[CANCER RESEARCH 58. 1391-1394. April l, 1998]
Advances in Brief
Down-Regulation of Homeobox Gene GBX2 Expression Inhibits Human Prostate Cancer Clonogenic Ability and Tumorigenicity1
Allen C. Gao, Wei Lou, and John T. Isaacs2
The Johns Hopkins Oncology Center, James Buchanan Brady Urological Institute, Department of Urologv, The Johns Hopkins University School of Medicine Baltimore Maryland 21231
Abstract of a homeobox domain region. The homeobox domain is a conserved structural motif of nucleotides encoding a 60-amino acid residue Previously, we have demonstrated that GBX genes, a homeobox-con- polypeptide sequence that recognizes and binds to specific DNA taining human family of DNA-binding transcription factors consisting of sequence motifs (2). In humans, as well as in mice, there are 39 class GBXl and GBX2, are overexpressed in a panel of human prostatic cancer cell lines (i.e., TSU-prl, PC3, DU145, and LNCaP) compared to normal I homeobox genes that are organized in four clusters designated A, B, prostate. In the present studies, specific primer sets were designed for C, and D. In humans, these clusters are located on chromosomes 7,17, reverse transcription-PCR detection of the expression of GBXl versus 12, and 2, respectively (4, 5). GBX2 in human prostate cancer. These studies demonstrated that the A number of homeobox genes have been found to be involved in GBX2 gene, but not the GBXl gene, is consistently overexpressed in this malignant processes in a series of cell lineages. These include Hox-7.1 panel of human prostate cancer cell lines compared to normal human (6), Hoxa-7, Hoxa-1, Hoxb-7, and Hoxc-8, as well as homeobox genes prostate. Using a quantitative-competitive PCR analysis, GBX2 mRNA Evx-1 and Cdx-1 (7). Expression of Hox-7.1 in myoblasts inhibits was expressed as 3 x HI1 copies//*g RNA in normal prostate tissue and terminal differentiation and induces cell transformation (6). Recently, 4 x IO4 copies/jig RNA in the immortalized normal neonatal prostate mutations in HOXDÌ3have been found in families affected by syn- epithelial cell line 267B-1, as compared to 6 x IO5, 5 x IO5, 3 x IO5, and 1 x IO5 copies/fig RNA in TSU-prl, DU145, LNCaP, and PC3 prostate polydactyly, an inherited human abnormality of the hands and feet (8, 9). Overexpression of homeobox genes (i.e., Hoxa-7, Hoxb-7, Hoxa-1, cancer cell lines, respectively. To examine the importance of GBX2 ex Hoxc-8, Evx-1, and Cdx-1) induces NIH 3T3 cell transformation and pression for prostate cancer malignancy, GBX2-overexpressing TSU-prl and PC3 human prostatic cancer cells were transacted with a eukaryotic tumorigenicity (7). Altered expression of the homeobox genes is expression vector containing an antisense GBX2 homeobox domain cDNA. observed also in various human carcinomas, including breast (10), Stable translatant clones with 5-10-fold decreased levels of GBX2 mRNA colon (11), rectal gastric (12), and lung (12). Recently, we demon expression were obtained. When tested in vitro, the clonogenic ability of strated that members of the GBX class of homeobox genes are over- the GBX2 antisense transfectants was reduced by approximately 50% in expressed in TSU-prl, PC3, LNCaP, and DU 145 metastatic prostate both cell lines. When implanted s.c. into nude mice, the tumorigenicity of cell lines compared to the normal prostate (13). The GBX class of the antisense GBX2 transfectants from both human prostatic cancer cell homeobox domain genes comprises six known members. These in lines was inhibited by more than 70% compared to the parental cells. clude GBX1 (14) and GBX2 (15, 16) in humans, Gbx-1 (17) and Gbx-2 These results suggest that expression of (¡HX2gene is required for ma (18) in mice, chicken Hox7 (19), and Xenopus laevis homeobox gene lignant growth of human prostate cells. xgbx-2 (GenBank accession no., U04867). This GBX family of ho meobox genes shares more than 97% sequence identity in the 60 Introduction amino acids of the homeobox domain (14, 15, 17, 18). In humans, the There is an urgent need for the development of prognostic indica GBXl and GBX2 genes are located on chromosomes 7q36 and 