Regulation of Autophagy Via PERK-eIF2α Effectively Relieve the Radiation Myelitis Induced by Iodine-125

Radiation myelitis is the most serious complication in clinical radiotherapy for spinal metastases. We previously showed that ¹²⁵I brachytherapy induced apoptosis of spinal cord neurons accompanied by autophagy. In this study, we further investigated the mechanism by which ¹²⁵I radiation triggered autophagy in neural cells. We found that autophagy induced by ¹²⁵I radiation was involved in endoplasmic reticulum (ER) stress and mainly dependent on PERK-eIF2α pathway. The expressions of LC3II, ATG12 and PI3K were significantly suppressed in PERK knockout neural cells. Meanwhile, the expressions of phosphorylated-Akt s473 and caspase3/8 all significantly increased in neural cells transfected with a PERK siRNA and which enhanced apoptosis of neurons after ¹²⁵I radiation. The results were consistent with that by MTT and Annexin-FITC/PT staining. In annimal model of banna pigs with radiation myelitis caused by ¹²⁵I brachytherapy, we have successfully decreased PERK expression by intrathecal administration of the lentivirus vector. The apoptosis rate was significantly higher than that in control group and which deteriorated radiation myelitis of banna pigs. Thus, autophagy caused by ¹²⁵I radiation was mainly as an attempt of cell survival at an early stage, but it would be a self-destructive process and promoted the process of apoptosis and necrosis radiated by ¹²⁵I for more than 72 hours. The study would be useful and helpful to maximize efficiency of radiation therapy in clinical therapy.     

Transforming Growth Factor-β1 Inhibits Trophoblast Cell Invasion by Inducing Snail-mediated Down-regulation of Vascular Endothelial-cadherin Protein

Human trophoblast cells express transforming growth factor-β (TGF-β) and TGF-β receptors. It has been shown that TGF-β1 treatment decreases the invasiveness of trophoblast cells. However, the molecular mechanisms underlying TGF-β1-decreased trophoblast invasion are still not fully understood. In the current study, we demonstrated that treatment of HTR-8/SVneo human trophoblast cells with TGF-β1 decreased cell invasion and down-regulated the expression of vascular endothelial cadherin (VE-cadherin). In addition, the inhibitory effect of TGF-β1 on VE-cadherin was confirmed in primary cultures of human trophoblast cells. Moreover, knockdown of VE-cadherin using siRNA decreased the invasiveness of HTR-8/SVneo cells and primary cultures of trophoblast cells. Treatment with TGF-β1 induced the activation of Smad-dependent signaling pathways and the expression of Snail and Slug. Knockdown of Smads attenuated TGF-β1-induced up-regulation of Snail and Slug and down-regulation of VE-cadherin. Interestingly, depletion of Snail, but not Slug, attenuated TGF-β1-induced down-regulation of VE-cadherin. Furthermore, overexpression of Snail suppressed VE-cadherin expression. Chromatin immunoprecipitation analyses showed the direct binding of Snail to the VE-cadherin promoter. These results provide evidence that Snail mediates TGF-β1-induced down-regulation of VE-cadherin, which subsequently contributed to TGF-β1-decreased trophoblast cell invasion.     

Dynamic Monitoring of Cellular Remodeling Induced by the Transforming Growth Factor-β1

The plasticity of differentiated adult cells could have a great therapeutic potential, but at the same time, it is characteristic of progression of serious pathological states such as cancer and fibrosis. In this study, we report on the application of a real-time noninvasive system for dynamic monitoring of cellular plasticity. Analysis of the cell impedance profile recorded as cell index using a real-time cell analyzer revealed its significant increase after the treatment of prostate epithelial cells with the transforming growth factor-β1. Changes in the cell index profile were paralleled with cytoskeleton rebuilding and induction of epithelial–mesenchymal transition and negatively correlated with cell proliferation. This novel application of such approach demonstrated a great potential of the impedance-based system for noninvasive and real-time monitoring of cellular fate.     

