Up-regulation of autophagy is a mechanism of resistance to chemotherapy and can be inhibited by pantoprazole to increase drug sensitivity.

BACKGROUND: Autophagy is a survival mechanism that allows recycling of cellular breakdown products, particularly in stressed cells. Here we evaluate the hypotheses that up-regulation of autophagy is a common mechanism of resistance to chemotherapy, and that drug resistance can be reversed by inhibiting autophagy with a proton pump inhibitor. METHODS: We exposed human PC3, LNCaP and MCF7 cells to seven clinically-used chemotherapy drugs ± pantoprazole, examined the up-regulation of autophagy and the effect on cellular proliferation by Western Blots, MTS assay and colony-forming assay. The distribution of drug effects and of autophagy was quantified in LNCaP tumor sections in relation to blood vessels and hypoxia by immunohistochemistry using γH2AX, cleaved caspase-3 and p62. RESULTS: All anticancer drugs led to up-regulation of autophagy in cultured tumor cells. Pantoprazole inhibited the induction of autophagy in a time- and dose-dependent manner, and sensitized cancer cells to the seven anti-cancer drugs. Treatment of LNCaP xenografts with paclitaxel induced both DNA damage and autophagy; autophagy was inhibited and markers of toxicity were increased by pantoprazole. CONCLUSIONS: Induction of autophagy is a general mechanism associated with resistance to anticancer drugs and that its inhibition is a promising therapeutic strategy to enhance the effects of chemotherapy and improve clinical outcomes.

Effect of pantoprazole to enhance activity of docetaxel against human tumour xenografts by inhibiting autophagy.

BACKGROUND: Autophagy allows recycling of cellular components and may facilitate cell survival after chemotherapy. Pantoprazole inhibits proton pumps and is reported to inhibit autophagy. Here we evaluate the effects of pantoprazole to modify cytotoxicity of the anticancer drug docetaxel, and underlying mechanisms. METHODS: Effects of docetaxel±pantoprazole were studied against wild-type and autophagy-deficient PC3 cells and against four human xenografts. Effects of pantoprazole on autophagy were evaluated by quantifying LC3-I, LC3-II and p62 proteins in western blots, and by fluorescent microscopy of cells transfected with RFP-GFP-LC3. The distribution of drug effects and of autophagy was quantified in tumour sections in relation to blood vessels and hypoxia by immunohistochemistry using γH2AX, cleaved caspase-3, Ki67 and LC3/ p62. RESULTS: Pantoprazole increased the toxicity of docetaxel in vitro, increased docetaxel-induced expression of γH2AX and cleaved caspase-3, and decreased Ki67 in tumour sections. Pantoprazole increased growth delay of four human xenografts of low, moderate and high sensitivity to docetaxel, with minimal increase in toxicity. Docetaxel led to increased autophagy throughout tumour sections. Pantoprazole inhibited autophagy, and effects of pantoprazole were reduced against genetically modified cells with decreased ability to undergo autophagy. CONCLUSIONS: Autophagy is a mechanism of resistance to docetaxel chemotherapy that may be modified by pantoprazole to improve therapeutic index.

Hypoxia signaling and the metastatic phenotype.

Conditions of poor oxygenation (hypoxia) are present in the majority of solid human tumors and are associated with poor patient prognosis due to both hypoxia-mediated resistance to treatment, and to hypoxia induced biological changes that promote increased malignancy, including metastasis. Tumor cells respond to hypoxia by activating several oxygen-sensitive signaling pathways that include the hypoxia inducible factor 1/2 (HIF1/2) signalling pathways and the unfolded protein response (UPR), which alter gene expression to promote adaptation and survival during hypoxic conditions. Furthermore, these hypoxia responsive pathways can lead to changes in gene expression and cellular phenotype that influence the potential of cancer cells to metastasize. However, the hypoxia-induced signaling events that promote tumor metastasis are still relatively poorly understood. Previous studies have largely focused on the contribution of the HIF signaling pathway to hypoxia-mediated metastasis. However, recent evidence demonstrates that hypoxic activation of the UPR is also an important mediator of metastasis.

Independent and functional validation of a multi-tumour-type proliferation signature.

