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[Cancer Research 65, 7320-7327, August 15, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Endothelin Axis Is a Target of the Lung Metastasis Suppressor Gene RhoGDI2

Brian Titus1, Henry F. Frierson, Jr.2, Mark Conaway3, Keith Ching5, Theresa Guise4, John Chirgwin4, Garret Hampton5 and Dan Theodorescu1

Departments of 1 Molecular Physiology and Biological Physics, 2 Pathology, 3 Health Evaluation Sciences, Division of Biostatistics, and 4 Medicine, Division of Endocrinology, University of Virginia, Charlottesville, Virginia; and 5 Genomics Institute of the Novartis Research Foundation, San Diego, California

Requests for reprints: Dan Theodorescu, Department of Urology, University of Virginia Health Sciences Center, Box 800422, Charlottesville, VA 22908. Phone: 434-924-0042; Fax: 434-982-3652; E-mail: dt9d{at}virginia.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Half of patients treated for locally advanced bladder cancer relapse with often fatal metastatic disease to the lung. We have recently shown that reduced expression of the GDP dissociation inhibitor, RhoGDI2, is associated with decreased survival of patients with advanced bladder cancer. However, the effectors by which RhoGDI2 affects metastasis are unknown. Here we use DNA microarrays to identify genes suppressed by RhoGDI2 reconstitution in lung metastatic bladder cancer cell lines. We identify such RNAs and focus only on those that also increase with tumor stage in human bladder cancer samples to discover only clinically relevant targets of RhoGDI2. Levels of endothelin-1 (ET-1), a potent vasoconstrictor, were affected by both RhoGDI2 reconstitution and tumor stage. To test the hypothesis that the endothelin axis is important in lung metastasis, lung metastatic bladder carcinoma cells were injected in mice treated with the endothelin receptor–specific antagonist, atrasentan, thereby blocking engagement of the up-regulated ET-1 ligand with its cognate receptor. Endothelin antagonism resulted in a dramatic reduction of lung metastases, similar to the effect of reexpressing RhoGDI2 in these metastatic cells. Taken together, these experiments show a novel approach of identifying therapeutic targets downstream of metastasis suppressor genes. The data also suggest that blockade of the ET-1 axis may prevent lung metastasis, a new therapeutic concept that warrants clinical evaluation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
An estimated 53,000 new cases of bladder cancer are diagnosed in the United States each year. Approximately 5% of these patients present with metastatic disease at diagnosis. However, an additional 50% of patients who are originally diagnosed with locally advanced disease later develop metastases, commonly to the lung, resulting in significant mortality among patients with bladder cancer (1, 2). The molecular basis of pulmonary metastases is not well understood but is likely to involve the progressive loss of suppressor genes and dominant gain of function mutations in critical pathways along steps of the metastatic cascade (3). Whereas in theory, the metastatic cascade could be interrupted at various points with equal potential for therapeutic benefit, only the steps that prevent the outgrowth of micrometastasis into a clinically life-threatening lesion are viable therapeutic targets. This is because the patients at greatest risk of metastatic disease are those diagnosed with clinically localized disease in which disseminated cancer cells may already be present in distant organs.

