Search

Background Autophagy plays an important role in tumour cell growth and survival and also promotes resistance to chemotherapy. Hence, autophagy has been targeted for cancer therapy. We previously reported that macrolide antibiotics including azithromycin (AZM) inhibit autophagy in various types of cancer cells in vitro. However, the underlying molecular mechanism for autophagy inhibition remains unclear. Here, we aimed to identify the molecular target of AZM for inhibiting autophagy. Methods We identified the AZM-binding proteins using AZM-conjugated magnetic nanobeads for high-throughput affinity purification. Autophagy inhibitory mechanism of AZM was analysed by confocal microscopic and transmission electron microscopic observation. The anti-tumour effect with autophagy inhibition by oral AZM administration was assessed in the xenografted mice model. Results We elucidated that keratin-18 (KRT18) and α/β-tubulin specifically bind to AZM. Treatment of the cells with AZM disrupts intracellular KRT18 dynamics, and KRT18 knockdown resulted in autophagy inhibition. Additionally, AZM treatment suppresses intracellular lysosomal trafficking along the microtubules for blocking autophagic flux. Oral AZM administration suppressed tumour growth while inhibiting autophagy in tumour tissue. Conclusions As drug-repurposing, our results indicate that AZM is a potent autophagy inhibitor for cancer treatment, which acts by directly interacting with cytoskeletal proteins and perturbing their dynamics.

PDF file - 1001KB, S1. Transformation of NIH3T3 cells expressing CD74-NRG1, EZR-ERBB4, or TRIM24-BRAF cDNAs. S2. Electropherogram of Sanger sequencing of cDNAs of novel fusion transcripts. S3. Schematic diagrams of genomic organization for NRG1, ERBB4, BRAF, and RET rearrangements. S4. Break-apart fluorescence in situ hybridization (FISH) for the NRG1 fusion. S5. Representative histological images obtained from fusion-positive IMAs.

PDF file - 115KB

Purpose: To identify druggable oncogenic fusions in invasive mucinous adenocarcinoma (IMA) of the lung, a malignant type of lung adenocarcinoma in which KRAS mutations frequently occur.Experimental Design: From an IMA cohort of 90 cases, consisting of 56 cases (62%) with KRAS mutations and 34 cases without (38%), we conducted whole-transcriptome sequencing of 32 IMAs, including 27 cases without KRAS mutations. We used the sequencing data to identify gene fusions, and then performed functional analyses of the fusion gene products.Results: We identified oncogenic fusions that occurred mutually exclusively with KRAS mutations: CD74-NRG1, SLC3A2-NRG1, EZR-ERBB4, TRIM24-BRAF, and KIAA1468-RET. NRG1 fusions were present in 17.6% (6/34) of KRAS-negative IMAs. The CD74-NRG1 fusion activated HER2:HER3 signaling, whereas the EZR-ERBB4 and TRIM24-BRAF fusions constitutively activated the ERBB4 and BRAF kinases, respectively. Signaling pathway activation and fusion-induced anchorage-independent growth/tumorigenicity of NIH3T3 cells expressing these fusions were suppressed by tyrosine kinase inhibitors approved for clinical use.Conclusions: Oncogenic fusions act as driver mutations in IMAs without KRAS mutations, and thus represent promising therapeutic targets for the treatment of such IMAs. Clin Cancer Res; 20(12); 3087–93. ©2014 AACR.

XLSX file - 22KB, Supplementary Table S1. RNA sequencing of 32 IMAs. Supplementary Table S2. PCR Primers used for RT-PCR screening.

PDF file - 168KB

PDF file - 41KB

Stable matching of neurotransmitters with their receptors is fundamental to synapse function, to achieve reliable and robust communication in neural circuits. Presynaptic neurotransmitters regulate selection of postsynaptic transmitter receptors. However, whether postsynaptic receptors regulate selection of presynaptic transmitters is unknown. Here we show that blockade of postsynaptic acetylcholine receptors at the neuromuscular junction leads to loss of the cholinergic phenotype in motor neurons and stabilization of an earlier, developmentally transient glutamatergic phenotype. Exogenous postsynaptic expression of GABAAreceptors leads to the stabilization of an earlier, developmentally transient GABAergic motor neuron phenotype. Both acetylcholine receptors and GABA receptors are linked to presynaptic neurons through trans-synaptic bridges. Knock-down of different components of these trans-synaptic bridges prevents stabilization of the cholinergic and GABAergic phenotypes. We conclude that this bidirectional communication enforces a match between transmitter and receptor and ensures the fidelity of synaptic transmission. Our findings suggest a role of dysfunctional transmitter receptors in neurological disorders that involve the loss of the presynaptic transmitter.

The proton pump inhibitor lansoprazole (LPZ) inhibits the growth of several cancer cell lines, including A549 and CAL 27. We previously reported that macrolide antibiotics such as azithromycin (AZM) and clarithromycin (CAM) potently inhibit autophagic flux and that combining AZM or CAM with the epidermal growth factor receptor inhibitors enhanced their antitumor effect against various cancer cells. In the present study, we conducted the combination treatment with LPZ and macrolide antibiotics against A549 and CAL 27 cells and evaluated cytotoxicity and morphological changes using cell proliferation and viability assays, flow cytometric analysis, immunoblotting, and morphological assessment. Combination therapy with LPZ and AZM greatly enhanced LPZ‑induced cell death, whereas treatment with AZM alone exhibited negligible cytotoxicity. The observed cytotoxic effect was not mediated through apoptosis or necroptosis. Transmission electron microscopy of A549 cells treated with the LPZ + AZM combination revealed morphological changes associated with necrosis and accumulated autolysosomes with undigested contents. Furthermore, the A549 cell line with ATG5 knockout exhibited complete inhibition of autophagosome formation, which did not affect LPZ + AZM treatment‑induced cytotoxicity, thus excluding the involvement of autophagy‑dependent cell death in LPZ + AZM treatment‑induced cell death. A549 cells treated with LPZ + AZM combination therapy retained the endosomal Alexa‑dextran for extended duration as compared to untreated control cells, thus indicating impairment of lysosomal digestion. Notably, lysosomal galectin‑3 puncta expression induced due to lysosomal membrane permeabilization was increased in cells treated with LPZ + AZM combination as compared to the treatment by either agent alone. Collectively, the present results revealed AZM‑induced autolysosome accumulation, potentiated LPZ‑mediated necrosis, and lysosomal membrane permeabilization, thus suggesting the potential clinical application of LPZ + AZM combination therapy for cancer treatment.

