International Journal of

Gastrointestinal Cancer Volume 33 • Number 1 • 2003 • ISSN 0169–4197

James L. Abbruzzese, MD, FACP Editor-in-Chief

ar m ch an ,R a ea Jo d, ur an na d D ls.c ow o nl m oa d

Special Issue Pancreatic Cancer: Bench to Bedside, edited by Douglas B. Evans, MD

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Humana Press IJGC 33/1 cvr (NEW)

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7/10/03, 12:22 PM

International Journal of Gastrointestinal Cancer, vol. 33, no. 1, 15–26, 2003 © Copyright 2003 by Humana Press Inc. All rights of any nature whatsoever reserved. 0169-4197/03/33:15–26/$25.00

Review Article

NF-κB in Pancreatic Cancer Guido M. Sclabas, Shuichi Fujioka, Christian Schmidt, Douglas B. Evans, and Paul J. Chiao* Departments of Surgical Oncology and Molecular Oncology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030

Abstract Although the genetic profile of pancreatic cancer is emerging as a result of much research, the role of specific genetic alterations that initiate tumorigenesis and produce its cardinal clinical features of locally aggressive growth, metastasis, and chemotherapy resistance remains unresolved. Recently, a number of studies have shown that the inhibition of constitutive NF-κB activation, one of the frequent molecular alterations in pancreatic cancer, inhibits tumorigenesis and metastasis. It also sensitizes pancreatic cancer cell lines to anticancer agent-induced apoptosis. Therefore because of the crucial role of NF-κB in pancreatic cancer, it is a potential target for developing novel therapeutic strategies for the disease. In vivo and in vitro models that mimic the tumorigenic phenotypes in the appropriate histological and molecular concert would be very useful for confirming the suspected role of the pancreatic cancer signature genetic lesions and better understanding the molecular basis of this disease. Key Words: NF-κB; IκBα; pancreatic cancer; tumorigenesis; metastasis; apoptosis; angiogenesis.

Introduction

(Ink4a/Arf) is at an intermediate stage in 71% of ductal lesions; inactivation of p53 is identified in 50 to 75% of pancreatic cancers; inactivation of Smad4/DPC4 is found in 50% of the cancers, and BRCA2 mutation occurs relatively late and in much lower frequency (4–9). NF-κB activity is found constitutively activated in about 70% of pancreatic cancers (10). These studies show that pancreatic cancer presents a quite consistent set of genetic alterations in contrast to most other adult cancers (11). The combination of cDNA microarray and cDNAbased CGH analyses can expand the list of signature mutations for pancreatic cancer. However, the role this genetic lesion profile plays in pancreatic cancer induction and metastasis is still unclear. This review focuses on the possible role of NF-κB, one of the recently identified molecular alterations in

A key point that has emerged from the analysis of mutations present in human pancreatic adenocarcinoma is that the cancer a unique profile of genetic and molecular alterations that distinguishes it from all other cancers (1,2). Genetically, pancreatic cancer is one of the better-characterized neoplasms (1,3). For example, it is known that HER-2/neu is overexpressed in about 90% of duct lesions and in 70% of invasive pancreatic adenocarcinoma; that point mutations in the K-ras gene exist in 45% of duct lesions and about 80–95% of pancreatic adenocarcinomas; inactivation of the p16 *Author to whom all correspondence and reprint requests should be addressed. E-mail: [email protected]

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16 pancreatic cancer, in the initiation or progression of pancreatic adenocarcinoma.

Oncogenic Activity of NF-κB NF-κB is a family of pleiotropic transcription factors that orchestrate the expression of a plethora of genes that play key roles in growth, oncogenesis, differentiation, apoptosis, tumorigenesis, and immune and inflammatory responses (12–14). Five members of mammalian NF-κB are described: NF-κB1 (p50 and its precursor p105), NF-κB2 (p52 and its precursor p100), c-Rel, RelA (p65), and RelB (15–18), each of which has a 300 residue-long Rel homology domain (RHD) (13,19–23). The C-terminal domains are responsible for dimerization with other Rel proteins, but sequence-specific interactions come primarily from loops in the N-terminal domain (24). Interaction of c-Rel, RelA (p65) and RelB with its inhibitors, referred to as IκB, results in inactive complexes in the cytoplasm by masking the nuclear localization signal, which is located at the C terminal end of the Rel homology domain (13,19–23). Currently, the inhibitor proteins IκBα, IκBβ, IκBγ, IκBε, Bcl-3, and the Drosophila protein Cactus are described and characterized (13,19–23). In most cell types, NF-κB proteins are sequestered in the cytoplasm in an inactive form through their noncovalent association with the inhibitor IκB (16). This association masks the nuclear localization signal of NF-κB, thereby preventing NF-κB nuclear translocation and DNA binding activity (25). NF-κB is activated through complex signaling cascades that are integrated by activation of IκB kinase complex (IKK) (26–30), which phosphorylates IκB bound to NF-κB complexes as its substrates (31). Consequently, NF-κB proteins are translocated into the nucleus, where they activate transcription of their target genes (13,20). One of the key target genes regulated by NF-κB is its inhibitor IκBα. A feedback inhibition pathway for control of IκBα gene transcription and downregulation of transient activation of NF-κB activity is described (32–34). The c-rel member of the Rel/NF-κB family of pleiotropic transcription factors was first identified as a cellular homolog of the v-rel oncogene from a highly oncogenic retrovirus (35). The v-Rel oncoprotein induces aggressive leukemias and lym-

International Journal of Gastrointestinal Cancer

Sclabas et al. phomas in chickens and transgenic mice and is able to transform avian lymphoid cells and fibroblasts, indicating the possibility that other members of Rel/NF-κB are oncogenes (36–38). Many reports demonstrated that members of the NF-κB and IκB families are involved in the development of cancer. Chromosomal amplification, overexpression and recurrent genomic rearrangement in the genes encoding c-Rel, Bcl-3, p105 (p50), and p100 (p52) are identified in many human hematopoietic cancers and several types of solid tumor, such as human non-small cell lung carcinomas (NSCLC) (39), squamous carcinomas of head and neck, and in adenocarcinomas of breast and stomach (40,41), thyroid carcinoma cell lines (42), colon, prostate, breast, bone, and brain cancer cells (43). Constitutive NF-κB activation has been found in many human hematopoietic malignancies and several types of solid tumors such as pancreas and breast cancers, as a result of mutations activating continuously upstream signaling kinases or inactivating inhibitory IκB proteins. For example, NF-κB constitutive activation was initially reported in Hodgkin’s disease as a direct consequence of mutations in the IκBα gene, which generate nonfunctional inhibitory proteins (44–46). Constitutive NF-κB activity is also detected in 93% of childhood acute lymphoblastic leukemia (C-ALL) (47). These results suggest a crucial role for NF-κB in leukemia cell survival. Constitutive activation of NF-κB is also emerging as a characteristic of various types of solid tumors, including breast (48–51), ovarian (43,48), colon (48), pancreatic (10), bladder (50), and prostate carcinomas (52–54), as well as in melanomas (55). In light of constitutive NF-κB activation found in approx 70% of pancreatic adenocarcinomas and the recent progress in elucidating its role in this disease, constitutive NF-κB activation in pancreatic cancer is reviewed in greater detail below.

