Integrins regulate hERG1 dynamics by girdin-dependent Gαi3: signaling and modeling in cancer cells.

The hERG1 potassium channel is aberrantly over expressed in tumors and regulates the cancer cell response to integrin-dependent adhesion. We unravel a novel signaling pathway by which integrin engagement by the ECM protein fibronectin promotes hERG1 translocation to the plasma membrane and its association with ß1 integrins, by activating girdin-dependent Gαi3 proteins and protein kinase B (Akt). By sequestering hERG1, ß1 integrins make it avoid Rab5-mediated endocytosis, where unbound channels are degraded. The cycle of hERG1 expression determines the resting potential (Vrest) oscillations and drives the cortical f-actin dynamics and thus cell motility. To interpret the slow biphasic kinetics of hERG1/ß1 integrin interplay, we developed a mathematical model based on a generic balanced inactivation-like module. Integrin-mediated cell adhesion triggers two contrary responses: a rapid stimulation of hERG1/ß1 complex formation, followed by a slow inhibition which restores the initial condition. The protracted hERG1/ß1 integrin cycle determines the slow time course and cyclic behavior of cell migration in cancer cells.

DNA supercoiling restricts the transcriptional bursting of neighboring eukaryotic genes.

DNA supercoiling has emerged as a major contributor to gene regulation in bacteria, but how DNA supercoiling impacts transcription dynamics in eukaryotes is unclear. Here, using single-molecule dual-color nascent transcription imaging in budding yeast, we show that transcriptional bursting of divergent and tandem GAL genes is coupled. Temporal coupling of neighboring genes requires rapid release of DNA supercoils by topoisomerases. When DNA supercoils accumulate, transcription of one gene inhibits transcription at its adjacent genes. Transcription inhibition of the GAL genes results from destabilized binding of the transcription factor Gal4. Moreover, wild-type yeast minimizes supercoiling-mediated inhibition by maintaining sufficient levels of topoisomerases. Overall, we discover fundamental differences in transcriptional control by DNA supercoiling between bacteria and yeast and show that rapid supercoiling release in eukaryotes ensures proper gene expression of neighboring genes.

Combination Therapy with a Bispecific Antibody Targeting the hERG1/ß1 Integrin Complex and Gemcitabine in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) represents an unmet medical need. Difficult/late diagnosis as well as the poor efficacy and high toxicity of chemotherapeutic drugs result in dismal prognosis. With the aim of improving the treatment outcome of PDAC, we tested the effect of combining Gemcitabine with a novel single chain bispecific antibody (scDb) targeting the cancer-specific hERG1/ß1 integrin complex. First, using the scDb (scDb-hERG1-ß1) in immunohistochemistry (IHC), Western blot (WB) analysis and immunofluorescence (IF), we confirmed the presence of the hERG1/ß1 integrin complex in primary PDAC samples and PDAC cell lines. Combining Gemcitabine with scDb-hERG1-ß1 improved its cytotoxicity on all PDAC cells tested in vitro. We also tested the combination treatment in vivo, using an orthotopic xenograft mouse model involving ultrasound-guided injection of PDAC cells. We first demonstrated good penetration of the scDb-hERG1-ß1 conjugated with indocyanine green (ICG) into tumour masses by photoacoustic (PA) imaging. Next, we tested the effects of the combination at either therapeutic or sub-optimal doses of Gemcitabine (25 or 5 mg/kg, respectively). The combination of scDb-hERG1-ß1 and sub-optimal doses of Gemcitabine reduced the tumour masses to the same extent as the therapeutic doses of Gemcitabine administrated alone; yielded increased survival; and was accompanied by minimised side effects (toxicity). These data pave the way for a novel therapeutic approach to PDAC, based on the combination of low doses of a chemotherapeutic drug (to minimize adverse side effects and the onset of resistance) and the novel scDb-hERG1-ß1 targeting the hERG1/ß1 integrin complex as neoantigen.

Quantifying how post-transcriptional noise and gene copy number variation bias transcriptional parameter inference from mRNA distributions.

