Nutrient Deprivation Stress Research Articles

15 - Lysosomal Dysfunction and Impaired Autophagy Exacerbate Hypoxia-Induced β-Cell Death and Islet Graft Failure

Autophagy delivers damaged organelles and protein aggregates to lysosomes for degradation during nutrient deprivation and cellular stress. Here, we examined the importance of autophagy and lysosomal homeostasis in β-cells under the stresses induced by hypoxia and islet transplantation.

Eukaryotic response to hypothermia in relation to integrated stress responses.

Eukaryotic cells respond to hypothermic stress through a series of regulatory mechanisms that preserve energy resources and prolong cell survival. These mechanisms include alterations in gene expression, attenuated global protein synthesis and changes in the lipid composition of the phospholipid bilayer. Cellular responses to hyperthermia, hypoxia, nutrient deprivation and oxidative stress have been comprehensively investigated, but studies of the cellular response to cold stress are more limited. Responses to cold stress are however of great importance both in the wild, where exposure to low and fluctuating environmental temperatures is common, and in medical and biotechnology settings where cells and tissues are frequently exposed to hypothermic stress and cryopreservation. This means that it is vitally important to understand how cells are impacted by low temperatures and by the decreases and subsequent increases in temperature associated with cold stress. Here, we review the ways in which eukaryotic cells respond to hypothermic stress and how these compare to the well-described and highly integrated stress response systems that govern the cellular response to other types of stress.

Genomic responses of DNA methylation and transcript profiles in zebrafish cells upon nutrient deprivation stress

Environmental stress such as nutrient deprivation across multiple fields in nature causes physiological and biochemical changes in organism. Understanding the potential epigenetic modulations to phenotypic variation upon nutrient deprivation stress is crucial for environmental assessments. Here, the methyl-cytosine at single-base resolution was mapped across the whole genome and the methylation patterns and methylation levels coordinated with transcript analysis were systemically elaborated in zebrafish embryonic fibroblast cells under serum starvation stress. The down-regulated genes mainly annotated to the pathways of DNA replication and cell cycle that were consistent with cell physiological changes. Vast differentially methylated regions were identified in genomic chromosome and showed enrichment in the intron and intergenic regions. In an integrated transcriptome and DNA methylation analyses, 135 negatively correlated genes were determined, wherein the hub genes of gins2, cdca5, fbxo5, slc29a2, suv39h1b, and zgc:174160 were predominant responsive to the nutrient condition changes. Besides, nutrient recovery and DNA methyltransferases inhibitor supplements partly rescued cell proliferation with decrease of DNA methylation and reactivation of several depressed genes, implying the possible intrinsic relationships among cell physiological state, mRNA expression, and DNA methylation. Collectively, current study proved the broad role of DNA methylation in governing cellular responses to nutrient deprivation and revealed the epigenetic risk of starvation stress in zebrafish.

Selenium Nanoparticle Protection of Fibroblast Stress: Activation of ATF4 and Bcl-xL Expression.

BackgroundIn recent years, selenium nanostructures have been researched due to their antibacterial properties, low toxicity to mammalian cells, and high biological efficacy. However, the clinical implementation of the use of selenium has received mixed results, and there is much work needed to improve the understanding of the biological mechanisms involved in the observed cellular responses.Materials and methodsIn this work, an investigation into the mechanistic pathways of selenium nanoparticles (SeNPs) in biological systems was conducted by studying the changes in gene expression of ATF4, Bcl-xL, BAD2, HSP70, and SOD2 in non-cancerous human dermal fibroblasts (HDF) under oxidative stress, nutrient deprivation stress, and no treatment (control) conditions.ResultsThis study revealed that SeNP incubation led to reduced internal reactive oxygen species (ROS) generation for all conditions tested, thus, providing a protective environment for HDF. At the stress conditions, the expression of ATF4 and Bcl-xL increased for cells treated with SeNP incubation, leading to attenuation of the cells under stress. These results also hint at reductive stress causing a detrimental impact to cell proliferation under routine cell passaging conditions.ConclusionIn summary, this study highlights some possible mechanistic pathways implicated in the action of SeNPs that warrant further investigation (specifically, reducing stress conditions for HDF) and continues to support the promise of SeNPs in a wide range of medical applications.

