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[Cancer Research 65, 1316-1324, February 15, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Increased Expression of the E3-Ubiquitin Ligase Receptor Subunit ßTRCP1 Relates to Constitutive Nuclear Factor-{kappa}B Activation and Chemoresistance in Pancreatic Carcinoma Cells

Susanne Müerköster1, Alexander Arlt1, Bence Sipos2, Maike Witt1, Maike Großmann1, Günter Klöppel2, Holger Kalthoff3, Ulrich R. Fölsch1 and Heiner Schäfer1

1 Laboratory of Molecular Gastroenterology and Hepatology, First Department of Medicine, 2 Department of Pathology, and 3 Molecular Oncology Research Group, Department of General Surgery, Kiel University, UKSH Campus-Kiel, Kiel, Germany

Requests for reprints: Heiner Schäfer, Laboratory of Molecular Gastroenterology and Hepatology, First Department of Medicine, University of Kiel, UKSH-Campus Kiel, Schittenhelmstraße 12, 24105 Kiel, Germany. Phone: 49-431-597-1266/1394; Fax: 49-431-597-1427; E-mail: hschaef{at}1med.uni-kiel.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The permanent activation of the transcription factor nuclear factor-{kappa}B (NF-{kappa}B) in pancreatic cancer cells is associated with a profound resistance towards chemotherapy. In the present study, we show that chemoresistant pancreatic cancer cell lines exhibiting constitutive NF-{kappa}B activity (i.e., PancTu-1, BxPc3, and Capan-1) express significantly elevated levels of the E3-ubiquitin ligase receptor subunit ßTRCP1, compared with pancreatic carcinoma cell lines lacking constitutive NF-{kappa}B activity and chemoresistance (i.e., PT45-P1 and T3M4). If transfected with ßTRCP1, PT45-P1 cells exhibit an elevated NF-{kappa}B activity and become less sensitive towards anticancer drug treatment (i.e., etoposide). Conversely, blockade of ßTRCP1 expression in PancTu-1 cells by transfection with a vector-expressed small interfering RNA reduces NF-{kappa}B activation and chemoresistance. In PancTu-1 cells, ßTRCP1 expression is inhibited, at least in part, by the interleukin-1 (IL-1) receptor(I) antagonist, whereas stimulation of PT45-P1 cells with IL-1ß resulted in an increased expression of ßTRCP1, and transfection of this cell line with ßTRCP1 induced IL-1ß secretion in a NF-{kappa}B–dependent fashion. Thus, via its close and mutual link to IL-1ß secretion, ßTRCP1 expression might substantially contribute to the persistent, IL-1ß–dependent activation of NF-{kappa}B in pancreatic carcinoma cells. In support of this, ßTRCP1 expression is detectable at considerable levels in a great number of pancreatic ductal adenocarcinoma specimens, along with an intense staining for activated NF-{kappa}B. Altogether, our findings of the elevated ßTRCP1 expression in pancreatic carcinoma cells pinpoint to another important mediator of constitutive NF-{kappa}B activation and thereby of chemoresistance.

Key Words: chemoresistance • pancreatic carcinoma • ubiquitin ligase • siRNA


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A sustained elevation of nuclear factor-{kappa}B (NF-{kappa}B) activity is an abundant phenomenon of malignant tumors including leukemias and lymphomas as well as solid tumors, such as prostate, colorectal, and pancreatic carcinoma (1–4). As a major consequence of this constitutive activation of NF-{kappa}B, tumor cells express a great variety of NF-{kappa}B target genes conferring considerable growth advantages, such as antiapoptotic (i.e., cIAP and Bcl-2) and cell cycle promoting (i.e., cyclinD) genes. In addition, permanent activation of NF-{kappa}B is associated with a profound resistance towards chemo- and radiotherapy (5, 6). In this way, pancreatic carcinoma represents a particularly threatening malignant disease inasmuch as pancreatic cancer cells are notoriously resistant to all known anticancer drugs such as gemcitabine, 5-fluorouracil, or etoposide (4, 7). However, from the close association of a chemoresistant phenotype and an elevated NF-{kappa}B activity, new concepts of chemosensitization have been proposed that rely on specific inhibition of NF-{kappa}B activation (5, 6). These concepts include substances such as proteasome inhibitors (i.e., MG132 and YPD431) or I{kappa}B kinase inhibitors (i.e., sulfasalazine; refs. 5–9). To confine the suitability of NF-{kappa}B as a new molecular target in anticancer therapy, a better understanding of the molecular mechanisms leading to constitutive NF-{kappa}B activation is still a major goal. Besides certain genetic alterations such as I{kappa}B gene mutations and Rel-gene amplifications (1–12), many signaling pathways upstream of the I{kappa}B kinase have been supposed to amplify NF-{kappa}B activation, including ras/raf1 (13), mitogen-activated protein kinase (14–16), or Akt/PKB (17).