Transcriptional rates are often estimated by fitting the distribution of mature mRNA numbers measured using smFISH (single molecule fluorescence in situ hybridization) with the distribution predicted by the telegraph model of gene expression, which defines two promoter states of activity and inactivity. However, fluctuations in mature mRNA numbers are strongly affected by processes downstream of transcription. In addition, the telegraph model assumes one gene copy but in experiments, cells may have two gene copies as cells replicate their genome during the cell cycle. While it is often presumed that post-transcriptional noise and gene copy number variation affect transcriptional parameter estimation, the size of the error introduced remains unclear. To address this issue, here we measure both mature and nascent mRNA distributions of GAL10 in yeast cells using smFISH and classify each cell according to its cell cycle phase. We infer transcriptional parameters from mature and nascent mRNA distributions, with and without accounting for cell cycle phase and compare the results to live-cell transcription measurements of the same gene. We find that: (i) correcting for cell cycle dynamics decreases the promoter switching rates and the initiation rate, and increases the fraction of time spent in the active state, as well as the burst size; (ii) additional correction for post-transcriptional noise leads to further increases in the burst size and to a large reduction in the errors in parameter estimation. Furthermore, we outline how to correctly adjust for measurement noise in smFISH due to uncertainty in transcription site localisation when introns cannot be labelled. Simulations with parameters estimated from nascent smFISH data, which is corrected for cell cycle phases and measurement noise, leads to autocorrelation functions that agree with those obtained from live-cell imaging.

Post-transplant de-novo renal phospholipidosis in a kidney transplant recipient: Fabry disease or something else?

Renal phospholipidosis is a rare cause of proteinuria and kidney dysfunction. We describe a kidney transplant recipient who presented with slowly rising serum creatinine, nephrotic range proteinuria, and lower extremity edema 10 years post transplant. He was diagnosed with renal phospholipidosis on the transplant kidney biopsy. Patient did not have prior history or current symptoms or signs of Fabry disease. Serum α-galactosidase level was normal. The etiology was suspected to be due to chronic use of sertraline, a previously reported cause of drug-induced renal phospholipidosis. Sertraline was discontinued, and proteinuria declined with stabilization of kidney function at 6-months follow-up.

Hemidesmosomes modulate force generation via focal adhesions.

Hemidesmosomes are specialized cell-matrix adhesion structures that are associated with the keratin cytoskeleton. Although the adhesion function of hemidesmosomes has been extensively studied, their role in mechanosignaling and transduction remains largely unexplored. Here, we show that keratinocytes lacking hemidesmosomal integrin α6ß4 exhibit increased focal adhesion formation, cell spreading, and traction-force generation. Moreover, disruption of the interaction between α6ß4 and intermediate filaments or laminin-332 results in similar phenotypical changes. We further demonstrate that integrin α6ß4 regulates the activity of the mechanosensitive transcriptional regulator YAP through inhibition of Rho-ROCK-MLC- and FAK-PI3K-dependent signaling pathways. Additionally, increased tension caused by impaired hemidesmosome assembly leads to a redistribution of integrin αVß5 from clathrin lattices to focal adhesions. Our results reveal a novel role for hemidesmosomes as regulators of cellular mechanical forces and establish the existence of a mechanical coupling between adhesion complexes.

Quantifying cellular forces and biomechanical properties by correlative micropillar traction force and Brillouin microscopy.

Cells sense and respond to external physical forces and substrate rigidity by regulating their cell shape, internal cytoskeletal tension, and stiffness. Here we show that the combination of micropillar traction force and noncontact Brillouin microscopy provides access to cell-generated forces and intracellular mechanical properties at optical resolution. Actin-rich cytoplasmic domains of 3T3 fibroblasts showed significantly higher Brillouin shifts, indicating a potential increase in stiffness when adhering on fibronectin-coated glass compared to soft PDMS micropillars. Our findings demonstrate the complementarity of micropillar traction force and Brillouin microscopy to better understand the relation between cell force generation and the intracellular mechanical properties.

Role of c-MET Inhibitors in Overcoming Drug Resistance in Spheroid Models of Primary Human Pancreatic Cancer and Stellate Cells.

