FLICE-like Inhibitory Protein Research Articles

Triple Silencing of HSP27, cFLIP, and CLU Genes Promotes the Sensitivity of Doxazosin-Induced Apoptosis in PC-3 Prostate Cancer Cells.

Background: This study investigated how the expression of heat shock protein 27 (HSP27), cellular FLICE-like inhibitory protein (cFLIP), and clusterin (CLU) affects the progression of cancer cells and their susceptibility to doxazosin-induced apoptosis. By silencing each of these genes individually, their effect on prostate cancer cell viability after doxazosin treatment was investigated. Methods: PC-3 prostate cancer cells were cultured and then subjected to gene silencing using siRNA targeting HSP27, cFLIP, and CLU, either individually, in pairs, or all together. Cells were then treated with doxazosin at various concentrations and their viability was assessed by MTT assay. Results: The study found that silencing the CLU gene in PC-3 cells significantly reduced cell viability after treatment with 25 µM doxazosin. In addition, the dual silencing of cFLIP and CLU decreased cell viability at 10 µM doxazosin. Notably, silencing all three genes of HSP27, cFLIP, CLU was most effective and reduced cell viability even at a lower doxazosin concentration of 1 µM. Conclusions: Taken together, these findings suggest that the simultaneous silencing of HSP27, cFLIP, and CLU genes may be a potential strategy to promote apoptosis in prostate cancer cells, which could inform future research on treatments for malignant prostate cancer.

Cleavage of cFLIP restrains cell death during viral infection and tissue injury and favors tissue repair.

Cell death coordinates repair programs following pathogen attack and tissue injury. However, aberrant cell death can interfere with such programs and cause organ failure. Cellular FLICE-like inhibitory protein (cFLIP) is a crucial regulator of cell death and a substrate of Caspase-8. However, the physiological role of cFLIP cleavage by Caspase-8 remains elusive. Here, we found an essential role for cFLIP cleavage in restraining cell death in different pathophysiological scenarios. Mice expressing a cleavage-resistant cFLIP mutant, CflipD377A, exhibited increased sensitivity to severe acute respiratory syndrome coronavirus (SARS-CoV)-induced lethality, impaired skin wound healing, and increased tissue damage caused by Sharpin deficiency. In vitro, abrogation of cFLIP cleavage sensitizes cells to tumor necrosis factor(TNF)-induced necroptosis and apoptosis by favoring complex-II formation. Mechanistically, the cell death-sensitizing effect of the D377A mutation depends on glutamine-469. These results reveal a crucial role for cFLIP cleavage in controlling the amplitude of cell death responses occurring upon tissue stress to ensure the execution of repair programs.

Mechanisms regulating the loss of Tregs in HUPO mice that develop spontaneous inflammatory arthritis

T regulatory cells (Tregs) are a potential therapeutic target in many autoimmune diseases including rheumatoid arthritis (RA). The mechanisms responsible for the maintenance of Tregs in chronic inflammatory conditions such as RA are poorly understood. We employed our mouse model of RA in which, the following deletion of Flice-like inhibitory protein in CD11c+ cells, CD11c-FLIP-KO (HUPO) mice develop spontaneous, progressive, erosive arthritis, with reduced Tregs, and the adoptive transfer of Tregs ameliorates the arthritis. HUPO thymic Treg development was normal, but peripheral of Treg Foxp3 was diminished mediated by reduction of dendritic cells and interleukin-2 (IL-2). During chronic inflammatory arthritis Tregs fail to maintain Foxp3, leading to non-apoptotic cell death and conversion to CD4+CD25+Foxp3- cells. Treatment with IL-2 increased Tregs and ameliorated the arthritis. In summary, reduced dendritic cells and IL-2 in the milieu of chronic inflammation, contribute to Treg instability, promoting HUPO arthritis progression, and suggesting a therapeutic approach in RA.

Prominin-1 deletion results in spermatogenic impairment, sperm morphological defects, and infertility in mice.

