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Experimental Therapeutics |
Chiron Corporation, Emeryville, California 94608 [V. E., S. H. L., L. I., L. G., C. G., E. G., M. W., P. D. G., J. H. F., V. C., F. R., R. G., J. E., S. L. A., W. J. F.], and Departments of Surgery [R. S. W.] and Obstetrics/Gynecology and Reproductive Sciences [R. N. T.], University of California San Francisco, California 94143-0556
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Originally discovered as a stimulator of vascular permeability, VEGF-A was subsequently found to be a mitogen and survival factor for endothelial cells through its interaction with two tyrosine kinase receptors, VEGFR-1 and VEGFR-2 (4 , 11, 12, 13) . The survival function is mediated through activation of the phosphatidylinositol 3'-kinase-Akt signaling pathway and up-regulation of Bcl-2 (14, 15, 16) as well as through up-regulation of inhibitors of apoptosis (IAP) family members (17) . VEGF-A expression is regulated at the level of transcription and translation in a complex fashion by numerous oncogenic and tumor suppressor pathways, conditions of hypoxia, and estradiol-receptor complexes (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29) .
In colon cancer, evidence from preclinical and clinical studies has shown the necessity of angiogenesis for tumor growth and metastasis. Furthermore, these studies implicate VEGF-A as the principal angiogenic factor (8 , 30) . The elegant work of Fearon and Vogelstein (31) has described the stepwise progression of the genetic lesions that are found in early adenoma and culminate in carcinoma and metastasis in colon cancer. In both the sporadic and familial adenomatosis polyposis forms of colorectal cancer, inactivation of the APC tumor suppressor gene initiates tumor formation. APC is a component of the Wnt signaling pathway and negatively regulates levels of ß-catenin, a critical mediator of the Wnt transcriptional response (32) .
Absence of functional APC results in the posttranslational stabilization of ß-catenin protein, its translocation into the nucleus, interaction with TCF/Lef family transcription factors, and changes in target gene expression including c-Myc (33)
, cyclin D1 (34)
, matrilysin (35)
, pArf (36)
, Connexin 43 (37)
, uPAR, c-jun and fra-1 (38)
, WISP, (39)
, fibronectin (40)
, PPAR
(41)
, and Naked (42)
.4
These genes contribute to the growth-promoting activities mediated by ß-catenin signaling.
Recent work suggests that Wnt/ß-catenin signaling regulates vessel development in normal and pathological conditions. Several studies have demonstrated the expression of Wnt ligands, Wnt receptors [frizzleds (Fz)], and Wnt inhibitors in vascular cells (43) . Moreover, mice deficient for Wnt-2 or Fz-5 have severe vascular defects, and mutations in human Fz-4 have been linked to familial exudative retinopathy, a hereditary disorder in which retinal angiogenesis is severely impaired (43, 44, 45) . ß-Catenin accumulation in the cytosol and nucleus has been observed in proliferating vessels of the human placenta and has been frequently observed in vascular cells during pathological angiogenesis (43) . A recent report described VEGF-A as a ß-catenin target gene in HeLa cells and colon cancer cells (46) .
To further explore the relationship between the Wnt/ß-catenin pathway and angiogenesis, we searched the promoters of genes encoding the proteins that participate in vessel formation for nucleotide sequences that recognize TCF/Lef. Notably, in the promoter for VEGF-A, we found seven TCF/Lef binding elements. This prompted us to investigate the relationship between VEGF-A levels and APC mutational status in human colon cancer samples and cancer cell lines and also to evaluate this relationship in the Min/+ mouse model of colorectal cancer. In human colon cancer cell lines, ß-catenin induced VEGF-A mRNA and protein expression. Our experiments thus confirm and expand the report that VEGF-A is a ß-catenin target gene (46) , emphasizing the importance of ß-catenin in the early and stepwise events of colon cancer neoangiogenesis.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Patient Samples.
Tissue samples were taken at surgery, with the informed consent of the patients, and frozen for subsequent cryosectioning and laser capture microdissection (50)
. All Institutional Review Board requirements were met.
Animals.
C57BL/6 Min/+ and C57BL6 wild-type littermates were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were housed 5 mice/cage in microisolator cages in an American Association of Laboratory Animal Care-accredited animal facility. Mice were fed special Purina laboratory animal diets (5K20). Mice received food and water ad libitum.
q-PCR.