Pancreatic stellate cells (PSCs) are a key component of tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) and contribute to drug resistance. c-MET receptor tyrosine kinase activation plays an important role in tumorigenesis in different cancers including PDAC. In this study, effects of PSC conditioned medium (PCM) on c-MET phosphorylation (by immunocytochemistry enzyme-linked immunosorbent assay (ELISA)) and drug response (by sulforhodamine B assay) were investigated in five primary PDAC cells. In novel 3D-spheroid co-cultures of cyan fluorescence protein (CFP)-firefly luciferase (Fluc)-expressing primary human PDAC cells and green fluorescence protein (GFP)-expressing immortalized PSCs, PDAC cell growth and chemosensitivity were examined by luciferase assay, while spheroids' architecture was evaluated by confocal microscopy. The highest phospho-c-MET expression was detected in PDAC5 and its subclone sorted for "stage specific embryonic antigen-4" (PDAC5 (SSEA4)). PCM of cells pre-incubated with PDAC conditioned medium, containing increased hepatocyte growth factor (HGF) levels, made PDAC cells significantly more resistant to gemcitabine, but not to c-MET inhibitors. Hetero-spheroids containing both PSCs and PDAC5 (SSEA4) cells were more resistant to gemcitabine compared to PDAC5 (SSEA4) homo-spheroids. However, c-MET inhibitors (tivantinib, PHA-665752 and crizotinib) were equally effective in both spheroid models. Experiments with primary human PSCs confirmed the main findings. In conclusion, we developed spheroid models to evaluate PSC-PDAC reciprocal interaction, unraveling c-MET inhibition as an important therapeutic option against drug resistant PDAC.

Repetitive switching between DNA-binding modes enables target finding by the glucocorticoid receptor.

Transcription factor mobility is a determining factor in the regulation of gene expression. Here, we have studied the intranuclear dynamics of the glucocorticoid receptor (GR) by using fluorescence recovery after photobleaching and single-molecule microscopy. First, we have described the dynamic states in which the GR occurs. Second, we have analyzed the transitions between these states by using a continuous-time Markov chain model and functionally investigated these states by making specific mutations in the DNA-binding domain. This analysis revealed that the GR diffuses freely through the nucleus and, once it leaves this free diffusion state, most often enters a repetitive switching mode. In this mode it alternates between slow diffusion as a result of brief nonspecific DNA-binding events, and a state of stable binding to specific DNA target sites. This repetitive switching mechanism results in a compact search strategy that facilitates finding of DNA target sites by the GR.This article has an associated First Person interview with the first author of the paper.

The Activity of Kv 11.1 Potassium Channel Modulates F-Actin Organization During Cell Migration of Pancreatic Ductal Adenocarcinoma Cells.

Cell migration exerts a pivotal role in tumor progression, underlying cell invasion and metastatic spread. The cell migratory program requires f-actin re-organization, generally coordinated with the assembly of focal adhesions. Ion channels are emerging actors in regulating cell migration, through different mechanisms. We studied the role of the voltage dependent potassium channel KV 11.1 on cell migration of pancreatic ductal adenocarcinoma (PDAC) cells, focusing on its effects on f-actin organization and dynamics. Cells were cultured either on fibronectin (FN) or on a desmoplastic matrix (DM) with the addition of a conditioned medium produced by pancreatic stellate cells (PSC) maintained in hypoxia (Hypo-PSC-CM), to better mimic the PDAC microenvironment. KV11.1 was essential to maintain stress fibers in a less organized arrangement in cells cultured on FN. When PDAC cells were cultured on DM plus Hypo-PSC-CM, KV11.1 activity determined the organization of cortical f-actin into sparse and long filopodia, and allowed f-actin polymerization at a high speed. In both conditions, blocking KV11.1 impaired PDAC cell migration, and, on cells cultured onto FN, the effect was accompanied by a decrease of basal intracellular Ca2+ concentration. We conclude that KV11.1 is implicated in sustaining pro-metastatic signals in pancreatic cancer, through a reorganization of f-actin in stress fibers and a modulation of filopodia formation and dynamics.

The microbiome of pancreatic cancer: from molecular diagnostics to new therapeutic approaches to overcome chemoresistance caused by metabolic inactivation of gemcitabine.

