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. Author manuscript; available in PMC: 2016 Apr 27.
Published in final edited form as: Mol Carcinog. 2008 Dec;47(12):947–955. doi: 10.1002/mc.20451

Wild-Type APC Regulates Caveolin-1 Expression in Human Colon Adenocarcinoma Cell Lines Via FOXO1a and C-myc

Upal K Basu Roy 1,2, Rebecca S Henkhaus 3, Natalia A Ignatenko 3,4,5, Jessica Mora 2, Kimberly E Fultz 6, Eugene W Gerner 2,3,4,5,*
PMCID: PMC4847746  NIHMSID: NIHMS766917  PMID: 18444242

Abstract

Genetic evidence suggests that caveolin-1, an essential component of membrane caveolae, acts as a tumor promoter in some, and a tumor suppressor in other cancers. The role of caveolin-1 in colon carcinogenesis is controversial. We report here, for the first time, that caveolin-1 is transcriptionally induced in colon cancer cells in response to conditional expression of a full length adenomatous polyposis coli (APC) gene. This induction of caveolin-1 by APC is mediated by both FOXO1a, a member of the Forkhead family of transcription factor, and c-myc. The FOXO1a protein, which is increased by wild-type APC expression, induces caveolin-1 promoter–reporter activity and binds directly to a FKHR consensus binding sequence in the caveolin-1 promoter. The c-myc protein, which is reduced in the presence of wild-type APC, acts to repress caveolin-1 expression by acting at non-E-box containing elements in the caveolin-1 promoter. These data predict that caveolin-1 protein expression would be decreased early in colonic carcinogenesis, which is associated with loss of wild-type APC. Our results would be consistent with the interpretation that caveolin-1 may have tumor suppressing functions during early stages of colon carcinogenesis.

Keywords: colon cancer, caveolin-1, APC, C-myc

INTRODUCTION

Ever since its discovery as a substrate for phosphorylation by the Rous sarcoma virus, the regulation and expression of caveolin-1 has intrigued biologists. Caveolin-1 is the primary protein involved in stabilizing caveolae, which are 50–100 nM flask-shaped plasma membrane invaginations [1]. Initial work on the transcriptional regulation of caveolin-1 was mainly carried out in murine (NIH-3T3 cells) fibroblasts. Engelman et al. [2] were the first to show that cellular transformation was accompanied by a decrease in caveolin-1 levels. They demonstrated that introduction of an activated H-RAS oncogene was sufficient to downregulate caveolin-1 mRNA levels. They also showed that this effect was not restricted to H-RAS, but was a phenomenon observed in NIH-3T3 cells transformed with other RAS isoforms. Also, caveolin-1 levels were downregulated in response to other oncogenes like SRC and V-ABL. Another study carried out by the same group established that caveolin-1 can be repressed by the myc isoforms, C-myc and N-myc in NIH-3T3 cells [3]. Razani et al. [4] further demonstrated that caveolin-1 levels can be suppressed by the E6 protein of the human papilloma virus (HPV). Another important observation of the study was that caveolin-1 downregulation was critical for E6-mediated transformation, since reexpression of caveolin-1 in E6-expressing NIH-3T3 cells was sufficient to rescue the transformed phenotype [4]. A hallmark of cancer cells is the ability to evade cellular senescence [5]. Work done by the Lisanti group showed that caveolin-1 is upregulated during cellular stresses like oxidative stress [6]. This is critical for the cells to undergo stress-mediated senescence. They speculated that cancer cells downregulate caveolin-1, and thereby acquire resistance to stress-mediated senescence. The studies mentioned, thus far, have been carried out in murine fibroblasts. Caveolin-1 expression is regulated differently in murine and human fibroblasts. This is evident from the study carried out by Sasai et al. [7], which shows that caveolin-1 downregulation is not observed in human fibroblasts transformed with an activated RAS oncogene, indicating that there are fundamental differences between rodent and human fibroblast transformation. As an extrapolation of their studies carried out in rodent fibroblasts, Lisanti and coworkers [8] proposed that caveolin-1 was a tumor suppressor since CAV1 is localized to a region, which is often deleted in many human cancers. Unfortunately, the regulation and expression of caveolin-1 in human cancers is far more complex. Caveolin-1 is upregulated in several tumors like bladder, urothelial, renal and prostate carcinoma [9]. In prostate cancer cells, caveolin-1 upregulation is mediated via protein kinase C ε (PKCε) [10]. Levels of caveolin-1 are reduced in tumors originating in the breast, cervix and ovary [9]. In breast cancer, caveolin-1 expression is down-regulated by the activity of oncogenes like the Neu tyrosine kinase, and the P-I3 kinase [11,12].

