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Mol Cancer Ther. 2004;3:687-691
© 2004 American Association for Cancer Research

Transcriptional targeting in renal cancer cell lines via the human CXCR4 promoter

Yosef S. Haviv2,3, Winan J. van Houdt1, Baogen Lu1, David T. Curiel1,2 and Zeng B. Zhu1,2

1 Division of Human Gene Therapy, Departments of Medicine, Pathology, and Surgery and 2 Gene Therapy Center, University of Alabama at Birmingham, Birmingham, Alabama and 3 Kidney Gene Therapy Program, Division of Nephrology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel

Requests for reprints: Yosef S. Haviv, Division of Nephrology, Hadassah-Hebrew University Medical Center, P.O. Box 12000, Jerusalem, Israel 91120. Phone: 972-2-6776881/3; Fax: 972-2-6434434. E-mail: yhaviv{at}hadassah.org.il


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Metastatic renal cell carcinoma (RCC) is often resistant to standard treatment, thereby requiring new therapeutic strategies. In this regard, tumor cell migration and metastasis have recently been shown to be regulated by chemokines and their respective receptors (e.g., SDF-1{alpha}/CXCR4). In the context of RCC, up-regulation of CXCR4 expression is closely related to the development of invasive cancer. Thus, we hypothesized that the CXCR4 pathway could be exploited for RCC targeting with gene therapy vectors. In this regard, targeting adenoviral vectors to tumor cells is critically dependent on tumor-specific gene expression. Toward the end of RCC tumor targeting, we evaluated the utility of the CXCR4 promoter in an adenoviral context. First, overexpression of CXCR4 was confirmed in several RCC cell lines. Next, an adenoviral vector was constructed, whereby the human CXCR4 promoter drives the expression of a reporter gene. We tested the activity of the CXCR4 promoter in vitro and in vivo in relevant models. Our data indicate that the human CXCR4 promoter is highly active in RCC cells but not in normal human cells. Finally, biodistribution studies in mice demonstrated dramatic repression of the CXCR4 promoter in the liver but not in the kidney. In conclusion, the unique activity of the CXCR4 promoter in RCC lines and its repression in normal human cells and in the murine liver underscore its potential utility as a novel candidate for transcriptional targeting of RCC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Metastatic renal cell carcinoma (RCC) has a poor prognosis, with a median survival of 8 months. RCC tumors are generally resistant to chemotherapy and radiotherapy, and while some patients respond to immunotherapy, the overall response rate is <20% (1, 2). Mortality in patients with RCC directly correlates with the metastatic stage of the disease (3). Therefore, the identification of new agents, specifically addressing the metastatic phase of RCC, merits a high priority in the treatment of advanced RCC. In this regard, emerging data suggest that chemokines and their receptors may play key roles in determining the site of metastases. Specifically, the SDF-1{alpha}/CXCR4 pathway has been implicated in the development of metastases or local invasion in the context of breast cancer (4), melanoma (5, 6), prostate cancer (7), ovarian cancer (8), glioblastoma (9), anaplastic thyroid carcinoma (10), and neuroblastoma (11). While CXCR4 is highly expressed in breast cancer cells and primary breast tumors, its unique ligand, SDF-1{alpha}, exhibits peak levels of expression in the tissues representing the metastatic sites of breast cancer (i.e., lymph nodes, bone marrow, lung, and liver; ref. 4). Of note, up-regulation of CXCR4 has recently been demonstrated to be an inherent feature of RCC, as mutation of the VHL growth suppressor gene results in overexpression of the hypoxia-inducible factor 1{alpha} and CXCR4 (12). In contrast, SDF-1{alpha} appears to be down-regulated in RCC tumors and in normal renal tissue (13, 14). This differential pattern may be one of the factors accounting for the finding that the kidney is not a major target organ for metastases from nonrenal tumors. Thus, the CXCR4 pathway is a rational approach for RCC targeting with novel gene therapy vectors.

