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Clinical Cancer Research 13, 1979, April 1, 2007. doi: 10.1158/1078-0432.CCR-06-1156
© 2007 American Association for Cancer Research
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Human Cancer Biology

ß-2-Microglobulin Is an Androgen-Regulated Secreted Protein Elevated in Serum of Patients with Advanced Prostate Cancer

Mitchell Gross, Irina Top, Isett Laux, Jonathan Katz, John Curran, Charles Tindell and David Agus

Authors' Affiliation: Louis Warschaw Prostate Cancer Center, Cedars-Sinai Medical Center, Los Angeles, California

Requests for reprints: Mitchell Gross, Louis Warschaw Prostate Cancer Center, Cedars-Sinai Medical Center, 8631 West Third Street, Suite 1001E, Los Angeles, CA 90048. Phone: 310-423-7600; Fax: 310-423-1998; E-mail: mitchell.gross{at}cshs.org.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: A better understanding of secreted proteins may lead to the discovery of new biomarkers, which, along with prostate-specific antigen (PSA), may be useful in the diagnosis and treatment of prostate cancer patients.

Experimental Design: Conditioned medium was collected from LNCaP cells following stimulation with methyltrienolone (R1881), 17ß-estradiol (estradiol), or interleukin-6 and analyzed for differential protein expression with surface-enhanced laser desorption/ionization-time of flight mass spectrometry. Quantitative reverse transcription-PCR, immunoblots, and ELISA were used to measure ß-2-microglobulin (B2M) message and protein levels in cells, conditioned medium, and serum.

Results: Surface-enhanced laser desorption/ionization-time of flight revealed that many peaks were induced or repressed following stimulation with R1881 or estradiol. A peak of interest centered at 11.8 kDa was chosen for additional analysis. Immunodepletion identified the peak of interest as B2M. Reverse transcription-PCR and immunoblots confirmed that PSA and B2M were induced by R1881. However, unlike PSA, B2M was not increased on stimulation with estradiol or interleukin-6. Human B2M is identified in the serum of mice bearing human prostate cancer xenograft. B2M is expressed in human prostate cancer cell lines and tissues. Serum B2M levels are elevated in patients with metastatic, androgen-independent prostate cancer.

Conclusions: B2M is a secreted protein expressed in prostate cancer, which is more specific for androgen stimulation than PSA under the conditions tested. Additional studies are warranted to explore if B2M is as useful marker for prostate cancer. Identification of proteins secreted from cancer cells in preclinical models may be a useful strategy for biomarker discovery.

Most serum biomarkers routinely used in the diagnosis and treatment of cancer are cell surface or secreted proteins expressed by both benign and malignant tissue. As these proteins are generally found at low abundance, highly sensitive tests (such as ELISAs) are required to measure biomarker levels in peripheral blood. However, the limited sensitivity and specificity of these tests often restricts their clinical utility. For example, prostate-specific antigen (PSA) is an androgen-regulated secreted protein that is expressed by both normal and malignant prostate epithelial cells, which is widely used as a serum marker for prostate cancer (1). Occult prostate cancers may be found in ~25% of patients with PSA levels in the generally accepted reference range (≤4 ng/mL; ref. 2). Most, but not all, patients with metastatic prostate cancer show elevated levels of PSA, which is often used as a marker to guide treatment decisions. However, the validity of PSA as a surrogate to predict overall survival in advanced prostate cancer remains controversial (3, 4). Therefore, additional biomarkers may be useful in the diagnosis and treatment of patients with prostate cancer.

