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Tissue transglutaminase-mediated chemoresistance in cancer cells
Drug Resistance Updates 10 (2007) 144–151
Tissue transglutaminase-mediated chemoresistance in cancer cells Amit Verma, Kapil Mehta ∗ Department of Experimental Therapeutics, Unit 362, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, United States Received 16 May 2007; received in revised form 8 June 2007; accepted 11 June 2007
Abstract Drug resistance and metastasis are major impediments for the successful treatment of cancer. A common feature among drug resistant and metastatic tumor cells is that they exhibit profound resistance to apoptosis. This property enables cancer cells not only to grow and survive in stressful environments (metastasis) but also to display resistance against many anticancer agents. Therefore, perturbation of the intrinsic apoptotic pathways of cancer cells will affect their ability to respond to chemotherapy and to metastasize and survive in distant sites. Recent studies have demonstrated that cancer cells and cancer cell lines selected for resistance against chemotherapeutic drugs or isolated from metastatic sites, express elevated levels of the multifunctional protein, tissue transglutaminase (TG2). TG2 is the most diverse and ubiquitous member of the transglutaminase family of proteins that is implicated to play a role in apoptosis, wound healing, cell migration, cell attachment, cell growth, angiogenesis, and matrix assembly. TG2 can associate with certain  members of the integrin family of proteins (1, 3, 4, and 5) and promote stable interaction between cells and the extracellular matrix (ECM), resulting in increased cell survival, cell migration, and invasion. Additionally, TG2 forms a ternary complex with IB/p65:p50 and results in constitutive activation of the nuclear transcription factor-B (NF-B). Moreover, TG2 expression in cancer cells leads to constitutive activation of the focal adhesion kinase (FAK) and its downstream PI3K/Akt survival pathway. Importantly, the inhibition of endogenous TG2 by small interfering RNA (siRNA) resulted in the reversal of drug resistance and the invasive phenotype. Conversely, ectopic expression of TG2 promoted cell survival, cell motility and invasive functions of cancer cells. This review discusses the current thinking and implications of increased TG2 expression in development of drug resistance and metastasis by cancer cells. © 2007 Elsevier Ltd. All rights reserved. Keywords: Invasion; Metastasis; Drug resistance; Focal adhesion kinase; -Integrins; NF-B; AKT; PTEN; Autophagy; Apoptosis
1. Introduction The ability of cancer cells to develop resistance to drugs poses a major clinical challenge in the successful treatment of cancer. “Virtually all of the roughly half-million annual cancer deaths in the United States can be said to have occurred because of chemotherapy failed” (Goldstein et al., 1989). Though it is generally accepted that the majority of cancers arise from a single precursor cell, a tumor is a not a collection of genetically identical cells. Genetic instability and accumulation of mutations are important hallmarks of cancer cells. This means that dividing cancer cells are able to acquire genetic and epigenetic changes that will favor their malignant phenotype as well as drug resistance (Richardson and Kaye, ∗
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2005). In addition to the acquired genetic and epigenetic changes, host factors like micro-tumor environment, tumor vascularity, drug bioavailability, metabolism, elimination and dose-dependent toxicity are some of the limitations, which play a role in the final outcome of conventional chemotherapy. For example the tumor vascularity may affect transit time of drugs within tumors and the interaction of tumor cell with the surrounding interstitial cells (Green et al., 1999). Also vascularity may dictate the tumor penetration of high molecular weight monoclonal antibodies and immunotoxins (Pluen et al., 2001). The classic approach to try to understand the molecular mechanisms of resistance to anticancer drugs has been to select cell lines that exhibit high level of drug resistance and then identify genes that might account for the resistance. As a result of this strategy mostly mechanisms that are able to confer relatively high levels of resistance have been identified,
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including decreased transporter-mediated uptake of watersoluble drugs, various mutations/alterations in genes involved in cell cycle, DNA repair mechanism and apoptosis; changes in metabolizing enzymes, drug target genes and/or drug efflux transporters, like P-glycoprotein, MRPs and BCRP (Assaraf, 2006; Szak´acs et al., 2006). The realization that most anticancer drugs might kill tumor cells by activating apoptotic pathways raised the notion that deregulation of apoptotic pathways may be a key determinant in the development of chemoresistance in tumor cells (Mashima and Tsuruo, 2005). Indeed, numerous alterations that confer resistance to apoptosis in cancer cells have been identified. For example, activation of pro-survival signal transduction pathways such as, those mediated by Ras, PI3K/Akt, or NF-B; inactivation of apoptotic pathways, e.g., due to mutation or silencing of p53, pRb, Bax, Bad, Apaf1, caspase-8 genes or overexpression of pro-survival proteins such as, Bcl-2, IAP, and FLIP are frequently observed in advanced cancers (Fesik, 2005). As a logical consequence many approaches to attempt improved cancer treatment involve combinations of cytotoxic agents with new molecular targeted agents. The rationale for such combination cancer therapies is based on the assumption that agents affecting molecular signaling pathways involved in apoptosis or other cell death mechanisms (Chen et al., 2006), migration and invasion (Sierra, 2005), angiogenesis (Broxterman and Georgopapadakou, 2005; Morabito et al., 2006), tumor cell signal transduction (Bloom and Kloog, 2005; Adjei, 2006), and protein turnover (Richardson et al., 2003; Nawrocki et al., 2005; Demarchi and Brancolini, 2005; Horton et al., 2006) will lead to synergistic tumor cell death. Detailed discussion on the mechanisms involved in drug resistance and approaches to overcome this problem are beyond the scope of this chapter but have been covered in the literature (Goldman, 2003; Liang et al., 2001; Smukste et al., 2006). Though all of these approaches seem logical and promising, however the understanding of cellular drug resistance behavior does not necessarily translate to understanding completely the resistance observed in clinical tumors, because tumors consist of a mixed population of cells which may not exhibit very high levels of drug resistance. Therefore, there is still a considerable interest to define novel resistance mechanisms as potential targets for novel compounds with greater effectiveness and for optimal drug combinations. This review will focus on recent data and understanding of the significance of increased tissue transglutaminase (TG2) expression in drug resistant (intrinsic or acquired) cancer cells as a potential novel anticancer drug target.