Spermatogenesis is a complex process orchestrated by several essential genes. Prominin-1 (Prom1/PROM1) is a gene that is expressed in the testis but with a poorly understood role in spermatogenesis. We used Prom1 knockout (Prom1 KO) mice to assess the role of Prom1 in spermatogenesis. To this end, we performed immunohistochemistry, immunofluorescence, western blotting, β-galactosidase staining, and apoptosis assay. Additionally, we analyzed the morphology of sperm and assessed litter sizes. We observed that PROM1 is localized to the dividing spermatocytes in seminiferous epithelial cells, sperm, and columnar epithelium in the epididymis. In the Prom1 KO testis, an aberrant increase in apoptotic cells and a decrease in proliferating seminiferous epithelial cells were observed. Cellular FLICE-like inhibitory protein (c-FLIP) and extracellular signal-regulated kinase 1/2 (ERK1/2) expression were also significantly decreased in Prom1 KO testis. In addition, a significantly increased number of epididymal spermatozoa with abnormal morphology and less motility was found in Prom1 KO mice. PROM1 maintains spermatogenic cell proliferation and survival via c-FLIP expression in the testis. It is also involved in sperm motility and fertilization potential. The mechanism underlying the effect of Prom1 on sperm morphology and motility remains to be identified.

Induction of Breast Cancer Cell Apoptosis by TRAIL and Smac Mimetics: Involvement of RIP1 and cFLIP.

Smac mimetics are a group of compounds able to facilitate cell death in cancer cells. TNF-related apoptosis-inducing ligand (TRAIL) is a death receptor ligand currently explored in combination with Smac mimetics. The molecular mechanisms determining if the combination treatment results in apoptosis are however not fully understood. In this study, we aimed to shed light on these mechanisms in breast cancer cells. Three breast cancer cell lines, MDA-MB-468, CAMA-1 and MCF-7, were used to evaluate the effects of Smac mimetic LCL-161 and TRAIL using cell death assays and Western blot. The combination treatment induces apoptosis and caspase-8 cleavage in MDA-MB-468 and CAMA-1 but not in MCF-7 cells and downregulation of caspase-8 blocked apoptosis. Downregulation, but not kinase inhibition, of receptor-interacting protein 1 (RIP1) suppressed apoptosis in CAMA-1. Apoptosis is preceded by association of RIP1 with caspase-8. Downregulating cellular FLICE-like inhibitory protein (c-FLIP) resulted in increased caspase cleavage and some induction of apoptosis by TRAIL and LCL-161 in MCF-7. In CAMA-1, c-FLIP depletion potentiated TRAIL-induced caspase cleavage and LCL-161 did not increase it further. Our results lend further support to a model where LCL-161 enables the formation of a complex including RIP1 and caspase-8 and circumvents c-FLIP-mediated inhibition of caspase activation.

Expression, purification, and characterization of c-FLIP tandem death effector domains from Escherichia coli

Cellular FLICE-like inhibitory protein (c-FLIP) regulates extrinsic apoptosis by controlling procaspase-8 activation through its tandem N-terminal death effector domains (DEDs). Here, we present the expression and purification of c-FLIP tandem DEDs (tDED) from Escherichia coli. We observed that the c-FLIPtDED maintains monomeric form under near-physiological pH condition in vitro. Our results also reveal a significant correlation between the pH conditions and the structure of c-FLIPtDED (F114A). The described methods and results would be helpful for follow-up study on the structural and functional of c-FLIP.

Beyond BCL-2 Inhibition in Acute Myloid Leukemia: Other Approaches to Leverage the Apoptotic Pathway.

BCL-2 inhibition has transformed the therapeutic landscape of acute myeloid leukemia (AML) but is not curative for the majority of patients. Consequently, there has been growing interest in targeting other facets of the apoptotic machinery to improve outcomes. These approaches include targeting the intrinsic and extrinsic apoptotic pathway, inducing apoptosis via p53 activation, and others. Targeting the intrinsic apoptotic pathway includes MCL-1 antagonism and BCL-xL inhibition. MCL-1 can be targeted via direct inhibitors as well as via indirect mechanisms to downregulate MCL-1 including inhibition of cyclin dependent kinases and Nedd8 activating enzyme. The extrinsic apoptotic pathway could be harnessed via inhibition of inhibitor of apoptosis proteins, agonism of the TNF-related apoptosis-inducing ligand receptors and inhibiting FLICE-like inhibitor protein. Approaches inducing p53-mediated apoptosis are being evaluated using inhibitors of MDM2, dual inhibitor of MDM2/X in TP53 wild-type AML and p53 reactivators in TP53-mutant myeloid disorders. Several such agents are in early clinical development and rationale combinations of these agents may help improving outcomes for patients with AML.