Serum-based protein profiling with mass spectrometry (MS) is a promising technology to improve the diagnosis and treatment for cancer patients (5). It is generally believed that changes in serum proteins reflect the complex interplay between malignant cells and host tissues and that protein profiles may elucidate molecular processes that will improve the care of patients with cancer. Surface-enhanced laser desorption/ionization-time of flight MS (SELDI-TOF MS) was one of the first MS-based profiling technologies applied to study serum from patients with cancer (6–8). SELDI-TOF uses substrate-coated chips to differentially retain species from complex protein mixtures based on chromatographic properties, such as hydrophobicity and binding to anionic, cationic, or metal affinity surfaces. The retained species are then analyzed via TOF MS. Although initial experiments have confirmed the reproducibility of SELDI-TOF, a major pitfall of this approach is the relatively low resolution, as a typical spectrum contains approximately 100 to 500 reproducible features (9). Technological improvements in bioinformatics and MS-based protein profiling may produce spectra with several orders of magnitude greater resolution (5). Another common criticism of SELDI-TOF–based approaches for serum diagnostics is that the "blind" determination of mass spectral features in blood is most likely to observe nonspecific, and possibly nonreproducible, secondary phenomenon than direct alterations induced by cancer cells (10). Direct analysis of secreted proteins from cancer cells may be a more productive approach to identify cancer biomarkers that can be observed in serum.

We explored the utility of low-resolution profiling of secreted species from cancer cells as a strategy for biomarker discovery. LNCaP cells are a well-established human prostate cancer cell line that exhibits androgen-sensitive growth and PSA secretion. SELDI-TOF MS was used to compare protein profiles in medium from LNCaP cells stimulated with androgen, estrogen, or interleukin-6 (IL-6). In this way, we sought to determine a profile of androgen-regulated proteins following both specific (androgen) and relatively nonspecific stimuli (estrogen and IL-6). Analysis of the MS data revealed that several peaks were differentially regulated depending on the stimulation conditions. Particular attention focused on an androgen-regulated peak of interest (POI) at 11.8 kDa. In contrast with PSA, the POI was specifically stimulated by androgen but not by estrogen or IL-6. We considered that the POI was ß-2-microglobulin (B2M), which was confirmed by immunodepletion studies. We show that B2M transcription and protein secretion were specifically stimulated by androgen, but not by 17ß-estradiol (estradiol) or IL-6, and confirm that B2M levels increase in conditioned medium following androgen stimulation of both LNCaP and 22RV1 androgen-responsive prostate cancer cell lines. Next, we explored the potential clinical relevance of these observations by examining B2M expression in prostate cancer. B2M was expressed in multiple cell lines derived from patients with prostate cancer, and human B2M was found in serum of mice bearing human prostate cancer xenografts. Immunohistochemistry showed that B2M is highly expressed in prostate acinar cells and suggests increased expression in malignant over nonmalignant cells. Lastly, we found serum B2M levels significantly elevated in patients with advanced prostate cancer. This work suggests that B2M may be explored as a progression marker in prostate cancer. In addition, we show that protein profiling can be applied to preclinical models to yield biomarkers that may be directly tested in vivo.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and treatments. Early-passage LNCaP and 22RV1 cells were maintained in RPMI 1640 in the presence of 10% fetal bovine serum (FBS; Omega Scientific, Tarzana, CA), 100 units/mL penicillin, and 100 µg/mL streptomycin. PC-3 and DU145 cells were similarly maintained in DMEM supplemented with FBS and penicillin/streptomycin. LNCaP and 22RV1 cells were washed in PBS and plated at ~75% confluence in the presence of phenol red–free RPMI 1640 supplemented with 0.1% charcoal-stripped FBS. Cells were then stimulated with methyltrienolone (R1881; NEN/Perkin-Elmer, Boston, MA), estradiol (Sigma, St. Louis, MO), IL-6 (R&D Systems, Minneapolis, MN), or vehicle controls for 48 h at the indicated doses. A single lot of charcoal-stripped FBS (Omega Scientific) was used for all experiments. Medium was collected and debris was immediately removed by centrifugation at 2,000 x g for 5 min. Aliquots were stored and frozen at –20°C for subsequent analysis. Viable cells were monitored by trypan blue exclusion (Life Technologies/Invitrogen, Carlsbad, CA). Commercially available ELISA kits were used to measure PSA (American Qualex Laboratories, San Clemente, CA) and B2M (Bio-Quant Laboratories, San Diego, CA and R&D Systems).

