Effective targeting of breast cancer by the inhibition of P-glycoprotein mediated removal of toxic lipid peroxidation byproducts from drug tolerant persister cells.

Therapy resistance has long been considered to occur through the selection of pre-existing clones equipped to survive and quickly regrow, or through the acquisition of mutations during chemotherapy. Here we show that following in vitro treatment by chemotherapy, epithelial breast cancer cells adopt a transient drug tolerant phenotype characterized by cell cycle arrest, epithelial-to-mesenchymal transition (EMT) and the reversible upregulation of the multidrug resistance (MDR) efflux transporter P-glycoprotein (P-gp). The drug tolerant persister (DTP) state is reversible, as cells eventually resume proliferation, giving rise to a cell population resembling the initial, drug-naïve cell lines. However, recovery after doxorubicin treatment is almost completely eliminated when DTP cells are cultured in the presence of the P-gp inhibitor Tariquidar. Mechanistically, P-gp contributes to the survival of DTP cells by removing reactive oxygen species-induced lipid peroxidation products resulting from doxorubicin exposure. In vivo, prolonged administration of Tariquidar during doxorubicin treatment holidays resulted in a significant increase of the overall survival of Brca1-/-;p53-/- mammary tumor bearing mice. These results indicate that prolonged administration of a P-gp inhibitor during drug holidays would likely benefit patients without the risk of aggravated side effects related to the concomitantly administered toxic chemotherapy. Effective targeting of DTPs through the inhibition of P-glycoprotein may result in a paradigm shift, changing the focus from countering drug resistance mechanisms to preventing or delaying therapy resistance.

Establishment and Characterization of a Brca1-/-, p53-/- Mouse Mammary Tumor Cell Line.

Breast cancer is the most commonly occurring cancer in women and the second most common cancer overall. By the age of 80, the estimated risk for breast cancer for women with germline BRCA1 or BRCA2 mutations is around 80%. Genetically engineered BRCA1-deficient mouse models offer a unique opportunity to study the pathogenesis and therapy of triple negative breast cancer. Here we present a newly established Brca1-/-, p53-/- mouse mammary tumor cell line, designated as CST. CST shows prominent features of BRCA1-mutated triple-negative breast cancers including increased motility, high proliferation rate, genome instability and sensitivity to platinum chemotherapy and PARP inhibitors (olaparib, veliparib, rucaparib and talazoparib). Genomic instability of CST cells was confirmed by whole genome sequencing, which also revealed the presence of COSMIC (Catalogue of Somatic Mutations in Cancer) mutation signatures 3 and 8 associated with homologous recombination (HR) deficiency. In vitro sensitivity of CST cells was tested against 11 chemotherapy agents. Tumors derived from orthotopically injected CST-mCherry cells in FVB-GFP mice showed sensitivity to cisplatin, providing a new model to study the cooperation of BRCA1-KO, mCherry-positive tumor cells and the GFP-expressing stromal compartment in therapy resistance and metastasis formation. In summary, we have established CST cells as a new model recapitulating major characteristics of BRCA1-negative breast cancers.

Characterization of calcium signals in human induced pluripotent stem cell-derived dentate gyrus neuronal progenitors and mature neurons, stably expressing an advanced calcium indicator protein.

Pluripotent stem cell derived human neuronal progenitor cells (hPSC-NPCs) and their mature neuronal cell culture derivatives may efficiently be used for central nervous system (CNS) drug screening, including the investigation of ligand-induced calcium signalization. We have established hippocampal NPC cultures derived from human induced PSCs, which were previously generated by non-integrating Sendai virus reprogramming. Using established protocols these NPCs were differentiated into hippocampal dentate gyrus neurons. In order to study calcium signaling without the need of dye loading, we have stably expressed an advanced calcium indicator protein (GCaMP6fast) in the NPCs using the Sleeping Beauty transposon system. We observed no significant effects of the long-term GCaMP6 expression on NPC morphology, gene expression pattern or neural differentiation capacity. In order to compare the functional properties of GCaMP6-expressing neural cells and the corresponding parental cells loaded with calcium indicator dye Fluo-4, a detailed characterization of calcium signals was performed. We found that the calcium signals induced by ATP, glutamate, LPA, or proteases - were similar in these two systems. Moreover, the presence of the calcium indicator protein allowed for a sensitive, repeatable detection of changes in calcium signaling during the process of neurogenesis and neuronal maturation.