We recently showed that the GDP dissociation inhibitor, RhoGDI2, is a metastasis suppressor in a bladder cancer lung metastasis model (4). Metastatic derivatives of the bladder carcinoma cell line, T24 (termed T24T), were shown to exhibit loss of RhoGDI2, which correlated with increasing pathologic cancer stage and grade. Reexpression of RhoGDI2 in these deficient cells led to a dramatic suppression of lung metastasis. The clinical importance of RhoGDI2 loss of function was validated by showing that reduced or absent RhoGDI2 expression is strongly correlated with the development of metastasis, resulting in decreased survival following therapy for locally advanced bladder cancer (5). Because targeted clinical restoration of RhoGDI2 function is currently impractical, we used our isogenic metastatic cell model to identify genes regulated downstream of RhoGDI2 activity, whose encoded proteins may be pharmaceutically tractable. Using DNA microarrays to monitor the changes in gene expression following restoration of RhoGDI2 expression, we identified several potentially targetable proteins, including the endothelin-1 ligand (ET-1), that were suppressed in the presence of RhoGDI2 protein. These results suggest that loss of RhoGDI2 during the clinical progression of bladder carcinoma may lead to the up-regulation of the endothelin axis. This finding was confirmed by examining the relationship between RhoGDI2 expression levels and those of ET-1 in human tumor samples and cell lines. Collectively, our findings indicate that adjuvant trials with endothelin antagonists may be considered for patients with advanced bladder cancer following therapy of the primary lesion. In addition, application of this approach to other metastasis suppressor genes could identify potential novel targets for therapy in other common malignancies (6, 7).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Gene expression profiling of bladder tumor cell lines and human bladder cancers. Isogenic T24T cells, bladder carcinoma–derived cell lines, primary human bladder carcinomas, and normal bladder tissues were profiled on HG-U133A GeneChip arrays (Affymetrix, Santa Clara, CA). For the previously described isogenic T24T cell pair containing vector or RhoGDI2 constructs (4), duplicate RNA samples were generated from independent cell cultures; RNA from the 41 human bladder cancer cell lines [acquired from American Type Culture Collection (Manassas, VA) or from Dr. Monica Liebert (M.D. Anderson Cancer Center, Houston, TX)] was generated from a single log-phase culture. Following approval of the University of Virginia Human Investigation Committee, 23 primary bladder cancer samples of varying pathologic stages and five samples of normal urothelium were obtained and evaluated by a genitourinary pathologist (H.F.F.). For the malignant samples, only areas containing >80% cancer cells were processed for RNA isolation. RNA was processed as previously described and hybridized to HG-U133A GeneChip arrays (Affymetrix). Image files were assessed for quality and artifacts and processed using Microarray Analysis Suite 5.0 (MAS 5.0, Affymetrix) using a scaling factor of 200.

Genes associated with RhoGDI2 expression in T24T cells were identified using the local-pooled error (LPE) test (8) with a false discovery rate (FDR) P < 0.05. Functional relationships among genes correlated with RhoGDI2 expression were analyzed using the ontology use in dCHIP1.3/ChipInfo (9, 10). The P value generated by this method indicates the strength of the association of gene clusters to the gene ontology terms or pathways, with P < 0.05 considered significant. We sought to identify genes encoding secreted proteins using two parallel approaches as previously described (11). To identify which of the 20 RhoGDI2-suppressed genes exhibited expression correlated with tumor stage, tumors with stage Ta were compared with tumors of stage T1 and above; these were then ranked according to fold change.

Neuromedin-U and endothelin axis evaluation in bladder cancer cells. T24T cells stably transfected and expressing RhoGDI2 transgene (T24T-RhoGDI2) or vector control (T24T-pcDNA3) were washed with PBS and total genomic RNA isolated and used as template for reverse transcription-PCR (RT-PCR) using primers for Neuromedin-U (NmU), ET-1, ET-1-converting enzyme, endothelin A receptor, and endothelin B receptor selected using OligoLite primer analysis software. Amplification was normalized to glyceraldehyde-3-phosphate dehydrogenase. A Qiagen One-step RT-PCR kit was used following the manufacturer's instructions. T24T, T24T-RhoGDI2, or T24T-pcDNA3 were plated and grown to 80% confluence in DMEM/F-12 + 5% fetal bovine serum. Then, the plates were washed with PBS and 14 mL DMEM/F-12 without serum was added. Cells were incubated in the above conditions for 36 hours. Medium was removed from the cells and analyzed by commercial ELISA assay (R&D Systems, Inc., Minneapolis, MN) according to the supplier's instructions.

