Oncogene (2003) 22, 1365–1370

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Inhibition of constitutive NF-jB activity by IjBaM suppresses tumorigenesis Shuichi Fujioka1, Guido M Sclabas1, Christian Schmidt1, Jiangong Niu2, Wayne A Frederick1, Qiang G Dong1, James L Abbruzzese3, Douglas B Evans1, Cheryl Baker4 and Paul J Chiao*,1,2,5 1 Department of Surgical Oncology, The University of Texas, Houston, TX 77030, USA; 2The Graduate School of Biomedical Sciences, The University of Texas, Houston, TX 77030, USA; 3Department of Gastrointestinal Medical Oncology, The University of Texas, Houston, TX 77030, USA; 4Department of Cancer Biology, The University of Texas, Houston, TX 77030, USA; 5Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, The University of Texas, Houston, TX 77030, USA

We have demonstrated that nuclear factor-jB (NF-jB) is constitutively activated in human pancreatic adenocarcinoma and human pancreatic cancer cell lines but not in normal pancreatic tissues or in immortalized, nontumorigenic pancreatic epithelial cells, suggesting that NF-jB plays a critical role in the development of pancreatic adenocarcinoma. To elucidate the role of constitutive NFjB activity in human pancreatic cancer cells, we generated pancreatic tumor cell lines that express a phosphorylation defective IjBa (S32, 36A) (IjBaM) that blocks NF-jB activity. In this study, we showed that inhibiting constitutive NF-jB activity by expressing IjBaM suppressed the tumorigenicity of a nonmetastatic human pancreatic cancer cell line, PANC-1, in an orthotopic nude mouse model. Immunohistochemical analysis showed that PANC-1-derived tumors expressed vascular endothelial growth factor (VEGF) and induced angiogenesis. Inhibiting NF-jB signaling by expressing IjBaM significantly reduced expression of Bcl-xL and Bcl-2. The cytokine-induced expression of VEGF and Interleukin-8 in PANC-1 cells is also decreased. Taken together, these results suggest that the inhibition of NF-jB signaling can suppress tumorigenesis of pancreatic cancer cells and that the NF-jB signaling pathway is a potential target for anticancer agents. Oncogene (2003) 22, 1365–1370. doi:10.1038/sj.onc.1206323 Keywords: IkBa; NF-kB; tumorigenesis; angiogenesis; pancreatic cancer

Nuclear factor-kB (NF-kB) is a family of pleiotropic transcription factors that regulate the transcription of a large number of genes that play key roles in embryonic development, lymphoid differentiation, apoptosis, and immune and inflammatory responses (Verma et al., *Correspondence: PJ Chiao, Departments of Molecular and Cellular Oncology and Surgical Oncology, Unit 107, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA; E-mail: [email protected] Received 7 October 2002; revised 10 December 2002; accepted 11 December 2002

1995; Baldwin, 1996; Gilmore et al., 1996). Several reports suggest that the members of the NF-kB family and their inhibitor, IkB family, are involved in the development of cancer (Sylla and Temin, 1986; Moore and Bose, 1988; Gilmore et al., 1996). For instance, the genes encoding c-Rel, Bcl-3, p105 (p50), and p100 (p52) are located at sites of recurrent genomic rearrangements in cancer (Ohno et al., 1990; Lu et al., 1991; Neri et al., 1991; Kitajima et al., 1992; Dejardin et al., 1995). We first reported that RelA, the p65 subunit of NF-kB, is constitutively activated in human pancreatic adenocarcinoma and human pancreatic cancer cell lines but not in normal pancreatic tissues or in immortalized, nontumorigenic pancreatic epithelial cells (Wang et al., 1999b). We previously found that NF-kB induces overexpression of urokinase plasminogen activator and that Bcl-xL is induced by NF-kB in pancreatic cancer cell lines (Wang et al., 1999a; Dong et al., 2002). Other studies showed that IkBaM-mediated inhibition of NFkB activity reduced angiogenesis and metastasis in an ovarian cancer cell line and a prostate cancer cell line and completely inhibited liver metastasis of a pancreatic cancer cell line in mice (Huang et al., 2000, 2001; Fujioka et al., 2002). The purpose of this study was to determine whether the inhibition of constitutive NF-kB activity would suppress tumorigenic phenotypes in pancreatic cancer cells. To determine the role of constitutive NF-kB activity in tumorigenesis, we constructed a retroviral vector encoding a Flag-tagged phosphorylation defective mutant of IkBa (S32, 36A) (IkBaM). The nonmetastatic human pancreatic tumor cell line PANC-1 was infected with retrovirus encoding Flag-tagged IkBaM or a control retrovirus and then selected for puromycin resistance (800 ng/ml). The puromycin-resistant clones of PANC-1 cells from each infection were pooled to establish PANC-1/IkBaM and PANC-1/Puro cell lines. Both cytoplasmic and nuclear extracts from pooled puromycin-resistant PANC-1 (PANC-1/puro) and PANC-1 cells expressing IkBaM (PANC-1/IkBaM) were prepared for subsequent analysis. Western blot analysis showed the expression of Flag-tagged IkBaM (Figure 1a). Constitutive NF-kB activity was found in

