Increasing evidence suggests the important role of metabolic reprogramming in the regulation of the innate inflammatory response, but the underlying mechanism remains unclear. Here, we provide evidence to support a novel role for the pyruvate kinase M2 (PKM2)-mediated Warburg effect, namely aerobic glycolysis, in the regulation of high mobility group box 1 (HMGB1) release. PKM2 interacts with hypoxia-inducible factor 1α (HIF1α) and activates the HIF-1α-dependent transcription of enzymes necessary for aerobic glycolysis in macrophages. Knockdown of PKM2, HIF1α, and glycolysis-related genes uniformly decreases lactate production and HMGB1 release. Similarly, a potential PKM2 inhibitor, shikonin, reduces serum lactate and HMGB1 levels and protects mice from lethal endotoxemia and sepsis. Collectively, these findings shed light on a novel mechanism for metabolic control of inflammation by regulating HMGB1 release and highlight the importance of targeting aerobic glycolysis in the treatment of sepsis and other inflammatory diseases.
Sepsis is caused by an overwhelming immune response to bacterial infection. The discovery of high mobility group box 1 (HMGB1) as a late mediator of lethal sepsis has prompted investigation into the development of new therapeutics which specifically target this protein. Here, we show that chloroquine, an anti-malarial drug, prevents lethality in mice with established endotoxemia or sepsis. This effect is still observed even if administration of chloroquine is delayed. The protective effects of chloroquine were mediated through inhibition of HMGB1 release in macrophages, monocytes, and endothelial cells, thereby preventing its cytokine-like activities. As an inhibitor of autophagy, chloroquine specifically inhibited HMGB1-induced Iκ-B degradation and NF-κB activation. These findings define a novel mechanism for the anti-inflammatory effects of chloroquine and also suggest a new potential clinical use for this drug in the setting of sepsis.
HMGB1; chloroquine; sepsis; autophagy; NF-κB; Beclin 1
Here we investigated the establishment of multicenter cooperative treatment groups in China, as well as radiotherapy compliance and effectiveness among children with renal tumors. Medical records were reviewed for 316 children with renal tumors diagnosed by a multicenter cooperative group from 14 hospitals in China from 1998 to 2012. Median patient age was 29.5 months (range, 2–173 months old), and male-to-female ratio was 1.4 : 1. After a median follow-up of 22 months (range, 1–177 months), five-year event-free survival rates were 72% overall; 76.1% for favorable histology (251 cases); 59% for unfavorable histology (27 cases); and 91%, 75%, 71%, 53%, and 48.5%, respectively for Stages I, II, III, IV, and V. Following standardized criteria, radiation therapy was indicated for 153 patients, among whom five-year event-free survival was 72.8% for the 95 who received radiation and 24% for the 58 patients who did not. Our results are reasonable but can be further improved and show the feasibility of a multicenter cooperative group model for childhood renal tumor treatment in China. Radiation therapy is important for stage III and IV patients but remains difficult to implement in some parts of China. Government management departments and medical professionals must pay attention to this situation. This clinical trial is registered with ChiCTR-PRCH-14004372.
Autophagy is a double-edged sword in tumorigenesis and plays an important role in the resistance of cancer cells to chemotherapy. S100A8 is a member of the S100 calcium-binding protein family and plays an important role in the drug resistance of leukemia cells, with the mechanisms largely unknown. Here we report that S100A8 contributes to drug resistance in leukemia by promoting autophagy. S100A8 level was elevated in drug resistance leukemia cell lines relative to the nondrug resistant cell lines. Adriamycin and vincristine increased S100A8 in human leukemia cells, accompanied with upregulation of autophagy. RNA interference-mediated knockdown of S100A8 restored the chemosensitivity of leukemia cells, while overexpression of S100A8 enhanced drug resistance and increased autophagy. S100A8 physically interacted with the autophagy regulator BECN1 and was required for the formation of the BECN1-PI3KC3 complex. In addition, interaction between S100A8 and BECN1 relied upon the autophagic complex ULK1-mAtg13. Furthermore, we discovered that exogenous S100A8 induced autophagy, and RAGE was involved in exogenous S100A8-regulated autophagy. Our data demonstrated that S100A8 is involved in the development of chemoresistance in leukemia cells by regulating autophagy, and suggest that S100A8 may be a novel target for improving leukemia therapy.
In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
LC3; autolysosome; autophagosome; flux; lysosome; phagophore; stress; vacuole
IFN1@ (interferon, type 1, cluster, also called IFNα) has been extensively studied as a treatment for patients with chronic myeloid leukemia (CML). The mechanism of anticancer activity of IFN1@ is complex and not well understood. Here, we demonstrate that autophagy, a mechanism of cellular homeostasis for the removal of dysfunctional organelles and proteins, regulates IFN1@-mediated cell death. IFN1@ activated the cellular autophagic machinery in immortalized or primary CML cells. Activation of JAK1-STAT1 and RELA signaling were required for IFN1@-induced expression of BECN1, a key regulator of autophagy. Moreover, pharmacological and genetic inhibition of autophagy enhanced IFN1@-induced apoptosis by activation of the CASP8-BID pathway. Taken together, these findings provide evidence for an important mechanism that links autophagy to immunotherapy in leukemia.
