High Autophagic Activity Research Articles

Simultaneously Monitoring Multiple Autophagic Processes and Assessing Autophagic Flux in Single Cells by In Situ Fluorescence Cross-Correlation Spectroscopy.

Autophagy is a widely conserved and multistep cellular catabolic process and maintains cellular homeostasis and normal cellular functions via the degradation of some harmful intracellular components. It was reported that high basal autophagic activity may be closely related to tumorigenesis. So far, the fluorescence imaging technique has been widely used to study autophagic processes, but this method is only suitable for distinguishing autophagosomes and autolysosomes. Simultaneously monitoring multiple autophagic processes remains a significant challenge due to the lack of an efficient detection method. Here, we demonstrated a new method for simultaneously monitoring multiple autophagic processes and assessing autophagic flux in single cells based on in situ fluorescence cross-correlation spectroscopy (FCCS). In this study, microtubule-associated protein 1A/1B-light chain 3B (LC3B) was fused with two tandem fluorescent proteins [mCherry red fluorescent protein (mCherry) and enhanced green fluorescent protein (EGFP)] to achieve the simultaneous labeling and distinguishing of multiple autophagic structures based on the differences in characteristic diffusion time (τD). Furthermore, we proposed a new parameter "delivery efficiency of autophagosome (DEAP)" to assess autophagic flux based on the cross correlation (CC) value. Our results demonstrate that FCCS can efficiently distinguish three autophagic structures, assess the induced autophagic flux, and discriminate different autophagy regulators. Compared with the commonly used fluorescence imaging technique, the resolution of FCCS remains unaffected by Brownian motion and fluorescent monomers in the cytoplasm and is well suitable to distinguishing differently colored autophagic structures and monitoring autophagy.

A PSD-95 peptidomimetic mitigates neurological deficits in a mouse model of Angelman syndrome.

Angelman Syndrome (AS) is a severe cognitive disorder caused by loss of neuronal expression of the E3 ubiquitin ligase UBE3A. In an AS mouse model, we previously reported a deficit in brain-derived neurotrophic factor (BDNF) signaling, and set out to develop a therapeutic that would restore normal signaling. We demonstrate that CN2097, a peptidomimetic compound that binds postsynaptic density protein-95 (PSD-95), a TrkB associated scaffolding protein, mitigates deficits in PLC-CaMKII and PI3K/mTOR pathways to restore synaptic plasticity and learning. Administration of CN2097 facilitated long-term potentiation (LTP) and corrected paired-pulse ratio. As the BDNF-mTORC1 pathway is critical for inhibition of autophagy, we investigated whether autophagy was disrupted in AS mice. We found aberrantly high autophagic activity attributable to a concomitant decrease in mTORC1 signaling, resulting in decreased levels of synaptic proteins, including Synapsin-1 and Shank3. CN2097 increased mTORC1 activity to normalize autophagy and restore hippocampal synaptic protein levels. Importantly, treatment mitigated cognitive and motor dysfunction. These findings support the use of neurotrophic therapeutics as a valuable approach for treating AS pathology.

Hematopoietic Stem Cells Depend upon Aggrephagy to Maintain Protein Homeostasis and Self-Renewal Activity

