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1 Department of Pathology, Baylor College of Medicine, and 2 Houston Department of Veterans Affairs Medical Center, Houston, Texas
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, a new family of regulators of FGF activity has been identified. Sprouty was originally identified as an antagonist of Breathless FGFR signaling during tracheal development in Drosophila (16) . Subsequent studies have shown that Sprouty inhibits signaling mediated by the FGFR and the epidermal growth factor receptor during eye development and oogenesis in Drosophila (17, 18, 19) . During Drosophila eye development, Sprouty seems to inhibit the activation of mitogen-activated protein kinase upstream of Ras function, whereas during wing development, it is reported to inhibit mitogen-activated protein kinase downstream of Ras function.
Four mammalian genes have been identified with sequence similarity to Drosophila sprouty (20) . The mammalian Sprouty family members are expressed in highly restricted patterns in the embryo in early development, and their expression shows a close correlation with known sites of FGF signaling (21, 22, 23) , which suggests that they may also function as negative regulators in FGF signaling during vertebrate embryonic development. All Sprouty proteins share a unique, highly conserved cysteine-rich domain at the COOH terminus, believed to be critical for targeting them to phosphatidylinositol (4,5-bisphosphate) in the plasma membrane, thus allowing their inhibitory role on the mitogen-activated protein kinase pathway (24 , 25) . The NH2-terminal portion of the Sprouty proteins is less conserved because it exhibits only 25–37% identity among the different mouse family members. These sequence differences could be responsible for the functional divergence among the Sprouty proteins. In vitro studies have demonstrated that after growth factor stimulation, Sprouty1 and Sprouty2 translocate to the plasma membrane, become tyrosine-phosphorylated, and interact with components of the Ras/mitogen-activated protein kinase and Ras/Raf/Erk pathways, such as Grb2 (26 , 27) and c-Cbl (28) , but the precise molecular mechanism by which the signal is blocked remains unknown. Tyrosine phosphorylation appears to be necessary for the ability of Sprouty to inhibit receptor tyrosine kinase-dependent Ras/Erk signaling while c-Cbl regulates the stability and hence the activity of Sprouty protein (27) . It is likely that Sprouty proteins can also act at additional stages of receptor tyrosine kinases signaling, because Sprouty2 has been shown to inhibit FGF-mediated extracellular-signal-regulated kinase activated at the level of Raf (29) , whereas Sprouty4 inhibits vascular endothelial growth factor receptor signaling upstream of Ras (30) . In contrast, epidermal growth factor receptor signaling is not reduced following expression of Sprouty2 or Sprouty4 (31) . It is thus conceivable that Sprouty proteins control receptor tyrosine kinase activation at different stages, with some additional regulatory mechanisms still unknown.
A search of the Unigene database3 and the Cancer Genome Anatomy Project database4 indicates that Sprouty cDNAs are present in cDNA libraries from many human tissues including the prostate, with Sprouty1 being the most abundant human Sprouty homologue expressed in human prostate. However, the role of Sprouty1 in human prostate cancer is not known, and little is known about alterations of regulatory molecules that may down-regulate growth factor signals in prostate cancer cells. An important consideration is that if Sprouty proteins can be up-regulated in prostate cancer by FGF stimulation, this would tend to inhibit any effects of FGFR activation in the neoplastic cells and negate the effects of the increased FGF expression in cancer tissues. To address this issue, we have investigated the expression of Sprouty1 in normal and neoplastic prostate tissues. We have found that Sprouty1 protein is decreased in prostate cancer cells when compared with matched normal epithelium in approximately 40% of prostate cancers and that there is a similar decrease in Sprouty1 mRNA by quantitative reverse transcription-PCR. We have also found that in prostate cancer cells, there is loss of the normal up-regulation of Sprouty1 mRNA in response to FGFs. The marked decrease in Sprouty1 expression in the human prostate cancer implies a loss of an important growth regulatory mechanism in prostate cancers that may potentiate the effects of increased FGF and FGFR expression in prostate cancer.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mutation Analysis of the Sprouty1 Coding Region.
