Xiaofang Sun

HMGB1 in Health and Disease

Complex genetic and physiological variations as well as environmental factors that drive emergence of chromosomal instability, development of unscheduled cell death, skewed differentiation, and altered metabolism are central to the pathogenesis of human diseases and disorders. Understanding the molecular bases for these processes is important for the development of new diagnostic biomarkers, and for identifying new therapeutic targets. In 1973, a group of non-histone nuclear proteins with high electrophoretic mobility was discovered and termed high-mobility group (HMG) proteins. The HMG proteins include three superfamilies termed HMGB, HMGN, and HMGA. High-mobility group box 1 (HMGB1), the most abundant and well-studied HMG protein, senses and coordinates the cellular stress response and plays a critical role not only inside of the cell as a DNA chaperone, chromosome guardian, autophagy sustainer, and protector from apoptotic cell death, but also outside the cell as the prototypic damage associated molecular pattern molecule (DAMP). This DAMP, in conjunction with other factors, thus has cytokine, chemokine, and growth factor activity, orchestrating the inflammatory and immune response. All of these characteristics make HMGB1 a critical molecular target in multiple human diseases including infectious diseases, ischemia, immune disorders, neurodegenerative diseases, metabolic disorders, and cancer. Indeed, a number of emergent strategies have been used to inhibit HMGB1 expression, release, and activity in vitro and in vivo. These include antibodies, peptide inhibitors, RNAi, anti-coagulants, endogenous hormones, various chemical compounds, HMGB1-receptor and signaling pathway inhibition, artificial DNAs, physical strategies including vagus nerve stimulation and other surgical approaches. Future work further investigating the details of HMGB1 localization, structure, post-translational modification, and identification of additional partners will undoubtedly uncover additional secrets regarding HMGB1s multiple functions.

Ferroptosis: Process and function

Ferroptosis is a recently recognized form of regulated cell death. It is characterized morphologically by the presence of smaller than normal mitochondria with condensed mitochondrial membrane densities, reduction or vanishing of mitochondria crista, and outer mitochondrial membrane rupture. It can be induced by experimental compounds (e.g., erastin, Ras-selective lethal small molecule 3, and buthionine sulfoximine) or clinical drugs (e.g., sulfasalazine, sorafenib, and artesunate) in cancer cells and certain normal cells (e.g., kidney tubule cells, neurons, fibroblasts, and T cells). Activation of mitochondrial voltage-dependent anion channels and mitogen-activated protein kinases, upregulation of endoplasmic reticulum stress, and inhibition of cystine/glutamate antiporter is involved in the induction of ferroptosis. This process is characterized by the accumulation of lipid peroxidation products and lethal reactive oxygen species (ROS) derived from iron metabolism and can be pharmacologically inhibited by iron chelators (e.g., deferoxamine and desferrioxamine mesylate) and lipid peroxidation inhibitors (e.g., ferrostatin, liproxstatin, and zileuton). Glutathione peroxidase 4, heat shock protein beta-1, and nuclear factor erythroid 2-related factor 2 function as negative regulators of ferroptosis by limiting ROS production and reducing cellular iron uptake, respectively. In contrast, NADPH oxidase and p53 (especially acetylation-defective mutant p53) act as positive regulators of ferroptosis by promotion of ROS production and inhibition of expression of SLC7A11 (a specific light-chain subunit of the cystine/glutamate antiporter), respectively. Misregulated ferroptosis has been implicated in multiple physiological and pathological processes, including cancer cell death, neurotoxicity, neurodegenerative diseases, acute renal failure, drug-induced hepatotoxicity, hepatic and heart ischemia/reperfusion injury, and T-cell immunity. In this review, we summarize the regulation mechanisms and signaling pathways of ferroptosis and discuss the role of ferroptosis in disease.

Activation of the p62-Keap1-NRF2 pathway protects against ferroptosis in hepatocellular carcinoma cells.

Ferroptosis is a recently recognized form of regulated cell death caused by an iron‐dependent accumulation of lipid reactive oxygen species. However, the molecular mechanisms regulating ferroptosis remain obscure. Here, we report that nuclear factor erythroid 2‐related factor 2 (NRF2) plays a central role in protecting hepatocellular carcinoma (HCC) cells against ferroptosis. Upon exposure to ferroptosis‐inducing compounds (e.g., erastin, sorafenib, and buthionine sulfoximine), p62 expression prevented NRF2 degradation and enhanced subsequent NRF2 nuclear accumulation through inactivation of Kelch‐like ECH‐associated protein 1. Additionally, nuclear NRF2 interacted with transcriptional coactivator small v‐maf avian musculoaponeurotic fibrosarcoma oncogene homolog proteins such as MafG and then activated transcription of quinone oxidoreductase‐1, heme oxygenase‐1, and ferritin heavy chain‐1. Knockdown of p62, quinone oxidoreductase‐1, heme oxygenase‐1, and ferritin heavy chain‐1 by RNA interference in HCC cells promoted ferroptosis in response to erastin and sorafenib. Furthermore, genetic or pharmacologic inhibition of NRF2 expression/activity in HCC cells increased the anticancer activity of erastin and sorafenib in vitro and in tumor xenograft models. Conclusion: These findings demonstrate novel molecular mechanisms and signaling pathways of ferroptosis; the status of NRF2 is a key factor that determines the therapeutic response to ferroptosis‐targeted therapies in HCC cells. (Hepatology 2016;63:173–184)

