Induction Of Autophagy In Response Research Articles

TRPML3/BK complex promotes autophagy and bacterial clearance by providing a positive feedback regulation of mTOR via PI3P

TRPML3 is a Ca2+/Na+ release channel residing in both phagophores and endolysosomal membranes. It is activated by PI3P and PI3,5P2. Its activity can be enhanced by high luminal pH and by replacing luminal Na+ with K+. Here, we report that big-conductance Ca2+-activated potassium (BK) channels form a positive feedback loop with TRPML3. Ca2+ release via TRPML3 activates BK, which in turn facilitates TRPML3-mediated Ca2+ release, potentially through removing luminal Na+ inhibition. We further show that TRPML3/BK and mammalian target of rapamycin (mTOR) form another positive feedback loop to facilitate autophagy induction in response to nutrient starvation, i.e., mTOR inhibition upon nutrient starvation activates TRPML3/BK, and this further reduces mTOR activity, thereby increasing autophagy induction. Mechanistically, the feedback regulation between TRPML3/BK and mTOR is mediated by PI3P, an endogenous TRPML3 activator that is enriched in phagophores and is up-regulated by mTOR reduction. Importantly, bacterial infection activates TRPML3 in a BK-dependent manner, and both TRPML3 and BK are required for mTOR suppression and autophagy induction responding to bacterial infection. Suppressing either TRPML3 or BK helps bacteria survival whereas increasing either TRPML3 or BK favors bacterial clearance. Considering that TRPML3/BK is inhibited by low luminal pH but activated by high luminal pH and PI3P in phagophores, we suggest that TRPML3/BK and mTOR form a positive feedback loop via PI3P to ensure efficient autophagy induction in response to nutrient deprivation and bacterial infection. Our study reveals a role of TRPML3-BK coupling in controlling cellular homeostasis and intracellular bacterial clearance via regulating mTOR signaling.

Crosstalk between endoplasmic reticulum and cytosolic unfolded protein response in tomato

Conditions that cause proteotoxicity like high temperature trigger the activation of unfolded protein response (UPR). The cytosolic (CPR) and endoplasmic reticulum (ER) UPR rely on heat stress transcription factor (HSF) and two members of the basic leucine zipper (bZIP) gene family, respectively. In tomato, HsfA1a is the master regulator of CPR. Here, we identified the core players of tomato ER-UPR including the two central transcriptional regulators, namely bZIP28 and bZIP60. Interestingly, the induction of ER-UPR genes and the activation of bZIP60 are altered in transgenic plants where HsfA1a is either overexpressed (A1aOE) or suppressed (A1CS), indicating an interplay between CPR and ER-UPR systems. Several ER-UPR genes are differentially expressed in the HsfA1a transgenic lines either exposed to heat stress or to the ER stress elicitor tunicamycin (TUN). The ectopic expression of HsfA1a is associated with higher tolerance against TUN. On the example of the ER-resident Hsp70 chaperone BIP3, we show that the presence of cis-elements required for HSF and bZIP regulation serves as a putative platform for the co-regulation of these genes by both CPR and ER-UPR mechanisms, in the case of BIP3 in a stimulatory manner under high temperatures. In addition, we show that the accumulation of HsfA1a results in higher levels of three ATG genes and a more sensitized induction of autophagy in response to ER stress which also supports the increased tolerance to ER stress of the A1aOE line. These findings provide a basis for the coordination of protein homeostasis in different cellular compartments under stress conditions.

Potential therapeutic effects and pharmacological evidence of sinomenine in central nervous system disorders.

Sinomenine is a natural compound extracted from the medicinal plant Sinomenium acutum. Its supplementation has been shown to present benefits in a variety of animal models of central nervous system (CNS) disorders, such as cerebral ischemia, intracerebral hemorrhage, traumatic brain injury (TBI), Alzheimer’s disease (AD), Parkinson’s disease (PD), epilepsy, depression, multiple sclerosis, morphine tolerance, and glioma. Therefore, sinomenine is now considered a potential agent for the prevention and/or treatment of CNS disorders. Mechanistic studies have shown that inhibition of oxidative stress, microglia- or astrocyte-mediated neuroinflammation, and neuronal apoptosis are common mechanisms for the neuroprotective effects of sinomenine. Other mechanisms, including activation of nuclear factor E2-related factor 2 (Nrf2), induction of autophagy in response to inhibition of protein kinase B (Akt)-mammalian target of rapamycin (mTOR), and activation of cyclic adenosine monophosphate-response element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF), may also mediate the anti-glioma and neuroprotective effects of sinomenine. Sinomenine treatment has also been shown to enhance dopamine receptor D2 (DRD2)-mediated nuclear translocation of αB-crystallin (CRYAB) in astrocytes, thereby suppressing neuroinflammation via inhibition of Signal Transducer and Activator of Transcription 3 (STAT3). In addition, sinomenine supplementation can suppress N-methyl-D-aspartate (NMDA) receptor-mediated Ca2+ influx and induce γ-aminobutyric acid type A (GABAA) receptor-mediated Cl− influx, each of which contributes to the improvement of morphine dependence and sleep disturbance. In this review, we outline the pharmacological effects and possible mechanisms of sinomenine in CNS disorders to advance the development of sinomenine as a new drug for the treatment of CNS disorders.

