The Mechanism Of DR5-induced Apoptosis In NSCLC Cells

Non-small cell lung carcinoma (NSCLC) is the most common cancer in the United States (>200,000cases/year), and carries a dismal5years15%survival. Surgery is available for a limited number of patients, and chemotherapy remains the mainstay of therapy for this disease. One part of this thesis is tried to figure out the mechanism of apoptosis in NSCLC cells induced by small molecular drugs, such as pemetrexed and salermide.There are two major apoptosis signalling pathways:the intrinsic mitochondria-mediated pathway and the extrinsic death receptor-induced pathway. The truncated form of the pro-apoptotic protein Bid serves as the cross-talk between these two pathways. Death Receptor5(DR5) is an important mediator of the extrinsic apoptotic signalling pathway. Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) binds DR5and preferentially induces apoptosis in transformed cells while sparing normal cells, in contradistinction to other mediators of programmed cell death such as TNF and FasL. DR5expression is inducible by cancer therapeutic and preventive agents, and up-regulation of DR5often explains induction of programmed cell death or augmentation of TRAIL-induced apoptosis. The trimeric activated DR5will recruit the adaptor molecule (FADD) and Pro-caspase-8to form death-inducing signaling complex (DISC). Caspase-8will be activated and lead to the activation of executioner caspases (caspase-3,-6, and-7) in type I cells. In type II cells, Bid was cleaved by the activated caspase-8and translocated from the cytoplasm to the mitochondria, leading to the apoptosis induced by mitochondria.In times of cellular stress, DR5expression can be induced without ligand-receptor interactions, resulting in a ligand-independent activation of the death receptor-mediated apoptotic signalling pathway. Some of the most powerful inducers of DR5transcription that circumvent TRAIL-DR5 interactions include high local concentrations of Sp1, AP1, p53, NFkB, YY1and C/EBP homologous protein (CHOP, also known as growth arrest and DNA damage gene153(GADD153)). CHOP is one of the most potent inducer of DR5and downstream apoptosis, and CHOP is frequently released during the endoplasmic reticulum (ER) stress response. CHOP is typically undetectable in physiological conditions; however, it is dramatically increased during periods of ER stress, resulting in cell cycle arrest and ultimately apoptosis.ER stress is one kind of signaling pathway in response to the stress. In non-stressed cells, Bip (the ER chaperone) binds to the luminal domains of the IRE1a, PERK and ATF6, which are the ER-stress sensors, remaining these proteins in an inactive state. When ER stress is induced by the accumulation of unfolded or misfolded proteins, Bip will combine with the proteins instead of binding with IRE1a, PERK and ATF6, which are major proteins of ER stress. The release of Bip leads to the activation of PERK by homodimerization and trans-autophosphorylation, which phosphorylates the translation-initiation factor elF2α and enhances the transcription of the ATF4mRNA. Some target genes, such as ATF3and CHOP, were induced by the activated ATF4.Both activating transcription factor3(ATF3) and activating transcription factor4(ATF4) belong to the activating transcription factor/cyclic AMP response element binding protein (ATF/CREB) family of basic region-leucine zipper (bZip) transcription factors. ATF3is an adaptive-response gene that participates in cellular processes by activating or repressing specific gene expression. Whereas previous studies have revealed that ATF4plays an important role in regulating CHOP, it is still unclear weather ATF3also plays an important role in CHOP, thereafter DR5regulation.Besides PERK, IRE1α and ATF6are also major effector proteins involved in ER stress. IRE1α dissociates with Bip and dimerization to induce the activity.26-base intron of XBP1mRNA will be removed by IRE1α and the active XBP1encodes the expression of ER stress target genes. In addition, the release of Bip allows ATF6to translocate to the Golgi apparatus and cleaved by the proteases S1P and S2P, resulting in the