The role of autophagy in microwave radiation induced toxicity in iPSC-derived cardiomyocytes

Objective: Macrophage infiltration in kidney is a major pathological feature of diabetic nephropathy (DN), which has been demonstrated associate with macrophages autophagy homeostasis. However, the relationships between autophagy and the infiltration response related of macrophages adhesion and migration are unknown. This study aims to investigate the impact of macrophages adhesion and migration by modulating autophagy.Methods: In vivo, rats were randomly distributed into control (NC) and DN groups. The pathological changes in renal tissue were assessed, and expression of CD68, LC3, P62 were analyzed. In vitro, RAW264.7 cells were divided into NC and high glucose (HG) groups. The capacity of macrophages adhesion migration and the expression of autophagy markers were observed with and without autophagy modulators (rapamycin, 3-methyladenine, chloroquine, and bafilomycin A1 for RAPA, 3-MA, CQ, BAFA). The macrophages autophagosome and the process of degradation and fusion of autophagosome-lysosome were observed by electron microscopy.Results: In vivo, renal injury is aggravated in diabetic rat compared with NC group. The autophagy level is inhibited in renal tissues of DN group with the increasing expression of CD68 and P62, while expression level of LC3 decreased (p < .05). In vitro, HG and 3-MA reduce the numbers of autophagosome of macrophages to inhibit autophagy level with decrease expression of LC3 and Beclin-1, but increase expression of P62, which promote the adhesion and migration capacity of macrophages (p < .05). Moreover, CQ and BAFA suppress autophagy level by inhibiting the process of autophagosome-lysosome degradation and fusion of macrophages, as well as the expression of LC3 and Beclin-1. We notice an increase expression of P62 by CQ and BAFA stimulation (p < .05). CQ and BAFA further facilitate the adhesion and migration capacity of macrophages. However, RAPA increases the numbers of macrophages autophagosome that inhibited by HG, resulting in a recovery of autophagy level with increase expression of LC3 and Beclin-1, whereas a reduction expression of P62, which lead to inhibition of adhesion and migration of macrophages induced by HG (p < .05)Conclusions: High glucose efficiently reduced the level of macrophage autophagy, following macrophages adhesion and migration enhanced when autophagy is suppressed. Activation of autophagosome improve the level of autophagy, but leading to a reduction of the macrophages adhesion and migration. While, inhibiting the process of degradation and fusion of autophagosome-lysosome suppress the level of autophagy and promote the macrophages adhesion and migration. These results indicate that high glucose may play an important role in macrophages adhesion and migration through modulating autophagy activities in diabetic nephropathy.

MiR-221/222 Promote Dexamethasone Resistance of Multiple Myeloma through Inhibition of Autophagy By Targeting ATG12

SIRT3 promotes metabolic maturation of human iPSC-derived cardiomyocytes via OPA1-controlled mitochondrial dynamics

Sa1687 Modeling Cholangiocarcinoma Desmoplasia for Rapidly Identifying Stromal Targeting Agents

