In July, the Beatson International Cancer Conference took place at The Beatson Institute for Cancer Research in Glasgow, UK. The Beatson Institute is a Cancer Research UK-funded centre which focuses on understanding cancer cell behaviour and developing new therapies and diagnostic tools. A wide variety of research is carried out at the institute, such as the work by Frezza et al. (2011), which was recently discussed in our blog. This meeting comprised of talks from both academic and industry professionals. For example, Dr Saul Rosenberg from Abbott Laboratories spoke about the development of the peptide mimetic ABT-737, which has been shown by Cash et al. (2011) to be effective in inducing apoptosis in FLCN^-/- cells. Professor Norbert Perrimon from Harvard Medical School also spoke about using genome-wide RNAi screens and TAP-tag pull-down assays to understand how signalling pathways in cells overlap. These techniques could also be used to help untangle the signalling networks involved in BHD syndrome.

More recently in September, the International Tuberous Sclerosis Complex (TSC) Research Conference was hosted by the Tuberous Sclerosis Association in Belfast, UK. Of particular interest were two talks which specifically focussed on BHD syndrome. The first, by Deirdre Donnelly of the TS Clinic in Belfast, introduced BHD to the audience and gave an overview of its clinical similarities and differences with TSC. The second by Dr Andrew Tee of Cardiff University discussed the dysregulation of HIF signalling, which is observed in both TSC and BHD syndrome. To learn more about Dr Tee’s BHD research, click here to be directed to his lab-profile from a previous blog entry.

Information gathered at this meeting was further supplemented by talks at the Second French scientific day on TSC in Paris in October. This study day was jointly organised by the Association Sclérose Tubéreuse de Bourneville and the French rare epilepsies and TSC reference centre at the Hôpital Necker, Paris. Here, the promising results of a number of clinical trials were presented by Professor David Franz from Cincinnati Children’s Hospital. These trials are largely based on the mTORC1 inhibitors rapamycin and everolimus, and they are thought to work through a pathway which is illustrated in our FLCN-associated signalling diagram.

For more information regarding forthcoming meetings that are relevant to BHD researchers and families, please do look at our regularly updated Conferences and Events page on BHDSyndrome.org.

• Cash TP, Gruber JJ, Hartman TR, Henske EP, & Simon MC (2011). Loss of the Birt-Hogg-Dubé tumor suppressor results in apoptotic resistance due to aberrant TGFβ-mediated transcription. Oncogene, 30 (22), 2534-46 PMID: 21258407 
• Frezza C, Zheng L, Folger O, Rajagopalan KN, MacKenzie ED, Jerby L, Micaroni M, Chaneton B, Adam J, Hedley A, Kalna G, Tomlinson IP, Pollard PJ, Watson DG, Deberardinis RJ, Shlomi T, Ruppin E, & Gottlieb E (2011). Haem oxygenase is synthetically lethal with the tumour suppressor fumarate hydratase. Nature, 477 (7363), 225-8 PMID: 21849978

HIF1α is a transcriptional regulator which plays an essential role in the cellular response to hypoxia. As discussed in last week’s blog, prolyl hydroxylases (PHDs) mark HIFα subunits for degradation, but HIF1α can also be regulated by reversible acetylation. Earlier work in a VHL-null human RCC cell line noted that histone deacetylases (HDACs), such as HDAC4 and HDAC6, are associated with this process (Qian et al., 2006). However, the mechanism by which HDAC4 regulates HIF1α was not completely clear. Now, a related study by Geng et al. (2011) suggests that HDAC4 inhibition increases HIF1α acetylation, which decreases HIF1α stability, and reduces HIF1-mediated gene expression in a prostate (C42B) and liver (Hep3Bc1) cancer cell line.

In this study, hypoxic Hep3Bc1 cells had significantly less HIF1α protein present after shRNA-mediated knockdown of HDAC4, with a similar result being obtained using CoCl₂ as a hypoxic mimic in C42B cells. The proteasome inhibitor MG132 was able to rescue this effect and maintain levels of HIF1α in CoCl₂-treated Hep3Bc1 cells after HDAC4 shRNA knockdown, and this HIF1α was also more acetylated. Using the protein synthesis inhibitor cyclohexamide, HIF1α also degraded faster in CoCl₂-treated Hep3Bc1 cells after HDAC4 shRNA knockdown, when compared to shRNA controls. In contrast, shRNA knockdown of HDAC1 and HDAC3 did not decrease HIF1α protein levels in hypoxic Hek293 cells. Co-immunoprecipitation experiments in Hek293T cells also showed that HIF1α interacts with HDAC4, and that HDAC4 (but not HDAC1) overexpression reduced the levels of acetylated HIF1α observed by western blot. Together, these experiments suggest that HDAC4 specifically influences both HIF1α acetylation and stability.