In this study, we hypothesized that the CXCR4 promoter could be employed for transcriptional targeting of RCC. To this end, we constructed an adenoviral vector, encoding a reporter gene under the control of the human CXCR4 promoter.

Our data indicate that the CXCR4 promoter may be a candidate for selective gene expression in human RCC cell lines and that it is repressed in the murine liver. Thus, the CXCR4 promoter emerges as a potential candidate for adenoviral-based targeting of RCC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Adenoviral Vectors
The minimal human CXCR4 promoter [–191 to +88, containing transcription start site (15), a kind gift of Dr. Nelson L. Michael, Division of Retrovirology, Walter Reed Army Institute of Research, Rockville, MD] (16) was inserted upstream of luciferase in an adenoviral backbone (Stratagene, La Jolla, CA) to construct AdGL3BCXCR4 using homologous recombination. As control adenoviral vectors, we used the isogenic AdFltLuc and AdCMVLuc as described previously (17). All viruses were titered by physical titration and plaque formation assays using 293 cells. Viral particle to infectious particle ratio was <35 for all vectors.

Cell Lines
The human kidney cancer lines A498, CAKI-1, and 786-0 were obtained from the American Type Culture Collection (Manassas, VA). The metastatic clones of the human RCC line SN12C were described previously (18). KU-19-20 and SN12C renal cancer cell lines were kindly provided by Profs. Masaaki Tachibana (Department of Urology, Tokyo Medical University, Tokyo, Japan) and Seiji Naito (Department of Urology, Kyushu University, Fukuoka, Japan), respectively. Primary, low-passage human keratinocytes were obtained from Clonetics (San Diego, CA), and primary, low-passage human mesothelial cells were obtained from Dr. Anna Kannerva (University of Alabama at Birmingham, Birmingham, AL). All cells were cultured in the recommended growth medium and maintained in 95% air-5% CO2 at 37°C.

Flow Cytometry
RCC cells were rinsed with PBS, harvested by incubating with 0.53 mmol/L EDTA in PBS, and resuspended in PBS containing 1% bovine serum albumin (Sigma Chemical Co., St. Louis, MO). For antibody incubation, 2 x 105 cells were incubated with rabbit anti-human CXCR4 antibody (1:80, StressGen, Victoria, British Columbia, Canada) for 1 hour at 4°C.

An isotype-matched normal mouse IgG1 (1:80) was used as a negative control. The cells were rinsed with PBS-bovine serum albumin and incubated with 1:100 dilution of FITC-labeled goat anti-rabbit IgG (Molecular Probes, Eugene, OR) for 1 hour at 4°C. After another PBS rinse, 2.6 µg/mL propidium iodide (Sigma Chemical) was added to sort out dead cells from the sample. After a final wash, labeled cells were fixed with 1% paraformaldehyde solution, and 104 live cells were analyzed by flow cytometry using the CellQuest software (BD Biosciences, Mountain View, CA). Relative mean fluorescence intensity was calculated as the ratio of the mean fluorescence intensity of the sample of interest to the mean fluorescence intensity of the corresponding negative control. The FITC-positive (live) cell population for each cell line was determined by gating cells incubated with 1% buffer only (negative control).

Luciferase Assay
Cells were seeded at 50,000 cells per 1 mL growth medium in 24-well plates. After 24 hours, cells were infected at three different multiplicities of infection (i.e., 80, 400, and 2,000) in 300 µL infection medium. After 1 hour, cells were washed and resuspended in growth medium. Twenty-four hours later, growth medium was removed and cells were lysed with 200 µL of lysis buffer (Promega, Madison, WI) and freeze-thawed once. Each sample (20 µL) was mixed with 100 µL of luciferase assay reagent (Promega) and measured for relative light units with Lumat LB9501 (Berthold, Nashua, NH). Standardization between cell lines and multiplicities of infections was accomplished by setting the values obtained with the cytomegalovirus promoter-driven luciferase as 100%. Data presented in Fig. 2 represent all three multiplicities of infections used in this study.