Introduction: Pancreatic cancer is a complex disease, with an extremely poor response to chemotherapy. Emerging evidence indicates that the tumor microenvironment (TME) might play an important role in mediating chemoresistance. Areas covered: The evaluated study by Geller and collaborators describes several bacterial species within pancreatic tumor tissues and TME and investigated their roles in gemcitabine chemoresistance. Intratumor bacteria express the enzyme cytidine deaminase (CDD), whose long form (CDDL) was shown to metabolize gemcitabine into its inactive metabolite. CDDL is mostly expressed by Gammaproteobacteria and this was among the most common species in pancreatic cancer tissues. Interestingly, mouse models of colorectal cancer injected with bacterial CDDL displayed a reduced response to gemcitabine, but this resistance was neutralized by the antibiotic ciprofloxacin. Expert Commentary: The increased knowledge on the microbiome in pancreatic tissues, as well as its role in chemoresistance, will provide innovative prognostic and therapeutic strategies.

A mechanopharmacology approach to overcome chemoresistance in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a highly chemoresistant malignancy. This chemoresistant phenotype has been historically associated with genetic factors. Major biomedical research efforts were concentrated that resulted in the identification of subtypes characterized by specific genetic lesions and gene expression signatures that suggest important biological differences. However, to date, these distinct differences could not be exploited for therapeutic interventions. Apart from these genetic factors, desmoplasia and tumor microenvironment have been recognized as key contributors to PDAC chemoresistance. However, while several strategies targeting tumor-stroma have been explored including drugs against members of the Hedgehog family, they failed to meet the expectations in the clinical setting. These unsatisfactory clinical results suggest that, an important link between genetics and the influence of tumor microenvironment on PDAC chemoresistance remains to be elucidated. In this respect, mechanobiology is an emerging multidisciplinary field that encompasses cell and developmental biology as well as biophysics and bioengineering. Herein we provide a comprehensive overview of the key players in pancreatic cancer chemoresistance from the perspective of mechanobiology, and discuss novel experimental avenues such as elastic micropillar arrays that could provide fresh insights for the development of mechanobiology-targeted therapeutic approaches (know as mechanopharmacology) to overcome anticancer drug resistance in pancreatic cancer.

The conformational state of hERG1 channels determines integrin association, downstream signaling, and cancer progression.

Ion channels regulate cell proliferation, differentiation, and migration in normal and neoplastic cells through cell-cell and cell-extracellular matrix (ECM) transmembrane receptors called integrins. K+ flux through the human ether-à-go-go-related gene 1 (hERG1) channel shapes action potential firing in excitable cells such as cardiomyocytes. Its abundance is often aberrantly high in tumors, where it modulates integrin-mediated signaling. We found that hERG1 interacted with the ß1 integrin subunit at the plasma membrane of human cancer cells. This interaction was not detected in cardiomyocytes because of the presence of the hERG1 auxiliary subunit KCNE1 (potassium voltage-gated channel subfamily E regulatory subunit 1), which blocked the ß1 integrin-hERG1 interaction. Although open hERG1 channels did not interact as strongly with ß1 integrins as did closed channels, current flow through hERG1 channels was necessary to activate the integrin-dependent phosphorylation of Tyr397 in focal adhesion kinase (FAK) in both normal and cancer cells. In immunodeficient mice, proliferation was inhibited in breast cancer cells expressing forms of hERG1 with impaired K+ flow, whereas metastasis of breast cancer cells was reduced when the hERG1/ß1 integrin interaction was disrupted. We conclude that the interaction of ß1 integrins with hERG1 channels in cancer cells stimulated distinct signaling pathways that depended on the conformational state of hERG1 and affected different aspects of tumor progression.

SIRT1-SIRT3 Axis Regulates Cellular Response to Oxidative Stress and Etoposide.

