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Molecular Cancer Research 5, 823-832, August 1, 2007. doi: 10.1158/1541-7786.MCR-06-0352
© 2007 American Association for Cancer Research
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Signaling and Regulation

Activated Signal Transducers and Activators of Transcription 3 Signaling Induces CD46 Expression and Protects Human Cancer Cells from Complement-Dependent Cytotoxicity

Ralf Buettner1, Mei Huang2, Tanya Gritsko2, Jim Karras3, Steve Enkemann2, Tania Mesa2, Sangkil Nam1, Hua Yu1 and Richard Jove1

1 Beckman Research Institute, City of Hope Comprehensive Cancer Center, Duarte, California; 2 H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida; and 3 Antisense Drug Discovery, Isis Pharmaceuticals, Carlsbad, California

Requests for reprints: Ralf Buettner, Molecular Medicine, Beckman Research Institute, City of Hope Comprehensive Cancer Center, Duarte, CA 91010. Phone: 626-256-4673; Fax: 626-256-8708. E-mail: rbuettner{at}coh.org


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
CD46 is one of the complement-regulatory proteins expressed on the surface of normal and tumor cells for protection against complement-dependent cytotoxicity. Cancer cells need to access the blood circulation for continued growth and metastasis, thus exposing themselves to destruction by complement system components. Previous studies have established that the signal transducers and activators of transcription 3 (STAT3) transcription factor is persistently activated in a wide variety of human cancer cells and primary tumor tissues compared with their normal counterparts. Using microarray gene expression profiling, we identified the CD46 gene as a target for activated STAT3 signaling in human breast and prostate cancer cells. The CD46 promoter contains two binding sites for activated STAT3 and mutations introduced into the major site abolished STAT3 binding. Chromatin immunoprecipitation confirms binding of STAT3 to the CD46 promoter. CD46 promoter activity is induced by activation of STAT3 and blocked by a dominant-negative form of STAT3 in luciferase reporter assays. CD46 mRNA expression is induced by interleukin-6 and by transient transfection of normal human epithelial cells with a persistently active mutant construct of STAT3, STAT3C. Furthermore, we show that inhibition of STAT3-mediated CD46 cell surface expression sensitizes DU145 prostate cancer cells to cytotoxicity in an in vitro complement lysis assay using rabbit anti-DU145 antiserum and rabbit complement. These results show that activated STAT3 signaling induces the CD46 promoter and protects human cancer cells from complement-dependent cytotoxicity, suggesting a potential mechanism whereby oncogenic signaling contributes to tumor cell evasion of antibody-mediated immunity. (Mol Cancer Res 2007;5(8):823–32)


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
The complement system is part of the innate immune system and comprises a set of proteins that is designed to eliminate bacteria and other invading cells (1). Complement components are present in blood and deposition of complement proteins, such as C3b or C4b, on cell surfaces can initiate direct lysis of cells via complement-dependent cytotoxicity in the presence or absence of surface-bound antibodies (1). Cancer cells frequently escape recognition and elimination by immune system components. This may be in part due to expression of membrane-bound complement-regulatory proteins (CRP), including CD55, CD59, and CD46, all of which are able to inactivate complement components (1-4). The expression of CRPs is proposed to protect cancer cells from complement-dependent cytotoxicity, especially when accessing the blood circulation during growth and metastasis. Although the role of CRPs in cancer remains to be determined, soluble forms of CD46 have been found to be present at slightly increased levels in the sera of cancer patients (5).

We and others have shown that signal transducers and activators of transcription (STAT) 3, a member of the STAT family of transcription factors, is constitutively activated with high frequency in many different human tumor cell lines and primary tumors compared with their normal counterparts (6-8). STAT3 activation has been shown to provide cancer cells with a growth and survival advantage (9-13). In addition, persistent STAT3 activation has been shown to promote tumor angiogenesis and metastasis (14-16). Recent studies also show a critical role of tumor cell STAT3 signaling in facilitating immune evasion by negatively regulating cellular and innate immune responses (17). In contrast, STAT1 has an opposing role in oncogenesis, and STAT1-null mice have an increased incidence of tumors due to defects in immune responses against tumor cells (18, 19). Thus, aberrant STAT signaling promotes oncogenesis through multiple mechanisms and proof-of-principle experiments have provided compelling evidence that activated STAT3 is a promising molecular target for cancer therapy (6, 20).