Mitochondrial Dysfunction Promotes Breast Cancer Cell Migration and Invasion through HIF1α Accumulation via Increased Production of Reactive Oxygen Species

Although mitochondrial dysfunction has been observed in various types of human cancer cells, the molecular mechanism underlying mitochondrial dysfunction mediated tumorigenesis remains largely elusive. To further explore the function of mitochondria and their involvement in the pathogenic mechanisms of cancer development, mitochondrial dysfunction clones of breast cancer cells were generated by rotenone treatment, a specific inhibitor of mitochondrial electron transport complex I. These clones were verified by mitochondrial respiratory defect measurement. Moreover, those clones exhibited increased reactive oxygen species (ROS), and showed higher migration and invasive behaviors compared with their parental cells. Furthermore, antioxidant N-acetyl cysteine, PEG-catalase, and mito-TEMPO effectively inhibited cell migration and invasion in these clones. Notably, ROS regulated malignant cellular behavior was in part mediated through upregulation of hypoxia-inducible factor-1 α and vascular endothelial growth factor. Our results suggest that mitochondrial dysfunction promotes cancer cell motility partly through HIF1α accumulation mediated via increased production of reactive oxygen species.     

ING1b negatively regulates HIF1α protein levels in adipose-derived stromal cells by a SUMOylation-dependent mechanism

Hypoxic niches help maintain mesenchymal stromal cell properties, and their amplification under hypoxia sustains their immature state. However, how MSCs maintain their genomic integrity in this context remains elusive, since hypoxia may prevent proper DNA repair by downregulating expression of BRCA1 and RAD51. Here, we find that the ING1b tumor suppressor accumulates in adipose-derived stromal cells (ADSCs) upon genotoxic stress, owing to SUMOylation on K193 that is mediated by the E3 small ubiquitin-like modifier (SUMO) ligase protein inhibitor of activated STAT protein γ (PIAS4). We demonstrate that ING1b finely regulates the hypoxic response by triggering HIF1α proteasomal degradation. On the contrary, when mutated on its SUMOylation site, ING1b failed to efficiently decrease HIF1α levels. Consistently, we observed that the adipocyte differentiation, generally described to be downregulated by hypoxia, was highly dependent on ING1b expression, during the early days of this process. Accordingly, contrary to what was observed with HIF1α, the absence of ING1b impeded the adipogenic induction under hypoxic conditions. These data indicate that ING1b contributes to adipogenic induction in adipose-derived stromal cells, and thus hinders the phenotype maintenance of ADSCs.Human mesenchymal stem/stromal cells (MSCs) are able to self-renew and differentiate into various cell types. Recently, MSCs have been developed as tools for tissue engineering and cell-based therapies¹ in particular owing to their trophic and immunosuppressive activities.² Conventionally, the bone marrow MSCs (BM-MSCs) and the adipose-derived stem/stromal cells (ADSCs) have constituted the main sources of MSCs for clinical use. These cells are expanded in vitro prior to their application; however, this long-term culture may allow the emergence of senescence and phenotypic alterations, rendering MSCs unsuitable for clinical purposes.³To overcome these issues, MSC culture in conditions mimicking hypoxic niches has been tested.⁴ Low O₂ tensions promote MSC growth, survival and maintain their self-renewing multipotent state.⁵ However, how hypoxia (1% O₂) affects MSC behavior is unclear. Responses to hypoxia are mainly mediated by hypoxia inducible factors (HIFs). HIF1, 2 and 3α subunits, are constitutively degraded in normoxia and stabilized in hypoxia. Consequently, when stabilized they can dimerize with HIF1β, and then translocate into the nucleus to modulate the expression of selected genes. HIF1α is highly expressed in MSCs, controls their metabolic fate and maintains them in an undifferentiated state.⁶ HIF1α has also been shown to delay the occurrence of senescence in MSCs, by repressing E2A and p21 expression.⁷The inhibitors of growth (ING) family genes act as readers of the epigenetic histone code. Among them, ING1 has been described as a type II tumor suppressor, regulating cell growth, DNA repair, apoptosis, chromatin remodeling and senescence.⁸ To some extent, ING1 and HIF might have opposite effects, (e.g. on tumor progression). Indeed, HIF1α, unlike ING1 that inhibits angiogenesis, promotes angiogenesis.⁹ Furthermore, p53, a well-known ING1b interactor, and HIF1α have been shown in several studies to have antagonistic effects. Following DNA damage, p53 induces apoptosis and inhibits survival of cells by reducing activity and levels of HIF1α.^{10, 11}So far, ING4 has been shown as the only ING protein to regulate the hypoxic response. Indeed, by interacting with HIF prolyl hydroxylase 2 (HPH-2), ING4 has been described to repress some HIF1α activities under hypoxic conditions.¹² Here, we show that ING1b accumulates in ADSCs following DNA damage in hypoxia. According to the opposing roles of ING1b and HIF1α, we hypothesized that ING1b could interfere with HIF1α and participate in the conservation of the genomic integrity of MSCs. Mechanistically, we found that ING1b interacted with HIF1α and promoted its proteasomal degradation in hypoxia. SUMOylation of ING1b played a role since the unSUMOylated form of ING1b was unable to trigger HIF1α degradation. The E3 small ubiquitin-like modifier (SUMO) ligase protein inhibitor of activated STAT protein γ (PIAS4) participated in HIF1α degradation and ING1b accumulation following a genotoxic stress in 1% O₂. ING1b, subsequently, took part in decreasing PIAS4 levels after DNA damage. Finally, we report that ING1b by decreasing HIF1α level modulated ADSC differentiation potential. These data indicate that ING1b, according to its SUMOylation status, regulates the hypoxic response by contributing to the HIF1α degradation, and therefore may impede HIF1α-related effects on the maintenance of ADSCs stem cell character.     

