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Endocrinology Vol. 145, No. 8 3769-3777
Copyright © 2004 by The Endocrine Society

Transcriptional Activation of the Insulin-Like Growth Factor I Receptor Gene by the Kruppel-Like Factor 6 (KLF6) Tumor Suppressor Protein: Potential Interactions between KLF6 and p53

Moran Rubinstein, Gila Idelman, Stephen R. Plymate, Goutham Narla, Scott L. Friedman and Haim Werner

Department of Clinical Biochemistry, Sackler School of Medicine, Tel Aviv University (M.R., G.I., H.W.), Tel Aviv 69978, Israel; Department of Medicine, University of Washington (S.R.P.), Seattle, Washington 98195; and Division of Liver Diseases, Mount Sinai School of Medicine (G.N., S.L.F.), New York, New York 10029

Address all correspondence and requests for reprints to: Dr. Haim Werner, Department of Clinical Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel. E-mail: hwerner{at}post.tau.ac.il.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The IGF system plays an important role in prostate cancer initiation and progression. Most of the biological actions of IGF-I and IGF-II are mediated by activation of the IGF-I receptor (IGF-IR). Evidence accumulated in recent years indicates that acquisition of the malignant phenotype is initially IGF-IR dependent, but progression toward metastatic stages is usually associated with a decrease in IGF-IR levels. The Kruppel-like factor 6 (KLF6) is a zinc finger-containing transcription factor that was shown to be mutated in a significant portion of prostate and other types of cancer. To examine the potential regulation of IGF-IR gene expression by KLF6, we measured KLF6 levels in prostate-derived cell lines displaying different levels of IGF-IR. The results of Western analysis showed that KLF6 levels were higher in nontumorigenic P69 cells expressing high IGF-IR levels than in metastatic M12 cells containing reduced IGF-IR levels. Transient coexpression of wild-type, but not mutated, KLF6 together with an IGF-IR promoter-luciferase reporter plasmid resulted in an approximately 3.4-fold stimulation of IGF-IR promoter activity. Furthermore, KLF6 expression induced a significant increment in endogenous IGF-IR levels. Deletion analysis of the IGF-IR promoter revealed that a cluster of four GC boxes located between nucleotides –399 and –331 mediates a significant portion of the transactivating effect of KLF6. KLF6, although unable to stimulate IGF-IR promoter activity in Sp1-null Drosophila-derived Schneider cells, significantly enhanced the effect of Sp1. To assess the potential interactions between KLF6 and p53 in the regulation of IGF-IR gene expression, transfections were performed in the colorectal cancer cell line HCT116+/+, which expresses p53, and its HCT116–/– derivative, which lacks p53. KLF6 exhibited an enhanced activity in p53-containing, compared with p53-null, cells. In addition, we were able to detect a physical interaction between KLF6 and p53. In summary, we have identified the IGF-IR gene as a novel downstream target for transcription factor KLF6. The regulation of IGF-IR gene expression by KLF6 may have significant implications in terms of cancer initiation and/or progression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE IGF-I RECEPTOR (IGF-IR) mediates the mitogenic, transforming, differentiating, and antiapoptotic actions of IGF-I and IGF-II (1, 2, 3). Most primary tumors and transformed cell lines display augmented numbers of IGF-IRs on their cell surface as well as increased levels of IGF-IR mRNA that confer upon the malignant cell an enhanced survival capacity. The involvement of the IGF-IR in transformation of prostate epithelium has been the subject of intensive investigation. Thus, acquisition of the malignant phenotype is initially IGF-IR dependent, but as the disease advances and the tumor becomes androgen independent, there is a significant decrease in IGF-IR mRNA and protein levels (4, 5). IGF-IR expression is also extinguished in a majority of human cancer bone marrow metastases (6), although a recent study showed sustained up-regulation of the IGF-IR in metastases (7). The molecular mechanisms that are responsible for regulation of the IGF-IR gene in prostate cancer, however, remain largely unidentified. It is, therefore, of considerable interest to further characterize the mechanisms underlying control of IGF-IR gene expression.

Molecular characterization of the IGF-IR gene regulatory region revealed that the promoter region lacks TATA or CAAT elements (8, 9). Transcription is initiated from a single start site contained within an initiator motif, a promoter element that directs accurate transcription initiation in the absence of a TATA box (10). Like many TATA-less promoters, the proximal 5'-flanking region of the IGF-IR gene is highly GC-rich and contains multiple binding sites for members of the Sp1 family of zinc finger transcription factors (11, 12). Analysis of physical and functional interactions of Sp1 at the IGF-IR promoter revealed that Sp1 is a potent transactivator of the IGF-IR gene. Thus, basal IGF-IR promoter activity was extremely low in Sp1-null, Drosophila-derived, Schneider cells, whereas cotransfection of an Sp1 expression vector significantly enhanced promoter activity (12, 13).

