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Endocrinology, doi:10.1210/en.2007-0844
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Endocrinology Vol. 149, No. 4 1890-1897
Copyright © 2008 by The Endocrine Society

Insulin-Like Growth Factor-I Regulates Krüppel-Like Factor-6 Gene Expression in a p53-Dependent Manner

Itay Bentov, Goutham Narla, Hagit Schayek, Kuhihara Akita, Stephen R. Plymate, Derek LeRoith, Scott L. Friedman and Haim Werner

Department of Human Molecular Genetics and Biochemistry (I.B., H.S., H.W.), Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel; Divisions of Liver Diseases (G.N., K.A., S.L.F.) and of Endocrinology and Diabetes (D.L.), Mount Sinai School of Medicine, New York, New York 10029; and Department of Medicine (S.R.P.), University of Washington, Seattle, Washington 98195

Address all correspondence and requests for reprints to: Haim Werner, Ph.D., Department of Human Molecular Genetics and 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
 
High-circulating IGF-I concentrations are associated with an increased risk for breast, prostate, and colorectal cancer. Krüppel-like factor-6 (KLF6) is a zinc finger tumor suppressor inactivated in prostate and other types of cancer. We have previously demonstrated that KLF6 is a potent transactivator of the IGF-I receptor promoter. The aim of the present study was to examine the potential regulation of KLF6 gene expression by IGF-I. The human colon cancer cell lines HCT116 +/+ and –/– (with normal and disrupted p53, respectively) were treated with IGF-I. Western blots, quantitative RT-PCR, and transfection assays were used to evaluate the effect of IGF-I on KLF-6 production. Signaling pathway inhibitors were used to identify the mechanisms responsible for regulation of KLF6 expression. Small interfering RNA against p53 and KLF6 was used to assess the role of p53 in regulation of KLF6 expression by IGF-I and to evaluate KLF6 involvement in cell cycle control. Results obtained showed that IGF-I stimulated KLF-6 transcription in cells with normal, but not disrupted, p53, suggesting that KLF6 is a downstream target for IGF-I action. Stimulation of KLF6 expression by IGF-I in a p53-dependent manner may constitute a novel mechanism of action of IGF-I, with implications in normal cell cycle progression and cancer biology.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE IGFs ARE a family of mitogenic growth factors, binding proteins, and receptors that are involved in normal growth and differentiation of most cells and organs. The vast majority of the biological actions of IGF-I and IGF-II are mediated by the IGF-I receptor (IGF-IR) (1). The IGF axis is involved in numerous pathological states, including disrupted growth conditions and cancer. Developmental abnormalities caused by defective IGF action have been suggested by animal models (2, 3, 4) as well as by epidemiological studies (5). In addition, studies have shown that high-circulating IGF-I concentrations are associated with an increased risk for breast, prostate, lung, and colorectal cancer (6, 7, 8). Although IGF-I, per se, is unable to elicit mutations or cellular transformation, once a malignant transformation has occurred, cell survival in transformed cells is strongly dependent on IGF-I action (9). Furthermore, IGF-I action can override the cellular signals of apoptosis (10). Thus, monoclonal antibodies directed against the human IGF-IR, as well as selective IGF-IR kinase inhibitors, diminished proliferation of several cancers and cancer-derived cell lines, including colon, breast, and others (11). IGF-I functions as a progression factor that is able to control cellular division by modulating specific events that occur mainly at the G1 phase (12). Specifically, IGF-I has stimulated the expression of a number of growth-regulated genes, including c-fos, cyclin-D1, twist, and others (13, 14, 15).