Blocking HIF1α activity eliminates hematological cancer stem cells

Selective targeting of cancer stem cells (CSCs) has the potential to prevent cancer relapse. Wang et?al. (2011) report that hypoxia-inducible factor 1α (HIF1α) represses Notch signaling to maintain CSC subsets from lymphoma, and that blocking HIF1α activity eliminates lymphoma and human acute myeloid leukemia (AML) CSCs.     

Autophagy Facilitates IFN-γ-induced Jak2-STAT1 Activation and Cellular Inflammation

Autophagy is regulated for IFN-γ-mediated antimicrobial efficacy; however, its molecular effects for IFN-γ signaling are largely unknown. Here, we show that autophagy facilitates IFN-γ-activated Jak2-STAT1. IFN-γ induces autophagy in wild-type but not in autophagy protein 5 (Atg5^−/−)-deficient mouse embryonic fibroblasts (MEFs), and, autophagy-dependently, IFN-γ induces IFN regulatory factor 1 and cellular inflammatory responses. Pharmacologically inhibiting autophagy using 3-methyladenine, a known inhibitor of class III phosphatidylinositol 3-kinase, confirms these effects. Either Atg5^−/− or Atg7^−/− MEFs are, independent of changes in IFN-γ receptor expression, resistant to IFN-γ-activated Jak2-STAT1, which suggests that autophagy is important for IFN-γ signal transduction. Lentivirus-based short hairpin RNA for Atg5 knockdown confirmed the importance of autophagy for IFN-γ-activated STAT1. Without autophagy, reactive oxygen species increase and cause SHP2 (Src homology-2 domain-containing phosphatase 2)-regulated STAT1 inactivation. Inhibiting SHP2 reversed both cellular inflammation and the IFN-γ-induced activation of STAT1 in Atg5^−/− MEFs. Our study provides evidence that there is a link between autophagy and both IFN-γ signaling and cellular inflammation and that autophagy, because it inhibits the expression of reactive oxygen species and SHP2, is pivotal for Jak2-STAT1 activation.     

Autophagy regulates myeloid cell differentiation by p62/SQSTM1-mediated degradation of PML-RARα oncoprotein

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 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.     

