TRAIL Mediated Signaling in Pancreatic Cancer

Mechanistically, multifaceted mechanisms including crosstalk of signaling pathways, genetic/epigenetic mutations, inactivation of tumor suppressors and activation of oncogenes in pancreatic cancer have gained tremendous appreciation in the past decade (Diamantidis et al., 2008). In the field of signaling pathways, it is getting sequentially more understandable that pancreatic cancer cells are difficult to target because of rewiring of intracellular signaling cascades. Linear and integrated signaling cascades also impair tumor necrosis factor alpha-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in pancreatic cancer cells. TRAIL mediated intracellular signaling has emerged as a deeply studied cascade in cancer cells (Röder et al., 2011; Arlt et al., 2013). TRAIL is a ligand and gained tremendous appreciation over the years because of its ability to selectively induce apoptosis in cancer cells. TRAIL signals through its receptors DR4 and DR5. Death-receptor signaling is initiated by ligandinduced receptor trimerization that results in formation of the death-inducing signaling complex (DISC). Death


Introduction
Mechanistically, multifaceted mechanisms including crosstalk of signaling pathways, genetic/epigenetic mutations, inactivation of tumor suppressors and activation of oncogenes in pancreatic cancer have gained tremendous appreciation in the past decade (Diamantidis et al., 2008).In the field of signaling pathways, it is getting sequentially more understandable that pancreatic cancer cells are difficult to target because of rewiring of intracellular signaling cascades.Linear and integrated signaling cascades also impair tumor necrosis factor alpha-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in pancreatic cancer cells.TRAIL mediated intracellular signaling has emerged as a deeply studied cascade in cancer cells (Röder et al., 2011;Arlt et al., 2013).TRAIL is a ligand and gained tremendous appreciation over the years because of its ability to selectively induce apoptosis in cancer cells.TRAIL signals through its receptors DR4 and DR5.Death-receptor signaling is initiated by ligandinduced receptor trimerization that results in formation of the death-inducing signaling complex (DISC).Death receptors have death domains (DD), which recruit FADD, to the receptor complex.Through death effector domains (DED) in FADD, recruitment and activation of the apoptosis-initiating caspase-8 occurs.Caspase-8 activates its downstream effector caspase-3.It is surprising to note that intrinsic pathway is triggered via an additional amplification loop that involves the cleavage of Bid by caspase-8 to truncated Bid (tBid).tBid moves into the mitochondrion to facilitate release of cytochrome c.Cytochrome c in concert with pro-caspase-9 and apoptotic protease activating factor (APAF) forms apoptosome.Bax and Bak are in an inactive state at the outer mitochondrial membrane however upon stimulation, pro-apoptotic proteins including Puma or Bim trigger activation of Bax and Bak via dissociation from anti-apoptotic Bcl-2 family.
It has previously been convincingly revealed using pancreatic cancer cell lines differentially expressingBcl-XL that TRAIL induced apoptosis was impaired in Bcl-XL overexpressing cancer cell lines (Panc-1 and PancTuI).However, enforced expression of a pro-apoptotic protein Bax or gene silencing of Bcl-XL induced apoptosis in TRAIL resistant cancer cells (Hinz et al., 2000).In vitro assays provided evidence that in five pancreatic cancer cell lines, TRAIL, DR4 and DR5 mRNA expression were detectable.Still TRAIL did not induce apoptosis in pancreatic cancer cells primarily because of overexpression of anti-apoptotic proteins (Ozawa et al., 2001).It is noteworthy that actinomycin improved TRAIL induced apoptosis in resistant cancer cells via targeting of cFLIP (Matsuzaki et al., 2001).Another contemporary study indicated that differentially high levels of decoy receptors also inhibited an efficient TRAIL induced apoptosis (Ibrahim et al., 2001).Increasingly it is being realized that TRAIL induced pro-survival signals in pancreatic cancer cells as evidenced byactivation of protein kinase C and NF-kappaB in TRAIL treated pancreatic adenocarcinoma cell line PancTuI.Synthetic agent mediated inhibition of NF-kappaB or transient transfection with a dominant negative mutant of IkappaBalpha induced apoptosis in TRAIL treated cancer cells (Trauzold et al., 2001).NEMO-binding domain (NBD) peptide is a novel selective NF-kappaB inhibitor and results revealed that NBD treated L3.6 cell line (TRAIL resistant) displayed TRAIL induced apoptosis (Thomas et al., 2002).TRAIL induced apoptosis was notably enhanced in gemcitabine treated SW1990 cancer cells.There was a marked increase in expression of Smac/DIABLO and cytochrome C (Zhao et al., 2011).