USP15 negatively regulates lung cancer progression through the TRAF6-BECN1 signaling axis for autophagy induction

TNF receptor-associated factor 6 (TRAF6)-BECN1 signaling axis plays a pivotal role in autophagy induction through ubiquitination of BECN1, thereby inducing lung cancer migration and invasion in response to toll-like receptor 4 (TLR4) stimulation. Herein, we provide novel molecular and cellular mechanisms involved in the negative effect of ubiquitin-specific peptidase 15 (USP15) on lung cancer progression. Clinical data of the TCGA and primary non-small cell lung cancer (NSCLC) patients (n = 41) revealed that the expression of USP15 was significantly downregulated in lung cancer patients. Importantly, USP15-knockout (USP15KO) A549 and USP15KO H1299 lung cancer cells generated with CRISPR-Cas9 gene-editing technology showed increases in cancer migration and invasion with enhanced autophagy induction in response to TLR4 stimulation. In addition, biochemical studies revealed that USP15 interacted with BECN1, but not with TRAF6, and induced deubiquitination of BECN1, thereby attenuating autophagy induction. Notably, in primary NSCLC patients (n = 4) with low expression of USP15, 10 genes (CCNE1, MMP9, SFN, UBE2C, CCR2, FAM83A, ETV4, MYO7A, MMP11, and GSDMB) known to promote lung cancer progression were significantly upregulated, whereas 10 tumor suppressor genes (FMO2, ZBTB16, FCN3, TCF21, SFTPA1B, HPGD, SOSTDC1, TMEM100, GDF10, and WIF1) were downregulated, providing clinical relevance of the functional role of USP15 in lung cancer progression. Taken together, our data demonstrate that USP15 can negatively regulate the TRAF6-BECN1 signaling axis for autophagy induction. Thus, USP15 is implicated in lung cancer progression.

Dietary Phosphorus Reduced Hepatic Lipid Deposition by Activating Ampk Pathway and Beclin1 Phosphorylation Levels to Activate Lipophagy in Tilapia Oreochromis niloticus

High-phosphorus diet (HPD) reduces lipid deposition and significantly influences lipid metabolism. However, the relevant mechanism is unknown. Herein, using widely-cultured teleost tilapia Oreochromis niloticus as the experimental animals, we found that HPD and Pi incubation reduced triglyceride (TG) content (P ≤ 0.05), suppressed lipogenesis, activated AMP-activated protein kinase (AMPK) pathway and autophagy (P ≤ 0.05), and increased fatty acid β-oxidation and lipolysis in tilapia liver and hepatocytes (P ≤ 0.05). Our further investigation indicated that Pi treatments activated the lipophagy and facilitated mitochondrial fatty acid β-oxidation, and according reduced TG deposition (P ≤ 0.05). Mechanistically, phosphorus increased the AMPKα1 phosphorylation level at S496 and Beclin1 phosphorylation at S90, and Beclin1 phosphorylation by AMPKα1 was required for phosphorus-induced lipophagy and lipolysis. Our study revealed a mechanism for Beclin1 regulation and autophagy induction in response to high-phosphorus diet, and provided novel evidences for the link between dietary phosphorus addition and lipolytic metabolism via the AMPK/Beclin1 pathway. Our results also suggested that AMPK should be the potential target for the prevention and control of lipid metabolic disorders. Overall, these results suggested that HPD reduced hepatic lipid deposition by activating AMPK pathway and Beclin1 phosphorylation levels to activate lipophagy, which provided potential targets for the prevention and control of fatty liver in fish.

Hepatitis B virus X Protein Promotes Liver Cancer Progression through Autophagy Induction in Response to TLR4 Stimulation.