Constitutive Activation of NF-κB in Pancreatic Cancer Our initial study demonstrated that NF-κB is constitutively activated in 67% (16 of 24) of human pancreatic adenocarcinoma, but not in normal pancreatic tissue, and in 69% (11of 16) of pancreatic adeno-

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NF-κB in Pancreatic Cancer carcinoma specimens in our recent follow-up study. NF-κB is also constitutively activated in 9 of 11 human pancreatic tumor cell lines, but not in immortalized nontumorigenic pancreatic cell lines. The constitutive NF-κB activity in some of the human pancreatic cancer cell lines can be further enhanced by TNF-α stimulation. Our data also showed that IκBα, a previously identified NF-κB-inducible gene, was overexpressed in human pancreatic tumor tissues and cell lines (10). NF-κB activation is inhibited by dominant negative mutants of IκBα, raf, and MAP kinase in the pancreatic tumor cell lines (10). These data are consistent with the possibility that NF-κB is activated in pancreatic tumor cells by the upstream signaling pathway involving Ras and MAP kinases. Recently, a number of reports provided supporting evidence for the constitutive NF-κB activity in various pancreatic cancer cell lines (56). Suppression of NF-κB activation correlated with inhibition of IL-6 and IL-8 secretion (57,58). Constitutive production of IL-8 and NF-κB activity were inhibited by curcumin treatment in the SUIT-2 human pancreatic cancer cell line (57). In the CAPAN-2 cell line, antioxidants inhibited both NF-κB activation and IL-6 and IL-8 secretion, whereas antioxidants generally failed to suppress both NF-κB activation and IL-6 and IL-8 secretion in CAPAN-1 cell line (58). Treatment with various NF-κB inhibitors such as Gliotoxin, MG132, and Sulfasalazine, or transfection with the IκBα super-repressor, strongly enhanced the apoptotic effects of VP16 or doxorubicin on resistant CAPAN-1 and 818-4 pancreatic cancer cells, indicating that the resistance of pancreatic carcinoma cells to chemotherapy is rather owing to their constitutive NF-κB activity than the transient induction of NF-κB by some anticancer drugs (59). These results suggest that blockade of NF-κB activity by well-established inhibitors efficiently reduces chemoresistance of pancreatic cancer cells and offers the potential for improved therapeutic strategies.

The Role of Constitutive NF-κB Activation Despite the identification of these specific genetic and molecular alterations, it was not possible to eluInternational Journal of Gastrointestinal Cancer

17 cidate their roles in pancreatic tumorigenesis and metastasis so far. The studies of this disease have unfortunately been hampered by a number of unique challenges. For instance, at the time of diagnosis, pancreatic cancer is usually at an advanced stage and has often metastasized. As a result, the stepwise tumorigenic progression has been inaccessible for study and the precursor cell types still remain an area of ongoing studies. Animal models that carry specific genetic alterations and resemble human pancreatic cancer and immortalized and nontumorigenic pancreatic cancer cell lines are lacking. To determine the role of constitutive NF-κB activity in pancreatic cancer and to specifically inhibit NF-κB activity, we constructed a retroviral vector that expressed a Flag-tagged IκBαM and infected several pancreatic cancer cell lines. The expression of Flag-tagged IκBαM suppressed the constitutive NF-κB activity completely in Panc-1 and AsPc-1 cells. Inhibiting constitutive NF-κB activity by IκBαM expression suppresses the formation of liver metastasis in the metastatic pancreatic tumor cell line, AsPc-1, and tumorigenesis in the non-metastatic pancreatic tumor cell line PANC-1, using an orthotopic nude mouse model (60,61). These findings further suggest that NF-κB plays a critical role in the development of pancreatic cancer. It still remains unclear why inhibition of NF-κB activation in AsPc-1 cells only suppressed metastasis but not tumor formation in pancreas, while inhibition of constitutive NF-κB activation in PANC-1 cells suppressed tumorigenesis. Several reports show that the PANC-1 cell line is nonmetastatic in the orthotopic nude mouse model (62–64). PANC-1 cells express wild-type Smad4 and a functional TGF-β signaling pathway, unlike other human pancreatic tumor cell lines (65). Reconstitution of Smad4 expression in a Smad4-null Hs667t human pancreatic cancer cell line reduced tumor formation by inhibiting angiogenesis (66). It is possible that Smad4 may reduce the expression of angiogenic factors and results in small, slow growing, and lower vascularized PANC-1 tumors with undetectable metastatic potential in the pancreas of nude mice. However, it remains unknown whether a functional TGF-β pathway reduces the metastatic potential of PANC-1 cells. It is also possible that constitutive NF-κB activity is required for tumorigenesis at the early stage of pancreatic cancer development. Oncogenic ras Volume 33, 2003

18 initiated p53-independent apoptotic response was inhibited through activation of NF-κB (19). This study suggests that NF-κB is required for ras mediated oncogenesis and ras-transformed cells are susceptible to apoptosis even in the absence of a functional p53 tumor suppressor gene. The transcription factors Stat3 and NF-κB induce increased Bcl-xL expression in the premalignant lesions and tumor cells from the EL-TGF-α and p53 null compound mutant mice. Functional analysis shows that blocking of both Stat3 and NF-κB together induces programmed cell death in murine pancreatic tumor cells. These findings indicate that apoptosis resistance precedes formation of invasive pancreatic cancer. Similarly, the activation of NF-κB appears to be an early event that occurs prior to the malignant transformation of human mammary epithelial cells in vitro and in rats treated with carcinogen in vivo (67). These findings support a model in which NF-κB may be involved early in the progression of breast epithelial cells towards malignancy. Another report showed that NF-κB activation correlates with the conversion of breast cancer cells to hormoneindependent growth, a characteristic of more aggressive and metastatic tumors (49). Abnormalities in apoptotic pathways may contribute to a variety of diseases, including cancer, autoimmunity, and degenerative disorders (68–72). Indeed, massive apoptosis of liver cells in embryos lacking relA suggests a role of NF-κB complexes in protecting cells from pro-apoptotic stimuli (73,74) and is supported by anti-apoptotic functions of NF-κB after TNF-α stimulation (75). Furthermore, the anti-apoptotic function of the v-Rel oncoprotein is essential for its oncogenic activity (76–78). Therefore, constitutive NF-κB activity plays an essential role in the initiation or progression of malignant transformation of pancreatic ductal epithelial cells by promoting tumor cell survival towards malignancy.

The NF-κB Downstream Target Genes Involved in Pancreatic Cancer NF-κB induces the expression of numerous genes encoding growth factors, cytokines, apoptotic and cell cycle regulators, reviewed in (19,21,79) (Fig. 1). The expression of the apoptosis inhibitors that are regulated by NF-κB (80) include c-IAP1, c-IAP2, Traf1, Traf2, A20, IEX-1L and the Bcl-2International Journal of Gastrointestinal Cancer

Sclabas et al.

