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Differential Expression of S100A2 and S100A4 During Progression of Human Prostate Adenocarcinoma

Sanjay Gupta, Tajamul Hussain, Gregory T. MacLennan, Pingfu Fu, Jigar Patel, Hasan Mukhtar

From the Departments of Urology, Pathology, Epidemiology, and Biostatistics, Case Western Reserve University and The Research Institute of University Hospitals of Cleveland, Cleveland, OH; Department of Dermatology, University of Wisconsin-Madison, Madison, WI.

Address reprint requests to Hasan Mukhtar, PhD, Department of Dermatology, University of Wisconsin, Medical School Center, Room B25, 1300 University Avenue, Madison, WI 53706; email: hmukhtar{at}wisc.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Purpose: To establish the clinical significance of calcium binding proteins S100A2 and S100A4 during progression of human prostate adenocarcinoma.

Patients and Methods: Expression pattern of S100A2 and S100A4 was determined in normal human prostate epithelial cells (NHPE); virally transformed prostate epithelial cells (PZ-HPV-7); several human prostate carcinoma cells (22Rv1, DU145, LNCaP, and PC3); tissue samples obtained during transuretheral prostatic resection from patients with benign prostate hyperplasia (BPH), prostatitis, and adenocarcinoma; and paraffin-embedded sections from pair-matched benign and cancer specimens of different tumor grade.

Results: High constitutive protein expression of S100A2 was observed in NHPE and PZ-HPV-7 cells, whereas its complete absence was observed in 22Rv1, DU145, LNCaP, and PC3 cells. Tissue samples of BPH and prostatitis exhibited higher mRNA and protein levels of S100A2 than low-grade cancer (Gleason score 6), whereas a complete loss was observed in high-grade cancer specimens (Gleason score > 6). Immunohistochemical analysis further confirmed high levels of S100A2 in benign tissues and a progressive loss with increasing tumor grade. The protein level of S100A4 was significantly higher in all carcinoma cells compared with NHPE and PZ-HPV-7 cells. The mRNA and protein level of S100A4 was significantly higher in high-grade cancer specimens compared with BPH, prostatitis, and low-grade cancer. The high levels of S100A4 observed in cancer tissue correlated with increasing tumor grade.

Conclusion: Loss of S100A2 and increased expression of S100A4 may be an important event during progression of prostate cancer in humans.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
CANCER OF the prostate gland (CaP) is one of the most common cancers in males in the United States.¹ Survival rate of these patients is poor, despite recent improvements in diagnostic and therapeutic techniques, because the majority of the patients have advanced disease at the time of diagnosis.^1–5 Posttreatment local and distant recurrence is still the major problem in the management of CaP.⁵ The genetic mechanisms of CaP development and progression are still largely unknown, and molecular data on CaP are relatively scarce and poorly understood.⁶ Predictive markers for CaP, especially those that are indicative of invasiveness of the disease, will be important for improving clinical management, outcome, and survival of these patients.

The S100 Ca²⁺-binding proteins have recently attracted considerable interest because of their differential expression in neoplastic compared to normal tissue, their involvement in metastatic processes, and the clustering organization of at least 17 S100 genes on human chromosome 1q21, a region frequently rearranged in several types of cancer.^7,8 S100 proteins have been shown to be involved in a variety of intracellular and extracellular functions including cell growth, cell-cell communication, energy metabolism, and intracellular signal transduction.^8,9 In recent years, S100A2 has become a gene of particular interest because of its cDNA coding identified in a search for novel tumor suppressor genes as a result of subtractive hybridization between normal and tumor-derived human mammary epithelial cells.^10,11 Studies have shown the regulation of transcriptional activation of S100A2 promoter by tumor suppressor gene p53.¹² Further studies have shown a marked downregulation of S100A2 in breast tumors compared with normal tissue.¹¹ This differential expression of S100A2 was not restricted to breast cancer but was also found in melanomas and in other types of cancer.^13–18 These data have led to the designation of S1000A2 as a tumor suppressor gene that might be generally downregulated in cells acquiring tumorigenic phenotype, indicating that this protein may play an important role in inhibiting cancer progression.

