SPHK1/sphingosine kinase 1-mediated autophagy differs between neurons and SH-SY5Y neuroblastoma cells

Although implicated in neurodegeneration, autophagy has been characterized mostly in yeast and mammalian non-neuronal cells. In a recent study, we sought to determine if SPHK1 (sphingosine kinase 1), implicated previously in macroautophagy/autophagy in cancer cells, regulates autophagy in neurons. SPHK1 synthesizes sphingosine-1-phosphate (S1P), a bioactive lipid involved in cell survival. In our study, we discovered that, when neuronal autophagy is pharmacologically stimulated, SPHK1 relocalizes to the endocytic and autophagic organelles. Interestingly, in non-neuronal cells stimulated with growth factors, SPHK1 translocates to the plasma membrane, where it phosphorylates sphingosine to produce S1P. Whether SPHK1 also binds to the endocytic and autophagic organelles in non-neuronal cells upon induction of autophagy has not been demonstrated. Here, we determined if the effect in neurons is operant in the SH-SY5Y neuroblastoma cell line. In both non-differentiated and differentiated SH-SY5Y cells, a short incubation of cells in amino acid-free medium stimulated the formation of SPHK1-positive puncta, as in neurons. We also found that, unlike neurons in which these puncta represent endosomes, autophagosomes, and amphisomes, in SH-SY5Y cells SPHK1 is bound only to the endosomes. In addition, a dominant negative form of SPHK1 was very toxic to SH-SY5Y cells, but cultured primary cortical neurons tolerated it significantly better. These results suggest that autophagy in neurons is regulated by mechanisms that differ, at least in part, from those in SH-SY5Y cells.     

XBP-1u suppresses autophagy by promoting the degradation of FoxO1 in cancer cells

Autophagy is activated to maintain cellular energy homeostasis in response to nutrient starvation. However, autophagy is not persistently activated, which is poorly understood at a mechanistic level. Here, we report that turnover of FoxO1 is involved in the dynamic autophagic process caused by glutamine starvation. X-box-binding protein-1u (XBP-1u) has a critical role in FoxO1 degradation by recruiting FoxO1 to the 20S proteasome. In addition, the phosphorylation of XBP-1u by extracellular regulated protein kinases1/2 (ERK1/2) on Ser61 and Ser176 was found to be critical for the increased interaction between XBP-1u and FoxO1 upon glutamine starvation. Furthermore, knockdown of XBP-1u caused the sustained level of FoxO1 and the persistent activation of autophagy, leading to a significant decrease in cell viability. Finally, the inverse correlation between XBP-1u and FoxO1 expression agrees well with the expression profiles observed in many human cancer tissues. Thus, our findings link the dynamic process of autophagy to XBP-1u-induced FoxO1 degradation.     

Analysis of the role of autophagy inhibition by two complementary human cytomegalovirus BECN1/Beclin 1-binding proteins

Autophagy is activated early after human cytomegalovirus (HCMV) infection but, later on, the virus blocks autophagy. Here we characterized 2 HCMV proteins, TRS1 and IRS1, which inhibit autophagy during infection. Expression of either TRS1 or IRS1 was able to block autophagy in different cell lines, independently of the EIF2S1 kinase, EIF2AK2/PKR. Instead, TRS1 and IRS1 interacted with the autophagy protein BECN1/Beclin 1. We mapped the BECN1-binding domain (BBD) of IRS1 and TRS1 and found it to be essential for autophagy inhibition. Mutant viruses that express only IRS1 or TRS1 partially controlled autophagy, whereas a double mutant virus expressing neither protein stimulated autophagy. A mutant virus that did not express IRS1 and expressed a truncated form of TRS1 in which the BBD was deleted, failed to control autophagy. However, this mutant virus had similar replication kinetics as wild-type virus, suggesting that autophagy inhibition is not critical for viral replication. In fact, using pharmacological modulators of autophagy and inhibition of autophagy by shRNA knockdown, we discovered that stimulating autophagy enhanced viral replication. Conversely, inhibiting autophagy decreased HCMV infection. Thus, our results demonstrate a new proviral role of autophagy for a DNA virus.     

