- Email: [email protected]
PDF in human prostate tumor cells
BBRC Biochemical and Biophysical Research Communications 305 (2003) 598–604 www.elsevier.com/locate/ybbrc
Dysregulated expression of MIC-1/PDF in human prostate tumor cells Dev Karan,a Siu-Ju Chen,a Sonny L. Johansson,b,c Ajay P. Singh,a Vishwas M. Paralkar,d Ming-Fong Lin,a,c,e and Surinder K. Batraa,b,c,* a
c
Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA b Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USA Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE USA d Pfizer Inc., Groton, CT, USA e Department of Surgery, Section of Urology, University of Nebraska Medical Center, Omaha, NE, USA Received 11 April 2003
Abstract As a part of the study to identify genes associated with hormone-refractory stage of human prostate cancer, we have recently identified several genetic and epigenetic changes that seem to be associated with the progression of androgen-sensitive to androgenindependent prostate tumor cells. In the present study, we report a novel gene, macrophage inhibitory cytokine-1 (MIC-1) also known as prostate derived factor (PDF), that was highly expressed in androgen-independent LNCaP-C81 cells and its metastatic variant LNCaP-Ln3 compared to androgen-sensitive LNCaP-C33 cells. The MIC-1/PDF expression was dysregulated (very low to non-detectable) in the androgen-independent PC3 and DU145 cells. Interestingly, serum factors demonstrated a differential regulation of MIC-1/PDF in the androgen-sensitive and the androgen-independent cells of LNCaP cells. Immunohistochemical analysis on 15 prostatic adenocarcinomas showed a weak staining in the benign prostatic glandular area (intensity score 2.38 0.25; n ¼ 13), while the immunoreactivity was significantly stronger (p < 0:05) in areas of adenocarcinoma (score 7.33 0.88; n ¼ 15). Altogether, these data suggest that the serum factors (including androgens and cytokines) might contribute to the regulation of the MIC-1/PDF gene that seems to be associated with the progression of prostate cancer. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: LNCaP cell model; MIC-1/PDF; Prostate cancer; Immunohistochemistry
Prostate cancer is the most prevalent malignancy among the male population of the United States. The lifetime risk for a man, in western society, to develop prostate cancer is about 30%, while the estimated deaths due to this disease are 10–11% [1]. Prostate cancer is initially localized and then progresses to an invasive, migratory, and metastatic disease. Localized prostate cancer is generally an androgen-dependent and is potentially curable by means of surgery or other modalities, such as radiation therapy [2]. When metastatic prostate cancer acquires androgen-independency, it becomes a life-threatening disease. Prostate cancer develops under the influence of androgens; therefore, the standard metastatic prostate cancer therapy is hormonal * Corresponding author. Fax: 1-402-559-6650. E-mail address: [email protected] (S.K. Batra).
manipulation after the patient has failed surgery or radiation therapy [3]. In about 70–80% cases, androgen deprivation therapy results in a dramatic regression of the androgen-responsive cancer cells but unfortunately, in the majority of patients the tumor ultimately becomes hormone-refractory, leading to a poor prognosis [4]. Our understanding of the underlying mechanisms that dictate prostate tumor progression and metastatic dissemination are still rudimentary. Identification of the differentially regulated genes during tumor progression from androgen-sensitive to androgen-independent (metastatic) stage may be an efficient way to evaluate their potential in the diagnosis and therapy of prostate cancer. A number of androgenresponsive genes have been identified using various strategies, and most recently, gene expression profiling of normal and cancerous specimens from prostate
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)00823-4
D. Karan et al. / Biochemical and Biophysical Research Communications 305 (2003) 598–604
cancer [5]. These studies have demonstrated that the proteins encoded by androgen-responsive genes are involved in a wide variety of cellular functions. Nonetheless, the biochemical and molecular mechanisms that control such type of cellular interaction during prostate organogenesis, morphogenesis, and functional differentiation remain undetermined. We have previously described several genetic and epigenetic changes that seem to be associated with the progression of androgen-sensitive to androgen-independent stage of prostate tumor [6–8], using in vitro LNCaP cell model [9,10]. From the cDNA microarray analysis, one of the identified genes, macrophage inhibitory cytokine-1 (MIC-1), exhibited a higher expression level in androgen-independent LNCaP-C81 cells as compared to androgen-sensitive LNCaP-C33 cells [7]. MIC-1 gene is also known with various names, such as prostate derived factor (PDF), placental transforming growth factor-b (PTGF-bÞ, placental bone morphogenetic protein (PLAB), growth differentiation factor-15 (GDF-15), and non-steroidal anti-inflammatory drugactivated gene (NAG-1). It is a divergent member of the TGF-b superfamily and was isolated from a differential screen for genes that were induced on macrophage activation [11]. The MIC-1/PDF gene is abundantly expressed in the placenta, while at low levels in prostate, liver, kidney, pancreas, and fetal brain [12]. Serum samples from patients with prostate cancer [13], colorectal cancer [14], pregnant women [15], and patients with cardiovascular disease [16] displayed an elevated level of the MIC-1/PDF protein, providing the significance of its association with the physiological processes of these conditions. Using in situ hybridization (ISH), the MIC-1/PDF gene was also shown to be aberrantly expressed in prostate tumors compared to normal prostate gland [17,18]. In the present study, we examined the expression of the MIC-1/PDF gene during progressive stages in prostate cancer cells. The secreted MIC-1/PDF protein was twoto three-fold higher in the androgen-independent LNCaP cells compared to hormone-sensitive early stage cancer cells. Additionally, serum factors demonstrated a differential regulation of MIC-1/PDF in the androgen-sensitive (C33) and the androgen-independent (C81) cells. Furthermore, a significantly higher (p < 0:05) amount of the MIC-1/PDF protein in prostatic adenocarcinoma as compared to benign prostatic glandular tissue was observed, suggesting the association of MIC-1/PDF gene with the progression and development of androgen-independent phenotype of prostate tumor cells.
