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Loss of NKX3.1 Expression in Human Prostate Cancers Correlates with Tumor Progression^1,2

Lombardi Cancer Center [C. B., H. J. V., R. S., G. S., E. P. G.] and Department of Pathology [E. E. L.], Georgetown University, Washington, DC 20007-2007; Cancer Genetics Branch, National Human Genome Research Institute, NIH, Bethesda, Maryland 20892-4470 [L. B., J. K., O-P. K.]; Institute for Pathology [L. B., N. W., G. S.] and Urologic Clinics [T. C. G.], University of Basel, Basel, Switzerland; Laboratory of Cancer Genetics, Tampere University Hospital, Tampere, Finland [P. K.]

	ABSTRACT

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NKX3.1 is a prostate-specific homeobox gene located on chromosome 8p21. In the mouse, Nkx3.1 has growth-suppressive and differentiating effects on prostatic epithelium. Mutations of the coding region of NKX3.1 were not found in human prostate cancer, failing to support the notion that NKX3.1 was a tumor suppressor gene. To study the expression of NKX3.1 protein in human tissues and prostate cancer, we derived a rabbit antiserum against purified recombinant NKX3.1. Among normal human tissues, NKX3.1 expression was seen in testis, in rare pulmonary mucous glands, and in isolated regions of transitional epithelium of the ureter. NKX3.1 was uniformly expressed in nuclei of normal prostate epithelial cells in 61 histological sections from radical prostatectomy specimens. We analyzed 507 samples of neoplastic prostate epithelium, most of which were contained on a tissue microarray that contained samples from different stages of prostatic neoplasia. We observed complete loss of NKX3.1 expression in 5% of benign prostatic hyperplasias, 20% of high-grade prostatic intraepithelial neoplasias, 6% of T1_a/b samples, 22% of T3/4 samples, 34% of hormone-refractory prostate cancers, and 78% of metastases. Our data show that NKX3.1 expression is highly, but not exclusively, specific for the prostate. Loss of NKX3.1 expression is strongly associated with hormone-refractory disease and advanced tumor stage in prostate cancer (P < 0.0001).

	INTRODUCTION

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NKX3.1 is a homeobox gene with prostate-specific expression in the adult (1) . NKX3.1 maps to chromosome 8p21, a region that undergoes LOH⁴ in 75% of prostate cancer specimens (2, 3, 4, 5, 6) . For this reason, NKX3.1 was a candidate target gene for disruption by the 8p21 LOH. However, mutational analysis failed to find any tumor-specific mutations of NKX3.1 in human prostate cancer tissues (2) . NKX3.1 has potent growth-suppressing and differentiating effects on prostatic epithelium. Mice heterozygous for targeted disruption of Nkx3.1 have abnormal prostate morphology with overgrown and dysplastic epithelium (7) . Disruption of prostate epithelial morphology and dysplasia is more severe in Nkx3.1-null mice (7) . The suggestion that gene dosage, and therefore the amount of protein, may be important for the growth-suppressor effects of NKX3.1 prompted us to study its expression in human prostate cancer specimens.

This report describes the derivation of an antiserum against purified recombinant NKX3.1 protein and the immunohistochemical expression of NKX3.1 in normal human tissues and in prostate cancer specimens. One report of NKX3.1 mRNA expression in human prostate cancer tissues described increased expression in prostate cancers compared with adjacent normal tissue (8) . Our data examining NKX3.1 protein expression support the opposite conclusion. We demonstrate that loss of the expression of this growth suppressor correlates with prostate tumor progression.

	MATERIALS AND METHODS

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Expression and Purification of NKX3.1 Recombinant Protein.
A 3'-truncated cDNA of wild-type NKX3.1, including nucleotides 1–581 and excluding the region that coded for the COOH-terminal region of the protein downstream from the homeodomain, was inserted into pMAL-C2g vector (New England Biolabs, Waltham, MA) at the SalI and EcoRI restriction sites. Fusion plasmid was transformed into BL-21-competent cells (Stratagene, La Jolla, CA). An overnight culture of bacteria containing the fusion plasmid was induced with 0.5 mM IPTG for 2 h. MBP⁹-NKX fusion protein was purified by affinity chromatography with amylose resin (New England Biolabs). Purified fusion protein was cleaved with 0.05 µg of genenase I (New England Biolabs) per 10 µg of fusion protein at room temperature for 24 h. Pure recombinant NKX3.1 was purified again by DEAE ion exchange chromatography.