Hepatitis B virus X (HBx) protein has been reported as a key protein regulating the pathogenesis of HBV-induced hepatocellular carcinoma (HCC). Recent evidence has shown that HBx is implicated in the activation of autophagy in hepatic cells. Nevertheless, the precise molecular and cellular mechanism by which HBx induces autophagy is still controversial. Herein, we investigated the molecular and cellular mechanism by which HBx is involved in the TRAF6-BECN1-Bcl-2 signaling for the regulation of autophagy in response to TLR4 stimulation, therefore influencing the HCC progression. HBx interacts with BECN1 (Beclin 1) and inhibits the association of the BECN1-Bcl-2 complex, which is known to prevent the assembly of the pre-autophagosomal structure. Furthermore, HBx enhances the interaction between VPS34 and TRAF6-BECN1 complex, increases the ubiquitination of BECN1, and subsequently enhances autophagy induction in response to LPS stimulation. To verify the functional role of HBx in liver cancer progression, we utilized different HCC cell lines, HepG2, SK-Hep-1, and SNU-761. HBx-expressing HepG2 cells exhibited enhanced cell migration, invasion, and cell mobility in response to LPS stimulation compared to those of control HepG2 cells. These results were consistently observed in HBx-expressed SK-Hep-1 and HBx-expressed SNU-761 cells. Taken together, our findings suggest that HBx positively regulates the induction of autophagy through the inhibition of the BECN1-Bcl-2 complex and enhancement of the TRAF6-BECN1-VPS34 complex, leading to enhance liver cancer migration and invasion.

Particulate β-glucan activates early and delayed phagosomal maturation and autophagy within macrophage in a NOX-2 dependent manner

AimsMacrophage is known to readily engulf any particulate material they encounter, including invading microbes and nano- or micro-particles. While recent studies show that some microparticles (MP) are immunogenic even without drug-cargo, the mechanism underlying this phenomenon is yet unclear. Phagocytosis induces NADPH oxidase-2 (NOX-2) mediated ROS generation that is reported to regulate antibacterial autophagy. We therefore, investigated the role of NOX-2 derived ROS in phagosomal maturation and autophagy induction in response to phagocytic uptake of two kinds of polymeric biodegradable and biocompatible microparticles: yeast-derived β-glucan particles (YDGP) and poly-(D, L-Lactic Acid) microparticles (PMP). Main methodsJ774A.1 macrophage wereas exposed to polymeric particles and the immune responses: ROS, phagosomal maturation and autophagy induction, were examined by assays including NBT, DCFH-DA, NADPH-Oxidase activity, Lysotracker and Acridine Orange. Further, the LC3 and NOX-2 expression were validated by RT-PCR, immunofluorescence assay and Western blotting. Antimicrobial activity of both MP was examined by CFU counting after administration to Mycobacterium tuberculosis and Salmonella typhimurium infected macrophage. Key findingsYDGP induces phagosomal maturation and acidic vesicle accumulation at 30 min and 24 h post-exposure, much more proficiently than that by PMP. YDGP exposure also induced NOX-2 dependent expression of light chain 3 (LC3-II), further confirmed as autophagy activation via autophagic flux assay with autophagolysosome inhibitor bafilomycin A1. Additionally, YDGP displayed superior anti-microbial activity than that by PMP. SignificanceThe induction of NOX-2-dependent autophagy and antimicrobial activity exhibited by particulate glucans has significant implications in harnessing these drug delivery vehicles as potential ‘value-added’ autophagy-mediated therapeutics in future.

Dietary Energy Modulation and Autophagy: Exploiting Metabolic Vulnerabilities to Starve Cancer.

Cancer cells experience unique and dynamic shifts in their metabolic function in order to survive, proliferate, and evade growth inhibition in the resource-scarce tumor microenvironment. Therefore, identification of pharmacological agents with potential to reprogram cancer cell metabolism may improve clinical outcomes in cancer therapy. Cancer cells also often exhibit an increased dependence on the process known as autophagy, both for baseline survival and as a response to stressors such as chemotherapy or a decline in nutrient availability. There is evidence to suggest that this increased dependence on autophagy in cancer cells may be exploitable clinically by combining autophagy modulators with existing chemotherapies. In light of the increased metabolic rate in cancer cells, interest is growing in approaches aimed at “starving” cancer through dietary and pharmacologic interventions that reduce availability of nutrients and pro-growth hormonal signals known to promote cancer progression. Several dietary approaches, including chronic calorie restriction and multiple forms of fasting, have been investigated for their potential anti-cancer benefits, yielding promising results in animal models. Induction of autophagy in response to dietary energy restriction may underlie some of the observed benefit. However, while interventions based on dietary energy restriction have demonstrated safety in clinical trials, uncertainty remains regarding translation to humans as well as feasibility of achieving compliance due to the potential discomfort and weight loss that accompanies dietary restriction. Further induction of autophagy through dietary or pharmacologic metabolic reprogramming interventions may enhance the efficacy of autophagy inhibition in the context of adjuvant or neo-adjuvant chemotherapy. Nonetheless, it remains unclear whether therapeutic agents aimed at autophagy induction, autophagy inhibition, or both are a viable therapeutic strategy for improving cancer outcomes. This review discusses the literature available for the therapeutic potential of these approaches.