IFN1@; autophagy; apoptosis; immunotherapy; chronic myeloid leukemia
microRNAs (miRNAs) are a class of small regulatory RNAs that regulate gene expression at the post-transcriptional level. miRNAs play important roles in the regulation of development, growth, and metastasis of cancer, and in determining the response of tumor cells to anticancer therapy. In recent years, they have also emerged as important regulators of autophagy, a lysosomal-mediated pathway that contributes to degradation of a cell's own components. Imatinib, a targeted competitive inhibitor of the BCR-ABL1 tyrosine kinase, has revolutionized the clinical treatment of chronic myelogenous leukemia (CML). We demonstrate that MIR30A-mediated autophagy enhances imatinib resistance against CML including primary stem and progenitor cells. MIR30A, but not MIR101, is a potent inhibitor of autophagy by selectively downregulating BECN1 and ATG5 expression in CML cells. MIR30A mimics, as well as knockdown of BECN1 and ATG5, increases intrinsic apoptotic pathways. In contrast, the antagomir-30A increases autophagy and inhibits intrinsic apoptotic pathways, confirming that autophagy serves to protect against apoptosis. Taken together, these data clarify some of the underlying molecular mechanisms of tyrosine kinase inhibitor-induced autophagy.
Atg5; autophagy; BCR-ABL tyrosine kinase; Beclin 1; chronic myelogenous leukemia; microRNA
Autophagy is a catabolic process critical to maintaining cellular homeostasis and responding to cytotoxic insult. Autophagy is recognized as “programmed cell survival” in contrast to apoptosis or programmed cell death. Upregulation of autophagy has been observed in many types of cancers and has been demonstrated to both promote and inhibit antitumor drug resistance depending to a large extent on the nature and duration of the treatment-induced metabolic stress as well as the tumor type. Cisplatin, doxorubicin and methotrexate are commonly used anticancer drugs in osteosarcoma, the most common form of childhood and adolescent cancer. Our recent study demonstrated that high mobility group box 1 protein (HMGB1)-mediated autophagy is a significant contributor to drug resistance in osteosarcoma cells. Inhibition of both HMGB1 and autophagy increase the drug sensitivity of osteosarcoma cells in vivo and in vitro. Furthermore, we demonstrated that the ULK1-FIP200 complex is required for the interaction between HMGB1 and BECN1, which then promotes BECN1-PtdIns3KC3 complex formation during autophagy. Thus, these findings provide a novel mechanism of osteosarcoma resistance to therapy facilitated by HMGB1-mediated autophagy and provide a new target for the control of drug-resistant osteosarcoma patients.
osteosarcoma; HMGB1; autophagy; apoptosis; chemotherapy
High mobility group box 1 (HMGB1) is a nuclear DNA-binding protein, which functions as Damage Associated Molecular Pattern molecule (DAMP) when released from cells under conditions of stress, such as injury and infection. Recent studies indicate that HMGB1 plays an important role in leukemia pathogenesis and chemotherapy resistance. Serum HMGB1 is increased in childhood acute lymphocytic leukemia as compared to healthy control and complete remission groups. Moreover, HMGB1 is a negative regulator of apoptosis in leukemia cells through regulation of Bcl-2 expression and caspase-3 activity. As a positive regulator of autophagy, intracellular HMGB1 interacts with Beclin 1 in leukemia cells leading to autophagosome formation. Additionally, exogenous HMGB1 directly induces autophagy and cell survival in leukemia cells. Experimental strategies that selectively target HMGB1 effectively reverse and prevent chemotherapy resistance in leukemia cells, suggesting that HMGB1 is a novel therapeutic target in leukemia.
HMGB1; leukemia; apoptosis; autophagy; chemotherapy
Autophagy and apoptosis are tightly regulated biological processes that are crucial for cell growth, development and tissue homeostasis. UVRAG (UV radiation resistance-associated gene), a mammalian homolog of yeast Vps38, activates the Beclin 1/PtdIns3KC3 (class III phosphatidylinositol-3-kinase) complex, which promotes autophagosome formation. Moreover, UVRAG promotes autophagosome maturation by recruiting class C Vps complexes (HOPS complexes) and Rab7 of the late endosome. We found that UVRAG has anti-apoptotic activity during tumor therapy through interactions with Bax. UVRAG inhibits Bax translocation from the cytosol to mitochondria during chemotherapy- or UV irradiation-induced apoptosis of human tumor cells. Moreover, deletion of the UVRAG C2 domain abolishes Bax binding and anti-apoptotic activity. These results suggest that, in addition to its previously recognized pro-autophagy activity in response to starvation, UVRAG has cytoprotective functions in the cytosol that control the localization of Bax in tumor cells exposed to apoptotic stimuli.
