GB2478556A - A composition for use in the treatment of Birt-Hogg-Dubô sydrome - Google Patents

Abstract

A composition comprises an inhibitor of glucose metabolism for use in the treatment of Birt-Hogg-Dube syndrome. The inhibitor of glucose metabolism may be a glycolytic inhibitor, such as 2-deoxy-D-glucose. Alternatively, the inhibitor may be an inhibitor of lactate dehydrogenase in pyruvate metabolism, such as oxamate.

Description

INHIBITORS OF GLUCOSE METABOLISM FOR USE IN THE TREATMENT OF

BIRT-HOGG-DUBE SYNDROME

This invention relates to drugs for use in the treatment of diseases. More particularly, this invention relates to compositions, including compositions comprising 2-deoxy-D- glucose and compositions comprising oxamate for use in the treatment of Birt-Hogg-Dubé syndrome.

Birt-Hogg-Dubé (BHD) syndrome is a dominantly inherited familial cancer syndrome associated with susceptibility to renal cell carcinoma (RCC). BHD is also associated with benign skin fibrofolliculomas (hamartomatous tumours of the hair follicle) and multiple lung cysts and spontaneous pneumothorax (Toro et al., J. Med. Genet. 2008; 45: 321-331). There is a 15 -30% occurrence of RCC within BHD patients, which include oncocytoma, chromophobe, papillary, clear cell, and oncocytic hybrid (see ref (Woodward, 2008)) renal cell carcinomas.

RCC is the fourteenth most common cancer in the UK. RCC is a heterogeneous disorder with a number of histopathological subtypes. One subtype is conventional clear cell RCC (ccRCC), which accounts for more than 75% of cases of RCC. Non-clear-cell forms of RCC comprise papillary (or chromophil) RCC, chromophobe tumours, oncocytoma, collecting duct carcinoma and the rare medullary carcinoma.

BHD-associated renal tumours are of variable histopathology but are often chromophobe RCC/oncocytoma.

BHD syndrome results from inactivating mutations in the folliculin (FLCN or BHD) gene (Nickerson et al., Cancer Cell 2002; 2: 157-164; Schmidt et al., Am J Hum Genet. 2005; 76: 1023-33; Lim et al., Hum Mutat. 2010 Jan 31(1):E1043-51) and renal tumours from BHD patients demonstrate somatic BHD loss.

Loss of heterozygosity of the BHD gene which encodes folliculin (FLCN also referred to as BHD) is a frequent event in renal cell carcinoma suggesting that folliculin (FLCN) can act as a classical "Knudson" tumour suppressor (Koo 2004).

Although it is considered that FLCN (the BHD gene product) represses cell growth, the role that FLCN plays in cancer progression and/or initiation is currently unresolved. Folliculin-interacting protein I (FNIP1) was discovered to interact with FLCN and this protein complex is phosphorylated in an mTOR (mammalian target of rapamycin) and AMP-dependent protein kinase (AMPK)-dependent manner (Baba et al, 2006).

In mice with kidney-targeted homozygous inactivation of BHD, renal tumours and cysts developed with activation of mTOR and the mTOR inhibitor rapamycin diminished kidney pathology and increased survival (Baba et al., J. NatI. Cancer Inst.

2008; 100: 140-1 54; Chen et al., PbS ONE 2008; 33: e3581). mTOR inhibitor drugs (e.g. Temsirolimus, Everolimus, etc) have shown promise as treatments for metastatic RCC (Molina and Motzer Clin Genitourin Cancer 2008 Dec; 6 Suppl 1:S7-13).

Under condition of reduced tissue oxygenation, hypoxia-inducible-factor (HIF) controls many processes including angiogenesis and cellular metabolism, as well as influencing cell proliferation and survival decisions. HIF1a and HIF2a are transcription factors that interact with HIF-13 (also known as aryl hydrocarbon receptor nuclear translocator (Arnt)) and as a heterodimer enhance expression of over 70 genes containing hypoxia response elements that are involved in angiogenesis, erythropoiesis, glucose metabolism, cell survival and metastasis (Semanza, 2004).

HIF-a proteins are rapidly degraded in an oxygen-dependent manner through hydroxylation of two proline residues within their oxygen-dependent degradation domain catalysed via HIF prolyl hydroxylases (Semenza, 2004).

Recent studies have implicated HIF as a factor involved in tumour growth in Von Hippel-Lindau (VHL) disease and tuberous sclerosis complex (TSC) (Liu, 2003).

VHL is an inherited genetic disorder that gives rise to RCC (Hemminki, 2002).

Higher levels of hypoxia-inducible factor (HIF)-mediated signalling are a feature attributed to tumour growth within VHL (Kaelin, 2005; Issacs, 2005). Proline hydroxylation leads to rapid degradation of H IF-a proteins which is dependent on the VHL tumour suppressor protein. VHL is a component of the E3 ubiquitin ligase complex that targets proline hydroxylated HIF-a proteins for degradation via the proteosome. Consequently, low oxygen or loss of VHL function leads to stabilization of HIF-a proteins and an upregulation of HIF-mediated gene expression (Semenza, 2004). VHL patients develop clear cell RCC which is caused by loss of function of the tumour suppressor protein VHL (Hemminki, 2002). TSC is also an inherited disorder that gives rise to RCC. TSC arises through mutations of either Tuberous Sclerosis Complex (TSC) 1 or 2 that function as a heterodimer to repress mTOR.

mTOR is a positive regulator of HIF, in an apparently multifaceted manner via the levels of gene expression, translation, protein stability and activity (see review Dunlop and Tee 2009).

Glycolysis is a central pathway in biological systems. Glycolysis is a sequence of reactions that converts glucose into pyruvate with concomitant production of ATP. In aerobic organisms, the pyruvate generated by glycolysis feeds into the citric acid cycle (also known as the Krebs or TCA cycle). The citric acid cycle together with the electron-transport chain harvest the energy stored in glucose. Pyruvate enters the mitochondria where the complete oxidation of pyruvate to carbon dioxide and water takes place. Under conditions where the oxygen supply is limited, pyruvate can instead be converted anaerobically to lactate. This however only releases a small amount of the energy stored in glucose.

The glycolysis pathway has been completely elucidated and includes ten reactions: Glucose is converted to glucose-6-phosphate, the reaction being catalysed by the enzyme hexokinase.

Glucose-6-phosphate is isomerised to fructose-6-phosphate by phosphoglucose isomerise.

Fructose-6-phosphate is phosphorylated to fructose 1,6-bisphosphate by phosphofructokinase.

Fructose 1,6-bisphosphate is converted to dihydroxyacetone phosphate and glyceraldehyde 3-phosophate by aldolase.

Dihydroxyacetone phosphate is isomerised to glyceraldehyde 3-phosphate by triose phosphate isomerise.

Glyceraldehyde 3-phosphate is converted to 1,3-bisphosphoglycerate by phosphoglycerate kinase.

1,3-Bisphosphoglycerate is converted to 3-phosphoglycerate by phosphoglycerate kin ase.

3-Phosphoglycerate is converted to 2-phosphoglycerate by phosphoglycerom utase.

2-Phosphoglycerate is converted to phosphoenolpyruvate by enolase.

Phosphoenolpyruvate is converted to pyruvate by pyruvate kinase.

Glycolysis generates a net gain of two molecules of ATP per molecule of glucose.

The net reaction is: Glucose + 2 P + 2 ADP + 2 NAD -2 pyruvate + 2 ATP + 2 NADH + 2 H + 2 H20 Under aerobic conditions, pyruvate can be converted to acetyl CoA which feeds into the citric acid cycle. NADH and FADH2 formed by glycolysis and the reactions of the citric acid cycle then undergo electron transfer to molecular oxygen, generating large amounts of ATP by oxidative phosphorylation.

