TSC2 mutations confer everolimus sensitivity in hepatocellular carcinomas

Hepatocellular carcinomas (HCCs) are the third leading cause of cancer deaths globally; frequently diagnosed only in the advanced stages and aggressive in nature. Although enhanced mTOR activity has a key role in HCC tumourigenesis, the EVOLVE-1 clinical trial of mTOR inhibitor everolimus found no associated improvement in overall survival (Zhu et al., 2014). However, everolimus is an effective treatment for tuberous sclerosis complex (TSC) manifestations, a rare disease associated with mutations in TSC1 and TSC2 that result in high mTOR activity. New research from Huynh et al. (2016) assessed the frequency of TSC2  loss in HCC and suggests this could predict a selective response to everolimus.

Expression of TSC2 was found to be markedly reduced in 5/9 HCC cell lines assessed (Cancer Cell line Encyclopedia collection). Huynh et al. confirmed the loss of TSC2 by immunoblot in four of these lines which showed associated increased mTOR activity and decreased AKT phosphorylation. The four TSC-null cell lines showed enhanced sensitivity, with greater inhibition of cell proliferation, to everolimus than four TSC-wildtype cell lines. Everolimus treatment also reversed the enhanced phosphorylation of S6K1, indicative of mTOR inhibition.

Huynh et al. then assessed the frequency of TSC2 mutations in patient derived xenografts. In 8/26 xenografts TSC2 was undetectable with associated increased S6K1 phosphorylation. When tumour-bearing mice where treated with 1mg/kg everolimus significant anti-tumour responses were seen in the TSC2-null tumours compared to those treated with vehicle only. In contrast the everolimus treated TSC-wildtype tumours showed little or no response indicative of selective efficacy rather than general toxicity. Treatment with everolimus also showed a dose-dependent reduction in S6K1 phosphorylation and increase in AKT activity.

Genetic sequencing of the TSC-null HCC cell lines and xenografts identified a range of mutations and deletions in 3/4 and 6/8 samples respectively. Epigenetic gene silencing was hypothesised to explain the loss of TSC2 in the remaining samples. To validate the existence of TSC2 mutations in primary tumours 13 HCC biopsies were also sequenced and three found to carry TSC2 mutations. Although sequencing can identify mutations it cannot always predict protein loss. Instead Huynh et al. developed an immunohistochemistry (IHC) assay, validated in the HCC cell lines and xenografts, where TSC2 protein levels could be quantified, and samples categorised as either TSC-null, TSC-low or TSC-wildtype. .

This IHC assay was then used to assess tumour samples from 139 patients enrolled on the EVOLVE-1 trial; eight samples were found to be TSC-null and seven to be TSC-low. Detailed assessment of overall survival in these patients determined that 6/10 treated with everolimus had an overall survival (OS) greater than the median for the whole trial: 9.53-32.72 months compared to 7.56 months. Three of the other patients receiving everolimus withdrew after 2-6 weeks treatment (OS 0.76-4.63 months). Comparatively those patients with TSC2 loss who received the placebo had a lower OS of 1.25-5.59 months. This prospective analysis supports the previous results suggesting that everolimus is more effective against tumours that lack TSC2.

Tumour heterogeneity can make treatment responses difficult to predict but understanding the molecular basis of tumourigenesis can help identify treatment targets. Everolimus has also been shown to be effective against other tumour types carrying TSC1 or TSC2 mutations including renal tumours (Voss et al., 2014), metastatic bladder cancer (Iyer et al., 2012) and thyroid tumours (Wagle et al., 2014). This highlights a need to design clinical trials, like basket and umbrella trials, that use the advances in genetic sequencing to stratify patients for a greater understanding of treatment efficacy.

This research also demonstrates how greater understanding of a protein associated with a rare inherited disease can affect the treatment of patients with a sporadic condition. Mutations associated with other cancer-predisposition conditions such as BHD and HLRCC have also been reported in sporadic tumours and could progress the development of more effective treatments based on cancer genetics rather than location.

  • Huynh H, Hao HX, Chan SL, Chen D, Ong R, Soo KC, Pochanard P, Yang D, Ruddy D, Liu M, Derti A, Balak MN, Palmer MR, Wang Y, Lee BH, Sellami D, Zhu AX, Schlegel R, & Huang A (2015). Loss of Tuberous Sclerosis Complex 2 (TSC2) Is Frequent in Hepatocellular Carcinoma and Predicts Response to mTORC1 Inhibitor Everolimus. Molecular cancer therapeutics, 14 (5), 1224-35 PMID: 25724664.
  • Iyer G, Hanrahan AJ, Milowsky MI, Al-Ahmadie H, Scott SN, Janakiraman M, Pirun M, Sander C, Socci ND, Ostrovnaya I, Viale A, Heguy A, Peng L, Chan TA, Bochner B, Bajorin DF, Berger MF, Taylor BS, Solit DB (2012). Genome sequencing identifies a basis for everolimus sensitivity. Science. 338(6104):221 PMID: 22923433.
  • Voss MH, Hakimi AA, Pham CG, Brannon AR, Chen YB, Cunha LF, Akin O, Liu H, Takeda S, Scott SN, Socci ND, Viale A, Schultz N, Sander C, Reuter VE, Russo P, Cheng EH, Motzer RJ, Berger MF, Hsieh JJ (2014). Tumor genetic analyses of patients with metastatic renal cell carcinoma and extended benefit from mTOR inhibitor therapy. Clin Cancer Res. 20(7):1955-64. PMID: 24622468.
  • Wagle N, Grabiner BC, Van Allen EM, Amin-Mansour A, Taylor-Weiner A, Rosenberg M, Gray N, Barletta JA, Guo Y, Swanson SJ, Ruan DT, Hanna GJ, Haddad RI, Getz G, Kwiatkowski DJ, Carter SL, Sabatini DM, Jänne PA, Garraway LA, Lorch JH (2014). Response and acquired resistance to everolimus in anaplastic thyroid cancer. N Engl J Med. 371(15):1426-33. PMID: 25295501.

HIF-2α regulates PD-L1 expression in RCC

Tumour cells can create immunosuppressive microenvironments by hijacking natural mechanisms such as PD-L1 expression to impair T-cell function. Several new immunotherapy treatments target the PD-1/PD-L1 pathway and have produced some long-lasting responses in patients (Motzer et al., 2015) but not all patients respond. High expression of PD-L1 in clear cell RCC (ccRCC) has been shown to correlate with metastasis and poor outcome (Thompson et al., 2007). New research from Messai et al. (2015) has assessed PD-L1 expression in ccRCC samples and cell lines carrying VHL mutations to identify meaningful correlations.

Messai et al. initially assessed PD-L1 expression in 32 ccRCC samples – 11 from VHL patients and 21 sporadic tumours – which had been sequenced for VHL gene mutations, loss of function (LOF) and loss of heterogeneity (LOH). Significantly higher PD-L1 expression was found in the tumours which had two altered VHL alleles (21/32), complete LOF (14/32) or had LOH at the VHL locus (23/32). This suggests a link between VHL status and PD-L1 expression in ccRCCs.

To further investigate the role of VHL in regulating PD-L1 expression Messai et al. reintroduced various mutated VHL constructs into VHL-deficient 786-O cells – these cells overexpress HIF-2α but not HIF-1α.  The different pVHL mutants resulted in a gradient expression of HIF-2α which positively correlated with PD-L1 mRNA and protein expression levels. In cells retransfected with wildtype VHL the expression of PD-L1 was significantly lower than those carrying VHL-mutants or no VHL constructs. This further confirms the correlation between VHL mutations and PD-L1 expression seen in the tumour samples.