The cellular responses of autophagy, apoptosis, and 5-methylcytosine level in zebrafish cells upon nutrient deprivation stress

Here we reported the stress responses of nutrient deprivation and extended observation of autophagy, apoptosis, and DNA methylation in zebrafish embryonic fibroblast (ZF4) cells. Our results showed that serum deprivation resulted in the changes of cell shape and adherent ability, the suppressed cell growth and viability, and the inhibited proliferation and cell cycle. Besides, the appearance of lysosome and autophagosome/autolysosome with significantly increased expression of mRNAs (ulk1a, becn1, atg12, sqstm1, maplc3, and lamp1) and proteins (Atg12, Becn1, Sqstm1, and Lamp1) indicate the autophagic activity was boosted at initial stage but relatively weakened at 48 h of serum starvation. When autophagy no longer mitigate for the stress, cell apoptosis detected by the mRNA expression of caspases, Bcl-2/Bax expression, and Annexin V/PI was gradually enhanced to execute the death plan upon prolonged starvation process. Furthermore, the methyl group metabolism was increased in accordance with autophagic activity and was suppressed by enhanced apoptotic activity. These data suggested that the recycle activity induced by autophagy could compensate the substrates and reactions of DNA transmethylation, which obviously increased 5-methylcytosine (5 mC) level in ZF4 cells. In summary, our results discovered the cellular responses under prolonged serum starvation stress and elaborated the switch from autophagy to apoptosis and corresponding correlation with 5 mC level changes in teleost fish in vitro.

Bacterial stress defense: the crucial role of ribosome speed.

In nature, bacteria are constantly adapting to various stressful conditions. Timely activation of stress response programs is crucial for bacteria to smoothly survive under stressful conditions. Stress response, demanding the de novo synthesis of many defense proteins, is generally activated at the transcriptional level by specific regulators. However, the effect of the global protein translational status on stress response has been largely overlooked. The translational capacity is limited by the number of translating ribosomes and the translational elongation rate. Recent work has shown that certain environmental stressors (e.g. oxidative stress) could severely compromise the stress response progress of bacteria by causing either slow-down or even complete stalling of the translational elongation process. The maintenance of ribosome elongation rate, being crucial for timely synthesis of stress defense proteins, becomes the physiological bottleneck that limits the survival of bacteria in some stressful conditions. Here, we briefly summarize some recent progress on the translational status of bacteria under two distinct stress conditions, nutrient deprivation and oxidative stress. We further discuss several important open questions on the translational regulation of bacteria during stress. The ribosome translation should be investigated in parallel with traditional transcriptional regulation in order to gain a better understanding on bacterial stress defense.

Cardiomyocyte damage control in heart failure and the role of the sarcolemma

The cardiomyocyte plasma membrane, termed the sarcolemma, is fundamental for regulating a myriad of cellular processes. For example, the structural integrity of the cardiomyocyte sarcolemma is essential for mediating cardiac contraction by forming microdomains such as the t-tubular network, caveolae and the intercalated disc. Significantly, remodelling of these sarcolemma microdomains is a key feature in the development and progression of heart failure (HF). However, despite extensive characterisation of the associated molecular and ultrastructural events there is a lack of clarity surrounding the mechanisms driving adverse morphological rearrangements. The sarcolemma also provides protection, and is the cell’s first line of defence, against external stresses such as oxygen and nutrient deprivation, inflammation and oxidative stress with a loss of sarcolemma viability shown to be a key step in cell death via necrosis. Significantly, cumulative cell death is also a feature of HF, and is linked to disease progression and loss of cardiac function. Herein, we will review the link between structural and molecular remodelling of the sarcolemma associated with the progression of HF, specifically considering the evidence for: (i) Whether intrinsic, evolutionary conserved, plasma membrane injury-repair mechanisms are in operation in the heart, and (ii) if deficits in key ‘wound-healing’ proteins (annexins, dysferlin, EHD2 and MG53) may play a yet to be fully appreciated role in triggering sarcolemma microdomain remodelling and/or necrosis. Cardiomyocytes are terminally differentiated with very limited regenerative capability and therefore preserving cell viability and cardiac function is crucially important. This review presents a novel perspective on sarcolemma remodelling by considering whether targeting proteins that regulate sarcolemma injury-repair may hold promise for developing new strategies to attenuate HF progression.

Abstract 3749: Metabolic reprogramming by GCN1 mediates metastatic reactivation and outgrowth at liver metastatic site in response to glutamine deprivation in pancreatic cancer