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  		Figure 2. The CXCR4 promoter is active in RCC cell lines. The RCC cell lines A498, SN12C, SN12C-MMP, 786-0, and CAKI-1 or the normal control primary mesothelial human cells and normal keratinocytes were infected in triplicates at multiplicity of infection of 400 with either AdGL3BCXCR4 or the isogenic control vectors AdCMVLuc or AdFltLuc. Gene expression was measured 24 hours after infection and is presented for the human CXCR4 and Flt-1 promoters as percentage of cytomegalovirus promoter activity in relative light units (RLU). *, P < 0.05 for repression of the CXCR4 promoter in the normal primary mesothelial cells and keratinocytes versus its activity in A498 RCC cells. M, primary human mesothelial cells; K, human keratinocytes.

 
Real-time Reverse Transcription-PCR
Total RNA was isolated from RCC cell lines or the control normal human cells using RNeasy Mini Kit (Qiagen, Valencia, CA). Isolated RNA was reverse transcribed into cDNA. To examine the expression of the gene, human CXCR4 mRNA was first identified and analyzed by standard reverse transcription-PCR (RT-PCR). This analysis was followed by characterization with real-time RT-PCR for semiquantitative evaluation of CXCR4 mRNA. Sense and antisense primers were designed from the cDNA sequence of the human CXCR4 gene (Genbank accession no. AF348491). The primers used were 5'-AACCAGCGGTTACCATGGAG-3' (sense, –14 to +6) and 5'-CTTCATGGAGTCATAGTCCCCTG-3' (antisense, +118 to +95). The amplified fragment was detected as an 88-nucleotide amplicon. GAPDH was used as reference gene. Control samples comprising distilled water or non-reverse-transcribed samples were negative for both CXCR4 and glyceraldehyde-3-phosphate dehydrogenase transcripts. Taqman analysis for real-time RT-PCR was used to quantify the mRNA levels. The Taqman probe used for semiquantitative RT-PCR was 5'-ACACTTCAGATAACTACACCGAGGAAATGGGC-3' (+63 to +83).

In vivo Gene Transfer
Male Sprague-Dawley mice (Harlan-Sprague-Dawley Inc., Indianapolis, IN) ages 6 to 8 weeks were used (n = 5). Experiments were approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee. AdCMVLuc or AdGL3BCXCR4 (5 x 108 plaque-forming units) were injected via the tail vein of the mice, and animals were sacrificed 2 days later. Organs were processed to determine luciferase activity, corrected for protein concentration as described previously (17).

Statistics
Data in this study were analyzed by the Wilcoxon test. Statistical significance was considered for P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of CXCR4 in RCC Cell Lines
To evaluate CXCR4 expression, we studied human CXCR4 transcripts in either RCC cell lines (A498, CAKI-1, 786-0, SN12C, and KU-19-20), the stably transformed 293 cell line derived from human embryonic renal tubular origin, or normal human keratinocytes and mesothelial cells. The SN12C, KU-19-20, and CAKI-1 cell lines were originally derived from metastatic tumors of renal cancer, while the A498 and 786-0 cell lines were derived from primary undifferentiated and clear cell renal carcinomas, respectively. Human CXCR4 mRNA transcripts were detected in four of five RCC lines tested but were practically undetectable in the normal control human cells (Fig. 1A). The A498 RCC cell line displayed the highest mRNA levels, while no CXCR4 expression was found in the 786-0 renal cancer cell line. CXCR4 expression in RCC lines was also confirmed at the protein level with fluorescence-activated cell sorting analysis (Fig. 1B). Thus, our studies indicate that CXCR4 is overexpressed in the majority of RCC lines evaluated.