Materials and methods Reagents. RPMI 1640 medium, FBS, penicillin–streptomycin, and trypsin were purchased from Mediatech (Herndon, VA). The charcoal/
599
dextran-treated FBS (c-FBS) was from Valley Biomedical (Winchester, VA). The random primers DNA labeling kit was from Invitrogen (Carlsbad, CA). All the reagents for reverse transcription-polymerase chain reaction (RT-PCR) were purchased from Fermentas (Hanover, MD). Protogel was purchased from the National Diagnostics. The protein molecular weight marker and the protein assay kit were obtained from Bio-Rad (Hercules, CA). Affinity purified anti-rabbit MIC-1/PDF antibody was described in a previous publication [12]. Polyclonal rabbit anti-human IGg and ECL reagents were purchased from Amersham Pharmacia Biotech (UK). All other required chemicals were purchased from Sigma (St. Louis, MO). Cell cultures and LNCaP cell model. Prostate tumor cell lines PC3, DU145, and PZ-HPV-7 were originally purchased from ATCC. The LNCaP cell model used in the present study was developed by Lin et al. [10] and was further characterized by Igawa et al. [9]. We utilized androgen-sensitive C33 and androgen-independent C81 cells of this LNCaP cell model. The LNCaP-Ln3 cells (obtained from Dr. Fidler, M.D. Anderson Cancer Center, TX) were generated by the repeated lymph node metastasis of the LNCaP cells grown orthotopically in the prostate gland of the athymic mice. The LNCaP-Ln3 cells showed low local aggressiveness, but significantly increased metastatic potential, less sensitive to androgen (in vitro and in vivo), secrete high levels of PSA, and have the ability to metastasize into the liver even after intrasplenic implantation [19]. Cells were maintained in RPMI 1640 medium supplemented with 5% FBS, 1% glutamine, and 1% penicillin– streptomycin, and were passaged weekly by trypsinization. Clinical samples. Human prostatic adenocarcinoma tissues were obtained from 15 patients. The tissue was fixed in 10% neutral formalin, embedded in paraffin and 5 lm sections were stained with hematoxylin and eosin for pathological evaluation. The combined Gleason grade and pathological stage was assessed by a Pathologist (Dr. Johansson) according to AJCC [20]. RNA isolation. Total RNA was extracted from PZ-HPV-7, LNCaP-C33, -C81, -Ln3, PC3, and DU145 cells by using guanidine isothiocyanate-cesium chloride ultracentrifugation method, as described previously [21]. RNA concentration was measured spectrophotometrically and its integrity was analyzed by electrophoresis on a formaldehyde agarose gel. Reverse transcription-polymerase chain reaction. Oligonucleotide primers for the MIC-1/PDF gene were synthesized from the published sequence (Accession No. AF019770). Total RNA (1 lg) from all the cell lines (PZ-HPV-7, LNCaP-C33, -C81, -Ln3, PC3, and DU145) was reverse transcribed using the SUPERSCRIPT II RNase H Reverse Transcriptase System. Samples were subjected to PCR amplification in a total reaction volume of 50 ll, containing 10 PCR buffer, 25 mM MgCl2, 10 mM dNTP, 5 pmol concentration of each primer, and 2.5 U of Taq DNA polymerase. The PCR was carried out in a programmable thermal controller (PTC-100, MJ Research, Watertown, MA). The reaction mixture was denatured at 94 °C for 3 min followed by 30 cycles at 94 °C for 45 s, annealing at 58 °C for 1 min, 72 °C for 1 min, and the final elongation was extended for an additional 15 min. The amplified PCR products were resolved electrophoretically on an agarose gel stained with ethidium bromide to verify size of the amplified product. PCR product was subcloned into pCR2.1 vector and the nucleotide sequence was verified. After digestion with restriction enzyme EcoRI, the insert was used to prepare the MIC-1/PDF probe for Northern analysis. Northern blot analysis. Total RNA (12 lg) was fractionated by gel electrophoresis on a 1.0% agarose gel, containing 0.66 M formaldehyde and transferred to nitrocellulose filter via capillary blotting. The cDNA probes were labeled with [32 P]dCTP using random primer labeling kit. Pre-hybridization and hybridization of the filter was carried out in a solution of 50% formamide, 5 SSPE, 5 DenhardtÕs reagent, 200 lg/ ml of sheared salmon sperm DNA, and a minimum of 106 cpm/ml of probe at 42 °C for 18 h. After hybridization, the filter was washed twice with 2 SSC and 1% SDS, and once with 0.5 SSC and 1% SDS solution at 55 °C for 15 min each. The filter was exposed to phosphor
600
D. Karan et al. / Biochemical and Biophysical Research Communications 305 (2003) 598–604
imaging screens and scanned with PhosphoImager (Molecular Dynamics, Sunnyvale, CA). Northern blot analysis for MIC-1/PDF gene was performed in triplicate from different sets of total RNA. Western blot analysis. Cells were plated at a density of 1 106 in T25 flask. After 48 h, the medium was replaced and further incubated for 48 h. Upon completion of the time period, the cultured media were collected and centrifuged (5000 rpm for 5 min at 4 °C) to remove the cell debris. Protein concentration was determined using the protein assay kit (Bio-Rad). About 30 lg of the secreted protein was resolved on SDS–PAGE (15%) and was transferred onto PVDF membrane (Millipore). After blocking with 5% non-fat dry milk in TBST (Trisbuffered saline containing 0.05% Tween 20) for 1 h, the blots were incubated for 3 h with polyclonal anti-human MIC-1/PDF antibody and then with corresponding secondary antibody in TBST for 1 h. All the incubations and washings were performed at room temperature. After several washes with TBST, the blots were incubated with ECL reagent, and were exposed to BioMax Film (Kodak), which was developed to analyze the specific band size. Immunocytometry. Tissue sections (5 lm) cut from the paraffin block were stained with the rabbit polyclonal anti-human MIC-1/PDF antibody for the MIC-1/PDF expression by immunohistochemical (IHC) technique with the appropriate positive and negative controls. The tumor tissue sections on the slide were deparaffinized using xylene and then graded alcohols. The tissue samples were washed three times with PBS and incubated with the diluted Vectastain Horse Normal Serum, (provided in the kit) in which the secondary antibody was raised, for 20 min to block the non-specific antigen–antibody immunoreactivity. The samples were incubated with 1:100 dilution of a stock solution of primary antibody at room temperature for 1 h. The IHC reaction was detected by using an ABC Elite Kit (Vector Laboratories) as per the manufacturerÕs instructions. A reddish-brown precipitate indicated positive immunoreactivity. The intensity of immunoreactivity of the MIC-1/PDF antigen was assessed by Dr. Johansson (pathologist). Staining intensity was graded on 0 to 3 scale i.e., 0 for no staining, 1+ for week immunoreactivity; 2+ for intermediate immunoreactivity; and 3+ for strong immunoreactivity. The extent of the staining scored as follows: <25% of tumor cells stained (1); 25–50% of the tumor cells stained positive (2); 50–75% of the tumor cells stained positive (3); and >75% of the tumor cells stained positive (4). Intensity and extent of staining scores were multiplied with the maximum score being 12.
Fig. 1. A representative of RT-PCR assay demonstrating the differential expression of MIC-1/PDF gene in a panel of prostatic tumor lines. PZ-HPV-7 (transformed epithelial cells from the normal prostate) and DU145 cells did not show any expression, while PC3 exhibits very low level of expression. GAPDH was used as an internal control.
pared to androgen-sensitive C33 cells. Further, the LNCaP-Ln3 cells exhibited an overexpression of the MIC-1/PDF mRNA transcript. The expression level was very weak in PC3 and non-detectable in PZ-HPV-7 and DU145 cell lines (Fig. 2). Western blot analysis, using an affinity-purified anti-human MIC-1/PDF antibody, showed a higher expression of the MIC-1/PDF protein in C81 cells, overexpression in LNCaP-Ln3 cells, and a very weak expression in PC3 cells, following a similar pattern
Results Differential expression of MIC-1/PDF mRNA and protein in human prostate cancer cell lines We used a panel of prostatic tumor cell lines to analyze the expression of the MIC-1/PDF gene both at the mRNA as well as the protein level. The expression level of the MIC-1/PDF gene in PZ-HPV-7, LNCaP-C33, -C81, -Ln3, PC3, and DU145 cells was evaluated by RTPCR. A specific band size of 936 bp for the MIC-1/PDF gene was observed in LNCaP-C33, -C81, and -Ln3 tumor cell lines (Fig. 1). PZ-HPV-7 (transformed epithelial) cells from the normal prostate gland and tumor cell line DU145 did not show the MIC-1/PDF gene expression, while a very low level of expression was detected in the PC3 cells. A similar observation was obtained using northern blot analysis confirming the differential expression of the MIC-1/PDF gene, which was significantly higher in androgen-independent C81 cells com-
Fig. 2. (A) Representative of Northern blot analysis showing the expression profile of MIC-1/PDF mRNA in a panel of human prostate cancer cell lines. To normalize the amount of total RNA in each sample, filter was re-hybridized with GAPDH probe as an internal control. (B) The bar diagram represents the intensity of the MIC-1/ PDF specific signal adjusted to that of GAPDH for each tested sample. Error bars indicate standard error from the triplicate values.
D. Karan et al. / Biochemical and Biophysical Research Communications 305 (2003) 598–604
601
Expression of the MIC-1/PDF under different culture conditions
Fig. 3. Expression profile of MIC-1/PDF secretion in different cell lines of human prostate cancer. The cells were seeded at a density of 1 106 in T-25 flasks in regular culture medium for 2 days. The media were replaced and the cells were further incubated for 48 h. At the given time point, cells were harvested and conditioned media were collected. An equal amount of proteins from the conditioned media from each cell line was fractionated by 15% SDS–PAGE and transferred onto the PVDF membrane. The blot was hybridized with anti-MIC-1/PDF antibody (upper panel) and then stained with Coomassie blue (lower panel) to demonstrate equal loading of proteins in all lanes.
obtained by RT-PCR and Northern blot analysis (Fig. 3). Altogether, these experiments showed a dysregulated expression pattern of the MIC-1/PDF level with the progression of prostate cancer.
