Identification of LDH-A as a therapeutic target for cancer cell killing via (i) p53/NAD(H)-dependent and (ii) p53-independent pathways.

Most cancer cells use aerobic glycolysis to fuel their growth. The enzyme lactate dehydrogenase-A (LDH-A) is key to cancer's glycolytic phenotype, catalysing the regeneration of nicotinamide adenine dinucleotide (NAD(+)) from reduced nicotinamide adenine dinucleotide (NADH) necessary to sustain glycolysis. As such, LDH-A is a promising target for anticancer therapy. Here we ask if the tumour suppressor p53, a major regulator of cellular metabolism, influences the response of cancer cells to LDH-A suppression. LDH-A knockdown by RNA interference (RNAi) induced cancer cell death in p53 wild-type, mutant and p53-null human cancer cell lines, indicating that endogenous LDH-A promotes cancer cell survival irrespective of cancer cell p53 status. Unexpectedly, however, we uncovered a novel role for p53 in the regulation of cancer cell NAD(+) and its reduced form NADH. Thus, LDH-A silencing by RNAi, or its inhibition using a small-molecule inhibitor, resulted in a p53-dependent increase in the cancer cell ratio of NADH:NAD(+). This effect was specific for p53(+/+) cancer cells and correlated with (i) reduced activity of NAD(+)-dependent deacetylase sirtuin 1 (SIRT1) and (ii) an increase in acetylated p53, a known target of SIRT1 deacetylation activity. In addition, activation of the redox-sensitive anticancer drug EO9 was enhanced selectively in p53(+/+) cancer cells, attributable to increased activity of NAD(P)H-dependent oxidoreductase NQO1 (NAD(P)H quinone oxidoreductase 1). Suppressing LDH-A increased EO9-induced DNA damage in p53(+/+) cancer cells, but importantly had no additive effect in non-cancer cells. Our results identify a unique strategy by which the NADH/NAD(+) cellular redox status can be modulated in a cancer-specific, p53-dependent manner and we show that this can impact upon the activity of important NAD(H)-dependent enzymes. To summarise, this work indicates two distinct mechanisms by which suppressing LDH-A could potentially be used to kill cancer cells selectively, (i) through induction of apoptosis, irrespective of cancer cell p53 status and (ii) as a part of a combinatorial approach with redox-sensitive anticancer drugs via a novel p53/NAD(H)-dependent mechanism.

Hypoxia modulates the activity of a series of clinically approved tyrosine kinase inhibitors.

BACKGROUND AND PURPOSE: Hypoxia in tumours is known to cause resistance to conventional chemotherapeutic drugs. In contrast, little is known about the effects of hypoxia on targeted anti-cancer drugs. This study evaluated the effect of hypoxia on a series of clinically approved tyrosine kinase inhibitors (TKIs). EXPERIMENTAL APPROACH: The effect of hypoxia (0.1% oxygen) on the activity of conventional cytotoxic drugs (5-fluorouracil, doxorubicin and vinblastine), the hypoxia-activated prodrug tirapazamine and 9 TKIs was determined in a panel of cell lines. Where hypoxia had a marked effect on chemosensitivity, Western blot analysis was conducted to determine the effect of hypoxia on target expression and the effect of TKIs on cell signalling response under aerobic and hypoxic conditions. KEY RESULTS: Three patterns of chemosensitivity were observed: resistance under hypoxia, equitoxic activity against hypoxic and aerobic cells, and preferential cytotoxicity to hypoxic cells. Significant hypoxia selectivity (independent of HIF1) was observed in the case of dasatinib and this correlated with the ability of dasatinib to inhibit phosphorylation of Src at tyrosine 530. Sorafenib was significantly less effective under hypoxic conditions but resistance did not correlate with hypoxia-induced changes in Raf/MEK/ERK signalling. CONCLUSIONS AND IMPLICATIONS: Hypoxia influences the activity of TKIs but in contrast to conventional cytotoxic drugs, preferential activity against hypoxic cells can occur. The search for hypoxia-targeted therapies has been long and fruitless and this study suggests that some clinically approved TKIs could preferentially target the hypoxic fraction of some tumour types.

The control of bone remodeling by neuropeptide Y receptors.

An important role for the neuropeptide Y receptor system in the regulation of bone formation was recently revealed with a significant elevation in trabecular bone formation and bone volume following germline or hypothalamus-specific deletion of neuropeptide Y2 receptors in mice. Subsequent studies have now demonstrated that this central pathway is distinct from that of the other centrally regulated bone formation pathway mediated by leptin. This review discusses these recent findings and outlines how these new pathways could translate into potential novel targets for the treatment of bone disease.

Retinoblastoma protein disrupts interactions required for RNA polymerase III transcription.