PKR and TLR3 trigger distinct signals that coordinate the induction of antiviral apoptosis

RIG-I-like receptors (RLRs), protein kinase R (PKR), and endosomal Toll-like receptor 3 (TLR3) sense viral non-self RNA and are involved in cell fate determination. However, the mechanisms by which intracellular RNA induces apoptosis, particularly the role of each RNA sensor, remain unclear. We performed cytoplasmic injections of different types of RNA and elucidated the molecular mechanisms underlying viral dsRNA-induced apoptosis. The results obtained revealed that short 5′-triphosphate dsRNA, the sole ligand of RIG-I, induced slow apoptosis in a fraction of cells depending on IRF-3 transcriptional activity and IFN-I production. However, intracellular long dsRNA was sensed by PKR and TLR3, which activate distinct signals, and synergistically induced rapid apoptosis. PKR essentially induced translational arrest, resulting in reduced levels of cellular FLICE-like inhibitory protein and functioned in the TLR3/TRIF-dependent activation of caspase 8. The present results demonstrated that PKR and TLR3 were both essential for inducing the viral RNA-mediated apoptosis of infected cells and the arrest of viral production.

Restoring TRAILR2/DR5-Mediated Activation of Apoptosis upon Endoplasmic Reticulum Stress as a Therapeutic Strategy in Cancer.

The uncontrolled proliferation of malignant cells in growing tumors results in the generation of different stressors in the tumor microenvironment, such as nutrient shortage, hypoxia and acidosis, among others, that disrupt endoplasmic reticulum (ER) homeostasis and may lead to ER stress. As a response to ER stress, both normal and tumor cells launch a set of signaling pathways known as the unfolded protein response (UPR) to restore ER proteostasis and maintain cell viability and function. However, under sustained ER stress, an apoptotic cell death process can be induced and this has been the subject of different review articles, although the role of the TRAIL-R2/DR5-activated extrinsic pathway of apoptosis has not yet been thoroughly summarized. In this Review, we provide an updated overview of the molecular mechanisms regulating cell fate decisions in tumor cells undergoing ER stress and discuss the role of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptor 2 (TRAIL-R2/DR5) in the final outcome of UPR signaling. Particularly, we focus on the mechanisms controlling cellular FLICE-like inhibitory protein (FLIP) levels in tumor cells undergoing ER stress, which may represent a potential target for therapeutic intervention in cancer.

Mangosteen Inhibits Growth and Survival of Cervical Cancer Cells.

Cervical cancer is the most common cancer of the female reproductive system. Late-stage cervical cancer treatment has been largely unsuccessful, and urgent anti-cancer therapy is needed. Mangosteen, a tropical fruit, has been studied and found to be rich in xanthones, known anti-cancer compounds. This study was designed to investigate the effect of mangosteen extract (ME) on SiHa cervical cancer cells and to explore the underlying molecular mechanisms. Clonogenic survival assay, Quick Cell Proliferation Assay, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining, and caspase-3 activity kits were used to investigate the in vitro role of ME treatment in SiHa cervical cancer cell growth. We further investigated the possible molecular mechanisms using RT-PCR. Statistical analysis was done with unpaired two-tailed Student's t-test and significance at p-value <0.05; each experiment was repeated three times. Our study found that the growth and proliferation of SiHa cervical cancer cells was inhibited by ME. ME also induced apoptosis in SiHa cervical cancer cells. The anti-proliferative effect of ME on cervical cancer cells was associated with statistically significant (p<0.05) down-regulation of the pro-proliferative molecules cyclin B, cyclin D and cyclin E. The pro-apoptotic effect of ME was associated with statistically significant (p<0.05) down-regulation of the anti-apoptotic molecules flice-like inhibitory protein (FLIP) and survivin. ME impedes the growth and survival of SiHa cervical cancer cells by down-regulating cyclin B, cyclin D, cyclin E as well as FLIP and survivin. ME may be a promising strategy for targeted cancer immunotherapy development.

