Pancreatic cancer (PC) is among the deadliest malignancies with the worst prognosis. Worldwide, nearly 230,000 new cases of PC are diagnosed every year, and almost the same number of patients die of the disease, underscoring the aggressive nature of this cancer [1]. Major reasons for poor survival of patients with PC are the absence of specific symptoms and lack of tests that would detect the disease early, rapid metastasis, and intrinsic resistance of cancer cells to chemo- and radiation therapies. Gemcitabine (Gemzar), the drug of choice for treating PC, shows minimal benefit in terms of patients' survival. Before we can develop new therapeutic strategies that would improve these dismal clinical outcomes, a more detailed understanding of the biology of PC progression is required. Chronic pancreatitis is considered to be the major risk factor in development of PC. Approximately 70% of chronic pancreatitis cases result from excessive alcohol consumption and are characterized by destruction of pancreatic parenchyma, infiltration of inflammatory cells, and tissue fibrosis, accompanied by impaired pancreatic exocrine and endocrine functions. Similarly, patients with hereditary pancreatitis, which arises as a result of mutation in the trypsinogen gene on chromosome 7, are at higher risk of developing pancreatic cancer [2]. Recent evidence suggests that endotoximia, which is due to increased gut permeability in alcoholics, may trigger inflammation in the pancreas tissue and contribute to the progression of chronic pancreatitis by activating pancreatic stellate cells (PSCs). Activation of PSCs could favor malignant transformation of pancreatic ductal cells, leading to dysplasia and cancer. Smoking is another risk factor that can contribute to the development of pancreatic cancer [3]. Cigarette smoke can induce ROS production and lead to inflammation and DNA damage due to continuous exposure to carcinogens.

The loss of tumor suppressor genes, along with mutations in proto-oncogenes, has been suggested to assist in the development of pancreatic cancer [4]. There are many mutations that have been reported in pancreatic cancer, K-ras being the most common [5], followed by the loss of p53 and DPC4/SMAD [6]. The K-ras mutation probably occurs early on and leads to uncontrolled cell growth. These genetic alterations can be induced by reactive oxygen species (ROS) released by activated PSCs and result in DNA strand breaks, sister chromatid exchanges, mutations, and DNA adduct formation [7]. ROS can also change the nucleotide structure and produce mismatched pairing during transcription. The major mutation caused by ROS involves the transversion of guanine to thiamine, which has been found in K-ras mutations [8].

PSCs play an important role in maintaining the homeostasis of the extracellular matrix (ECM) within the pancreas by regulating its synthesis and degradation. During pancreatic injury, however, these cells are activated and secrete cytokines like transforming growth factor (TGF)-β, resulting in an autocrine loop for cell activation. Moreover, activated PSCs acquire a myofibroblast-like phenotype and synthesize and secrete excessive amounts of the ECM proteins such as collagen, laminin, and fibronectin, which comprise fibrous tissue.

In recent years, molecular mechanisms and pathways that contribute to the transformation of PSCs from the quiescent to the activated state have been areas of intense research. Several signaling pathways (e.g., JAK-STAT, MAPK, NF-κB, AP-1, etc.) have been implicated in PSCs activation [9]. In this context, recent observations that activated PSCs, PanINs [10] and advanced-stage pancreatic cancer [11,12] express elevated levels of the pro-inflammatory protein tissue transglutaminase (TG2), in conjunction with the earlier observations that TG2-catalyzed protein crosslinking reactions play an important role in ECM stabilization [13], are of particular importance. This review will discuss the implications of TG2 expression in inflammation and its possible significance in the initiation, progression and pathogenesis of pancreatic cancer.