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Figure 1 Effect of IkBaM expression on constitutive and TNF-a-induced NF-kB activity. (a) Flag-IkBaM/puror and pRetro/puror control retroviruses were generated and infections were performed as previously described (Naviaux et al., 1996). Pooled puromycinresistant control cells and the cells that expressed Flag-tagged IkBaM were used for subsequent analyses. Western blot analysis using whole cell protein extracts with a rabbit antibody against IkBa (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was performed as previously described (Grau et al., 1997). (b) Electrophoretic mobility shift assay (EMSA) was performed to determine NF-kB DNAbinding activity in PANC-1/puro and PANC-1/IkBaM cells as previously described (Chiao et al., 1994). A probe containing the Oct-1 motif was used as a control for quality and quantity of cell extracts, (c) Specificity of the NF-kB DNA-binding activity, as determined by competition and supershift experiments in PANC-1/puro cells. Arrows indicate the migration of the induced NF-kB DNA-binding complexes. Migration of the free probe is not shown. SS, supershifted band; NS, nonspecific band. Western blot analysis of the expression of Bcl-xL (d), Bcl-2 (e), and Bcl-xs (f) in PANC-1/puro and PANC-1/IkBaM cells. Equal loading of protein extracts was determined by probing the same membrane filter with anti-b-actin antibody

the nuclear extracts from PANC-1/puro cells using electrophoretic mobility shift assay (EMSA) (Figure 1b), consistent with our previous report that NF-kB is constitutively activated in human pancreatic adenocarcinoma cells (Wang et al., 1999b). NF-kB DNA binding activity is completely inhibited in PANC-1/IkBaM cells (Figure 1b). Competition and supershift assays showed the presence of RelA and p50 in the NF-kB binding activity in PANC-1/puro cells (Figure 1c). Expression of antiapoptotic NF-kB downstream target genes, bcl-2 and bcl-xL, are inhibited in PANC-1/IkBaM cells (Figure 1d, e), which is consistent with our previous reports (Dong et al., 2002; Fujioka et al., 2002). These results suggest that the expression of these and other NF-kB downstream target genes is decreased through the IkBaM-mediated inhibition of NF-kB activity. The expression of the proapoptotic gene bcl-xS, the alterOncogene

native spliced form of bcl-xL, does not decrease (Figure 1f). It is possible that the half-life of bcl-xS mRNA or protein is much longer as a number of reports suggest that bcl-xL and bcl-xS are differentially regulated at post-transcriptional and post-translational stages (Chang et al., 1997; Klingler et al., 1997; Tsai et al., 2000; Parborell et al., 2002). To determine whether the inhibition of NF-kB suppresses the tumorigenic phenotype of PANC-1/puro cells, we performed an in vivo experiment using an orthotopic nude mouse model. Six weeks after injection of PANC-1/puro and PANC-1/IkBaM into the pancreas of nude mice, all the mice injected with PANC-1 and PANC-1/puro cells became sick. Pathologic examination was performed to determine the extent of tumor formation and metastasis. As shown in Figure 2a, 100% (10/10) of animals injected with PANC-1/puro cells