Tissue Expression
It has previously been reported that TRAIL-R3 expression was low and TRAIL-R4 expression ranged from moderate to high levels.However weakness of the study was limited number of cases studied (Liao et al., 2001).

Molecular Basis of TRAIL Resistance
It has previously been persuasively revealed that constitutively activated NFKB, but not TRAIL induced NF-kappaB modulated apoptosis.Panc-1 cells have constitutively activated NFKB and it has been experimentally verified that introduction of IκB-α superrepressor considerably reduced NF-kB DNA-binding activity (Braeuer et al., 2006).Shikonin, a naphthoquinone derivative has been shown to repress constitutive as well as gemcitabine-induced activation of NF-kB and NF-kBtarget genes.Mice xenografted with pancreatic cancer cells displayed decreased proliferation, decreased microvessel density and increased apoptosis upon treatment with Wang et al. (2014).α-Mangostin, an antioxidant derived from the Garcinia mangostana L. Expression of pNF-kB/ p65Ser552 was also found to be inhibited in α-Mangostin treated pancreatic cancer cell lines (Hafeez et al., 2014).Wogonin, a naturally occurring flavone was noted to suppress cFLIP in Capan-1 cell line thus restoring TRAIL induced apoptosis.Moreover, DR5 was upregulated in wogonin treated Capan-1 cancer cells (Ding et al., 2012).Zyflamend is a formulation of 10 standardized herbal extracts and reported to be effective in restoration of apoptosis in cancer cells.Zyflamend triggered expression of DR5 via CCAAT/enhancer-binding proteinhomologous protein (CHOP).Mechanistically it was shown that DR5 promoter region contained a CHOP binding site.Cancer cells transfected with DR5 promoter plasmids, containing either a wild-type or mutated CHOP binding site, revealed that Zyflamendupregulated expression of DR5 in cancer cells transfected with wildtype CHOP binding site containing plasmids (Kim et al., 2012a).Pro-survival signaling induced in TRAIL treated cancer cells has added another layer of information into the molecular biology of TRAIL induced transduction cascade.TRAIL-induced expression of uPA and IL-8 was noted to be severely impaired in DR4 and DR5 blocked Colo357 cells and Panc89 cells.Colo357 cells stably transfected with TRAF2, upon treatment with TRAIL induced upregulation of uPA and IL-8.Similar results were noted in Colo357 cells transfected with Bcl-xl (Zhou et al., 2013).
MUC5AC, a secretory mucin expressed frequently in cancer cells and inoculating nude mice with MUC5AC silenced SW1990 cells did not induce tumor growth.Additionally it was shown that MUC5AC silenced SW1990 cells displayed remarkably enhanced TRAIL induced apoptosis (Hoshi et al., 2013).Mesothelin affinity for MUC16 has recently been utilized for targeting of MUC16 overexpressing pancreatic cancer cells.TRAILbased fusion protein Meso-TR3 was designed by insertion of cDNA of mesothelin to the 5'-end of a TR3 expression plasmid.Construct was expressed in mammalian 293T cells as a fusion protein.BxPC3 pancreatic cancer cells treated with Meso-TR3 displayed considerably enhanced apoptotic rate (Garg et al., 2014).
AEG35156/GEM 640, a second-generation antisense oligonucleotide against XIAP has shown notable efficacy against Panc-1 pancreatic carcinoma cells.TRAIL induced apoptosis was substantially enhanced in Panc-1 cells treated with AEG35156 (LaCasse et al., 2006).Subsequent study has shown that gene silencing strategy against XIAP also improved TRAIL mediated apoptosis.Mechanistically it was reported that targeted inhibition of XIAP in Bcl-2 overexpressing cancer cells potentiated TRAIL induced apoptosis.Xenografting PancTu1 cells transduced with XIAP shRNA in nude mice induced regression of tumor in combination with TRAIL (Vogler et al., 2008).
Gimatecan, a potent lipophilic camptothecin has earlier been tested for efficacy in combination with CpGoligodeoxynucleotides in pancreatic cancer xenograft (Petrangolini et al., 2008).
Benzyl isothiocyanate (BITC) is reported to induce apoptosis in pancreatic cancer cells.In vitro studies revealed that it triggered intrinsic pathway via activation of Bid.BITC also potentiated TRAIL induced apoptosis DOI:http://dx.doi.org/10.7314/APJCP.2014.15.15.5977TRAIL Mediated Signaling in Pancreatic Cancer via inhibition of Rac1 in pancreatic ductal carcinoma cells CFPAC-1 (Basu and Haldar, 2009).Confluence of information suggests that gene silencing of c-FLIP(L) and c-FLIP(S) restored apoptosis in pancreatic cancer cells.More interestingly cells reconstructed with c-FLIP induced TRAIL resistant phenotype.It is relevant to mention that 5-fluorouracil (5-FU), cisplatin or gemcitabine induced apoptosis via inhibition of c-FLIP and enforced expression of c-FLIP impaired drug induced efficacy (Haag et al., 2011).Triptolide, a diterpene triepoxide improved TRAIL mediated apoptosis in pancreatic cancer cells via upregulation of DR5 and downregulation of c-FLIP (Chen et al., 2014).
There is a direct piece of evidence suggesting that Bcl-xl knockdown BxPC-3 cells transduced with empty vector demonstrated notably enhanced apoptotic rate following GSK-3i or TRAIL exposure.However, Bclxl knockdown cells reconstituted with Bcl-xl, indicated partial reversal of GSK-3 inhibitor and TRAIL-induced apoptosis.Mechanistically it was reported that GSK-3i treatment resulted in an increased SIRT1 and HDAC3 loading at BCL2L1 promoter that consequently resulted in formation of repressive chromatin (Zhang et al., 2014).Embelin, isolated from the fruit of Embelia ribes has been shown to inhibit growth of AsPC-1 xenografts in Balb C nude mice.EMT was also notably repressed in xenografted nude mice.Tissue analysis of xenografted mice revealed that Embelin triggered upregulation of DR4 and DR5 Huang et al, 2014.Garlic oil has been shown to exert its inhibitory effects on proliferation of AsPC-1, PANC-1, and Mia PaCa-2 cells Lan et al, 2013.