The Kruppel-like factor 6 (KLF6; Zf9, core promoter binding protein) is a ubiquitous transcription factor that includes a proline- and serine-rich N-terminal activation domain, and three C2H2 zinc finger motifs at its C-terminal domain (14, 15). The KLF6 gene is located at 10p, a chromosomal region that is deleted in a large portion of sporadic prostate cancers (16, 17). Using specific microsatellite markers flanking KLF6, some of us have demonstrated that of a collection of 22 prostate tumor samples, 77% displayed loss of heterozygosity (LOH) of the KLF6 locus (18). Furthermore, 71% of tumor specimens exhibiting LOH had mutations in the remaining KLF6 allele. No mutations were seen in the patient’s normal adjacent prostate tissue or in germline DNA from unaffected individuals. More recent data suggested a potential role for KLF6 in tumorigenesis of other tissues, including nasopharyngeal carcinomas, colorectal cancer, and astrocytic gliomas (19, 20, 21, 22).

Although the mechanism of action and the biological role of KLF6 have not yet been established, it has been demonstrated that KLF6 interacts with GC boxes located in putative target promoters, including those of the keratin 4 (23), TGF-ß1 (24), 47-kDa heat shock protein (25), inducible nitric oxide synthase (26), and endoglin (27) genes. Furthermore, the expression of KLF6 in NIH-3T3 cells resulted in a significant decrease in DNA synthesis that was associated with increased expression of p21, a negative regulator of the G1/S transition, and enhanced activity of a cotransfected p21 promoter-luciferase reporter plasmid (18). In addition, results of electrophoretic mobility shift assays demonstrated that the transactivating effect of KLF6 was associated with specific binding to GC boxes located in the p21 promoter region.

In view of the important roles of tumor suppressor KLF6 and the IGF-IR in the etiology of prostate cancer and to extend our previous observations on regulation of IGF-IR gene expression by transcription factors of the Sp and Kruppel-like family, we have examined the potential transcriptional regulation of the IGF-IR gene by KLF6. The results obtained indicate that wild-type, but not tumor-derived mutated, KLF6 has a stimulatory effect on IGF-IR gene expression. The transactivating effect of KLF6 seems to involve functional interactions with the Sp1 zinc finger protein and with tumor suppressor p53. Impaired activation of the IGF-IR gene by a defective KLF6 may have profound implications in terms of cancer initiation and/or progression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures
The Chinese hamster ovary (CHO) cell line was obtained from the American Type Culture Collection (Manassas, VA). Cells were grown in Ham’s F-12 nutrient mixture supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and 50 µg/ml gentamicin sulfate. P69 and M12 prostate-derived cell lines were provided by Dr. Joy L. Ware (Medical College of Virginia, Richmond, VA). The P69 line was obtained by immortalization of prostate epithelial cells with simian virus 40 T antigen, and M12 cells were derived by injection of P69 cells into athymic nude mice and serial reimplantation of tumor nodules into nude mice (28). P69 and M12 cells were cultured in RPMI 1640 medium supplemented with 10 ng/ml epidermal growth factor, 0.1 nM dexamethasone, 5 µg/ml insulin, 5 µg/ml transferrin, and 5 ng/ml selenium. The human colorectal cancer cell lines HCT116+/+, which expresses wild-type p53, and HCT116–/–, in which the p53 gene has been disrupted by targeted homologous recombination, were provided by Dr. Bert Vogelstein (Johns Hopkins University School of Medicine, Baltimore, MD) (29). HCT116 cells were grown in McCoy’s 5A medium with 10% FBS. Drosophila Schneider cells were grown in Schneider’s Drosophila medium containing 10% FBS, 2 mM glutamine, and 20 µg/ml gentamicin sulfate. Schneider cells were grown at room temperature in tightly closed, 80-cm2 flasks. Cells were plated at a density of 1.5 x 106 cells/ml in 100-mm dishes 24 h before transfection.

Plasmids and DNA transfections
For transient cotransfection experiments an IGF-IR promoter luciferase reporter construct was employed that includes 476 bp of 5'-flanking and 640 bp of 5'-untranslated regions of the IGF-IR gene [p(–476/+640)luciferase (LUC)]. The promoter activity of this genomic fragment and the location of Sp1 binding sites (GC boxes) and of a CT box have been previously described (12, 13, 30). Transient transfections were also performed using deleted reporter constructs that include 188 or 40 bp of the IGF-IR 5'-flanking region [p(–188/+640)LUC and p(–40/+640)LUC, respectively]. Site-directed mutagenesis of the CT box was carried out directly within p(–476/+640)LUC, using a Transformer site-directed mutagenesis kit (Clontech Laboratories, Palo Alto, CA), as previously described (12). This mutation replaced the 24-bp homopurine/homopyrimidine motif that extends from +434 to +458 in the 5'-untranslated region with a nonspecific DNA sequence of equal size. The mutation in the resulting p(–476/+640{Delta}CT)LUC plasmid was confirmed by DNA sequencing.