Activation of Mechanistic Target of Rapamycin (mTOR) in Human Endothelial Cells Infected with Pathogenic Spotted Fever Group Rickettsiae.

Attributed to the tropism for host microvascular endothelium lining the blood vessels, vascular inflammation and dysfunction represent salient features of rickettsial pathogenesis, yet the details of fundamentally important pathogen interactions with host endothelial cells (ECs) as the primary targets of infection remain poorly appreciated. Mechanistic target of rapamycin (mTOR), a serine/threonine protein kinase of the phosphatidylinositol kinase-related kinase family, assembles into two functionally distinct complexes, namely mTORC1 (Raptor) and mTORC2 (Rictor), implicated in the determination of innate immune responses to intracellular pathogens via transcriptional regulation. In the present study, we investigated activation status of mTOR and its potential contributions to host EC responses during Rickettsia rickettsii and R. conorii infection. Protein lysates from infected ECs were analyzed for threonine 421/serine 424 phosphorylation of p70 S6 kinase (p70 S6K) and that of serine 2448 on mTOR itself as established markers of mTORC1 activation. For mTORC2, we assessed phosphorylation of protein kinase B (PKB or Akt) and protein kinase C (PKC), respectively, on serine 473 and serine 657. The results suggest increased phosphorylation of p70 S6K and mTOR during Rickettsia infection of ECs as early as 3 h and persisting for up to 24 h post-infection. The steady-state levels of phospho-Akt and phospho-PKC were also increased. Infection with pathogenic rickettsiae also resulted in the formation of microtubule-associated protein 1A/1B-light chain 3 (LC3-II) puncta and increased lipidation of LC3-II, a response significantly inhibited by introduction of siRNA targeting mTORC1 into ECs. These findings thus yield first evidence for the activation of both mTORC1 and mTORC2 during EC infection in vitro with Rickettsia species and suggest that early induction of autophagy in response to intracellular infection might be regulated by this important pathway known to function as a central integrator of cellular immunity and inflammation.

Class I PI3K Is Required for HSC Differentiation

Adult hematopoietic stem cells (HSCs) are a rare and unique population of stem cells that reside in the bone marrow, where they undergo self-renewal and differentiation to maintain the blood system. The maintenance of a proper balance between HSC self-renewal and differentiation requires growth factors, cytokines, and chemokines, most of which activate the phosphoinositide 3-kinase/Protein Kinase B (PI3K/AKT) signaling pathway. Pathologic activation of the AKT pathway is frequently observed in tumors, making it a desirable target for cancer treatment. Since several PI3K inhibitors are now in clinical use, it is critical to determine the roles of PI3K in adult HSCs. However, the specific roles of PI3K in HSC function are poorly understood. Hematopoietic cells express three Class IA catalytic PI3K isoforms (P110α, β, and δ), which can all transduce growth factor and cytokine signals, and can compensate for one another in some cell types. Individual Class 1A PI3K isoforms have unique functions in mature hematopoietic lineages, but are dispensable for HSC function. To uncover the potentially redundant roles of PI3K isoforms in HSCs, we have generated a triple knockout (TKO) mouse model with conditional deletion of p110α and p110β in hematopoietic cells using MX1-Cre, and germline deletion of p110δ. TKO mice develop pancytopenia, which is also observed upon transplantation of TKO bone marrow. Competitive repopulation assays reveal a defect in long-term multi-lineage chimerism. Surprisingly, loss of Class 1A PI3K causes significant expansion of donor-derived long-term (Lin-cKit+Flk2-CD150+CD48-) and short-term (Lin-cKit+Flk2-CD150-CD48-) HSCs in the bone marrow, but not committed progenitors. This phenotype could not be explained by alterations in HSC cell cycling or apoptosis in TKO HSCs. TKO transplant recipients also have dysplastic features in the bone marrow. Methylcellulose plating assays of TKO bone marrow revealed a relative increase in granulocyte erythroid macrophage megakaryocyte (GEMM) colonies and extended serial replating, suggesting increased self-renewal. Thus, our data are consistent with impaired HSC differentiation upon deletion of all Class IA PI3K isoforms, which leads to dysplastic changes. RNA sequencing of sorted long-term HSCs from the bone marrow of TKO transplant recipients revealed the enrichment of human and mouse HSC signatures, and the downregulation of DNA repair gene sets and RNA splicing gene sets in TKO HSCs. Interestingly, we also observed downregulation of autophagy gene sets in TKO HSCs. Macroautophagy has been shown to be essential for the maintenance of HSC metabolism and self-renewal. Analysis of the autophagosomal marker LC3-II in TKO HSCs revealed a decrease in autophagy upon growth factor deprivation. Surprisingly, we observed an increase in MTOR activation in TKO cKit+ bone marrow cells via compensatory signaling through the MAPK pathway. Given that MTOR is a known negative regulator of autophagy, this is consistent with the observed autophagy decrease in TKO HSCs. Additionally, we found that autophagy can still be induced in TKO HSCs with the MTOR inhibitor rapamycin. Furthermore, rapamycin treatment impairs serial replating of TKO bone marrow cells. In conclusion, we found that inactivation of all Class 1A PI3 kinases leads to impaired HSC differentiation, likely due to a defect in autophagy induction in response to growth factor deprivation. Disclosures No relevant conflicts of interest to declare.