SELDI-TOF MS analysis. Conditioned medium was spotted and analyzed using protein chip arrays basically according to the manufacturer's instructions (Ciphergen, Fremont, CA). Briefly, immobilized metal affinity chip arrays were charged with 0.1 mol/L cupric sulfate and then equilibrated with high-stringency binding buffer [0.1 mol/L sodium acetate (pH 4.5), 0.5 mol/L NaCl, 0.1% Triton X-100]. Samples from each experiment were normalized for protein concentration and spotted onto protein chip arrays (approximately 500-700 ng total protein) followed by sinapinic acid (3,5-dimethoxy-4-hydroxycinnamic acid; Ciphergen) as the matrix. The chips were analyzed with the Ciphergen ProteinChip Reader (model PBSII). Mass spectral data from each spot were acquired in the SELDI quantitation mode between 5 and 200 kDa. Data were analyzed using ProteinChip Software 3.2.1 (Ciphergen). Spectra from at least two independent samples for each set of stimulation conditions were obtained in duplicate or triplicate. Samples were normalized by total ion current and compared against vehicle-treated controls. The Biomarker Wizard application was used to detect and quantify MS features. Peak detection was done with a first-pass discrimination at a signal/noise ratio of 5 and a second-pass peak selection with a 0.3% mass window. Peak heights were compared for each group and presented as a fold change in the stimulated versus unstimulated, and a P value was assigned via nonparametric calculations according to the proprietary software. Significance was arbitrarily defined as P ≤ 0.10.

Immunoblotting and immunodepletion. Samples of conditioned medium or tissue culture lysates were standardized for protein concentration and resolved on a 4% to 20% gradient polyacrylamide-SDS gel (Bio-Rad Laboratories, Hercules, CA) and analyzed by standard immunoblotting procedures. Monoclonal anti-B2M and human-specific anti-B2M (goat) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). ELISA plates provided a convenient solid-phase support system for immunodepletion studies. Conditioned medium from LNCaP cells was incubated at room temperature for 2 h in wells coated with anti-B2M antibody or anti-PSA antibody (as a control). Immunoblot and SELDI-TOF analysis of the immunodepleted medium was done as described above.

Real-time quantitative PCR. RNA was isolated from LNCaP cultures using Trizol reagent (Life Technologies/Invitrogen) and reverse transcribed using Taqman One-Step RT-PCR Master Mix Reagents kit (Applied Biosystems, Foster City, CA). Primers and probes were designed to detect B2M (Genbank: NM004048) and PSA (Genbank: 001648). Specific oligonucleotides used were as follows: B2M, 5'-cgcgctactctctct-3' (forward), 5'-ggatgaaacccagacac-3' (reverse), and 5'-aggctatccagcgtactccaaagattcagg-3' (probe); PSA, 5'-acgtggattggtgctgca-3' (forward), 5'-tgggaatgcttctcgcactc-3' (reverse), and 5'-cctgtctcggattgtgggaggctg-3' (probe). At least one oligonucleotide in each set was designed to span an exon boundary to minimize potential signal from contaminating genomic DNA. A commercially available detector set for the glyceraldehyde-3-phosphate dehydrogenase gene (Applied Biosystems) was used as an endogenous control. All other oligonucleotides were purchased from Biosource (Camarillo, CA).

Real-time quantitative reverse transcription-PCRs were done in a 384-well plate with 100 ng of total RNA run in triplicate for each sample. Baseline and threshold values were set accordingly via the SDS 2.1 interface. Transcript abundance was quantified by the comparative CT method using glyceraldehyde-3-phosphate dehydrogenase as the calibrator.

Prostate tumor xenografts. CWR22R tumors were maintained by passage in 8- to 10-week-old nude athymic BALB/c mice obtained from Charles River Breeding Laboratories (Wilmington, MA) and maintained in pressurized ventilated cages at the Cedars-Sinai Medical Center vivarium (Los Angeles, CA) as described previously (11, 12). Female mice were injected with equal amount of tumor together with Matrigel (Collaborative Research, Bedford, MA) and RPMI 1640 (American Type Culture Collection, Manassas, VA) and allowed to grow for 18 days. Final tumor volume (mean ± SD) was 935 ± 325 mm3. Animals with and without tumors were sacrificed, and blood was obtained by intracardiac injection and placed into a serum-separating tube. The samples were allowed to clot for 30 min at room temperature and then spun at 2,000 x g. Serum was separated and stored at –80°C until analysis.