Total genomic DNA was extracted from 26 prostate tissue samples [including 23 radical prostatectomies (all 70% or more cancer), 1 metastasis, and 2 benign tissues from radical prostatectomies] as described previously (32)
. The DNAs were used in standard PCR reaction conditions with three sets of primers to amplify the entire Sprouty1 coding region. Set 1 primers (SF1): forward, 5'-ACGAGCACAGACACACAAG-3', and reverse, 5'-CAACCCACCTCCAAAAATCA-3'; set 2 primers (SF2): forward, 5'-CCTTCTTTGGATAGCCGTCA-3', and reverse, 5'-CCCTTCAAGTCATCCACAATC-3'; set 3 primers (SF3): forward, 5'-AGGACCCCAGCATCATTGTA-3', and reverse, 5'-GTGGCTTGTGTGTCTGTGCT-3'. The nucleotide positions for the amplification products as given by the GenBank accession no. (XM_036349) are 137–628, 394–843, and 605-1317. The PCR products were purified and sequenced using the respective set of primers for each product.
Preparation, Quantification, and Dilution of DNA Standards.
The Sprouty1 plasmid, keratin-18 plasmid (ATCC MGC-9348), and ß-actin plasmid (ATCC MGC-10559) were prepared using the Qiagen Maxi-prep Kit (Qiagen, Valencia, CA). The FGF2 and FGF7 plasmids have been described previously (33)
. Quantification of plasmid was performed spectrophotometrically. The measurements of the plasmid concentration were done in duplicate and then converted to copy number. A dilution series of each plasmid (109-101 copies) was used as a DNA standard for real-time PCR.
Primer Design and Synthesis for Real-Time PCR.
Oligonucleotide primers for Sprouty1 were forward, 5'-TGTCCGAAAAGGATTTCAGATGC-3', and reverse, 5'-ACTGCCACTGCCA TGTTGAT-3'; for ß-actin were forward, 5'-AGCACGGCATCGTCA CCAACT-3', and reverse 5'-TGGCTGGGGTGTTGAAGGTCT-3'; for keratin 18 were forward, 5'-AGGGCTCAGATCT-TCGCAAAT-3', and reverse, 5'-GTCATCAATGACCTTGCGGAG-3'; for FGF2 were forward, 5'-CCACTTCAAGGACCCCAAG-3', and reverse, 5'-ATAGCCAGGTAACGGTTAGC-3'; and for FGF7 were forward, 5'-CCTTCTGCCTGTTGATTTATGG-3', and reverse, 5'-GTTGCTGTGACGCTGTTTG-3'. Primers were carefully designed to cross exon/intron regions and to avoid the formation of primer-dimers, hairpins, and self complementarity. The nucleotide positions for the amplification products as given per the GenBank accession nos. are 275–373, 458–622, 256–435, 539–721, and 26–209 for Sprouty1 (XM_036349), Keratin 18 (BC020982), ß-actin (BC004251), FGF2 (NM_0020006), and FGF7 (S81661), respectively.
cDNA Synthesis and Quantitative Real-Time PCR.
Total RNA extracted from cells and tissues using TRIzoL Reagent (Invitrogen) was used in first-strand DNA (cDNA) synthesis using Invitrogen SuperScript first-strand synthesis system for reverse transcription-PCR and according to the manufacturer’s protocol. Real-time PCR was carried out in a Bio-Rad iCycler real-time thermal cycler (Hercules, CA) as described previously (34)
and incorporating the following optimized PCR reaction conditions. The amplification of Sprouty1 was carried out as follows: a 3 min hot start at 95°C; followed by 40 cycles of denaturation at 95°C for 15 s; and annealing at 61°C for 30 s. The amplification protocol for FGF2 was the same as for Sprouty1 except that annealing was done at 63.5°C. The amplification protocol for FGF7, ß-actin, and keratin 18 was carried out as follows: a 3 min hot start at 95°C; followed by 40 cycles of denaturation at 95°C for 30 s; annealing at 56°C for 20 s; and a 72°C extension for 30 s. Each experiment was done in duplicate. The threshold cycle (Ct) values in log linear range representing the detection threshold values were used for quantitation and expressed as copy numbers based on a standard curve generated using plasmid DNA.