2. TG2 and apoptosis TG2, also referred to as cytosolic or type II transglutaminase, is the most diverse member of the transglutaminase family of enzymes (Chen and Mehta, 1999; Fesus and
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Piacentini, 2002; Griffin et al., 2002; Mehta, 2005). In addition to catalyzing calcium-dependent posttranslational modification of proteins, TG2 can bind and hydrolyze guanosine triphosphate (GTP) with an affinity similar to those of the ␣ subunits of large heterodimeric G proteins and small Ras-type G proteins (Stephens et al., 2004). The calcium-dependent protein cross-linking reactions of TG2 are implicated in cell differentiation, receptor-mediated endocytosis, cell adhesion, and induction of apoptosis. In addition, TG2/Gh is involved in coupling the ␣1 b- and ␣1 d-adrenoreceptors, thromboxane, and oxytocin receptors to phospholipase C, thereby mediating inositol phosphate production in response to the agonist-induced activation. In addition, TG2 can function as a protein disulfide isomerase, ATPase and as a protein kinase (Mishra and Murphy, 2006; Lorand and Graham, 2003; Hasegawa et al., 2003; Mastroberardino et al., 2006), the major component of insulin-like growth factor-binding protein-3 kinase (Mishra and Murphy, 2004). Although, predominantly a cytosolic protein, TG2 can be secreted outside the cell (by yet unknown mechanism), where it stabilizes the ECM by making the matrix resistant to mechanical and proteolytic degradation (Aeschlimann and Thomazy, 2000). Similarly, in association with other proteins, TG2 can translocate to the nucleus and cell membranes. Its localization (membrane, cytosol, and nuclear) and the functional configuration (cross-linking, GTP-binding, PDI, kinase, etc.) are two major determinants in regulating cell survival or cell death signaling pathways. An initial link between TG2 and apoptosis was proposed by Fesus et al. (1987) based on their observation that leadinduced hypertrophy of the liver in rats was associated with an increased expression of TG2. Since then, many reports have supported the involvement of TG2 in apoptosis (Piacentini et al., 2005 and the references therein). In particular, cells undergoing apoptosis show increased expression of TG2 and its inhibition by means of an anti-sense approach results in a decreased propensity of cells to die (Oliverio et al., 1999). Despite the evidence supporting the involvement of TG2 in apoptosis its exact physiologic significance remains elusive. We do know that the cross-linking function of TG2, which requires millimolar concentrations of calcium, is intimately involved in its pro-apoptotic functions. In the presence of high calcium concentrations, TG2 cross-links cellular proteins and induces their polymerization and the formation of detergent-insoluble structures. The protein scaffolds thus formed stabilize the structure of the dying cell before its clearance by phagocytosis, thereby preventing the release of intracellular components and a subsequent inflammatory response. It is likely that TG2 remains inactive under normal conditions; however, under extreme stressful conditions such as hypoxia, growth factor deprivation as well as treatment with chemotherapeutic drugs, the calcium homeostasis in the cell is perturbed and latent TG2 is converted to active crosslinking configuration, resulting in extensive cross-linking of cellular proteins. Interestingly, on the basis of studies performed in TG2−/− mice, it was recently suggested that TG2
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is required for the phagocytosis of apoptotic bodies (Szondy et al., 2003). This function of TG2 probably depends on its ability to activate TGF-, a cytokine that plays an essential role in phagocytosis. Interestingly, the expression of TG2 and the occurrence of apoptosis do not completely overlap, as directly evidenced by TG2−/− mice that do not display any phenotype that could be ascribed to perturbed apoptosis (De Laurenzi and Melino, 2001; Nanda et al., 2001). However, the possibility that another member of the family of transglutaminases compensates for the loss of TG2 in these mice cannot be ruled out. Moreover, several actively dividing cancer cell types that express resistance to apoptosis, are known to express high basal levels of TG2. More recent studies have provided direct evidence that the increased expression of TG2 can prolong cell survival by preventing apoptosis (Antonyak et al., 2003; Boehm et al., 2002; Verma et al., 2006). Eventually it became apparent that the activation of the pro-apoptotic or anti-apoptotic functions of TG2 depended upon its location within the cell (Milakovic et al., 2004). The available data suggest that a cytosolic fraction of TG2 is involved in inducing alterations that lead to apoptosis, whereas, when in the nucleus, TG2 can interact with and result in the transamidation of pRb which protects cells from apoptotic insults (Milakovic et al., 2004). In addition, in cell membranes, TG2 can associate with integrins and provide a binding site for fibronectin by simultaneously binding to  integrins and fibronectin through a gelatin-binding domain (42-kDa fragment, module I6 II1,2 ,I7–9 ) (Akimov et al., 2000).