Posttranslational modification of autophagy-related proteins in macroautophagy

Macroautophagy is an intracellular catabolic process involved in the formation of multiple membrane structures ranging from phagophores to autophagosomes and autolysosomes. Dysfunction of macroautophagy is implicated in both physiological and pathological conditions. To date, 38 autophagy-related (ATG) genes have been identified as controlling these complicated membrane dynamics during macroautophagy in yeast; approximately half of these genes are clearly conserved up to human, and there are additional genes whose products function in autophagy in higher eukaryotes that are not found in yeast. The function of the ATG proteins, in particular their ability to interact with a number of macroautophagic regulators, is modulated by posttranslational modifications (PTMs) such as phosphorylation, glycosylation, ubiquitination, acetylation, lipidation, and proteolysis. In this review, we summarize our current knowledge of the role of ATG protein PTMs and their functional relevance in macroautophagy. Unraveling how these PTMs regulate ATG protein function during macroautophagy will not only reveal fundamental mechanistic insights into the regulatory process, but also provide new therapeutic targets for the treatment of autophagy-associated diseases.

Autophagy promotes ferroptosis by degradation of ferritin

ABSTRACT Macroautophagy/autophagy is an evolutionarily conserved degradation pathway that maintains homeostasis. Ferroptosis, a novel form of regulated cell death, is characterized by a production of reactive oxygen species from accumulated iron and lipid peroxidation. However, the relationship between autophagy and ferroptosis at the genetic level remains unclear. Here, we demonstrated that autophagy contributes to ferroptosis by degradation of ferritin in fibroblasts and cancer cells. Knockout or knockdown of Atg5 (autophagy-related 5) and Atg7 limited erastin-induced ferroptosis with decreased intracellular ferrous iron levels, and lipid peroxidation. Remarkably, NCOA4 (nuclear receptor coactivator 4) was a selective cargo receptor for the selective autophagic turnover of ferritin (namely ferritinophagy) in ferroptosis. Consistently, genetic inhibition of NCOA4 inhibited ferritin degradation and suppressed ferroptosis. In contrast, overexpression of NCOA4 increased ferritin degradation and promoted ferroptosis. These findings provide novel insight into the interplay between autophagy and regulated cell death.

HSPB1 as a novel regulator of ferroptotic cancer cell death.

Ferroptosis is an iron-dependent form of non-apoptotic cell death, but its molecular mechanism remains largely unknown. Here, we demonstrate that heat shock protein beta-1 (HSPB1) is a negative regulator of ferroptotic cancer cell death. Erastin, a specific ferroptosis-inducing compound, stimulates heat shock factor 1 (HSF1)-dependent HSPB1 expression in cancer cells. Knockdown of HSF1 and HSPB1 enhances erastin-induced ferroptosis, whereas heat shock pretreatment and overexpression of HSPB1 inhibits erastin-induced ferroptosis. Protein kinase C-mediated HSPB1 phosphorylation confers protection against ferroptosis by reducing iron-mediated production of lipid reactive oxygen species. Moreover, inhibition of the HSF1–HSPB1 pathway and HSPB1 phosphorylation increases the anticancer activity of erastin in human xenograft mouse tumor models. Our findings reveal an essential role for HSPB1 in iron metabolism with important effects on ferroptosis-mediated cancer therapy.

PKM2-dependent glycolysis promotes NLRP3 and AIM2 inflammasome activation

Sepsis, severe sepsis and septic shock are the main cause of mortality in non-cardiac intensive care units. Immunometabolism has been linked to sepsis; however, the precise mechanism by which metabolic reprogramming regulates the inflammatory response is unclear. Here we show that aerobic glycolysis contributes to sepsis by modulating inflammasome activation in macrophages. PKM2-mediated glycolysis promotes inflammasome activation by modulating EIF2AK2 phosphorylation in macrophages. Pharmacological and genetic inhibition of PKM2 or EIF2AK2 attenuates NLRP3 and AIM2 inflammasomes activation, and consequently suppresses the release of IL-1β, IL-18 and HMGB1 by macrophages. Pharmacological inhibition of the PKM2–EIF2AK2 pathway protects mice from lethal endotoxemia and polymicrobial sepsis. Moreover, conditional knockout of PKM2 in myeloid cells protects mice from septic death induced by NLRP3 and AIM2 inflammasome activation. These findings define an important role of PKM2 in immunometabolism and guide future development of therapeutic strategies to treat sepsis.

