Role Of Autophagy In Response Research Articles

Prenatal diethylhexylphthalate exposure disturbs adult Leydig cell function via epigenetic downregulation of METTL4 expression in male rats

Prenatal exposure to diethylhexyl phthalate (DEHP) has been linked with a decline in testosterone levels in adult male rats, but the underlying mechanism remains unclear. We investigated the potential epigenetic regulation, particularly focusing on N6-methyladenosine (m6A) modification, as a possible mechanism. Dams were gavaged with DEHP (0, 10, 100, and 750 mg/kg/day) from gestational day 14 to day 21. The male offspring were examined at the age of 56 days. Prenatal DEHP administration at 750 mg/kg/day caused a decline in testosterone concentrations, an elevation in follicle-stimulating hormone, a downregulated expression of CYP11A1 HSD3B2, without affecting Leydig cell numbers. Interestingly, Methyltransferase Like 4 (METTL4), an m6A methyltransferase, was downregulated, while there were no changes in METTL3 and METTL14. Moreover, CYP11A1 showed m6A reduction in response to prenatal DEHP exposure. Additionally, METTL4 expression increased postnatally, peaking in adulthood. Knockdown of METTL4 resulted in the downregulation of CYP11A1 and HSD3B2 and an increase in SCARB1 expression. Furthermore, the increase in autophagy protection in adult Leydig cells induced by prenatal DEHP exposure was not affected by 3-methyladenosine (3MA) treatment, indicating a potential protective role of autophagy in response to DEHP exposure. In conclusion, prenatal DEHP exposure reduces testosterone by downregulating CYP11A1 and HSD3B2 via m6A epigenetic regulation and induction of autophagy protection in adult Leydig cells as a response to DEHP exposure.

The Cytoprotective Role of Autophagy in Response to BRAF-Targeted Therapies.

BRAF-targeted therapies are widely used for the treatment of melanoma patients with BRAF V600 mutations. Vemurafenib, dabrafenib as well as encorafenib have demonstrated substantial therapeutic activity; however, as is the case with other chemotherapeutic agents, the frequent development of resistance limits their efficacy. Autophagy is one tumor survival mechanism that could contribute to BRAF inhibitor resistance, and multiple studies support an association between vemurafenib-induced and dabrafenib-induced autophagy and tumor cell survival. Clinical trials have also demonstrated a potential benefit from the inclusion of autophagy inhibition as an adjuvant therapy. This review of the scientific literature relating to the role of autophagy that is induced in response to BRAF-inhibitors supports the premise that autophagy targeting or modulation could be an effective adjuvant therapy.

Cytoprotective, Cytotoxic and Cytostatic Roles of Autophagy in Response to BET Inhibitors.

The bromodomain and extra-terminal domain (BET) family inhibitors are small molecules that target the dysregulated epigenetic readers, BRD2, BRD3, BRD4 and BRDT, at various transcription-related sites, including super-enhancers. BET inhibitors are currently under investigation both in pre-clinical cell culture and tumor-bearing animal models, as well as in clinical trials. However, as is the case with other chemotherapeutic modalities, the development of resistance is likely to constrain the therapeutic benefits of this strategy. One tumor cell survival mechanism that has been studied for decades is autophagy. Although four different functions of autophagy have been identified in the literature (cytoprotective, cytotoxic, cytostatic and non-protective), primarily the cytoprotective and cytotoxic forms appear to function in different experimental models exposed to BET inhibitors (with some evidence for the cytostatic form). This review provides an overview of the cytoprotective, cytotoxic and cytostatic functions of autophagy in response to BET inhibitors in various tumor models. Our aim is to determine whether autophagy targeting or modulation could represent an effective therapeutic strategy to enhance the response to these modalities and also potentially overcome resistance to BET inhibition.

Therapeutic Potential for Targeting Autophagy in ER+ Breast Cancer.