Sequestosome 1 (p62) is a multifunctional adapter protein involved in various physiological functions, such as selective autophagy and oxidative stress response. Hence, aberrant expression and defective regulation of p62 are thought to lead to the onset of various diseases, including cancer. The expression of p62 has been shown to be increased in breast cancer tissues, and is correlated with a poor prognosis. However, the role of p62 in the breast cancer pathophysiology is still unclear. Here, we aimed to analyze the effect of changes in p62 expression on breast cancer cell lines. DNA microarray analysis revealed that the expression of progesterone receptor (PR), which is one of the indices for the classification of breast cancer subtypes, was markedly suppressed by forced expression of p62. The protein expression of PR was also decreased by forced expression of p62, but increased by knockdown of p62. Moreover, we found that p62 knockdown induced the protein expression of argonaute 2 (AGO2). Luciferase reporter assay results showed that the gene expression of PR was promoted by AGO2. Furthermore, results revealed that overexpression of AGO2 partially rescued the decrease in PR expression induced by forced expression of p62. Collectively, our findings indicated that p62 accumulation suppressed the expression of AGO2, which in turn decreased the expression of PR, suggesting that p62 may serve as a marker of aggressive breast cancer and poor prognosis. Moreover, the p62-AGO2-PR axis was identified as a crucial signaling cascade in breast cancer progression.

Aims/Introduction Taste receptors, T1rs and T2rs, and the taste‐selective G‐protein, α‐gustducin, are expressed outside the taste‐sensing system, such as enteroendocrine L cells. Here, we examined whether α‐gustducin also affects nutrition sensing and insulin secretion by pancreatic β‐cells. Materials and Methods The expression of α‐gustducin and taste receptors was evaluated in β‐cell lines, and in rat and mouse islets either by quantitative polymerase chain reaction or fluorescence immunostaining. The effects of α‐gustducin knockdown on insulin secretion and on cyclic adenosine monophosphate and intracellular Ca2+ levels in rat INS‐1 cells were estimated. Sucralose (taste receptor agonist)‐induced insulin secretion was investigated in INS‐1 cells with α‐gustducin suppression and in islets from mouse disease models. Results The expression of Tas1r3 and α‐gustducin was confirmed in β‐cell lines and pancreatic islets. Basal levels of cyclic adenosine monophosphate, intracellular calcium and insulin secretion were significantly enhanced with α‐gustducin knockdown in INS‐1 cells. The expression of α‐gustducin was decreased in high‐fat diet‐fed mice and in diabetic db/db mice. Sucralose‐induced insulin secretion was not attenuated in INS‐1 cells with α‐gustducin knockdown or in mouse islets with decreased expression of α‐gustducin. Conclusions α‐Gustducin is involved in the regulation of cyclic adenosine monophosphate, intracellular calcium levels and insulin secretion in pancreatic β‐cells in a manner independent of taste receptor signaling. α‐Gustducin might play a novel role in β‐cell physiology and the development of type 2 diabetes., The expression of α‐gustducin in pancreatic islets decreases with high‐fat diet feeding and obesity.

Sensory information in the brain is organized into spatial representations, including retinotopic, somatotopic, and tonotopic maps, as well as ocular dominance columns. The spatial representation of sensory inputs is thought to be a fundamental organizational principle that is important for information processing. Topographic maps are plastic throughout an animal's life, reflecting changes in development and aging of brain circuitry, changes in the periphery and sensory input, and changes in circuitry, for instance in response to experience and learning. Here, we review mechanisms underlying the role of activity in the development, stability and plasticity of topographic maps, including recent work suggesting that the spatial information in the visual field, and the resulting spatiotemporal patterns of activity, provide instructive cues that organize visual projections.

The autophagy‑lysosome system allows cells to adapt to environmental changes by regulating the degradation and recycling of cellular components, and to maintain homeostasis by removing aggregated proteins and defective organelles. Cyclin G‑associated kinase (GAK) is involved in the regulation of clathrin‑dependent endocytosis and cell cycle progression. In addition, a single nucleotide polymorphism at the GAK locus has been reported as a risk factor for Parkinson's disease. However, the roles of GAK in the autophagy‑lysosome system are not completely understood, thus the present study aimed to clarify this. In the present study, under genetic disruption or chemical inhibition of GAK, analyzing autophagic flux and observing morphological changes of autophagosomes and autolysosomes revealed that GAK controlled lysosomal dynamics via actomyosin regulation, resulting in a steady progression of autophagy. GAK knockout (KO) in A549 cells impaired autophagosome‑lysosome fusion and autophagic lysosome reformation, which resulted in the accumulation of enlarged autophagosomes and autolysosomes during prolonged starvation. The stagnation of autophagic flux accompanied by these phenomena was also observed with the addition of a GAK inhibitor. Furthermore, the addition of Rho‑associated protein kinase (ROCK) inhibitor or ROCK1 knockdown mitigated GAK KO‑mediated effects. The results suggested a vital role of GAK in controlling lysosomal dynamics via maintaining lysosomal homeostasis during autophagy.

Pancreatic cancer is one of the leading causes of cancer-related mortality and has the lowest 5-year survival rate. Therefore, novel strategies are urgently required to treat pancreatic cancer. Pancreatic ductal adenocarcinoma (PDAC) cells rely on enhanced lysosomal function for survival and proliferation to facilitate the degradation of contents accumulated via autophagy and macropinocytosis. Previously, we have reported that the combination of epidermal growth factor receptor/HER2 inhibitor lapatinib and sphingosine analog fingolimod (FTY720) confers a significant cytostatic effect in lung cancer cells. In the present study, the combined effects of these drugs on PDAC cell lines, BxPC-3, KP-4, PANC-1 and MIA PaCa-2, were examined. It was observed that FTY720 enhanced the lapatinib-induced cytotoxic effect and caused non-canonical and lysosome-dependent death in PDAC cells. Lapatinib and FTY720 induced lysosomal swelling and inhibited lysosomal acidification. Combination treatment with lapatinib and FTY720 increased lysosomal membrane permeability, induced mitochondrial depolarization, induced endoplasmic reticulum stress and disturbed intracellular calcium homeostasis. Additionally, the cytotoxic effect of lapatinib was enhanced by hydroxychloroquine or the CDK4/6 inhibitor abemaciclib, both of which induce lysosomal dysfunction. Collectively, these results indicated that the lysosome-targeted drug combination induces multiple organelle dysfunction and exerts a marked cytotoxic effect in PDAC cells.

