Role of hypoxia and autophagy in MDA‐MB‐231 invasiveness

Survival strategies adopted by tumor cells in response to a hypoxic stress include activation of hypoxia‐inducible factor 1 (HIF‐1) and autophagy. However, the importance and the function of each molecular response is not well defined. In the present study, we investigated invasiveness, migration, matrix metalloproteinases (MMPs) activity, and cell survival of MDA‐MB‐231 cells under normoxia, hypoxia, and hypoxia/reoxygenation (H/R). Moreover, to assess the importance of hypoxia and autophagy on the parameters studied, cells were either left untreated or treated with Chetomin (a selective inhibitor of HIF‐1α) or trifluoperazine (TFP, an activator of autophagy). We found that hypoxia and H/R stimulated invasiveness and migration of MDA‐MB‐231 cells with an increased MMP‐2 activity. Chetomin and TFP differently regulated the cellular behavior under the oxygenation conditions studied. In fact, Chetomin was most effective in inhibiting cell invasion, MMPs activity, and cell survival under hypoxia but not normoxia or H/R. By contrast, TFP inhibition of cell invasion, migration, and cell survival was independent from oxygenation conditions. TFP‐induced autophagy was inhibited by light chain protein 3 (LC3) silencing or 3‐methyladenine (3MA) treatment. In fact, LC3‐silenced cells were able to invade in the presence of TFP without any GATE16 processing and p62 degradation. Immunofluorescence assay showed that LC3 silencing inhibited TFP‐induced autophagosome formation. However, we also showed that both TPF treatment and LC3 silencing caused cytoskeleton impairments suggesting a possible interaction between LC3 and cytoskeleton components. In conclusion, our study shows that hypoxia and autophagy by acting on common (HIF‐1α) or separate (MMPs, cytoskeleton) targets differently regulate cell invasion, MMPs activity, and survival. J. Cell. Physiol. 223: 359–368, 2010. © 2010 Wiley‐Liss, Inc.     

Benzyl isothiocyanate causes FoxO1-mediated autophagic death in human breast cancer cells

Benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables, inhibits growth of breast cancer cells but the mechanisms underlying growth inhibitory effect of BITC are not fully understood. Here, we demonstrate that BITC treatment causes FoxO1-mediated autophagic death in cultured human breast cancer cells. The BITC-treated breast cancer cells (MDA-MB-231, MCF-7, MDA-MB-468, BT-474, and BRI-JM04) and MDA-MB-231 xenografts from BITC-treated mice exhibited several features characteristic of autophagy, including appearance of double-membrane vacuoles (transmission electron microscopy) and acidic vesicular organelles (acridine orange staining), cleavage of microtubule-associated protein 1 light chain 3 (LC3), and/or suppression of p62 (p62/SQSTM1 or sequestosome 1) expression. On the other hand, a normal human mammary epithelial cell line (MCF-10A) was resistant to BITC-induced autophagy. BITC-mediated inhibition of MDA-MB-231 and MCF-7 cell viability was partially but statistically significantly attenuated in the presence of autophagy inhibitors 3-methyl adenine and bafilomycin A1. Stable overexpression of Mn-superoxide dismutase, which was fully protective against apoptosis, conferred only partial protection against BITC-induced autophagy. BITC treatment decreased phosphorylation of mTOR and its downstream targets (P70s6k and 4E-BP1) in cultured MDA-MB-231 and MCF-7 cells and MDA-MB-231 xenografts, but activation of mTOR by transient overexpression of its positive regulator Rheb failed to confer protection against BITC-induced autophagy. Autophagy induction by BITC was associated with increased expression and acetylation of FoxO1. Furthermore, autophagy induction and cell growth inhibition resulting from BITC exposure were significantly attenuated by small interfering RNA knockdown of FoxO1. In conclusion, the present study provides novel insights into the molecular circuitry of BITC-induced cell death involving FoxO1-mediated autophagy.     

