Clinical development of cancer therapeutics that target metabolism.

Glucose and glutamine metabolism in cancer cells are markedly elevated relative to non-transformed normal cells. This metabolic reprogramming enables the production of adenosine triphosphate and the anabolic precursors needed for survival, growth and motility. The recent observations that mutant oncogenic proteins and the loss of tumor suppressors activate key metabolic enzymes suggest that selective inhibition of these enzymes may yield effective cancer therapeutics with acceptable toxicities. In support of this concept, pre-clinical studies of small molecule antagonists of several metabolic enzymes in tumor-bearing mice have demonstrated reasonable therapeutic indices. We will review the rationale for targeting metabolic enzymes as a strategy to treat cancer and will detail the results of several recent clinical trials of metabolic inhibitors in advanced cancer patients.

6-Phosphofructo-2-kinase (PFKFB3) promotes cell cycle progression and suppresses apoptosis via Cdk1-mediated phosphorylation of p27.

The control of glucose metabolism and the cell cycle must be coordinated in order to guarantee sufficient ATP and anabolic substrates at distinct phases of the cell cycle. The family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) are well established regulators of glucose metabolism via their synthesis of fructose-2,6-bisphosphate (F2,6BP), a potent allosteric activator of 6-phosphofructo-1-kinase (Pfk-1). PFKFB3 is overexpressed in human cancers, regulated by HIF-1α, Akt and PTEN, and required for the survival and growth of multiple cancer types. Although most functional studies of the role of PFKFB3 in cancer progression have invoked its well-recognized function in the regulation of glycolysis, recent observations have established that PFKFB3 also traffics to the nucleus and that its product, F2,6BP, activates cyclin-dependent kinases (Cdks). In particular, F2,6BP stimulates the Cdk-mediated phosphorylation of the Cip/Kip protein p27 (threonine 187), which in turn results in p27's ubiquitination and proteasomal degradation. As p27 is a potent suppressor of the G1/S transition and activator of apoptosis, we hypothesized that the known requirement of PFKFB3 for cell cycle progression and prevention of apoptosis may be partly due to the ability of F2,6BP to activate Cdks. In this study, we demonstrate that siRNA silencing of endogenous PFKFB3 inhibits Cdk1 activity, which in turn stabilizes p27 protein levels causing cell cycle arrest at G1/S and increased apoptosis in HeLa cells. Importantly, we demonstrate that the increase in apoptosis and suppression of the G1/S transition caused by siRNA silencing of PFKFB3 expression is reversed by co-siRNA silencing of p27. Taken together with prior publications, these observations support a model whereby PFKFB3 and F2,6BP function not only as regulators of Pfk-1 but also of Cdk1 activity, and therefore serve to couple glucose metabolism with cell proliferation and survival in transformed cells.

Control of glutamine metabolism by the tumor suppressor Rb.

Retinoblastoma (Rb) protein is a tumor suppressor that is dysregulated in a majority of human cancers. Rb functions to inhibit cell cycle progression in part by directly disabling the E2F family of cell cycle-promoting transcription factors. Because the de novo synthesis of multiple glutamine-derived anabolic precursors is required for cell cycle progression, we hypothesized that Rb also may directly regulate proteins involved in glutamine metabolism. We examined glutamine metabolism in mouse embryonic fibroblasts (MEFs) isolated from mice that have triple knock-outs (TKO) of all three Rb family members (Rb-1, Rbl1 and Rbl2) and found that loss of global Rb function caused a marked increase in (13)C-glutamine uptake and incorporation into glutamate and tricarboxylic acid cycle (TCA) intermediates in part via upregulated expression of the glutamine transporter ASCT2 and the activity of glutaminase 1 (GLS1). The Rb-controlled transcription factor E2F-3 altered glutamine uptake by direct regulation of ASCT2 mRNA and protein expression, and E2F-3 was observed to associate with the ASCT2 promoter. We next examined the functional consequences of the observed increase in glutamine uptake and utilization and found that glutamine exposure potently increased oxygen consumption, whereas glutamine deprivation selectively decreased ATP concentration in the Rb TKO MEFs but not the wild-type (WT) MEFs. In addition, TKO MEFs exhibited elevated production of glutathione from exogenous glutamine and had increased expression of gamma-glutamylcysteine ligase relative to WT MEFs. Importantly, this metabolic shift towards glutamine utilization was required for the proliferation of Rb TKO MEFs but not for the proliferation of the WT MEFs. Last, addition of the TCA cycle intermediate α-ketoglutarate to the Rb TKO MEFs reversed the inhibitory effects of glutamine deprivation on ATP, GSH levels and viability. Taken together, these studies demonstrate that the Rb/E2F cascade directly regulates a major energetic and anabolic pathway that is required for neoplastic growth.