The expression of caveolin-1 in colon carcino-genesis is still contested. Several groups have shown that caveolin-1 is downregulated in human colon cancers [13]. Work done by Bender et al. [13] revealed that caveolin-1 downregulation is necessary for colon cancer progression, and ectopic expression of caveolin-1 in caveolin-1 deficient cells was sufficient to reduce tumor growth in a SCID mouse model system. Using methylation-specific PCR, Lin et al. [14] showed that the caveolin-1 gene promoter is methylated at CpG islands, leading to gene silencing and decreased caveolin-1 expression in sporadic colorectal cancer cases. However, studies carried out by other groups have shown that caveolin-1 levels are, in fact, increased in colon cancer samples. Using immunohistochemistry (IHC), Fine et al. [15] demonstrated that caveolin-1 expression is elevated in colon adenocarcinoma samples, in comparison to normal colonic epithelial tissue. Their studies were further corroborated in findings by Patlolla et al. [16], which showed that caveolin-1 expression is increased in experimentally induced colon tumors in a murine model system. Thus, there is still a controversy surrounding the expression of caveolin-1 in colorectal cancer.

As far as regulation of expression of caveolin-1 in colorectal cancer is concerned, it seems that caveolin-1 transcriptional control is mainly exerted by tumor suppressors. Peroxisome proliferator activated receptor (PPARγ) is a tumor suppressor, often downregulated in colon tumors [17]. Liganddependant activation of PPARγ upregulates caveolin-1 expression in human-colon tumor derived cells [18]. In another study, it was shown that caveolin-1 expression is transcriptionally controlled by a member of the forkhead (FKHR) family of transcription factors, FOXO3a in human colon cancer cells [19]. Timme et al. demonstrated in Rat1A cells that caveolin-1 is negatively regulated by C-myc. Acting as a repressor, C-myc binds not at the canonical E-box sequence, but at a sequence termed the INR (initiator) [20,21]. It has been demonstrated that expression of wild-type adenomatous polyposis coli (APC) restricts expression of C-myc [22].

In a cDNA microarray study carried out by our laboratory using human colon cancer cells, caveolin-1 was upregulated in response to a wild-type APC gene. In the present study, we have evaluated the mechanisms by which this gene regulates caveolin-1 expression in human colon cancer cells. We have also discussed the implications of these findings in colon cancer progression and how this might resolve earlier conflicting findings regarding caveolin-1 expression in colon carcinogenesis.

MATERIALS AND METHODS

Cell Culture

The HT29-APC and HT29-β-gal cells were a kind gift from Dr. Bert Vogelstein and were maintained in McCoy's 5A medium supplemented with 10% fetal bovine serum (FBS), 1% Penicillin–Streptomycin–Glutamine and 600 μg/mL Hygromycin B [23]. For the induction of the full length APC gene in HT29-APC cells, ZnCl2 (300 μM) was added for the indicated time periods as described in figure legends. The HCT116 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA) and were maintained in McCoy's 5A medium supplemented with 10% FBS and 1% Penicillin–Streptomycin. The SW480-Mock and SW480-APC cells have been previously described [24]. All cells were grown at 37°C in a humidified incubator with 5% carbon dioxide.