Ex Vivo Infection of Human Placental Explants by Trypanosoma cruzi Reveals a microRNA Profile Similar to That Seen in Trophoblast Differentiation.

Congenital Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is responsible for 22.5% of new cases each year. However, placental transmission occurs in only 5% of infected mothers and it has been proposed that the epithelial turnover of the trophoblast can be considered a local placental defense against the parasite. Thus, Trypanosoma cruzi induces cellular proliferation, differentiation, and apoptotic cell death in the trophoblast, which are regulated, among other mechanisms, by small non-coding RNAs such as microRNAs. On the other hand, ex vivo infection of human placental explants induces a specific microRNA profile that includes microRNAs related to trophoblast differentiation such as miR-512-3p miR-515-5p, codified at the chromosome 19 microRNA cluster. Here we determined the expression validated target genes of miR-512-3p and miR-515-5p, specifically human glial cells missing 1 transcription factor and cellular FLICE-like inhibitory protein, as well as the expression of the main trophoblast differentiation marker human chorionic gonadotrophin during ex vivo infection of human placental explants, and examined how the inhibition or overexpression of both microRNAs affects parasite infection. We conclude that Trypanosoma cruzi-induced trophoblast epithelial turnover, particularly trophoblast differentiation, is at least partially mediated by placenta-specific miR-512-3p and miR-515-5p and that both miRNAs mediate placental susceptibility to ex vivo infection of human placental explants. Knowledge about the role of parasite-modulated microRNAs in the placenta might enable their use as biomarkers, as prognostic and therapeutic tools for congenital Chagas disease in the future.

The deubiquitinase Usp27x as a novel regulator of cFLIPL protein expression and sensitizer to death-receptor-induced apoptosis

Death receptors are transmembrane proteins that can induce the activation of caspase-8 upon ligand binding, initiating apoptosis. Recent work has highlighted the great molecular complexity of death receptor signalling, in particular through ubiquitination/deubiquitination. We have earlier defined the deubiquitinase Ubiquitin-Specific Protease 27x (Usp27x) as an enzyme capable of stabilizing the pro-apoptotic Bcl-2 family member Bim. Here, we report that enhanced expression of Usp27x in human melanoma cells leads to the loss of cellular FLICE-like inhibitory protein (cFLIP) and sensitizes to Tumor necrosis factor receptor 1 (TNF-R1) or Toll-like receptor 3 (TLR3)-induced extrinsic apoptosis through enabling enhanced processing of caspase-8. The loss of cFLIPL upon overexpression of Usp27x was not due to reduced transcription, could be partially counteracted by blocking the ubiquitin proteasome system and was independent of the known cFLIPL destabilizing ubiquitin E3-ligases Itch and DTX1. Instead, Usp27x interacted with the E3-ligase TRIM28 and reduced ubiquitination of TRIM28. Reduction of cFLIPL protein levels by Usp27x-induction depended on TRIM28, which was also required for polyI:C-induced cell death. This work defines Usp27x as a novel regulator of cFLIPL protein expression and a deubiquitinase in fine tuning death receptor signalling pathways to execute apoptosis.

Ethyl pyruvate attenuates isoproterenol-induced myocardial infarction in rats: Insight to TNF-α-mediated apoptotic and necroptotic signaling interplay