Krüppel-like factor-6 (KLF6), originally named Zf9 or COPEB, is a zinc finger nuclear protein that belongs to the Krüppel-like family of transcription factors. All of the members of this family contain three C2H2 zinc fingers at the C-terminal domain, and recognize GC or CACCC motifs in responsive promoters (16). KLF6 mRNA is broadly expressed in numerous cell types and at various developmental stages (17). KLF6 has been shown to regulate the transcription of genes involved in growth, proliferation, and response to injury. The list of KLF6 targets includes, among others, pregnancy specific glycoprotein 5, TGF-β1, TGF-β receptor, leukotriene C4 synthase, urokinase plasminogen activator, inducible nitric oxide synthase, collagen {alpha}1, and c-Jun (18, 19, 20, 21, 22). This rapidly growing list clearly illustrates the versatility of KLF6. The role of KLF6 as a tumor suppressor was emphasized by studies showing that the KLF6 gene undergoes inactivation in prostate (23), colon (24), and other types of cancer. In prostate cancer, for example, reduced KLF6 expression levels correlated with the increased likelihood of recurrence (25, 26). Analysis of the KLF6 promoter revealed a high sequence homology in mammals, including the presence of a conserved array of positive cis-acting elements. Furthermore, KLF6 gene transcription is under the control of a TATA box-independent initiation mechanism (27). Although KLF6 is quickly induced after exogenous stimulation by serum or phorbol esters (22), little information is available regarding the secreted and cellular factors involved in regulation of KLF6 gene expression.

A potential link between KLF6 and the IGF axis was recently provided by studies showing that KLF6 is a potent transactivator of the IGF-IR gene promoter (28). Specifically, our previous studies demonstrated that expression of wild-type, but not mutated, KLF6 stimulated IGF-IR promoter activity and endogenous IGF-IR levels. This effect of KLF6 was mapped to a GC-rich fragment in the proximal IGF-IR promoter region, containing four Sp1 binding sites. In addition, the effect of KLF6 on IGF-IR levels was largely reduced in p53-null cells, suggesting that the p53 pathway has a crucial role in the interactions between KLF6 and the IGF-IR. Given the involvement of KLF6 and the IGF system in cell cycle progression, as well as in the etiology of colorectal carcinoma and other neoplasms, we hypothesized that the regulatory mechanisms controlling IGF-IR and KLF6 expression may be tightly interrelated. The present study was designed to investigate the potential regulation of KLF6 gene expression by IGF-I and the impact of p53 status on this interaction. Results obtained indicate that the KLF6 gene is a bona fide target for IGF-I action. Thus, IGF-I stimulated KLF6 gene transcription in cells with normal, but not disrupted, p53, and, furthermore, KLF6 disruption resulted in increased apoptosis. Therefore, the results of our study challenge the concept that KLF6 is a classical tumor suppressor and suggest a dual function for this protein. Combined, our data suggest that dysregulated expression of the KLF6 gene by increased IGF-I levels in preneoplastic conditions may have important consequences in terms of cancer initiation and/or progression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures
The human colon cancer cell lines HCT116 p53 +/+ and HCT116 p53 –/– [with normal and disrupted p53, respectively (29)] were maintained in McCoy’s medium with 10% fetal calf serum, 2 mM glutamine, and 50 µg/ml gentamicin sulfate. Cells were grown in monolayers and treated with IGF-I when a confluency of 60% was reached. HCT116 cells were provided by Dr. Bert Vogelstein (Johns Hopkins University School of Medicine, Baltimore, MD).

Western immunoblots
Cells were serum starved overnight and then incubated with increasing doses of IGF-I for 2 h. After incubation, cells were harvested, and whole cell lysates were prepared. Protein content of the lysates was determined using the BCA protein assay (Pierce, Rockford, IL). Samples were subjected to 10% SDS-PAGE, followed by electrophoretic transfer of the proteins to nitrocellulose membranes. After blocking with 3% BSA in 20 mM Tris-HCl (pH 7.5), 135 mM NaCl, and 0.1% Tween 20, blots were incubated with a polyclonal antibody against Zf9 (R-173; Santa Cruz Biotechnology, Santa Cruz, CA), washed extensively with 20 mM Tris-HCl (pH 7.5), 135 mM NaCl, and 0.1% Tween 20, and incubated with a horseradish peroxidase-conjugated secondary antibody. Proteins were detected using the SuperSignal West Pico Chemiluminescent Substrate (Pierce). In addition, blots were probed with antibodies against Erk1, phospho-Erk1/2 (Thr202/Tyr204), Akt, phospho-Akt (Ser473), p21, p53, poly(ADP-ribose) polymerase (PARP), and actin. Antibodies were obtained from Cell Signaling (Beverly, MA). The MEK1/2-specific inhibitor U0126 was obtained from Calbiochem (San Diego, CA), and the PI3K inhibitor LY294002 was from Sigma-Aldrich (St. Louis, MO).