3. TG2, cell survival and drug resistance Recently, the expression of TG2 has been found to be selectively elevated in cancer cells that exhibit resistance to anticancer drugs or have been isolated from metastatic sites (Mehta, 1994; Mehta et al., 2004, 2006; Fok et al., 2006). Irrespective of the source and type, cancer cells selected for resistance against chemotherapeutic drugs expressed very high levels of TG2 when compared with the parental drugsensitive cell line from which they were derived (Chen et al., 2004; Herman et al., 2006; Verma et al., 2006; Mangala et al., 2007). The mechanistic significance of the TG2 expression is discussed below. 3.1. Interaction of TG2 with integrins Depending on the cell type, 10–20% of 1-integrins on the cell surface can exist as a complex with TG2 in a 1:1 ratio (Akimov et al., 2000; Akimov and Belkin, 2001). Recently, we identified other members of -integrins (3, 4- and 5-) that could form stable complex with TG2 in cancer cell membranes (Herman et al., 2006; Mangala et al., 2007). Integrins are a family of cell-surface proteins that serve as receptors for ECM proteins (fibronectin, vitronectin, laminin, collagen, etc.) and can influence several
aspects of cancer cell behavior, including motility, invasion, growth, and survival in response to their binding to ECM ligands. In normal cells the interaction of integrins with the ECM is a highly regulated phenomenon and ascertains controlled growth, survival and migration of normal cells. Alternatively, certain proteins can associate with integrins and enhance their affinity for the ECM ligands. First such interaction was identified between integrin 3 and integrinassociated protein (IAP), also referred to as CD47 (Brown and Frazier, 2001). Depending on the nature of interacting protein, these interactions can alter the binding ability and signaling functions of integrins. In view of this, our observation that TG2 is associated with -integrins (Mangala et al., 2007; Herman et al., 2006; Fok et al., 2006) may have important implications in terms of deregulated proliferation, survival and migratory functions of cancer cells. The association of TG2 with integrins is known to occur primarily at extracellular domains of integrins and promotes their interaction with the ECM ligands such as, fibronectin, collagen, and vitronectin (Akimov et al., 2001). Importantly, the interaction between TG2 and integrin is independent of the cross-linking activity of TG2 and results in increased cell adhesion, migration, and activation of the downstream survival signaling pathways (Akimov et al., 2001; Herman et al., 2006). 3.2. Role of TG2 in FAK/PI3K/Akt pathway activation TG2 regulates focal adhesion kinase (FAK), a key mediator in cell-survival signaling pathways. Thus, TG2 expression in cancer cells induced constitutive activation of FAK and its downstream PI3K/Akt pathway (Verma et al., 2006). We demonstrated that TG2 expression augmented the autophosphorylation of FAK (Y397), the first step in FAK activation pathway. Although, the precise mechanism by which TG2 affects the autophosphorylation of FAK is not yet clear, the observation that TG2 physically associates with FAK suggests that such interaction may induce a conformational change in the FAK protein and lead to its autophosphorylation. It is well established that the autophosphorylation of FAK is followed by the recruitment of Src kinase that phosphorylates other tyrosine sites on FAK (Y407 , Y576 , Y577 , Y861 , and Y925 ) leading to the activation of several downstream signaling pathways such as, RAS/ERK, PI3K/Akt, and Crk/Dock180/Rac (McLean et al., 2005). Among various pathways regulated by FAK, TG2-induced activation of FAK leads to the activation of the PI3K/Akt pathway. Adding further to the complexity of the role of TG2 in PI3K/Akt pathway regulation, we recently observed a novel function of TG2 in regulating PTEN expression. TG2 inversely regulates the expression of PTEN. PTEN is known for its tumor suppressive functions such as inhibition of cell growth, invasion, migration and focal adhesions (Tamura et al., 1998), primarily by dephosphorylation and inactivation of signaling proteins, such as Akt and FAK. Gene deletion and mutation of PTEN are observed frequently in various
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human cancers. These genetic changes, however, are not necessarily the only way to inactivate PTEN during tumorigenesis but some subtle changes induced by posttranslational modifications can also affect the stability and degradation of PTEN, which have profound effect on the tumorigenesis. Indeed, a recent report demonstrated that the oncogenic protein NEDD4-1 acts like E3 ubiquitin ligase for PTEN and promotes its degradation via ubiquitin-proteasomal pathway (Wang et al., 2007). Very little is known about the regulation and stability of PTEN, except that phosphorylation can affect the stability of PTEN in certain cell types (Vazquez et al., 2000, 2001; Okahara et al., 2004). There are reports suggesting that phosphorylation of PTEN by casein kinase 2 (CK2) is important for the stability of PTEN protein in the face of proteasome-mediated degradation. In separate study, it was shown that phosphorylation of the PTEN tail could negatively regulate PTEN stability (Vazquez et al., 2000). Phosphorylation of PTEN by CK2 prevents its interaction with MAGI-2 and its consequent recruitment into a high molecular weight complex (PTEN-associated complex). In line with these reports, our observations suggest that TG2 expression could affect the stability of PTEN protein by preventing its phosphorylation at ser380 (Vazquez et al., 2000; Torres and Pulido, 2001) and promoting the degradation. TG2 inhibits the phosphorylation of PTEN at site serine 380 and results in increased degradation of PTEN by ubiquitin/proteasome pathway. Thus, TG2 has two complementary ways of promoting the activation of FAK/PI3K/Akt pathway, one by direct phosphorylation of FAK and the second by inhibiting the phosphatase activity of PTEN resulting in constitutively activate PI3K/Akt pathway. 3.3. Role of TG2 in NF-κB activation The transcription factor NF-B plays an important role in regulating genes that are involved in controlling cell growth, apoptosis, and metastatic functions (Baeuerle and Baltimore, 1996; Bours et al., 2000; Karin et al., 2002). Constitutive activation of NF-B has been observed in various cancers and its role in drug resistance and metastasis has been envisaged (Bours et al., 1994; Bargou et al., 1997; Mori et al., 1999; Pahl, 1999; Mukhopadhyay et al., 2001). TG2 expression contributes to constitutive activation of NF-B in a classical IKK-independent manner. The expression of TG2 in various cancer cell types is associated with constitutive activation of NF-B (Mann et al., 2006). Forced expression of TG2 in low TG2-expressing BxPC-3 as well as in high endogenous TG2-expressing Panc28 pancreatic cancer cells, showed high NF-B activity that was significantly inhibited by TG2 enzyme inhibitors but not by dominantnegative IB␣. Conversely, knockdown of endogenous TG2 expression by siRNA in Panc28 cells resulted in strong inhibition of NF-B. Interestingly, in situ activation of the TG2 transamidating activity by calcium ionophore A23187 treatment, caused strong activation of NF-B, while inhibition of the activity by enzyme-specific inhibitors such as, 5-