Sirtuins are conserved NAD+ -dependent deacylases. SIRT1 is a nuclear and cytoplasmic sirtuin involved in the control of histones a transcription factors function. SIRT3 is a mitochondrial protein, which regulates mitochondrial function. Although, both SIRT1 and SIRT3 have been implicated in resistance to cellular stress, the link between these two sirtuins has not been studied so far. Here we aimed to unravel: i) the role of SIRT1-SIRT3 axis for cellular response to oxidative stress and DNA damage; ii) how mammalian cells modulate such SIRT1-SIRT3 axis and which mechanisms are involved. Therefore, we analyzed the response to different stress stimuli in WT or SIRT1-silenced cell lines. Our results demonstrate that SIRT1-silenced cells are more resistant to H2 O2 and etoposide treatment showing decreased ROS accumulation, γ-H2AX phosphorylation, caspase-3 activation and PARP cleavage. Interestingly, we observed that SIRT1-silenced cells show an increased SIRT3 expression. To explore such a connection, we carried out luciferase assays on SIRT3 promoter demonstrating that SIRT1-silencing increases SIRT3 promoter activity and that such an effect depends on the presence of SP1 and ZF5 recognition sequences on SIRT3 promoter. Afterwards, we performed co-immunoprecipitation assays demonstrating that SIRT1 binds and deacetylates the transcription inhibitor ZF5 and that there is a decreased interaction between SP1 and ZF5 in SIRT1-silenced cells. Therefore, we speculate that acetylated ZF5 cannot bind and sequester SP1 that is free, then, to increase SIRT3 transcription. In conclusion, we demonstrate that cells with low SIRT1 levels can maintain their resistance and survival by increasing SIRT3 expression. J. Cell. Physiol. 232: 1835-1844, 2017. © 2016 Wiley Periodicals, Inc.

New views and insights into intracellular trafficking of drug-delivery systems by fluorescence fluctuation spectroscopy.

Biomaterials in the nanometer size range can be engineered for site-specific delivery of drugs after injection into the blood circulation. However, translation of such nanomedicines from the bench to the bedside is still hindered by many extracellular and intracellular barriers. To realize the concept of targeted drug delivery with nanomedicines, research groups are studying intensively the extra- and intra-cellular mechanisms involved as a response to the physicochemical properties of the nanomedicines. In this review, we highlight the contributions of fluorescence fluctuations spectroscopy techniques to better understand, and in turn to bypass, the major hurdles to therapeutic delivery, focusing mostly on the intracellular dynamics of drug-delivery systems.

The role of cytoskeleton networks on lipid-mediated delivery of DNA.

BACKGROUND: Lipid-mediated delivery of DNA is hindered by extracellular and intracellular barriers that significantly reduce the transfection efficiency of synthetic nonviral vectors. RESULTS: In this study we investigated the role of the actin and microtubule networks on the uptake and cytoplasmic transport of multicomponent cationic liposome-DNA complexes in CHO-K1 live cells by means of confocal laser scanning microscopy and 3D single particle tracking. Treatment with actin (latrunculin B)- and microtubule-disrupting (nocodazole) reagents indicated that intracellular trafficking of complexes predominantly involves microtubule-dependent active transport. We found that the actin network has a major effect on the initial uptake of complexes, while the microtubule network is mainly responsible for the subsequent active transportation to the lysosomes. CONCLUSION: Collectively, a strategy to improve the efficiency of lipid gene vectors can be formulated. We could find a lipid formulation that allows the nanoparticles to avoid the microtubule pathway to lysosomes.

Intracellular trafficking of cationic liposome-DNA complexes in living cells.

Three-dimensional single-particle tracking (SPT) was used to calculate the mean square displacement (MSD) and the diffusion coefficients of multicomponent cationic liposome-DNA complexes (lipoplexes) in CHO-K1 living cells. In untreated (NT) control cells, we found that the intracellular lipoplex motion was either directed or Brownian with active transportation being definitely more frequent (more than 70%) than Brownian diffusion. The MSD analysis was supported by the calculation of the three-dimensional asphericity, A3, which was close to unity, denoting the preponderant occurrence of movement along a direction. To elucidate the role of the cytoskeleton structure in the lipoplex trafficking, cells were treated with cytoskeleton (actin microfilaments and microtubules) polymerization inhibitors (latrunculin B and nocodazole, respectively). When cells were treated with inhibitors, the lipoplex movement tended towards a random walk at the expense of directed motion. The disassembly of microtubules had a stronger effect on the reduction of directional movement than that of actin microfilaments. Relevance of the results for enhanced gene delivery is discussed.