Another member of this family of proteins is S100A4, which has been associated with invasion and metastasis of cancer cells.¹⁹ S100A4 is frequently overexpressed in metastatic tumors as well as in normal cells with uninhibited movement, such as macrophages, neutrophils, and T-lymphocytes.²⁰ S100A4 has been shown to be upregulated in transformed cells²¹ and in some forms of cancer including breast,²² ovary,²³ thyroid,²⁴ lung,²⁵ esophageal squamous cell carcinoma,²⁶ gastric,²⁷ and colon.²⁸ Amplification or overexpression of S100A4 in breast cancer patients reflects an increased metastatic potential that is prognostically significant and is closely correlated with a fatal outcome.^22,29 S100A4 has been shown to be primarily expressed in 44% of foci of carcinoma within colon adenoma specimens and 94% of colon carcinoma specimens.²⁸ It is activated in 25% of cases of esophageal squamous cell carcinoma.²⁶ These studies have shown that S100A4 may play an important role in invasion and metastasis of some types of human cancers. In addition, an inverse relationship between S100A2 gene expression and cancer progression has been observed.^10–12 There are no reports concerning the clinical significance of S100A2 and S100A4 in human prostate adenocarcinoma.

In this study, using several normal and human prostate carcinoma cells, tissue obtained during transuretheral prostatic resection from patients with benign prostate hyperplasia (BPH), prostatitis, and adenocarcinoma, and a panel of 25 unique pair-matched specimens of benign and cancer tissue obtained from the same individuals, as well as additional unmatched benign and malignant prostate specimens, we examined the role of S100A2 and S100A4 expression in progression of human prostate adenocarcinoma. Here, for the first time we demonstrate the loss of S100A2 and increased expression of S100A4 protein during progression of CaP in humans. This study supports a role for the use of S100A2 and S100A4 protein as markers in the clinical management of CaP.

	PATIENTS AND METHODS

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Cell Lines
NHPE cells were obtained from Clonetics (Walkersville, MD), and PZ-HPV-7, 22Rv1, DU145, PC3, and LNCaP cells were obtained from ATCC (Manassas, VA). The cells were cultured in an appropriate culture medium in a humidified atmosphere of 95% air/5% CO₂ in an incubator.

Tissue Samples
Tissue samples were obtained from patients with BPH, prostatitis, and adenocarcinoma who underwent transuretheral resection of prostate at University Hospitals of Cleveland (Cleveland, OH). Immediately after resection, samples of about 10 mm in diameter were snap frozen in liquid nitrogen and stored at -80°C until further use. In addition, paraffin-embedded sections (4 µm) of 25 unique pair-matched specimens of benign and cancer tissue obtained from the same individuals, as well as 11 unmatched benign prostate specimens and 12 unmatched prostate cancers, were obtained from the Tissue Procurement Core Facility of the Comprehensive Cancer Center at the University Hospitals of Cleveland. The Gleason grade and score of adenocarcinoma specimens were assigned by an experienced surgical pathologist. These studies were approved by the institutional review board at Case Western Reserve University.

RNA Isolation and Reverse Transcription
Total RNA was isolated from benign and cancer tissue using the RNeasy Total RNA isolation kit (Qiagen, Valencia, CA) and was checked for its purity and concentration as previously described.³⁰ Two micrograms of total RNA was reverse transcribed using murine MLV-reverse transcriptase and oligo dT^12–18 primer from GIBCO-BRL (Life Technologies Inc., Gaithersburg, MD) for cDNA synthesis.

