FADD-like Interleukin-1β-converting Enzyme Research Articles

Immune checkpoint blockade therapy mitigates systemic inflammation and affects cellular FLIP-expressing monocytic myeloid-derived suppressor cells in non-progressor non-small cell lung cancer patients

ABSTRACT Cancer cells favor the generation of myeloid cells with immunosuppressive and inflammatory features, including myeloid-derived suppressor cells (MDSCs), which support tumor progression. The anti-apoptotic molecule, cellular FLICE (FADD-like interleukin-1β-converting enzyme)-inhibitory protein (c-FLIP), which acts as an important modulator of caspase-8, is required for the development and function of monocytic (M)-MDSCs. Here, we assessed the effect of immune checkpoint inhibitor (ICI) therapy on systemic immunological landscape, including FLIP-expressing MDSCs, in non-small cell lung cancer (NSCLC) patients. Longitudinal changes in peripheral immunological parameters were correlated with patients’ outcome. In detail, 34 NSCLC patients were enrolled and classified as progressors (P) or non-progressors (NP), according to the RECIST evaluation. We demonstrated a reduction in pro-inflammatory cytokines such as IL-8, IL-6, and IL-1β in only NP patients after ICI treatment. Moreover, using t-distributed stochastic neighbor embedding (t-SNE) and cluster analysis, we characterized in NP patients a significant increase in the amount of lymphocytes and a slight contraction of myeloid cells such as neutrophils and monocytes. Despite this moderate ICI-associated alteration in myeloid cells, we identified a distinctive reduction of c-FLIP expression in M-MDSCs from NP patients concurrently with the first clinical evaluation (T1), even though NP and P patients showed the same level of expression at baseline (T0). In agreement with the c-FLIP expression, monocytes isolated from both P and NP patients displayed similar immunosuppressive functions at T0; however, this pro-tumor activity was negatively influenced at T1 in the NP patient cohort exclusively. Hence, ICI therapy can mitigate systemic inflammation and impair MDSC-dependent immunosuppression.

Efficient recruitment of c-FLIPL to the death-inducing signaling complex leads to Fas resistance in natural killer-cell lymphoma.

Activation‐induced cell death (AICD) mediated by the Fas/Fas ligand (FasL) system plays a key role in regulating immune response. Although normal natural killer (NK) cells use this system for their homeostasis, malignant NK cells seem to disrupt the process. Extranodal NK/T‐cell lymphoma, nasal type (ENKL) is a rare but fatal disease, for which novel therapeutic targets need to be identified. We confirmed that ENKL‐derived NK cell lines NK‐YS and Hank1, and primary lymphoma cells expressed procaspase‐8/FADD‐like interleukin‐1β‐converting enzyme (FLICE) modulator and cellular FLICE‐inhibitory protein (c‐FLIP), along with Fas and FasL. Compared with Fas‐sensitive Jurkat cells, NK‐YS and Hank1 showed resistance to Fas‐mediated apoptosis in spite of the same expression levels of c‐FLIP and the death‐inducing signaling complex (DISC) formation. Unexpectedly, the long isoform of c‐FLIP (c‐FLIPL) was coimmunoprecipitated with Fas predominantly in both ENKL‐derived NK cell lines after Fas ligation. Indeed, c‐FLIPL was more sufficiently recruited to the DISC in both ENKL‐derived NK cell lines than in Jurkat cells after Fas ligation. Knockdown of c‐FLIPL per se enhanced autonomous cell death and restored the sensitivity to Fas in both NK‐YS and Hank1 cells. Although ENKL cells are primed for AICD, they constitutively express and efficiently utilize c‐FLIPL, which prevents their Fas‐mediated apoptosis. Our results show that c‐FLIPL could be a promising therapeutic target against ENKL.

Su Yang Decoction induces human colon carcinoma cell apoptosis by activating caspases.

