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Tumor Biology |
Department of Molecular and Cellular Biology [W. J. H., N. M. G.], Scott Department of Urology [N. M. G.], and Department of Pathology [R. J. B.], Baylor College of Medicine, Houston, Texas 77030; The Johns Hopkins Oncology Center, Brady Urological Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287 [C. F. H.]; and Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia 30321 [J. W. S.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(HIF-1), vascular endothelial growth factor (VEGF), and the cognate receptors VEGFR1 and VEGFR2 were characterized. Immunohistochemical and in situ analyses of prostate tissue specimens derived from the spontaneous autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP) model identified a distinct early angiogenic switch consistent with the expression of PECAM-1, HIF-1, and VEGFR1 and the recruitment of new vasculature to lesions representative of high-grade prostatic epithelial neoplasia (PIN). During progression of prostate cancer, the intraductal microvessel density (IMVD) was also observed to increase as a function of tumor grade. Immunoblot and in situ analyses further demonstrated a distinct late angiogenic switch consistent with decreased expression of VEGFR1, increased expression of VEGFR2, and the transition from a differentiated adenocarcinoma to a more poorly differentiated state. Analysis of clinical prostate cancer specimens validated the predictions of the TRAMP model. This resolution of prostate cancer-associated angiogenesis into distinct early and late molecular events establishes the basis for a "progression-switch" model to explain how the targets of antiangiogenic therapy might change as a function of tumor progression. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Investigations into the molecular basis of tumor vascularization have previously demonstrated that tumors express a number of autocrine and paracrine factors that activate or otherwise facilitate this process. These include VEGF,3 basic FGF (FGF-2), acidic FGF (FGF-1), matrix metalloproteinases, insulin-like growth factor I, and angiopoietin-1 (3) . VEGF and the closely related proteins VEGF-B and VEGF-C are very potent proangiogenic factors that are expressed by several types of tumors (4, 5, 6, 7) . Expression of VEGF has been shown in prostate cells of normal, benign, and malignant phenotypes (8, 9, 10) . For example, in a study of patients with either BPH or organ-confined prostate cancer, no statistically significant difference was observed in the levels of urinary VEGF between these groups and the normal controls (11) . In the Dunning R3327 PAP tumor model VEGF, VEGFR1, and VEGFR2 mRNA levels were elevated compared with normal ventral prostate (12) . However, VEGF was readily detected in the serum of mice harboring orthotopic grafts of the PC-3 M and DU145 prostate cancer cell lines (13) . Although tumor cells may express VEGF, expression of the cognate receptors VEGFR1 and VEGFR2 is generally believed to be restricted to endothelial cells.
Identification of the molecular mechanisms regulating expression of VEGF, VEGFR1, and VEGFR2 is a subject of intense investigation. For example, hypoxic conditions have been demonstrated to regulate expression of VEGF, VEGFR1, and VEGFR2 (14)
. VEGF and VEGFR1 were both observed to be up-regulated by the transcription factor HIF-1 (15, 16, 17)
. Furthermore, a HIF-2 response element was identified in the regulatory region of the VEGFR2 gene (18)
. A number of peptide and steroid hormones associated with prostate cancer growth such as basic FGF2, insulin-like growth factor I, and androgens have also been shown to regulate expression of VEGF (19, 20, 21)
.
The mechanism of VEGFR signal transduction is complex. Many signaling proteins have been shown to be associated with the activated VEGFR1, including: SHP-2, p27, Grb2, PLC
, Crk, and NCK (22)
. However VEGFR1 does not appear to signal through the MAPK cascade or induce endothelial cell proliferation, yet it appears to be important in endothelial cell migration (23)
. On the other hand, VEGFR2 is believed to signal through both the MAPK cascade to induce endothelial cell proliferation as well as the PI3'K to activate an antiapoptotic pathway (24
, 25) .
Equally important to the activators of the angiogenic phenotype are the molecules that inhibit this process. In fact, it has been proposed that a tumor will become vascularized as a consequence of stochastic events that disrupt any balance that exists between the activators and inhibitors (2)
. Molecules that posses antiangiogenic properties, such as thrombospondin-1, IFN /ß, tissue inhibitor of metalloproteinase-1, angiopoietin-2, endostatin and angiostatin, are currently being isolated, characterized, and exploited as potential therapeutics (3)
. It is obvious that a better understanding of the temporal and spatial patterns of expression of molecules that regulate this process will be required to develop more effective diagnostics and therapeutics.