Fig. 1. NF-κB inducers and downstream target genes. Many factors can induce NF-κB. These include cytokines (i.e., IL-1, TNF-α), growth factors (i.e., EGF), proapoptotic stimuli (i.e., Doxycycline, Taxol, UV irradiation), and hydrogen peroxide. Once NF-κB is active, it induces downstream target genes that encode IκBα, cytokines (TNF-α, IL-1), antiapoptotic proteins, such as Bcl-xL (a member of the Bcl-2 family), proteins involved in metastasis (i.e., urokinase-like plasminogen activator [uPA] and matrix metalloproteinases [i.e., MMP9]), and proteins involved in cell-cycle arrest (i.e., p21Cip1).

homologs Bfl-1/A1 and Bcl-x (81–89). A number of reports show that NF-κB is a direct activator of bcl-xL expression (90–93) and suggest that apoptotic inhibitors such as Bcl-xL play a key role in NF-κB mediated antiapoptotic signaling cascades in pancreas cancers (10,94–100). However, the role of most of these factors in tumors, that display deregulated NF-κB activity, remains to be elucidated. In certain cell types, the constitutive NF-κB activity activates the expression of genes important for invasion and metastasis and is associated with advanced stages of oncogenesis, supporting its role in tumor progression. These include angiogenic factors like vascular endothelial growth factor (VEGF). VEGF is one of the most important and potent inducers and mediators of angiogenesis (101). A hallmark of cancer (102) bigger than 1 mm3 is angiogenesis (103,104) and involvement of NF-κB is under debate (105,106). The exact mechanisms underlying NFκB function in angiogenesis remain unclear. Recently published data may illustrate the complex signaling pathways mediated by EGFR (107–109) and an Volume 33, 2003

NF-κB in Pancreatic Cancer involvement of NF-κB in VEGF regulation (110–114). However, how angiogenesis is controlled and which factors or pathways are involved needs to be further elucidated. Moreover, constitutive NF-κB activity also activates the expression of genes encoding proteolytic enzymes such as matrix metalloproteinases, urokinase plasminogen activator (uPA) and cell adhesion molecules such as ICAM-1 (79), which are important for invasion and metastasis. uPA is significantly increased in most breast cancer cell lines that contain constitutively active NF-κB, is required for intravasation, and is associated with poor prognosis (115). This supports a role for NF-κB in metastasis. The function of NF-κB signaling in tumorigenesis and angiogenesis has been illustrated in AsPc-1 and PANC-1 human pancreatic cancer cell lines. IκBαM-mediated inhibition of constitutive NF-κB activity substantially inhibited the expression of key antiapoptotic genes bcl-xL and bcl-2, and of major proangiogenic molecules VEGF and IL-8, suggesting that the antiapoptotic potential of the tumor cells and neoplastic angiogenesis may be decreased. Our results suggest that constitutive NF-κB activity, found in 70% of pancreatic cancers, plays an important role in pancreatic tumorigenesis. Our study also implies that constitutive NF-κB activity induces overexpression of its downstream target genes such as bcl-xL, bcl-2, VEGF, and IL-8, which may mediate its cardinal features of locally aggressive growth and resistance to therapeutically induced apoptosis.

The Signaling Cascades for Activating NF-κB in Pancreatic Cancer NF-κB is activated by many distinct stimuli, including pro-inflammatory cytokines, such as TNF-α and IL-1 (13,14). Potent activators, like tumor necrosis factor-α (TNF-α) or interleukin 1 (IL-1) induce rapid degradation of IκB within minutes, exposing the nuclear localization sequence of NF-κB, which results in translocation to the nucleus (13,19–23). Pro-inflammatory cytokine-induced NF-κB activation is biphasic, consisting of a rapid and transient phase mediated through IκBα followed by a delayed and persistent phase mediated through IκBβ (116). Recent studies have shown that biphasic NF-κB actiInternational Journal of Gastrointestinal Cancer

19 vation is induced in response to LPS stimulation in an animal model and in various cell types, and is important for the host defense, underlying the proand antiinflammatory function of NO (117–119). Phosphorylation of IκBα on serine-residues thirty-two and thirty-six is the critical step in activation of NF-κB from NF-κB: IκBα complexes. The responsible kinase was named IκB kinase (IKK) and contains three polypeptides: IKK1, IKK2, and NF-κB Essential Modulator (NEMO). Deletion studies revealed that IKK1 is dispensable, whereas IKK2 and NEMO are necessary for pro-inflammatory cytokine-mediated activation of NF-κB (120–126). Genetic studies revealed that NEMO is the essential modulatory element of the IKK complex (123, 124,127). Several genetic analyses show that the two related kinases IKK1 and IKK2 have distinct roles and cannot be substituted for each other’s function (120–122). The IKK2 subunit, but not IKK itself, serves as the target for phosphorylation-dependent activation by other, as-yet-unidentified protein kinases involved in pro-inflammatory signaling (22,128). Although several members of the MAP3K family, such as MEKK1, MEKK2, MEKK3, transforming growth factor-β-activating kinase 1 (TAK1), and NIK can activate IKK and NF-κB when overexpressed (129–133), recent genetic studies revealed that MEKK1 and NIK are not involved in NF-κB activation induced by most stimuli, including TNF-α (134,135). Consistent with the report that mutations in dtak1 reveal a conserved function for MAPKK in NF-κB regulation (136), biochemical analyses showed that TAK1 may function as ubiquitindependent kinase of IKK (137). The signaling cascades that regulate IKK activity may be also cell type dependent (138). One genetic study revealed that PKCξ is not required for IKK activation in embryonic fibroblasts, but PKCξ is required for IKK activation in lungs (139). Another report showed that MEKK3 is necessary in early TNF-α-induced NF-κB activation (140). However, the underlying molecular mechanisms are unknown. Other pathways for NF-κB activation are phosphorylation of tyrosine residue forty-two of IκBα, followed by dissociation from NF-κB, or degradation of IκBα, induced by ultraviolet light, independent of its phosphorylation (13,19–23). Another activation pathway for NF-κB has been discovered. It is described that IKK1 is necessary Volume 33, 2003

20 for NF-κB2 p100 processing (141,142). After removing its IκB like C-terminus, the active N-terminal half, NF-κB2 (p52), function as transcription factor. Claudio et al. reported that NF-κB2–/– mice have defects in B-cell development, similarly to BAFF –/– (B-cell activating factor) mice (143) and demonstrated that BAFF-induced processing of NF-κB2 p100 is dependent on BAFF-R, NF-κB inducing kinase (NIK)(134), but independent of NEMO. This report demonstrates that NF-κB2 p100 processing is dependent on IKK1. Another line of evidence comes from Dejardin et al. Using LTβR–/– (lymphotoxin β receptor) mice, they describe IKK1-dependent LTβR-induced genes (144). Many human tumors frequently contain mutations in ras genes. NF-κB is a critical downstream mediator of cell transformation mediated by oncogenic ras (145,146). In this context, the transcriptional activity of RelA is significantly increased in Rastransformed cells as compared to non-transformed cells (145). It is thus reasonable to expect that activation of the Ras pathway in human tumors may yet be another means to induce constitutive NF-κB activity and oncogenesis. In agreement with this hypothesis, RelA has been observed to be constitutively activated in 67% of pancreatic adenocarcinomas and the human K-ras oncogene to be frequently mutated in pancreatic cancer (10). Epidermal growth factor receptor (EGFR) is overexpressed in 30% to 50% of human pancreatic tumors (147–149) and simultaneous overexpression of EGFR and its principal ligands, EGF and TGF-α, is associated with enhanced tumor growth and metastasis (150,151). Several studies have shown that EGFR can activate NF-κB in various human cancer cell lines (152). There is also evidence that transactivation of EGFR by TNF-α leads to NF-κB activation (153) and that a high level of EGFR expression is optimal for EGF-induced NF-κB activation (152). However, the signals generated by EGFR that lead to NF-κB activation remain unknown (154–157). The cumulative data suggest that the overexpression of EGFR in human pancreatic adenocarcinoma cell lines may be critical to the mediation of constitutive NF-κB activity and resistance to apoptosis. Pancreatic cancer, as many other malignant tumors, exhibit autocrine- or paracrine-stimulated growth (158). Besides EGFR also cytokines are reported to play an important role. Elevated basal NF-κB activity involving an IL-1β mediated autocrine mechaInternational Journal of Gastrointestinal Cancer

