Mutant p53 promotes RCP-dependent chemoresistance coinciding with increased delivery of P-glycoprotein to the plasma membrane.

TP53 is the most frequently mutated gene in cancers. Mutations lead to loss of p53 expression or expression of a mutant protein. Mutant p53 proteins commonly lose wild-type function, but can also acquire novel functions in promoting metastasis and chemoresistance. Previously, we uncovered a role for Rab-coupling protein (RCP) in mutant p53-dependent invasion. RCP promotes endosomal recycling and signalling of integrins and receptor tyrosine kinases. In a screen to identify novel RCP-interacting proteins, we discovered P-glycoprotein (P-gp). Thus, we hypothesised that mutant p53 could promote chemoresistance through RCP-dependent recycling of P-gp. The interaction between RCP and P-gp was verified endogenously and loss of RCP or mutant p53 rendered cells more sensitive to cisplatin and etoposide. In mutant p53 cells we detected an RCP-dependent delivery of P-gp to the plasma membrane upon drug treatment and decreased retention of P-gp substrates. A co-localisation of P-gp and RCP was seen in mutant p53 cells, but not in p53-null cells upon chemotherapeutic exposure. In conclusion, mutant p53 expression enhanced co-localisation of P-gp and RCP to allow for rapid delivery of P-gp to the plasma membrane and increased resistance to chemotherapeutics.

Rab11-FIP1/RCP Functions as a Major Signalling Hub in the Oncogenic Roles of Mutant p53 in Cancer.

Rab11-FIP1 is a Rab effector protein that is involved in endosomal recycling and trafficking of various molecules throughout the endocytic compartments of the cell. The consequence of this can be increased secretion or increased membrane expression of those molecules. In general, expression of Rab11-FIP1 coincides with more tumourigenic and metastatic cell behaviour. Rab11-FIP1 can work in concert with oncogenes such as mutant p53, but has also been speculated to be an oncogene in its own right. In this perspective, we will discuss and speculate upon our observations that mutant p53 promotes Rab11-FIP1 function to not only promote invasive behaviour, but also chemoresistance by regulating a multitude of different proteins.

Persistence of Epigenomic Effects After Recovery From Repeated Treatment With Two Nephrocarcinogens.

The discovery of the epigenetic regulation of transcription has provided a new source of mechanistic understanding to long lasting effects of chemicals. However, this information is still seldom exploited in a toxicological context and studies of chemical effect after washout remain rare. Here we studied the effects of two nephrocarcinogens on the human proximal tubule cell line RPTEC/TERT1 using high-content mRNA microarrays coupled with miRNA, histone acetylation (HA) and DNA methylation (DM) arrays and metabolomics during a 5-day repeat-dose exposure and 3 days after washout. The mycotoxin ochratoxin A (OTA) was chosen as a model compound for its known impact on HA and DM. The foremost effect observed was the modulation of thousands of mRNAs and histones by OTA during and after exposure. In comparison, the oxidant potassium bromate (KBrO3) had a milder impact on gene expression and epigenetics. However, there was no strong correlation between epigenetic modifications and mRNA changes with OTA while with KBrO3 the gene expression data correlated better with HA for both up- and down-regulated genes. Even when focusing on the genes with persistent epigenetic modifications after washout, only half were coupled to matching changes in gene expression induced by OTA, suggesting that while OTA causes a major effect on the two epigenetic mechanisms studied, these alone cannot explain its impact on gene expression. Mechanistic analysis confirmed the known activation of Nrf2 and p53 by KBrO3, while OTA inhibited most of the same genes, and genes involved in the unfolded protein response. A few miRNAs could be linked to these effects of OTA, albeit without clear contribution of epigenetics to the modulation of the pathways at large. Metabolomics revealed disturbances in amino acid balance, energy catabolism, nucleotide metabolism and polyamine metabolism with both chemicals. In conclusion, the large impact of OTA on transcription was confirmed at the mRNA level but also with two high-content epigenomic methodologies. Transcriptomic data confirmed the previously reported activation (by KBrO3) and inhibition (by OTA) of protective pathways. However, the integration of omic datasets suggested that HA and DM were not driving forces in the gene expression changes induced by either chemical.