Increased HIF-2α signalling resulting from VHL loss plays a role in tumourigenesis altering cellular metabolism and increasing angiogenesis. To determine the role of HIF-2α in the correlation between VHL and PD-L1 expression Messai et al. used siRNA to knockdown HIF-2α in 786-0 and A498 cells (also VHL-deficient); resulting in reduced PD-L1 mRNA, protein and surface expression. In addition overexpression of HIF-2α in cells with functional VHL results in increased PD-L1 expression. This suggests that PD-L1 expression is being regulated by HIF-2α signalling, which is mediated by VHL status.

Analysis of the PD-L1 promoter identified several putative Hypoxia Response Elements (HRE). Based on ChIP and luciferase assays, HIF-2α specifically binds to HRE4 and is transcriptionally active. Previously HIF-1α was shown to regulate PD-L1 expression in tumour infiltrating MDSCs, also through binding to HRE4 (Norman et al., 2014). To determine if HIF-1α has a similar role in ccRCC cells Messai et al. assessed PD-L1 expression in RRC4 cells which overexpress both HIF-1α and HIF-2α.  Knockdown of either HIF-1α or HIF-2α, or both resulted in a decrease in PD-L1 expression supporting a direct role for HIF signalling in PD-L1 regulation.

Further studies will be needed to determine the validity of VHL mutation status as a biomarker for PD-L1 expression and if there is any correlation to immunotherapy responses. Increased HIF signalling is generally associated with tumourigenesis and is also seen in BHD tumours and cell lines (Preston et al., 2011, Nishii et al., 2013). Greater understanding of the role of HIF signalling and PD-L1 expression in response to various treatments including immunotherapies could help identify the optimal combination or sequential treatment plans for a range of RCC patients.

  • Messai Y, Gad S, Noman MZ, Le Teuff G, Couve S, Janji B, Kammerer SF, Rioux-Leclerc N, Hasmim M, Ferlicot S, Baud V, Mejean A, Mole DR, Richard S, Eggermont AM, Albiges L, Mami-Chouaib F, Escudier B, & Chouaib S (2015). Renal Cell Carcinoma Programmed Death-ligand 1, a New Direct Target of Hypoxia-inducible Factor-2 Alpha, is Regulated by von Hippel-Lindau Gene Mutation Status. European urology PMID: 26707870.
  • Motzer RJ, Rini BI, McDermott DF, Redman BG, Kuzel TM, Harrison MR, Vaishampayan UN, Drabkin HA, George S, Logan TF, Margolin KA, Plimack ER, Lambert AM, Waxman IM, Hammers HJ (2015). Nivolumab for Metastatic Renal Cell Carcinoma: Results of a Randomized Phase II Trial. J Clin Oncol. 33(13):1430-7. PMID: 25452452.
  • Nishii T, Tanabe M, Tanaka R, Matsuzawa T, Okudela K, Nozawa A, Nakatani Y, Furuya M (2013). Unique mutation, accelerated mTOR signaling and angiogenesis in the pulmonary cysts of Birt-Hogg-Dubé syndrome. Pathol Int. 63(1):45-55. PMID: 23356225.
  • Noman MZ, Desantis G, Janji B, Hasmim M, Karray S, Dessen P, Bronte V, Chouaib S (2014). PD-L1 is a novel direct target of HIF-1α, and its blockade under hypoxia enhanced MDSC-mediated T cell activation. J Exp Med. 211(5):781-90. PMID: 24778419.
  • Preston RS, Philp A, Claessens T, Gijezen L, Dydensborg AB, Dunlop EA, Harper KT, Brinkhuizen T, Menko FH, Davies DM, Land SC, Pause A, Baar K, van Steensel MA, Tee AR (2011). Absence of the Birt-Hogg-Dubé gene product is associated with increased hypoxia-inducible factor transcriptional activity and a loss of metabolic flexibility. Oncogene. 30(10):1159-73. PMID: 21057536.
  • Thompson RH, Dong H, Kwon ED (2007). Implications of B7-H1 expression in clear cell carcinoma of the kidney for prognostication and therapy. Clin Cancer Res. 13(2 Pt 2):709s-715s. Review. PMID: 17255298.




Annual review 2015

With the end of the year fast approaching, we thought we would use this week’s blog to review the studies we’ve particularly enjoyed writing about.

In the spring Iribe et al. characterised the expression patterns in BHD renal tumours with Furuya et al. focusing on FLCN and GPNMB expression. The development of a screening panel for inherited tumours associated with FLCN mutations, rather than sporadic tumours, would enable pathologists to identify BHD patients earlier. This would help ensure they receive optimal future monitoring and treatment.

In June Chen et al. published details of a new tissue specific FLCN-deficient mouse model which develops bilateral renal cysts and tumours within one year. These mice live significantly longer than other kidney-specific FLCN knockout mice enabling longer term study of tumourigenesis. The tumourigenic potential of renal cyst cells from another mouse models was confirmed by Wu et al.  this year; after in vitro culture cells formed into sarcomatoid RCCs.

Tumours derived in both of these mouse models responded to treatment with the mTOR inhibitor rapamycin. Based on studies like these a stage II clinical trial of Everolimus (a rapamycin derivative) in BHD patients with RCC was announced in July.

In the autumn studies in the C. elegans BHD model identified a role for FLCN-1 in modulating resistance to hyperosmotic stress. Possik et al. determined that flcn-1 knockout worms could resist hyperosmotic stress due to increased glycogen accumulation and rapid osmolyte production. As such glycogen deposits could have dual roles in tumourigenesis as an energy source and protection from stress.

Towards the end of the year two groups reported on the structure and function of the yeast orthologues of FLCN and FNIP1/2 – Lst7 and Lst4. Lst4 was confirmed as a DENN-family protein by Pacitto et al. who used X-ray crystallography to solve the 3D structure. The role of Lst7-Lst4 in stimulating TORC1 activity was further elucidated by Peli-Gulli et al. – Lst7-Lst4 complexes recapitulate the reported function of FLCN-FNIP2 at the lysosomal membrane where they act as a GAP to stimulate mTOR signalling.

A recurring theme in BHD research this year has been using CT screening for pulmonary cysts to identify potential BHD patients in pneumothorax and RCC patients. Several reviews of imaging in cystic lung diseases including BHD have also helped raise awareness (Ha et al., 2015, Gupta et al., 2015, Richards et al., 2015, Ferreira Francisco et al., 2015).

Early in the year Johannesma et al. identified FLCN mutations in 7.5% of primary spontaneous pneumothorax (PSP) patients – all of whom had pulmonary cysts below the carina. Ding et al. also identified and mapped large intragenic FLCN deletions in PSP families with characteristic pulmonary cysts. More recently Johannesma et al. screened RCC patients for pulmonary cysts to determine if they could identify BHD patients; no new patients were identified but the presence of multiple cysts in the lower regions of RCC patient lungs, especially those with a family history, could still be key in differential diagnosis.

In addition to the research in September Professor Gennady Bratslavsky and Dr Mehdi Mollapour organised and ran the very successful Sixth BHD Symposium and First International Upstate Kidney Cancer Symposium in New York. Summaries of the scientific & clinical and patient & family member sessions are available online.

These topics are just a selection of those published in 2015, and we at the BHD Foundation are very much looking forward to seeing how the field develops in 2016. We wish all our readers a very Happy New Year.