Following surgery and chemoradiation, ~50% of patients with locally advanced head and neck tumors experience relapse within the first two years, with a poor prognosis. Therefore, a novel therapeutic approach is required. The aim of the present study was to investigate the effect of combination treatment with the proteasome inhibitor bortezomib (BTZ), and ricolinostat (RCS), a specific inhibitor of histone deacetylase 6 (HDAC6), on CAL27 and Detroit562 head and neck cancer cells. BTZ and RCS exhibited cytotoxicity in a dose- and time-dependent manner. Simultaneous treatment with BTZ and RCS resulted in the synergistic enhancement of non-apoptotic cell death and autophagy. The receptor-interacting serine/threonine-protein kinase 1 (RIPK1) inhibitor, necrostatin, but not the autophagy inhibitor, 3-methyladenine, attenuated the cytotoxicity of combined BTZ and RCS treatment. Thus, necroptosis [type-III programmed cell death (PCD)], but not autophagic cell death (type-II PCD), appeared to contribute to the pronounced cytotoxicity. However, no phosphorylation of RIPK1 or mixed lineage kinase domain-like protein was detectable in response to BTZ or RCS. Furthermore, RCS induced α-tubulin acetylation and inhibited BTZ-induced aggresome formation along with endoplasmic reticulum stress loading. Combined treatment with BTZ and RCS enhanced the production of reactive oxygen species (ROS). The ROS scavenger, N-acetyl cysteine, abrogated the increase in cytotoxicity. These results suggest the potential therapeutic value of the dual targeting of the proteasome and HDCA6 for head and neck cancers through the induction of necroptosis-like cell death along with ROS generation.

Partial hepatectomy (PH) induces estradiol production, and then hepatocyte proliferation. Estradiol may play a role in triggering hepatocyte proliferation after PH. In this study, estradiol was injected to the Estrogen Receptor alpha (ERα) or ERβ KO mice. No increased hepatocyte proliferation was observed in ERα KO mice, indicates that ERα is involved in estradiol-induced hepatocyte proliferation. The ERα and ERβ KO mice are sterile, hence it is impossible to study ERα and ERβ function during pregnancy when the estrogen levels are highest. Using conditional mutagenesis technique, we made ERα hepatocyte KO mice, which are fertile. We used these mice for analyzing the hepatocyte ERα function during pregnancy. However, in the control mice, the maternal hepatocyte was proliferated higher in late pregnancy, but no pregnancy-induced hepatocyte proliferation was observed in KO mice. Hence, we conclude that the maternal hepatocyte ERα is involved in estradiol-induced hepatocyte proliferation in late pregnancy.

Cancer cells use autophagy for growth, survival, and cytoprotection from chemotherapy. Therefore, autophagy inhibitors appear to be good candidates for cancer treatment. Our group previously reported that macrolide antibiotics, especially azithromycin (AZM), have potent autophagy inhibitory effects, and combination treatment with tyrosine kinase inhibitors or proteasome inhibitors enhances their anti–cancer activity. In this study, we evaluated the effect of combination therapy with DNA‐damaging drugs and AZM in non–small‐cell lung cancer (NSCLC) cells. We found that the cytotoxic activities of DNA‐damaging drugs, such as doxorubicin (DOX), etoposide, and carboplatin, were enhanced in the presence of AZM in NSCLC cell lines, whereas AZM alone exhibited almost no cytotoxicity. This enhanced cell death was dependent on wild‐type‐p53 status and autophagosome‐forming ability because TP53 knockout (KO) and ATG5‐KO cells attenuated AZM‐enhanced cytotoxicity. DOX treatment upregulated lysosomal biogenesis by activating TFEB and led to lysosomal membrane damage as assessed by galectin 3 puncta assay and cytoplasmic leakage of lysosomal enzymes. In contrast, AZM treatment blocked autophagy, which resulted in the accumulation of lysosomes/autolysosomes. Thus, the effects of DOX and AZM were integrated into the marked increase in damaged lysosomes/autolysosomes, leading to prominent lysosomal membrane permeabilization (LMP) for apoptosis induction. Our data suggest that concomitant treatment with DNA‐damaging drugs and AZM is a promising strategy for NSCLC treatment via pronounced LMP induction., In this paper, we showed that coadministration of DNA‐damaging drugs and azithromycin (AZM), a macrolide antibiotic, enhanced cytotoxicity due to increased lysosomal membrane permeabilization (LMP). Because DNA‐damaging drugs induce LMP, cancer cells activate autophagy to remove damaged lysosomes by lysophagy. However, AZM inhibited lysophagy, as well as autophagy, resulting in the prominent cytoplasmic accumulation of damaged lysosomes for induction of LMP‐mediated apoptosis in non–small‐cell lung cancer cells.

BRCA1 is a well-studied tumor suppressor involved in the homologous repair of DNA damage, whereas PINK1, a mitochondrial serine/threonine kinase, is known to be involved in mitochondrial quality control. Genetic mutations of PINK1 and Parkin cause autosomal recessive early-onset Parkinson’s disease. We found that in breast cancer cells, the mitochondrial targeting reagents, which all induce mitochondrial depolarization along with PINK1 upregulation, induced proteasomal BRCA1 degradation. This BRCA1 degradation was dependent on PINK1, and BRCA1 downregulation upon mitochondrial damage caused DNA double-strand breaks. BRCA1 degradation was mediated through the direct interaction with the E3 ligase Parkin. Strikingly, BRCA1 and PINK1/Parkin expression were inversely correlated in cancerous mammary glands from breast cancer patients. BRCA1 knockdown repressed cancer cell growth, and high BRCA1 expression predicted poor relapse-free survival in breast cancer patients. These observations indicate a novel mechanism by which mitochondrial damage is transmitted to the nucleus, leading to BRCA1 degradation.

