Rescue of tropomyosin deficiency in Drosophila and human cancer cells by synaptopodin reveals a role of tropomyosin α in RhoA stabilization.

Tropomyosins are widespread actin-binding proteins that influence numerous cellular functions including actin dynamics, cell migration, tumour suppression, and Drosophila oocyte development. Synaptopodin is another actin-binding protein with a more restricted expression pattern in highly dynamic cell compartments such as kidney podocyte foot processes, where it promotes RhoA signalling by blocking the Smurf1-mediated ubiquitination of RhoA. Here, we show that synaptopodin has a shorter half-life but shares functional properties with the highly stable tropomyosin. Transgenic expression of synaptopodin restores oskar mRNA localization in Drosophila oocytes mutant for TmII, thereby rescuing germline differentiation and fertility. Synaptopodin restores stress fibres in tropomyosin-deficient human MDA-MB 231 breast cancer cells and TPMα-depleted fibroblasts. Gene silencing of TPMα but not TPMß causes loss of stress fibres by promoting Smurf1-mediated ubiquitination and proteasomal degradation of RhoA. Functionally, overexpression of synaptopodin or RhoA(K6,7R) significantly reduces MDA-MB 231 cell migration. Our findings elucidate RhoA stabilization by structurally unrelated actin-binding proteins as a conserved mechanism for regulation of stress fibre dynamics and cell motility in a cell type-specific fashion.

Combined niclosamide with cisplatin inhibits epithelial-mesenchymal transition and tumor growth in cisplatin-resistant triple-negative breast cancer.

Women with triple-negative breast cancer have worse prognosis compared to other breast cancer subtypes. Acquired drug resistance remains to be an important reason influencing triple-negative breast cancer treatment efficacy. A prevailing theory postulates that the cancer resistance and recurrence results from a subpopulation of tumor cells with stemness program, which are often insensitive to cytotoxic drugs such as cisplatin. Recent studies suggested that niclosamide, an anti-helminthic drug, has potential therapeutic activities against breast cancer stem cells, which prompts us to determine its roles on eliminating cisplatin-resistant cancer cells. Hence, we established a stable cisplatin-resistant MDA-MB-231 cell line (231-CR) through continuously exposure to increasing concentrations of cisplatin (5-20 µmol/l). Interestingly, 231-CR exhibited properties associated to epithelial-mesenchymal transition with enhanced invasion, preserved proliferation, increased mammosphere formation, and reduced apoptosis compared to naive MDA-MB-231 sensitive cells (231-CS). Importantly, niclosamide or combination with cisplatin inhibited both 231-CS and 231-CR cell proliferation in vitro. In addition, niclosamide reversed the EMT phenotype of 231-CR by downregulation of snail and vimentin. Mechanistically, niclosamide treatment in combination with or without cisplatin significantly inhibited Akt, ERK, and Src signaling pathways. In vivo study showed that niclosamide or combination with cisplatin could repress the growth of xenografts originated from either 231-CS or 231-CR cells, with prominent suppression of Ki67 expression. These findings suggested that niclosamide might serve as a novel therapeutic strategy, either alone or in combination with cisplatin, for triple-negative breast cancer treatment, especially those resistant to cisplatin.

Impact of the F508del mutation on ovine CFTR, a Cl- channel with enhanced conductance and ATP-dependent gating.