To further analyze the expression pattern of the MIC1/PDF protein, we used three different culture conditions [serum free, 5% charcoal treated-FBS (c-FBS), and 5% FBS culture medium]. In androgen-sensitive LNCaP-C33 cells, the secreted level of MIC-1/PDF protein was significantly higher in the conditioned medium obtained from the cells maintained with 5% FBS compared to the media from the cells containing 5% cFBS and serum free medium. On the other hand, in androgen-independent LNCaP-C81 cells, both the culture media containing 5% c-FBS and 5% FBS showed a higher expression of the secreted MIC-1/PDF protein as compared to the cells maintained in serum free medium, however, the secretion level was maximum in case of 5% FBS (Fig. 4). Distribution of the MIC-1/PDF protein in human tumor tissues The MIC-1/PDF expression was studied in 15 specimens of human prostatic adenocarcinomas by IHC. The staining pattern and intensity was different in the areas
Table 1 Level of expression of the MIC-1/PDF antigen by immunoperoxidase staining in benign and adenocarcinomatous prostatic tissues Fig. 4. Effect of culture conditions on the expression levels of the secreted MIC-1/PDF protein in androgen-sensitive (LNCaP-C33) and -independent (LNCaP-C81) cells. The cells were seeded for 2 days as detailed in Fig. 3. Cells were then washed and fed with fresh media, serum free, 5% c-FBS or 5% FBS. After 48 h, cells were harvested and an equal amount of proteins from supernatant was fractionated on 15% SDS–PAGE and transferred onto the PVDF membrane for blotting with anti-MIC-1/PDF antibody.
Type of lesion
n
Benign prostatic glandular tissue Prostatic adenocarcinoma
13
Staining score (m SE) 9 2.38 0.25 > =
15
> 7.33 0.88 ;
t test
p < 0:05
n, number of specimens; m, mean; and SE, standard error. * Significant level (<0.05) with t test.
Fig. 5. A representative immunohistochemical analysis of the paraffin-embedded tissue sections of the benign prostatic glandular tissue (A) and prostatic adenocarcinoma (B).
602
D. Karan et al. / Biochemical and Biophysical Research Communications 305 (2003) 598–604
with benign prostatic glandular tissue and prostatic adenocarcinomas (Table 1). For simplicity, we calculated the intensity score by multiplying the intensity of staining with the extent of staining for a particular lesion. Out of 15 specimens, 13 showed low to moderate immunoreactivity in the areas of benign prostatic glandular tissue with an overall intensity score of 2.38 0.25. We could not find any benign tissue area in two samples. On the other hand, a strong immunoreactivity was detected for the MIC-1/PDF antigen in prostate cancer areas in all the specimens with an intensity score of 7.33 0.88. Overall, immunoreactivity for the MIC-1/PDF antigen was significantly higher (p < 0:05) in prostatic adenocarcinomas as compared to benign prostatic glandular tissue (Fig. 5). Since prostate cancer is multifocal and heterogeneous, there was a variability in the intensity of staining.
Discussion Development of an androgen-independent phenotype from androgen-sensitive cancer is a major challenge in the treatment of prostate cancer. The treatment and diagnosis may be improved significantly by the identification and characterization of the molecules that are regulated differentially during the development of the metastatic hormone-refractory phenotype. Recently, by using a step-wise progression of in vitro LNCaP cell model for prostate cancer that exhibits the conditions analogous to be seen in the patients, we identified through microarray gene filter that the MIC-1/PDF mRNA transcript is highly expressed in androgen-independent LNCaP-C81 cells [7]. The MIC-1/PDF gene, located on chromosomal position 19p13.1, is a divergent member of the TGF-b superfamily. It shares only a 25% overall sequence identity to the TGF-b family members, but shows the typical characteristics of the TGF-b superfamily including, signal peptide, consensus cleavage signal for the processing of mature protein, and a cysteine knot of seven residues at the carboxyl terminal. It is synthesized as a 62-kDa intracellular protein, but secreted as a 25kDa disulfide-linked dimeric protein after cleavage [22]. We investigated the expression of MIC-1/PDF both at the mRNA as well as the protein level in a panel of prostatic tumor cell lines. The MIC-1/PDF gene expression was low to non-detectable in the areas of benign prostatic glandular tissue and the transformed epithelial cells of the normal prostate (immortal PZHPV-7 cells), whereas it was overexpressed in tumor areas and in androgen-independent cells (LNCaP-C81 and LNCaP-Ln3). Further, low to undetectable levels of MIC-1/PDF in PC3 and DU145 cells compared to LNCaP cells might be associated with the differential expression of androgen receptor (AR). It is known that
the LNCaP cells are positive for AR, whereas the PC3 cells demonstrated a very low level of AR and DU 145 cells have no detectable AR [23,24]. These results indicated that at the hormone-refractory stage of prostate tumor AR might contribute to the dysregulation of MIC-1/PDF. Hence, the elevated level of mRNA as well as protein in a metastatic variant cell line suggests that the MIC-1/PDF gene may play a crucial role in the development of androgen-independent phenotype of human prostate tumor. However, further experimental studies are required to elucidate the pathological significance of higher secretion of the MIC-1/PDF protein in association with tumor development as well as its dysregulation in conjunction with androgen receptor. Androgens are critical for the normal development and differentiation of the prostate gland [3]. Most of the biological effects of androgens are mediated via AR, a nuclear transcription factor, upon ligand binding influence the transcription of androgen-responsive genes [25–27]. It is interesting to note that the treatment of androgen-sensitive (C33) and -independent (C81) cells with different culture conditions [serum free, steroid reduced 5% FBS (c-FBS), and 5% FBS in RPMI 1640 phenol red-free medium] demonstrated the regulation of MIC-1/PDF protein secretion by serum factors, including androgens. Feeding the C33 cells with 5% FBS exhibited higher secretion of the MIC-1/PDF protein compared to the cells treated with serum-free media or with 5% c-FBS, suggesting its regulation by steroids including androgens at the androgen-sensitive stage of the tumor. On the other hand, induction of the MIC-1/ PDF protein in C81 cells either treated with 5% FBS or 5% c-FBS indicated that its secretion might be regulated by growth factors and/or cytokines at the advanced stage of prostate cancer. Therefore, it would be of further interest to analyze the hormonal regulation of MIC-1/PDF in human prostate tumor to better understand its involvement with the hormone-refractory phenotype. A study in rat model has revealed that this gene is highly regulated by androgens, as orchitectomy resulted in a remarkable decrease in prostate size along with decrease in MIC-1/PDF expression level compared to those of control animals [12]. Its prostatic expression and regulation suggest that this gene may play a decisive role for the androgen-driven differentiation of prostatic tissue. A significantly higher expression of MIC-1/PDF in prostate tumor specimens indicated its involvement with the advancement of the disease. However, it was not possible to correlate the MIC-1/PDF immunostaining pattern with the tumor grade (Gleason grade) because (1) most of the specimens were of high grade (7–9 Gleason score) and (2) the number of specimens was too small for such a correlative analysis. We observed an enhanced pattern of MIC-1/PDF expression, however, with the progression of prostate tumor. Furthermore, a
D. Karan et al. / Biochemical and Biophysical Research Communications 305 (2003) 598–604
study on paired samples (four paired samples) from human prostate cancer specimens, using RT-PCR analysis, showed higher expression of the MIC-1/PDF mRNA in cancerous tissues compared to normal samples (data not shown). In conclusion, MIC-1/PDF showed a higher expression with the progression of prostate tumor both in the archival specimens and in a progressive in vitro LNCaP cell model. Our data suggest that MIC-1/PDF could be of specific importance in prostate cancer due to (1) its association with the progression of prostate tumor, (2) its regulation by steroids, including androgens at the androgen-sensitive stage and by cytokines and/or growth factors at the advanced stage correlating with the conditions in the cancer patients, and (3) its dysregulation at the hormone-refractory stage, following the expression pattern of androgen receptor. In addition, MIC-1/PDF has structural motifs similar to a TGF-b superfamily member that supports the hypothesis for its role in tumorigenesis. TGF-b is known to suppress immune surveillance, facilitate tumor invasion, and promote the development of metastases when abundantly expressed in tumors of epithelial origin [28]. Further studies are needed, however, to confirm its biological significance in association with carcinogenesis and tumor development, and whether MIC-1/PDF can serve as an useful biomarker in an androgen-independent phenotype of prostate cancer.
