Molecular Mechanisms Of Autophagy And Apoptosis Regulated By Endoplasmic Reticulum Stress Related Gene DDIT3in Human Cancer Cells

Cancer has become one of the most deadly diseases, which severely threats human beings’ life span. The.cancer incidence in our country is even more pessimistic. The world registered14million new cancer cases and8.2million deaths in2012, and the numbers for China were3.07million and2.2million respectively, according to the World Cancer Report2014. China accounted for about21.9percent of the world’s new cancer cases in2012and26.8percent of cancer deaths globally. Lung cancer remains the most common and deadliest cancer in the world, with an estimated1.8million new cases and1.59million deaths in2012. More than one-third of such cases occurred in China. The chemotherapy treatment has become an important measure to treat cancer for now, but it can not be denied that the clinically therapeutic drugs exist problems of strong side effects and induce drug-resistance easily. Therefore, to find more safe and effective drugs is evidently important.The endoplasmic reticulum (ER) is an important organelle for the synthesis, folding and secretion of proteins in eukaryotic cells. Either intrinsic or extrinsic stimulus will lead to the folding dysfunction of ER and thus lead to endoplasmic reticulum stress, one cellular stress state. Endoplasmic reticulum stress can clear away the misfolded proteins in ER. If endoplasmic reticulum stress is not reservible, it will cause cellular malfuntion, which leads to the cell death. More and more evidence has revealed that the endoplasmic reticulum stress exists widely in various tumors, and is closely related to the survival of cancer cells and their resistance to anti-tumor therapy. Thus it is the hotspot of current research to design and develop anti-cancer drugs in view of the endoplasmic reticulum stress signaling pathway.Autophagy and apoptosis are two important processes to regulate the fate of cancer cells, but the relationship between them remains unclear. In the tumor cells, endoplasmic reticulum stress may influence the survival or apoptosis through autophagy, or induce apoptosis directly. But how endoplasmic reticulum stress regulates autophagy and apoptosis in human cancer cells to determine cellular fates remains largely elusive. Therefore, to study the regulation of endoplasmic reticulum stress to autophagy and apoptosis in tumor cells has its evident theoretical significance and practicable applied value in design and development of targeted chemotherapeutic drugs.The accumulation of unfolded proteins in the ER cause endoplasmic reticulum stress. These unfolded proteins make the ER molecular chaperon HSPA5disassociate from EIF2AK3, ERN1and ATF6. Upon activation of the ERN1, unconventional splicing initiated by ERN1removes a26-nucleotide intron from unspliced mammalian XBP1mRNA. And the spliced mRNA experiences the translational box frameshift and becomes transcription factor XBP1S (X box binding protein1splicing) with transcription activity. XBP1, as a transcription factor, can up-regulate the expression of genes involved in endoplasmic reticulum associated degredation (ERAD), maintain the quality control of the correct folding of protein in ER and keep the balance of ER in the oxidative and reductive states. In the meanwhile, ERN1can recruit TRAF2and ASK1genes to activate p38and JNK MAPK signaling pathways. EIF2AK3mediates the phosphorylation of transcription initiation factor eIF2a and inhibits protein translation to make cells to enter the temporary’stationary phase1. In this condition, transcription factor ATF4is translated selectively in a cap-independent fashion. ATF4can upregulates the genes involved in the maintainance of ER homeostasis. After released from HSPA5, ATF6translocates to golgi apparatus, where it is spliced by site1protease (SIP) and site2protease (S2P). The spliced fragments, acting as transcription factors, are involved in upregulating the expressions of several genes associated with ERAD and ER homeostasis. In the UPR process, DDIT3(c/EBP homologous protein, CHOP) is the target gene of EIF2AK3, ATF6, and ERN1. CHOP, which is identified as the first member of C/EBPs (CCAAT/enhancer binding proteins), performs its action as a dominant negative inhibitor. CHOP is also called GADD153(growth arrest-and DNA damaged-inducible gene153), DDIT3(DNA damage inducible transcript3) and C/EBPξ. C/EBPs regulate many genes which are widely involved in immunity, cellular differentiation, etc. There are six members of C/EBP family identified, C/EBPα, C/EBPβ, C/EBPγ, C/EBPε and DDIT3. DDIT3can interacts with one of the C/EBP family members or interacts with one of the JUN/FOS family members to form a heterodimer, to regulate the transcription of genes induced in the stress processes as transcription factor. DDIT3protein is composed of two known functional domains, an N-terminal transcriptional activation domain and a C-terminal basic-leucine