Abstract Introduction: Disseminated tumor cells in the circulation upon extravasation into a secondary organ like the liver encounter hostile conditions like nutrient deprivation and oxidative stress and may undergo dormancy or growth arrest. The molecular drivers and mechanisms that enable disseminated tumor cells to overcome such adverse growth conditions and revert to proliferative stage are unknown. Method: To identify these molecular drivers, a retroviral cDNA library was generated from a highly metastatic pancreatic cell line (M-4964Liv) derived from a KPC mouse which forms macroscopic liver lesions upon splenic injection in mice. The cDNA library was then transduced into a dormant pancreatic KPC cells (D-4964Liv) cells which does not give liver lesions upon splenic injection in mice. This methodology will allow all the cDNA’s from the metastatic cells to express in the dormant cells but only those cDNA’s which can trigger the dormant cells to outgrow will give rise to a metastatic liver lesion. Results: The screening strategy led to the identification of general control of amino-acid synthesis 1-like 1 (GCN1L1). The human homologue GCN1 has been shown to regulate the activation of mammalian amino acid sensor general control non-derepressible 2 (GCN2) under amino acid starvation in budding yeast but how GCN1 regulates metastatic reactivation is not known. We show that GCN1 is activated under physiologic glutamine levels in the liver which is around 0.5 mM compared to standard tissue culture levels of 2-4 mM. Mechanistic interrogation revealed that GCN1 activates both the mammalian amino acid sensors: the general control nonderepressible 2 (GCN2) and the mechanistic target of rapamycin complex 1 (mTORC1) leading to metastatic outgrowth in vivo. Contrary, knockdown of GCN1 dramatically impairs the activation of GCN2 and mTORC1 and abrogates liver metastatic outgrowth upon splenic injection in immunocompetent mice. Conclusions: These results suggest that under physiologic glutamine levels, induction of GCN1 activates two different pathways: One pathway impinges on ATF4 and the other on mTORC1. While ATF4 activation promotes metabolic homeostasis by inducing de novo amino acid synthesis, the activation of mTOR pathway ensures re-entry in to the cell cycle leading to metastatic reactivation and outgrowth. Citation Format: Surajit Sinha, Jonathan Hernandez, Reed Ayabe, Michael Wach, Samantha Ruff, Alok Ranjan, Kirsten Remmert, Imani Alexander. Metabolic reprogramming by GCN1 mediates metastatic reactivation and outgrowth at liver metastatic site in response to glutamine deprivation in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3749.

Abstract 3596: The combinatory effect of bevacizumab and metabolic inhibitors on angiogenesis and autophagy in gastrointestinal cancer cells

Abstract Background: Bevacizumab is anti-VEGF drug that has shown therapeutic. However, treatment with bevacizumab has been associated to the development of resistance in several types of cancer. Glycolysis has been considered as a potential target for anti-cancer therapies and several molecules that target this pathway have been developed, including 2-Deoxy-D-glucose (2-DG) and 3-Bromopyruvate (3-BP). Autophagy is a process that degrades cellular organelles and proteins and maintains cellular biosynthesis during nutrient deprivation or metabolic stress. Since anti-angiogenic therapy and metabolic inhibitors both lead to nutrient deprivation in cancers, we investigated how a combination therapy would affect angiogenesis and autophagy in gastrointestinal cancer cells. Methods: AGS and Caco-2 cells were treated with glycolysis inhibitors 2-DG (4 mM) and 3-BP (20 µM), in the presence or in the absence of bevacizumab at 100 µg/ml. Cell proliferation was assessed using tetrazolium salt. VEGF protein levels were quantified by ELISA. Autophagy and apoptosis markers’ expression was assessed by quantitative real time PCR. Tube formation by human umbilical vein endothelial cells (HUVECs) was assessed using ECMatrix™. Results: Our results showed that combining bevacizumab to 2-DG decreased AGS and Caco-2 cells’ proliferation rates compared to each of these compounds alone. Interestingly, VEGF quantification showed that 2-DG increased VEGF secretion, in contrary to 3-BP. Moreover, our results showed that 3-BP reduced tube formation by HUVECs and amplified bevacizumab’s anti-angiogenic effect when combined together. In autophagy markers’ assessment, our results showed that 2-DG, 3-BP, and bevacizumab treatment increased FOXO1, FOXO3, LC3II, and HIF-1α expression. However, Beclin-1 expression, a protein implicated in the autophagic programmed cell death, decreased following bevacizumab treatment while it was upregulated by 2-DG and 3-BP. Hence, it was important to evaluate apoptotic markers’ expression, caspase-3 and BAX. Our results showed that bevacizumab decreased caspase-3 and BAX expression while their expression was upregulated by 2-DG and that its combination to bevacizumab reestablished their levels. Conclusions: Taken together, our results suggest that 2-DG and 3-BP, both improve cancer cells response to bevacizumab treatment by increasing its antiangiogenic outcome and reversing its anti-apoptotic effect. Moreover, it was interesting to find that bevacizumab treatment decreased Beclin-1, caspase-3, and BAX expression, suggesting a possible implication of autophagy in the development of resistance to bevacizumab. Hence, further studies are required in order to uncover the means by which bevacizumab modulates cancer cells autophagy and metabolism, and the effect of these changes on cancer cells survival. Citation Format: Nadine Mahfouz, Rita Ammoury, Mayssam Moussa, Charbel Khalil, Joe Aoun, George Hilal. The combinatory effect of bevacizumab and metabolic inhibitors on angiogenesis and autophagy in gastrointestinal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3596.