Production of NKX3.1 Polyclonal Antibody.
Twenty-five µg of purified NKX3.1 recombinant protein in TiterMax adjuvant emulsion (CytRx Corporation, Norcross, GA) were inoculated into New Zealand White rabbits. The total volume of the initial inoculation was 400 µl, and a 200-µl boost was administered 3–4 weeks later. Rabbits were test bled 3–4 weeks after the initial inoculation and after the second boost. Rabbit anti-NKX3.1 antibody was purified by affinity chromatography by successive passes through BL-21-MBP CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech, Uppsala Sweden) followed NKX3.1 CNBr-activated Sepharose 4B.

Immunohistochemical Staining.
Deparaffinized tissue sections were preheated in 10 mM sodium citrate solution for 20 min in a Black and Decker vegetable steamer. NKX3.1 antibody diluted 1:1000 in blocking buffer (1:70 dilution of goat serum in PBS) was incubated on slides for 1 h at room temperature. Sections were then incubated with 1:200 diluted biotinylated secondary antibody (Vector Labs, Burlingame, CA) for 30 min and ABC solution (Vector Labs) for another 30 min. VIP peroxidase substrate (Vector Labs) was used to stain tissues, which were then counterstained with methyl green.

Tissues.
Sections of normal human tissues were obtained from the Lombardi Cancer Center Histopathology and Tissue Shared Resource. Prostate tissue specimens for normal tissue and the 30 prostate cancers in the validation set came from the Lombardi Cancer Center Histopathology and Tumor Core Facility. These specimens were collected at the time of RP and therefore represent specimens from clinical stage I and II prostate cancers. All histological diagnoses were confirmed by staining parallel sections with H&E. Specimens were reviewed by one of us (E. L.) for purposes of assigning Gleason grades.

Tissue Microarrays.
The prostate tissue microarray was constructed as described previously (9) . Briefly, core tissue biopsies (diameter, 0.6 mm) were taken from paraffin-embedded prostate tumors (donor blocks) and precisely arrayed into a new recipient paraffin block (35 x 20 mm) with a custom-built precision instrument (Beecher Instruments, Silver spring, MD). After the array block was constructed, multiple 4-µm sections were cut with a microtome using an adhesive-coated tape sectioning system (Instrumedics, Hackensack, NJ). Formalin-fixed and paraffin-embedded tumor and benign control specimens were obtained from the archives of the Institutes for Pathology, University of Basel (Basel, Switzerland), the Cantonal Institute for Pathology (Liestal, Switzerland), and the Tampere University Hospital (Tampere, Finland). The tissue microarray initially contained 632 specimens from all stages of tumor progression. The presence of tissue conforming to the histopathological category assigned in the original assembly was verified by review of an H&E-stained section within 50 µm of the section stained for NKX3.1; this review identified 477 tissue core specimens that were included in the analysis. Tissue samples included BPH as control (n = 43); primary tumors with stage T1_a/b according to International Union Against Cancer criteria (10) , incidentally discovered after transurethral resection for presumed BPH (n = 109); clinically localized tumors obtained from RP specimens (clinical stage T2; n = 110); primary, locally advanced tumors (clinical stage T3/4) treated by transurethral resection (n = 27); distant metastases collected from autopsies of patients who had died from end-stage metastatic prostate cancer (n = 35); and 108 local recurrences after hormonal therapy failure, including 65 transurethral resections from living patients and 43 specimens obtained from autopsies. Tumor grading on the original tissue sections was performed according to Gleason (11) . The array also included 54 cores from high grade PIN lesions; however, because of the focal nature of PIN, we verified the H&E staining of each sample on the array and identified only 20 as clearly showing high-grade PIN in the tissue core specimens on the array.

Statistical Methods.
Specimens were available from 30 radical prostatectomies. These specimens were assessed for Gleason score and NKX3.1 expression to determine whether NKX3.1 expression differed among specimens with at least one Gleason grade 4 compared with those with both grades <4. The prostate tissue samples available for tissue microarray analysis were ordered by increasing disease severity for the following classifications: BPH, PIN, T1 tumors, RP specimens, and T3/4 tumors. Specimens available from HR samples and metastatic disease represented more severe disease than the previously mentioned tissues, but their position in severity status relative to each other was unknown. Of primary interest was whether there is a decrease in NKX3.1 expression with increasing disease status. Two separate questions were addressed. The first was whether a trend in NKX3.1 expression is present with disease status BPH through T3/4 in the order listed above, with HR tumors as the most severe disease status. The second question was similar, except that metastatic disease rather than HR tumors was the most severe disease. Of additional interest was whether the combined group of T1_a/b and RP tissues differed from T3/4 and whether it differed from metastatic tumors. These questions were tested using a Jonkheere-Terpstra test as implemented in StatXact (Cytel). Unless specified below, all tests were considered significant if P was <0.05. To control for the two tests using HR or metastatic tumors as the sixth tissue type, the decrease in NKX3.1 expression was considered significant if the two-sided P was <0.025. Specific pairwise comparisons with BPH through T3/4 were performed for HR or metastatic tumors provided the overall test was significant. Similarly, the two comparisons of T1_a/b with either T3/4 or metastatic tissue were considered significant for P < 0.025.