TORC1 regulates autophagy induction in response to proteotoxic stress in yeast and human cells

Misfolded and aggregated proteins are eliminated to maintain protein homeostasis. Autophagy contributes to the removal of protein aggregates. However, if and how proteotoxic stress induces autophagy is poorly understood. Here we show that proteotoxic stress after treatment with azetidine-2-carboxylic acid (AZC), a toxic proline analog, induces autophagy in budding yeast. AZC treatment attenuated target of rapamycin complex 1 (TORC1) activity, resulting in the dephosphorylation of Atg13, a key factor of autophagy. By contrast, AZC treatment did not affect target of rapamycin complex 2 (TORC2). Proteotoxic stress also induced TORC1 inactivation and autophagy in fission yeast and human cells. This study suggested that TORC1 is a conserved key factor to cope with proteotoxic stress in eukaryotic cells.

20(S)-ginsenoside Rg3 sensitizes human non-small cell lung cancer cells to icotinib through inhibition of autophagy

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR–TKIs) have become a standard therapy for non-small cell lung cancer (NSCLC) patients with sensitive mutations. However, acquired resistance inevitably emerges after a median of 6–12 months. It has been demonstrated that autophagy plays an important role in EGFR–TKI resistance. 20(S)-ginsenoside Rg3 (Rg3) is proposed to sensitize the cancer cells to chemotherapy by inhibiting autophagy. We examined the ability of Rg3 to inhibit autophagy and increase the sensitivity of NSCLC cells to icotinib. We show that the induction of autophagy in response to icotinib contributes to the development of icotinib resistance. Rg3 is capable of inhibiting autophagic flux and enhancing the sensitivity of NSCLC cells to icotinib. The resistance to icotinib could also be reversed through Rg3-induced autophagy inhibition. Autophagy inhibition by Rg3 increases the therapeutic response in both icotinib-sensitive and icotinib-resistant NSCLC cells with an EGFR-activating mutation and may be an effective new treatment strategy for this disease.

AMPK Promotes Xenophagy through Priming of Autophagic Kinases upon Detection of Bacterial Outer Membrane Vesicles

The autophagy pathway is an essential facet of theinnate immune response, capable of rapidly targeting intracellular bacteria. However, the initial signaling regulating autophagy induction in response to pathogens remains largely unclear. Here, we report that AMPK, an upstream activator of the autophagy pathway, is stimulated upon detection of pathogenic bacteria, before bacterial invasion. Bacterial recognition occurs through the detection of outer membrane vesicles. We found that AMPK signaling relieves mTORC1-mediated repression of the autophagy pathway in response to infection, positioning the cell for a rapid induction of autophagy. Moreover, activation of AMPK and inhibition of mTORC1 in response to bacteria is not accompanied by an induction of bulk autophagy. However, AMPK signaling is required for the selective targeting of bacteria-containing vesicles by the autophagy pathway through the activation of pro-autophagic kinase complexes. These results demonstrate a key role for AMPK signaling in coordinating the rapid autophagic response to bacteria.