Northern Blot Analysis.
A multiple tissue Northern blot (MTN Blot II) containing polyadenylated RNAs isolated from human adult tissues was obtained from Clontech (Palo Alto, CA). Northern hybridization was performed at 68°C in 10 ml of PerfectHyb Plus hybridization solution (Sigma, St. Louis, MO). The blot was prehybridized in the above buffer for 30 min. Hybridization was done for 1 h by adding 50 ng of full-length Sprouty1 or ß-actin cDNA fragment that were radioactively labeled with [
-32P]dCTP using a RadPrime Labeling Kit (Invitrogen) and included at a concentration of 1 x 109 cpm/ml. Blots were washed according to the manufacturer’s protocol, and signals were visualized by autoradiography.
Tissue Microarrays and Immunohistochemistry.
The tissue microarrays used to study Sprouty1 expression in clinically localized prostate cancer have been described previously (35)
. In brief, three 0.6-mm cores of cancer and uninvolved prostate tissue were obtained from radical prostatectomy specimens and used to construct tissue microarrays. Patients received no adjuvant therapy such as radiation or hormonal therapy. Other patient characteristics were as described previously. A total of 511 of the original 640 cancers were evaluable, with some cases lost due to depletion of tumor or technical artifacts, and of these, 407 had matched evaluable normal tissue. Immunohistochemistry was performed as described previously (36)
. Antigen retrieval was performed for 30 min in a rice cooker in 10 mM citrate buffer (pH 6.0). Endogenous biotin and peroxidase were blocked using appropriate kits from Vector Laboratories (Burlingame, CA) according to the manufacturer’s protocol. Rabbit polyclonal anti-Sprouty1 antibody (Upstate Biotechnology, Lake Placid, NY) was incubated with each tissue array section at 5 ng/ml at 4°C overnight followed by the avidin-biotin peroxidase complex procedure (Vector Laboratories) and counterstaining with hematoxylin as described previously. Slides were then scanned using a Bliss automated slide scanner system to produce high-resolution digital images. Staining was evaluated in the normal and prostate cancer epithelial cells as described previously. Staining intensity was graded as absent (0), weak (1+), intermediate (2+), or strong (3+). The extent of staining was estimated and scored as follows: no staining (0); 1–33% of cell stained (1+); 34–66% of cell stained (2+); or 67–100% of cells stained (3+). The staining index for each case was then calculated by multiplying the average intensity score for the three cores by the average percentage score for the three cores, yielding a 10-point tumor staining index ranging from 0 (no staining) to 9 (extensive, strong staining) for each case.
Western Blotting.
Total protein was extracted from cells using protein lysis buffer as described previously. For Western blots, 30 mg of protein extract/lane were electrophoresed, transferred to nitrocellulose membrane (Hybond ECL; Amersham Biosciences, Piscataway, NJ), and incubated overnight with a 1:100 dilution of anti-Sprouty1 goat polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or a 1:5000 dilution of anti-ß-actin mouse monoclonal antibody (Sigma). Membranes were washed and treated with bovine antigoat IgG (1:5000; Santa Cruz Biotechnology) or rat antimouse IgG secondary antibody conjugated to horseradish peroxidase (1:2000 dilution; Southern Biotechnology Associates, Birmingham, AL) for Sprouty1 and ß-actin, respectively. The antigen-antibody reaction was visualized using an enhanced chemiluminescence (ECL) assay (Amersham Biosciences) and exposed to ECL film (Amersham Biosciences). Western blot signals were quantified using NucleoVision imaging station (Nucleotech, San Carlos, CA).