Acknowledgments This work was supported, in part, by grants from the National Cancer Institute (CA 78590, CA 88184), the Nebraska Health Department/Eppley Cancer Center LB 595, the Cattleman Ball Association, and the Nebraska Research Initiative Program. We acknowledge the NCI Cancer Center Support Grant (P30 CA36727) to UNMC for tumor tissues and CHTN Western Division, Case Western Reserve University, Cleveland, OH, for providing prostatic tissues. We also thank Dr. Fidler, TX, for the LNCaP-Ln3 cells, Mr. Erik Moore and Ms. Fen-Fen Lin for their technical assistance.
References [1] A. Jemal, A. Thomas, T. Murray, M. Thun, Cancer statistics 2002, CA Cancer J. Clin. 52 (2002) 23–47. [2] M.D. Sadar, M. Hussain, N. Bruchovsky, Prostate cancer: molecular biology of early progression to androgen independence, Endocr. Relat. Cancer 6 (1999) 487–502. [3] G.R. Cunha, A.A. Donjacour, P.S. Cooke, S. Mee, R.M. Bigsby, S.J. Higgins, Y. Sugimura, The endocrinology and developmental biology of the prostate, Endocr. Rev. 8 (1987) 338–362. [4] R.F. Gittes, Carcinoma of the prostate, N. Engl. J. Med. 324 (1991) 236–245. [5] H. Qi, Y. Labrie, J. Grenier, A. Fournier, C. Fillion, C. Labrie, Androgens induce expression of SPAK, a STE20/SPS1-related kinase, in LNCaP human prostate cancer cells, Mol. Cell Endocrinol. 182 (2001) 181–192.
603
[6] D. Karan, B.M. Schmied, B.J. Dave, U.A. Wittel, M.F. Lin, S.K. Batra, Decreased androgen-responsive growth of human prostate cancer is associated with increased genetic alterations, Clin. Cancer Res. 7 (2001) 3472–3480. [7] D. Karan, D.L. Kelly, A. Rizzino, M.F. Lin, S.K. Batra, Expression profile of differentially-regulated genes during progression of androgen-independent growth in human prostate cancer cells, Carcinogenesis 23 (2002) 967–975. [8] D. Karan, M.F. Lin, S.L. Johansson, S.K. Batra, Current status of the molecular genetics of human prostatic adenocarcinomas, Int. J. Cancer 103 (2003) 285–293. [9] T. Igawa, F.F. Lin, M.S. Lee, D. Karan, S.K. Batra, M.F. Lin, Establishment and characterization of androgen-independent human prostate cancer LNCaP cell model, Prostate 50 (2002) 222–235. [10] M.F. Lin, T.C. Meng, P.S. Rao, C. Chang, A.H. Schonthal, F.F. Lin, Expression of human prostatic acid phosphatase correlates with androgen-stimulated cell proliferation in prostate cancer cell lines, J. Biol. Chem. 273 (1998) 5939–5947. [11] M.R. Bootcov, A.R. Bauskin, S.M. Valenzuela, A.G. Moore, M. Bansal, X.Y. He, H.P. Zhang, M. Donnellan, S. Mahler, K. Pryor, B.J. Walsh, R.C. Nicholson, W.D. Fairlie, S.B. Por, J.M. Robbins, S.N. Breit, MIC-1, a novel macrophage inhibitory cytokine, is a divergent member of the TGF-b superfamily, Proc. Natl. Acad. Sci. USA 94 (1997) 11514–11519. [12] V.M. Paralkar, A.L. Vail, W.A. Grasser, T.A. Brown, H. Xu, S. Vukicevic, H.Z. Ke, H. Qi, T.A. Owen, D.D. Thompson, Cloning and characterization of a novel member of the transforming growth factor-b/bone morphogenetic protein family, J. Biol. Chem. 273 (1998) 13760–13767. [13] J.B. Welsh, L.M. Sapinoso, S.G. Kern, D.A. Brown, T. Liu, A.R. Bauskin, R.L. Ward, N.J. Hawkins, D.I. Quinn, P.J. Russell, R.L. Sutherland, S.N. Breit, C.A. Moskaluk, H.F. Frierson Jr., G.M. Hampton, Large-scale delineation of secreted protein biomarkers overexpressed in cancer tissue and serum, Proc. Natl. Acad. Sci. USA 100 (2003) 3410–3415. [14] P. Buckhaults, C. Rago, B. St Croix, K.E. Romans, S. Saha, L. Zhang, B. Vogelstein, K.W. Kinzler, Secreted and cell surface genes expressed in benign and malignant colorectal tumors, Cancer Res. 61 (2001) 6996–7001. [15] A.G. Moore, D.A. Brown, W.D. Fairlie, A.R. Bauskin, P.K. Brown, M.L. Munier, P.K. Russell, L.A. Salamonsen, E.M. Wallace, S.N. Breit, The transforming growth factor-b superfamily cytokine macrophage inhibitory cytokine-1 is present in high concentrations in the serum of pregnant women, J. Clin. Endocrinol. Metab. 85 (2000) 4781–4788. [16] D.A. Brown, S.N. Breit, J. Buring, W.D. Fairlie, A.R. Bauskin, T. Liu, P.M. Ridker, Concentration in plasma of macrophage inhibitory cytokine-1 and risk of cardiovascular events in women: a nested case-control study, Lancet 359 (2002) 2159–2163. [17] R. Thomas, L.D. True, P.H. Lange, R.L. Vessella, Placental bone morphogenetic protein (PLAB) gene expression in normal, premalignant and malignant human prostate: relation to tumor development and progression, Int. J. Cancer 93 (2001) 47–52. [18] J.B. Welsh, L.M. Sapinoso, A.I. Su, S.G. Kern, J. Wang-Rodriguez, C.A. Moskaluk, H.F. Frierson Jr., G.M. Hampton, Analysis of gene expression identifies candidate markers and pharmacological targets in prostate cancer, Cancer Res. 61 (2001) 5974–5978. [19] C.A. Pettaway, S. Pathak, G. Greene, E. Ramirez, M.R. Wilson, J.J. Killion, I.J. Fidler, Selection of highly metastatic variants of different human prostatic carcinomas using orthotopic implantation in nude mice, Clin. Cancer Res. 2 (1996) 1627–1636. [20] I.D. Fleming, J.S. Cooper, D.E. Henson, R.V.P. Hutter, B.J. Kennedy, G.P. Murphy, B. OÕSullivan, L.H. Sobin, J.W. Yarbro, in: AJCC Cancer Staining Manual, fifth edition, Lippincot Williams and Wilkins, Philadelphia, 1997, pp. 1–294. [21] S.K. Batra, H.F. Kern, A.J. Worlock, R.S. Metzgar, M.A. Hollingsworth, Transfection of the human Muc 1 mucin gene into
604
D. Karan et al. / Biochemical and Biophysical Research Communications 305 (2003) 598–604
a poorly differentiated human pancreatic tumor cell line, Panc1: integration, expression and ultrastructural changes, J. Cell Sci. 100 (1991) 841–849. [22] W.D. Fairlie, A.G. Moore, A.R. Bauskin, P.K. Russell, H.P. Zhang, S.N. Breit, MIC-1 is a novel TGF-rbeta superfamily cytokine associated with macrophage activation, J. Leukoc. Biol. 65 (1999) 2–5. [23] R. Garcia-Arenas, F.F. Lin, D. Lin, L.P. Jin, C.C. Shih, C. Chang, M.F. Lin, The expression of prostatic acid phosphatase is transcriptionally regulated in human prostate carcinoma cells, Mol. Cell Endocrinol. 111 (1995) 29–37. [24] D.L. Lin, M.C. Whitney, Z. Yao, E.T. Keller, Interleukin-6 induces androgen responsiveness in prostate cancer cells through
[25]
[26]
[27]
[28]
up-regulation of androgen receptor expression, Clin. Cancer Res. 7 (2001) 1773–1781. Z. Culig, H. Klocker, G. Bartsch, A. Hobisch, Androgen receptors in prostate cancer, Endocr. Relat. Cancer 9 (2002) 155–170. B.J. Feldman, D. Feldman, The development of androgenindependent prostate cancer, Nat. Rev. Cancer 1 (2001) 34–45. M.E. Grossmann, H. Huang, D.J. Tindall, Androgen receptor signaling in androgen-refractory prostate cancer, J. Natl. Cancer Inst. 93 (2001) 1687–1697. E.R. Barrack, TGF b in prostate cancer: a growth inhibitor that can enhance tumorigenicity, Prostate 31 (1997) 61–70.