Reagents and Antibodies

All chemicals and reagents were of the highest grade. Zinc chloride and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO). G418 sulfate was purchased from CellGro, Mediatech Inc. (Lawerence, KS). Lipofectamine 2000, Hygromycin B and all media were from Invitrogen (Carlsbad, CA). The primary antibodies used were caveolin-1 (Santa Cruz Biotechnology, Santa Cruz, CA), FOXO1a (Cell Signaling Technology, Inc., Danvers, MA), FOXO3a (Cell Signaling Technology, Inc.), APC (Calbiochem, San Diego, CA) and myc (Upstate, Lake Placid, NY). The secondary antibodies for immunoblots with primary antibodies from Cell Signaling Technology were from Cell Signaling Technology, Inc. All other secondary antibodies were purchased from Santa Cruz Biotechnology. The APC Western was performed as per the manufacturer's protocol.

cDNA Microarray Study

cDNA microarray analysis was performed as previously described [25]. The RNA concentration was quantified and used for cDNA microarray analysis.

Western Blotting

Cells (2 × 106 cells/100 mm plate) were plated for 48 h. They were lysed in radio-immunoprecipitation assay (RIPA) buffer with protease inhibitors (10 μg/mL Aprotinin, 10 μg/mL phenyl–methyl–sulfonyl chloride (PMSF) and 50 μM sodium orthovanadate). Samples were kept on ice for 30 min, followed by centrifugation at 14 000 rpm for 10 min. Supernatants were collected and protein concentration was determined with the Bio-Rad DC protein assay (Bio-Rad, Hercules, CA). Fifty micrograms of protein were resolved on a 12.5% SDS–PAGE gel and transferred overnight to a Hybond-C nitrocellulose membrane, at 4°C. The next day, the membrane was blocked in Blotto A [5% nonfat dry milk in Tris-buffered saline with 0.1% Tween-20 (TBST)] for 1 h at room temperature. Membranes were probed with antibodies in blocking buffer for 2 h at room temperature. Alternatively, primary antibodies from Cell Signaling were diluted in 5% bovine serum albumin (BSA) in TBST, overnight at 4°C. After washing with TBST three times, the membrane was probed with horse-radish peroxidase (HRP)-conjugated secondary antibody, washed and protein was detected with an enhanced chemiluminescence detection reagent (Amersham Biosciences Corp., Piscataway, NJ). All blots were then stripped with Pierce Restore Western Blot Stripping Buffer (Pierce, Rockford, IL) as described by the manufacturer and reprobed with appropriate protein loading controls.

For APC Western blotting, cells (2.5 × 106 cells/100 mm plate) were grown for 24 h and then treated with 300 μM ZnCl2 for 24 h. Cells were then lysed in the SDS loading buffer. Lysates (120 μg) were resolved on a 5% SDS–PAGE gel and transferred overnight to a Hybond-C nitrocellulose membrane, at 4°C. The next day, the membrane was blocked in Blotto A for 1 h at room temperature and incubated with APC antibody (1:75 dilution, EMD Chemicals, Inc., San Diego, CA) in blocking buffer overnight at 4°C. After washing with TBST three times, the membrane was probed with anti-mouse HRP-conjugated secondary antibody for 1 h (1:2000 dilution), washed and the APC protein was detected with an enhanced chemiluminescence detection reagent (Amersham Biosciences Corp.).

RNA Extraction and Semi-Quantitative RT-PCR

For total cellular RNA extraction, cells (2.5 × 106) were plated in 100 mm plates. Cells were harvested by trypsinization after 48 h of plating. Total cellular RNA was extracted using the Qiagen RNAEASY Kit (# 74104, Qiagen, Valencia, CA) using manufacturer's instructions. Reverse transcription was performed on 1 μg of total RNA with random primers and the Reverse Transcription System kit (Promega Corp., Madison, WI). The cDNA template (100 ng) obtained from the above reaction was amplified using specific primers and puReTaq™Ready-To-Go™PCR Beads (Amersham Biosciences Corp.). For caveolin-1 RT-PCR analysis, 200 ng of cDNA was used as template. The PCR products were then resolved on agarose gels and photographed using the Kodak 1D Imaging software. PCR primers used, along with their Tm and amplicon sizes for various genes were: Caveolin-1 F- 5′ TCA ACC GCG ACC CTA AAC ACC 3′ R- 5′ TGA AAT AGC TCA GAA GAG ACA T 3′ (Tm = 60°C, 561 bp), FOXO1a (FKHR) F- 5′ GAC GCC GTG CTA CTC GTT 3′ R- 5′ CGG TTC ATA CCC GAG GTG (Tm—51°C, 490 bp) [26], GAPDH F- 5′-TGG TAT CGT GGA AGG ACT CAT GAC-3′R- 5′-AGT CCA GTG AGC TTC CCG TTC AGC-3′ (Tm—50–60°C, 181 bp). All PCR primers were synthesized through Invitrogen Custom Primer Synthesis.