The complex nature of the VEGF signaling axis and the inherent interactions between epithelium, stroma, and endothelium have made the angiogenic switch difficult to characterize during the natural history of clinical prostate cancer. Hence, we have chosen to examine the molecular changes in the VEGF signaling axis in the autochthonous spontaneous TRAMP model. Briefly, TRAMP mice express a PB-SV40 early gene (T/t antigen; Tag) construct under prostate specific control of the minimal rat probasin promoter and display mild to severe hyperplasia of the prostate epithelium, resembling PIN by 6–12 weeks of age (26)
. Well-differentiated neoplasia is generally observed in TRAMP mice between 10 and 16 weeks of age, and between 18 and 24 weeks of age, all of the mice will display primary tumors and metastases to distant sites (27
, 28)
. The restricted temporal and spatial pattern of prostate cancer progression in TRAMP affords a unique window of opportunity for investigation of the earliest molecular events of the disease. By investigating angiogenesis in the TRAMP model, we now demonstrate that the angiogenic switch is, in fact, a series of sequential molecular events that resolve to a distinct early "initiation event" and a later "progression event." The initiation event corresponds to the expression of HIF-1 and VEGFR1, whereas the progression event corresponds to the expression of VEGFR2 and the transition from a differentiated adenocarcinoma to a more poorly differentiated state. Subsequent analysis of clinical prostate cancer specimens was used to confirm and validate the predictions of the TRAMP model. Taken together, these data establish the basis for a "progression-switch" model to explain how the targets of antiangiogenic therapy change as a function of tumor progression and to ascertain that VEGFR2 is a logical target for intervention therapy.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Clinical Prostate Samples.
Prostate tissue was obtained from the S.P.O.R.E. prostate tissue bank at Baylor College of Medicine. The clinical specimens represented Gleason grades 5–7, and normal sections from each patient were used as controls.
Western Blot Analysis.
Total cell lysates were prepared by tissue homogenization in RIPA buffer [50 mM Tris (pH 8.0), 1% Triton X-100, 150 mM NaCl, 0.02% sodium azide, 5 µg/ml leupeptin, 5 µg/ml pepstatin, 1 mM phenylmethanesulfonyl fluoride]. Approximately 40 µg of protein from each tumor sample was denatured in loading buffer [100 mM Tris (pH 6.8), 0.01% ß-mercaptoethanol, 20% glycerol, 4% SDS] by boiling for 10 min and loaded onto a 7.5 or 12% SDS-polyacrylamide gel. Proteins were separated by electrophoresis and then transferred to Immobilon-P membrane (Millipore, Bedford, MA). The filters were blocked for 1 h in TBST [0.1 M Tris (pH 7.4), 1.5 N NaCl, 0.5% Tween 20], 5% nonfat dry milk, and 2% BSA. When appropriate, blots were incubated overnight at 4°C in 3% nonfat dry milk in TBST with a rabbit polyclonal antibody specific for VEGF diluted 1:400 (Ab-1; Neomarkers, Fremont, CA), or a rabbit polyclonal antibody specific for VEGFR1/Flt-1 diluted 1:100 (C-17; Santa Cruz, Santa Cruz, CA), or a rabbit polyclonal antibody VEGFR2/Flk-1 diluted 1:1000 (N-931; Santa Cruz), or a monoclonal antibody specific for ß-Actin diluted 1:5000 (AC-74, Sigma). After several washes in 0.5% nonfat dry milk in TBST, filters were incubated with either horseradish peroxidase-conjugated -rabbit or -mouse (Amersham, Piscataway, NJ) IgG antibody diluted 1:5000 for 1 h at room temperature in 3% nonfat dry milk in TBST. After several washes with 0.5% nonfat dry milk the filters were developed with enhanced chemiluminescent (ECL) detection system (Pierce, Rockford, IL) according to the manufacturer’s recommended protocol and exposed to X-Ray film (XAR-1; Eastman Kodak, Rochester, NY). The Western analysis was scored as either positive for a band present at the appropriate size or negative when there was no evidence of a band at the appropriate size; all of the blots were performed in duplicate, and each contained at least one positive and one negative sample.
Immunohistochemistry.