Total RNA was prepared from cell lysates using the RNeasy total RNA isolation kit (Qiagen Inc., Chatsworth, CA). Using a reverse transcription reaction kit (Perkin-Elmer, Boston, MA), 1 µg of total RNA was reverse transcribed. Ten percent of the reverse transcription reaction was amplified by q-PCR using the LightCycler System (Roche, Indianapolis, IN), and the product was quantified using a standard curve that calculated each cycle number at which the amplification of the product was in the linear phase. This value was normalized to the value of the internal standard GAPDH for each analysis. In the case of patient samples, RNA was extracted from laser capture microdissected cells from matched tumor and normal biopsies and amplified using established procedures to accurately retain relative abundance of mRNAs (51)
. Normalization was to glucuronidase synthase as described previously (42)
. For the VEGF-A mRNA, q-PCR was performed using primers as described by Liu et al. (52)
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Mutational Status of APC, ß-Catenin, and Ras in Patient Samples.
The mutational status of ß-catenin and K-Ras was measured by single strand conformational polymorphism using total tumor RNA, as described previously (42)
. This gel analysis allows mutant RNA to be separated from wild type and is thus more sensitive than PCR and sequencing for detecting small percentages of mutant RNA in the tumor sample. Two hundred ng of total RNA from colon cancer patient samples were reverse transcribed and PCR amplified using the primers described previously for ß-catenin (42)
and the forward (K-Ras-F, 5'-AGAGGCCTGCTGAAAATG-3') and reverse (K-Ras-R, 5'-TTCCTGTAGGAATCCTCTATTGTT-3') primers for K-Ras.
To detect truncations in APC, 200 ng of total tumor RNA from colon tissue samples were reverse-transcribed with primer RT-APC (5'-GTATGGTTACAGATGAGGTTTTTCC-3') and amplified (Advantage GC-rich kit; CLONTECH) with primers as described previously (42) . The PCR products were transcribed in vitro and translated with the T7-coupled reticulocyte lysate system (Promega) in the presence of [35S]methionine and [35S]cysteine to detect translation products after SDS-PAGE and autoradiography (42 , 53) .
ELISA.
VEGF-A and bFGF protein in tissues and cell lysates was quantified using a commercially available ELISA kit (R&D Systems, Minneapolis, MN). Protein concentration of each sample was determined using Bio-Rad (Hercules, CA) protein assay reagent. Preparation of small intestine homogenates from Min/+ and wild-type mice involved freezing the dissected tissue in liquid nitrogen followed by homogenization in ice-cold lysis buffer (1% NP40, 0.5% sodium deoxycholate, and 0.1% SDS in PBS with a protease inhibitor mixture).
Immunohistochemistry.
Small intestine and colon from Min/+ mice and normal litter mates were cleaned of feces with saline, cut open, and laid flat, marking the proximal and distal ends of the organs. Samples were rolled, placed into a histology cassette, and fixed for 4–6 h in 10% neutral buffered formalin. Samples were then processed and embedded in paraffin blocks.
Sections (3–4 µm) sections were cut and floated onto glass slides, baked and deparaffinized in xylene, and rehydrated in graded alcohols. Antigen retrieval was performed using Citrate Plus buffer (Biogenex, San Ramon, CA) in a Biocare steamer. Cooled samples were stained using a Ventana Medical Systems NEXUS automated stainer. Slides were treated for 32 min with appropriate primary antibody [VEGF-A-20 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) at 1:100 dilution, ß-catenin antibody (B19022; Chemicon, Temecula, CA) at 1:500 dilution, or rabbit antibody to vWF (DAKO Carpinteria, CA) at 1:1000 dilution]. Samples were treated for 32 min with secondary antibody [goat antirabbit F(ab')2 biotinylated antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) at 1:100 dilution]. The signal was resolved with a streptavidin-horseradish peroxidase conjugate, 3,3'-diaminobenzidine, and heavy metal enhancement with copper sulfate (Ventana DAB detection reagents; Ventana Medical Systems, Tucson, AZ). Slides were counterstained with hematoxylin and bluing reagent (Ventana Medical Systems). Negative control samples were exposed to rabbit 
-globulin (Jackson ImmunoResearch Laboratories Inc.) at concentrations matching concentrations of primary antibodies and stained as described. All antibodies were diluted in 1x PBS/0.5% BSA (IgG-free fraction)/0.01% thiomersol (pH 7.4).
Cell Fractionation and Western Blotting.