Semiquantitative PCR for S100A2 and S100A4
Two microliters of the RT product was subjected to polymerase chain reaction (PCR) in a 25-µL reaction mixture containing 10 mmol/L TrisHCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl₂, 0.2 mmol/L dNTP, 2.5 units of Amplitaq polymerase, and 40 pmol of each primer for S100A2 (forward, 5'-CCAAGAGGGCGACAAGTTCAA-3'; reverse, 5'-CATGGCAGGGAGTCAAGAGTTC-3') and S100A4 (forward, 5'-GAGGGTGACAAGTTCAAGCTC-3'; reverse, 5'-GGAGGGCCCCAGCTGGCAGA-3') obtained from GIBCO-BRL (Gaithersburg, MD). The cDNA was amplified with an initial denaturation at 94°C for 2 minutes followed by the sequential cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 65°C for 1 minute for 23 cycles, with final extension at 65°C for 5 minutes. A constitutively expressed GAPDH gene (forward, 5'-TGAAGGTCGGAGTCAAGCGATTTGGT-3'; reverse, 5'-CATGTGGGCCATGAGGTCCACCAC-3') was coamplified to confirm the equal loading of the RT product. The conditions for PCR cycles were same as in the case of S100A2 and S100A4. Ten-microliter aliquots of PCR products were electrophoresed on 1.5% agarose gel and visualized by ethidium bromide staining under UV transilluminator. Negative control of S100A2 and S100A4 RT-PCR that contained no reverse transcriptase showed no PCR products.

Western Blot Analysis
The total cell lysate was prepared in cold RIPA buffer (0.15 mmol/L NaCl, 0.05 mmol/L Tris[HCl] pH 7.3, 1% Triton X-100, 1% sodium deoxycholate), and protein concentration was determined by DC Bio-Rad assay as previously described.^31,32 For Western blot analysis, 25 µg protein was resolved over 4% - 20% polyacrylamide gels (Novex, Carlsbad, CA) and transferred to a nitrocellulose membrane. The blot was blocked in blocking buffer (5% nonfat dry milk/1% Tween 20; in 20 mmol/L TBS, pH 7.6) for 2 hours at room temperature, incubated with appropriate monoclonal antibody (human reactive S100A2 and S100A4, Transduction Laboratories, Lexington, KY, and Dako Corp, Carpenteria, CA) in blocking buffer for 2 hours at room temperature or overnight at 4°C followed by incubation with appropriate secondary antibody horseradish peroxidase conjugate (Amersham, Piscataway, NJ), and detected by ECL-chemiluminescence and autoradiography using XAR-5 film (Eastman Kodak, Rochester, NY).

Immunohistochemistry
Thick paraffin-embedded sections (4 µM) from benign and cancer tissue was deparaffinized, rehydrated, and immersed in 0.3% H₂O₂ for 20 minutes to block endogenous peroxidase activity. The sections were permeabilized in TNB-BB (100 mmol/L Tris [pH 7.5], 150 mmol/L NaCl, 0.5% blocking agent, 0.3% Triton-X, and 0.2% saponin) and incubated in primary antibody of S100A2 and S100A4, obtained from Dako at the dilution of 1:50 and 1:200, respectively, overnight at 4°C. Control sections were incubated with antisera in the presence of 10-fold excess of S100A2 and S100A4 protein or with isotype-matched IgG normal goat serum. After washing three times in Tris buffered-saline, sections were incubated for 2 hours at room temperature with appropriate biotinylated antibody, followed by incubation with peroxidase-conjugated streptavidin. Immunoreactive complexes were detected using 3,3'-diaminobenzidene. Slides were then counterstained in haemotoxylin, mounted in crystal mount, and cover slipped in 50:50 xylene/Permount as described previously.^31,32

Evaluation of S100 Immunostaining
The immunostained sections were examined independently by three of the authors (S.G., T.H., and G.T.M) using light microscopy.³² Positive staining for basal cells and smooth muscle cells in the benign specimens provided a positive control for S100 staining. The intensity of staining was graded semiquantitatively, and each specimen was assigned a score on a scale from 0 to 3 (0, negative), 1, weak; 2, moderate; and 3, strong. The immunoreactive score was determined by the percentage of positive cells and the staining intensity.

