In our last conference summary, it was noted that a number of clinical trials are underway to find an effective treatment for tuberous sclerosis complex (TSC). These trials use the mTORC1 inhibitors rapamycin and everolimus to compensate for mutations in the TSC1 and TSC2 genes. The results of one such trial have recently been reported by Dabora et al. (2011), and they show that treatment with rapamycin (which is also known as sirolimus) causes the regression of a number of tumours in TSC, such as kidney angiomyolipomas. Furthermore, VEGF-D was identified as a novel biomarker which could be used to monitor the size of these kidney angiomyolipomas over time.

The primary aim of this trial was to evaluate the effect of daily rapamycin treatment on kidney angiomyolipomas, and 36 adults (with at least one TSC-associated kidney angiomyolipoma) were recruited at six clinical sites across the US. In this study, it could be seen that almost half of the patients involved had a decrease in tumour size during the first 52 weeks of treatment. This treatment was reasonably tolerated, and no patients had progressive disease or complete regression. 13 subjects were then kept on rapamycin for a further year, and the difference between the numbers of patients showing a response, versus stable and progressive disease, was significant when compared to those not treated for an additional year.

As a secondary objective, it was noted that there was regression of TSC-associated liver angiomyolipomas and brain tumours (known as subependymal giant cell astrocytomas). Additionally, stable lung function was observed in those with TSC/LAM, and subjective improvements in TSC-associated skin lesions were also seen. However, the authors did not observe any significant changes in TSC-associated tubers, subependymal nodules or seizures.

As a whole, the results from Dabora et al. (2011) correlate well with similar mTOR inhibitor studies (Bissler et al., 2008; Krueger et al., 2010; Davies et al., 2011; McCormack et al., 2011). In addition, the authors noted a novel correlation between the levels of VEGF-D and kidney angiomyolipoma size. Accordingly, this biomarker could be used to monitor kidney angiomyolipomas during the treatment of TSC and in future clinical trials. Could a similar biomarker be found for BHD syndrome?

In summary, the results from this study indicate that rapamycin could be used to treat certain symptoms of TSC. It should be noted that pre-clinical rodent models have been crucial in enabling such studies to get to this stage, and a number of BHD models have been developed which could be utilised in a similar manner. However, the functional role of FLCN in mTOR signalling is still subject to some debate, as several recent publications have reported differing impacts on phosphorylated ribosomal protein S6 (an indicator of mTOR activation) when FLCN expression is reduced (click here for more information). Nevertheless, a clinical trial testing topical rapamycin for the treatment of fibrofolliculomas is currently under way – for more details about this and other trials which may be applicable to those with BHD syndrome, please do visit our clinical trials page on BHDSyndrome.org.

• Bissler JJ, McCormack FX, Young LR, Elwing JM, Chuck G, et al. (2008) Sirolimus for angiomyolipoma in tuberous sclerosis complex or lymphangioleiomyomatosis. N Engl J Med 358: 140–151. PMID: 18184959
• Dabora SL, Franz DN, Ashwal S, Sagalowsky A, DiMario FJ Jr, Miles D, Cutler D, Krueger D, Uppot RN, Rabenou R, Camposano S, Paolini J, Fennessy F, Lee N, Woodrum C, Manola J, Garber J, Thiele EA. (2011) Multicenter phase 2 trial of sirolimus for tuberous sclerosis: kidney angiomyolipomas and other tumors regress and VEGF- D levels decrease. PLoS One. 6(9): e23379. PMID: 21915260
• Davies DM, de Vries PJ, Johnson SR, McCartney DL, Cox JA, et al. (2011) Sirolimus Therapy for Angiomyolipoma in Tuberous Sclerosis and Sporadic Lymphangioleiomyomatosis: A Phase 2 trial. Clin Cancer Res. 17(12): 4071-81. PMID: 21525172
• Krueger DA, Care MM, Holland K, Agricola K, Tudor C, et al. (2010) Everolimus for subependymal giant-cell astrocytomas in tuberous sclerosis. New England Journal of Medicine 363: 1801–1811. PMID: 21047224
• McCormack FX, Inoue Y, Moss J, Singer LG, Strange C, et al. (2011) Efficacy and Safety of Sirolimus in Lymphangioleiomyomatosis. N Engl J Med. 364(17): 1595-606. PMID: 21410393

Hypoxia inducible factor (HIF) regulates processes such as cell proliferation and metabolism, and it has been implicated in tumour growth in several disorders such as VHL and TSC. Preston et al. (2010) demonstrated that HIF-1α activity was increased in FLCN-null cells and that there was an increase in the transcription of HIF target genes such as VEGF and GLUT1. This suggests that tumourigenesis in BHD syndrome may also result from the dysregulation of HIF.