Hematopoietic stem cells (HSCs) regenerate blood and immune cells throughout life. To preserve their health and longevity, HSCs are particularly dependent on maintaining protein homeostasis (proteostasis). HSCs maintain proteostasis partly by sustaining low protein synthesis rates that limit the biogenesis of misfolded proteins. Nevertheless, HSCs ultimately accumulate misfolded proteins that must be eliminated to preserve stem cell fitness. However, how HSCs purge misfolded proteins to maintain proteostasis is mostly unknown. We found that in contrast to most cell types that utilize the proteasome to degrade misfolded proteins, HSCs depend on aggrephagy, a selective form of autophagy, to maintain proteostasis in vivo. Young adult HSCs exhibit unusually high autophagic activity, and genetically disabling autophagy results in significant accumulation of protein aggregates and impaired HSC function. Furthermore, we determined that mouse and human HSCs preferentially express Bag3, a critical stress response gene that promotes aggrephagy and transport of misfolded proteins to aggresomes. Aggresomes are cytosolic inclusion bodies containing protein aggregates that typically form under stress conditions to help maintain proteostasis. Surprisingly, we found that the vast majority of HSCs contain aggresomes, even under steady state conditions in vivo. Conditional deletion of Bag3 causes HSCs to accumulate protein aggregates and impairs their self-renewal activity in vivo. Finally, we determined that old adult HSCs exhibit diminished autophagic flux and become increasingly reliant on the proteasome to degrade misfolded proteins. Overall, these studies demonstrate that protein degradation pathways are uniquely configured in HSCs to preserve proteostasis and fitness, and that disruptions in proteostasis network activity contribute to cellular and molecular changes in HSCs during aging.

Iron regulatory protein (IRP)-mediated iron homeostasis is critical for neutrophil development and differentiation in the bone marrow.

Iron is mostly devoted to the hemoglobinization of erythrocytes for oxygen transport. However, emerging evidence points to a broader role for the metal in hematopoiesis, including the formation of the immune system. Iron availability in mammalian cells is controlled by iron-regulatory protein 1 (IRP1) and IRP2. We report that global disruption of both IRP1 and IRP2 in adult mice impairs neutrophil development and differentiation in the bone marrow, yielding immature neutrophils with abnormally high glycolytic and autophagic activity, resulting in neutropenia. IRPs promote neutrophil differentiation in a cell intrinsic manner by securing cellular iron supply together with transcriptional control of neutropoiesis to facilitate differentiation to fully mature neutrophils. Unlike neutrophils, monocyte count was not affected by IRP and iron deficiency, suggesting a lineage-specific effect of iron on myeloid output. This study unveils the previously unrecognized importance of IRPs and iron metabolism in the formation of a major branch of the innate immune system.

Abstract 2549: Immune microenvironment landscape shows treatment-specific differences in rectal cancer patients

Abstract Introduction: Neoadjuvant therapy is the backbone of modern rectal cancer treatment. More intensive therapy leads to higher complete responses, which means an organ-preservation approach can be used. Insights in the biology of tumor response are essential for a successful implementation of such strategies, as different treatments may lead to specific tumor responses. In this study we aim to explore treatment-specific responses of tumor microenvironment and how these are related to patient outcome. Methods: Patients with adenocarcinoma of the rectum who had received one of five different treatment regimens between 2015-2019 were included; no neo-adjuvant therapy (NT), neo-adjuvant chemotherapy (CT), neo-adjuvant radiochemotherapy (RCT), neo-adjuvant radiotherapy with a long interval (LRT) or short interval (SRT). In order to minimize heterogeneity we excluded cT1 tumors, cT4 tumors as well as stage IV rectal cancers. Consecutive slides were stained for HE, LC3-II (autophagy stain) and a multiplex immunofluorescence VECTRA panel for CD8, CD3, CD20, Foxp3, CD11c and pan-CK AE 1/3. Tumor response was scored by two independent investigators. Results: A total of 80 rectal cancer patients were included. Median age was slightly biased, with older patients in the SRT groups and younger patients in the CT group (p=0.03). Less regression and more lymph-node involvement was seen among the CT group, while higher angioinvasion and lymph node involvement was seen in the SRT group. A fragmented pattern of response was more prevalent in CT and RCT treated patients (>73% of cases), whereas a shrinkage pattern of response was predominant in RTL treated patients (64% of cases). Furthermore, autophagic activity in tumor cells was increased in RCT treated patients, especially compared to NT and CT. When analyzing the tumor immunophenotype, differences were found according to specific treatment regimens. RCT treated patients seemed to have lower stromal CD4+ cells, Foxp3+ cells and CD11c+ cells. The latter were found to be especially decreased in treatments will longer intervals (CT, RCT, LTR) compared to those with no treatment or immediate surgery (NT and SRT). The specific relations within the network of immune cells and how these relate to autophagy and the patterns of response are currently being evaluated. Thus, further results will follow shortly. Conclusions: We demonstrated treatment-specific differences in the immune microenvironment landscape of rectal cancer patients. We found higher autophagic activity in patients who received RT treatment, especially significant in the RCT group. Lower immune cell density was observed in longer wait intervals, stressing the importance of immune recovery. Understanding the immune landscape after a specific therapy will shed light on to future treatment decisions, as using immunotherapy in combination with established treatments is the future of personalized therapy. Citation Format: Cristina Graham Martinez, Yari F. H. C. Barella, Sonay Kus Öztürk, Mark A. Gorris, Corrie A. Marijnen, Iris D. Nagtegaal. Immune microenvironment landscape shows treatment-specific differences in rectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2549.