Novel targets and interaction partners of mutant p53 Gain-Of-Function.

In many human cancers p53 expression is lost or a mutant p53 protein is expressed. Over the past 15 years it has become apparent that a large number of these mutant p53 proteins have lost wild type function, but more importantly have gained functions that promote tumorigenesis and drive chemo-resistance, invasion and metastasis. Many researchers have investigated the underlying mechanisms of these Gain-Of-Functions (GOFs) and it has become apparent that many of these functions are the result of mutant p53 hijacking other transcription factors. In this review, we summarize the latest research on p53 GOF and categorize these in light of the hallmarks of cancer as presented by Hannahan and Weinberg.

Transcriptomics hit the target: Monitoring of ligand-activated and stress response pathways for chemical testing.

High content omic methods provide a deep insight into cellular events occurring upon chemical exposure of a cell population or tissue. However, this improvement in analytic precision is not yet matched by a thorough understanding of molecular mechanisms that would allow an optimal interpretation of these biological changes. For transcriptomics (TCX), one type of molecular effects that can be assessed already is the modulation of the transcriptional activity of a transcription factor (TF). As more ChIP-seq datasets reporting genes specifically bound by a TF become publicly available for mining, the generation of target gene lists of TFs of toxicological relevance becomes possible, based on actual protein-DNA interaction and modulation of gene expression. In this study, we generated target gene signatures for Nrf2, ATF4, XBP1, p53, HIF1a, AhR and PPAR gamma and tracked TF modulation in a large collection of in vitro TCX datasets from renal and hepatic cell models exposed to clinical nephro- and hepato-toxins. The result is a global monitoring of TF modulation with great promise as a mechanistically based tool for chemical hazard identification.

Mechanism of cisplatin proximal tubule toxicity revealed by integrating transcriptomics, proteomics, metabolomics and biokinetics.

Cisplatin is one of the most widely used chemotherapeutic agents for the treatment of solid tumours. The major dose-limiting factor is nephrotoxicity, in particular in the proximal tubule. Here, we use an integrated omics approach, including transcriptomics, proteomics and metabolomics coupled to biokinetics to identify cell stress response pathways induced by cisplatin. The human renal proximal tubular cell line RPTEC/TERT1 was treated with sub-cytotoxic concentrations of cisplatin (0.5 and 2 µM) in a daily repeat dose treating regime for up to 14 days. Biokinetic analysis showed that cisplatin was taken up from the basolateral compartment, transported to the apical compartment, and accumulated in cells over time. This is in line with basolateral uptake of cisplatin via organic cation transporter 2 and bioactivation via gamma-glutamyl transpeptidase located on the apical side of proximal tubular cells. Cisplatin affected several pathways including, p53 signalling, Nrf2 mediated oxidative stress response, mitochondrial processes, mTOR and AMPK signalling. In addition, we identified novel pathways changed by cisplatin, including eIF2 signalling, actin nucleation via the ARP/WASP complex and regulation of cell polarization. In conclusion, using an integrated omic approach together with biokinetics we have identified both novel and established mechanisms of cisplatin toxicity.

Application of RPTEC/TERT1 cells for investigation of repeat dose nephrotoxicity: A transcriptomic study.

The kidney is a major target organ for toxicity. Incidence of chronic kidney disease (CKD) is increasing at an alarming rate due to factors such as increasing population age and increased prevalence of heart disease and diabetes. There is a major effort ongoing to develop superior predictive models of renal injury and early renal biomarkers that can predict onset of CKD. In the EU FP7 funded project, Predict-IV, we investigated the human renal proximal tubule cells line, RPTEC/TERT1 for their applicability to long term nephrotoxic mechanistic studies. To this end, we used a tiered strategy to optimise dosing regimes for 9 nephrotoxins. Our final testing protocol utilised differentiated RPTEC/TERT1 cells cultured on filter inserts treated with compounds at both the apical and basolateral side, at concentrations not exceeding IC10, for 14 days in a 24 h repeat application. Transepithelial electrical resistance and supernatant lactate were measured over the duration of the experiments and genome wide transcriptomic profiles were assayed at day 1, 3 and 14. The effect of hypoxia was investigated for a subset of compounds. The transcriptomic data were analysed to investigate compound-specific effects, global responses and mechanistically informative signatures. In addition, several potential clinically useful renal injury biomarkers were identified.