A KLF6 expression vector was constructed by inserting the full-length KLF6 cDNA into the EcoRI and XbaI sites of pCI-neo (18). An expression vector encoding the X137 truncation mutant of KLF6 was generated by site-directed mutagenesis of pCI-neo-KLF6 using the QuikChange kit (Stratagene, La Jolla, CA) (18). Expression plasmids containing actin promoter-driven KLF6/Zf9 (pPacZf9) and Sp1 (pPacSp1) have been previously described (12, 15). A wild-type p53 expression vector was provided by Dr. Edward Mercer (Thomas Jefferson University, Philadelphia, PA).

CHO and HCT116 cells were transfected with 0.8 µg of the p(–476/+640)LUC plasmid and increasing amounts of the KLF6 expression vector (pCI-neo-KLF6), along with 0.4 µg of a ß-galactosidase expression plasmid [cytomegalovirus plasmid ß (pCMVß), Clontech Laboratories] using Polyfect (Qiagen, Hilden, Germany), Metafectene (Biontex Laboratories, Munich, Germany), or Jet-PEI (Polyplus, Illkirch, France) transfection reagents. The total amount of transfected DNA was kept constant using pCI-neo DNA. Cells were harvested 48 h after transfection, and luciferase and ß-galactosidase activities were measured as previously described (13). Promoter activities were expressed as luciferase values normalized for ß-galactosidase activity. Schneider cells were transfected with calcium phosphate as previously described (12). In previous studies we found that the large amounts of ß-galactosidase plasmid required to obtain detectable ß-galactosidase activity in Schneider cells severely impaired Sp1 activation of IGF-IR promoter constructs. Therefore, luciferase data were normalized per micrograms of protein in each sample, as measured using the Bradford reagent (Bio-Rad Laboratories, Hercules, CA).

Western immunoblots
Cells were harvested with ice-cold PBS containing 5 mM EDTA and lysed in a buffer containing 150 mM NaCl, 20 mM HEPES (pH 7.5), 1% Triton X-100, 2 mM EDTA, 2 mM EGTA, 1 mM phenylmethylsulfonylfluoride, 2 µg/ml aprotinin, 1 mM leupeptin, 1 mM pyrophosphate, 1 mM vanadate, and 1 mM dithiothreitol. Protein content was determined using the Bradford reagent. Samples were electrophoresed through 10% SDS-PAGE, followed by blotting of the proteins onto nitrocellulose membranes. After blocking with 2.5% skim milk in T-TBS [20 mM Tris-HCl (pH 7.5), 135 mM NaCl, and 0.1% Tween 20], blots were incubated with rabbit polyclonal antihuman IGF-IR {alpha}-subunit or ß-subunit antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), washed with T-TBS, and incubated with a horseradish peroxidase-conjugated secondary antibody. Proteins were detected using the SuperSignal West Pico Chemiluminescent Substrate (Pierce Chemical Co., Rockford, IL). In addition, blots were probed with antibodies against KLF6/Zf9 (R-173; Santa Cruz Biotechnology, Inc.) and actin.

Physical interactions between KLF6 and p53
HCT116–/– cells were transiently transfected with 3 µg each of a KLF6 expression vector (or empty pCI-neo) and a hemagglutinin (HA)-tagged p53 expression vector (or empty pcDNA3-HA) (31), using the FuGene-6 reagent (Roche, Indianapolis, IN). The pcDNA3-HA-p53 plasmid was provided by Dr. William G. Kaelin (Harvard Medical School, Boston, MA). Forty-eight hours after transfection, formaldehyde was added to the cultures to a final concentration of 1% for 10 min at room temperature. At the end of the incubation period, cells were washed twice and harvested using ice-cold PBS. Pelleted cells were resuspended in a 1% sodium dodecyl sulfate-containing buffer, incubated on ice for 10 min, and sonicated with three sets of 10-sec pulses. Cell extracts were then immunoprecipitated using 1 µg of an anti-HA antibody (HA.11, Covance, Inc., Princeton, NJ) for 18 h at 4 C, followed by incubation with 30 µl protein A-G agarose for an additional 2 h. Immunoprecipitates were electrophoresed through 10% SDS-PAGE and immunoblotted with a KLF6 antibody. Membranes were then washed, and proteins were detected as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Activation of the IGF-IR constitutes a basic requirement for progression through the cell cycle. In addition, overexpression of the IGF-IR gene is a typical hallmark in most types of cancer. Certain malignancies, including prostate tumors, display high IGF-IR levels and activity at early stages of the disease, whereas progression to advanced, metastatic stages is associated with a significant decrease in IGF-IR gene expression (4). Although regulation of IGF-IR gene expression is primarily achieved at the transcriptional level (32), the specific transcription factors involved in pathological regulation of the IGF-IR gene have not yet been identified. KLF6 has been characterized as a candidate tumor suppressor whose mutation was correlated with the etiology of prostate, colorectal, and other cancers (18, 19, 21, 22). To begin to address the potential involvement of KLF6 in IGF-IR gene regulation, we employed the P69 and M12 prostate epithelial-derived cell lines. As previously shown, the poorly tumorigenic P69 cell line expresses high IGF-IR levels, whereas the tumorigenic and metastatic M12 derivative exhibits significantly reduced IGF-IR values (33). Western blot analysis using a KLF6 antibody revealed that KLF6 expression was approximately 2-fold higher in P69 than in M12 cells, whereas IGF-IR levels were 2.24-fold higher (Fig. 1Go).