Autophagy Contributes to Metabolic Reprogramming and Therapeutic Resistance in Pancreatic Tumors.

Metabolic reprogramming is a feature of cancers for which recent research has been particularly active, providing numerous insights into the mechanisms involved. It occurs across the entire cancer process, from development to resistance to therapies. Established tumors exhibit dependencies for metabolic pathways, constituting vulnerabilities that can be targeted in the clinic. This knowledge is of particular importance for cancers that are refractory to any therapeutic approach, such as Pancreatic Ductal Adenocarcinoma (PDAC). One of the metabolic pathways dysregulated in PDAC is autophagy, a survival process that feeds the tumor with recycled intracellular components, through both cell-autonomous (in tumor cells) and nonautonomous (from the local and distant environment) mechanisms. Autophagy is elevated in established PDAC tumors, contributing to aberrant proliferation and growth even in a nutrient-poor context. Critical elements link autophagy to PDAC including genetic alterations, mitochondrial metabolism, the tumor microenvironment (TME), and the immune system. Moreover, high autophagic activity in PDAC is markedly related to resistance to current therapies. In this context, combining autophagy inhibition with standard chemotherapy, and/or drugs targeting other vulnerabilities such as metabolic pathways or the immune response, is an ongoing clinical strategy for which there is still much to do through translational and multidisciplinary research.

The propagation of HSV-1 in high autophagic activity

Autophagy is an intracellular process involving double-membrane vacuoles that ultimately merge with the lysosomes and play a key role in the inhibition of herpessimplex virus 1 (HSV-1) proliferation. This virus is an agent of some lethal neuronal diseases like encephalitis. HSV-1 requires the expression of its latent proteins, such as ICP34.5, to promote cell infection, which disrupts the autophagy process. In this study, we aimed to evaluate the effect of autophagy induction on HSV-1 replication in host cells at the early and late stages of its replication. Furthermore, we explored the consequences of autophagy induction before and after cell infection.Cells were transfected through Beclin-1-expressing plasmids. Autophagy induction was performed with microtubule-associated protein 1 light chain 3 (LC3-II) as an autophagosome formation marker by using flow cytometry. In the first stage, HSV-1 was inoculated into transfected cells 18 hours post-transfection. Next, viral DNA was extracted 18 and 48 hours post-infection, and eventually viral copies per milliliter were calculated through real-time polymerase chain reaction (PCR). For the second stage, the plasmid containing Beclin-1 was transfected to the cells following virus inoculation to examine the influence of autophagy induction after cell infection.Study results have shown that in neuroblastoma cells autophagy activation reduces virus yield from 4×10 5 copies/ml (control sample) to 9×10 4 copies/ml at 24 h postinfection and viral load after 48 h declines up to 1×10 6 copies/ml, which is less than that of the control sample about 5 logs. However, in HeLa cells, we observed a significant reduction in autophagy induction with reducing HSV-1 propagation. Despite these results, HSV-1 proliferation in both cell types increased and these viruses were able to maintain their ability to propagate even in high autophagic activity. Hyperactivation of autophagy can only slow the rate of virus replication. This study may provide new insight into the effect of autophagy on HSV-1 replication.