Introduction: High recurrence rates, drug resistance after initial response to chemotherapy, and overall poor prognosis along with the limited treatment options make triple-negative breast cancers (TNBCs) a major clinical challenge. Autophagy, an evolutionary conserved degradation and recycling process, has been shown to function as an adaptive survival response to chemotherapy. Previous studies have indicated higher expression of autophagy markers in TNBCs compared to other breast cancer subtypes, as well as their dependence on autophagy for survival. Our laboratory has also shown in xenograft models an enhanced effectiveness of chemotherapy for the treatment of TNBC when given in combination with autophagy inhibition (AI). These results support TNBCs as a good candidate for AI to improve efficacy of existing therapeutic regimens. However, currently available agents for AI in cancer patients have limited effectiveness, and development of more potent autophagy inhibitors (AIs) is underway. Objective: Develop and test new tools for more potent AI in vivo. Experimental Design: We are employing in vitro models using TNBC lines MDA-MB-231 and SUM159PT, as well as their derivatives R8 and R75, resistant to Epirubicin (EPI) and other anthracyclines. We are evaluating effects of various AIs, including lysosomotropic agents HCQ and lys05, siRNAs and shRNAs targeting autophagy-related (Atg) proteins, and small molecule inhibitors (under development) of ATG4B protein. In vivo xenograft mouse models of MDA-MB-231 and R8 are being used to evaluate the effects of combinatorial therapy with EPI and AI. Methods: For the assessment of autophagy levels before and after AI we used autophagy flux (degradative completion of autophagy) assays. We evaluated the effects of chemotherapy alone and in combination with AIs on parent and resistant sub-lines by assessing their proliferation. For in vivo studies, TNBC cells are injected subcutaneously in Rag2M mice. Treatment with EPI, AI, or their combination is administered after tumor formation. Treatment efficacy is evaluated by tumor volume measurements; tumors are also assessed for the expression of autophagy markers. Results: Our in vitro experiments showed enhanced cytotoxicity of lys05 compared to HCQ either alone or in combination with EPI. However, the use of lys05 in vivo gives contradictory results and requires further evaluation. AI targeting ATG4B, using shRNA-inducible monoclonal cell lines derived from MDA-MB-231 cells and novel small molecule inhibitors of ATG4B, significantly affected cancer cell proliferation in vitro, and is currently being investigated in vivo. Conclusion: Novel approaches to AI may serve as useful tools to assess the effects of AI in vitro and in vivo. Our preliminary results suggest that more potent AIs may improve the effectiveness of treatment of TNBC. Supported by CIHR GPG102167 and CIHR/AVON OBC127216. Citation Format: Svetlana Bortnik, Suganthi Chittaranjan, Jing Xu, Wieslawa H. Dragowska, Jianghong An, Adrienne Kyle, Nancy E. Go, Lubomir Vezenkov, Courtney Choutka, Amy Leung, Suzana Kovacic, Damien Bosc, Karen Gelmon, Marcel Bally, Steven Jones, Robert Young, Sharon Gorski. The effectiveness of autophagy inhibition in sensitizing triple-negative breast cancer cells to chemotherapy. [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 2902. doi:10.1158/1538-7445.AM2015-2902