A common pathological hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis, is cytoplasmic mislocalization and aggregation of nuclear RNA-binding protein TDP-43. Perry disease, which displays inherited atypical parkinsonism, is a type of TDP-43 proteinopathy. The causative gene DCTN1 encodes the largest subunit of the dynactin complex. Dynactin associates with the microtubule-based motor cytoplasmic dynein and is required for dynein-mediated long-distance retrograde transport. Perry disease-linked missense mutations (e.g., p.G71A) reside within the CAP-Gly domain and impair the microtubule-binding abilities of DCTN1. However, molecular mechanisms by which such DCTN1 mutations cause TDP-43 proteinopathy remain unclear. We found that DCTN1 bound to TDP-43. Biochemical analysis using a panel of truncated mutants revealed that the DCTN1 CAP-Gly-basic supradomain, dynactin domain, and C-terminal region interacted with TDP-43, preferentially through its C-terminal region. Remarkably, the p.G71A mutation affected the TDP-43-interacting ability of DCTN1. Overexpression of DCTN1G71A, the dynactin-domain fragment, or C-terminal fragment, but not the CAP-Gly-basic fragment, induced cytoplasmic mislocalization and aggregation of TDP-43, suggesting functional modularity among TDP-43-interacting domains of DCTN1. We thus identified DCTN1 as a new player in TDP-43 cytoplasmic-nuclear transport, and showed that dysregulation of DCTN1-TDP-43 interactions triggers mislocalization and aggregation of TDP-43, thus providing insights into the pathological mechanisms of Perry disease and other TDP-43 proteinopathies.

The Xenopus tadpole visual system exhibits an extraordinary extent of developmental and visual experience-dependent plasticity, establishing sophisticated neuronal response properties that guide essential survival behaviors. The external development and access to the developing visual circuit of Xenopus tadpoles make them an excellent experimental system in which to elucidate plastic changes in neuronal properties and their capacity to encode information about the visual scene. The temporal structure of neural activity encodes a significant amount of information, access to which requires recording methods with high temporal resolution. Conversely, elucidating changes in the temporal structure of neural activity requires recording over extended periods. It is challenging to maintain patch clamp recordings over extended periods and Ca(++) imaging has limited temporal resolution. Extracellular recordings have been used in other systems for extended recording, however spike amplitudes in the developing Xenopus visual circuit are not large enough to be captured by distant electrodes. Here we describe a juxtacellular tetrode recording method for continuous long-term recordings from neurons in intact tadpoles, which can also be exposed to diverse visual stimulation protocols. Electrode position in the tectum is stabilized by the large contact area in the tissue. Contamination of the signal from neighboring neurons is minimized by the tight contact between the glass capillaries and the dense arrangement of neurons in the tectum. This recording method enables analysis of developmental and visual experience-dependent plastic changes in neuronal response properties at higher temporal resolution and over longer periods than current methods.

Tyrosine kinase inhibitors (TKIs) induce autophagy in many types of cancer cells. We previously reported that gefitinib (GEF) and imatinib (IMA) induce autophagy in epidermal growth factor receptor (EGFR) knock-out A549 and non-BCR-ABL-expressing leukemia cell lines, respectively. This evidence suggests that TKI-induced autophagy is independent of the original target molecules. The present study compared the autophagy-inducing abilities of various TKIs, regardless of their targets, by quantitative autophagy flux assay. We established stable clones expressing the GFP-LC3-mCherry-LC3ΔG plasmid in A549, PC-9, and CAL 27 cell lines and assessed autophagy inducibility by monitoring the fluorescent ratios of GFP-LC3 to mCherry-LC3ΔG using an IncuCyte live cell imaging system during exposure to TKIs viz; GEF, osimertinib (OSI), lapatinib (LAP), lenvatinib (LEN), sorafenib (SOR), IMA, dasatinib (DAS), and tivantinib (TIV). Among these TKIs, DAS, GEF, and SOR exhibited prominent autophagy induction in A549 and PC-9 cells. In CAL 27 cells, IMA, SOR, and LEN, but not GEF, TIV, or OSI, exhibited autophagy induction. In the presence of azithromycin (AZM), which showed an inhibitory effect on autophagy flux, TKIs with prominent autophagy inducibility exhibited enhanced cytotoxicity via non-apoptotic cell death relative to effects of TKI alone. Therefore, autophagy inducibility of TKIs differed in the context of cancer cells. However, once induced, they appeared to have cytoprotective functions. Thus, blocking TKI-induced autophagy with AZM may improve the therapeutic effect of TKIs in cancer cells., Highlights • Tyrosine kinase inhibitors (TKIs) induce autophagy regardless of their main target. • This autophagy inducibility is partially determined in the context of cancer cells. • Azithromycin (AZM) has an inhibitory effect on autophagy. • Blocking TKI-induced autophagy with AZM enhances their cytotoxicity in cancer cells. • This enhanced cytotoxicity is mediated through non-apoptotic cell death.