In this report, we applied microarray gene expression profiling to identify novel STAT3-regulated genes that may contribute to malignancy. We show that the CD46 promoter is regulated by STAT3 in cancer cells harboring constitutive STAT3 DNA-binding activity. In particular, we show that blockade of STAT3 activity inhibits CD46 mRNA expression in human breast and prostate cancer cells. Furthermore, we identify two STAT3-binding sites in the CD46 gene promoter region and show that STAT3 activity is required for CD46 promoter up-regulation. Using chromatin immunoprecipitation assay, we show that STAT3 binds to the CD46 promoter. We also show that CD46 mRNA can be induced in normal human epithelial cells by interleukin (IL)-6 or by transient transfection with a persistently active mutant form of STAT3, STAT3C. Moreover, inhibition of CD46 mRNA and cell surface protein expression through blockade of STAT3 signaling in DU145 human prostate cancer cells suggests a role for CD46 in protection of DU145 cells from antibody-mediated complement-dependent cytotoxicity as measured by a complement lysis assay. Taken together, these results provide one potential molecular mechanism by which activated STAT3 signaling may directly contribute to tumor cell evasion of antibody-mediated immunity.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
Inhibition of Activated STAT3 Blocks CD46 mRNA Expression in Human Cancer Cells
Previous studies in breast and prostate cancer cell lines have shown that STAT3 is constitutively activated (7, 8). To identify new STAT3-regulated genes, we inhibited STAT3 signaling with antisense oligonucleotides in the human prostate cancer cell line DU145 and the human breast cancer cell line MDA-MB-435s. Figure 1 shows the effect of STAT3 antisense oligonucleotides on total and phosphorylated STAT3 protein expression (Fig. 1A, Western blot) and STAT3 DNA-binding activity [Fig. 1B, electrophoretic mobility shift assay (EMSA)] in DU145 cells treated for 24 h. Similar results were obtained for MDA-MB-435s cells (data not shown). Microarray analysis was done using the human genome Affymetrix HG-U133A GeneChip. When compared with control oligonucleotide–treated cells, analysis of the microarray data revealed that, at 24 h after transfection with 250 nmol/L STAT3 antisense oligonucleotides, CD46 is a candidate STAT3-regulated gene in both cell lines (Fig. 1C). As expected, STAT3 antisense oligonucleotides reduce expression of STAT3 mRNA in all cases. Real-time PCR analysis confirmed that STAT3 regulates CD46 mRNA expression in DU145 cells (Fig. 1D).


Figure 1
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  		FIGURE 1. Analysis of STAT3 and CD46 expression in human prostate and breast cancer cells treated with STAT3 antisense oligonucleotides. A. Western blot. DU145 prostate cancer cells were treated for 24 h with 250 nmol/L of control oligonucleotides or 150 to 250 nmol/L of STAT3 antisense oligonucleotides. Total protein was harvested and equal amounts of total protein were loaded on a 10% SDS-polyacrylamide gel, electrophoresed, and immunoblotted for total STAT3, phosphorylated STAT3 (phospho-Stat3; Tyr705), and ß-actin protein. B. EMSA. DU145 prostate cancer cells were treated as mentioned above. Nuclear protein was harvested and equal amounts of nuclear protein were preincubated with a 32P-labeled DNA oligonucleotide containing a STAT3-binding site (hSIE, see EMSA protocol) before separation by nondenaturating PAGE and autoradiographic detection. CO, control oligonucleotide; AS, STAT3 antisense oligonucleotide. C. Microarray analysis. DU145 prostate cancer cells (top) and MDA-MB-435 breast cancer cells (bottom) were treated for 24 h with 250 nmol/L of STAT3 antisense or control oligonucleotides. Twenty-four hours after transfection, total RNA was isolated, processed, and hybridized to Affymetrix HG-U133A GeneChips. Columns, average signal intensity of three (DU145) or two (MDA-MB-435) independent experiments. For each gene, data for two different Affymetrix probe sets are presented. The signal intensity of control oligonucleotide–treated cells was set to 100%. D. Real-time PCR analysis. RNA/cDNA samples prepared from DU145 cells treated for 24 h with LipofectAMINE Plus (LF), 250 nmol/L of control oligonucleotides, or 250 nmol/L of STAT3 antisense oligonucleotides were measured for CD46 mRNA expression using real-time PCR with gene-specific primers and fluorescent-labeled probe. RNA expression was normalized to 18S rRNA, and RNA expression of LipofectAMINE Plus–treated samples was set to 100%. n = 3 independent experiments.

 
Activated STAT3 Binds to the Human CD46 Promoter
We next investigated whether activated STAT3 can bind to the CD46 promoter. The human CD46 promoter contains two potential STAT3-binding sites, CD46/–920 and CD46/–450, based on the consensus sequence TT[N4/5]AA (–450 and –920 represent the nucleotide position of the 5' T residue in the STAT3 consensus sequence TT[N4/5]AA in both potential binding sites relative to the first A in the translational start site ATG; ref. 21). Double-stranded 32P-radiolabeled DNA oligonucleotides for both sites were used in an EMSA to detect STAT3 DNA-binding activity. Both probes as well as the positive control probe hSIE, an artificial high-affinity variant of the sis-inducible element that binds STAT3 and STAT1, were preincubated with 5 µg nuclear protein extract derived from DU145 cells and subjected to native gel electrophoresis.