Dysregulated expression of MIC-1/PDF in human prostate tumor cells Dev Karan,a Siu-Ju Chen,a Sonny L. Johansson,b,c Ajay P. Singh,a Vishwas M. Paralkar,d Ming-Fong Lin,a,c,e and Surinder K. Batraa,b,c,* a
c
Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA b Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USA Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE USA d Pfizer Inc., Groton, CT, USA e Department of Surgery, Section of Urology, University of Nebraska Medical Center, Omaha, NE, USA Received 11 April 2003
Abstract As a part of the study to identify genes associated with hormone-refractory stage of human prostate cancer, we have recently identified several genetic and epigenetic changes that seem to be associated with the progression of androgen-sensitive to androgenindependent prostate tumor cells. In the present study, we report a novel gene, macrophage inhibitory cytokine-1 (MIC-1) also known as prostate derived factor (PDF), that was highly expressed in androgen-independent LNCaP-C81 cells and its metastatic variant LNCaP-Ln3 compared to androgen-sensitive LNCaP-C33 cells. The MIC-1/PDF expression was dysregulated (very low to non-detectable) in the androgen-independent PC3 and DU145 cells. Interestingly, serum factors demonstrated a differential regulation of MIC-1/PDF in the androgen-sensitive and the androgen-independent cells of LNCaP cells. Immunohistochemical analysis on 15 prostatic adenocarcinomas showed a weak staining in the benign prostatic glandular area (intensity score 2.38 0.25; n ¼ 13), while the immunoreactivity was significantly stronger (p < 0:05) in areas of adenocarcinoma (score 7.33 0.88; n ¼ 15). Altogether, these data suggest that the serum factors (including androgens and cytokines) might contribute to the regulation of the MIC-1/PDF gene that seems to be associated with the progression of prostate cancer. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: LNCaP cell model; MIC-1/PDF; Prostate cancer; Immunohistochemistry
Prostate cancer is the most prevalent malignancy among the male population of the United States. The lifetime risk for a man, in western society, to develop prostate cancer is about 30%, while the estimated deaths due to this disease are 10–11% [1]. Prostate cancer is initially localized and then progresses to an invasive, migratory, and metastatic disease. Localized prostate cancer is generally an androgen-dependent and is potentially curable by means of surgery or other modalities, such as radiation therapy [2]. When metastatic prostate cancer acquires androgen-independency, it becomes a life-threatening disease. Prostate cancer develops under the influence of androgens; therefore, the standard metastatic prostate cancer therapy is hormonal * Corresponding author. Fax: 1-402-559-6650. E-mail address: [email protected] (S.K. Batra).
manipulation after the patient has failed surgery or radiation therapy [3]. In about 70–80% cases, androgen deprivation therapy results in a dramatic regression of the androgen-responsive cancer cells but unfortunately, in the majority of patients the tumor ultimately becomes hormone-refractory, leading to a poor prognosis [4]. Our understanding of the underlying mechanisms that dictate prostate tumor progression and metastatic dissemination are still rudimentary. Identification of the differentially regulated genes during tumor progression from androgen-sensitive to androgen-independent (metastatic) stage may be an efficient way to evaluate their potential in the diagnosis and therapy of prostate cancer. A number of androgenresponsive genes have been identified using various strategies, and most recently, gene expression profiling of normal and cancerous specimens from prostate
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)00823-4
D. Karan et al. / Biochemical and Biophysical Research Communications 305 (2003) 598–604
cancer [5]. These studies have demonstrated that the proteins encoded by androgen-responsive genes are involved in a wide variety of cellular functions. Nonetheless, the biochemical and molecular mechanisms that control such type of cellular interaction during prostate organogenesis, morphogenesis, and functional differentiation remain undetermined. We have previously described several genetic and epigenetic changes that seem to be associated with the progression of androgen-sensitive to androgen-independent stage of prostate tumor [6–8], using in vitro LNCaP cell model [9,10]. From the cDNA microarray analysis, one of the identified genes, macrophage inhibitory cytokine-1 (MIC-1), exhibited a higher expression level in androgen-independent LNCaP-C81 cells as compared to androgen-sensitive LNCaP-C33 cells [7]. MIC-1 gene is also known with various names, such as prostate derived factor (PDF), placental transforming growth factor-b (PTGF-bÞ, placental bone morphogenetic protein (PLAB), growth differentiation factor-15 (GDF-15), and non-steroidal anti-inflammatory drugactivated gene (NAG-1). It is a divergent member of the TGF-b superfamily and was isolated from a differential screen for genes that were induced on macrophage activation [11]. The MIC-1/PDF gene is abundantly expressed in the placenta, while at low levels in prostate, liver, kidney, pancreas, and fetal brain [12]. Serum samples from patients with prostate cancer [13], colorectal cancer [14], pregnant women [15], and patients with cardiovascular disease [16] displayed an elevated level of the MIC-1/PDF protein, providing the significance of its association with the physiological processes of these conditions. Using in situ hybridization (ISH), the MIC-1/PDF gene was also shown to be aberrantly expressed in prostate tumors compared to normal prostate gland [17,18]. In the present study, we examined the expression of the MIC-1/PDF gene during progressive stages in prostate cancer cells. The secreted MIC-1/PDF protein was twoto three-fold higher in the androgen-independent LNCaP cells compared to hormone-sensitive early stage cancer cells. Additionally, serum factors demonstrated a differential regulation of MIC-1/PDF in the androgen-sensitive (C33) and the androgen-independent (C81) cells. Furthermore, a significantly higher (p < 0:05) amount of the MIC-1/PDF protein in prostatic adenocarcinoma as compared to benign prostatic glandular tissue was observed, suggesting the association of MIC-1/PDF gene with the progression and development of androgen-independent phenotype of prostate tumor cells.