Su Yang Decoction (SYD) is a popular healthcare product comprised of various brassicaceous vegetables known to exhibit anticancer activity. However, the effects of SYD on tumor growth, following its combination with brassicaceous vegetables into a compound formula, require further investigation. In the present study, a quality control of SYD was subjected to high-performance liquid chromatography for the quantitative and qualitative determination of sulforaphane, the main anticancer component of SYD. SYD inhibited colon cancer cell proliferation in a dose- and time-dependent manner and induced G1 phase arrest in colon cancer HT-29 cell lines. In addition, SYD triggered caspase-mediated apoptosis in a dose-dependent manner and induced the cleavage of poly (ADP-ribose) polymerase, tumor necrosis factor superfamily member 10, X-linked inhibitor of apoptosis, and truncated BH3 interacting domain death agonist. Furthermore, the expression of FADD-like interleukin-1β-converting enzyme (FLICE)-like inhibitory proteins (FLIPs) and long isoform of FLICE-inhibitory protein was reduced by SYD and the direct targeting of cellular-FLIP with small interfering RNA inhibited colon cancer cell proliferation and decreased the SYD concentration required for proliferation inhibition. SYD treatment was also associated with the translocation of proapoptotic BCL2 associated X, apoptosis regulator to the mitochondria and the release of cytochrome c from the mitochondria to the cytosol. The aforementioned results indicate that SYD exerts anti-colorectal cancer effects through an underlying mechanism that may involve caspase activation.

인체 방광암세포에서 histone deacetylase 억제제인 sodium butyrate이 TRAIL에 의한 apoptosis 유도에 미치는 영향

Anti-Aging Research Center and Blue-Bio Industry RIC, Dongeui U niversity, Busan 614-71, KoreaReceived October 26, 2015/Revised November 12, 2015/Accepted November 13, 2015The tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is considered a promising anticancer agent due to its unique ability to induce cancer cell death having only negligible effects on normal cells. However, many cancer cells tend to be resistant to TRAIL. In this study, we inves-tigated the effects and molecular mechanisms of sodium butyrate (SB), a histone deacetylase inhibitor, in sensitizing TRAIL-induced apoptosis in 5637 human bladder cancer cells. Our results indicated that co-treatment with SB and TRAIL significantly increased the apoptosis induction, compared with treat-ment with either agent alone. Co-treatment with SB and TRAIL effectively increased the cell-surface expression of death receptor (DR) 5, but not DR4, which was associated with the inhibition of cellular Fas-associated death domain (FADD)-like interleukin-1β-converting enzyme (FLICE) inhibitory protein (c-FLIP). Furthermore, the activation of caspases (caspase-3, -8 and -9) and degradation of poly(ADP- ribose) were markedly increased in 5637 cells co-treated with SB and TRAIL; however, the synergistic effect was perfectly attenuated by caspase inhibitors. We also found that combined treatment with SB and TRAIL effectively induced the expression of pro-apoptotic Bax, cytosolic cytochrome c and cleave Bid to truncated Bid (tBid), along with down-regulation of anti-apoptotic Bcl-xL expression. These re-sults collectively suggest that a combined regimen of SB plus TRAIL may offer an effective therapeutic strategy for safely and selectively treating TRAIL-resistant bladder cancer cells.Keywords :

Suppression of c‑FLIPL promotes JNK activation in malignant melanoma cells.

The up‑regulation of cellular Fas‑associated death domain‑like interleukin‑1β‑converting enzyme (FLICE)‑like inhibitory protein (c‑FLIP) has been reported in various tumor types, and has been previously shown to be associated with the clinicopathological features of melanoma. To assess its potential role in cancer therapy, the present study evaluated the effects of short hairpin (sh)RNAs of different c‑FLIP isoforms on cellular proliferation and c‑Jun N‑terminal kinase (JNK) signaling. Human c‑FLIP shRNA plasmids were constructed and transfected into the A875 melanoma cell line. It was observed that c‑FLIP shRNA exhibited strong inhibitory effects against the levels of phosphorylated‑JNK and inhibited cellular proliferation in A875 cells. Thus, this indicated that c‑FLIP long form shRNA serves a specific inhibitory role in cellular proliferation through inducing the activation of the JNK pathway in A875 cells. The present study provided insight into the development of RNAi based therapies for melanoma.

FasL expression in cardiomyocytes activates the ERK1/2 pathway, leading to dilated cardiomyopathy and advanced heart failure.