BACKGROUND: Previously we demonstrated that an mRNA signature reflecting cellular proliferation had strong prognostic value. As clinical applicability of signatures can be controversial, we sought to improve our marker's clinical utility by validating its biological relevance, reproducibility in independent data sets and applicability using an independent technique. METHODS: To facilitate signature evaluation with quantitative PCR (qPCR) a novel computational procedure was used to reduce the number of signature genes without significant information loss. These genes were validated in different human cancer cell lines upon serum starvation and in a 168 xenografts panel. Analyses were then extended to breast cancer and non-small-cell lung cancer (NSCLC) patient cohorts. RESULTS: Expression of the qPCR-based signature was dramatically decreased under starvation conditions and inversely correlated with tumour volume doubling time in xenografts. The signature validated in breast cancer (hazard ratio (HR)=1.63, P<0.001, n=1820) and NSCLC adenocarcinoma (HR=1.64, P<0.001, n=639) microarray data sets. Lastly, qPCR in a node-negative, non-adjuvantly treated breast cancer cohort (n=129) showed that patients assigned to the high-proliferation group had worse disease-free survival (HR=2.25, P<0.05). CONCLUSION: We have developed and validated a qPCR-based proliferation signature. This test might be used in the clinic to select (early-stage) patients for specific treatments that target proliferation.

Robust prognostic value of a knowledge-based proliferation signature across large patient microarray studies spanning different cancer types.

Tumour proliferation is one of the main biological phenotypes limiting cure in oncology. Extensive research is being performed to unravel the key players in this process. To exploit the potential of published gene expression data, creation of a signature for proliferation can provide valuable information on tumour status, prognosis and prediction. This will help individualizing treatment and should result in better tumour control, and more rapid and cost-effective research and development. From in vitro published microarray studies, two proliferation signatures were compiled. The prognostic value of these signatures was tested in five large clinical microarray data sets. More than 1000 patients with breast, renal or lung cancer were included. One of the signatures (110 genes) had significant prognostic value in all data sets. Stratifying patients in groups resulted in a clear difference in survival (P-values <0.05). Multivariate Cox-regression analyses showed that this signature added substantial value to the clinical factors used for prognosis. Further patient stratification was compared to patient stratification with several well-known published signatures. Contingency tables and Cramer's V statistics indicated that these primarily identify the same patients as the proliferation signature does. The proliferation signature is a strong prognostic factor, with the potential to be converted into a predictive test. Furthermore, evidence is provided that supports the idea that many published signatures track the same biological processes and that proliferation is one of them.

Poorer outcome in stromal HIF-2 alpha- and CA9-positive colorectal adenocarcinomas is associated with wild-type TP53 but not with BNIP3 promoter hypermethylation or apoptosis.

Stromal expression of hypoxia inducible factor 2 alpha (HIF-2 alpha) and carbonic anhydrase 9 (CA9) are associated with a poorer prognosis in colorectal cancer (CRC). Tumour cell death, regulated by a hypoxic stromal microenvironment, could be of importance in this respect. Therefore, we correlated apoptosis, TP53 mutational status and BNIP3 promoter hypermethylation of CRC cells with HIF-2 alpha- and CA9-related poor outcome. In a series of 195 CRCs, TP53 mutations in exons 5-8 were analysed by direct sequencing, and promoter hypermethylation of BNIP3 was determined by methylation-specific PCR. Expressions of HIF-2 alpha, CA9, p53, BNIP3 and M30 were analysed immunohistochemically. Poorer survival of HIF-2 alpha and CA9 stromal-positive CRCs was associated with wild-type TP53 (P=0.001 and P=0.0391), but not with BNIP3 methylation. Furthermore, apoptotic levels were independent of the TP53 status, but lower in unmethylated BNIP3 CRCs (P=0.004). It appears that wild-type TP53 in CRC cells favours the progression of tumours expressing markers for hypoxia in their stroma, rather than in the epithelial compartment. Preserved BNIP3 function in CRC cells lowers apoptosis, and may thus be involved in alternative cell death pathways, such as autophagic cell death. However, BNIP3 silencing in tumour cells does not impact on hypoxia-driven poorer prognosis. These results suggest that the biology of CRC cells can be modified by alterations in the tumour microenvironment under conditions of tumour hypoxia.

Dose- and time-dependent changes in gene expression in human glioma cells after low radiation doses.