Ovarian cancer (OvCa) is the 5th leading cause of cancer-related mortalities among women in the US. The majority of patients diagnosed with OvCa are initially responsive to first-line chemotherapy; however, 75% will suffer a recurrence characterized by progressive drug resistance. Overcoming chemoresistance is a major obstacle in treating patients with OvCa. Autophagy is a conserved biological process involved in multiple hematologic and solid malignancies, including OvCa, and is a potential target for overcoming chemoresistance. Autophagy has a context-dependent role in cancer, where, initially, it offers a protective role by preventing the initiation of tumorigenesis and, subsequently, is used by the tumor for survival. A number of chemotherapeutics, including cisplatin, are potent inducers of autophagy; taken together, these indicate a significant role for autophagy in cancer. The role of autophagy in OvCa is additionally complex, with increased levels of major autophagic components associated with contradictory outcomes: increased levels of Beclin-1 and LC3-II correlate with low grade and increased survival, yet increased levels of p62 in the cytoplasm correlate with advanced stage and decreased survival. Further, inhibition of autophagy by pharmacologic treatment or knockdown of key autophagic components induces OvCa cell death and re-sensitizes CP-resistant cells to cisplatin. These results indicate that autophagy has a nonobvious role in chemoresistance, which may be distinguished by increased basal autophagy and/or a more acute autophagic response. In the present study, we investigated whether chemoresistant OvCa cells are more susceptible to autophagy modulation relative to sensitive cells, and if this increased sensitivity was due to higher levels of basal autophagy occurring in resistant cells or due to a more robust autophagic response. First, to evaluate the effect of autophagy on chemosensitivity, MTTs were performed with cisplatin and in combination with autophagy activation or inhibition in two pairs of isogenic cisplatin (CP)-resistant OvCa cell lines: A2780 and CP70; OVCAR-3 and CP_OVCAR-3, an isogenic line developed using intermittent exposure to cisplatin [IC20] over 180 days. Inhibition of autophagy enhanced CP sensitivity in both pairs of cell lines, though resistant cells were more sensitive to autophagic inhibition with a greater increase in cisplatin sensitivity. Conversely, autophagy induction decreased sensitivity to cisplatin in the sensitive lines though not in the resistant lines. Beclin-1 and p62 protein were increased in resistant lines relative to the parental lines. Next, we measured autophagic flux to compare levels of basal autophagy. Increased LC3 turnover and p62 degradation in resistant lines indicated increased basal autophagic flux in resistant lines. Autophagy induction was measured temporally to determine the maximal turnover rate and earliest time of response. Resistant lines had no difference in interval between treatment and start of autophagy after treatment, but did reach a greater level of autophagy flux compared to sensitive lines. These results indicate there is an increase in basal autophagy rather than an increase in response in resistant cells relative to sensitive cells, which in turn contributes to a greater level of autophagy achieved by resistant cells. Finally, we investigated the role of autophagy inhibition in OvCa patient-derived cell lines from our biobank of 400+ cell lines that span CP sensitivities. There was a synergistic cytotoxic response with co-treatment of cisplatin and autophagy inhibition, and, as with the isogenic models of CP-resistance, patient-derived lines that were less sensitive to cisplatin were more susceptible to autophagy inhibition. We are currently examining the basal levels of autophagy in this cohort. These results indicate that inhibition of autophagy is a promising strategy to overcome chemoresistance in OvCa patients. Citation Format: Thomas R. Silvers, Peter R. Dottino, John A. Martignetti, Brad R. Evans. Increased basal autophagy decreases sensitivity to cisplatin in chemoresistant ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B75.

Intracellular accumulation of altered proteins, including p62 and ubiquitinated proteins, is the basis of most neurodegenerative disorders. The relationship among the accumulation of altered proteins, autophagy, and spinal cord dysfunction by cervical spondylotic myelopathy has not been clarified. We examined the expression of p62 and autophagy markers in the chronically compressed spinal cord of tiptoe-walking Yoshimura mice. In addition, we examined the expression and roles of p62 and autophagy in hypoxic neuronal cells. Western blot analysis showed the accumulation of p62, ubiquitinated proteins, and microtubule-associated protein 1 light chain 3 (LC3), an autophagic marker, in the compressed spinal cord. Immunohistochemical examinations showed that p62 accumulated in neurons, axons, astrocytes, and oligodendrocytes. Electron microscopy showed the expression of autophagy markers, including autolysosomes and autophagic vesicles, in the compressed spinal cord. These findings suggest the presence of p62 and autophagy in the degenerated compressed spinal cord. Hypoxic stress increased the expression of p62, ubiquitinated proteins, and LC3-II in neuronal cells. In addition, LC3 turnover assay and GFP-LC3 cleavage assay showed that hypoxic stress increased autophagy flux in neuronal cells. These findings suggest that hypoxic stress induces accumulation of p62 and autophagy in neuronal cells. The forced expression of p62 decreased the number of neuronal cells under hypoxic stress. These findings suggest that p62 accumulation under hypoxic stress promotes neuronal cell death. Treatment with 3-methyladenine, an autophagy inhibitor decreased the number of neuronal cells, whereas lithium chloride, an autophagy inducer increased the number of cells under hypoxic stress. These findings suggest that autophagy promotes neuronal cell survival under hypoxic stress. Our findings suggest that pharmacological inducers of autophagy may be useful for treating cervical spondylotic myelopathy patients.

Autophagy is required for the promoting effect of angiogenic factor with G patch domain and forkhead-associated domain 1 (AGGF1) in retinal angiogenesis.