As shown in Fig. 2A , activated STAT3 binds to hSIE (lane 1) as well as to both sequences derived from the CD46 promoter (lanes 3 and 5). Preincubation of the probe/nuclear extract mix with anti-STAT3 antibodies confirmed that the protein complex bound to these sites contains STAT3 ("supershifted" bands; lanes 2, 4, and 6). CD46/–450 shows much stronger STAT3 DNA-binding activity compared with CD46/–920 and binds to STAT3 with similar affinity as the high-affinity STAT3 DNA-binding element hSIE. Furthermore, we used nonradioactive CD46/–450 DNA oligonucleotides to compete with the radiolabeled hSIE probe for binding of activated STAT3. As shown in Fig. 2B (top), a 100-fold molar excess of unlabeled CD46/–450 is sufficient to compete with 32P-hSIE for STAT3 protein binding. Deletions introduced into CD46/–450, which eliminate the STAT3 consensus binding sequence (TTCCCggAA -> TTggAA), completely abolish STAT3 DNA-binding activity even at 10,000-fold molar excess as shown by lack of competition (Fig. 2B, bottom).


Figure 2
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  		FIGURE 2. EMSA analysis of STAT3 DNA-binding sites in the human CD46 promoter and chromatin immunoprecipitation. Two candidate STAT3 DNA-binding sites in the human CD46 promoter (positions –920 and –450, relative to the translation initiation site) were identified. Sense and antisense strands for each site were synthesized, annealed, 32P labeled, and incubated with 5 µg of DU145 nuclear extract as a source of activated STAT3. The high-affinity STAT3-binding oligonucleotide hSIE was used as a positive control. *, "supershifting" anti-STAT3 antibodies were added to the reaction to confirm the presence of active STAT3. A. EMSA. DU145 nuclear extract was incubated with the following 32P-labeled double-stranded oligonucleotides: hSIE (lanes 1 and 2), CD46/–920 (lanes 3 and 4), and CD46/–450 (lanes 5 and 6). B. Lanes 1 to 7, competition EMSA. DU145 nuclear extract was incubated with 32P-labeled double-stranded hSIE oligonucleotide. Unlabeled, wild-type (wt; top), or mutated (mut; bottom) CD46/–450 oligonucleotide was then added to the reaction at the same molar concentration (lanes 3 in top and bottom) or in 10- to 104-fold molar excess (lanes 4-7 in top and bottom) to compete with hSIE for STAT3 binding. SS, supershift with anti-STAT3 antibodies. C. Chromatin immunoprecipitation assay. DU145 cells possess constitutively active STAT3 and were used for chromatin immunoprecipitation assay. Briefly, after cross-linking histones to DNA by formaldehyde, the cells were sonicated to shear DNA to lengths between 200 and 1,000 bp. A portion of this sample was used as positive control in subsequent PCRs (input material). The remaining sample was incubated with anti-STAT3, anti-STAT5, or without antibody overnight, and the antibodies were immunoprecipitated using protein A-agarose. After several washes, the histone-DNA complex was reverse cross-linked and all samples were subjected to PCR using specific primers that surround the STAT3-binding site at position –450 in the human CD46 promoter. Result after nested PCR.

 
To show whether activated STAT3 binds to the CD46 promoter in vivo, we did chromatin immunoprecipitation assay using DU145 cells. Immunoprecipitations were done with antibodies for total STAT3 and total STAT5. After reverse cross-linking of the precipitated DNA/histone complex, the purified DNA was subjected to PCR using primers that surround the major STAT3-binding site (–450) in the CD46 promoter as shown in Fig. 2A (lane 5). Figure 2C shows the result after nested PCR and the upper band represents the DNA region surrounding site –450. These data confirm that activated STAT3 can bind to the CD46 promoter.

Activated STAT3 Induces CD46 Promoter Activity
To determine whether activated STAT3 can induce CD46 promoter activity, we did reporter gene studies using NIH3T3 mouse fibroblast cells transiently transfected with a reporter construct consisting of the human CD46 promoter linked to a luciferase gene. Cells were cotransfected with plasmids encoding either activated viral Src (v-Src), a dominant-negative form of STAT3 (STAT3ß), or both. As shown in Fig. 3A , NIH3T3 cells, which express low levels of endogenous STAT3 activity, almost completely lose basal (column 1) CD46 promoter activity when cotransfected with the dominant-negative STAT3 construct (column 2). Cotransfection with v-Src, an inducer of STAT3 activation in these cells (22, 23), increases the CD46 promoter activity ~2-fold (column 3). Cotransfection with both v-Src and dominant-negative STAT3 significantly decreased CD46 promoter activity (column 4) even below basal levels. Furthermore, as shown in Fig. 3B, deletion of the STAT3 DNA-binding site at position –450 (see also Fig. 2B) completely prevented v-Src/STAT3–mediated luciferase induction (column 4). Taken together, the data thus far provide evidence that STAT3 activity is required for CD46 mRNA expression and that STAT3-mediated CD46 up-regulation occurs at the level of promoter regulation.