Sclabas et al. nism is described in the two chemoresistant pancreatic cancer cell lines A818-4 and PancTu-1. In these cells, blockade of NF-κB disrupts the IL-1β mediated amplification loop and the accompanying chemoresistance, giving further evidence that in pancreatic cancer cells, NF-κB is important in mediating cell signals responsible for chemoresistance (159). The data further suggest that IL-1β may play an important role in constitutive NF-κB activity in these cells. Similar findings are also reported in other cell types. IL-1 as an important autocrine growth factor in raf-induced transformation is described in NIH3T3 cells (160). The importance of cytokines in NF-κB activation is shown in a study using small interfering RNAs against IKK1, IKK2 and the upstream regulatory kinase TAK1. In this study both IKK1 and IKK2 are important for cytokine-induced NF-κB activation, and MAP3K TAK1 is found to be critical for TNF-α-induced NF-κB activation (161). Another study reports the importance of MAPK signaling cascades in NIK-induced constitutive activation of NF-κB in melanoma cells (162).

Conclusion Of the many unanswered questions concerning the roles of various signature genetic alterations in pancreatic cancer, this review focused on the potentially important function of NF-κB in pancreatic cancer. A recent National Cancer Institute Pancreatic Cancer Progress Review Group identified the crucial questions and challenges facing pancreatic cancer research and called for insightful study of pancreatic cancer biology. The identification of signature genetic and molecular alterations in pancreatic adenocarcinoma was an important first step in this direction providing conceptual framework to guide the future research in this disease. The development of pancreatic cancer-relevant in vitro and in vivo models that induce tumorigenic transformation, pancreatic cancer, and metastasis on the appropriate histological and molecular background would be very useful to confirm the suspected role of the pancreatic cancer signature genetic and molecular alterations (Fig. 2). The elucidation of the roles of these genetic alterations in the induction and progression of pancreatic cancer and the delineation of complexities of intersecting signaling cascades that control cellular growth, apoptosis, and differentiation will provide a better understanding of the molecuVolume 33, 2003

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Fig. 2. Involvement of NF-κB in cellular transformation. Oncogenes such as Her2/neu and p21K-Ras and environmental carcinogens can activate NF-κB. NF-κB positively influences signaling pathways involving the tumor suppressor protein p53, anti-apoptotic members of the Bcl-2 family (Bcl-xL), and factors involved in metastasis (IL-8 and VEGF). In contrast, NF-κB negatively influences TGF-β signal transduction at the level of the SMAD4 signaling intermediates. Independent of these functions, NF-κB may also be involved in cell-cycle control via ERK/p16Ink4a. Little is known about the connection between NF-κB, environmental carcinogens, and DNA repair genes.

lar basis of this disease and serve as a guide to design new early detection and therapeutic strategies.

Acknowledgments We thank Betty Notzon and Pat Thomas for editorial assistance.

References 1. Hruban RH, Wilentz RE, Kern SE. Genetic progression in the pancreatic ducts. Am J Pathol 2000;156:1821–1825. 2. Hruban RH, Goggins M, Parsons J, Kern SE. Progression model for pancreatic cancer. Clin Cancer Res 2000;6: 2969–2972. 3. Hruban RH, Iacobuzio-Donahue C, Wilentz RE, Goggins M, Kern SE. Molecular pathology of pancreatic cancer. Cancer J 2001;7:251–258. 4. Almoguera C, Shibata D, Forrester K, Martin J, Arnheim N, Perucho M. Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell 1988;53: 549–554. 5. Hall PA, Hughes CM, Staddon SL, Richman PI, Gullick WJ, Lemoine NR. The c-erbB-2 proto-oncogene in human pancreatic cancer. J Pathol 1990;161:195–200. 6. Schutte M, Hruban RH, Geradts J, et al. Abrogation of the Rb/p16 tumor-suppressive pathway in virtually all pancreatic carcinomas. Cancer Res 1997;57:3126–3130. International Journal of Gastrointestinal Cancer

7. Caldas C, Hahn SA, da Costa LT, et al. Frequent somatic mutations and homozygous deletions of the p16 (MTS1) gene in pancreatic adenocarcinoma. Nat Genet 1994;8:27–32. 8. Barton CM, Staddon SL, Hughes CM, et al. Abnormalities of the p53 tumour suppressor gene in human pancreatic cancer. Br J Cancer 1991;64:1076–1082. 9. Wilentz RE, Su GH, Dai JL, et al. Immunohistochemical labeling for dpc4 mirrors genetic status in pancreatic adenocarcinomas:a new marker of DPC4 inactivation. Am J Pathol 2000;156:37–43. 10. Wang W, Abbruzzese JL, Evans DB, Larry L, Cleary KR, Chiao PJ. The nuclear factor-κB RelA transcription factor is constitutively activated in human pancreatic adenocarcinoma cells. Clin Cancer Res 1999;5:119–127. 11. Jaffee EM, Hruban RH, Canto M, Kern SE. Focus on pancreas cancer. Cancer Cell 2002;2:25–28. 12. Baeuerle PA, Baltimore D. IκB: a specific inhibitor of the NF-κB transcription factor. Science 1988;242:540–546. 13. Verma IM, Stevenson JK, Schwarz EM, Van Antwerp D, Miyamoto S. Rel/NF-κB/IκB family: intimate tales of association and dissociation. Genes Dev 1995;9:2723–2735. 14. Baeuerle PA, Baltimore D. NF-κB: ten years after. Cell 1996;87:13–20. 15. Sen R, Baltimore D. Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell 1986;46: 705–716. 16. Sen R, Baltimore D. Inducibility of κ immunoglobulin enhancer-binding protein NF-κB by a posttranslational mechanism. Cell 1986;47:921–928. Volume 33, 2003