Regulation of miRNA by DR5 in Pancreatic Cancer Cells
Recently emerging hints have started to scratch the surface of new role of death receptor as a negative regulator of miRNA.It has lately been shown that DR5 is located in nucleus of pancreatic cancer cells.It has also been experimentally verified that Drosha and DGCR8 are associated with DR5 in cancer cells as evidenced by immunoprecipitation of nuclear DR5.Sequential methodologies including gene silencing of DR5 indicated that DR5 silenced pancreatic cancer cells did not display an increase in the levels of pri-let-7.However, processing activity of Drosha towards pri-let-7 was substantially enhanced underscoring the fact that DR5 interaction with Drosha considerably impaired its activity (Haselmann et al., 2014).

Preclinical Studies
It has lately been shown in an orthotopic model of a sensitive tumor that there was a considerably reduced primary and metastatic tumor burden in TRAIL treated mice.More importantly it was revealed that site of tumor engraftment did not alter the inherent sensitivity of patient xenografts to TRAIL (Sharma et al., 2014).
Pancreatic cancer cells frequently express EMMPRIN, a membrane-bound glycoprotein.Experimentally verified data revealed that EMMPRIN regulated expression of Matrix metalloproteinases, thus inducing degradation of extracellular matrix components.SCID mice xenografted with MIA PaCa-2 and PANC-1 cancer cells were treated with anti-EMMPRIN antibody and results revealed that it exerted its inhibitory effects on cell proliferation and endothelial cell densities.More interestingly, anti-EMMPRIN antibody and monomeric monoclonal antibody TRA-8 were used in combination in xenografted mice and considerably inhibited tumor growth in mice (Kim et al., 2012b).Monoclonal antibodies against DR4 (HGS-ETR1, mapatumumab) or DR5 (HGS-ETR2, lexatumumab) were found to be effective when used as combination therapy in MiaPaCa2 cells.Mapatumumab efficiently induced regression of tumor growth in xenografted mice (Stadel et al., 2010).Oral administration of phenethyl isothiocyanate inhibited tumor growth of MIAPaca2 cancer cells in xenografted mice (Stan et al., 2013).
GANT-61 (Gli transcription factor inhibitor) has been shown to exert its inhibitory effects on tumor growth via upregulation of DR4 and DR5 as evidenced by tumor tissues derived from NOD/SCID IL2Rγ null mice (Fu et al., 2013).
It is now well known that epithelial cell adhesion molecule (EpCAM) is frequently overexpressed by pancreatic cancer cells.XenograftingEpCAMpositive BxPc-3 cancer cells in immunodeficient mice induced tumor growth, both in absence of TRAIL transfected lymphocytes.However, EpCAM-positive BxPc-3 cancer cells did not establish tumor in TRAIL transfected lymphocytes bearing mice.More importantly, intraperitoneally injected EpCAMxCD3 Bispecific antibody substantially inhibited tumor formation in xenografted mice (Groth et al., 2012).It has recently been shown that a multipronged approach, consisting of atorvastatin, celecoxib and tipifarnib considerably reduced growth of the tumors in SCID mice xenografted with pancreatic cancer cells (Ding et al., 2014).PARP inhibitor (Olaparib) and the pan-Bcl-2 inhibitor (Obatoclax) represented anti-tumor activity in xenografted mice (Chen et al., 2014).Triptolide, isolated from Tripteryglum wilfordii was noted to significantly reduce tumor growth in mice (Liu and Cui, 2013).