However, in pancreatic carcinoma cells other mechanisms seem also to be involved (18). From recent studies, evidence accumulated that the autocrine and juxtacrine actions of certain cytokines, such as interleukin-1ß (IL-1ß), confer permanent NF-{kappa}B activation in pancreatic carcinoma along with a profound chemoresistance (19, 20). Furthermore, other molecular and cellular conditions are conceivable as a prerequisite for the maintenance of high NF-{kappa}B activation. This particularly includes molecules involved in the second step of NF-{kappa}B activation—the degradation of I{kappa}B{alpha}. As a consequence of the signal induced I{kappa}B kinase-mediated phosphorylation of I{kappa}B{alpha} [i.e., in response to tumor necrosis factor-{alpha} (TNF{alpha}) or IL-1ß], the inhibitory protein of NF-{kappa}B becomes rapidly polyubiquitinated and then degraded by the 26S proteasome (21). The substrate-specific polyubiquitination of phospho-I{kappa}B{alpha} represents the rate-limiting step in the signal-driven turnover of I{kappa}B and is executed by the E3-ubiquitin ligase multi-protein complex, also known as Skp1-Cullin-F-box protein complex (22). The substrate specificity of I{kappa}B{alpha} polyubiquitination depends on the 67 kDa F-box protein ßTRCP1 (ß-transducin repeat–containing protein) and its closely related homologue ßTRCP2/homologue of Slimb1 (ßTRCP2/HOS; ref. 23). These two proteins bring phospho-I{kappa}B{alpha} into the right position for polyubiquitination via the RING E3-ligase Rbx. Whereas HOS is particularly expressed in the cytoplasm (24), ßTRCP1 is predominantly expressed in the nucleus (24, 25) where it is part of the recently described intranuclear regulation of the I{kappa}B{alpha} shuttling (reviewed in ref. 26). An elevated expression of these F-box proteins may cause a permanent derepression of NF-{kappa}B activation, as reported for pancreatic cancer cells (7).

We herein investigated the role of ßTRCP1 in the constitutive NF-{kappa}B activation in pancreatic cancer cell lines and the development of chemoresistance in these cells. Our findings indicate an important contribution of ßTRCP1 expression to the NF-{kappa}B-dependent chemoresistance, and therefore allow a better understanding of this particular phenotype.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Lines and Culture. The human pancreatic carcinoma cell lines PancTu-1, BxPc3, Capan-1, T3M4, and PT45-P1 and their handling were described previously (7, 27, 28). Cells were kept in culture (37°C, 5% CO2, 85% humidity) using RPMI-40 (PAA Laboratories, Cölbe, Germany) supplemented with 1% glutamine (Life Technologies, Eggenstein, Germany) and 10% FCS (Biochrom KG, Berlin, Germany).

Reagents. Etoposide was from Bristol-Myers-Squibb (Munich, Germany). TNF{alpha}, bovine serum albumin, sulfasalazine, and Leptomycin B were purchased from Sigma-Aldrich Chemie (Steinheim, Germany). Recombinant human IL-1ß and human IL-1 receptor(I) antagonist were obtained from R&D Systems (Wiesbaden, Germany). MG132 was from Biomol (Hamburg, Germany) and Annexin V/propidium iodide (PI) was from Biocarta (Hamburg, Germany).

Generation of ßTRCP1 siRNA and ßTRCP1 Expression Vectors. Synthetic ssDNA oligonucleotides encoding duplexed siRNA targeting to ßTRCP1 mRNA (position 441-461): 5'-tcgagtgtcattaccaacatgggcacataagcccatgttggtaatgacacttttt-3'/5'-ctagaaaaagtgtcattaccaacatgggcttatgtgcccatgttggtaatgacac-3', or encoding a scrambled control RNA: 5'-tcgagtgtcattaccaacatgggcacataagtgtcattaccaacatgggcttttt-3'/5'-ctagaaaaagcccatgttggtaatgacacttatgtgcccatgttggtaatgacac-3', both flanked by restriction sites for 5'SalI/3'XbaI, were annealed and then ligated into the pSHH1 expression vector (Imgenex). The coding region of ßTRCP1 mRNA (Genbank accession no. Y14153) from PancTu-1 cells was cloned via reverse transcription-PCR (RT-PCR; forward/reverse primers: 5'-agtggcctcggcgattatggac-3', position 54/5'-ggtcagtgtatggttatttatctgg-3', position 1,796) into the pCR3.1 expression vector (Invitrogen, Karlsruhe, Germany). All constructs were checked by automated DNA-sequencing (AbiPrism 3770).

Cell Transfection. PT45-P1 and PancTu-1 cells, seeded into six-well plates (2.5 x 105 cells/well), were grown overnight, followed by mono- or cotransfection with 5 µL/well of DIMRIE reagent (Invitrogen) and 1 µg/well of the following plasmids: pcDNA3.1-ßTRCP1, pcDNA3.1-{Delta}NI{kappa}B{alpha} (I{kappa}B{alpha} superrepressor; ref. 7), pcDNA3.1-lacZ, pSHH1-ßTRCP1-siRNA, pSHH1-control-siRNA, and pSHH1-empty. Upon transfection for 8 to 16 hours, 1 mL medium containing 20% FCS was added and cells were left untreated for at least 8 hours before their further handling.

Preparation of Nuclear and Cytoplasmic Extracts. Hypotonic lysis of cells and subsequent fractionation into cytoplasmic and nuclear extracts were done as described (29, 30). The protein content of each fraction was determined by the Dc-Protein assay (Bio-Rad Laboratories, Munich, Germany), and the quality of the separation in nuclear and cytoplasmic protein was checked by a commercial enzyme assay (Sigma-Aldrich) for lactate dehydrogenase (LDH) activity following the instructions of the manufacturer. LDH activity was almost completely retained in the cytoplasmic fraction (20-30 milliunits/µg protein), whereas no significant LDH contamination was found in the nuclear material (<1 milliunit/µg protein). For comparative Western blot analysis of nuclear and cytoplasmic extracts, stimulatory protein 1 and LDH, respectively, were used as protein loading control.

Gel-Shift and Supershift Assay. Nuclear extracts were incubated with the {gamma}32P-labeled oligonucleotide 5'-AGTTGAGGGGACTTTCCCAGGC-3' containing a consensus NF-{kappa}B binding site (Promega, Mannheim, Germany). After incubation at room temperature (30 minutes), samples were separated by gel electrophoresis at 100 V, 4°C. Gels were dried and exposed to X-ray Hyperfilm (Amersham, Freiburg, Germany). For supershift assays, p65/p50 antibodies (Santa Cruz, Heidelberg, Germany) were added (1 hour, 4°C) before electrophoresis.