	RESULTS

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Recombinant NKX3.1 was made as an MBP fusion protein in Escherichia coli. Cleavage of the fusion protein with genenase yielded electrophoretically pure NKX3.1 (Fig. 1A) . Antibody to MBP did not react with cleaved NKX3.1, indicating complete cleavage of NKX3.1 from the fusion protein (Fig. 1B) . Rabbit antiserum derived against purified recombinant NKX3.1 reacted only with NKX3.1 on Western blot and not with either E. coli proteins or MBP (Fig. 1C) . The antiserum recognized 32-kDa NKX3.1 in TSU-Pr1 cells transfected with an NKX3.1 expression plasmid, but detected no proteins in TSU-Pr1 cells because they express <1/100 the level of NKX3.1 mRNA found in LNCaP cells (Fig. 1D) .⁵ The induction of NKX3.1 mRNA by androgen treatment of LNCaP cells has been described and was reflected in the induction of NKX3.1 protein after R1881 treatment of LNCaP cells (Fig. 1D ; Refs. 1 , 12 ).

View larger version (19K): [in this window] [in a new window]   Fig. 1. Antibody to recombinant NKX3.1. A, Coomassie blue staining of samples from purification of recombinant protein. Lane 1, total E. coli protein extract prior to IPTG induction; Lane 2, total E. coli protein extract after IPTG induction, Lane 3, NKX3.1/MBP fusion protein from amylose column; Lane 4, DEAE-Sepharose-purified fusion protein cleaved with genenase I; Lane 5, DEAE-Sepharose-purified MBP after genenase I digestion of fusion protein; Lane 6, DEAE-Sepharose-purified NKX3.1 after genenase I digestion of fusion protein. B, Western blot of gel as in A, using anti-MBP. C, Western blot of gel as in A, using purified rabbit antibody to NKX3.1. D, Western blot with anti-NKX3.1. Contents of the lanes are labeled.

We detected expression of NKX3.1 in testis, confirming the results for mRNA expression (Fig. 2, A and B) . We also noted expression of NKX3.1 in rare mucous glands of the lung (Fig. 2, C and D) . Lastly, we found expression of NKX3.1 in groups of ureteral epithelial cells periodically spaced along the lumen of the ureter (Fig. 2, E and F) . Bladder transitional epithelium contained rare single cells with nuclear staining (not shown). We found no expression in tissues that contained blood cells, including bone marrow and spleen. Nonmalignant prostatic epithelial cells had uniformly positive nuclear staining for NKX3.1. This was seen in 61 RP specimens (Fig. 2, G and H) .

View larger version (91K): [in this window] [in a new window]   Fig. 2. Immunohistochemical staining of normal human tissues with anti-NKX3.1. Sections were cut in parallel and stained with either H&E (A, C, E, and G) or anti-NKX3.1 (B, D, F, and H). Images A–F were captured digitally using x400 microscopic magnification; G and H, were captured digitally using x63 magnification. A and B, normal testis seminiferous tubules. C and D, pulmonary mucous gland. E and F, ureteral transitional epithelium. G and H, prostate gland. IHC, immunohistochemistry.

Neoplastic prostate epithelium was found to display three different patterns of immunostaining for NKX3.1 expression. Many samples stained uniformly for NKX3.1. Some samples stained heterogeneously, with some malignant cells stained and adjacent cells not stained. Some samples displayed no staining for NKX3.1. In samples in which malignant cells did not express NKX3.1, adjacent normal epithelial cells were invariably positive, providing an internal control for the quality of the specimen. The patterns of staining are shown in Fig. 3 . For the purposes of analyzing the 477 microarray samples and the 30 sections, uniform staining was awarded a score of 2, heterogeneous staining a score of 1, and samples that did not stain were scored 0.

