stabilizing p53 (Jones et al., 2005), p53 is known to enhance autophagy and apoptosis in response to cellular stress (Crighton et al., 2006). However, no differences in B-cell numbers were observed between p53-sufficient or deficient Fnip1 mutants in this study. Lack of Fnip1 caused a block in B-cell development and although autophagy was increased, its inhibition, via deletion of the autophagy protein Atg3, failed to rescue the phenotype, suggesting that enhanced autophagy alone is not sufficient to block B-cell development.

In summary, the study supports other reports showing a non-redundant role for FNIP1 in B-cell development (Baba et al., 2012), skeletal muscle position (Reyes et al., 2015) and cardiac function (Hasumi et al., 2015) and it provides evidence that FNIP1 is an endogenous negative regulator of AMPK. Supporting this idea, previous studies have reported that FLCN repressed AMPK in C. elegans and mammalian cells (Possik et al., 2014; Yan et al., 2014). In these models, absence of FLCN resulted in AMPK activation, which induced autophagy. However, these models oppose the suggestion that autophagy is reduced in BHD-associated tumour tissue (Dunlop et al., 2014).

Authors are unsure how the lack of Fnip1 causes a block in B-cell development and it remains to be investigated if another AMPK-regulated pathway or a different pathway is responsible for the phenotype. It is therefore crucial to pursue future investigations on the role of AMPK and the FLCN/FNIP complex in BHD.

  • Siggs OM, Stockenhuber A, Deobagkar-Lele M, Bull KR, Crockford TL, Kingston BL, Cornall RJ. (2016). Mutation of Fnip1 is associated with B-cell deficiency, cardiomyopathy, and elevated AMPK activity. Proceedings of the National Academy of Sciences of the United States of America. PMID: 27303042


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FLCN activates mTORC1 by maintaining lysosomal leucine level

The intracellular amino acid pool within the lysosome has been shown to activate the mTORC1 signaling pathway (Zoncu et al., 2011; Jewell et al., 2013). However, how the sequester of the signaling molecules within the lysosome occurs remains poorly understood. New research from Wu et al. (2016) shows that the suppression of FLCN, a tumour suppressor gene associated with the Birt-Hogg-Dubé (BHD) syndrome, controls mTORC1 activity by modulating the lysosomal leucine levels. FLCN exerts this new function by regulating the accumulation of the amino acid transporter PAT1 on the lysosome surface.

Previous work by the authors using a Drosophila model showed that fly FLCN mutants had a growth defects and did not reach adulthood. Interestingly, the growth defects were rescued by supplementation with high levels of leucine, but not other amino acids, in the medium. Adding an inhibitor of mTOR to the leucine-rich medium failed to rescue the slow growth phenotype (Liu et al., 2013). In mammalian cell systems, FLCN was necessary to anchor mTORC1 and this function was performed at the lysosome (Petit et al., 2013; Tsun et al., 2013). In the present study, Wu et al. (2016) show in human embryonic kidney (HEK) 293 cells, that depletion of FLCN suppresses mTORC1 activity. Addition of high amounts of leucine, but not other tested amino acids, to the medium reverses this down-regulation of mTORC1 in FLCN-depleted cells. Starving cells and then adding either basal or high levels of leucine to the medium shows that depletion of FLCN sensitizes cells to the available leucine for mTORC1 induction. The authors confirm work by previous groups (Petit et al., 2013; Tsun et al., 2013) showing that FLCN is localized on the lysosome in a nutrient-dependent manner. They show by western blotting that leucine is reduced in lysosomes from cells lacking FLCN and that this effect is reversed by supplementation with high levels of leucine in the medium. This suggests that FLCN plays an important role keeping the leucine level in lysosome but this can alternatively be achieved by increasing the amount of leucine supplied to the environment to keep the leucine signal strong enough to activate mTORC1. To test if ectopic FLCN would sequester more leucine to the lysosome, the authors generate a cell line overexpressing FLCN (FLCN-HA). In starving conditions, leucine levels and mTORC1 activation are decreased in wild-type cells. In FLCN-HA cells this reduction is much less pronounced suggesting that ectopic FLCN promotes the accumulation of leucine within the lysosome, enabling cells to resist to leucine shortages in the environment and facilitating mTORC1 activation. Similar results were shown in a Drosophila model. FLCN is located on the lysosome and it seems to regulate the efflux of leucine. This process requires it to be loaded onto membrane-anchored transporters. PAT1, a lysosome-associated membrane protein whose overexpression promotes the release of amino acids from lysosome and inhibits mTORC1 (Zoncu et al., 2011) seemed a good candidate to be involved in the FLCN-mediated mTORC1 signaling. After confirming that overexpression of PAT1 in HEK293 cells inhibits mTORC1 the authors show that both the addition of high levels of leucine to the medium and the overexpression of FLCN prevent this inhibitory effect. In addition, depletion of PAT1 using siRNA inhibits the starvation-induced decrease of mTORC1 activation and this effect is counteracted by co-depletion of FLCN. These results led to the conclusion that FLCN and PAT1 antagonize each other to control mTORC1 pathway. In starvation, cells recruit PAT1 to the lysosome to recycle the luminal storage, resulting in a decreased leucine level. However, authors show that this recruitment of PAT1 can be inhibited by ectopic FLCN resulting in the maintenance of the lysosome level. Immunoprecipitation experiments show that FLCN may regulate the localization of PAT1 through direct interactions.