  • Chen J, Huang D, Rubera I, Futami K, Wang P, Zickert P, Khoo SK, Dykema K, Zhao P, Petillo D, Cao B, Zhang Z, Si S, Schoen SR, Yang XJ, Zhou M, Xiao GQ, Wu G, Nordenskjöld M, Tauc M, Williams BO, Furge KA, Teh BT. Disruption of tubular Flcn expression as a mouse model for renal tumor induction. Kidney Int. 2015 Nov;88(5):1057-69. PMID: 26083655.
  • Ding Y, Zhu C, Zou W, Ma D, Min H, Chen B, Ye M, Pan Y, Cao L, Wan Y, Zhang W, Meng L, Mei Y, Yang C, Chen S, Gao Q, Yi L. FLCN intragenic deletions in Chinese familial primary spontaneous pneumothorax. Am J Med Genet A. 2015 May;167A(5):1125-33. PMID: 25807935.
  • Ferreira Francisco FA, Soares Souza A Jr, Zanetti G, Marchiori E. Multiple cystic lung disease. Eur Respir Rev. 2015 Dec;24(138):552-64. PMID: 26621970.
  • Furuya M, Hong SB, Tanaka R, Kuroda N, Nagashima Y, Nagahama K, Suyama T, Yao M, Nakatani Y. Distinctive expression patterns of glycoprotein non-metastatic B and folliculin in renal tumors in patients with Birt-Hogg-Dubé syndrome. Cancer Sci. 2015 Mar;106(3):315-23. PMID: 25594584.
  • Gupta N, Vassallo R, Wikenheiser-Brokamp KA, McCormack FX. Diffuse Cystic Lung Disease. Part II. Am J Respir Crit Care Med. 2015 Jul 1;192(1):17-29. Review. PMID: 25906201.
  • Ha D, Yadav R, Mazzone PJ. Cystic lung disease: systematic, stepwise diagnosis. Cleve Clin J Med. 2015 Feb;82(2):115-27. Review. PMID: 25897602.
  • Iribe Y, Kuroda N, Nagashima Y, Yao M, Tanaka R, Gotoda H, Kawakami F, Imamura Y, Nakamura Y, Ando M, Araki A, Matsushima J, Nakatani Y, Furuya M. Immunohistochemical characterization of renal tumors in patients with Birt-Hogg-Dubé syndrome. Pathol Int. 2015 Mar;65(3):126-32. PMID: 25597876.
  • Johannesma PC, Reinhard R, Kon Y, Sriram JD, Smit HJ, van Moorselaar RJ, Menko FH, Postmus PE; Amsterdam BHD working group. Prevalence of Birt-Hogg-Dubé syndrome in patients with apparently primary spontaneous pneumothorax. Eur Respir J. 2015 Apr;45(4):1191-4. PMID: 25537564.
  • Johannesma PC, Houweling AC, Menko FH, van de Beek I, Reinhard R, Gille JJ, van Waesberghe JT, Thunnissen E, Starink TM, Postmus PE, van Moorselaar RJ. Are lung cysts in renal cell cancer (RCC) patients an indication for FLCN mutation analysis? Fam Cancer. 2015b Nov 24. [Epub ahead of print] PMID: 26603437.
  • Schmidt LS, & Linehan WM (2015). Clinical Features, Genetics and Potential Therapeutic Approaches for Birt-Hogg-Dubé Syndrome. Expert opinion on orphan drugs, 3 (1), 15-29 PMID: 26581862
  • Pacitto A, Ascher DB, Wong LH, Blaszczyk BK, Nookala RK, Zhang N, Dokudovskaya S, Levine TP, Blundell TL. Lst4, the yeast Fnip1/2 orthologue, is a DENN-family protein. Open Biol. 2015 Dec;5(12). pii: 150174. PMID: 26631379.
  • Péli-Gulli MP, Sardu A, Panchaud N, Raucci S, De Virgilio C. Amino Acids Stimulate TORC1 through Lst4-Lst7, a GTPase-Activating Protein Complex for the Rag Family GTPase Gtr2. Cell Rep. 2015 Oct 6;13(1):1-7. PMID: 26387955.
  • Possik E, Ajisebutu A, Manteghi S, Gingras MC, Vijayaraghavan T, Flamand M, Coull B, Schmeisser K, Duchaine T, van Steensel M, Hall DH, Pause A. FLCN and AMPK Confer Resistance to Hyperosmotic Stress via Remodeling of Glycogen Stores. PLoS Genet. 2015 Oct 6;11(10):e1005520. PMID: 26439621.
  • Richards JC, Lynch DA, Chung JH. Cystic and nodular lung disease. Clin Chest Med. 2015 Jun;36(2):299-312, ix. Review. PMID: 26024606.
  • Wu M, Si S, Li Y, Schoen S, Xiao GQ, Li X, Teh BT, Wu G, Chen J. Flcn-deficient renal cells are tumorigenic and sensitive to mTOR suppression. Oncotarget. 2015 Oct 20;6(32):32761-73. PMID: 26418749.

New HLRCC patient-derived cell line to model papillary RCC

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a rare genetic condition caused by mutations in fumarate hydratase (FH). HLRCC patients are at risk of developing type 2 papillary renal cell carcinoma (pRCC2) which typically has an early onset with high metastatic potential. Existing targeted treatments have very limited response rates in both primary and metastatic pRCC2 tumours. Developing more effective treatments relies on preclinical models such as the new FH-deficient cell line derived by Perrier-Trudova et al., (2015).

This cell line, NCCFH1, was derived from the pleural fluid of a pRRC2 lung metastasis in a 17 year old patient who, despite initially responding to sunitinib, died 19 months after diagnosis due to disease progression. The germline mutation in FH was a small deletion in exon 8 – c.1162delA – that results in a frameshift and protein truncation, with a loss of heterozygosity in the tumours and NCCFH1. Perrier-Trudova et al. confirmed a reduction in FH mRNA levels with no protein detected by western blot and no FH-enzymatic activity.

Loss-of-function mutations in the FH enzyme result in accumulation of fumarate and subsequent activation of oncogenetic pathways including upregulation of HIF-1α signalling. Previously two other FH-deficient cell lines have been derived from patient samples: UOK 262 from a renal metastasis (Yang et al., 2010) and UOK 268 from a primary renal tumour (Yang et al., 2012). Both of these cells lines display the Warburg effect with reduced oxidative phosphorylation, increased glucose-dependence and deregulated metabolism typical of cancer cells. UOK 262 and the healthy human kidney line HK-2 were used as controls during the characterisation of the NCCFH1 line.

To assess oxidative phosphorylation and glycolysis rates Perrier-Trudova et al. measured basal oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). HK-2 cells have an OCR of 130-140pMole/min (30,000 cells), significantly higher than in NCCFH1 (10pMole/min) and UOK 262 (<20pMole/min) identifying them as respiratory-deficient cell lines. Both FH-deficient cells lines had an OCR/ECAR ratio that was remarkably lower than HK-2 cells indicative of dependency on glycolysis. This dependency was confirmed using proliferation assay in media containing 0.5-4.5g/litre glucose. Whilst HK-2 cells survived and proliferated in as low as 0.5-1g/litre glucose, the FH-deficient cell lines required 2g/litre glucose for optimal proliferation. NCCFH1 showed lower proliferation compared to UOK 262 at 1-1.5g/litre glucose suggestive of a higher glucose dependency.

Using microarrays Perrier-Trudova et al. identified over 200 genes that are differentially regulated in the FH-deficient cell lines compared to HK-2. Functional annotation clustering analysis identified that the loss of FH alters expression of genes associated with AKT/ERK signalling, apoptosis, metabolism, ubiquitin-proteosome pathways, extracellular matrix activity and GTPase binding activity. Overexpression of one of the upregulated genes, AKR1B10, has previously been reported as a predominant feature of pRCC2 tumours (Ooi et al., 2011). Accordingly both NCCFH1 and UOK 262 showed significantly more AKR1B10 mRNA than HK-2 cells.