Figure 3
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  		FIGURE 3. STAT3 activity is required for CD46 promoter activity. A. NIH3T3 cells were transiently transfected for 4 h with a plasmid containing the CD46 promoter (position –600 to +76) driving expression of the firefly luciferase reporter gene (pGL2-basic). As indicated, NIH3T3 cells were cotransfected with expression vectors for dominant-negative STAT3ß (column 2), v-Src (column 3), or both (column 4). Luciferase activity was measured 48 h after start of transfection. B. NIH3T3 cells were transiently transfected for 4 h with either wild-type (columns 1 and 2) or mutated CD46 reporter (three-nucleotide deletions within the STAT3-binding site at position –450; columns 3 and 4) in the presence or absence of v-Src vector. In all experiments, ß-galactosidase activity was determined to normalize for transfection efficiency. Data represent each a total of three independent experiments. C. Relative expression of human CD46 mRNA as measured by quantitative real-time PCR of RNA/cDNA prepared from normal human epithelial cells and DU145 cells after stimulation with 10 ng/mL IL-6 (3 h) or transient transfection with a constitutive active STAT3 mutant, STAT3C, or control plasmid (pCDNA3) for 2 d. For Western blotting of IL-6–treated cells, samples were harvested 10 min after treatment. PrEC, prostate epithelial cells; HMEC, human mammary epithelial cells. The relative expression of control-treated samples was set to 1. Real-time PCR results are representative of two independent experiments.

 
To address the question whether up-regulation of STAT3 DNA-binding activity can induce CD46 mRNA expression, normal human prostate epithelial cells and DU145 cells were stimulated with 10 ng/mL IL-6 for 3 h. Normal human mammary epithelial cells were transiently transfected with the constitutively active STAT3 mutant construct STAT3C for 2 days. RNA was isolated, transcribed into cDNA, and subjected to real-time PCR using primers and probe specific for human CD46 mRNA. As shown in Fig. 3C, IL-6 stimulation of both human prostate epithelial cells and DU145 cells induces CD46 mRNA expression. Moreover, transient transfection of human mammary epithelial cells with STAT3C induces CD46 mRNA expression independent of cytokine stimulation. For Western blotting of IL-6–treated cells, samples were harvested 10 min after treatment. These results show that STAT3 can activate CD46 mRNA expression.

Inhibition of STAT3 Blocks CD46 Cell Surface Protein Expression
We determined whether CD46 mRNA down-regulation by direct STAT3 inhibition results in loss of CD46 cell surface protein expression. First, we compared two different anti-CD46 antibodies for specificity toward CD46 (Fig. 4 ). For this experiment, DU145 cells were treated with either 100 nmol/L control small interfering RNA (siRNA) or CD46 siRNA. At 42 h after transfection, cells were harvested and preincubated with 300 µg/mL total mouse IgG (to block Fc receptors) followed by cell surface staining using the FITC-labeled anti-CD46 antibodies E4.3 and 122 as well as the corresponding FITC-labeled isotype control antibodies. Dead cells were identified and excluded from analysis through TOPRO-3 staining. Figure 4A and B shows the results of flow cytometry analysis. More than 95% of control-treated cells stained CD46 positive using either anti-CD46 antibody (Fig. 4A and B, top right). Treatment with CD46 siRNA reduced the number of CD46-positive cells to ~20% using either antibody (Fig. 4A and B, bottom right). The isotype controls did not show any FITC-positive cells. Therefore, because both antibodies show similar results, we conclude that these antibodies are specific for CD46. We used the anti-CD46 E4.3 antibody clone for subsequent analyses.


Figure 4
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  		FIGURE 4. Blockade of STAT3 signaling inhibits CD46 cell surface expression. A and B. Specificity of anti-CD46 antibodies. DU145 cells were treated for 42 h with 100 nmol/L of either CD46 or control siRNA. Cells were then harvested and the cell surface was stained using either the FITC-labeled anti-CD46 antibody E4.3 (A) or the FITC-labeled anti-CD46 antibody 122 (B). The corresponding FITC-labeled isotype controls are indicated on the left sides of A and B. C. Flow cytometry. DU145 prostate cancer cells were treated for 18 h with suboptimal concentrations (200 nmol/L, to prevent apoptosis) of STAT3 antisense or control oligonucleotides before flow cytometric analysis of CD46 cell surface protein expression. Left, IgG2a-FITC isotype controls; right, CD46-FITC–stained cells treated with STAT3 antisense (red) or control oligonucleotides (green). Number of CD46-FITC–positive cells: 90% (control sample) and 60% (antisense sample). Median intensity of CD46-FITC–positive cells: 450 (control sample) and 270 (antisense sample). D. STAT3 DNA-binding analysis (EMSA). The 32P-hSIE probe was incubated with 5 µg of nuclear extract from DU145 cells treated as mentioned above. LF/SS, nuclear extract from LipofectAMINE Plus–treated cells preincubated with anti-STAT3 antibodies for supershift. *, supershifted STAT3 complex.

 
To avoid accumulation of apoptotic cells due to STAT3 inactivation (7), DU145 cells were treated for only 18 h with suboptimal concentrations (200 nmol/L) of STAT3 antisense or control oligonucleotides followed by cell surface staining with either anti-CD46-FITC E4.3 or IgG2a-FITC isotype control antibodies. As shown in Fig. 4C, results from flow cytometric analysis (only living cells were gated) reveal that, in comparison with control-treated cells (green), 30% of the antisense-treated cells (red) have completely lost CD46 cell surface protein expression. Moreover, the remaining CD46-positive cells have further decreased CD46 expression by 40% (median intensity). Figure 4D shows the results from EMSA analysis using nuclear extracts prepared from the same experiment and incubated with 32P-hSIE. As expected, the suboptimal concentration of STAT3 antisense oligonucleotides used in this experiment only partially inhibited STAT3 DNA-binding activity (Fig. 4D, lane 3).