ELISA-Based Nuclear Factor-{kappa}B Assay. Additionally to gel-shift assays, an ELISA-based kit was used for quantitative detection of NF-{kappa}B activity (TransAM NF-{kappa}B kit, Active Motif Europe, Rixensart, Belgium). For each sample, 20 µL of nuclear extracts (= 5 µg protein) were used according to the instructions of the manufacturer. For the detection of activated NF-{kappa}B, antibodies against the p65/RelA or p50 subunit (Santa Cruz) were used, followed by a secondary antibody conjugated to horseradish peroxidase. The colorimetric readout (450 nm) was done with an ELISA plate-reader.

Interleukin-1ß ELISA. To measure human IL-1ß secretion, cell culture supernatants were precleared by centrifugation (10,000 rpm, 10 minutes) and submitted to the Quantikine-HS human IL-1ß immunoassay (R&D Systems) following the instructions of the manufacturer. IL-1ß concentrations were normalized to the cell numbers determined in parallel.

Measurement of Apoptosis. Cells (2-5 x 105) were stained with Annexin V/PI following the instructions of the manufacturer and analyzed by fluorescence flow cytometry (GalaxyArgon Plus; DakoCytomation, Hamburg, Germany) using the FLOMAX software. Cells exhibiting high specific Annexin V staining were regarded as apoptotic.

Immunoprecipitation and Western Blotting. Mock- or ßTRCP1-transfected PT45-P1 cells (2 x 105) were resuspended in 500 µL of 50 mmol/L Tris-HCl (pH 7.4), 1% (v/v) Triton X-100, 150 mmol/L NaCl, 1 mmol/L EDTA, 0.1 mmol/L phenylmethylsulfonyl fluoride (TNET buffer), 0.1 mmol/L Na3VO4 supplemented with protease inhibitor cocktail (Complete-Mini/EDTA-free, Roche, Mannheim, Germany), and lysed by sonification (3 x 10 seconds). Then, lysates were cleared by centrifugation (microfuge, 13°000 rpm for 10 minutes, 4°C) and adjusted to equal protein concentrations using the Dc-Protein assay (Bio-Rad Laboratories). Afterwards, lysates were preadsorbed to protein A-agarose beads at room temperature for 1 hour and the resulting supernatant was incubated overnight at 4°C with anti-I{kappa}B{alpha}-agarose conjugate (C21, Santa Cruz). The beads were recovered by centrifugation, washed five times with TNET buffer, and prepared for electrophoresis in 50 µL SDS sample buffer (95°C, 5 minutes). For Western blotting, nuclear extracts or total cell lysates in SDS sample buffer (without bromphenol blue) were prepared and protein concentrations were determined. Ten micrograms of protein were used of each sample and an appropriate volume of SDS sample buffer containing 0.2 mg/mL bromphenol blue and 2.5% ß-mercaptoethanol was added. These samples or the immunoprecipitated proteins were run on SDS-PAA gels (4-20% gradient) and electroblotted as described previously (18). For the detection of ßTRCP1 and ßTRCP2/HOS, a polyclonal goat antibody (N15 and C20, Santa Cruz) was used at a concentration of 1 µg/mL in 0.05% Tween 20 in TBS (TBST) containing 2% nonfat milk powder (2% Skim-TBST). Polyclonal antibodies were also used for the detection of phospho-I{kappa}B{alpha} (Cell Signaling, Frankfurt, Germany) and I{kappa}B{alpha} (C21, Santa Cruz) at 0.1 and 0.4 µg/mL in 5% bovine serum albumin-TBST, respectively, and polyubiquitinated I{kappa}B{alpha} was detected with a monoclonal polyubiquitin antibody (clone P4D1, Santa Cruz) at 0.2 µg/mL in 2% Skim-TBST. As control of equal protein load, an {alpha}-tubulin monoclonal antibody or a LDH monoclonal antibody (Sigma-Aldrich) was used at 0.01 µg/mL or a stimulatory protein 1 antibody (Santa Cruz) at 0.2 µg/mL in 5% bovine serum albumin-TBST. All antibodies were incubated overnight at 4°C. For the detection of these primary antibodies, anti-goat (Santa Cruz), anti-mouse, and anti-rabbit horseradish peroxidase-linked antibodies (Cell Signaling) were used at a dilution of 1:2,000 in 5% Skim-TBST at room temperature for 1 hour.

Reverse Transcription-PCR. Total RNA was isolated and reverse transcribed into single-stranded cDNA as described (31). PCR for the detection of ßTRCP1 was conducted as follows: 2 µL of cDNA were submitted to thermal cycling (95°C, 2 minutes/95°C, 60 seconds; 58°C, 30 seconds; 72°C 30 seconds; for 25 cycles/72°C, 10 minutes) using 1.5 units of Taq polymerase (Invitrogen) and 0.5 µmol/L forward/reverse primers (5'-agtggcctcggcgattatggac-3', position 54/5'-ggtcagtgtatggttatttatctgg-3', position 1,796). For control, ß-actin was analyzed with RT-PCR amplimer-primers (BD Clontech, Heidelberg, Germany). PCR products were separated by 8% PAGE and visualized by ethidium bromide.