The sphingosine kinase 1 inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole induces proteasomal degradation of sphingosine kinase 1 in mammalian cells

Sphingosine kinase 1 (SK1) is an enzyme that catalyzes the phosphorylation of sphingosine to produce the bioactive lipid sphingosine 1-phosphate (S1P). We demonstrate here that the SK1 inhibitor, SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole) induces the proteasomal degradation of SK1 in human pulmonary artery smooth muscle cells, androgen-sensitive LNCaP prostate cancer cells, MCF-7 and MCF-7 HER2 breast cancer cells and that this is likely mediated by ceramide as a consequence of catalytic inhibition of SK1 by SKi. Moreover, SK1 is polyubiquitinated under basal conditions, and SKi appears to increase the degradation of SK1 by activating the proteasome. In addition, the proteasomal degradation of SK1a and SK1b in androgen-sensitive LNCaP cells is associated with the induction of apoptosis. However, SK1b in LNCaP-AI cells (androgen-independent) is less sensitive to SKi-induced proteasomal degradation and these cells are resistant to SKi-induced apoptosis, thereby implicating the ubiquitin-proteasomal degradation of SK1 as an important mechanism controlling cell survival.     

TPT1 (tumor protein,translationally-controlled 1) negatively regulates autophagy through the BECN1 interactome and an MTORC1-mediated pathway

TPT1/TCTP (tumor protein, translationally-controlled 1) is highly expressed in tumor cells, known to participate in various cellular activities including protein synthesis, growth and cell survival. In addition, TPT1 was identified as a direct target of the tumor suppressor TP53/p53 although little is known about the mechanism underlying the anti-survival function of TPT1. Here, we describe a role of TPT1 in the regulation of the MTORC1 pathway through modulating the molecular machinery of macroautophagy/autophagy. TPT1 inhibition induced cellular autophagy via the MTORC1 and AMPK pathways, which are inhibited and activated, respectively, during treatment with the MTOR inhibitor rapamycin. We also found that the depletion of TPT1 potentiated rapamycin-induced autophagy by synergizing with MTORC1 inhibition. We further demonstrated that TPT1 knockdown altered the BECN1 interactome, a representative MTOR-independent pathway, to stimulate autophagosome formation, via downregulating BCL2 expression through activating MAPK8/JNK1, and thereby enhancing BECN1-phosphatidylinositol 3-kinase (PtdIns3K)-UVRAG complex formation. Furthermore, reduced TPT1 promoted autophagic flux by modulating not only early steps of autophagy but also autophagosome maturation. Consistent with in vitro findings, in vivo organ analysis using Tpt1 heterozygote knockout mice showed that autophagy is enhanced because of haploinsufficient TPT1 expression. Overall, our study demonstrated the novel role of TPT1 as a negative regulator of autophagy that may have potential use in manipulating various diseases associated with autophagic dysfunction.     

Regulation of glycolytic metabolism by autophagy in liver cancer involves selective autophagic degradation of HK2 (hexokinase 2)

Impaired macroautophagy/autophagy and high levels of glycolysis are prevalent in liver cancer. However, it remains unknown whether there is a regulatory relationship between autophagy and glycolytic metabolism. In this study, by utilizing cancer cells with basal or impaired autophagic flux, we demonstrated that glycolytic activity is negatively correlated with autophagy level. The autophagic degradation of HK2 (hexokinase 2), a crucial glycolytic enzyme catalyzing the conversion of glucose to glucose-6-phosphate, was found to be involved in the regulation of glycolysis by autophagy. The Lys63-linked ubiquitination of HK2 catalyzed by the E3 ligase TRAF6 was critical for the subsequent recognition of HK2 by the autophagy receptor protein SQSTM1/p62 for the process of selective autophagic degradation. In a tissue microarray of human liver cancer, the combination of high HK2 expression and high SQSTM1 expression was shown to have biological and prognostic significance. Furthermore, 3-BrPA, a pyruvate analog targeting HK2, significantly decreased the growth of autophagy-impaired tumors in vitro and in vivo (p < 0.05). By demonstrating the regulation of glycolysis by autophagy through the TRAF6- and SQSTM1-mediated ubiquitination system, our study may open an avenue for developing a glycolysis-targeting therapeutic intervention for treatment of autophagy-impaired liver cancer.     