For the analysis of cytosolic ß-catenin, HCT116 cells were lysed in hypotonic buffer with protein inhibitors and passed through a 27G1/2-guage needle. After centrifugation at 6,000 rpm for 10 min, supernatants were further clarified in an ultracentrifuge (Optima TLX; Beckman) at 10,000 rpm for 35 min at 4°C. After normalization for protein, aliquots of lysate were subjected to Western analysis with a ß-catenin monoclonal antibody (Transduction Laboratories, Lexington, KY).
Statistical Analysis.
Data are reported as mean ± SD of multiple experiments. Differences between the laser microdissected, matched normal and malignant specimens were compared using paired Student’s t tests. The unpaired Student’s t test was used to compare VEGF-A mRNA levels in liver metastases with a pool of normal colon tissue and in the Min/+ and wild-type (control) mouse experiments. Correlations between APC and K-Ras mutational status and VEGF-A or VEGF-D gene expression were estimated using a dichotomous model and analyzed by the 
2 test. An arbitrary ratio cutoff of >2.0 for tumor tissue/normal tissue mRNA was selected (Figs. 2
and 3)
. All experiments were repeated a minimum of three times unless otherwise stated. Two-tailed tests with P < 0.05 were accepted as significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Refs. 34
, 35
, and 54
). Thus, whereas the probability of uncovering a ß-catenin/TCF binding consensus sequence by chance is 1 for every 2048 nucleotides, the promoter regions of VEGF-A and KGF each contained at least three such binding sites. Of these, the human VEGF-A gene was particularly noteworthy; it contains four consensus binding sites (sites 1, 2, 5, and 7) and an additional three putative, partial sites (sites 3, 4, and 6) over a 3500-nucleotide stretch that includes the promoter and 5'-untranslated region (Fig. 1B)
. These findings strongly suggest that VEGF-A is a ß-catenin target gene.
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). Of the 20 primary lesions investigated, 17 harbored APC gene deletions, and in all samples, ß-catenin was wild type (Fig. 2B)
. Of these, 11 expressed elevated levels of VEGF-A mRNA when normalized to glucuronidase synthase mRNA (as an internal control) and using an arbitrary cutoff ratio for tumor tissue/normal tissue mRNA of >2.0. By this analysis, none of the three lesions with wild-type APC was positive for elevated VEGF-A levels. Using a conservative 2 analysis and acknowledging that there are only three samples with wild-type APC, the results indicate that lesions harboring APC mutations have statistically higher levels of VEGF-A gene expression than lesions with wild-type APC (2 = 4.31; P = 0.038). Of the five primary tumors showing mutations in both APC and K-Ras, there was no evidence of a significant cumulative effect on VEGF-A mRNA levels (Fig. 2A
; 2 = 0.61; P = 0.436). The explanation for these latter data awaits analysis of more patient specimens.
However, in liver metastases, VEGF-A mRNA levels were elevated in 20 independent patients when compared with a pool of normal colon tissue from 20 patients [autologous tissue was not available (4.70 ± 2.03-fold; P = 0.0001, unpaired t test)], with no correlations to the APC status of the patient (Fig. 3, A and B)
. It should be noted that in none of the samples analyzed (primary tumors and metastases) was the ß-catenin gene mutated.
ß-Catenin Regulates VEGF-A in the APC Min/+ Mouse Model.
To determine the role of ß-catenin in the regulation of VEGF-A during colon cancer progression, we used the Min mouse model (56)
. The Min/+ mouse strain, heterozygous for a nonsense mutation in the murine APC gene at codon 850 (57)
, permits the study of factors that regulate the transition between precancerous and neoplastic growth. VEGF-A protein levels were quantified from small intestine and serum of Min/+ mice and wild-type littermates by ELISA assays. VEGF-A protein levels were increased in samples from Min/+ mice relative to wild-type littermates at 5 months but not at 1 month of age (Fig. 4A)
. Moreover, levels of bFGF were not augmented at 5 months in the Min/+ mice (data not shown).