Immunohistochemistry. For ßTRCP1 and NF-{kappa}B immunostaining, 26 ductal pancreatic carcinoma and five normal pancreatic tissue samples obtained from surgical specimens were used, according to a protocol approved by the ethics committee of the University Hospitals, Kiel (Permission no. 110/99). Samples were snap frozen in a mixture of isopentane and dry ice and stored at –80°C. Five-micrometer cryostat sections were cut, air dried, and fixed in acetone at room temperature for 10 minutes, then incubated with 0.03% hydrogen peroxide (DakoCytomation) four times for 20 minutes. Intrinsic avidin and biotin were blocked with the Avidin/Biotin-Blocking Kit (Vector Laboratories/Alexis, Grünberg, Germany). Serum blocking and detection were done with a peroxidase-based rabbit Vectastain Kit (Vector Laboratories). Rabbit polyclonal anti-ßTRCP1 antibody (C18, Santa Cruz) was applied in 3 µg/mL concentration for 1 hour at room temperature. For negative control, anti ßTRCP1 antibody was coincubated with 5-fold weight excess of specific blocking peptide (Santa Cruz) for 1 hour before immunostaining. A mouse p65/RelA antibody (Chemicon, Hofheim, Germany), recognizing the unmasked, activated NF-{kappa}B-subunit p65, was applied in 25 µg/mL concentration for 1 hour at room temperature and developed with mouse Envision Kit (DakoCytomation). The primary antibody was omitted for negative control. After staining, sections were counterstained in 50% haemalaun (Merck, Darmstadt, Germany) and mounted with glycerol gelatin. Immunohistochemical stains were evaluated by a semiquantitative method. ßTRCP1 and NF-{kappa}B expression in tumor cells was rated as mild (<10% of cells stained), moderate (10-50%), or strong (>50%).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Elevated ßTRCP1 Expression Levels in Pancreatic Carcinoma Cell Lines Exhibiting Constitutive Nuclear Factor-{kappa}B Activation. By means of Western blotting, ßTRCP1 expression was analyzed in various pancreatic carcinoma cell lines. Nuclear extracts from chemoresistant Capan-1, BxPc3, and PancTu-1 cells (7) exhibiting high constitutive NF-{kappa}B activity (Fig. 1A) were compared with those from chemosensitive PT45-P1 and T3M4 cells lacking constitutive NF-{kappa}B activity (Fig. 1A). As shown in Fig. 1B, a strong signal for ßTRCP1 expression, corresponding to a 67 kDa protein, was detectable in Capan-1, BxPc3, and PancTu-1 cells. The specificity of this band was verified by antibody incubation in the presence of an excess of a blocking peptide (data not shown) as well as by transfection of ßTRCP1 yielding a protein of identical size (see below). In striking contrast, ßTRCP1 expression was not detectable at significant levels in PT45-P1 and T3M4 cells (Fig. 1B).



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  		Figure 1. Expression levels of ßTRCP1 in pancreatic carcinoma cell lines exhibiting distinct basal NF-{kappa}B activities. Nuclear extracts of the human pancreatic carcinoma cell lines Capan-1, BxPc3, PanCTu-1, PT45-P1, and T3M4 were submitted to (A) gel shift assay, using a labelled specific consensus oligonucleotide for NF-{kappa}B binding, or (B) Western blotting for the detection of ßTRCP1 (antibody N-15, Santa Cruz). The detection of {alpha}-tubulin was used as a control for equal protein load. Representative results from three independent experiments are shown.

 
Effect of Increased ßTRCP1 Expression on Nuclear Factor-{kappa}B Activity and Chemoresistance in PT45-P1 Cells. PT45-P1 cells lacking significant ßTRCP1 expression were transfected with an expression vector for ßTRCP1 or an empty vector (mock). As shown by Western blot (Fig. 2A), a 67 kDa protein was detected in nuclear extracts from ßTRCP1 transfectants, similar in size to the protein identified in the resistant cell lines described above. To check whether ßTRCP1 expression in PT45-P1 cells confers increased NF-{kappa}B activity, gel-shift assays were done. As shown in Fig. 2B, ßTRCP1-transfected PT45-P1 cells exhibited a significantly stronger NF-{kappa}B binding activity than mock-transfected or untransfected PT45-P1 cells. Similarly, an ELISA-based NF-{kappa}B binding assay (TransAM, ActiveMotif), specifically detecting DNA-bound p65/p50, revealed an increased level of activated NF-{kappa}B in ßTRCP1-transfected cells compared with mock transfectants (Fig. 2C), supporting the data revealed by gel-shift assay.



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  		Figure 2. Effect of increased ßTRCP1 expression on NF-{kappa}B activity and chemoresistance in PT45-P1 cells. PT45-P1 cells [untransfected (ut)] were transfected with the empty vector (mock) or with ßTRCP1. Nuclear extracts from these cells were submitted to (A) Western blotting for the detection of ßTRCP1 or (B) NF-{kappa}B gel-shift/supershift assay. Representative results from three independent experiments are shown. C and D, PT45-P1 cells were mock-transfected or transfected with ßTRCP1, either alone or in combination with pCMV-lacZ (as control) or pcDNA3.1-{Delta}N-I{kappa}B{alpha} (I{kappa}BSR). C, mock- or ßTRCP1-transfected PT45-P1 cells were either left untreated (w/o) or treated with 0.5 mmol/L sulfasalazine (24 hours). Nuclear extracts from these cells and from I{kappa}BSR/lacZ cotransfectants were analyzed by ELISA-based NF-{kappa}B binding assay [columns, mean (n = 4); bars, SD]. D, mock- or ßTRCP1-transfected PT45-P1 cells were treated with etoposide (24 hours) or without, either alone or after preincubation (–1 hour) with 0.5 mmol/L sulfasalazine. PT45-P1 cells cotransfected with ßTRCP1/mock together with I{kappa}BSR/lacZ were treated without or with 20 µg/mL etoposide for 24 hours. Apoptosis was determined by Annexin V/PI staining and subsequent flow cytometry. Data are presented as etoposide-induced apoptosis (%) over basal [columns, mean (n = 4); bars, SD].