Visualization of Calcium Dynamics in Kidney Proximal Tubules.

Intrarenal changes in cytoplasmic calcium levels have a key role in determining pathologic and pharmacologic responses in major kidney diseases. However, cell-specific delivery of calcium-sensitive probes in vivo remains problematic. We generated a transgenic rat stably expressing the green fluorescent protein-calmodulin-based genetically encoded calcium indicator (GCaMP2) predominantly in the kidney proximal tubules. The transposon-based method used allowed the generation of homozygous transgenic rats containing one copy of the transgene per allele with a defined insertion pattern, without genetic or phenotypic alterations. We applied in vitro confocal and in vivo two-photon microscopy to examine basal calcium levels and ligand- and drug-induced alterations in these levels in proximal tubular epithelial cells. Notably, renal ischemia induced a transient increase in cellular calcium, and reperfusion resulted in a secondary calcium load, which was significantly decreased by systemic administration of specific blockers of the angiotensin receptor and the Na-Ca exchanger. The parallel examination of in vivo cellular calcium dynamics and renal circulation by fluorescent probes opens new possibilities for physiologic and pharmacologic investigations.

Inhibition of PHLDA3 expression in human superoxide dismutase 1-mutant amyotrophic lateral sclerosis astrocytes protects against neurotoxicity.

Pleckstrin homology-like domain family A-member 3 (PHLDA3) has recently been identified as a player in adaptive and maladaptive cellular stress pathways. The outcome of pleckstrin homology-like domain family A-member 3 signalling was shown to vary across different cell types and states. It emerges that its expression and protein level are highly increased in amyotrophic lateral sclerosis (ALS) patient-derived astrocytes. Whether it orchestrates a supportive or detrimental function remains unexplored in the context of neurodegenerative pathologies. To directly address the role of pleckstrin homology-like domain family A-member 3 in healthy and ALS astrocytes, we used overexpression and knockdown strategies. We generated cultures of primary mouse astrocytes and also human astrocytes from control and ALS patient-derived induced pluripotent stem cells harbouring the superoxide dismutase 1 mutation. Then, we assessed astrocyte viability and the impact of their secretome on oxidative stress responses in human stem cell-derived cortical and spinal neuronal cultures. Here, we show that PHLDA3 overexpression or knockdown in control astrocytes does not significantly affect astrocyte viability or reactive oxygen species production. However, PHLDA3 knockdown in ALS astrocytes diminishes reactive oxygen species concentrations in their supernatants, indicating that pleckstrin homology-like domain family A-member 3 can facilitate stress responses in cells with altered homeostasis. In support, supernatants of PHLDA3-silenced ALS and even control spinal astrocytes with a lower pleckstrin homology-like domain family A-member 3 protein content could prevent sodium arsenite-induced stress granule formation in spinal neurons. Our findings provide evidence that reducing pleckstrin homology-like domain family A-member 3 levels may transform astrocytes into a more neurosupportive state relevant to targeting non-cell autonomous ALS pathology.

Dynamic ABCG2 expression in human embryonic stem cells provides the basis for stress response.

ABCG2 is a plasma membrane multidrug transporter with an established role in the cancer drug-resistance phenotype. This protein is expressed in a variety of tissues, including several types of stem cell. Although ABCG2 is not essential for life, knock-out mice were found to be hypersensitive to xenobiotics and had reduced levels of the side population of hematopoietic stem cells. Previously we have shown that ABCG2 is present in human embryonic stem cell (hESC) lines, with a heterogeneous expression pattern. In this study we examined this heterogeneity, and investigated whether it is related to stress responses in hESCs. We did not find any difference between expression of pluripotency markers in ABCG2-positive and negative hESCs; however, ABCG2-expressing cells had a higher growth rate after cell separation. We found that some harmful conditions (physical stress, drugs, and UV light exposure) are tolerated much better in the presence of ABCG2 protein. This property can be explained by the transporter function which eliminates potential toxic metabolites accumulated during stress conditions. In contrast, mild oxidative stress in hESCs caused rapid internalization of ABCG2, indicating that some environmental factors may induce removal of this transporter from the plasma membrane. On the basis of these results we suggest that a dynamic balance of ABCG2 expression at the population level has the advantage of enabling prompt response to changes in the cellular environment. Such actively maintained heterogeneity might be of evolutionary benefit in protecting special cell types, including pluripotent stem cells.