Increase in the apoptotic molecule Fas ligand (FasL) in serum and cardiomyocytes has been shown to be associated with progressive dilated cardiomyopathy (DCM) and congestive heart failure (CHF) in humans. However, the underlying mechanism(s) of FasL-related deterioration of heart function remain obscure. The aim of the present study is to determine roles of myocardial FasL in the activation of alternative pathways such as extracellular-signal-regulated kinase 1/2 (ERK1/2), inflammation or fibrosis and to identify effective treatments of progressive DCM and advanced CHF. Transgenic mice with cardiomyocyte-specific overexpression of FasL were investigated and treated with an ERK1/2 inhibitor (U-0126), losartan (los), prednisolone (pred) or placebo. Morpho-histological and molecular studies were subsequently performed. FasL mice showed significantly higher mortality compared with wild-type (WT) littermates due to DCM and advanced CHF. Prominent perivascular and interstitial fibrosis, increased interleukin secretion and diffuse CD3-positive cell infiltration were evident in FasL hearts. Up-regulation of the short form of Fas-associated death domain (FADD)-like interleukin 1β-converting enzyme (FLICE) inhibitory protein (s-FLIP), RIP (receptor-interacting protein) and ERK1/2 and down-regulation of transforming growth factor beta 1 (TGFβ1) and nuclear factor-κB (NF-κB) was determined in the myocardium, whereas expression of ERK1/2, periostin (Postn) and osteopontin increased in cardiac fibroblasts. U-0126 and los increased CHF survival by 75% compared with pred and placebo groups. U-0126 had both anti-fibrotic and anti-apoptotic effects, whereas los reduced fibrosis only. Myocardial FasL expression in mice activates differential robust fibrotic, apoptotic and inflammatory responses via ERK1/2 in cardiomyocytes and cardiac fibroblasts inducing DCM and CHF. Blocking the ERK1/2 pathway prevented progression of FasL-induced DCM and CHF by reducing fibrosis, inflammation and apoptosis in the myocardium.

Aloperine executes antitumor effects against multiple myeloma through dual apoptotic mechanisms.

BackgroundAloperine, a natural alkaloid constituent isolated from the herb Sophora alopecuroides displays anti-inflammatory properties in vitro and in vivo. Our group previously demonstrated that aloperine significantly induced apoptosis in colon cancer SW480 and HCT116 cells. However, its specific target(s) remain to be discovered in multiple myeloma (MM) and have not been investigated.MethodsHuman myeloma cell lines (n = 8), primary myeloma cells (n = 12), drug-resistant myeloma cell lines (n = 2), and animal models were tested for their sensitivity to aloperine in terms of proliferation and apoptosis both in vitro and in vivo, respectively. We also examined the functional mechanisms underlying the apoptotic pathways triggered by aloperine.ResultsAloperine induced MM cell death in a dose- and time-dependent manner, even in the presence of the proliferative cytokines interleukin-6 and insulin-like growth factor I. Mechanistic studies revealed that aloperine not only activated caspase-8 and reduced the expression of FADD-like interleukin-1β-converting enzyme (FLICE)-like inhibitory protein long (FLIPL) and FLICE-inhibitory proteins (FLIPS) but also activated caspase-9 and decreased the expression of phosphorylated (p)-PTEN. Moreover, co-activation of the caspase-8/cellular FLICE-inhibitory protein (cFLIP)- and caspase-9/p-PTEN/p-AKT-dependent apoptotic pathways by aloperine caused irreversible inhibition of clonogenic survival. Aloperine induce more MM apoptosis with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or borterzomib. A U266 xenograft tumor model and 5T33 MM cells recapitulated the antitumor efficacy of aloperine, and the animals displayed excellent tolerance of the drug and few adverse effects.ConclusionsAloperine has multifaceted antitumor effects on MM cells. Our data support the clinical development of aloperine for MM therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-015-0120-x) contains supplementary material, which is available to authorized users.

The Histone Deacetylase Inhibitor Trichostatin A Sensitizes Human Renal Carcinoma Cells to TRAIL-Induced Apoptosis through Down-Regulation of c-FLIPL.

Histone acetylation plays a critical role in the regulation of transcription by altering the structure of chromatin, and it may influence the resistance of some tumor cells to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) by regulating the gene expression of components of the TRAIL signaling pathway. In this study, we investigated the effects and molecular mechanisms of trichostatin A (TSA), a histone deacetylase inhibitor, in sensitizing TRAIL-induced apoptosis in Caki human renal carcinoma cells. Our results indicate that nontoxic concentrations of TSA substantially enhance TRAIL-induced apoptosis compared with treatment with either agent alone. Cotreatment with TSA and TRAIL effectively induced cleavage of Bid and loss of mitochondrial membrane potential (MMP), which was associated with the activation of caspases (-3, -8, and -9) and degradation of poly (ADP-ribose) polymerase (PARP), contributing toward the sensitization to TRAIL. Combined treatment with TSA and TRAIL significantly reduced the levels of the cellular Fas-associated death domain (FADD)-like interleukin-1β-converting enzyme (FLICE) inhibitory protein (c-FLIP), whereas those of death receptor (DR) 4, DR5, and FADD remained unchanged. The synergistic effect of TAS and TRAIL was perfectly attenuated in c-FLIPL-overexpressing Caki cells. Taken together, the present study demonstrates that down-regulation of c-FLIP contributes to TSA-facilitated TRAIL-induced apoptosis, amplifying the death receptor, as well as mitochondria-mediated apoptotic signaling pathways.