Phospho-Erk1/2 evaluation in RhoGDI2-transfected cells stimulated with Endothelin-1. HOst, T24T-RhoGDI2, or T24T-pcDNA3 were serum starved overnight. Before lysis, cells were subjected to a 20-minute exposure of vehicle or varying concentrations of ET-1 (Sigma, St. Louis, MO). HOst cells were obtained from Cambrex (Walkersville, MD) and are Normal Human Osteoblasts (http://www.cambrex.com). Cells were then washed on ice in BioPlex Cell Wash Buffer followed by addition of 300 µL of BioPlex Lysis Buffer (containing phenylmethylsulfonyl fluoride and BioPlex phosphatase inhibitors), scraped and agitated on a microplate shaker. Protein concentration was determined by bicinchoninic acid assay buffer (Pierce, Rockford, IL) and allowed to incubate with phospho-Erk1/2-coupled beads (Bio-Rad, Richmond, CA) overnight. The lysate/bead mixture was then washed and exposed to phospho-Erk1/2 detection antibody and then allowed to incubate with Streptavidin-PE for 10 minutes, in the dark. Captured lysates were washed again and resuspended in BioPlex Resuspension Buffer before analysis. All assay samples were run in multiple biological replicates and tested in duplicate.

In vitro and in vivo growth and metastasis assays. Growth and colony formation in soft agar of T24T-RhoGDI2 or T24T-pcDNA3cells was analyzed ± atrasentan using techniques previously described (4, 12). S.c. tumorigenicity in 6-week-old nude mice was evaluated as described (13) with 5 x 106 cells in 0.1 mL of SFM. For experimental metastasis, mice were given an i.v. lateral tail vein injection with 106 tumor cells suspended in 0.1 mL of SFM as described (13). At the time of euthanasia, the lungs were removed by dissection away from adjacent organs and examined grossly and microscopically as described (4). RhoGDI2 immunohistochemistry on lung samples was carried out and scored as described (5) without knowledge of the experimental groups (atrasentan or vehicle/pcDNA3 or RhoGDI2) by the pathologist (H.F.F). The presence, number, and size of metastatic deposits were evaluated. For both s.c. tumorigenicity and experimental metastasis experiments, animals were separated into two groups: group 1, pcDNA3 transfected cells and group 2, pcDNA3 + atrasentan. Mice were treated with either atrasentan (5 mg/kg/d) in their drinking water or not (control water) for 12 weeks. This dose of atrasentan has previously been shown to give effective serum concentrations of the drug in nude mice (14). In vivo experiments were carried out twice or more and results pooled for the statistical analysis.

Statistical methods. Expression levels were transformed to the natural log scale to stabilize the variance. The Pearson correlation coefficient was used to assess the relationship between the log ratios of ET-1 to RhoGDI2 and NmU to RhoGDI2. F tests were used to assess trends in the fold changes in HOst, pcDNA3, and RhoGDI2 with the addition of varying levels of ET-1. {chi}2 tests were used to compare the proportion of mice developing metastases in the pcDNA3, RhoGDI2 and the pcDNA3 + atrasentan groups. For each group, exact 95% confidence intervals based on the binomial distribution were used for the proportion of mice with metastases.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Previous microarray analysis of a nonmetastatic human bladder cancer cell line, T24 and a lineage-related lung metastatic variant, T24T, revealed the loss of expression of several genes, including the GDP dissociation inhibitor, RhoGDI2. Following reexpression in T24T cells, RhoGDI2 functions as a potent suppressor of lung metastasis (4). We have also shown that RhoGDI2 gene expression correlates with metastatic competence in bladder carcinoma–derived cell xenografts and have shown here that RhoGDI2 protein expression is also decreased as a function of increased metastatic capability in a series of human bladder cell lines (Fig. 1A). To begin to understand the mechanism by which RhoGDI2 suppresses metastasis, we reasoned that genes downstream of RhoGDI2 activity in bladder cancer patients may be down-regulated by reexpression of RhoGDI2 in RhoGDI2-deficient, metastatic cells. To determine the identity of these genes, stable T24T derivative bladder cancer cells were selected with empty vector or a construct expressing RhoGDI2 (Fig. 1A). RNA from these cells was hybridized to a microarray (11) containing ~21,500 human genes. Sixty-three and 40 genes were up-regulated and down-regulated by >2-fold, respectively, in RhoGDI2-expressing cells. Analysis of the data with respect to gene sequence and GO annotations indicated that several of these encode putatively secreted proteins that may be involved in autocrine or paracrine signaling (Table 1). An ontological view of these genes (Table 2) revealed that many are involved in cytoskeletal organization providing potential insight into the mechanism of action of RhoGDI2.