Development of an in vitro renal epithelial disease state model for xenobiotic toxicity testing.

There is a growing impetus to develop more accurate, predictive and relevant in vitro models of renal xenobiotic exposure. As part of the EU-FP7, Predict-IV project, a major aim was to develop models that recapitulate not only normal tissue physiology but also aspects of disease conditions that exist as predisposing risk factors for xenobiotic toxicity. Hypoxia, as a common micro-environmental alteration associated with pathophysiology in renal disease, was investigated for its effect on the toxicity profile of a panel of 14 nephrotoxins, using the human proximal tubular epithelial RPTECT/TERT1 cell line. Changes in ATP, glutathione and resazurin reduction, after 14 days of daily repeat exposure, revealed a number of compounds, including adefovir dipivoxil with enhanced toxicity in hypoxia. We observed intracellular accumulation of adefovir in hypoxia and suggest decreases in the efflux transport proteins MRP4, MRP5, NHERF1 and NHERF3 as a possible explanation. MRP5 and NHERF3 were also down-regulated upon treatment with the HIF-1 activator, dimethyloxalylglycine. Interestingly, adefovir dependent gene expression shifted from alterations in cell cycle gene expression to an inflammatory response in hypoxia. The ability to investigate aspects of disease states and their influence on renal toxin handling is a key advantage of in vitro systems developed here. They also allow for detailed investigations into mechanisms of compound toxicity of potential importance for compromised tissue exposure.

Expression of xenobiotic transporters in the human renal proximal tubule cell line RPTEC/TERT1.

The kidney is a major target for drug-induced injury, primarily due the fact that it transports a wide variety of chemical entities into and out of the tubular lumen. Here, we investigated the expression of the main xenobiotic transporters in the human renal proximal tubule cell line RPTEC/TERT1 at an mRNA and/or protein level. RPTEC/TERT1 cells expressed OCT2, OCT3, OCTN2, MATE1, MATE2, OAT1, OAT3 and OAT4. The functionality of the OCTs was demonstrated by directional transport of the fluorescent dye 4-Di-1-ASP. In addition, P-glycoprotein activity in RPTEC/TERT1 cells was verified by fluorescent dye retention in presence of various P-glycoprotein inhibitors. In comparison to proliferating cells, contact inhibited RPTEC/TERT1 cells expressed increased mRNA levels of several ABC transporter family members and were less sensitive to cyclosporine A. We conclude that differentiated RPTEC/TERT1 cells are well suited for utilisation in xenobiotic transport and pharmacokinetic studies.

Interleukin-19 as a translational indicator of renal injury.

Accurate detection and prediction of renal injury are central not only to improving renal disease management but also for the development of new strategies to assess drug safety in pre-clinical and clinical testing. In this study, we utilised the well-characterised and differentiated human renal proximal tubule cell line, RPTEC/TERT1 in an attempt to identify markers of renal injury, independent of the mechanism of toxicity. We chose zoledronate as a representative nephrotoxic agent to examine global transcriptomic alterations using a daily repeat bolus protocol over 14 days, reflective of sub-acute or chronic injury. We identified alterations in targets of the cholesterol and mevalonate biosynthetic pathways reflective of zoledronate specific effects. We also identified interleukin-19 (IL-19) among other inflammatory signals such as SERPINA3 and DEFB4 utilising microarray analysis. Release of IL-19 protein was highly induced by an additional four nephrotoxic agents, at magnitudes greater than the characterised marker of renal injury, lipocalin-2. We also demonstrate a large increase in levels of IL-19 in urine of patients with chronic kidney disease, which significantly correlated with estimated glomerular filtration rate levels. We suggest IL-19 as a potential new translational marker of renal injury.

Evidence for a role of claudin 2 as a proximal tubular stress responsive paracellular water channel.