Histopathology. Prostate tissue obtained following transrectal ultrasound-guided biopsies for prostate cancer was selected to represent Gleason patterns 3, 4, and 5 and analyzed in a deidentified manner. Immunohistochemical analysis for B2M expression was done with polyclonal rabbit anti-human B2M (DakoCytomation, Carpinteria, CA) according to the manufacturer's instructions. Signals were visualized with DAKO EnVision/horseradish peroxidase kit and counterstained with hematoxylin.

Measurement of B2M in human serum. Serum samples from 42 patients were collected from patients with prostate cancer seen during routine clinical care at the Louis Warschaw Prostate Cancer Center at Cedars-Sinai Medical Center. Serum from 14 unaffected individuals (siblings, children, or spouses of patients) was similarly collected to be used as controls. The protocols for collection and analysis of the samples were approved by the Institutional Review Board as part of an ongoing repository of patient-derived samples and clinical information for prostate cancer patients. Samples were stored at –80°C until analysis. B2M in human serum was determined using the Quantikine IVD human B2M immunoassay (R&D Systems) according to the manufacturer's instruction.

Statistics. Unless otherwise specified, a two-sided, paired Student's t test was used to determine statistical significance. Fold change is expressed as mean ± 1 SD normalized to untreated controls for each set of conditions. A difference between groups of P < 0.05 was considered significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Minimum serum model of androgen-, estrogen-, and IL-6–dependent proliferation and PSA secretion. We hypothesized that proteomic analysis of medium obtained from prostate cancer cells may yield potential biomarkers. A potential pitfall of protein profiling with SELDI-TOF is low sensitivity caused by highly abundant, invariant proteins and the low dynamic range of the instrument (5). To minimize this problem, we sought to use a minimal serum model that would preserve steroid and cytokine growth responses in LNCaP cells. We found that 0.1% charcoal-stripped FBS supported physiologically relevant growth responses (Fig. 1A ). LNCaP cells were grown in the presence of 1 nmol/L R1881, 10 nmol/L estradiol, 10 ng/mL IL-6, or vehicle control for 48 h. Total viable cells were counted by trypan blue exclusion. Stimulation with 1 nmol/L R1881 results in ~2-fold increase in cell number after 48 h, a growth response that is comparable with the basal growth rate in complete medium (10% FBS). In comparison, 10 nmol/L estradiol and 10 ng/mL IL-6 both supported less of a proliferative response (~1.5-fold). Measurement of secreted PSA was used to confirm that the minimal serum conditions also supported the androgen-regulated protein secretion. PSA levels in medium from LNCaP cells grown in the presence of 1 nmol/L R1881, 10 nmol/L estradiol, 10 ng/mL IL-6, or vehicle control were analyzed by ELISA (Fig. 1B). The results show that PSA increased ~5-fold with R1881, 2.5-fold by estradiol, and 1.5-fold by IL-6. These results confirm that the minimal serum conditions supported androgen-, estrogen-, and IL-6–dependent growth and PSA secretion that approximates the basal growth conditions of these cells in vitro.


Figure 1
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  		Fig. 1. Cell growth and PSA secretion in response to R1881, estradiol, and IL-6 stimulation. A, LNCaP cells were plated at equal density in 0.1% charcoal-stripped FBS followed by stimulation for 48 h by 1 nmol/L R1881 (R), 10 nmol/L estradiol (E2), 10 ng/mL IL-6, or vehicle control. Maximal growth response of LNCaP cells in the presence of 10% FBS is shown for comparison. Total viable cells were counted in triplicate by trypan blue exclusion for each data point. Columns, fold change relative to untreated controls; bars, SD. B, PSA secretion in medium from cultures as described in (A) was measured in duplicate by ELISA. Columns, fold change of the treated samples over vehicle-treated controls; bars, SD. *, P < 0.05, for all comparisons versus unstimulated controls.