Serum- and glucocorticoid-regulated kinase 1 (SGK1) induction by the EWS/NOR1(NR4A3) fusion protein

The NR4A3 nuclear receptor (also known as NOR1) is involved in tumorigenesis by the t(9;22) chromosome translocation encoding the EWS/NOR1 fusion protein found in approximately 75% of all cases of extraskeletal myxoid chondrosarcomas (EMC). Several observations suggest that one role of EWS/NOR1 in tumorigenesis may be to deregulate the expression of specific target genes. We have shown previously that constitutive expression of EWS/NOR1 in CFK2 fetal rat chondrogenic cells induces their transformation as measured by growth beyond confluency and growth in soft agar. To identify genes regulated by the fusion protein in this model, we have generated a CFK2 cell line in which the expression of EWS/NOR1 is controlled by tetracycline. Using the differential display technique, we have identified the serum- and glucocorticoid-regulated kinase 1 (SGK1) mRNA as being up-regulated in the presence of EWS/NOR1. Co-immunocytochemistry confirmed over-expression of the SGK1 protein in cells expressing EWS/NOR1. Significantly, immunohistochemistry of 10 EMC tumors positive for EWS/NOR1 showed that all of them over-express the SGK1 protein in contrast to non-neoplastic cells in the same biopsies and various other sarcoma types. These results strongly suggest that SGK1 may be a genuine in vivo target of EWS/NOR1 in EMC.     

Attenuation of TNFSF10/TRAIL-induced apoptosis by an autophagic survival pathway involving TRAF2- and RIPK1/RIP1-mediated MAPK8/JNK activation

Although it is known that tumor necrosis factor-related apoptosis-inducing ligand (TNFSF10/TRAIL) induces autophagy, the mechanism by which autophagy is activated by TNFSF10 is still elusive. In this report, we show evidence that TRAF2- and RIPK1-mediated MAPK8/JNK activation is required for TNFSF10-induced cytoprotective autophagy. TNFSF10 activated autophagy rapidly in cancer cell lines derived from lung, bladder and prostate tumors. Blocking autophagy with either pharmacological inhibitors or siRNAs targeting the key autophagy factors BECN1/Beclin 1 or ATG7 effectively increased TNFSF10-induced apoptotic cytotoxicity, substantiating a cytoprotective role for TNFSF10-induced autophagy. Blocking MAPK8 but not NFκB effectively blocked autophagy, suggesting that MAPK8 is the main pathway for TNFSF10-induced autophagy. In addition, blocking MAPK8 effectively inhibited degradation of BCL2L1/Bcl-xL and reduction of the autophagy-suppressing BCL2L1–BECN1complex. Knockdown of TRAF2 or RIPK1 effectively suppressed TNFSF10-induced MAPK8 activation and autophagy. Furthermore, suppressing autophagy inhibited expression of antiapoptosis factors BIRC2/cIAP1, BIRC3/cIAP2, XIAP and CFLAR/c-FLIP and increased the formation of TNFSF10-induced death-inducing signaling complex (DISC). These results reveal a critical role for the MAPK8 activation pathway through TRAF2 and RIPK1 for TNFSF10-induced autophagy that blunts apoptosis in cancer cells. Thus, suppression of MAPK8-mediated autophagy could be utilized for sensitizing cancer cells to therapy with TNFSF10.     

Attenuation of TNFSF10/TRAIL-induced apoptosis by an autophagic survival pathway involving TRAF2- and RIPK1/RIP1-mediated MAPK8/JNK activation

Although it is known that tumor necrosis factor-related apoptosis-inducing ligand (TNFSF10/TRAIL) induces autophagy, the mechanism by which autophagy is activated by TNFSF10 is still elusive. In this report, we show evidence that TRAF2- and RIPK1-mediated MAPK8/JNK activation is required for TNFSF10-induced cytoprotective autophagy. TNFSF10 activated autophagy rapidly in cancer cell lines derived from lung, bladder and prostate tumors. Blocking autophagy with either pharmacological inhibitors or siRNAs targeting the key autophagy factors BECN1/Beclin 1 or ATG7 effectively increased TNFSF10-induced apoptotic cytotoxicity, substantiating a cytoprotective role for TNFSF10-induced autophagy. Blocking MAPK8 but not NFκB effectively blocked autophagy, suggesting that MAPK8 is the main pathway for TNFSF10-induced autophagy. In addition, blocking MAPK8 effectively inhibited degradation of BCL2L1/Bcl-xL and reduction of the autophagy-suppressing BCL2L1–BECN1complex. Knockdown of TRAF2 or RIPK1 effectively suppressed TNFSF10-induced MAPK8 activation and autophagy. Furthermore, suppressing autophagy inhibited expression of antiapoptosis factors BIRC2/cIAP1, BIRC3/cIAP2, XIAP and CFLAR/c-FLIP and increased the formation of TNFSF10-induced death-inducing signaling complex (DISC). These results reveal a critical role for the MAPK8 activation pathway through TRAF2 and RIPK1 for TNFSF10-induced autophagy that blunts apoptosis in cancer cells. Thus, suppression of MAPK8-mediated autophagy could be utilized for sensitizing cancer cells to therapy with TNFSF10.     