Inhibition of STAT3 Sensitizes DU145 Prostate Cancer Cells to Antibody-Mediated Complement-Dependent Cytotoxicity
Expression of CD46 was previously shown to inhibit complement activation and to protect cells from complement-dependent cytotoxicity (24-27). We determined whether CD46 mRNA down-regulation by direct STAT3 inhibition results in sensitization of DU145 prostate cancer cells to antibody-mediated complement-dependent cytotoxicity. For complement lysis assays, DU145 cells were seeded in 96-well plates and incubated with control or STAT3 antisense oligonucleotides at suboptimal concentrations so as not to induce apoptosis, as mentioned above (200 nmol/L, 18 h). The cells were then preincubated for 1 h on ice with heat-inactivated normal rabbit serum or, as a source of antibodies, with heat-inactivated serum from rabbits immunized with DU145 cells. Serum was subsequently washed off and replaced with 7.5% standard rabbit complement. After 2 h of incubation at 37°C, the extent of cell death was measured by lactate dehydrogenase release.

As shown in Fig. 5A , cotreatment of cells with rabbit anti-DU145 serum plus complement, but not the antiserum alone, induces low levels of cell death in control oligonucleotide–treated cells in a dose-dependent manner. Importantly, the cytotoxic effect was significantly more pronounced in the STAT3 antisense–treated sample (Fig. 5B), indicating that enhanced STAT3 activity leading to induction of CD46 cell surface protein expression protects DU145 cells from antibody-mediated complement-dependent cytotoxicity. Cotreatment of the cells with normal rabbit serum plus complement using the same concentrations as above did not induce cell death in either STAT3 antisense–treated or control oligonucleotide–treated samples (data not shown). Substitution of 7.5% standard rabbit complement with 100% human serum (nonheat inactivated) had similar effects on cytotoxicity (data not shown). These results show that DU145 cells are susceptible to antibody-mediated complement-dependent cytotoxicity when STAT3 signaling and CD46 expression are inhibited.


Figure 5
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  		FIGURE 5. Inhibition of STAT3 signaling sensitizes DU145 cells to antibody-mediated complement-dependent cytotoxicity. DU145 prostate cancer cells were treated for 18 h with suboptimal concentrations (200 nmol/L) of control (A) or STAT3 antisense (B) oligonucleotides as in Fig. 4. The cells were then placed on ice and prechilled, and heat-inactivated rabbit anti-DU145 antiserum was added to the cells for 1 h at concentrations indicated. Gray columns, antibody-mediated complement-dependent cytotoxicity was initiated by addition of fresh standard rabbit complement to a final concentration of 7.5%. Cells were incubated for 2 h at 37°C. Amount of lactate dehydrogenase was then measured in cell supernatant before and after lysis of cells with lysis buffer. The ratio of lactate dehydrogenase in supernatant before and after addition of lysis buffer was calculated to determine the degree of cytotoxicity. Replacement of rabbit anti-DU145 antiserum with serum from the same animal before intravenous injection of DU145 cells did not induce cytotoxicity (data not shown). One representative result (in quadruplicate) of a total of three independent experiments.

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
In this report, we identify the CD46 gene as a target of activated STAT3 signaling in human breast and prostate cancer cell lines by microarray gene expression profiling. CD46 reporter gene experiments and DNA-binding assays provide evidence that STAT3-mediated CD46 expression occurs through binding of STAT3 to the CD46 promoter. The newly identified strong STAT3 DNA-binding site is located at position –450 in the human CD46 promoter (28). Chromatin immunoprecipitation assay suggests that activated STAT3 binds to this site in the CD46 promoter. Blockade of STAT3-mediated CD46 expression using STAT3 antisense oligonucleotides decreases CD46 cell surface protein expression and renders DU145 prostate cancer cells susceptible to cytotoxicity in an antibody-mediated complement-dependent lysis assay in vitro. These results raise the possibility that STAT3 may contribute to the protection of cancer cells from complement system attack in vivo at least in part through up-regulation of the CRP CD46. However, we cannot exclude the possibility that STAT3 may regulate other genes that confer resistance to complement, such as the CRPs CD55 and CD59.

Previous studies show that cellular immune responses against tumor cells are modulated by STAT signaling (20). In particular, STAT3 activation in tumor cells inhibits the expression of proinflammatory cytokines and chemokines while at the same time promoting production of factors that down-regulate dendritic cell functional maturation (17). Consequently, persistent STAT3 signaling in tumor cells leads to inhibition of T-cell responses, thereby facilitating evasion of cellular immunity (17). By contrast, STAT1 enhances immune responses against tumor cells through IFN signaling (18, 19). Our finding that tumor STAT3 activity promotes CD46 expression provides the first molecular evidence that oncogenic signaling may directly contribute to tumor cell evasion of antibody-mediated immunity.