A novel small molecule antagonist of choline kinase-α that simultaneously suppresses MAPK and PI3K/AKT signaling.

Choline kinase-α expression and activity are increased in multiple human neoplasms as a result of growth factor stimulation and activation of cancer-related signaling pathways. The product of choline kinase-α, phosphocholine, serves as an essential metabolic reservoir for the production of phosphatidylcholine, the major phospholipid constituent of membranes and substrate for the production of lipid second messengers. Using in silico screening for small molecules that may interact with the choline kinase-α substrate binding domain, we identified a novel competitive inhibitor, N-(3,5-dimethylphenyl)-2-[[5-(4-ethylphenyl)-1H-1,2,4-triazol-3-yl]sulfanyl] acetamide (termed CK37) that inhibited purified recombinant human choline kinase-α activity, reduced the steady-state concentration of phosphocholine in transformed cells, and selectively suppressed the growth of neoplastic cells relative to normal epithelial cells. Choline kinase-α activity is required for the downstream production of phosphatidic acid, a promoter of several Ras signaling pathways. CK37 suppressed mitogen-activated protein kinase and phosphatidylinositol 3-kinase/AKT signaling, disrupted actin cytoskeletal organization, and reduced plasma membrane ruffling. Finally, administration of CK37 significantly decreased tumor growth in a lung tumor xenograft mouse model, suppressed tumor phosphocholine, and diminished activating phosphorylations of extracellular signal-regulated kinase and AKT in vivo. Together, these results further validate choline kinase-α as a molecular target for the development of agents that interrupt Ras signaling pathways, and indicate that receptor-based computational screening should facilitate the identification of new classes of choline kinase-α inhibitors.

Mseleni joint disease: a potential model of epigenetic chondrodysplasia.

OBJECTIVE: In this paper past research on the natural history of Mseleni joint disease, a crippling endemic osteoarthritis, its socio-economic impacts, the demographics, diet, geology and the genetic background of affected people are reviewed. In addition, some new research ideas are suggested to continue the search for etiological avenues for this disease such as stable isotope analysis and epigenetic mechanisms. RESULTS: Mseleni joint disease is a chondrodysplasia first described in 1970. It is geographically confined to a remote area in the Maputaland region in northern Kwazulu Natal, South Africa. This disease affects most joints but primarily those of the hip; it is a progressive condition beginning with pain and stiffness until the patient's ability to walk becomes compromised. Mseleni joint disease is characterized by two distinct abnormalities, protrusio acetabuli that mainly affects females and increases in frequency with age, and hip dysplasia that is more frequent with age. Much research has been conducted on the people with the disease and their surrounding environment. CONCLUSION: Despite intensive investigations into the etiology of Mseleni joint disease, it remains unknown. As a result the examination of epigenetic mechanisms and stable isotope analysis of teeth are suggested as a means of providing information on the etiology of the disease. These methods can also be applied to other chondroplasias of unknown etiology.

Selective inhibition of choline kinase simultaneously attenuates MAPK and PI3K/AKT signaling.