Mitochondria of highly metastatic breast cancer cell line MDA-MB-231 exhibits increased autophagic properties

Autophagy is a cellular housekeeping process that removes damaged or unwanted cellular components and recycles them to build new constituents. It is essential for tumor growth under adverse environment. Mitochondria play an important role in the formation of autophagosome and its subsequent docking and fusion with lysosome. To understand the contribution of mitochondria to the regulation of homeostatic autophagy in cancer cells, we used the transmitochondrial cytoplasmic hybrid (cybrid) model. Cybrid system allowed us to compare mitochondria from different cell types including highly metastatic breast cancer cell line MDA-MB-231 (c231), less metastatic breast cancer cell lines: MDA-MB-436 (c436) and MDA-MB-468 (c468), as well as non-cancerous mammary epithelial cell MCF-10A (c10A) in a defined nuclear background. The c231 exhibited lower LC3-II levels but higher ratio of LC3-II/LC3-I than c436, c468 and c10A. In addition, c231 displayed more punctate LC3-positive cells and had lower levels of sequestosome 1 (p62/SQSTM1) than other cybrids. These suggested that mitochondria could contribute to the increased autophagy and autophagic flux in metastatic cancer. This increased autophagy was found to be non-selective autophagy instead of selective mitophagy since LC3 puncta in c231 did not co-localize with mitochondria labeled by Mitotracker red or Tomm 20. The promotion of mitochondrial permeability transition (MPT) in c231 also contributed to increased autophagy. Block of MPT by the inhibition of low-conductance stage of MPT pores resulted in a decrease of LC3 puncta in c231. These results suggested that mitochondria from highly metastatic breast cancer cell line MDA-MB-231 can promote homeostatic autophagy of cancer through opening low-conductance MPT pores.     

Differences in the starvation-induced autophagy response in MDA-MB-231 and MCF-7 breast cancer cells

Breast cancer is a heterogeneous disease with distinct subtypes that have made targeted therapy of breast cancer challenging. Previous studies have demonstrated that an altered autophagy capacity can influence the development of breast cancer. However, the molecular differences in starvation-induced autophagic responses in MDA-MB-231 and MCF-7 cells have not been fully elucidated. In this study, we found that an increase of LC3B-II protein expression level and a decrease of the p62 protein expression level in both cells treated by Earle’s balanced salt solution. Meanwhile, we observed an increase of autophagosome using transmission electron microscopy and an enhancement in the green fluorescence intensity of LC3B protein by confocal microscopy. Furthermore, we detected the expression of 13 autophagy-related (ATG) genes and 11 autophagy signaling pathway-related genes using qPCR. Among 13 ATG genes, we found that 6 genes were up-regulated in treated MDA-MB-231 cells, while 4 genes were up-regulated and 1 gene was down-regulated in treated MCF-7 cells. In addition, among 11 autophagy signaling pathway-related genes, 7 genes were up-regulated in treated MDA-MB-231 cells, while 5 genes were up-regulated and 1 gene was down-regulated in treated MCF-7 cells. These findings suggest that the autophagic response to starvation was different in the two treated cell lines, which will contribute to further study on the molecular mechanism of starvation-induced autophagy and improve the targeted therapy of breast cancer.     

Crystal structure of bacterial cell-surface alginate-binding protein with an M75 peptidase motif

Receptor activator of NF-κB (RANK) and RANK ligand (RANKL) are known to play an important role in the development and progression of breast cancer. However, the mechanisms by which stimuli regulate the expression of RANK and RANKL in breast cancer cells are largely unknown. In this study, we show that hypoxia, a common feature of malignant tumors, can enhance the expression of RANK and RANKL mRNA and protein in MDA-MB-231 and MCF-7 breast cancer cells. In addition, we found that hypoxia induced hypoxia-inducible factor-1 alpha (HIF-1α) and phosphorylation of Akt, resulting in upregulation of RANK and RANKL expression; HIF-1α-targeted siRNA and PI3K-Akt inhibitor abrogated this upregulation in MDA-MB-231 cells. Furthermore, we also observed that hypoxia accelerated RANKL-mediated cell migration, which was inhibited following HIF-1α knockdown and PI3K-Akt inhibition. Thus, we provide evidence that hypoxia upregulates RANK and RANKL expression and increases RANKL-induced cell migration via the PI3K/Akt-HIF-1α pathway.     