active ATF6fragment (ATF6p50), which will translocate to the nucleus and activate the transcription of ER stress target genes.A central step in the execution of apoptosis is the activation of caspases, which are widely present as inactive forms. Cellular FLICE-inhibitory protein (c-FLIP) is the pivotal protein that negatively modulates the caspase cascade. Specifically, caspase-8is activated by death receptors through Fas associated death domain (FADD) binding of the death inducing signalling complex (DISC). Thus, the primary role of c-FLIP is a specific inhibitor of death receptor-mediated apoptosis. Accordingly, down-regulation of c-FLIP confers sensitivity to death receptor-induced apoptosis. Although multiple splicing isoforms of c-FLIP mRNA have been reported, c-FLIPL and c-FLIPs, are major splicing variants detectable at the protein level and have been extensively characterised.Pemetrexed is a clinically available anti-folate therapeutic agent used in combination with cisplatin for the management of patients with malignant pleural mesothelioma and advanced non-small cell lung cancer. Pemetrexed inhibits three enzymes in purine and pyrimidine synthesis necessary for precursor DNA nucleotides which in turn disrupts growth and survival of normal and cancer cells. The mechanism by which pemetrexed induces apoptosis remains largely uncharacterised. In the current study, we examined the downstream effect of pemetrexed in inducing apoptosis in lung cancer cells. We showed that pemetrexed induced apoptosis via up-regulation of Death Receptor5(DR5), an important death receptor for tumour necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL). In addition, we discovered a synergistic effect of combination pemetrexed and recombinant TRAIL in inducing apoptosis. Modulating DR5induction by small interfering RNA abrogated the ability of pemetrexed to induce apoptosis. In addition, silencing of C/EBP homologous protein (CHOP) expression reduced DR5expression, demonstrating that the transcriptional factor CHOP has a pivotal role on DR5 up-regulation following pemetrexed treatment. In addition, enforced expression of cellular FLICE-inhibitory protein (c-FLIP), a known inhibitor of caspase8, protected neoplastic cells from apoptosis despite pemetrexed and/or TRAIL therapy. This work demonstrates the efficacy and mechanistic underpinnings of pemetrexed-induced apoptosis, and they suggest pemetrexed may have clinical utility when used in combination with TRAIL for the management of patients with lung cancer.Another chemical we use is salermide, a reverse amide compound, which inhibits Sirtuin1(Sirt1) and Sirtuin2(Sirt2), has been shown to induce apoptosis in human cancer cells. The mechanism underlying cellular apoptotic signalling by salermide remains unclear. In this study, we show that salermide up-regulates the expression of death receptor5(DR5) in human non-small cell lung cancer (NSCLC) cells. Blocking DR5expression by gene silencing technology results in a decrease in activated forms of several pro-apoptotic proteins (caspase-8, caspase-9, caspase-3, PARP). Increasing DR5protein expression correlates with salermide-induced apoptosis in human NSCLC cells. We discovered that IRE-1a, Bip, activating transcription factor3(ATF3), activating transcription factor4(ATF4) and C/EBP homologous protein (CHOP) are induced by salermide, which suggests that DR5-dependent apoptosis is induced by endoplasmic reticulum stress. Moreover, knockdown of Sirt1and Sirt2expression resulted in up-regulation of ATF4, CHOP and DR5. Transfected NSCLC cells with ATF4, ATF3or CHOP siRNA results in a decline in proapoptotic proteins (such as caspase-8, caspase-9, caspase-3and PARP) despite salermide treatment. We demonstrate that salermide induces expression of ATF4, and ATF4up-regulates ATF3and subsequently modulates CHOP. This suggests that DR5is modulated by the ATF4-ATF3-CHOP axis in NSCLC after Sirt112inhibition or salermide treatment, indicating the importance of DR5up-regulation in apoptosis induced by Sirt1/2inhibition and elucidates the underlying mechanism in human NSCLC cells. Another research focus in my thesis is about the development