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1993). IL-8 can induce proliferation and migration of human umbilical vein endothelial cells and can stimulate vascularization in a rat cornea assay (Strieter et al., 1992). Recently, it was demonstrated that NF-kB activation is obligatory for retinal angiogenesis (Yoshida et al., 1999). To determine whether PANC-1/puro cell-derived tumors express NF-kB target genes, such as VEGF, we performed immunohistochemical staining to analyse the expression of VEGF and the extent of angiogenesis in the pancreatic tissues obtained from the mice injected with PANC-1/puro cells. As shown in Figure 3, much higher levels of VEGF expression and a significantly higher level of intratumoral microvessel formation were found in the tumors derived from PANC-1 cells than in the adjacent pancreatic tissues. Because cytokines including TNF-a have been suggested to play an important role in tumor angiogenesis and cancer progression (Leibovich et al., 1987; Passaniti et al., 1992; Leek et al., 1996; Shono et al., 1996; Yancopoulos et al., 2000; Xu et al., 2001), we investigated the role of NF-kB activity in regulating TNF-a-mediated VEGF and IL-8 expression. As shown Figure 2 IkBaM-mediated suppression of tumorigenicity in an orthotopic nude mouse model. (a) One million viable PANC-1 and PANC-1/IkBaM cells suspended in 50 ml phosphate-buffered saline (PBS) were injected into the pancreatic parenchyma of female athymic BALB/c nude mice at 8 weeks of age (Charles River Laboratories, Inc. Wilmington, MA, USA). Six weeks after injection, all mice were killed, and the degrees of tumorigenesis and metastasis were investigated. The incidence of tumorigenesis, ascitic formation, jaundice, liver, or peritoneal metastasis is shown in a graph. (b) Summary of the weights of tumors resected from mice injected with PANC-1 cells

presented pancreatic tumors with no metastasis in the liver or other organs, whereas none of 10 mice injected with PANC-1/IkBaM cells showed pancreatic tumor formation, implying that constitutive RelA/NF-kB activity induces tumorigenicity. In addition, 100% (5/ 5) mice injected with PANC-1 cells developed pancreatic tumor as those observed in the mice injected with PANC-1/puro (data not shown). It is now well established that angiogenesis is essential for the growth of both primary tumors and metastatic lesions both of which require an adequate blood supply (Folkman et al., 1989; Folkman, 1990; Folkman, 1992). Tumor angiogenesis is, in part, regulated by angiogenic factors that are produced and secreted by tumor cells. Vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) are two positive regulators of endothelial cells that have been identified (Yoshimura et al., 1987; Liotta et al., 1991; Ferrara, 1995). VEGF binds to the specific transmembrane tyrosine kinase receptors KDR/ flk-1 and flt-1, which are selectively expressed on vascular endothelial cells, thus stimulating the growth of endothelial cells (Millauer et al., 1993). Subsequent studies have revealed that IL-8 triggers angiogenesis in vivo via mechanisms that are mediated by direct stimulation of endothelial cell growth or by indirect leukocyte-dependent effects (Koch et al., 1992; Hu et al.,

Figure 3 Immunohistochemical staining of pancreatic tumors derived from orthotopically injected pancreatic cancer cells. (a) Detection of VEGF expression by a polyclonal anti-VEGF antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). (b) Detection of CD31 by a monoclonal anti-CD31 antibody (Pharmingen, San Diego, CA, USA). Paraffin-embedded sections (for VEGF staining) and frozen sections (for CD-31 staining) were obtained from marginal regions of resected mouse pancreas and the tumors derived from implanted PANC-1/puro human pancreatic cancer cell lines. Immunohistochemical staining was performed as previously described (Grau et al., 1997) Oncogene