Hypoxia Induces Autophagic Cell Death through Hypoxia-Inducible Factor 1α in Microglia

As phagocytic cells of central nervous system, excessive activation or cell death of microglia is involved in a lot of nervous system injury and degenerative disease, such as stroke, epilepsy, Parkinson''s disease, Alzheimer''s disease. Accumulating evidence indicates that hypoxia upregulates HIF-1α expression leading to cell death of microglia. However, the exact mechanism of cell death induced by hypoxia in microglia is not clear. In the current study, we showed that hypoxia induced cell death and autophagy in microglia. The suppression of autophagy using either pharmacologic inhibitors (3-methyladenine, bafilomycin A1) or RNA interference in essential autophagy genes (BECN1 and ATG5) decreased the cell death induced by hypoxia in microglia cells. Moreover, the suppression of HIF-1α using either pharmacologic inhibitors (3-MA, Baf A1) or RNA interference decreased the microglia death and autophagy in vitro. Taken together, these data indicate that hypoxia contributes to autophagic cell death of microglia through HIF-1α, and provide novel therapeutic interventions for cerebral hypoxic diseases associated with microglia activation.     

Hypoxia signals autophagy in tumor cells via AMPK activity, independent of HIF-1, BNIP3, and BNIP3L

Macroautophagy (called autophagy hereafter) is a catabolic process activated by various types of stress, most notably by nutrient deprivation. The autophagic degradation of intracellular macromolecules provides metabolic support for the cell; however, this physiological process can also initiate a form of cell death (type 2 programmed cell death). Here we report that oxygen deprivation can activate the autophagic pathway in human cancer cell lines. We observed that hypoxia induced distinct cellular changes characteristic of autophagy such as an increase in cytoplasmic acidic vesicles, and processing and cellular localization of microtubule-associated protein-1 light chain 3. Oxygen deprivation-induced autophagy did not require nutrient deprivation, hypoxia-inducible factor-1 (HIF-1) activity, or expression of the HIF-1 target gene BNIP3 (Bcl-2 adenovirus E1a nineteen kilodalton interacting protein 3) or BNIP3L (BNIP3 like protein). Hypoxia-induced autophagy involved the activity of 5'-AMP-activated protein kinase (AMPK). Finally, we determined that cells lacking the autophagy gene ATG5 were unable to activate the autophagic machinery in hypoxia, had decreased oxygen consumption and increased glucose uptake under hypoxia, had increased survival in hypoxic environments, and exhibited accelerated growth as xenografted tumors. Together, these findings suggest that the autophagic degradation of cellular macromolecules contributes to the energetic balance governed by AMPK, and that suppression of autophagy in transformed cells can increase both resistance to hypoxic stress and tumorigenicity.     

Hypoxia induces autophagic cell death in apoptosis-competent cells through a mechanism involving BNIP3

Hypoxia (lack of oxygen) is a physiological stress often associated with solid tumors. Hypoxia correlates with poor prognosis since hypoxic regions within tumors are considered apoptosisresistant. Autophagy (cellular "self digestion") has been associated with hypoxia during cardiac ischemia and metabolic stress as a survival mechanism. However, although autophagy is best characterized as a survival response, it can also function as a mechanism of programmed cell death. Our results show that autophagic cell death is induced by hypoxia in cancer cells with intact apoptotic machinery. We have analyzed two glioma cell lines (U87, U373), two breast cancer cell lines (MDA-MB-231, ZR75) and one embryonic cell line (HEK293) for cell death response in hypoxia (<1% O(2)). Under normoxic conditions, all five cell lines undergo etoposide-induced apoptosis whereas hypoxia fails to induce these apoptotic responses. All five cell lines induce an autophagic response and undergo cell death in hypoxia. Hypoxia-induced cell death was reduced upon treatment with the autophagy inhibitor 3-methyladenine, but not with the caspase inhibitor z-VAD-fmk. By knocking down the autophagy proteins Beclin-1 or ATG5, hypoxia-induced cell death was also reduced. The pro-cell death Bcl-2 family member BNIP3 (Bcl-2/adenovirus E1B 19kDainteracting protein 3) is upregulated during hypoxia and is known to induce autophagy and cell death. We found that BNIP3 overexpression induced autophagy, while expression of BNIP3 siRNA or a dominant-negative form of BNIP3 reduced hypoxia-induced autophagy. Taken together, these results suggest that prolonged hypoxia induces autophagic cell death in apoptosis-competent cells, through a mechanism involving BNIP3.     