A human proteogenomic-cellular framework identifies KIF5A as a modulator of astrocyte process integrity with relevance to ALS.

Genome-wide association studies identified several disease-causing mutations in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). However, the contribution of genetic variants to pathway disturbances and their cell type-specific variations, especially in glia, is poorly understood. We integrated ALS GWAS-linked gene networks with human astrocyte-specific multi-omics datasets to elucidate pathognomonic signatures. It predicts that KIF5A, a motor protein kinesin-1 heavy-chain isoform, previously detected only in neurons, can also potentiate disease pathways in astrocytes. Using postmortem tissue and super-resolution structured illumination microscopy in cell-based perturbation platforms, we provide evidence that KIF5A is present in astrocyte processes and its deficiency disrupts structural integrity and mitochondrial transport. We show that this may underly cytoskeletal and trafficking changes in SOD1 ALS astrocytes characterised by low KIF5A levels, which can be rescued by c-Jun N-terminal Kinase-1 (JNK1), a kinesin transport regulator. Altogether, our pipeline reveals a mechanism controlling astrocyte process integrity, a pre-requisite for synapse maintenance and suggests a targetable loss-of-function in ALS.

P-Glycoprotein Activity at Diagnosis Does Not Predict Therapy Outcome and Survival in Canine B-Cell Lymphoma.

Various mechanisms are known to be involved in the development of multidrug resistance during cancer treatment. P-glycoprotein (P-gp) decreases the intracellular concentrations of cytotoxic drugs by an energy-dependent efflux mechanism. The aim of this study was to investigate the predictive value of P-gp function based on the evaluation of P-gp activity in tumor cells obtained from canine B-cell lymphoma patients at diagnosis. P-gp function of 79 immunophenotyped canine lymphoma samples was determined by flow cytometry using the Calcein assay. Dogs were treated with either the CHOP or the L-CHOP protocol, a subset of relapsed patients received L-asparaginase and lomustine rescue treatments. Among the 79 dogs, the median overall survival time was 417 days, and the median relapse-free period was 301 days. 47 percent of the samples showed high P-gp activity, which was significantly higher in Stage IV cancer patients compared to Stage II + III and V. Whereas staging was associated with major differences in survival times, we found that the intrinsic P-gp activity of tumor cells measured at diagnosis is not predictive for therapy outcome. Further studies are needed to identify the intrinsic and acquired resistant mechanisms that shape therapy response and survival in B-cell canine lymphoma patients.

Comparison of Different Clinical Chemotherapeutical Agents' Toxicity and Cell Response on Mesenchymal Stem Cells and Cancer Cells.

Mesenchymal stem cells (MSCs) or fibroblasts are one of the most abundant cell types in the tumor microenvironment (TME) exerting various anti- and pro-apoptotic effects during tumorigenesis, invasion, and drug treatment. Despite the recently discovered importance of MSCs in tumor progression and therapy, the response of these cells to chemotherapeutics compared to cancer cells is rarely investigated. A widely accepted view is that these naive MSCs have higher drug tolerance than cancer cells due to a significantly lower proliferation rate. Here, we examine the differences and similarities in the sensitivity of MSCs and cancer cells to nine diverse chemotherapy agents and show that, although MSCs have a slower cell cycle, these cells are still sensitive to various drugs. Surprisingly, MSCs showed similar sensitivity to a panel of compounds, however, suffered fewer DNA double-stranded breaks, did not enter into a senescent state, and was virtually incapable of apoptosis. Our results suggest that MSCs and cancer cells have different cell fates after drug treatment, and this could influence therapy outcome. These findings could help design drug combinations targeting both MSCs and cancer cells in the TME.