DNA transfections
For transient transfection experiments, three KLF6 promoter deletion fragments containing 2200, 1100, and 500 bp were subcloned upstream of a luciferase reporter gene. The construction of these plasmids has been described elsewhere (Yea, S., G. Narla, X. Zhao, R. Garg, S. Tal-Kremer, E. Hod, A. Villanueva, J. Loke, M. Tarocchi, K. Akita, S. Shirasawa, T. Sasazuki, J. A. Martignetti, J. M. Llovet, S. L. Friedman, submitted for publication). Briefly, human genomic DNA served as a template for genomic PCR. PCR products were subcloned into a TOPO cloning plasmid (Invitrogen, Carlsbad, CA) by TA cloning, and sequenced to confirm that no mutations had occurred in the process. Inserts were excised by enzymatic digestion and ligated into the pGL3-basic luciferase vector (Promega, Madison, WI). Cells were seeded in six-well plates the day before transfection, and transfected with 0.8 µg of the indicated KLF6 promoter construct, along with 0.2 µg of a β-galactosidase expression plasmid (pCMVβ; Clontech, Palo Alto, CA), using the Metafectene reagent (Biontex Laboratories GmbH, Munich, Germany). After transfection, cells were incubated in the absence or presence of IGF-I for 48 h, after which cells were harvested, and luciferase and β-galactosidase were measured, as described previously (30).

Real-time quantitative-PCR
Total cellular RNA was extracted using the RNeasy Mini kit (QIAGEN, Valencia, CA). RNA purity and integrity were assessed by spectrophotometric analysis and by denaturing agarose-MOPS gel. One microgram of total RNA was reverse transcribed using the Superscript First-Strand Synthesis System for RT-PCR (Invitrogen). Real-time quantitative-PCR was performed using the following primers on an ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA): KLF6 forward, 5'-CGGACGCACACAGGAGAAAA-3', and KLF6 reverse, 5'-CGGTGTGCTTTCGGAAGTG-3'; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward, 5'-CAATGACCCCTTCATTGACC-3', and GAPDH reverse, 5'-GATCTCGCTCCTGGAAGATG-3'.

All experiments were done in duplicate and normalized to GAPDH mRNA. Fluorescent signals were analyzed during each of 40 cycles consisting of denaturation (95 C, 15 sec), annealing (55 C, 15 sec), and extension (72 C, 30 sec). Relative quantitation was calculated using the comparative threshold cycle method (CT) (as described in the User Bulletin). CT indicates the fractional cycle number at which the amplified gene amounts to a fixed threshold within the linear phase of amplification. Agarose gel electrophoresis of PCR products of KLF6 and GAPDH was used to confirm homogeneity of the DNA products.

Small interfering RNA (siRNA) p53 silencing
HCT116 cells were seeded in six-well plates the day before transfection, and transfected with 1 µg pSUPER-p53 vector and pBabe-puro plasmid, using the Metafectene reagent. The pSUPER-p53 vector was provided by Dr. Reuven Agami (Netherlands Cancer Institute, Amsterdam, The Netherlands) (31). Cells were selected with 1 µg/ml puromycin for 5 d. Cells were then serum starved overnight and incubated with IGF-I for 2 h, after which cells were harvested, and lysates were prepared for Western immunoblots as described previously.

siRNA KLF6 silencing
The sequence of siRNA for KLF6 consists of a sequence derived from exon 2: sense 5'-GGAGAAAAGCCUUACAGAUTT-3' and antisense 3'-TTCCUCUUUUCGGAAUGUCUA-5'. The double-strand oligonucleotide was cloned and expressed in the pSuper plasmid. A pSuper vector expressing an irrelevant protein, pSuper-Luciferase, was used as a control as described (31). HCT116 cells were seeded in six-well plates the day before transfection, and transfected with 3 µg KLF6 siRNA or control vector, using Metafectene. Cells were harvested after an additional 24 h, and prepared for Western immunoblots and cell cycle analysis.