Autophagy in cancer associated fibroblasts promotes tumor cell survival: Role of hypoxia,HIF1 induction and NFκB activation in the tumor stromal microenvironment

Recently, using a co-culture system, we demonstrated that MCF7 epithelial cancer cells induce oxidative stress in adjacent cancer-associated fibroblasts, resulting in the autophagic/lysosomal degradation of stromal caveolin-1 (Cav-1). However, the detailed signaling mechanism(s) underlying this process remain largely unknown. Here, we show that hypoxia is sufficient to induce the autophagic degradation of Cav-1 in stromal fibroblasts, which is blocked by the lysosomal inhibitor chloroquine. Concomitant with the hypoxia-induced degradation of Cav-1, we see the upregulation of a number of well-established autophagy/mitophagy markers, namely LC3, ATG16L, BNIP3, BNIP3L, HIF-1α and NFκB. In addition, pharmacological activation of HIF-1α drives Cav-1 degradation, while pharmacological inactivation of HIF-1 prevents the downregulation of Cav-1. Similarly, pharmacological inactivation of NFκB—another inducer of autophagy—prevents Cav-1 degradation. Moreover, treatment with an inhibitor of glutathione synthase, namely BSO, which induces oxidative stress via depletion of the reduced glutathione pool, is sufficient to induce the autophagic degradation of Cav-1. Thus, it appears that oxidative stress mediated induction of HIF1- and NFκB-activation in fibroblasts drives the autophagic degradation of Cav-1. In direct support of this hypothesis, we show that MCF7 cancer cells activate HIF-1α- and NFκB-driven luciferase reporters in adjacent cancer-associated fibroblasts, via a paracrine mechanism. Consistent with these findings, acute knockdown of Cav-1 in stromal fibroblasts, using an siRNA approach, is indeed sufficient to induce autophagy, with the upregulation of both lysosomal and mitophagy markers. How does the loss of stromal Cav-1 and the induction of stromal autophagy affect cancer cell survival? Interestingly, we show that a loss of Cav-1 in stromal fibroblasts protects adjacent cancer cells against apoptotic cell death. Thus, autophagic cancer-associated fibroblasts, in addition to providing recycled nutrients for cancer cell metabolism, also play a protective role in preventing the death of adjacent epithelial cancer cells. We demonstrate that cancer-associated fibroblasts upregulate the expression of TIGAR in adjacent epithelial cancer cells, thereby conferring resistance to apoptosis and autophagy. Finally, the mammary fat pads derived from Cav-1 (−/−) null mice show a hypoxia-like response in vivo, with the upregulation of autophagy markers, such as LC3 and BNIP3L. Taken together, our results provide direct support for the “autophagic tumor stroma model of cancer metabolism”, and explain the exceptional prognostic value of a loss of stromal Cav-1 in cancer patients. Thus, a loss of stromal fibroblast Cav-1 is a biomarker for chronic hypoxia, oxidative stress and autophagy in the tumor microenvironment, consistent with its ability to predict early tumor recurrence, lymph node metastasis and tamoxifen-resistance in human breast cancers. Our results imply that cancer patients lacking stromal Cav-1 should benefit from HIF-inhibitors, NFκB-inhibitors, anti-oxidant therapies, as well as autophagy/lysosomal inhibitors. These complementary targeted therapies could be administered either individually or in combination, to prevent the onset of autophagy in the tumor stromal compartment, which results in a “lethal” tumor microenvironment.Key words: caveolin-1, autophagy, BNIP3, cancer-associated fibroblasts, HIF1, hypoxia, LC3, mitophagy, NFκB, oxidative stress, predictive biomarker, TIGAR, tumor stroma     