Cell Culture.
The human prostate cancer cell lines PC3, DU145 and LNCaP and the immortalized normal prostate epithelial cell lines PNT1a and PNT2 were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (Invitrogen). Primary epithelial and stromal cultures were established from normal peripheral zone tissue from radical prostatectomy specimens as described previously (33)
. For Western blotting studies, prostate cancer cell lines were incubated in primary epithelial growth medium for 24 h before collection.
Cell Transfection.
For stable transfections, PC3 or LNCaP cells were seeded at 5 x 106 cells/100-mm dish and transfected with 10 µg of Sprouty1 construct (pcDNA-Sprouty1) or vector only (pcDNA3.1) using LipofectAMINE 2000 transfection reagent (Invitrogen) according to the manufacturer’s protocol. Two days after transfection, cells were selected in medium containing Geneticin (Sigma) at a final concentration of 400 and 200 µg/ml for LNCaP and PC3 cells, respectively. After 14 days into the selection, individual Geneticin-resistant colonies were fixed with 10% formalin and stained with crystal violet, and the colonies visible to the naked eye were counted. For transient transfection, LNCaP cells were plated at 5 x 104 cells/60-mm dish and transfected with 2 µg of Sprouty1 plasmid or vector only using LipofectAMINE 2000 transfection reagent (Invitrogen) according to the manufacturer’s protocol. After 24, 48, or 72 h, cells were trypsinized and counted using a Coulter counter. A second transfected plate was used to collect protein extract for Western blotting at the same time.
Cell Sorting of LNCaP Cells Transfected with GFP Constructs.
LNCaP cells were plated at 5 x 106 cells/100-mm dish and transfected with either 24 µg of pEGFP (BD Biosciences Clontech) alone or 6 µg of pEGFP with 18 µg of the Sprouty1 plasmid using LipofectAMINE 2000 transfection reagent (Invitrogen). After 24 h, GFP-positive cells were sorted by single color-flow cytometry using Epics-Alpra flow cytometer (Beckman Coulter) and plated into complete growth medium. After 24, 48, or 72 h, cells were trypsinized and counted using a Coulter counter.
FGF2 Induction Studies.
Primary epithelial, LNCaP, and PC3 cells were placed in serum-free medium for 24 h. Cells were refed with serum-free medium with 1% insulin, transferrin, and selenium (Sigma) with or without 25 ng/ml recombinant FGF-2 (R&D Systems, Minneapolis, MN) and incubated at 37°C for different time points. Cells were then harvested for either RNA or protein extraction. RNA extracted was used in real-time quantitative PCR, and protein extraction was used in Western blot analysis as described above.
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RESULTS |
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Analysis of Sprouty1 Expression by Immunohistochemical Analysis of Tissue Microarrays.