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Figure 4 IkBaM-mediated inhibition of expression of NF-kB downstream target genes. (a) Electrophoretic mobility shift assay (EMSA) was performed to determine NF-kB DNA-binding activity in PANC-1/puro and PANC-1/IkBaM cells stimulated with TNFa (10 ng/ml) for 5, 15, and 30 min, as previously described (Chiao et al., 1994). A probe containing the Oct-1 motif was used as a control for quality and quantity of cell extracts. Expression of VEGF and IL-8 stimulated with TNF-a as indicated were analysed by Northern blot (b) and Western blot analyses (c) as previously described (Chiao et al., 1994). Equal loading of mRNA and protein samples was determined by reprobing the same membrane filter with a cDNA probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and anti-b-actin antibody, respectively. kB (d), VEGF (e), and IL-8 (f) luciferase reporter gene activities in PANC-1/puro and PANC-1/ IkBaM cells were determined. A measure of 1 mg of control HIV-kB, 1.5 kb VEGF, or 0.27-kb IL-8 promoter reporter-gene constructs was cotransfected into the cells with an internal control, p-TK Renilla luciferase (pRL-TK), using the lipotransfection method (FuGENE 6, Roche, Indianapolis, IN, USA). The activities of both Firefly and Renilla luciferase were determined 48 h after transfection using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). The fold-increases in luciferase activity were calculated relative to the Luciferase activity of an internal control. Data represent the mean 7s.e. from three different experiments performed in triplicate

Oncogene

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in Figure 4a, NF-kB DNA binding activity was induced by stimulation with TNF-a (10 ng/ml) in PANC-1/puro, whereas TNF-a stimulated NF-kB DNA binding activity was inhibited in PANC-1/ IkBaM cells. A probe containing the Oct-1 motif was used as a control for quality and quantity of cell extracts. VEGF mRNA was induced following stimulation with TNF-a and peaked at 1 h in PANC-1/puro cells (Figure 4b, lanes 2 and 3). The TNF-a-induced expression of VEGF was inhibited by IkBaM (Figure 4b, lanes 7–10). These results were consistent with the Western blot analysis as shown in Figure 4c. Similarly, IL-8 expression was induced in response to TNF-a stimulation and reaches to its peak of expression at 1 h in PANC-1/puro cells (Figure 4b). In contrast to observed IkBaMmediated inhibition of VEGF expression in response to TNF-a, IkBaM only partially inhibited TNF-ainduced IL-8 expression (Figure 4b and c, lanes 7–10), implying the involvement of alternative signaling cascades that regulate IL-8 gene expression. Interestingly, Figure 4c shows that induction of IL-8 protein production by TNF-a occurs at 3 h in PANC-1/puro cells and 6 h in PANC-1/IkBaM cells. Taken together, the results in Figure 4 show that TNF-a induced IL-8 protein synthesis is delayed comparing with TNF-ainduced IL-8 mRNA expression in Figure 4b, suggesting that IL-8 expression is also regulated at translational level. This finding is consistent with an early report that VEGF-/VPF-induced IL-8 expression is further regulated translationally even although IL-8 mRNA was increased through activation of NF-kB to maximal level after 1 h of VEGF/VPF treatment of the human brain microvascular endothelial cells (Lee et al., 2002). Altogether, these results indicate that TNF-a-mediated expression of VEGF and IL-8 is NF-kB-dependent. However, the induction of TNF-a-mediated IL-8 mRNA expression is only partially inhibited or delayed by IkBaM. To further determine the role of NF-kB activity in regulating the expression of VEGF and IL-8, we analysed activities of the 1.5-kb VEGF promoter and 0.27-kb IL-8 promoter luciferase reporter-gene constructs (Mukaida et al., 1990; Fukumura et al., 1998). The VEGF and IL-8 promoter reporter-gene constructs, and kB control reporter-gene plasmid were transiently transfected into PANC-1/puro and PANC-1/IkBaM cells. As shown in Figure 4d, IkBaM expression inhibited constitutive NF-kB activation as determined by kB control luciferase reporter-gene activity. IkBaM expression significantly reduced IL-8 and VEGF promoter-mediated reporter-gene transcriptional activities in PANC-1 cells (Figure 4e and f). Taken together, these results show that IkBaM-mediated inhibition of constitutive NF-kB activity downregulated VEGF and IL-8 expression in PANC-1 cells, suggesting that NF-kBinducible expression of VEGF, IL-8 and other NF-kB downstream target genes is involved in pancreatic tumorigenesis. In this study, we investigated the function of NF-kB signaling on the tumorigenesis and angiogenesis of PANC-1/puro human pancreatic cancer cell lines.