Cell cycle analysis by fluorescence-activated cell sorting
For fluorescence-activated cell sorting analysis, cells were washed three times with cold PBS, trypsinized with 1 ml trypsin-EDTA, centrifuged, and washed twice in PBS. Cells were resuspended in 1 ml PBS, exposed to propidium iodide, and then analyzed using a FACSort Flow Cytometer (Becton Dickinson, Mountain View, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
KLF6 has been identified as a zinc finger transcription factor with tumor suppressor activity. Mutations in the KLF6 gene have been associated with the etiology of a number of malignancies, including colon carcinoma. To investigate whether the biological actions of IGF-I are associated with modulation of KLF6 gene expression, human colon carcinoma HCT116 p53 +/+ cells were serum starved overnight and then stimulated with increasing concentrations of IGF-I (0–50 ng/ml) for 2 h. KLF6 expression was assessed by Western immunoblotting using a specific antibody. As shown in Fig. 1Go, IGF-I caused a relatively small, although significant and consistent, increase in KLF6 levels at doses of 25–50 ng/ml (28–35% stimulation; P < 0.05 in three independent experiments).


Figure 1
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  		FIG. 1. Dose-dependent effect of IGF-I on KLF6 levels. A, Serum-starved HCT116 p53 +/+ cells were stimulated with increasing concentrations of IGF-I for 2 h. After incubation, cells were lysed as indicated in Materials and Methods, and equal amounts of protein (50 µg) were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. KLF6 levels were determined using a polyclonal antibody against Zf9/KLF6. Blots were reprobed with an actin antibody. Shown are the results of a typical experiment, repeated three times with similar results. B, The bar graph represents the densitometric scanning of the KLF6 bands normalized to the corresponding actin bands of the Western blot shown in A. A value of 100% was given to the optical density of untreated cells.

 
To study the signaling pathway(s) involved in the IGF-I-induced stimulation of KLF6 expression, HCT116 cells were serum starved overnight and then exposed to IGF-I in the presence of inhibitors specific for the MAPK pathway (Erk1/2 inhibitor UO126, at 0.5 mM) and the PI3K pathway (Akt inhibitor LY294002, at 5 mM). As shown in Fig. 2Go (left panel), UO126 suppressed the IGF-I-induced Erk1/2 phosphorylation, and LY294002 suppressed the IGF-I-induced Akt phosphorylation. No changes in total levels of Erk1/2 and Akt were noticed. Likewise, no change was seen in actin level. Suppression of Erk1/2 and Akt phosphorylation were not associated with a significant reduction of the IGF-I-induced elevation in KLF6 levels. Similarly, combined treatment with both inhibitors did not abolish the effect of IGF-I (data not shown).


Figure 2
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  		FIG. 2. Evaluation of the signaling pathways involved in the IGF-I-induced KLF6 up-regulation in HCT116 p53 +/+ and –/– cells. A, Serum-starved HCT116 p53 +/+ (left panel) and –/– (right panel) cells were pretreated with the MEK1/2 inhibitor UO126 (UO) (0.5 mM) or with the Akt inhibitor LY 294002 (5 mM) for 2 h and then incubated with or without IGF-I for an additional 2 h. Cell lysates were electrophoresed, transferred to membranes, and KLF6 levels were determined by Western blotting. After stripping, blots were reprobed with an actin antibody. For assessment of the intracellular signaling pathway(s) involved in the IGF-I-induced KLF6 up-regulation, membranes were probed with antibodies against phospho- and total Erk1/2, and phospho- and total Akt. B, Scanning densitometry of the KLF6 bands normalized to the corresponding actin bands. A value of 100% was given to the optical density of untreated cells. Note the different scales in the y-axes. LY, LY294002. UO, UO126.