Plasmids and Transfection

The human caveolin-1 promoter region, corresponding to −737 to −37 in the promoter region, cloned into the PGL2 vector was a kind gift from Dr. Vijay Shah [27]. This promoter was used in the C-myc study experiments. The human caveolin-1 promoter cloned into the PGL2 vector, with the putative FKHR binding sites spanning the region between −2080 and −1569, was obtained from Dr. Burgering [19]. This promoter was used in the FOXO1a study. The C-myc expression vector was constructed by cloning C-myc cDNA into a pcDNA 3.1 vector (Invitrogen) using standard cloning procedures. The Renilla-TK plasmid was purchased from Promega Corp., and used as a transfection efficiency control in all promoter–reporter transfection experiments. For transfection with promoter–reporter constructs, 3.0 × 105 cells were plated per well, in 6-well plates. The next day, cells were transfected with Lipofectamine 2000 transfection reagent, as described by the manufacturer. Typically, cells were transfected with 2 mg of promoter and 0.01 μg of Renilla-TK vector. For studies with C-myc expression vector, 2 μg of C-myc vector was transfected with 2 mg of promoter and 0.01 μg of Renilla-TK vector. The HT29-APC and HT29-β-Gal cells were transfected as previously described [22]. Twenty-four hours after transfection, fresh medium with zinc was added to the cells. The next day, the cells were lysed in Passive Lysis Buffer from the Dual Luciferase Assay kit (Promega Corp.) and dual luciferase activity was measured, as described by the manufacturer, using a TD-20/20 Luminometer.

Chromatin Immuniprecipitation (ChIP) Assay

HT29-APC and HT29-β-Gal cells (2 × 106 cells/100 mm plate) were plated and next day, treated with zinc chloride (ZnCl2) at a final concentration of 300 μM. Cells were harvested 6 and 24 h after the addition of zinc and ChIP assay was carried out using the ChIP assay kit (Catalog No: 53006) from Active Motif (Carlsbad, CA). The antibodies used for the analysis were FOXO1a and FOXO3a. The primers used to amplify the caveolin-1 promoter region have been previously described [19]. They amplify the putative FKHR binding regions in the caveolin-1 promoter.

Transfection of HCT116 and HT29 Cells With FOXO1a siRNA

Dharmacon siGENOME SMARTpool anti-FOXO1A siRNA and negative control siRNA (catalog numbers L-003006-00 and D-001820-01, respectively) were prepared as follows: The lyophilized sequences were resuspended in the manufacturer's siRNA Suspension Buffer to a concentration of 20 μM. The suspension was incubated at 90°C for 1 min, then at 37°C for 60 min to disrupt aggregates formed during lyophilization. Both siRNA sequences were used at a final concentration of 75 nM during cell transfection. Sixteen to eighteen hours preceding transfection, 2.0 × 105 cells/well were plated in 6-well plates in DMEM with 10% FBS and no antibiotics. Just prior to transfection, cells were rinsed 3 with saline. Cells were then incubated in Opti-MEM with siRNA and Lipofectamine2000 (Invitrogen; 12 μl/well) for 6 h. After the 6 h, an equal volume of Opti-MEM plus 10% FBS was added to each well. Cell lysates and RNA were collected 48 h posttransfection.

Densitometric Quantification of RT-PCR Gels and Western Blots

Densitometric analysis of RT-PCR gels and Western blots was performed using Scion Imaging Software, available at www.scioncorp.com.