Simple SummaryWhile ER+ breast cancer is generally considered to have a better prognosis than other breast cancer subtypes, relapse may nevertheless occur years after diagnosis and treatment. Despite initially responding to treatment, 30–40% of tumors acquire resistance to treatment that contributes to disease recurrence, metastasis, and ultimately, death. In the case of the individual estrogen antagonists or aromatase inhibitors, the autophagy induced by these agents is largely cytoprotective. However, whether autophagy inhibition will prove to be a useful strategy for improving outcomes for current combination therapeutic strategies awaits further studies.While endocrine therapy remains the mainstay of treatment for ER-positive, HER2-negative breast cancer, tumor progression and disease recurrence limit the utility of current standards of care. While existing therapies may allow for a prolonged progression-free survival, however, the growth-arrested (essentially dormant) state of residual tumor cells is not permanent and is frequently a precursor to disease relapse. Tumor cells that escape dormancy and regain proliferative capacity also tend to acquire resistance to further therapies. The cellular process of autophagy has been implicated in the adaptation, survival, and reactivation of dormant cells. Autophagy is a cellular stress mechanism induced to maintain cellular homeostasis. Tumor cells often undergo therapy-induced autophagy which, in most contexts, is cytoprotective in function; however, depending on how the autophagy is regulated, it can also be non-protective, cytostatic, or cytotoxic. In this review, we explore the literature on the relationship(s) between endocrine therapies and autophagy. Moreover, we address the different functional roles of autophagy in response to these treatments, exploring the possibility of targeting autophagy as an adjuvant therapeutic modality together with endocrine therapies.

Is autophagy induction by PARP inhibitors a target for therapeutic benefit?

PARP inhibitors have proven to be effective in conjunction with conventional therapeutics in the treatment of various solid as well as hematologic malignancies, particularly when the tumors are deficient in DNA repair pathways. However, as the case with other chemotherapeutic agents, their effectiveness is often compromised by the development of resistance. PARP inhibitors have consistently been reported to promote autophagy, a process that maintains cellular homeostasis and acts as an energy source by the degradation and reutilization of damaged subcellular organelles and proteins. Autophagy can exhibit different functional properties, the most prominent being cytoprotective. In addition, both cytotoxic and non-protective functions forms have also been identified. In this review, we explore the available literature regarding the different roles of autophagy in response to clinically-used PARP inhibitors, highlighting the possibility of targeting autophagy as an adjuvant therapy to potentially increase the effectiveness of PARP inhibition and to overcome the development of resistance.

Identification of Autophagy-Related Genes in the Potato Psyllid, Bactericera cockerelli and Their Expression Profile in Response to 'Candidatus Liberibacter Solanacearum' in the Gut.

Simple SummaryIn North America, the bacterial plant pathogen ‘Candidatus Liberibacter solanacearum’ (Lso) infects solanaceous plants. Currently, Lso haplotypes, LsoA and LsoB are transmitted by potato psyllid, Bactericera cockerelli (Šulc). Because these bacteria are transmitted in a circulative and persistent manner, the gut of the psyllid is the first organ they encounter and could be a barrier to its transmission. Therefore, it is important to understand the molecular mechanisms involved in Lso acquisition and transmission. This study explored if an autophagic response was triggered in response to LsoA and/or LsoB in the gut of the adult potato psyllid. The results showed that Lso may induce the autophagic response in the adult psyllid gut since the majority of autophagy-related genes (ATGs) are sensitive and responsive to the exposure or infection of both LsoA and LsoB. Therefore, this study represents a stepping-stone towards understanding the molecular mechanisms involved in Lso transmission.Autophagy, also known as type II programmed cell death, is a cellular mechanism of “self-eating”. Autophagy plays an important role against pathogen infection in numerous organisms. Recently, it has been demonstrated that autophagy can be activated and even manipulated by plant viruses to facilitate their transmission within insect vectors. However, little is known about the role of autophagy in the interactions of insect vectors with plant bacterial pathogens. ‘Candidatus Liberibacter solanacearum’ (Lso) is a phloem-limited Gram-negative bacterium that infects crops worldwide. Two Lso haplotypes, LsoA and LsoB, are transmitted by the potato psyllid, Bactericera cockerelli and cause damaging diseases in solanaceous plants (e.g., zebra chip in potatoes). Both LsoA and LsoB are transmitted by the potato psyllid in a persistent circulative manner: they colonize and replicate within psyllid tissues. Following acquisition, the gut is the first organ Lso encounters and could be a barrier for transmission. In this study, we annotated autophagy-related genes (ATGs) from the potato psyllid transcriptome and evaluated their expression in response to Lso infection at the gut interface. In total, 19 ATGs belonging to 17 different families were identified. The comprehensive expression profile analysis revealed that the majority of the ATGs were regulated in the psyllid gut following the exposure or infection to each Lso haplotype, LsoA and LsoB, suggesting a potential role of autophagy in response to Lso at the psyllid gut interface.