22 17. Lenardo MJ, Baltimore D. NF-κB: a pleiotropic mediator of inducible and tissue-specific gene control. Cell 1989; 58:227–229. 18. Lenardo MJ, Fan CM, Maniatis T, Baltimore D. The involvement of NF-κB in β-interferon gene regulation reveals its role as widely inducible mediator of signal transduction. Cell 1989;57:287–294. 19. Siebenlist U, Franzoso G, Brown K. Structure, regulation and function of NF-κB. Annu Rev Cell Biol 1994;10:405–455. 20. Baldwin AS, Jr. The NF-κB and IκB proteins: new discoveries and insights. Annu Rev Immunol 1996;14:649–683. 21. Ghosh S, May MJ, Kopp EB. NF-κB and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol 1998;16:225–260. 22. Karin M, Ben-Neriah Y. Phosphorylation meets ubiquitination: the control of NF-κB activity. Annu Rev Immunol 2000;18:621–663. 23. Ghosh S, Karin M. Missing pieces in the NF-κB puzzle. Cell 2002;109 Suppl:S81–96. 24. Jacobs MD, Harrison SC. Structure of an IκBα/NF-κB complex. Cell 1998;95:749–758. 25. Baeuerle PA, Baltimore D. A 65-kD subunit of active NF-κB is required for inhibition of NF-κB by IκB. Genes Dev 1989;3:1689–1698. 26. Brown K, Gerstberger S, Carlson L, Franzoso G, Siebenlist U. Control of IκBα proteolysis by site-specific, signal-induced phosphorylation. Science 1995;267: 1485–1488. 27. Chen ZJ, Parent L, Maniatis T. Site-specific phosphorylation of IκBα by a novel ubiquitination-dependent protein kinase activity. Cell 1996;84:853–862. 28. Regnier CH, Song HY, Gao X, Goeddel DV, Cao Z, Rothe M. Identification and characterization of an IκB kinase. Cell 1997;90:373–383. 29. Zandi E, Rothwarf DM, Delhase M, Hayakawa M, Karin M. The IκB kinase complex (IKK) contains two kinase subunits, IKKα and IKKβ, necessary for IκB phosphorylation and NF-κB activation. Cell 1997;91:243–252. 30. DiDonato JA, Hayakawa M, Rothwarf DM, Zandi E, Karin M. A cytokine-responsive IκB kinase that activates the transcription factor NF-κB. Nature 1997;388:548–554. 31. Zandi E, Chen Y, Karin M. Direct phosphorylation of IκB by IKKα and IKKβ: discrimination between free and NF-κB-bound substrate. Science 1998;281:1360–1363. 32. Chiao PJ, Miyamoto S, Verma IM. Autoregulation of IκBα activity. Proc Natl Acad Sci U S A 1994;91:28–32. 33. Sun SC, Ganchi PA, Ballard DW, Greene WC. NF-κB controls expression of inhibitor IκBα: evidence for an inducible autoregulatory pathway. Science 1993;259:1912–1915. 34. Rice NR, Ernst MK. In vivo control of NF-κB activation by IκBα. EMBO J 1993;12:4685–4695. 35. Gilmore TD. The Rel/NF-κB signal transduction pathway: introduction. Oncogene 1999;18:6842–6844. 36. Sylla BS, Temin HM. Activation of oncogenicity of the c-rel proto-oncogene. Mol Cell Biol 1986;6:4709–4716. 37. Moore BE, Bose HR, Jr. Transformation of avian lymphoid cells by reticuloendotheliosis virus. Mutat Res 1988; 195:79–90.

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Sclabas et al. 38. Carrasco D, Cheng J, Lewin A, et al. Multiple hemopoietic defects and lymphoid hyperplasia in mice lacking the transcriptional activation domain of the c-Rel protein. J Exp Med 1998;187:973–984. 39. Mukhopadhyay T, Roth JA, Maxwell SA. Altered expression of the p50 subunit of the NF-κB transcription factor complex in non-small cell lung carcinoma. Oncogene 1995; 11:999–1003. 40. Mathew S, Murty VV, Dalla-Favera R, Chaganti RS. Chromosomal localization of genes encoding the transcription factors, c-rel, NF-κBp50, NF-κBp65, and lyt-10 by fluorescence in situ hybridization. Oncogene 1993;8: 191–193. 41. Motokura T, Arnold A. PRAD1/cyclin D1 protooncogene: genomic organization, 5′ DNA sequence, and sequence of a tumor-specific rearrangement breakpoint. Genes Chromosomes Cancer 1993;7:89–95. 42. Visconti R, Cerutti J, Battista S, et al. Expression of the neoplastic phenotype by human thyroid carcinoma cell lines requires NF-κB p65 protein expression. Oncogene 1997;15:1987–1994. 43. Bours V, Dejardin E, Goujon-Letawe F, Merville MP, Castronovo V. The NF-κB transcription factor and cancer: high expression of NF-κB- and IκB-related proteins in tumor cell lines. Biochem Pharmacol 1994;47:145–149. 44. Cabannes E, Khan G, Aillet F, Jarrett RF, Hay RT. Mutations in the IκBα gene in Hodgkin’s disease suggest a tumour suppressor role for IκBα. Oncogene 1999;18:3063–3070. 45. Krappmann D, Emmerich F, Kordes U, Scharschmidt E, Dorken B, Scheidereit C. Molecular mechanisms of constitutive NF-κB/Rel activation in Hodgkin/Reed-Sternberg cells. Oncogene 1999;18:943–953. 46. Wood KM, Roff M, Hay RT. Defective IκBα in Hodgkin cell lines with constitutively active NF-κB. Oncogene 1998;16:2131–2139. 47. Kordes U, Krappmann D, Heissmeyer V, Ludwig WD, Scheidereit C. Transcription factor NF-κB is constitutively activated in acute lymphoblastic leukemia cells. Leukemia 2000;14:399–402. 48. Dejardin E, Deregowski V, Chapelier M, et al. Regulation of NF-κB activity by IκB-related proteins in adenocarcinoma cells. Oncogene 1999;18:2567–2577. 49. Nakshatri H, Bhat-Nakshatri P, Martin DA, Goulet RJ, Jr., Sledge GW, Jr. Constitutive activation of NF-κB during progression of breast cancer to hormone-independent growth. Mol Cell Biol 1997;17:3629–3639. 50. Sumitomo M, Tachibana M, Ozu C, et al. Induction of apoptosis of cytokine-producing bladder cancer cells by adenovirus-mediated IκBα overexpression. Hum Gene Ther 1999;10:37–47. 51. Sovak MA, Bellas RE, Kim DW, et al. Aberrant nuclear factor-κB/Rel expression and the pathogenesis of breast cancer. J Clin Invest 1997;100:2952–2960. 52. Herrmann JL, Beham AW, Sarkiss M, et al. Bcl-2 suppresses apoptosis resulting from disruption of the NF-κB survival pathway. Exp Cell Res 1997;237:101–109. 53. Sumitomo M, Tachibana M, Nakashima J, et al. An essential role for nuclear factor κ B in preventing TNF-α-induced

Volume 33, 2003

NF-κB in Pancreatic Cancer

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

cell death in prostate cancer cells. J Urol 1999;161: 674–679. Suh J, Payvandi F, Edelstein LC, et al. Mechanisms of constitutive NF-κB activation in human prostate cancer cells. Prostate 2002;52:183–200. Devalaraja MN, Wang DZ, Ballard DW, Richmond A. Elevated constitutive IκB kinase activity and IκB-α phosphorylation in Hs294T melanoma cells lead to increased basal MGSA/GRO-α transcription. Cancer Res 1999; 59:1372–1377. Nawrocki ST, Bruns CJ, Harbison MT, et al. Effects of the proteasome inhibitor PS-341 on apoptosis and angiogenesis in orthotopic human pancreatic tumor xenografts. Mol Cancer Ther 2002;1:1243–1253. Hidaka H, Ishiko T, Furuhashi T, et al. Curcumin inhibits interleukin 8 production and enhances interleukin 8 receptor expression on the cell surface:impact on human pancreatic carcinoma cell growth by autocrine regulation. Cancer 2002;95:1206–1214. Blanchard JA, 2nd, Barve S, Joshi-Barve S, Talwalker R, Gates LK, Jr. Antioxidants inhibit cytokine production and suppress NF-κB activation in CAPAN-1 and CAPAN-2 cell lines. Dig Dis Sci 2001;46:2768–2772. Arlt A, Vorndamm J, Breitenbroich M, et al. Inhibition of NF-κB sensitizes human pancreatic carcinoma cells to apoptosis induced by etoposide (VP16) or doxorubicin. Oncogene 2001;20:859–868. Fujioka S, Sclabas GM, Schmidt C, et al. Function of nuclear factor κB in pancreatic cancer metastasis. Clin Cancer Res 2003;9:346–354. Fujioka S, Sclabas GM, Schmidt C, et al. Inhibition of constitutive NF-κB activity by IκBαM suppresses tumorigenesis. Oncogene 2003;22:1365–1370. Teraoka H, Sawada T, Nishihara T, et al. Enhanced VEGF production and decreased immunogenicity induced by TGF-β1 promote liver metastasis of pancreatic cancer. Br J Cancer 2001;85:612–617. Mohammad RM, Adsay NV, Philip PA, Pettit GR, Vaitkevicius VK, Sarkar FH. Bryostatin 1 induces differentiation and potentiates the antitumor effect of Auristatin PE in a human pancreatic tumor (PANC-1) xenograft model. Anticancer Drugs 2001;12:735–740. Sawai H, Yamamoto M, Okada Y, et al. Alteration of integrins by interleukin-1α in human pancreatic cancer cells. Pancreas 2001;23:399–405. Grau AM, Zhang L, Wang W, et al. Induction of p21waf1 expression and growth inhibition by transforming growth factor β involve the tumor suppressor gene DPC4 in human pancreatic adenocarcinoma cells. Cancer Res 1997; 57:3929–3934. Schwarte-Waldhoff I, Volpert OV, Bouck NP, et al. Smad4/DPC4-mediated tumor suppression through suppression of angiogenesis. Proc Natl Acad Sci U S A 2000; 97:9624–9629. Kim DW, Sovak MA, Zanieski G, et al. Activation of NF-κB/Rel occurs early during neoplastic transformation of mammary cells. Carcinogenesis 2000;21: 871–879.