Human ALS/FTD brain organoid slice cultures display distinct early astrocyte and targetable neuronal pathology.

Amyotrophic lateral sclerosis overlapping with frontotemporal dementia (ALS/FTD) is a fatal and currently untreatable disease characterized by rapid cognitive decline and paralysis. Elucidating initial cellular pathologies is central to therapeutic target development, but obtaining samples from presymptomatic patients is not feasible. Here, we report the development of a cerebral organoid slice model derived from human induced pluripotent stem cells (iPSCs) that recapitulates mature cortical architecture and displays early molecular pathology of C9ORF72 ALS/FTD. Using a combination of single-cell RNA sequencing and biological assays, we reveal distinct transcriptional, proteostasis and DNA repair disturbances in astroglia and neurons. We show that astroglia display increased levels of the autophagy signaling protein P62 and that deep layer neurons accumulate dipeptide repeat protein poly(GA), DNA damage and undergo nuclear pyknosis that could be pharmacologically rescued by GSK2606414. Thus, patient-specific iPSC-derived cortical organoid slice cultures are a reproducible translational platform to investigate preclinical ALS/FTD mechanisms as well as novel therapeutic approaches.

Applying a "double-feature" promoter to identify cardiomyocytes differentiated from human embryonic stem cells following transposon-based gene delivery.

Human embryonic stem (HuES) cells represent a new potential tool for cell-therapy and gene-therapy applications. However, these approaches require the development of efficient, stable gene delivery, and proper progenitor cell and tissue separation methods. In HuES cell lines, we have generated stable, enhanced green fluorescent protein (EGFP)-expressing clones using a transposon-based (Sleeping Beauty) system. This method yielded high percentage of transgene integration and expression. Similarly to a lentiviral expression system, both the undifferentiated state and the differentiation pattern of the HuES cells were preserved. By using the CAG promoter, in contrast to several other constitutive promoter sequences (such as CMV, elongation factor 1alpha, or phosphoglycerate kinase), an exceptionally high EGFP expression was observed in differentiated cardiomyocytes. This phenomenon was independent of the transgene sequence, methods of gene delivery, copy number, and the integration sites. This "double-feature" promoter behavior, that is providing a selectable marker for transgene expressing undifferentiated stem cells, and also specifically labeling differentiated cardiomyocytes, was assessed by transcriptional profiling. We found a positive correlation between CAG promoter-driven EGFP transcription and expression of cardiomyocyte-specific genes. Our experiments indicate an efficient applicability of transposon-based gene delivery into HuES cells and provide a novel approach to identify differentiated tissues by exploiting a nontypical behavior of a constitutively active promoter, thereby avoiding invasive drug selection methods.

Celecoxib Prevents Doxorubicin-Induced Multidrug Resistance in Canine and Mouse Lymphoma Cell Lines.

BACKGROUND: Treatment of malignancies is still a major challenge in human and canine cancer, mostly due to the emergence of multidrug resistance (MDR). One of the main contributors of MDR is the overexpression P-glycoprotein (Pgp), which recognizes and extrudes various chemotherapeutics from cancer cells. METHODS: To study mechanisms underlying the development of drug resistance, we established an in vitro treatment protocol to rapidly induce Pgp-mediated MDR in cancer cells. Based on a clinical observation showing that a 33-day-long, unplanned drug holiday can reverse the MDR phenotype of a canine diffuse large B-cell lymphoma patient, our aim was to use the established assay to prevent the emergence of drug resistance in the early stages of treatment. RESULTS: We showed that an in vitro drug holiday results in the decrease of Pgp expression in MDR cell lines. Surprisingly, celecoxib, a known COX-2 inhibitor, prevented the emergence of drug-induced MDR in murine and canine lymphoma cell lines. CONCLUSIONS: Our findings suggest that celecoxib could significantly improve the efficiency of chemotherapy by preventing the development of MDR in B-cell lymphoma.

Generation of multidrug resistant human tissues by overexpression of the ABCG2 multidrug transporter in embryonic stem cells.