Epigenetic regulation of gene expression has been implicated in the pathogenesis of obesity and type 2 diabetes. However, detailed information, such as key transcription factors in pancreatic beta cells that mediate environmental effects, is not yet available. To analyse genome-wide cis-regulatory profiles and transcriptome of pancreatic islets derived from a diet-induced obesity (DIO) mouse model, we conducted chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) of histone H3 lysine 27 acetylation (histone H3K27ac) and high-throughput RNA sequencing. Transcription factor-binding motifs enriched in differential H3K27ac regions were examined by de novo motif analysis. For the predicted transcription factors, loss of function experiments were performed by transfecting specific siRNA in INS-1, a rat beta cell line, with and without palmitate treatment. Epigenomic and transcriptional changes of possible target genes were evaluated by ChIP and quantitative RT-PCR. After long-term feeding with a high-fat diet, C57BL/6J mice were obese and mildly glucose intolerant. Among 39,350 islet cis-regulatory regions, 13,369 and 4610 elements showed increase and decrease in ChIP-Seq signals, respectively, significantly associated with global change in gene expression. Remarkably, increased H3K27ac showed a distinctive genomic localisation, mainly in the proximal-promoter regions, revealing enriched elements for nuclear respiratory factor 1 (NRF1), GA repeat binding protein α (GABPA) and myocyte enhancer factor 2A (MEF2A) by de novo motif analysis, whereas decreased H3K27ac was enriched for v-maf musculoaponeurotic fibrosarcoma oncogene family protein K (MAFK), a known negative regulator of beta cells. By siRNA-mediated knockdown of NRF1, GABPA or MEF2A we found that INS-1 cells exhibited downregulation of fatty acid β-oxidation genes in parallel with decrease in the associated H3K27ac. Furthermore, in line with the epigenome in DIO mice, palmitate treatment caused increase in H3K27ac and induction of β-oxidation genes; these responses were blunted when NRF1, GABPA or MEF2A were suppressed. These results suggest novel roles for DNA-binding proteins and fatty acid signalling in obesity-induced epigenomic regulation of beta cell function. The next-generation sequencing data in the present study were deposited at ArrayExpress. RNA-Seq: Dataset name: ERR2538129 (Control), ERR2538130 (Diet-induced obesity) Repository name and number: E-MTAB-6718 - RNA-Seq of pancreatic islets derived from mice fed a long-term high-fat diet against chow-fed controls. ChIP-Seq: Dataset name: ERR2538131 (Control), ERR2538132 (Diet-induced obesity) Repository name and number: E-MTAB-6719 - H3K27ac ChIP-Seq of pancreatic islets derived from mice fed a long-term high-fat diet (HFD) against chow-fed controls.

The D-type cyclin (CCND)-cyclin-dependent kinase 4/6 (CDK4/6) complex has been implicated in multiple myeloma development. We investigated the biological activity of CDK4/6 inhibitor abemaciclib on cell growth and survival in three myeloma cell lines, KMS-12-PE, RPMI 8226, and IM-9. Abemaciclib inhibited myeloma cell growth in a dose-dependent manner in all cell lines, with significant differences seen at a concentration of 320 nM. Treatment with 1 μM abemaciclib increased the fraction of cells in the G0/G1 phase and decreased the fraction in the S-G2/M phases. Further, treatment with abemaciclib at a concentration of 3.2 μM or more showed apparent cytocidal activity accompanied with cytoplasmic vacuolization against myeloma cells. Importantly, abemaciclib induced autophagy in a dose-dependent manner in all three cell lines. These results indicate that the CCND-CDK4/6 complex is closely tied to myeloma cell growth and survival.

In the cell cycle, the G1/S transition is controlled by the cyclin‐dependent kinase (CDK) 4/6‐cyclin D complex. Constitutive activation of CDK4/6 dysregulates G1/S transition, leading to oncogenic transformation. We found that 3 CDK4/6 inhibitors, abemaciclib, ribociclib, and palbociclib, exerted a cytocidal effect as well as a cytostatic effect at the G1 phase in cancer cell lines, including A549 human non–small cell lung cancer cells. Among these inhibitors, abemaciclib exhibited the most potent cytotoxic effect. The cell‐death phenotype induced by abemaciclib, which entailed formation of multiple cytoplasmic vacuoles, was not consistent with apoptosis or necroptosis. Abemaciclib blocked autophagic flux, resulting in accumulation of autophagosomes, however vacuole formation and cell death induced by abemaciclib were independent of autophagy. In addition, methuosis, a cell‐death phenotype characterized by vacuole formation induced by excessive macropinocytosis, was excluded because the vacuoles did not incorporate fluorescent dextran. Of note, both formation of vacuoles and induction of cell death in response to abemaciclib were inhibited by vacuolar‐type ATPase (V‐ATPase) inhibitors such as bafilomycin A1 and concanamycin A. Live‐cell imaging revealed that the abemaciclib‐induced vacuoles were derived from lysosomes that expanded following acidification. Transmission electron microscopy revealed that these vacuoles contained undigested debris and remnants of organelles. Cycloheximide chase assay revealed that lysosomal turnover was blocked by abemaciclib. Furthermore, mTORC1 inhibition along with partial lysosomal membrane permeabilization occurred after abemaciclib treatment. Together, these results indicate that, in cancer cells, abemaciclib induces a unique form of cell death accompanied by swollen and dysfunctional lysosomes., We found that abemaciclib, a CDK4/6 inhibitor, exerted a potent cytocidal effect in cancer cell lines with an atypical cell death phenotype, which entailed formation of multiple cytoplasmic vacuoles, and both the formation of vacuoles and the induction of cell death were inhibited by V‐ATPase inhibitors. Precise live‐cell imaging and transmission electron microscopy revealed that these vacuoles were derived from lysosomes that expanded following acidification and contained undigested debris and remnants of organelles. Thus we here propose a unique form of cell death accompanied by swollen and dysfunctional lysosomes by abemaciclib treatment.

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase and mutations in this gene are major drivers of lung cancer development. EGFR tyrosine kinase inhibitors (TKIs) are standard first‑line therapies for patients with advanced non‑small cell lung cancer (NSCLC) with activating EGFR mutations, but are not effective in patients with wild‑type EGFR. In the present study, the cytotoxic effects of various TKIs against EGFR were investigated in wild‑type NSCLC cells as single treatments or in combination with Fingolimod (FTY720), which has been approved for treating multiple sclerosis and has cytotoxic effects against several tumor cell lines. It was found that the combined treatment with TKIs lapatinib (Lap) or sorafenib (Sor) and FTY720 synergistically suppressed the viability of the NSCLC cell lines A549 and H596. Additionally, FTY720 inhibited lysosomal acidification and suppressed autophagy flux. Immunoblotting and reverse transcription‑quantitative polymerase chain reaction showed that FTY720 combined with Lap or Sor, enhanced endoplasmic reticulum (ER) stress loading and cell cycle arrest in A549 cells. The enhancement of ER stress loading and cell cycle arrest induced by combined treatment with Lap or Sor and FTY720, which was associated with the cytotoxicity induced by the combination of these drugs. These findings suggested that FTY720 improved TKI therapy in NSCLC patients with wild‑type EGFR, by sensitizing NSCLC cells to TKIs.