International Journal of Gastrointestinal Cancer

23 68. Strasser A, O′Connor L, Dixit VM. Apoptosis signaling. Annu Rev Biochem 2000;69:217–245. 69. Wang X. The expanding role of mitochondria in apoptosis. Genes Dev 2001;15:2922–2933. 70. Martin SJ. Destabilizing influences in apoptosis: sowing the seeds of IAP destruction. Cell 2002;109:793–796. 71. Rathmell JC, Thompson CB. Pathways of apoptosis in lymphocyte development, homeostasis, and disease. Cell 2002;109Suppl:S97–107. 72. Johnstone RW, Ruefli AA, Lowe SW. Apoptosis: a link between cancer genetics and chemotherapy. Cell 2002; 108:153–164. 73. Beg AA, Baltimore D. An essential role for NF-κB in preventing TNF-α-induced cell death. Science 1996;274: 782–784. 74. Beg AA, Sha WC, Bronson RT, Ghosh S, Baltimore D. Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-κB. Nature 1995;376: 167–170. 75. Van Antwerp DJ, Martin SJ, Kafri T, Green DR, Verma IM. Suppression of TNF-α-induced apoptosis by NF-κB. Science 1996;274:787–789. 76. Chen C, Agnes F, Gelinas C. Mapping of a serine-rich domain essential for the transcriptional, antiapoptotic, and transforming activities of the v-Rel oncoprotein. Mol Cell Biol 1999;19:307–316. 77. White DW, Roy A, Gilmore TD. The v-Rel oncoprotein blocks apoptosis and proteolysis of IκB-α in transformed chicken spleen cells. Oncogene 1995;10:857–868. 78. Zong WX, Farrell M, Bash J, Gelinas C. v-Rel prevents apoptosis in transformed lymphoid cells and blocks TNFα-induced cell death. Oncogene 1997;15:971–980. 79. Pahl HL. Activators and target genes of Rel/NF-κB transcription factors. Oncogene 1999;18:6853–6866. 80. Barkett M, Gilmore TD. Control of apoptosis by Rel/ NF-κB transcription factors. Oncogene 1999;18: 6910–6924. 81. Chu ZL, McKinsey TA, Liu L, Gentry JJ, Malim MH, Ballard DW. Suppression of tumor necrosis factor-induced cell death by inhibitor of apoptosis c-IAP2 is under NF-κB control. Proc Natl Acad Sci U S A 1997;94: 10,057–10,062. 82. Grumont RJ, Rourke IJ, Gerondakis S. Rel-dependent induction of A1 transcription is required to protect B cells from antigen receptor ligation-induced apoptosis. Genes Dev 1999;13:400–411. 83. Krikos A, Laherty CD, Dixit VM. Transcriptional activation of the tumor necrosis factor alpha-inducible zinc finger protein, A20, is mediated by κB elements. J Biol Chem 1992;267:17,971–17,976. 84. Wang CY, Mayo MW, Korneluk RG, Goeddel DV, Baldwin AS, Jr. NF-κB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science 1998;281:1680–1683. 85. Wang CY, Cusack JC, Jr., Liu R, Baldwin AS, Jr. Control of inducible chemoresistance: enhanced anti-tumor therapy through increased apoptosis by inhibition of NF-κB. Nat Med 1999;5:412–417.

Volume 33, 2003

24 86. Wu MX, Ao Z, Prasad KV, Wu R, Schlossman SF. IEX-1L, an apoptosis inhibitor involved in NF-κB-mediated cell survival. Science 1998;281:998–1001. 87. You M, Ku PT, Hrdlickova R, Bose HR, Jr. ch-IAP1, a member of the inhibitor-of-apoptosis protein family, is a mediator of the antiapoptotic activity of the v-Rel oncoprotein. Mol Cell Biol 1997;17:7328–7341. 88. Zong WX, Edelstein LC, Chen C, Bash J, Gelinas C. The prosurvival Bcl-2 homolog Bfl-1/A1 is a direct transcriptional target of NF-κB that blocks TNFα-induced apoptosis. Genes Dev 1999;13:382–387. 89. Lee HH, Dempsey PW, Parks TP, Zhu X, Baltimore D, Cheng G. Specificities of CD40 signaling: involvement of TRAF2 in CD40-induced NF-κB activation and intercellular adhesion molecule-1 upregulation. Proc Natl Acad Sci U S A 1999;96:1421–1426. 90. Chen C, Edelstein LC, Gelinas C. The Rel/NF-κB family directly activates expression of the apoptosis inhibitor Bcl-xL. Mol Cell Biol 2000;20:2687–2695. 91. Sevilla L, Zaldumbide A, Pognonec P, Boulukos KE. Transcriptional regulation of the bcl-x gene encoding the anti-apoptotic Bcl-xL protein by Ets, Rel/NF-κB, STAT and AP1 transcription factor families. Histol Histopathol 2001;16:595–601. 92. Khoshnan A, Tindell C, Laux I, Bae D, Bennett B, Nel AE. The NF-κB cascade is important in Bcl-xL expression and for the anti-apoptotic effects of the CD28 receptor in primary human CD4+ lymphocytes. J Immunol2000;165:1743–1754. 93. Cao W, Zhang Y, Zhang D, Zou P. Effect of antisense oligodeoxynucleotide directed to NF-κB-RelA on Bcl-XL mRNA in extended drug resistance leukemia cell line HL-60/E6. J Tongji Med Univ 2001;21:32–34. 94. Yang J, Richmond A. Constitutive IκB kinase activity correlates with nuclear factor-κB activation in human melanoma cells. Cancer Res 2001;61:4901–4909. 95. Wang W, Abbruzzese JL, Evans DB, Chiao PJ. Overexpression of urokinase-type plasminogen activator in pancreatic adenocarcinoma is regulated by constitutively activated RelA. Oncogene 1999;18:4554–4563. 96. Darieva ZA, Pospelov VA, Pospelova TV. Transcription factor NF-κB/RelA are constitutively activated and localized in the cell nuclei of E1A+ cHa-Ras transformants. Tsitologiia 1999;41:622–627. 97. Slater AF, Kimland M, Jiang SA, Orrenius S. Constitutive nuclear NF-κB/rel DNA-binding activity of rat thymocytes is increased by stimuli that promote apoptosis, but not inhibited by pyrrolidine dithiocarbamate. Biochem J 1995;312(Pt 3):833–838. 98. Mufson RA, Myers C, Turpin JA, Meltzer M. Phorbol ester reduces constitutive nuclear NF-κB and inhibits HIV-1 production in mature human monocytic cells. J Leukoc Biol 1992;52:637–644. 99. Yeh PY, Chuang SE,Yeh KH, Song YC, Ea CK, Cheng AL. Increase of the resistance of human cervical carcinoma cells to cisplatin by inhibition of the MEK to ERK signaling pathway partly via enhancement of anticancer druginduced NF-κB activation. Biochem Pharmacol 2002; 63:1423–1430.