Tissues procured at necropsy were fixed in 4% paraformaldehyde for 4 h and then transferred to 70% ethanol and embedded in paraffin, and 5-µm sections were cut and mounted on ProbeOn-Plus slides (Fisher, Houston TX). Slides were hydrated through xylene and graded alcohol and equilibrated in PBS. Antigen retrieval was performed with 10 µg/ml proteinase K (Amresco, Solon, OH) at 37°C for 10 min. Endogenous peroxidases were quenched with 3% H2O2 in methanol. Nonspecific binding was blocked with Power Block (BioGenex, San Ramon, CA) according to manufacturer’s recommendations. When appropriate, slides were incubated with a rat monoclonal antibody specific for CD31/PECAM-1 (PharMingen, San Diego, CA) at a 1:50 dilution. Immunodetection of HIF-1 was performed essentially as described previously (29)
. All of the slides were subsequently washed several times in PBS with 0.1% Tween 20 and were incubated with a 1:100 dilution of biotin-conjugated goat antirat IgG (PharMingen, San Diego, CA) or 1:2000 dilution of biotin-conjugated goat antirabbit IgG (Vector Laboratories, Burlingame, CA) for 1 h at room temperature. Immunoreactive species were detected with the Vectastain Elite ABC immunoperoxidase system according to the manufacturer’s recommendations, (Vector Laboratories, Burlingame, CA). Sections were counterstained with methyl green, dehydrated through graded alcohol into xylene, and mounted under glass coverslips.
IMVD.
To determine IMVD, sections were stained with an antibody to CD31/PECAM-1 as described above. The number of intraductal vessels was determined by counting three high-power (x40) fields of the highest vascular density. The averaged IMVD for each specimen was determined and statistical analysis was performed by nonparametric ANOVA multiple comparisons with tumor grade using Fisher’s least significant difference. Interductal vessels were considered to be normal vasculature.
Mouse VEGF Immunoassay.
The Quantikine M Mouse VEGF immunoassay (R&D Systems, Minneapolis, MN) was used to determine serum levels of VEGF according to the manufacturer’s recommendations. Briefly, blood samples collected from TRAMP mice and nontransgenic littermates prior to sacrifice were allowed to clot for several hours and serum fraction was recovered after centrifugation. Serum was stored at -20°C. All of the sera were diluted 5-fold in supplied diluent, and the immunoassay was performed with supplied standards and controls. Statistical analysis was performed using nonparametric ANOVA multiple comparisons.
In Situ Hybridization.
To detect the VEGF axis by in situ hybridization, 33P-antisense and -sense RNA probes were synthesized from linearized plasmids containing subcloned cDNA fragments. To generate the probes total RNA was first isolated from 12-day mouse embryos using Trizol (Life Technologies, Grand Island, NY) according to the manufacturer’s recommendations. All of the probes were then generated by reverse transcription-PCR using 1 µg of total RNA as template essentially as described previously (30)
. The VEGF probe (213-bp), pVEGF-213, was prepared with primers that amplified within exons 1–5 that are shared between all splice forms (forward, 5'-ATGGACGTCTACCAGCGAAG; and reverse, 5'-GCTTTGGTGAGGTTTGATCC), corresponding to bp 213–426 of GenBank accession number M95200 (31)
. The VEGF-B-probe (215 bp), pVEGF-B-215, was amplified with primers (forward, 5'-CCAGAAGAAAGTGGTGCCAT; and reverse, 5'-ATGAGGATCTGCATTCGGAC) corresponding to bp 285–500 of GenBank accession number MMU43836 (32)
. The VEGF-C-probe (290 bp), pVEGF-C-290, was amplified with primers (forward, 5'-TGTGTCCAGCGTAGATGAGC; and reverse, 5'-CCACATCTGTAGACGGACAC) corresponding to bp 339–629 of GenBank accession number U73620 (33)
. The sVEGFR1/sFlt-1 probe (316 bp), psVEGFR1–316, was amplified with primers (forward, 5'-AAATTTAAAAGCAGGAGGATT; and reverse, 5'-TTGCTGCTATGAAGCAGAAGA) corresponding to bp 2265–2581 of GenBank accession number D88690 (34)
. The VEGFR1/Flt-1 probe (268 bp), pVEGFR1–268, was amplified with primers (forward, 5'-AACAGCACGCTGTTTATTGAA; and reverse, 5'-ATCTGGGTCCATAATGATTGA) corresponding to bp 2166–2434 of GenBank accession number L07297 (35)
. The VEGFR2/KDR probe (427 bp), pVEGFR2–427, was amplified with primers (forward, 5'-TTTGGAAACCTATCAACTTAC; and reverse, 5'-GGACAGGAACAAATTATCTCCAT) corresponding to bp 4085–4512 of GenBank accession number S53103 (36)