 
SELDI-TOF analysis of conditioned medium identifies a peak at 11.8 kDa, which is up-regulated by androgen but not by estradiol or IL-6. SELDI-TOF was used to identify MS features, which varied according to growth stimuli. Preliminary experiments optimized affinity chip conditions using cationic, hydrophobic, and metal-binding surfaces. The results show that maximal MS features appeared with the copper-coated immobilized metal affinity chip conditions, which were then used for subsequent experiments (Fig. 2A ; data not shown). LNCaP cells were plated at equal concentrations in the presence of minimal serum and stimulated with 1 nmol/L R1881, 10 nmol/L estradiol, 10 ng/mL IL-6, or vehicle control for 48 h. Medium was harvested and spotted in duplicate or triplicate onto copper-coated immobilized metal affinity chips. Samples were grouped according to stimulation condition and compared with vehicle-stimulated controls for each set of stimulation conditions. Approximately 100 to 300 features were identified and compared for each set of experimental conditions by the ProteinChip software. The fold change for each peak in the stimulated versus unstimulated medium was compared, and features with P ≤ 0.1 were considered significant for a preliminary analysis. The results show that R1881 stimulation resulted in a 2- to 3-fold elevation of six distinct peaks at 11.8, 12.0, 28.7, 28.8, 40.2, and 49.5 kDa (Fig. 2B). Estradiol led to 2- to 4-fold increases in six peaks at 28.9, 29.1, 48.7, 91.0, 93.3, and 114 kDa and 2-fold decreases in peaks at 11.9 and 14.2 kDa (Fig. 2C). No reproducible changes in SELDI-TOF profiles were observed with IL-6 stimulation (data not shown). We conclude that divergent growth stimuli produce distinct SELDI-TOF profiles in conditioned medium.


Figure 2
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  		Fig. 2. SELDI-TOF MS analysis of conditioned medium according to treatment conditions. A, representative SELDI-TOF spectra. Spectra were obtained in the quantitative mode for the range 5 to 200 kDa optimized for 5 to 60 kDa. Spectra were normalized for total ion current. Portion of the spectra covering the mass range of 5 to 20 kDa. Arrows, identify POI at 11.8 kDa. B and C, MS features differentially present in conditioned medium from unstimulated cultures (white columns) versus medium from cultures stimulated with 5 nmol/L R1881 (B, black columns) or 10 nmol/L estradiol (C, black columns). Samples from at least two independent experiments were pooled and analyzed using Biomarker Wizard 3.0. Columns, normalized peak intensities; bars, SD.

 
To validate the SELDI-TOF approach, a peak at 11.8 kDa was chosen for further analysis. The 11.8-kDa POI was the most abundant peak, which was constitutively present in all cultures and exhibited the greatest fold change on androgen stimulation (Fig. 2A). We specifically investigated how expression of the POI changed following each of the stimulation conditions tested. We found that the POI increased ~3.5-fold following stimulation with R1881, but no changes were observed following stimulation with estradiol or IL-6 (Fig. 3A ). Further experiments explored the dose-dependent changes in the POI in response to androgen stimulation. Supraphysiologic doses of androgen are known to inhibit the expression of many androgen-regulated genes, including PSA (13). We observed a biphasic dose response curve for the POI in relation to R1881 dose (Fig. 3B). The POI reached maximal levels between 1 and 5 nmol/L R1881 with subsequent inhibition seen between 10 and 100 nmol/L R1881. A similar dose response curve was observed for PSA secretion (data not shown). These results suggested that the POI was an androgen-responsive secreted protein.


Figure 3
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  		Fig. 3. Changes in the POI in response to R1881, estradiol, and IL-6. A, conditioned medium from LNCaP cultures following 48 h of stimulation with 5 nmol/L R1881, 10 nmol/L estradiol, and 10 ng/mL IL-6 was collected and analyzed by SELDI-TOF MS. The POI at 11.8 kDa was quantified. B, conditioned medium was collected following stimulation with 0.1 to 100 nmol/L R1881. *, P < 0.05, versus unstimulated sample group.