The potential evasion of immune surveillance in mucosa associated lymphoid tissue lymphoma by DcR2-mediated up-regulation of nuclear factor-κB

This study investigated expression profiles of tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) pathway components and mechanisms underlying TRAIL-induced apoptosis in mucosa associated lymphoid tissue (MALT) lymphoma. Genetic aberrations including translocations and trisomies were assessed by reverse transcription polymerase chain reaction and fluorescence in situ hybridization. Expression of TRAIL, death receptors 4 and 5, decoy receptors 1 and 2, and FADD-like interleukin-1β-converting enzyme (FLICE) inhibitory protein was analyzed by immunohistochemistry. All 32 patients under study showed some alterations in TRAIL pathway mainly involving loss of death receptors (37.5%), gain of decoy receptors (3.1%) or both (59.4%). Decoy receptor 2 (DcR2) was highly expressed in patients with normal cytogenetic status as compared to those with cytogenetic aberrations (p = 0.005). Moreover, DcR2 expression correlated significantly with nuclear factor-κB (NF-κB) expression (R = 0.372, p = 0.047). High expression of DcR2 in patients with normal cytogenetic status and its significant correlation with NF-κB expression provides a potential clue to evasion of immune surveillance in cytogenetically normal MALT lymphomas.

Metformin sensitizes human bladder cancer cells to TRAIL-induced apoptosis through mTOR/S6K1-mediated downregulation of c-FLIP

Metformin, an oral antidiabetic agent, has been reported to potentiate chemotherapeutic-induced cytotoxicity. In this study, we investigated the effects and molecular mechanisms of metformin in sensitizing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human bladder cancer cells. Metformin alone did not induce apoptosis, but markedly potentiated TRAIL-induced apoptosis in 253J and RT4 bladder cancer cells. To elucidate the underlying mechanism, we examined the modulatory effects of metformin on the key components of the TRAIL signaling pathway and found that metformin did not alter the expression levels of death receptor 4 (DR4) and death receptor 5 (DR5), but significantly reduced the cellular Fas-associated death domain (FADD)-like interleukin-1β-converting enzyme (FLICE) inhibitory protein (c-FLIP) levels, contributing toward the sensitization to TRAIL. Further experiments showed that metformin did not affect the mRNA level, proteasomal degradation, and protein stability of c-FLIPL. However, metformin inhibited the mTOR/S6K1 pathway in 253J and RT4 cells, which usually regulates protein translation; moreover, knockdown of S6K1 effectively reduced the levels of c-FLIPL, indicating that metformin downregulates c-FLIP through inhibition of the mTOR/S6K1 pathway. In addition, AMP-activated protein kinase (AMPK) inhibitor compound C did not prevent the inhibitory effects of metformin on the mTOR/S6K1 pathway and metformin-mediated sensitization to TRAIL. Taken together, our results indicate that metformin sensitizes human bladder cancer cells to TRAIL-induced apoptosis through downregulation of c-FLIP, which is mediated by the mTOR/S6K1 pathway, but independent of AMPK; furthermore, these findings provide a rationale for the combined application of metformin with TRAIL in the treatment of bladder cancer.

Evasion of TNF-α-mediated apoptosis by hepatitis C virus.