We have used DNA microarrays to identify changes in gene expression in cells of the radioresistant human glioma cell lines T98G and U373 after low radiation doses (0.2-2 Gy). Using Bayesian linear models, we have identified a set of genes that respond to low doses of radiation; furthermore, a hypothesis-driven approach to data analysis has allowed us to identify groups of genes with defined non-linear dose responses. Specifically, one of the cell lines we have examined (T98G) shows increased radiosensitivity at low doses (low-dose hyper-radiosensitivity, HRS); thus we have also assessed sets of genes whose dose response mirrors this survival pattern. We have also investigated a time course for induction of genes over the period when the DNA damage response is expected to occur. We have validated these data using quantitative PCR and also compared genes up-regulated in array data to genes present in the polysomal RNA fraction after irradiation. Several of the radioresponsive genes that we describe code for proteins that may have an impact on the outcome of irradiation in these cells, including RAS homologues and kinases involved in checkpoint signaling, so understanding their differential regulation may suggest new ways of altering radioresistance. From a clinical perspective these data may also suggest novel targets that are specifically up-regulated in gliomas during radiotherapy treatments.

Efficacy of gene therapy-delivered cytosine deaminase is determined by enzymatic activity but not expression.

The potential utility of tumour-selective 5-fluorouracil treatment using attenuated Salmonella serovar typhimurium recombinant for cytosine deaminase (TAPET-CD) has been documented in experimental settings. The present data demonstrate that in vivo (19)F-magnetic resonance spectroscopy measurements allow the outcome prediction of this prokaryotic-based therapy, demonstrating the necessity of non-invasive real-time imaging techniques for treatment monitoring.

Repeated cycles of Clostridium-directed enzyme prodrug therapy result in sustained antitumour effects in vivo.

The unique properties of the tumour microenvironment can be exploited by using recombinant anaerobic clostridial spores as highly selective gene delivery vectors. Although several recombinant Clostridium species have been generated during the past decade, their efficacy has been limited. Our goal was to substantially improve the prospects of clostridia as a gene delivery vector. Therefore, we have assessed a series of nitroreductase (NTR) enzymes for their capacity to convert the innocuous CB1954 prodrug to its toxic derivative. Among the enzymes tested, one showed superior prodrug turnover characteristics. In addition, we established an efficient gene transfer procedure, based on conjugation, which allows for the first time genetic engineering of Clostridium strains with superior tumour colonisation properties with high success rates. This conjugation procedure was subsequently used to create a recombinant C. sporogenes overexpressing the isolated NTR enzyme. Finally, analogous to a clinical setting situation, we have tested the effect of multiple consecutive treatment cycles, with antibiotic bacterial clearance between cycles. Importantly, this regimen demonstrated that intravenously administered spores of NTR-recombinant C. sporogenes produced significant antitumour efficacy when combined with prodrug administration.

Low-dose hyper-radiosensitivity: a consequence of ineffective cell cycle arrest of radiation-damaged G2-phase cells.

This review highlights the phenomenon of low-dose hyper- radiosensitivity (HRS), an effect in which cells die from excessive sensitivity to small single doses of ionizing radiation but become more resistant (per unit dose) to larger single doses. Established and new data pertaining to HRS are discussed with respect to its possible underlying molecular mechanisms. To explain HRS, a three-component model is proposed that consists of damage recognition, signal transduction and damage repair. The foundation of the model is a rapidly occurring dose-dependent pre-mitotic cell cycle checkpoint that is specific to cells irradiated in the G2phase. This checkpoint exhibits a dose expression profile that is identical to the cell survival pattern that characterizes HRS and is probably the key control element of low-dose radiosensitivity. This premise is strengthened by the recent observation coupling low- dose radiosensitivity of G2-phase cells directly to HRS. The putative role of known damage response factors such as ATM, PARP, H2AX, 53BP1 and HDAC4 is also included within the framework of the HRS model.

An association between the radiation-induced arrest of G2-phase cells and low-dose hyper-radiosensitivity: a plausible underlying mechanism?

The survival of asynchronous and highly enriched G1-, S- and G2-phase populations of Chinese hamster V79 cells was measured after irradiation with 60Co gamma rays (0.1-10 Gy) using a precise flow cytometry-based clonogenic survival assay. The high-dose survival responses demonstrated a conventional relationship, with G2-phase cells being the most radiosensitive and S-phase cells the most radioresistant. Below 1 Gy, distinct low-dose hyper-radiosensitivity (HRS) responses were observed for the asynchronous and G2-phase enriched cell populations, with no evidence of HRS in the G1- and S-phase populations. Modeling supports the conclusion that HRS in asynchronous V79 populations is explained entirely by the HRS response of G2-phase cells. An association was discovered between the occurrence of HRS and the induction of a novel G2-phase arrest checkpoint that is specific for cells that are in the G2 phase of the cell cycle at the time of irradiation. Human T98G cells and hamster V79 cells, which both exhibit HRS in asynchronous cultures, failed to arrest the entry into mitosis of damaged G2-phase cells at doses less than 30 cGy, as determined by the flow cytometric assessment of the phosphorylation of histone H3, an established indicator of mitosis. In contrast, human U373 cells that do not show HRS induced this G2-phase checkpoint in a dose-independent manner. These data suggest that HRS may be a consequence of radiation-damaged G2-phase cells prematurely entering mitosis.