Pharmacokinetic and Pharmacodynamic Assessment of Hydroxychloroquine in Breast Cancer.

Hydroxychloroquine (HCQ) is being tested in a number of human clinical trials to determine the role of autophagy in response to standard anticancer therapies. However, HCQ pharmacodynamic (PD) responses are difficult to assess in patients, and preclinical studies in mouse models are equivocal with regard to HCQ exposure and inhibition of autophagy. Here, pharmacokinetic (PK) assessment of HCQ in non-tumor-bearing mice after intraperitoneal dosing established 60 mg/kg as the human equivalent dose of HCQ in mice. Autophagy inhibition, cell proliferation, and cell death were assessed in two-dimensional (2D) cell culture and three-dimensional tumor organoids in breast cancer. Mice challenged with breast cancer xenografts were then treated with 60 mg/kg HCQ via intraperitoneal dosing, and subsequent PK and PD responses were assessed. Although autophagic flux was significantly inhibited in cells irrespective of autophagy-dependence status, autophagy-dependent tumors had decreased cell proliferation and increased cell death at earlier time points compared with autophagy-independent tumors. Overall, this study shows that 2D cell culture, three-dimensional tumor organoids, and in vivo studies produce similar results, and in vitro studies can be used as surrogates to recapitulate in vivo antitumor responses of HCQ. SIGNIFICANCE STATEMENT: Autophagy-dependent tumors but not autophagy-independent tumors have decreased cell proliferation and increased cell death after single-agent hydroxychloroquine treatment. However, hydroxychloroquine causes decreased autophagic flux regardless of autophagy status, suggesting its clinical efficacy in the context of autophagy inhibition.

Autophagy deficiency exacerbates acute lung injury induced by copper oxide nanoparticles

Copper oxide nanoparticles (CuONPs) are one of the widely used metal nanoparticles in the industrial and commercial fields. Autophagy is an intracellular degradation system that delivers cytoplasmic constituents to the lysosome and has been linked to nanoparticles-induced toxicity. In particular, the roles of autophagy in response to CuONPs have been explored in vitro, although the conclusions are controversial. To clarify the role of autophagy in CuONPs-induced acute lung injury, microtubule-associated protein 1 light chain 3 beta (Map1lc3b or lc3b) knockout mice and their corresponding wild type mice are applied. Our results showed that single-dose intratracheal instillation of CuONPs with dosages of 1.25, 2.5 or 5 mg/kg caused acute lung injury 3 days after treatment in a dose-dependent manner, as evidenced by deteriorative lung histopathology, more infiltration of macrophage cells, increased oxidative stress and copper ions. Loss of lc3b resulted in aggravated lung injury induced by CuONPs, which was probably due to the blockade of mitophagy and consequently the accumulation of aberrant mitochondria with overloaded copper ions. Our study provides the first in vivo evidence that autophagy deficiency exacerbates CuONPs-induced acute lung injury, and highlights that targeting autophagy is a meaningful strategy against CuONPs-associated respiratory toxicity.

Hydrogen peroxide mediates spermidine-induced autophagy to alleviate salt stress in cucumber

ABSTRACT Autophagy, an evolutionally conserved cellular degradation process, plays critical roles in plant development and stress response. Despite the wealth of information on the vital role of autophagy in responses to environmental stresses, little is known about the regulation of autophagy. In this study, we demonstrated that spermidine (Spd), a kind of polyamine, was involved in the regulation of salt tolerance through activating the expression of ATG (autophagy-related) genes and the formation of autophagosomes in cucumber under salt stress. Furthermore, NADPH oxidase-derived apoplastic H2O2-mediated Spd-induced salt tolerance and autophagy. Exogenous Spd significantly increased the tolerance to salt stress and inhibited the accumulation and ubiquitination of insoluble proteins. Foliar application of Spd promoted the transcript levels of ATG genes and autophagosomes formation. Besides, Spd induced the expression of RBOH (respiratory burst oxidase homolog), and the accumulation of H2O2 both in leaves and roots. However, either pretreatment with dimethylthiourea (DMTU, an H2O2 scavenger) or diphenyleneiodonium chloride (DPI, an inhibitor of NADPH oxidase) reduced Spd-induced accumulation of apoplastic H2O2. Importantly, Spd-induced salt tolerance and autophagy were compromised when plants were pretreated with DMTU or DPI. Furthermore, the silencing of ATG4 and ATG7 reduced Spd-induced salt tolerance and autophagosomes formation. Taken together, these results revealed that RBOH-dependent H2O2 mediated the Spd-induced autophagy and salt tolerance in cucumber. Abbreviations: Asat: light-saturated rate of CO2 assimilation; ATG: autophagy-related; DCF-DA: 2, 7-dichlorofluorescein diacetate; DMTU: dimethylthiourea; DPI: diphenyleneiodonium chloride; DW: dry weight; EL: electrolyte leakage; FW: fresh weight; Fv/Fm: the maximum quantum yield of photosystem II; GFP: green fluorescent protein; MDC: monodansylcadaverine; PDS: phytoene desaturase; PE: phosphatidylethanolamine; PLD: phospholipase D; RBOH: respiratory burst oxidase homolog; ROS: reactive oxygen species; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SIN1: salt induced NAC1; Spd: spermidine; TOR: target of rapamycin; VIGS: virus-induced gene silencing.