Materials and methods Reagents. RPMI 1640 medium, FBS, penicillin–streptomycin, and trypsin were purchased from Mediatech (Herndon, VA). The charcoal/
599
dextran-treated FBS (c-FBS) was from Valley Biomedical (Winchester, VA). The random primers DNA labeling kit was from Invitrogen (Carlsbad, CA). All the reagents for reverse transcription-polymerase chain reaction (RT-PCR) were purchased from Fermentas (Hanover, MD). Protogel was purchased from the National Diagnostics. The protein molecular weight marker and the protein assay kit were obtained from Bio-Rad (Hercules, CA). Affinity purified anti-rabbit MIC-1/PDF antibody was described in a previous publication [12]. Polyclonal rabbit anti-human IGg and ECL reagents were purchased from Amersham Pharmacia Biotech (UK). All other required chemicals were purchased from Sigma (St. Louis, MO). Cell cultures and LNCaP cell model. Prostate tumor cell lines PC3, DU145, and PZ-HPV-7 were originally purchased from ATCC. The LNCaP cell model used in the present study was developed by Lin et al. [10] and was further characterized by Igawa et al. [9]. We utilized androgen-sensitive C33 and androgen-independent C81 cells of this LNCaP cell model. The LNCaP-Ln3 cells (obtained from Dr. Fidler, M.D. Anderson Cancer Center, TX) were generated by the repeated lymph node metastasis of the LNCaP cells grown orthotopically in the prostate gland of the athymic mice. The LNCaP-Ln3 cells showed low local aggressiveness, but significantly increased metastatic potential, less sensitive to androgen (in vitro and in vivo), secrete high levels of PSA, and have the ability to metastasize into the liver even after intrasplenic implantation [19]. Cells were maintained in RPMI 1640 medium supplemented with 5% FBS, 1% glutamine, and 1% penicillin– streptomycin, and were passaged weekly by trypsinization. Clinical samples. Human prostatic adenocarcinoma tissues were obtained from 15 patients. The tissue was fixed in 10% neutral formalin, embedded in paraffin and 5 lm sections were stained with hematoxylin and eosin for pathological evaluation. The combined Gleason grade and pathological stage was assessed by a Pathologist (Dr. Johansson) according to AJCC [20]. RNA isolation. Total RNA was extracted from PZ-HPV-7, LNCaP-C33, -C81, -Ln3, PC3, and DU145 cells by using guanidine isothiocyanate-cesium chloride ultracentrifugation method, as described previously [21]. RNA concentration was measured spectrophotometrically and its integrity was analyzed by electrophoresis on a formaldehyde agarose gel. Reverse transcription-polymerase chain reaction. Oligonucleotide primers for the MIC-1/PDF gene were synthesized from the published sequence (Accession No. AF019770). Total RNA (1 lg) from all the cell lines (PZ-HPV-7, LNCaP-C33, -C81, -Ln3, PC3, and DU145) was reverse transcribed using the SUPERSCRIPT II RNase H Reverse Transcriptase System. Samples were subjected to PCR amplification in a total reaction volume of 50 ll, containing 10 PCR buffer, 25 mM MgCl2, 10 mM dNTP, 5 pmol concentration of each primer, and 2.5 U of Taq DNA polymerase. The PCR was carried out in a programmable thermal controller (PTC-100, MJ Research, Watertown, MA). The reaction mixture was denatured at 94 °C for 3 min followed by 30 cycles at 94 °C for 45 s, annealing at 58 °C for 1 min, 72 °C for 1 min, and the final elongation was extended for an additional 15 min. The amplified PCR products were resolved electrophoretically on an agarose gel stained with ethidium bromide to verify size of the amplified product. PCR product was subcloned into pCR2.1 vector and the nucleotide sequence was verified. After digestion with restriction enzyme EcoRI, the insert was used to prepare the MIC-1/PDF probe for Northern analysis. Northern blot analysis. Total RNA (12 lg) was fractionated by gel electrophoresis on a 1.0% agarose gel, containing 0.66 M formaldehyde and transferred to nitrocellulose filter via capillary blotting. The cDNA probes were labeled with [32 P]dCTP using random primer labeling kit. Pre-hybridization and hybridization of the filter was carried out in a solution of 50% formamide, 5 SSPE, 5 DenhardtÕs reagent, 200 lg/ ml of sheared salmon sperm DNA, and a minimum of 106 cpm/ml of probe at 42 °C for 18 h. After hybridization, the filter was washed twice with 2 SSC and 1% SDS, and once with 0.5 SSC and 1% SDS solution at 55 °C for 15 min each. The filter was exposed to phosphor
600
D. Karan et al. / Biochemical and Biophysical Research Communications 305 (2003) 598–604
imaging screens and scanned with PhosphoImager (Molecular Dynamics, Sunnyvale, CA). Northern blot analysis for MIC-1/PDF gene was performed in triplicate from different sets of total RNA. Western blot analysis. Cells were plated at a density of 1 106 in T25 flask. After 48 h, the medium was replaced and further incubated for 48 h. Upon completion of the time period, the cultured media were collected and centrifuged (5000 rpm for 5 min at 4 °C) to remove the cell debris. Protein concentration was determined using the protein assay kit (Bio-Rad). About 30 lg of the secreted protein was resolved on SDS–PAGE (15%) and was transferred onto PVDF membrane (Millipore). After blocking with 5% non-fat dry milk in TBST (Trisbuffered saline containing 0.05% Tween 20) for 1 h, the blots were incubated for 3 h with polyclonal anti-human MIC-1/PDF antibody and then with corresponding secondary antibody in TBST for 1 h. All the incubations and washings were performed at room temperature. After several washes with TBST, the blots were incubated with ECL reagent, and were exposed to BioMax Film (Kodak), which was developed to analyze the specific band size. Immunocytometry. Tissue sections (5 lm) cut from the paraffin block were stained with the rabbit polyclonal anti-human MIC-1/PDF antibody for the MIC-1/PDF expression by immunohistochemical (IHC) technique with the appropriate positive and negative controls. The tumor tissue sections on the slide were deparaffinized using xylene and then graded alcohols. The tissue samples were washed three times with PBS and incubated with the diluted Vectastain Horse Normal Serum, (provided in the kit) in which the secondary antibody was raised, for 20 min to block the non-specific antigen–antibody immunoreactivity. The samples were incubated with 1:100 dilution of a stock solution of primary antibody at room temperature for 1 h. The IHC reaction was detected by using an ABC Elite Kit (Vector Laboratories) as per the manufacturerÕs instructions. A reddish-brown precipitate indicated positive immunoreactivity. The intensity of immunoreactivity of the MIC-1/PDF antigen was assessed by Dr. Johansson (pathologist). Staining intensity was graded on 0 to 3 scale i.e., 0 for no staining, 1+ for week immunoreactivity; 2+ for intermediate immunoreactivity; and 3+ for strong immunoreactivity. The extent of the staining scored as follows: <25% of tumor cells stained (1); 25–50% of the tumor cells stained positive (2); 50–75% of the tumor cells stained positive (3); and >75% of the tumor cells stained positive (4). Intensity and extent of staining scores were multiplied with the maximum score being 12.
Fig. 1. A representative of RT-PCR assay demonstrating the differential expression of MIC-1/PDF gene in a panel of prostatic tumor lines. PZ-HPV-7 (transformed epithelial cells from the normal prostate) and DU145 cells did not show any expression, while PC3 exhibits very low level of expression. GAPDH was used as an internal control.
pared to androgen-sensitive C33 cells. Further, the LNCaP-Ln3 cells exhibited an overexpression of the MIC-1/PDF mRNA transcript. The expression level was very weak in PC3 and non-detectable in PZ-HPV-7 and DU145 cell lines (Fig. 2). Western blot analysis, using an affinity-purified anti-human MIC-1/PDF antibody, showed a higher expression of the MIC-1/PDF protein in C81 cells, overexpression in LNCaP-Ln3 cells, and a very weak expression in PC3 cells, following a similar pattern
Results Differential expression of MIC-1/PDF mRNA and protein in human prostate cancer cell lines We used a panel of prostatic tumor cell lines to analyze the expression of the MIC-1/PDF gene both at the mRNA as well as the protein level. The expression level of the MIC-1/PDF gene in PZ-HPV-7, LNCaP-C33, -C81, -Ln3, PC3, and DU145 cells was evaluated by RTPCR. A specific band size of 936 bp for the MIC-1/PDF gene was observed in LNCaP-C33, -C81, and -Ln3 tumor cell lines (Fig. 1). PZ-HPV-7 (transformed epithelial) cells from the normal prostate gland and tumor cell line DU145 did not show the MIC-1/PDF gene expression, while a very low level of expression was detected in the PC3 cells. A similar observation was obtained using northern blot analysis confirming the differential expression of the MIC-1/PDF gene, which was significantly higher in androgen-independent C81 cells com-
Fig. 2. (A) Representative of Northern blot analysis showing the expression profile of MIC-1/PDF mRNA in a panel of human prostate cancer cell lines. To normalize the amount of total RNA in each sample, filter was re-hybridized with GAPDH probe as an internal control. (B) The bar diagram represents the intensity of the MIC-1/ PDF specific signal adjusted to that of GAPDH for each tested sample. Error bars indicate standard error from the triplicate values.
D. Karan et al. / Biochemical and Biophysical Research Communications 305 (2003) 598–604
601
Expression of the MIC-1/PDF under different culture conditions
Fig. 3. Expression profile of MIC-1/PDF secretion in different cell lines of human prostate cancer. The cells were seeded at a density of 1 106 in T-25 flasks in regular culture medium for 2 days. The media were replaced and the cells were further incubated for 48 h. At the given time point, cells were harvested and conditioned media were collected. An equal amount of proteins from the conditioned media from each cell line was fractionated by 15% SDS–PAGE and transferred onto the PVDF membrane. The blot was hybridized with anti-MIC-1/PDF antibody (upper panel) and then stained with Coomassie blue (lower panel) to demonstrate equal loading of proteins in all lanes.
obtained by RT-PCR and Northern blot analysis (Fig. 3). Altogether, these experiments showed a dysregulated expression pattern of the MIC-1/PDF level with the progression of prostate cancer.