PARP1 and Poly(ADP-ribosyl)ation Signaling during Autophagy in Response to Nutrient Deprivation

Autophagy is considered to be the primary degradative pathway that takes place in all eukaryotic cells. Morphologically, the autophagy pathway refers to a process by which cytoplasmic portions are delivered to double-membrane organelles, called autophagosomes, to fuse with lysosomes for bulk degradation. Autophagy, as a prosurvival mechanism, can be stimulated by different types of cellular stress such as nutrient deprivation, hypoxia, ROS, pH, DNA damage, or ER stress, promoting adaptation of the cell to the changing and hostile environment. The functional relevance of autophagy in many diseases such as cancer or neurodegenerative diseases remains controversial, preserving organelle function and detoxification and promoting cell growth, although in other contexts, autophagy could suppress cell expansion. Poly(ADP-ribosyl)ation (PARylation) is a covalent and reversible posttranslational modification (PTM) of proteins mediated by Poly(ADP-ribose) polymerases (PARPs) with well-described functions in DNA repair, replication, genome integrity, cell cycle, and metabolism. Herein, we review the current state of PARP1 activation and PARylation in starvation-induced autophagy.

Nutrient deprivation and lysosomal stress induce activation of TFEB in retinal pigment epithelial cells

BackgroundInduction of lysosomal function and autophagy is regarded as an adaptive mechanism in response to cellular stress. The transcription factor EB (TFEB) has been identified as a master regulator of lysosomal function and autophagy. TFEB is a member of the microphthalmia family of bHLH-LZ transcription factors that includes other members such as micropthalmia-associated transcription factor (MITF), TFE3, and TFEC. TFEB controls lysosome biogenesis and autophagy by upregulation of a family of genes belonging to the Coordinated Lysosomal Expression and Regulation (CLEAR) network. Here, we investigated the expression of TFEB in cells subjected to nutrient deprivation and lysosomal stress. We studied transcriptional induction of TFEB-regulated genes in response to nutrient deprivation and lysosomal stress in retinal pigment epithelial (RPE) cells. Furthermore, we also investigated the induction of autophagy and lysosomal genes upon overexpression of constitutively active form of TFEB.MethodsExpression of TFEB and MITF protein levels were evaluated in cells subjected to prolonged periods of nutrient deprivation. mRNA levels of the CLEAR network genes was measured by quantitative real time PCR (qRT-PCR) analysis in cells deprived of nutrients, treated with ammonium chloride and upon overexpression of constitutively active TFEB. Immunostaining with LC3 antibody was used to measure autophagy flux. Labeling with lysoTracker dye was used to assess lysosomes.ResultsOur results show that nutrient deprivation increases protein levels of TFEB and MITF in ARPE-19 cells. Nutrient stress induces the expression of lysosomal (LAMP1, CTSD MCOLN1, SGSH) and autophagy (BECN1) genes. Lysosomal stress also increases the expression of lysosomal (ATP6V0A1 and LAMP1) and autophagy (p62 and BECN1) genes. Our results show that overexpression of constitutively active TFEB also induces the expression of CLEAR network genes.ConclusionsCollectively, these observations suggest that nutrient stress induces the protein expression of both MITF and TFEB in ARPE-19 cells. TFEB-regulated transcriptional program plays an important role in adaptive response of cells during both nutrient and lysosomal stress.

TRB3 regulates skeletal muscle mass in food deprivation-induced atrophy.

Tribbles 3 (TRB3) is a pseudokinase that has been found in multiple tissues in response to various stress stimuli, such as nutrient deprivation and endoplasmic reticulum (ER) stress. We recently found that TRB3 has the potential to regulate skeletal muscle mass at the basal state. However, it has not yet been explored whether TRB3 regulates skeletal muscle mass under atrophic conditions. Here, we report that food deprivation for 48 h in mice significantly reduces muscle mass by ∼15% and increases TRB3 expression, which is associated with increased ER stress. Interestingly, inhibition of ER stress in C2C12 myotubes reduces food deprivation-induced expression of TRB3 and muscle-specific E3-ubiquitin ligases. In further in vivo experiments, muscle-specific TRB3 transgenic mice increase food deprivation-induced muscle atrophy compared with wild-type (WT) littermates presumably by the increased proteolysis. On the other hand, TRB3 knockout mice ameliorate food deprivation-induced atrophy compared with WT littermates by preserving a higher protein synthesis rate. These results indicate that TRB3 plays a pivotal role in skeletal muscle mass regulation under food deprivation-induced muscle atrophy and TRB3 could be a pharmaceutical target to prevent skeletal muscle atrophy.-Choi, R. H., McConahay, A., Silvestre, J. G., Moriscot, A. S., Carson, J. A., Koh, H.-J. TRB3 regulates skeletal muscle mass in food deprivation-induced atrophy.