Claudins are the major proteins of the tight junctions and the composition of claudin subtypes is decisive for the selective permeability of the paracellular route and thus tissue specific function. Their regulation is complex and subject to interference by several factors, including oxidative stress. Here we show that exposure of cultured human proximal tubule cells (RPTEC/TERT1) to the immunosuppressive drug cyclosporine A (CsA) induces an increase in transepithelial electrical resistance (TEER), a decrease in dome formation (on solid growth supports) and a decrease in water transport (on microporous growth supports). In addition, CsA induced a dramatic decrease in the mRNA for the pore forming claudins -2 and -10, and the main subunits of the Na(+)/K(+) ATPase. Knock down of claudin 2 by shRNA had no discernable effect on TEER or dome formation but severely attenuated apical to basolateral water reabsorption when cultured on microporous filters. Generation of an osmotic gradient in the basolateral compartment rescued water transport in claudin 2 knock down cells. Inhibition of Na(+)/K(+) ATPase with ouabain prevented dome formation in both cell types. Taken together these results provide strong evidence that dome formation is primarily due to transcellular water transport following a solute osmotic gradient. However, in RPTEC/TERT1 cells cultured on filters under iso-osmotic conditions, water transport is primarily paracellular, most likely due to local increases in osmolarity in the intercellular space. In conclusion, this study provides strong evidence that claudin 2 is involved in paracellular water transport and that claudin 2 expression is sensitive to compound induced cellular stress.

Delineation of the key aspects in the regulation of epithelial monolayer formation.

The formation, maintenance, and repair of epithelial barriers are of critical importance for whole-body homeostasis. However, the molecular events involved in epithelial tissue maturation are not fully established. To this end, we investigated the molecular processes involved in renal epithelial proximal-tubule monolayer maturation utilizing transcriptomic, metabolomic, and functional parameters. We uncovered profound dynamic alterations in transcriptional regulation, energy metabolism, and nutrient utilization over the maturation process. Proliferating cells exhibited high glycolytic rates and high transcript levels for fatty acid synthesis genes (FASN), whereas matured cells had low glycolytic rates, increased oxidative capacity, and preferentially expressed genes for beta oxidation. There were dynamic alterations in the expression and localization of several adherens (CDH1, -4, and -16) and tight junction (TJP3 and CLDN2 and -10) proteins. Genes involved in differentiated proximal-tubule function, cilium biogenesis (BBS1), and transport (ATP1A1 and ATP1B1) exhibited increased expression during epithelial maturation. Using TransAM transcription factor activity assays, we could demonstrate that p53 and FOXO1 were highly active in matured cells, whereas HIF1A and c-MYC were highly active in proliferating cells. The data presented here will be invaluable in the further delineation of the complex dynamic cellular processes involved in epithelial cell regulation.

Application of integrated transcriptomic, proteomic and metabolomic profiling for the delineation of mechanisms of drug induced cell stress.

High content omic techniques in combination with stable human in vitro cell culture systems have the potential to improve on current pre-clinical safety regimes by providing detailed mechanistic information of altered cellular processes. Here we investigated the added benefit of integrating transcriptomics, proteomics and metabolomics together with pharmacokinetics for drug testing regimes. Cultured human renal epithelial cells (RPTEC/TERT1) were exposed to the nephrotoxin Cyclosporine A (CsA) at therapeutic and supratherapeutic concentrations for 14days. CsA was quantified in supernatants and cellular lysates by LC-MS/MS for kinetic modeling. There was a rapid cellular uptake and accumulation of CsA, with a non-linear relationship between intracellular and applied concentrations. CsA at 15µM induced mitochondrial disturbances and activation of the Nrf2-oxidative-damage and the unfolded protein-response pathways. All three omic streams provided complementary information, especially pertaining to Nrf2 and ATF4 activation. No stress induction was detected with 5µM CsA; however, both concentrations resulted in a maximal secretion of cyclophilin B. The study demonstrates for the first time that CsA-induced stress is not directly linked to its primary pharmacology. In addition we demonstrate the power of integrated omics for the elucidation of signaling cascades brought about by compound induced cell stress.

Oxidative stress induced by potassium bromate exposure results in altered tight junction protein expression in renal proximal tubule cells.