. All of the primers were synthesized by Genosys, Woodlands, TX, or Life Technologies, Inc., with SacI and EcoRI or KpnI and BamHI sites added to the 5'-end of the forward and reverse primers, respectively. The cDNA products were directionally cloned into KpnI- and SacI-restricted pBluescript KS-(Stratagene, Inc.). Confirmation of the sequence and insert orientation was determined by sequence analysis (Molecular Genetics Core Facility, University of Texas-Houston Medical School). To prepare 33P-probes, the plasmids were linearized with EcoRI or BamHI restriction enzymes, and T7 or T3 polymerases were used to synthesize the antisense and sense probes, respectfully. Before hybridization, paraffin sections were hydrated in xylene and graded alcohol and equilibrated into PBS. Slides were treated with proteinase K (20 µg/ml, 15 min) and 4% paraformaldehyde (20 min) and were allowed to hybridize with RNA probes (3.5 x 106 cpm/section) at 42°C overnight in hybridization solution. Slides were washed in 2x SSC/15 mM ß-mercaptoethanol (20 min), 2x SSC/RNase (40 µg/ml, 15 min), 0.1x SSC (42°C, 15 min), and 0.1x SSC (room temp, 15 min), dehydrated through graded alcohols, and dipped in Kodak NTB2 emulsion (Kodak, Rochester NY). Emulsion was developed after 5–7 days, antisense and sense of each probe were developed after equal time of emulsion, and slides were counterstained with H&E. Slides were then dehydrated through graded alcohol into xylene and mounted under glass coverslips.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, vasculature was observed to be exclusively interductal in the dorsolateral prostates of nontransgenic mice at all ages (6, 12, 18, and 24 weeks). In contrast, we observed two distinct and different types of vascularization in the PIN lesions of TRAMP mice. In low-grade PIN, the vasculature was predominantly interductal (Fig. 1B)
; however, vasculature was observed to become increasingly arching the edge of the duct and progressively intraductal within epithelial clusters concomitant with the appearance of the high-grade PIN lesions (Fig. 1C)
. In the moderately and poorly differentiated TRAMP tumors, the vasculature was observed to be scattered within the tumor (Fig. 1, D and E)
, with the most obvious blood vessels appearing in the most poorly differentiated tumors (Fig. 1E)
. These observations demonstrate that an early angiogenic event temporally correlates with the transition between low- and high-grade PIN.
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, intraductal microvessels were not detected in prostate samples obtained from nontransgenic mice. In contrast, only 18% (3 of 17) of high-grade PIN samples had no detectable IMVD, whereas 35% (6 of 17) of high-grade PIN samples had an IMVD between 1 and 10, and 47% (8 of 17) had an IMVD >10. When compared with nontransgenic littermates, the increase in IMVD observed in well-, moderately, and poorly differentiated and androgen-independent tumors was statistically significant (P < 0.005). Furthermore, the increase in IMVD was found to be a function of progression. The poorly differentiated tumors had a significant increase in IMVD when compared with low-grade PIN, high-grade PIN, and well-differentiated tumors (P < 0.005). In addition, the androgen-independent tumors derived from castrated mice demonstrated a significant increase in IMVD compared with all of the other stages, including the poorly differentiated tumors isolated from intact mice (P < 0.0001).
Temporally and Spatially Restricted Expression of VEGF.
To further characterize changes in the VEGF axis at the molecular level during progression of prostate cancer, in situ hybridization analysis was performed, in which the VEGF riboprobe was designed across the first 5 exons to detect all known isoforms of VEGF. As shown in Fig. 2
, VEGF mRNA was not detected in the prostate sections obtained from nontransgenic mice (Fig. 2D)
, whereas transcripts were detected in samples representing PIN, and well- and poorly differentiated prostate cancers (Fig. 2, E and F)
, compared with sense riboprobe controls (Fig. 2, G, H, and I)
. In contrast, VEGF-B mRNA was readily detectable in all of the samples examined (data not shown). Transcripts encoding VEGF-C were detected in only 1 of 2 poorly differentiated tumors (data not shown).
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, expression of the predominant isoforms VEGF-121 and VEGF-165 were detected by immunoblotting in an extract prepared from a 12-days-post-conception mouse embryo control. However, these isoforms were not detected in the extracts prepared from dorsolateral or ventral prostate of adult mice nor in the samples prepared from PIN lesions or from well-differentiated or moderately differentiated tumors. In contrast, VEGF-165 was readily detectable in samples prepared from the poorly differentiated tumors of intact and castrated mice.
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, significantly elevated levels of serum VEGF were detected in mice bearing poorly differentiated or androgen-independent tumors as compared with nontransgenic animals or animals bearing PIN or well- or moderately differentiated lesions (P < 0.01). There was also a strong correlation between serum VEGF levels and IMVD (F, 7.296; R2, 0.139; P < 0.01), which indicated that VEGF expression is associated with a high IMVD index. It is interesting to note, that 35% (5 of 14) animals bearing a poorly differentiated or androgen-independent tumor had levels of serum VEGF between 0 and 20 pg/ml, well within the normal range. Hence, these observations also raise the possibility that other angiogenic factors, independent of VEGF, could be facilitating tumor growth in these animals.