A negative feedback regulation of MTORC1 activity by the lysosomal Ca2+ channel MCOLN1 (mucolipin 1) using a CALM (calmodulin)-dependent mechanism

Macroautophagy/autophagy is an evolutionarily conserved pathway that is required for cellular homeostasis, growth and survival. The lysosome plays an essential role in autophagy regulation. For example, the activity of MTORC1, a master regulator of autophagy, is regulated by nutrients within the lysosome. Starvation inhibits MTORC1 causing autophagy induction. Given that MTORC1 is critical for protein synthesis and cellular homeostasis, a feedback regulatory mechanism must exist to restore MTORC1 during starvation. However, the molecular mechanism underlying this feedback regulation is unclear. In this study, we report that starvation activates the lysosomal Ca²⁺ release channel MCOLN1 (mucolipin 1) by relieving MTORC1's inhibition of the channel. Activated MCOLN1 in turn facilitates MTORC1 activity that requires CALM (calmodulin). Moreover, both MCOLN1 and CALM are necessary for MTORC1 reactivation during prolonged starvation. Our data suggest that lysosomal Ca²⁺ signaling is an essential component of the canonical MTORC1-dependent autophagy pathway and MCOLN1 provides a negative feedback regulation of MTORC1 to prevent excessive loss of MTORC1 function during starvation. The feedback regulation may be important for maintaining cellular homeostasis during starvation, as well as many other stressful or disease conditions.     

Regulatory role of sphingosine kinase and sphingosine-1-phosphate receptor signaling in progenitor/stem cells

Balanced sphingolipid signaling is important for the maintenance of homeostasis. Sphingolipids were demonstrated to function as structural components, second messengers, and regulators of cell growth and survival in normal and disease-affected tissues. Particularly, sphingosine kinase 1 (SphK1) and its product sphingosine-1-phosphate (S1P) operate as mediators and facilitators of proliferation-linked signaling. Unlimited proliferation (selfrenewal) within the regulated environment is a hallmark of progenitor/stem cells that was recently associated with the S1P signaling network in vasculature, nervous,muscular, and immune systems. S1P was shown to regulate progenitor-related characteristics in normal and cancerstemcells(CSCs) viaG-protein coupled receptorsS1Pn(n=1 to 5). The SphK/S1P axis is crucially involved in the regulation of embryonic development of vasculature and the nervous system, hematopoietic stem cell migration, regeneration of skeletal muscle, and development of multiple sclerosis. The ratio of the S1P receptor expression, localization, and specific S1P receptoractivated downstream effectors influenced the rate of selfrenewal and should be further explored as regeneration related targets. Considering malignant transformation,it is essential to control the level of self-renewal capacity.Proliferation of the progenitor cell should be synchronized with differentiation to provide healthy lifelong function of blood, immune systems, and replacement of damaged ordead cells. The differentiation-related role of SphK/S1P remains poorly assessed. A few pioneering investigations exploredpharmacologicaltoolsthattargetsphingolipid signaling and can potentially confine and direct self-renewal towards normal differentiation. Further investigation is required to test the role of the SphK/S1P axis in regulation of self-renewal and differentiation.     

Daintain/AIF-1强化了HepG2细胞顺铂耐药性的产生

目的：探讨炎症因子Daintain/AIF-1对肝癌细胞耐药性产生的影响。方法：利用MTT法测定耐药HepG2细胞株的IC50,流式细胞术测定耐药细胞株期凋亡率,HPLC方法检测隔耐药细胞株胞内顺铂的外排。结果：Daintain/AIF-1提高了HepG2耐药细胞株的IC50;再次受到相同剂量顺铂的攻击时,Daintain/AIF-1与顺铂联合运用构建的耐药细胞株凋亡率明显下降;Daintain/AIF-1促进了耐药细胞株胞内顺铂的外排。结论：此研究表明Daintain/AIF-1通过影响胞内顺铂的外排而促进了肝癌细胞对顺铂耐药性的产生。     

Involvement of catalase in the apoptotic mechanism induced by apigenin in HepG2 human hepatoma cells

Apigenin has been reported to inhibit proliferation of cancer cells; however, the mechanism underlying its action is not completely understood. Here, we evaluated the effects of apigenin on the levels of expression and activity of antioxidant enzymes, and the involvement of ROS in the mechanism of cell death induced by apigenin in HepG2 human hepatoma cells. Upon treatment with apigenin, HepG2 cells displayed a reduction in cell viability in a dose- and time-dependent manner, and some morphological changes. In addition, apigenin treatment induced ROS generation and significantly decreased the mRNA levels and activity of catalase and levels of intracellular GSH. On the other hand, apigenin treatment did not alter the expression or activity levels of other antioxidant enzymes. Addition of exogenous catalase significantly reduced the effects of apigenin on HepG2 cell death. We also demonstrated that HepG2 cells are more sensitive to apigenin-mediated cell death than are primary cultures of mouse hepatocytes, suggesting a differential toxic effect of this agent in tumor cells. Our results suggest that apigenin-induced apoptosis in HepG2 cells may be mediated by a H₂O₂-dependent pathway via reduction of the antioxidant defenses.     