Subsequent mass spectrometric analysis indicated that the first 30 amino acids of HIF1α may be acetylated, and site-directed mutagenesis of all 5 lysines in this region produced a HIF1α mutant that had significantly lower levels of acetylation than wild-type HIF1α. Overexpression of HDAC4 in Hek293T cells had no effect on the acetylation and protein levels of the HIF1α mutant, and HDAC4 shRNA knockdown in CoCl₂-treated Hek293 cells also had no effect on this mutant’s protein levels. The HIF1α mutant was also resistant to HDAC inhibitor (SAHA)-mediated degradation in Hek293T cells, further emphasising the importance of these residues in the regulation of HIF1α.

Using qRT-PCR, it could be seen that the hypoxic upregulation of the HIF1α-target genes VEGFA, LDHA and GLUT1 was inhibited by shRNA knockdown of HDAC4 when compared to shRNA controls in both cancer cell lines. Genes such as LDHA and GLUT1 are involved in glycolysis, and accordingly, shRNA knockdown of HDAC4 in Hep3Bc1 and C42B cells significantly reduced hypoxia-induced lactate production when compared to controls. Additionally, after HDAC4 shRNA knockdown, Hep3Bc1 cells grew slower under long-term hypoxia, and hypoxic C42B cells were also more sensitive to the cytotoxic agent docetaxel. This latter result could have therapeutic implications, as HIF signalling has been shown to play a role in tumourigenesis in a number of disorders, including BHD syndrome (Preston et al., 2010).

Additionally, although the functional implications of HIF1α lysine acetylation are yet to be fully elucidated, it is useful to remember that HDACs and their inhibitors do not only augment transcription through chromatin regulation (as observed by Cash et al., 2011), but also through the direct modulation of transcription factors themselves.

• Cash TP, Gruber JJ, Hartman TR, Henske EP, & Simon MC (2011). Loss of the Birt-Hogg-Dubé tumor suppressor results in apoptotic resistance due to aberrant TGFβ-mediated transcription. Oncogene, 30 (22), 2534-46. PMID: 21258407
• Geng H, Harvey CT, Pittsenbarger J, Liu Q, Beer TM, Xue C, Qian DZ (2011). HDAC4 regulates HIF1{alpha} lysine acetylation and cancer cell response to hypoxia. J Biol Chem. Sep 14. [Epub ahead of print]. PMID: 21917920
• 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
• Qian DZ, Kachhap SK, Collis SJ, Verheul HM, Carducci MA, Atadja P, Pili R (2006). Class II histone deacetylases are associated with VHL-independent regulation of hypoxia-inducible factor 1 alpha. Cancer Res. Sep 1; 66(17): 8814-21. PMID: 16951198

Mutations in Fumarate Hydratase (FH) cause HLRCC, a kidney cancer syndrome related to BHD. FH is an enzyme involved in the TCA cycle and its deficiency results in the accumulation of fumarate within the cell. This accumulation leads to increased levels of HIF, via the inhibition of prolyl hydroxylases (PHDs), the enzymes that mark HIFα subunits for degradation. It is believed that increased HIF promotes tumourigenesis in FH-deficient cells.

FH-deficient cells are known to exhibit the Warburg effect, a phenomenon in which cancer cells shift to a high rate of glycolysis rather than oxidative phosphorylation. Tong et al. (2011) have recently investigated the metabolic state of the HLRCC cell line UOK262 and the effect of this glycolytic shift. They found that IRPs, proteins which are activated by cytosolic iron depletion, had increased activity in UOK262 cells, suggesting low levels of iron. PHDs require iron for their activity and therefore the low level of iron in FH-deficient cells could be an additional mechanism leading to increased levels of HIF.