TG2 is a multifunctional protein, which in addition to catalyzing calcium-dependent crosslinking of proteins, can bind and hydrolyze GTP [14]. TG2 has been implicated in a number of pathological processes such as hepatic injury, degenerative disorders of neurons, myocardial hypertrophy, celiac disease, and tissue fibrosis [15]. Many biological functions of TG2 are attributed to its ability to catalyze calcium-dependent protein crosslinking reactions. However, in recent years evidence has accumulated indicating that TG2 can also function as a cell-signaling scaffold protein and regulate protein stability, cell functions and gene regulation in a context-dependent manner [16-19]. Though predominantly a cytosolic protein, TG2 can also translocate to the nucleus, cell membrane, or mitochondria and can even be secreted outside the cell.

Under physiological conditions, TG2 exists as a catalytically inactive intracellular protein due to low Ca²⁺ and high inhibitory concentrations of GTP/GDP [20]. In this form, TG2 acts as a scaffold protein and results in the activation of some critical cell survival signaling pathways (NF-κB, Akt, focal adhesion kinase) [16,19,21,22]. However, under extreme conditions of cellular stress or trauma, loss in Ca²⁺ homeostasis can activate intracellular TG2 resulting in crosslinking of cellular proteins and apoptosis or necrotic death of cells [23,24] (Figure 1). Thus, distinct activation of status of TG2 (compact versus extended) in a cell may be a reflection of the fact that TG2 functions as a double-edged sword in cancer cells, conferring advantage or disadvantage to the tumor.

Inflammation (derived from the Latin word ‘inflammare’ to set on fire) is a complex biological response triggered by harmful stimuli. Inflammation involves such multiple events as cell migration, cell proliferation, synthesis and stabilization of the ECM, neovascularization, and apoptosis, all of which are needed for successful wound healing and tissue repair. Acute inflammation also referred to as therapeutic or physiologic inflammation is an initial stage of inflammation (innate immunity) mediated through activation of the immune system; it helps the body ward off infections and lasts for only a short period. If inflammation persists for a long period of time, it becomes chronic and is referred to as pathological inflammation [25]. Indeed, chronic inflammation has been linked to most chronic illnesses, including cancer, cardiovascular diseases, diabetes, obesity, pulmonary diseases and neurologic diseases [25].

Several studies have implicated TG2 in wound healing and inflammation [26–28]. Cytokines and growth factors secreted during early phases of cell injury regulate TG2 expression. For example, pro-inflammatory cytokine transforming growth factor (TGF)-β1 enhances TG2 gene expression through a response element located 868 bases upstream of its translational initiation site [29]. TG2, in turn, binds to cell surface TGFβ1 and promotes its conversion to the biologically active form, thus creating a positive feedback loop. Similarly, TNF-α induces TG2 synthesis by activating NF-κB [30] and TG2 activates NF-κB [31], thus creating an autocrine loop. TG2 expression can also be enhanced by IL-1 [32] and IL-6 [33]. In response to cutaneous injury, TG2 expression and activity are increased at sites of neovascularization and in endothelial cells, skeletal muscle cells, and macrophages infiltrating wounds in the border between healthy and injured tissue [34].

In vivo studies using TG2-deficient mice revealed that these mice are better protected from lipopolysaccharide-induced septic shock than their wild-type counterparts [35]. The protective effect was associated with decreased NF-κB activation, decreased neutrophil recruitment into the kidney and peritoneum, and reduced damage to renal and myocardial tissues in TG2-deficient mice. These observations imply that TG2 expression plays a role in promoting the inflammation-induced pathogenesis of septic shock. Moreover, TG2-deficient mice have been used to determine whether TG2 plays a protective or pathologic role during liver injury. Results from these studies suggested that TG2-deficient mice were more prone to death caused by carbon tetrachloride- or alcohol-induced liver injury than the control mice indicating a protective role for TG2 in liver injury [36]. The protection of TG2 expressing mice was associated with increased inflammatory response and increased ECM accumulation. Studies to determine the involvement of TG2 in renal fibrosis also revealed that TG2-deficient mice are less susceptible to kidney damage than their normal counterparts [37]. A protective effect was associated with reduced infiltration of macrophages and myofibroblasts, decreased collagen synthesis and reduced TGF-β activation in TG2-deficient mice.