Monoclonal antibodies (mAb) specific for cancer cells are being evaluated as cancer therapeutics due to their potential to directly induce apoptosis and initiate complement-dependent cytotoxicity or complement-dependent cellular cytotoxicity. Because a successful tumor needs to access the blood circulation for continued growth and metastasis, utilization of the complement system that is present primarily in blood and other body fluids offers the potential for mAb cancer immunotherapy based on antibody-mediated complement-dependent cytotoxicity or complement-dependent cellular cytotoxicity to target aggressive, metastasizing tumors. Indeed, in experimental animal models, cancer cell–directed mAb immunotherapy has provided encouraging results (3, 29). The therapeutic potential of cancer immunotherapy using mAbs, however, is limited due to the ability of cancer cells to resist complement-dependent cytotoxicity in part through expression of one or more CRPs, including CD46, CD55, and CD59.

One possible approach to enhance mAb cancer immunotherapy is to decrease expression levels of membrane-bound CRPs. Several reports have shown that cytokines, such as IL-1ß, tumor necrosis factor-{alpha}, and transforming growth factor-ß1, alter CRP expression levels in cancer cells (30, 31). However, the effects on CRP expression are not consistent and are frequently dependent on the cell type investigated. Furthermore, the use of cytokines may also alter CRP expression on nontumor cells, potentially making them susceptible to complement-dependent attacks. Therefore, down-regulation of CRPs specifically in tumor cells could be a desirable strategy in combination with mAb cancer immunotherapy. This is the first report to show inhibition of one CRP, CD46, through blockade of its expression at the gene level.

A critical role of STAT3 in tumor cell survival, proliferation, angiogenesis, and metastasis has been well established (9-16, 32). Recent studies also showed the important role of STAT3 activation in evasion of cellular immune responses (17). Our finding that activated STAT3 is required for CD46 expression in cancer cells expands the utilization of STAT3 inhibition as a target for development of novel anticancer drugs. These results open new avenues for investigating the role of STAT3-mediated CD46 expression in complement resistance and mAb cancer immunotherapy. Furthermore, the antitumor activities of STAT3-specific inhibitors currently under development (33-35) may be enhanced through increased recognition and attack of tumor cells by the complement system. Because STAT3 is normally tightly regulated under physiologic conditions, targeting of persistently active STAT3 in tumor cells offers a potentially promising new approach to attack cancer cells on multiple fronts.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Cancer Cell Lines, Normal Cells, and Cell Culture
DU145 human prostate cancer cells, MDA-MB-435s human breast cancer cells, and NIH3T3 mouse fibroblast cells were obtained from the American Type Culture Collection and maintained in RPMI 1640/10% fetal bovine serum (DU145), DMEM/10% fetal bovine serum (MDA-MB-435s), or DMEM/10% bovine calf serum (NIH3T3) supplemented with 50 units/mL penicillin and 50 µg/mL streptomycin. Normal human mammary epithelial cells and normal prostate epithelial cells were obtained from Lonza and maintained in complete mammary epithelial growth medium or prostate epithelial cell basal medium supplemented with growth factors as described by the supplier (Lonza).

Microarray Analysis
For microarray analysis, total RNA was extracted 24 h after transfection using Trizol (Invitrogen) and further purified with RNeasy (Qiagen). RNA was processed, as per the supplier's instructions (Affymetrix), and hybridized on Affymetrix HG-U133A human genome arrays that contain >22,000 probe sets representing ~14,500 mostly known genes. Fluorescence was measured using Affymetrix GeneArray 2500 GeneChip scanner, and the data were analyzed using Affymetrix Microarray Suite 5.0 software. All microarray experiments were done at least in duplicate independent experiments, and the results are presented for each probe set as average fold change in RNA expression.

Quantitative Real-time PCR
For real-time PCR, total RNA was isolated as mentioned above. Before reverse transcription, as per the supplier's instructions (SuperScript Preamplification System, Invitrogen), 1 µg of total RNA was digested with DNase I, amplification grade (Invitrogen). Primers and Fam/Tamra fluorescent-labeled probes were designed with PrimerExpress software (Applied Biosystems) and obtained from Eurogentec. For each primer/probe set, one of the oligonucleotides was positioned over an intron/exon boundary to minimize the possibility of amplifying genomic DNA. Oligonucleotide sequences for CD46 were as follows: probe, 5'-CgACATTTgACCACTTTACACTCTggAgCA-3'; forward primer, 5'-TggTgACAATTCAgTgTggAgTC-3'; and reverse primer, 5'-CCTgATATCTgTTTTCCATTTTCgA-3'. The primer/probe set for 18S rRNA (Vic/Tamra labeled) was used for normalization (Applied Biosystems). PCR was done on the ABI 7700 Sequence Detection System, and data were analyzed using the "standard curve" analysis method as described in User Bulletin 2 (Applied Biosystems).