The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection

Candida albicans is an opportunistic fungus that is part of the normal microflora commonly found in the human digestive tract and the normal mucosa or skin of healthy individuals. However, in immunocompromised individuals, it becomes a serious health concern and a threat to their lives and is ranked as the leading fungal infection in humans worldwide. As existing treatments for this infection are non-specific or under threat of developing resistance, there is a dire necessity to find new targets for designing specific drugs to defeat this fungus. Some authors reported the presence of the transglutaminase activity in Candida and Saccharomyces, but its identity remains unknown. We report here the phenotypic effects produced by the inhibition of transglutaminase enzymatic activity with cystamine, including growth inhibition of yeast cells, induction of autophagy in response to damage caused by cystamine, alteration of the normal yeast division pattern, changes in cell wall, and inhibition of the yeast-to-mycelium transition. The latter phenomenon was also observed in the C. albicans ATCC 26555 strain. Growth inhibition by cystamine was also determined in other Candida strains, demonstrating the importance of transglutaminase in these species. Finally, we identified enolase 1 as the cell wall protein responsible for TGase activity. After studying the inhibition of enzymatic activities with anti-CaEno1 antibodies and through bioinformatics studies, we suggest that the enolase and transglutaminase catalytic sites are localized in different domains of the protein. The aforementioned data indicate that TGase/Eno1 is a putative target for designing new drugs to control C. albicans infection.

Abstract 3307: Induction of autophagy as a mechanism of therapeutic resistance in head and neck cancer

Abstract Background: In the curative setting for head and neck cancer (HNC) a commonly used treatment is radiation combined with the anti-EGFR monoclonal antibody cetuximab (CTX). However, despite decades of research into improved treatments, therapeutic resistance remains a major challenge for this malignancy with roughly 40% of patients developing recurrent disease. We are studying the molecular mechanisms by which radiation and cetuximab induce autophagy and how this process can be modulated to improve the efficacy of therapy. Methods: We evaluated a panel of both human papillomavirus (HPV) positive and negative HNC cell lines for autophagic response to both CTX treatment and ionizing radiation (XRT). Induction of autophagy was detected by immunoblot flux assays for LC3 and p62 and by immunofluorescent staining of autophagic vesicles. siRNA knockdown of EGFR, LAMPT4B, ULK1, Beclin1 and others was used to probe the involvement of different signaling molecules. The addition of specific autophagy inhibitors, such as the ULK1 inhibitor SBI-0206965, to CTX or XRT treatment was tested to determine whether the reduction of autophagic response reduced cell survival in a clonogenic survival assay. Induction of apoptosis was analyzed by immunoblot against cleaved Caspase and PARP and AnnexinV staining. Results: Flux assays revealed that both CTX and XRT induced autophagy in a time and dose dependent manner. Immunofluorescent staining of LC3 to identify autophagic vesicles showed that a relatively small fraction of the total cell population is able to induce this response. Specific siRNA knockdown of EGFR and LAMPT4B was able to abrogate the induction of autophagy in response to both CTX and XRT. Involvement of alternative downstream pathways involving either ULK1 and Beclin1 or p53 in response to either treatment is under investigation. To determine whether blockade of cytoprotective autophagy can help overcome therapeutic resistance, we tested specific autophagy inhibitors in combination with either CTX or XRT treatment. The addition of the ULK1 inhibitor to CTX or XRT induced apoptosis as shown by caspase activity, AnnexinV staining, and reduced cell survival in clonogenic assays. Conclusions: Autophagy may play a critical and protective role in how head and neck cancers respond to therapeutic stress. The addition of specific autophagy inhibitors to standard treatments may provide a way to overcome resistance to therapy. Citation Format: Jaimee C. Eckers, Adam D. Swick, Tyler Fowler, Justin Skiba, Kwangok P. Nickel, Randall J. Kimple. Induction of autophagy as a mechanism of therapeutic resistance in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3307. doi:10.1158/1538-7445.AM2017-3307

Abstract 4501: CEBPD is an early endoplasmic reticulum stress response gene implicated in breast cancer cell survival