zipper (bZIP) domain consisting of a basicamino-acid-rich DNA-binding region followed by a leucine zipper dimerization motif. It has been reported that DDIT3, acting as a transcriptional factor, can mediate the transcription of BBC3, BCL2L11and TNFRSF10B, which are involved in ER stress induced apoptosis. The recent study suggests that DDIT3can also regulate many autophagy associated genes directly or indirectly to induce autopahgy, for example, DDIT3can act as a transcriptional factor and upregulate the expressions of TRIB3and ATG5to mediate autophagy, which plays a prosurvival role. Therefore, DDIT3may affect cells’ fate by regulating apoptosis and autophagy, two mutually antagonistic biological processes. However, it is still unclear that whether DDIT3can induce autophagy through other pathways or whether DDIT3plays important roles in regulating the relationship between apoptosis and autophagy.Above all, this study aims at exploring the mechanisms underlying ER stress related gene DDIT3regulating autophagy and apoptosis, and the relationship between autophagy and apoptosis inducing by DDIT3. We hope that our study can provide new evidence about the regulation mechanisms of ER stress in autophagy and apoptosis, and may provide basis for the design and development of anticancer drug.1. Molecular mechanisms of autophagy regulated by ER stress related gene DDIT3in human cancer cells 1.1Molecular mechanisms of autophagy regulated by DDIT3acting as a transcription factor in human cancer cellsSalinomycin is perhaps the first promising compound that was discovered through high throughput screening in cancer stem cells. This novel agent can selectively eliminate breast and other cancer stem cells, though the mechanism of action remains unclear. Exploring the molecular mechanisms underlying it will help us to develop new anti-cancer drugs. In this study, we discovered that salinomycin could upregulate the expression of MAP1LC3B and increase the average number of EGFP-MAP1LC3B puncta per cell. In addition, we examined whether salinomycin could induce the autophagic flux in cancer cells. Cotreatment with salinomycin and autophagy inhibitors, such as3-MA and LY294002, or genetic inhibition of autophagy by knocking down either the ATG5or ATG7gene decreased MAP1LC3B-Ⅱ formation and reduced EGFP-MAP1LC3B puncta. In contrast, coincubation with salinomycin and autophagy inhibitors, such as bafilomycin A1or chloroquine, increased MAP1LC3B-II formation and EGFP-MAP1LC3B puncta, indicating that salinomycin induces autophagy in human lung cancer cells. In our study, we demonstrated that salinomycin could upregulate ER stress-related proteins such as phospho-EIF2A, ATF4, DDIT3in both a time-dependent and dose-dependent manner in human NSCLC cells. Furthermore, using RNA interference against ATF4or DDIT3and inhibitor of ER stress (4-PBA) in combination with salinomycin, we confirmed that the EIF2A-ATF4-DDIT3axis was the crucial mediator of salinomycin-induced autophagy. Moreover, our data demonstrate that salinomycin reduces activation of AKT1as well as its downstream substrate MTOR. Taken together, we demonstrated that salinomycin stimulated endoplasmic reticulum stress and mediated autophagy via the ATF4-DDIT3/CHOP-TRIB3-AKT1-MTOR axis. Moreover, we found that the autophagy induced by salinomycin played a prosurvival role in human NSCLC cells and attenuated the apoptotic cascade. To verify whether salinomycin also plays an important role in cancer stem-like cells, we infected A549cells with the shCDHl lentivirus to inhibit CDH1expression, then we treated A549/shCDHl cells with salinomycin for indicated times and detected the expressions of autophagy-related genes and apoptosis-related genes by western blot analysis. The data demonstrated that salinomycin triggered more apoptosis and less autophagy in A549cells in which CDH1expression was inhibited, suggesting that the inhibition of autophagy might represent a promising strategy to target cancer stem cells. In conclusion, these findings provide evidence that combination treatment with salinomycin and pharmacological autophagy inhibitors will be an effective therapeutic strategy for eliminating cancer cells as well as cancer stem cells.1.2Molecular mechanisms of autophagy regulated by DDIT3via interaction with ATG5in human cancer cellsIt has been reported that DDIT3acts as transcription factor and upregulates the expressions of TRIB3and ATG5, both of which can mediate autophagy in human caner cells. However, whether DDIT3also induces autophagy in a novel mechanism remains elusive. In this study, we demonstrated that ATG5is regulated by DDIT3in three distinct mechanisms. Firstly, we confirmed that DDIT3could upregulate the expression of ATG5in A549, H157, H1792and293FT cells, which may verify that DDIT3acts as the transcription factor of ATG5and promotes the expression of ATG5. Secondly, the results of immunoprecipitation assay indicated that DDIT3could interact with ATG5, ATG12-ATG5conjugate