Autophagy and Its Regulators in Response to Stress in Plants.

To survive in stressful conditions, plants have developed multiple strategies to relieve damage. One of the strategies is to clear the damaged protein and organelles. Autophagy is a highly conservative degradation process, which refers to the recycling of damaged protein and organelles. Over the past decades, increasing evidence has revealed the important roles of autophagy in response to stress conditions, and many factors have been revealed involved in the sophisticated regulation of the autophagy signaling pathway. However, the accurate regulation pathway of the autophagy pathway is largely unknown. The current review proposes how stress-response factors respond to stress conditions involved in regulating the autophagy signaling pathway. In short, clarifying the regulating pathway of autophagy in response to stress conditions is beneficial to plant breeding.

Substrate stiffness differentially impacts autophagy of endothelial cells and smooth muscle cells

Stiffening of blood vessels is one of the most important characteristics in the process of many cardiovascular pathologies such as atherosclerosis, angiosteosis, and vascular aging. Increased stiffness of the vascular extracellular matrix drives artery pathology and alters phenotypes of vascular cell. Understanding how substrate stiffness impacts vascular cell behaviors is of great importance to the biomaterial design in tissue engineering, regenerative medicine, and medical devices. Here we report that changing substrate stiffness has a significant impact on the autophagy of vascular endothelial cells (VECs) and smooth muscle cells (VSMCs). Interestingly, our findings demonstrate that, with the increase of substrate stiffness, the autophagy level of VECs and VSMCs showed differential changes: endothelial autophagy levels reduced, leading to the reductions in a range of gene expression associated with endothelial function; while, autophagy levels of VSMCs increased, showing a transition from contractile to the synthetic phenotype. We further demonstrate that, by inhibiting cell autophagy, the expressions of endothelial functional gene were further reduced and the expression of VSMC calponin increased, suggesting an important role of autophagy in response of the cells to the challenge of microenvironment stiffness changing. Although the underlying mechanism requires further study, this work highlights the relationship of substrate stiffness, autophagy, and vascular cell behaviors, and enlightening the design principles of surface stiffness of biomaterials in cardiovascular practical applications.

Repurposing Ziyuglycoside II Against Colorectal Cancer via Orchestrating Apoptosis and Autophagy.

Effective chemotherapy drugs for colorectal cancer remain a challenge. In this research, Ziyuglycoside II (Ziyu II), exhibits considerable antitumor activity against CRC cells both in vitro and in vivo. The results showed that Ziyu II induced apoptosis through the accumulation of reactive oxygen species (ROS), which was necessary for Ziyu II to inhibit colorectal cancer cells. Intriguingly, The treatment of Ziyu II triggered complete autophagic flux in CRC cells. Inhibition of autophagy partially reversed Ziyu II-induced growth inhibition, demonstrating a cytotoxic role of autophagy in response to Ziyu II-treated. Mechanism indicated that Ziyu II-induced autophagy by inhibiting Akt/mTOR pathway. Akt reactivation partially reduced Ziyu II-induced LC3-II turnover and LC3 puncta accumulation. Especially, Ziyu II improves the sensitivity of 5-fluorouracil which is the first-line chemotherapy drug in colorectal cancer cells. This research provides novel insight into the molecular mechanism of Ziyu II’s anti-proliferation, including apoptosis and autophagy, and lays a foundation for the potential application of Ziyu II in clinical CRC treatment.