The conversion of pyruvate to lactate does not generate as much energy but can be used as an alternative pathway for the metabolism of glucose without using oxygen as the final electron acceptor. The overall reaction is: Glucose + 2 P + 2 ADP -2 lactate + 2 ATP + 2 H20 The production of lactate regenerates NAD from NADH. Lactate must be converted back to pyruvate before it can be metabolised.

Anaerobic glycolysis is the process by which the normal pathway of glycolysis is routed to produce lactate (rather than feeding pyruvate into the citric acid cycle) and is a result of low oxygen levels preventing mitochondria from carrying out oxidative phosphorylation to produce ATP.

Aerobic glycolysis is a term used to denote the production of lactate from glucose in the presence of oxygen. Cells that produce lactate even though they have enough oxygen can be described as using aerobic glycolysis.

The reaction steps in glucose metabolism are shown diagrammatically in Figure 9.

Renal cell carcinoma (RCC) has traditionally been considered to be largely resistant to radiotherapy and in vitro studies have shown that renal cancer cells are among the least radiosensitive of human cell types. Furthermore, the majority of advanced RCC tumours have proved to be resistant to cytotoxic agents and therefore chemotherapy has had a very limited role in the treatment of metastatic renal cancer. Surgical resection is therefore currently the preferred treatment for locally confined RCC and can often achieve a cure in the earlier stages of RCC.

Patients with BHD syndrome are typically offered renal imaging to facilitate early detection of RCC. However, some patients may only be diagnosed after presentation with advanced RCC. Treatment of metastatic RCC is challenging for both familial and sporadic cases. Although occasional patients may respond to immunotherapy with the cytokines interferon and interleukin-2, recently treatment with targeted therapies to HIF downstream targets (e.g. Sunitinib, Sorafenib, Bevacizumab, etc) and the mTOR pathway (e.g. Temsirolimus, Everolimus) has emerged as the most frequent management strategy. However these agents, whilst prolonging life, are not cytotoxic and so the identification of alternative targeted therapies would be a significant advance.

Summary of Invention

According to the present invention there is now provided a composition comprising an inhibitor of glucose metabolism for use in the treatment of Birt-Hogg-Dube syndrome.

According to another aspect of the invention there is provided a composition comprising an inhibitor of glucose metabolism for use in the inhibition of growth of BHD-null renal cell carcinoma cells.

According to a yet further aspect of the invention there is provided a composition comprising an inhibitor of glucose metabolism for use in the treatment of renal cell carcinoma associated with BHD gene inactivation.

According to a yet further aspect of the invention there is provided a composition comprising an inhibitor of glucose metabolism for use in the differential growth inhibition of BHD-null cells over BHD-wild type cells.

Understanding the biological differences between normal and BHD-null cells is essential for the design and development of drugs with selective activity against BHD-null cells (i.e. preferential killing of BHD-null cells without significant toxicity to normal cells).

We have now found through our studies, as explained herein, that BHD-null cells have a higher dependency on glucose metabolism than normal BHD-wild type cells.

This dependence on a single fuel for metabolic production of ATP is known as loss of "metabolic flexibility".

It is known that some cancer cells can alter their metabolism to favour aerobic glycolysis over oxidative phosphorylation. In this way cancers can meet their bioenergetic and biosynthetic demands and elicit a proliferative advantage. This effect is known as the "Warburg Effect". Proliferating cancer cells showing the Warburg Effect consume glucose at a high rate and release L-lactate and not 002.

Although an increase in aerobic glycolysis is seen in a number of types of cancer cells, it is not seen in all cancer cell types. It is not understood what triggers the Warburg effect in cancer cells and it has not been shown that there is any general link established between gene mutation and triggering of the Warburg effect. Since not all cancer types show an increase in aerobic glycolysis, it is not known whether a particular cancer type can be treated by trying to inhibit glycolysis, unless the activity of the glucose metabolism pathway of a particular cancer cell type has been studied and understood.

We have surprisingly found that by inhibiting glucose metabolism, the growth of BHD-null cells can be inhibited. The invention therefore provides a new way of selectively targeting BHD-null cells and therefore provides a new treatment for BHD syndrome.

Compositions according to the invention may comprise an optional pharmaceutically acceptable carrier, diluent or excipient, including combinations thereof.

The compositions may be for human or animal usage in human and veterinary medicine and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier or excipient. Acceptable carriers or diluents for therapeutic use are well known in the art. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to the carrier, excipient or diluent, any suitable binder(s), lubricant(s), suspending agent (s), coating agent(s) or solubilising agent(s).

The present invention provides compositions comprising inhibitors of all aspects of glucose metabolism. The term "inhibiting" means decreasing, slowing or stopping.

The invention provides compositions comprising inhibitors of enzymes of glucose metabolism. An inhibitor of such an enzyme is a substance which reduces, attenuates, decreases or eliminates the expression and/or activity of such an enzyme. Expression in this context is used to refer to any of the steps of transcription and translation. Activity of the enzyme in this context is used to refer to enzymatic activity of a polypeptide encoded by a gene involved in glucose metabolism. An inhibitor may exert inhibition via any mechanism.

A suitable inhibitor may be capable of inhibiting hexokinase, phosphoglucose isomerase, phosphofructokinase, aldolase, triose phosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase or pyruvate kinase. A suitable inhibitor may be capable of inhibiting more than one enzyme within glucose metabolism, for example two, three, four or more enzymes involved in glucose metabolism.

The term glucose metabolism as used herein refers to not only the conversion of glucose to pyruvate, but also the conversion of pyruvate in anaerobic and aerobic glycolysis, and also the uptake of glucose by cells.

The invention provides not only compositions comprising inhibitors of the conversion of glucose and sequential metabolites to other intermediates in the glucose metabolism pathways, but also inhibitors of glucose uptake by cells. The invention provides inhibition of glucose metabolism by, for example, down regulation of the glucose transporter GLUT-i.

As used herein, "growth" means increase in size or proliferation or both. Therefore "growth" means both cell growth (increase in cell mass with cells getting larger) and cell proliferation (cells increasing in number via cell division). Thus a composition of the invention can inhibit a tumour cell from becoming larger and/or can prevent the tumour cell from dividing and replicating and increasing the number of tumour cells.

Preferably, the inhibitor of glucose metabolism is a glycolytic inhibitor.

The term glycolytic inhibitors as used herein refers to inhibitors that inhibit one or more of the steps involved in the conversion of glucose to pyruvate.

The glycolytic inhibitor may be a competitive inhibitor of glucose metabolism.

A competitive inhibitor can be bound by an enzyme in place of the substrate. The enzyme cannot bind the inhibitor and the substrate at the same time. A competitive inhibitor may bind in the active site of the enzyme and therefore prevent the binding of the substrate. A competitive inhibitor diminishes the rate of catalysis by reducing the proportion of enzyme molecules bound to a substrate.

Preferably, the glycolytic inhibitor is 2-deoxy-D-glucose (2DG) or a pharmaceutically acceptable salt or solvate thereof. 2-Deoxy-D-glucose is a glucose analogue lacking a hydroxyl group on carbon 2. The 2-hydroxyl group is replaced by hydrogen so that it cannot undergo further glycolysis. 2DG is also taken up by glucose transporters, thereby inhibiting the transport of glucose.

Preferably, the inhibitor of glucose metabolism is an inhibitor of pyruvate metabolism.

Pyruvate is converted to lactate as an alternative to conversion to acetyl-CoA and entry into the citric acid cycle. We have surprisingly found that BHD-null cells utilise this pathway even in the presence of oxygen, thereby preferentially utilising glucose for aerobic glycolysis rather than oxidative phosphorylation.