Chromatin Immunoprecipitation
Chromatin immunoprecipitation was done using a kit from Upstate as described by the supplier. Briefly, DNA of 2 million DU145 cells was cross-linked to protein with formaldehyde. Cellular lysate was obtained by scraping followed by pulse ultrasonication to shear cellular DNA. Immunoprecipitations were done with the following antibodies (1.0 µg): anti-total STAT3 (C20, Santa Cruz Biotechnology) and anti-total STAT5 (Santa Cruz Biotechnology). Subsequently, cross-links were reversed, and bound DNA was purified using a PCR product purification kit from Qiagen. The first PCR was done using the following primers specific for human CD46 promoter: forward primer 1, 5'-CAAgTAAgggCCCAggCAgTCC-3'; reverse primer 1, 5'-CCCTggCggAACCTAACAgAACAg-3'. These primers correspond to nucleotide positions –592 to –571 and –297 to –274. For nested PCR, forward primer 2 (5'-ggTgCTAgCCCACggTgACC-3') and reverse primer 2 (5'-CCTACCATCCCggAAgCCTgAg-3') were used. These primers correspond to nucleotide positions –530 to –510 and –358 to –336.

Western Blotting
For STAT3 analysis by Western blotting, cells were washed in 1x PBS and lysed in TGH buffer (1% Triton X-100, 10% glycerol, 50 mmol/L NaCl, 50 mmol/L HEPES, 1 mmol/L EGTA, 1% sodium deoxycholate, 1 mmol/L sodium vanadate, 1 mmol/L phenylmethylsulfonyl fluoride, 50 µg/mL antipain, 0.5 µg/mL leupeptin, 2 µg/mL aprotinin). Protein amount was determined using the Bio-Rad Protein Assay reagent (Bio-Rad), and equal amounts (50 µg) of total protein were loaded in each lane of a 10% SDS-polyacrylamide gel. The proteins were transferred to nitrocellulose membrane, washed with PBS/0.1% Tween 20, and incubated in 1x PBS/5% milk overnight with anti-STAT3 antibody (C20). The membrane was then washed with PBS/0.1% Tween 20, incubated for 1 h at room temperature with alkaline phosphatase–linked antirabbit secondary antibodies, and visualized using SuperSignal West Pico Reagent (Pierce). For detection of ß-actin, the blot was stripped with stripping buffer [2% SDS, 64 mmol/L Tris (pH 6.7), 0.7% ß-mercaptoethanol] and reblotted with anti-ß-actin (Sigma) for 1 h at room temperature followed by incubation with alkaline phosphatase–linked antimouse secondary antibody.

Antisense and siRNA Oligonucleotides
STAT3 antisense (5'-gCTCCAgCATCTgCTgCTTC-3') and random control oligonucleotides (equal chance of any of the four nucleotides being incorporated at any position) were synthesized using phosphorothioate chemistry (Isis Pharmaceuticals). To increase stability and hybridization affinity, all oligonucleotides were synthesized with 2'-O-methoxyethyl modification of the five terminal nucleotides (italicized; ref. 36). Transfections were carried out by the LipofectAMINE Plus method as described by the supplier (Life Technologies). Briefly, 2 x 106 DU145 or MDA-MB-435s cells were seeded in 10-cm tissue culture plates 24 h before transfection. Cells were transfected for 3 h with LipofectAMINE Plus only, with LipofectAMINE Plus/STAT3 control oligonucleotides, or with LipofectAMINE Plus/STAT3 antisense oligonucleotides. The final concentration of the oligonucleotides was 100 to 250 nmol/L as indicated. Transfection was terminated by aspirating the supernatant, washing the cells once with PBS, and adding fresh serum–containing medium. At times indicated, nonadherent cells were washed off and the remaining cells were harvested for CD46 cell surface staining, EMSA analysis, or RNA preparation. For transfection with siRNA, 1 x 105 DU145 cells were seeded in each well of a 12-well cell culture plate 24 h before transfection. Cells were then transfected with either CD46 siRNA or control siRNA duplexes (Dharmacon) using RiboJuice transfection reagent as per the supplier's instructions (Novagen). The final concentration of the siRNAs was 100 nmol/L. At 42 h after transfection, nonadherent cells were washed off and the remaining cells were harvested for CD46 cell surface staining.

CD46 Reporter Construct, Reporter Gene Assay, and Transient Transfection
For reporter gene assays, a genomic DNA fragment from nucleotides –600 to +76 of the human CD46 promoter gene (nucleotide position +1 in the CD46 promoter construct represents the ATG translational start site) was cloned upstream of a promoterless firefly luciferase reporter gene (pGL2-basic; Promega). NIH3T3 cells (3 x 105 in 60-mm dishes) were transiently transfected with the CD46 reporter gene construct using the LipofectAMINE Plus method described above and cotransfected with either viral Src (v-Src) expression vector or dominant-negative STAT3 (STAT3ß) expression vector as indicated. All transfection reactions contained a total of 6.0 µg DNA, including 2.0 µg of pGL2-CD46/luciferase, 2.0 µg v-Src, 2.0 µg STAT3ß, and 10 ng ß-galactosidase expression vector (internal control for normalization of transfection efficiency). To normalize for the total amount of DNA transfected, 2.0 or 4.0 µg of control empty vector (pCDNA3) were used. After 4 h, transfection medium was removed, cells were washed twice in PBS, and fresh serum–containing medium was added for another 44 h. At 48 h after the start of transfection, cells were lysed and luciferase and ß-galactosidase activities were measured as described previously (23). A CD46 reporter containing a mutated STAT3 DNA-binding site at position –450 (Fig. 3B) was generated by site-directed mutagenesis (Invitrogen) using specific, high-performance liquid chromatography–purified primers with a three-nucleotide deletion within the STAT3 consensus binding sequence (for sequence information, see EMSA protocol below). The final amount of transfected DNA in the reporter gene assay using the mutated CD46 reporter was 4 µg. For transient transfection of normal human mammary epithelial cells, 2 µg of STAT3C vector (kind gift of Dr. Beverly Barton, University of Medicine & Dentistry of New Jersey, New Jersey Medical School, Newark, NJ) or 2 µg of pCDNA3 vector were used.