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  		Figure 1. A, expression of RhoGDI2 protein in human bladder cell lines as a function of clinical and xenograft behavior. i, cell lysates of bladder cancer cell lines were prepared and Westerns probed with anti-RhoGDI2 antibody carried out as described (4). Xenograft behavior is indicated using color-coded lines. ii, protein expression of T24T cells stably transfected and expressing RhoGDI2 transgene or vector control (pcDNA3). B, ET-1 and NmU mRNA expression as a function of human bladder cancer stage. A set of 23 primary carcinomas (five Ta, five T1, six T2, three T3, and four T4) was macrodissected and areas comprised of >80% viable carcinoma was used for expression profiling using the Affymetrix HG-U133A array as described. Columns, mean Affymetrix expression levels as a function of stage; bars, SD. Both Et-1 (P = 0.004) and NmU (P = 0.001), which show significant increases in expression levels across stage, assuming a linear trend in the ln(expression level). C, expression of ET-1, NmU, and endothelin receptor A (ETa) in RhoGDI2- and vector-transfected cells. RT-PCR was done with primers specific for NmU and ET-1 as described in Materials and Methods. The template used was RNA isolated from T24T-RhoGDI2 and T24T-pcDNA3 cells. D, expression ET-1 and NmU as a function of RhoGDI2 in bladder cancer cell lines. Forty-one human bladder cell lines were analyzed on oligonucleotide arrays for the expression levels of RhoGDI2, NmU, and ET-1. Ratio of NmU to RhoGDI2 expression (X-axis); ratio of endothelin to RhoGDI2 expression (Y-axis). Regression line is shown. Correlation = 0.85; 95% confidence interval, 0.78-0.90; P < 0.001.

 

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  		Table 1. Gene expression differences found with Affymetrix HG-U133A GeneChip array analysis of pcDNA- and RhoGDI2-transfected metastatic human bladder cancer cell lines

 

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  		Table 2. Gene Ontology Analysis based on Affymetrix HG-U133A GeneChip array analysis of pcDNA- and RhoGDI2-transfected metastatic human bladder cancer cell lines

 
To determine which of the 40 down-regulated genes in stable RhoGDI2 T24T transfectants may be clinically relevant targets, we searched for genes whose expression was increased as a function of tumor stage in a series of 20 human bladder cancers using microarray analysis. Tumor tissue from lung metastases is not available from bladder cancer patients. This is primarily because removal of metastases in patients with bladder cancer is not required for routine medical care. Therefore, we evaluated gene expression from primary human bladder carcinomas according to pathologic stage, as the frequency of metastasis robustly increases with higher tumor stage (15–17). Whereas 28 genes were up-regulated in association with increasing stage, only two of these were also down-regulated by stable RhoGDI2 expression in the cell line model (Table 1). The relationship between expression of these two genes and tumor stage is shown in Fig. 1B. In the T24T cell line system, the most strongly down-regulated mRNA in response to RhoGDI2 was ET-1, which was decreased 9.5-fold (Table 1), whereas NmU was decreased 3-fold. These microarray expression levels were independently verified by RT-PCR analysis (Fig. 1C). As ET-1 is a secreted factor that requires enzymatic processing to generate the mature active form by endothelin-converting enzyme (ECE), we tested conditioned media from both RhoGDI2 stably transfected and vector control cell lines for the presence of active ET-1 protein. In RhoGDI2-expressing cells, ET-1 was present at a concentration of 7 pg/mL, whereas in vector control cells, ET-1 was present at 69 pg/mL, showing that RhoGDI2 affects the active levels of this secreted peptide.

To further explore the relationship between RhoGDI2 expression with that of ET-1 and NmU in bladder cancer, microarray data from 41 human bladder cancer cell lines were viewed as a logarithmic plot of the ratio of ET-1 to RhoGDI2 versus ratio of NmU to RhoGDI2. The log-linear relationship suggests a codependent relationship between RhoGDI2 and both ET-1 and NmU expression (Fig. 1D; correlation between log ratios, 0.85; 95% confidence interval, 0.78-0.90; P < 0.001). Taken together with data from the RhoGDI2 transfection studies in T24T cells and the human bladder carcinoma data, this finding independently supports and generalizes the importance of RhoGDI2 regulation of ET-1 and NmU in human bladder cancer.