Metallothionein-1G facilitates sorafenib resistance through inhibition of ferroptosis

Hepatocellular carcinoma (HCC) is a major cause of cancer‐related death worldwide and currently has the fastest rising incidence of all cancers. Sorafenib was originally identified as an inhibitor of multiple oncogenic kinases and remains the only approved systemic therapy for advanced HCC. However, acquired resistance to sorafenib has been found in HCC patients, which results in poor prognosis. Here, we show that metallothionein (MT)‐1G is a critical regulator and promising therapeutic target of sorafenib resistance in human HCC cells. The expression of MT‐1G messenger RNA and protein is remarkably induced by sorafenib but not other clinically relevant kinase inhibitors (e.g., erlotinib, gefitinib, tivantinib, vemurafenib, selumetinib, imatinib, masitinib, and ponatinib). Activation of the transcription factor nuclear factor erythroid 2‐related factor 2, but not p53 and hypoxia‐inducible factor 1‐alpha, is essential for induction of MT‐1G expression following sorafenib treatment. Importantly, genetic and pharmacological inhibition of MT‐1G enhances the anticancer activity of sorafenib in vitro and in tumor xenograft models. The molecular mechanisms underlying the action of MT‐1G in sorafenib resistance involve the inhibition of ferroptosis, a novel form of regulated cell death. Knockdown of MT‐1G by RNA interference increases glutathione depletion and lipid peroxidation, which contributes to sorafenib‐induced ferroptosis. Conclusion: These findings demonstrate a novel molecular mechanism of sorafenib resistance and suggest that MT‐1G is a new regulator of ferroptosis in HCC cells. (Hepatology 2016;64:488‐500)

Generation of induced pluripotent stem cells from human β-thalassemia fibroblast cells

Induced pluripotent stem (iPS) cells have recently been generated by directly introducing several transcription factors into differentiated human somatic cells, and these iPS cells show great similarities to embryo-derived ES cells [1-3]. Moreover, patient-specific iPS cells have recently been generated, and these studies provided hopes for patients with genetic and degenerative diseases [4, 5]. β-thalassemia is an inherited blood disorder that is characterized by reduced synthesis of hemoglobin subunit beta (hemoglobin β-chain). Individuals with thalassemia major (also called Cooleys anemia) have severe anemia and hepatosplenomegaly; without treatment , affected children have severe failure to thrive and a shortened life expectancy. Even with transfusion and chelation therapy treatments, the life span of patients with thalassemia major can only be extended for a limited time. More importantly, β-thalassemia patients are widely distributed throughout the southern part of China, and this genetically inherited disease has threatened millions of peoples lives for decades with no effective treatment available. Here, we report that we have successfully generated β-thalassemia-specific iPS cells, which may pave the way to optimize life span-extending treatments for patients with β-thalassemia major. We used a previously published protocol [6] to generate β-thalassemia patient-specific iPS cells. Retroviruses containing human OCT3/4, SOX2, KLF4, and c-MYC were introduced into fibroblast cells derived from a ho-mozygous β-thalassemia individual. To monitor infection efficiency, a GFP-expressing plasmid pMXs-GFP was used as a control. The single-gene infection efficiency reached approximately 80%-90%, as determined by GFP expression. Six days after transduction, the cells were harvested by trypsinization and plated onto mitomycin C-treated MEF feeder cells at a density of 1×10 5 cells per 100-mm dish. Two days later, the medium was replaced with the one typically used for human ES (hES) cell culture that was supplemented with 10 ng/ml basic fibro-blast growth factor. Approximately 2 weeks after the transduced cells were plated on the feeder cells, two kinds of colonies appeared. Some were granulated colonies that were not similar in morphology to hES cells, whereas the others closely resembled hES cell colonies. These colonies exhibited a flat and tightly packed morphology with sharp edges, and had a high nucleus/cytoplasm ratio and large nucleoli. From each plate, approximately 10 to 50 hES-like colonies were obtained, and these colonies were individually isolated and mechanically passaged around 25 days after infection. These cells were passaged every 3-5 days, and they displayed morphologies and growth rates that are similar to those of hES cells …

HMGB1-dependent and -independent autophagy.

HMGB1 (high mobility group box 1) is a multifunctional, ubiquitous protein located inside and outside cells that plays a critical role in various physiological and pathological processes including cell development, differentiation, inflammation, immunity, metastasis, metabolism, and death. Increasing evidence demonstrates that HMGB1-dependent autophagy promotes chemotherapy resistance, sustains tumor metabolism requirements and T cell survival, prevents polyglutamine aggregates and excitotoxicity, and protects against endotoxemia, bacterial infection, and ischemia-reperfusion injury in vitro or in vivo. In contrast, HMGB1 may not be required for autophagy in some organs such as the liver and heart. Understanding HMGB1-dependent and -independent autophagy in more detail will provide insight into the integrated stress response and guide HMGB1-based therapeutic intervention.