Preferably, the inhibitor of pyruvate metabolism is an inhibitor of lactate dehydrogenase.

Lactate dehydrogenase catalyses the conversion of pyruvate to lactate with concomitant conversion of NADH to NAD.

More preferably, the inhibitor of pyruvate metabolism is oxamate or a pharmaceutically acceptable salt or solvate thereof.

Oxamate is the ion of amino oxoacetic acid (aminooxoacetate). Aminooxoacetate has formula NH200000. Oxamate inhibits lactate dehydrogenase. Oxamate is available as aminooxoacetic acid sodium salt (NH200000Na) which is also know as oxalic acid monoamide sodium salt and oxamic acid sodium salt.

The invention also provides a method of treating Birt-Hogg-Dubé syndrome comprising administering to a subject a composition comprising an inhibitor of glucose metabolism.

The "subject" can include domesticated animals, such as cats, dogs etc., livestock (e.g. cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g. mouse, rabbit, rat, guinea pig etc.) and birds. Preferably, the subject is a mammal such as a primate, and more preferably a human.

The composition may further comprise a pharmaceutically acceptable carrier.

Suitably the composition is administered in amount that is effective to treat Birt-Hogg-Dubé syndrome in a subject. In general an "effective amount" of a compound is that amount needed to achieve the desired result or results.

A composition comprising a compound of the instant invention may be administered to a subject by any of a number of routes of administration including, for example, orally (for example drenches as in aqueous or non-aqueous solutions or suspension, tablets, boluses, powders, granules, pastes for application to the tongue); sublingually, anally, rectally, or vaginally (for example as a pessary, cream or foam); parenterally (including intramuscularly, intravenously, subcutaneously or intrathecally as for example a sterile solution or suspension); nasally; intraperitoneally; subcutaneously; transdermally (for example as a patch applied to the skin); or topically (for example as a cream, ointment or spray applied to the skin). The compound may also be formulated for inhalation.

In particular, the invention provides a method of inhibiting growth of BHD-null renal cell carcinoma cells comprising administering to a subject a composition comprising an inhibitor of glucose metabolism.

The invention also provides a method of treating renal cell carcinoma associated with BHD inactivation comprising administering to a subject a composition comprising an inhibitor of glucose metabolism.

The invention also provides a method of differentially inhibiting the growth of BHD-null cells over BHD-wild type cells comprising administering to a subject a composition comprising an inhibitor of glucose metabolism.

The invention will now be described by way of example with reference to the figures, in which: Figure 1 shows BHD negatively regulates HIF-induced gene expression during hypoxia. The mRNA levels of (a) BNIP3, (b) CCND1, (c) VEGF-A and (d)G6PD1 were compared within BHD (U0K257-2) and BHD (U0K257) cells treated overnight with 50 nM Rapamycin under normoxia (21 %) and hypoxia (1 %), where indicated, by qRT-PCR. mRNA levels were standardised against 3-Actin and fold activation was compared to BHD cells under normoxia. n = 3. , p < 0.05 when comparing BHD and BHD cells under hypoxia and normoxia and rapamycin treatment. (e) Western blot analysis were carried out on cell lysates prepared from BHD and BHD cells under normoxia (21 %) and hypoxia (1 %), where indicated. Protein levels of BHD, VEGF-A, BNIP3, CCND1, GLUT1 and 3-actin (as loading control) were determined.

Figure 2 show increased transcriptional activity of HIF1a in BHD cells. (a) The mRNA and (b) protein levels of HIF1a were compared within BHD (U0K257-2) and BHD (U0K257) cells after 18 h of normoxia (21 %) or hypoxia (1 %), where indicated, by RT-PCR. mRNA levels were standardised against 3-Actin and fold activation was compared to BHD cells under normoxia. n = 3. (c) BHD cells transiently transfected with a H IF-inducible luciferase reporter and either empty pRK7 or Flag-BHD vector (where indicated) were maintained at 1 % 02 for 18 h. Lysates were analyzed for luciferase fluorescence to determine the transcriptional activity of HIF. n=3. , p<0.05. Protein levels of Flag-tagged BHD were determined.

Figure 3 shows increased levels of HIF target proteins. Paraffin embedded samples were obtained from a chromophobe renal carcinoma from a patient with BHD. There is strong and specific staining with antibodies directed against the HIF targets BNIP3 (a, b), GLUT1 (c, d) and VEGF-A (e, f). Magnification is x 400 for all panels. Note the different staining patterns for BNIP3 and GLUT1 in chromophobe carcinoma compared to unaffected tissue. For VEGF, staining is much more intense in the tumor than in normal tissue but the intracellular distribution seems to be more diffuse.

In panels C and D, erythrocytes are clearly stained, serving as a positive internal control. In panels E and F, vascular endothelium does the same for VEGF.

Figure 4 shows increased enzyme activity levels of pyruvate kinase and lactate dehydrogenase within BHD cells. (a) Diagram of glucose and fatty acid metabolism depicting aerobic and anaerobic respiration. Enzyme activity assays were conducted in order to compare activity levels of (b) Hexokinase, (c) Pyruvate Kinase, (d) Lactate Dehyd rogenase, (e) 3-hyd roxyacyl-C0A dehydrogenase, (f) Citrate Synthase, (g) Malate Dehydrogenase within BHD (U0K257), BHD (U0K257-2) and HEK-293 cells. n = 3. , p < 0.01; , p < 0.05 when comparing enzyme activity in BHD and BHD cells.

Figure 5 shows inactivation of PDH in BHD cells. Western blot analysis were carried out on cell lysates prepared from BHD (U0K257-2) and BHD (U0K257) cells after 18 h of normoxia (21%) or hypoxia (1%), with and without 50 nM rapamycin, where indicated. Protein levels of BHD, rpS6, phosphorylated rpS6 at Ser235 and Ser236, Akt, phosphorylated Akt at Thr308, PDH, phosphorylated PDH at Ser293, PDK1 and 3-actin (employed as a loading control) where determined.

Figure 6 shows BHD cells utilise L-lactate as a metabolic fuel (a) The extracellular acidification rate (ECAR) was determined in the BHD (U0K257-2) and BHD (U0K257) cells under normoxia. (b) Western blot analyses were performed to determine protein levels of BHD, MCT1, MCT4, GLUT1 and GLUT4 from lysates prepared from BHD and BHD grown in normoxia. 3-Actin was used as a loading control. Densitometry analyses were also performed on (c) MCT1, and (d) MCT4. n = 4. , p < 0.05; , p < 0.01 relative to BHD and BHD cells. (e) Oxygen consumption was also measured in BHD and BHD cells treated with 5, 10, 15, and 20 mM L-lactate. n = 3. , p < 0.05 relative to oxygen consumption within BHD cells at 5 mM and 15 mM L-lactate.

Figure 7 shows proliferation of the BHD cells is selectively inhibited with 2-deoxyglucose. (a) BHD (U0K257-2) and (b) BHD (U0K257) were treated with 0 mM, 1.25 mM, 2.5 mM, 5 mM, 10 mM and 25 mM 2-deoxyglucose. The percentage of the original number of cells present in each cell line was then determined after 24, 48 and 72 h. n = 3. (c) % number of cells of BHD and BHD cells after 72 h of treatment with 2.5 mM, 5 mM and 10mM 2-deoxyglucose from figure 7A and 7B were directly compared. n = 3. p < 0.05; p < 0.01.

Figure 8 shows proliferation of the BHD cells is selectively inhibited with oxamate.

(a) BHD and (b) BHD were treated with different concentrations of oxamate. The percentage of the original number of cells present in each cell line was then determined after 24,48 and 72 h. Figure 9 is a schematic diagram showing the reaction steps in glucose metabolism.