The current study investigated the prophylactic effect of ethyl pyruvate (EP) in Isoproterenol (ISO) - induced myocardial infarction (MI). Ethyl pyruvate (EP) was given at a dose of 100 mg/kg i.p for 7 days, while isoproterenol (ISO) was administered at a dose of 10 mg/kg s.c. on the 6th and 7th days to induce MI. All parameters were assessed 24 and 48 h following treatment. Interestingly, EP pre-treatment significantly improved ISO-induced hemodynamic alterations and remarkably ameliorated serum levels of cardiac injury markers, Cardiac Troponin I (cTnI) and Cardiac Creatine Kinase (CK-MB). Also, EP notably suppressed levels of oxidative stress markers, total antioxidants (TAO) and malondialdehyde (MDA) as compared to ISO-treated group. Cardioprotective effects of EP were confirmed by histopathological examination. Moreover, EP remarkably attenuated ISO-induced elevation in Tumor Necrosis Factor Alpha (TNF-α) and Nuclear factor kappa-B p65 (NF-κB) expression, along with Interleukin-6 (IL-6), Monocyte chemoattractant protein 1 (MCP-1) and Inducible nitric oxide synthase (i-NOS) levels. Also, EP significantly diminished expression of apoptotic markers; caspase 8, cleaved caspase 3 and apoptotic regulator; cellular FLICE-like inhibitory protein (cFLIP). Finally, EP notably mitigated necroptotic mediators, phosphorylated receptor-interacting serine/threonine protein kinase 1 and 3 (p-RIPK1 and p-RIPK3), phosphorylated mixed lineage kinase domain-like protein (p-MLKL) and heat shock protein 70 (HSP 70) expression as compared to the ISO-treated group. Our study was the first to investigate the effect of EP on the necroptotic signaling. Taken together, EP conferred its cardioprotective effect against ISO-induced MI partially through mitigation of TNF-α and its downstream inflammatory, apoptotic and necroptotic signaling pathways.

An engineered construct of cFLIP provides insight into DED1 structure and interactions

Cellular FLICE-like inhibitory protein (cFLIP) is a member of the Death Domain superfamily with pivotal roles in many cellular processes and disease states, including cancer and autoimmune disorders. In the context of the death-inducing signaling complex (DISC), cFLIP isoforms regulate extrinsic apoptosis by controlling procaspase-8 activation. The function of cFLIP is mediated through a series of protein-protein interactions, engaging the two N-terminal death effector domains (DEDs). Here, we solve the structure of an engineered DED1 domain of cFLIP using solution nuclear magnetic resonance (NMR) and we define the interaction with FADD and calmodulin, protein-protein interactions that regulate the function of cFLIP in the DISC. cFLIP DED1 assumes a canonical DED fold characterized by six α helices and is able to bind calmodulin and FADD through two separate interfaces. Our results clearly demonstrate the role of DED1 in the cFLIP/FADD association and contribute to the understanding of the assembly of DISC filaments.

Oncogenic signaling inhibits c-FLIPL expression and its non-apoptotic function during ECM-detachment

Inhibition of programmed cell death pathways is frequently observed in cancer cells where it functions to facilitate tumor progression. However, some proteins involved in the regulation of cell death function dichotomously to both promote and inhibit cell death depending on the cellular context. As such, understanding how cell death proteins are regulated in a context-dependent fashion in cancer cells is of utmost importance. We have uncovered evidence that cellular FLICE-like Inhibitory Protein (c-FLIP), a well-known anti-apoptotic protein, is often downregulated in tumor tissue when compared to adjacent normal tissue. These data argue that c-FLIP may have activity distinct from its canonical role in antagonizing cell death. Interestingly, we have discovered that detachment from extracellular matrix (ECM) serves as a signal to elevate c-FLIP transcription and that oncogenic signaling blocks ECM-detachment-induced c-FLIP elevation. In addition, our data reveal that downregulation of c-FLIP promotes luminal filling in mammary acini and that c-FLIP overexpression in cancer cells inhibits colony formation in cells exposed to ECM-detachment. Taken together, our study reveals an unexpected, non-apoptotic role for c-FLIP during ECM-detachment and raises the possibility that c-FLIP may have context-dependent roles during tumorigenesis.