The endothelin axis is composed of several members that can be expressed by both cancer cells and host cells resulting in both autocrine and paracrine interactions favoring growth and angiogenesis (18). Hence, we sought to evaluate this axis by examining the microarray data and confirming these findings by RT-PCR analysis. These data showed that vector-transfected T24T cells express mRNAs for the two endothelin receptors, endothelin A receptor (ETaR) and endothelin B receptor (ETbR), as well as the ECE. Although mRNA expression of these three genes was uniformly lower in RhoGDI2-transfected cells, this did not reach statistical significance (data not shown). Because we had previously established that ET-1 is produced in the medium conditioned by the vector-transfected cells, we sought to determine whether this ET-1 plays a role in cell growth in vitro and in vivo, using the selective ETaR antagonist, atrasentan. ETaR blockade was shown to have no effect on growth in monolayer culture (Fig. 2A) or to affect the anchorage-independent colony forming ability of RhoGDI2-stable or empty vector transfected cells (Fig. 2B). In addition, no difference in local tumor growth was observed following s.c. injection of immunocompromised mice with vector and RhoGDI2-transfected T24T cells and treated with atrasentan or vehicle for 8 weeks.



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  		Figure 2. A, role of the ETaR antagonist atrasentan on the in vitro growth monolayer of T24T cells. Growth curves in monolayer culture of T24T-RhoGDI2 and T24T-pcDNA3 cells treated with the selective ETaR antagonist atrasentan or vehicle for 5 days. Point, average measurements of five replicate wells; bars, SD. Representative of assays carried out three independent times. B, role of ETaR antagonist atrasentan on colony formation in agar of T24T cells. Soft agar cultures were analyzed after 3 weeks growth in agarose with either two different concentrations of atrasentan or control water. C, phospho-Erk1/2 evaluation in RhoGDI2-transfected cells in response to ET-1 stimulation. HOst, T24T-RhoGDI2, and T24T-pcDNA3 cells were serum starved overnight. Before lysis, cells were subjected to a 20-minute exposure of vehicle or varying concentrations of ET-1. Lysates and analysis was prepared according to Bio-Rad Bio-Plex Phosphoprotein testing instructions as described in Materials and Methods. Results are pooled data from two biological replicates with duplicate samples in each. Significant changes in expression compared to vehicle control are observed with HOst (P < 0.001) and pcDNA (P < 0.001) but not with RhoGDI2 (P = 0.73). Columns, means; bars, SD.

 
ET-1 is known to induce cell proliferation and differentiation through multiple G-protein-linked signaling systems (14). Studies have also shown that ET-1 induces a biphasic activation of p21ras, which subsequently activates the extracellular signal-regulated kinase (Erk) cascade (19). Our cumulative data above shows that blockade of the autocrine endothelin axis does not have an effect on in vitro growth or colony formation in bladder cancer cells, suggesting the possibility that these cells are insensitive to ET-1 by virtue of receptor loss or defects in signaling. However, as shown above (Fig. 1C), these cells do express the ET-1 receptor, ETaR. Evaluation of the effect of ET-1 on Erk phosphorylation revealed sensitivity of these cells to ET-1. In contrast, reexpression of RhoGDI2 resulted in a marked blockade of this effect, suggesting that this protein blocks the intracellular signaling pathways downstream of the ETaR (Fig. 2C). This finding is particularly relevant because it suggests that RhoGDI2 may block paracrine tumor-host crosstalk in distant organs such as the lung, where metastasis occurs. We tested this hypothesis below.

The endothelin axis, specifically ET-1, is known to be important for the regulation of multiple biological processes including angiogenesis in the normal lung (14, 20). Furthermore, ET-1 is a known angiogenic factor responsible for the tumor vasculature of cancers that metastasize to the lung (14, 21). Taken together with the results presented above, we reasoned that the paracrine effects of pulmonary ET-1-coupled with the autocrine effects of tumor ET-1 stimulate the growth of T24T cells in the lung. Conversely, by their decreased expression and lack of responsiveness to ET-1, T24T cells with reconstituted RhoGDI2 can no longer form metastases.