N-Acetylcysteine Protects Against Hypoxia Mimetic-Induced Autophagy by Targeting the HIF-1α Pathway in Retinal Ganglion Cells

Hypoxia-induced retinal ganglion cell (RGC) death has been proposed to be the critical event in the pathophysiology of glaucoma. Therefore, delaying or halting RGC degeneration, known as neuroprotection, is a novel and promising approach with potential clinical applications for treating glaucoma. In this study, we investigate hypoxia-induced cell death of RGCs and the underlying mechanisms of N-acetylcysteine (NAC) as a neuroprotectant. To establish a model for chemical hypoxia-induced cell death, RGC-5 cells were treated with the hypoxia mimetic cobalt chloride (CoCl₂). Following CoCl₂ exposure, significant levels of apoptotic and autophagic cell death were observed in RGC-5 cells, evidenced by lysosome dysfunction and autophagosome formation. Pretreating RGC-5 cells with NAC significantly counteracted the autophagic cell death. NAC-mediated neuroprotection was attributed to the direct scavenging of reactive oxygen species and was mediated by targeting the hypoxia-inducible factor-1?? pathway via the BNIP3 and PI3K/Akt/mTOR pathways. These results provide insights into the degeneration of RGCs and present a potential clinical application for NAC as a neuroprotectant.     

Autophagy regulates myeloid cell differentiation by p62/SQSTM1-mediated degradation of PML-RARα oncoprotein

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 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.     

The real face of HIF1α in the tumor process

It is well known that the hypoxia-inducible factor 1 α (HIF1α) is detectable as adaptive metabolic response to hypoxia. However, HIF1/HIF1α is detectable even under normoxic conditions, if the metabolism is altered, e.g., high proliferation index. Importantly, both hypoxic metabolism and the Warburg effect have in common a decrease of the intracellular pH value.In our interpretation, HIF1α is not directly accumulated by hypoxia, but by a process which occurs always under hypoxic conditions, a decrease of the intracellular pH value because of metabolic imbalances. We assume that HIF1α is a sensitive controller of the intracellular pH value independently of the oxygen concentration. Moreover, HIF1α has its major role in activating genes to eliminate toxic metabolic waste products (e.g., NH₃/NH₄+) generated by the tumor-specific metabolism called glutaminolysis, which occur during hypoxia, or the Warburg effect. For that reason, HIF1α appears as a potential target for tumor therapy to disturb the pH balance and to inhibit the elimination of toxic metabolic waste products in the tumor cells.     

The real face of HIF1α in the tumor process

It is well known that the hypoxia-inducible factor 1 α (HIF1α) is detectable as adaptive metabolic response to hypoxia. However, HIF1/HIF1α is detectable even under normoxic conditions, if the metabolism is altered, e.g., high proliferation index. Importantly, both hypoxic metabolism and the Warburg effect have in common a decrease of the intracellular pH value.

In our interpretation, HIF1α is not directly accumulated by hypoxia, but by a process which occurs always under hypoxic conditions, a decrease of the intracellular pH value because of metabolic imbalances. We assume that HIF1α is a sensitive controller of the intracellular pH value independently of the oxygen concentration. Moreover, HIF1α has its major role in activating genes to eliminate toxic metabolic waste products (e.g., NH₃/NH₄+) generated by the tumor-specific metabolism called glutaminolysis, which occur during hypoxia, or the Warburg effect. For that reason, HIF1α appears as a potential target for tumor therapy to disturb the pH balance and to inhibit the elimination of toxic metabolic waste products in the tumor cells.     

Targeting HIF1α eliminates cancer stem cells in hematological malignancies

Molecular targeting of cancer stem cells (CSCs) has therapeutic potential for efficient treatment of cancer, although relatively few specific targets have been identified so far. Hypoxia-inducible factor (HIF) was recently shown to regulate the tumorigenic capacity of glioma stem cells under hypoxic conditions. Surprisingly, we found that, under normoxia, HIF1α signaling was selectively activated in the stem cells of mouse lymphoma and human acute myeloid leukemia (AML). HIF1a shRNA and HIF inhibitors abrogated the colony-forming unit (cfu) activity of mouse lymphoma and human AML CSCs. Importantly, the HIF-inhibitor echinomycin efficiently eradicated mouse lymphoma and serially transplantable human AML in xenogeneic models by preferential elimination of CSCs. Hif1α maintains mouse lymphoma CSCs by repressing a negative feedback loop in the Notch pathway. Taken together, our results demonstrate an essential function of Hif1α-Notch interaction in maintaining CSCs and provide an effective approach to target CSCs for therapy of hematological malignancies.     