To further analyze the expression pattern of the MIC1/PDF protein, we used three different culture conditions [serum free, 5% charcoal treated-FBS (c-FBS), and 5% FBS culture medium]. In androgen-sensitive LNCaP-C33 cells, the secreted level of MIC-1/PDF protein was significantly higher in the conditioned medium obtained from the cells maintained with 5% FBS compared to the media from the cells containing 5% cFBS and serum free medium. On the other hand, in androgen-independent LNCaP-C81 cells, both the culture media containing 5% c-FBS and 5% FBS showed a higher expression of the secreted MIC-1/PDF protein as compared to the cells maintained in serum free medium, however, the secretion level was maximum in case of 5% FBS (Fig. 4). Distribution of the MIC-1/PDF protein in human tumor tissues The MIC-1/PDF expression was studied in 15 specimens of human prostatic adenocarcinomas by IHC. The staining pattern and intensity was different in the areas
Table 1 Level of expression of the MIC-1/PDF antigen by immunoperoxidase staining in benign and adenocarcinomatous prostatic tissues Fig. 4. Effect of culture conditions on the expression levels of the secreted MIC-1/PDF protein in androgen-sensitive (LNCaP-C33) and -independent (LNCaP-C81) cells. The cells were seeded for 2 days as detailed in Fig. 3. Cells were then washed and fed with fresh media, serum free, 5% c-FBS or 5% FBS. After 48 h, cells were harvested and an equal amount of proteins from supernatant was fractionated on 15% SDS–PAGE and transferred onto the PVDF membrane for blotting with anti-MIC-1/PDF antibody.
Type of lesion
n
Benign prostatic glandular tissue Prostatic adenocarcinoma
13
Staining score (m SE) 9 2.38 0.25 > =
15
> 7.33 0.88 ;
t test
p < 0:05
n, number of specimens; m, mean; and SE, standard error. * Significant level (<0.05) with t test.
Fig. 5. A representative immunohistochemical analysis of the paraffin-embedded tissue sections of the benign prostatic glandular tissue (A) and prostatic adenocarcinoma (B).
602
D. Karan et al. / Biochemical and Biophysical Research Communications 305 (2003) 598–604
with benign prostatic glandular tissue and prostatic adenocarcinomas (Table 1). For simplicity, we calculated the intensity score by multiplying the intensity of staining with the extent of staining for a particular lesion. Out of 15 specimens, 13 showed low to moderate immunoreactivity in the areas of benign prostatic glandular tissue with an overall intensity score of 2.38 0.25. We could not find any benign tissue area in two samples. On the other hand, a strong immunoreactivity was detected for the MIC-1/PDF antigen in prostate cancer areas in all the specimens with an intensity score of 7.33 0.88. Overall, immunoreactivity for the MIC-1/PDF antigen was significantly higher (p < 0:05) in prostatic adenocarcinomas as compared to benign prostatic glandular tissue (Fig. 5). Since prostate cancer is multifocal and heterogeneous, there was a variability in the intensity of staining.
Discussion Development of an androgen-independent phenotype from androgen-sensitive cancer is a major challenge in the treatment of prostate cancer. The treatment and diagnosis may be improved significantly by the identification and characterization of the molecules that are regulated differentially during the development of the metastatic hormone-refractory phenotype. Recently, by using a step-wise progression of in vitro LNCaP cell model for prostate cancer that exhibits the conditions analogous to be seen in the patients, we identified through microarray gene filter that the MIC-1/PDF mRNA transcript is highly expressed in androgen-independent LNCaP-C81 cells [7]. The MIC-1/PDF gene, located on chromosomal position 19p13.1, is a divergent member of the TGF-b superfamily. It shares only a 25% overall sequence identity to the TGF-b family members, but shows the typical characteristics of the TGF-b superfamily including, signal peptide, consensus cleavage signal for the processing of mature protein, and a cysteine knot of seven residues at the carboxyl terminal. It is synthesized as a 62-kDa intracellular protein, but secreted as a 25kDa disulfide-linked dimeric protein after cleavage [22]. We investigated the expression of MIC-1/PDF both at the mRNA as well as the protein level in a panel of prostatic tumor cell lines. The MIC-1/PDF gene expression was low to non-detectable in the areas of benign prostatic glandular tissue and the transformed epithelial cells of the normal prostate (immortal PZHPV-7 cells), whereas it was overexpressed in tumor areas and in androgen-independent cells (LNCaP-C81 and LNCaP-Ln3). Further, low to undetectable levels of MIC-1/PDF in PC3 and DU145 cells compared to LNCaP cells might be associated with the differential expression of androgen receptor (AR). It is known that
the LNCaP cells are positive for AR, whereas the PC3 cells demonstrated a very low level of AR and DU 145 cells have no detectable AR [23,24]. These results indicated that at the hormone-refractory stage of prostate tumor AR might contribute to the dysregulation of MIC-1/PDF. Hence, the elevated level of mRNA as well as protein in a metastatic variant cell line suggests that the MIC-1/PDF gene may play a crucial role in the development of androgen-independent phenotype of human prostate tumor. However, further experimental studies are required to elucidate the pathological significance of higher secretion of the MIC-1/PDF protein in association with tumor development as well as its dysregulation in conjunction with androgen receptor. Androgens are critical for the normal development and differentiation of the prostate gland [3]. Most of the biological effects of androgens are mediated via AR, a nuclear transcription factor, upon ligand binding influence the transcription of androgen-responsive genes [25–27]. It is interesting to note that the treatment of androgen-sensitive (C33) and -independent (C81) cells with different culture conditions [serum free, steroid reduced 5% FBS (c-FBS), and 5% FBS in RPMI 1640 phenol red-free medium] demonstrated the regulation of MIC-1/PDF protein secretion by serum factors, including androgens. Feeding the C33 cells with 5% FBS exhibited higher secretion of the MIC-1/PDF protein compared to the cells treated with serum-free media or with 5% c-FBS, suggesting its regulation by steroids including androgens at the androgen-sensitive stage of the tumor. On the other hand, induction of the MIC-1/ PDF protein in C81 cells either treated with 5% FBS or 5% c-FBS indicated that its secretion might be regulated by growth factors and/or cytokines at the advanced stage of prostate cancer. Therefore, it would be of further interest to analyze the hormonal regulation of MIC-1/PDF in human prostate tumor to better understand its involvement with the hormone-refractory phenotype. A study in rat model has revealed that this gene is highly regulated by androgens, as orchitectomy resulted in a remarkable decrease in prostate size along with decrease in MIC-1/PDF expression level compared to those of control animals [12]. Its prostatic expression and regulation suggest that this gene may play a decisive role for the androgen-driven differentiation of prostatic tissue. A significantly higher expression of MIC-1/PDF in prostate tumor specimens indicated its involvement with the advancement of the disease. However, it was not possible to correlate the MIC-1/PDF immunostaining pattern with the tumor grade (Gleason grade) because (1) most of the specimens were of high grade (7–9 Gleason score) and (2) the number of specimens was too small for such a correlative analysis. We observed an enhanced pattern of MIC-1/PDF expression, however, with the progression of prostate tumor. Furthermore, a
D. Karan et al. / Biochemical and Biophysical Research Communications 305 (2003) 598–604
study on paired samples (four paired samples) from human prostate cancer specimens, using RT-PCR analysis, showed higher expression of the MIC-1/PDF mRNA in cancerous tissues compared to normal samples (data not shown). In conclusion, MIC-1/PDF showed a higher expression with the progression of prostate tumor both in the archival specimens and in a progressive in vitro LNCaP cell model. Our data suggest that MIC-1/PDF could be of specific importance in prostate cancer due to (1) its association with the progression of prostate tumor, (2) its regulation by steroids, including androgens at the androgen-sensitive stage and by cytokines and/or growth factors at the advanced stage correlating with the conditions in the cancer patients, and (3) its dysregulation at the hormone-refractory stage, following the expression pattern of androgen receptor. In addition, MIC-1/PDF has structural motifs similar to a TGF-b superfamily member that supports the hypothesis for its role in tumorigenesis. TGF-b is known to suppress immune surveillance, facilitate tumor invasion, and promote the development of metastases when abundantly expressed in tumors of epithelial origin [28]. Further studies are needed, however, to confirm its biological significance in association with carcinogenesis and tumor development, and whether MIC-1/PDF can serve as an useful biomarker in an androgen-independent phenotype of prostate cancer.
Acknowledgments This work was supported, in part, by grants from the National Cancer Institute (CA 78590, CA 88184), the Nebraska Health Department/Eppley Cancer Center LB 595, the Cattleman Ball Association, and the Nebraska Research Initiative Program. We acknowledge the NCI Cancer Center Support Grant (P30 CA36727) to UNMC for tumor tissues and CHTN Western Division, Case Western Reserve University, Cleveland, OH, for providing prostatic tissues. We also thank Dr. Fidler, TX, for the LNCaP-Ln3 cells, Mr. Erik Moore and Ms. Fen-Fen Lin for their technical assistance.