Statistical Analysis
All measures were summarized as means ± SD. Graphical summaries of the distributions of measure were made using boxplots. Measures were examined for the appropriateness of a normality assumption by density estimation (data not shown). Associations of categoric variables were evaluated using the Fisher’s exact test. Comparisons of means within groups were made using the Kruskal-Wallis test (the nonparametric version of ANOVA). Sample correlations were estimated using Spearman’s rank correlation.³³ All tests were two-sided and conducted at the alpha = 0.05 significance level. All analyses were performed with the SAS (Statistical Analysis System, version 6.12, Cary NC) software.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
As a first attempt toward identifying the expression of S100A2 and S100A4 in CaP progression, we performed immunoblot analysis for these proteins in several human prostate carcinoma cells and compared the results with those from normal and virally transformed human prostate epithelial cells. As shown in Fig 1A, the immunoblot analysis for S100A2 protein exhibited a high constitutive expression in NHPE and PZ-HPV-7 cells, whereas a complete absence was observed in human prostate carcinoma 22Rv1, DU145, LNCaP, and PC3 cells. In sharp contrast, the level of S100A4 protein was significantly higher in all carcinoma cells compared with NHPE and PZ-HPV-7 cells (Fig 1B).

View larger version (50K): [in this window] [in a new window]   Fig 1. Expression of (A) S100A2 and (B) S100A4 protein in normal human prostate epithelial cells (NHPE; lane 1), virally transformed prostate epithelial cells (PZ-HPV-7; lane 2), and human prostate carcinoma 22Rv1 (lane 3), DU145 (lane 4), LNCaP (lane 5), and PC3 (lane 6) cells.

View larger version (45K): [in this window] [in a new window]   Fig 2. Expression of S100A2 and S100A4 by (A) mRNA performing RT-PCR, and (B) protein by Western blot in tissue samples from patients with BPH (lanes 2 and 3), prostatitis (lanes 4 and 5), low-grade (lanes 6 and 7), and high-grade prostate cancer (lanes 8 and 9). Lane 1, positive control (nontumor prostate tissue), M-marker.

View larger version (27K): [in this window] [in a new window]   Fig 3. Boxplots for (A) S100A2 and (B) S100A4 based on staining pattern in benign and prostate cancer specimens. The intensity of staining was graded semiquantitatively by assigning a score to each tissue specimen. NS, nonsignificant. Brackets, Min-max; gray boxes, 25%–75%; black bars, median value.

View larger version (157K): [in this window] [in a new window]   Fig 4. Immunostaining for S100A2 in representative samples of benign tissue obtained after separating cancer tissue from prostate cancer patients. S100A2 expression was moderate to strong and was primarily expressed in basal cells (A, B) and smooth muscle cells (C, D) (arrows; magnification x200).

View larger version (159K): [in this window] [in a new window]   Fig 5. Immunostaining for S100A2 protein in representative samples from cancer tissue of different Gleason score obtained from prostate cancer patients. A progressive loss of S100A2 protein was observed in cancer specimens with increasing tumor grade (A–D; arrows; magnification x200).

The staining pattern of S100A2 protein was further compared in low-grade (Gleason score 6) and high-grade (Gleason score > 6) cancer specimens. The mean S100A2 expression in low-grade cancer tissue was 0.56 ± 0.70 (mean ± SD), whereas it was further diminished to 0.26 ± 0.45 (mean ± SD; P < .19) in high-grade cancer specimens, representing a loss during cancer progression. In low-grade cancer specimens, moderate staining was observed in two of 18 specimens, weak expression was observed in six of 18, and 10 of 18 samples did not exhibit staining for S100A2 protein. Similarly, five of 19 high-grade cancer specimens exhibited weak staining for S100A2 protein, whereas 14 of 19 samples did not exhibit staining for S100A2 protein (Table 1). These results indicate a decrease of S100A2 protein expression during progression of human prostate adenocarcinoma.