4-cholesten-3-one suppresses lung adenocarcinoma metastasis by regulating translocation of HMGB1, HIF1α and Caveolin-1

Metastasis is a great challenge in lung adenocarcinoma (ADC) therapy. Cholesterol has been implicated in ADC metastasis. 4-cholesten-3-one, as cholesterol metabolite and analog, can substitute membrane cholesterol and increase membrane fluidity. In this study, we explored the possibility that 4-cholesten-3-one inhibited ADC metastasis. Low-dose 4-cholesten-3-one significantly restrained ADC cells migration and invasion with little effects on cells viabilities. Further investigation showed that 4-cholesten-3-one promoted ROS generation, which transiently activated AMPKα1, increased HIF1α expression, reduced Bcl-2 expression and caused autophagy. AMPKα1 knockdown partly suppressed 4-cholesten-3-one-induced autophagy but, neither prevented 4-cholesten-3-one-induced upregulation of HIF1α or downregulation of Bcl-2. 4-cholesten-3-one-induced autophagy facilitated the release of HMGB1 from nuclei to cytoplasm, blocking nuclear translocation of HIF1α and activation of MMP2 and MMP9. Also, 4-cholesten-3-one induced time-dependent phosphorylation of caveolin-1, Akt and NF-κB. With increasing treatment time, 4-cholesten-3-one accelerated caveolin-1 internalization, but reduced the phosphorylation of Akt and NF-κB, and inhibited the expression of snail and twist. These data suggested that 4-cholesten-3-one could be a potential candidate for anti-metastasis of lung adenocarcinoma.Lung cancer is the leading cause of cancer-related death globally, with an estimated incidence of 1.3 million new cases every year.¹ Lung adenocarcinoma (ADC) is the main common subtype of lung cancer. The high mortality of ADC is mainly attributed to early metastasis.² Because metastasis is a complex and multistep process, the molecular mechanisms of ADC metastasis remain largely unknown.Malignant cells migration is an early event of metastasis, followed by intravasation, survival in the circulation, extravasation and colonization at distant target organs.³ Recent studies have found the connection between cholesterol metabolism and metastases.^{4, 5, 6} Diet-induced hypercholesterolemia accelerates prostate cancer metastases to lymph node, lung and bones.⁷ Inhibition of cholesterol biosynthesis hampers the metastases of colon carcinoma and pancreatic ADC.^{8, 9} The cholesterol metabolite 27-hydroxycholesterol promotes breast cancer metastasis by activating liver X receptor.¹⁰ Cholesterol as an essential component of lipid rafts impacts diverse signaling molecules that mediate multiple biological functions, such as cell survival and death.¹¹ A series of evidences have confirmed that the depletion of membrane cholesterol disrupts lipid rafts, resulting in cell apoptosis.^{12, 13, 14} Elevated cholesterol level has been found in various tumors, including prostate, lung, acute myeloid leukemia and breast cancer,^{15, 16, 17, 18} especially in chemoresistant tumors.^{19, 20} Cholesterol accumulation in solid tumors promotes the proliferation, differentiation and migration of tumor cells by mediating cellular surface molecules, such as caveolin-1 translocation.²¹ Depletion of membrane cholesterol suppresses the phosphorylation of Akt and ERK.^{22, 23} Furthermore, membrane cholesterol depletion also restrains the expression of BCL-2 family members.²⁴ Thus, dysregulated cholesterol metabolism is likely to be implicated in tumor metastases through related signaling pathway.Our previous results suggest that cholesterol oxidation by cholesterol oxidase from Bordetella species (COD-B) promotes the irreversible apoptosis of ADC cells.²⁵ COD-B as a microbial flavoprotein can oxidize cholesterol to 4-cholesten-3-one. In this study, we further investigated whether 4-cholesten-3-one influenced ADC metastasis. We evidenced that low-dose 4-cholesten-3-one inhibited ADC migration in vitro and metastasis in vivo by inducing the translocations of HMGB1, HIF1α and caveolin-1. Our data demonstrated that translocations of HMGB1 and HIF1α had key roles in ADC metastasis.