Inhibition of constitutive NF-kB activity completely suppressed tumor formation from the nonmetastatic human pancreatic cancer cell line PANC-1 in an orthotopic nude mouse model. Furthermore, IkBaM expression substantially inhibited expression of key antiapoptotic genes, bcl-xL and bcl-2, and 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-kB activity, found in 70% of pancreatic cancers, plays an important role in pancreatic tumorigenesis. Our study also implies that constitutive NF-kB 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. Several reports show that the PANC-1 cell line is nonmetastatic in the orthotopic nude mouse model (Mohammad et al., 2001; Sawai et al., 2001; Teraoka et al., 2001). PANC-1 cells express wild-type Smad4 and a functional TGF-b signaling pathway, unlike other human pancreatic tumor cell lines (Grau et al., 1997). Reconstitution of Smad4 expression in a Smad4-null Hs667t human pancreatic cancer cell line reduced tumor formation by inhibiting angiogenesis (Schwarte-Waldhoff et al., 2000), implying that Smad4 may reduce the expression of angiogenic factors and result 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-b pathway reduces the metastatic potential of PANC-1 cells. Human pancreatic cancer has a very poor prognosis, even after curative resection, and is currently the fourth leading cause of cancer death in the United States (Jemal et al., 2002). The overall 5-year survival rate continues to be dismal, at 1–3% (Bramhall et al., 1995). Most patients with pancreatic cancer have locally advanced, unresectable disease or metastasis at the time of diagnosis (Breslin et al., 2001). Currently, chemotherapy, radiation therapy, and surgery are largely ineffective in treating this disease (Breslin et al., 2001). Recent studies suggest that VEGF is the best-validated target for antiangiogenic therapies to inhibit angiogenesis and tumor growth in pancreatic cancer (Grisham et al., 1999; Schwarte-Waldhoff et al., 2000; Tsuzuki et al., 2001). Our findings provide important implications for the therapeutic usefulness of NF-kB inhibition in antiangiogenic therapeutic strategies.

Acknowledgements We thank Mariann Crapanzano for editorial assistance. This work was supported in part by grants CA73675, CA78778, and CA 75517 from the National Cancer Institute (NCI), and a grant from the Lockton Fund for Pancreatic Cancer Research. WAF is a recipient of the NCI T32 Training Grant Fellowship, and GMS is a recipient of a Fellowship of the Cancer League of Bern, Switzerland. Oncogene

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Sciences, The University of Texas, Houston, TX 77030, USA; 3Department of Gastrointestinal Medical ..... studies suggest that VEGF is the best-validated target.

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Jun 29, 2005 - amplifier (Astro-Med, West Warwick, RI; for hook recordings) or a Get- ting 5A microelectrode amplifier (Getting Instruments, San Diego, CA) and digitized (16 bits; sampling rate, 10 kHz; Digidata 1320A; Molecular. Devices, Palo Alto,

Augmentation of induced visual gamma activity by ...
The current study investigated the modulation of visually induced gamma band oscillations by .... explaining artefactual data points in subjects where electrodes had .... this burst was not present in the time±frequency maps generated by.

Representation of Concurrent Stimuli by Population Activity in Visual ...
Dec 23, 2009 - How do neuronal populations represent concurrent stimuli? .... data set (Figures 1D and 1E) and an average of 98.5% of the variance in all nine ...

Management of synchronized network activity by highly ...
Sep 9, 2008 - Management of synchronized network activity by highly active neurons. This article has been ..... acquisition software (Multi Channel Systems, version 3.2.20). ...... findings, while taking into account the observations reported.