2q37, tors to predict which prostate cancer patient with apparently localized respectively (14). Using RNA in situ hybridization analysis of mouse disease actually has clinically undetectable micrometastatic cancer embryos, expression of mouse Gbx2 RNA, which is a homologue to requiring additional systemic treatment (1). The down-regulation of human GBX2, occurs during gastrulation and neurulation (20). Gbx2 expression of a series of metastatic suppressor genes encoding cell RNA is expressed in the spinal cord, hindbrain, optic vesicle, and surface proteins (e.g., KAI1, CD44, and E-cadherin) is a candidate for mandibular arch (20), suggesting that Gbx2 expression is associated such a prognostic indicator (1). This has raised the issue of the with the development of the nervous system. In a more recent study, mechanism for such coordinated down-regulation during the acquisi GBX2 was identified as a transcription factor, the expression of which in myeloblasts is regulated by the v-Myb oncoprotein encoded by the tion of metastatic ability by prostate cancer cells. One class of possible avian myeloblastosis virus (21). This latest study demonstrated that master regulators for such repression is the homeobox family of transcription factors. Homeobox genes comprise a large family en virally induced expression of GBX2 in myeloblasts resulted in en coding positive and negative transcription factors of the helix-turn- hanced autocrine growth stimulation (21). helix motif, the expression of which regulates body plan and pattern In this report, we determined the levels of expression of GBXl versus GBX2 in normal human prostate versus human prostate cancer formation during development and various aspects of cell regulation cell lines. In addition, GBX2 expression in TSU-prl and PC3 human and differentiation (2, 3). Characteristic of these genes is the presence prostate cancer cell lines was decreased by expression of GBX2 homeobox domain cDNA in the antisense orientation to test what Received 11/19/97; accepted 2/16/98. effect this down-regulation has on the clonogenic ability and tumor 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 igenicity of these prostate cancer cells. 18 U.S.C. Section 1734 solely to indicate this fact. ' Supported by Specialized Programs of Research Excellence Grant CA 58236 (Na Materials and Methods tional Cancer Institute). 2 To whom requests for reprints should be addressed, at The Johns Hopkins Oncology Cells. LNCaP. DU 145, TSU-prl. and PC3 human prostate cancer cell lines Center, James Buchanan Brady Urological Institute, Department of Urology, The Johns Hopkins University School of Medicine, Baltimore, MD 21231. Fax: 410-955-0840. were maintained in RPMI 1640 with 10% fetal bovine serum as described 1391
Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1998 American Association for Cancer Research. CBX2 EXPRESSION IN PROSTATE CANCER previously (13). 267B-1 (22), an immortalized normal human neonatal pros- RNA and in reverse transcriptase efficiency in RT reactions were minimized tatic epithelial line isolated as a single colony of pRSV-T-transfected NP-2s by normalizing data in comparison to the expression of the housekeeping gene cells, was maintained in P4-8F medium (Biological Research Faculty and GAPDH. Facility, Inc., Ijamsville, MD). Clonogenic Assay. The anchorage-dependent clonogenic ability, expressed Construction of Antisense GBX Homeobox Domain. The cDNA of the as the percentage of cells capable of producing growing colonies on tissue GBX homeobox domain was generated by RT'-PCR of RNA from TSU-prl culture plastic plates, was performed as described previously (26). cells using degenerate oligonucleotide primers to the homeobox domain as Tumorigenicity Assay. Five-week-old adult male nude mice (Charles River Breeding Laboratories. Boston. MA) received a s.c. injection of 1 X IO6 described previously (13). The GBX homeobox domain was ligated into pCDNAS.l^ vector (Invitrogen. San Diego. CA), and the plasmid containing TSU-prl cells or PC3 antisense transfectants or a s.c. injection of vector alone the GBX homeobox domain in the antisense