Abbreviations used in the diagram are: HK, hexokinase; PGI, phosphoglucose isomerase; PFK, phosphofructokinase; TPI, triosephosphate isomerase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; PK, pyruvate kinase; PDH: pyruvate dehydrogenase; LDH:lactate dehydrogenase.

Figure 10 shows HIF activity is enhanced upon knockdown of BHD. Total protein levels of HIF1a and a-BHD during normoxia (21% 02) and hypoxia (1% 02) and rapamycin treatment were analysed for ACHN cells and ACHN cells that have had BHD stably knocked down with BHD shRNA (referred to as BHD in the figure) (Figure lOa). HIF-la activity was monitored by luciferase assay (Figure lOb).

Protein levels of GLUT1 and VEGF-A were determined (Figures 1 Oc, 1 Od).

Examples

We performed experiments to investigate whether inhibitors of the glucose metabolism pathway, such as 2-deoxy-D-glucose and oxamate, could be used to treat BHD.

Results Cells lacking BHD have elevated levels of HIF1a and HIF2a-driven gene expression To examine whether the transcriptional activity of HIF was upregulated within the BHD cells (from a UOK-257 cell line, a cell line that was derived from a patient with BHD), we compared the levels of HIF1a and HIF2a gene expression within the BHD cells to a stably generated U0K257-2 cell line that re-express wild-type BHD (Baba, 2006) (now referred to as BHD). To do this we analysed the mRNA levels of genes that are transcribed by HIF1a (BNIP3 (Figure 1A); Guo, 2001), HIF2a, (CCND1 (Figure 1B); Maxwell, 2005), or both (VEGF (Figure 10); Maxwell, 2005). Loss of BHD markedly increases the transcription of mRNAs that are regulated by both HIF1a and HIF2a under conditions of hypoxia. Furthermore, there is an elevated level of HIF1a and HIF2z-mediated gene expression under normoxic conditions.

Treatment of BHD cells with the specific mTOR inhibitor, rapamycin, inhibited expression of BNIP3 (Figure 1A) and CCND1 (Figure 1B) under hypoxic conditions suggesting that mTOR plays a role in hypoxia-mediated signalling in BHD cells. We did not observe any significant change in the levels of VEGF-A mRNA upon rapamycin treatment in the BHD cells (Figure 10). Of interest, the mRNA levels of BNIP3 (Figure 1A), CCND1 (Figure 1B) and VEGF-A (Figure 10), were very low in the BHD cells in both normoxic and hypoxic conditions.

A mechanism to maintain cellular redox homeostasis during hypoxia is through HIF-dependent glucose-6-Phosphate Dehydrogenase 1 (G6PD1) gene expression (Gao, 2004). Glucose-6-Phosphate Dehydrogenase 1 (G6PD1) is the rate-limiting enzyme within the pentose phosphate pathway (PPP, also referred to as the hexose-monophosphate shunt) that is induced upon oxygen stress. The PPP maintains the level of co-enzyme nicotinamide adenine dinucleotide phosphate (NADPH), the primary reducing agent within mammalian cells and in so doing helps maintain cellular redox homeostasis (Gao, 2004). Of interest, we observe a higher level of G6PD1 mRNA within the BHD cells when compared to the BHD cells (Figure 1 D).

The mRNA levels of G6PD1 under normoxic conditions were not further enhanced when the BHD cells were cultured in low oxygen suggesting that G6PD1 is maximally transcribed by HIF under normoxic conditions.

The protein levels of the HIFa transcriptional targets VEGF-A, BNIP3, CCND1, and GLUT1 were compared between the BHD cells and the BHD cells via western blotting (Figure 1E). We observed an increase in the protein levels of VEGF, BNIP3, CCND1 and GLUT1 in the BHD cells (lane 4) compared to the BHD cells (lane 3) under hypoxic conditions. Even under normoxia, there is an increase in the levels of BNIP3, VEGF-A and GLUT1 in the BHD cells (lane 2 compared to lane 1). These results show that the levels of HIF1a and HIF2a-regulated proteins are increased in this BHD patient renal tumour cell line.

Loss of BHD in U0K257 does not affect the mRNA or protein levels of HIF1a To determine whether BHD regulates HIF1a at the level of its gene-expression, we analysed the mRNA levels of HIF1a (Figure 2A) within the U0K257 cell lines. Our data showed no significant differences in the levels of HIF1a mRNA between the BHD and BHD cells under both normoxic and hypoxic conditions (Figure 2A). We next analysed the protein level of HIF1a under the same conditions (Figure 2B) and observed that hypoxia caused a marked accumulation of HIF1a protein within both the BHD and BHD cell lines. However there were no obvious differences between the cells. We then utilized a HIF-induced luciferase reporter construct to measure the transcriptional activity of HIF (Figure 20). In this assay, we observed a high level of HIF activity in the BHD null U0K257 cells (lane 1, Figure 20) which was dramatically repressed in the rescue experiment after BHD was re-expressed (lane 2, Figure 20). Collectively, this data suggests that over-expression of BHD does not affect HIF1a protein levels via gene-expression or protein stability but rather BHD impairs the transcriptional activity of HIF1a.

HIF gene expression is upregulated in a BHD tumour Immunohistochemical examination of a chromophobe renal carcinoma from a patient with BHD supports the in vitro data using antibodies against BNIP3, GLUT1 and VEGF (Figure 3). We observed staining patterns within the carcinoma that differed from those in unaffected parts of the kidney. For BNIP3, staining of tumour cells was prominent but uneven, being more pronounced near the cell membrane (Figure 3A).

This deviates from the pattern observed in unaffected tissue, where diffuse staining is observed in tubules (Figure 3B). Tumour cells showed intense GLUT1 staining, again concentrated near the membrane (Figure 30) whereas unaffected tubules displayed weaker and diffuse staining (Figure 3D). Likewise, tumour cells demonstrated intense, uneven but diffuse VEGF staining (Figure 3E). Unaffected tissue did not stain appreciably above background levels (Figure 3F).

Analysis of metabolic enzyme activities within U0K257 cells is suggestive of an altered metabolic phenotype that reflects the Warburg' Effect We hypothesized that the high level of HIF-mediated gene expression within the U0K257 cell line would alter their metabolism. To explore this notion, we analysed the activity of a series of metabolic enzymes involved in glucose metabolism, fatty acid oxidation and the Krebs cycle (also known as the tricarboxylic acid cycle). The enzymes examined are depicted in Figure 4A and were analysed from cells cultured in normoxic conditions. We did not observe any significant differences in hexokinase activity between HEK293 cells (the cell line we use to standardise these metabolic assays) and the BHD and BHD cell lines (Figure 4B). Hexokinase regulates the first step of glycolysis (phosphorylation of glucose to form glucose-6-phosphate) which is necessary to maintain a positive influx of glucose via glucose transporters such as GLUT1 (Robey, 2005). We observed a marked increase in the activity of pyruvate kinase (Figure 40) and lactate dehydrogenase (LDH) (Figure 4D) in cells devoid of BHD. These higher levels of pyruvate kinase and LDH activity within BHD cells would encourage the production of L-lactate. Indeed, the glycolytic enzyme, LDH-A is known to be induced by oxygen stress (Firth, 1994). We also saw a significant increase of 3-hydroxyacyl-CoA dehydrogenase (HOAD) activity which suggests that fatty acid oxidation might be also upregulated in BHD cells (Figure 4E). Analysis of two Kreb cycle enzymes, citrate synthase (Figure 4F) and malate dehydrogenase (Figure 4G) showed no marked difference of activity between the BHD and BHD cells and their activity was comparable to that of HEK293 cells. The data presented here suggest that BHD cells possess a metabolic profile that parallels the observation that Otto Warburg made of cancer cells (i.e. the BHD cells show the Warburg effect', as described above, which is a phenomenon where cancerous cells convert D-glucose to L-lactate rather than utilize oxidative phosphorylation even when oxygen levels are adequate (Warburg, 1956)).