International Journal of Gastrointestinal Cancer

Sclabas et al. 100. Dong QG, Sclabas GM, Fujioka S, et al. The function of multiple IκB : NF-κB complexes in the resistance of cancer cells to Taxol-induced apoptosis. Oncogene 2002;21: 6510–6519. 101. Niu G, Wright KL, Huang M, et al. Constitutive Stat3 activity up-regulates VEGF expression and tumor angiogenesis. Oncogene 2002;21:2000–2008. 102. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57–70. 103. Laham RJ, Simons M, Sellke F. Gene transfer for angiogenesis in coronary artery disease. Annu Rev Med 2001;52: 485–502. 104. Zetter BR, PhD. Angiogenesis and tumor metastasis. Annu Rev Med 1998;49:407–424. 105. Sasaki H, Zhu L, Fukuda S, Maulik N. Inhibition of NF-κB activation by pyrrolidine dithiocarbamate prevents in vivo hypoxia/reoxygenation-mediated myocardial angiogenesis. Int J Tissue React 2000;22:93–100. 106. Sasaki H, Ray PS, Zhu L, Otani H, Asahara T, Maulik N. Hypoxia/reoxygenation promotes myocardial angiogenesis via an NF-κB-dependent mechanism in a rat model of chronic myocardial infarction. J Mol Cell Cardiol 2001; 33:283–294. 107. Yu DH, Scorsone K, Hung MC. Adenovirus type 5 E1A gene products act as transformation suppressors of the neu oncogene. Mol Cell Biol 1991;11:1745–1750. 108. Yu D, Jing T, Liu B, et al. Overexpression of ErbB2 blocks Taxol-induced apoptosis by upregulation of p21Cip1, which inhibits p34Cdc2 kinase. Mol Cell 1998;2: 581–591. 109. Tan M, Jing T, Lan KH, et al. Phosphorylation on tyrosine-15 of p34(Cdc2) by ErbB2 inhibits p34(Cdc2) activation and is involved in resistance to taxol-induced apoptosis. Mol Cell 2002;9:993–1004. 110. Bancroft CC, Chen Z, Yeh J, et al. Effects of pharmacologic antagonists of epidermal growth factor receptor, PI3K and MEK signal kinases on NF-κB and AP-1 activation and IL-8 and VEGF expression in human head and neck squamous cell carcinoma lines. Int J Cancer 2002;99:538–548. 111. Wang Z, Castresana MR, Newman WH. Reactive oxygen and NF-κB in VEGF-induced migration of human vascular smooth muscle cells. Biochem Biophys Res Commun 2001;285:669–674. 112. Ohm JE, Carbone DP. VEGF as a mediator of tumorassociated immunodeficiency. Immunol Res 2001;23: 263–272. 113. Bancroft CC, Chen Z, Dong G, et al. Coexpression of proangiogenic factors IL-8 and VEGF by human head and neck squamous cell carcinoma involves coactivation by MEK-MAPK and IKK-NF-κB signal pathways. Clin Cancer Res 2001;7:435–442. 114. Sasaki H, Ray PS, Zhu L, Galang N, Maulik N. Oxidative stress due to hypoxia/reoxygenation induces angiogenic factor VEGF in adult rat myocardium: possible role of NF-κB. Toxicology 2000;155:27–35. 115. Newton TR, Patel NM, Bhat-Nakshatri P, Stauss CR, Goulet RJ, Jr., Nakshatri H. Negative regulation of trans-

Volume 33, 2003

NF-κB in Pancreatic Cancer

116.

117.

118.

119.

120.

121.

122.

123.

124.

125.

126.

127.

128.

129.

130.

activation function but not DNA binding of NF-κB and AP-1 by IκBβ1 in breast cancer cells. J Biol Chem 1999; 274:18,827–18,835. Thompson JE, Phillips RJ, Erdjument-Bromage H, Tempst P, Ghosh S. IκB-β regulates the persistent response in a biphasic activation of NF-κB. Cell 1995;80: 573–582. Connelly L, Palacios-Callender M, Ameixa C, Moncada S, Hobbs AJ. Biphasic regulation of NF-κB activity underlies the pro- and anti-inflammatory actions of nitric oxide. J Immunol 2001;166:3873–3881. Han Y, Brasier AR. Mechanism for biphasic rel A· NF-κB1 nuclear translocation in Tumor Necrosis Factor αstimulated hepatocytes. J Biol Chem 1997;272: 9825–9832. Han SJ, Ko HM, Choi JH, et al. Molecular mechanisms for LPS-induced biphasic activation of NF-κB. J Biol Chem 2002;277:44,715–44,721. Tanaka M, Fuentes ME, Yamaguchi K, et al. Embryonic lethality, liver degeneration, and impaired NF-κB activation in IKK-β-deficient mice. Immunity 1999;10:421–429. Li Q, Lu Q, Hwang JY, et al. IKK1-deficient mice exhibit abnormal development of skin and skeleton. Genes Dev 1999;13:1322–1328. Li Q, Estepa G, Memet S, Israël A, Verma IM. Complete lack of NF-κB activity in IKK1 and IKK2 doubledeficient mice: additional defect in neurulation. Genes Dev 2000;14:1729–1733. Schmidt-Supprian M, Bloch W, Courtois G, et al. NEMO/IKKγ-deficient mice model incontinentia pigmenti. Mol Cell 2000;5:981–992. Rudolph D, Yeh WC, Wakeham A, et al. Severe liver degeneration and lack of NF-κB activation in NEMO/IKKγ-deficient mice. Genes Dev 2000;14: 854–862. Orange JS, Brodeur SR, Jain A, et al. Deficient natural killer cell cytotoxicity in patients with IKK-γ/NEMO mutations. J Clin Invest 2002;109:1501–1509. Huber MA, Denk A, Peter RU, Weber L, Kraut N, Wirth T. The IKK-2/IκBα /NF-κB pathway plays a key role in the regulation of CCR3 and eotaxin-1 in fibroblasts. A critical link to dermatitis in IκBα-deficient mice. J Biol Chem 2002;277:1268–1275. Makris C, Godfrey VL, Krahn-Senftleben G, et al. Female mice heterozygous for IKKγ/NEMO deficiencies develop a dermatopathy similar to the human X-linked disorder incontinentia pigmenti. Mol Cell 2000;5: 969–979. Karin M. How NF-κB is activated: the role of the IκB kinase (IKK) complex. Oncogene 1999;18: 6867–6874. Woronicz JD, Gao X, Cao Z, Rothe M, Goeddel DV. IκB kinase-β: NF-κB activation and complex formation with IκB kinase-α and NIK. Science 1997;278:866–869. Ling L, Cao Z, Goeddel DV. NF-κB-inducing kinase activates IKK-α by phosphorylation of Ser-176. Proc Natl Acad Sci U S A 1998;95:3792–3797.