View larger version (80K): [in this window] [in a new window]   Fig. 3. Immunohistochemical staining of prostatic tissue and tissue microarrays with anti-NKX3.1. A, examples of prostate cancer specimens from tissue microarrays stained with H&E (left) and corresponding section stained with anti-NKX3.1 (right). Staining patterns observed were diffuse staining (2), heterogeneous staining (1), or no staining (0). All tissues were scored according to this scheme, and the data are summarized in Table 1 . B, parallel sections from a Gleason score 3 + 3 prostate cancer specimen stained with either H&E (left) or anti-NKX3.1 (right). Images were captured at x100. NKX3.1 expression was absent in the adenocarcinoma (upper left of slide) and present in hypertrophic glands (lower right of slide). IHC, immunohistochemistry.

The series of 30 RP specimens analyzed as conventional sections cut from paraffin blocks gave nearly identical results to the distribution of staining patterns found among the RP specimens in the tissue microarray. The microarray RP samples gave a nearly identical distribution of staining scores compared to these 30 paraffin block samples; therefore, the staining of conventional samples validated the tissue microarray data and confirmed that a fraction of early-stage prostate cancers lose expression of NKX3.1.

The 30 samples from paraffin blocks were subjected to histological grading (11) . In contrast to the relationship between loss of NKX3.1 staining and prostate tumor progression, we found no relationship between NKX3.1 staining scores and Gleason scores in the 30 RP blocks. The distribution of NKX3.1 staining results over the range of tumor grades as measured by Gleason score is shown in Table 2 . Samples were compared across all scores and compared as groups with at least one grade 4 versus both grades <4. There was no evidence indicating that patients with lower NKX3.1 expression had higher Gleason scores (P = 0.611; Ref. 13 ).

	DISCUSSION

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In the survey of 61 tissue sections, we found no example of nonmalignant prostatic glands failing to stain for NKX3.1. In the array, two BPH specimens displayed no staining. We cannot state at this time whether this represents a background of tissues that failed to stain for technical reasons or a subset of prostatic hyperplasia with true loss of NKX3.1 expression. Analysis of PIN samples revealed that more than half had reduced or absent NKX3.1 expression. Therefore, NKX3.1 may also play a role in the development of prostate cancer. A larger number of PIN lesions need to be analyzed to elucidate the role of NKX3.1 in prostate cancer development.

The finding that NKX3.1 expression was lost most often in metastases is consistent with the notion that metastatic disease is the most dedifferentiated state of prostate cancer. It may also be that NKX3.1 expression is under the control of prostate stromal cells. In murine tissue recombinants of neonatal epithelium and mesenchyme from the urogenital sinus, only tissues that underwent prostatic differentiation expressed Nkx3.1 (7) . On the other hand, the quality of the specimens, particularly from autopsies, could have resulted in sample degradation and diminished ability to detect NKX3.1. The mechanism of modulating NKX3.1 expression in human prostate cancer remains to be elucidated. It has not been determined whether NKX3.1 undergoes LOH in those tissues that display LOH at 8p21. Therefore, it is possible that loss of a single NKX3.1 allele as a result of LOH at 8p21 could down-regulate NKX3.1 expression. Because NKX3.1 is a differentiating protein, its expression may be regulated by gene methylation. We presently are characterizing the upstream sequences of NKX3.1 to identify regions that may be targets for gene silencing by methylation. Methylation is an important mechanism for loss of differentiated functions in human cancers such as diminished estrogen-receptor expression in breast cancer (15 , 16) . It remains to be determined whether promoter methylation plays a role in the down-regulation of NKX3.1 expression in prostate cancer.

The survey of NKX3.1 expression in normal tissues underscores the high degree of prostate specificity in the expression pattern of this protein. The role of NKX3.1 in the function of extraprostatic cells where it was found, bronchial mucous glands, testis, and ureter, is unknown. Nkx3.1 is not expressed in murine testis, and the Nkx3.1 (-/-) mice were fertile. There was no obvious ureteral or pulmonary pathology attributed to loss of Nkx3.1 in the gene-deleted mice (7) . If the only apparent action of NKX3.1 is as a prostate-specific repressor, the gene may have application in prostate-specific gene therapy. The potential for application of gene therapy to the treatment of prostate cancer is under active investigation. There may be advantages to the use of suppressor genes with limited tissue-specific effects to minimize toxicity of gene therapy to other organs. Whether ectopic expression of NKX3.1 in organs other than the prostate will have any functional ramifications remains to be shown. In addition, because of its tissue-specific expression in the adult, the NKX3.1 promoter is a potentially useful determinant for prostate-specific expression of exogenous genes. The probasin promoter has been quite useful in generating a murine prostate cancer model by driving organ-specific expression of the SV40 T antigen (17, 18, 19) . Whether the Nkx3.1 promoter will have similar effects remains to be shown. Early in murine development, Nkx3.1 expression occurs in many regions of the embryo and may play a noncritical role in the development of other organs (20 , 21) . Lastly, the NKX3.1 promoter may have applications in tissue-specific gene therapy of prostatic disease. The expression of NKX3.1 in other tissues shown in this report will help to identify potential organs for side effects of treatments targeted to the prostate by the NKX3.1 promoter.