Macrophages are among the first cells that are recruited at inflammatory sites. In response to the inflammatory cytokines and other pro-inflammatory factors, macrophages accumulate large amounts of TG2 protein at these sites [38]. Generally, in intracellular sites, TG2 attains a folded or compact conformation (due to low calcium and high GTP levels) and remains catalytically inactive. However, upon its release in the extracellular environment (by passive release or due to cell death) TG2 acquires a catalytically active or extended conformation and thus catalyzes irreversible crosslinking of the ECM proteins (Figure 1). TG2-catalyzed crosslinking of ECM proteins via N′ε(γ-glutamyl)-lysine isopeptide bonds represents an important step in maturation and stabilization of such ECM proteins, such as collagen and fibronectin and is critical for the wound healing. Indeed, N′ε(γ-glutamyl)-lysine crosslinks, which are undetectable in normal liver tissue, become prominent in fibrotic livers of patients with a variety of chronic liver diseases [36]. In addition, the crosslinking ability of TG2 appears to be crucial for the activation of TGF-β, the most fibrogenic cytokine [39]. In many tissues, enhanced TG2 activity, which results from cell damage due to chronic inflammation, is required for TGF-β activation via crosslinking of large latent complexes to the cell surface, or to fibronectin and other ECM components through the latent TGF-β binding protein portion. Amine donor substrates, such as putrescine and cystamine, which act as competitive inhibitors for TG2 in its crosslinking reaction, have been shown to protect against CCl4-induced fibrosis [40]. Thus, chronic expression of TG2 may promote cell survival and permit accumulation of oncogenic mutations, resulting in cell transformation and tumor progression (Figure 1).

Pancreatic carcinoma is believed to develop from histologically identifiable intra-ductal lesions known as PanINs that follow a series of structural, cytological, and genetic premalignant changes. Cheung et al. [10] recently observed that 60% of preneoplastic lesions (PanIN-1, 2, and 3) in tumor samples from patients expressed elevated TG2 levels. In a preliminary study using tissue samples from transgenic mice carrying pancreas-specific oncogenic K-ras^g12d mutation, we observed a similar increase in TG2 expression in early/late-stage PanINs and in invasive carcinoma (Figure 2). Similarly, gene analysis of tumor samples revealed that TG2 is among the highest expressed genes in pancreatic cancer [12]. Moreover, our own studies demonstrated that basal expression of TG2 is significantly high in PC cell lines as well as in tumor samples from PC patients than in normal ducts (P < 0.0001) [11]. Importantly, high TG2 expression in tumor samples is associated with nodal metastasis (P = 0.017) and lymphovascular invasion (P = 0.045) [11]. These observations suggest that aberrant expression of TG2 occurs early in pre-neoplastic lesions in the pancreas and increases as the disease progresses. Using the gain- and loss-of-function approaches, several studies have supported a role of TG2 in drug resistance and metastasis of PC cells. Thus, high-TG2-expressing Panc-28 PC cells showed minimal growth inhibitory effect (IC₅₀ =10 μM) in response to gemcitabine treatment when compared with the TG2-deficient BxPC3 cells (IC₅₀ = 0.1 μM). As is the case for PC cells, the development of drug resistance in breast cancer, melanoma, lung carcinoma, and ovarian carcinoma has been associated with increased TG2 expression [16]. Downregulation of TG2 by antisense or siRNA resulted in reversal of drug resistance in these cells.

Cell motility and invasion processes are intimately related to metastasis. TG2 expression has been linked to enhanced invasive potential of PC cells in a Matrigel invasion assay. High-TG2-expressing Panc-28 cells showed three-fold higher invasion than the low-TG2-expressing BxPC3 cells.

Moreover, downregulation of TG2 by siRNA dramatically compromised the ability of Panc-28 cells to invade through Matrigel transwell inserts. Conversely, the infection of low-TG2-expressing BxPC-3 cells with TG2-adenoviral construct rendered the cells four-fold more invasive than non-infected or adenovirus-alone-infected cells [11].