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biotinamido pentylamine (BPA) and monodansylcadaverine (MDC), attenuated NF-B activation. Thus, TG2 expression and its cross-linking function are critical for NF-B activation. Indeed, IkB␣ has been shown to be a good substrate for TG2-catalyzed cross-linking reaction. Thus, in vitro incubation of IkB␣ protein with purified TG2 and calcium could effectively catalyze the cross-linking of IkB␣ into high molecular weight polymers (Lee et al., 2004). Importantly, the polymeric form of IB␣ had much lower binding affinity for p65:p50 complex (Lee et al., 2004) suggesting that TG2-mediated posttranslational modification of IkB␣ may hamper its ability to associate with p65:p50 complex resulting in constitutively active NF-B. In a recent report, Kim et al. (2006) observed that inhibition of TG2 activity attenuated NF-B activation and reversed the sensitivity of drug-resistant breast cancer cells to doxorubicin. Based on these observations, these authors concluded that TG2 expression and its cross-linking function are critical for NFB activation and conferring drug resistance phenotype on cancer cells. We observed a similar reversal in drug resistance in drug-resistant MCF-7 cells in response to the down regulation of endogenous TG2 by siRNA (Herman et al., 2006). TG2 can directly associate with p65:p50 complex in the cytoplasm and with p65 in the nucleus. The significance of this association is still not clear. We speculate that TG2 association with p65:p50 complex could mitigate the binding of IB␣ to NF-B complex in the cytoplasm, contributing to NF-B constitutive activation. Under certain conditions, TG2 can also serve as a kinase and its serine–threonine kinase activity can phosphorylate histones and p53 (Mishra et al., 2006). Because p65 undergoes phosphorylation by various kinases at Ser536 site (Bohuslav et al., 2004; Douillette et al., 2006), it is likely that p65 serves as substrate for TG2 kinase activity. In addition, it is also possible that TG2 being a bulky protein (80 kDa), when complexed with p65 could cause stearic hindrance/conformational changes and permit preferential binding of NF-B (p50:p65) complex only to high affinity/selective promoters resulting in differential transcriptional regulation and expression of target proteins that are involved in drug resistance and metastasis. Currently, we are investigating these and other possibilities in our laboratory to determine the potential of TG2 as a target for inhibiting constitutive activation of NF-B and promoting sensitivity of cancer cells to chemotherapeutic drugs. 3.4. TG2 promotes cell survival and chemoresistance TG2 expression can regulate autophagy; the type II programmed cell death. Autophagy is a highly regulated form of programmed cell death that plays a crucial role in various physiologic processes such as, development, homeostasis, elimination of unwanted cells and carcinogenesis. Cancer cells exhibit defective autophagic cell death compared to their normal counterparts (Edinger and Thompson, 2003), which
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Fig. 1. TG2 down regulation inhibits pancreatic tumor growth in vivo. (A) Growth curve of Panc28/cl.10 (TG2-shRNA stably transfected Panc28 clone) and Panc28/empty vector xenografts, growing subcutaneously in nude mice over 5 weeks. (B) The tumor volume of Panc28/cl.10 and Panc28/empty vector-induced xenografts after 6 weeks of implantation. Inset, representative tumor-bearing mice after 6 weeks (upper panel) of tumor implantation and the size of resected tumors 6 weeks post-implantation (lower panel).
may contribute to chemoresistance. Autophagic cell death is caspase-independent and is characterized by autophagosomes formation that surrounds various cellular organelles such as Golgi complexes, polyribosomes, and endoplasmic reticulum (Kondo et al., 2005). Subsequently, autophago-
somes merge with lysosomes and digest the organelles, leading to cell death (Kroemer and Jaattela, 2005). A recent observation made by our group showed that inhibition of TG2 expression by siRNA resulted in induction of autophagy in pancreatic cancer cells and eventual cell death (Akar
Fig. 2. Schematic representation of TG2 in activation of cell survival signaling FAK and its downstream PI3K/AKT and NF-B pathways. Association of TG2 with integrins (e.g., 1, 3, 4, 5) can increase their avidity for extracelluar matrix proteins, such as fibronectin (FN) and laminin and result in the activation of downstream cell survival pathways. TG2 can also affect FAK phosphorylation directly by associating with FAK molecule or indirectly by promoting PTEN degradation via ubiquitin-proteasomal pathway. Either collectively or individually, activation of these signaling molecules in the PI3K/Akt axis leads to its constitutive activation. Thus, TG2-mediated activation of Akt results in phosphorylation of various downstream substrates and activation of MDM2, NF-B and inhibition of FKHR and BAD resulting in increased cell survival and chemoresistance. On the other hand, TG2 by cross-linking IB␣ (inhibitor of kappa B alpha) can induce dissociation of NF-B/IB␣ complex resulting in constitutive activation NF-B in non-classical IKK independent manner.
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et al., 2007). Notably inhibition of PKC␦ activity by rottlerin or its downregulation by PKC␦ siRNA was associated with a parallel decrease in TG2 expression and induction of autophagy in pancreatic cancer cells. These observations suggest that PKC␦ is an upstream regulator of TG2 expression and therefore targeting PKC␦, TG2 or both, may represent an effective approach to treat pancreatic cancer patients. Understanding the significance of TG2 in cancer cells led us to initiate an in vivo study in a nude mouse model (unpublished data). Our experiments revealed that knocking-down TG2 in human pancreatic tumor cell line (Panc28) cause significant decrease in tumor growth and tumor volume as shown in Fig. 1A and B. Further experiments to investigate the possibility of reversing gemcitabine resistance by targeting TG2 in pancreatic cancer cells have shown very promising results. We used the liposome-based RNA interference (TG2 siRNA)-mediated gene silencing therapeutic approach to silence TG2 in orthotopic human pancreatic tumors. Established orthotopic Panc28 tumors were treated with either gemcitabine (i.p.) alone or in combination with liposomal TG2 siRNA (i.v.). The tumor growth and metastasis in the combination group, compared with the groups treated with gemcitabine (i.p.), liposomal TG2 siRNA (i.v.) and liposomal control siRNA (i.v.), alone were markedly reduced. Combination of gemcitabine and TG2 siRNA as compared to other treatment groups resulted in a synergistic effect in inhibiting the tumor growth and metastasis in the nude mice (unpublished data). These initial results suggest that TG2 is a good target and RNA interference mediated gene silencing of TG2 could be a promising therapeutic approach for improved therapy outcome in advanced stages of pancreatic cancer.
4. Conclusions In conclusion, the observations of elevated TG2 expression in drug-resistant cancer cells have raised great interest in understanding its contributions to the development of drug resistance. TG2 expression results in a constitutive activation of FAK/PI3K/AKT and NF-B cell-survival signaling pathways as summarized in Fig. 2. Either collectively or individually, these pathways confer an apoptosis-resistant phenotype and inhibit autophagy in cancer cells, thereby enabling tumor cells not only to display resistance against many anticancer drugs but also to proliferate and survive successfully in stressful environments in distant tissues (metastasis). Therefore, TG2 may be an attractive target for overcoming drug resistance in cancer cells.