Temporally and Spatially Restricted Expression of VEGFR1 and VEGFR2 Isoforms.
Expression of VEGFR1 mRNA was detected with a riboprobe designed to recognize the alternatively spliced exon, thus making it specific for the full-length receptor, in samples representing PIN and well-differentiated tumors (Fig. 4E)
compared with sense riboprobe controls (Fig. 4, G, H, and I)
but not in prostate samples from nontransgenic mice (Fig. 4D)
or in poorly differentiated tumors (Fig. 4F)
. Expression of VEGFR1 mRNA in the epithelial cells of PIN lesions and well-differentiated tumors supported our observations on the localization of VEGFR1 by immunohistochemical analysis (data not shown). Although transcripts encoding the sVEGFR1 were detected in normal prostate and samples representing PIN and well-differentiated prostate cancer, sVEGFR1 transcripts were detected in only 1 (50%) of 2 poorly differentiated tumors (data not shown). Transcripts encoding VEGFR2 were easily detected in poorly differentiated TRAMP tumors (Fig. 4L)
compared with sense riboprobe control (Fig. 4O)
. VEGFR2 mRNA was also detectable in normal prostate (Fig. 4J)
. Hence, the in situ analysis demonstrates that epithelial cells in PIN lesions and in well-differentiated tumors expressed VEGFR1, whereas the majority of VEGFR2 expression appears to be restricted to endothelial cells that are representative of more advanced prostate cancer.
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in general agreement with our in situ analysis. It is interesting to note that the sVEGFR1 was detected only in the high-grade tumors (Fig. 5B)
. Similarly, expression of VEGFR2 was primarily detectable in the high-grade tumors (Fig. 5C)
. Although expression of VEGFR2 was also detectable in normal prostate, it was surprising to note that VEGFR2 expression was not detectable in PIN or in well-differentiated or moderately differentiated lesions (Fig. 5C)
. The expression of the short form of VEGFR2 (sVEGFR2) was readily detectable in moderately, poorly differentiated, and androgen-independent tumors (Fig. 5D)
. On the basis of this analysis, there appears to be a profound and invariant switch between the expression of VEGFR1 and VEGFR2 during the progression of prostate cancer that correlates with the transition from a differentiated to a poorly differentiated disease.
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Expression Is an Early Angiogenic Event in Prostate Cancer. has been shown previously to regulate the expression of VEGF and VEGFR1. Hence, we examined the expression of HIF-1 during the progression of prostate cancer in the TRAMP model. As shown in Fig. 6
, HIF-1 was detected by immunohistochemistry in PIN lesions and in well-differentiated and poorly differentiated tumors (Fig. 6, B, C, and D)
but not in the prostate glands of nontransgenic mice (Fig. 6A)
. It is interesting to note that the samples representative of PIN lesions with the highest level of VEGF and VEGFR1 mRNA expression also expressed substantial levels of HIF-1 in the nucleus (Fig. 6B)
. Therefore, it appears that expression of HIF-1 correlates with, and perhaps precedes, the expression of VEGFR1 and VEGF mRNA in PIN lesions.
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, there were no significant changes observed in the level of VEGF expression in the cancer samples [21 (70%) of 30] when compared with the normal samples [19 (63%) of 30]. However there was a significant decrease in VEGFR1 expression in the cancer samples [21 (70%) of 30] when compared with the normal samples [28 (93%) of 30; P < 0.05]. Even more striking was the increase in VEGFR2 expression in the cancer samples [25 (83%) of 30] when compared with the normal samples [11 (37%) of 30; P < 0.0001). It is also interesting to note that 8 (27%) of 30 of this collection of similarly matched cancer samples lost VEGFR1 expression whereas 15 (50%) of 30 gained VEGFR2 expression when compared with the matched normal samples. This analysis of human prostate specimens not only validates the predictive nature of the TRAMP model but also validates VEGF receptor switching as a molecular marker of prostate cancer progression.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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On the basis of our observations, we propose that there are, in fact, two angiogenic events consistent with the progression of TRAMP and clinical prostate cancer. As shown in Fig. 7
, the early angiogenic "initiation switch" correlates expression of HIF1- and VEGFR1 in addition to the recruitment and elaboration of intraductal vasculature in PIN lesions. Because HIF-1 has previously been shown to initiate the transcription of VEGF and VEGFR1 mRNA (16
, 17)
, it was not surprising to note that these transcripts were also found to be expressed in PIN. Hence, these observations are consistent with previous reports showing HIF-1 to be expressed in preneoplastic lesions and in human prostate cancer (29)