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  		FIG. 1. Expression of endogenous IGF-IR and KLF6 in P69 and M12 prostate epithelial-derived cell lines. Untransfected M12 and P69 cells were lysed in the presence of protease inhibitors, as indicated in Materials and Methods. Equal amounts of protein (100 µg) were separated by 10% SDS-PAGE, transferred to nitrocellulose filters, and blotted with anti-IGF-IR (upper panel), anti-KLF6 (medium panel), and antiactin (lower panel) antibodies. The positions of the 135-kDa IGF-IR {alpha}-subunit and approximately 35–37-kDa KLF6 proteins are indicated. The bar graph represents the densitometric scanning of three independent experiments. {blacksquare}, M12 cells; {square}, P69 cells. Shown are the mean ± SEM. *, P < 0.01 vs. M12 cells.

 
To examine the mechanism(s) responsible for regulation of IGF-IR gene expression by KLF6, transient cotransfection experiments were performed in CHO cells using a KLF6 expression vector together with the reporter plasmid p(–476/+640)LUC, which contains most of the proximal region of the IGF-IR promoter. The results of coexpression experiments are presented in Fig. 2AGo. KLF6 induced a dose-dependent increase in IGF-IR promoter activity, with maximal stimulation occurring with 0.1 µg expression vector (337 ± 32% activation). Remarkably, KLF6 displayed a very potent activity even at input doses as low as 10 ng DNA (260 ± 23% stimulation). At higher DNA doses, the transactivation effect of KLF6 was significantly diminished, although a stimulatory effect was observed at each concentration studied. To assess the effect of a tumor-derived mutant form of KLF6 (X137; C to A substitution at codon 3315, within the KLF6 transactivation domain, that results in the introduction of a premature stop codon), cotransfections were performed using an expression vector encoding the truncated KLF6/X137 protein. As shown in Fig. 2BGo, KLF6/X137 was unable to stimulate IGF-IR gene transcription.



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  		FIG. 2. Regulation of IGF-IR promoter activity by KLF6. The p(–476/+640)LUC reporter plasmid (0.8 µg) was cotransfected into CHO cells with increasing amounts of wild-type (A, {blacksquare}) or truncated (X137; B, ) KLF6 expression vectors along with 0.4 µg of the pCMVß plasmid using the Polyfect transfection reagent. Luciferase and ß-galactosidase activities were measured after 48 h. Luciferase values, normalized for ß-galactosidase, are expressed as a percentage of the luciferase activity of the reporter plasmid in the absence of KLF6 vector. Experiments were performed between three and five times, each time in duplicate. Shown are the mean ± SEM. *, P < 0.01 vs. controls.

 
To examine whether the transactivating effect of KLF6 was associated with corresponding changes in the levels of endogenous IGF-IR protein, Western blot analysis was performed. For this purpose, CHO cells were transiently transfected with 8 µg wild-type or mutant KLF6 expression vectors (or empty pCI-neo plasmid), and after 48 h cells were lysed as described in Materials and Methods. Cell lysates (50 µg) were electrophoresed through 10% SDS-PAGE, followed by blotting of the proteins onto nitrocellulose membranes. Western immunoblotting with an IGF-IR antibody revealed that KLF6, but not KLF6/X137, induced an approximately 1.3-fold increase in mature IGF-IR levels and an approximately 1.8-fold increase in IGF-IR precursor levels (Fig. 3AGo). No change was seen in the levels of actin. To verify that both wild-type and mutant forms of KLF6 were expressed in the cells, lysates were prepared from CHO cells that were transfected with increasing amounts of the appropriate expression vector, and KLF6 abundance was assessed by Western analysis. As shown in Fig. 3BGo, both wild-type and mutant KLF6 proteins were produced by the cells, with apparent molecular weights of approximately 37-kDa (lanes 2 and 3) and approximately 21-kDa (lanes 4 and 5), respectively.