orientation downstream from the (controls). Fifty days after the TSU-prl injection and 60 days after the PC3 cytomegalovirus promoter was selected for further study. For transfection, injection, the mice were sacrificed and the tumors were weighed. TSU-prl and PC3 cells were transfected with this antisense GBX or vector Statistical Analysis. Values are expressed as means ± SE. Statistical control plasmid by a lipofectamine (Life Technologies, Inc., Gaithersburg, significance was calculated by one-way ANOVA. followed by the Newman- MD) protocol according to the manufacturer's instructions. Single transfectant Keuls test for multiple comparison, with P < 0.05 being considered signifi colonies were isolated and maintained in the presence of G418 as described cant. previously (23). RNA Preparation and Northern Blot Analysis. Total cellular RNA was Results and Discussion isolated and Northern blot analysis was performed as described previously GBX2 but not GBX1 Is Overexpressed in Prostatic Cancer. We (13). Normal human prostate RNA was purchased from Clontech Laboratories. previously demonstrated that GBX genes are consistently overex- Inc. (Palo Alto, CA). Twenty fig of total RNA were used for the Northern blot. pressed in a series of human prostatic cancer cell lines (i.e., TSU-prl, Hybridization was performed with GBX homeobox domain cDNA probe (13) labeled with [a-12P]dCTP by random primer method according to the manu PCS, DU 145. and LNCaP) compared to normal human prostate using facturer's instructions (Amersham Corp., Arlington Heights, IL). a cDNA probe recognizing the GBX homeobox domain region (13). RT-PCR Assay. Five ;u,gof total RNA were added to 0.5 fig of oligode- Because GBX1 shares high homology (81%) with GBX2 cDNAs in oxythymidylate|,_1K primer and brought to a final volume of 20 fil. The the homeobox domain region, this probe does not differentiate be primer-RNA mixture was combined with 200 units of Moloney murine tween the expression of GBX1 and GBX2. To semiquantitatively leukemia virus reverse transcriptase. and the reaction was performed according compare the level of GBX1 versus GBX2 expression, RT-PCR anal to the manufacturer's instructions (Life Technologies). Samples were stored at -20°C. Oligonucleotide primers specific for GBX1 (forward. 5'-AAG- ysis was performed using two distinct oligonucleotide primers de CTAAAGGGAAGCCTGGGGACT-3'; reverse, 5'-CATTTGTTGGTGCTG- signed specifically to amplify either GBX1 or GBX2, but not both. As GCTCCGCAC-3') and GBX2 (forward, 5'- CACCACGTCTACGGGCAA- shown in Fig. 1, all of the human prostate cancer cell lines (i.e., PC3. GAAC-3'; reverse, 5'-AGCTGCTGATGCTGACTTCTGA-3') were designed LNCaP, DU 145, and TSU-prl) and normal human prostate express according to the published sequences (14, 15) so that each would amplify GBX 1; however, the expression of GBX 1 is not consistently higher in specifically GBX I or GBX2 cDNAs. The predicted PCR products for GBX I the prostate cancers compared to normal human prostate. These re and GBX2 are 369 and 309 bp. respectively. The PCR was performed on the sults suggest that, whereas normal prostate and prostate cancer con cDNAs with a denaturing temperature of 94°Cfor 45 s. an annealing temper sistently express GBX1, GBX1 overexpression is not required for ature of 68°C(GBX1 ) or 65°C(GBX2) for 30 s, and an extension temperature prostate malignancy. In contrast, the GBX2 gene is overexpressed in of 72°Cfor 1 min for 35 cycles. As a control for RNA integrity, a separate all of the human prostate cancer cell lines compared to normal portion of cDNA was amplified using primers specific for human GAPDH (forward, 5'-CCTGGTCACCAGGGCTGCCT-3'; reverse, 5'-TACCAC- prostate (Fig. 1), suggesting that overexpression of the GBX2 gene is CACTTCTGCGGTCA-3') according to the GAPDH sequences (24). The consistently associated with prostate cancer. To determine this more predicted PCR product for GAPDH was 214 bp. PCR products were separated precisely, the absolute amounts of GBX2 expression in these malig by electrophoresis on a 1.5% agarose gel, and DNA fragments were visualized nant cell lines and in