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is indicative of a hypoxic response and hypoxia has been shown to induce translation of VEGF mRNA through a 5'-untranslated region internal ribosome entry site (38
, 39)
. This indicates that VEGF expression in prostate cancer under hypoxic conditions may be regulated through mechanisms including, but not limited to, transcription, mRNA stability, and translation. Elucidation of the nature of this regulation will require additional studies at the molecular level. In contrast, VEGFR1 mRNA and protein were both readily observed in the endothelial and tumor epithelial cells in low-grade tumors. Although VEGFR1 has generally been considered to be endothelial cell-specific, there are a number of recent reports of VEGFR1 expression in mammary carcinomas, glioblastomas, squamous cell carcinoma of the head and neck, and prostate carcinomas (40, 41, 42)
. In addition to the early initiation switch, our data also provide evidence for a later progression switch consistent with the high level expression of VEGF protein in the prostatic tissues and serum of TRAMP mice harboring advanced, poorly differentiated, and androgen-independent tumors. This observation also suggests that the prostate is a source of VEGF and that tumor growth is at least partially dependent on this expression. Furthermore, our data suggest that prostatic VEGF likely is secreted only after the tumor is no longer organ confined. The correlation between serum VEGF, IMVD, and the transition of a differentiated tumor to a poorly differentiated tumor also supports a causal relationship between serum VEGF and tumor progression.
Consistent with the progression switch is the observation that expression of VEGFR2 in the vasculature is concomitant with the loss of VEGFR1 and an increase in IMVD in poorly differentiated high-grade tumors (Fig. 7)
. This is consistent with the functions ascribed to these receptors. Although VEGFR1 is important in endothelial cell migration, the receptor does not appear to signal through MAPK nor induce endothelial cell proliferation (23)
. In contrast, VEGFR2 has been demonstrated to signal through MAPK to induce endothelial cell proliferation and through PI3'K imparting an antiapoptotic function in endothelial cells (24
, 25) . Hence, the molecular changes observed in clinical and TRAMP prostate cancer progression relate to the specific biochemical properties of the various components of the VEGF axis and demonstrate that these components, in fact, represent specific therapeutic targets.
The similar trends observed in the temporal pattern of VEGFR expression in the clinical and TRAMP specimens validate the predictive nature of such mouse models of human cancer. Although the VEGF receptor switch was not as clearly defined in the clinical samples, these studies underscore how difficult it is to resolve the heterogeneity inherent in clinical disease. Nevertheless, to our knowledge this is the first report demonstrating an angiogenic initiation switch that correlates with PIN in prostate cancer in an animal model. Furthermore, we have also demonstrated that a second progression switch is a function of the differential expression of VEGFR1 and VEGFR2. Studies are currently underway to determine whether therapies designed to target these specific molecules will prove efficacious in a preclinical trail. Lastly, we have demonstrated that the data obtained with the mouse model can indeed be predicative of the molecular events operating in human disease.
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ACKNOWLEDGMENTS |
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staining, Caroline Castile and Rhonda Chaplin for support with animal husbandry, and Alvenia Daniels for secretarial support. |
FOOTNOTES |
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1 This study was supported by National Cancer Institute Grant CA64851 (to N. M. G.), Specialized Program of Research Excellence (SPORE) CA58204 (to N. M. G.), and an award from the Scott Department of Urology Prostate Cancer Research Initiative (to N. M. G. and W. J. H.). 
2 To whom requests for reprints should be addressed, at Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, M626A, Houston, TX 77030. Phone: (713) 798-3819; Fax: (713) 798-8012; E-mail: normang{at}bcm.tmc.edu
3 The abbreviations used are: VEGF, vascular endothelial growth factor; VEGFR, VEGF eceptor; FGF, fibroblast growth factor; BPH, benign prostatic hypertrophy; HIF, hypoxia-induced factor; MAPK, mitogen-activated protein kinase; PBS, phosphate-buffered saline; PI3'K, phosphotidyl inositol 3' kinase; TBST, Tris-buffered saline with Tween; TRAMP, transgenic adenocarcinoma of the mouse prostate; PIN, prostatic intraepithelial neoplasia; PECAM, platelet endothelial cell adhesion molecule; IMVD, intraductal microvessel density; sVEGFR1, soluble form of VEGFR1.
Received 8/24/00. Accepted 1/12/00.