Autophagy Dynamics and Modulation in a Rat Model of Renal Ischemia-Reperfusion Injury.

Renal ischemia-reperfusion (IR) injury leading to cell death is a major cause of acute kidney injury, contributing to morbidity and mortality. Autophagy counteracts cell death by removing damaged macromolecules and organelles, making it an interesting anchor point for treatment strategies. However, autophagy is also suggested to enhance cell death when the ischemic burden is too strong. To investigate whether the role of autophagy depends on the severity of ischemic stress, we analyzed the dynamics of autophagy and apoptosis in an IR rat model with mild (45 min) or severe (60 min) renal ischemia. Following mild IR, renal injury was associated with reduced autophagy, enhanced mammalian target of rapamycin (mTOR) activity, and apoptosis. Severe IR, on the other hand, was associated with a higher autophagic activity, independent of mTOR, and without affecting apoptosis. Autophagy stimulation by trehalose injected 24 and 48 h prior to onset of severe ischemia did not reduce renal injury markers nor function, but reduced apoptosis and restored tubular dilation 7 days post reperfusion. This suggests that trehalose-dependent autophagy stimulation enhances tissue repair following an IR injury. Our data show that autophagy dynamics are strongly dependent on the severity of IR and that trehalose shows the potential to trigger autophagy-dependent repair processes following renal IR injury.

Loss of BRUCE reduces cellular energy level and induces autophagy by driving activation of the AMPK-ULK1 autophagic initiating axis.

Autophagy is an intracellular catabolic system. It delivers cellular components to lysosomes for degradation and supplies nutrients that promote cell survival under stress conditions. Although much is known regarding starvation-induced autophagy, the regulation of autophagy by cellular energy level is less clear. BRUCE is an ubiquitin conjugase and ligase with multi-functionality. It has been reported that depletion of BRUCE inhibits starvation-induced autophagy by blockage of the fusion step. Herein we report a new function for BRUCE in the dual regulation of autophagy and cellular energy. Depletion of BRUCE alone (without starvation) in human osteosarcoma U2OS cells elevated autophagic activity as indicted by the increased LC3B-II protein and its autophagic puncta as well as further increase of both by chloroquine treatment. Such elevation results from enhanced induction of autophagy since the numbers of both autophagosomes and autolysosomes were increased, and recruitment of ATG16L onto the initiating membrane structure phagophores was increased. This concept is further supported by elevated lysosomal enzyme activities. In contrast to starvation-induced autophagy, BRUCE depletion did not block fusion of autophagosomes with lysosomes as indicated by increased lysosomal cleavage of the GFP-LC3 fusion protein. Mechanistically, BRUCE depletion lowered the cellular energy level as indicated by both a higher ratio of AMP/ATP and the subsequent activation of the cellular energy sensor AMPK (pThr-172). The lower energy status co-occurred with AMPK-specific phosphorylation and activation of the autophagy initiating kinase ULK1 (pSer-555). Interestingly, the higher autophagic activity by BRUCE depletion is coupled with enhanced cisplatin resistance in human ovarian cancer PEO4 cells. Taken together, BRUCE depletion promotes induction of autophagy by lowering cellular energy and activating the AMPK-ULK1-autophagy axis, which could contribute to ovarian cancer chemo-resistance. This study establishes a BRUCE-AMPK-ULK1 axis in the regulation of energy metabolism and autophagy, as well as provides insights into cancer chemo-resistance.

Signal regulatory protein α protects podocytes through promoting autophagic activity.