 
To elucidate whether overexpression of ßTRCP1 influences the sensitivity towards anticancer drugs, PT45-P1 cells were transfected with ßTRCP1 and apoptosis was determined by Annexin V/PI staining and subsequent fluorescence flow cytometry. As shown in Fig. 2D, mock-transfected PT45-P1 cells exhibited strong apoptosis (up to 50% apoptotic cells) on treatment with the anticancer drug etoposide (20 g/mL, 24 hours). In contrast, PT45-P1 cells transfected with ßTRCP1 became much less sensitive towards anticancer drug-induced apoptosis (20-25% apoptotic cells). To verify that this desensitization by ßTRCP1 is NF-{kappa}B–dependent, PT45-P1 cells were either cotransfected with the I{kappa}B{alpha} superrepressor or were pretreated with the I{kappa}B kinase inhibitor sulfasalazine before etoposide incubation. As shown in Fig. 2D, the decreased sensitivity of ßTRCP1 transfectants in response to etoposide treatment (20-25% apoptotic cells) was almost completely reversed by sulfasalazine (>45% apoptotic cells) and strongly inhibited by the I{kappa}B{alpha} superrepressor (35-40% apoptotic cells). In parallel, significant suppression of the elevated NF-{kappa}B activity in ßTRCP1-transfected PT45-P1 cells by sulfasalazine or the I{kappa}B superrepressor could be verified by the ELISA-based NF-{kappa}B binding assay (Fig. 2C).

Enhanced Turnover of I{kappa}B{alpha} in PT45-P1 Cells Transfected with ßTRCP1. To elucidate whether the expression of ßTRCP1 affects I{kappa}B{alpha} expression, transfected PT45-P1 cells were treated with TNF{alpha} and MG132, or not. Western blot analysis of total cell extracts from these cells (Fig. 3A) revealed significantly lower levels of I{kappa}B{alpha} in PT45-P1 cells transfected with ßTRCP1 than in mock transfectants. This effect was already seen in untreated cells and was much more pronounced in cells treated with the proteasome inhibitor MG132 alone (45 minutes) or with TNF{alpha} alone (15 minutes), or in cells preincubated with MG132 (30 minutes) and treated with TNF{alpha} (15 minutes). The corresponding levels of phospho-I{kappa}B{alpha} were similarly reduced in ßTRCP1 transfectants. Whereas in untreated cells no significant amount of phospho-I{kappa}B{alpha} could be detected, a fairly detectable phospho-I{kappa}B{alpha} band was present in MG132-treated cells, and this band was significantly weaker in ßTRCP1-transfected PT45-P1 cells than in mock transfectants. The amount of phospho-I{kappa}B{alpha} in PT45-P1 cells treated with TNF{alpha} alone or with the combination of MG132 and TNF{alpha} was also lower if ßTRCP1 was overexpressed. A similar ßTRCP1-dependent pattern of the I{kappa}B{alpha} turnover has been recently found in 293T cells (22) or in murine cells (32).



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  		Figure 3. Enhanced turnover of I{kappa}B{alpha} and phospho-I{kappa}B{alpha} in PT45-P1 cells expressing ßTRCP1. PT45-P1 cells were mock- or ßTRCP1-transfected, followed by incubation with TNF{alpha} (30 ng/mL, 15 minutes), with MG132 (5 µmol/L, 45 minutes), or with TNF{alpha} upon preincubation (30 minutes) with MG132. A, total cell lysates were submitted to Western blotting using I{kappa}B{alpha}, phospho-I{kappa}B{alpha}, and tubulin antibodies. B, total cell lysates were immunoprecipitated with I{kappa}B{alpha} antibodies, submitted to Western blotting, and probed with I{kappa}B{alpha} or polyubiquitin antibodies; Ig-hc, immunoglobuline heavy-chain. C, mock- or ßTRCP1-transfected PT45-P1 cells were incubated with Leptomycin B (LmB; 50 ng/mL, 16 hours) or not. Cytosolic and nuclear extracts were submitted to Western blotting for the detection of I{kappa}B{alpha}. The detection of LDH and stimulatory protein 1 was used as control for the purity of cytoplasmic and nuclear extracts and for equal protein load. The results shown are representative for two independent experiments.

 
Next, total cellular lysates from transfected PT45-P1 cells were submitted to immunoprecipitation using an anti-I{kappa}B{alpha} conjugate. Again, distinct levels of phospho-I{kappa}B{alpha} and I{kappa}B{alpha} were detectable in the immunoprecipitated material (Fig. 3B). In ßTRCP1 transfectants, the level of phospho-I{kappa}B{alpha} already detectable in the presence of MG132 alone—indicating intrinsic I{kappa}B{alpha}-phosphorylation in unstimulated PT45-P1 cells—was significantly lower than in mock transfectants. In the presence of both MG132 and TNF{alpha}, strongly suppressed levels of phospho-I{kappa}B{alpha} were observed in PT45-P1 cells expressing ßTRCP1 in comparison with mock-transfected cells. To further elucidate the influence of ßTRCP1 transfection on I{kappa}B{alpha} polyubiquitination, the immunoprecipitated protein from MG132-treated cells (either alone or together with TNF{alpha}) was probed with a polyubiquitin-specific antibody. As shown in Fig. 3B, the amount of polyubiquitinated protein exhibiting high molecular weight (>100 kDa) was rather low in mock-transfected PT45-P1 cells treated with MG132 alone, whereas under these conditions a strong ubiquitin positive protein signal was detectable in ßTRCP1 transfectants. This higher level of polyubiquitinated high molecular weight protein was also seen in ßTRCP1-transfected PT45-P1 cells subject to MG132/TNF{alpha} coincubation, producing a much stronger polyubiquitination pattern in both transfectants.