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  		FIG. 3. Effect of KLF6 on endogenous IGF-IR gene expression. A, CHO cells were transfected with 8 µg wild-type or mutant (X137) KLF6 expression vectors (or empty pCI-neo) using the Jet-PEI reagent, and after 48 h, cells were lysed in the presence of protease inhibitors. Equal amounts of protein (50 µg) were electrophoresed through 10% SDS-PAGE, transferred to nitrocellulose filters, and blotted with an anti-IGF-IR antibody. The positions of the 97-kDa IGF-IR ß-subunit and the approximately 205-kDa IGF-IR precursor are denoted. Membranes were reprobed with an actin antibody. The bar graph represents the densitometric scanning of the IGF-IR bands normalized to the corresponding actin bands. Bars are the mean ± SEM (n = 3 independent experiments). *, P < 0.01 vs. controls. B, CHO cells were transfected with 1.5 or 3 µg KLF6 (lanes 2 and 3) or KLF6/X137 (lanes 4 and 5) expression vectors (or empty vector, lane 1), and after 48 h, the abundance of the approximately 37-kDa wild-type and approximately 21-kDa truncated forms was assessed by Western blotting using an anti-Zf9/KLF6 antibody.

 
To more precisely map the IGF-IR promoter region responsible for mediating the effect of KLF6, cotransfections were performed in CHO cells using the reporter plasmids p(–188/+640)LUC and p(–40/+640)LUC (Fig. 4AGo) along with the KLF6 expression vector. Construct p(–188/+640)LUC lacks a cluster of four GC boxes localized between nucleotides –399 and –331 that appears to mediate the majority of Sp1 activation of the promoter (12). Two additional GC boxes are located at nucleotides (nt) –259/–253 and –193/–188, whereas no consensus GC boxes are located between nt –188 and –40. The transactivating effect of KLF6 on the p(–188/+640)LUC construct was significantly reduced compared to the p(–476/+640)LUC plasmid (165.4 ± 13.1% stimulation vs. 286.8 ± 49% at 50 ng DNA; Fig. 4BGo). No further reduction was seen with the p(–40/+640)LUC construct (195 ± 34% activation). Electrophoretic mobility shift assay analysis with nuclear extracts of KLF6-transfected cells, however, failed to reveal a specific KLF6 binding to a labeled fragment contained within the promoter region shown to mediate the effect of KLF6 (data not shown).



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  		FIG. 4. Mapping of KLF6-responsive regions in the IGF-IR promoter. A, Schematic representation of reporter constructs. Plasmids p(–476/+640)LUC, p(–188/+640)LUC, and p(–40/+640)LUC contain, respectively, 476, 188, and 40 bp of the 5'-flanking region ({square}) and 640 bp of the 5'-untranslated region ({blacksquare}) of the IGF-IR gene, fused to a luciferase cDNA (LUC). The transcription start site is denoted by an arrow. The luciferase cDNA is not shown to scale. {diamondsuit}, Consensus GC elements; {bullet}, the CT motif. B, CHO cells were cotransfected with 0.8 µg of the indicated reporter plasmid along with 0.05 µg of the KLF6 expression vector ({square}) or empty pCI-neo ({blacksquare}) and 0.4 µg pCMVß, as indicated in Fig. 1Go. Results are the mean ± SEM of three to five experiments, performed in duplicate. *, P < 0.01; #, P < 0.05 (vs. cells transfected with empty vector).

 
The fact that a significant level of KLF6 stimulation was retained in the p(–40/+640)LUC construct suggested that additional elements in the 5'-untranslated region can also mediate the response to KLF6. We have previously identified a homopurine/homopyrimidine motif or CT box (3'-CCTCCTCCTCCTCGGCCTCCTCCCC-5') located at nt +434 to +458 that was important for Sp1 activation of the IGF-IR promoter (Fig. 4AGo) (12). To examine the contribution of the CT element in KLF6 activation of the promoter, a mutation was made that replaced the 24-bp homopurine/homopyrimidine box with a DNA sequence of similar size. Results of cotransfection experiments using wild-type [p(–476/+640)LUC] and mutant [p(–476/+640{Delta}CT)LUC] reporter constructs indicated that both plasmids were stimulated to a similar extent by a coexpressed KLF6 expression vector (not shown). Hence, these results indicate that, unlike Sp1, the CT box is not critical for KLF6 activation of the IGF-IR gene.