KEY POINTS: Malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR), a gated pathway for chloride movement, causes the common life-shortening genetic disease cystic fibrosis (CF). Towards the development of a sheep model of CF, we have investigated the function of sheep CFTR. We found that sheep CFTR was noticeably more active than human CFTR, while the most common CF mutation, F508del, had reduced impact on sheep CFTR function. Our results demonstrate that subtle changes in protein structure have marked effects on CFTR function and the consequences of the CF mutation F508del. ABSTRACT: Cross-species comparative studies are a powerful approach to understanding the epithelial Cl(-) channel cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF). Here, we investigate the single-channel behaviour of ovine CFTR and the impact of the most common CF mutation, F508del-CFTR, using excised inside-out membrane patches from transiently transfected CHO cells. Like human CFTR, ovine CFTR formed a weakly inwardly rectifying Cl(-) channel regulated by PKA-dependent phosphorylation, inhibited by the open-channel blocker glibenclamide. However, for three reasons, ovine CFTR was noticeably more active than human CFTR. First, single-channel conductance was increased. Second, open probability was augmented because the frequency and duration of channel openings were increased. Third, with enhanced affinity and efficacy, ATP more strongly stimulated ovine CFTR channel gating. Consistent with these data, the CFTR modulator phloxine B failed to potentiate ovine CFTR Cl(-) currents. Similar to its impact on human CFTR, the F508del mutation caused a temperature-sensitive folding defect, which disrupted ovine CFTR protein processing and reduced membrane stability. However, the F508del mutation had reduced impact on ovine CFTR channel gating in contrast to its marked effects on human CFTR. We conclude that ovine CFTR forms a regulated Cl(-) channel with enhanced conductance and ATP-dependent channel gating. This phylogenetic analysis of CFTR structure and function demonstrates that subtle changes in structure have pronounced effects on channel function and the consequences of the CF mutation F508del.

Exploiting species differences to understand the CFTR Cl- channel.

The anion channel cystic fibrosis transmembrane conductance regulator (CFTR) is a unique ATP-binding cassette (ABC) transporter. CFTR plays a pivotal role in transepithelial ion transport as its dysfunction in the genetic disease cystic fibrosis (CF) dramatically demonstrates. Phylogenetic analysis suggests that CFTR first appeared in aquatic vertebrates fulfilling important roles in osmosensing and organ development. Here, we review selectively, knowledge of CFTR structure, function and pharmacology, gleaned from cross-species comparative studies of recombinant CFTR proteins, including CFTR chimeras. The data argue that subtle changes in CFTR structure can affect strongly channel function and the action of CF mutations.

Infiltrating S100A8+ myeloid cells promote metastatic spread of human breast cancer and predict poor clinical outcome.

The mechanisms by which breast cancer (BrC) can successfully metastasize are complex and not yet fully understood. Our goal was to identify tumor-induced stromal changes that influence metastatic cell behavior, and may serve as better targets for therapy. To identify stromal changes in cancer-bearing tissue, dual-species gene expression analysis was performed for three different metastatic BrC xenograft models. Results were confirmed by immunohistochemistry, flow cytometry, and protein knockdown. These results were validated in human clinical samples at the mRNA and protein level by retrospective analysis of cohorts of human BrC specimens. In pre-clinical models of BrC, systemic recruitment of S100A8+ myeloid cells-including myeloid-derived suppressor cells (MDSCs)-was promoted by tumor-derived factors. Recruitment of S100A8+ myeloid cells was diminished by inhibition of tumor-derived factors or depletion of MDSCs, resulting in fewer metastases and smaller primary tumors. Importantly, these MDSCs retain their ability to suppress T cell proliferation upon co-culture. Secretion of macrophage inhibitory factor (MIF) activated the recruitment of S100A8+ myeloid cells systemically. Inhibition of MIF, or depletion of MDSCs resulted in delayed tumor growth and lower metastatic burden. In human BrC specimens, increased mRNA and protein levels of S100A8+ infiltrating cells are highly associated with poor overall survival and shorter metastasis free survival of BrC patients, respectively. Furthermore, analysis of nine different human gene expression datasets confirms the association of increased levels of S100A8 transcripts with an increased risk of death. Recruitment of S100A8+ myeloid cells to primary tumors and secondary sites in xenograft models of BrC enhances cancer progression independent of their suppressive activity on T cells. In clinical samples, infiltrating S100A8+ cells are associated with poor overall survival. Targeting these molecules or associated pathways in cells of the tumor microenvironment may translate into novel therapeutic interventions and benefit patient outcome.

Simultaneous analysis of tumor and stromal gene expression profiles from xenograft models.

Identifying the gene expression alterations that occur in both the tumor and stroma is essential to understanding tumor biology. We have developed a dual-species microarray analysis method that allows the dissection of both tumor and stromal gene expression profiles from xenograft models, based on limited interspecies cross-hybridization on Illumina gene expression beadchips. This methodology allows for simultaneous genome-wide analysis of gene expression profiles of both tumor cells and the associated stromal tissue.