normal prostate were determined via competitive and photographed under UV light by ethidium bromide staining. PCR. In competitive RT-PCR, a constant amount of reverse-tran Cloning and Sequencing of the GBX1 and GBX2 PCR Products. Both scribed RNA is mixed and coamplified with variable known amounts of the PCR products (i.e.. GBX1 and GBX2) were excised from the agarose gel of synthetic competitor that contains the same primer template se and directly cloned into the TA cloning vector (Invitrogen). The cloned PCR quences as the target but differs from the target cDNA for the products were confirmed by sequencing using the double-stranded DNA cycle sequencing system (Life Technologies) according to the manufacturer's in structions. LNCaP TSU-prl PC3 DU145 NP Quantitative PCR Analysis. GBX2 RNA expression analysis was per formed using a quantitative competitive PCR method (25) with a synthetic competitor that differed from the GBX2 cDNA by having a small internal 309 pb- GBX2 deletion. The GBX2 competitor containing a 79-bp deletion was generated by PCR amplification of TA vector containing the GBX2 PCR product, produced as described in the preceding section. Competitive PCRs were performed in a total volume of 20 fd using a constant amount of cDNA that was coamplified with serial dilutions containing known numbers of copies of the competitor. The cDNA was produced via RT as described above for the RT-PCR assay. 369 bp. GBX1 PCR conditions were the same as described above. The two products are amplified with the same efficiency because of the high similarity. The PCR products were separated by electrophoresis in a 3% agarose gel. and the bands were visualized by ethidium bromide staining and quantified by densitometric scanning. The absolute amount of the target cDNA expressed as copies/fig of starting RNA was quantified on the basis of the amount of competitor needed 214 bp- GAPDH to produce an equivalent PCR signal. Differences in the amount of starting
Fig. 1. RT-PCR analysis of GBX 1 and GBX2 expression in human prostate cancer ' The abbreviations used are: RT, reverse transcription: GAPDH. glyceraldehyde-3- cells. NP. normal prostate (purchased from Clontech). Human GAPDH primers were used phosphale dehydrogenase. in a parallel PCR reaction to confirm RNA integrily. 1392
Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1998 American Association for Cancer Research. CSX2 EXPRESSION IN PROSTATE CANCER a 5-10-fold decrease in the levels of GBX2 expression (Table 1), as GBX2 measured by competitive PCR analysis (Fig. 2ß).The number of romnetitior GBX2 molecules in the parentaland vector control transfectantis " 1 X 105copies//xg of RNA compared to 1-2 X 104copies//ng of RNA in the PC3 antisense clones, whereas the number of GBX2 molecules 1000 100 10 l 0.1 in the TSU parental and vector control is 6 X 10s copies/fig RNA x IO4 copies / (Ig RNA compared to 8-10 X IO4copies/jag RNA in the TSU antisense clones. Effect of Antisense GBX2 Homeobox Domain on Clonogenic Ability and in Vivo. Tumorigenicparental,vector control, and anti- B sense transfectants were cultured under identical conditions, and their clonogenic ability was determined in vitro. All of the antisense trans AS3 AS2 ASI Vector fectants from both cell lines had a significantly decreased (approxi mately 50%) clonogenic ability (i.e., decreased percentage of initially GBX2 inoculated cells capable of producing continuously growing clones) compared to the vector controls (Table 1). The GBX2 mRNA expres competitor sion in the antisense clones in both cell lines was only reduced by 5-10-fold compared to their parental cells, but GBX2 mRNA expres Fig. 2. Competitive PCR analysis of GBX2 mRNA expression. The amplified products were separated by 3% agarose gel electrophoresis, and the ratio of cDNA:competitor was sion was not eliminated (Table 1). This may explain why a more quantified by densitometric analysis. The differences in the amount of starting RNA and complete inhibition of their clonogenic ability was not observed. To reverse transcriptase efficiency in diverse samples were minimized by normalizing data in test