Aerobic glycolysis is favoured within UOK257cells lacking BHD Pyruvate lies at the intersection of two glycolytic pathways: as a substrate for LDH enabling L-lactate production or as a substrate of the PDH reaction to generate the Krebs cycle entry molecule, acetyl CoA (see Figure 4A). Under low oxygen, PDH is typically inhibited via phosphorylation by PDK1. As a consequence of PDH phosphorylation, pyruvate is preferentially converted to lactic acid by LDH (Wigfield, 2008) and entry of acetyl-CoA from fatty acid oxidation is preferred. To examine whether the activity of PDH was affected in BHD cells we analysed the protein expression of PDK1 and levels of PDH phosphorylation (Figure 5). Of interest, we observed a robust increase in the levels of PDK1 in the BHD cells, when compared to the BHD cells under conditions of both normoxia (Figure 5, compare lane 1 and 3) and hypoxia (Figure 5, compare lane 5 and 7). 13-actin and PDH total protein expression are shown as protein loading controls. In accordance with the higher levels of PDK1 protein levels, PDH phosphorylation was markedly enhanced in the BHD cells (Figure 5, lanes 3 and 7) indicating that PDH is inhibited in these cells. We also analysed the phosphorylation of both ribosomal protein S6 (rpS6) and Akt (also known as protein kinase B). rpS6 is routinely employed to determine relative levels of signal transduction through the mTOR/70 kDa ribosomal protein S6 kinase 1 (S6K1) signalling pathway. As previously reported, the level of rpS6 phosphorylation is elevated in the BHD cells (Figure 5, lane 3), indicating that they have a higher basal level of mTOR signalling (Hartman, 2009) (Proc NatI Acad Sci U S A. 2009 Nov 3;106(44):18722-7. Epub 2009 Oct 22. Hasumi Y et al). Analysis of rpS6 phosphorylation revealed that rapamycin effectively repressed mTOR signalling within these BHD cells (Figure 5, lane 4). Under hypoxia, which is known to potently repress mTOR via multiple negative signalling feedback loops (Liu, 2006), we were only able to detect a modest level of rpS6 phosphorylation within the BHD cells (Figure 5, lane 7). We also observe an elevated level of Thr308 phosphorylation of Akt which suggests that phosphoinositide-dependent kinase-1 activity within these BHD cells is also elevated (Figure 5, lane 3 and 7). Our data reveals that there is an increase in both the activity of LDH (Figure 4D) and phosphorylation of PDH within the BHD cells (Figure 5), which implies that pyruvate might be converted more readily to L-lactate. If this is the case, the energy demands of the cell would have to be produced by either anaerobic glycolysis or by a shift towards fat derived acetyl-CoA.

LJOK257 cells utilise L-lactate as a metabolic fuel To test whether anaerobic glycolysis is increased in the BHD cells, we next sought to determine whether these cells produced more lactate through facilitated diffusion.

Surprisingly, the extracellular acidification rate, a measure of glycolytic rate and lactate production, was not different between the cells (Figure 6A). Of interest, the BHD cells had significantly more of the monocarboxylate transporter 1 (MCT1) associated with L-lactate influx and only marginally more of the efflux transporter MCT4 (Figure 6B, C and D). Consistent with these findings, the BHD cells were better able to uptake and oxidize lactate when exogenous lactate was added to the media (Figure 6E). These data suggest that the metabolic shift is not towards anaerobic glycolysis since lactate can be taken up and oxidised.

HIF activity is enhanced upon knockdown of BHD To examine effects when BHD expression is present or absent we utilised ACHN cells, which express BHD at endogenous levels, and ACHN cells that have had BHD stably knocked down with BHD shRNA (termed ACHN(BHD-). ACHN is a human renal carcinoma cell line. We analysed total protein levels of HIFict during normoxia (21 % 02) and hypoxia (1 % 02) (Figure lOa). We found that the basal protein level of H1F1a during normoxia was slightly higher in the ACHN(BHD-) cells when compared to the wild-type ACHN cells (Figure lOa, compare lanes 3 and 4, to lanes 1 and 2).

The protein level of HIF1a was increased after the cells were subjected to hypoxia (Figure lOa, lanes 5 to 8) but there was no significant differences between the levels of HIF1a protein between wild-type ACHN and ACHN(BHD-) cells. As expected, the ACHN(BHD-) cells had almost complete knock down of the BHD protein (Figure lOa, compare lanes 7 and 8 to lanes 5 and 6). Interestingly, even with similar levels of HIF1a protein in both the wild-type ACHN and ACHN(BHD-) cells after hypoxia, there was a higher level of activity of HIF1a in the ACHN cells lacking BHD (Figure lOb), which was partially sensitive to inhibition of mTOR with rapamycin. Consistent with a higher level of activity of HIF1a in cell lacking BHD, the ACHN(BHD-) also had higher levels of mRNA of HIF gene targets, GLUT1 (Figure lOc) and VEGF-A (Figure lOd).

These findings reveal that loss of BHD is able to enhance the activity of HIF1a during hypoxia where this enhanced activity is not through accumulation of more HIF1a protein, but is rather through enhanced activity of the HIF1a to function as a transcription factor.

The observations here are very different to how loss of VHL causes upregulation of HIF1a. Loss of VHL results in increased stability of HIF1a (Semenza, 2004).

Consequently, this accumulation of HIF1a in cells causes higher level of HIF1a-mediated gene expression even when cells are growing in normoxia. What we observe in these BHD-cells is quite different to what occurs in VHL null cells, in that that there is a higher level of HIF1a activity that is not due to an accumulation of more HIF1a protein.

2-deoxy-D-qlucose selectively prevents proliferation of the BHD cells Our pyruvate kinase, GLUT1, and G6PD1 data suggest that the U0K257 renal-derived cell line from the BHD patient has adapted its metabolism to possibly use fatty acid-derived acetyl-CoA or to increase the production and utilisation of the glycolytic intermediate L-lactate. To determine which hypothesis was more likely, we tested the dependency of BHD cells on glucose by competitively blocking glycolysis using 2-deoxy-D-glucose and examined cell proliferation (Figure 7A and 7B).

Consistent with a glycolytic requirement, the BHD cells were more sensitive to 2-deoxy-D-glucose (1C50 at 48h: 2.5mM) than the BHD cells (1C50 at 48h: 10mM) and show significant reduction to cell proliferation after 72h of treatment (Figure 7C). This suggests that the BHD cells have a higher dependence on glucose as a fuel. This inability to shift fuel oxidation to match availability is know as a loss of "metabolic flexibility" (Galgani, 2008).

Oxamate selectively impairs growth of BHD cells (U0K257) We tested the dependency of BHD cells to grow by competitively blocking pyruvate metabolism using oxamate and examined cell proliferation (Figure 8A and 8B). The BHD cells were more sensitive to oxamate than the BHD cells.

Materials and methods Antibodies and Other Biochemicals Anti-Folliculin antibodies were kindly provided by Dr Arnim Pause, McGill University, Canada. Anti-PDK1, Anti-Beta-Actin, Anti-GLUT1 and anti-VEGF-A antibodies were bought from Abcam (Cambridge, United Kingdom). Anti-phospho-ribsosomal protein S6 (Thr308, Ser476), anti-ribosomal protein S6, anti-phospho-AKT (Thr308, Ser476), total anti-Akt antibodies, anti-phospho Acetyl Choline carboxylase and total and phospho AMPKa antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-phospho-Pyruvate Dehyd rogenase (Th r306, Ser473) and anti-Pyruvate Dehydrogenase antibodies were purchased from Santa Cruz Biotechnology, inc. (Santa Cruz, CA. USA) and anti-BNIP3 antibodies and all other reagents used if not otherwise stated was obtained from Sigma-Aldrich. Rapamycin was purchased from Calbiochem/Merck (Beeston, Nottingham, UK). The N-terminal Flag-tagged BHD vector was a kind gift from Dr. Laura S. Schmitt (Bethesda, MD USA, described in (Baba, 2006)).