To compare the potency of KLF6 to that of Sp1 in transcriptional regulation of the IGF-IR gene, both zinc finger proteins were expressed in Drosophila Schneider cells along with the p(–476/+640)LUC reporter. The rationale for using Schneider cells lies in the fact that these cells lack endogenous Sp/KLF factors and therefore provide an optimal background for this type of experiments (11). As previously demonstrated, Sp1 induced a strong stimulation of IGF-IR promoter activity (~39-fold induction) (12, 34). KLF6, on the other hand, was unable to enhance IGF-IR gene transcription in this specific cellular environment (Fig. 5Go). Coexpression of both zinc finger proteins, however, resulted in synergistic transactivation of the IGF-IR promoter (~65-fold stimulation).



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  		FIG. 5. Comparison of the transactivating effects of KLF6 and Sp1 in Drosophila Schneider cells. Schneider cells were cotransfected with 5 µg of the IGF-IR promoter construct p(–476/+640)LUC along with 300 ng actin promoter-driven KLF6 (pPacZf9) and/or Sp1 (pPacSp1) expression vectors, using the calcium phosphate method. LUC activity was measured after 48 h. A value of 1 was given to the promoter activity in the absence of expression vectors. Shown are the results (mean ± SEM) of a typical experiment, repeated three times. *, P < 0.01 vs. cells transfected with Sp1 alone.

 
Although previous studies showed that KLF6 up-regulated p21 (WAF1/CIP1) in a p53-independent manner (18), we undertook a series of experiments aimed at establishing whether KLF6 and p53 functionally cooperate in the specific context of IGF-IR gene regulation. The P69 and M12 prostate-derived cell lines express wild-type p53 in equivalent concentrations (Plymate, S. R., unpublished observations). Therefore, we chose to perform cotransfections in the human colorectal cancer cell lines HCT116+/+, containing wild-type p53, and HCT116–/–, in which the p53 gene has been disrupted by targeted homologous recombination. In these cells the stimulatory effect of KLF6 was significantly reduced in p53-null ({blacksquare}) compared with p53-expressing ({square}) cells (149 ± 6% vs. 235 ± 9% at 10 ng DNA; 180 ± 9% vs. 283 ± 33% at 50 ng DNA) (Fig. 6Go). Mutant KLF6/X137 had no effect in either the absence (

) or the presence ({square}) of p53. Western blot analysis revealed no differences in endogenous IGF-IR and KLF6 levels between untransfected HCT116+/+ and HCT116–/– cells. However, expression of KLF6 induced a 1.5- to 2-fold increase in IGF-IR levels in HCT116+/+ cells (data not shown).



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  		FIG. 6. Effect of p53 background on KLF6 action. Human colorectal cancer cell lines HCT116+/+, containing wild-type p53, and HCT116–/–, lacking p53, were cotransfected with 0.8 µg of the p(–476/+640)LUC reporter plasmid and increasing amounts of wild-type or truncated (X137) KLF6 expression vectors along with 0.4 µg pCMVß, using the Metafectene reagent. Promoter activities are expressed as luciferase normalized to ß-galactosidase levels. A value of 100% was given to the IGF-IR promoter activity in the absence of expression vectors. The figure shows the results of three experiments, performed in duplicate dishes. {blacksquare}, HCT116–/– cells transfected with KLF6; {square}, HCT116+/+ cells transfected with KLF6; , HCT116–/– cells transfected with KLF6/X137; {square}, HCT116+/+ cells transfected with KLF6/X137. *, P < 0.001 vs. control cells transfected with empty pCI-neo; #, P < 0.01 vs. p53-lacking cells at the same dose of expression vector.

 
To more rigorously examine the potential cooperation between KLF6 and p53 in modulation of IGF-IR gene expression, HCT116–/– cells were transfected with 4 µg each of expression vectors encoding KLF6, p53, or both (or empty vectors), and IGF-IR levels were measured by Western blotting. As shown in Fig. 7Go (upper panel), transfection of KLF6 alone had no effect on endogenous IGF-IR levels in a p53-null background. However, the combined expression of both KLF6 and p53 vectors induced an approximately 1.5-fold increase in IGF-IR levels and an approximately 5.6-fold increase in IGF-IR precursor levels. Interestingly, coexpression of KLF6 and p53 induced a small reduction in p53 levels (second panel, lane 4).



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  		FIG. 7. Cooperation between KLF6 and p53 in stimulation of IGF-IR gene expression. HCT116–/– cells were transfected with 4 µg each of expression vectors encoding KLF6, p53, or both, using the FuGene-6 reagent. Cells were lysed after 48 h, and 50 µg protein were separated through 10% SDS-PAGE gels. The resolved proteins were transferred to nitrocellulose membranes and blotted with IGF-IR, p53, KLF6, and actin antibodies.