The Integrated Stress Response Activity Marks Stem Cells in Normal Hematopoiesis and Leukemia

Lifelong maintenance of the blood system requires equilibrium between clearance of damaged hematopoietic stem cells (HSCs) and long-term survival of the HSC pool. Perturbations of cellular homeostasis such as nutrient deprivation, irradiation, and endoplasmic reticulum stress can result in HSC loss. However, HSCs must survive low-level stressors in order to sustain lifelong replenishment of the hematopoietic system. It is poorly understood how human HSCs balance apoptosis with survival in the context of basal stress, and how adaptive signalling is regulated in leukemia stem cells (LSCs). The Integrated Stress Response (ISR) is an adaptive pathway that can protect cells against stressors such as ROS, nutrient deprivation and misfolded proteins.To assess the expression levels of key ISR pathway components, we analyzed the proteome of purified human HSCs and progenitor cells from cord blood (CB). Quantitative label-free mass spectrometry revealed lower expression of eIF2α, eIF2β and eIF2γ subunits in HSCs compared to downstream progenitors. Furthermore, activated-transcription factor 4 (ATF4) mRNA is highly expressed in HSCs compared to progenitors. Similar to our findings in normal CB cells, analysis of acute myeloid leukemia (AML) patient samples revealed lower protein levels of eIF2α, eIF2β and eIF2γ in phenotypically primitive (CD34+CD38-) compared to differentiated (CD34+CD38+) AML cell populations. These results suggest that primitive cells in normal hematopoiesis and AML are primed for ISR activation.To assess ISR activity in human HSPCs, we used an ATF4 lentiviral reporter (ATF4rep) that measures ISR-induced ATF4 translation. We subjected ATF4rep-transduced CD34+ CB cells to hypoxia and amino acid deprivation, and found that valine depletion strongly induced ATF4rep activity. ATF4rep upregulation was abolished in the presence of an eIF2αS52A mutant that cannot be phosphorylated. Furthermore, knockdown of eIF2α, eIF2β or eIF2γ subunits in CD34+ CB cells increased ATF4rep activity. Thus, low levels of eIF2α, eIF2β or eIF2γ result in efficient ATF4 translation, and nutrient deprivation upregulates ATF4 through eIF2α phosphorylation.We assessed the effect of ATF4 upregulation on CB cell proliferation and survival. Following knockdown of ATF4 mRNA in CD34+ CB cells, the cells were incubated in valine deficient media to induce translational upregulation of ATF4. Valine depletion of shCTRL-transduced cells for 2 days did not affect proliferation or apoptosis, as measured by EdU incorporation or Annexin-V. In contrast, valine depletion of shATF4-transduced cells resulted in decreased proliferation (2-fold, P = 0.0004) and increased apoptosis (4-fold, P < 0.0001,). Thus, ATF4 promotes survival of primitive CD34+ CB cells undergoing valine depletion.We performed in vivo xenograft studies to examine the ISR activity in the best available setting to approximate homeostatic conditions for human HSPCs. Transplantation of ATF4rep-transduced CB cells showed that human HSPCs in the mouse bone marrow maintained a 2.4-fold higher ATF4rep activity compared to downstream progenitors (P = 0.0002). ATF4rep activity further declined in mature monocytes, granulocytes and B-cells (13-fold, P < 0.0001). To determine if high ISR activity is associated with improved HSC function, we transplanted lin- CB cells expressing high ATF4rep activity (GFP-high) and low ATF4rep activity (GFP-low) into mice. The level of engraftment as well as the number of engrafted mice was increased from GFP-high cells compared to GFP-low cells (P = 0.001). The hierarchical structure of normal hematopoiesis is partially maintained in AML. We evaluated ATF4rep expression in the malignant hierarchy and found that 4/5 patient samples had higher ATF4rep expression in CD34+ cells compared to CD34- cells. Furthermore, serial transplantation of ATF4rep-transduced cells showed higher engraftment from GFP-high compared to GFP-low cells ( P < 0.0001). Thus, primary human AML cells that possess high ISR activity are enriched for LSC function.Our data establish that the adaptive ISR pathway plays a key role in maintaining homeostasis of normal and malignant stem cells. We show that Amino acid deprivation activates the ISR in human HSPCs resulting in ATF4-dependent pro-survival signals. In an unperturbed state, HSCs are in a state of primed ISR activity, mechanistically maintained by eIF2 scarcity and high ATF4 levels. DisclosuresNo relevant conflicts of interest to declare.