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). In these samples, no nuclear ß-catenin was detected. In sharp contrast, in 5-month-old Min/+ mice, ß-catenin was detected in both the nucleus and cytoplasm (Fig. 4C)
. VEGF-A immunohistochemistry in normal mice revealed low-intensity, uniform expression in enterocytes as well as in specific regions of the crypts with somewhat higher intensity (Fig. 4B)
. Immunolocalization of VEGF-A in the Min/+ mouse was more diffusely distributed, with some enterocytes showing very intense staining, particularly within polyps. Overall, the level of VEGF-A appeared greater in the Min/+ mice compared with wild-type mice, corroborating the ELISA data. Furthermore, VEGF-A appeared to infiltrate the entire polyp, perhaps contributing to the genesis of a more expansive vasculature (Fig. 4C)
. Staining of normal tissue with vWF showed the expected sporadic occurrence of well-defined blood vessels in the cellular lamina propria of the villi, adjacent to some crypts, with some larger vessels being present in the surrounding connective tissue including the muscularis mucosa (Fig. 4B)
. In 5-month-old Min/+ mice, the intensity of vWF staining was increased compared with the wild-type controls. The tight, focused staining that typified vessels in normal animals was seen to be more diffuse, indicating a distinct change in vessel morphology of Min/+ mice (Fig. 4, B and C)
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ß-Catenin Induces VEGF-A Expression in Normal Colon Epithelial Cells.
To test whether ß-catenin can directly induce VEGF-A expression, an early-passage human colon epithelial cell line, NCM460, derived from normal colon mucosa (47)
, was transfected with cDNA encoding a mutant form of ß-catenin, S37A. This mutant is resistant to phosphorylation-dependent ubiquitination, resulting in its stabilized expression (59)
. Forty-eight h after transfection of a S37A-ß-catenin cDNA plasmid, VEGF-A mRNA levels were quantified in the NCM460 cells using real-time q-PCR. In a representative experiment, after transfection of S37A-ß-catenin, a 280% increase in VEGF-A mRNA and a 240% increase in cyclin D1 mRNA (a known ß-catenin target gene; Ref. 34
) were observed (Fig. 5, A and B)
. Fig. 5A
is the quantitation of the PCR products from the Light cycler using the standard curve. The products of the reaction are shown on the gel in Fig. 5B
. Overexpression of ß-catenin also produced an increase in cellular VEGF-A protein, comparable with the change seen in VEGF-A mRNA in the same experiment (Fig. 5C)
. These observations indicate that in normal colon epithelial cells, transcriptional control of VEGF-A appears to be rate limiting, a situation comparable with estradiol-stimulated expression of VEGF-A in endometrial cells (29)
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with a concurrent reduction in ß-catenin signaling monitored using a ß-catenin reporter assay (data not shown). A significant reduction in ß-catenin protein was also seen by Western analysis in the ß-catenin antisense ODN-treated HCT116 cells, relative to cells transfected with reverse control ODN (Fig. 6B)
. In an earlier report, transfection of cells with the same ß-catenin antisense ODN used here inhibited expression of axin 2 and human Nkd, whereas the reverse control ODN failed to inhibit their expression (42)
. ß-Catenin antisense ODN transfection of HCT116 cells significantly down-regulated VEGF-A mRNA (60 ± 6%; P < 0.05), as measured by quantitative PCR (Fig. 6A)
. c-Myc, another known ß-catenin target gene, was also down-regulated by the ß-catenin antisense ODN treatment (33)
. Similar results were obtained in SW620 colon cancer cells (data not shown).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Given that APC is a tumor suppressor in human colon cancer and that its mutation enhances ß-catenin-regulated gene expression, we wished to determine whether ß-catenin could be involved in the regulation of angiogenesis-related genes. Strikingly, analysis of the promoter for VEGF-A revealed the presence of seven potential ß-catenin/TCF consensus binding sites, pointing to a direct interaction between two of the recognized players involved in colon carcinogenesis. These observations are consistent with the transcription data reported by Zhang et al. (46) , who showed that VEGF-A promoter activity could be stimulated by oncogenic ß-catenin in HeLa cells.