Abstract 1342: Development and preclinical evaluation of unique first-in-class small molecule inhibitors of the anti-apoptotic protein FLIP

Abstract In cancer, evasion of cell death is a fundamental cause of resistance to therapy, prompting the development of therapeutics that reactivate cell death pathways such as Bcl-2 family inhibitors and IAP antagonists. The apoptosis modulator Cellular FLICE-like inhibitory protein (FLIP) is a non-redundant inhibitor of caspase-8 activation and is the only human pseudo-caspase. Caspase-8 is the initiator caspase for the extrinsic apoptotic pathway and is now recognized as the molecular “switch” that controls the 3 major forms of programmed cell death: apoptosis, necroptosis and pyroptosis. As such, methods to selectively activate caspase-8 in appropriate disease contexts represent an exciting new therapeutic paradigm. FLIP is frequently overexpressed in solid and haematological cancers where it is associated with poor prognosis and chemo- and radio-resistance. Moreover, by regulating caspase-8 activity, FLIP is a key determinant of cell death induced by death ligands such as TRAIL expressed by immune effector cells. Thus, targeting FLIP's interaction with caspase-8 represents a unique therapeutic opportunity for enhancing standard-of-care anti-cancer therapies and promoting anti-tumor immunity.We report the discovery and characterisation of small molecule first-in-class selective inhibitors capable of disrupting FLIP's interaction with procaspase-8 in human cancer cells. These small molecule inhibitors induce caspase-8-dependent cell death as single agents and dramatically enhance apoptosis induced by recombinant TRAIL and 2nd generation multivalent TRAIL-R2 agonists in the nM concentration range. KRAS mutant non-small cell lung cancer (NSCLC) was identified as a major sensitive disease setting for FLIP inhibitors with single digit nM activity in vitro in several models and single agent in vivo efficacy. FLIP inhibitors also demonstrated in vitro efficacy in combination with KRAS G12C inhibitors in KRAS G12C mutant NSCLC and in vitro and in vivo efficacy in combination with the 3rd generation EGFR inhibitor Osimertinib in EGFR mutant NSCLC. We have also observed single agency nM efficacy in lymphoma and leukemia models and significant efficacy in combination with standard-of-care chemotherapeutics in KRAS mutant colorectal and pancreatic cancers. In summary, we have identified novel FLIP-targeted activators of caspase-8 with a unique mechanism-of-action and the potential for use in the treatment of several human cancers and leukemias either as a single agent, or in combination with standard-of-care chemotherapeutics and other clinically-relevant targeted agents. AcknowledgementsThis work was supported by a Seeding Drug Discovery award from the Wellcome Trust. Citation Format: Catherine A. Higgins, Jennifer Fox, Jamie Roberts, Declan Doherty, Trevor Perrior, Ray Boffey, Tim Harrison, Daniel B. Longley. Development and preclinical evaluation of unique first-in-class small molecule inhibitors of the anti-apoptotic protein FLIP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1342.

Abstract 1980: Non-canonical regulation and function of anti-apoptotic protein c-FLIP

Abstract The metastasis of cancer cells to distant sites accounts for over 90% of cancer related deaths. In order to metastasize, cancer cells must be able to survive during extracellular matrix (ECM) detachment, namely by blocking anoikis (a caspase-dependent cell death) and restoring metabolic deficiencies. Such barriers during tumorigenesis are abrogated through activation of oncogenic signaling, and/or inhibition of tumor suppressors. Elucidating the signaling pathways involved in cancer cell metastasis is a crucial step in designing effective therapeutic interventions. Surprisingly, our investigation has revealed that cellular FLICE-like Inhibitory Protein (c-FLIP), a protein well known for its pro-survival function, instead antagonizes the viability of ECM-detached cancer cells. Additionally, we demonstrated that signaling emanating from oncogenic growth factor receptors results in the downregulation of c-FLIP expression providing additional evidence that cancer cells may benefit from diminished FLIP levels. Interestingly, this repression of c-FLIP expression by activated oncogenes is a consequence of PI3K signaling. Our study also suggests that low c-FLIP levels are beneficial in cancer cells due to capacity of c-FLIP to induce β-catenin degradation. Taken together, our study has revealed an unexpected role for c-FLIP in restricting the viability of tumor cells and raises the idea that the c-FLIP may have context-dependent pro- and anti-cell death roles during tumorigenesis. Citation Format: Matyas Abel Tsegaye, Kyle McGeehan, Mati Nemera, Zachary T. Schafer. Non-canonical regulation and function of anti-apoptotic protein c-FLIP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1980.