Summary: Studies on visual experience-dependent plasticity can benefit tremendously from experimental protocols in which sensory stimulation is precisely controlled for extended periods over which neuronal, circuit, and behavioral plasticity occurs. Small vertebrates, such as Xenopus tadpoles and zebrafish, are excellent systems for studying brain plasticity. Here, we present a detailed protocol to perform controlled visual stimulation for extended time periods. These methods have been used to study structural plasticity induced by temporally controlled visual stimulation in Xenopus tadpoles.For further details on the use and execution of this protocol, please refer to Hiramoto and Cline (2014, 2020).

The ubiquitin-proteasome and autophagy-lysosome pathways are two major self-digestive systems for cellular proteins. Ubiquitinated misfolded proteins are degraded mostly by proteasome. However, when ubiquitinated proteins accumulate beyond the capacity of proteasome clearance, they are transported to the microtubule-organizing center (MTOC) along the microtubules to form aggresomes, and subsequently some of them are degraded by the autophagy-lysosome system. We previously reported that macrolide antibiotics such as azithromycin and clarithromycin block autophagy flux, and that concomitant treatment with the proteasome inhibitor bortezomib (BZ) and macrolide enhances endoplasmic reticulum (ER) stress-mediated apoptosis in breast cancer cells. As ubiquitinated proteins are concentrated at the aggresome upon proteasome failure, we focused on the microtubule as the scaffold of this transport pathway for aggresome formation. Treatment of metastatic breast cancer cell lines (e.g., MDA-MB-231 cells) with BZ resulted in induction of aggresomes, which immunocytochemistry detected as a distinctive eyeball-shaped vimentin-positive inclusion body that formed in a perinuclear lesion, and that electron microscopy detected as a sphere of fibrous structure with some dense amorphous deposit. Vinorelbine (VNR), which inhibits microtubule polymerization, more effectively suppressed BZ-induced aggresome formation than paclitaxel (PTX), which stabilizes microtubules. Combined treatment using BZ and VNR, but not PTX, enhanced the cytotoxic effect and apoptosis induction along with pronounced ER stress loading such as upregulation of GRP78 and CHOP/GADD153. The addition of azithromycin to block autophagy flux in the BZ plus VNR-containing cell culture further enhanced the cytotoxicity. These data suggest that suppression of BZ-induced aggresome formation using an inhibitory drug such as VNR for microtubule polymerization is a novel strategy for meta-static breast cancer therapy.

The tumor suppressor BRCA1 protein has been implicated in hereditary breast and ovarian cancer syndrome when its gene is mutated. Among the many functions of BRCA1 including DNA repair, transcriptional regulation, cell cycle checkpoint, apoptosis, chromatin remodeling, and centrosome replication, DNA double-strand breaks repair by homologous recombination (HR) is one of the most important. BRCA1-associated tumors increase DNA instability and become sensitive to the Poly (ADP-ribose) polymerase (PARP) inhibitor. It is well known that PTEN-induced kinase 1 (PINK1) and Parkin are involved in mitochondrial quality control and a variety of mutations in these genes causes early-onset Parkinson's disease. In the present study, we report a novel degradation mechanism for BRCA1 protein in response to mitochondrial damage. While investigating the role of BRCA1 in mitophagy using a breast cancer cell line, we found that BRCA1 protein is rapidly degraded by the mitochondrial targeting reagents, which induce mitochondrial depolarization. The degradation was mediated by the ubiquitin-proteasome system through the direct interaction with the E3 ligase Parkin upon PINK1 upregulation in response to the mitochondrial damage. Moreover, BRCA1 knockdown repressed cancer cell growth. Immunostaining the specimens from breast cancer patients revealed higher BRCA1 and lower PINK1/Parkin expression in their mammary glands. This result correlates with the analysis using the mRNA expression data set from TCGA database. Additionally, BRCA1 expression inversely correlated with PINK1/Parkin expression in the case of relapse-free survival in breast cancer patients. Thus, these findings demonstrated the unanticipated physiological functions of BRCA1 for maintaining cancer cell growth. Overall, our study shows that: 1) Degradation of BRCA1 due to PINK1-Parkin activity through the ubiquitin-proteasome system occurs in response to mitochondrial damage. 2) BRCA1 promotes the growth of breast cancer cells. 3) Immunostaining of patient specimens and cancer genome data set analysis revealed higher BRCA1 expression with lower PINK1/Parkin expression was observed in the cancerous mammary glands. Moreover, recent reports have suggested that BRCA1 is involved in the pathogenesis of Alzheimer's disease. Thus, transmission of mitochondrial damage to the nucleus causing nuclear DNA double-strand breaks via the PINK1-Parkin-BRCA1 axis may not only be seen in the field of oncology, but also in various other fields including neurodegenerative diseases. Citation Format: Kana Miyahara, Naoharu Takano, Yumiko Yamada, Hiromi Kazama, Mayumi Tokuhisa, Hirotsugu Hino, Koji Fujita, Edward Barroga, Masaki Hiramoto, Hiroshi Handa, Masahiko Kuroda, Takashi Ishikawa, Keisuke Miyazawa. BRCA1 degradation in response to mitochondrial damage in breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 296.