Choline is an essential anabolic substrate for the synthesis of phospholipids. Choline kinase phosphorylates choline to phosphocholine that serves as a precursor for the production of phosphatidylcholine, the major phospholipid constituent of membranes and substrate for the synthesis of lipid signaling molecules. Nuclear magnetic resonance (NMR)-based metabolomic studies of human tumors have identified a marked increase in the intracellular concentration of phosphocholine relative to normal tissues. We postulated that the observed intracellular pooling of phosphocholine may be required to sustain the production of the pleiotropic lipid second messenger, phosphatidic acid. Phosphatidic acid is generated from the cleavage of phosphatidylcholine by phospholipase D2 and is a key activator of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/AKT survival signaling pathways. In this study we show that the steady-state concentration of phosphocholine is increased by the ectopic expression of oncogenic H-Ras(V12) in immortalized human bronchial epithelial cells. We then find that small interfering RNA (siRNA) silencing of choline kinase expression in transformed HeLa cells completely abrogates the high concentration of phosphocholine, which in turn decreases phosphatidylcholine, phosphatidic acid and signaling through the MAPK and PI3K/AKT pathways. This simultaneous reduction in survival signaling markedly decreases the anchorage-independent survival of HeLa cells in soft agar and in athymic mice. Last, we confirm the relative importance of phosphatidic acid for this pro-survival effect as phosphatidic acid supplementation fully restores MAPK signaling and partially rescues HeLa cells from choline kinase inhibition. Taken together, these data indicate that the pooling of phosphocholine in cancer cells may be required to provide a ready supply of phosphatidic acid necessary for the feed-forward amplification of cancer survival signaling pathways.

Brief communication: Minimally invasive bone sampling method for DNA analysis.

Obtaining a bone sample for DNA analysis has traditionally been a destructive practice, which has resulted in reluctance on behalf of curators for skeletal collections to allow invasive testing. A novel minimally invasive bone sampling method for DNA analysis is presented here. This method uses a conventional hand drill wherein the bone sample is extracted from the intercondylar fossa of the femur; it does not interfere with any known anthropometric landmarks and only leaves a small hole on the surface of the bone. The temperature of the drill is documented and it was established due to the minor increase in temperature, that this should not affect the molecular integrity of the sample. This method is easily replicated and is suitable for both human and other animal skeletal material and can be applied to rare specimens with little risk.

A high resolution SEM study of the effects of RU486, used as a postcoital contraceptive, on the rat uterus during early pregnancy.

During the window of receptivity, a narrow range of time under the control of the ovarian hormones progesterone and oestrogen, when a blastocyst can attach to the uterine surface, the plasma membrane of the uterine epithelial cells undergoes a remarkable change in structure, known as 'the plasma membrane transformation' of early pregnancy. RU486, the controversial abortion drug (Mifegyne), acts as a progesterone receptor antagonist, resulting in transcriptionally inactive progesterone receptors. In view of this, a change in the well-documented sequences of the plasma membrane transformation is postulated. This study therefore aims to investigate the effects of RU486 on this sequence of events in the implantation and non-implantation sites of the rat uterus. In both RU486 treated and control animals, on days 4.5, 5.5 and 6.5 of pregnancy, scanning electron microscopy revealed a distinct pattern of folding of the uterine surface in non-implantation sites. In contrast, folding was not observed within the implantation sites. These results indicate that surface alterations are probably not under the control of progesterone signalling. The lack of folding at the implantation sites possibly ensures maximum close contact between the blastocyst and the maternal tissue thus promoting implantation. During early pregnancy, specifically on day 5.5, the microvilli of the uterine epithelial cells in the treated animals were more dense than those in the untreated animals. Such microvilli are characteristic of the uterine epithelial cells of a uterus under-stimulated by hormones. Flattening of the apical cell borders usually seen at the time of blastocyst attachment and implantation was not observed following RU486 treatment. Large apical protrusions were observed in the RU486 treated animals only, possibly linked in some way to apoptosis. The antiprogestin properties of RU486 may further elucidate the progesterone effects associated with early pregnancy.

The relationship between alkaline phosphatase activity and intracellular lipid accumulation in murine 3T3-L1 cells and human preadipocytes.