Hepatitis C virus (HCV) often causes chronic infection in humans, although the mechanisms for viral chronicity are not clearly understood. Tumor necrosis factor-α (TNF-α)-mediated apoptosis is a key element in a host organism's defense inhibiting viral spread and persistence. HCV has evolved mechanisms that antagonize host cell death signals so that virus propagation can continue unabated in infected cells. HCV core protein blocks TNF-α-mediated apoptosis signaling and inhibits caspase-8 activation by sustaining the expression of cellular FADD-like interleukin-1β-converting enzyme (FLICE)-like inhibitory protein (c-FLIP). HCV core protein also blocks TNF-induced proteolytic cleavage of the death substrate poly (SDP-ribose) polymerase from its native 116-kDa protein to the characteristic 85-kDa polypeptide. A decrease in endogenous c-FLIP by specific small-interfering RNA induces TNF-α-mediated apoptotic cell death and caspase-8 activation. However, HCV core neither affects the association between TNF receptor 1 (TNFR1) and TNFR1-associated death domain protein (TRADD) nor TRADD-Fas-associated death domain protein (FADD) and procaspase-8. Thus, HCV core protein appears to play a role in the inhibition of TNF-α-mediated cell death. This chapter describes methods to identify inhibitory mechanism of HCV for TNF-α-mediated apoptosis.

Elevation of c-FLIP in Castrate-Resistant Prostate Cancer Antagonizes Therapeutic Response to Androgen Receptor–Targeted Therapy

To characterize the importance of cellular Fas-associated death domain (FADD)-like interleukin 1β-converting enzyme (FLICE) inhibitory protein (c-FLIP), a key regulator of caspase-8 (FLICE)-promoted apoptosis, in modulating the response of prostate cancer cells to androgen receptor (AR)-targeted therapy. c-FLIP expression was characterized by immunohistochemical analysis of prostatectomy tissue. The functional importance of c-FLIP to survival and modulating response to bicalutamide was studied by molecular and pharmacologic interventions. c-FLIP expression was increased in high-grade prostatic intraepithelial neoplasia and prostate cancer tissue relative to normal prostate epithelium (P < 0.001). Maximal c-FLIP expression was detected in castrate-resistant prostate cancer (CRPC; P < 0.001). In vitro, silencing of c-FLIP induced spontaneous apoptosis and increased 22Rv1 and LNCaP cell sensitivity to bicalutamide, determined by flow cytometry, PARP cleavage, and caspase activity assays. The histone deacetylase inhibitors (HDACi), droxinostat and SAHA, also downregulated c-FLIP expression, induced caspase-8- and caspase-3/7-mediated apoptosis, and increased apoptosis in bicalutamide-treated cells. Conversely, the elevated expression of c-FLIP detected in the CRPC cell line VCaP underpinned their insensitivity to bicalutamide and SAHA in vitro. However, knockdown of c-FLIP induced spontaneous apoptosis in VCaP cells, indicating its relevance to cell survival and therapeutic resistance. c-FLIP reduces the efficacy of AR-targeted therapy and maintains the viability of prostate cancer cells. A combination of HDACi with androgen deprivation therapy may be effective in early-stage disease, using c-FLIP expression as a predictive biomarker of sensitivity. Direct targeting of c-FLIP, however, may be relevant to enhance the response of existing and novel therapeutics in CRPC.

Abstract B4: Elevated expression of c-FLIP in castrate-resistant prostate cancer antagonizes response to androgen receptor-targeted therapy

Abstract Cellular Fas-associated death domain (FADD)-like interleukin 1β-converting enzyme (FLICE) inhibitory protein (c-FLIP) is a key inhibitor of caspase 8 (FLICE)-promoted apoptosis. We have previously shown c-FLIP to be a transcriptional target of the androgen receptor (AR) and NF-κB. Accordingly, we proposed that cells with elevated expression of c-FLIP may remain viable and withstand androgen-deprivation. Expression of c-FLIP was assessed in prostatectomy tissue while in vitro experiments were performed to determine its importance in regulating response to the androgen receptor (AR)-targeted therapeutic bicalutamide. c-FLIP expression was increased in pre-malignant prostatic intra-epithelial neoplasia (PIN) (P&amp;lt;0.001) and each of Gleason 3 and Gleason 4 prostate cancer (CaP) tissue (P&amp;lt;0.001) relative to normal prostate epithelium. Maximal expression of c-FLIP was detected in castrate-resistant prostate cancer (CRPC) (P&amp;lt;0.001 relative to Gleason 3/4 cancer. In vitro, siRNA-mediated silencing of c-FLIP in 22Rv1 and LNCaP cells induced spontaneous apoptosis and increased their response to bicalutamide, determined by flow cytometry, PARP cleavage and caspase activity assays. Two histone deacetylase inhibitors (HDACi), droxinostat and SAHA, were also shown to down-regulate c-FLIP expression, resulting in caspase-8 and caspase-3/7 mediated apoptosis. HDACi also potentiated the sensitivity of 22Rv1 cells to bicalutamide, while maximal apoptosis induction in LNCaP cells was not changed in the presence of bicalutamide. The combination of SAHA and bicalutamide promoted cell-specific inhibition of the AR or NF-κB signaling pathways consistent with decreased transcription of c-FLIP in these cells. Conversely, the elevated expression of c-FLIP and Bcl-2 was consistent with the insensitivity of the castrate-resistant VCaP cell line to SAHA in vitro, and the sub-optimal performance of this class of agents in castrate-resistant patients. Therefore, HDACi may prove more effective in enhancing androgen-deprivation therapy (ADT) in early-stage disease. Moreover, c-FLIP expression may serve as a predictive biomarker for androgen-deprivation strategies including bicalutamide. Citation Format: Clare McCourt, Pamela Maxwell, Roberta Mazzucchelli, Manuel Salto-Tellez, Joe O'Sullivan, Rodolfo Montironi, Marina Scarpelli, Daniel Longley, David Waugh. Elevated expression of c-FLIP in castrate-resistant prostate cancer antagonizes response to androgen receptor-targeted therapy [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B4.