We next examined the S100A4 protein expression in benign and cancer specimens. The boxplots for S100A4 exhibited a significant difference in the protein expression between benign and cancer tissue (Fig 3B). The average scoring pattern for S100A4 in the benign tissue was 0.64 ± 0.72 (mean ± SD), whereas it was significantly higher 2.11 (± 0.84 [mean ± SD; P < .0001]) in cancer tissue, representing a 3.3-fold increase. The expression for S100A4 was faint in the cytoplasm of the benign specimens but moderate to strong in the cytoplasm of cancer tissue. The cells predominantly stained for S100A4 are lymphocyte and smooth muscle cells in the stroma (Fig 6). The staining pattern observed for S100A4 in benign specimen was high in three of 36 specimens, and weak in 16 of 36, and 17 of 36 samples did not exhibit staining for S100A4 protein (Table 1). In contrast, a progressive increase of S100A4 was observed in cancer specimens (Fig 7). In cancer tissue, high S100A4 staining pattern was observed in 28 of 37 specimens, weak pattern was observed in eight specimens of 37, and one of 37 samples did not exhibit any S100A4 protein (Table 1).

View larger version (165K): [in this window] [in a new window]   Fig 6. Immunostaining for S100A4 in representative samples of benign tissue obtained after separating tumor tissue from prostate cancer patients. Weak expression of S100A4 was observed, which was primarily expressed in smooth muscle cells in stromal region (A–D; arrows; magnification x200).

View larger version (164K): [in this window] [in a new window]   Fig 7. Immunostaining for S100A4 protein in representative samples from cancer tissue of different Gleason score obtained from prostate cancer patients. A progressive increase of S100A4 protein was observed in cancer specimens with increasing tumor grade (A–D; arrows; magnification x200).

The staining pattern of S100A4 protein was further compared in low-grade and high-grade cancer specimens. The mean S100A4 expression in low-grade cancer tissue was 1.94 ± 0.94 (mean ± SD), whereas it increased to 2.26 ± 0.73 (mean ± SD; P < .31) in high-grade cancer tissue, indicating an increase of this protein during cancer progression. In low-grade cancer specimens, moderate to strong staining was observed in 12 of 18 specimens, weak staining in five of 18, and one of 18 samples did not exhibit staining for S100A4 protein. Similarly, 16 of 19 high-grade cancer specimens exhibited moderate to strong staining for S100A4 protein, and weak staining was exhibited in three of 19 samples (Table 1). This indicates a progressive increase of S100A4 protein expression with increasing tumor grade in human prostate adenocarcinoma (Fig 7).

We next examined the association between the S100A2 and S100A4 protein expression in benign and tumor tissues. The Spearman correlation between S100A2 and S100A4 was found to be -0.54 (P < .0001) based on total specimen. For pair-matched specimens, the Spearmen correlation between the two proteins is -0.50 (P < .0003). This analysis indicates an inverse correlation between S100A2 and S100A4 protein expression during progression of human prostate adenocarcinoma.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
The expression levels of S100A2 and S100A4 genes, which it has been proposed are involved in tumor development and metastases for some cancers, were examined in normal and prostate carcinoma cell lines, tissue specimens involved by BPH, prostatitis, and benign and malignant prostate tissue. The strong expression of S100A2 in NHPE and PZ-HPV-7 cells and its complete absence in prostate carcinoma cells indicate that this gene product may be involved in suppression of human prostate adenocarcinoma. In contrast, S100A4 protein expression was significantly higher in prostate carcinoma cells, whereas low constitutive level was observed in NHPE and PZ-HPV-7 cells. These results provide for the first time evidence that loss of S100A2 and increased expression of S100A4 may be indicative of cancer progression in human prostate adenocarcinoma.