Cell Culture U0K257 and U0K257-2 cells that were a kind gift from Dr. Laura S. Schmitt (Bethesda, MD USA) were cultured in Dulbecco's Modified Eagle's Medium supplemented with 10 % (vlv) foetal calf serum, 100 U/mI penicillin and 100 pg/mI streptomycin (Gibco, Paisley, UK). U0K257-2 and U0K257 cells were incubated at either 21 % or 1 % oxygen either with or without 50 nM rapamycin treatment. After 48 h, these cells were harvested. U0K257 is the only RCC cell line that has been derived from a patient with BHD and harbours a germline FLCN frameshift mutation (c.l285dupC) (predicted, in the absence of nonsense mediated mRNA decay, to lead to premature protein truncation (p.His429ProfsX27) (Yang et al., Cancer Genet Cytogenet. 2008 Jan 15;180(2):100-9).

mRNA extraction and reverse transcription Cells were first washed in phosphate buffered saline (PBS), then lysed from the 6 cm2 plates using 0.5 ml RNAprotect® Cell Reagent (Qiagen, West Sussex, United Kingdom). RNA was then extracted using a RNeasy Plus mini kit (Qiagen, West Sussex, United Kingdom). Cell lysates were homogenised using Qiashredder tubes during the RNA extraction procedure as described in the manufacturer's protocol.

Real Time Quantitative PCR Total RNA from each sample (1 pg) was transcribed into cDNA using Quantitect reverse transcription kit (Qiagen, West Sussex, United Kingdom) in a thermal cycler (Applied Biosystems, California, USA). The sequences of the VEGF-A I-Il primers used were Forward 5'-CTGCTGTCTTGGGTGCATTG-3'; Reverse 5'-TTCACAATTTGTTGTGCTGTAG-3' as described in (Roland et al., 2000). All other primer sets were purchased from Qiagen, who have to right to with hold primer sequence information. Quantitative Real Time PCR reactions were conducted in 96 well plates using appropriate primer assays and Sybr Green PCR Master mix (Qiagen, West Sussex, United Kingdom). Assays were performed as follows: initial denaturation step (95 °C, 15 mm), 40 cycles of denaturation.(94 °C, 15 sec), annealing step (55 °C, 30 sec), extension step (72 °C, 40 sec). The amplification products were quantified during the extension step in the 40th cycle. The results were then determined using the ddCT method, and normalised first to 13-Actin. A dissociation step was performed, which verified that only one PCR product was produced with each primer set and shows their specificity. The correct size of PCR products were also verified via resolution on a 2% polyacrylamide gel. The expected size of the amplified products was approximately 70 for VEGFa, 104 bp for 13-Actin (Catalogue Number QT01680476), 73 bp for BNIP3 (Catalogue Number QT000241 78), 96 bp for CCND1 (Catalogue Number QT00495285), 91 bp for G6PD1 (Catalogue number QT00071596), and 104 bp for HIFict (Catalogue Number QT00083664). The information given above is in accordance to the minimum information for publication of real time quantitative PCR data as described in (Bustin et al., 2009).

Western Blotting Cells were washed in PBS and then lysed from the 6 cm2 plates using lysis buffer (20 mM Tris, 135 mM NaCI, 5 % (vlv) glycerol, 50 mM NaF and 0.1 % (vlv) triton X-100, pH 7.5 supplemented with complete mini protease inhibitor cocktail (Roche Diagnostics Ltd. Burgess Hill, United Kingdom) and 1 mM Dithiothreitol (DTT)) at 4 00. Following centrifugation at 13,000 rpm for 8 mm at 4 00 the samples were prepared in x4 NuPAGE® LDS Sample buffer (Invitrogen, Paisley, United Kingdom) with 25 mM DTT and boiled at 70 °C for 10 mm. For preparation of HIFIa samples, cells were lysed directly into 62.5mM Tris-HCI (pH 6.8), 2 % (wlv) SDS, 10 % (vlv) glycerol, 50 mM DTT, and 0.1 (wlv) bromophenol blue followed by pulse sonication and then subsequently boiled at 95 00 for 5 mm. Samples were then separated by electrophoresis using the NuPAGE® gel system (Invitrogen, Paisley, United Kingdom). Proteins were transferred to Polyvinylidene Difluoride membranes (Millipore, South West, United Kingdom), blocked in 5 % (wlv) dry milk powder/Tris buffered saline 0.1 % (vlv) tween (TBS-T), then probed using the required primary antibody and Horse Radish Peroxidse-conjugated secondary antibody in TBST.

Proteins were visualized using Enhanced Chemiluminescent solution and Hyperfilm (both from GE Healthcare, Buckinghamshire, United Kingdom). All western blots shown are representative of at least three independent experiments.

Luciferase Assay U0K257 cells were transfected with either pRK7 empty vector or Flag-tagged BHD with the firefly luciferase reporter pGL2-TK-HRE plasmid (a gift from G. Melillo (National Cancer Institute at Frederick, Maryland) using Lipofectamine 2000 transfection reagent (Invitrogen, Paisley, United Kingdom) according to the manufacturer's protocol. The pGL2-TK-HRE plasmid was generated by subcloning three hypoxia response elements (5'-GTGACTACGTGCTGCCTAG-3') from the inducible nitric-oxide synthase promoter into the promoter region of the pGL2-TK vector as previously described (Rapisarda, A., Uranchimeg, B., Scudiero, D. A., Selby, M., Sausville, E. A., Shoemaker, R. H., and Melillo, G. (2002) Cancer Res. 62, 4316-4324). Media was changed on cells 4 h post-transfection then after 24 h post transfection the cells were wash in PBS then lysed in lysis buffer (20 mM Tris, 135 mM NaCI, 5 % (vlv) glycerol, 50 mM NaF and 0.1 % (vlv) triton X-100, pH 7.5 supplemented with complete mini protease inhibitor cocktail (Roche Diagnostics Ltd. Burgess Hill, United Kingdom) and 1 mM DTT) at 4 00 Following centrifugation at 13,000 rpm for 8 mm at 4 00 20 il of each sample was then injected with 50 il Luciferase Reagent (20 mM HEPES (pH 7.7), 5 mM Mg504, 1 mM d-luciferin, and 2mM ATP). The protein amount in each sample was determined using the Bradford assay. Levels of Luciferase present were determined via a luminometer 2 s and 10 s after initial injection.