The ABCG2 multidrug transporter provides resistance against various endo- and xenobiotics, and protects the stem cells against toxins and stress conditions. We have shown earlier that a GFP-tagged version of ABCG2 is fully functional and may be used to follow the expression, localization and function of this transporter in living cells. In the present work we have overexpressed GFP-ABCG2, driven by a constitutive (CAG) promoter, in HUES9 human embryonic stem cells. Stem cell clones were generated to express the wild-type and a substrate-mutant (R482G) GFP-ABCG2 variant, by using the Sleeping Beauty transposon system. We found that the stable overexpression of these transgenes did not change the pluripotency and growth properties of the stem cells, nor their differentiation capacity to hepatocytes or cardiomyocytes. ABCG2 overexpression provided increased toxin resistance in the stem cells, and protected the derived cardiomyocytes against doxorubicin toxicity. These studies document the potential of a stable ABCG2 expression for engineering toxin-resistant human pluripotent stem cells and selected stem cell derived tissues.

Enthalpy/entropy driven activation of the first interquinone electron transfer in bacterial photosynthetic reaction centers embedded in vesicles of physiologically important phospholipids.

The thermodynamics and kinetics of light-induced electron transfer in bacterial photosynthetic RCs are sensitive to physiologically important lipids (phosphatidylcholine, cardiolipin and phosphatidylglycerol) in the environment. The analysis of the temperature-dependence of the rate of the P(+)Q(A)(-)Q(B)-->P(+)Q(A)Q(B)(-) interquinone electron transfer revealed high enthalpy change of activation in zwitterionic or neutral micelles and vesicles and low enthalpy change of activation in vesicles constituted of negatively charged phospholipids. The entropy change of activation was compensated by the changes of enthalpy, thus the free energy change of activation ( approximately 500 meV) did not show large variation in vesicles of different lipids.

Identification and Validation of Compounds Selectively Killing Resistant Cancer: Delineating Cell Line-Specific Effects from P-Glycoprotein-Induced Toxicity.

Despite significant progress, resistance to chemotherapy is still the main reason why cancer remains a deadly disease. An attractive strategy is to target the collateral sensitivity of otherwise multidrug resistant (MDR) cancer. In this study, our aim was to catalog various compounds that were reported to elicit increased toxicity in P-glycoprotein (Pgp)-overexpressing MDR cells. We show that the activity of most of the serendipitously identified compounds reported to target MDR cells is in fact cell-line specific, and is not influenced significantly by the function of Pgp. In contrast, novel 8-hydroxyquinoline derivatives that we identify in the National Cancer Institute (NCI) drug repository possess a robust Pgp-dependent toxic activity across diverse cell lines. Pgp expression associated with the resistance of the doxorubicin-resistant Brca1-/-;p53-/- spontaneous mouse mammary carcinoma cells could be eliminated by a single treatment with NSC57969, suggesting that MDR-selective compounds can effectively revert the MDR phenotype of cells expressing Pgp at clinically relevant levels. The discovery of new MDR-selective compounds shows the potential of this emerging technology and highlights the 8-hydroxyquinoline scaffold as a promising starting point for the development of compounds targeting the Achilles heel of drug-resistant cancer. Mol Cancer Ther; 16(1); 45-56. ©2016 AACR.

Pegylated liposomal formulation of doxorubicin overcomes drug resistance in a genetically engineered mouse model of breast cancer.

Success of cancer treatment is often hampered by the emergence of multidrug resistance (MDR) mediated by P-glycoprotein (ABCB1/Pgp). Doxorubicin (DOX) is recognized by Pgp and therefore it can induce therapy resistance in breast cancer patients. In this study our aim was to evaluate the susceptibility of the pegylated liposomal formulation of doxorubicin (PLD/Doxil®/Caelyx®) to MDR. We show that cells selected to be resistant to DOX are cross-resistant to PLD and PLD is also ineffective in an allograft model of doxorubicin-resistant mouse B-cell leukemia. In contrast, PLD was far more efficient than DOX as reflected by a significant increase of both relapse-free and overall survival of Brca1-/-;p53-/- mammary tumor bearing mice. Increased survival could be explained by the delayed onset of drug resistance. Consistent with the higher Pgp levels needed to confer resistance, PLD administration was able to overcome doxorubicin insensitivity of the mouse mammary tumors. Our results indicate that the favorable pharmacokinetics achieved with PLD can effectively overcome Pgp-mediated resistance, suggesting that PLD therapy could be a promising strategy for the treatment of therapy-resistant breast cancer patients.