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Tissue transglutaminase-mediated chemoresistance in cancer cells Amit Verma, Kapil Mehta ∗ Department of Experimental Therapeutics, Unit 362, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, United States Received 16 May 2007; received in revised form 8 June 2007; accepted 11 June 2007
Abstract Drug resistance and metastasis are major impediments for the successful treatment of cancer. A common feature among drug resistant and metastatic tumor cells is that they exhibit profound resistance to apoptosis. This property enables cancer cells not only to grow and survive in stressful environments (metastasis) but also to display resistance against many anticancer agents. Therefore, perturbation of the intrinsic apoptotic pathways of cancer cells will affect their ability to respond to chemotherapy and to metastasize and survive in distant sites. Recent studies have demonstrated that cancer cells and cancer cell lines selected for resistance against chemotherapeutic drugs or isolated from metastatic sites, express elevated levels of the multifunctional protein, tissue transglutaminase (TG2). TG2 is the most diverse and ubiquitous member of the transglutaminase family of proteins that is implicated to play a role in apoptosis, wound healing, cell migration, cell attachment, cell growth, angiogenesis, and matrix assembly. TG2 can associate with certain  members of the integrin family of proteins (1, 3, 4, and 5) and promote stable interaction between cells and the extracellular matrix (ECM), resulting in increased cell survival, cell migration, and invasion. Additionally, TG2 forms a ternary complex with IB/p65:p50 and results in constitutive activation of the nuclear transcription factor-B (NF-B). Moreover, TG2 expression in cancer cells leads to constitutive activation of the focal adhesion kinase (FAK) and its downstream PI3K/Akt survival pathway. Importantly, the inhibition of endogenous TG2 by small interfering RNA (siRNA) resulted in the reversal of drug resistance and the invasive phenotype. Conversely, ectopic expression of TG2 promoted cell survival, cell motility and invasive functions of cancer cells. This review discusses the current thinking and implications of increased TG2 expression in development of drug resistance and metastasis by cancer cells. © 2007 Elsevier Ltd. All rights reserved. Keywords: Invasion; Metastasis; Drug resistance; Focal adhesion kinase; -Integrins; NF-B; AKT; PTEN; Autophagy; Apoptosis
1. Introduction The ability of cancer cells to develop resistance to drugs poses a major clinical challenge in the successful treatment of cancer. “Virtually all of the roughly half-million annual cancer deaths in the United States can be said to have occurred because of chemotherapy failed” (Goldstein et al., 1989). Though it is generally accepted that the majority of cancers arise from a single precursor cell, a tumor is a not a collection of genetically identical cells. Genetic instability and accumulation of mutations are important hallmarks of cancer cells. This means that dividing cancer cells are able to acquire genetic and epigenetic changes that will favor their malignant phenotype as well as drug resistance (Richardson and Kaye, ∗
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2005). In addition to the acquired genetic and epigenetic changes, host factors like micro-tumor environment, tumor vascularity, drug bioavailability, metabolism, elimination and dose-dependent toxicity are some of the limitations, which play a role in the final outcome of conventional chemotherapy. For example the tumor vascularity may affect transit time of drugs within tumors and the interaction of tumor cell with the surrounding interstitial cells (Green et al., 1999). Also vascularity may dictate the tumor penetration of high molecular weight monoclonal antibodies and immunotoxins (Pluen et al., 2001). The classic approach to try to understand the molecular mechanisms of resistance to anticancer drugs has been to select cell lines that exhibit high level of drug resistance and then identify genes that might account for the resistance. As a result of this strategy mostly mechanisms that are able to confer relatively high levels of resistance have been identified,
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including decreased transporter-mediated uptake of watersoluble drugs, various mutations/alterations in genes involved in cell cycle, DNA repair mechanism and apoptosis; changes in metabolizing enzymes, drug target genes and/or drug efflux transporters, like P-glycoprotein, MRPs and BCRP (Assaraf, 2006; Szak´acs et al., 2006). The realization that most anticancer drugs might kill tumor cells by activating apoptotic pathways raised the notion that deregulation of apoptotic pathways may be a key determinant in the development of chemoresistance in tumor cells (Mashima and Tsuruo, 2005). Indeed, numerous alterations that confer resistance to apoptosis in cancer cells have been identified. For example, activation of pro-survival signal transduction pathways such as, those mediated by Ras, PI3K/Akt, or NF-B; inactivation of apoptotic pathways, e.g., due to mutation or silencing of p53, pRb, Bax, Bad, Apaf1, caspase-8 genes or overexpression of pro-survival proteins such as, Bcl-2, IAP, and FLIP are frequently observed in advanced cancers (Fesik, 2005). As a logical consequence many approaches to attempt improved cancer treatment involve combinations of cytotoxic agents with new molecular targeted agents. The rationale for such combination cancer therapies is based on the assumption that agents affecting molecular signaling pathways involved in apoptosis or other cell death mechanisms (Chen et al., 2006), migration and invasion (Sierra, 2005), angiogenesis (Broxterman and Georgopapadakou, 2005; Morabito et al., 2006), tumor cell signal transduction (Bloom and Kloog, 2005; Adjei, 2006), and protein turnover (Richardson et al., 2003; Nawrocki et al., 2005; Demarchi and Brancolini, 2005; Horton et al., 2006) will lead to synergistic tumor cell death. Detailed discussion on the mechanisms involved in drug resistance and approaches to overcome this problem are beyond the scope of this chapter but have been covered in the literature (Goldman, 2003; Liang et al., 2001; Smukste et al., 2006). Though all of these approaches seem logical and promising, however the understanding of cellular drug resistance behavior does not necessarily translate to understanding completely the resistance observed in clinical tumors, because tumors consist of a mixed population of cells which may not exhibit very high levels of drug resistance. Therefore, there is still a considerable interest to define novel resistance mechanisms as potential targets for novel compounds with greater effectiveness and for optimal drug combinations. This review will focus on recent data and understanding of the significance of increased tissue transglutaminase (TG2) expression in drug resistant (intrinsic or acquired) cancer cells as a potential novel anticancer drug target.