In this report, we extended the studies of Zhang et al. (46)
that described VEGF-A as a ß-catenin target gene in cell lines. Our findings indicate that in primary human colon tumors, increased VEGF-A expression correlated with mutations in the APC tumor suppressor gene that activates ß-catenin signaling. Of 17 tumors with a proven APC mutation, 11 showed an elevation greater than 2-fold. Laser capture microdissection permitted accurate sampling of malignant and normal control tissue in these tumors. Such a correlation was not observed in laser capture microdissected human liver metastases, in which VEGF-A levels were increased regardless of the APC status. Perhaps in this latter situation, genetic instability and tumor progression activate other of the multiple pathways that regulate VEGF-A, overriding the contribution made by the ß-catenin pathway in primary lesions (see "Introduction" and Fig. 7
). Although only a small number of patient tumors were analyzed, laser capture microdissection of malignant and matched autologous normal tissue gave the study an additional level of precision and statistical power. Seventeen of the tumors (85%) tested in this analysis had an APC functional deletion, in accord with the published values for colorectal patients in which APC is mutated in 85% of sporadic colorectal cancers and is detected as a germ-line mutation in >95% of familial adenomatosis polyposis colorectal patients (55)
. We did not detect any point mutations in the NH2-terminal domain of ß-catenin itself (32)
. To our knowledge, this is the first report to correlate VEGF-A expression and APC/ß-catenin genotype in the same human colon cancer samples and suggests that ß-catenin-regulated VEGF-A production may be pivotal in the early stages of colon cancer. Other pathways that regulate VEGF-A production may have more prominence at later stages of the disease.
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. It is unclear why the elevation in VEGF-A was apparent at 5 months and not at 1 month, given that the mice harbor the APC mutation from birth. Immunohistochemistry of the intestinal polyps from the Min/+ mice clearly showed ß-catenin staining in the nuclei with regions of VEGF-A-positive staining throughout the polyp. This correlated with an expansive vasculature within the polyp that exhibited an altered morphology compared with the vessels seen in intestine from wild-type mice (Fig. 4, B and C)
. This is consistent with numerous reports describing tumor vessels as leaky and unstable (65)
.
In vitro experiments with colon cell lines transfected with a stabilized, cytosolic ß-catenin (S37A) resulted in the production of augmented levels of VEGF-A mRNA and protein (Fig. 5, A–C)
. Similarly, a reduction in these levels was seen when the cell lines were transfected with antisense ß-catenin ODN (Fig. 6A)
. These results support reporter assays measuring VEGF-A promoter activity in response to stabilized cytosolic ß-catenin in cancer cell lines (46)
.
The data described here suggest an intimate link between ß-catenin and VEGF-A production in human colon cancer samples and that this early event may initiate angiogenesis. It is relevant to consider the regulation of VEGF-A by ß-catenin described here within the context of the temporal progression of colon cancer (31) . However, another early event, activation of the cyclooxygenase-2 pathway, also produces an increase in VEGF-A that is ß-catenin independent (48 , 66) . Subsequent to these steps are the activation of Ras and inactivation of p53, events that in combination with hypoxia activate a transcriptional program that includes an increase in VEGF-A (25 , 55 , 67) . Additionally, we have shown that cytoplasmic levels of ß-catenin are increased in response to hypoxia.5 The multitude of pathways that increase VEGF-A levels provide compelling evidence of its importance at every stage during tumor progression.
However, translation of therapeutics targeting the VEGF pathway in the clinic remains a challenge for the future (68 , 69) . Several possibilities may account for this. One recent report suggests that the genetic make-up of the tumor cells can profoundly influence the response to antiangiogenic therapy. A combination of vinblastine with an antibody directed against VEGFR-2 was less effective in tumor xenografts derived from HCT116 cells lacking p53 compared with their isogenic counterparts with intact p53 (70) . Furthermore, when the p53 pathway is activated, the oncogenic capabilities of ß-catenin are diminished (36 , 71) . Thus, there is selective pressure for tumors to inactivate p53 to sustain activation of the Wnt/ß-catenin pathway. In light of these findings as well as the emerging importance of Wnt/ß-catenin signaling in the neovasculature, therapies that focus on disrupting this pathway may be beneficial in the field of oncology.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Both authors contributed equally to this work. 
2 To whom requests for reprints should be addressed, at Chiron Corporation, Building 4.6, 4560 Horton Street, Emeryville, CA 94608. Phone: (510) 923-4041; E-mail: wendy_fantl{at}chiron.com
3 The abbreviations used are: VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; APC, adenomatous polyposis coli; q-PCR, quantitative reverse transcription-PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; bFGF, basic fibroblast growth factor; TBE, TCF binding element; Min, multiple intestinal neoplasia; vWF, von Willebrand factor; ODN, oligodeoxynucleotide; Lef, lymphoid enhancer factor.
4 http://www.stanford.edu/
rnusse/pathways/targets.html.
5 V. Easwaran and W. J. Fantl, unpublished data.
Received 8/14/02. Accepted 4/11/03.
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R.A |