PI3K independent activation of mTORC1 as a target in lapatinib-resistant ERBB2+ breast cancer cells.

Therapies targeting the ERBB2 receptor, including the kinase inhibitor lapatinib (Tykerb, GlaxoSmithKline), have improved clinical outcome for women with ERBB2-amplified breast cancer. However, acquired resistance to lapatinib remains a significant clinical problem, and the mechanisms governing resistance remain poorly understood. We sought to define molecular alterations that confer an acquired lapatinib resistance phenotype in ER-/ERBB2+ human breast cancer cells. ERBB2-amplified SKBR3 breast cancer cells were rendered resistant to lapatinib via culture in increasing concentrations of the drug, and molecular changes associated with a resistant phenotype were interrogated using a collaborative enzyme-enhanced immunoassay platform and immunoblotting techniques for detection of phosphorylated signaling cascade proteins. Interestingly, despite apparent inactivation of the PI3K/AKT signaling pathway, resistant cells exhibited constitutive activation of mammalian target of rapamycin complex 1 (mTORC1) and were highly sensitive to mTOR inhibition with rapamycin and the dual PI3K/mTOR inhibitor NVP-BEZ235. These data demonstrate a role for downstream activation of mTORC1 in the absence of molecular alterations leading to PI3K/AKT hyperactivation as a potential mechanism of lapatinib resistance in this model of ERBB2+ breast cancer and support the rationale of combination or sequential therapy using ERBB2 and mTOR-targeting molecules to prevent or target resistance to lapatinib. Moreover, our data suggest that assessment of mTOR substrate phosphorylation (i.e., S6) may serve as a more robust biomarker to predict sensitivity to mTOR inhibitors in the context of lapatinib resistance than PI3K mutations, loss of PTEN and p-AKT levels.

Whole genome in vivo RNAi screening identifies the leukemia inhibitory factor receptor as a novel breast tumor suppressor.

Cancer is caused by mutations in oncogenes and tumor suppressor genes, resulting in the deregulation of processes fundamental to the normal behavior of cells. The identification and characterization of oncogenes and tumor suppressors has led to new treatment strategies that have significantly improved cancer outcome. The advent of next generation sequencing has allowed the elucidation of the fine structure of cancer genomes, however, the identification of pathogenic changes is complicated by the inherent genomic instability of cancer cells. Therefore, functional approaches for the identification of novel genes involved in the initiation and development of tumors are critical. Here we report the first whole human genome in vivo RNA interference screen to identify functionally important tumor suppressor genes. Using our novel approach, we identify previously validated tumor suppressor genes including TP53 and MNT, as well as several novel candidate tumor suppressor genes including leukemia inhibitory factor receptor (LIFR). We show that LIFR is a key novel tumor suppressor, whose deregulation may drive the transformation of a significant proportion of human breast cancers. These results demonstrate the power of genome wide in vivo RNAi screens as a method for identifying novel genes regulating tumorigenesis.

Simultaneous quantification of 12 different nucleotides and nucleosides released from renal epithelium and in human urine samples using ion-pair reversed-phase HPLC.

Nucleotides and nucleosides are not only involved in cellular metabolism but also act extracellularly via P1 and P2 receptors, to elicit a wide variety of physiological and pathophysiological responses through paracrine and autocrine signalling pathways. For the first time, we have used an ion-pair reversed-phase high-performance liquid chromatography ultraviolet (UV)-coupled method to rapidly and simultaneously quantify 12 different nucleotides and nucleosides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine, uridine triphosphate, uridine diphosphate, uridine monophosphate, uridine, guanosine triphosphate, guanosine diphosphate, guanosine monophosphate, guanosine): (1) released from a mouse renal cell line (M1 cortical collecting duct) and (2) in human biological samples (i.e., urine). To facilitate analysis of urine samples, a solid-phase extraction step was incorporated (overall recovery rate ≥ 98 %). All samples were analyzed following injection (100 µl) into a Synergi Polar-RP 80 Å (250 × 4.6 mm) reversed-phase column with a particle size of 10 µm, protected with a guard column. A gradient elution profile was run with a mobile phase (phosphate buffer plus ion-pairing agent tetrabutylammonium hydrogen sulfate; pH 6) in 2-30 % acetonitrile (v/v) for 35 min (including equilibration time) at 1 ml min(-1) flow rate. Eluted compounds were detected by UV absorbance at 254 nm and quantified using standard curves for nucleotide and nucleoside mixtures of known concentration. Following validation (specificity, linearity, limits of detection and quantitation, system precision, accuracy, and intermediate precision parameters), this protocol was successfully and reproducibly used to quantify picomolar to nanomolar concentrations of nucleosides and nucleotides in isotonic and hypotonic cell buffers that transiently bathed M1 cells, and urine samples from normal subjects and overactive bladder patients.