To further investigate whether the reduced level of I{kappa}B{alpha} in ßTRCP1-expressing PT45-P1 cells is the result of an enhanced nuclear turnover, mock or ßTRCP1 transfectants were treated with Leptomycin B, an inhibitor of the nuclear exporter Crm-1, and the distribution of I{kappa}B{alpha} in nuclear and cytoplasmic fractions was analyzed. The purity of these fractions was confirmed by excluding significant LDH contamination in nuclear extracts and stimulatory protein 1 contaminations in cytoplasmic samples (30, 33). As shown by Western blotting (Fig. 3C), after Leptomycin B treatment an increase in the amount of I{kappa}B{alpha} in nuclear extracts from both transfectants occurred and, in turn, a decrease in cytosolic extracts. Of note, in ßTRCP1-transfected PT45-P1 cells, the amount of nuclear I{kappa}B{alpha} was significantly lower than in mock-transfected cells, whereas this decrease in the amount of I{kappa}B{alpha} was not seen in cytosolic extracts upon Leptomycin B treatment.

Effect of Disrupted ßTRCP1 Expression on Nuclear Factor-{kappa}B Activity and Chemoresistance in PancTu-1 Cells. Next, it was elucidated whether disruption of ßTRCP1 expression in PancTu-1 cells affects the elevated basal NF-{kappa}B activity and the chemoresistance of this cell line. For this purpose, an expression vector for a ßTRCP1-siRNA was generated and transfected into PancTu-1 cells. Western blot analysis (Fig. 4A) revealed that in total cellular extracts from these cells, the amount of the 67 kDa ßTRCP1 protein was strongly reduced in comparison with PancTu-1 cells expressing a control-siRNA or with mock-transfected cells. In contrast, the smaller ßTRCP-variant ßTRCP2/HOS (58 kDa) was not affected by the ßTRCP1-siRNA, thus underscoring its target specificity. The effect of disrupted ßTRCP1 on NF-{kappa}B activity in PancTu-1 cells was further checked by gel-shift assay. As shown in Fig. 4B, the intensity of the detectable NF-{kappa}B-DNA complex was significantly decreased in PancTu-1 cells transfected with ßTRCP1-siRNA compared with mock-transfected PancTu-1 cells or with cells transfected with the control-siRNA. This decreased NF-{kappa}B binding activity in ßTRCP1-siRNA-transfected PancTu-1 cells was also verified by the ELISA-based NF-{kappa}B binding assay (Fig. 4C). As an obvious consequence of the disrupted ßTRCP1 expression in PancTu-1 cells, the chemoresistance of this cell line was significantly decreased. As shown in Fig. 4D, PancTu-1 cells expressing the ßTRCP1-siRNA exhibited a significantly higher rate of apoptosis in response to etoposide treatment (30% apoptotic cells) than transfectants with control-siRNA (16% apoptotic cells) or mock-transfected PancTu-1 cells (14% apoptotic cells).



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  		Figure 4. Effect of disrupted ßTRCP1 expression on NF-{kappa}B activity and chemoresistance in PancTu-1 cells. PancTu-1 cells were transfected either with an empty control vector (mock), with a control-siRNA, or with the ßTRCP1-siRNA construct. A, Western blot analysis was done with total cell lysates using ßTRCP1 and ßTRCP2/HOS antibodies (N15 and C20, respectively; Santa Cruz). The detection of tubulin was used as a control for equal protein load. Nuclear extracts were used for the detection of NF-{kappa}B-DNA binding activity by (B) gel-shift assay and by (C) the ELISA-based NF-{kappa}B assay. The results shown are representative for two independent experiments. D, the same transfectants were treated with etoposide (24 hours) or not, and apoptosis was determined by Annexin V/PI staining and subsequent flow cytometry [columns, mean (n = 4); bars, SD].

 
Interrelation of IL-1ß Secretion and ßTRCP1 expression in PancTu-1 and PT45-P1 Cells. Owing to the elevated NF-{kappa}B activity in some pancreatic carcinoma cells recently shown to be related to the autocrine action of IL-1ß, we elucidated the effect of IL-1ß on ßTRCP1 expression. As shown by Western blot (Fig. 5A, left) and RT-PCR analysis (Fig. 5B, left), treatment of PancTu-1 cells with the IL-1 receptor(I) antagonist significantly reduced the expression of ßTRCP1 within 16 hours, along with a loss of NF-{kappa}B activity and chemoprotection, as has been shown recently (19). This mode of ßTRCP1 expression was not affected by sulfasalazine (result not shown), indicating its independence from NF-{kappa}B. In addition, treatment of PT45-P1 cells with IL-1ß (10 ng/mL) increased ßTRCP1 protein levels within 16 hours (Fig. 5A, right). This increased expression was also seen on the mRNA level, as shown by RT-PCR analysis (Fig. 5B, right). Within 6 hours after IL-1ß administration, the ßTRCP1 mRNA content was elevated in PT45-P1 cells and this increase persisted for at least 18 hours. Under these conditions, PT45-P1 gained an elevated NF-{kappa}B activity and significant protection from anticancer drug-induced apoptosis, as shown recently (7, 18). Furthermore, transfection of PT45-P1 cells with ßTRCP1 markedly elevated the release of IL-1ß in comparison with mock-transfected cells (Fig. 5C), an effect that was inhibited by sulfasalazine treatment, confirming NF-{kappa}B dependency. In turn, the increase of NF-{kappa}B binding activity by ßTRCP1 in PT45-P1 cells could be partially inhibited by the IL-1 receptor(I) antagonist (Fig. 5D), thus pinpointing to the NF-{kappa}B-induced autocrine action of IL-1ß, as described previously (19). If incubated with the IL-1 receptor(I) antagonist, the protection of PT45-P1 cells against etoposide induced apoptosis by ßTRCP1 (<25% apoptotic cells) was also diminished (>35% apoptotic cells), as shown in Fig. 5E.