 
Identification of 11.8-kDa POI as B2M. Several studies have examined androgen-dependent transcription responses in LNCaP cells (14–19). Inspection of lists of androgen-dependent genes led us to hypothesize that the identity of the POI was B2M. B2M is expressed with a known molecular weight of 11.8 kDa in cell culture medium and was identified as an androgen-induced gene in three of six studies, which examined genome-wide expression patterns in LNCaP cells (14, 20). A recent study identified B2M among many other proteins induced by androgen from LNCaP cells using a quantitative proteomic approach (21). Moreover, the presence of a functional androgen response element in the promoter region of B2M was recently confirmed by chromatin immunoprecipitation (22).

We used a solid-phase immunodepletion approach to specifically test if the POI was B2M. We found that ELISA plates coated with monoclonal anti-B2M antibody provided a convenient substrate to specifically immunodeplete B2M from conditioned medium. Wells coated with anti-PSA antibody served as a negative control. Conditioned medium from LNCaP cells was incubated with wells coated with anti-B2M or anti-PSA antibodies (Fig. 4 ). We observed ~75% depletion of B2M after a single round of incubation in B2M-coated wells (Fig. 4A). The level of the POI was monitored by SELDI-TOF MS. We observed that the POI was decreased by ~75% by immunodepletion with B2M antibody (Fig. 4B). The results show that the peak at 11.8 kDa is specifically depleted by B2M antibody in parallel with the depletion in B2M protein. Therefore, we conclude that the POI at 11.8 kDa is B2M.


Figure 4
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  		Fig. 4. Immunodepletion shows POI is B2M. Conditioned medium was incubated in a plate coated with monoclonal antibody against B2M or PSA as a control. Medium was analyzed for B2M expression by immunoblotting (A) or SELDI-TOF MS quantitation of the 11.8-kDa POI (B). *, P < 0.05, immunodepleted sample versus control.

 
Regulation of B2M in LNCaP and 22RV1 cells. Identification of the POI allowed us to explore the androgen-specific regulation of B2M transcription and protein expression. Total RNA from LNCaP cells stimulated with R1881, estradiol, IL-6, or vehicle control was analyzed by quantitative reverse transcription-PCR. The results show that B2M message is up-regulated ~2.5-fold by androgen, whereas no significant change was noted for estradiol or IL-6 (Fig. 5A ). Further, we wanted to confirm the androgen-dependent secretion of B2M at the protein level in our system. Conditioned medium from LNCaP cells stimulated by R1881, estradiol, and IL-6 was analyzed for expression of B2M and PSA by immunoblot (Fig. 5B). The results show that B2M is specifically induced by R1881 but not by estradiol or IL-6. PSA expression was induced under all three conditions (see Fig. 1B).


Figure 5
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  		Fig. 5. B2M expression is specifically regulated by androgen and not by estradiol or IL-6. A, representative real-time reverse transcription-PCR analysis of B2M mRNA expression in LNCaP cells. The absolute B2M mRNA expression was normalized to glyceraldehyde-3-phosphate dehydrogenase. Columns, average of three measurements relative to expression in unstimulated control cells; bars, SD. B, bottom, expression of B2M in conditioned medium from LNCaP cells stimulated with 1 nmol/L R1881, 10 nmol/L estradiol, and 10 ng/mL IL-6 analyzed by immunoblotting; top, blots were stripped and analyzed for expression of PSA by immunoblotting. C, B2M levels measured by ELISA from LNCaP and 22RV1 cells stimulated with 1 nmol/L R1881. Fold change versus unstimulated controls is presented for each cell line. *, P < 0.05, versus unstimulated control group.

 
22RV1 cells express a mutant form of the androgen receptor and are weakly responsive to androgen (23). B2M levels were measured by ELISA following stimulation with R1881 in conditioned medium from LNCaP and 22RV1 cells (Fig. 5C). LNCaP cells exhibited a 2.5-fold increase and 22RV1 cells exhibited a 1.5-fold increase in B2M levels in this assay. This result confirms that B2M expression is androgen responsive in both the LNCaP and 22RV1 cells.