Hypoxia as a target for combined modality treatments.

There is overwhelming evidence that solid human tumours grow within a unique micro-environment. This environment is characterised by an abnormal vasculature, which leads to an insufficient supply of oxygen and nutrients to the tumour cells. These characteristics of the environment limit the effectiveness of both radiotherapy and chemotherapy. Measurement of the oxygenation status of human tumours has unequivocally demonstrated the importance of this parameter on patient prognosis. Tumour hypoxia has been shown to be an independent prognostic indicator of poor outcome in prostate, head and neck and cervical cancers. Recent laboratory and clinical data have shown that hypoxia is also associated with a more malignant phenotype, affecting genomic stability, apoptosis, angiogenesis and metastasis. Several years ago, scientists realised that the unique properties within the tumour micro-environment could provide the basis for tumour-specific therapies. Efforts that are underway to develop therapies that exploit the tumour micro-environment can be categorised into three groups. The first includes agents that exploit the environmental changes that occur within the micro-environment such as hypoxia and reduced pH. This includes bioreductive drugs that are specifically toxic to hypoxic cells, as well as hypoxia-specific gene delivery systems. The second category includes therapies designed to exploit the unique properties of the tumour vasculature and include both angiogenesis inhibitors and vascular targeting agents. The final category includes agents that exploit the molecular and cellular responses to hypoxia. For example, many genes are induced by hypoxia and promoter elements from these genes can be used for the selective expression of therapeutic proteins in hypoxic tumour cells. An overview of the various properties ascribed to tumour hypoxia and the current efforts underway to exploit hypoxia for improving cancer treatment will be discussed.

Sensitivity of mammalian cells expressing mutant ubiquitin to protein-damaging agents.

There is convincing evidence from studies in yeast that a functional ubiquitin/proteasome pathway is required to degrade misfolded or oxidatively damaged proteins but for technical reasons, it has been difficult to perform comparable studies in mammalian cells. To investigate the possibility that the ubiquitin/proteasome pathway is cytoprotective for mammalian cells, we have introduced epitope-tagged wild-type ubiquitin or dominant-negative mutant versions of ubiquitin into mouse HT4 neuroblastoma cells. Cells expressing mutant versions of ubiquitin were found to be sensitive to cadmium, an agent that causes oxidative damage to cellular components, and to canavanine, an amino acid analog that generates misfolded proteins. The greatest sensitivity to canavanine was observed in cells expressing a mutant version of ubiquitin unable to support the formation of Lys(48) linkages. Substrates of the proteasome were found to accumulate in these cells, suggesting a general deficit in proteolysis. Our data suggest that defects in the ubiquitin-mediated proteolytic system predispose mammalian cells to the toxic effects of abnormal protein.

Transcription factor HIF-1 is a necessary mediator of the pasteur effect in mammalian cells.

The ability to respond to differential levels of oxygen is important to all respiring cells. The response to oxygen deficiency, or hypoxia, takes many forms and ranges from systemic adaptations to those that are cell autonomous. Perhaps the most ancient of the cell-autonomous adaptations to hypoxia is a metabolic one: the Pasteur effect, which includes decreased oxidative phosphorylation and an increase in anaerobic fermentation. Because anaerobic fermentation produces far less ATP than oxidative phosphorylation per molecule of glucose, increased activity of the glycolytic pathway is necessary to maintain free ATP levels in the hypoxic cell. Here, we present genetic and biochemical evidence that, in mammalian cells, this metabolic switch is regulated by the transcription factor HIF-1. As a result, cells lacking HIF-1alpha exhibit decreased growth rates during hypoxia, as well as decreased levels of lactic acid production and decreased acidosis. We show that this decrease in glycolytic capacity results in dramatically lowered free ATP levels in HIF-1alpha-deficient hypoxic cells. Thus, HIF-1 activation is an essential control element of the metabolic state during hypoxia; this requirement has important implications for the regulation of cell growth during development, angiogenesis, and vascular injury.

Cell cycle progression and radiation survival following prolonged hypoxia and re-oxygenation.