Enzyme assays The enzyme assay protocol where carried out as described in Suarez, 1986 where the cells were lysed directly into homogenization buffer (20 mM Hepes (pH 7.4), 2 mM EDTA, 0.1 % (vlv) triton X-100, and 10 mM DTT. Lysates were pulse sonicated and centrifugation at 13,000 rpm for 8 mm at 4 00. Protein levels were quantified by using a Bradford assay. For the Hexokinase, Pyruvate kinase and LDH enzyme assays the samples were assayed in 50 mM imidazole-HCI (pH 7.4) under the following conditions. Hexokinase: 5 mM glucose (omitted for control), 1 mM ATP, 5 mM MgCI2, 5 mM DTT, 0.5 mM NADP, and excess glucose-6-phosphate dehydrogenase (GGPDH). Pyruvate kinase: 5 mM phospho(enol)pyruvate (omitted for control), 5 mM ADP, 0.15 mM NADH, 10 mM MgC12, 100 mM KCI, 0.02 mM fructose 1,6-bisphosphate, 5 mM DTT, excess LDH. LDH assay: 4 mM pyruvate (omitted for control), 0.15 mM NADH, 5 mM DTT. For the Citrate synthase, Malate dehydrogenase and HOAD enzyme assays, samples were assayed in 50 mM Tris-CI (pH 8.0). Citrate synthase: 0.5 mM oxaloacetate (omitted for control), 0.3 mM acetyl CoA, 0.1 mM 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Malate dehydrogenase: 50 mM imidazole-HCI (pH 7.4), 10 mM oxaloacetate (omitted for control), 0.15 mM NADH, 5 mM DTT. HOAD: 50 mM imidazole-HCI (pH 7.4), 0.1 mM acetoacetyl CoA (omitted for control), 0.15 mM NADH, 1 mM EDTA, 5 mM DTT. Enzyme activity (U.mgP1; U = 1 umol substrate converted to product per minute) was determined from the maximum rate of change in absorbance at 340 nm (NADH-linked assays) or 412 nm (DNTB-linked assays) at 37 00 using a Beckman Coulter DU650 spectrophotometer. Results were normalised to the respective enzyme activity measured in parallel in HEK293 cells.

Immunohistochemistry Immunohistochemical staining was performed on paraffin embedded samples of a chromophobe renal cell carcinoma (samples obtained during total nephrectomy).

After deparaffination with xylene and rehydration, sections were incubated in 3 % (wlv) hydrogen peroxide (H202) diluted in methanol to inactivate endogenous peroxidases. Antigen retrieval was done by microwave treatment using citrate buffer (pH 6). Sections were then incubated for 60 mm with the following primary antibodies: polyclonal rabbit anti-GLUT1 (dilution 1:200), monoclonal mouse anti-BNIP3 (dilution 1:400) and monoclonal mouse anti-VEGF-A (dilution 1:150). After washing the samples, antibody visualization was performed with PowerVision (ImmunoVision Technology, Brisbane, CA, USA) using an HRP-conjugated second antibody that was incubated for 30 mm. Tissue was then counterstained with hematoxylin, dehydrated and coverslipped. Renal clear cell carcinoma was used as positive control tissue for BNIP3. Erythrocyte membranes and vascular endothelial cells were used as internal positive controls for GLUT1 and VEGF-A, respectively.

Negative control specimens, without the primary antibody, were included in all experiments.

2-deoxy-D-qlucose proliferation assay Cells were counted and 5 x i03 cells were plated per well of a black 96 well plate (one plate for each time point). 0.5 mM, 1 mM, 2.5 mM, 5 mM, 10 mM, 25 mM 2-deoxy-D-glucose were added 4 h after cells were plated and the plates incubated at 37 °C for 24, 48 or 72 h. At the required time point, media was removed from the cells and the plates frozen at -80 °C. Following thawing, 2O0il Cyquant mix, prepared as described in the CyQUANT® Cell Proliferation Assay (Invitrogen, Paisley, United Kingdom) manufacturer's handbook, was added to each well. Fluorescence was then measured using a fluorometer at 485 nm and 520 nm.

Conclusions

The Warburg effect is not apparent in all cancerous cells. However, the studies we have carried out show that U0K257 cells emulate Warburg's observation in that the U0K257 cells appear to utilize glucose for aerobic glycolysis rather than oxidative phosphorylation.

The higher expression levels of the HIF gene target, GLUT1 (Figure 1E, 3C and 6B) would enhance glucose uptake (Figure 5). Inhibition of pyruvate dehydrogenase via phosphorylation by PDK1 (Figure 5) would block pyruvate's entry into the Kreb's cycle, while the elevated level of lactate dehydrogenase activity within the cells (Figure 4) would increase L-lactate production (Wigfield, 2008). Although secretion of L-lactate is a marker of glycolysis, we did not observe any noticeable difference in the extracellular acidification rate (Figure 6A) or an increase of L-lactate secretion in U0K257 cells under normoxia (data not shown). The lack of L-lactate accumulation within culture media could be due to an increase of MCT1 expression (Figure 6B and 6C), which is known to regulate L-lactate uptake (Garcia, 1995), as well as favoured entry of glucose into the pentose phosphate pathway (Figure 1 D). Supporting higher uptake of L-lactate by MCT1, we do observe a high capacity for these U0K257 cells to oxidise L-lactate (Figure 6E). Recent studies revealed that L-lactate can act as a major source of fuel in adequately oxygenated tumour cells (Sonveaux, 2008). L-lactate from cells within the hypoxic core of a tumour is consequently taken up and oxidised by the surrounding oxygenated tumour cells. Utilization of L-lactate within the periphery of tumours as the main metabolic fuel might increase the amount of glucose that reaches and feeds the hypoxic tumour core. Such a mechanism would provide a better environment for tumour growth and expansion.

BHD is considered a tumour suppressor, but it is currently unclear how BHD functions to repress cell growth. Folliculin-interacting protein 1 was discovered to interact with folliculin and this protein complex is phosphorylated in an mTOR and AMP-dependent protein kinase (AMPK)-dependent manner (Baba et al, 2006). This evidence implies that FLCN is a downstream cell signalling component of both mTOR and AMPK. In the context of other inherited genetic disorders that give rise to RCC, HIF plays a pivotal role for tumour progression in VHL (Kaelin, 2005), HLRCC (Issacs, 2005) and TSC (Liu, 2003). Our data suggests that aberrant activation of HIF plays a role in cancer progression of renal tumours in BHD patients. Our data is not only particular to U0K257 cells, which are derived from a clear cell carcinoma and have chromosomal aberrations. Our immunohistochemical data show that our findings are broadly applicable. In a chromophobe carcinoma from a patient with Birt-Hogg-Dube syndrome, we find robustly increased levels of the HIF targets BNIP3, GLUT1 and VEGF, paralleling our findings in the U0K257 cells. Therefore, our findings are relevant to multiple BHD tumour types. It is of interest to note the pronounced membranous localization of the GLUT1 staining, which likely reflects the cells' increased glycolytic potential. Of note, BNIP3's expression pattern has changed in the tumour compared to unaffected tissue. BNIP3 does have a transmembrane domain and has a pro-apoptotic function. In the latter context, BNIP3 translocates to mitochondria (van de Velde et al, 2000). However, BNIP3 has emerging functions in regulating autophagy and may therefore well have protective functions during hypoxia. The apparent shift in staining intensity from cytoplasm to the cell membrane is a quite intriguing observation in this respect and might be consistent with BNIP3 fulfilling a protective rather than apoptotic function in the chromophobe carcinoma.

It is well appreciated that HIF1 and HIF2 are critical players in promoting tumour progression by driving gene expression that leads to cellular changes in energy metabolism, cell survival, angiogenesis, glucose transport, and metastasis (Semenza, 2004). When BHD expression is restored in U0K257 cells we see a robust inhibition of HIF-mediated gene expression (Figure 1) and HIF activity (Figure 20) implying that BHD negatively regulates HIF. Although it is unlikely that BHD modulates HIF directly, the high level of HIF-mediated gene expression observed in U0K257 cells is partially accounted for by the absence of BHD. Given that BHD is implicated in downstream signalling from AMPK (Baba et al, 2006), a known modulator of HIF and energy metabolism (Treins, 2006), it is plausible that loss of BHD might dysregulate signalling through AMPK and consequently activate HIF.