Effect of Annexin A5 on CFTR: regulated traffic or scaffolding?

Previous studies have implicated annexins in regulating ion channels and in particular annexin A5 (AnxA5) in the traffic of the cystic fibrosis transmembrane conductance regulator (CFTR). In the present study, we further investigated the role of AnxA5 in regulating CFTR function and intracellular trafficking in both Xenopus oocytes and mammalian cells. Although we could confirm the previously reported CFTR/AnnxA5 interaction, we found that in oocytes AnxA5 inhibits CFTR-mediated whole-cell membrane conductance presumably by a mechanism independent of PDZ-binding domain at the C-terminus of CFTR but protein kinase C (PKC)-dependent and results from either endocytosis activation and/or exocytosis block. In contrast, in human cells, co-expression of AnxA5 augmented CFTR whole-cell currents, an effect that was independent of CFTR PDZ-binding domain. We conclude that annexin A5 has multiple effects on CFTR, so that the net effect observed is cell system-dependent. Nevertheless, both effects observed here are consistent with the described role of annexins forming scaffolding platforms at cell membranes, thus contributing to a decrease in their dynamics. Finally, we could not confirm that AnxA5 overexpression rescues traffic/function of the most frequent disease-causing mutant F508del-CFTR, thus concluding that AnxA5 is not a promising tool for correction of the F508del-CFTR defect.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in Human Lung Microvascular Endothelial Cells Controls Oxidative Stress, Reactive Oxygen-Mediated Cell Signaling and Inflammatory Responses.

BACKGROUND: Perturbation of endothelial function in people with cystic fibrosis (CF) has been reported, which may be associated with endothelial cell expression of the cystic fibrosis transmembrane conductance regulator (CFTR). Previous reports indicate that CFTR activity upregulates endothelial barrier function, endothelial nitric oxide synthase (eNOS) expression and NO release, while limiting interleukin-8 (IL-8) release, in human umbilical vein endothelial cells (HUVECs) in cell culture. In view of reported microvascular dysfunction in people with CF we investigated the role of CFTR expression and activity in the regulation of oxidative stress, cell signaling and inflammation in human lung microvascular endothelial cells (HLMVECs) in cell culture. METHODS: HLMVECs were cultured in the absence and presence of the CFTR inhibitor GlyH-101 and CFTR siRNA. CFTR expression was analyzed using qRT-PCR, immunocytochemistry (IHC) and western blot, and function by membrane potential assay. IL-8 expression was analyzed using qRT-PCR and ELISA. Nrf2 expression, and NF-κB and AP-1 activation were determined using IHC and western blot. The role of the epidermal growth factor receptor (EGFR) in CFTR signaling was investigated using the EGFR tyrosine kinase inhibitor AG1478. Oxidative stress was measured as intracellular ROS and hydrogen peroxide (H2O2) concentration. VEGF and SOD-2 were measured in culture supernatants by ELISA. RESULTS: HLMVECs express low levels of CFTR that increase following inhibition of CFTR activity. Inhibition of CFTR, significantly increased intracellular ROS and H2O2 levels over 30 min and significantly decreased Nrf2 expression by 70% while increasing SOD-2 expression over 24 h. CFTR siRNA significantly increased constitutive expression of IL-8 by HLMVECs. CFTR inhibition activated the AP-1 pathway and increased IL-8 expression, without effect on NF-κB activity. Conversely, TNF-α activated the NF-κB pathway and increased IL-8 expression. The effects of TNF-α and GlyH-101 on IL-8 expression were additive and inhibited by AG1478. Inhibition of both CFTR and EGFR in HLMVECs significantly increased VEGF expression. The antioxidant N-acetyl cysteine significantly reduced ROS production and the increase in IL-8 and VEGF expression following CFTR inhibition. CONCLUSION: Functional endothelial CFTR limits oxidative stress and contributes to the normal anti-inflammatory state of HLMVECs. Therapeutic strategies to restore endothelial CFTR function in CF are warranted.