High autophagic activity in podocytes, terminally differentiated cells which serve as main components of the kidney filtration barrier, is essential for podocyte survival under various challenges. How podocytes maintain such a high level of autophagy, however, remains unclear. Here we report that signal regulatory protein α (SIRPα) plays a key role in promoting podocyte autophagy. Unlike other glomerular cells, podocytes strongly express SIRPα, which is, however, downregulated in patients with focal segmental glomerulosclerosis and mice with experimental nephropathy. Podocyte SIRPα levels are inversely correlated with the severity of podocyte injury and proteinuria but positively with autophagy. Compared to wild-type littermates, Sirpa-deficient mice display greater age-related podocyte injury and proteinuria and develop more rapid and severe renal injury in various models of experimental nephropathy. Mechanistically, podocyte SIRPα strongly reduces Akt/GSK-3β/β-catenin signaling, leading to an increase in autophagic activity. Our findings thus demonstrate a critical protective role of SIRPα in podocyte survival via maintaining autophagic activity.

The good, the bad and the autophagosome: exploring unanswered questions of autophagy-dependent cell death.

The recent discovery of autosis as a variant of autophagy-dependent cell death has challenged the conventional understanding of cell death and programmed cell death in cellular decision making. In contrast to previous accounts of distinct cell death modalities, autosis occurs with high autophagic activity, in the absence of apoptotic and necrotic markers and yet is not fully regulated by typical autophagy markers. Given the metabolic importance of autophagic responses and the extensive cross-talk with both apoptosis and necrosis signalling, the classical and morphotype-driven characterization of cell death as pre-determined subroutines is being increasingly called into question. Furthermore, the conflicting evidence with regards to cell death induction through autophagy modulation in various cancer models highlights the lack of consensus over the extent to which autophagy assists in cell death ontrol and whether it is capable of being a bona fide lethal process. This review evaluates the evidence and context of autophagy-dependent cell death and delineates the role of an autophagic flux threshold associated with 'lethal' and 'non-lethal' autophagy and its role in autosis control. In doing so, cancer treatment avenues will be explored with regards to precision modulation of tumour autophagic flux to ascertain whether autosis induction may present a novel therapeutic strategy.

Ultrastructure of vegetative cells and resting cysts, and live observations of the encystation and excystation processes in Diophrys oligothrix Borror, 1965 (Protista, Ciliophora).

Ciliated protists can form cysts to resist unfavorable environmental conditions and then excyst when environmental conditions become favorable. This study used electron and light microscopy to investigate the structure of vegetative cells and resting cysts, as well as the encysting and excysting processes of Diophrys oligothrix. For the first time, ampules were revealed beneath the pellicle in the genus Diophrys, and their extrusome types differed between Diophrys species. Membrane-packed discs of diverse shapes were found in the cytoplasm just beneath the pellicle around the cytopharynx and were separated by rows of microtubule units. Beneath the discs, some double-layer microtubule structures were detected as well. During encystment, the ventral ciliature was folded in a ventral cavity of the cell, and the caudal cirri were retracted directly into the cyst in a separate cavity on the dorsal side. In the resting cysts, high autophagic activity occurred, possibly including digestion of membrane-packed discs and ampules. Two macronuclear nodules kept their basic shape, although the chromatin aggregation and fusion region were observed in ultrathin sections. The cyst wall contained two layers, namely, the ectocyst, and endocyst. In mature cysts, basal bodies and ciliary shafts were observed, demonstrating that D. oligothrix forms non-kinetosome-resorbing cysts. The process of excystment occurred in two modes, either with or without participation of a contractile vacuole.

Abstract 5026: Alcohol enriches therapy-resistant cancer cells with high autophagy, CD44 expression and tumor initiating capability