Autophagy in cancer associated fibroblasts promotes tumor cell survival

Recently, using a co-culture system, we demonstrated that MCF7 epithelial cancer cells induce oxidative stress in adjacent cancer-associated fibroblasts, resulting in the autophagic/lysosomal degradation of stromal caveolin-1 (Cav-1). However, the detailed signaling mechanism(s) underlying this process remain largely unknown. Here, we show that hypoxia is sufficient to induce the autophagic degradation of Cav-1 in stromal fibroblasts, which is blocked by the lysosomal inhibitor chloroquine. Concomitant with the hypoxia-induced degradation of Cav-1, we see the upregulation of a number of well-established autophagy/mitophagy markers, namely LC3, ATG16L, BNIP3, BNIP3L, HIF-1α and NFκB. In addition, pharmacological activation of HIF-1α drives Cav-1 degradation, while pharmacological inactivation of HIF-1 prevents the downregulation of Cav-1. Similarly, pharmacological inactivation of NFκB-another inducer of autophagy-prevents Cav-1 degradation. Moreover, treatment with an inhibitor of glutathione synthase, namely BSO, which induces oxidative stress via depletion of the reduced glutathione pool, is sufficient to induce the autophagic degradation of Cav-1. Thus, it appears that oxidative stress mediated induction of HIF1- and NFκB-activation in fibroblasts drives the autophagic degradation of Cav-1. In direct support of this hypothesis, we show that MCF7 cancer cells activate HIF-1α- and NFκB-driven luciferase reporters in adjacent cancer-associated fibroblasts, via a paracrine mechanism. Consistent with these findings, acute knock-down of Cav-1 in stromal fibroblasts, using an siRNA approach, is indeed sufficient to induce autophagy, with the upregulation of both lysosomal and mitophagy markers. How does the loss of stromal Cav-1 and the induction of stromal autophagy affect cancer cell survival? Interestingly, we show that a loss of Cav-1 in stromal fibroblasts protects adjacent cancer cells against apoptotic cell death. Thus, autophagic cancer-associated fibroblasts, in addition to providing recycled nutrients for cancer cell metabolism, also play a protective role in preventing the death of adjacent epithelial cancer cells. We demonstrate that cancer-associated fibroblasts upregulate the expression of TIGAR in adjacent epithelial cancer cells, thereby conferring resistance to apoptosis and autophagy. Finally, the mammary fat pads derived from Cav-1 (-/-) null mice show a hypoxia-like response in vivo, with the upregulation of autophagy markers, such as LC3 and BNIP3L. Taken together, our results provide direct support for the "Autophagic Tumor Stroma Model of Cancer Metabolism," and explain the exceptional prognostic value of a loss of stromal Cav-1 in cancer patients. Thus, a loss of stromal fibroblast Cav-1 is a biomarker for chronic hypoxia, oxidative stress and autophagy in the tumor microenvironment, consistent with its ability to predict early tumor recurrence, lymph node metastasis and tamoxifen-resistance in human breast cancers. Our results imply that cancer patients lacking stromal Cav-1 should benefit from HIF-inhibitors, NFκB-inhibitors, anti-oxidant therapies, as well as autophagy/lysosomal inhibitors. These complementary targeted therapies could be administered either individually or in combination, to prevent the onset of autophagy in the tumor stromal compartment, which results in a "lethal" tumor microenvironment.     