Abstract CEBPD (C/EBP delta) is a member of the CCAAT-enhancer binding protein (C/EBP) family of transcription factors characterized by a b-Zip domain that mediates dimerization and DNA binding. CEBPD is induced in response to acute stressors such as cytokine stimulation, bacterial lipopolysaccharide (LPS), corticosteroids, radiation and hypoxia. We have previously reported that CEBPD has dual functions in breast cancer by both attenuating or enhancing oncogenic pathways depending on context (Balamurugan and Sterneck, 2013, Mendoza-Villanueva et al., 2016). Recent studies reveal that elevated Endoplasmic Reticulum (ER) stress is associated with the pathology of several diseases including cancer. Limiting supply of nutrients and oxygen in growing tumor cells disrupts the protein folding homeostasis resulting in activation of the unfolded protein response (UPR). The UPR includes pathways that support adaptation to stress, and that are also implicated in promoting malignant features and therapy resistance in breast cancer. Hence, we investigated a possible role for CEBPD in this stress response pathway. Using breast cancer cell lines as model systems, we found that CEBPD is dynamically regulated in response to chemical and physiological inducers of ER stress. Kinetic analyses indicate that CEBPD induction is concurrent with the activation of the UPR effectors XBP1s and ATF4 suggesting a potential role in stress adaptation. Examination of the global transcriptional prolife of the Thapsigargin-induced stress response in MDA MB-231 cells indicated that CEBPD activates genes involved in cytokine and chemokine production/signaling, immune cell recruitment as well as angiogenesis pathways. While XBP1s has been previously ascribed to activating these pathways, the levels of activated XBP1 were not affected by CEBPD silencing. This is the first report of a similar role for CEBPD in the ER stress response and warrants the investigation of possible co-operation of CEBPD and XBP1 in the UPR pathway. Further, we show that knock down of CEBPD in cancer cells increased PARP cleavage and susceptibility to ER stress induced cell death. CEBPD deficient cells also displayed reduced LC3II accumulation, suggesting impaired autophagy induction in response to ER stress. Taken together, our data suggests that CEBPD is a novel proximal effector of the ER stress response in breast cancer cells. Activation of this transcription factor regulates tumor promoting inflammatory and angiogenic signaling pathways. Additionally, CEBPD expression confers a survival advantage to cancer cells encountering ER stress. Therefore, we propose that targeting CEBPD may sensitize cancer cells to ER stress inducing therapeutics. Citation Format: Namratha Sheshadri, Shikha Sharan, Esta Sterneck. CEBPD is an early endoplasmic reticulum stress response gene implicated in breast cancer cell survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4501. doi:10.1158/1538-7445.AM2017-4501

Nicotinamide protects hepatocytes against palmitate-induced lipotoxicity via SIRT1-dependent autophagy induction

Lipotoxicity induced by saturated fatty acids (SFAs) plays a pathological role in the development of non-alcoholic fatty liver disease (NAFLD); however, the exact mechanism remains to be clearly elucidated. Palmitate is the most abundant SFA in the circulation and major lipotoxic inducer. Accumulating evidence supports that autophagy induction is protective against palmitate-induced cell death in a variety of cell types, including hepatocytes. Nicotinamide is the amide form of nicotinic acid (vitamin B3, Niacin) and a dietary supplementation as a source of vitamin B3. We previously reported that nicotinamide endowed hepatocytes resistance to palmitate-induced ER stress via up-regulating SIRT1, with cAMP/PKA/CREB pathway activation being a fundamental mechanism. This study was undertaken to investigate the potential anti-lipotoxic effect of nicotinamide and to elucidate underlying mechanism(s). Our data demonstrated that nicotinamide supplementation protected hepatocytes against palmitate-induced cell death. Mechanistic investigations revealed that nicotinamide supplementation activated autophagy in hepatocytes. Importantly, we showed that the anti-lipotoxic property of nicotinamide was abolished by autophagy inhibitors, suggesting that autophagy induction plays a mechanistic role in nicotinamide's anti-lipotoxic effect. Furthermore, we showed that SIRT1 inhibition blunted autophagy induction in response to nicotinamide supplementation and similarly abrogated the anti-lipotoxic effect conferred by nicotinamide supplementation. In conclusion, our data suggest that nicotinamide protects against palmitate-induced hepatotoxicity via SIRT1-dependent autophagy induction and that nicotinamide supplementation may represent a therapeutic choice for NAFLD.

Autophagy impacts on oxaliplatin-induced hepatocarcinoma apoptosis via the IL-17/IL-17R-JAK2/STAT3 signaling pathway.

The interleukin (IL)-17/IL-17 receptor (IL-17R) complex has been shown to be important for the regulation of inflammation; however, its role in the regulation of tumor processes has recently emerged as a research focus. The present study demonstrated that oxaliplatin was able to increase the levels of IL-17/IL-17R in hepatocellular carcinoma (HCC) patients and cells lines, and that it had important roles in reducing the susceptibility of the cells to oxaliplatin-induced apoptosis. Furthermore, the expression of autophagy-related proteins was induced by IL-17/IL-17R and autophagy was shown to induce resistance to oxaliplatin in HCC. In addition, the janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway was shown to be an important pathway in the induction of autophagy in response to oxaliplatin. Autopjhagy was inhibited by 3-methyladenine and JAK2/STAT3 signaling was blocked by AG490, which induced apoptosis in SMMC7721 cells treated with oxaliplatin. The results of the present study may help to elucidate the mechanism underlying the role of IL-17/IL-17R-induced autophagy in the chemoresistance of HCC, as well as help to establish and develop measures to overcome chemoresistance in HCC.