Extensive astrocyte synchronization advances neuronal coupling in slow wave activity in vivo.

Slow wave activity (SWA) is a characteristic brain oscillation in sleep and quiet wakefulness. Although the cell types contributing to SWA genesis are not yet identified, the principal role of neurons in the emergence of this essential cognitive mechanism has not been questioned. To address the possibility of astrocytic involvement in SWA, we used a transgenic rat line expressing a calcium sensitive fluorescent protein in both astrocytes and interneurons and simultaneously imaged astrocytic and neuronal activity in vivo. Here we demonstrate, for the first time, that the astrocyte network display synchronized recurrent activity in vivo coupled to UP states measured by field recording and neuronal calcium imaging. Furthermore, we present evidence that extensive synchronization of the astrocytic network precedes the spatial build-up of neuronal synchronization. The earlier extensive recruitment of astrocytes in the synchronized activity is reinforced by the observation that neurons surrounded by active astrocytes are more likely to join SWA, suggesting causality. Further supporting this notion, we demonstrate that blockade of astrocytic gap junctional communication or inhibition of astrocytic Ca2+ transients reduces the ratio of both astrocytes and neurons involved in SWA. These in vivo findings conclusively suggest a causal role of the astrocytic syncytium in SWA generation.

A convenient method to pre-screen candidate guide RNAs for CRISPR/Cas9 gene editing by NHEJ-mediated integration of a 'self-cleaving' GFP-expression plasmid.

The efficacies of guide RNAs (gRNAs), the short RNA molecules that bind to and determine the sequence specificity of the Streptococcus pyogenes Cas9 nuclease, to mediate DNA cleavage vary dramatically. Thus, the selection of appropriate target sites, and hence spacer sequence, is critical for most applications. Here, we describe a simple, unparalleled method for experimentally pre-testing the efficiencies of various gRNAs targeting a gene. The method explores NHEJ-cloning, genomic integration of a GFP-expressing plasmid without homologous arms and linearized in-cell. The use of 'self-cleaving' GFP-plasmids containing universal gRNAs and corresponding targets alleviates cloning burdens when this method is applied. These universal gRNAs mediate efficient plasmid cleavage and are designed to avoid genomic targets in several model species. The method combines the advantages of the straightforward FACS detection provided by applying fluorescent reporter systems and of the PCR-based approaches being capable of testing targets in their genomic context, without necessitating any extra cloning steps. Additionally, we show that NHEJ-cloning can also be used in mammalian cells for targeted integration of donor plasmids up to 10 kb in size, with up to 30% efficiency, without any selection or enrichment.

The importance of drug transporters in human pluripotent stem cells and in early tissue differentiation.

INTRODUCTION: Drug transporters are large transmembrane proteins which catalyse the movement of a wide variety of chemicals, including drugs as well as xeno- and endobiotics through cellular membranes. The major groups of these proteins include the ATP-binding cassette transporters which in eukaryotes work as ATP-fuelled drug 'exporters' and the Solute Carrier transporters, with various transport directions and mechanisms. AREAS COVERED: In this review, we discuss the key ATP-binding cassette and Solute Carrier drug transporters which have been reported to contribute to the function and/or protection of undifferentiated human stem cells and during tissue differentiation. We review the various techniques for studying transporter expression and function in stem cells, and the role of drug transporters in foetal and placental tissues is also discussed. We especially focus on the regulation of transporter expression by factors modulating cell differentiation properties and on the function of the transporters in adjustment to environmental challenges. EXPERT OPINION: The relatively new and as yet unexplored territory of transporters in stem cell biology may rapidly expand and bring important new information regarding the metabolic and epigenetic regulation of 'stemness' and the early differentiation properties. Drug transporters are clearly important protective and regulatory components in stem cells and differentiation.