To determine whether Sprouty1 protein is decreased in clinically localized prostate cancers, we analyzed a total of 407 prostate cancers and matched normal peripheral zone tissues by immunohistochemistry of prostate cancer and prostate tissue microarrays. The tissue microarrays used were obtained from the Baylor prostate cancer SPORE and have been described previously (35)
. These arrays contain 0.6-mm tissue cores from cancers in triplicate as well as non-neoplastic peripheral zone tissue cores (also in triplicate) from patients undergoing radical prostatectomy. In normal prostate, the Sprouty1 protein is expressed in epithelial and smooth muscle cells, with some staining of stromal fibroblasts (Fig. 1, A and B)
. Sprouty1 expression in normal epithelium was variable. The vast majority of cases showed moderate to strong staining in the normal epithelium as shown in Fig. 1
, although some tissues had only minimal staining. Prostate cancer epithelium also had quite variable expression. Some prostate cancer had minimal expression of Sprouty1 (Fig. 1, C--E)
. Other cancers had moderate to strong expression of Sprouty1 protein (Fig. 1F)
. To quantitatively compare the expression of Sprouty1 in normal and neoplastic prostatic epithelium, we quantitated the expression of Sprouty1 based on methodology described previously (35)
. In brief, stained slides were digitized, and staining was scored both for extent of staining (scale of 0–3) and intensity of staining (scale of 0–3). A staining index was calculated from the average extent of staining score for the three cores multiplied by the average staining intensity score. The mean staining index of Sprouty1 in normal epithelium was significantly higher than in the cancer cells (P = 0.045, Wilcoxon’s signed ranks test). Perhaps more meaningfully, given the variability in Sprouty1 expression, we also compared the staining index in normal and cancer cells from the same patient. Overall 39% of cancer had a lower staining index than that of benign tissues from the same patient. However, it should be noted that a significant fraction of prostate cancers had higher expression of Sprouty1 in cancer cells than in normal epithelium. Therefore, although decreased Sprouty1 expression is seen in a substantial fraction of prostate cancers, loss of Sprouty1 expression is clearly not required in all prostate cancers.
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. The expression of Sprouty1 in both normal prostate and cancer tissues was variable, presumably reflecting both random variability in tissue composition and variable expression per cell (as seen in the immunohistochemistry studies). Sprouty1 expression was about 70% higher on average in normal prostate tissues (6.60 ± 2.0, SE) compared with prostate cancers (3.82 ± 1.1, SE), despite the fact that FGFs are significantly up-regulated in the vast majority of prostate cancers. Of the 20 cancers analyzed, 16 had Sprouty1 mRNA levels lower than the mean Sprouty1 mRNA level in normal tissue. The difference in Sprouty1 mRNA level between normal and cancer tissue was statistically significant (P < 0.035, t test). Similar results were obtained when Sprouty1 expression was normalized using keratin 18 mRNA, which is expressed exclusively by epithelium (Fig. 2B)
. Expression of Sprouty1 normalized for epithelial content was almost 2-fold higher in normal tissues when compared with cancers (3.8 ± 0.9 versus 2.0 ± 0.2), and this difference was again statistically significant (P < 0.02, t test).
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Another possible explanation for decreased expression of Sprouty1 mRNA in the cancer tissues is decreased expression of FGFs in a subset of the prostate cancers. Our laboratory has shown previously that FGF2 and FGF7 are expressed in the stromal cells of cancer tissues and that FGF2 protein is approximately 2.5-fold higher in prostate cancer tissues, whereas FGF7 protein levels are similar in normal and prostate cancer tissues (5) . We therefore compared the expression of FGF2 and FGF7 mRNA by quantitative reverse transcription-PCR in cancer tissues with Sprouty1 expression below the mean value for all cancers (average 1.2 Sprouty1 transcripts/102 K18 transcript) to those with Sprouty1 expression above the mean (average 3.22 Sprouty1 transcripts/102 K18 transcript). There was no significant difference in expression of FGF2 between these two groups (2.31 ± 0.8 versus 2.61 ± 0.9 FGF2 transcripts/103 ß-actin transcripts; mean ± SE, n = 10). It should be noted that both groups have higher FGF2 mRNA transcript levels than a set of nine normal peripheral zone tissues analyzed at the same time (1.3 FGF2 transcripts/103 ß-actin transcripts). Similarly, FGF7 transcript levels were not significantly different between the two groups (0.77 ± 0.3 versus 0.50 ± 0.2 transcripts/103 ß-actin transcripts; mean ± SE, n = 10). Thus it is unlikely that the decreased Sprouty1 mRNA expression in many of the cancer tissues in vivo reflects decreases in FGF ligand concentration in the cancer tissues.
Effect of FGF2 on Sprouty1 Expression in Normal and Neoplastic Prostate Epithelial Cells.