Abstract A106: Induction of synthetic lethality in mutant KRAS cells for lung cancer prevention and therapy

Abstract Lung cancer is the leading cause of cancer death in both men and women in the United States, with a low five-year survival rate despite improved treatment strategies. Mutations and activation of KRAS occur frequently in non-small cell lung cancers (NSCLCs) and are associated with higher risk of drug-resistance, tumor recurrence, and poor prognosis. Currently, there are no effective therapies for targeting mutant KRAS and there is a great need to develop such a strategy. We have previously discovered a synthetic lethal interaction among TNF-related apoptosis-inducing ligand (TRAIL), the second mitochondria-derived activator of caspase Smac/DIABLO (Smac), and mutant KRAS in premalignant lung epithelial cells, which allows for chemoprevention of KRAS-driven lung cancer by the combination of TRAIL and Smac mimic [Cancer Prev Res (Phila). 2011 May;4(5):666–73.]. Here we demonstrated that this synthetic lethal interaction also exists in malignant lung cancer cells. We showed that 3 out of 4 lung cancer cell lines harboring mutant KRAS displayed high sensitivity to the combination of TRAIL and Smac mimic and KRAS wild type lung cancer cell lines were resistant to the treatment. Expression of mutant KRAS in KRAS wild type lung cancer cells sensitized these cells to TRAIL and Smac mimic and knocking down of mutant KRAS in mutant KRAS expressing cells resulted in resistance to the treatment. Furthermore, we showed that TRAIL and Smac mimic treatment inhibited xenograft tumor growth in mutant KRAS dependent manner. Mechanistically, we found that mutant KRAS repressed cellular FADD-like interleukin 1β–converting enzyme (FLICE)-like inhibitory protein (c-FLIP) expression through the activation of Erk/mitogen-activated protein kinase (MAPK)-mediated activation of c-Myc. Mutant KRAS also enhanced the secretion of Prostate Apoptosis Response-4 (Par4) to promote TRAIL-induced apoptosis. Our study provided a potential new approach for lung cancer chemoprevention and therapy based on synthetic lethality that targets mutant KRAS for apoptosis using the combination of and Smac mimic. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A106.

Lung-Cancer Chemoprevention by Induction of Synthetic Lethality in Mutant KRAS Premalignant Cells In Vitro and In Vivo