Based on survival assays NCCFH1 and UOK 262 cells show limited toxicity at 72 hours when incubated with 10μM of the commonly used sunitinib, everolimus and temsirolimus, as well as 12 other cytotoxic and cytostatic drugs. Although significant toxicity was seen when FH-deficient cells were incubated with mitoxantrone, epirubicin, topotecan and bortezomib, normal HK-2 cells showed similar levels of toxicity.

Similar to BHD, HLRCC is a rare condition and consequently there are relatively few pRCC2 patients available for clinical trials. It is therefore important to have reliable and representative in vitro cellular models that can be used to increase understanding of tumourigenesis and to develop new treatments. This new FH-deficient cell line provides another preclinical model, with a different FH mutation, for hereditary pRRC2.


  • Ooi A, Wong JC, Petillo D, Roossien D, Perrier-Trudova V, Whitten D, Min BW, Tan MH, Zhang Z, Yang XJ, Zhou M, Gardie B, Molinié V, Richard S, Tan PH, Teh BT, Furge KA (2011). An antioxidant response phenotype shared between hereditary and sporadic type 2 papillary renal cell carcinoma. Cancer Cell. 20(4):511-23. PMID: 22014576.
  • Perrier-Trudova V, Huimin BW, Kongpetch S, Huang D, Ong P, LE Formal A, Poon SL, Siew EY, Myint SS, Gad S, Gardie B, Couvé S, Foong YM, Choudhury Y, Poh J, Ong CK, Toh CK, Ooi A, Richard S, Tan MH, & Teh BT (2015). Fumarate Hydratase-deficient Cell Line NCCFH1 as a New In Vitro Model of Hereditary Papillary Renal Cell Carcinoma Type 2. Anticancer research, 35 (12), 6639-53 PMID: 26637880.
  • Yang Y, Valera VA, Padilla-Nash HM, Sourbier C, Vocke CD, Vira MA, Abu-Asab MS, Bratslavsky G, Tsokos M, Merino MJ, Pinto PA, Srinivasan R, Ried T, Neckers L, Linehan WM (2010). UOK 262 cell line, fumarate hydratase deficient (FH-/FH-) hereditary leiomyomatosis renal cell carcinoma: in vitro and in vivo model of an aberrant energy metabolic pathway in human cancer. Cancer Genet Cytogenet. 196(1):45-55. PMID: 19963135.
  • Yang Y, Valera V, Sourbier C, Vocke CD, Wei M, Pike L, Huang Y, Merino MA, Bratslavsky G, Wu M, Ricketts CJ, Linehan WM (2012). A novel fumarate hydratase-deficient HLRCC kidney cancer cell line, UOK268: a model of the Warburg effect in cancer. Cancer Genet. 205(7-8):377-90 PMID: 22867999.


Yeast FNIP1/2 orthologue Lst4 confirmed as DENN-family protein

Solving the crystal structure of FLCN and subsequent bioinformatics studies identified FLCN, FNIP1 and FNIP2 as DENN-family proteins (Nookala et al., 2012, Zhang et al., 2012). The yeast orthologues of FLCN and FNIP1/2 are proposed to be Lst7 and Lst4 respectively. Pacitto et al. (2015) have now solved the crystal structure of Kluyveromyces lactis Lst4, confirming it to be a structural DENN-family protein and functional FNIP orthologue.

Using X-ray crystallography Pacitto et al. solved the structure of the N-terminal longin domain of Lst4 (residues 58-226) to 2.14 Å (PDB ID: 4ZY8). This showed the classic longin domain architecture of a core five-strand β-sheet with a single α-helix on the concave side and two α-helices on the convex side, identifying Lst4 as a DENN-family protein. K. lactis Lst4 is slightly more compact that the more commonly used Saccharomyces cerevisiae protein but there is high conservation of the α-helices and β-sheet strands surface amino acids.

To further validate Lst4 as an orthologue of FNIP1/2 Pacitto et al. demonstrated that Lst4 interacts with Lst7 forming a 1:1 heterodimer similar to FLCN-FNIP1/2. In humans, the DENN domain of FLCN interacts with FNIP1 (Baba et al. 2006). Lst7 does not contain a DENN domain, so Pacitto et al. used truncated C-terminal Lst4 proteins to show that the interaction with Lst7 is dependent on the Lst4 DENN domain. The authors found that this binding was not dependent on the unstructured insertion found in the Lst4 DENN domain, suggesting this region could have an alternative function, potentially in regulating the Lst7-Lst4 complex. Similar insertions of unknown function are found in the longin and DENN domains of human FNIP1/2.

As the Lst7-Lst4 complex contains two longin domains but only one DENN domain, Pacitto et al. propose that the interaction between the Lst7-longin domain and Lst4-DENN domain results in the formation of a functional DENN protein. The Lst4-longin domain is then available for other interactions potentially related to the propagation of DENN-signalling or could, like FNIP1, interact with the AMPK-orthologue Snf1.

The Lst7-Lst4 complex also recapitulates the relocation of FLCN-FNIP1/2 complexes to the lysosomal membranes under starvation conditions. The role of Lst7-Lst4 in TORC1 activity in response to amino acids was recently further elucidated by Péli-Gulli et al. (2015) who reported it as a GAP for the GTPase Gtr2. FLCN-FNIP2 has also been reported to function as GAPs for RagC in mammals (Tsun et al., 2012). However, DENN-family proteins typically function as GEFs for Rab proteins and the FLCN-FNIP1 complex has been reported to act as a GEF for RagA/B GTPases at the lysosomal membrane (Petit et al., 2013). The lack of Lst7-Lst4 GEF activity could be due to the absence of a second DENN domain or as Pacitto et al. suggest the binding of Lst-7 to the DENN-domain of Lst-4 could block the more characteristic DENN-protein GEF activity. Further studies are required to understand the roles of these complexes as GEFs and GAPs in TORC1 signalling and potentially with other GTPases.

The FLCN-FNIP1/2 complexes are unique as dimers of two DENN-family proteins and further studies are needed to clarify their structural arrangements and any impacts this has on function. The confirmation that the yeast orthologues Lst7 and Lst4 share both structure and function with FLCN and FNIP1/2 supports the use of yeast as a eukaryotic model for BHD. Further studies could also help to explain the roles of these proteins and also the impact of FLCN mutations on function. A more detailed understanding of the roles played by FLCN and associated proteins could aid the development of new treatments.


  • Baba M, Hong SB, Sharma N, Warren MB, Nickerson ML, Iwamatsu A, Esposito D, Gillette WK, Hopkins RF 3rd, Hartley JL, Furihata M, Oishi S, Zhen W, Burke TR Jr, Linehan WM, Schmidt LS, Zbar B (2006). Folliculin encoded by the BHD gene interacts with a binding protein, FNIP1, and AMPK, and is involved in AMPK and mTOR signaling. Proc Natl Acad Sci U S A. 103(42):15552-7. PMID: 17028174.
  • Nookala RK, Langemeyer L, Pacitto A, Ochoa-Montaño B, Donaldson JC, Blaszczyk BK, Chirgadze DY, Barr FA, Bazan JF, Blundell TL (2012). Crystal structure of folliculin reveals a hidDENN function in genetically inherited renal cancer. Open Biol. 2(8):120071. PMID: 22977732.
  • Pacitto A, Ascher DB, Wong LH, Blaszczyk BK, Nookala RK, Zhang N, Dokudovskaya S, Levine TP, & Blundell TL (2015). Lst4, the yeast Fnip1/2 orthologue, is a DENN-family protein. Open biology, 5 (12) PMID: 26631379.
  • Péli-Gulli MP, Sardu A, Panchaud N, Raucci S, De Virgilio C (2015). Amino Acids Stimulate TORC1 through Lst4-Lst7, a GTPase-Activating Protein Complex for the Rag Family GTPase Gtr2. Cell Rep. 13(1):1-7. PMID: 26387955.
  • Petit CS, Roczniak-Ferguson A, Ferguson SM (2013). Recruitment of folliculin to lysosomes supports the amino acid-dependent activation of Rag GTPases. J Cell Biol. 202(7):1107-22. PMID: 24081491.
  • Tsun ZY, Bar-Peled L, Chantranupong L, Zoncu R, Wang T, Kim C, Spooner E, Sabatini DM (2013). The folliculin tumor suppressor is a GAP for the RagC/D GTPases that signal amino acid levels to mTORC1. Mol Cell. 52(4):495-505. PMID: 24095279.
  • Zhang D, Iyer LM, He F, Aravind L (2012). Discovery of Novel DENN Proteins: Implications for the Evolution of Eukaryotic Intracellular Membrane Structures and Human Disease. Front Genet. 3:283. PMID: 23248642.