We next examined the expression of Sprouty1 protein in vitro in normal primary prostatic epithelial cells and prostate cancer cell lines. Consistent with our in vivo data, the three commonly used prostate cancer cell lines (LNCaP, DU145, and PC3) all expressed lower levels of Sprouty1 protein, as determined by Western blotting, than did the normal epithelial cells (Fig. 3)
. It should be noted that these three cell lines express FGFs in an autocrine fashion and so would be expected to express higher levels of Sprouty1 than normal epithelial cells if FGFs are inducing Sprouty1 expression as in other systems. To investigate whether regulation of Sprouty1 expression in response to FGF stimulation is altered in the prostate cancer cells, we examined the effect of FGF2 stimulation on Sprouty1 mRNA expression in vitro. Fig. 4A
shows that when primary epithelial cells were stimulated with FGF2, there was a 5-fold increase in Sprouty1 expression within 30 min. However, this expression was rapidly down-regulated to below basal level in 1 h. There was a subsequent increase in Sprouty1 expression after 2 h that was sustained, again peaking within 24 h, suggesting that there is a biphasic increase in expression of Sprouty1 in response to FGF2 stimulation. In contrast to primary epithelial cells, LNCaP and PC3 cells did not show induction of Sprouty1 expression in response to FGF2 stimulation. In fact, there was a slight down-regulation of Sprouty1 at 30 min or 2 h after FGF stimulation in PC3 and LNCaP cells, respectively. This result indicates that, unlike normal prostate cells, Sprouty1 expression in prostatic cancer cells is no longer up-regulated by FGF2. To determine whether Sprouty1 mRNA correlates with protein level, we investigated the effects of FGF2 stimulation on Sprouty1 protein expression in vitro. Because basal sprouty1 expression in PC3 cells is much lower than primary cells (Fig. 3)
, we have normalized expression in each cell line to basal expression in that cell type in normal growth medium to facilitate comparison of changes in expression, but absolute expression of Sprouty1 in PC3 cells is far lower than in the primary epithelial cells. Fig. 4B
shows that when primary cells were stimulated with FGF2, there was a gradual and a rather sustained increase in Sprouty1 protein expression that was 3-fold higher than basal Sprouty1 expression in normal growth medium by 24 h. In contrast, with the primary cells, PC3 cells did not show induction of Sprouty1 protein expression in response to FGF2 stimulation. Rather, there was a decrease in Sprouty1 protein expression within 30 min before returning to basal level at 24 h. The initial decrease in Sprouty1 protein could be a direct result of proteosomal degradation mediated by c-Cbl after growth factor signaling, as described for Sprouty proteins in other systems (27
, 37, 38, 39)
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The Effect of Sprouty1 Expression in Human Prostate Cancer Cells.