Downregulation of TG2 in PC cells has been associated with autophagic death (type II programmed cell death). Thus, transfection of Panc-28 or Capan-1 cells with TG2 siRNA (but not control siRNA) induced massive accumulation of autophagic vacuoles, as revealed by phase-contrast microscopy [41]. The vacuoles were highly acidic, as determined by acridine orange staining, and were accompanied by the accumulation of microtubule-associated light chain (LC3) protein, hallmarks of cells undergoing autophagy. Transmission electron microscopy (TEM) revealed the formation of autophagosomes with cellular organelles in TG2 siRNA-transfected Panc-28 cells. In addition to autophagic vesicles, TEM images also revealed the merging of autophagic vesicles with lysosomes and mitochondria, suggesting lysosomal-mediated degradation of cellular organelles. These results suggested that TG2 expression protects PC cells from autophagic death. Although the role of autophagy in cell death has been controversial, recent reports have noted that prolonged autophagy can lead to cell death. Indeed, the autophagy regulator gene, BECN1, is considered to be a haploinsufficient tumor suppressor gene that induces autophagy when overexpressed. From these results, it is reasonable to believe that downregulation of TG2 in PC cells blocks cell survival signaling and thus leads to autophagic death. Taken together, these results suggested a novel role for TG2 in protecting PC cells from cell death.

Based on the information discussed in preceding sections, we concluded that inhibition of TG2 could be a promising therapeutic target for reversing intrinsic resistance of pancreatic cancer cells to chemotherapeutic drugs. Using DOPC liposomes as the delivery system, we showed that TG2 siRNA could effectively downregulate TG2 expression in orthotopically growing pancreatic tumors in a nude mouse model and inhibit their growth and metastatic spread [50]. Downregulation of TG2 rendered pancreatic tumors sensitive to gemcitabine treatment.

To determine potential mechanisms underlying the antitumor effect of TG2 siRNA-DOPC, we examined its effect on several biological end-points, including cell proliferation (Ki-67), angiogenesis (CD31), and apoptosis (TUNEL). More than 90% reduction in Ki-67 expression was evident (P < 0.001) in tumors obtained from mice given TG2 siRNA-DOPC alone or in combination with gemcitabine. Tumors obtained from mice given gemcitabine alone or control siRNA-DOPC consistently showed high levels of Ki-67 staining [50]. We also evaluated the blood vessel density in tumors obtained from mice treated with control siRNA-DOPC, gemcitabine, TG2- siRNA-DOPC or a combination of gemcitabine plus TG2-siRNA-DOPC. We found a significant decrease in the mean blood vessel density in tumors recovered from mice that received TG2 siRNA-DOPC or gemcitabine alone. Densitometric analysis showed a significant difference in the mean microvascular density (P < 0.0013) in the mice that received TG2 siRNA-DOPC plus gemcitabine. Finally, we evaluated apoptosis in orthotopic tumors using TUNEL assay. Pancreatic tumors treated with gemcitabine alone or in combination with TG2 siRNA-DOPC showed significant increases in the numbers of apoptotic cells. No differences in the apoptotic index in TG2 siRNA-DOPC-treated tumors were evident, and the extent of apoptosis in this group was similar to that in the control siRNA-DOPC group.

In conclusion, our studies have provided the proof-of-concept that therapeutic delivery of TG2-specific siRNA to pancreatic tumors can be achieved using nanoliposomes. Two recent reports have documented a similar effect of liposomal delivery of siRNA to silence the expression of the oncoprotein EphA2 [68] and FAK [69]. Using this approach, both studies documented encouraging responses to the treatment of ovarian tumors in mice. Thus, the significance of elevated TG2 expression and its role in promoting the drug resistance and metastatic phenotype can be rapidly translated into the clinical setting for treatment of aggressive forms of cancer, including pancreatic cancer.