Role of autophagy in response of epithelial ovarian cancer cells to cisplatin treatment and cisplatin resistance

Ovarian cancer survival rate is inversely associated with the extent of tumor metastasis. One of the main treatment approaches against ovarian cancer is employment of platinum based therapies, including cisplatin. Majority of ovarian cancer patients develop cisplatin resistance. We aimed to investigate roles for macroautophagy in response of epithelial ovarian cancer cells to cisplatin, including changes in cell motility, as well as in development of cisplatin resistance. Cisplatin treatment induced autophagy in Caov-3 cells in vitro, as well as resulted in increased cell motility. Pharmacologic inhibition of autophagy by wortmannin eliminated the effect of cisplatin on cell motility. We further selected Caov-3 cells with acquired cisplatin resistance and observed elevated baseline expression of autophagy markers in the resistant cells. Our data indicate a role for autophagy in development of cisplatin resistance by the EOC cells, as well as a potential role for cisplatin-induced autophagy in ovarian tumor metastasis.

MCT1 relieves osimertinib-induced CRC suppression by promoting autophagy through the LKB1/AMPK signaling

Colorectal cancer (CRC) is one of the most frequently diagnosed cancers worldwide. Development of novel chemotherapeutics is still required to enable successful treatment and improve survival for CRC patients. Here, we found that osimertinib (OSI) exhibits potent anti-CRC effects by inducing apoptosis, independent of its selective inhibitory activity targeting the EGFR T790M mutation. Intriguingly, OSI treatment triggers autophagic flux in CRC cells. Inhibition of autophagy markedly augments OSI-induced apoptosis and growth inhibition in CRC cells, suggesting a protective role of autophagy in response to OSI treatment. Mechanistically, OSI upregulates the expression of monocarboxylate transporter 1 (MCT1) and subsequently activates LKB1/AMPK signaling, leading to autophagy induction in CRC cells. Notably, OSI significantly exaggerates the sensitivity of CRC cells to the first-line drugs 5-fluorouracil or oxaliplatin. Taken together, our study unravels a novel mechanism of OSI-mediated protective autophagy involving MCT1/LKB1/AMPK signaling, and suggests the use of OSI as a potential agent for clinical CRC treatment.

Autophagy: New Insights into Mechanisms of Action and Resistance of Treatment in Acute Promyelocytic leukemia.

Autophagy is one of the main cellular catabolic pathways controlling a variety of physiological processes, including those involved in self-renewal, differentiation and death. While acute promyelocytic leukemia (APL) cells manifest low levels of expression of autophagy genes associated with reduced autophagy activity, the introduction of all-trans retinoid acid (ATRA)—a differentiating agent currently used in clinical settings—restores autophagy in these cells. ATRA-induced autophagy is involved in granulocytes differentiation through a mechanism that involves among others the degradation of the PML-RARα oncoprotein. Arsenic trioxide (ATO) is another anti-cancer agent that promotes autophagy-dependent clearance of promyelocytic leukemia retinoic acid receptor alpha gene (PML-RARα) in APL cells. Hence, enhancing autophagy may have therapeutic benefits in maturation-resistant APL cells. However, the role of autophagy in response to APL therapy is not so simple, because some autophagy proteins have been shown to play a pro-survival role upon ATRA and ATO treatment, and both agents can activate ETosis, a type of cell death mediated by the release of neutrophil extracellular traps (ETs). This review highlights recent findings on the impact of autophagy on the mechanisms of action of ATRA and ATO in APL cells. We also discuss the potential role of autophagy in the development of resistance to treatment, and of differentiation syndrome in APL.