Alkaline phosphatase (ALP) is expressed in 3T3-L1 preadipocytes, and its activity increases during adipogenesis. The purpose of this study was to determine whether ALP activity could be used as a measure of intracellular lipid accumulation in human preadipocytes and 3T3-L1 cells and which of the factors that induce adipogenesis are responsible for stimulating ALP activity. Adipogenesis was initiated in 3T3-L1 cells by incubation with differentiation medium containing insulin, dexamethasone, and 3-isobutyl-1-methylxanthine. The effect of leaving out each of the differentiation medium components was studied. Adipogenesis was also assessed in human preadipocytes and 3T3-L1 cells in the presence of the ALP inhibitor histidine. ALP activity was measured using an automated colorimetric assay and intracellular lipid accumulation was measured using the lipid-specific dye oil red O. Removal of insulin or dexamethasone from the differentiation medium had little effect on either ALP activity or lipid accumulation in 3T3-L1 cells, while removal of IBMX blocked both. Histidine inhibited ALP activity and adipogenesis in human preadipocytes and 3T3-L1 cells. Pearson univariate correlation analysis demonstrated strong correlations between ALP activity and lipid accumulation in human preadipocytes (r=0.78, n=69) and in 3T3-L1 cells (r=0.92, n=27). These data suggest that ALP and fat storage are tightly linked during preadipocyte maturation and that the measurement of ALP activity may be a novel technique for the quantification of intracellular lipid accumulation that is more sensitive and rapid than currently used methods.

The effect of alkaline phosphatase inhibitors on intracellular lipid accumulation in preadipocytes isolated from human mammary tissue.

BACKGROUND: A previous study has demonstrated that alkaline phosphatase (AP) may play a role in the control of intracellular lipid accumulation in the rodent preadipocyte cell line, 3T3-L1. The present study investigated whether AP may have a similar function in preadipocytes isolated from human mammary gland tissue. METHODS: Preadipocyte maturation was induced in the presence or absence of the tissue non-specific AP inhibitors levamisole and histidine, and the tissue-specific AP inhibitor PheGlyGly. Cellular AP activity and adipogenesis were both assessed at 0 and 12 days post-induction of differentiation. RESULTS: After differentiation, AP activity increased 5.1 +/- 1.3-fold in the absence and 8.9 +/- 2.8-fold (P < 0.05) in the presence of levamisole. However, adipogenesis increased 1.95 +/- 0.11-fold in the absence but only 1.36 +/- 0.06-fold (P < 0.001) in the presence of levamisole. There was a 4.2 +/- 2.2-fold increase in AP activity in the absence and a 0.51 +/- 0.46-fold (P < 0.05) decrease in the presence of histidine. Adipogenesis increased 2.09 +/- 0.35-fold in the absence of histidine but only 1.22 +/- 0.30-fold (P < 0.05) in the presence of histidine. PheGlyGly had no effects. Fluorescent microscopy showed AP activity was localized to the triglyceride-containing droplets of the cell. CONCLUSION: This is the first study to show that tissue non-specific AP inhibitors can block adipogenesis in human preadipocytes.

Alkaline phosphatase is involved in the control of adipogenesis in the murine preadipocyte cell line, 3T3-L1.

OBJECTIVE: As alkaline phosphatase may play a role in cell differentiation, our aim was to study the possible role of this enzyme in the differentiation of preadipocytes (3T3-L1 cells) into adipocytes. RESEARCH METHODS AND PROCEDURES: 3T3-L1 cells were grown in medium containing insulin, dexamethasone and IBMX to induce adipogenesis. Adipogenesis was measured using the triglyceride-specific dye, oil red O at 0, 3, 7 and 11 days after initiation of adipogenesis in the presence or absence of the alkaline phosphatase inhibitors, levamisole, histidine and Phe-Gly-Gly. Intracellular localisation of the enzyme was detected using ELF-phosphatase, a fluorescent substrate and alkaline phosphatase gene expression was assessed using RT-PCR. RESULTS: Alkaline phosphatase activity was detected in untransformed cells (1.91+/-0.62 mU/mg protein) and activity increased 11.5+/-1.4-fold after 11 days treatment with transformation medium and 5.3+/-0.3-fold in transformation medium containing levamisole (p<0.05). Triglyceride content of cells increased 3.1+/-0.2-fold after 11 days treatment with transformation medium and 2.1+/-0.3-fold in the presence of levamisole (p<0.005). Histidine inhibited adipogenesis and alkaline phosphatase to a greater extent than did levamisole, but Phe-Gly-Gly had no effect on these variables. Alkaline phosphatase was localised around the lipid droplets of the cells. Gene expression of alkaline phosphatase increased during adipogenesis. DISCUSSION: This study demonstrates that tissue-nonspecific alkaline phosphatase is present in 3T3-L1 cells and that it may play a role in the control of adipogenesis.