UVRAG; Bax; apoptosis; autophagy; mitochondria; tumor therapy
PML-RARα oncoprotein is a fusion protein of promyelocytic leukemia (PML) and the retinoic acid receptor-α (RARα) and causes acute promyelocytic leukemias (APL). A hallmark of all-trans retinoic acid (ATRA) responses in APL is PML-RARα degradation, which promotes cell differentiation. Here, we demonstrated that autophagy is a crucial regulator of PML-RARα degradation. Inhibition of autophagy by short hairpin (sh) RNA that target essential autophagy genes such as ATG1, ATG5 and PI3KC3, and by autophagy inhibitors (e.g., 3-methyladenine), blocked PML-RARα degradation and subsequently granulocytic differentiation of human myeloid leukemic cells. In contrast, rapamycin, the mTOR kinase inhibitor, enhanced autophagy and promoted ATRA-induced PML-RARα degradation and myeloid cell differentiation. Moreover, PML-RARα co-immunoprecipitated with the ubiquitin-binding adaptor protein p62/SQSTM1, which is degraded through autophagy. Furthermore, knockdown of p62/SQSTM1 inhibited ATRA-induced PML-RARα degradation and myeloid cell differentiation. The identification of PML-RARα as a target of autophagy provides new insight into the mechanism of action of ATRA and its specificity for APL.
autophagy; differentiation; oncoprotein; leukemia; degradation; PML-RARa; p62/SQSTM1
Damage-associated molecular pattern molecules (DAMPs) are cellularly derived molecules that can initiate and perpetuate immune responses following trauma, ischemia and other types of tissue damage in the absence of pathogenic infection. High mobility group box 1 (HMGB1) is a prototypical DAMP and is associated with the hallmarks of cancer. Recently we found that HMGB1 release after chemotherapy treatment is a critical regulator of autophagy and a potential drug target for therapeutic interventions in leukemia. Overexpression of HMGB1 by gene transfection rendered leukemia cells resistant to cell death; whereas depletion or inhibition of HMGB1 and autophagy by RNA interference or pharmacological inhibitors increased the sensitivity of leukemia cells to chemotherapeutic drugs. HMGB1 release sustains autophagy as assessed by microtubule-associated protein 1 light chain 3 (LC3) lipidation, redistribution of LC3 into cytoplasmic puncta, degradation of p62 and accumulation of autophagosomes and autolysosomes. Moreover, these data suggest a role for HMGB1 in the regulation of autophagy through the PI3KC3-MEKERK pathway, supporting the notion that HMGB1-induced autophagy promotes tumor resistance to chemotherapy.
DAMP; autophagy; HMGB1; chemotherapy resistance; leukemia; PI3KC3; ERK
Neuroblastoma is a pediatric malignancy characterized by tremendous clinical heterogeneity, in which some tumors are extremely aggressive while others spontaneously differentiate into benign forms. Because the degree of differentiation correlates with prognosis, and because differentiating agents such as retinoic acid (RA) have proven to decrease mortality, much effort has been devoted to identifying critical regulators of neuroblastoma differentiation in the cellular microenvironment, including cues encoded in the extracellular matrix (ECM). While signaling between tumor cells and the ECM is classically regarded to be based purely on biochemical recognition of ECM ligands by specific cellular receptors, a number of recent studies have made it increasingly clear that the biophysical properties of the ECM may also play an important role in this cross-talk. Given that RA-mediated neuroblastoma differentiation is accompanied by profound changes in cell morphology and neurite extension, both of which presumably rely upon mechanotransductive signaling systems, it occurred to us that mechanical cues from the ECM might also influence RA-mediated differentiation, which in turn might regulate clinically-relevant aspects of neuroblastoma biology. In this study, we tested this hypothesis by subjecting a series of neuroblastoma culture models to ECM microenvironments of varying mechanical stiffness and examined the regulatory role of ECM stiffness in proliferation, differentiation, and expression of tumor markers. We find that increasing ECM stiffness enhances neuritogenesis and suppresses cell proliferation. Remarkably, increasing ECM stiffness also reduces expression of N-Myc, a transcription factor involved in multiple aspects of oncogenic proliferation that is used for evaluating prognosis and clinical grading of neuroblastoma. Furthermore, the addition of RA enhances all of these effects for all ECM stiffnesses tested. Together, our data strongly support the notion that the mechanical signals from the cellular microenvironment influence neuroblastoma differentiation and do so synergistically with RA. These observations support further investigation of the role of microenvironmental mechanical signals in neuroblastoma proliferation and differentiation and suggest that pharmacological agents that modulate the underlying mechanotransductive signaling pathways may have a role in neuroblastoma therapy.