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H. Ishizaki, T. Tsunoda, S. Wada, M. Yamauchi, M. Shibuya, and H. Tahara Inhibition of tumor growth with antiangiogenic cancer vaccine using epitope peptides derived from human vascular endothelial growth factor receptor 1. Clin. Cancer Res., October 1, 2006; 12(19): 5841 - 5849. [Abstract] [Full Text] [PDF]
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 K S Kimbro and J W Simons Hypoxia-inducible factor-1 in human breast and prostate cancer. Endocr. Relat. Cancer, September 1, 2006; 13(3): 739 - 749. [Abstract] [Full Text] [PDF]
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 K. J. Higgins, S. Liu, M. Abdelrahim, K. Yoon, K. Vanderlaag, W. Porter, R. P. Metz, and S. Safe Vascular Endothelial Growth Factor Receptor-2 Expression Is Induced by 17{beta}-Estradiol in ZR-75 Breast Cancer Cells by Estrogen Receptor {alpha}/Sp Proteins Endocrinology, July 1, 2006; 147(7): 3285 - 3295. [Abstract] [Full Text] [PDF]
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A. M. Houghton, J. L. Grisolano, M. L. Baumann, D. K. Kobayashi, R. D. Hautamaki, L. C. Nehring, L. A. Cornelius, and S. D. Shapiro Macrophage elastase (matrix metalloproteinase-12) suppresses growth of lung metastases. Cancer Res., June 15, 2006; 66(12): 6149 - 6155. [Abstract] [Full Text] [PDF]
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L. M. Brubaker, E. Bullitt, C. Yin, T. Van Dyke, and W. Lin Magnetic Resonance Angiography Visualization of Abnormal Tumor Vasculature in Genetically Engineered Mice Cancer Res., September 15, 2005; 65(18): 8218 - 8223. [Abstract] [Full Text] [PDF]
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 H. S. Rugo, R. S. Herbst, G. Liu, J. W. Park, M. S. Kies, H. M. Steinfeldt, Y. K. Pithavala, S. D. Reich, J. L. Freddo, and G. Wilding Phase I Trial of the Oral Antiangiogenesis Agent AG-013736 in Patients With Advanced Solid Tumors: Pharmacokinetic and Clinical Results J. Clin. Oncol., August 20, 2005; 23(24): 5474 - 5483. [Abstract] [Full Text] [PDF]
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W. J. Huss, D. R. Gray, N. M. Greenberg, J. L. Mohler, and G. J. Smith Breast Cancer Resistance Protein-Mediated Efflux of Androgen in Putative Benign and Malignant Prostate Stem Cells Cancer Res., August 1, 2005; 65(15): 6640 - 6650. [Abstract] [Full Text] [PDF]
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V. Kaushal, P. Mukunyadzi, R. A. Dennis, E. R. Siegel, D. E. Johnson, and M. Kohli Stage-Specific Characterization of the Vascular Endothelial Growth Factor Axis in Prostate Cancer: Expression of Lymphangiogenic Markers Is Associated with Advanced-Stage Disease Clin. Cancer Res., January 15, 2005; 11(2): 584 - 593. [Abstract] [Full Text] [PDF]
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J. Pandey, A. Bannout, and D. L. Wendell The Edpm5 locus prevents the 'angiogenic switch' in an estrogen-induced rat pituitary tumor Carcinogenesis, October 1, 2004; 25(10): 1829 - 1838. [Abstract] [Full Text] [PDF]
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L. A. Maddison, B. W. Sutherland, R. J. Barrios, and N. M. Greenberg Conditional Deletion of Rb Causes Early Stage Prostate Cancer Cancer Res., September 1, 2004; 64(17): 6018 - 6025. [Abstract] [Full Text] [PDF]
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H. Li, W. L. Gerald, and R. Benezra Utilization of Bone Marrow-Derived Endothelial Cell Precursors in Spontaneous Prostate Tumors Varies with Tumor Grade Cancer Res., September 1, 2004; 64(17): 6137 - 6143. [Abstract] [Full Text] [PDF]
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S. S. W. Ng, G. R. MacPherson, M. Gutschow, K. Eger, and W. D. Figg Antitumor Effects of Thalidomide Analogs in Human Prostate Cancer Xenografts Implanted in Immunodeficient Mice Clin. Cancer Res., June 15, 2004; 10(12): 4192 - 4197. [Abstract] [Full Text] [PDF]
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S. F. Shariat, V. A. Anwuri, D. J. Lamb, N. V. Shah, T. M. Wheeler, and K. M. Slawin Association of Preoperative Plasma Levels of Vascular Endothelial Growth Factor and Soluble Vascular Cell Adhesion Molecule-1 With Lymph Node Status and Biochemical Progression After Radical Prostatectomy J. Clin. Oncol., May 1, 2004; 22(9): 1655 - 1663. [Abstract] [Full Text] [PDF]