 
Finally, we examined the potential physical interactions between KLF6 and p53 using a modified chromatin immunoprecipitation method. Specifically, HCT116–/– cells were cotransfected with 3 µg of a KLF6 expression vector (or empty pCI-neo) along with 3 µg of an HA-tagged p53 vector (or empty pcDNA3-HA). After 48 h, transfected cells were treated with formaldehyde to a final concentration of 1% for 10 min to induce cross-linking between interacting proteins. Cells were harvested, lysates were immunoprecipitated with an anti-HA antibody, and electrophoresed through 10% SDS-PAGE. After electrophoresis, complexes were transferred to nitrocellulose membranes and blotted with an anti-KLF6 antibody. As shown in Fig. 8Go, immunoblotting with the KLF6 antibody identified KLF6 in anti-HA immunoprecipitates of cells that were transfected with KLF6 and pcDNA3-HA-p53 (lane 4), but not in cells transfected with KLF6 and empty pcDNA3-HA (lane 2).



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  		FIG. 8. Physical interaction between KLF6 and p53. HCT116–/– cells were transfected with 3 µg each of expression vectors encoding KLF6 (or empty pCI-neo) and pcDNA3-HA-p53 (or empty pcDNA3-HA). After 48 h cells were treated with formaldehyde (final concentration, 1%) for 10 min at room temperature, lysed in the presence of protease inhibitors, sonicated, and immunoprecipitated with an HA antibody. Precipitates were loaded onto 10% SDS-PAGE gels, electrophoresed, and transferred to nitrocellulose membranes that were blotted with an anti-KLF6 antibody. I.P., Immunoprecipitation; I.B., immunoblotting.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The involvement of the IGF-IR in the initiation and/or progression of prostate cancer has been the subject of extensive investigation. Contradictory reports, however, have been presented regarding the pattern of expression of the IGF-IR throughout the various stages of the disease. Thus, although progression to androgen independence in prostate cancer xenografts was associated with a significant increase in IGF-IR mRNA expression (compared with the original androgen-dependent tumors) (35), levels of expression were much higher in the nonmetastatic prostate epithelial cell line P69, compared with its metastatic derivative, the M12 cell line (33). Furthermore, although IGF-IR mRNA levels were shown to be largely suppressed in bone marrow metastases (6), other studies reported a persistent expression of the IGF-IR gene in prostate metastases (7). Whereas these conflicting results may arise from the large heterogeneity of the tumors, sample selection, analytical techniques used, etc., the seemingly paradoxical results may reflect the ability of the IGF-IR to mediate both differentiative and proliferative effects. The capacity of the IGF-IR to favor a particular biological pathway is dictated by multiple cellular and extracellular factors, many of whom remain to be identified.

This study identifies the IGF-IR gene as a potential downstream target for transcription factor KLF6 and suggests a potential functional link between these important players in the etiology of a subset of prostate and other types of cancer. Consistent with the postulated tumor suppressor role of KLF6, two recent studies have shown that KLF6 is inactivated in cases of prostate cancer, although the extent of LOH incidence differed between the studies (28% vs. 77% of the cases) (18, 36). Loss of function of the KLF6 gene resulted from three different events, including allelic loss, mutation, and gene silencing (37). Interestingly, epigenetic modifications such as promoter methylation may lead to KLF6 silencing in cases of esophageal squamous cell carcinoma (38). In addition to prostate tumors, mutations in the KLF6 gene were reported in neurally derived cancers, including glioblastomas (11.8% of the cases) and astrocytomas (7%) (21), and in colorectal cancers (22).

The IGF-IR promoter has been characterized as a highly G-C-rich (>75%), initiator type of promoter. Multiple GC boxes, which constitute binding elements for zinc finger-containing transcription factors, were mapped to its proximal region (i.e. the region located immediately upstream of the transcription initiation site). Similar to Sp1, another member of the Sp/KLF family of zinc finger transcription factors, KLF6 exhibited a potent transactivating effect toward the IGF-IR promoter, even at very low input doses of expression vector (12). On the other hand, a prostate cancer-derived truncated mutant lacking the DNA-binding and part of the activation domains of KLF6 had no effect on IGF-IR promoter activity. Comparison of the effects of KLF6 with those of Sp1 revealed that KLF6, unlike Sp1, was unable to stimulate IGF-IR promoter activity in Sp/KLF-null Drosophila-derived Schneider cells. Coexpression of both zinc finger proteins, however, enhanced IGF-IR promoter activity in a synergistic fashion, suggesting that Sp1 and KLF6 exhibit different mechanisms of action. Furthermore, these results seem to imply that the transactivating potentials of KLF6 and Sp1 toward the IGF-IR promoter depend, to a significant extent, on the cellular context.