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  		Figure 1. Expression of CXCR4 in RCC lines. A, Real-time RT-PCR for CXCR4 expression in RCC or control normal human cells. Total RNA was extracted from 1 x 106 cells of either the RCC cell lines A498, CAKI-1, SN12C, KU-19-20, and 786-0 or primary mesothelial human cells and normal keratinocytes. Real-time RT-PCR reactions for transcripts of CXCR4 and the control housekeeping GAPDH gene were performed under identical conditions. B, Flow cytometry histograms for cell surface expression of CXCR4 in RCC cell lines. The RCC cell lines 786-0, A498, SN12C, and CAKI-1 were incubated with either anti-human CXCR4 antibody (shaded histograms) or the control isotype-matched antibody (gray line) followed by incubation with FITC-labeled secondary antibody.

 
The CXCR4 Promoter Is Active in RCC Cell Lines
Having established the expression of CXCR4 in RCC lines, we proceeded to evaluate the activity of the CXCR4 promoter in an adenoviral backbone. To this end, we infected RCC and control cells with either AdGL3BCXCR4 or the isogenic control vectors AdFltLuc or AdCMVLuc, differing only in the respective Flt-1 and cytomegalovirus promoter sequences driving the firefly luciferase gene. AdFltLuc was selected as the control virus because the Flt-1 gene is also up-regulated in RCC, but not to the extent of CXCR4 (14). In all RCC cell lines expressing CXCR4, the CXCR4 promoter was highly active in the adenoviral backbone (Fig. 2). Furthermore, in the highly metastatic SN12C-MMP RCC cell line clone, the CXCR4 promoter activity was 10 times higher than cytomegalovirus. In all RCC cell lines, the activity of the CXCR4 promoter was superior to Flt-1. Of note, in the CXCR4-negative RCC cell line 786-0, the activity of the CXCR4 promoter activity was substantially lower than in the CXCR4-positive RCC cells, compatible with relative specificity of the promoter. Furthermore, the CXCR4 promoter was repressed in the normal human cells examined (e.g., keratinocytes and mesothelial cells; Fig. 2). Thus, in the RCC and normal cell lines examined, the CXCR4 promoter appears promising for transcriptionally targeted transgene expression.

The CXCR4 Promoter Is Repressed in the Murine Liver
Because systemic administration may be required to address advanced-stage RCC, we studied the activity of the CXCR4 promoter in vivo in a murine model. Specifically, because liver toxicity often compromises the utility of adenoviral-based gene therapy, we were interested in the activity of the CXCR4 promoter in the kidney relative to the liver. Our in vivo studies show that the CXCR4 promoter activity, normalized to the cytomegalovirus promoter, was 137-fold lower in the murine liver relative to the kidneys (Fig. 3). Of all the visceral organs, the murine kidneys showed the highest CXCR4 promoter activity followed by the lungs (Fig. 3). These findings agree with the patterns of CXCR4 expression in the murine lungs and kidneys (19, 20). Thus, the CXCR4 promoter meets a major requirement from potential candidates for transcriptional targeting of cancer (i.e., down-regulation in the liver).



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  		Figure 3. The CXCR4 promoter is repressed in the murine liver. Male BALB/c mice (n = 5) ages 6 to 8 weeks were injected with 5 x 108 plaque-forming units of AdGL3BCXCR4 or AdCMVLuc via the tail vein. Animals were sacrificed 2 days later and organs were processed to determine luciferase activity. The luciferase activity in organs harvested from mice injected with AdGL3BCXCR4 was normalized to luciferase activity in the same organs from animals injected with AdCMVLuc. *, P < 0.05 for repression of the human CXCR4 promoter activity in murine organs versus the liver.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The CXCR4/SDF-1{alpha} axis appears to be involved in the acquisition of invasive and metastatic phenotypes in several cancer types (4-7). Therefore, up-regulation of CXCR4 expression by cancer cells may have an ominous impact on survival. However, the expression of metastasis-associated chemokines may vary in distinct tumors. For example, while many breast and rhabdomyosarcoma cells express CXCR4 consistently, some melanoma, lung, and hepatocellular cancer cells do not express CXCR4 (4, 21, 22). Thus, the respective chemokine receptor expression profile should be evaluated for each tumor type. In the context of RCC, compelling evidence indicates that CXCR4 expression is tumor specific (12-14).