PURPOSE: To investigate cell cycle progression and radiation survival following prolonged hypoxia and re-oxygenation. MATERIALS AND METHODS: NHIK 3025 human cervical carcinoma cells were exposed to extremely hypoxic conditions (<4ppm O2) for 20 h and then re-oxygenated. The subsequent cell cycle progression was monitored by analysing cell cycle distribution at different time-points after re-oxygenation using two-dimensional flowcytometry. The clonogenic survival after a 3.6 Gy X-ray dose was also measured at each of these time-points. The measured radiation survival was compared with theoretical predictions based on cell cycle distribution and the radiation age response of the cells. RESULTS: Following re-oxygenation the cells resumed cell cycle progression, completed S-phase, and then accumulated in G2. Non-clonogenic cells remained permanently arrested in G2, while the remainder of the cells completed mitosis after a few hours delay. The radiation survival of the hypoxia-pretreated cell population remained lower than for an exponentially growing control population for the investigated 50h of re-oxygenation. However, following 7 h of re-oxygenation, the radiation survival of the hypoxia-treated cell population correlated well with theoretically predicted values based on cell cycle distribution and radiation age response. CONCLUSIONS: The work demonstrates that prolonged hypoxia followed by re-oxygenation results in a G2 delay similar to that observed after DNA damage. Furthermore, chronic hypoxia results in decreased radiation survival for at least 50h following the reintroduction of oxygen. The hypoxia-induced radiosensitization following 7 h of re-oxygenation could in large part be explained by the synchronous cell cycle progression that occurred.

Mitochondrial dysfunction after aerobic exposure to the hypoxic cytotoxin tirapazamine.

Tirapazamine (TPZ) is a bioreductive drug that exhibits a high degree of selective toxicity toward hypoxic cells, and at doses that are used clinically, little or no cell killing is observed in aerobic cells. Nonetheless, the effects of TPZ on aerobic tissues are still responsible for the dose limitations on the clinical administration of this drug. Clinical side effects include fatigue, muscle cramping, and reversible ototoxicity. We have investigated TPZ-induced changes in the mitochondria in aerobically exposed cells as a potential mediator of these side effects. Our data show that aerobic administration of TPZ at clinically relevant doses results in a profound loss in the mitochondrial membrane potential (MMP). We show that loss in the MMP occurs in a variety of cell lines in vitro and also occurs in muscle tissues in vivo. The loss in MMP is temporary because recovery occurs within 2 h. TPZ is directly metabolized within mitochondria to a DNA-damaging form, and this metabolism leads to both the cell-killing effects of TPZ on aerobic cells at high doses and to the loss in MMP at clinically relevant doses. Using cell lines derived from genetically modified mice with a targeted deletion in manganese superoxide dismutase, we have further distinguished the phenotypic effects of TPZ in mitochondria at high toxic doses versus those at clinically relevant doses. We have investigated several potential mechanisms for this TPZ-induced loss in MMP. Our results indicate no change in the rate of cellular respiration in TPZ-treated cells. This implies that the loss in MMP results from an inability of the inner mitochondrial membrane to sustain a potential across the membrane after TPZ treatment. Incubation of cells with an inhibitor of the mitochondrial permeability transition suggests that the loss of MMP may result from the regulated opening of a large mitochondria channel.

A p53 and apoptotic independent role for p21waf1 in tumour response to radiation therapy.

Loss of p21 in human cancer cells results in checkpoint failure, induction of polyploidy and subsequent apoptosis following DNA damage. Tumours in immunodeficient mice derived from cells lacking p21 are also more sensitive to ionizing radiation than their wild-type counterparts. Abrogation of p53 in the p21+/+ parental cells results in an in vitro phenotype that is indistinguishable from that of the p21 knockout cells. Thus, the in vitro phenotype resulting from loss of p21 is consistent with its well-established role in the p53/p21 damage response pathway. However, despite the similar in vitro phenotype, p21+/+ cells with abrogated p53 show no evidence of the sensitivity observed in the p21-/- cells when grown as tumours in immunodeficient mice. The increased radio-sensitization stabilization of p21-/- tumours is also unrelated to the increase in apoptosis observed in these tumours following radiation treatment. Apoptosis in the p21-/- tumours was significantly reduced by expression of bcl-2 without any corresponding change in the overall response of the tumour. Similarly, abrogation of p53 in the p21+/+ tumours substantially increased radiation-induced apoptosis within the tumours without increasing their radiation sensitivity. Dissociation of these in vivo and in vitro phenotypes indicates that p21 participates in a novel in vivo specific damage response pathway that is distinct from its role in the p53 pathway, and therefore that it may be an effective therapeutic target for cancer therapy.