Downregulation of miR-17-92a cluster promotes autophagy induction in response to celastrol treatment in prostate cancer cells

Celastrol has potential application for the treatment of prostate cancer. However it causes autophagy as a protective response in prostate and other types of cancers, thus unveiling the underlying mechanisms may benefit its future application. In the present study, we demonstrate that the miR-17-92a cluster plays a negative role in celastrol induced-autophagy. Dissection of miR-17-92a cluster revealed the role of miR-17 seed family (miR-20a and miR-17) in autophagy inhibition in the context of prostate cancer cells. Autophagy-related gene ATG7 was validated as a target of miR-17 seed family by dual-luciferase assay and qPCR. Celastrol induced autophagy was inhibited by miR-20a or miR-17, while the inhibitory effects were rescued in the presence of pcDNA-ATG7 lacking 3′ UTR, demonstrating that these two members target ATG7 to inhibit celastrol-induced autophagy. As celastrol degrades androgen receptor (AR), a key transcription factor in prostate cancer cells, we further investigated whether AR affected miR-17-92a expression in prostate cancer cells. AR binding sites were found in the promoter and two introns of miR-17-92a. In addition, higher expression levels of miR-17-92a were observed in AR positive cells compared with AR negative cells. Ectopic expression of AR could enhance the expression of miR-17-92a cluster in AR-negative prostate cancer cells while knockdown of AR decreased miR-17-92a expression in AR-positive cells, demonstrating the regulation of AR on miR-17-92a transcription. In summary, our results demonstrate that celastrol downregulates AR and its target miR-17-92a, leading to autophagy induction in prostate cancer cells.

Abstract 2129: Induction of autophagy as a mechanism of therapeutic resistance in head and neck cancer

Abstract Background and purpose: Despite decades of research into improved treatments for head and neck cancer, therapeutic resistance remains a major challenge for this malignancy, with roughly 40% of patients developing recurrent disease. Among the plethora of intrinsic and acquired mechanisms of resistance, recent evidence has suggested the cellular process of autophagy may be an additional contributor. This stress response, which normally functions during periods of nutrient deprivation to maintain cellular homeostasis, may have the unintended consequence of protecting tumor cells from the cytotoxic effects of chemotherapy and radiotherapy. We show that autophagy mediates resistance to chemotherapies targeting the EGFR and mTORC pathways in addition to radiotherapy in several head and neck cancer models, begin to elucidate the mechanism of this resistance, and demonstrate that the addition of a specific autophagy inhibitor can block cytoprotective autophagy. Methods and results: Building on well established evidence that mTORC inhibition can directly induce autophagy, recent work has established a novel mechanism by which kinase inactive EGFR resulting from either growth factor starvation or anti-EGFR antibody (cetuximab) can upregulate this response via an interaction with LAMPT4B. Treatment of a panel of HNC cell lines with either serum starvation or cetuximab produced increased formation of LC3-B puncta, a marker for autophagasome formation, by IF staining, and was confirmed by Western blot for increased LC3-B flux and decreased p62. SiRNA knockdown of either EGFR or LAMPT4B in HNC cells abrogated the induction of autophagy in response to either serum starvation or cetuximab treatment. We have also shown that radiation induces autophagy in a time and dose dependent manner. The mechanism of radiation-induced autophagy remains a subject of study; preliminary data suggest that both EGFR and LAMPT4B play a role as siRNA knockdown of those mRNAs reduces the autophagic response. To determine whether blockade of cytoprotective autophagy can help overcome therapeutic resistance, we have tested specific autophagy inhibitors in combination with either chemotherapy or radiation. The novel ULK1 inhibitor SBI-0206965 successfully blocks the induction of autophagy in a panel of HNC lines. More compellingly, while neither SBI-0206965 nor the NEDD4 inhibitor Heclin, impacted cell growth in untreated cells, both were effective in reducing cell survival following treatment with radiation, cetuximab, or mTORC inhibitor (AZD8055). Summary: These pre-clinical studies have established the proof of concept for the cytoprotective effect of autophagy in response to anti-cancer treatments including EGFR or mTORC inhibition and radiotherapy. Further we have identified the addition of specific autophagy inhibitors to standard treatments as a potential strategy to overcome this mechanism of resistance. Citation Format: Adam D. Swick, Tan Xiaojun, Tyler Fowler, Kwangok Nickel, Richard A. Anderson, Randall J. Kimple. Induction of autophagy as a mechanism of therapeutic resistance in head and neck cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2129.