In addition, the results of deletion analysis experiments revealed that a cluster of four GC boxes located between nt –399 and –331 appears to mediate a significant portion of KLF6 activation of this promoter. We were unable, however, to demonstrate a specific binding of KLF6 to this particular fragment. Because this region was previously shown to bind Sp1 with relatively high affinity (12, 34), our results are consistent with a model in which KLF6 binds (and, potentially, stabilizes) Sp1, thus enhancing its ability to bind and transactivate the IGF-IR promoter. Interestingly, a CT box in the 5'-untranslated region of the promoter, whose presence was critical for Sp1 transactivation of the IGF-IR promoter (12), had no major role in KLF6 action. Physical interactions between Sp1 and KLF6/Zf9 have been reported in the transcriptional activation of TGF-ß1 during stellate cell activation as well as in regulation of the endoglin promoter in response to vascular injury (24, 27). Together with our data, these studies underscore the important role of Sp/KLF family members in cell growth and, furthermore, suggest the existence of a transcriptional network that allows the fine-tuning of gene expression of target gene promoters (11). The participation of other DNA elements in stimulation of IGF-IR gene expression by KLF6 cannot be discounted. Furthermore, additional mechanisms, such as a KLF6-induced increase in IGF-IR mRNA stability or translational efficiency, may also contribute to the increase in IGF-IR expression.

In addition to its DNA sequence-dependent effects, the results of the present study suggest that a significant portion of the biological actions of KLF6 may result from protein-protein interactions with tumor suppressor p53. Specifically, we demonstrated that in HCT116+/+ cells, expressing a wild-type p53, the transactivating effect of KLF6 was approximately 1.6-fold higher than that in p53-null HCT116–/– cells. In addition, coexpression of KLF6 and p53 induced a significant and consistent increase in endogenous IGF-IR and IGF-IR precursor levels, whereas the expression of KLF6 alone had no major effect. We may speculate that the enhanced capacity of KLF6 to stimulate IGF-IR gene expression in the presence of p53 results from its physical interaction with this protein, which may lead to an increased stability of the KLF6 molecule. These results are particularly intriguing in view of our early results showing that wild-type p53, in itself, strongly suppressed IGF-IR promoter activity, whereas a number of tumor-derived mutated p53 molecules stimulated IGF-IR gene transcription (39). The specific KLF6 and p53 domains involved in protein-protein interactions remain to be identified.

Consistent with these results, we recently demonstrated that the inhibitory activity of tumor suppressor WT1 (a member of the early growth response family of zinc finger transcription factors whose mutation was linked to the etiology of Wilms’ tumor) was significantly enhanced in the presence of p53 (40). Similarly, tumor suppressor BRCA1 (breast cancer susceptibility gene) was able to suppress IGF-IR promoter activity in the presence of wild-type, but not mutant, p53 (41). Unlike the cooperative effect between p53 and KLF6 described in the present paper, we have previously reported that Sp1 counteracted the inhibitory effect of p53 on IGF-IR promoter in a dose-dependent manner (42). Together these data indicate that the IGF-IR gene promoter constitutes a molecular target to a number of transcription factors with tumor suppressor activity (32). The level of expression of the IGF-IR gene is the net result of complex interactions involving multiple DNA-binding as well as non-DNA-binding nuclear proteins.

In summary, we have demonstrated that the transcription factor KLF6 is an important activator of the IGF-IR gene. Regulation of the expression of this gene occurs primarily through a cluster of GC boxes in the 5'-flanking region. In addition, the ability of KLF6 to efficiently activate the IGF-IR promoter depends to a significant extent on the cellular status of p53. In the specific context of prostate cancer, we may assume that the decrease in IGF-IR gene expression that occurs in aggressive metastatic stages may result at least in part from impaired activation of the IGF-IR promoter by mutant KLF6. The concentration of cell surface IGF-I-binding sites in malignant cells will ultimately have a significant impact on cell proliferation, differentiation, and sensitivity to apoptosis.


   Acknowledgments
 
We thank Drs. D. Beitner-Johnson, J. L. Ware, E. Mercer, W. G. Kaelin, and B. Vogelstein for providing cell lines and reagents.


   Footnotes
 
This work was supported by National Institutes of Health (NIH) Grant R01-DK-37340, Department of Defense Grant PC020770, and the Bendheim Foundation (to S.L.F.) and by NIH Grants R01-DK-52683 and P01-CA-85859 (to S.R.P.).

Abbreviations: CHO, Chinese hamster ovary; CMV, cytomegalovirus; FBS, fetal bovine serum; HA, hemagglutinin; IGF-IR, IGF-I receptor; KLF6, Kruppel-like factor 6; LOH, loss of heterozygosity; LUC, luciferase; nt, nucleotide.

Received February 10, 2004.

Accepted for publication April 27, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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