Protective effects of autophagy against blue light-induced retinal degeneration in aged mice

The aim of this study was to explore the role of autophagy in response to blue light damage in aged mice and in human retinal pigmented epithelium (hRPE) cells. Blue light damage to the retina was induced in 10-month-old (10 mo) C57 mice and hRPE cells. Flash electroretinography was used to assess retinal function. Retinal structure changes were observed by electron microscopy. Western blot was conducted to determine the expression levels of the following proteins: cleaved caspase-3, p38 mitogen-activated protein kinases, protein kinase R-like endoplasmic reticulum kinase (PERK), autophagy marker light chain 3 (LC3), P62, and Beclin-1. On day 1 after light damage to the 10 mo mice, retinal function was changed. The latent periods of a-wave and b-wave were delayed, and amplitude was reduced. The electron microscopy results revealed mitochondria damage in the retinal pigmented epithelium and a disorganized photoreceptor outer segment (OS). PERK, LC3, and Beclin-1 were upregulated, whereas P62 was not. On day 5 after the blue light damage, restoration of electroretinography and OS was observed. PERK, LC3, and Beclin-1 were downregulated, whereas P62 was not. Protein changes in vitro were consistent with in vivo. The present study provided structural and functional evidence that autophagy plays an important role in the response to blue lightinduced retinal damage.

P-58 - The chondroprotective function of autophagy against acute exogenous oxidative insult in osteoarthritic chondrocytes is impaired

It has been reported that oxidative stress (OS) is involved in the pathogenesis of osteoarthritis (OA) and that defective autophagy/mitophagy is accompanying the OA phenotype. In this study, we analyzed the effect of acute exogenous oxidative insult in control and OA chondrocytes. We analyzed the mRNA and protein levels of genes (Atg5, Beclin-1 and LC3) known to be involved in the formation of the autophagosome in control and OA cells treated with sublethal concentrations of hydrogen peroxide (H2O2). Cells were allowed to recover and the same analyses were performed. Under these conditions morphological differences in the mitochondria of control and OA cells were also assessed. Our results showed that the expression of these genes was significantly reduced in OA cells under normal conditions. Furthermore, in control cells mRNA and protein levels of these genes increased as a result of the OS and decreased back to their original levels 24 h after the treatment. On the contrary, their levels remained high in OA cells even after 24 h. Also, a significantly higher number of impaired mitochondria were observed in OA cells as compared to control cells at all time points. These data offer further understanding on the role of autophagy in response to OS in the onset and progression of OA.

Gastrin activates autophagy and increases migration and survival of gastric adenocarcinoma cells

BackgroundThe peptide hormone gastrin exerts a growth-promoting effect in both normal and malignant gastrointestinal tissue. Gastrin mediates its effect via the cholecystokinin 2 receptor (CCKBR/CCK2R). Although a substantial part of the gastric adenocarcinomas express gastrin and CCKBR, the role of gastrin in tumor development is not completely understood. Autophagy has been implicated in mechanisms governing cytoprotection, tumor growth, and contributes to chemoresistance. This study explores the role of autophagy in response to gastrin in gastric adenocarcinoma cell lines.MethodsImmunoblotting, survival assays and the xCELLigence system were used to study gastrin induced autophagy. Chemical inhibitors of autophagy were utilized to assess the role of this process in the regulation of cellular responses induced by gastrin. Further, knockdown studies using siRNA and immunoblotting were performed to explore the signaling pathways that activate autophagy in response to gastrin treatment.ResultsWe demonstrate that gastrin increases the expression of the autophagy markers MAP1LC3B-II and SQSTM1 in gastric adenocarcinoma cells. Gastrin induces autophagy via activation of the STK11-PRKAA2-ULK1 and that this signaling pathway is involved in increased migration and cell survival. Furthermore, gastrin mediated increase in survival of cells treated with cisplatin is partially dependent on induced autophagy.ConclusionThis study reveals a novel role of gastrin in the regulation of autophagy. It also opens up new avenues in the treatment of gastric cancer by targeting CCKBR mediated signaling and/or autophagy in combination with conventional cytostatic drugs.

Abstract 2084: Pharmacokinetic and pharmacodynamic assessment of autophagy inhibition following hydroxychloroquine in mice