Abstract Introduction: Esophageal squamous cell carcinoma (ESCC) is the deadliest of all human cancers with alcohol (EtOH) as a major risk factor. Malignant properties of ESCC have been attributed to ESCC cells with high CD44 expression (CD44H) and autophagy; however, the pathogenic role of EtOH in ESCC remains elusive Methods: With a goal of translation in personalized medicine, we analyzed therapy-naïve ESCC tumor biopsies from patients and ESCC cell lines in three-dimensional (3D) organoids for functional characterization of ESCC cells following exposure to 5-fluorouracil (5FU) or EtOH ex vivo. Tumor growth was assessed in EtOH-fed mice carrying xenograft tumors with two independent ESCC cell lines in the presence or absence of 4-methylpyrazole (4MP), an alcohol dehydrogenase inhibitor. 3D organoids and tumors were analyzed by flow cytometry and immunostaining for CD44, Ki67, p53 and autophagic vesicle (AV) content. Results: ESCC patient biopsies contained CD44H cells with increased AV content. ESCC 3D organoids were grown successfully from 11 out of 16 tumors (68.8%) and passaged with recapitulation of the histopathology, proliferation, p53 and CD44 expression and autophagy present in the original in situ tumors. Successful organoid formation was significantly associated with poor chemoradiation therapy response (progressive and stable diseases, n=10 vs. partial response, n=6). In 3D organoids surviving 5FU treatment, CD44H cells with high autophagic activity was found to be enriched. Pharmacological autophagy flux inhibition by chloroquine augmented 5FU-mediated cytotoxicity in 3D ESCC organoids. 5FU-resistant CD44H cells were more capable of forming 3D organoids compared to bulk populations of ESCC cells. EtOH promoted expansion of CD44H cells with significantly increased proliferation and organoid formation rate upon serial passages, suggesting the increased self-renewal of CD44H cells. In xenograft tumors, alcohol drinking not only promoted tumor growth but increased the intratumoral CD44H cell content which was antagonized by 4MP. Moreover, pharmacological autophagy flux impairment depleted CD44H cells in xenograft tumors. Conclusions: The novel single cell-based 3D ESCC organoid system may serve as a highly efficient platform to explore the role of alcohol and other environmental factors as well as cancer therapeutics and therapy resistance mechanisms in conjunction with morphological and functional assays with implications for translation in personalized medicine. Citation Format: Koji Tanaka, Takashi Kijima, Prasanna Modayur Chandramouleeswaran, Kelly A Whelan, Yuta Kasagi, Masataka Shimonosono, Haruna Furukawa, Takeo Hara, Tomoki Makino, Makoto Yamasaki, Andres J Klein-Szanto, Shoji Natsugoe, Masaki Mori, Yuichiro Doki, Hiroshi Nakagawa. Alcohol enriches therapy-resistant cancer cells with high autophagy, CD44 expression and tumor initiating capability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5026.

Stat3‐Atg5 signal axis inducing autophagy to alleviate hepatic ischemia‐reperfusion injury

In performing our experiment, impaired autophagy increased hepatocellular damage during the reperfusion period. It was demonstrated by the effect of blocking autophagy using bafilomycin A1 or knocking Atg5 gene out reduces the anti-apoptotic effect of Stat3. Here we focus on the role of signal transducer and activator of transcription 3 (Stat3) in regulating autophagy to alleviate hepatic IRI. We found that Stat3 was up-regulated during hepatic IRI and was associated with an activation of the autophagic signaling pathway. This increased Stat3 expression, which was allied with high autophagic activity, alleviated liver damage to IR, an effect which was abrogated by Stat3 epletion as demonstrated in both in vivo and in vitro methods. The levels of Atg5 protein were decreased when Stat3 was inhibited by HO 3867 or siStat3. We conclude that Stat3 appeared to exert a pivotal role in hepatic IRI, by activating autophagy to alleviate hepatic IRI, and Atg5 was required for this process. The identification of this novel pathway, that links expression levels of Stat3 with Atg5-mediated autophagy, may provide new insights for the generation of novel protective therapies directed against hepatic IRI.