In summary, this study shows a new role for FLCN in modulating the leucine level in the lysosome and how important this mechanism is to regulate mTORC1 signaling pathway. Previous work by Preston et al. (2011) on the FLCN deficient BHD patient-derived renal tumour cell line (UOK257) showed that these cells have a higher dependency on glucose metabolism demonstrating a loss of ‘metabolic flexibility’. The present work sheds light into how cells sense the available nutrients in their environment and helps to appreciate the context-dependent relations between FLCN and mTORC1, crucial for the advancement in BHD syndrome research where targeting metabolic pathways may be used therapeutically to treat BHD-associated kidney lesions.

Wu X, Zhao L, Chen Z, Ji X, Qiao X, Jin Y, & Liu W (2016). FLCN Maintains the Leucine Level in Lysosome to Stimulate mTORC1. PloS one, 11 (6) PMID: 27280402

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BHD syndrome and thyroid conditions

Dong et al., (2016) have recently reported two BHD syndrome patients also affected with papillary thyroid cancer. Lesions were bilateral and multifocal and small lymph node metastases occurred. Due to the small number of patients in the study the authors are unsure whether thyroid cancer in BHD patients is susceptible to exhibiting bilaterally and lymph node metastasis. However, they suggest considering thyroidectomy and prophylactic lymph node dissection for thyroid cancer patients with BHD. They also strongly recommend neck ultrasound for BHD patients and their families and suggest a large-scale investigation be conducted to evaluate the prevalence of thyroid cancer or nodules in patients with BHD.

So far, there is no evidence associating BHD and thyroid conditions.

The original BHD patients were found in a family in which six of nine siblings had hereditary thyroid medullary carcinoma (Birt et al. 1977). However, it was subsequently shown that these six patients also displayed a mutation in the RET proto-oncogene which might have caused the thyroid carcinoma (Toro et al., 1999). Several cases of thyroid cancer have been reported in BHD patients in the literature. Gunji et al. (2007) identify thyroid carcinoma in one of five unrelated BHD cases studied where the patient also had a familial history of thyroid cancer. Fahmy et al. (2007), Toro et al. (2008), Kunogi et al. (2010) and Yamada et al. (2014) describe another five cases of thyroid cancer in BHD patients. In all cases the authors did not attribute the thyroid tumour to BHD or investigate a link between the two. Benusiglio et al., 2014 describe a BHD patient with thyroid cancer where a FLCN mutation was identified both in the patient’s blood DNA and thyroid tumour DNA, this provides a molecular basis for this association, however, alternative genetic lesions or causes of thyroid cancer were not ruled out, so no association between the patient’s BHD and thyroid cancer could be shown. In a previous study Warren et al. (2004) did not find any FLCN mRNA expression in normal thyroid.

In another report, mutation of FLCN, TSC2 and TP53 were found in a case of anaplastic thyroid cancer treated with Everolimus that had a near-complete response for 18 months, at which point the tumour acquired resistance to treatment due to mutation in the mTOR gene (Wagle et al., 2014). This suggests that somatic FLCN mutation, and other genes that activate mTOR, can cause additional types of cancer to renal cell carcinoma (RCC) and make tumours resistant to treatments with mTOR inhibitors. A population-based study in the USA shows that patients with thyroid cancer have an increase in prevalence of subsequent RCC, and in patients with RCC, there is an increase in the prevalence of subsequent thyroid cancer (Van Fossen et al., 2013). This bidirectional association could be explained by shared genetic and/or environmental risk factors or treatment effects. Thyroid cancer has also been found with a greater frequency in the Cowden syndrome, familial adenomatous polyposis, and in multiple endocrine neoplasia type 2A (Cetta et al., 2000; Liaw et al., 1997; Biscolla et al., 2004).

The only study that attempts to identify a link between BHD and thyroid conditions is a five-year prospective study of twenty-two patients from ten unrelated French families with BHD (Kluger et al. 2010).  The authors identified thyroid nodules or cysts by ultrasonography in thirteen of twenty BHD patients. None of the affected individuals had thyroid carcinomas or a familial history of thyroid cancer. Overall, nine of the ten families affected by BHD with germline FLCN mutations included individuals with thyroid nodules. The high prevalence of thyroid nodules in the BHD patients in this study is suggestive, but the lack of a control group limits assessment of the significance of the results. Benhammou et al. (2011) also identified thyroid pathology in four of eleven BHD patients studied: three had hypothyroidism and one had a thyroid nodule. Mikesell et al., (2014) report another case of a BHD patient with thyroid nodules. Hypothyroidism and Hashimoto’s thyroiditis were noted in two other cases (Nadershahi et al., 1997; Khoo et al., 2002). There are also reports of multinodular goiter in association with BHD (Drummond et al., 2002; Welsch et al., 2005; Zeibek et al,. 2013; Mikesell et al., 2014). A parathyroid adenoma was diagnosed in one BHD patient of the Kluger et al. (2010) study, and previously in another BHD patient by Chung et al. (1996).

There is currently insufficient evidence to associate thyroid cancer and other thyroid conditions with BHD. However, all the studies mentioned suggest a possible link between the two that should be considered for future research.

Dong L, Gao M, Hao WJ, Zheng XQ, Li YG, Li XL, & Yu Y (2016). Case Report of Birt-Hogg-Dubé Syndrome: Germline Mutations of FLCN Detected in Patients With Renal Cancer and Thyroid Cancer. Medicine, 95 (22) PMID: 27258496

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