PG-Priming Enhances Doxorubicin Influx to Trigger Necrotic and Autophagic Cell Death in Oral Squamous Cell Carcinoma.

Synergistic effects between natural compounds and chemotherapy drugs are believed to have fewer side effects with equivalent efficacy. However, the synergistic potential of prodigiosin (PG) with doxorubicin (Dox) chemotherapy is still unknown. This study explores the synergistic mechanism of PG and Dox against oral squamous cell carcinoma (OSCC) cells. Three OSCC cell lines were treated with different PG/Dox combinatory schemes for cytotoxicity tests and were further investigated for cell death characteristics by cell cycle flow cytometry and autophagy/apoptosis marker labelling. When OSCC cells were pretreated with PG, the cytotoxicity of the subsequent Dox-treatment was 30% higher than Dox alone. The cytotoxic efficacy of PG-pretreated was found better than those of PG plus Dox co-treatment and Dox-pretreatment. Increase of Sub-G1 phase and caspase-3/LC-3 levels without poly (ADP-ribose) polymeras (PARP) elevation indicated both autophagy and necrosis occurred in OSCC cells. Dox flux after PG-priming was further evaluated by rhodamine-123 accumulation and Dox transporters analysis to elucidate the PG-priming effect. PG-priming autophagy enhanced Dox accumulation according to the increase of rhodamine-123 accumulation without the alterations of Dox transporters. Additionally, the cause of PG-triggered autophagy was determined by co-treatment with endoplasmic reticulum (ER) stress or AMP-activated protein kinase (AMPK) inhibitor. PG-induced autophagy was not related to nutrient deprivation and ER stress was proved by co-treatment with specific inhibitor. Taken together, PG-priming autophagy could sensitize OSCC cells by promoting Dox influx without regulation of Dox transporter. The PG-priming might be a promising adjuvant approach for the chemotherapy of OSCC.

The putative transcription factor CaMaf1 controls the sensitivity to lithium and rapamycin and represses RNA polymerase III transcription in Candida albicans.

Maf1 is a repressor of RNA polymerase (Pol) III transcription for tRNA. Nutrient deprivation and environmental stress repress Pol III transcription through Maf1 in Saccharomyces cerevisiae. The sole Candida albicans homolog CaMaf1 is a protein of 380 amino acids with conserved domains and motifs of the eukaryotic Maf1 family. Here, we show that C. albicans cells lacking CaMAF1 show elevated levels of tRNA. Deletion of CaMAF1 increases the sensitivity of C. albicans cells to lithium cation and SDS as well as tolerance to rapamycin and azole. In addition, deletion of CaMAF1 reduces the level of filamentation and alters the surface morphology of colonies. CaMaf1 is localized in the nucleus of log-phase growing cells. However, a dynamic change of subcellular localization of CaMaf1 exists during serum-induced morphological transition, with CaMaf1 being localized in the nuclei of cells with germ tubes and short filaments but outside of the nuclei of cells with long filaments. In addition, CaMaf1 is required for rapamycin-induced repression of CaERG20, encoding the farnesyl pyrophosphate synthetase involved in ergosterol biosynthesis. Therefore, CaMaf1 plays a role as a general repressor of Pol III transcription in C. albicans.

Abstract 2035: Knockdown of TGFβ-activated ATF4 inhibits triple negative breast cancer metastases independently of cellular stress