Interferon regulatory factor-1 activates autophagy to aggravate hepatic ischemia-reperfusion injury via the P38/P62 pathway in mice

Increasing evidence has linked autophagy to a detrimental role in hepatic ischemia- reperfusion (IR) injury (IRI). Here we focus on the role of interferon regulatory factor-1 (IRF-1) in regulating autophagy to aggravate hepatic IRI. We found that IRF-1 was up-regulated during hepatic IRI and was associated with an activation of the autophagic signaling. This increased IRF-1 expression, which was allied with high autophagic activity, amplified liver damage to IR, an effect which was abrogated by IRF-1 depletion. Moreover, IRF-1 contributed to P38 induced autophagic and apoptotic cell death, that can play a key role in liver dysfunction. The levels of P62 mRNA and protein were increased when P38 was activated and decreased when P38 was inhibited by SB203580. We conclude that IRF-1 functioned as a trigger to activate autophagy via P38 activation and that P62 was required for this P38-mediated autophagy. IRF-1 appears to exert a pivotal role in hepatic IRI, by predisposing hepatocytes to activate an autophagic pathway. Such an effect promotes autophagic cell death through the P38/P62 pathway. The identification of this novel pathway, that links expression levels of IRF-1 with autophagy, may provide new insights for the generation of novel protective therapies directed against hepatic IRI.

Evidence of Placental Autophagy during Early Pregnancy after Transfer of In Vitro Produced (IVP) Sheep Embryos.

Pregnancies obtained by Assisted Reproductive Technologies (ART) are associated with limited maternal nutrient uptake. Our previous studies shown that in vitro culture of sheep embryos is associated with vascularization defects in their placentae and consequent reduction of embryo growth. Autophagy is a pro-survival cellular mechanism triggered by nutrient insufficiency. Therefore, the goal of our present study was to determine if autophagy is involved in early placental development after transfer of in vitro produced (IVP) embryos. To do this, placentae obtained following transfer of IVP sheep embryos were compared with placentae obtained after natural mating (control—CTR). The placentae were collected on day 20 post-fertilization and post-mating, respectively, and were analyzed using molecular (qPCR), ultrastructural and histological/immunological approaches. Our results show drastically increased autophagy in IVP placentae: high levels of expression (p<0.05) of canonical markers of cellular autophagy and a high proportion of autophagic cells (35.08%; p<0.001) were observed. We conclude that high autophagic activity in IVP placentae can be a successful temporary counterbalance to the retarded vasculogenesis and the reduction of foetal growth observed in pregnancies after transfer of IVP embryos.

MiR-409-3p sensitizes colon cancer cells to oxaliplatin by inhibiting Beclin-1-mediated autophagy.

The chemoresistance of colon cancer cells limits the efficacy of chemotherapy. miR-409-3p has been shown to be downregulated in various types of cancer. In the present study, we examined the role of miR-409-3p in colon cancer as well as the effects of miR‑409-3p on the sensitivity of colon cancer cells to oxaliplatin. The expression of miR-409 was significantly downregulated in the human colon cancer cell lines compared with the normal colon epithelial cells. Importantly, the miR-409-3p expression levels were lower in human colon cancer patient samples than in normal colon tissues. Moreover, we observed a negative correlation between the miR‑409-3p levels and resistance to oxaliplatin: the oxaliplatin-resistant colon cancer cells exhibited significantly downregulated miR‑409-3p levels, but higher autophagic activity than the oxaliplatin-sensitive cells. Using bioinformatics analysis, we predicted that miR‑409-3p miRNA binds to the key autophagy gene encoding Beclin-1. Our findings indicated that the overexpression of miR‑409-3p inhibited Beclin-1 expression and autophagic activity by binding to the 3'-untranslated region of Beclin-1 mRNA. In addition, the overexpression of miR‑409-3p enhanced the chemosensitivity of the oxaliplatin-sensitive and oxaliplatin-resistant colon cancer cells. The restoration of Beclin-1 abrogated these effects of miR‑409-3p. In a xenograft model using nude mice, we examined the effects of miR‑409-3p on tumor growth during chemotherapy. miR‑409-3p overexpression sensitized the tumor to chemotherapy, while inhibiting chemotherapy-induced autophagy in a manner dependent on Beclin-1. The findings of our study suggest that miR-409-3p is capable of enhancing the chemosensitivity of colon cancer cells by inhibiting Beclin-1-mediated autophagy.