In this study, we confirmed overexpression of CXCR4 in RCC cell lines and employed the CXCR4 promoter for adenoviral-based transcriptional targeting of RCC. This approach resulted in dramatically high levels of reporter gene expression in RCC cells. Furthermore, relative specificity to cancer cells was demonstrated by repression of the CXCR4 promoter in the normal cells examined. Potentially, the specificity of the CXCR4 promoter may be even further enhanced with the full-length 5' flanking region harboring repressor sequences but likely at the expense of potency of the promoter (15).

While the CXCR4 promoter appears conceptually attractive for targeting several other types of metastatic tumors, our in vitro data indicate that the CXCR4 promoter is an excellent candidate for RCC targeting regardless of the original metastatic phenotype. In this regard, in the SN12C and CAKI-1 cell lines, both derived from metastatic RCC tumors, CXCR4 expression and CXCR4 promoter activity were both inferior to the A498 cell line derived from a primary RCC tumor.

In contrast, in the highly metastatic SN12C-MMP RCC cell line, the CXCR4 promoter is highly active. However, further studies are required to determine whether the CXCR4 promoter activity correlates with the primary tumor or metastatic origin of the RCC cells.

Previous studies have shown a gradient for SDF-1{alpha} expression in organs targeted by metastases (4). Thus, the finding that distinctly low expression of SDF-1{alpha} is detected in the human kidney (4) may account for the infrequent metastases from other cancer types to the kidney. In contrast, because the metastatic patterns of breast cancer and RCC are similar, de novo expression of CXCR4 in RCC cells may not be random but may rather be an inherent feature of RCC (12) and one of the attributes accounting for metastasis of RCC cells to other organs, expressing high levels of SDF-1{alpha}, such as the bone marrow, liver, and lungs (4, 6, 13, 14).

Our biodistribution studies in mice indicated that in the context of an adenoviral backbone, the CXCR4 promoter is repressed in the liver but not in the murine kidneys. Previous studies showed abundant expression of CXCR4 in murine and bovine adult kidneys (19-21, 23) but not in human kidneys (4, 9, 20, 22, 24). Therefore, the significant activity of the human CXCR4 promoter in the murine kidneys does not necessarily indicate that it is active in normal human renal tissues. Because systemic administration of adenoviral vectors in both humans and mice results in significant hepatotoxicity, promoter activity in the liver is of critical importance. In this regard, both murine and human CXCR4 homologues are either not expressed (9, 18-20) or down-regulated (24) in the liver. Thus, based on the dramatically low activity of the CXCR4 promoter in the liver, the CXCR4 promoter may be a good candidate to mitigate hepatic toxicity.

In conclusion, we have identified the human CXCR4 promoter as a candidate for RCC targeting. Based on the implication of the CXCR4/SDF-1{alpha} axis in cancer metastases and possibly also in RCC, gene therapy strategies for transcriptional targeting based on the human CXCR4 promoter may be a meaningful approach in the context of RCC.


   Acknowledgments
 
We thank Minghui Wang for assistance with real-time PCR.


   Footnotes
 
Grant support: Solomon Papper, M.D., 2003 Young Investigator Grant of the National Kidney Foundation (Y. S. Haviv) and NIH grants RO1 HL67962, R01 CA86881, RO1 AG021875, RO1 CA090547, and P50 CA89019 (D.T. Curiel).

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 2/23/04; revised 4/ 7/04; accepted 4/15/04.


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 Introduction
 Materials and Methods
 Results
 Discussion
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