Several HIF inhibitors have previously been described, such as Trichostatin A (Kong et al., 2006) and 17-AAG (Isaacs et al., 2002). Trichostatin A is a HDAC inhibitor which inhibits the acetylation of HIF (see this previous blog post) whereas 17-AAG inhibits the molecular chaperone Hsp90, which leads to the destabilisation of HIF. Barliya et al. (2011) have recently described another HIF-1α inhibitor called hypericin, which accelerates the degradation of HIF-1α through a unique mechanism and looks promising as an anti-cancer therapy.

Hypericin-mediated HIF-1α degradation was shown in two human tumour cell lines, U87-MG glioblastoma and RCC-C2VHL^-/- renal cell carcinoma, and also a non-malignant ARPE19 retinal pigment epithelial cell line. The reduction in HIF-1α protein levels was shown to occur independently of cellular oxygen levels, VHL and the ubiquitination-proteasome pathway. A cathepsin-B inhibitor prevented HIF-1α degradation by hypericin, suggesting cathepsin-B-mediated degradation was occurring. Hypericin is known to reduce intracellular pH, which creates the optimal conditions for cathepsin activity. Barliya et al. confirmed this by stabilising the pH and observing less HIF-1α degradation.

Hypericin was previously reported to induce forced poly-ubiquitination of Hsp90, leading to its enhanced degradation and therefore also the destabilisation and degradation of Hsp90 client proteins (Blank et al., 2003). As HIF-1α is transported into the nucleus via Hsp90, the authors investigated how HIF is affected by the hypericin-mediated degradation of Hsp90. As expected, the degradation of Hsp90 prevents HIF-1α transport into the nucleus. Therefore hypericin has two effects on HIF: enhancing its degradation by activating cathepsin-B and also preventing its nuclear localisation by ubiquitination of Hsp90.

The degradation of HIF-1α prevents HIF binding to DNA. In the presence of hypericin, reduced binding of HIF to the VEGF and GLUT1 promoter regions was observed. A luciferase assay also showed reduced VEGF promoter activity in hypoxic conditions in the presence of hypericin.

A Phase I/II clinical trial using hypericin for the treatment of glioblastoma multiforme and anaplastic astrocytoma has been completed, showing promising results. Barliya et al. now suggest hypericin should be evaluated as a treatment in other cancers in which high levels of HIF cause tumourigenesis. It would certainly be interesting to study the effect of hypericin in FLCN-null cells.

• 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. Absence of the Birt-Hogg-Dubé gene product is associated with increased hypoxia-inducible factor transcriptional activity and a loss of metabolic flexibility. Oncogene. 2011 Mar 10;30(10):1159-73. PMID:21057536
• Barliya T, Mandel M, Livnat T, Weinberger D, Lavie G. Degradation of HIF-1alpha under Hypoxia Combined with Induction of Hsp90 Polyubiquitination in Cancer Cells by Hypericin: a Unique Cancer Therapy. PLoS One. 2011;6(9):e22849. Epub 2011 Sep 19. PMID:21949677
• Blank M, Mandel M, Keisari Y, Meruelo D, Lavie G. Enhanced ubiquitinylation of heat shock protein 90 as a potential mechanism for mitotic cell death in cancer cells induced with hypericin. Cancer Res. 2003 Dec 1;63(23):8241-7. PMID:14678981
• Kong X, Lin Z, Liang D, Fath D, Sang N, Caro J. Histone deacetylase inhibitors induce VHL and ubiquitin-independent proteasomal degradation of hypoxia-inducible factor 1alpha. Mol Cell Biol. 2006 Mar;26(6):2019-28. PMID:16507982
• Isaacs JS, Jung YJ, Mimnaugh EG, Martinez A, Cuttitta F, Neckers LM. Hsp90 regulates a von Hippel Lindau-independent hypoxia-inducible factor-1 alpha-degradative pathway. J Biol Chem. 2002 Aug 16;277(33):29936-44. PMID:12052835

www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.