Biomarkers in diagnosis, prognosis and treatment of RCC

Tumour biomarkers are measurable changes in cancer cells that could be used to improve available therapies. The identification of early biomarkers could increase early diagnosis rates and provide insight into tumour biology including aggressiveness. In addition tumour subtype-specific biomarkers could help identify the treatments most likely to be effective and also be used to measure response. The search for biomarkers in renal cell carcinoma (RCC) is an active field, with various types of potential biomarker reported (reviewed in Mickley et al., 2015).

The majority of renal tumours are clear cell RCC associated with the loss of VHL protein due to mutation or promoter hypermethylation. Consequently, most current treatments target disrupted downstream pathways such as VEGF signalling. Choueiri et al., (2008) found that VHL status could be a prognostic as well as a predicative biomarker as patients lacking VHL responded better to anti-VEGF therapies. The presence of driver MET mutations in papillary RCC is also highly predictive of response to MET/VEGFR2 inhibitor foretanib (Choueiri et al., 2013). Serum VEGF levels can then act as a pharmacodynamic biomarker. Other potential diagnostic biomarkers of RCC – mTOR, Akt, S6K and PTEN – are not specific to RCC pathology but could be used in combination with other biomarkers to identify key tumourigenic pathways.

Natural genetic variations have also been identified as prognostic biomarkers for existing treatments. In patients treated with sunitinib certain SNPs in either CYP3A5, NR1I3 or ABCB1 improved progression free survival (PFS) (van der Veldt et al., 2011) but two SNPs in VEGFR3 were associated with decreased PFS (Garcia-Donas et al., 2011). In patients receiving pazopanib SNPs in either IL8 or HIF1A were associated with reduced PFS (Xu et al., 2011). Identification of such variations could help streamline treatment choices.

Epigenetic changes due to CpG island methylation could also be prognostic biomarkers. Wei et al. (2015) identified five regions of variable methylation close to the PITX1, FOXE3, TWF2, EHBP1L1 and RIN1 genes that could help predict overall survival with patients categorised as either low-risk or high-risk. Changes in methylation are common in cancer cells and PITX1, TWF2 and RIN1 are periphery genes associated with a diverse network of other genes and signalling pathways associated with tumourigenesis.

Another class of molecules proposed as diagnostic and prognostic biomarkers are miRNAs which, through modifying post-transcription translation, have roles in several key tumourigenic pathways. Varied expression of several different individual, combinations and panels of circulating miRNAs have been proposed for early screening and prognosis assessments (Gu et al., 2015, Wang et al., 2015). Confirmation of these biomarkers could enable less invasive detection of primary and relapse tumours quickly.

Most biomarker studies have been single studies on relatively small patient populations, and significantly larger studies would be better suited to verify widely-applicable biomarkers. Kidney Cancer Scotland recently announced funding for Dr Grant Stewart at the University of Edinburgh and Professor David Harris at St Andrews’ University to search for therapeutic biomarkers in over 900 patient samples in the SCOTRRCC database.

It is hoped that the identification of suitable biomarkers will vastly increase treatment response rates and reduce RCC mortality through earlier diagnosis and more effective treatment. Biomarkers might also identify novel therapeutic pathways for research, particularly in less common cancers. However such biomarkers would need to be validated in independent cohorts and be easily applicable to the clinical setting, preferable from easily accessible samples. More research is required before every RCC patient can receive personalised treatment, but ongoing research and new technologies continue to advance the field.


  • Choueiri TK, Vaziri SA, Jaeger E, Elson P, Wood L, Bhalla IP, Small EJ, Weinberg V, Sein N, Simko J, Golshayan AR, Sercia L, Zhou M, Waldman FM, Rini BI, Bukowski RM, Ganapathi R (2008). von Hippel-Lindau gene status and response to vascular endothelial growth factor targeted therapy for metastatic clear cell renal cell carcinoma. J Urol. 180(3):860-6. PMID: 18635227.
  • Choueiri TK, Vaishampayan U, Rosenberg JE, Logan TF, Harzstark AL, Bukowski RM, Rini BI, Srinivas S, Stein MN, Adams LM, Ottesen LH, Laubscher KH, Sherman L, McDermott DF, Haas NB, Flaherty KT, Ross R, Eisenberg P, Meltzer PS, Merino MJ, Bottaro DP, Linehan WM, Srinivasan R (2013). Phase II and biomarker study of the dual MET/VEGFR2 inhibitor foretinib in patients with papillary renal cell carcinoma. J Clin Oncol. 31(2):181-6. PMID: 23213094.
  • Garcia-Donas J, Esteban E, Leandro-García LJ, Castellano DE, del Alba AG, Climent MA, Arranz JA, Gallardo E, Puente J, Bellmunt J, Mellado B, Martínez E, Moreno F, Font A, Robledo M, Rodríguez-Antona C (2011). Single nucleotide polymorphism associations with response and toxic effects in patients with advanced renal-cell carcinoma treated with first-line sunitinib: a multicentre, observational, prospective study. Lancet Oncol. 12(12):1143-50. PMID: 22015057.
  • Gu L, Li H, Chen L, Ma X, Gao Y, Li X, Zhang Y, Fan Y, Zhang X (2015). MicroRNAs as prognostic molecular signatures in renal cell carcinoma: a systematic review and meta-analysis. Oncotarget. 6(32):32545-60. PMID: 26416448.
  • Mickley A, Kovaleva O, Kzhyshkowska J, & Gratchev A (2015). Molecular and immunologic markers of kidney cancer-potential applications in predictive, preventive and personalized medicine. The EPMA journal, 6 PMID: 26500709.
  • van der Veldt AA, Eechoute K, Gelderblom H, Gietema J, Guchelaar HJ, van Erp NP, van den Eertwegh AJ, Haanen JB, Mathijssen RH, Wessels JA (2011). Genetic polymorphisms associated with a prolonged progression-free survival in patients with metastatic renal cell cancer treated with sunitinib. Clin Cancer Res. 17(3):620-9. PMID: 21097692.
  • Wang C, Hu J, Lu M, Gu H, Zhou X, Chen X, Zen K, Zhang CY, Zhang T, Ge J, Wang J, Zhang C (2015). A panel of five serum miRNAs as a potential diagnostic tool for early-stage renal cell carcinoma. Sci Rep. 5:7610. PMID: 25556603.
  • Wei JH, Haddad A, Wu KJ, Zhao HW, Kapur P, Zhang ZL, Zhao LY, Chen ZH, Zhou YY, Zhou JC, Wang B, Yu YH, Cai MY, Xie D, Liao B, Li CX, Li PX, Wang ZR, Zhou FJ, Shi L, Liu QZ, Gao ZL, He DL, Chen W, Hsieh JT, Li QZ, Margulis V, Luo JH (2015). A CpG-methylation-based assay to predict survival in clear cell renal cell carcinoma. Nat Commun. 6:8699. PMID: 26515236.
  • Xu CF, Bing NX, Ball HA, Rajagopalan D, Sternberg CN, Hutson TE, de Souza P, Xue ZG, McCann L, King KS, Ragone LJ, Whittaker JC, Spraggs CF, Cardon LR, Mooser VE, Pandite LN (2011). Pazopanib efficacy in renal cell carcinoma: evidence for predictive genetic markers in angiogenesis-related and exposure-related genes. J Clin Oncol. 29(18):2557-64. PMID: 21576632.