The retinoblastoma protein (RB) has been shown to suppress RNA polymerase (Pol) III transcription in vivo (R. J. White, D. Trouche, K. Martin, S. P. Jackson, and T. Kouzarides, Nature 382:88-90, 1996). This regulation involves interaction with TFIIIB, a multisubunit factor that is required for the expression of all Pol III templates (C. G. C. Larminie, C. A. Cairns, R. Mital, K. Martin, T. Kouzarides, S. P. Jackson, and R. J. White, EMBO J. 16:2061-2071, 1997; W.-M. Chu, Z. Wang, R. G. Roeder, and C. W. Schmid, J. Biol. Chem. 272:14755-14761, 1997). However, it has not been established why RB binding to TFIIIB results in transcriptional repression. For several Pol II-transcribed genes, RB has been shown to inhibit expression by recruiting histone deacetylases, which are thought to decrease promoter accessibility. We present evidence that histone deacetylases exert a negative effect on Pol III activity in vivo. However, RB remains able to regulate Pol III transcription in the presence of the histone deacetylase inhibitor trichostatin A. Instead, RB represses by disrupting interactions between TFIIIB and other components of the basal Pol III transcription apparatus. Recruitment of TFIIIB to most class III genes requires its binding to TFIIIC2, but this can be blocked by RB. In addition, RB disrupts the interaction between TFIIIB and Pol III that is essential for transcription. The ability of RB to inhibit these key interactions can explain its action as a potent repressor of class III gene expression.

RNA polymerase III transcription factor TFIIIC2 is overexpressed in ovarian tumors.

Most transformed cells display abnormally high levels of RNA polymerase (pol) III transcripts. Although the full significance of this is unclear, it may be fundamental because healthy cells use two key tumor suppressors to restrain pol III activity. We present the first evidence that a pol III transcription factor is overexpressed in tumors. This factor, TFIIIC2, is a histone acetyltransferase that is required for synthesis of most pol III products, including tRNA and 5S rRNA. TFIIIC2 is a complex of five polypeptides, and mRNAs encoding each of these subunits are overexpressed in human ovarian carcinomas; this may explain the elevated TFIIIC2 activity that is found consistently in the tumors. Deregulation in these cancers is unlikely to be a secondary response to rapid proliferation, because there is little or no change in TFIIIC2 mRNA levels when actively cycling cells are compared with growth-arrested cells in culture. Using purified factors, we show that raising the level of TFIIIC2 is sufficient to stimulate pol III transcription in ovarian cell extracts. The data suggest that overexpression of TFIIIC2 contributes to the abnormal abundance of pol III transcripts in ovarian tumors.

RNA polymerase III transcription factor IIIB is a target for repression by pocket proteins p107 and p130.

RNA polymerase III (Pol III) transcription is subject to repression by the retinoblastoma protein RB, both in vitro and in vivo (R. J. White, D. Trouche, K. Martin, S. P. Jackson, and T. Kouzarides, Nature 382:88-90, 1996). This is achieved through a direct interaction between RB and TFIIIB, a multisubunit factor that is required for the expression of all Pol III templates (C. G. C. Larminie, C. A. Cairns, R. Mital, K. Martin, T. Kouzarides, S. P. Jackson, and R. J. White, EMBO J. 16:2061-2071, 1997; W.-M. Chu, Z. Wang, R. G. Roeder, and C. W. Schmid, J. Biol. Chem. 272:14755-14761, 1997). p107 and p130 are two closely related proteins that display 30 to 35% identity with the RB polypeptide and share some of its functions. We show that p107 and p130 can both repress Pol III transcription in transient transfection assays or when added to cell extracts. Pull-down assays and immunoprecipitations using recombinant components demonstrate that a subunit of TFIIIB interacts physically with p107 and p130. In addition, endogenous TFIIIB is shown by cofractionation and coimmunoprecipitation to associate stably with both p107 and p130. Disruption of this interaction in vivo by using the E7 oncoprotein of human papillomavirus results in a marked increase in Pol III transcription. Pol III activity is also deregulated in fibroblasts derived from p107 p130 double knockout mice. We conclude that TFIIIB is targeted for repression not only by RB but also by its relatives p107 and p130.

Sedative effects and impaired learning and recall after single oral doses of lorazepam.

Seven healthy subjects received oral placebo, 1.5 mg lorazepam, or 3.0 mg lorazepam in a single-dose, three-way crossover study. Plasma lorazepam concentrations and subjects' self-rated sedative effects were evaluated at multiple points during 24 hours after each dose. Information acquisition and recall was studied by use of a 16-item word list at 3 and 24 hours after dosing. Lorazepam plasma concentrations were proportional to dose. Self-rated sedation was maximal 2 to 3 hours after lorazepam dosing, persisted for 8 hours, and was dose dependent in intensity; no significant sedation occurred with placebo. At 3 hours after placebo dosing, subjects learned a mean 96% of words presented during six trials; this was reduced to 79% and 62% after lorazepam, 1.5 and 3.0 mg, respectively (F = 6.2; P less than 0.02). Twenty-four hours after placebo, subjects recalled 92% of words presented the previous day, then improved to 99% after six relearning trials. After 1.5 and 3.0 mg lorazepam, however, only 52% and 44% of words were initially recalled from the previous day. Thus single oral doses of lorazepam within the therapeutic range produce dose-dependent sedation and impairment of information acquisition and recall.