Thank you very much for taking the time to complete our blog survey. We had a good response with many positive comments. In particular, we are pleased to hear that so many of you found the blog informative with regards to the current state of BHD research. Nevertheless, do feel free to comment or ask questions about any blog post by filling in the form below it – the BHD Research blog is freely accessible to all, and you can read all posts and comment on an existing post without registering. Additionally, if at any stage you would like to give us more feedback about the blog or any other aspect of BHDSyndrome.org, please do not hesitate to email your comments to contact@bhdsyndrome.org. We positively encourage discourse within the BHD community and will endeavour to respond to any specific queries as soon as possible.

Within our blog survey, we did receive some specific comments and questions, but we are unable to reply directly as it was an anonymous questionnaire. However, do feel free to post these within our Forum topic entitled Living with BHD Syndrome. It is also possible to email contact@BHDSyndrome.org with any questions that you would like posted anonymously on the Forum. Here you can connect with the international BHD community and share as much or as little as you wish. Further information can also be found in the For Families section of the website, where you can learn more about the range of symptoms of BHD syndrome, as well as their diagnosis and treatment in our information pamphlets.

Alternatively, come join us at the Fourth BHD Symposium in Cincinnati, USA on 28^th – 30^th March 2012. Here you can meet the scientists and clinicians studying BHD syndrome, and hear about their latest work first-hand. Further information regarding the Symposium will be available in the near future, so do check back soon!

Additionally, the winner of the survey draw for a £500 in-kind contribution (or local currency equivalent) towards attendance at the Fourth BHD Symposium will be contacted by email by 1st December 2011.

www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.

In this month’s lab-profile we introduce Professor Maurice van Steensel, a Professor of genetic dermatology at the University Hospital Maastricht. Professor van Steensel leads a research group studying the role of FLCN in BHD syndrome and he is also a founding member of the European BHD Consortium. As a clinician, Professor van Steensel is in an excellent position to understand and connect both the clinical and basic science aspects of BHD syndrome.

Professor van Steensel’s research lab, which includes senior post-doc Dr Barry Coull and PhD students Tijs Claessens, Lieke Gijezen, Monique Luijten and Marigje Vernooij, study the role of FLCN in cellular signalling. Their data suggests that FLCN detects signals from outside the cell, which leads to changes in intracellular signalling pathways. Work is on-going to unravel this signalling function of FLCN and to understand how it is dysregulated in BHD syndrome. The van Steensel lab works closely with Dr Andrew Tee’s lab at Cardiff University, collaborating to produce the publication in Oncogene last year which describes the effect of FLCN loss on HIF signalling (Preston et al., 2010). This paper has been discussed in a previous blog post.

As well as investigating FLCN’s function in the cell, Professor van Steensel’s lab is trying to identify effective therapeutics to treat BHD syndrome. A clinical trial testing topical rapamycin for the treatment of fibrofolliculomas is currently on-going. More information about this can be found on the ClinicalTrials.gov website.

In May, Professor van Steensel chaired the Scientific Organising Committee for the Third BHD Symposium in Maastricht. The 2-day symposium, attended by over 80 scientists and patients from around the world, was a huge success. Reports from the meeting can be found here and here. During the symposium, Professor van Steensel took some time to film a video interview for BHDSyndrome.org. Watch the interview to learn more about the research carried out in the van Steensel lab. The audio-only and transcript files are also available for this interview.

The following publications further highlight the work of Professor van Steensel:

• 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. Absence of the Birt-Hogg-Dubé gene product is associated with increased hypoxia-inducible factor transcriptional activity and a loss of metabolic flexibility. Oncogene. 2011 Mar 10;30(10):1159-73. PMID:21057536
• Claessens T, Weppler SA, van Geel M, Creytens D, Vreeburg M, Wouters B, van Steensel MA. Neuroendocrine carcinoma in a patient with Birt-Hogg-Dubé syndrome. Nat Rev Urol. 2010 Oct;7(10):583-7. PMID:20842188
• Menko FH, van Steensel MA, Giraud S, Friis-Hansen L, Richard S, Ungari S, Nordenskjöld M, Hansen TV, Solly J, Maher ER; European BHD Consortium. Birt-Hogg-Dubé syndrome: diagnosis and management. Lancet Oncol. 2009 Dec;10(12):1199-206. PMID:19959076