Summary of recent kidney cancer clinical trials

Kidney tumours, if detected early enough, can often be removed surgically without the need for further drug treatments. However, if the primary tumour metastasises traditional chemotherapies and radiotherapies become ineffective and patient survival is limited. In recent years there have been great advances in treatments for metastatic renal cell carcinoma (mRCC) with several targeted treatments now available. However, these targeted treatments show variable response rates and efficacy. This blog summarises recent results from clinical trials assessing new treatments.

The standard first-line treatment for mRCC is an anti-angiogenic and anti-proliferative tyrosine kinase inhibitor (TKI) such as sunitinib or sorafenib. In a recent phase II trial sunitinib was used in combination with trebananib – a peptide-Fc fusion protein that acts on additional pathways to further limit angiogenesis. The combination treatment of trebananib and sunitinib showed an increased response rate and longer progression free survival (PFS) (Atkins et al., 2015).However, it also showed increased toxicity compared to sunitinib alone, with a higher percentage of severe adverse events.

In patients where tumours become resistant to the first-line TKI treatment it is common to use an mTOR inhibitor such as everolimus for the second-line treatment. Recently two trials have suggested that novel TKIs Cabozantinib and Lenvatinib could be provide more effective treatments. Patients on cabozantinib – which acts on VEGFR, MET, RET, KIT and AXL signalling – showed a 42% reduction in progression, extended PFS, and a greater rate of tumour reduction (Choueiri et al., 2015). It is also being trialled as a first line treatment and in combination with other treatments. Lenvatinib – which acts on VEGFR, FGFR, RET, KIT and PDGFR signalling – also enhanced PFS and overall survival (OS) in combination with everolimus compared to everolimus treatment alone (Motzer et al., 2015).

An alternative treatment strategy is immunotherapy which enables the body’s own immune system to more efficiently target tumour cells. Nivolumab is a PD-1 inhibitor that restores T-cell immune activity. In comparison to everolimus as a second line treatment, nivolumab was recently reported to increase overall survival and have a greater impact on tumour shrinkage. Patients receiving nivolumab also had a lower rate of severe adverse events (Motzer et al., 2015b).

Finally it was recently announced that Dr W. Marston Linehan is leading a phase II trial of Berg’s drug BPM 31510 at the NIH. BPM 31510 modulates mitochondrial metabolic networks to reverse the Warburg effect characteristic of tumour cells. Preclinical and early trials results show solid tumour reduction and stable disease with no reported severe adverse events.

The development of new and more efficient treatments for kidney cancer is an active field. Ongoing basic research into the biology of tumourigenesis will enable more specific and targeted drugs to be produced. Being able to select patient cohorts that are more likely to respond to a particular treatment would reduce unnecessary and ineffective treatments which can have severe side effects. This relies on the identification of biomarkers which will be discussed in a further blog post.

  • Atkins MB, Gravis G, Drosik K, Demkow T, Tomczak P, Wong SS, Michaelson MD, Choueiri TK, Wu B, Navale L, Warner D, Ravaud A (2015). Trebananib (AMG 386) in Combination With Sunitinib in Patients With Metastatic Renal Cell Cancer: An Open-Label, Multicenter, Phase II Study. J Clin Oncol. 20;33(30):3431-8. PMID: 26304872
  • Choueiri TK, Escudier B, Powles T, Mainwaring PN, Rini BI, Donskov F, Hammers H, Hutson TE, Lee JL, Peltola K, Roth BJ, Bjarnason GA, Géczi L, Keam B, Maroto P, Heng DY, Schmidinger M, Kantoff PW, Borgman-Hagey A, Hessel C, Scheffold C, Schwab GM, Tannir NM, Motzer RJ; METEOR Investigators (2015). Cabozantinib versus Everolimus in Advanced Renal-Cell Carcinoma. N Engl J Med. 5;373(19):1814-23. PMID: 26406150
  • Motzer RJ, Hutson TE, Glen H, Michaelson MD, Molina A, Eisen T, Jassem J, Zolnierek J, Maroto JP, Mellado B, Melichar B, Tomasek J, Kremer A, Kim HJ, Wood K, Dutcus C, Larkin J (2015). Lenvatinib, everolimus, and the combination in patients with metastatic renal cell carcinoma: a randomised, phase 2, open-label, multicentre trial. Lancet Oncol. 16(15):1473-82. PMID: 26482279
  • Motzer RJ, Escudier B, McDermott DF, George S, Hammers HJ, Srinivas S, Tykodi SS, Sosman JA, Procopio G, Plimack ER, Castellano D, Choueiri TK, Gurney H, Donskov F, Bono P, Wagstaff J, Gauler TC, Ueda T, Tomita Y, Schutz FA, Kollmannsberger C, Larkin J, Ravaud A, Simon JS, Xu LA, Waxman IM, Sharma P, & CheckMate 025 Investigators (2015). Nivolumab versus Everolimus in Advanced Renal-Cell Carcinoma. The New England journal of medicine, 373 (19), 1803-13 PMID: 26406148

FLCN modifies the cytoplasmic translocation and aggregation of TDP-43

TDP-43 is a DNA/RNA binding protein whose cytoplasmic aggregation is associated with neuronal death in ALS and frontotemporal lobar degeneration (FTLD). TDP-43 has multiple cellular functions and shuttles between the nucleus and cytoplasm. However, in ALS and FTLD nuclear clearance of TDP-43 results in increased cytoplasmic localisation – a precursor to TDP-43 aggregation and stress granule formation. The mechanisms that regulate TDP-43 transport are not well understood but new research from Xia et al. (2015) has uncovered a role for FLCN in its nuclear export and the formation of stress granules.

TDP-43 is ubiquitously expressed and under normal conditions the majority of the protein is located within cell nucleus, only being sequestered to the cytoplasm under stress conditions. When TDP-43 is overexpressed in HEK293 the majority of cells show predominately nuclear localisation with a small percentage of cells showing cytoplasmic or diffuse localisation. However, concurrent overexpression of GFP-FLCN results in increased mislocalisation of TDP-43 to the cytoplasm. Xia et al. found this was due to enhancing FLCN-dependent nuclear export of TDP-43 rather than disruption of nuclear import. siRNA knockdown of FLCN results in reduced TDP-43 transport to the cytoplasm, even under stress conditions.

In the FLCN overexpression cells TDP-43 and GFP-FLCN colocalise in cytoplasmic punctate structures. Co-immunoprecipitation assays determined that FLCN and TDP-43, both wild type and mutant forms, directly interact in an RNA-independent manner. Xia et al. then used deletion mutation assays to map the interaction sites to FLCN residues 202-299 and TDP-43 RNA Recognition Motif 1 (RRM1).