To test this hypothesis, we injected stably transfected T24T vector or RhoGDI2 overexpressing cells into the tail veins of ten 8-week-old nude mice, which were subsequently exposed to drinking water containing atrasentan or vehicle. After 8 weeks, the lungs were inspected grossly and microscopically (Fig. 3A). Fifty-three percent of the mice receiving the stable T24T-empty vector cells developed lung metastases. This was reduced to 5% by treatment with atrasentan. Mice exposed to RhoGDI2-overexpressing cells had a 20% incidence of lung metastases. In control mice, the tumor size of metastases ranged from 0.8 to 6 mm compared with 1.3 to 1.5 mm for metastases of RhoGDI2-transfected cells and 0.8 to 1.0 mm size of metastases in atrasentan-treated mice. Among the mice who developed at least one metastasis, the mean number of metastases per mouse was 25.3 (range, 20-80) in the control group, 3.4 (range, 0-6) for those with RhoGDI2-overexpressing cells, and 1.0 (range, 0-2) in the group treated with atrasentan. Overall, this indicates that the effect of atrasentan in suppressing metastasis mediated by the loss of RhoDGI2 is similar to reexpression of the gene itself. Careful microscopic examination of cancer cell morphology, percent tumor necrosis and location of metastatic foci relative to the vasculature did not differ between groups (Fig. 3B, i and ii; data not shown). Immunohistochemical analyses of lung metastases in mice with RhoGDI2-transfected T24T cells using an anti-RhoGDI2 antibody (Fig. 3B, iii) showed that ~70% to 90% of the tumor cells expressed the protein. This observation suggests that the cells may have developed compensatory mechanisms to overcome the metastatic suppression by RhoGDI2 by loss of downstream mediators of RhoGDI2 function or other pathways.



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  		Figure 3. A, in vivo lung metastasis in mice injected with bladder cancer cells. i, Mice in each of two groups were injected s.c. with T24T-RhoGDI2 and T24T-pcDNA3 cells. After randomization, one group of mice received atrasentan in their drinking water, the other group received vehicle. Animals were euthanized at 12 weeks and lungs examined grossly and then processed for microscopy and immunohistochemistry as described in Materials and Methods. ii, a plot of incidence of lung metastasis from four independent experiments. Columns, means; bars, SD. The groups are significantly different with respect to the proportion of mice with lung metastases (P < 0.0001, {chi}2 test). B, histologic appearance of lung metastases: H&E-stained sections [H&E, x40 (i) and x200 (ii)] and immunohistochemical (iii) staining for RhoGDI2 in pulmonary metastases obtained from RhoGDI2-transfected T24T bladder cancer cells injected i.v. via tail vein. Avidin-biotin immunoperoxidase technique described in Materials and Methods is shown at x400 magnification. C, diagrammatic representation of the autocrine and paracrine effects of RhoGDI2 on the endothelin axis in bladder cancer lung metastasis. Abbreviation: preproendothelin-1, pET-1.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We previously identified RhoGDI2 as a putative suppressor of metastasis in an isogenic model of bladder cancer metastasis. The clinical importance of this observation was underscored by the fact that reduced RhoGDI2 expression was found to be associated with decreased survival for patients with bladder cancer. In the present study, we postulated that RhoGDI2 works as a brake on the expression of prometastatic genes and sought to identify such genes in a combined analysis of the T24/T24t cell model and primary human bladder carcinomas. Because clinical reconstitution of RhoGDI2 protein is currently impractical, we sought to discover clinically druggable proteins or pathways downstream of RhoGDI2. We required these genes to be up-regulated following the loss of RhoGDI2 protein from the cells and be capable of being inhibited by small molecule antagonists or functionally antagonistic antibodies.