The simian varicella virus uracil DNA glycosylase and dUTPase genes are expressed in vivo, but are non-essential for replication in cell culture.

Neurotropic herpesviruses express viral deoxyuridine triphosphate nucleotidohydrolase (dUTPase) and uracil DNA glycosylase (UDG) enzymes which may reduce uracil misincorporation into viral DNA, particularly in neurons of infected ganglia. The simian varicella virus (SVV) dUTPase (ORF 8) and UDG (ORF 59) share 37.7% and 53.9% amino acid identity, respectively, with varicella-zoster virus (VZV) homologs. Infectious SVV mutants defective in either dUTPase (SVV-dUTPase(-)) or UDG (SVV-UDG(-)) activity or both (SVV-dUTPase(-)/UDG(-)) were constructed using recA assisted restriction endonuclease cleavage (RARE) and a cosmid recombination system. Loss of viral dUTPase and UDG enzymatic activity was confirmed in CV-1 cells infected with the SVV mutants. The SVV-dUTPase(-), SVV-UDG(-), and SVV-dUTPase(-)/UDG(-) mutants replicated as efficiently as wild-type SVV in cell culture. SVV dUTPase and UDG expression was detected in tissues derived from acutely infected animals, but not in tissues derived from latently infected animals. Further studies will evaluate the pathogenesis of SVV dUTPase and UDG mutants and their potential as varicella vaccines.

Electrophoretic cytopathology resolves ERBB2 forms with single-cell resolution.

In addition to canonical oncoproteins, truncated isoforms and proteolysis products are implicated in both drug resistance and disease progression. In HER2-positive breast tumors, expression of truncated HER2 isoforms resulting from alternative translation and/or carboxy-terminal fragments (CTFs) resulting from proteolysis (collectively, t-erbB2) have been associated with shortened progression-free survival of patients. Thus, to advance clinical pathology and inform treatment decisions, we developed a high-selectivity cytopathology assay capable of distinguishing t-erbB2 from full-length HER2 expression without the need for isoform-specific antibodies. Our microfluidic, single-cell western blot, employs electrophoretic separations to resolve full-length HER2 from the smaller t-erbB2 in each ~28 pL single-cell lysate. Subsequently, a pan-HER2 antibody detects all resolved HER2 protein forms via immunoprobing. In analysis of eight breast tumor biopsies, we identified two tumors comprised of 15% and 40% t-erbB2-expressing cells. By single-cell western blotting of the t-erbB2-expressing cells, we observed statistically different ratios of t-erbB2 proteins to full-length HER2 expression. Further, target multiplexing and clustering analyses scrutinized signaling, including ribosomal S6, within the t-erbB2-expressing cell subpopulation. Taken together, cytometric assays that report both protein isoform profiles and signaling state offer cancer classification taxonomies with unique relevance to precisely describing drug resistance mechanisms in which oncoprotein isoforms/fragments are implicated.

Hydrogel Pore-Size Modulation for Enhanced Single-Cell Western Blotting.

Pore-gradient microgel arrays enable thousands of parallel high-resolution single-cell protein electrophoresis separations for targets accross a wide molecular mass (25-289 kDa), yet within 1 mm separation distances. Dual crosslinked hydrogels facilitate gel-pore expansion after electrophoresis for efficient and uniform immunoprobing. The photopatterned, light-activated, and acid-expandable hydrogel underpins single-cell protein analysis, here for oncoprotein-related signaling in human breast biopsy.