C-FLIP regulates autophagy by interacting with Beclin-1 and influencing its stability

c-FLIP (cellular FLICE-like inhibitory protein) protein is mostly known as an apoptosis modulator. However, increasing data underline that c-FLIP plays multiple roles in cellular homoeostasis, influencing differently the same pathways depending on its expression level and isoform predominance. Few and controversial data are available regarding c-FLIP function in autophagy. Here we show that autophagic flux is less effective in c-FLIP−/− than in WT MEFs (mouse embryonic fibroblasts). Indeed, we show that the absence of c-FLIP compromises the expression levels of pivotal factors in the generation of autophagosomes. In line with the role of c-FLIP as a scaffold protein, we found that c-FLIPL interacts with Beclin-1 (BECN1: coiled-coil, moesin-like BCL2-interacting protein), which is required for autophagosome nucleation. By a combination of bioinformatics tools and biochemistry assays, we demonstrate that c-FLIPL interaction with Beclin-1 is important to prevent Beclin-1 ubiquitination and degradation through the proteasomal pathway. Taken together, our data describe a novel molecular mechanism through which c-FLIPL positively regulates autophagy, by enhancing Beclin-1 protein stability.

Cellular FLICE-like inhibitory protein (cFLIP) critically maintains apoptotic resistance in human lens epithelial cells

The present study aims to understand the mechanism of the lens epithelial cell’s strong anti-apoptotic capacity and survival in the mature human lens that, on the one hand, maintains lens transparency over several decades, while on the other hand, increases the risk of posterior capsule opacification (PCO). Here we compared FHL124 cells and HeLa cells, spontaneously immortalized epithelial cell lines derived from the human lens and cervical cancer cells, respectively, of their resistance to TNFα-mediated cell death. TNFα plus cycloheximide (CHX) triggered almost all of HeLa cell death. FHL124 cells, however, were unaffected and able to block caspase-8 activation as well as prevent caspase-3 and PARP-1 cleavage. Interestingly, despite spontaneous NFκB and AP-1 activation and upregulation of multiple cell survival/anti-apoptotic genes in both cell types, only FHL124 cells were able to survive the TNFα challenge. After screening and comparing the cell survival genes, cFLIP was found to be highly expressed in FHL124 cells and substantially upregulated by TNFα stimulation. FHL124 cells with a mild cFLIP knockdown manifested a profound apoptotic response to TNFα stimulus similar to HeLa cells. Most importantly, we confirmed these findings in an ex vivo lens capsular bag culture system. In conclusion, our results show that cFLIP is a critical gene that is regulating lens epithelial cell survival.

H2O2 mediated FLIP and XIAP down-regulation involves increased ITCH expression and ERK-Akt crosstalk in imatinib resistant Chronic Myeloid Leukemia cell line K562

Regulation of anti-apoptotic protein FLICE-like inhibitory protein (FLIP) and X-linked inhibitor of apoptosis protein (XIAP) remains a crucial step in the cell fate determination and thus targeting these anti-apoptotic proteins could be a viable strategy for the treatment of cancer. However the regulation of FLIP and XIAP is not very well established till date. Here we have shown that ROS decreased XIAP and FLIP by activation of ubiquitin-proteasomal pathway in imatinib resistant K562 cells. Activation of the components of MAPK pathway, ERK and JNK, played a crucial role in XIAP and FLIP degradation because ectopic expression or knock down of ERK and JNK changed the pattern of ROS mediated down-regulation of these two proteins. We have also found that JNK and ERK differentially regulates FLIP and XIAP, respectively. Moreover, our data suggests that activated ERK decreased Akt phosphorylation and thus its binding to and stabilization of XIAP. On the other hand, JNK activation increased E3 ubiquitin ligase ITCH expression and its binding to FLIP which leads to its degradation. Thus, we have, for the first time elucidated that ROS mediated ERK-Akt crosstalk regulates XIAP. We have also shown for the first time that ROS regulates ITCH expression which controls FLIP degradation.