Lung cancer is the leading cause of cancer death in both men and women in the United States, with a low 5-year survival rate despite improved treatment strategies. These data underscore the great need for effective chemoprevention of this cancer. Mutations and activation of KRAS occur frequently in, and are thought to be a primary driver of the development of, non-small cell lung cancers (NSCLC) of the adenocarcinoma subtype. In this study, we developed a new approach for the chemoprevention of NSCLC involving specific targeting of apoptosis in mutant KRAS cells. This approach is based on a synthetic lethal interaction among TNF-related apoptosis-inducing ligand (TRAIL), the second mitochondria-derived activator of caspase Smac/DIABLO (Smac), and KRAS. Mutational activation of KRAS modulated the expression of TRAIL receptors by upregulating death receptors and downregulating decoy receptors. Furthermore, oncogenic KRAS repressed cellular FADD-like interleukin 1β-converting enzyme (FLICE)-like inhibitory protein (c-FLIP) expression through activation of Erk/mitogen-activated protein kinase (MAPK)-mediated activation of c-Myc. Smac overcame KRAS-induced cell-survival signaling by antagonizing X-linked inhibitor of apoptosis protein (XIAP). Therefore, the combination of TRAIL and a small molecule mimic of Smac induced apoptosis specifically in mutant KRAS cells without harming normal cells. We further showed that short-term, intermittent in vivo treatment with TRAIL and Smac mimic induced apoptosis in tumor cells and reduced tumor burden in a murine model of KRAS-induced lung cancer. These results reflect the potential benefit of a selective therapeutic approach for the chemoprevention of NSCLC.

FLIPL induces caspase 8 activity in the absence of interdomain caspase 8 cleavage and alters substrate specificity

Caspase 8 is an initiator caspase that is activated by death receptors to initiate the extrinsic pathway of apoptosis. Caspase 8 activation involves dimerization and subsequent interdomain autoprocessing of caspase 8 zymogens, and recently published work has established that elimination of the autoprocessing site of caspase 8 abrogates its pro-apoptotic function while leaving its proliferative function intact. The observation that the developmental abnormalities of caspase 8-deficient mice are shared by mice lacking the dimerization adapter FADD (Fas-associated death domain) or the caspase paralogue FLIP(L) [FLICE (FADD-like interleukin 1β-converting enzyme)-inhibitory protein, long form] has led to the hypothesis that FADD-dependent formation of heterodimers between caspase 8 and FLIP(L) could mediate the developmental role of caspase 8. In the present study, using an inducible dimerization system we demonstrate that cleavage of the catalytic domain of caspase 8 is crucial for its activity in the context of activation by homodimerization. However, we find that use of FLIP(L) as a partner for caspase 8 in dimerization-induced activation rescues the requirement for intersubunit linker proteolysis in both protomers. Moreover, before processing, caspase 8 in complex with FLIP(L) does not generate a fully active enzyme, but an attenuated species able to process only selected natural substrates. Based on these results we propose a mechanism of caspase 8 activation by dimerization in the presence of FLIP(L), as well as a mechanism of caspase 8 functional divergence in apoptotic and non-apoptotic pathways.

Model-based dissection of CD95 signaling dynamics reveals both a pro- and antiapoptotic role of c-FLIPL

Cellular FADD-like interleukin-1beta-converting enzyme inhibitory proteins (c-FLIPs; isoforms c-FLIP long [c-FLIP(L)], c-FLIP short [c-FLIP(S)], and c-FLIP Raji [c-FLIP(R)]) regulate caspase-8 activation and death receptor (DR)-induced apoptosis. In this study, using a combination of mathematical modeling, imaging, and quantitative Western blots, we present a new mathematical model describing caspase-8 activation in quantitative terms, which highlights the influence of c-FLIP proteins on this process directly at the CD95 death-inducing signaling complex. We quantitatively define how the stoichiometry of c-FLIP proteins determines sensitivity toward CD95-induced apoptosis. We show that c-FLIP(L) has a proapoptotic role only upon moderate expression in combination with strong receptor stimulation or in the presence of high amounts of one of the short c-FLIP isoforms, c-FLIP(S) or c-FLIP(R). Our findings resolve the present controversial discussion on the function of c-FLIP(L) as a pro- or antiapoptotic protein in DR-mediated apoptosis and are important for understanding the regulation of CD95-induced apoptosis, where subtle differences in c-FLIP concentrations determine life or death of the cells.

Forced Expression of Methionine Adenosyltransferase 1A in Human Hepatoma Cells Suppresses in Vivo Tumorigenicity in Mice