There are different mechanisms of dysregulated HIF signalling between inherited genetic disorders such as VHL, HLRCC and TSC. Enhanced stability of HIF-a proteins is the main contributing factor of cancer progression in both VHL (Bratslavsky, 2007) and HLRCC (Issacs, 2005), while an upregulation of mTOR signalling is the main driving influence of HIF in TSC. BHD has also been implicated with regulating Akt and mTOR (Baba, 2006; Hasumi, 2009), which are signalling pathways known to potently activate HIF (Hudson, 2002). It is possible that this higher basal level of Akt and mTOR signalling within the U0K257 cells (Figure 5) promotes the activity of HIF. In line with this notion, inhibition of mTOR with rapamycin treatment was able to impair hypoxia driven gene-expression of BNIP3 (Figure 1A), CCND1 (Figure 1 B) and G6PD1 (Figure 1 D) within these U0K257 cells.

Our study reveals that U0K257 cells have a higher dependency for glucose metabolism. By targeting glycolysis with 2-deoxy-D-glucose, which cannot be fully metabolised and thereby competitively blocks glycolysis, we were able to selectively inhibit the growth of U0K257 cells (Figure 7). This dependence on a single fuel for metabolic production of ATP is known as a loss of "metabolic flexibility." A loss of metabolic flexibility is a common feature of insulin resistance. However, in the case of insulin resistance the cells are unable to increase lipid metabolism in response to increased lipid supply. In BHD cells, there is a similar inability to increase lipid metabolism when glycolytic production of lactate is lost. Therefore, impairment of glycolytic production of lactate by inhibiting glycolysis using 2-deoxy-D-glucose can be used for the treatment of BHD-associated lesions renal carcinomas. From the new understanding of glucose metabolism in BHD cells shown by our studies, it will be understood that other glycolytic inhibitors, that inhibit the conversion of glucose to pyruvate, can be also used to treat Birt-Hogg-Dubé syndrome.

By targeting glucose metabolism with oxamate, which is a competitive inhibitor of lactate dehydrogenase (LDH), we were able to selectively inhibit the growth of U0K257 cells (Figure 8). Oxamate is a structural analogue of pyruvate, and binds to and inhibits LDH. Oxamate competes with enzymes that use pyruvate as a substrate, and therefore competitively inhibits LDH, gluconogenesis and pyruvate entry into the mitochondria. These data show that impairment of glycolytic production of lactate by inhibiting pyruvate metabolism using 2-deoxy-D-glucose or another pyruvate metabolism inhibitor can be used for the treatment of BHD-associated lesions renal carcinomas.

We have now demonstrated that the targeting of metabolic pathways in BHD cells using inhibitors of glucose metabolism (including inhibitors of glucose metabolism yet to be discovered) can be used for the treatment of (unresectable or metastasized) renal cancer in BHD syndrome.

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It will be understood that various details of the invention may be changed without departing from the scope of the invention. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation -the invention being defined by the claims.

Claims (35)

1. CLAIMS1. A composition comprising an inhibitor of glucose metabolism for use in the treatment of Birt-Hogg-Dube syndrome.
2. 2. A composition according to claim 1 wherein the inhibitor of glucose metabolism is a glycolytic inhibitor.
3. 3. A composition according to claim 2 wherein the glycolytic inhibitor is a competitive inhibitor of glucose metabolism.
4. 4. A composition according to claim 2 or claim 3 wherein the glycolytic inhibitor is 2-deoxy-D-glucose or a pharmaceutically acceptable salt or solvate thereof.
5. 5. A composition according to claim 1 wherein the inhibitor of glucose metabolism is an inhibitor of pyruvate metabolism.
6. 6. A composition according to claim 5 wherein the inhibitor of pyruvate metabolism is a competitive inhibitor of pyruvate metabolism.
7. 7. A composition according to claim 5 or 6 wherein the inhibitor of pyruvate metabolism is an inhibitor of lactate dehydrogenase.
8. 8. A composition according to any one of claims 5 to 7 wherein the inhibitor of pyruvate metabolism is oxamate or a pharmaceutically acceptable salt or solvate thereof.
9. 9. A composition comprising an inhibitor of glucose metabolism for use in the inhibition of growth of BHD-null renal cell carcinoma cells.
10. 10. A composition according to claim 9 wherein the inhibitor of glucose metabolism is a glycolytic inhibitor.
11. 11. A composition according to claim 10 wherein the glycolytic inhibitor is a competitive inhibitor of glucose metabolism.
12. 12. A composition according to claim 10 or claim 11 wherein the glycolytic inhibitor is 2-deoxy-D-glucose or a pharmaceutically acceptable salt or solvate thereof.
13. 13. A composition according to claim 9 wherein the inhibitor of glucose metabolism is an inhibitor of pyruvate metabolism.
14. 14. A composition according to claim 13 wherein the inhibitor of pyruvate metabolism is a competitive inhibitor of pyruvate metabolism.
15. 15. A composition according to claim 13 or 14 wherein the inhibitor of pyruvate metabolism is an inhibitor of lactate dehydrogenase.
16. 16. A composition according to any one of claims 13 to 15 wherein the inhibitor of pyruvate metabolism is oxamate or a pharmaceutically acceptable salt or solvate thereof.
17. 17. A composition comprising an inhibitor of glucose metabolism for use in the treatment of renal cell carcinoma associated with BHD gene inactivation.
18. 18. A composition according to claim 17 wherein the inhibitor of glucose metabolism is a glycolytic inhibitor.
19. 19. A composition according to claim 18 wherein the glycolytic inhibitor is a competitive inhibitor of glucose metabolism.
20. 20. A composition according to claim 18 or 19 wherein the glycolytic inhibitor is 2-deoxy-D-glucose or a pharmaceutically acceptable salt or solvate thereof.
21. 21. A composition according to claim 17 wherein the inhibitor of glucose metabolism is an inhibitor of pyruvate metabolism.
22. 22. A composition according to claim 21 wherein the inhibitor of pyruvate metabolism is a competitive inhibitor of pyruvate metabolism.
23. 23. A composition according to claim 21 or 22 wherein the inhibitor of pyruvate metabolism is an inhibitor of lactate dehydrogenase.
24. 24. A composition according to any one of claims 21 to 21 wherein the inhibitor of pyruvate metabolism is oxamate or a pharmaceutically acceptable salt or solvate thereof.
25. 25. A composition comprising an inhibitor of glucose metabolism for use in the differential growth inhibition of BHD-null cells over BHD-wild type cells.
26. 26. A composition according to claim 25 wherein the inhibitor of glucose metabolism is a glycolytic inhibitor.
27. 27. A composition according to claim 26 wherein the glycolytic inhibitor is a competitive inhibitor of glucose metabolism.
28. 28. A composition according to claim 26 or claim 27 wherein the glycolytic inhibitor is 2-deoxy-D-glucose or a pharmaceutically acceptable salt or solvate thereof.
29. 29. A composition according to claim 25 wherein the inhibitor of glucose metabolism is an inhibitor of pyruvate metabolism.
30. 30. A composition according to claim 29 wherein the inhibitor of pyruvate metabolism is a competitive inhibitor of pyruvate metabolism.
31. 31. A composition according to claim 29 or 30 wherein the inhibitor of pyruvate metabolism is an inhibitor of lactate dehydrogenase.
32. 32. A composition according to any one of claims 29 to 31 wherein the inhibitor of pyruvate metabolism is oxamate or a pharmaceutically acceptable salt or solvate thereof.
33. 33. A composition according to any of claims 1, 9, 17 or 25 wherein the inhibitor of glucose metabolism is an inhibitor of an enzyme involved in glucose metabolism.
34. 34. A composition according to claim 33 wherein the inhibitor of glucose metabolism is an inhibitor of hexokinase, pyruvate kinase or lactate dehydrogenase.
35. 35. A composition substantially as herein described and illustrated.