To ascertain the biological effect of Sprouty1 expression in human prostate cancer cells, pcDNA-Sprouty1 (encoding the full length of Sprouty1 sequence) was transfected into the human prostate cancer cell lines LNCaP and PC3, and transfected cells were selected in Geneticin. Only rare colonies were observed in both the LNCaP and PC3 cells transfected with the Sprouty1 plasmid, whereas numerous colonies were observed when PC3 and LNCaP were transfected with the vector only plasmid (Fig. 5)
. The inhibition of colony formation by Sprouty1 was more than 99%, suggesting that sustained overexpression of Sprouty1 has a markedly deleterious effect on prostate cancer cells proliferation and/or survival. To determine whether more modest, transient expression of Sprouty1 could also inhibit prostate cancer cell proliferation, we analyzed proliferation of LNCaP cells after transient transfection of a Sprouty1 expression plasmid. Sprouty1 expression levels were analyzed on the same cells by Western blotting. Despite the modest increase in Sprouty1 expression under these conditions (Fig. 6A)
, there was a profound decrease in proliferation in the cells transfected with the Sprouty1 expression construct (Fig. 6B)
. To confirm that the inhibition of growth is due to Sprouty1 expression, we repeated the transient transfection assay this time by cotransfecting the Sprouty1 plasmid with a vector containing GFP. Cells that were GFP positive were sorted and used in cell proliferation analysis. Fig. 6C
shows that LNCaP cells transfected with the Sprouty1 plasmid had a profound decrease in proliferation when compared with the GFP only transfection, which is consistent with our initial observation. Thus in prostate cancer cells, either sustained or transiently increased Sprouty1 expression markedly inhibits proliferation, which is similar to observations made in other systems (30
, 41
, 42)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In human prostate cancer tissues, there is up-regulation of FGFs when compared with uninvolved prostate. We have shown previously that the tissue content of FGF2 is increased more than 2-fold in prostate cancer tissue compared with control prostate, whereas FGF7 is present at essentially equal levels (5) . FGF9 is also present at equal levels in normal and neoplastic prostate tissue based on ELISA assay.5 Finally, FGF6 is expressed as an autocrine growth factor in 40% of prostate cancers (6) , and the majority of prostate cancers express FGF8 in a similar manner (7, 8, 9) . Thus, the decreased Sprouty1 expression seen in 40% of cancers cannot be due to loss of FGF ligands in these cases. Loss of Sprouty1 expression may give rise to unrestrained signal transduction by FGFs that could result in increased proliferation (2 , 6) and/or decreased cell death (43) in prostate cancer and potentiate the effects of increased FGFs and FGFRs in prostate cancer. We have also seen that some prostate cancers have increased Sprouty1 expression. These cancers must have other alterations that allow them to resist the negative growth regulatory effects of Sprouty1 that were seen in LNCaP cells, which have very low basal Sprouty1 expression. The nature of these alterations is currently under investigation.
We have found that Sprouty1 expression in prostate cancer cells in vitro is no longer up-regulated by FGF2. This could be due to decreased transcription, for example, secondary to alterations of trans-acting factors, such as loss of essential transcription factors or up-regulation of negative regulatory factors, or it could be a consequence of increased mRNA degradation. The loss of expression of Sprouty1 in prostate cancer in vivo could also be due to alterations in the gene itself, such as deletion or methylation. The Sprouty1 gene maps to chromosome 4q27.6 Comparative genomic hybridization shows loss of this region in 23% of prostate cancers examined (44) , so it is possible that in some cases, decreased Sprouty1 expression could be due to hemi- or homozygous deletion of the Sprouty1 locus. Methylation has been shown to be involved in loss of gene expression in prostate cancer (45) . Systematic studies of Sprouty1 promoter methylation and correlation with gene expression in prostate cancers in vivo will need to be carried out to exclude this possibility. Additional work is currently under way seeking to understand the molecular mechanisms that lead to decreased Sprouty1 mRNA in prostate cancer.
In summary, there is considerable evidence showing up-regulation of FGFs in prostate cancer based on studies in animal models, human tissues, and human prostate cancer cell lines. Sprouty1, an inhibitor of FGF signal transduction, is decreased in approximately 40% of clinically localized prostate cancers and may lead to the unrestrained signal transduction by FGFs and hence tumor progression. Because Sprouty1 may inhibit the transduction of many growth factor signals, it could be an attractive target to explore for drug intervention or gene therapies of prostate cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Michael Ittmann, Research Service, Houston Veterans Affairs Medical Center, 2002 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 791-1414, extension 4008; Fax: (713) 794-7938; E-mail: mittmann{at}bcm.tmc.edu
3 Internet address: www.ncbi.nlm.nih.gov/UniGene. 
4 Internet address: www.ncbi.nlm.nih.gov/ncicgap.
5 M. Ittman, unpublished data.
6 Internet address: www.ncbi.nlm.nih.gov/LocusLink.
Received 12/ 2/03. Revised 4/15/04. Accepted 5/ 5/04.
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