Abstract Hydroxychloroquine (HCQ) is being tested in a number of human clinical trials to determine the role of autophagy in response to standard anticancer therapies. However, preclinical studies in mouse models are equivocal with regard to HCQ exposure (pharmacokinetics) and inhibition of autophagy (pharmacodynamics) in various tissues. Understanding HCQ pharmacokinetics (PK) and pharmacodynamics (PD) in mice allows for a comparison to human PK as reported in recent trials and as such the subsequent PD associated with autophagy inhibition in tissues. In this study, female BALB/c mice were treated with a single intraperitoneal dose of 20, 40, or 80 mg/kg HCQ and tissues and whole blood collected at 3, 6, 12, 24, 48 and 72 hours. Levels of HCQ and desethylhydroxychloroquine (dHCQ), an active metabolite, in whole blood and tissues were determined via a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. Non-compartmental analysis (NCA) measured exposure, as determined by area under the drug concentration versus time curve (AUC0-inf), to both HCQ and dHCQ was dose proportional in whole blood, liver and brain. The maximum concentration (Cmax) of HCQ was 1.3, 3.1 and 4.7 μg/mL and 20.6, 41.7 and 114.7 μg/mL in whole blood and liver, respectively, following 20, 40 and 80 mg/kg. Brain exposure was far less with Cmax achieving 272, 717 and 2220 ng/mL. In general, the time of maximal concentration (Tmax) of HCQ and dHCQ was observed at 3 hr or 6 hr, regardless of dose and location measured. PD assessment for autophagy inhibition was determined by western blotting. The LC3 protein, which is involved in membrane dynamics during autophagy, was visualized and the ratio of LC3-II to tubulin was measured. In the liver, autophagy was inhibited approximately two-fold following all doses at 24 and 48 hr compared to controls. Autophagy was inhibited in both the gut and kidney at 24 and 48 hr at all doses. In the brain, autophagy inhibition was not dose related and may be a result of low HCQ levels. These results demonstrate that a dose of 20 mg/kg is sufficient to inhibit autophagy in most tissues investigated; however, while PK parameters appear to be dose proportional, increased tissue drug levels does not necessarily equate to increased PD effects/autophagy inhibition. In addition, the data establishes a dose of 40 mg/kg in mice results in Cmax and AUC0-inf of HCQ equivalent to that observed in human clinical trials targeting autophagy modulation following a dose of 600 mg daily. Studies are ongoing to assess this relationship in solid tumors. Citation Format: Kristen M. Jackson, Ryan J. Hansen, Daniel L. Gustafson. Pharmacokinetic and pharmacodynamic assessment of autophagy inhibition following hydroxychloroquine in mice. [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 2084.

Abstract 1013: Understanding the molecular pathways underlying radio-sensitization of non-small cell lung cancer (NSCLC) by vitamin D (EB1089)

Abstract Ionization radiation (IR) is an integral component of lung cancer treatment. However, disease recurrence represents a significant barrier to effective therapy. One explanation for the difficulties associated with treatment of NSCLC could be the promotion of autophagy, a cytoprotective signaling pathway that controls the degradation and recycling of cellular components. Cytoprotective autophagy may be one of the primary causes for the development of resistance to standard therapies. In recently published studies (Sharma et al, Autophagy), we showed that the vitamin D analog, EB 1089, leads to conversion of the cytoprotective autophagy induced by radiation alone to a novel ‘cytostatic’ form of autophagy. While pharmacological and genetic inhibition of autophagy increased sensitivity to radiation, interference with autophagy reversed the radio-sensitization by EB 1089 (or Vitamin D3) in H460 and A549 NSCLC cells. These studies demonstrated a switch between the cytoprotective and cytostatic roles of autophagy in response to radiation alone and the combination of EB 1089 (or Vitamin D3) and radiation. AMPK, an energy sensor, has been shown to be involved in regulating autophagy via the AMPK-mTOR pathway. We observed an increase in phosphorylated AMPK (and ULK-1, which is downstream of AMPK) with the combination treatment of IR and EB 1089. Both pharmacological and genetic inhibition of AMPK, reverted the radio-sensitization effects of EB 1089, implicating AMPK in radio-sensitization. Studies conducted with NSCLC cell lines both wt and mutant or null in p53 indicated that functional p53 was required to radio-sensitize tumor cells with the combination treatment. p53 appears to promote AMPK upregulation via sestrin. Finally, studies to determine the potential involvement of NF-kB in radiation sensitization showed degradation of IKB-α in response to IR alone that was suppressed by EB 1089, indicating NF-kB's involvement in the initiation of cytoprotective autophagy by radiation Overall, these studies suggest that the use of vitamin D or a vitamin D analogue could increase sensitization to radiation treatment for lung cancer. Citation Format: Khushboo Sharma, Theresa Thekkudan, David Gewirtz, Hisashi Harada, Frank Torti, Suzy Torti. Understanding the molecular pathways underlying radio-sensitization of non-small cell lung cancer (NSCLC) by vitamin D (EB1089). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1013. doi:10.1158/1538-7445.AM2015-1013