Nuclear Extract Preparation and EMSA
For the detection of DNA-binding activity of STAT3 by EMSA, nuclear protein extracts were prepared using high-salt extraction as described previously (8). Nuclear protein (5 µg) from DU145 cells was incubated with 32P-radiolabeled dsDNA oligonucleotides using a high-affinity variant of the sis-inducible element (hSIE; sense strand, 5'-AgCTTCATTTCCCTgAAATCCCTA-3') derived from the c-fos gene promoter, which binds activated STAT3 and STAT1 proteins (22, 37). In addition, wild-type/mutated oligonucleotide probes derived from the CD46 gene promoter (STAT3 consensus DNA-binding sequence italicized) were as follows: wild-type CD46/–920 (sense strand, 31-mer), 5'-gCATTTATTgCTTCCAggAATTTggCACTTA-3'; wild-type CD46/–450 (sense strand, 31-mer), 5'-gACTCCCgAATTCCCggAAACTATTACCAAA-3'; and mutated CD46/–450 (sense strand, 28-mer, three-nucleotide deletion within the STAT3 consensus binding sequence), 5'-gACTCCCgAATTggAAACTATTACCAAA-3'. Where indicated in the text, unlabeled oligonucleotides derived from the CD46 promoter were used to compete with 32P-hSIE for STAT3 binding. Anti-STAT3 polyclonal antibodies (C20X, Santa Cruz Biotechnology) were used to identify STAT3 in "supershift" assays. For use in supershift assays, 1 µL of the concentrated STAT3 antibodies was preincubated with nuclear protein for 20 min at room temperature before the addition of radiolabeled probe (30 min, 30°C) and separation by nondenaturating PAGE and autoradiographic detection.

Flow Cytometric Analysis
CD46 cell surface–positive cancer cells were measured and quantified by flow cytometry using the FACSCalibur instrument (Becton Dickinson PharMingen). Briefly, cells were harvested with trypsin, washed with PBS, and incubated for 1 h with CD46-FITC (Santa Cruz Biotechnology) or FITC isotype control antibodies (Santa Cruz Biotechnology) in staining buffer (PBS, 0.5% bovine serum albumin) in the dark. The samples were washed thrice in staining buffer and propidium iodide or TOPRO-3 was added 15 min before analysis for exclusion of dead cells. Flow cytometric results are expressed as percentage FITC-positive cells and as median FITC intensity.

Antibody-Mediated Complement-Dependent Lysis Assay
DU145 prostate cancer cells were treated with a suboptimal concentration (200 nmol/L) of STAT3 antisense or control oligonucleotides. After completion of the initial 3-h transfection step, cells were harvested and seeded in 96-well plates at 10,000/100 µL/well. Eighteen hours later, the cells were incubated on ice for 1 h with normal rabbit serum or with serum from rabbits immunized with DU145 cells (Rockland Immunochemicals) at concentrations indicated in the text. For the production of rabbit anti-DU145 serum as a source of antibodies, rabbits were initially injected intravenously with 10 x 106 living DU145 cells. An identical booster injection was done 14 days later. Normal rabbit serum ("prebleed," harvested 4 days before the initial injection of cancer cells) as well as rabbit anti-DU145 serum that was collected 24 days after the initial injection into the same animal were heat inactivated for 30 min at 57°C before use in the complement lysis assay. Antibodies not bound to DU145 cells were washed off with PBS (4 x 200 µL) and replaced by addition of 7.5% rabbit standard complement (Cedarlane). After 2 h of incubation at 37°C, lactate dehydrogenase release into the cell supernatant as well as total lactate dehydrogenase content were measured at 490 nm using the CytoTox96 assay system as recommended by the supplier (Promega).


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank members of our laboratories and colleagues for stimulating discussions. We thank Beverly Barton for providing the Stat3C-GFP construct.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Grant support: NIH grants CA55652 and CA82533 (R. Jove) and Dr. Mildred Scheel Stiftung fuer Krebsforschung (R. Buettner).

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 10/18/06; revised 4/22/07; accepted 5/ 2/07.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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H. D. Lewis, A. Winter, T. F. Murphy, S. Tripathi, V. N. Pandey, and B. E. Barton
STAT3 inhibition in prostate and pancreatic cancer lines by STAT3 binding sequence oligonucleotides: differential activity between 5' and 3' ends
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