B2M expression in human prostate cancer models. We surveyed common human prostate cancer cell lines to determine relative expression of B2M. As shown in Fig. 6A , B2M is expressed in multiple prostate cancer cell lines. An increase in expression was noted for the two androgen-independent lines (PC-3 and DU145) versus androgen-sensitive lines (LNCaP and RV1) examined.


Figure 6
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  		Fig. 6. B2M expression in mice bearing a human prostate cancer xenograft. A, lysates obtained from prostate cancer cell lines were normalized for protein concentration, and expression of B2M was examined by immunoblotting. B, equal amounts of purified recombinant hB2M and mouse B2M (mB2M; 75 ng) were resolved and analyzed by immunoblot with human-specific and nonspecific (human/mouse cross-reactive) antibodies against B2M. C, equal volumes of sera from matched naive and tumor-bearing animals were analyzed by immunoblot with hB2M-specific antibody. D, B2M levels in serum of patients with localized (Local; n = 15), metastatic (Mets.) noncastrate (n = 5), and metastatic castrate (n = 17) prostate cancer were examined by ELISA. Level of B2M observed in serum from unaffected controls (n = 14). Points, mean values; bars, SD. *, P < 0.05, metastatic castrate versus control.

 
As B2M is highly expressed and secreted by prostate cancer cells in tissue culture, we sought to examine if a human-specific form of B2M (hB2M) can be found in serum from mice bearing human prostate cancer xenografts. The CWR22R xenograft is a commonly used model of androgen-independent prostate cancer (24). First, we showed that a commercially available antibody is specific for hB2M. Equal amounts of recombinant hB2M and mouse B2M (kindly provided by Magda Ivanova and David Eisenberg, University of California, Los Angeles, CA) were analyzed by immunoblot with human-specific and nonspecific (human/mouse cross-reactive) antibodies (Fig. 6B; ref. 25). The results show that anti-hB2M preferentially recognizes hB2M. Next, we examined if hB2M was present in mice bearing the CWR22R xenografts. Immunoblots of lysates and secreted proteins from CWR22R tumors showed that hB2M was expressed in this model (data not shown). Equal volumes of serum (2 µL) from mice bearing CWR22R xenografts or naive mice were analyzed by immunoblot with anti-hB2M (Fig. 6C). The results show that hB2M is present in sera from mice bearing the human CWR22R xenografts.

B2M is expressed in tissue and serum from prostate cancer patients. Following the identification of B2M as an androgen-regulated secreted protein, we sought to explore if B2M was expressed in serum and tissue in prostate cancer patients.

Serum levels of B2M were analyzed from patients and unaffected related controls (Fig. 6D). The sample set included patients with localized prostate cancer (n = 15), noncastrate metastatic (n = 5), castrate metastatic (immediately before cytotoxic chemotherapy; n = 17), and normal controls (n = 14). We observed B2M levels (mean ± SD) of 2.3 ± 0.9, 2.3 ± 0.6, and 2.7 ± 1.1 µg/mL in patients with localized, noncastrate metastatic, and castrate metastatic prostate cancer, respectively. In contrast, B2M was measured as 1.9 ± 0.6 µg/mL in serum obtained from spouses and family members of affected. In pairwise comparison, B2M level was found to be significantly elevated in patients with castrate metastatic prostate cancer versus controls (P < 0.05). These results show that B2M levels are significantly elevated in patients with metastatic prostate cancer progressive despite androgen deprivation therapy.

Immunohistochemistry was used to explore B2M expression in normal and malignant prostate tissue (Fig. 7 ). A collection of prostate biopsies was chosen to represent major histologic grades (Gleason patterns 3, 4, and 5) and normal glands and stained with a commercially available B2M antibody. We found a low level of B2M expression in the apical acinar cells of normal glands with a relative absence of expression in basal cells. Interestingly, we found increased expression of B2M in all grades of prostate cancer examined, including intense staining of individual infiltrating malignant cells in a Gleason 5 pattern. We conclude that B2M is expressed in benign and malignant prostate epithelial cells.