2. TG2 and apoptosis TG2, also referred to as cytosolic or type II transglutaminase, is the most diverse member of the transglutaminase family of enzymes (Chen and Mehta, 1999; Fesus and
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Piacentini, 2002; Griffin et al., 2002; Mehta, 2005). In addition to catalyzing calcium-dependent posttranslational modification of proteins, TG2 can bind and hydrolyze guanosine triphosphate (GTP) with an affinity similar to those of the ␣ subunits of large heterodimeric G proteins and small Ras-type G proteins (Stephens et al., 2004). The calcium-dependent protein cross-linking reactions of TG2 are implicated in cell differentiation, receptor-mediated endocytosis, cell adhesion, and induction of apoptosis. In addition, TG2/Gh is involved in coupling the ␣1 b- and ␣1 d-adrenoreceptors, thromboxane, and oxytocin receptors to phospholipase C, thereby mediating inositol phosphate production in response to the agonist-induced activation. In addition, TG2 can function as a protein disulfide isomerase, ATPase and as a protein kinase (Mishra and Murphy, 2006; Lorand and Graham, 2003; Hasegawa et al., 2003; Mastroberardino et al., 2006), the major component of insulin-like growth factor-binding protein-3 kinase (Mishra and Murphy, 2004). Although, predominantly a cytosolic protein, TG2 can be secreted outside the cell (by yet unknown mechanism), where it stabilizes the ECM by making the matrix resistant to mechanical and proteolytic degradation (Aeschlimann and Thomazy, 2000). Similarly, in association with other proteins, TG2 can translocate to the nucleus and cell membranes. Its localization (membrane, cytosol, and nuclear) and the functional configuration (cross-linking, GTP-binding, PDI, kinase, etc.) are two major determinants in regulating cell survival or cell death signaling pathways. An initial link between TG2 and apoptosis was proposed by Fesus et al. (1987) based on their observation that leadinduced hypertrophy of the liver in rats was associated with an increased expression of TG2. Since then, many reports have supported the involvement of TG2 in apoptosis (Piacentini et al., 2005 and the references therein). In particular, cells undergoing apoptosis show increased expression of TG2 and its inhibition by means of an anti-sense approach results in a decreased propensity of cells to die (Oliverio et al., 1999). Despite the evidence supporting the involvement of TG2 in apoptosis its exact physiologic significance remains elusive. We do know that the cross-linking function of TG2, which requires millimolar concentrations of calcium, is intimately involved in its pro-apoptotic functions. In the presence of high calcium concentrations, TG2 cross-links cellular proteins and induces their polymerization and the formation of detergent-insoluble structures. The protein scaffolds thus formed stabilize the structure of the dying cell before its clearance by phagocytosis, thereby preventing the release of intracellular components and a subsequent inflammatory response. It is likely that TG2 remains inactive under normal conditions; however, under extreme stressful conditions such as hypoxia, growth factor deprivation as well as treatment with chemotherapeutic drugs, the calcium homeostasis in the cell is perturbed and latent TG2 is converted to active crosslinking configuration, resulting in extensive cross-linking of cellular proteins. Interestingly, on the basis of studies performed in TG2−/− mice, it was recently suggested that TG2
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is required for the phagocytosis of apoptotic bodies (Szondy et al., 2003). This function of TG2 probably depends on its ability to activate TGF-, a cytokine that plays an essential role in phagocytosis. Interestingly, the expression of TG2 and the occurrence of apoptosis do not completely overlap, as directly evidenced by TG2−/− mice that do not display any phenotype that could be ascribed to perturbed apoptosis (De Laurenzi and Melino, 2001; Nanda et al., 2001). However, the possibility that another member of the family of transglutaminases compensates for the loss of TG2 in these mice cannot be ruled out. Moreover, several actively dividing cancer cell types that express resistance to apoptosis, are known to express high basal levels of TG2. More recent studies have provided direct evidence that the increased expression of TG2 can prolong cell survival by preventing apoptosis (Antonyak et al., 2003; Boehm et al., 2002; Verma et al., 2006). Eventually it became apparent that the activation of the pro-apoptotic or anti-apoptotic functions of TG2 depended upon its location within the cell (Milakovic et al., 2004). The available data suggest that a cytosolic fraction of TG2 is involved in inducing alterations that lead to apoptosis, whereas, when in the nucleus, TG2 can interact with and result in the transamidation of pRb which protects cells from apoptotic insults (Milakovic et al., 2004). In addition, in cell membranes, TG2 can associate with integrins and provide a binding site for fibronectin by simultaneously binding to  integrins and fibronectin through a gelatin-binding domain (42-kDa fragment, module I6 II1,2 ,I7–9 ) (Akimov et al., 2000).