References [1] A. Jemal, A. Thomas, T. Murray, M. Thun, Cancer statistics 2002, CA Cancer J. Clin. 52 (2002) 23–47. [2] M.D. Sadar, M. Hussain, N. Bruchovsky, Prostate cancer: molecular biology of early progression to androgen independence, Endocr. Relat. Cancer 6 (1999) 487–502. [3] G.R. Cunha, A.A. Donjacour, P.S. Cooke, S. Mee, R.M. Bigsby, S.J. Higgins, Y. Sugimura, The endocrinology and developmental biology of the prostate, Endocr. Rev. 8 (1987) 338–362. [4] R.F. Gittes, Carcinoma of the prostate, N. Engl. J. Med. 324 (1991) 236–245. [5] H. Qi, Y. Labrie, J. Grenier, A. Fournier, C. Fillion, C. Labrie, Androgens induce expression of SPAK, a STE20/SPS1-related kinase, in LNCaP human prostate cancer cells, Mol. Cell Endocrinol. 182 (2001) 181–192.
603
[6] D. Karan, B.M. Schmied, B.J. Dave, U.A. Wittel, M.F. Lin, S.K. Batra, Decreased androgen-responsive growth of human prostate cancer is associated with increased genetic alterations, Clin. Cancer Res. 7 (2001) 3472–3480. [7] D. Karan, D.L. Kelly, A. Rizzino, M.F. Lin, S.K. Batra, Expression profile of differentially-regulated genes during progression of androgen-independent growth in human prostate cancer cells, Carcinogenesis 23 (2002) 967–975. [8] D. Karan, M.F. Lin, S.L. Johansson, S.K. Batra, Current status of the molecular genetics of human prostatic adenocarcinomas, Int. J. Cancer 103 (2003) 285–293. [9] T. Igawa, F.F. Lin, M.S. Lee, D. Karan, S.K. Batra, M.F. Lin, Establishment and characterization of androgen-independent human prostate cancer LNCaP cell model, Prostate 50 (2002) 222–235. [10] M.F. Lin, T.C. Meng, P.S. Rao, C. Chang, A.H. Schonthal, F.F. Lin, Expression of human prostatic acid phosphatase correlates with androgen-stimulated cell proliferation in prostate cancer cell lines, J. Biol. Chem. 273 (1998) 5939–5947. [11] M.R. Bootcov, A.R. Bauskin, S.M. Valenzuela, A.G. Moore, M. Bansal, X.Y. He, H.P. Zhang, M. Donnellan, S. Mahler, K. Pryor, B.J. Walsh, R.C. Nicholson, W.D. Fairlie, S.B. Por, J.M. Robbins, S.N. Breit, MIC-1, a novel macrophage inhibitory cytokine, is a divergent member of the TGF-b superfamily, Proc. Natl. Acad. Sci. USA 94 (1997) 11514–11519. [12] V.M. Paralkar, A.L. Vail, W.A. Grasser, T.A. Brown, H. Xu, S. Vukicevic, H.Z. Ke, H. Qi, T.A. Owen, D.D. Thompson, Cloning and characterization of a novel member of the transforming growth factor-b/bone morphogenetic protein family, J. Biol. Chem. 273 (1998) 13760–13767. [13] J.B. Welsh, L.M. Sapinoso, S.G. Kern, D.A. Brown, T. Liu, A.R. Bauskin, R.L. Ward, N.J. Hawkins, D.I. Quinn, P.J. Russell, R.L. Sutherland, S.N. Breit, C.A. Moskaluk, H.F. Frierson Jr., G.M. Hampton, Large-scale delineation of secreted protein biomarkers overexpressed in cancer tissue and serum, Proc. Natl. Acad. Sci. USA 100 (2003) 3410–3415. [14] P. Buckhaults, C. Rago, B. St Croix, K.E. Romans, S. Saha, L. Zhang, B. Vogelstein, K.W. Kinzler, Secreted and cell surface genes expressed in benign and malignant colorectal tumors, Cancer Res. 61 (2001) 6996–7001. [15] A.G. Moore, D.A. Brown, W.D. Fairlie, A.R. Bauskin, P.K. Brown, M.L. Munier, P.K. Russell, L.A. Salamonsen, E.M. Wallace, S.N. Breit, The transforming growth factor-b superfamily cytokine macrophage inhibitory cytokine-1 is present in high concentrations in the serum of pregnant women, J. Clin. Endocrinol. Metab. 85 (2000) 4781–4788. [16] D.A. Brown, S.N. Breit, J. Buring, W.D. Fairlie, A.R. Bauskin, T. Liu, P.M. Ridker, Concentration in plasma of macrophage inhibitory cytokine-1 and risk of cardiovascular events in women: a nested case-control study, Lancet 359 (2002) 2159–2163. [17] R. Thomas, L.D. True, P.H. Lange, R.L. Vessella, Placental bone morphogenetic protein (PLAB) gene expression in normal, premalignant and malignant human prostate: relation to tumor development and progression, Int. J. Cancer 93 (2001) 47–52. [18] J.B. Welsh, L.M. Sapinoso, A.I. Su, S.G. Kern, J. Wang-Rodriguez, C.A. Moskaluk, H.F. Frierson Jr., G.M. Hampton, Analysis of gene expression identifies candidate markers and pharmacological targets in prostate cancer, Cancer Res. 61 (2001) 5974–5978. [19] C.A. Pettaway, S. Pathak, G. Greene, E. Ramirez, M.R. Wilson, J.J. Killion, I.J. Fidler, Selection of highly metastatic variants of different human prostatic carcinomas using orthotopic implantation in nude mice, Clin. Cancer Res. 2 (1996) 1627–1636. [20] I.D. Fleming, J.S. Cooper, D.E. Henson, R.V.P. Hutter, B.J. Kennedy, G.P. Murphy, B. OÕSullivan, L.H. Sobin, J.W. Yarbro, in: AJCC Cancer Staining Manual, fifth edition, Lippincot Williams and Wilkins, Philadelphia, 1997, pp. 1–294. [21] S.K. Batra, H.F. Kern, A.J. Worlock, R.S. Metzgar, M.A. Hollingsworth, Transfection of the human Muc 1 mucin gene into
604
D. Karan et al. / Biochemical and Biophysical Research Communications 305 (2003) 598–604
a poorly differentiated human pancreatic tumor cell line, Panc1: integration, expression and ultrastructural changes, J. Cell Sci. 100 (1991) 841–849. [22] W.D. Fairlie, A.G. Moore, A.R. Bauskin, P.K. Russell, H.P. Zhang, S.N. Breit, MIC-1 is a novel TGF-rbeta superfamily cytokine associated with macrophage activation, J. Leukoc. Biol. 65 (1999) 2–5. [23] R. Garcia-Arenas, F.F. Lin, D. Lin, L.P. Jin, C.C. Shih, C. Chang, M.F. Lin, The expression of prostatic acid phosphatase is transcriptionally regulated in human prostate carcinoma cells, Mol. Cell Endocrinol. 111 (1995) 29–37. [24] D.L. Lin, M.C. Whitney, Z. Yao, E.T. Keller, Interleukin-6 induces androgen responsiveness in prostate cancer cells through
[25]
[26]
[27]
[28]
up-regulation of androgen receptor expression, Clin. Cancer Res. 7 (2001) 1773–1781. Z. Culig, H. Klocker, G. Bartsch, A. Hobisch, Androgen receptors in prostate cancer, Endocr. Relat. Cancer 9 (2002) 155–170. B.J. Feldman, D. Feldman, The development of androgenindependent prostate cancer, Nat. Rev. Cancer 1 (2001) 34–45. M.E. Grossmann, H. Huang, D.J. Tindall, Androgen receptor signaling in androgen-refractory prostate cancer, J. Natl. Cancer Inst. 93 (2001) 1687–1697. E.R. Barrack, TGF b in prostate cancer: a growth inhibitor that can enhance tumorigenicity, Prostate 31 (1997) 61–70.