To study the role of microRNA (miR)-30d-5p in high glucose-induced podocyte injury. Podocytes were hyperglycated with 30 mmol/L glucose, transfected with miR-30d-5p inhibitor and mimic, and then treated with 1 mg/mL 3-methyladenine (3-MA). The transfection efficiency of miR-30d-5p was quantified by reverse transcription PCR. Apoptosis was detected by flow cytometry. The expressions of nephrin, microtubule-associated protein light chain (LC) 3Ⅱ/LC3Ⅰ, P62, autophagy-related gene (ATG) 5, PTEN induced putative kinase (PINK) 1 and Parkin gene (PARK2) were detected by Western blotting. The mito-chondrial membrane potential was detected by JC-1 fluorescent probe, and adenosine triphosphate (ATP) content in cells was detected by relevant kits. Under high glucose induction, podocyte apoptosis increased, miR-30d-5p and P62 expressions were upregulated, while nephrin, ATG5, PINK1, PARK2 and LC3Ⅱ/LC3Ⅰ expressions decreased (all P<0.01). MiR-30d-5p inhibitor reversed the effect of high glucose on apoptosis, and the expression of ATG5, PINK1, PARK2, nephrin, LC3Ⅱ/LC3Ⅰ and P62 (all P<0.01). High glucose induced loss of mitochondrial membrane potential and ATP content in podocytes, while inhibition of miR-30d-5p increased them. Autophagy inhibitors 3-MA and miR-30d-5p mimics reversed the effects of miR-30d-5p inhibition on apoptosis, autophagy and mitochondrial function of podocytes induced by high glucose (all P<0.05). Inhibition of miR-30d-5p may promote mitochondrial autophagy (mitophagy) by promoting the expression of ATG5, PINK1, PARK2 and alleviating high glucose-induced podocyte damage.

To explore the expression of the RNA-binding protein tristetraprolin in lung adenocarcinoma cells and its molecular mechanism for inhibiting autophagy. Quantitative real-time PCR and Western blotting were performed to detect the expression of autophagy-related genes (including Beclin1, LC3-Ⅱ/LC3-Ⅰ and SQSTM1/p62) in cultured lung adenocarcinoma cells at 24, 48 and 72 h after transient transfection with a tristetraprolin-overexpressing plasmid and the empty plasmid. The effects of transfection with the tristetraprolin-overexpressing plasmid and empty plasmids in the presence or absence of tumor necrosis factor-α (TNF-α) on the expressions of nuclear factor-κB (NF-κB) p65, c-rel, and p50 were examined in lung adenocarcinoma cells using immunofluorescence assay and Western blotting. The cells were also transfected with the IκBα-mut plasmid and the tristetraprolin-overexpressing plasmid, either alone or in combination, and the changes in the expressions of tristetraprolin and autophagy-related genes were detected using RT-qPCR and Western blotting. The expressions of tristetraprolin were significantly reduced at both the mRNA and protein levels in lung adenocarcinoma cells (P &lt; 0.001). Overexpression of tristetraprolin in the cells significantly lowered the expressions of autophagy-related genes Beclin1 and the ratio of LC3-Ⅱ/LC3-Ⅰ at the mRNA and protein levels (P &lt; 0.001), obviously lowered the expressions of NF-κB p65 and c-rel, and almost totally blocked the nuclear translocation of NF-κB p65 and c-rel (P &lt; 0.05); the expression of p50, however, did not undergo significant changes in response to tristetraprolin overexpression (P &gt; 0.05). The inhibitory effect of tristetraprolin overexpression on autophagy was abrogated by transfection of the cells with IκBα-mut plasmid, which blocked the NF-κB signaling pathway. Co-transfection of the cells with IκBα-mut also attenuated the inhibitory effect of tristetraprolin overexpression on Beclin1 and the LC3-Ⅱ/LC3-Ⅰ ratio at both the mRNA and protein levels (P &lt; 0.05). The expression of tristetraprolin is low in lung adenocarcinoma cells. Tristetraprolin overexpression causes inhibition of autophagy in lung adenocarcinoma cells possibly by blocking NF-κB p65 and c-rel nuclear translocation.