Autophagy is a self-digestive cellular mechanism that enables survival under nutrient depleted conditions. In normal cells, autophagy has been known to prevent tumorigenesis by removal of genotoxicity-causing mitochondria, as well as transformed cells. In contrast, once cancer has been established, autophagy appears to play a cytoprotective role to adapt to the insufficiently vascularized hypoxic and low nutrient environment; and also works for metastasis and therapeutic resistance. Therefore, autophagic inhibitors could be suitable candidates for cancer therapeutics. However, the only two autophagic inhibitors clinically available at present are chloroquine (CQ) and hydroxychloroquine (HCQ). We previously reported that azithromycin (AZM), a macrolide antibiotic, has an inhibitory effect on autophagy and that combined administration of AZM with various anti-cancer drugs, such as a tyrosine kinase inhibitor and a proteasomal inhibitor, appears to enhance the anti-cancer effect. With an aim to explore the clinical application of AZM for cancer therapy, we attempted to identify the molecular target of AZM for autophagy inhibition. High throughput affinity purification using AZM-conjugated magnetic nano-beads was used to identify several candidates for the AZM-binding protein involved in autophagy inhibition from human NSCLC-derived A549 cell lysates. Knockdown of one of the candidate genes resulted in a prominent accumulation of autophagosomes and lysosomes, leading to autophagy inhibition. Fluorescence microscopic observation of A549 cells expressing GFP-LC3 and LAMP1-mCherry markers suggested that AZM suppressed the fusion between autophagosomes and lysosomes. Comparison of autophagy inhibitory capacity using the GFP-LC3/RFP-LC3ΔG fluorescent reporter system revealed an almost equivalent autophagic inhibition between AZM and HCQ but less cytotoxicity by itself at the same concentrations. Suppression of lysosomal acidification was not observed upon AZM administration as same as in the case of HCQ administration. However, unlike HCQ, AZM did not increase the levels of HIF-1α and phospho-ERK1/2. This indicates the existence of different autophagy inhibitory mechanisms for AZM and HCQ. In the A549 murine xenograft model, daily oral administration of AZM (100 μg/g/day) significantly suppressed tumor cell growth. These results from the present study encourage the use of AZM as an autophagy inhibitor for cancer therapy. Citation Format: Naoharu Takano, Yumiko Yamada, Mayumi Tokuhisa, Hirotsugu Hino, Masaki Hiramoto, Keisuke Miyazawa. Identification of the molecular target of azithromycin as an autophagy inhibitor and its potential application in cancer therapeutics [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1227.

Summary Spatial representations of the sensory world are important for brain function. Timing is an essential component of sensory information. Many brain circuits transform the temporal sequence of input activity into spatial maps; however, the mechanisms underlying this transformation are unclear. Different N-methyl-D-aspartate receptor (NMDAR) response magnitudes result in synaptic potentiation or depression. We asked whether NMDAR response magnitude also affects the transformation of temporal information into directional spatial maps. We quantified retinotectal axon branch dynamics in Xenopus optic tectum in response to temporal sequences of visual stimulation. Reducing NMDAR responses by 50% inverts the spatial distribution of branch dynamics along the rostrocaudal axis in response to temporal patterns of input, suggesting that the magnitude of NMDAR signaling encodes the temporal sequence of inputs and translates the temporal code into a directional spatial map using structural plasticity-based branch dynamics. We discuss how this NMDAR-dependent decoding mechanism retrieves spatial information from sequential afferent activity., Graphical Abstract, Highlights • NMDAR response magnitude encodes the temporal sequence of inputs • NMDAR mechanism decodes spatial information from sequential input activity • NMDAR attenuation inverts the temporal to spatial transformation • NMDAR activity alters the spatial distribution of dynamic and stable branches, Developmental Neuroscience; Sensory Neuroscience ; Techniques in Neuroscience

Excess stress caused by accumulation of misfolded proteins inside the endoplasmic reticulum (ER) lumen can cause cells to undergo apoptosis. Misfolded proteins exported from ER to cytoplasm are ubiquitinated and mostly degraded by the proteasome, but can also be destroyed by autophagy mediated by the docking proteins p62 and NBR1. When misfolded proteins accumulate beyond the capacity of these clearance systems, they are transported to the microtubule organization center to form aggresomes, which are also degraded by autophagy. Together, these phenomena suggest the existence of a coordinated intracellular network for coping with the accumulation of misfolded proteins. Thus, rational inhibition of this network system might enhance killing of cancer cells subjected to pronounced ER stress loading. Based on this rationale, we sought to establish a quantitative assay for monitoring ER stress loading. MDA-MB231 cells stably transfected with the ERAI-Venus vector exhibited a strong XBP1 splicing signal in response to ER stress. Using the IncuCyte cell imaging system, we monitored the fluorescence intensity of XBP1-Venus, normalized against cell density, as an ER stress indicator. This parameter correlated closely with other reporters of unfolded protein responses. Assessment of the XBP1-Venus signal during exposure to various drug combinations revealed that simultaneous inhibition of the proteasome, autophagy, and aggresome formation led to more effective ER stress loading and higher cytotoxicity than inhibition of only two components. Our data suggest that this monitoring system is a useful tool for designing effective drug combinations for ER stress loading in cancer therapy.

Pancreatic cancer is one of the most difficult types of cancer to treat because of its high mortality rate due to chemotherapy resistance. We previously reported that combined treatment with gefitinib (GEF) and clarithromycin (CAM) results in enhanced cytotoxicity of GEF along with endoplasmic reticulum (ER) stress loading in non-small cell lung cancer cell lines. An epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) such as GEF induces autophagy in a pro-survival role, whereas CAM inhibits autophagy flux in various cell lines. Pronounced GEF-induced cytotoxicity therefore appears to depend on the efficacy of autophagy inhibition. In the present study, we compared the effect on autophagy inhibition among such macrolides as CAM, azithromycin (AZM), and EM900, a novel 12-membered non-antibiotic macrolide. We then assessed the enhanced GEF-induced cytotoxic effect on pancreatic cancer cell lines BxPC-3 and PANC-1. Autophagy flux analysis indicated that AZM is the most effective autophagy inhibitor of the three macrolides. CAM exhibits an inhibitory effect but less than AZM and EM900. Notably, the enhancing effect of GEF-induced cytotoxicity by combining macrolides correlated well with their efficient autophagy inhibition. However, this pronounced cytotoxicity was not due to upregulation of apoptosis induction, but was at least partially mediated through necroptosis. Our data suggest the possibility of using macrolides as 'chemosensitizers' for EGFR-TKI therapy in pancreatic cancer patients to enhance non-apoptotic tumor cell death induction.