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 G. Hopfl, O. Ogunshola, and M. Gassmann HIFs and tumors--causes and consequences Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2004; 286(4): R608 - R623. [Abstract] [Full Text] [PDF]
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D. R. Gray, W. J. Huss, J. M. Yau, L. E. Durham, E. S. Werdin, W. K. Funkhouser Jr., and G. J. Smith Short-Term Human Prostate Primary Xenografts: An in Vivo Model of Human Prostate Cancer Vasculature and Angiogenesis Cancer Res., March 1, 2004; 64(5): 1712 - 1721. [Abstract] [Full Text] [PDF]
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K. W. Freeman, R. D. Gangula, B. E. Welm, M. Ozen, B. A. Foster, J. M. Rosen, M. Ittmann, N. M. Greenberg, and D. M. Spencer Conditional Activation of Fibroblast Growth Factor Receptor (FGFR) 1, but not FGFR2, in Prostate Cancer Cells Leads to Increased Osteopontin Induction, Extracellular Signal-regulated Kinase Activation, and in Vivo Proliferation Cancer Res., October 1, 2003; 63(19): 6237 - 6243. [Abstract] [Full Text] [PDF]
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N. Polnaszek, B. Kwabi-Addo, L. E. Peterson, M. Ozen, N. M. Greenberg, S. Ortega, C. Basilico, and M. Ittmann Fibroblast Growth Factor 2 Promotes Tumor Progression in an Autochthonous Mouse Model of Prostate Cancer Cancer Res., September 15, 2003; 63(18): 5754 - 5760. [Abstract] [Full Text] [PDF]
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 C. Colnot, Z. Thompson, T. Miclau, Z. Werb, and J. A. Helms Altered fracture repair in the absence of MMP9 Development, September 1, 2003; 130(17): 4123 - 4133. [Abstract] [Full Text] [PDF]
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 W. J. Huss, R. J. Barrios, and N. M. Greenberg SU5416 Selectively Impairs Angiogenesis to Induce Prostate Cancer-specific Apoptosis Mol. Cancer Ther., July 1, 2003; 2(7): 611 - 616. [Abstract] [Full Text] [PDF]
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S. B. Shappell, S. J. Olson, S. E. Hannah, S. Manning, R. L. Roberts, N. Masumori, M. Jisaka, W. E. Boeglin, V. Vader, D. S. Dave, et al. Elevated Expression of 12/15-Lipoxygenase and Cyclooxygenase-2 in a Transgenic Mouse Model of Prostate Carcinoma Cancer Res., May 1, 2003; 63(9): 2256 - 2267. [Abstract] [Full Text] [PDF]
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 J.-H. Park, J. E. Walls, J. J. Galvez, M. Kim, C. Abate-Shen, M. M. Shen, and R. D. Cardiff Prostatic Intraepithelial Neoplasia in Genetically Engineered Mice Am. J. Pathol., August 1, 2002; 161(2): 727 - 735. [Abstract] [Full Text] [PDF]
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P. Sweeney, T. Karashima, S.-J. Kim, D. Kedar, B. Mian, S. Huang, C. Baker, Z. Fan, D. J. Hicklin, C. A. Pettaway, et al. Anti-Vascular Endothelial Growth Factor Receptor 2 Antibody Reduces Tumorigenicity and Metastasis in Orthotopic Prostate Cancer Xenografts via Induction of Endothelial Cell Apoptosis and Reduction of Endothelial Cell Matrix Metalloproteinase Type 9 Production Clin. Cancer Res., August 1, 2002; 8(8): 2714 - 2724. [Abstract] [Full Text] [PDF]
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J. A. Tuxhorn, S. J. McAlhany, T. D. Dang, G. E. Ayala, and D. R. Rowley Stromal Cells Promote Angiogenesis and Growth of Human Prostate Tumors in a Differential Reactive Stroma (DRS) Xenograft Model Cancer Res., June 1, 2002; 62(11): 3298 - 3307. [Abstract] [Full Text] [PDF]
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 F. TOSETTI, N. FERRARI, S. DE FLORA, and A. ALBINI Angioprevention': angiogenesis is a common and key target for cancer chemopreventive agents FASEB J, January 1, 2002; 16(1): 2 - 14. [Abstract] [Full Text] [PDF]
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 R. Bos, P. J. van Diest, and E. van der Wall RESPONSE: Re: Levels of Hypoxia-Inducible Factor-1{alpha} During Breast Carcinogenesis J Natl Cancer Inst, August 1, 2001; 93(15): 1177 - 1177. [Full Text] [PDF]
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