Fatty Acid Synthase (FASN), a key enzyme of de novo lipid synthesis, is significantly up regulated in colorectal cancer (CRC) and its activity is associated with poor prognosis. Recent studies suggest that FASN plays a crucial role in the survival of cancer cells by reprogramming metabolic pathways to maintain energy homeostasis under conditions of metabolic stress. However, the mechanisms of metabolic adaptation of cancer cells regulated by FASN to promote their survival are not yet fully understood. Cancer cells use autophagy as one of the survival strategies under metabolic stress. The purpose of our study was to determine the role of aberrant activation of FASN in regulation of autophagy in vitro and in human CRC. METHODS. Expression of p62 and LC3 (autophagy markers), pAMPK, and Cyclin D was assessed in CRC cell lines with altered expression of FASN cultured in normal, serum free medium and/or detached conditions by Western blot and confocal microscopy. Human specimens, including matching cancer, normal colon and liver metastasis tissues from 19 patients were analyzed for expression of FASN, p62, and pAMPK by immunohistochemistry (IHC). RESULTS. We demonstrated that under metabolic stress conditions overexpression of FASN is associated with a decrease in autophagy as shown by accumulation of p62, a marker of inhibition of autophagy, and a decrease in LC3A, an autophagosome marker, in CRC cell lines. Under energy stress conditions, overexpression of FASN is also associated with an increase in Cyclin D and a decrease in activation of pAMPK. Statistical analysis of IHC staining showed that expression of FASN, p62, and pAMPK is significantly higher in cancer and metastatic tissues as compared to normal colon tissues. Using Spearman's rank correlation, we determined moderate correlation between expression of FASN and p62 in human primary CRC and liver metastasis. Similar results were obtained using Western blot analysis on human normal colon and matching cancer tissues. CONCLUSIONS. Our data suggest that overexpression of FASN is protective during metabolic stress conditions by shifting metabolism towards higher energy production as indicated by activity of pAMPK. Furthermore, we have identified a novel link between FASN and p62, and showed that, under metabolic stress conditions, overexpression of FASN is associated with inhibition of autophagy. However, further studies are require to understand how FASN-mediated up regulation of p62 contributes to tumorigenesis. The differential expression of FASN in normal versus cancer cells makes de novo lipogenesis a desirable target for therapeutic intervention. Understanding the role of FASN in regulation of survival and aggressive behavior of cancer cells will uncover new therapeutic approaches in CRC. Citation Format: Yekaterina Y. Zaytseva, Jennifer W. Harris, Ji Tae Kim, Tianyan Gao, Eun Y. Lee, Heidi L. Weiss, B Mark Evers. Overexpression of Fatty Acid Synthase is associated with inhibition of autophagy under conditions of metabolic stress in colorectal cancer cells. [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 1151. doi:10.1158/1538-7445.AM2015-1151

Exercise-mediated autophagy in cardiovascular diseases.

Left ventricular non-compaction (LVNC), is a hereditary cardiomyopathy with limited treatments. Our previous study linked phosphodiesterase 4D interacting protein (PDE4DIP) to LVNC development. To explore the functional role of PDE4DIP activation in regulating cell polarity, skeleton, and energy metabolism, and to elucidate its mechanisms driving LVNC development, bioinformatics analysis was performed to compare its expression in LVNC patients and normal subjects. Overexpression and knockdown of PDE4DIP were constructed in H9C2 cells and neonatal Sprague–Dawley rat primary cardiomyocytes, respectively. Electron microscopy, MitoTracker-Green staining, and an ATP kit were employed to assess mitochondria's morphology and functional status. Real-time quantitative PCR, western blotting, and immunofluorescence assays were employed to detect the expression of cell polarity-, skeleton-, and Rho-ROCK signaling-related genes and proteins. Cell scratching and CCK-8 assays were employed to detect cell migration and proliferation abilities of H9C2, respectively. We found that PDE4DIP expression was increased in the LVNC-derived human-induced pluripotent stem cell-derived cardiomyocytes compared with normal subjects. Furthermore, overexpression of PDE4DIP induced cytoskeletal disorganization, decreased ATP content and cell migration, and increased cell proliferation and mitochondrial vacuolation. Moreover, the knockdown of PDE4DIP promoted cytoskeleton formation and contributed to increased ATP content and elevated cell migration. Mechanically, overexpression of PDE4DIP inhibited cell polarity-, skeleton-, and Rho-ROCK signaling-related genes and proteins, which could be increased by knockdown of PDE4DIP, suggesting that a critical regulation of PDE4DIP to Rho-ROCK pathway. This discovery suggests that PDE4DIP contributes to the development of LVNC by regulating cell polarity, skeleton, and energy metabolism through the Rho-ROCK pathway.