Clinical Trials
Tigatuzumab, a humanized version of the agonistic murine monoclonal antibody TRA-8 has been tested for efficacy in chemotherapy-naive patients.Phase 2 trial evaluated the efficacy of combinatorial approach consisting of tigatuzumab and gemcitabine in 62 chemotherapy-naive patients and results revealed that it was well tolerated (Forero-Torres et al., 2013).

The Role of Nanotechnology on Antitumor Activity of TRAIL
To overcome the resistance of many cancer cells to TRAIL-induced apoptosis, wide ranging natural and synthetic agents have shown potential.Matsuzaki et al. (2001) showed that the combination of TRAIL and actinomycin D prompted apoptosis in different human pancreatic cancer cell lines, including TRAIL-resistant cells.Likewise, Bai et al. (2005) studied a TRAIL-based triple chemotherapy to overcome the chemoresistance in pancreatic cancer cells.The combination of geldanamycin, PS-341, and TRAIL dramatically enhances TRAIL cytotoxic effects and could be a novel therapeutic strategy for pancreatic cancer.
Nanotechnology approaches have been widely used in medical research, medical imaging, and clinical applications.In an attempt to gain a greater and specific antitumor activity of TRAIL, the research on nanocarriers encapsulating TRAIL (Lim et al., 2011) or TRAILencoding plasmid (Mangipudi et al., 2009;Miao et al., 2013) gained a special attention over the last decade.The delivery of TRAIL and classical antitumor drugs using different kinds of nanotechnological approaches have also been investigated as a promising strategy to achieve a greater and more effective killing activity against cancer cells (Guo et al., 2011;2012;Lee et al., 2011a;2011b).
As mentioned, researchers are constantly looking for new strategies to gain a more efficient cancer treatment by using specific ligands with selective affinity to cancer cells.Different strategies including encapsulating TRAIL alone or in combination with other antitumor drugs have been studied to kill tumor cells in different kinds of cancer.Intratumoral injections of PEG-TRAIL (50μg) in mice xenografted with Mia Paca-2 cancer cells exerted inhibitory effects on tumor formation (Byeon et al., 2013).It is interesting to note that PEGylated TRAIL has further been tested using a specific delivery system consisting of four-arm PEG cross-linked hyaluronic acid (HA) hydrogels.The results revealed that antitumor activity of TRAIL in mice xenografted with Mia Paca-2 was remarkably higher because of stability of PEGylated TRAIL in HA hydrogels (Byeon et al., 2014).Bae et al. (2012) designed human serum albumin (HSA) nanoparticles with the surface modified by TRAIL and transferrin, and containing doxorubicin, which were demonstrated to have a significant in vitro killing activity against different cancer cell lines.Among these cells, the authors studied the cytotoxic and apoptotic activities of the nanoparticles on CAPAN-1, a metastatic pancreatic cancer cell line.These nanoparticles were also tested in HCT 116-xenografted nu/nu mice and localization of HSA-NPs was noted in tumors.
Therefore, in view of the need for more efficacious treatments, together with the numerous advantages reported regarding the use of nanomaterials in clinical applications, the delivery of TRAIL-using different nanotechnological approaches to treat pancreatic cancer demands more investigation.

Concluding Remarks
The potential of TRAIL to selectively induce apoptosis in cancer cells is clear and already proven in many studies.Likewise, nano-based drug delivery systems are emerging and promising technologies that have the ability to specifically deliver chemotherapeutics to cancer cells.Numerous studies have demonstrated that drug-loaded nanomaterials are more active to kill cancer cells than free antitumor drugs, which might result in better outcomes of clinical treatment protocols.Therefore, TRAIL encapsulation in nanodevices is particularly encouraging in an attempt to achieve an increased antitumor activity.
However, despite the merits that make TRAIL a promising apoptotic agent, it has drawbacks such as rapid clearance from the systemic circulation and short biological half-life.To overcome these problems, the nanoencapsulation of TRAIL has become a promising strategy to achieve a greater apoptotic activity.Furthermore, combination of nano-based drug delivery devices containing TRAIL and other chemotherapeutic agent is more likely to reach enhanced antitumor activity.
Altogether, the growing research on nanotechnology and the need for innovative formulations that can enhance the biological performance of biomolecules with known antitumor activity, highlight TRAIL-nanovectors as potential new approaches to treat many kinds of tumors, including the pancreatic cancer that have a high lethality level.In conclusion, it can be stated that TRAIL delivery needs further improvement using nanotechnological strategies.