the effect of down-regulation of the GBX2 expression on the comparison to the expression of the GAPDH gene. A, GBX2 mRNA expression in PC3 cells. A constant amount of reverse-transcribed RNA was coamplified with a serial tumorigenicity of the TSU-prl and PC3 human prostate cancer cells, dilution of the OBX2 synthetic competitor. B. GBX2 mRNA expression in PC3 vector stable transfectants expressing the antisense plasmids were inoculated control and three representative transfectants with GBX antisense cDNA. For each sample, a constant amount of reverse-transcribed RNA was coamplified with a constant s.c. into nude mice. As controls, parental and vector-alone transfec amount of the competitor. tants were also injected. As shown in Table 2, all of the animals that received injection of either TSU-prl or PC3 antisense GBX2 trans presence of a small insertion/deletion or a restriction site, such that fectants showed a more than 70% suppression of their tumor weight two products are easily distinguishable by gel electrophoresis. The compared to those that received injection of the parental cells. In two products are amplified with the same efficiency because of the contrast, no significant tumor suppression for either cancer was ob high similarity. The absolute amount of the target cDNA is quantified served in the controls treated with Neo vector alone. on the basis of the amount of competitor needed to produce an In summary, we have demonstrated that overexpression of GBX2 equivalent PCR signal. Fig. 2A is an example of GBX2 expression in but not GBXI gene consistently occurs in human prostate cancer cell PC3 cells detected by competitive PCR. The amount of GBX2 ex lines. In addition, using an antisense approach, the present studies pression in TSU-prl, LNCaP, DU 145, PC3, and normal human pros provide direct evidence that GBX2 gene overexpression is an impor tate tissue is 6 x IO5, 5 x IO5. 3 x IO5, 1 x IO5, and 3 X IO3 tant requirement for the clonogenic ability and tumorigenicity of copies/¿¿gofRNA, respectively.
The mRNA for the normal human prostate used for these analyses Table 1 In vitro clonogenic ability of PC3 and TSU-prl parental, vector, and their contains not only glandular epithelial cells, but also stromal cells (i.e., GBX antisense transfectants fibroblasts, smooth muscle cells, and so on). To allow a more direct Percentage inhibition of No. of copies of comparison to a pure population of human prostate epithelial cells, Cell clones Clonogenic ability (%) clonogenic ability" GBX2//j.g RNA XIO51 similar competitive PCR analyses were performed using mRNA from PC3parentalPC3 ±214 the immortalized 267B-1 normal neonatal prostate epithelial cells. vectorPC3-ASIPC3-AS2PC3-AS3TSU-prl±37±2*6±3"5 XIO52X IO41 The 267B-1 normal prostate cell line was found to be nontumorigenic XIO41.4X in vivo (i.e., no palpable tumors were detected at 3 months post-s.c. ±2"21 IO46X injection of 3 x IO6cells into nude mice). These normal prostate cells parentalTSU ±420 IO56X IO51 vectorTSU-ASITSU-AS2TSU-AS316±511 expressed 3 X IO4copies of GBX2//xg RNA. These results demon ±3*12 XIO51 strated that when cancer cell lines are directly compared, GBX2 ±4*13 XIO58 mRNA is overexpressed by 3-20-fold compared to normal prostate ±4*1256*63*69*548*43"38"1 X IO4 " Inhibition versus parental cells epithelial cells. h P < 0.05. Construction and Expression of GBX2 Homeobox Domain in the Antisense Orientation. To directly test whether GBX2 gene Table 2 In vivo characteristics of TSU-prl and PC} párenla!, vector, anil GBX overexpression is critically involved in determining the malignant antisense transfectants ability of human prostate cancer cells and is not just an epiphenom- weight (mg) inhibition of tumorweight"2(17
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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1998 American Association for Cancer Research. Down-Regulation of Homeobox Gene GBX2 Expression Inhibits Human Prostate Cancer Clonogenic Ability and Tumorigenicity
Allen C. Gao, Wei Lou and John T. Isaacs
Cancer Res 1998;58:1391-1394.
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