The authors found that levels of the iron transporter DMT1 were extremely low in UOK262 cells, thereby explaining the reduced iron level and activation of IRPs. It was found that AMPK levels were reduced, which is caused by the glycolytic shift, and that this leads to the reduced DMT1 levels, revealing a role of AMPK in the regulation of intracellular iron metabolism. AMPK is known to stabilise p53, and so the authors then investigated whether p53 had an effect on DMT1. It was found that reduced p53 decreased DMT1 levels, suggesting AMPK regulates DMT1 expression through its effect on p53.

Then the authors compared the levels of AMPK, DMT1 and HIF-1α protein in UOK262 cells against the levels in a VHL-deficient cell line. In contrast to the UOK262 cells, VHL-deficient cells had higher levels of AMPK and DMT1 and lower levels of HIF-1α. This suggests that disease-specific therapies could be developed. Preston et al. (2010) showed that FLCN-null cells have increased levels of AMPK, suggesting BHD cells exhibit a metabolic profile more akin to VHL cells than HLRCC cells.

This new study correlates with the recent paper by Frezza et al. (2011) (discussed in a previous blog post) who found that haem metabolism was altered in FH-deficient cells and that the levels of three genes involved in haem degradation were increased. Haem degradation releases the bound iron, and therefore may be a mechanism used to increase the intracellular iron when levels are low.

More research is required to fully understand the metabolic profile of FH-deficient cells and how this can be exploited to develop effective therapies for HLRCC. Additionally, understanding how the metabolic profile of HLRCC cells differs from that of other inherited kidney cancer syndromes will help unravel the biology behind this class of diseases and perhaps lead to disease-specific therapies.

• Tong WH, Sourbier C, Kovtunovych G, Jeong SY, Vira M, Ghosh M, Romero VV, Sougrat R, Vaulont S, Viollet B, Kim YS, Lee S, Trepel J, Srinivasan R, Bratslavsky G, Yang Y, Linehan WM, Rouault TA. The Glycolytic Shift in Fumarate-Hydratase-Deficient Kidney Cancer Lowers AMPK Levels, Increases Anabolic Propensities and Lowers Cellular Iron Levels. Cancer Cell. 2011 Sep 13;20(3):315-27. PMID:21907923
• Frezza C, Zheng L, Folger O, Rajagopalan KN, MacKenzie ED, Jerby L, Micaroni M, Chaneton B, Adam J, Hedley A, Kalna G, Tomlinson IP, Pollard PJ, Watson DG, Deberardinis RJ, Shlomi T, Ruppin E, Gottlieb E. Haem oxygenase is synthetically lethal with the tumour suppressor fumarate hydratase. Nature. 2011 Aug 17;477(7363):225-8. PMID:21849978
• 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

As mentioned in our latest newsletter, several new interviews were filmed at the Third BHD Symposium, and have been posted to BHDSyndrome.org.

Dr Derek Lim’s work at the University of Birmingham has been described previously, including his contribution to the Folliculin Mutation Database (Lim et al., 2010) and the UK BHD patient registry. The FLCN mutation database is a comprehensive and up-to-date database of sequence variation in the FLCN gene, which is hosted by LOVD and freely available to all. Within it, you can observe the types and frequencies of different FLCN mutations, as well as a graphical summary of the entire dataset.  There are currently 139 unique FLCN mutations listed in the database from both published and unpublished work, and together these mutations could be useful in identifying potential genotype-phenotype correlations.

As part of the UK BHD registry, Dr Lim visits UK-based BHD patients and monitors their symptoms. This could help identify trends among BHD patients and assess the age of onset of certain symptoms, which collectively could be used to refine an appropriate surveillance programme for BHD syndrome.

To learn more about this and Dr Lim’s other work as a Clinical Research Fellow, as well as his career path, views on this year’s Symposium and thoughts regarding the future of BHD research, watch Dr Lim’s video interview, with its associated transcript and audio-only files.

In the coming months, more videos will also be added to BHDSyndrome.org, so do come back soon and let us know what you think!

• Lim, D., Rehal, P., Nahorski, M., Macdonald, F., Claessens, T., Van Geel, M., Gijezen, L., Gille, J., Giraud, S., Richard, S., van Steensel, M., Menko, F., & Maher, E. (2010). A new locus-specific database (LSDB) for mutations in the folliculin (FLCN) gene. Human Mutation, 31 (1) DOI: 10.1002/humu.21130