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine, the principal methyl donor, and is encoded by MAT1A and MAT2A in mammals. Normal liver expresses MAT1A, which is silenced in hepatocellular carcinoma. We have shown that hepatoma cells overexpressing MAT1A grew slower, but whether this is also true in vivo remains unknown. To investigate the effect of overexpressing MAT1A on in vivo tumorigenesis, we generated stable transfectants of Huh7 cells overexpressing either MAT1A or empty vector. Real-time PCR and Western blotting were used to measure expression, and BALB/c nude mice were injected subcutaneously with untransfected or Huh7 cells transfected with empty or MAT1A expression vector to establish tumors. Tumor properties such as proliferation, angiogenesis, and apoptosis were compared, and microarray analysis was performed. Huh7 cells overexpressing MAT1A had higher S-adenosylmethionine levels but lower bromodeoxyuridine incorporation than control cells. Tumor growth rates and weights were lower in MAT1A transfected tumors. In addition, microvessel density and CD31 and Ki-67 staining were lower in MAT1A transfected tumors than control tumors, whereas the apoptosis index was higher in MAT1A-transfected tumors. Forced expression of MAT1A induced genes related to apoptosis and tumor suppression and lowered expression of cell growth and angiogenesis proteins. Our data demonstrate in vivo overexpression of MAT1A in liver cancer cells can suppress tumor growth. They also suggest inducing MAT1A expression might be a strategy to treat hepatocellular carcinoma.

The role of c-FLIP L in ovarian cancer: Chaperoning tumor cells from immunosurveillance and increasing their invasive potential

Objective In the current study, we aimed to investigate the role of the long isoform of cellular Fas-associated death domain-like interleukin-1β-converting enzyme (FLICE)-like inhibitory protein (c-FLIP L) in ovarian cancer (OC) development by using RNA interference (RNAi) in vitro and in vivo. Methods TRAIL-resistant human OC cell lines were genetically manipulated by RNAi-mediated suppression of c-FLIP L. Subsequently, the genetic alteration that was introduced into the various OC cell lines was characterized in vitro and in vivo. Results We previously showed that about 40% of OC patients express high levels of c-FLIP L, and that natural killer (NK) cells mediated immunosurveillance in OC. In the present study, we observed that the knockdown of c-FLIP L in human OC cell lines not only enhanced their sensitivity to TRAIL-mediated apoptosis, but also inhibited their migratory phenotype in a TRAIL-dependent manner in vitro. Shutdown of c-FLIP L in OC cells significantly decreased tumor development by induction of apoptosis and reduction of proliferation in vivo. Importantly, the knockdown of c-FLIP L particularly inhibited the invasion of OC cells into the peritoneal cavity, which might be due to high expression of TRAIL by NK cells and NK-cell mediated immunosurveillance. Conclusion These data demonstrate that c-FLIP L exhibits multiple functions in OC cells: first by concomitantly evading the natural immunity mediated by TRAIL-induced cell death, and second by augmenting cell motility and invasion in vivo. Our findings indicate that c-FLIP L regulates sensitivity of OC to TRAIL-mediated apoptosis and offers possible therapeutical implications for OC in the future.

Fas/CD95 down-regulation in lymphoma cells through acquired alkyllysophospholipid resistance: partial role of associated sphingomyelin deficiency

The ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) induces apoptosis in S49 mouse lymphoma cells. A variant cell line, S49AR, made resistant to ALP, was found previously to be impaired in ALP uptake via lipid-raft-mediated endocytosis. In the present paper, we report that these cells display cross-resistance to Fas/CD95 ligation [FasL (Fas ligand)], and can be gradually resensitized by prolonged culturing in the absence of ALP. Fas and ALP activate distinct apoptotic pathways, since ALP-induced apoptosis was not abrogated by dominant-negative FADD (Fas-associated protein with death domain), cFLIP(L) [cellular FLICE (FADD-like interleukin 1beta-converting enzyme)-inhibitory protein long form] or the caspase 8 inhibitor Z-IETD-FMK (benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone). ALP-resistant cells showed decreased Fas expression, at both the mRNA and protein levels, in a proteasome-dependent fashion. The proteasome inhibitor MG132 partially restored Fas expression and resensitized the cells to FasL, but not to ALP. Resistant cells completely lacked SM (sphingomyelin) synthesis, which seems to be a unique feature of the S49 cell system, having very low SM levels in parental cells. Lack of SM synthesis did not affect cell growth in serum-containing medium, but retarded growth under serum-free (SM-free) conditions. SM deficiency determined in part the resistance to ALP and FasL. Exogenous short-chain (C12-) SM partially restored cell-surface expression of Fas in lipid rafts and FasL sensitivity, but did not affect Fas mRNA levels or ALP sensitivity. We conclude that the acquired resistance of S49 cells to ALP is associated with down-regulated SM synthesis and Fas gene transcription and that SM in lipid rafts stabilizes Fas expression at the cell surface.