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  		Figure 5. Interrelation of IL-1ß secretion and ßTRCP1 expression in PancTu-1 and PT45-P1 cells. A and B, PancTu-1 cells were treated with 125 ng/mL human IL-1 receptor(I) antagonist (IL1RA) or not (w/o), and PT45-P1 cells were treated with 10 ng/mL human IL-1ß, or not. A, nuclear extracts were submitted to Western blotting or (B) total RNA prepared on the indicated periods was submitted to RT-PCR using specific ßTRCP1 primers and ß-actin primers as control. Representative results from three independent experiments are shown. C, PT45-P1 cells, mock- or ßTRCP1-transfected, were either left untreated or treated with sulfasalazine (0.5 mmol/L, 24 hours). The amount of IL-1ß (pg human IL-1ß/106 cells) in the supernatants of these cells was determined. D, PT45-P1 cells, mock- or ßTRCP1-transfected, were treated with the IL-1 receptor(I) antagonist (18 hours) or not, and nuclear extracts were analyzed by the ELISA-based NF-{kappa}B assay. E, mock- or ßTRCP1-transfected PT45-P1 cells were treated with etoposide or without followed by administration of the IL-1 receptor(I) antagonist 8 hours thereafter, or not (w/o). Apoptosis was determined 24 hours after etoposide administration by Annexin V/PI staining and subsequent flow cytometry [C-E, columns, mean (n = 4); bars, SD].

 
ßTRCP1 Expression Is Abundantly Present in Human Pancreatic Ductal Adenocarcinoma Specimens. To further investigate whether pancreatic carcinoma is characterized by an increased ßTRCP1 expression, tissue sections of snap frozen surgical material from pancreatic cancer patients were analyzed by immunohistochemistry. In 17 of 26 analyzed specimens, pancreatic ductal adenocarcinomas exhibited significant ßTRCP1 expression, as shown by the intensive and epitope-specific immunostaining with the ßTRCP1 antibody (Fig. 6A). In 11 of these positively tested carcinomas, ßTRCP1 expression was more abundant, as indicated by the dense immunostaining of 10% to 50% or even >50% of the tumor cells. In six tumor specimens, the density of ßTRCP1 immunostaining was lower, as indicated by the detection of <10% positive tumor cells, and in nine tumor specimens no significant ßTRCP1 immunostaining was detectable. In comparison, all carcinoma specimens were positively tested for activated NF-{kappa}B (Fig. 6C), and more than 90% of these carcinomas were strongly stained (>50% of the tumor cells) with the antibody recognizing the unmasked, I{kappa}B-freed, p65/RelA subunit of activated NF-{kappa}B. In normal pancreatic tissue, no expression of ßTRCP1 was found at all and activated NF-{kappa}B was detected in several cases in pancreatic acini (data not shown).



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  		Figure 6. Immunohistochemical stain of ßTRCP1 and NF-{kappa}B in human pancreatic ductal adenocarcinomas. A representative immunohistochemical staining shows (A) strong ßTRCP1 immunoreactivity in tumor cells; (B) negative control by preabsorption of the ßTRCP1 antibody with a specific blocking peptide before staining; (C) strong immunoreactivity for activated NF-{kappa}B (unmasked p65/RelA) in tumor cells; and (D) negative control of the NF-{kappa}B stain by omitting the primary antibody.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Pancreatic ductal adenocarcinoma is characterized by an extremely malignant phenotype and a desperately poor prognosis. Mostly (>80%), the disease is diagnosed in an advanced stage and the only therapeutic options then rely on palliative chemotherapy treatments (34, 35). The major limitation in the efficacy of chemotherapy is the innate and acquired resistance of pancreatic carcinoma cells to apoptosis-inducing anticancer drugs. In this context, constitutive activation of NF-{kappa}B is of particular importance for a chemoresistant phenotype, as shown for the pancreatic ductal carcinoma cell lines PancTu-1, BxPc3, and Capan-1 (7, 18) .

Our present study addressed the mechanisms of this constitutive NF-{kappa}B activation. We were able to show that chemoresistant pancreatic carcinoma cell lines are characterized by a significantly elevated expression of the E3-ubiquitin ligase receptor subunit ßTRCP1 (Fig. 1). This protein is of particular importance for the activation of NF-{kappa}B as it recruits phosphorylated I{kappa}B{alpha} to the ubiquitin conjugating E3-ligase (SCF) complex, followed by the polyubiquitination of I{kappa}B{alpha} and its rapid degradation by the 26S proteasome (21, 22). As shown for PancTu-1 cells, down-regulation of ßTRCP1 expression by siRNA caused a significant decrease in NF-{kappa}B activity and an increased sensitivity towards anticancer drugs such as etoposide (Fig. 4). Conversely, in those pancreatic carcinoma cell lines lacking high ßTRCP1 expression, constitutive NF-{kappa}B activation, and chemoresistance (i.e., PT45-P1 cells), transfection with ßTRCP1 strongly increased the NF-{kappa}B activity and conferred a more chemoresistant phenotype (Fig. 2). Preceding this altered phenotype, the turnover of I{kappa}B{alpha} along with its polyubiquitination was enhanced in ßTRCP1-transfected PT45-P1 cells (Fig. 3), indicating a reduced NF-{kappa}B repression. Consequently, inhibition of these molecular events (i.e., by sulfasalazine or the I{kappa}B{alpha} superrepressor) abrogated the NF-{kappa}B-dependent chemoresistance induced by ßTRCP1 (Fig. 2).