International Journal of Gastrointestinal Cancer

25 131. Lee FS, Peters RT, Dang LC, Maniatis T. MEKK1 activates both IκB kinase α and IκB kinase β. Proc Natl Acad Sci U S A 1998;95:9319–9324. 132. Zhao Q, Lee FS. Mitogen-activated protein kinase/ERK kinase kinases 2 and 3 activate nuclear factor-κB through IκB kinase-α and IκB kinase-β. J Biol Chem 1999;274: 8355–8358. 133. Ninomiya-Tsuji J, Kishimoto K, Hiyama A, Inoue J, Cao Z, Matsumoto K. The kinase TAK1 can activate the NIK-IκB as well as the MAP kinase cascade in the IL-1 signalling pathway. Nature 1999;398:252–256. 134. Yin L, Wu L, Wesche H, et al. Defective lymphotoxinbeta receptor-induced NF-κB transcriptional activity in NIK-deficient mice. Science 2001;291:2162–2165. 135. Xia Y, Makris C, Su B, et al. MEK kinase 1 is critically required for c-Jun N-terminal kinase activation by proinflammatory stimuli and growth factor-induced cell migration. Proc Natl Acad Sci U S A 2000;97: 5243–5248. 136. Vidal S, Khush RS, Leulier F, Tzou P, Nakamura M, Lemaitre B. Mutations in the Drosophila dTAK1 gene reveal a conserved function for MAPKKKs in the control of rel/NF-κB-dependent innate immune responses. Genes Dev 2001;15:1900–1912. 137. Wang C, Deng L, Hong M, Akkaraju GR, Inoue J, Chen ZJ. TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature 2001;412:346–351. 138. Li Q, Verma IM. NF-κB regulation in the immune system. Nat Rev Immunol 2002;2:725–734. 139. Leitges M, Sanz L, Martin P, et al. Targeted disruption of the ξPKC gene results in the impairment of the NF-κB pathway. Mol Cell 2001;8:771–780. 140. Yang J, Lin Y, Guo Z, et al. The essential role of MEKK3 in TNF-induced NF-κB activation. Nat Immunol 2001; 2:620–624. 141. Xiao G, Harhaj EW, Sun SC. NF-κB-inducing kinase regulates the processing of NF-κB2 p100. Mol Cell 2001; 7:401–409. 142. Senftleben U, Cao Y, Xiao G, et al. Activation by IKKα of a second, evolutionary conserved, NF-κB signaling pathway. Science 2001;293:1495–1499. 143. Schiemann B, Gommerman JL, Vora K, et al. An essential role for BAFF in the normal development of B cells through a BCMA-independent pathway. Science 2001; 293:2111–2114. 144. Dejardin E, Droin NM, Delhase M, et al. The Lymphotoxin-β receptor induces different patterns of gene expression via two NF-κB pathways. Immunity 2002; 17:525. 145. Finco TS, Westwick JK, Norris JL, Beg AA, Der CJ, Baldwin AS, Jr. Oncogenic Ha-Ras-induced signaling activates NF-κB transcriptional activity, which is required for cellular transformation. J Biol Chem 1997;272: 24,113–24,116. 146. Mayo MW, Wang CY, Cogswell PC, et al. Requirement of NF-κB activation to suppress p53-independent apoptosis induced by oncogenic Ras. Science 1997;278: 1812–1815.

Volume 33, 2003

26 147. Korc M, Meltzer P, Trent J. Enhanced expression of epidermal growth factor receptor correlates with alterations of chromosome 7 in human pancreatic cancer. Proc Natl Acad Sci U S A 1986;83:5141–5144. 148. Salomon DS, Brandt R, Ciardiello F, Normanno N. Epidermal growth factor-related peptides and their receptors in human malignancies. Crit Rev Oncol Hematol 1995; 19:183–232. 149. Uegaki K, Nio Y, Inoue Y, et al. Clinicopathological significance of epidermal growth factor and its receptor in human pancreatic cancer. Anticancer Res 1997;17: 3841–3847. 150. Yamanaka Y, Friess H, Kobrin MS, Buchler M, Beger HG, Korc M. Coexpression of epidermal growth factor receptor and ligands in human pancreatic cancer is associated with enhanced tumor aggressiveness. Anticancer Res 1993; 13:565–569. 151. Korc M, Chandrasekar B, Yamanaka Y, Friess H, Buchler M, Beger HG. Overexpression of the epidermal growth factor receptor in human pancreatic cancer is associated with concomitant increases in the levels of epidermal growth factor and transforming growth factor α. J Clin Invest 1992;90:1352–1360. 152. Habib AA, Chatterjee S, Park SK, Ratan RR, Lefebvre S, Vartanian T. The epidermal growth factor receptor engages receptor interacting protein and nuclear factor-κB (NF-κB)-inducing kinase to activate NF-κB. Identification of a novel receptor-tyrosine kinase signalosome. J Biol Chem 2001;276:8865–8874. 153. Hirota K, Murata M, Itoh T, Yodoi J, Fukuda K. Redoxsensitive transactivation of epidermal growth factor receptor by tumor necrosis factor confers the NF-κB activation. J Biol Chem 2001;276:25,953–25,958.

International Journal of Gastrointestinal Cancer

Sclabas et al. 154. Sun L, Carpenter G. Epidermal growth factor activation of NF-κB is mediated through IκBα degradation and intracellular free calcium. Oncogene 1998;16:2095–2102. 155. Biswas DK, Cruz AP, Gansberger E, Pardee AB. Epidermal growth factor-induced nuclear factor κB activation: A major pathway of cell-cycle progression in estrogenreceptor negative breast cancer cells. Proc Natl Acad Sci U S A 2000;97:8542–8547. 156. Habib AA, Hognason T, Ren J, Stefansson K, Ratan RR. The epidermal growth factor receptor associates with and recruits phosphatidylinositol 3-kinase to the plateletderived growth factor β receptor. J Biol Chem 1998; 273:6885–6891. 157. Obata H, Biro S, Arima N, et al. NF-κB is induced in the nuclei of cultured rat aortic smooth muscle cells by stimulation of various growth factors. Biochem Biophys Res Commun 1996;224:27–32. 158. Mendelsohn J, Baselga J. The EGF receptor family as targets for cancer therapy. Oncogene 2000;19:6550–6565. 159. Arlt A, Vorndamm J, Muerkoster S, et al. Autocrine production of interleukin 1β confers constitutive nuclear factor κB activity and chemoresistance in pancreatic carcinoma cell lines. Cancer Res 2002;62:910–916. 160. Vale T, Ngo TT, White MA, Lipsky PE. Raf-induced transformation requires an interleukin 1 autocrine loop. Cancer Res 2001;61:602–607. 161. Takaesu G, Surabhi RM, Park KJ, Ninomiya-Tsuji J, Matsumoto K, Gaynor RB. TAK1 is critical for IκB kinasemediated activation of the NF-κB pathway. J Mol Biol 2003;326:105–115. 162. Dhawan P, Richmond A. A novel NF-κB-inducing kinaseMAPK signaling pathway up-regulates NF-κB activity in melanoma cells. J Biol Chem 2002;277:7920–7928.

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