The TDP-43 punctate structures are associated with the lysosomes and colocalise with ubiquitination and autophagy markers. TDP-43 colocalises with the stress granule marker G3BP1 only when GFP-FLCN is present to induce cytoplasmic TDP-43 localisation. In FLCN-depleted cells under stress TDP-43 dissociates from stress granules and then returns to the nucleus after conditions return to normal. The stress granules in these cells were smaller than those in control cells suggesting a role for TDP-43 in regulating the size of stress granules. As FLCN does not colocalise with G3BP1 it is most likely only indirectly impacting stress granule formation by mediating the cytoplasmic accumulation of TDP-43.

This research suggests a role for FLCN in enhancing the translocation of TDP-43 into the cytoplasm ahead of aggregation and stress granule production – although the exact mechanisms are unknown. Whilst interesting, these results are based on in vitro overexpression assays in a single, non-neuronal cell type. To understand the impact of this role for FLCN in neuronal pathology in ALS and FLTD further research is required to assess the interactions of endogenous TDP-43 and FLCN (Warren et al., 2004) in neuronal cell lines and ex vivo human samples. Additional research using kidney, lung and skin cells would be required to determine any pathological impacts in BHD patients. Studying endogenous proteins, using FLCN-specific antibodies, would also avoid the risk of the GFP tag affecting FLCN structure or function.

Increased AMPK signalling in motor neuron cell lines has also been shown to enhance nuclear clearing of TDP-43 (Liu et al., 2015). However, TDP-43 was found not to be a direct substrate of AMPK. As FLCN binds AMPK it is possible that FLCN is mediating the TDP-43 cytoplasmic localisation reported in response to changes in AMPK activity, and that depletion of FLCN could reduce this mislocalisation. TDP-43 was also recently linked to the alternative splicing of FNIP1 (De Conti et al., 2015) but further research is required to determine the impact of these interactions and any variations in isoform production on either ALS, FTLD or BHD pathology.


  • De Conti L, Akinyi MV, Mendoza-Maldonado R, Romano M, Baralle M, Buratti E (2015). TDP-43 affects splicing profiles and isoform production of genes involved in the apoptotic and mitotic cellular pathways. Nucleic Acids Res. Oct 15;43(18):8990-9005. PMID: 26261209.
  • Liu YJ, Ju TC, Chen HM, Jang YS, Lee LM, Lai HL, Tai HC, Fang JM, Lin YL, Tu PH, Chern Y (2015). Activation of AMP-activated protein kinase α1 mediates mislocalization of TDP-43 in amyotrophic lateral sclerosis. Hum Mol Genet. Feb 1;24(3):787-801. PMID: 25256353.
  • Warren MB, Torres-Cabala CA, Turner ML, Merino MJ, Matrosova VY, Nickerson ML, Ma W, Linehan WM, Zbar B, Schmidt LS (2004). Expression of Birt-Hogg-Dubé gene mRNA in normal and neoplastic human tissues. Mod Pathol. Aug;17(8):998-1011. PMID: 15143337.
  • Xia Q, Wang G, Wang H, Hu Q, & Ying Z (2015). Folliculin, a tumor suppressor associated with Birt-Hogg-Dubé (BHD) syndrome, is a novel modifier of TDP-43 cytoplasmic translocation and aggregation. Human molecular genetics PMID: 26516189.

In response to amino acids yeast FLCN-FNIP orthologues Lst7-Lst4 stimulate TORC1 activity

In eukaryotic cells TORC1 signalling has a key role in controlling cell growth in response to nutritional status. Folliculin (FLCN) and the FNIP proteins regulate mTORC signalling via interactions with Rag family GTPases (Petit et al., 2013, Tsun et al., 2012). Recently Péli-Gulli et al. (2015) reported that the yeast orthologues of FLCN and FNIP, Lst7 and Lst4, form a heterodimer that acts as a GTPase Activating Protein (GAP) for yeast Rag family GTPase Gtr2 . Lst4-Lst7 is the first GAP identified for Gtr2.

Rag family GTPases are heterodimers that cycle between an active and inactive state. In the active stimulating state, GTP is bound to RagA or RagB in mammals, or Gtr1 in yeast, and GDP is bound to RagC or RagD in mammals, or Gtr2 in yeast. Regulating and maintaining these GTP/GDP associations relies on interplay between distinct Guanine nucleotide Exchange Factors (GEFs) and GAPs.

During amino acid starvation TORC1 activity is vastly reduced, and only upregulated upon readdition of amino acids. Péli-Gulli et al. found that loss of either or both Lst4 and Lst7 similarly reduced TORC1 activity after amino acid readdition indicative of a shared biological role in amino acid stimulation of TORC1. Using mutant, GTP-locked variants of Gtr1 and Gtr2 it was determined that Lst4 and Lst7 specifically act upstream of Gtr2.

Péli-Gulli et al. used co-immunoprecipitation assays to confirm direct binding between Lst4 and Lst7.  They showed that, under normal conditions, the Lst4-Lst7 heterodimer is mostly cytoplasmic, but that during amino acid starvation is rapidly recruited to the vacuolar membrane where it is adjacent to, but not associated with, Gtr2. Following amino acid readdition, Lst-4-LSt7 interacts with Gtr2, TORC1 is activated, and Lst4-Lst7 is released from the membrane (Figure 1). Additional TORC1 activity inhibits Lst4-Lst7 localisation to the vacuolar membrane, creating a negative feedback loop. Lst4-Lst7 is a Gtr2-specific GAP and cannot stimulate Gtr1 GTP hydrolysis.

Yeast GTPase3

Figure 1: In yeast cells Lst7-Lst4 is recruited to the vacuolar membrane during amino acid starvation. Following readdition of amino acids Lst7-Lst4 act as a GAP for the GTPase Gtr2 stimulating hydrolysis of bound GTP to GDP. The Rag GTPases can then stimulate TORC1 activity. The Lst7-Lst4 heterodimer is then released into the cytoplasm.

Interestingly, in mammalian cells FLCN helps to regulate both RagA/B and RagC. Petit et al., (2013) reported that FLCN-FNIP1 complexes preferentially bind to GDP-loaded RagA/B, acting as a GEF to stimulate mTORC1 signalling. In contrast Tsun et al., (2012) reported that FLCN-FNIP2 complexes acts as a GAP for RagC to induce mTOR signalling (Figure 2).

Mammal GTPase3


Figure 2: In mammalian cells FLCN-FNIP2 is recruited to the vacuolar membrane during amino acid starvation. Following readdition of amino acids FLCN-FNIP2 act as a GAP for the GTPase RagC stimulating hydrolysis of bound GTP to GDP. FLCN-FNIP1 are a GEF for the GTPase RagA or RagB facilitating the release GDP and binding of GTP. The Rag GTPases can then stimulate mTORC1 activity.

The distinction between FNIP1 and FNIP2 in these complexes could explain the different functions with specific Rag family GTPases. In addition different FLCN domains might be associated with different functions: the C-terminal of FLCN contains a DENN domain (Nookala et al., 2012), a domain typically found in GEFs, whilst Tsun et al. showed that it was the N-terminal domains of FLCN that were required for GAP activity. As the yeast FLCN orthologue Lst7 does not contain the DENN domain, and Lst4 is the only identified FNIP orthologue, it may be only the role for FLCN-FNIP as a RagC/Gtr2 GAP that is conserved.

More research is required to determine how these roles for FLCN and FNIP in regulating amino acid induced TORC1 signalling are related to BHD pathology. Further understanding of the impact of FLCN loss on this regulation and the effects on cell growth could identify new research avenues and therapeutic targets.