3. TG2, cell survival and drug resistance Recently, the expression of TG2 has been found to be selectively elevated in cancer cells that exhibit resistance to anticancer drugs or have been isolated from metastatic sites (Mehta, 1994; Mehta et al., 2004, 2006; Fok et al., 2006). Irrespective of the source and type, cancer cells selected for resistance against chemotherapeutic drugs expressed very high levels of TG2 when compared with the parental drugsensitive cell line from which they were derived (Chen et al., 2004; Herman et al., 2006; Verma et al., 2006; Mangala et al., 2007). The mechanistic significance of the TG2 expression is discussed below. 3.1. Interaction of TG2 with integrins Depending on the cell type, 10–20% of 1-integrins on the cell surface can exist as a complex with TG2 in a 1:1 ratio (Akimov et al., 2000; Akimov and Belkin, 2001). Recently, we identified other members of -integrins (3, 4- and 5-) that could form stable complex with TG2 in cancer cell membranes (Herman et al., 2006; Mangala et al., 2007). Integrins are a family of cell-surface proteins that serve as receptors for ECM proteins (fibronectin, vitronectin, laminin, collagen, etc.) and can influence several
aspects of cancer cell behavior, including motility, invasion, growth, and survival in response to their binding to ECM ligands. In normal cells the interaction of integrins with the ECM is a highly regulated phenomenon and ascertains controlled growth, survival and migration of normal cells. Alternatively, certain proteins can associate with integrins and enhance their affinity for the ECM ligands. First such interaction was identified between integrin 3 and integrinassociated protein (IAP), also referred to as CD47 (Brown and Frazier, 2001). Depending on the nature of interacting protein, these interactions can alter the binding ability and signaling functions of integrins. In view of this, our observation that TG2 is associated with -integrins (Mangala et al., 2007; Herman et al., 2006; Fok et al., 2006) may have important implications in terms of deregulated proliferation, survival and migratory functions of cancer cells. The association of TG2 with integrins is known to occur primarily at extracellular domains of integrins and promotes their interaction with the ECM ligands such as, fibronectin, collagen, and vitronectin (Akimov et al., 2001). Importantly, the interaction between TG2 and integrin is independent of the cross-linking activity of TG2 and results in increased cell adhesion, migration, and activation of the downstream survival signaling pathways (Akimov et al., 2001; Herman et al., 2006). 3.2. Role of TG2 in FAK/PI3K/Akt pathway activation TG2 regulates focal adhesion kinase (FAK), a key mediator in cell-survival signaling pathways. Thus, TG2 expression in cancer cells induced constitutive activation of FAK and its downstream PI3K/Akt pathway (Verma et al., 2006). We demonstrated that TG2 expression augmented the autophosphorylation of FAK (Y397), the first step in FAK activation pathway. Although, the precise mechanism by which TG2 affects the autophosphorylation of FAK is not yet clear, the observation that TG2 physically associates with FAK suggests that such interaction may induce a conformational change in the FAK protein and lead to its autophosphorylation. It is well established that the autophosphorylation of FAK is followed by the recruitment of Src kinase that phosphorylates other tyrosine sites on FAK (Y407 , Y576 , Y577 , Y861 , and Y925 ) leading to the activation of several downstream signaling pathways such as, RAS/ERK, PI3K/Akt, and Crk/Dock180/Rac (McLean et al., 2005). Among various pathways regulated by FAK, TG2-induced activation of FAK leads to the activation of the PI3K/Akt pathway. Adding further to the complexity of the role of TG2 in PI3K/Akt pathway regulation, we recently observed a novel function of TG2 in regulating PTEN expression. TG2 inversely regulates the expression of PTEN. PTEN is known for its tumor suppressive functions such as inhibition of cell growth, invasion, migration and focal adhesions (Tamura et al., 1998), primarily by dephosphorylation and inactivation of signaling proteins, such as Akt and FAK. Gene deletion and mutation of PTEN are observed frequently in various
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human cancers. These genetic changes, however, are not necessarily the only way to inactivate PTEN during tumorigenesis but some subtle changes induced by posttranslational modifications can also affect the stability and degradation of PTEN, which have profound effect on the tumorigenesis. Indeed, a recent report demonstrated that the oncogenic protein NEDD4-1 acts like E3 ubiquitin ligase for PTEN and promotes its degradation via ubiquitin-proteasomal pathway (Wang et al., 2007). Very little is known about the regulation and stability of PTEN, except that phosphorylation can affect the stability of PTEN in certain cell types (Vazquez et al., 2000, 2001; Okahara et al., 2004). There are reports suggesting that phosphorylation of PTEN by casein kinase 2 (CK2) is important for the stability of PTEN protein in the face of proteasome-mediated degradation. In separate study, it was shown that phosphorylation of the PTEN tail could negatively regulate PTEN stability (Vazquez et al., 2000). Phosphorylation of PTEN by CK2 prevents its interaction with MAGI-2 and its consequent recruitment into a high molecular weight complex (PTEN-associated complex). In line with these reports, our observations suggest that TG2 expression could affect the stability of PTEN protein by preventing its phosphorylation at ser380 (Vazquez et al., 2000; Torres and Pulido, 2001) and promoting the degradation. TG2 inhibits the phosphorylation of PTEN at site serine 380 and results in increased degradation of PTEN by ubiquitin/proteasome pathway. Thus, TG2 has two complementary ways of promoting the activation of FAK/PI3K/Akt pathway, one by direct phosphorylation of FAK and the second by inhibiting the phosphatase activity of PTEN resulting in constitutively activate PI3K/Akt pathway. 3.3. Role of TG2 in NF-κB activation The transcription factor NF-B plays an important role in regulating genes that are involved in controlling cell growth, apoptosis, and metastatic functions (Baeuerle and Baltimore, 1996; Bours et al., 2000; Karin et al., 2002). Constitutive activation of NF-B has been observed in various cancers and its role in drug resistance and metastasis has been envisaged (Bours et al., 1994; Bargou et al., 1997; Mori et al., 1999; Pahl, 1999; Mukhopadhyay et al., 2001). TG2 expression contributes to constitutive activation of NF-B in a classical IKK-independent manner. The expression of TG2 in various cancer cell types is associated with constitutive activation of NF-B (Mann et al., 2006). Forced expression of TG2 in low TG2-expressing BxPC-3 as well as in high endogenous TG2-expressing Panc28 pancreatic cancer cells, showed high NF-B activity that was significantly inhibited by TG2 enzyme inhibitors but not by dominantnegative IB␣. Conversely, knockdown of endogenous TG2 expression by siRNA in Panc28 cells resulted in strong inhibition of NF-B. Interestingly, in situ activation of the TG2 transamidating activity by calcium ionophore A23187 treatment, caused strong activation of NF-B, while inhibition of the activity by enzyme-specific inhibitors such as, 5-