Precedence for the inhibition of cellular activities downstream of tumor suppressor gene loss is limited but informative. A key example is the constitutive activation of AKT and mammalian target of rapamycin (mTOR) in PTEN-deficient cells. Inhibition of mTOR kinase by rapamycin has been shown to lead to a loss of cell viability, and there is evidence that inhibitors of AKT phosphorylation may have the same potential (22). Although not particularly tractable from a therapeutic stand point, up-regulation of E2F proteins following loss of pRB and stabilization (23) of ß-catenin and therefore ß-catenin/TCF-4-mediated transcription are also important examples of up-regulated genes and pathways that become activated following the loss of tumor suppressor genes. Here, we show that the inhibition of the endothelin axis, mediated by the blockade of up-regulated functional ET-1 ligand, leads to metastatic suppression in cells deficient for RhoGDI2. Indeed, inhibition of the endothelin axis is particularly attractive, because we find no evidence for loss of organismal or tissue viability.

The endothelin ligand has been shown to regulate vascular tone, tissue differentiation, cell proliferation, hormone production, cell invasion, angiogenesis, and bone remodeling (18). ET-1 also has potent effects on cells in the skeleton (18) and lung (24), two important sites of bladder cancer metastasis (15). In addition to such direct effects, ET-1 may indirectly increase vascular endothelial growth factor (VEGF) and induce hypoxia-inducible factor 1 {alpha} (25). ET-1 and VEGF stimulate one another (26), resulting in proliferation of endothelial and vascular smooth muscle cells thus promoting tumor growth. Previous work also shows a role for tumor-secreted ET-1 in skeletal metastases from breast and prostate cancers (14) and supports a cyclical model in which tumor-secreted ET-1 stimulates bone cells, in turn providing a fertile microenvironment for metastases (27). A similar cycle could occur in other in endothelin-responsive tissues, such as the lung and kidney (24, 28) and may be the primary driving force for lung metastasis. Further evidence for this model comes from the observation that neither RhoGDI2 reconstitution nor atrasentan treatment altered primary tumor growth, suggesting that both act at the metastatic site, likely in breaking paracrine signaling (Fig. 3C).

The molecular relationship between ET-1 and RhoGDI2 shown here is highly significant, because of the existence of clinically tested, orally active small molecule ETA antagonists such as atrasentan with good clinical safety profiles (14). In fact, 39 patients, 30 of whom had prostate cancer, were treated in a recently reported dose escalation trial. The most common adverse events were rhinitis, headache, and peripheral edema indicate that atrasentan is well tolerated, with no dose-limiting adverse events observed up to 95 mg (29). Furthermore, in other studies, quality of life was not adversely affected by atrasentan (30).

As yet, we do not know how RhoGDI2 suppresses ET-1 expression. RhoGDI2 may have similar targets to those modulated by RhoGDI1, such as RhoA, which affects the cytoskeleton and influences metastatic disease progression. RhoGDI2 may also prevent activation of RhoA and RhoB (31), which stimulate the ET-1 promoter (32). Interestingly, levels of RhoA and RhoC have been reported as predictors of metastasis for patients with bladder cancer (33, 34). Our data suggests that ET-1 blockade may interfere with tumor-host interactions in the lung, thus endothelin receptor antagonists, such as atrasentan. Such drugs could reduce the development of lung metastases following resection of advanced bladder cancers by blocking the growth of a micrometastasis, which during its early developmental phase, is highly dependent on the host organ ET-1 for growth. Furthermore, because ET-1 stimulates smooth muscle cells (35), ETA antagonists might also inhibit muscle invasive primary bladder cancer, which could reduce patient morbidity from radical cystectomy. Such ETA antagonism could be tested in a model of muscle-invasive disease, which we have described (36, 37).

In conclusion, we have applied a novel approach to identify druggable targets which become activated following the loss of RhoGDI2 expression. We provide strong support for this concept in vivo by showing that an ETA antagonist effectively decreased lung metastases in a bladder cancer animal model thus validating ET-1 as a novel therapeutic target in lung metastasis. These findings suggest that clinical inhibition of ET-1 activity might be considered in clinical trials of patients with bladder cancer seeking to reduce the frequency of relapse with pulmonary metastasis.


   Acknowledgments
 
Grant support: NIH grant CA075115.

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.

Received 4/25/05. Revised 5/31/05. Accepted 6/14/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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