Niclosamide inhibits epithelial-mesenchymal transition and tumor growth in lapatinib-resistant human epidermal growth factor receptor 2-positive breast cancer.

Acquired resistance to lapatinib, a human epidermal growth factor receptor 2 kinase inhibitor, remains a clinical problem for women with human epidermal growth factor receptor 2-positive advanced breast cancer, as metastasis is commonly observed in these patients. Niclosamide, an anti-helminthic agent, has recently been shown to exhibit cytotoxicity to tumor cells with stem-like characteristics. This study was designed to identify the mechanisms underlying lapatinib resistance and to determine whether niclosamide inhibits lapatinib resistance by reversing epithelial-mesenchymal transition. Here, two human epidermal growth factor receptor 2-positive breast cancer cell lines, SKBR3 and BT474, were exposed to increasing concentrations of lapatinib to establish lapatinib-resistant cultures. Lapatinib-resistant SKBR3 and BT474 cells exhibited up-regulation of the phenotypic epithelial-mesenchymal transition markers Snail, vimentin and α-smooth muscle actin, accompanied by activation of nuclear factor-кB and Src and a concomitant increase in stem cell marker expression (CD44(high)/CD24(low)), compared to naive lapatinib-sensitive SKBR3 and BT474 cells, respectively. Interestingly, niclosamide reversed epithelial-mesenchymal transition, induced apoptosis and inhibited cell growth by perturbing aberrant signaling pathway activation in lapatinib-resistant human epidermal growth factor receptor 2-positive cells. The ability of niclosamide to alleviate stem-like phenotype development and invasion was confirmed. Collectively, our results demonstrate that lapatinib resistance correlates with epithelial-mesenchymal transition and that niclosamide inhibits lapatinib-resistant cell viability and epithelial-mesenchymal transition. These findings suggest a role of niclosamide or derivatives optimized for more favorable bioavailability not only in reversing lapatinib resistance but also in reducing metastatic potential during the treatment of human epidermal growth factor receptor 2-positive breast cancer.

Voltage-dependent gating of the cystic fibrosis transmembrane conductance regulator Cl- channel.

When excised inside-out membrane patches are bathed in symmetrical Cl--rich solutions, the current-voltage (I-V) relationship of macroscopic cystic fibrosis transmembrane conductance regulator (CFTR) Cl- currents inwardly rectifies at large positive voltages. To investigate the mechanism of inward rectification, we studied CFTR Cl- channels in excised inside-out membrane patches from cells expressing wild-type human and murine CFTR using voltage-ramp and -step protocols. Using a voltage-ramp protocol, the magnitude of human CFTR Cl- current at +100 mV was 74 +/- 2% (n = 10) of that at -100 mV. This rectification of macroscopic CFTR Cl- current was reproduced in full by ensemble currents generated by averaging single-channel currents elicited by an identical voltage-ramp protocol. However, using a voltage-step protocol the single-channel current amplitude (i) of human CFTR at +100 mV was 88 +/- 2% (n = 10) of that at -100 mV. Based on these data, we hypothesized that voltage might alter the gating behavior of human CFTR. Using linear three-state kinetic schemes, we demonstrated that voltage has marked effects on channel gating. Membrane depolarization decreased both the duration of bursts and the interburst interval, but increased the duration of gaps within bursts. However, because the voltage dependencies of the different rate constants were in opposite directions, voltage was without large effect on the open probability (Po) of human CFTR. In contrast, the Po of murine CFTR was decreased markedly at positive voltages, suggesting that the rectification of murine CFTR is stronger than that of human CFTR. We conclude that inward rectification of CFTR is caused by a reduction in i and changes in gating kinetics. We suggest that inward rectification is an intrinsic property of the CFTR Cl- channel and not the result of pore block.

Strategies to investigate the mechanism of action of CFTR modulators.

The malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is associated with a wide spectrum of disease. In the search for modulators of CFTR, pharmaceutical agents have been identified that (i) act indirectly by regulating the protein kinases and phosphatases, which control CFTR, and (ii) interact directly with CFTR. Some agents modulate CFTR by altering the function of the nucleotide-binding domains (NBDs) that control channel gating, whereas others inhibit CFTR by preventing Cl- flow through the channel pore. Knowledge of CFTR modulators might lead to new understanding of the CFTR Cl- channel, its physiological role and malfunction in disease.