A hypoxia-induced decrease of either MICA/B or Hsp70 on the membrane of tumor cells mediates immune escape from NK cells

Recent findings suggest that hypoxia of the tumor microenvironment contributes to immune escape from natural killer (NK) cell-mediated cytotoxicity. Heat shock protein 70 (Hsp70) and the stress-regulated major histocompatibility class I chain-related protein A and B (MICA/B) both serve as ligands for activated NK cells when expressed on the cell surface of tumor cells. Herein, we studied the effects of hypoxia and hypoxia-inducible factor-1α (HIF-1α) on the membrane expression of these NK cell ligands in H1339 with high and MDA-MB-231 tumor cells with low basal HIF-1α levels and its consequences on NK cell-mediated cytotoxicity. We could show that a hypoxia-induced decrease in the membrane expression of MICA/B and Hsp70 on H1339 and MDA-MB-231 cells, respectively, is associated with a reduced sensitivity to NK cell-mediated lysis. A knockdown of HIF-1α revealed that the decreased surface expression of MICA/B under hypoxia is dependent on HIF-1α in H1339 cells with high basal HIF-1α levels. Hypoxia and HIF-1α did not affect the MICA/B expression in MDA-MB-231 cells but reduced the Hsp70 membrane expression which in turn also impaired NK cell recognition. Furthermore, we could show that the hypoxia-induced decrease in membrane Hsp70 is independent of HIF-1α in MDA-MB-231. Our data indicate that hypoxia-induced downregulation of both NK cell ligands MICA/B and Hsp70 impairs NK cell-mediated cytotoxicity, whereby only MICA/B appears to be regulated by HIF-1α.     

Protein kinase C inhibits autophagy and phosphorylates LC3

During autophagy, the microtubule-associated protein light chain 3 (LC3), a specific autophagic marker in mammalian cells, is processed from the cytosolic form (LC3-I) to the membrane-bound form (LC3-II). In HEK293 cells stably expressing FLAG-tagged LC3, activation of protein kinase C inhibited the autophagic processing of LC3-I to LC3-II induced by amino acid starvation or rapamycin. PKC inhibitors dramatically induced LC3 processing and autophagosome formation. Unlike autophagy induced by starvation or rapamycin, PKC inhibitor-induced autophagy was not blocked by the PI-3 kinase inhibitor wortmannin. Using orthophosphate metabolic labeling, we found that LC3 was phosphorylated in response to the PKC activator PMA or the protein phosphatase inhibitor calyculin A. Furthermore, bacterially expressed LC3 was directly phosphorylated by purified PKC in vitro. The sites of phosphorylation were mapped to T6 and T29 by nanoLC-coupled tandem mass spectrometry. Mutations of these residues significantly reduced LC3 phosphorylation by purified PKC in vitro. However, in HEK293 cells stably expressing LC3 with these sites mutated either singly or doubly to Ala, Asp or Glu, autophagy was not significantly affected, suggesting that PKC regulates autophagy through a mechanism independent of LC3 phosphorylation.     

自噬抑制剂氯奎对低氧诱导肺动脉平滑肌细胞增殖的影响

目的：探讨自噬抑制刺氯喹（cQ）在低氧（hypoxia）调节肺动脉平滑肌细胞（PASMCs）增殖中的作用。方法：将体外培养的大鼠PASMCs分为4组：正常对照组、1％低氧组、50μmol／L氯喹＋1％低氧组、50ttmol／L氯喹组。MTF方法检测各组的PASMCs增殖率;MDC染色检测细胞自噬空泡的变化;Westernblot方法检测微管相关蛋白轻链3（LC3）蛋白的表达变化;划痕法检测细胞迁移的变化。结果：与对照组比较,氯喹组的PASMCs细胞增殖率无明显变化。与对照组比较,1％低氧组PASMCs增殖率明显增加,细胞内出现大量自噬空泡,细胞迁移速度明显增加。细胞LC3-Ⅱ蛋白表达增强。与1％低氧组比较,氯喹与低氧联合作用时细胞自噬空泡的积聚以及rE3．II蛋白表达增强,但细胞增殖率和迁移明显降低。结论：低氧激活自噬过程并促进了PASMCs增殖和迁移,而自噬抑制剂氯喹在一定程度上通过抑制自噬进程,达到抑制肺动脉平滑肌细胞增殖和迁移的作用。     

HIF-1α/FOXO1 axis regulated autophagy is protective for β cell survival under hypoxia in human islets