 
To assess whether the elevation of KLF6 expression was mediated at the level of transcription, HCT116 p53 +/+ cells were transiently transfected with a luciferase reporter gene under the control of the KLF6 promoter. The KLF6 promoter construct, extending 500-bp upstream of the major transcription start site, was used for this purpose. Transfected cells were incubated in the absence or presence of IGF-I for 2 h, after which cells were harvested, and luciferase and β-galactosidase activities were measured. Results obtained show that IGF-I induced a 2- to 3-fold increase in promoter activity (P = 0.01) (Fig. 3Go). Interestingly, both UO126 and LY294002 inhibitors abrogated the IGF-I-induced increase in KLF-6 promoter activity.


Figure 3
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  		FIG. 3. Regulation of KLF6 promoter activity by IGF-I. HCT116 p53 +/+ cells were transfected with 0.8 µg of a KLF6 promoter-luciferase reporter plasmid, along with 0.2 µg of a β-galactosidase control vector, using the Metafectene reagent. Transfected cells were incubated with 50 ng/ml IGF-I (or left untreated) in the absence or presence of the MEK1/2 inhibitor UO126 (0.5 mM) or the Akt inhibitor LY 294002 (5 mM). After 48 h, cells were harvested, and the levels of luciferase and β-galactosidase were measured. Promoter activities are expressed as luciferase values normalized for β-galactosidase levels. A value of 100% was given to the promoter activity in control, untreated cells. Results are mean ± SD of three independent experiments, performed in triplicate dishes. *, P = 0.01 vs. control.

 
We next evaluated the effect of p53 status on the IGF-I-stimulated KLF6 expression. The rationale for this experiment was the fact that IGF-IR levels and IGF-I action are dependent on p53 status. Specifically, wild-type p53 inhibited, whereas mutated p53 up-regulated, IGF-IR expression (32). In addition, the stimulatory effect of KLF6 on IGF-IR promoter activity also depended on the presence of an intact p53 (28). For this purpose, the HCT116 p53 –/– cell line, in which the p53 gene was disrupted by homologous recombination, was serum starved overnight and then stimulated with IGF-I (50 ng/ml) for 2 h. Western blot analysis revealed that IGF-I was unable to stimulate KLF6 expression in the HCT116 p53 –/– cell line (Fig. 2Go, right panel). To evaluate whether the differential effects of IGF-I on KLF6 levels in p53-containing and p53-null cells were associated with corresponding changes in KLF6 mRNA levels, HCT116 p53 +/+ and HCT116 p53 –/– cells were serum starved overnight and then treated with IGF-I for 30 min, after which total RNA was extracted. One microgram of RNA was reverse transcribed, and KLF6 and GAPDH mRNA levels were assessed by quantitative RT-PCR. Results obtained indicate that IGF-I induced a 40% increase in KLF6 mRNA levels in HCT116 p53 +/+ cells (P < 0.05), whereas no change was seen in HCT116 p53 –/– cells (Fig. 4Go).


Figure 4
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  		FIG. 4. Quantitative RT-PCR evaluation of the IGF-I-induced KLF6 mRNA regulation in HCT116 p53 +/+ and –/– cells. Serum-starved HCT116 p53 +/+ and HCT116 p53 –/– cells were incubated with or without IGF-I (50 ng/ml) for 30 min. Total RNA was extracted, 1 µg was reverse transcribed, and real-time quantitative-PCR was performed using specific KLF6 primers. Relative quantitation was calculated using the CT. Results are mean ± SD of five independent experiments. A value of 100% was assigned to the KLF6 mRNA levels in untreated HCT116 p53 +/+ cells.

 
To identify the KLF6 promoter region responsible for mediating the effect of IGF-I, luciferase reporter constructs spanning 500, 1100, and 2200-bp upstream of the major transcription start site were transfected into HCT116 p53 +/+ and –/– cells. Transfected cells were incubated in the absence or presence of IGF-I for 48 h, after which cells were harvested, and luciferase and β-galactosidase activities were measured. Results obtained showed that IGF-I caused a significant elevation of KLF6 promoter activity in HCT116 p53 +/+ cells transfected with all three constructs (P < 0.05) (Fig. 5AGo). No change in luciferase activity was observed in IGF-I-treated empty PGL3 basic vector-transfected cells. In contrast to p53-containing cells, IGF-I did not increase KLF6 promoter activity in p53-null cells (Fig. 5BGo).