of Kaposi sarcoma (KS), which occurs frequently in people living in Equatorial Africa. It is also a common cancer diagnosed in human immunodeficiency virus (HIV) carriers or patients under immunosuppression. KS is typically found on the skin, mouth, gastrointestinal tract, and lymph node. Growth rates and size of the tumor vary among patients, and can be life-threatening. Kaposi sarcoma-associated herpesvirus (KSHV, also referred to as HHV-8) has been identified as the infectious agent responsible for KS. The incidence of KS in HIV-infected individuals in the United States has been decreasing due to effective highly active antiretroviral therapy (HAART). However, it remains high morbidity and mortality in AIDS patients and some sub-Saharan African countries.KSHV encodes a viral G-protein-coupled receptor (vGPCR) that has been shown to be sufficient to induce neoplastic transformation of endothelial cells. vGPCR has high basal signaling activity, which can be further enhanced via ligands like CXCL1(chemokine (C-X-C motif) ligand1) and CXCL3(chemokine (C-X-C motif) ligand3). Mouse models, such as transgenic mice expressing vGPCR and endothelial-specific vGPCR gene transduction (TIE2-tva transgenic mice), have revealed that the vGPCR-induced tumors are remarkably similar to KS, suggesting vGPCR initiates KS. However, the precise molecular mechanism involved in KSHV-induced Kaposi sarcoma is still unclear.A complicated signaling network is mediated by vGPCR, which contributes to the oncogenic transform of endothelial cells. For example, Pl3Ky is activated by vGPCR and has an important role in vGPCR-induced sarcomagenesis, while NFkB plays a role in vGPCR-induced paracrine neoplasia. In addition, vGPCR dramatical amplification of VEGF secretion by avtivation of AKT, ERK, p38and IKKp, results in the TSC/mTOR activation and HIF upregulation. Furthermore, Heterotrimeric G-proteins are required to relay GPCR signals to downstream effectors. It has been reported small G protein Rac1is activated by vGPCR. Repressing Rac1expression blocks the induction of the NFκB, AP-1, and NFAT activity and inhibits vGPCR sarcomagenesis in vivo. Moreover, expression of Rac1is sufficient to generate KS-like tumors in mouse. Furthermore, the endothelial cell-specific animal model reveals that deletion of Rac1disrupts the endothelial cell function and vascular development.The Hippo tumor-suppressor pathway is fundamental in regulating organ size, and dysregulation promotes cell proliferation and tumorigenesis. Within the Hippo pathway, MST1/2kinases phosphorylate and activate the Lats1/2kinases, which in turn phosphorylate and inhibit the transcription co-activators YAP/TAZ. YAP and TAZ are transcription co-activators with oncogenic potential and their inhibition represents the major functional output of the Hippo pathway. Lats dependent phosphorylation inhibits YAP/TAZ by inducing nuclear exclusion and proteasome dependent degradation. On the contrary, dephosphorylated YAP/TAZ localize to the cell nucleus associating with TEAD family transcriptional factors (TEAD1-4) to stimulate gene expression, particularly genes involved in cell survival and proliferation. Extensive studies have demonstrated that YAP/TAZ play important roles in cancer development. For example, transgenic YAP expression in mice induces tissue overgrowth and tumorigenesis.Increased YAP/TAZ expression and/or nuclear localization are frequently observed in multiple human cancers. However, the mechanisms leading to YAP/TAZ activation in human cancers are largely unknown. In this report, we demonstrate that YAP and TAZ expression is highly elevated in Kaposi sarcoma. KSHV-encoded vGPCR signals through the Hippo pathway to induce oncogenic transformation. Also, downregulation of YAP/TAZ suppresses vGPCR-induced tumorigenesis, indicating a pivotal role of the Hippo pathway in the KS development.In summary, the thesis clarifies the molecular mechanism of apoptosis in NSCLC cells induced by small molecular drugs. Endoplasmic reticulum stress may be critical in this process. In Kaposi sarcoma, we find YAP/TAZ are activated by KSHV and contribute to KSHV-induced tumorigenesis.