The existence of a family of S100 proteins has been demonstrated, and as many as 17 human S100 proteins have been identified.^7–9 The S100 proteins are structurally related and share a high degree of homology, particularly in the regions that code for the two EF-hand motifs, which create the Ca²⁺ binding domains.⁹ Knowledge of the biologic functions of S100 protein is limited, but some of them have been postulated to participate in signal transduction pathways regulating cell cycle progression and differentiation.^8,9 The precise functions of these genes are not known. The S100A2 gene (known as S100L and CaN19) encodes a 99–amino acid protein, which is located on chromosome 1q21, in a region that is frequently rearranged in a number of human cancers.^7,10 S100A2 is preferentially expressed in normal cells and is regulated during the cell cycle progression and by the tumor suppressor gene p53.¹² Loss of S100A2 has been shown to be associated with the development and progression of some human cancers.^13–18 Previous studies on cell lines and tissues found high S100A2 expression in normal breast,¹¹ uterine cervix,³⁴ skin,³⁵ oral mucosa,³⁶ and lung¹⁶ cells and low expression to complete loss in cancer cells. In recent years, S100A2 has also been proposed to be a class II tumor suppressor gene because of its loss of expression in a large number of tumors, which is believed to influence the regulation of genes that are important for normal growth and differentiation.^16,37 Studies have indicated that the loss of function of the S100A2 gene may be caused by selective hypermethylation with the promoter region that leads to inactivation of the most tumor suppressor genes.³⁸ Our studies indicate that diminished expression of S100A2 in human prostate adenocarcinoma is a potential predictor of prognosis. It is important to emphasize here that diminution of S100A2 expression was observed in prostate cancer but not in nonmalignant conditions of the prostate (BPH and prostatitis).

Another member of S100 family of proteins is S100A4 (known as p9Ka and MTS1), which acts in cell cycle progression, cell motility, and modulating intercellular adhesion and invasive properties.^19,20 The S100A4 gene has been linked to invasion and metastasis of cancer cells and has been shown to be upregulated in a number of human cancers.^20,39 Recent studies have shown that breast cancers expressing high levels of S100A4 have a significantly worse prognosis than breast cancers negative for S100A4.^22,29 Studies have shown that increased expression of S100A4 is associated with metastatic potential in some human cancers.^22,26–29 Another recent study has shown that overexpression of S100A4 may be sufficient to induce a metastatic phenotype in human mammary tumor cells.⁴⁰ Studies have also shown that S100A4 exerts its metastatic effect by influencing matrix metalloproteinases and their endogenous inhibitors, tissue inhibitors of metalloproteinase, in a way that facilitates extracellular matrix destruction.⁴¹ Recent reports indicate that in human smooth muscle cells, S100A4 interacts with the sarcoplasmic reticulum and with actin stress fibers in a Ca²⁺-dependent manner, resulting in the regulation of cell deformability and morphology.⁴² In cancer cells, S100A4 activates the nonmuscle myosin that participates in cellular motility.^19,20 Similar reports indicate that overexpression of S100A4 correlates with the in vitro invasive potential of glioma cells⁴³ and breast cancer,^22,29 conferring enhanced metastatic ability. In our study, moderate to strong expression of S100A4 protein was observed in cancer tissue and increased with tumor grade. Because of a limited number of specimens, our results could not establish a significant inverse correlation between S100A2 and S100A4 protein expression during prostate cancer progression. Additional studies with a large sample size of various tumor grades are required to validate these findings.

In conclusion, this study indicates that diminution of S100A2 and enhancement of S100A4 expression in prostate neoplasia is associated with increasing biologic aggressiveness, based on Gleason grading. Thus, the combined analysis of S100A2 and S100A4 in tissue biopsies may serve as a prognostic marker in cases of adenocarcinoma and may ultimately lead to monitoring therapeutic response during prostate cancer treatment protocols.

	NOTES

 
Supported by the funds from United States Public Health Services Grants RO1 CA78809, American Institute for Cancer Research Grants 00A030, and Department of Defense Grant DAMD 17-00-1-0527.

S.G. and T.H. contributed equally to this work.

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Submitted March 5, 2002; accepted September 4, 2002.

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