Figure 5
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  		FIG. 5. Regulation of KLF6 promoter activity by IGF-I in HCT116 p53 +/+ and –/– cells. HCT116 p53 +/+ (A) and HCT 116 p53 –/– (B) cells were transfected with 0.8 µg KLF6 promoter-luciferase reporter deletion constructs, including 500, 1100, and 2200-bp upstream of the transcription start site, or an empty PGL3-basic vector, along with 0.2 µg of a β-galactosidase vector. Transfected cells were incubated with 50 ng/ml IGF-I (or left untreated, for control purposes). After 48 h, cells were harvested, and the levels of luciferase and β-galactosidase were measured. Promoter activities are expressed as luciferase values normalized for β-galactosidase levels. A value of 100% was given to the basal promoter activity elicited by the KLF6-Luc –2.2-kb reporter construct in each cell line. Results are mean ± SD of three independent experiments, performed in triplicate dishes. *, P < 0.05 vs. respective control.

 
To corroborate that p53 activity is essential for the IGF-I-induced stimulation of KLF6 gene expression, HCT116 p53 +/+ cells were transfected with a p53-specific siRNA along with a puromycin resistance plasmid. Cells were selected by daily addition of puromycin (1 µg/ml) beginning the day after transfection. Cells transfected solely with the resistance plasmid served as a control. After selection, cells were serum starved overnight and then treated with IGF-I for 2 h. p53, p21, and KLF6 levels were assessed by Western immunoblotting. As shown in Fig. 6Go, transfection with the p53 siRNA caused a marked reduction in p53 expression. Expression of p21, one of the main targets for p53 action, was also decreased, although not to the same extent as p53. Actin levels were unchanged. Consistent with the results obtained in HCT116 p53 –/– cells, p53 silencing in HCT116 p53 +/+ cells abrogated the IGF-I-stimulated increase in KLF6 expression (Fig. 6Go).


Figure 6
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  		FIG. 6. p53 silencing with a specific siRNA abrogates the IGF-I-induced KLF6 up-regulation. A, HCT116 p53 +/+ cells were transfected with 1 µg of a p53-specific siRNA and a puromycin resistance plasmid. Cells were selected with puromycin (1 µg/ml daily, for 5 d). Cells were then serum starved overnight and incubated with or without IGF-I for 2 h. Cell lysates were electrophoresed, transferred to membranes, and probed with antibodies against p53, p21, KLF6, and actin. B, Scanning densitometry of the KLF6 bands normalized to the corresponding actin bands in control and siRNA p53-transfected cells. Open bars represent untreated cells, and solid bars are IGF-I-treated cells.

 
Finally, we investigated the possible biological significance of KLF6 expression in the regulation of apoptosis. For this purpose, HCT116 cells were transfected with a KLF6 siRNA or a control vector. Cells were then serum starved overnight, and apoptosis was assessed using flow cytometry as well as PARP cleavage. Transfection of the KLF6 siRNA markedly reduced KLF6 expression as compared with cells transfected with a control vector (Fig. 7AGo). KLF6 silencing was associated with a drastic decrease in the ratio between full-length PARP (~116 kDa) and an approximate 85-kDa band, which represents a cleavage product of PARP and which serves as a marker of apoptosis. These results are consistent with an antiapoptotic role for KLF6. Actin levels were not changed. Flow cytometric measurements of propidium iodide-stained cells revealed a similar pattern. Thus, KLF6 siRNA caused a 40% increase (P < 0.05) in the population of G0 Sub G1 (apoptotic cells) (Fig. 7BGo). Relative populations of G1, S, and G2M cells were not different between groups (data not shown).