Abstract Background. Triple negative breast cancer (TNBC) is a very aggressive form of breast cancer which is characterized by a poor survival rate and high incidence of metastases. The integrated stress response (ISR) is activated under stress conditions (hypoxia, nutrient deprivation, or endoplasmic reticulum stress) and reduces the protein synthesis through phospho-eIF2α/ATF4 (activating transcription factor 4) to regulate cell fate. ATF4, which is overexpressed in breast cancer including TNBC, regulates tumor growth, autophagy, drug resistance, and metastasis during ISR through PERK and GCN2 pathways. We have reported enhanced ATF4 expression in unstressed MCF10A cells treated with TGFβ1. Here, we investigate the potential TGFβ-mediated stress-independent control of ATF4 activity and its impact on the TNBC-associated metastasis. Methods. SUM159PT and BT549 TNBC cell lines were treated with human recombinant TGFβ1 (10 ng/ml) and the TGFBRI kinase inhibitor LY2157299 (5µM) for 72h. Effects of TGFβ on ATF4 expression were assessed by silencing SMAD2/3 and SMAD4 with TGFβ1 for 24h in SUM159PT and BT549 cells. Changes in ATF4 expression by IRS were assessed after PERK, GCN2, PKR, HRI, and eIF2α inhibition with TGFβ1 for 72h in SUM159PT, BT549 and MDA-MB-231 cell lines. ATF4 expression levels were determined by RT-PCR and/or western blot. Effects on TGFβ-induced metastasis were analyzed by ATF4 knockdown for 48h by using two different siRNA sequences (#1 and #2) following treatment with TGFβ1 for 24h. Migration and invasion were performed by wound healing and transwell assays, respectively. Results were compared to a scrambled siRNA as negative control (SCR). Results. Our results show that ATF4 expression (at RNA and protein levels) was abrogated by LY2157299 upon TGFβ activation, suggesting that ATF4 is active downstream of TGFBRI. This result was supported by further SMAD2/3 and SMAD4 knockdown following treatment with TGFβ1, which was correlated with decreased ATF4 expression. Additionally, we assessed whether ATF4 inhibition can reduce the TGFβ-induced metastatic properties of TNCB cell lines. We found that ATF4 depletion inhibited migration and invasiveness in the three cell lines tested. ATF4 is known to exert a pro-metastatic role downstream of ISR through PERK and GCN2 pathways. Our results demonstrate that, upon treatment with TGFβ1, knockdown of the IRS mediators PERK, PKR, GCN2, HRI, and eIF2α did not correlate with a consistent decrease of ATF4 levels in the three cell lines. Conclusion. In conclusion, our results show for the first time that ATF4 is a downstream target of the canonical TGFβ/SMAD pathway in a ISR-independent fashion, and its depletion correlates with an inhibition of the TGFβ1-mediated migration and invasion of TNBC cell lines. Therefore, ATF4 may represent a therapeutic target in TNBC patients with active TGFβ signaling pathway. Citation Format: Jenny C. Chang, Alberto Ramirez, Maria P. Molina, Francisca E. Cara, Wei Qian, Wen Chen, Anthony J. Kozielski, Roberto R. Rosato, Juan A. Marchal, Jose A. Lorente, Pedro Sanchez-Rovira, Sergio Granados-Principal. Knockdown of TGFβ-activated ATF4 inhibits triple negative breast cancer metastases independently of cellular stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2035.

Autophagy and acetaminophen-induced hepatotoxicity.

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug. APAP overdose can induce acute liver injury in humans, which is responsible for approximately 50% of total cases of acute liver failure in the United States and some European countries. Currently, the metabolism of APAP in the body has been extensively investigated; however, the exact mechanisms for APAP hepatotoxicity are not well understood. Recent studies have shown that mitochondrial dysfunction, oxidative stress and inflammatory responses play a critical role in the pathogenesis of APAP hepatotoxicity. Autophagy is a catabolic machinery aimed at recycling cellular components and damaged organelles in response to a variety of stimuli, such as nutrient deprivation and toxic stress. Increasing evidence supports that autophagy is involved in the pathophysiological process of APAP-induced liver injury. In this review, we summarized the changes of autophagy in the liver following APAP intoxication and discussed the role and its possible mechanisms of autophagy in APAP hepatotoxicity. Furthermore, this review highlights the crosstalk between mitophagy, oxidative stress and inflammation in APAP-induced liver injury and presents some possible molecular mechanisms by which activated autophagy protects against APAP-induced liver injury.

The unfolded protein response as a target for anticancer therapeutics

The endoplasmic reticulum (ER) is an essential organelle in eukaryotic cells, responsible for protein synthesis, folding, sorting, and transportation. ER stress is initiated when the unfolded or misfolded protein load exceeds the capacity of the ER to properly fold protein. Tumor microenvironmental conditions, such as nutrient deprivation, hypoxia, and oxidative stress perturb protein folding and trigger chronic ER stress. Cancer cells can tolerate mild ER stress, however, persistent and severe ER stress kills cancer cells by inducing their autophagy, apoptosis, necroptosis, or immunogenic cell death. Based on this rationale, many drugs have been developed for triggering irremediable ER stress in cancer cells by targeting various processes in the secretory pathway. This review discusses the mechanisms of protein targeting to the ER, the key signaling cassettes that are involved in the ER stress response, and their correlation with cancer formation and progression. Importantly, this review discusses current experimental and FDA approved anti-cancer drugs that induce ER stress, and emerging targets within the secretory pathway for the development of new anticancer drugs.