Although recent genome-wide association studies (GWAS) have been extremely successful, it remains a big challenge to functionally annotate disease‑associated single nucleotide polymorphisms (SNPs), as the majority of these SNPs are located in non‑coding regions of the genome. In this study, we described a novel strategy for identifying the proteins that bind to the SNP‑containing locus in an allele‑specific manner and successfully applied this method to SNPs in the type 2 diabetes mellitus susceptibility gene, potassium voltage‑gated channel, KQT‑like subfamily Q, member 1 (KCNQ1). DNA fragments encompassing SNPs, and risk or non‑risk alleles were immobilized onto the novel nanobeads and DNA‑binding proteins were purified from the nuclear extracts of pancreatic β cells using these DNA‑immobilized nanobeads. Comparative analysis of the allele-specific DNA-binding proteins indicated that the affinities of several proteins for the examined SNPs differed between the alleles. Nuclear transcription factor Y (NF‑Y) specifically bound the non‑risk allele of the SNP rs2074196 region and stimulated the transcriptional activity of an artificial promoter containing SNP rs2074196 in an allele‑specific manner. These results suggest that SNP rs2074196 modulates the affinity of the locus for NF‑Y and possibly induces subsequent changes in gene expression. The findings of this study indicate that our comparative method using novel nanobeads is effective for the identification of allele‑specific DNA‑binding proteins, which may provide important clues for the functional impact of disease‑associated non‑coding SNPs.

The maintenance of the intracellular level of amino acids is crucial for cellular homeostasis. This is carried out via the regulation of both the influx from the extracellular environment and the recycling of intracellular resources. Since epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors, including gefitinib (GEF) have been reported to induce the apoptosis of several cancer cell lines, in the present study, we examined whether the cytotoxic effects of GEF are further enhanced under amino acid starvation (AAS) culture conditions. Under AAS culture conditions, the cell killing effect of GEF was synergistically pronounced in the EGFR-expressing cell lines, namely, CAL 27, Detroit 562, A549 and PANC-1 cells compared with those treated with either GEF or AAS alone. The addition of essential amino acids, but not non-essential amino acids to the cell culture medium resulted in the cancellation of this pronounced cytotoxicity. The knockdown of L-type amino acid transporter 1 (LAT-1) by siRNA also enhanced GEF-induced cytotoxicity. Therefore, the shortage of the intracellular amino acid pool appears to determine the sensitivity to GEF. Notably, this enhanced cytotoxicity is not mediated by the induction of apoptosis, but is accompanied by the pronounced induction of autophagy. The presence of necrostatin-1, an inhibitor of receptor-interacting serine/threonine-protein kinase 1 (RIPK‑1), but not that of Z-VAD-fmk, attenuated the cytotoxic effects of GEF under AAS culture conditions. Electron microscopy demonstrated that the CAL 27 cells treated with GEF under AAS culture conditions exhibited swelling of the cytosol and organelles with an increased number of autophagosomes and autolysosomes, but without chromatin condensation and nuclear fragmentation. Autophagic cell death was excluded as the inhibition of autophagy did not attenuate the cytotoxicity. These results strongly suggest the induction of necroptosis in response to GEF under AAS culture conditions. However, we could not detect any phosphorylation of RIPK-1 and mixed lineage kinase domain like pseudokinase (MLKL), as well as any necrosome formation. Therefore, the enhanced cytotoxic effect of GEF under AAS culture conditions is thought to be mediated by atypical necroptosis.

Although genome-wide association studies have shown that potassium voltage-gated channel subfamily Q member 1 (KCNQ1) is one of the genes that is most significantly associated with type 2 diabetes mellitus (T2DM), functionally annotating disease-associated single nucleotide polymorphisms (SNPs) remains a challenge. Recently, our group described a novel strategy to identify proteins that bind to SNP-containing loci in an allele-specific manner. The present study successfully applied this strategy to investigate rs163184, a T2DM susceptibility SNP located in the intronic region of KCNQ1. Comparative analysis of DNA-binding proteins revealed that the binding activities for the genomic region containing SNP rs163184 differed between alleles for several proteins, including Sp3 and Lsd1/Kdm1a. Sp3 preferentially bound to the non-risk rs163184 allele and stimulated transcriptional activity in an artificial promoter containing this region. Lsd1/Kdm1a was identified to be preferentially recruited to the non-risk allele of the rs163184 region and reduced Sp3-dependent transcriptional activity in the artificial promoter. In addition, expression of the nearby cyclin‑dependent kinase inhibitor 1C (CDKN1C) gene was revealed to be upregulated after SP3 knockdown in cells that possessed non-risk alleles. This suggests that CDKN1C is potentially one of the functional targets of SNP rs163184, which modulates the binding activity of the locus for Sp3 and Lsd1/Kdm1a.

The inhibitory effects of macrolide antibiotics including clarithromycin (CAM) on autophagy flux have been reported. Although a macrolide antibiotic exhibits no cytotoxicity, its combination with bortezomib (BZ), a proteasome inhibitor, for the simultaneous blocking of the ubiquitin (Ub)-proteasome and autophagy-lysosome pathways leads to enhanced multiple myeloma (MM) cell apoptosis induction via stress overloading of the endoplasmic reticulum (ER). As misfolded protein cargo is recruited by histone deacetylase 6 (HDAC6) to dynein motors for aggresome transport, serving to sequester misfolded proteins, we further investigated the cellular effects of targeting proteolytic pathways and aggresome formation concomitantly in MM cells. Pronounced apoptosis was induced by the combination of vorinostat [suberoylanilide hydroxamic acid (SAHA); potently inhibits HDAC6] with CAM and BZ compared with each reagent or a 2-reagent combination. CAM/BZ treatment induced vimentin positive-aggresome formation along with the accumulation of autolysosomes in the perinuclear region, whereas they were inhibited in the presence of SAHA. The SAHA/CAM/BZ combination treatment maximally upregulated genes related to ER stress including C/EBP homologous protein (CHOP). Similarly to MM cell lines, enhanced cytotoxicity with CHOP upregulation following SAHA/CAM/BZ treatment was shown by a wild-type murine embryonic fibroblast (MEF) cell line; however, a CHOP-deficient MEF cell line almost completely canceled this pronounced cytotoxicity. Knockdown of HDAC6 with siRNA exhibited further enhanced CAM/BZ-induced cytotoxicity and CHOP induction along with the cancellation of aggresome formation. Targeting the integrated networks of aggresome, proteasome, and autophagy is suggested to induce efficient ER stress-mediated apoptosis in MM cells.