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biotinamido pentylamine (BPA) and monodansylcadaverine (MDC), attenuated NF-B activation. Thus, TG2 expression and its cross-linking function are critical for NF-B activation. Indeed, IkB␣ has been shown to be a good substrate for TG2-catalyzed cross-linking reaction. Thus, in vitro incubation of IkB␣ protein with purified TG2 and calcium could effectively catalyze the cross-linking of IkB␣ into high molecular weight polymers (Lee et al., 2004). Importantly, the polymeric form of IB␣ had much lower binding affinity for p65:p50 complex (Lee et al., 2004) suggesting that TG2-mediated posttranslational modification of IkB␣ may hamper its ability to associate with p65:p50 complex resulting in constitutively active NF-B. In a recent report, Kim et al. (2006) observed that inhibition of TG2 activity attenuated NF-B activation and reversed the sensitivity of drug-resistant breast cancer cells to doxorubicin. Based on these observations, these authors concluded that TG2 expression and its cross-linking function are critical for NFB activation and conferring drug resistance phenotype on cancer cells. We observed a similar reversal in drug resistance in drug-resistant MCF-7 cells in response to the down regulation of endogenous TG2 by siRNA (Herman et al., 2006). TG2 can directly associate with p65:p50 complex in the cytoplasm and with p65 in the nucleus. The significance of this association is still not clear. We speculate that TG2 association with p65:p50 complex could mitigate the binding of IB␣ to NF-B complex in the cytoplasm, contributing to NF-B constitutive activation. Under certain conditions, TG2 can also serve as a kinase and its serine–threonine kinase activity can phosphorylate histones and p53 (Mishra et al., 2006). Because p65 undergoes phosphorylation by various kinases at Ser536 site (Bohuslav et al., 2004; Douillette et al., 2006), it is likely that p65 serves as substrate for TG2 kinase activity. In addition, it is also possible that TG2 being a bulky protein (80 kDa), when complexed with p65 could cause stearic hindrance/conformational changes and permit preferential binding of NF-B (p50:p65) complex only to high affinity/selective promoters resulting in differential transcriptional regulation and expression of target proteins that are involved in drug resistance and metastasis. Currently, we are investigating these and other possibilities in our laboratory to determine the potential of TG2 as a target for inhibiting constitutive activation of NF-B and promoting sensitivity of cancer cells to chemotherapeutic drugs. 3.4. TG2 promotes cell survival and chemoresistance TG2 expression can regulate autophagy; the type II programmed cell death. Autophagy is a highly regulated form of programmed cell death that plays a crucial role in various physiologic processes such as, development, homeostasis, elimination of unwanted cells and carcinogenesis. Cancer cells exhibit defective autophagic cell death compared to their normal counterparts (Edinger and Thompson, 2003), which
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Fig. 1. TG2 down regulation inhibits pancreatic tumor growth in vivo. (A) Growth curve of Panc28/cl.10 (TG2-shRNA stably transfected Panc28 clone) and Panc28/empty vector xenografts, growing subcutaneously in nude mice over 5 weeks. (B) The tumor volume of Panc28/cl.10 and Panc28/empty vector-induced xenografts after 6 weeks of implantation. Inset, representative tumor-bearing mice after 6 weeks (upper panel) of tumor implantation and the size of resected tumors 6 weeks post-implantation (lower panel).
may contribute to chemoresistance. Autophagic cell death is caspase-independent and is characterized by autophagosomes formation that surrounds various cellular organelles such as Golgi complexes, polyribosomes, and endoplasmic reticulum (Kondo et al., 2005). Subsequently, autophago-
somes merge with lysosomes and digest the organelles, leading to cell death (Kroemer and Jaattela, 2005). A recent observation made by our group showed that inhibition of TG2 expression by siRNA resulted in induction of autophagy in pancreatic cancer cells and eventual cell death (Akar
Fig. 2. Schematic representation of TG2 in activation of cell survival signaling FAK and its downstream PI3K/AKT and NF-B pathways. Association of TG2 with integrins (e.g., 1, 3, 4, 5) can increase their avidity for extracelluar matrix proteins, such as fibronectin (FN) and laminin and result in the activation of downstream cell survival pathways. TG2 can also affect FAK phosphorylation directly by associating with FAK molecule or indirectly by promoting PTEN degradation via ubiquitin-proteasomal pathway. Either collectively or individually, activation of these signaling molecules in the PI3K/Akt axis leads to its constitutive activation. Thus, TG2-mediated activation of Akt results in phosphorylation of various downstream substrates and activation of MDM2, NF-B and inhibition of FKHR and BAD resulting in increased cell survival and chemoresistance. On the other hand, TG2 by cross-linking IB␣ (inhibitor of kappa B alpha) can induce dissociation of NF-B/IB␣ complex resulting in constitutive activation NF-B in non-classical IKK independent manner.
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et al., 2007). Notably inhibition of PKC␦ activity by rottlerin or its downregulation by PKC␦ siRNA was associated with a parallel decrease in TG2 expression and induction of autophagy in pancreatic cancer cells. These observations suggest that PKC␦ is an upstream regulator of TG2 expression and therefore targeting PKC␦, TG2 or both, may represent an effective approach to treat pancreatic cancer patients. Understanding the significance of TG2 in cancer cells led us to initiate an in vivo study in a nude mouse model (unpublished data). Our experiments revealed that knocking-down TG2 in human pancreatic tumor cell line (Panc28) cause significant decrease in tumor growth and tumor volume as shown in Fig. 1A and B. Further experiments to investigate the possibility of reversing gemcitabine resistance by targeting TG2 in pancreatic cancer cells have shown very promising results. We used the liposome-based RNA interference (TG2 siRNA)-mediated gene silencing therapeutic approach to silence TG2 in orthotopic human pancreatic tumors. Established orthotopic Panc28 tumors were treated with either gemcitabine (i.p.) alone or in combination with liposomal TG2 siRNA (i.v.). The tumor growth and metastasis in the combination group, compared with the groups treated with gemcitabine (i.p.), liposomal TG2 siRNA (i.v.) and liposomal control siRNA (i.v.), alone were markedly reduced. Combination of gemcitabine and TG2 siRNA as compared to other treatment groups resulted in a synergistic effect in inhibiting the tumor growth and metastasis in the nude mice (unpublished data). These initial results suggest that TG2 is a good target and RNA interference mediated gene silencing of TG2 could be a promising therapeutic approach for improved therapy outcome in advanced stages of pancreatic cancer.
4. Conclusions In conclusion, the observations of elevated TG2 expression in drug-resistant cancer cells have raised great interest in understanding its contributions to the development of drug resistance. TG2 expression results in a constitutive activation of FAK/PI3K/AKT and NF-B cell-survival signaling pathways as summarized in Fig. 2. Either collectively or individually, these pathways confer an apoptosis-resistant phenotype and inhibit autophagy in cancer cells, thereby enabling tumor cells not only to display resistance against many anticancer drugs but also to proliferate and survive successfully in stressful environments in distant tissues (metastasis). Therefore, TG2 may be an attractive target for overcoming drug resistance in cancer cells.
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