Tribbles 3 Regulates Skeletal Muscle Mass in Fasting‐induced Atrophy

Tribbles homolog 3 (TRB3) is a pseudokinase that has been found in multiple tissues, including skeletal muscle, in response to various stress stimuli, such as insulin resistance, nutrient deprivation, and endoplasmic reticulum stress. TRB3 has been mainly known as a negative regulator of Akt, which is an essential protein kinase to control protein synthesis and breakdown. Recently, our lab has demonstrated that TRB3 decreases protein synthesis and increases protein degradation in mouse skeletal muscle at basal state. However, it still remains to be determined whether TRB3 regulates skeletal muscle mass under atrophic conditions. Here, we hypothesized that TRB3 has a detrimental effect on skeletal muscle mass under 48hr fasting‐induced atrophy. In order to test the hypothesis, we utilized C57BL/6 wild type (WT) mice and fasted them for 48hr to induce skeletal muscle atrophy. Our fasting strategy reduced body weight by 20% and effectively decreased skeletal muscle mass by 15–25% compared to fed controls in multiple muscles (TA; 26%, EDL; 26%, GAS;14%, all P&lt;0.05). In addition, TRB3 protein (60%) and mRNA (49%, P&lt;0.05) expression was significantly increased in fasted muscles, which was associated with elevated atrogin−1 (750%, P&lt;0.01) and MuRF‐1 (1840%, P&lt;0.001) mRNA expression. Next, we analyzed Akt and its downstream proteins associated with protein synthesis and degradation. Phosphorylation of Akt (T308) was significantly decreased after 48hr fasting (37%, P&lt;0.05) and FOXO1 (69%, P&lt;0.01) and FOXO3a (47%, P=0.10) were activated whereas protein synthesis, mTOR and p70S6K1, was not affected. Next, we fasted muscle‐specific TRB3 transgenic (TG) and WT mice for 48hr in order to determine if TRB3 overexpression worsens the fasting‐induced atrophy. Body weight was significantly reduced by 22% after 48hr fasting in both WT and TG groups. Interestingly, we found a significant reduction in muscle mass in TG mice compared to WT (WT vs. TG in EDL; P&lt;0.05, in GAS; P=0.07). Further, Akt and its downstream proteins were determined in EDL muscle. In TG mice, Akt phosphorylation (T308) was significantly decreased after fasting (18%, P&lt;0.05) in line with suppressed S6K1 (10%, P=0.065) and activated FOXO1 (76%, P&lt;0.001) and FOXO3a (32%, P=0.068) signaling. Additionally, protein synthesis rate was assessed by the incorporation of puromycin into nascent peptide chains. 48hr fasting significantly decreased protein synthesis rate in both WT and TG (P&lt;0.01) but the degree of reduction was prominent in TG mice compared to WT (P=0.08). Lastly, we studied 48hr fasting‐induced atrophy in TRB3 knockout (KO) mice to determine if the deletion of TRB3 could prevent fasting‐induced atrophy. Body weight was significantly decreased by 20% in both WT and KO groups. Although both genotypes significantly reduced body weight, KO mice interestingly preserved ~10% more muscle mass in fasted TA, EDL, and SOL compared to fasted WT muscles (TA; 8.5%, EDL;15%, P&lt;0.05, SOL;3.6%). These data suggest that TRB3 could be a key regulator of protein synthesis and breakdown through the Akt/mTOR/FOXO signaling under 48hr fasting‐induced atrophy.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Genetic analysis of the liverwort Marchantia polymorpha reveals that R2R3MYB activation of flavonoid production in response to abiotic stress is an ancient character in land plants.

The flavonoid pathway is hypothesized to have evolved during land colonization by plants c. 450Myr ago for protection against abiotic stresses. In angiosperms, R2R3MYB transcription factors are key for environmental regulation of flavonoid production. However, angiosperm R2R3MYB gene families are larger than those of basal plants, and it is not known whether the regulatory system is conserved across land plants. We examined whether R2R3MYBs regulate the flavonoid pathway in liverworts, one of the earliest diverging land plant lineages. We characterized MpMyb14 from the liverwort Marchantia polymorpha using genetic mutagenesis, transgenic overexpression, gene promoter analysis, and transcriptomic and chemical analysis. MpMyb14 is phylogenetically basal to characterized angiosperm R2R3MYB flavonoid regulators. Mpmyb14 knockout lines lost all red pigmentation from the flavonoid riccionidin A, whereas overexpression conferred production of large amounts of flavones and riccionidin A, activation of associated biosynthetic genes, and constitutive red pigmentation. MpMyb14 expression and flavonoid pigmentation were induced by light- and nutrient-deprivation stress in M.polymorpha as for anthocyanins in angiosperms. MpMyb14 regulates stress-induced flavonoid production in M.polymorpha, and is essential for red pigmentation. This suggests that R2R3MYB regulated flavonoid production is a conserved character across land plants which arose early during land colonization.