Agrobacterium tumefaciens ExoR represses succinoglycan biosynthesis and is required for biofilm formation and motility.

The ubiquitous plant pathogen Agrobacterium tumefaciens attaches efficiently to plant tissues and abiotic surfaces and can form complex biofilms. A genetic screen for mutants unable to form biofilms on PVC identified disruptions in a homologue of the exoR gene. ExoR is a predicted periplasmic protein, originally identified in Sinorhizobium meliloti, but widely conserved among alphaproteobacteria. Disruptions in the A. tumefaciens exoR gene result in severely compromised attachment to abiotic surfaces under static and flow conditions, and to plant tissues. These mutants are hypermucoid due to elevated production of the exopolysaccharide succinoglycan, via derepression of the exo genes that direct succinoglycan synthesis. In addition, exoR mutants have lost flagellar motility, do not synthesize detectable flagellin and are diminished in flagellar gene expression. The attachment deficiency is, however, complex and not solely attributable to succinoglycan overproduction or motility disruption. A. tumefaciens ExoR can function independently of the ChvG-ChvI two component system, implicated in ExoR-dependent regulation in S. meliloti. Mutations that suppress the exoR motility defect suggest a branched regulatory pathway controlling succinoglycan synthesis, motility and biofilm formation.

c-FLIP gene silencing eliminates tumor cells in breast cancer xenografts without affecting stromal cells.

Cellular FLICE-like inhibitory protein (c-FLIP) is an inhibitor of death receptor-mediated apoptosis and exerts its anti-apoptotic function by blocking the activation of caspase-8. We recently showed that the siRNA-mediated knockdown of c-FLIP in MCF-7 breast cancer cells growing in vitro triggered apoptosis. The aim of this study was to determine if the in vivo knockdown of c-FLIP in MCF-7 breast cancer xenografts affected tumor viability. Immunohistochemical detection of c-FLIP in the tumor sections revealed that the knockdown of c-FLIP eliminated the neoplastic cells within the breast cancer xenografts without affecting the normal stromal and fibroblastic cells. These results indicate that c-FLIP is required for breast cancer growth and is a relevant therapeutic target for the treatment of breast cancer.

RNA interference in cancer: targeting the anti-apoptotic protein c-FLIP for drug discovery.

The silencing of genes by RNA interference (RNAi) has identified proteins involved in the resistance of cancers to chemotherapeutic drugs. Resistance is associated with defects in the apoptotic signaling pathways. In this article, we examine using RNAi to target the anti-apoptotic protein cellular FLICE-like inhibitory protein (c-FLIP) for drug development.

Etoposide induces protein kinase Cdelta- and caspase-3-dependent apoptosis in neuroblastoma cancer cells.

In this report, we reveal that etoposide inhibits the proliferation of SK-N-AS neuroblastoma cancer cells and promotes protein kinase Cdelta (PKCdelta)- and caspase-dependent apoptosis. Etoposide induces the caspase-3-dependent cleavage of PKCdelta to its active p40 fragment, and active PKCdelta triggers the processing of caspase-3 by a positive-feedback mechanism. Treatment of cells with the caspase-3-specific inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone or caspase-3-specific small interacting RNA (siRNA) prevented the etoposide-induced activation of caspase-8 and inhibited apoptosis. The silencing of the caspase-2 or caspase-8 genes using siRNAs did not affect the etoposide-induced processing of caspase-3, indicating that these caspases lie downstream of caspase-3 in this signaling pathway. Furthermore, the etoposide-induced processing of caspase-2 required the expression of caspase-8, and the etoposide-mediated processing of caspase-8 required the expression of caspase-2, indicating that these two caspases activate each other after etoposide treatment. We also observed that etoposide-mediated apoptosis was decreased by treating the cells with the caspase-6-specific inhibitor benzyloxycarbonyl-Val-Glu(OMe)-Ile-Asp-(OMe)-fluoromethyl ketone and that caspase-6 was activated by a caspase-8-dependent mechanism. Finally, we show that rottlerin blocks etoposide-induced apoptosis by inhibiting the PKCdelta-mediated activation of caspase-3 and by degrading caspase-2, which prevents caspase-8 activation. Our results add important insights into how etoposide mediates apoptotic signaling and how targeting these pathways may lead to the development of novel therapeutics for the treatment of neuroblastomas.

Human beta-galactoside alpha-2,3-sialyltransferase (ST3Gal III) attenuated Taxol-induced apoptosis in ovarian cancer cells by downregulating caspase-8 activity.

Taxol triggers apoptosis in a variety of cancer cells, but it also upregulates cytoprotective proteins and/or pathways that compromise its therapeutic efficacy. In this report, we found that Taxol treatment resulted in caspase-8-dependent apoptosis in SKOV3 human ovarian cancer cells. Moreover, Taxol-induced apoptosis was associated with caspase-3 activation. Interestingly, Taxol treatment upregulated alpha-2,3-sialyltransferase (ST3Gal III) expression and forced expression of ST3Gal III attenuated Taxol-induced apoptosis. Furthermore, ST3Gal III overexpression inhibited Taxol-triggered caspase-8 activation, indicating that ST3Gal III upregulation produces cellular resistance to Taxol and hence reduces the efficacy of Taxol therapy.

The Xpc gene markedly affects cell survival in mouse bone marrow.