  • Nookala RK, Langemeyer L, Pacitto A, Ochoa-Montaño B, Donaldson JC, Blaszczyk BK, Chirgadze DY, Barr FA, Bazan JF, Blundell TL (2012). Crystal structure of folliculin reveals a hidDENN function in genetically inherited renal cancer. Open Biol. Aug;2(8):120071. PMID: 22977732.
  • Péli-Gulli MP, Sardu A, Panchaud N, Raucci S, & De Virgilio C (2015). Amino Acids Stimulate TORC1 through Lst4-Lst7, a GTPase-Activating Protein Complex for the Rag Family GTPase Gtr2. Cell reports, 13 (1), 1-7 PMID: 26387955.
  • Petit CS, Roczniak-Ferguson A, Ferguson SM (2013). Recruitment of folliculin to lysosomes supports the amino acid-dependent activation of Rag GTPases. J Cell Biol. Sep 30;202(7):1107-22. PMID: 24081491.
  • Tsun ZY, Bar-Peled L, Chantranupong L, Zoncu R, Wang T, Kim C, Spooner E, Sabatini DM (2013). The folliculin tumor suppressor is a GAP for the RagC/D GTPases that signal amino acid levels to mTORC1. Mol Cell. Nov 21;52(4):495-505. PMID: 24095279.

Pleural covering as an alternative treatment for recurrent pneumothorax

Most BHD patients develop pulmonary cysts and although only 30-35% will suffer a pneumothorax the recurrence rate is very high (Toro et al., 2008). The standard treatment for recurrent pneumothorax is pleurodesis, sometimes accompanied with pleurectomy, which attaches the lung surface to the chest wall thereby reducing the risk of further air leaks. An alternative treatment pioneered by several independent groups of researchers and clinicians in Japan is pleural covering which reinforces the surface of the lung without attachment to the chest wall.

Every year in Japan over 12,000 patients with cystic lung diseases undergo surgery – mostly related to recurrent or intractable pneumothoraces.  An average post-operative recurrence rate of 4-20% highlighted the need for alternative treatments.  Pleurodesis is effective and widely used, but permanently attaching the lungs to the chest wall can reduce respiratory function and make subsequent thoracic surgery more difficult, so alternative non-adhesive treatments were of interest to clinicians and patients (Kurihara et al., 2010).

The pleural covering technique uses a bio-absorbable mesh – either regenerative oxidized cellulose (ROC) mesh or polyglycolic acid (PGA) felt – placed over the surface of the lung and attached using fibrin glue (Ueda et al., 2009, Kurihara et al., 2010). The mesh is absorbed into the pleura reinforcing the remaining lung tissue and unruptured cysts, and sealing air leaks. ROC membranes are less likely to adhere to the chest wall and are associated with fewer post-operative recurrences, compared to PGA felt (Uramoto & Tanaka, 2014).

The covering procedure was originally only used to reinforce excision and staple lines following bulbectomies, and found to reduce bullae regrowth at the staple lines and post-operative recurrence (Sakamoto et al., 2004). More extensive lung covering in LAM patients, to reinforce existing cysts, was found to prevent recurrent pneumothoraces without adhesion to the chest wall or reduction in lung function (Kurihara et al., 2008, Noda et al., 2010).

Data presented by Professor Kurihara at the Fifth BHD Symposium in 2013 (5th BHD Symposium Abstract 14) highlighted the efficacy of pleural covering in BHD patients – 45/46 patients treated with bilateral pleural covering had no post-operative pneumothoraces (<56-month follow-up). Additionally Okada et al., (2015) and Ebana et al., (2015) published pleural covering case studies detailing the treatment of four patients who have suffered no further pneumothoraces (<32 month follow-up). Removing all pulmonary cysts in BHD patients would significantly reduce lung function. Therefore instead the ruptured and thin-walled bulging cysts can be removed before reinforcing both the excision sites and remaining unruptured cysts (Okada et al., 2015).

Pleural covering has also been successful in a range of other cystic lung diseases including bronchiolitis obliterans, pulmonary eosinophilic granulomas (Noda et al., 2011), and AAT-deficiency (Kusu et al., 2012). Although currently not a common practice outside Japan, as more long-term case studies are reported and the efficacy of the treatment becomes clearer pleural covering could become a viable alternative to pleurodesis worldwide for patients suffering from recurrent pneumothorax. In particular the lack of adhesion to the chest wall would be advantageous in patients who are likely to require subsequent surgeries to the lungs, heart or oesophagus.

  • Ebana H, Otsuji M, Mizobuchi T, Kurihara M, Takahashi K, & Seyama K (2015). Pleural Covering Application for Recurrent Pneumothorax in a Patient with Birt-Hogg-Dubé Syndrome. Annals of thoracic and cardiovascular surgery : official journal of the Association of Thoracic and Cardiovascular Surgeons of Asia PMID: 26370712.
  • Kurihara M, Seyama K, Kumasaka T (2009). Preventing LAM Patients from Recurrent Pneumothorax – An Innovative Surgical Method without Adhesion: Total Pleural Covering Technique (TPC). ATS International Conference 2009 Abstract.
  • Kurihara M, Kataoka H, Ishikawa A, Endo R (2010). Latest treatments for spontaneous pneumothorax. Gen Thorac Cardiovasc Surg. Mar;58(3):113-9. PMID: 20349299.
  • Kusu T, Nakagiri T, Minami M, Shintani Y, Kadota Y, Inoue M, Sawabata N, Okumura M (2012). Null allele alpha-1 antitrypsin deficiency: case report of the total pleural covering technique for disease-associated pneumothorax. Gen Thorac Cardiovasc Surg. Jul;60(7):452-5. PMID: 22544422.
  • Noda M, Okada Y, Maeda S, Sado T, Sakurada A, Hoshikawa Y, Endo C, Kondo T (2010). An experience with the modified total pleural covering technique in a patient with bilateral intractable pneumothorax secondary to lymphangioleiomyomatosis. Ann Thorac Cardiovasc Surg. Dec;16(6):439-41. PMID: 21263428.
  • Noda M, Okada Y, Maeda S, Sado T, Sakurada A, Hoshikawa Y, Endo C, Kondo T (2011). A total pleural covering technique in patients with intractable bilateral secondary spontaneous pneumothorax: Report of five cases. Surg Today. Oct;41(10):1414-7. PMID: 21922367.
  • Okada A, Hirono T, Watanabe T, Hasegawa G, Tanaka R, Furuya M (2015). Partial pleural covering for intractable pneumothorax in patients with Birt-Hogg-Dubé Syndrome. Clin Respir J. Jun 15. PMID: 26073198.
  • Sakamoto K, Takei H, Nishii T, Maehara T, Omori T, Tajiri M, Imada T, Takanashi Y (2004). Staple line coverage with absorbable mesh after thoracoscopic bullectomy for spontaneous pneumothorax. Surg Endosc. Mar;18(3):478-81. PMID: 14752657.
  • Toro JR, Wei MH, Glenn GM, Weinreich M, Toure O, Vocke C, Turner M, Choyke P, Merino MJ, Pinto PA, Steinberg SM, Schmidt LS, Linehan WM (2008). BHD mutations, clinical and molecular genetic investigations of Birt-Hogg-Dubé syndrome: a new series of 50 families and a review of published reports. J Med Genet. 208 Jun;45(6):321-31. Review. PMID: 18234728.
  • Ueda S, Isogami K, Kobayashi S (2009). [Uncomplicated covering technique for preventing the recurrence in the thoracoscopic surgery for pneumothorax]. Kyobu Geka. May;62(5):381-4. PMID: 19425378.
  • Uramoto H, Tanaka F (2014). What is an appropriate material to use with a covering technique to prevent the recurrence of spontaneous pneumothorax? J Cardiothorac Surg. Apr 29;9:74. PMID: 24775221.