Umbilical Cord-Derived Mesenchymal Stem Cells Suppress Autophagy of T Cells in Patients with Systemic Lupus Erythematosus via Transfer of Mitochondria.

Aberrant autophagy played an important role in the pathogenesis of autoimmune diseases, especially in systemic lupus erythematosus (SLE). In this study, we showed that T cells from SLE patients had higher autophagic activity than that from healthy controls. A correlation between autophagic activity and apoptotic rate was observed in activated T cells. Moreover, activation of autophagy with rapamycin increased T cell apoptosis, whereas inhibition of autophagy with 3-MA decreased T cell apoptosis. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) could inhibit respiratory mitochondrial biogenesis in activated T cells to downregulate autophagy and consequently decrease T cell apoptosis through mitochondrial transfer and thus may play an important role in SLE treatment.

Estrogen receptor α regulates non-canonical autophagy that provides stress resistance to neuroblastoma and breast cancer cells and involves BAG3 function.

Breast cancer is a heterogeneous disease and approximately 70% of newly diagnosed breast cancers are estrogen receptor (ER) positive. Out of the two ER types, α and β, ERα is the only ER that is detectable by immunohistochemistry in breast cancer biopsies and is the predominant subtype expressed in breast tumor tissue. ER-positive tumors are currently treated with anti-hormone therapy to inhibit ER signaling. It is well known that breast cancer cells can develop endocrine resistance and resistance to anti-hormone therapy and this can be facilitated via the autophagy pathway, but so far the description of a detailed autophagy expression profile of ER-positive cancer cells is missing. In the present study, we characterized tumor cell lines ectopically expressing ERα or ERβ as well as the breast cancer-derived MCF-7 cell line endogenously expressing ERα but being ERβ negative. We could show that ERα-expressing cells have a higher autophagic activity than cells expressing ERβ and cells lacking ER expression. Additionally, for autophagy-related gene expression we describe an ERα-specific ‘autophagy-footprint' that is fundamentally different to tumor cells expressing ERβ or lacking ER expression. This newly described ERα-mediated and estrogen response element (ERE)-independent non-canonical autophagy pathway, which involves the function of the co-chaperone Bcl2-associated athanogene 3 (BAG3), is independent of classical mammalian target of rapamycin (mTOR) and phosphatidylinositol 3 kinase (PI3K) signaling networks and provides stress resistance in our model systems. Altogether, our study uncovers a novel non-canonical autophagy pathway that might be an interesting target for personalized medicine and treatment of ERα-positive breast cancer cells that do not respond to anti-hormone therapy and classical autophagy inhibitors.

Autophagy is Required for the Maintenance of Liver Progenitor Cell Functionality

Background: Liver progenitor cells (LPCs) are bipotent stem cells existing in the adult liver, which could be activated upon massive liver injury and contribute to liver regeneration. However, mechanisms of maintenance of LPC functionality remain poorly understood. Previous studies found that autophagy was required for the self-renewal and differentiation of several tissue stem cells. Methods: The study compared the level of autophagic activity in LPCs and differentiated hepatocytes. Then, autophagic activity was inhibited in LPCs by lentivirus-mediated autophagy-related gene 5 or Beclin 1 knockdown. Clonogenic assay, cell viability assays, hepatic differentiation assay, and senescence analysis were conducted to assess the role of autophagy in regulating self-renewal, hepatic differentiation and senescence of LPCs. Results: We observed high autophagic activity in LPCs compared with differentiated hepatocytes. We found that inhibition of autophagy impaired the self-renewal, proliferation, and hepatic differentiation capability of LPCs under normal cultural condition, but had little impact on cell viability. Interestingly, while wild-type LPCs remained rarely affected by the toxin, etoposide, inhibition of autophagy induced the senescent phenotype of LPCs. Overexpression of Beclin 1 in Beclin 1-knockdown LPCs restored the functionality of stem cells. Conclusion: Our findings indicate that autophagy may function as a critical regulator of LPC functionality under both physiological and pathological condition.