Statistical Analysis

All statistical analysis was carried out using Microsoft Excel Software and the paired Student's t-test was used to establish significance. In all figures shown, a * indicated a P-value of <0.05.

RESULTS

Induction of a Full Length APC Gene in an APC-Deficient Colon Tumor-Derived Cell Line Induces Caveolin-1 mRNA and Protein Expression

The HT29 cells are a human colon adenocarcinoma cell line, which express a truncated APC gene. The HT29-APC and HT29-β-gal cells are isogenic clones of the HT29 cells, which are stably transfected with a full length APC or a β-galacatosidase gene (as a vector control) respectively, under the control of a zinc-inducible metallothionein promoter [23]. We have previously shown that addition of ZnCl2 leads to induction of a full length APC gene in the HT29-APC cells [22]. Caveolin-1 was found to be a transcriptional target of the APC tumor suppressor gene in a cDNA microarray study done in our laboratory (4.853-fold induction in response to a wild-type APC gene in HT29-APC cells, P-value 0.0245). In order to validate the results obtained in the cDNA microarray study, caveolin-1 mRNA and protein expression was studied after induction of the full length APC protein. There was a dramatic increase in caveolin-1 mRNA in HT29-APC cells, after the induction of a full length APC gene, as shown in Figure 1A. Caveolin-1 protein level was significantly upregulated in response to a full length APC gene, as shown in Figure 1B. This confirmed the cDNA microarray results. However, in order to confirm whether the effect of APC expression was restricted to the HT29-APC cells, we used another cell line SW480 (expressing truncated APC) and its isogenic clone, which is transfected with a full length APC gene [24]. As shown in Supplementary Information Figure 1, expression of a full length in the SW480 cells leads to an increase in caveolin-1 mRNA levels.

Figure 1.

Figure 1

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Induction of wild-type APC gene in the HT29-APC cells induces caveolin-1 mRNA and protein expression. (A) Cells (2.5 × 106 cells/100 mm plate) were grown for 24 h and then treated with 300 μM ZnCl2 for 24 h. RNA was extracted and analyzed as described in Materials and Methods Section (upper panel). Caveolin-1 mRNA was quantified by semi-quantitative RT-PCR . The figure is representative of three independent experiments, which were quantified by densitometry using Scion Image software. In the figure, * indicates P < 0.05. (B) Cells (2.5 × 106 cells/100 mm plate) were grown for 24 h and then treated with 300 μM ZnCl2 for 48 h. For protein expression analysis, cells were lysed in RIPA buffer after 48 h of ZnCl2 treatment. Lysates were resolved on a 12.5% SDS–PAGE gel and probed for caveolin-1 by immunoblotting as described in Materials and Methods Section (upper panels). The figure is representative of three independent experiments, which were quantified by densitometry using Scion Image software (lower panel). In the figure, * indicates P < 0.05.

APC Induces Caveolin-1 mRNA Via Inducing the Activator, FOXO1a

Previous studies have revealed that the FKHR family of transcription factors can induce caveolin-1 in colon cancer cells [19]. In the same cDNA microarray study with a full length APC gene in the HT29-APC cells, FOXO1a (FKHR) was identified as a target of the APC tumor suppressor gene, and is upregulated in response to a wild-type APC gene (3.458-fold induction in response to a wild-type APC gene in HT29-APC cells, P-value 0.0013). We first validated whether induction of a full length APC gene in the HT29 colon tumor cells lead to a concurrent increase in FOXO1a mRNA and protein levels, and found that zinc treatment leads to an increase in FOXO1a expression in the HT29-cells (Figure 2A and B). We did not see any change in expression levels of another member of the FKHR family, FOXO3a (Supplementary Information Figure 2). In order to test whether FOXO1a is involved in APC-dependent upregulation of caveolin-1, HT29-APC and HT29-β-gal cells were transfected with a caveolin-1 promoter reporter construct, which encompasses the distal caveolin-1 promoter region (−2080 to −1569), and contains a previously characterized FKHR-binding site at position −1814 [19]. After induction of a wild-type APC gene with zinc treatment, there was a significant increase in the caveolin-1 promoter reporter activity, as compared to the untreated cells (Figure 3A). As a final confirmatory test to determine whether APC-dependent upregulation of caveolin-1, in part, is mediated via the induction of FOXO1, we performed a Chromatin Immunoprecipitation Assay (ChIP) using a FOXO1 antibody. The PCR primers were designed to amplify the FKHR-binding site at position −1814 upstream of the transcription start site. ChIP assay revealed that there is an amplification of the 511 bp fragment, corresponding to the caveolin-1 promoter sequence encompassing the FKHR binding site (Figure 3B). No PCR product was detected in the control samples, and very little product was seen in the HT29-β-gal cells, indicating that the sequence amplified, does indeed, correspond to the region containing the FKHR-binding site. We confirmed the effect of FOXO1a on caveolin-1 expression by knocking down FOXO1a expression in HCT116 cells (these cells expression high levels of caveolin-1) [16]. We found that reduction in FOXO1a expression leads to a concurrent decrease in caveolin-1 expression (Figure 3C).