The patch-clamp and planar lipid bilayer techniques: powerful and versatile tools to investigate the CFTR Cl- channel.

Using the patch-clamp (PC) and planar lipid bilayer (PLB) techniques the molecular behaviour of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel can be visualised in real-time. The PC technique is a highly powerful and versatile method to investigate CFTR's mechanism of action, interaction with other proteins and physiological role. Using the PLB technique, the structure and function of CFTR can be investigated free from the influence of other proteins. Here we discuss how these techniques are employed to investigate the CFTR Cl- channel with special emphasis on its permeation, conduction and gating properties.

Loop diuretics are open-channel blockers of the cystic fibrosis transmembrane conductance regulator with distinct kinetics.

BACKGROUND AND PURPOSE: Loop diuretics are widely used to inhibit the Na(+), K(+), 2Cl(-) co-transporter, but they also inhibit the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. Here, we investigated the mechanism of CFTR inhibition by loop diuretics and explored the effects of chemical structure on channel blockade. EXPERIMENTAL APPROACH: Using the patch-clamp technique, we tested the effects of bumetanide, furosemide, piretanide and xipamide on recombinant wild-type human CFTR. KEY RESULTS: When added to the intracellular solution, loop diuretics inhibited CFTR Cl(-) currents with potency approaching that of glibenclamide, a widely used CFTR blocker with some structural similarity to loop diuretics. To begin to study the kinetics of channel blockade, we examined the time dependence of macroscopic current inhibition following a hyperpolarizing voltage step. Like glibenclamide, piretanide blockade of CFTR was time and voltage dependent. By contrast, furosemide blockade was voltage dependent, but time independent. Consistent with these data, furosemide blocked individual CFTR Cl(-) channels with 'very fast' speed and drug-induced blocking events overlapped brief channel closures, whereas piretanide inhibited individual channels with 'intermediate' speed and drug-induced blocking events were distinct from channel closures. CONCLUSIONS AND IMPLICATIONS: Structure-activity analysis of the loop diuretics suggests that the phenoxy group present in bumetanide and piretanide, but absent in furosemide and xipamide, might account for the different kinetics of channel block by locking loop diuretics within the intracellular vestibule of the CFTR pore. We conclude that loop diuretics are open-channel blockers of CFTR with distinct kinetics, affected by molecular dimensions and lipophilicity.

The microRNA -23b/-27b cluster suppresses the metastatic phenotype of castration-resistant prostate cancer cells.

MicroRNAs (miRs) are small, endogenous, non-coding RNAs that regulate the stability and/or translation of complementary mRNA targets. MiRs have emerged not only as critical modulators of normal physiologic processes, but their deregulation may significantly impact prostate and other cancers. The expression of miR-23b and miR-27b, which are encoded by the same miR cluster (miR-23b/-27b), are downregulated in metastatic, castration-resistant tumors compared to primary prostate cancer and benign tissue; however, their possible role in prostate cancer progression is unknown. We found that ectopic expression of miR-23b/-27b in two independent castration-resistant prostate cancer cell lines resulted in suppression of invasion and migration, as well as reduced survival in soft agar (a measure of anoikis). However, there was no effect of miR-23b/-27b on cell proliferation suggesting that these miRs function as metastasis (but not growth) suppressors in prostate cancer. Conversely, inhibition of miR-23b/-27b in the less aggressive androgen-dependent LNCaP prostate cancer cell line resulted in enhanced invasion and migration also without affecting proliferation. Mechanistically, we found that introduction of miR-23b/-27b in metastatic, castration-resistant prostate cancer cell lines resulted in a significant attenuation of Rac1 activity without affecting total Rac1 levels and caused increased levels of the tumor suppressor E-cadherin. Inhibition of these miRs had the opposite effect in androgen-dependent LNCaP cells. These results suggest that miR-23b/-27b are metastasis suppressors that might serve as novel biomarkers and therapeutic agents for castration-resistant disease.