ßTRCP1 is supposed to be involved in the intranuclear derepression of NF-{kappa}B activity (24, 25) that mainly affects the shuttling of free I{kappa}B{alpha} between the cytoplasm, where it prevents NF-{kappa}B from nuclear entry, and the nucleus where it retrieves nuclear NF-{kappa}B back to the cytoplasm. However, evidence accumulated indicating that even I{kappa}B{alpha}-bound NF-{kappa}B enters the nucleus, either via a nuclear localization signal of I{kappa}B{alpha} or an incompletely masked p50 subunit (26, 36, 37). However, this NF-{kappa}B/I{kappa}B{alpha} complex is unable to stay in the nucleus, due to being retrieved from there by the nuclear export sequence of I{kappa}B{alpha} (26, 38), unless intranuclear I{kappa}B{alpha} is recruited by ßTRCP1 to the resident nuclear polyubiquitination complex, followed by its proteasomal degradation in the nucleus (24, 33). Another F-box protein, designated HOS (23), is predominantly expressed in the cytoplasm and is regarded as mediator of cytoplasmic polyubiquitination of I{kappa}B{alpha} and I{kappa}Bß. In those pancreatic carcinoma cell lines investigated in the present study, HOS (ßTRCP2) expression was less different between chemoresistant and chemosensitive cell lines than ßTRCP1 expression (data not shown), suggesting that the constitutive NF-{kappa}B activation seen in chemoresistant cells is rather due to the forced derepression of NF-{kappa}B activity in the nucleus by ßTRCP1. This is supported by the finding that the enhanced I{kappa}B{alpha} turnover was more prominent in the nucleus of ßTRCP1-transfected PT45-P1 cells subject to Leptomycin B treatment (Fig. 3C). Such an intranuclear derepression requires a preceding signal that induces I{kappa}B{alpha} phosphorylation. We have recently shown that PancTu-1 cells as well as several other pancreatic carcinoma cell lines sharing the chemoresistant and NF-{kappa}B positive phenotype possess an autocrine activation loop involving IL-1ß (19, 20) . It can be assumed that IL-1ß releases a discrete NF-{kappa}B activating signal that is maintained at a higher level by ßTRCP1. Intriguingly, the elevated ßTRCP1 expression in PancTu-1 cells is to some extent related to the autocrine action of IL-1ß as shown by the blocking effect of the IL-1 receptor(I) antagonist (Fig. 5A and B). In further support of this notion, exogenously added IL-1ß induces ßTRCP1 expression in PT45-P1 cells (Fig. 5A and B). Obviously, by inducing ßTRCP1 expression, IL-1ß contributes to a persistent NF-{kappa}B activation that, in turn, maintains IL-1ß expression. Even in unstimulated PT45-P1 cells lacking prior IL-1ß secretion but exhibiting significant I{kappa}B{alpha} phosphorylation (i.e., due to an amplified ras/raf1 pathway; Fig. 3A), ßTRCP1 overexpression alone is sufficient for inducing constitutive NF-{kappa}B activation and chemoresistance. In this way, ßTRCP1 is essentially involved in a potent loop of amplified NF-{kappa}B activation, due to a reduced NF-{kappa}B repression on the one hand (Figs. 2 and 3) and an increased secretion of cytokines such as IL-1ß on the other hand (Fig. 5A-C). Thereby, ßTRCP1 might participate in the development of chemoresistance and, possibly, also in pancreatic carcinogenesis.

An involvement of ßTRCP in tumorigenesis has been recently proposed for skin, gastric, and colon cancer (39–41). In addition, deregulated ßTRCP expression has been linked to other tumor promoting pathways such as down-regulation of the disc large tumor suppressor (hDlg; ref. 42). The implication of ßTRCP1 in tumorigenesis of pancreatic cancer and development of chemoresistance is underscored by the finding that a great number of tumor sections from pancreatic carcinoma patients exhibit increased levels of ßTRCP1 expression, as we showed in this study by immunohistochemical analysis of resected tumor tissues. Being reminiscent of the cell lines described above, significant ßTRCP1 expression was detected in 65% of the tumor specimens along with strong staining for activated NF-{kappa}B (Fig. 6). This was particularly seen in all low differentiated G3 tumors, whereas normal pancreatic tissue did not express ßTRCP1 at all. Furthermore, chemoresistant pancreatic carcinoma cell lines as well as pancreatic ductal adenocarcinoma tissues have recently been shown to express IL-1ß quite abundantly (19, 20). Altogether, these findings provide new insights into the molecular mechanism by which pancreatic carcinoma cells develop chemoresistance in a NF-{kappa}B–dependent fashion. As key elements, autocrine IL-1ß secretion (i.e., induced by stroma-tumor interactions; ref. 20) and ßTRCP1-mediated derepression of NF-{kappa}B confer a persistent NF-{kappa}B activation and thereby resistance to anticancer drugs.


   Acknowledgments
 
Grant support: German Research Society (DFG-Scha677/7-2) and Kiel Medical Faculty grant IZKF/B9.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 5/10/04. Revised 9/10/04. Accepted 12/10/04.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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