Figure 2.

Figure 2

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Induction of wild-type APC gene in the HT29-APC cells induces FOXO1a mRNA and protein. (A) Cells (2.5 × 106 cells/100 mm plate) were grown for 24 h and then treated with 300 μM ZnCl2 for 6 h. FOXO1a mRNA was quantified by semi-quantitative RT-PCR as described in Materials and Methods Section (upper panel). The figure is representative of three independent experiments, which were quantified by densitometry using Scion Image software (lower panel). In the figure, * indicates P < 0.05. (B) Cells (2.5 × 106 cells/100 mm plate) were grown for 24 h and then treated with 300 μM ZnCl2 for 24 h. For protein expression analysis, cells were lysed in RIPA buffer and lysates were resolved on a 12.5% SDS–PAGE gel and probed for FOXO1a by immunoblotting as described in Materials and Methods Section (upper panels). The figure is representative of three independent experiments, which were quantified by densitometry using Scion Image software (lower panel). In the figure, * indicates P < 0.05.

Figure 3.

Figure 3

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Induction of wild-type APC gene in the HT29-APC cells induces caveolin-1 promoter reporter activity and direct binding of FOXO1a to the caveolin-1 promoter region, whereas FOXO1a knockdown reduces caveolin-1 expression. (A) A caveolin-1 promoter reporter construct with the consensus FKHR binding site (from −2080 to −1569 bp) (FOXO) was transfected in the HT29 cells. Following 48 h of ZnCl2 treatment, luciferase activity was measured as described in Materials and Methods Section. The figure is representative of three independent experiments. In the figure, * indicates P < 0.05. (B) ChIP analysis was carried out according to Materials and Methods Section using FOXO1a antibody, and the caveolin-1 promoter region was amplified. (C) HCT116 and HT29 cells (2.0 × 105 cells/well in 6-well plates) were grown for 16–18 h and then transfected with FOXO1a siRNA as described in Materials and Methods Section. Following transfection, FOXO1a mRNA was quantified by semi-quantitative RT-PCR as described in Materials and Methods Section. The figure is representative of three independent experiments.

Induction of Caveolin-1 Gene Expression in Colon Cancer Cells by APC Is, in Part, Through Decreased C-myc Expression

The C-myc oncogene is a transcriptional target of the APC tumor suppressor [22]. In the cDNA micro-array study carried out by our laboratory using HT29-APC cells, it was identified as a downstream target of the APC gene [22]. We first validated whether induction of a full length APC gene in the HT29 colon tumor cells lead to a concurrent decrease in C-myc protein. As shown in Figure 4A, induction of the wild-type APC gene leads to a decrease in C-myc protein levels. In order to test whether C-myc is involved in caveolin-1 expression, HT29-APC and HT29-β-gal cells were transfected with a caveolin-1 promoter–reporter construct, which spans the sequence −737 to −37 in the caveolin-1 promoter region. It has been shown previously, that this sequence contains the putative initiator elements (INRs) that are known to be binding sites for myc and consequent myc-dependent suppression of gene expression [3]. As seen in Figure 4B, induction of wild-type APC gene leads to a significant increase in the caveolin-1 promoter reporter activity. In order to confirm whether this effect was mediated by an APC-dependent decrease in myc expression, a C-myc expression vector was cotransfected with the caveolin-1 promoter reporter. We verified the C-myc over-expression in these cells by immuno-blot analysis (Figure 4C). Figure 4D demonstrates that expression of C-myc protein abrogates the APC-dependent caveolin-1 promoter reporter activity. There is no effect of C-myc expression in the HT29-β-gal cells, indicating that the effect if mediated via expression of a full length APC gene.