β cells suffer from hypoxia due to the rapid metabolic rate to supply insulin production. Mechanistic study of β cell survival under hypoxia may shed light on the β cell mass loss in type 2 diabetes mellitus (T2DM). Here, we found that the expressions of LC3 and p62/SQSTM1, two key autophagy regulators, were significantly higher in β cells than that in non-β endocrine cells in both non-diabetic and T2DM human pancreases, and the autophagy process was accelerated upon Cobalt Chloride (CoCl2) treatment in ex vivo cultured primary human islets. Meanwhile, CoCl2 induced the upregulation of FOXO1 in human islets, where HIF-1α played a key role. CoCl2 treatment caused the increase of β cell apoptosis, yet inhibiting autophagy by Chloroquine or by FOXO1 knockdown further aggravated apoptosis, suggesting that FOXO1-regulated autophagy is protective for β cell survival under hypoxia. Immunofluorescence staining showed that LC3 and p62/SQSTM1 expressions were significantly decreased in T2DM patients and negatively correlated with HbA1c, indicating that the autophagy capacity of β cells is impaired along with the progression of the disease. Our study revealed that HIF-1α/FOXO1 regulated autophagy benefits β cell survival under hypoxia and autophagy dysregulation may account for β cell mass loss in T2DM.Brief summaryOur study revealed that HIF-1α/FOXO1 regulated autophagy benefits β cell survival under hypoxia and autophagy dysregulation may account for β cell mass loss in T2DM.     

SLC9A3R1 stimulates autophagy via BECN1 stabilization in breast cancer cells

Autophagy, a self-catabolic process, has been found to be involved in abrogating the proliferation and metastasis of breast cancer. SLC9A3R1 (solute carrier family 9, subfamily A [NHE3, cation proton antiporter 3], member 3 regulator 1), a multifunctional scaffold protein, is involved in suppressing breast cancer cells proliferation and the SLC9A3R1-related signaling pathway regulates the activation of autophagy processes. However, the precise regulatory mechanism and signaling pathway of SLC9A3R1 in the regulation of autophagy processes in breast cancer cells remains unknown. Here, we report that the stability of BECN1, the major component of the autophagic core lipid kinase complex, is augmented in SLC9A3R1-overexpressing breast cancer MDA-MB-231 cells, subsequently stimulating autophagy by attenuating the interaction between BECN1 and BCL2. Initially, we found that SLC9A3R1 partially stimulated autophagy through the PTEN-PI3K-AKT1 signaling cascade in MDA-MB-231 cells. SLC9A3R1 then attenuated the interaction between BECN1 and BCL2 to stimulate the autophagic core lipid kinase complex. Further findings revealed that SLC9A3R1 bound to BECN1 and subsequently blocked ubiquitin-dependent BECN1 degradation. And the deletion of the C-terminal domain of SLC9A3R1 resulted in significantly reduced binding to BECN1. Moreover, the lack of C-terminal of SLC9A3R1 neither reduced the ubiquitination of BECN1 nor induced autophagy in breast cancer cells. The decrease in BECN1 degradation induced by SLC9A3R1 resulted in the activity of autophagy stimulation in breast cancer cells. These findings indicate that the SLC9A3R1-BECN1 signaling pathway participates in the activation of autophagy processes in breast cancer cells.     

HIF-1 Regulation of Chondrocyte Apoptosis: Induction of the Autophagic Pathway

The goal of our investigation was to explore the mechanism by which hypoxia regulates growth plate chondrocyte survival. At low O2 tension, chondrocytes were refractory to a staurosporine (i.e., apoptosis-inducing) challenge. To determine whether hypoxic survival was due to the expression of HIF-1, we evaluated the response of HIF silenced cells to staurosporine. Both, silenced cells and control chondrocytes were equally sensitive to the apoptogen challenge. To learn if resistance was mediated by the proteins of the autophagic pathway, we examined the expression of Beclin 1 and LC3. Both proteins were present in the growth plate as well as in N1511 chondrocytes. Moreover, silencing of Beclin 1 resulted in enhanced chondrocyte death. Thus, this gene served to maintain chondrocyte survival activity. Besides serving a cytoprotective role, it is known that autophagy can function in cell death. Accordingly, to ascertain if autophagy might also sensitize cells to apoptosis, we activated autophagy and examined viability following exposure to an apoptogen. Treatment with the autophagy inhibitor 3-methyladenine rendered the chondrocytes refractory to killing, suggesting that sustained autophagy promoted cell death. We next examined expression of BID and caspase-8. When autophagy was suppressed, chondrocytes promoted caspase-8 activation and activated BID. Finally, we explored the relationship between HIF-1 and Beclin 1. We noted a decrease in Beclin 1 expression and loss of caspase-8 activation in HIF silenced cells and Beclin 1-Bcl-2 association was maintained upon serum starvation. This study indicates that HIF-1 serves to regulate both autophagy and apoptosis.     