Figure 7
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  		Fig. 7. B2M expression in normal and malignant human prostate. A and B, representative images show moderate staining in normal luminal acinar cells with relative absence of staining in basal cells. Moderate cytoplasmic staining is noted in Gleason pattern 3 adenocarcinoma cells (C) increased relative to adjacent nonmalignant acinus (D). E and F, intense cytoplasmic staining is noted in adenocarcinoma Gleason patterns 4 and 5, including individual infiltrating cells.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
This article presents a proteomic-based approach that identifies B2M as an androgen-regulated protein directly secreted from prostate cancer cells. We compared profiles of secreted protein following stimulation by androgen, estradiol, and IL-6 in an androgen-sensitive prostate cancer model with SELDI-TOF MS. Comparison of the profiles revealed numerous MS features, which were reproducibly changed by R1881 and estradiol, whereas no significant changes were observed with IL-6. A POI at 11.8 kDa was chosen for further analysis. The POI was increased ~3.5-fold on stimulation with R1881 but not significantly changed on stimulation with estradiol or IL-6. We identified the POI as B2M by immunodepletion. Next, we explored the regulation of B2M in several prostate cancer models. In contrast with PSA, we show that B2M transcription was increased following stimulation with R1881 but not with estradiol or IL-6. We confirmed that B2M protein expression was also specifically up-regulated by androgen in both the LNCaP and 22RV1 models. Finally, we confirmed that B2M protein levels are increased in the sera of mice bearing prostate cancer xenografts and in tissue and sera of patients with prostate cancer.

These results are of particular interest toward the goal of discovering biomarkers for prostate cancer. Although PSA is the most widely used marker for the diagnosis and treatment of prostate cancer, it is highly expressed in both benign and malignant prostate epithelium. Therefore, elevation in serum PSA may occur from the growth of either benign or malignant prostate tissue. B2M has been used as a biomarker for other cancers, especially lymphoid malignancies, such as non–Hodgkin's lymphoma and multiple myeloma, consistent with the observation that the major source of serum B2M in normal patients is lymphatic tissue (26). As B2M is cleared by the kidneys, B2M levels also reflect renal function. In addition, as B2M may also be part of the acute-phase response, nonspecific elevation in B2M may occur as a result of other immune stimulation, such as acute viral infection (26). There is a single report of B2M as a biomarker for prostate cancer (20). These investigators observed increased urine B2M in patients with advanced prostate cancer associated with shortened overall survival in patients with bone metastatic disease. Our data support this general observation in that serum B2M levels are elevated in patients with metastatic prostate cancer. Taken together, these data suggest that B2M may be explored as a marker of immune activation and/or tumor expansion in patients with prostate cancer.

Of particular relevance to our observation are reports on the autocrine and paracrine role of B2M in epithelial malignancies. In tissue culture, B2M is mitogenic for prostate cancer cells and osteoblasts (27, 28). More recently, B2M has been identified as an autocrine factor that stimulates expression of bone-specific proteins in prostate cancer cells (29). Moreover, overexpression of B2M promotes rapid growth and inhibition of B2M promotes regression of prostate cancer xenografts.

Our results suggest that additional investigation in the role of B2M for more defined patients with prostate cancer warrants further investigation. Further, protein profiling may be used to find novel biomarkers secreted from cancer cells in preclinical models with direct biological relevance to patients.


   Acknowledgments
 
We thank Parag Mallick and Anjali Jain for helpful discussions and review of the manuscript; Laura Kelley, Robert Damoiseaxu, Kamy Missaghi, Isett Laux, and Charles Tindell for technical assistance; Magda Ivanova and David Eisenberg for providing reagents; and Kathy Kozak, Robin Farias-Eisner, Steven Wong, and Dennis Slamon for use of equipment.


   Footnotes
 
Grant support: Phase One Foundation (M. Gross and D. Agus) and NIH grant DK64379 (M. Gross).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 5/15/06; revised 11/20/06; accepted 1/ 8/07.


   References
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 Abstract
 Materials and Methods
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