Figure 7
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  		FIG. 7. KLF6 silencing with a specific siRNA leads to enhanced apoptosis. HCT116 p53 +/+ cells were transfected with a KLF6 siRNA, or a control vector. A, Cells were serum starved overnight, after which cell lysates were prepared, electrophoresed, transferred to membranes, and probed with antibodies against PARP, KLF6, and actin. B, Apoptosis was also assessed using flow cytometry. Results show the percentage of cells at G0 Sub G1 (apoptotic cells). Results are mean ± SD of four independent experiments, performed in triplicate dishes.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Large epidemiological studies have suggested that high-circulating IGF-I concentrations are associated with an increased risk for breast, prostate, lung, and colorectal cancers (33). Several mechanisms have been proposed to explain the possible role of the IGF system in the initiation and/or progression of neoplasia (34). Although IGF-I increased chromosomal fragility under experimental conditions (35), it is usually considered to be nongenotoxic. In addition, when nude mice were implanted with colon cancer cells, expression of a dominant-negative truncated IGF-IR resulted in reduced tumor progression (36), suggesting that once a malignant transformation has occurred, cell survival in transformed cells depends, to a large extent, on IGF-I action (37).

Expression of IGF-I by hepatic stellate cells reduces fibrogenesis and enhances regeneration after liver injury. In addition, IGF-I also acts as a negative feedback to restrict stellate cell activation (38). We hypothesized that IGF-I, a potent activator of proliferation, would cause an increase in KLF6 expression. KLF6 was originally described as a gene that is rapidly induced in response to nonspecific stimuli such as liver damage or hemorrhage (39). Furthermore, KLF6 levels are very high in the placenta (17) and are quickly induced after injury (40). Embryonic mutations of the KLF6 gene are a lethal condition (41). Combined, these data suggest that KLF6 has an important role in tissue growth, repair, and proliferation. However, more recently, KLF6 was suggested to act as a tumor suppressor. The list of malignancies in which a defective KLF6 gene has been described is rapidly growing, and it includes prostate, colon, gastric, head and neck, and hepatocellular cancers (23, 24, 42, 43, 44).

Results obtained in the present study suggest that KLF6 is a novel downstream target for IGF-I action. Thus, IGF-I induced a modest stimulation of KLF6 gene expression (i.e. a 2-fold increase in KLF6 promoter activity, a 40% increase in KLF6 mRNA levels, and a 30% increase in KLF6 protein levels). Although the extent of this effect is of relative low magnitude, it is known that small changes in IGF-I levels are often largely amplified and may lead to biologically relevant downstream activities. In addition, it is reasonable to assume that IGF-I-induced activities may, over a long period of time (as is the case in the development and propagation of neoplastic processes), induce major biological effects. Alternatively, the small increment in KLF6 protein levels, but not KLF6 promoter activity, after IGF-I stimulation is compatible with the possibility that basal KLF6 levels are elevated in HCT116 cells, and, therefore, the net effect of exogenous IGF-I is difficult to discern.

Furthermore, our data suggest that abrogation of KLF6 action by siRNA induced apoptosis. This observation contradicts a previous study in which induction of KLF6 induced apoptosis (45). This discrepancy can be attributed to the different cell lines used (colon vs. lung), different endogenous KLF6 levels, and treatment conditions. Moreover, differences may result from potentially distinct activities elicited by different alternatively spliced forms of KLF6 (23). These observations may also imply that short-term IGF-I-induced stimulation of KLF6 has a protective role in acute injury of normal cells, with ensuing induction of cellular repair and growth. On the other hand, prolonged stimulation may be deleterious in that it may facilitate the propagation of a neoplastic process. Combined, our data are consistent with a model in which concerted antiapoptotic signals of KLF6 and IGF-I promote proliferation and growth. However, prolonged stress may uncover KLF6’s tumor suppressor role, conferring upon cell protection from neoplastic transformation. The discrepancy between the effect of the PI3K and MAPK pathway inhibitors in promoter assays compared with their lack of effect in Western immunoblots may be explained by the different time frames of the assays (2 d in the case of transient transfections vs. 2 h in Westerns). However, we cannot discard the possibility that IGF-I affects KLF6 protein levels by controlling posttranscriptional regulatory steps.