Daintain/AIF-1促进肝癌细胞的增殖与迁移

目的：探讨炎症因子Daintain/AIF-1在肝癌发生发展进程中的作用。方法：利用结晶紫染色方法测定HepG2细胞的增殖,流式细胞术测定细胞周期分布,western blot方法检测相关周期表达蛋白,Transwell方法检测HepG2细胞的迁移。结果：在此研究中我们发现Daintain/AIF-1通过上调周期相关蛋白cyclinD1和cdk4的表达以及增加Rb的磷酸化,加快了HepG2细胞周期的进程,从而促进了HepG2细胞的增殖,另外我们发现Daintain/AIF-1也促进了HepG2细胞的迁移。结论：此研究表明Daintain/AIF-1参与了肝癌的发生发展进程,更进一步证明了炎症因子与癌症的发生发展密不可分。     

Persistent nuclear accumulation of protein kinase CK2 during the G1-phase of the cell cycle does not depend on the ERK1/2 pathway but requires active protein synthesis

Protein kinase CK2 and phosphorylated ERK1/2 accumulated in nucleus after serum stimulation of quiescent HepG2 cells. Nonetheless, phospho-ERK1/2 accumulated mainly in the nuclease-extracted fraction (NE) whereas the increases in nuclear CK2 (either CK2alpha or CK2beta) occurred initially in the nuclease-resistant fraction (NR). Transient decreases in CK2 were observed in cytoplasm and NE in the first 3h but thereafter they either reverted (cytoplasm) or increased above the control (NE). CK2 levels in both NE and NR were high in cells arrested at G1/S. Maximal nuclear accumulation of CK2 was blocked by cycloheximide but little affected by PD98059, SB203580 or apigenin, all of which affected nuclear phopho-ERK1/2. Thus, nuclear accumulation of CK2 during G1 phase is independent of ERK1/2 pathway. Although this process may initially relay on intracellular redistribution of the preexisting enzyme, active protein synthesis is required to attain maximal nuclear CK2 levels.     

PAWR-mediated suppression of BCL2 promotes switching of 3-azido withaferin A (3-AWA)-induced autophagy to apoptosis in prostate cancer cells

An active medicinal component of plant origin with an ability to overcome autophagy by inducing apoptosis should be considered a therapeutically active lead pharmacophore to control malignancies. In this report, we studied the effect of concentration-dependent 3-AWA (3-azido withaferin A) sensitization to androgen-independent prostate cancer (CaP) cells which resulted in a distinct switching of 2 interrelated conserved biological processes, i.e. autophagy and apoptosis. We have observed 3 distinct parameters which are hallmarks of autophagy in our studies. First, a subtoxic concentration of 3-AWA resulted in an autophagic phenotype with an elevation of autophagy markers in prostate cancer cells. This led to a massive accumulation of MAP1LC3B and EGFP-LC3B puncta coupled with gradual degradation of SQSTM1. Second, higher toxic concentrations of 3-AWA stimulated ER stress in CaP cells to turn on apoptosis within 12 h by elevating the expression of the proapoptotic protein PAWR, which in turn suppressed the autophagy-related proteins BCL2 and BECN1. This inhibition of BECN1 in CaP cells, leading to the disruption of the BCL2-BECN1 interaction by overexpressed PAWR has not been reported so far. Third, we provide evidence that pawr-KO MEFs exhibited abundant autophagy signs even at toxic concentrations of 3-AWA underscoring the relevance of PAWR in switching of autophagy to apoptosis. Last but not least, overexpression of EGFP-LC3B and DS-Red-BECN1 revealed a delayed apoptosis turnover at a higher concentration of 3-AWA in CaP cells. In summary, this study provides evidence that 3-AWA is a strong anticancer candidate to abrogate protective autophagy. It also enhanced chemosensitivity by sensitizing prostate cancer cells to apoptosis through induction of PAWR endorsing its therapeutic potential.