Potassium bromate (KBrO(3)) is an oxidising agent that has been widely used in the food and cosmetic industries. It has shown to be both a nephrotoxin and a renal carcinogen in in vivo and in vitro models. Here, we investigated the effects of KBrO(3) in the human and rat proximal tubular cell lines RPTEC/TERT1 and NRK-52E. A genome-wide transcriptomic screen was carried out from cells exposed to a sub-lethal concentration of KBrO(3) for 6, 24 and 72 h. Pathway analysis identified "glutathione metabolism", "Nrf2-mediated oxidative stress" and "tight junction (TJ) signalling" as the most enriched pathways. TJ signalling was less impacted in the rat model, and further studies revealed low transepithelial electrical resistance (TEER) and an absence of several TJ proteins in NRK-52E cells. In RPTEC/TERT1 cells, KBrO(3) exposure caused a decrease in TEER and resulted in altered expression of several TJ proteins. N-Acetylcysteine co-incubation prevented these effects. These results demonstrate that oxidative stress has, in conjunction with the activation of the cytoprotective Nrf2 pathway, a dramatic effect on the expression of tight junction proteins. The further understanding of the cross-talk between these two pathways could have major implications for epithelial repair, carcinogenesis and metastasis.

Transcriptomic alterations induced by Ochratoxin A in rat and human renal proximal tubular in vitro models and comparison to a rat in vivo model.

Ochratoxin A (OTA) is a widely studied compound due to its role in renal toxicity and carcinogenicity. However, there is still no consensus on the exact mechanisms of toxicity or carcinogenicity. In the current study, we analysed the effect of OTA on three human renal proximal tubular models (human primary, RPTEC/TERT1 and HK-2 cells) and two rat renal proximal tubular models (rat primary and NRK-52E cells). Global transcriptomics analysis at two exposure times was performed to generate a set of 756 OTA sensitive genes. This gene set was then compared in more detail across all models and additionally to a rat in vivo renal cortex model. The results demonstrate a well-conserved response across all models. OTA resulted in deregulation of a number of pathways including cytoskeleton, nucleosome regulation, translation, transcription, ubiquitination and cell cycle pathways. Interestingly, the oxidative stress activated Nrf2 pathway was not enriched. These results point to an epigenetic action of OTA, perhaps initiated by actin binding as the actin remodelling gene, advillin was the highest up-regulated in all models. The largest model differences were observed between the human and the rat in vitro models. However, since the human in vitro models were more similar to the rat in vivo model, it is more likely that these differences are model-specific rather than species-specific per se. This study demonstrates the usefulness of in vitro cell culture models combined with transcriptomic analysis for the investigation of mechanisms of toxicity and carcinogenicity. In addition, these results provide further evidence supporting a non-genotoxic mechanism of OTA-induced carcinogenicity.

Lactate is an ideal non-invasive marker for evaluating temporal alterations in cell stress and toxicity in repeat dose testing regimes.

Technological developments are driving in vitro methods towards integrated "omic" strategies. However, there is still an over reliance on classical viability assays for dose range finding. Such assays are not readily suited to the investigation of subtle alterations in cell function and most require termination of the experiment, which makes it difficult to monitor temporal alterations in repeat-dose long term exposure experiments. To this end, we investigated the use of lactate production as a marker of cell stress in long term repeat dose experiments. We conducted daily exposures to eight compounds at five concentrations for 14 days on human renal proximal tubular cells (RPTEC/TERT1), human hepatoma cells (HepaRG) and mouse fibroblasts (BALB-3T3) cells. Compounds were chosen from a training set used in the 7th EU Framework project Predict-IV and consisted of amiodarone, diclofenac, troglitazone, cadmium chloride, cephaloridine, cidofovir, cyclosporine A and buflomedil. At days 1, 3, 7 and 14, lactate was measured in the supernatant medium. At day 14, cells were assayed for resazurin reduction capability and subsequently lysed in methanol for ATP determination. Compound-induced loss of viability was comparable across all cell lines. For all cell types, when cell viability was compromised at day 14, lactate production was induced during the treatment period. In some situations, lactate also fell below control values, indicating cell death. Thus, temporal alterations in supernatant lactate provides information on the time and concentration of stress induction and the time and concentration where cell death becomes the dominant factor. Supernatant lactate production is a simple, cheap and non-invasive parameter. Since many molecular pathways converge on the glycolytic pathway, enhanced lactate production may be considered as a global marker of sub-lethal injury and thus an ideal marker for investigating temporal alterations in long term repeat dose testing in vitro regimes.