In a previous study, we established that KLF6 is a strong transactivator of the IGF-IR gene and that regulation of gene expression by KLF6 occurs primarily though a cluster of four Sp1 sites located in the IGF-IR gene 5'-flanking region (28). Together with our previous results, data are consistent with a model in which IGF-I stimulates KLF6 production. KLF6, in turn, enhances IGF-IR gene biosynthesis, a key step in cell cycle progression. These results suggest the possibility of a "feedback" loop that controls the expression and action of the IGF system and KLF6 in a coordinated fashion. It is reasonable to assume that dynamic control of transcription factor KLF6 may allow this protein to engage in both proliferative and inhibitory types of activities. Of interest, a similar paradigm was recently reported for tumor suppressor BRCA1 (46). Thus, IGF-I and IGF-II were shown to stimulate BRCA1 gene transcription, and BRCA1 mRNA and protein levels in breast cancer-derived cells. BRCA1, in turn, induced an approximate 50% reduction in IGF-IR promoter activity and endogenous IGF-IR levels (47). As in the case of the KLF6-IGF-I interplay, these results are consistent with coordinated regulation of the cellular levels of BRCA1 and cell-surface concentrations of IGF-IRs.

The p53 protein is a multifunctional transcription factor that regulates the expression of genes involved in cell cycle control, apoptosis, and DNA repair. The p53 pathway is activated in response to an extensive variety of cellular stress signals, and various reports identified a convergence between the p53 and IGF-I signaling pathways (48, 49). Association between the p53 and IGF-I pathways can occur at several levels. Thus, p53 regulates PTEN expression (50), whereas PTEN and inhibitors of Akt signaling up-regulated p53 expression (51). The ubiquitin ligase, Mdm2, is of primary importance in regulation of p53 activity (52). IGF-I induced p53 degradation in an Mdm2-dependent manner (53), whereas Mdm2 physically associates with IGF-IR and causes IGF-IR ubiquitination and degradation (54). Because mutations in the p53 gene are the most common genetic alteration in human neoplasia, we speculated that p53 status might play an important role in the ability of IGF-I to control KLF6 expression. The finding that IGF-I did not stimulate KLF6 levels in p53-null cells suggests that the effect of the growth factor requires an intact p53-signaling pathway. Furthermore, this result is consistent with previous data showing that KLF6 was able to transactivate the IGF-IR gene in p53-containing, but not in p53-null, cells (28). In addition, coimmunoprecipitation assays revealed a physical interaction between KLF6 and p53, and it has been suggested that this interaction enhances KLF6 stability. We may assume that in cells, including a mutated p53, IGF-I cannot stimulate the protective action of KLF6, leading to increased tumorogenicity. In summary, we have identified KLF6 as a novel downstream target for IGF-I action. Dysregulated expression of the KLF6 gene as a result of aberrant IGF-I signaling may have important consequences in terms of cancer initiation and/or progression.


   Acknowledgments
 
We thank Drs. Bert Vogelstein and Reuven Agami for providing cell lines and reagents, and Ms. Hila Seti, for statistical analyses of the data.


   Footnotes
 
This work was supported by grants from the U.S.-Israel Binational Science Foundation (to H.W. and S.R.P), and the Cooperation Program in Cancer Research of the Deutsches Krebsforschungszentrum and Israel’s Ministry of Science and Technology (to H.W.).

This work was performed in partial fulfillment of the requirements for a Ph.D. degree by I.B. in the Sackler Faculty of Medicine, Tel Aviv University.

Disclosure Statement: The authors have nothing to disclose.

First Published Online January 3, 2008

Abbreviations: CT, Comparative threshold cycle method; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IGF-IR, IGF-I receptor; KLF6, Krüppel-like factor-6; PARP, poly(ADP-ribose) polymerase; siRNA, small interfering RNA.

Received June 25, 2007.

Accepted for publication December 27, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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