Idarubicin induces mTOR-dependent cytotoxic autophagy in leukemic cells

We investigated if the antileukemic drug idarubicin induces autophagy, a process of programmed cellular self-digestion, in leukemic cell lines and primary leukemic cells. Transmission electron microscopy and acridine orange staining demonstrated the presence of autophagic vesicles and intracellular acidification, respectively, in idarubicin-treated REH leukemic cell line. Idarubicin increased punctuation/aggregation of microtubule-associated light chain 3B (LC3B), enhanced the conversion of LC3B-I to autophagosome-associated LC3B-II in the presence of proteolysis inhibitors, and promoted the degradation of the selective autophagic target p62, thus indicating the increase in autophagic flux. Idarubicin inhibited the phosphorylation of the main autophagy repressor mammalian target of rapamycin (mTOR) and its downstream target p70S6 kinase. The treatment with the mTOR activator leucine prevented idarubicin-mediated autophagy induction. Idarubicin-induced mTOR repression was associated with the activation of the mTOR inhibitor AMP-activated protein kinase and down-regulation of the mTOR activator Akt. The suppression of autophagy by pharmacological inhibitors or LC3B and beclin-1 genetic knockdown rescued REH cells from idarubicin-mediated oxidative stress, mitochondrial depolarization, caspase activation and apoptotic DNA fragmentation. Idarubicin also caused mTOR inhibition and cytotoxic autophagy in K562 leukemic cell line and leukocytes from chronic myeloid leukemia patients, but not healthy controls. By demonstrating mTOR-dependent cytotoxic autophagy in idarubicin-treated leukemic cells, our results warrant caution when considering combining idarubicin with autophagy inhibitors in leukemia therapy.     

HIF-1 regulation of chondrocyte apoptosis: induction of the autophagic pathway

The goal of our investigation was to explore the mechanism by which hypoxia regulates growth plate chondrocyte survival. At low O2 tension, chondrocytes were refractory to a staurosporine (i.e., apoptosis-inducing) challenge. To determine whether hypoxic survival was due to the expression of HIF-1, we evaluated the response of HIF silenced cells to staurosporine. Both, silenced cells and control chondrocytes were equally sensitive to the apoptogen challenge. To learn if resistance was mediated by the proteins of the autophagic pathway, we examined the expression of Beclin 1 and LC3. Both proteins were present in the growth plate as well as in N1511 chondrocytes. Moreover, silencing of Beclin 1 resulted in enhanced chondrocyte death. Thus, this gene served to maintain chondrocyte survival activity. Besides serving a cytoprotective role, it is known that autophagy can function in cell death. Accordingly, to ascertain if autophagy might also sensitize cells to apoptosis, we activated autophagy and examined viability following exposure to an apoptogen. Treatment with the autophagy inhibitor 3-methyladenine rendered the chondrocytes refractory to killing, suggesting that sustained autophagy promoted cell death. We next examined expression of BID and caspase-8. When autophagy was suppressed, chondrocytes promoted caspase-8 activation and activated BID. Finally, we explored the relationship between HIF-1 and Beclin 1. We noted a decrease in Beclin 1 expression and loss of caspase-8 activation in HIF silenced cells and Beclin 1-Bcl-2 association was maintained upon serum starvation. This study indicates that HIF-1 serves to regulate both autophagy and apoptosis.