and ATG16L1, respectively. In addition, the results of immunofluorescence assay suggested that DDIT3can colocalize with endogenous ATG5and ATG16L1, respectively. Furthermore, we confirmed that DDIT3induces the interaction between ATG12-ATG5conjugate and ATG16L1, which promotes the autophagosome formation. Thirdly, we found that ATG5interacts with DDIT3via its C-terminal region (aa194-274). Moreover, we demonstrated that overexpression of DDIT3could inhibit ATG5from cleavage by Calpain into truncated ATG5(24kDa), which indicates that the interaction between DDIT3and ATG5may protect ATG5from cleavage by Calpain. We also verified that overexpression of DDIT3could slow down the degradation of ATG5, indicating that the interaction between them may maintain the stability of ATG5. In turn, overexpression of ATG5also slowed down the degradation of DDIT3, which suggested that the interaction may promote the stability of DDIT3. In addition, we demonstrated that ATG5could translocate into nucleus after treatment of TG, cisplatin, doxorubicin and etoposide. We also discovered that ATG5played an important roles in DDIT3-dependent transactiviy of TNFRSF10A and TNFRF10B. In sum, our findings indicated that DDIT3could induce autophagy via upregulation of ATG5, or via interaction with ATG5, ATG12-ATG5conjugate and ATG16L to promote autophagosome formation or through sustaining the stability of ATG5. In addition, we also demonstrated that ATG5 could translocate into nucleus and promote DDIT3-dependent transactiviy. These data may enrich our understandings of the mechanisms underlying autophagy and apoptosis induced by DDIT3. The interaction between ATG5and DDIT3may trigger novel crosstalk between autophagy and apoptosis.2. Molecular mechanisms of apoptosis regulated by ER stress related gene DDIT3in human cancer cells2.1Molecular mechanisms of apoptosis mediated by DDIT3via upregulation of TNFRSF10Aand TNFRSF10B in human cancer cellsTumor necrosis factor receptor superfamily member10a (TNFRSF10A; also called DR4or TRAIL-R1) and member10b (TNFRSF10B; also called DR5or TRAIL-R2) are two cell surface receptors which bind to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and mediate the extrinsic pathway of apoptosis. It has been reported that TNFRSF10A and TNFRSF10B up-regulation can enhance the apoptosis mediated by ER stress inducers. It has been reported that DDIT3acts as a transcription factor to enhance TNFRSF10B expression and triggers ER stress-induced apoptosis. However, more details underlying the regulation of TNFRSF10B by DDIT3have not been explored. In addition, whether TNFRSF10A, another important death receptor, is also regulated by DDIT3and mediates ER stress-induced apoptosis remains elusive. In this study, we found that DDIT3and TNFRSF10A expressions were upregulated by ER stress inducers, such as TG and Tm, in A549, H1792and H460cells, respectively. In addition, we demonstrated that DDIT3overexpression causes TNFRSF10A induction in the indicated four human lung cancer cell lines, while TNFRSF10A induction was inhibited in DDIT3-konckdown cells after treatment with ER stress inducers, suggesting that TNFRSF10A expression is regulated in a DDIT3-dependent manner. To verify whether DDIT3may act as a transcription factor and induce TNFRSF10A expression through binding to the TNFRSF10A promoter, we predicted and confirmed that there were two putative binding sites located at-1636/-1625and-371/-364in the TNFRSF10A promoter, respectively, and one putative AP-1binding site located in located at-304/-298in TNFRSF10A promoter region, which is the most important among them after our verification. Furthermore, we found that DDIT3interacts directly with phospho-JUN and the DDIT3/phospho-JUN heterodimer binds to the AP-1binding site (-304/-298) within the TNFRSFIOA promoter region. In addition, we confirmed that KAT2A physically interacted with DDIT3. Importantly, knockdown of KAT2A evidently downregulated both the expressions of TNFRSF10A and TNFRSF10B and dramatically decreased luciferase activity of the cells transfected with luciferase reporter plasmid containing AP-1binding site (-304/-298) of the TNFRSF10A promoter and luciferase activity of the cells transfected with luciferase reporter plasmid containing DDIT3binding site (-276/-264) of the TNFRSF10B promoter. Chromatin immunoprecipitation (ChlP) assays showed that KAT2A may participate in KAT2A/DDIT3/phospho-JUN complex or KAT2A/DDIT3complex and acetylate H3K9/K14to further promote the transcription of TNFRSF10A and TNFRSF10B, respectively. Moreover, we verified that KAT2A is recruited by DDIT3at the DDIT3binding site located at-276A264in TNFRSF10B promoter region and mediates H3K9/K14acetylation to further enhance TNFRSF10B transcription. Our findings highlight two novel mechanisms that underlie ER stress-induced TNFRSF10A and TNFRSF10B expression and apoptosis, which will be helpful to elucidate the mechanisms by which anticancer drugs mediates induces apoptosis.