	ACKNOWLEDGMENTS

 
The Lombardi Cancer Center Histopathology and Tissue Shared Resource provided normal human tissues. We are particularly grateful to Baljit Singh for assistance. Microscopy was done in the Lombardi Cancer Center Microscopy and Imaging Shared Resource.

	FOOTNOTES

 
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.

¹ This work was supported by grants from the NIH (CA78327) and the United States Army (DAMD17-98-1-8484) to E. P. G. and was assisted by Shared Resources of the Lombardi Cancer Center through a National Cancer Institute Cancer Center Support Grant. L. B. was supported by the Swiss National Science Foundation (81BS-052807) and the CaPCURE Foundation, and P. K. by the Academy of Finland and the Tampere University Hospital Foundation.

² The authors of this report made the following contributions: C. B. isolated and purified the recombinant protein, made the antibody, developed the immunohistochemical technique, and stained the histologic sections. L. B. helped develop the tissue microarray technology and constructed the prostate cancer tissue microarray. H. J. V. made the expression vectors for the recombinant fusion protein. R. S. performed the statistical analysis. E. E. L. did the Gleason scoring of histologic samples. N. W. supported tissue microarray construction and histologic review of specimens. G. S. contributed to the development of tissue microarray technology and coordinated the tissue collection and the microarray facility in Basel. T. C. G. helped to select the specimens for the array and provided important clinical information. P. K. collected specimens of hormone-refractory prostate cancer and supporting clinical information. J. K. and O-P. K. developed the tissue microarray technology for high-density arraying of the clinical prostate specimens. E. P. G. directed the research on NKX3.1, performed the review of NKX3.1 staining and scoring, analyzed the data, and prepared the figures.

³ To whom requests for reprints should be addressed, at Lombardi Cancer Center, Georgetown University, 3800 Reservoir Road, N.W., Washington, DC 20007-2007. Phone: (202) 687-2207; Fax: (202) 784-1229; E-mail: Gelmanne{at}gunet.georgetown.edu

⁴ The abbreviations used are: LOH, loss of heterozygosity; IPTG, isopropyl-1-thio-ß-D-galactopyranoside; MBP, maltose-binding protein; RP, radical prostatectomy; BPH, benign prostatic hyperplasia; PIN, prostatic intraepithelial neoplasia; HR, hormone-refractory.

⁵ H. J. Voeller and E. P. Gelmann, unpublished data.

⁶ H. J. Voeller and E. P. Gelmann, unpublished data.

Received 3/17/00. Accepted 9/ 1/00.

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				S. L. Zheng, J.-h. Ju, B.-l. Chang, E. Ortner, J. Sun, S. D. Isaacs, J. Sun, K. E. Wiley, W. Liu, M. Zemedkun, et al. Germ-Line Mutation of NKX3.1 Cosegregates with Hereditary Prostate Cancer and Alters the Homeodomain Structure and Function Cancer Res., January 1, 2006; 66(1): 69 - 77. [Abstract] [Full Text] [PDF]

				S. R. Payne and C. J. Kemp Tumor suppressor genetics Carcinogenesis, December 1, 2005; 26(12): 2031 - 2045. [Abstract] [Full Text] [PDF]

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				E. Asatiani, W.-X. Huang, A. Wang, E. Rodriguez Ortner, L. R. Cavalli, B. R. Haddad, and E. P. Gelmann Deletion, Methylation, and Expression of the NKX3.1 Suppressor Gene in Primary Human Prostate Cancer Cancer Res., February 15, 2005; 65(4): 1164 - 1173. [Abstract] [Full Text] [PDF]

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				H. Qi, C. Fillion, Y. Labrie, J. Grenier, A. Fournier, L. Berger, M. El-Alfy, and C. Labrie AIbZIP, a Novel bZIP Gene Located on Chromosome 1q21.3 That Is Highly Expressed in Prostate Tumors and of Which the Expression Is Up-Regulated by Androgens in LNCaP Human Prostate Cancer Cells Cancer Res., February 1, 2002; 62(3): 721 - 733. [Abstract] [Full Text] [PDF]

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M. Schoenberg Fejzo and D. J. Slamon Frozen Tumor Tissue Microarray Technology for Analysis of Tumor RNA, DNA, and Proteins Am. J. Pathol., November 1, 2001; 159(5): 1645 - 1650. [Abstract] [Full Text]