Figure 4.

Figure 4

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Wild-type APC gene decreases C-myc expression and increase caveolin-1 promoter reporter activity in the HT29-APC cells. (A) Cells (2.5 × 106 cells/100 mM plate) were grown for 24 h and then treated with 300 μM ZnCl2 for 24 h. They were then lysed in RIPA buffer. Lysates were resolved on a 12.5% SDS–PAGE gel and probed for C-myc by immunoblotting (upper panels). The figure is representative of three independent experiments, which were quantified by densitometry using Scion Image software (lower panel). In the figure, * indicates P < 0.05. (B) Cells (0.5 × 106 cells/well in 6-plate) were grown for 24 h and transfected with a caveolin-1 promoter–reporter plasmid, spanning the caveolin-1 promoter region between −737 and −37 bp. The next day, Zn was added. After 48 h of ZnCl2 addition, cells were lysed and luciferase activity was measured as in Materials and Methods Section. The figure is representative of three independent experiments. In the figure, * indicates P < 0.05. (C) Cells were transfected with a C-myc expression vector, as described in Materials and Methods Section. The next day, ZnCl2 was added. After 48 h of ZnCl2 addition, cells were lysed in RIPA buffer. Lysates were resolved on a 12.5% SDS–PAGE gel and probed for C-myc by immunoblotting. The figure is representative of three independent experiments. (D) Cells were transfected with both a caveolin-1 promoter–reporter plasmid, described in B, and with a C-myc expression vector. The next day, ZnCl2 was added. After 48 h of ZnCl2 addition, cells were lysed and luciferase activity was measured as in Materials and Methods Section. *P < 0.05.

DISCUSSION

Results presented in the present study indicate that caveolin-1 expression is influenced by the APC tumor suppressor gene in colon cancer cells. Loss of wild-type APC is associated with a decrease in caveolin-1 protein, which occurs via a mechanism affecting caveolin-1 transcription and involves the FOXO1a and c-myc transcription factors. The APC tumor suppressor gene is lost or mutated in nearly 90% of sporadic colonic adenomas [28], indicating that loss of wild-type APC is an early event in colonic carcinogenesis. Thus, our data would predict that caveolin-1 protein levels would decrease early in this process. Our prediction is consistent with the findings of Bender et al. [13], which showed that caveolin-1 protein was decreased in human colon cancers. Further, this group showed that ectopic expression of caveolin-1 reduced tumorigenicity of colon cancer cells when implanted in SCID mice. Furthermore, our results are also consistent with the results of Lin et al. [14], who reported that the caveolin-1 promoter activity was reduced by methylation and correlated caveolin-1 gene silencing with loss of caveolin-1 protein expression in human colon cancer tissues.

However, our findings are not consistent with two other reports, which found caveolin-1 protein levels increasing in colon cancers [15,16]. As noted earlier, caveolin-1 expression is influenced by several signaling pathways. Specific PKC isoforms appear to enhance caveolin-1 expression in prostate cancer cells [10]. RAS isoforms also regulate caveolin-1 expression [2]. In human colon cells, activated K-RAS is associated with an increase in caveolin-1 RNA and protein expression (manuscript in preparation). Since a progressive accumulation of mutations in a number of genes, including APC and K-RAS, is known to occur during colon carcinogenesis [29], determination of the specific genetic makeup of colon cancers may be needed in order to understand changes in caveolin-1 which occur during the transitions from normal colonic mucosa to adenomas to invasive cancer and subsequently metastatic colon cancer.