The XPC protein (encoded by the xeroderma pigmentosum Xpc gene) is a key DNA damage recognition factor that is required for global genomic nucleotide excision repair (G-NER). In contrast to transcription-coupled nucleotide excision repair (TC-NER), XPC and G-NER have been reported to contribute only modestly to cell survival after DNA damage. Previous studies were conducted using fibroblasts of human or mouse origin. Since the advent of Xpc-/- mice, no study has focused on the bone marrow of these mice. We used carboplatin to induce DNA damage in Xpc-/- and strain-matched wild-type mice. Using several independent methods, Xpc-/- bone marrow was approximately 10-fold more sensitive to carboplatin than the wild type. Importantly, 12/20 Xpc-/- mice died while 0/20 wild-type mice died. We conclude that G-NER, and XPC specifically, can contribute substantially to cell survival. The data are important in the context of cancer chemotherapy, where Xpc gene status and G-NER may be determinants of response to DNA-damaging agents including carboplatin. Additionally, altered cell cycles and altered DNA damage signalling may contribute to the cell survival end point.

c-FLIP knockdown induces ligand-independent DR5-, FADD-, caspase-8-, and caspase-9-dependent apoptosis in breast cancer cells.

Cellular-FLICE inhibitory protein (c-FLIP) is an inhibitor of apoptosis downstream of the death receptors Fas, DR4, and DR5, and is expressed as long (c-FLIP(L)) and short (c-FLIP(S)) splice forms. We found that the knockdown of c-FLIP using small interfering RNA (siRNA) triggered ligand-independent caspase-8- and -9-dependent spontaneous apoptosis and decreased the proliferation of MCF-7 breast cancer cells. Further analysis revealed that an apoptotic inhibitory complex (AIC) comprised of DR5, FADD, caspase-8, and c-FLIP(L) exists in MCF-7 cells, and the absence of c-FLIP(L) from this complex induces DR5- and FADD-mediated caspase-8 activation in the death inducing signaling complex (DISC). c-FLIP(S) was not detected in the AIC, and using splice form-specific siRNAs we showed that c-FLIP(L) but not c-FLIP(S) is required to prevent spontaneous death signaling in MCF-7 cells. These results clearly show that c-FLIP(L) prevents ligand-independent death signaling and provides direct support for studying c-FLIP as a relevant therapeutic target for breast cancers.

Cellular FLICE-like inhibitory protein (C-FLIP): a novel target for cancer therapy.

Cellular FLICE-like inhibitory protein (c-FLIP) has been identified as a protease-dead, procaspase-8-like regulator of death ligand-induced apoptosis, based on observations that c-FLIP impedes tumor necrosis factor-alpha (TNF-alpha), Fas-L, and TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by binding to FADD and/or caspase-8 or -10 in a ligand-dependent fashion, which in turn prevents death-inducing signaling complex (DISC) formation and subsequent activation of the caspase cascade. c-FLIP is a family of alternatively spliced variants, and primarily exists as long (c-FLIP(L)) and short (c-FLIP(S)) splice variants in human cells. Although c-FLIP has apoptogenic activity in some cell contexts, which is currently attributed to heterodimerization with caspase-8 at the DISC, accumulating evidence indicates an anti-apoptotic role for c-FLIP in various types of human cancers. For example, small interfering RNAs (siRNAs) that specifically knocked down expression of c-FLIP(L) in diverse human cancer cell lines, e.g., lung and cervical cancer cells, augmented TRAIL-induced DISC recruitment, and thereby enhanced effector caspase stimulation and apoptosis. Therefore, the outlook for the therapeutic index of c-FLIP-targeted drugs appears excellent, not only from the efficacy observed in experimental models of cancer therapy, but also because the current understanding of dual c-FLIP action in normal tissues supports the notion that c-FLIP-targeted cancer therapy will be well tolerated. Interestingly, Taxol, TRAIL, as well as several classes of small molecules induce c-FLIP downregulation in neoplastic cells. Efforts are underway to develop small-molecule drugs that induce c-FLIP downregulation and other c-FLIP-targeted cancer therapies. In this review, we assess the outlook for improving cancer therapy through c-FLIP-targeted therapeutics.

Cellular FLICE-like inhibitory protein (c-FLIP): a novel target for Taxol-induced apoptosis.

It is known that by binding to the FAS-associated death domain (FADD) protein and/or caspases-8 and -10 at the level of the death-inducing signaling complex (DISC), cellular FLICE-like inhibitory protein (c-FLIP) can prevent apoptosis triggered by death-inducing ligands. We investigated whether the c-FLIP splice variants, c-FLIP long [c-FLIP(L)] and c-FLIP short [c-FLIP(S)], play a role in Taxol-induced apoptosis. Our results showed that low Taxol concentrations triggered caspase-8- and caspase-10-dependent apoptosis in the CCRF-HSB-2 human lymphoblastic leukemia cell line, and induced the down-regulation of c-FLIP(S) and c-FLIP(L). Taxol decreased the expression of c-FLIP by a post-transcriptional and caspase-independent mechanism. To explore the distinct functions of the c-FLIP variants in Taxol-induced apoptosis, we transfected the cells with expression vectors carrying c-FLIP(L) and c-FLIP(S) in the sense orientation or c-FLIP(S) in the antisense orientation [c-FLIP(S)-AS]. Caspases-8 and -10 were more efficiently activated in the c-FLIP(S)-AS strain treated with 5-50nM Taxol, which revealed that c-FLIP regulates Taxol-induced apoptosis by interacting with these caspases. Furthermore, our data showed that increased expression of c-FLIP(L) or c-FLIP(S) reduced apoptosis at 5-50nM Taxol concentrations suggesting that both isoforms of c-FLIP prevent Taxol-induced apoptosis. These results revealed that Taxol induces apoptosis by down-regulating c-FLIP(S) and c-FLIP(L) expression.