Statins are effective drugs in reducing cardiovascular morbidity and mortality by inhibiting cholesterol synthesis. These effects are primarily beneficial for the patient's vascular system. A significant number of statin users suffer from muscle complaints probably due to mitochondrial dysfunction, a mechanism that has recently been elucidated. This has raised our interest in exploring the effects of statins on cardiac muscle cells in an era where the elderly and patients with poorer functioning hearts and less metabolic spare capacity start dominating our patient population. Here, we investigated the effects of statins on human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-derived CMs). hiPSC-derived CMs were exposed to simvastatin, atorvastatin, rosuvastatin, and cerivastatin at increasing concentrations. Metabolic assays and fluorescent microscopy were employed to evaluate cellular viability, metabolic capacity, respiration, intracellular acidity, and mitochondrial membrane potential and morphology. Over a concentration range of 0.3-100µM, simvastatin lactone and atorvastatin acid showed a significant reduction in cellular viability by 42-64%. Simvastatin lactone was the most potent inhibitor of basal and maximal respiration by 56% and 73%, respectively, whereas simvastatin acid and cerivastatin acid only reduced maximal respiration by 50% and 42%, respectively. Simvastatin acid and lactone and atorvastatin acid significantly decreased mitochondrial membrane potential by 20%, 6% and 3%, respectively. The more hydrophilic atorvastatin acid did not seem to affect cardiomyocyte metabolism. This calls for further research on the translatability to the clinical setting, in which a more conscientious approach to statin prescribing might be considered, especially regarding the current shift in population toward older patients with poor cardiac function.

Esophageal adenocarcinoma (EAC) is a highly lethal cancer type with an overall poor survival rate. Twenty to thirty percent of EAC overexpress the human epidermal growth factor receptor 2 (Her2), a transmembrane receptor tyrosine kinase promoting cell growth and proliferation. Patients with Her2 overexpressing breast and gastroesophageal cancer may benefit from Her2 inhibitors. Therapy resistance, however, is well documented. Since autophagy, a lysosome-dependent catabolic process, is implicated in cancer resistance mechanisms, we tested whether autophagy modulation influences Her2 inhibitor sensitivity in EAC. Her2-positive OE19 EAC cells showed an induction in autophagic flux upon treatment with the small molecule Her2 inhibitor Lapatinib. Newly generated Lapatinib-resistant OE19 (OE19 LR) cells showed increased basal autophagic flux compared to parental OE19 (OE19 P) cells. Based on these results, we tested if combining Lapatinib with autophagy inhibitors might be beneficial. OE19 P showed significantly reduced cell viability upon double treatment, while OE19 LR were already sensitive to autophagy inhibition alone. Additionally, Her2 status and autophagy marker expression (LC3B and p62) were investigated in a treatment-naïve EAC patient cohort (n = 112) using immunohistochemistry. Here, no significant correlation between Her2 status and expression of LC3B and p62 was found. Our data show that resistance to Her2-directed therapy is associated with a higher basal autophagy level, which is not per se associated with Her2 status. Therefore, we propose that autophagy may contribute to acquired resistance to Her2-targeted therapy in EAC, and that combining Her2 and autophagy inhibition might be beneficial for EAC patients.