In our study, we found that introduction of a wild-type APC gene in an APC-deficient colon tumor cell line induces caveolin-1 expression at the level of transcription. Van den Heuvel et al. [19] have shown that caveolin-1 is regulated by the FKHR group of transcription factors. Though their work showed the role of FOXO3a in induction of caveolin-1 in human colon cancer cells, it is plausible that other FKHR family members might have the same effect. This is because all members of this family bind to the same consensus DNA sequence, TTGTTTAC [30]. Using a distal caveolin-1 promoter reporter construct, we saw that induction of the full length APC protein led to an increase in the activation of the promoter. This promoter reporter construct has an FKHR canonical binding site at position −1814 upstream of the start of transcription [19]. To confirm whether the increase in promoter activity was because of binding of FOXO1a, we performed a ChIP assay which revealed that FOXO1a bound to the TTGTTTAC site in the caveolin-1 promoter region. Thus, APC might transcriptionally regulate caveolin-1 expression in human colon cancer cells via induction of the activator FOXO1a. However, this might not be the only mechanism of caveolin-1 induction via APC. The C-myc oncogene has been shown to be a transcriptional target of the APC tumor suppressor [22]. It is known to repress caveolin-1 transcription by binding to the INR elements present in the caveolin-1 promoter region [3]. These are pyrimidine rich regions to which myc isoforms can bind to and repress transcription. Our work shows that APC-dependent upregulation of caveolin-1 may, in part, be via the downregulation of C-myc. The myc-dependent repression of caveolin-1 has also been reported in prostate cancer cells [21]. However, further studies showing direct binding of myc protein to the INR elements in the caveolin-1 promoter region need to be done to verify the effect of C-myc in APC-dependent caveolin-1 regulation. Though we have evaluated two mechanisms by which APC regulates caveolin-1 expression, there might be other pathways involved. PPARg can upregulate caveolin-1 in human colon tumor-derived cells [18]. Several groups have shown that PPARγ can act as a tumor suppressor in an APC-wild-type background [31]. Several groups have reported methylation-dependent silencing of caveolin-1 in colon cancer [14]. We saw that treatment of HT29 cells with a methylation inhibitor, 5′-Aza-deoxycytidine did not affect caveolin-1 expression in HT29 cells (Supplementary Information Figure 3). Thus, APC exerts different effects at two different regions of the promoter: at the FOXO1a binding site in −2080 to −1569, and through myc in the region between −737 and −37.

Our work reveals that caveolin-1 is transcriptionally regulated by the APC tumor suppressor in colon carcinogenesis. The APC tumor suppressor is mutated in as many as 85% of diagnosed colorectal cancer cases, and is often considered to be an initiating mutation [32]. This has implications in tumorigenesis. We have recently shown that caveolin-1 is a negative regulator of polyamine uptake by colon cancer cells [33]. Caveolin-1 has been shown to induce cellular senescence [6]. Caveolin-1 reexpression in a colon cancer cell line devoid of caveolin-1 expression, the HT29 cells, decreases its tumor forming capacity [13]. Loss of the APC tumor suppressor is also associated with an evasion of senescence [34]. Thus, caveolin-1 might be a mediator of APC-dependent cellular senescence and other APC-dependent anti-tumorigenic phenotypes. Through differential regulation by the APC tumor suppressor, caveolin-1 might be an important regulator of APC-dependent tumor suppression in colon cancer.

Supplementary Material

Supplementary Information

ACKNOWLEDGMENTS

The authors wish to thank David E. Stringer, Dr. H. Yerushalmi, Karen Kachel and Nathaniel S. Rial for assistance with experimental set-up. This work was supported by NIH/NCI grants CA95060 and CA72008.

Abbreviations

APC

adenomatous polyposis coli

FBS

fetal bovine serum

Footnotes

This article contains supplementary material, which may be viewed at the Molecular Carcinogenesis website at http://www.interscience.wiley.com/jpages/0899-1987/suppmat/index.html.

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